WO2024146553A1

WO2024146553A1 - Antibodies against cd47, method for preparing the same, and use thereof

Info

Publication number: WO2024146553A1
Application number: PCT/CN2024/070331
Authority: WO
Inventors: Qin Mei; Fagen HU; Yunying CHEN; Jijie Gu
Original assignee: Wuxi Biologics Shanghai Co Ltd; Wuxi Biologics Ireland Ltd
Current assignee: Wuxi Biologics Shanghai Co Ltd; Wuxi Biologics Ireland Ltd
Priority date: 2023-01-04
Filing date: 2024-01-03
Publication date: 2024-07-11
Anticipated expiration: 2025-07-04

Abstract

It provides anti-CD47 antibodies comprising a heavy chain variable region comprising CDR1-3 represented by SEQ ID NOs: 1-3, respectively. Preferably, the antibodies are single domain antibodies, more preferably humanized single domain antibodies. It also provides a nucleic acid molecule encoding the heavy chain variable region or a heavy chain of the antibodies, a vector comprising the nucleic acid molecule, a host cell comprising said nucleic acid molecule or vector, and a pharmaceutical composition comprising at least one of the antibodies for use in treatment of CD47-related diseases.

Description

ANTIBODIES AGAINST CD47, METHOD FOR PREPARING THE SAME, AND USE THEREOF

Priority Information

The present application claims the priority of PCT/CN2023/070471 filed on January 4, 2023, which is incorporated herein by reference.

Field of Invention

This application generally relates to antibodies. More specifically, the application relates to antibodies against CD47, a method for preparing the same, and the use thereof. Particularly, the antibody against CD47 is a single domain antibody.

Sequence listing

The instant application contains a sequence listing and is hereby incorporated by reference in its entirety.

Background of Invention

Cluster of differentiation 47 (CD47) , also known as integrin-associated protein (IAP) , is a ～50 kDa immunoglobulin superfamily membrane protein that is a ubiquitous cell surface glycoprotein expressed on most normal cell types. CD47 interacts with its ligand, signal regulatory protein alpha (SIRPα) expressed on macrophages, and then sending an anti-phagocytic or “don’ t eat me” signal to macrophages and therefore evading immune surveillance [1] . Analysis of various malignant tumors reveals that CD47 is overexpressed on acute myelocytic leukemia (AML) , non-Hodgkin’s lymphoma (NHL) , breast cancer, non-small-cell lung cancer (NSCC) and ovarian cancer cells, and the increased CD47 expression correlated with a worse clinical prognosis. These data indicated CD47 may serve as a new immune checkpoint for cancer therapy by blocking CD47-SIRPα interaction and switching-off “don’ t eat me” signal. Several anti-CD47 monoclonal antibodies (mAbs) have achieved effective macrophage involved phagocytosis against AML, NHL, breast cells, and ovarian cells. Besides, anti-CD47 mAb combined with approved antibodies (anti-tumor-associated antigen) or using dual-targeting bispecific antibodies have efficiently enhanced anti-tumor activity [2-4] . Based on these preclinical studies, more than seven anti-CD47 mAbs and three SIRPα fusion proteins or combo therapies are in active phase I or II clinical trials, covering human hematological malignancies and solid tumors.

However, no single-domain anti-CD47 antibody has been available so far. A single domain antibody (sdAb) , is an antibody containing a single heavy chain variable domain. Like IgG antibodies, it is capable of selectively binding specific antigens, but the molecular weight of single domain antibody is much smaller than IgG antibodies. The first single domain antibody is modified from the heavy chain antibody found in camelid, and the heavy chain antibody found in camelid is also referred to as VHH antibody. At present, most of the studies for single domain antibodies are based on the heavy chain variable domain.

Single domain antibodies may have many advantages. For example, they have high solubility, good thermal stability and tissue permeability, and some single domain antibodies are also resistant to the degradation of papain and the like due to the presence of intramolecular disulfide bonds. Furthermore, single domain antibodies can be produced in many kinds of host cells such as yeast, plant and mammalian cells, and expressed in high amounts, making them extremely cost effective. Single domain antibodies, by virtue of their numerous advantages, have made them promising in a variety of biotechnology and medical fields. Currently the first single domain antibody drug from Ablackynx has been marketed.

Since more and more research findings have showed targeting CD47-SIRPα signal axis could serve as a new immune checkpoint in cancer immunotherapy, and may have potent anti-tumors ability either with single or combination therapy making CD47 a universal target in multiple human malignant cancers, there is a need to develop anti-CD47 antibodies having good specificity and improved efficacy, especially single-domain anti-CD47 antibodies.

Summary of Invention

These and other objectives are provided for by the present invention which, in a broad sense, is directed to compounds, methods, compositions and articles of manufacture that provide antibodies with improved efficacy. The benefits provided by the present invention are broadly applicable in the field of antibody therapeutics and diagnostics and may be used in conjunction with antibodies that react with a variety of targets.

The present invention provides anti-CD47 antibodies, preferably single-domain antibodies, more preferably humanized single-domain antibodies, that specifically bind to human and monkey CD47. It also provides methods of cell panning and protein panning, nucleic acid molecules encoding the anti-CD47 antibodies, vectors and host cells used for the expression of anti-CD47 antibodies. The invention further provides the methods for validating the function of antibodies in vitro and in vivo. The antibodies of the invention provide a potent agent for treating multiple diseases and improving clinical prognosis.

The present invention aims to develop a humanized single-domain antibody with the right binding affinity to human and monkey CD47, which is capable of blocking CD47-SIRPαinteraction activity, inducing potent antibody dependent tumor cells phagocytosis in-vitro and in-vivo studies, and minimizing potential side effect on RBC system by reducing or without RBCs binding and hemagglutination activity.

In some aspects, the invention provides an isolated anti-CD47 antibody, or an antigen-binding portion thereof.

In some embodiments, the anti-CD47 antibody or the antigen-binding portion thereof comprises a heavy chain variable region comprising:

(a) CDR1 comprising SEQ ID NO: 1, or an amino acid sequence having at least 90%sequence identity with SEQ ID NO: 1, or an amino acid sequence differing from SEQ ID NO: 1 by an amino acid addition, deletion or substitution of not more than 1 amino acid;

(b) CDR2 comprising SEQ ID NO: 2, or an amino acid sequence having at least 85%or 90%sequence identity with SEQ ID NO: 2, or an amino acid sequence differing from SEQ ID NO: 2 by amino acid addition, deletion or substitution of not more than 2 amino acids, and

(c) CDR3 comprising SEQ ID NO: 3.

In some embodiments, the anti-CD47 antibody is a single domain antibody, preferably a humanized single domain antibody.

In some embodiments, the anti-CD47 antibody is a monoclonal antibody.

In some embodiments, the anti-CD47 antibody or the antigen-binding portion thereof comprises a heavy chain variable region comprising SEQ ID NO: 4 or 5, or an amino acid sequence having at least 80%, 85%, 90%, 95%or 99%sequence identity with SEQ ID NO: 4 or 5 while maintaining similar binding specificity as SEQ ID NO: 4 or 5, or an amino acid sequence differing from SEQ ID NO: 4 or 5 by amino acid addition, deletion or substitution of one or more (e.g., not more than 15, e.g., 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1) amino acids while maintaining similar binding specificity as SEQ ID NO: 4 or 5.

In some embodiments, the anti-CD47 antibody or the antigen-binding portion thereof further comprises a Fc region, preferably human IgG1 Fc region or human IgG4 Fc region. In some embodiments, the Fc region is human IgG4 Fc region with mutation (s) , such as S228P and/or YTE mutation.

In some embodiments, the anti-CD47 antibody or the antigen-binding portion thereof consists of an amino acid sequence represented by SEQ ID NO: 7 or 9, or an amino acid sequence having at least 80%, 85%, 90%, 95%or 99%sequence identity with SEQ ID NO: 7 or 9 while maintaining similar binding specificity as SEQ ID NO: 7 or 9, or an amino acid sequence differing from SEQ ID NO: 7 or 9 by amino acid addition, deletion or substitution of one or more (e.g., not more than 30, e.g., 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1) amino acids while maintaining similar binding specificity as SEQ ID NO: 7 or 9.

(a) a CDR1 comprising or consisting of SEQ ID NO: 1;

(b) a CDR2 comprising or consisting of SEQ ID NO: 2; and

(c) a CDR3 comprising or consisting of SEQ ID NO: 3.

In some embodiments, the invention comprises an isolated antibody or the antigen-binding portion thereof which competes binding for the same epitope with the anti-CD47 antibody or the antigen-binding portion thereof as defined above.

In some aspects, the invention is directed to a conjugate comprising the anti-CD47 antibody or the antigen-binding portion thereof as defined above and one or more moieties conjugated to the antibody or the antigen-binding portion thereof.

In some embodiments, the one or more moieties in the conjugate may be pharmacokinetic modifying moieties, purification moieties, moieties for targeted delivery, half-life improving moieties, or cytotoxic moieties. For example, Monomethyl auristatin E (MMAE) can be used as the moiety conjugated to the antibody. Those skilled in the art can select suitable moieties which will be conjugated to the anti-CD47 antibody according to the requirements in practice.

In some aspects, the invention is directed to an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the heavy chain variable region of the anti-CD47 antibody as disclosed herein.

In some embodiments, the invention is directed to an isolated nucleic acid molecule, comprising nucleic acid sequence encoding the heavy chain of the anti-CD47 antibody as disclosed herein.

In some aspects, the invention is directed to a vector comprising the nucleic acid molecule encoding the heavy chain variable region or the heavy chain of the anti-CD47 antibody or antigen-binding portion thereof as disclosed herein.

In some embodiments, the vector is an expression vector.

In some aspects, the invention is directed to a host cell comprising the isolated nucleic acid molecule or the recombinant vector as disclosed herein.

In some embodiments, the host cell may be selected from, but not limited to, bacterial cells, fungi cells, insect cells, plant cells or mammalian cells. For example, the bacterial cells may be E. coli cells or any other bacterial cells commonly used in the art, the fungi cells may be yeast cells, or any other fungi cells commonly used in the art.

In some aspects, the invention is directed to a pharmaceutical composition comprising at least one antibody or antigen-binding portion thereof as disclosed herein and a pharmaceutically acceptable carrier.

In some aspects, the invention is directed to a kit comprising a container which comprises an least one anti-CD47 antibody or antigen-binding portion thereof as disclosed herein, and an instruction.

In some embodiments, the kit is used for preventing or treating CD47-related diseases in a subject.

In some embodiments, the kit is used for diagnosing CD47-related diseases in a subject.

In some embodiments, the kit may further comprise other pharmaceutical formulations for combined therapy.

In some aspects, the invention is directed to a method for preparing an anti-CD47 antibody or antigen-binding portion thereof, comprising expressing the anti-CD47 antibody or antigen-binding portion thereof in the host cell as described herein, and isolating the anti-CD47 antibody or antigen-binding portion thereof from the host cell, e.g., a culture of the host cell.

In some aspects, the invention is directed to a method for preventing or treating CD47-related diseases in a subject, comprising administering the anti-47 antibody or antigen-binding portion thereof as disclosed herein or the pharmaceutical composition as disclosed herein to the subject.

In some embodiments, the CD47-related diseases are characterized by (over) expression of CD47 on diseased cells, and may be hematologic diseases or solid tumors, for example, but not limited to acute myelocytic leukemia (AML) , non-Hodgkin’s lymphoma (NHL) , breast cancer, non-small-cell lung cancer (NSCC) , ovarian cancer, myelodysplastic syndrome, brain cancer, squamous cell carcinoma of the head and neck, and advanced malignant tumors.

In some embodiments, the method further comprises administrating other pharmaceutical formulations or other therapies for combined therapy.

In some embodiments, the anti-47 antibody or antigen-binding portion thereof or the pharmaceutical composition as disclosed herein is combined with a chemotherapy or a radiotherapy.

In some aspects, the invention is directed to a method for diagnosing CD47-related diseases in a subject, comprising: measuring the amount of CD47 in a sample from the subjected by using the anti-CD47 antibody or antigen-binding portion thereof in the host cell as described herein.

In some embodiments, the method for diagnosing CD47-related diseases further comprises: comparing the measured amount of CD47 in the sample with a threshold value, wherein the threshold value represents an average amount of CD47 in healthy subjects.

In some aspects, the invention is directed to a method for improving clinical prognosis in a subject in need thereof, comprising administering the anti-47 antibody or antigen-binding portion thereof as disclosed herein or the pharmaceutical composition as disclosed herein to the subject.

In some aspects, the invention is directed to a method for increasing tumor cells phagocytosis in a subject, comprising administering the anti-47 antibody or antigen-binding portion thereof as disclosed herein or the pharmaceutical composition as disclosed herein to the subject.

In some embodiments, the invention is directed to a method for increasing tumor cells phagocytosis in vitro, comprising contacting the tumor cells with the anti-47 antibody or antigen-binding portion thereof as disclosed herein or the pharmaceutical composition as disclosed herein.

In some aspects, the invention is directed to a method for inhibiting growth of tumor cells in a subject, comprising administering an effective amount of the anti-47 antibody or antigen-binding portion thereof or the pharmaceutical composition as disclosed herein to the subject.

In some aspects, the invention is directed to a method for reducing tumor cell metastasis in a subject, comprising administering an effective amount of the anti-47 antibody or antigen-binding portion thereof or the pharmaceutical composition as disclosed herein to the subject.

In some embodiments, the subject is a mammal, preferably a human.

In some aspects, the invention is directed to the use of the anti-47 antibody or antigen-binding portion thereof as disclosed herein in manufacture of a medicament for preventing or treating CD47-related diseases.

In some aspects, the invention is directed to the use of the anti-47 antibody or antigen-binding portion thereof as disclosed herein in manufacture of a diagnostic agent for diagnosing CD47-related diseases in a subject.

In some aspects, the invention is directed to the use of the anti-47 antibody or antigen-binding portion thereof as disclosed herein in manufacture of a medicament for improving clinical prognosis in a subject.

In some aspects, the invention is directed to the use of the anti-47 antibody or antigen-binding portion thereof as disclosed herein in manufacture of a medicament for increasing tumor cells phagocytosis in a subject.

In some aspects, the invention is directed to the use of the anti-47 antibody or antigen-binding portion thereof as disclosed herein in manufacture of a medicament for inhibiting growth of tumor cells in a subject.

In some aspects, the invention is directed to the use of the anti-47 antibody or antigen-binding portion thereof as disclosed herein in manufacture of a medicament for reducing tumor cell metastasis in a subject.

In some aspects, the invention is directed to kits or devices and associated methods that employ the anti-47 antibody or antigen-binding portion thereof as disclosed herein, and pharmaceutical compositions as disclosed herein, which are useful for prevention and/or treatment of CD47-related diseases. To this end, the present invention preferably provides an article of manufacture useful for treating such CD47-related diseases, comprising a receptacle containing the anti-47 antibody or antigen-binding portion thereof as disclosed herein and instructional materials for using the anti-47 antibody or antigen-binding portion thereof as disclosed herein to treat, ameliorate or prevent CD47-related diseases or progression or recurrence thereof.

The foregoing is a summary and thus contains, by necessity, simplifications, generalizations, and omissions of detail; consequently, those skilled in the art will appreciate that the summary is illustrative only and is not intended to be in any way limiting. Other aspects, features, and advantages of the methods, compositions and/or devices and/or other subject matter described herein will become apparent in the teachings set forth herein. The summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. Further, the contents of all references, patents and published patent applications cited throughout this application are incorporated herein in entirety by reference.

Brief Description of Figures

Figure 1 shows the binding activity of the antibodies of the present invention to human CD47 (A) and cynomolgus monkey CD47 (B) , as measured by FACS.

Figure 2 shows the binding activity of the antibodies of the present invention to tumor cells, Raji cell (A) and MDA-MB-231 cell (B) , as measured by FACS.

Figure 3 shows the binding activity of the antibodies of the present invention to human RBCs, as measured by FACS.

Figure 4 shows the hemagglutination activity of the antibodies of the present invention on RBCs.

Figure 5 shows the blocking activity of the antibodies of the present invention on human CD47-expressing stable cells (A) and Jurkat tumor cells (B) .

Figure 6 shows the human CD47 binding kinetics curve of W3456-P5R1-1C1-z19-uIgG4V1, as measured by SPR.

Figures 7 shows the thermal stability of the antibodies W3456-P5R1-1C1-z19-uIgG1 (A) and W3456-P5R1-1C1-z19-uIgG4V1 (B) .

Figures 8 shows the stability of the antibodies W3456-P5R1-1C1-z19-uIgG4V1 (A) and W3456-P5R1-1C1-z19-uIgG1 (B) in human serum.

Figure 9 shows phagocytic activity of the antibodies against Raji cells (A) , A375 cells (B) , and human red blood cells (C) .

Figure 10 shows the ADCC (A) and CDC ability (B) against Raji tumor cells of the antibodies of the present application.

Figure 11 shows the body weight changes (A) and tumor volume changes (B) of CB-17 SCID mice of Raji B lymphatic cancer model during days 0-28 after treatment with the antibodies.

Figure 12 shows the body weight changes (A) and tumor volume changes (B) of CB-17 SCID mice of MDA-MB-231 human triple negative breast cancer model during days 0-32 after treatment with the antibodies.

Figure 13 shows the serum concentration of the antibodies W3456-P5R1-1C1-z19-uIgG4V1 and W3456-P5R1-1C1-z19-uIgG1 in cynomolgus monkey PK study.

Figure 14 shows hematology of RBC, HGB, RET and PLT values change after dosing.

Figure 15 shows T and B cells count were monitored after dosing.

Detailed Description of Invention

While the present invention may be embodied in many different forms, disclosed herein are specific illustrative embodiments thereof that exemplify the principles of the invention. It should be emphasized that the present invention is not limited to the specific embodiments illustrated. Moreover, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms “a” , “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a protein” includes a plurality of proteins; reference to “a cell” includes mixtures of cells, and the like. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “comprising” , as well as other forms, such as “comprises" and “comprised” , is not limiting. In addition, ranges provided in the specification and appended claims include both end points and all points between the end points.

Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Abbas et al., Cellular and Molecular Immunology, 6^th ed., W.B. Saunders Company (2010) ; Sambrook J. &Russell D. Molecular Cloning: A Laboratory Manual, 3^rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000) ; Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John &Sons, Inc. (2002) ; Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998) ; and Coligan et al., Short Protocols in Protein Science, Wiley, John &Sons, Inc. (2003) . The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Moreover, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Definitions

In order to better understand the invention, the definitions and explanations of the relevant terms are provided as follows.

The term “antibody” or “Ab” , as used herein, generally refers to a Y-shaped tetrameric protein comprising two heavy (H) and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Light chains of an antibody may be classified into κ and λ light chain. Heavy chains may be classified into μ, δ, γ, α and ε, which define isotypes of an antibody as IgM, IgD, IgG, IgA and IgE, respectively. In a light chain and a heavy chain, a variable region is linked to a constant region via a “J” region of about 12 or more amino acids, and a heavy chain further comprises a “D” region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V_H) and a heavy chain constant region (C_H) . A heavy chain constant region consists of 3 domains (C_H1, C_H2 and C_H3) . Each light chain consists of a light chain variable region (V_L) and a light chain constant region (C_L) . V_H and V_L region can further be divided into hypervariable regions (called complementary determining regions (CDR) ) , which are interspaced by relatively conservative regions (called framework region (FR) ) . Each V_H and V_L consists of 3 CDRs and 4 FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminal to C-terminal. The variable region (V_H and V_L) of each heavy/light chain pair forms antigen binding sites, respectively. Distribution of amino acids in various regions or domains follows the definition in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991) ) , or Chothia &Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al., (1989) Nature 342: 878-883. Antibodies may be of different antibody isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype) , IgA1, IgA2, IgD, IgE or IgM antibody.

The term “antigen-binding portion” or “antigen-binding fragment” of an antibody, which can be interchangeably used in the context of the application, refers to polypeptides comprising fragment (s) of a full-length antibody, which retain the ability of specifically binding to an antigen that the full-length antibody specifically binds to, and/or compete with the full-length antibody for binding to the same antigen. Generally, see Fundamental Immunology, Ch. 7 (Paul, W., ed., the second edition, Raven Press, N. Y. (1989) , which is incorporated herein by reference for all purposes. Antigen binding fragments of an antibody may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of an intact antibody. Under some conditions, antigen binding fragments include Fab, Fab', F (ab') ₂, Fd, Fv, dAb and complementary determining region (CDR) fragments, single chain antibody (e.g., scFv) , chimeric antibody, diabody and such polypeptides that comprise at least part of antibody sufficient to confer the specific antigen binding ability on the polypeptides. Antigen binding fragments of an antibody may be obtained from a given antibody (e.g., the monoclonal anti-human CD47 antibody provided in the instant application) by conventional techniques known by a person skilled in the art (e.g., recombinant DNA technique or enzymatic or chemical cleavage methods) , and may be screened for specificity in the same manner by which intact antibodies are screened.

The term “asingle domain antibody (sdAb) ” , as used herein, refers to an antibody containing a single heavy chain variable domain. The first single domain antibody is modified from the heavy chain antibody found in camelid, and the heavy chain antibody found in camelid is also referred to as VHH antibody. Like IgG antibodies, it is capable of selectively binding specific antigens, but the molecular weight of single domain antibody is much smaller than IgG antibodies.

The term “monoclonal antibody” or “mAb” , as used herein, refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope.

The term “human antibody” or “fully human antibody” , as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) . However, the term “human antibody” , as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The term “human monoclonal antibody” , as used herein, refers to antibodies displaying a single binding specificity, which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences.

The term “humanized antibody” is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a Camelidae animal or a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

The term “chimeric antibody” , as used herein, refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

The term “recombinant antibody” , as used herein, refers to an antibody that is prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal that is transgenic for another species’ immunoglobulin genes, antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of immunoglobulin gene sequences to other DNA sequences.

The term “anti-CD47 antibody” or “CD47 antibody, as used herein, refers to an antibody, as defined herein, capable of binding to a CD47, for example, a CD47 expressing or even overexpressing on various malignant tumors.

The term “CD47” is an abbreviation of “Cluster of differentiation 47” , and is also known as “integrin-associated protein (IAP) ” , which are used interchangeably herein.

The term “Ka” , as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term “Kd” as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction. Kd values for antibodies can be determined using methods well established in the art. The term “K_D” as used herein, is intended to refer to the dissociation constant of a particular antibody-antigen interaction, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and is expressed as a molar concentration (M) . A preferred method for determining the Kd of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as asystem.

The term “high affinity” for an IgG antibody, as used herein, refers to an antibody having a K_D of 1 x 10^-8 M or less, more preferably 5 x 10^-9 M or less, even more preferably 1x10^-9 M or less, even more preferably 5 x 10^-10 M or less and even more preferably 1 x 10^-10 M or less for a target antigen, for example, a CD47.

The term “EC₅₀” , as used herein, which is also termed as “half maximal effective concentration” refers to the concentration of a drug, antibody or toxicant which induces a response halfway between the baseline and maximum after a specified exposure time. In the context of the application, EC₅₀ is expressed in the unit of “nM” .

The term “compete for binding” , as used herein, refers to the interaction of two antibodies in their binding to a binding target. A first antibody competes for binding with a second antibody if binding of the first antibody with its cognate epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody, can, but need not, be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope. However, where each antibody detectably inhibits the binding of the other antibody with its cognate epitope, whether to the same, greater, or lesser extent, the antibodies are said to “cross-compete” with each other for binding of their respective epitope (s) .

The ability of “inhibit binding” , as used herein, refers to the ability of an antibody or antigen-binding fragment thereof to inhibit the binding of two molecules (e.g., CD47 and anti-CD47 antibody) to any detectable level. In certain embodiments, the binding of the two molecules can be inhibited at least 50%by the antibody or antigen-binding fragment thereof. In certain embodiments, such an inhibitory effect may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

The term “epitope” , as used herein, refers to a portion on antigen that an immunoglobulin or antibody specifically binds to. “Epitope” is also known as “antigenic determinant” . Epitope or antigenic determinant generally consists of chemically active surface groups of a molecule such as amino acids, carbohydrates or sugar side chains, and generally has a specific three-dimensional structure and a specific charge characteristic. For example, an epitope generally comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique steric conformation, which may be “linear” or “conformational” . See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996) . In a linear epitope, all the interaction sites between a protein and an interaction molecule (e.g., an antibody) are present linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction sites span over amino acid residues that are separate from each other in a protein. Antibodies may be screened depending on competitiveness of binding to the same epitope by conventional techniques known by a person skilled in the art. For example, study on competition or cross-competition may be conducted to obtain antibodies that compete or cross-compete with each other for binding to antigens. High-throughput methods for obtaining antibodies binding to the same epitope, which are based on their cross-competition, are described in an international patent application WO 03/48731.

The term “isolated” , as used herein, refers to a state obtained from natural state by artificial means. If a certain “isolated” substance or component is present in nature, it is possible because its natural environment changes, or the substance is isolated from natural environment, or both. For example, a certain un-isolated polynucleotide or polypeptide naturally exists in a certain living animal body, and the same polynucleotide or polypeptide with a high purity isolated from such a natural state is called isolated polynucleotide or polypeptide. The term “isolated” excludes neither the mixed artificial or synthesized substance nor other impure substances that do not affect the activity of the isolated substance.

The term “isolated antibody” , as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds a CD47 is substantially free of antibodies that specifically bind antigens other than CD47) . An isolated antibody that specifically binds human CD47 may, however, have cross-reactivity to other antigens, such as CD47 from other species. Moreover, an isolated antibody can be substantially free of other cellular material and/or chemicals.

The term “vector” , as used herein, refers to a nucleic acid vehicle which can have a polynucleotide inserted therein. When the vector allows for the expression of the protein encoded by the polynucleotide inserted therein, the vector is called an expression vector. The vector can have the carried genetic material elements expressed in a host cell by transformation, transduction, or transfection into the host cell. Vectors are well known by a person skilled in the art, including, but not limited to plasmids, phages, cosmids, artificial chromosome such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC) ; phage such as λ phage or M13 phage and animal virus. The animal viruses that can be used as vectors, include, but are not limited to, retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus) , pox virus, baculovirus, papillomavirus, papova virus (such as SV40) . A vector may comprise multiple elements for controlling expression, including, but not limited to, a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element and a reporter gene. In addition, a vector may comprise origin of replication.

The term “host cell” , as used herein, refers to a cellular system which can be engineered to generate proteins, protein fragments, or peptides of interest. Host cells include, without limitation, cultured cells, e.g., mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters) such as CHO, BHK, NSO, SP2/0, YB2/0; or human tissues or hybridoma cells, fungi cells such as yeast cells, bacterial cells such as E. coli cells, and insect cells, and cells comprised within a transgenic animal or cultured tissue. The term encompasses not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term “host cell. ”

The term “identity” , as used herein, refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (i.e., an “algorithm” ) . Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A.M., ed. ) , 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W., ed. ) , 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds. ) , 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds. ) , 1991, New York: M. Stockton Press; and Carillo et al, 1988, SIAMJ. Applied Math. 48: 1073.

The term “immunogenicity” , as used herein, refers to ability of stimulating the formation of specific antibodies or sensitized lymphocytes in organisms. It not only refers to the property of an antigen to stimulate a specific immunocyte to activate, proliferate and differentiate so as to finally generate immunologic effector substance such as antibody and sensitized lymphocyte, but also refers to the specific immune response that antibody or sensitized T lymphocyte can be formed in immune system of an organism after stimulating the organism with an antigen. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce the generation of an immune response in a host depends on three factors, properties of an antigen, reactivity of a host, and immunization means.

The term “transfection” , as used herein, refers to the process by which nucleic acids are introduced into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include but not limited to lipid transfection and chemical and physical methods such as electroporation. A number of transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52: 456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al, 1981, Gene 13: 197.

The term “SPR” or “surface plasmon resonance” , as used herein, refers to and includes an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J. ) . For further descriptions, seeU., et al. (1993) Ann. Biol. Clin. 51: 19-26; U., et al. (1991) Biotechniques 11: 620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8: 125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198: 268-277.

The term “fluorescence-activated cell sorting” or “FACS” , as used herein, refers to a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell (FlowMetric. “Sorting Out Fluorescence Activated Cell Sorting” . Retrieved 2017-11-09. ) . Instruments for carrying out FACS are known to those of skill in the art and are commercially available to the public. Examples of such instruments include FACS Star Plus, FACScan and FACSort instruments from Becton Dickinson (Foster City, Calif. ) Epics C from Coulter Epics Division (Hialeah, Fla. ) and MoFlo from Cytomation (Colorado Springs, Colo. ) .

The term “antibody-dependent cell-mediated cytotoxicity” or “ADCC” , as used herein, refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991) . To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998) .

The term “complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) , may be performed.

The term “subject” may include any kind of mammals, for example, but not limited to, human or nonhuman mammals, preferably humans. The term “nonhuman mammals” may include Cynomolgus macaques, Camelidae, rodents and the like.

The term “cancer” , as used herein, refers to any or a tumor or a malignant cell growth, proliferation or metastasis-mediated, solid tumors and non-solid tumors such as leukemia, that initiate a medical condition.

The term “treatment” , “treating” or “treated” , as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal, in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, regression of the condition, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis, prevention) is also included. For cancer, “treating” may refer to dampen or slow the tumor or malignant cell growth, proliferation, or metastasis, or some combination thereof. For tumors, “treatment” includes removal of all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.

The term “a (therapeutically) effective amount” , as used herein, pertains to that amount of an active compound, or a material, composition or dosage form comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen. For instance, the “an effective amount” , when used in connection with treatment of CD47-related diseases or conditions, refers to an anti-CD47 antibody or antigen-binding portion thereof in an amount or concentration effective to treat the said diseases or conditions.

The term “prevent” , “prevention” or “preventing” , as used herein, with reference to a certain disease condition in a mammal, refers to preventing or delaying the onset of the disease, or preventing the manifestation of clinical or subclinical symptoms thereof.

The term “pharmaceutically acceptable” , as used herein, means that the vehicle, diluent, excipient and/or salts thereof, are chemically and/or physically is compatible with other ingredients in the formulation, and the physiologically compatible with the recipient.

As used herein, the term “apharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient pharmacologically and/or physiologically compatible with a subject and an active agent, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) , and includes, but is not limited to pH adjuster, surfactant, adjuvant and ionic strength enhancer. For example, the pH adjuster includes, but is not limited to, phosphate buffer; the surfactant includes, but is not limited to, cationic, anionic, or non-ionic surfactant, e.g., Tween-80^TM; the ionic strength enhancer includes, but is not limited to, sodium chloride.

As used herein, the term “adjuvant” refers to a non-specific immunopotentiator, which can enhance immune response to an antigen or change the type of immune response in an organism when it is delivered together with the antigen to the organism or is delivered to the organism in advance. There are a variety of adjuvants, including, but not limited to, aluminium adjuvants (for example, aluminum hydroxide) , Freund’s adjuvants (for example, Freund’s complete adjuvant or Freund’s incomplete adjuvant) , coryne bacterium parvum, lipopolysaccharide, cytokines, and the like. Freund's adjuvant is the most commonly used adjuvant in animal experiments now. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

Anti-CD47 Antibodies and Antigen-Binding portion thereof

In some aspects, the invention provides an isolated anti-CD47 antibody or an antigen-binding portion thereof.

In the context of the application, the “antibody” may include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, and humanized antibodies, CDR grafted antibodies, human antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies, including muteins and variants thereof; and derivatives thereof including Fc fusions and other modifications, and any other immunoreactive molecule so long as it exhibits preferential association or binding with a CD47 protein. Moreover, unless dictated otherwise by contextual constraints the term further comprises all classes of antibodies (i.e., IgA, IgD, IgE, IgG, and IgM) and all subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) . In an embodiment, the antibody is a single domain antibody. In a preferred embodiment, the antibody is a monoclonal antibody. In a more preferred embodiment, the antibody is a humanized monoclonal antibody. In a specific embodiment, the antibody is a single domain antibody, preferably a humanized single domain antibody.

Generation of Transformants Producing Monoclonal Antibodies of the Invention

Antibodies of the invention also can be produced in a host cell transformed with a recombinant DNA by using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (e.g., Morrison, S. (1985) Science 229: 1202) . In one embodiment, DNA encoding partial or full-length heavy chain and/or light chain obtained by standard molecular biology techniques is inserted into one or more expression vectors such that the genes are operatively linked to transcriptional and translational regulatory sequences. In this context, the term "operatively linked" is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.

The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, e.g., in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990) ) . Exemplary regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) , Simian Virus 40 (SV40) , adenovirus, (e.g., the adenovirus major late promoter (AdMLP) and polyoma. Alternatively, nonviral regulatory sequences can be used, such as the ubiquitin promoter or β-globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe et al. (1988) Mol. Cell. Biol. 8: 466-472) . The expression vector and expression control sequences are chosen to be compatible with the expression host cell used.

The antibody light chain gene and/or the antibody heavy chain gene can be inserted into the same or separate expression vectors. In some embodiments, the variable regions are used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the CH segment (s) within the vector and the VL segment is operatively linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein) .

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665 and 5,179,017) . For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Selectable marker genes may include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection) .

For expression of the light and heavy chains, the expression vector (s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. It is possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells, for example, mammalian host cells, which can assemble and secrete a properly folded and immunologically active antibody.

Mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, used with a DHFR selectable marker, e.g., as described in R.J. Kaufman and P.A. Sharp (1982) J. Mol. Biol. 159: 601-621) , NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma cells, another expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338, 841. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

Anti-CD47 antibodies with certain properties

The anti-CD47 antibodies of the invention are characterized by particular functional features or properties. In some embodiments, the isolated anti-CD47antibody or the antigen-binding portion thereof has one or more of the following properties:

(a) binding human CD47 with high affinity (e.g., with a K_D of 5.56 x 10^-11 M or less) ;

(b) specifically binding to tumor cells expressing or overexpressing human CD47 in a dose dependent manner;

(c) inducing potent antibody dependent tumor cells phagocytosis in vitro and in vivo;

(d) minimizing potential side effect on RBC system by reducing or without RBCs binding and hemagglutination activity;

(e) having good thermal stability and/or serum stability; and/or

(f) having no cross-reactivity to human antigens other than CD47, for example, CD147, PD-1, and CTLA4, and the like.

Anti-CD47 antibodies comprising CDRs with sequence identity to specific sequences

In some embodiments, the isolated anti-CD47 antibody or the antigen-binding portion thereof comprises a heavy chain variable region comprising:

(a) CDR1 comprising SEQ ID NO: 1 or an amino acid sequence having at least 90%sequence identity with SEQ ID NO: 1;

(b) CDR2 comprising SEQ ID NO: 2 or an amino acid sequence having at least 85%or 90%sequence identity with SEQ ID NO: 2, and

(c) CDR3 comprising SEQ ID NO: 3.

The assignment of amino acids to each CDR may be in accordance with the numbering schemes provided by Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5^th Ed. ) , US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, unless otherwise noted.

Variable regions and CDRs in an antibody sequence can be identified according to general rules that have been developed in the art (as set out above, such as, for example, the Kabat numbering system) or by aligning the sequences against a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary databases of antibody sequences are described in, and can be accessed through, the “Abysis" website at www. bioinf. org. uk/abs (maintained by A. C. Martin in the Department of Biochemistry &Molecular Biology University College London, London, England) and the VBASE2 website at www. vbase2. org, as described in Retter et al., Nucl. Acids Res., 33 (Database issue) : D671 -D674 (2005) . Preferably sequences are analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT and the Protein Data Bank (PDB) with structural data from the PDB. See Dr.Andrew C. R. Martin's book chapter Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13: 978-3540413547, also available on the website bioinforg. uk/abs) . The Abysis database website further includes general rules that have been developed for identifying CDRs which can be used in accordance with the teachings herein. Unless otherwise indicated, all CDRs set forth herein are derived according to the Abysis database website as per Kabat. Unless otherwise indicated, CDR boundaries for antibodies are defined or identified by the conventions of Kabat (Kabat E. A. et al., National Institutes of Health, Bethesda, Md. (1991) ) and IMGT (the international ImMunoGeneTics database, http: //www. imgt. org) in the present disclosure.

The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988) ) which has been incorporated into the ALIGN program (version 2.0) , using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percentage of identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970) ) which has been incorporated into the GAP program in the GCG software package (available at http: //www. gcg. com) , using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

Additionally or alternatively, the protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215: 403-10. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the antibody molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25 (17) : 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www. ncbi. nlm. nih. gov.

In other embodiments, the CDR amino acid sequences can be at least 85%or at least 90%identical to the respective sequences set forth above. As an illustrative example, the antibody may comprise a heavy chain variable region CDR1 with at least 90%sequence identity with SEQ ID NO:1, a heavy chain variable region CDR2 with at least 80%or 90%sequence identity with SEQ ID NO: 2, or CDR3 of SEQ ID NO: 3.

Anti-CD47 antibodies comprising CDRs with amino acid addition, deletion and/or substitution

(a) CDR1 comprising SEQ ID NO: 1 or an amino acid sequence differing from SEQ ID NO: 1 by an amino acid addition, deletion or substitution of not more than 1 amino acid;

(b) CDR2 comprising SEQ ID NO: 2 or an amino acid sequence differing from SEQ ID NO: 2 by amino acid addition, deletion or substitution of not more than 2 amino acids, and

(c) CDR3 comprising SEQ ID NO: 3.

In some embodiments, the CDRs of the isolated anti-CD47 antibody or the antigen-binding portion thereof contain a conservative substitution of not more than 1 amino acid. The term “conservative substitution” , as used herein, refers to amino acid substitutions which would not disadvantageously affect or change the essential properties of a protein/polypeptide comprising the amino acid sequence. For example, a conservative substitution may be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions wherein an amino acid residue is substituted with another amino acid residue having a similar side chain, for example, a residue physically or functionally similar (such as, having similar size, shape, charge, chemical property including the capability of forming covalent bond or hydrogen bond, etc. ) to the corresponding amino acid residue. The families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having alkaline side chains (for example, lysine, arginine and histidine) , amino acids having acidic side chains (for example, aspartic acid and glutamic acid) , amino acids having uncharged polar side chains (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan) , amino acids having nonpolar side chains (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine) , amino acids having β-branched side chains (such as threonine, valine, isoleucine) and amino acids having aromatic side chains (for example, tyrosine, phenylalanine, tryptophan, histidine) . Therefore, a corresponding amino acid residue is preferably substituted with another amino acid residue from the same side-chain family. Methods for identifying amino acid conservative substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993) ; Kobayashi et al., Protein Eng. 12 (10) : 879-884 (1999) ; and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997) , which are incorporated herein by reference) .

Anti-CD47 antibodies comprising CDRs

(a) a CDR1 comprising SEQ ID NO: 1;

(b) a CDR2 comprising SEQ ID NO: 2; and

(c) a CDR3 comprising SEQ ID NO: 3.

In a specific embodiment, the isolated anti-CD47 antibody or the antigen-binding portion thereof comprises a heavy chain variable region comprising:

(a) a CDR1 consisting of SEQ ID NO: 1;

(b) a CDR2 consisting of SEQ ID NO: 2; and

(c) a CDR3 consisting of SEQ ID NO: 3.

In some embodiments, the isolated anti-CD47 antibody is a single domain antibody.

Anti-CD47 antibodies comprising a heavy chain variable region

(i) SEQ ID NO: 4 or 5;

(ii) an amino acid sequence having at least 80%, 85%, 90%, 95%or 99%sequence identity with SEQ ID NO: 4 or 5 while maintaining similar binding specificity as SEQ ID NO: 4 or 5; or

(iii) an amino acid sequence differing from SEQ ID NO: 4 or 5 by amino acid addition, deletion or substitution of one or more (e.g., not more than 15, e.g., 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1) amino acids while maintaining similar binding specificity as SEQ ID NO: 4 or 5.

In a specific embodiment, the isolated anti-CD47 antibody or the antigen-binding portion thereof comprises a heavy chain variable region of SEQ ID NO: 4.

In a specific embodiment, the isolated anti-CD47antibody or the antigen-binding portion thereof comprises a heavy chain variable region of SEQ ID NO: 5.

In other embodiments, the amino acid sequences of the heavy chain variable region can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%identical to SEQ ID NO: 4 or 5. As an illustrative example, the antibody may comprise a heavy chain variable region with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%sequence identity to SEQ ID NO: 5.

In some further embodiments, the isolated antibody or the antigen-binding portion thereof may contain conservative substitution or modification of amino acids in the variable regions of the heavy chain. It is understood in the art that certain conservative sequence modification can be made which do not remove antigen binding. See, e.g., Brummell et al. (1993) Biochem 32: 1180-8; de Wildt et al. (1997) Prot. Eng. 10: 835-41; Komissarov et al. (1997) J. Biol. Chem. 272: 26864-26870; Hall et al. (1992) J. Immunol. 149: 1605-12; Kelley and O’ Connell (1993) Biochem. 32: 6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10: 341-6 and Beers et al. (2000) Clin. Can. Res. 6: 2835-43.

As described above, the term “conservative substitution” , as used herein, refers to amino acid substitutions which would not disadvantageously affect or change the essential properties of a protein/polypeptide comprising the amino acid sequence. For example, a conservative substitution may be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions wherein an amino acid residue is substituted with another amino acid residue having a similar side chain, for example, a residue physically or functionally similar (such as, having similar size, shape, charge, chemical property including the capability of forming covalent bond or hydrogen bond, etc. ) to the corresponding amino acid residue. The families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having alkaline side chains (for example, lysine, arginine and histidine) , amino acids having acidic side chains (for example, aspartic acid and glutamic acid) , amino acids having uncharged polar side chains (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan) , amino acids having nonpolar side chains (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine) , amino acids having β-branched side chains (such as threonine, valine, isoleucine) and amino acids having aromatic side chains (for example, tyrosine, phenylalanine, tryptophan, histidine) . Therefore, a corresponding amino acid residue is preferably substituted with another amino acid residue from the same side-chain family. Methods for identifying amino acid conservative substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993) ; Kobayashi et al., Protein Eng. 12 (10) : 879-884 (1999) ; and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997) , which are incorporated herein by reference) .

Anti-CD47 antibodies consisting of full-length amino acid sequence

In some embodiments, the isolated anti-CD47 antibody or the antigen-binding portion thereof is a single domain antibody, and consists of a full-length amino acid sequence represented by SEQ ID NO: 7 or 9, or an amino acid sequence having at least 80%, 85%, 90%, 95%or 99%sequence identity with SEQ ID NO: 7 or 9 while maintaining similar binding specificity as SEQ ID NO: 7 or 9, or an amino acid sequence differing from SEQ ID NO: 7 or 9 by amino acid addition, deletion or substitution of one or more (e.g., not more than 30, e.g., 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1) amino acids while maintaining similar binding specificity as SEQ ID NO: 7 or 9.

Nucleic Acid Molecules Encoding Antibodies of the Invention

In some aspects, the invention is directed to an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the heavy chain variable region of the isolated anti-CD47 antibody as disclosed herein.

In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence encoding the heavy chain of the isolated anti-CD47 antibody as disclosed herein.

Nucleic acids of the invention can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below) , cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques) , a nucleic acid encoding such antibodies can be recovered from the gene library.

The isolated nucleic acid encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding nucleic acid to another DNA molecule encoding heavy chain constant regions (CH1, CH2 and CH3) . The sequences of human heavy chain constant region genes are known in the art (see e.g., Kabat et al. (1991) , supra) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but more preferably is an IgG1 or IgG4 constant region.

The isolated nucleic acid encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see e.g., Kabat et al., supra) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. In preferred embodiments, the light chain constant region can be a kappa or lambda constant region.

Once DNA fragments encoding VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a VL-or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term “operatively linked” , as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

In some embodiments, the isolated anti-CD47 antibody is a single domain antibody, and the invention is directed to an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the heavy chain variable region of the isolated anti-CD47 antibody as disclosed herein.

In some specific embodiments, the isolated nucleic acid molecule encodes the heavy chain variable region of the isolated anti-CD47 antibody and comprises a nucleic acid sequence selected from the group consisting of:

(A) a nucleic acid sequence that encodes a heavy chain variable region as set forth in SEQ ID NO: 4 or 5;

(B) a nucleic acid sequence as set forth in SEQ ID NO: 6; or

(C) a nucleic acid sequence that hybridized under high stringency conditions to the complementary strand of the nucleic acid sequence of (A) or (B) .

For example, the nucleic acid molecule may consist of SEQ ID NO: 6. Alternatively, the nucleic acid molecule share an at least 80% (e.g. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 6. In some specific embodiments, the percentage of identity is derived from the degeneracy of the genetic code, and the encoded protein sequences remain unchanged.

In some specific embodiments, the isolated nucleic acid molecule encodes the heavy chain of the isolated anti-CD47 antibody and comprises a nucleic acid sequence selected from the group consisting of:

(A) a nucleic acid sequence that encodes a heavy chain as set forth in SEQ ID NO: 7 or 9;

(B) a nucleic acid sequence as set forth in SEQ ID NO: 8 or 10; or

Exemplary high stringency conditions include hybridization at 45℃ in 5X SSPE and 45%formamide, and a final wash at 65℃ in 0.1 X SSC. It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described Ausubel, et al. (Eds. ) , Protocols in Molecular Biology, John Wiley &Sons (1994) , pp. 6.0.3 to 6.4.10. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe. The hybridization conditions can be calculated as described in Sambrook, et al, (Eds. ) , Molecular Cloning: A laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989) , pp. 9.47 to 9.51.

Pharmaceutical Compositions

In some aspects, the invention is directed to a pharmaceutical composition comprising at least one anti-CD47 antibody or antigen-binding portion thereof as disclosed herein and a pharmaceutically acceptable carrier.

Components of the compositions

The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or a drug. The pharmaceutical compositions of the invention also can be administered in a combination therapy with, for example, another immune-stimulatory agent, anti-cancer agent, an antiviral agent, or a vaccine. A pharmaceutically acceptable carrier can include, for example, a pharmaceutically acceptable liquid, gel or solid carriers, an aqueous medium, a non-aqueous medium, an anti-microbial agent, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispersing agent, a chelating agent, a diluent, adjuvant, excipient or a nontoxic auxiliary substance, other known in the art various combinations of components or more.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrating agents, buffers, preservatives, lubricants, flavorings, thickening agents, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrin. Suitable anti-oxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercapto glycerol, thioglycolic acid, Mercapto sorbitol, butyl methyl anisole, butylated hydroxy toluene and/or propyl gallate. As disclosed in the present invention, the compositions containing an antibody or an antigen-binding fragment of the present invention comprise one or more anti-oxidants such as methionine, reducing potential oxidation of the antibody or antigen binding fragment thereof. The oxidation reduction may prevent or reduce a decrease in binding affinity, thereby enhancing antibody stability and extended shelf life. Thus, in some embodiments, the present invention provides a composition comprising one or more antibodies or antigen binding fragment thereof and one or more anti-oxidants such as methionine. The present invention further provides a variety of methods, wherein an antibody or antigen binding fragment thereof is mixed with one or more anti-oxidants, such as methionine, so that the antibody or antigen binding fragment thereof can be prevented from oxidation, to extend their shelf life and/or increased activity.

To further illustrate, pharmaceutical acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, nonaqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80^TM) , sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid) , ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

Administration, Formulation and Dosage

The pharmaceutical composition of the invention may be administered in vivo, to a subject in need thereof, by various routes, including, but not limited to, oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or otherwise by implantation or inhalation. The subject compositions may be formulated into preparations in solid, semi-solid, liquid, or gaseous forms; including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols. The appropriate formulation and route of administration may be selected according to the intended application and therapeutic regimen.

Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.

Formulations suitable for parenteral administration (e.g., by injection) , include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) , in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate) . Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Similarly, the particular dosage regimen, including dose, timing and repetition, will depend on the particular individual and that individual's medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc. ) .

Frequency of administration may be determined and adjusted over the course of therapy, and is based on reducing the number of proliferative or tumorigenic cells, maintaining the reduction of such neoplastic cells, reducing the proliferation of neoplastic cells, or delaying the development of metastasis. In some embodiments, the dosage administered may be adjusted or attenuated to manage potential side effects and/or toxicity. Alternatively, sustained continuous release formulations of a subject therapeutic composition may be appropriate.

It will be appreciated by one of skill in the art that appropriate dosages can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action that achieve the desired effect without causing substantial harmful or deleterious side-effects.

In general, the antibody or the antigen binding portion thereof of the invention may be administered in various ranges. These include about 5 μg/kg body weight to about 100 mg/kg body weight per dose; about 50 μg/kg body weight to about 5 mg/kg body weight per dose; about 100 μg/kg body weight to about 10 mg/kg body weight per dose. Other ranges include about 100 μg/kg body weight to about 20 mg/kg body weight per dose and about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose. In certain embodiments, the dosage is at least about 100 μg/kg body weight, at least about 250 μg/kg body weight, at least about 750 μg/kg body weight, at least about 3 mg/kg body weight, at least about 5 mg/kg body weight, at least about 10 mg/kg body weight.

In any event, the antibody or the antigen binding portion thereof of the invention is preferably administered as appropriate to subjects in need thereof. Determination of the frequency of administration may be made by persons skilled in the art, such as an attending physician based on considerations of the condition being treated, age of the subject being treated, severity of the condition being treated, general state of health of the subject being treated and the like.

In certain preferred embodiments, the course of treatment involving the antibody or the antigen-binding portion thereof of the instant invention will comprise multiple doses of the selected drug product over a period of weeks or months. More specifically, the antibody or the antigen-binding portion thereof of the instant invention may be administered once every day, every two days, every four days, every week, every ten days, every two weeks, every three weeks, every month, every six weeks, every two months, every ten weeks or every three months. In this regard, it will be appreciated that the dosages may be altered or the interval may be adjusted based on patient response and clinical practices.

Dosages and regimens may also be determined empirically for the disclosed therapeutic compositions in individuals who have been given one or more administration (s) . For example, individuals may be given incremental dosages of a therapeutic composition produced as described herein. In selected embodiments, the dosage may be gradually increased or reduced or attenuated based respectively on empirically determined or observed side effects or toxicity. To assess efficacy of the selected composition, a marker of the specific disease, disorder or condition can be followed as described previously. For cancer, these include direct measurements of tumor size via palpation or visual observation, indirect measurement of tumor size by x-ray or other imaging techniques; an improvement as assessed by direct tumor biopsy and microscopic examination of the tumor sample; the measurement of an indirect tumor marker (e.g., PSA for prostate cancer) or a tumorigenic antigen identified according to the methods described herein, a decrease in pain or paralysis; improved speech, vision, breathing or other disability associated with the tumor; increased appetite; or an increase in quality of life as measured by accepted tests or prolongation of survival. It will be apparent to one of skill in the art that the dosage will vary depending on the individual, the type of neoplastic condition, the stage of neoplastic condition, whether the neoplastic condition has begun to metastasize to other location in the individual, and the past and concurrent treatments being used.

Compatible formulations for parenteral administration (e.g., intravenous injection) will comprise the antibody or antigen-binding portion thereof as disclosed herein in concentrations of from about 10 μg/ml to about 100 mg/ml. In certain selected embodiments, the concentrations of the antibody or the antigen binding portion thereof will comprise 20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml, 100 μg/ml, 200 μg/ml, 300, μg/ml, 400 μg/ml, 500 μg/ml, 600 μg/ml, 700 μg/ml, 800 μg/ml, 900 μg/ml or 1 mg/ml. In other preferred embodiments the concentrations of the antibody or the antigen binding portion thereof will comprise 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 8 mg/ml, 10 mg/ml, 12 mg/ml, 14 mg/ml, 16 mg/ml, 18 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 35 mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml or 100 mg/ml.

Applications of the Invention

The antibodies, antibody compositions and methods of the present invention have numerous in vitro and in vivo utilities involving, for example, blocking CD47-SIRPα interaction activity, inducing potent antibody dependent tumor cells phagocytosis in vitro and in vivo. For example, these molecules can be administered to cells in culture, in vitro or ex vivo, or to human subjects, e.g., in vivo, to enhance immunity in a variety of situations. The immune response can be modulated, for instance, augmented, stimulated or up-regulated.

Preferred subjects include human patients in need of enhancement of an immune response. The methods are particularly suitable for treating human patients having a disorder that can be treated by augmenting an immune response (e.g., tumor cells phagocytosis) . In a particular embodiment, the methods are particularly suitable for treatment of CD47-related diseases (e.g., cancer) in vivo. To achieve antigen-specific enhancement of immunity, the anti-CD47 antibodies can be administered together with an antigen of interest or the antigen may already be present in the subject to be treated (e.g., a tumor-bearing or virus-bearing subject) . When anti-CD47 antibodies are administered together with another agent, the two can be administered in either order or simultaneously.

Prevention or Treatment of CD47-related diseases

In some aspects, the present invention provides a method for preventing or treating CD47-related diseases in a subject, which comprises administering to the subject (for example, a human) a therapeutically effective amount of the anti-CD47 antibody or antigen-binding portion thereof as disclosed herein.

A variety of cancers where CD47 is implicated, whether malignant or benign and whether primary or secondary, may be treated or prevented with a method provided by the disclosure. The cancers may be solid cancers or hematologic malignancies. Examples of CD47-related diseases may be hematologic diseases or solid tumors, for example, but not limited to acute myelocytic leukemia (AML) , non-Hodgkin’s lymphoma (NHL) , breast cancer, non-small-cell lung cancer (NSCC) , ovarian cancer, myelodysplastic syndrome, brain cancer, squamous cell carcinoma of the head and neck, and advanced malignant tumors.

The anti-CD47 antibody or the antigen-binding portion thereof may be used alone as a monotherapy, or may be used in combination with chemotherapies or radiotherapies.

Combined use with chemotherapies

The anti-CD47 antibody or the antigen-binding portion thereof may be used in combination with an anti-cancer agent, a cytotoxic agent or chemotherapeutic agent.

The term “anti-cancer agent” or “anti-proliferative agent” means any agent that can be used to treat a cell proliferative disorder such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, debulking agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapies, radiation therapy and anti-metastatic agents and immunotherapeutic agents. It will be appreciated that, in selected embodiments as discussed above, such anti-cancer agents may comprise conjugates and may be associated with the disclosed site-specific antibodies prior to administration. More specifically, in certain embodiments selected anti-cancer agents will be linked to the unpaired cysteines of the engineered antibodies to provide engineered conjugates as set forth herein. Accordingly, such engineered conjugates are expressly contemplated as being within the scope of the instant invention. In other embodiments, the disclosed anti-cancer agents will be given in combination with site-specific conjugates comprising a different therapeutic agent as set forth above.

As used herein the term “cytotoxic agent” means a substance that is toxic to the cells and decreases or inhibits the function of cells and/or causes destruction of cells. In certain embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, small molecule toxins or enzymatically active toxins of bacteria (e.g., Diptheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcal enterotoxin A) , fungal (e.g., α-sarcin, restrictocin) , plants (e.g., abrin, ricin, modeccin, viscumin, pokeweed anti-viral protein, saporin, gelonin, momoridin, trichosanthin, barley toxin, Aleurites fordii proteins, dianthin proteins, Phytolacca mericana proteins (PAPI, PAPII, and PAP-S) , Momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, mitegellin, restrictocin, phenomycin, neomycin, and the tricothecenes) or animals, (e.g., cytotoxic RNases, such as extracellular pancreatic RNases; DNase I, including fragments and/or variants thereof) .

For the purposes of the instant invention a “chemotherapeutic agent” comprises a chemical compound that non-specifically decreases or inhibits the growth, proliferation, and/or survival of cancer cells (e.g., cytotoxic or cytostatic agents) . Such chemical agents are often directed to intracellular processes necessary for cell growth or division, and are thus particularly effective against cancerous cells, which generally grow and divide rapidly. For example, vincristine depolymerizes microtubules, and thus inhibits cells from entering mitosis. In general, chemotherapeutic agents can include any chemical agent that inhibits, or is designed to inhibit, a cancerous cell or a cell likely to become cancerous or generate tumorigenic progeny (e.g., TIC) . Such agents are often administered, and are often most effective, in combination, e.g., in regimens such as CHOP or FOLFIRI.

Examples of anti-cancer agents that may be used in combination with the site-specific constructs of the present invention (either as a component of a site specific conjugate or in an unconjugated state) include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, ethylenimines and methylamelamines, acetogenins, a camptothecin, bryostatin, callystatin, CC-1065, cryptophycins, dolastatin, duocarmycin, eleutherobin, pancratistatin, a sarcodictyin, spongistatin, nitrogen mustards, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, esperamicin, chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogues, purine analogs, androgens, anti-adrenals, folic acid replenisher such as frolinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, an epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, polysaccharide complex (JHS Natural Products, Eugene, OR) , razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine) ; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ( “Ara-C” ) ; cyclophosphamide; thiotepa; taxoids, chloranbucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, vinblastine; platinum; etoposide (VP-16) ; ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) , topoisomerase inhibitor RFS 2000; difluorometlhylornithine; retinoids; capecitabine; combretastatin; leucovorin; oxaliplatin; inhibitors of PKC-alpha, Raf, H-Ras, EGFR and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, and anti-androgens; as well as troxacitabine (a 1, 3-dioxolane nucleoside cytosine analog) ; antisense oligonucleotides, ribozymes such as a VEGF expression inhibitor and a HER2 expression inhibitor; vaccines, rIL-2; topoisomerase 1 inhibitor; rmRH; Vinorelbine and Esperamicins and pharmaceutically acceptable salts, acids or derivatives of any of the above.

Combined use with radiotherapies

The present invention also provides for the combination of the anti-CD47 antibody or the antigen-binding portion thereof with radiotherapy (i.e., any mechanism for inducing DNA damage locally within tumor cells such as gamma-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions and the like) . Combination therapy using the directed delivery of radioisotopes to tumor cells is also contemplated, and the disclosed conjugates may be used in connection with a targeted anti-cancer agent or other targeting means. Typically, radiation therapy is administered in pulses over a period of time from about 1 to about 2 weeks. The radiation therapy may be administered to subjects having head and neck cancer for about 6 to 7 weeks. Optionally, the radiation therapy may be administered as a single dose or as multiple, sequential doses.

Pharmaceutical packs and kits

Pharmaceutical packs and kits comprising one or more containers and an instruction, each of the containers comprising one or more doses of the anti-CD47 antibody or the antigen-binding portion thereof are also provided. In an embodiment, the instruction may comprise the information for instructing the users how to use the kit, for example, information about the dosage of the component (s) , the route and frequency of the administration, the indications to be treated by the kit, and the like. In certain embodiments, a unit dosage is provided wherein the unit dosage contains a predetermined amount of a composition comprising, for example, the anti-CD47 antibody or the antigen-binding portion thereof, with or without one or more additional agents. For other embodiments, such a unit dosage is supplied in single-use prefilled syringe for injection. In still other embodiments, the composition contained in the unit dosage may comprise saline, sucrose, or the like; a buffer, such as phosphate, or the like; and/or be formulated within a stable and effective pH range. Alternatively, in certain embodiments, the conjugate composition may be provided as a lyophilized powder that may be reconstituted upon addition of an appropriate liquid, for example, sterile water or saline solution. In certain preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. Any label on, or associated with, the container (s) indicates that the enclosed conjugate composition is used for treating the neoplastic disease condition of choice.

The present invention also provides kits for producing single-dose or multi-dose administration units of site-specific conjugates and, optionally, one or more anti-cancer agents. The kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic and contain a pharmaceutically effective amount of the disclosed conjugates in a conjugated or unconjugated form. In other preferred embodiments, the container (s) comprise a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle) . Such kits will generally contain in a suitable container a pharmaceutically acceptable formulation of the engineered conjugate and, optionally, one or more anti-cancer agents in the same or different containers. The kits may also contain other pharmaceutically acceptable formulations for combined therapy. For example, in addition to the antibody or the antigen-binding portion thereof of the invention such kits may contain any one or more of a range of anti-cancer agents such as chemotherapeutic or radiotherapeutic drugs; anti-angiogenic agents; anti-metastatic agents; targeted anti-cancer agents; cytotoxic agents; and/or other anti-cancer agents.

More specifically the kits may have a single container that contains the anti-CD47 antibody or the antigen-binding portion thereof, with or without additional components, or they may have distinct containers for each desired agent. Where combined therapeutics are provided for conjugation, a single solution may be pre-mixed, either in a molar equivalent combination, or with one component in excess of the other. Alternatively, the conjugates and any optional anti-cancer agent of the kit may be maintained separately within distinct containers prior to administration to a patient. The kits may also comprise a second/third container means for containing a sterile, pharmaceutically acceptable buffer or other diluent such as bacteriostatic water for injection (BWFI) , phosphate-buffered saline (PBS) , Ringer's solution and dextrose solution.

When the components of the kit are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, with a sterile aqueous or saline solution being particularly preferred. However, the components of the kit may be provided as dried powder (s) . When reagents or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container.

As indicated briefly above, the kits may also contain a means by which to administer the anti-CD47 antibody or the antigen-binding portion thereof and any optional components to a patient, e.g., one or more needles, I. V. bags or syringes, or even an eye dropper, pipette, or other such like apparatus, from which the formulation may be injected or introduced into the animal or applied to a diseased area of the body. The kits of the present invention will also typically include a means for containing the vials, or such like, and other component in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials and other apparatus are placed and retained.

Summary of Sequence Listing

Appended to the instant application is a sequence listing comprising a number of nucleic acid and amino acid sequences. The following Table A, B and C provides a summary of the included sequences.

Two illustrative antibodies as disclosed herein, which are humanized anti-CD47 single domain antibodies and are also monoclonal antibodies, are designated as “W3456-P5R1-1C1-z19-uIgG4V1” and “W3456-P5R1-1C1-z19-uIgG1” , respectively.

Table A. CDR amino acid sequences of heavy chain

Table B. Heavy chain variable region

Table C. Full-length sequence

Examples

The present invention, thus generally described, will be understood more readily by reference to the following Examples, which are provided by way of illustration and are not intended to be limiting of the instant invention. The Examples are not intended to represent that the experiments below are all or the only experiments performed.

Materials and Methods

1. Commercial materials

The commercial materials used in the examples are listed in Table 1.

Table 1. Commercial materials

2. Antigen protein, BMK and CD47-expressing cell line generation

2.1 Production of Antigens

Human CD47 (NP_001768.1, NCBI) and cynomolgus monkey CD47 (XP_005548289.1, NCBI) extracellular domain (ECD) genes with human or mouse Fc-tag were cloned into expression vector. The plasmid was transfected into EXpi293 cells according to manufacturer’s instructions (Expi293F Transfection Kit, Invitrogen) . The cells were cultured in an incubator at 37℃, 8%CO₂ and then Harvest the supernatant after 5 days culturing to purify proteins using Protein A column and SEC column.

2.2 Production of Benchmark Antibodies

DNA sequences encoding the variable regions of anti-CD47 antibodies BMK2 and BMK8 (see Table 2) were separately cloned into expression vectors with the constant region of human IgG4, the human IgG4 heavy chain constant region was modified to incorporate the Ser228Pro substitution to reduce the rate of Fab arm exchange. Then, the plasmid was transfected into EXpi293 cells according to manufacturer’s instructions (Expi293F Transfection Kit, Invitrogen) . The cells were cultured in an incubator at 37℃, 8%CO₂ and then the supernatant was collected after 5 days of culturing. The proteins were purified using Protein A column and SEC column.

Benchmark antibodies “W345-BMK2-IgG4K. SP” and “W345-BMK8” were generated and applied as controls in the following examples. They were also referred to as W345-BMK2 and W345-BMK8 herein, respectively.

2.3 Cell Pool/Line Generation

The full length gene of human (NP_001768.1, NCBI) or monkey CD47 (XP_005548289.1, NCBI) was cloned into an expression vector for development of cell line. Briefly, the CHO-K1 cells at 70-90%confluents were transfected with human or monkey CD47 full length plasmid using lipofectamine 2000 reagent. The transfected cells were cultured in an incubator at 37℃, 5%CO2. 24 hours after transfection, blasticidin at a final concentration of 2-10 μg/mL was used to select the stable pool. Then, the positive pool cells were subcloned by limited dilution. Single clone was picked and tested by FACS using anti-CD47 antibodies.

3. Materials Code and Abbreviations

The materials code and abbreviations used in the disclosure are listed in Tables 2 and 3.

Table 2. Materials code

Table 3. Abbreviations

Example 1. Generation of VHH Antibodies

1.1 Animal immunization and serum titer detection

To induce a humoral immune response directed towards CD47 in camelid animals, the animals were subcutaneously injected with human CD47 (NP_942088) or mouse CD47 (Q61735) extra-cellular domain (ECD) proteins for 7 doses at 1 to 3 weeks intervals. The dose ranged from 100 μg to 400 μg per injection. The animal blood was collected before and after immunization and serum titers against target proteins were monitored by ELISA according to general ELISA procedures.

1.2 Phage library construction

50 ml blood samples were collected at 6-7 days after the last two injections, respectively. Peripheral blood mononuclear cells (PBMCs) were purified by density gradient centrifugation on Ficoll-Paque PLUS (GE Healthcare, Little Chalfont, UK) , resulting in the isolation of approximately 8×10⁷ PBMCs. Total RNA was extracted from PBMCs and transcribed into cDNA using oligo-dT primers and SuperScript III First-Strand Synthesis SuperMix System (Invitrogen, Carlsbad, CA, USA) according to the manufacturers’ recommendations.

The purified cDNA was used as template to amplify the VHH gene repertoire for phage library construction. Briefly, two phage libraries were constructed from the same blood sample. One library was constructed by using the VHH repertoire genes amplified by a nested PCR and the other library by a direct PCR.

The first round of PCR of the nested PCR used signal peptide domain specific primers and CH2 domain specific primers, which resulted in PCR fragments of approximately 900 bp (representing conventional IgGs) and 700 bp (representing heavy-chain IgGs that lack a CH1 domain) . The two classes of heavy chain encoding genes were then size-separated on agarose gels and the genes encoding heavy-chain only IgG were purified by QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) . The purified fragments were used as template to amplify the VHH repertoire with the use of framework1 (FR1) and framework4 (FR4) specific primer pairs. This amplification procedure introduced a Sfi I restriction site at the 5’ end of FR1 and a Not I restriction site at the 3’ end of FR4. The repertoire of PCR-amplified VHH genes of about 300-400 bp were loaded on agarose gels and purified by QIAquick Gel Extraction Kit. The purified fragments were then treated with Sfi I and Not I and purified by QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) . The VHH gene fragments were finally ligated in phagemid vector pFL249 and electrotransformed into E. coli TG1 (K12, Δ (lac-pro) , supE, thi, hsdD5/F’ traD36, proA+B+, lacIq, lacZΔM15) . After transformation, the TG1 cells were cultured in SOC medium with shaking at 200 rpm for 1 h, then the E. coli TG1 were plated onto plates containing solid 2YT medium supplemented with 100 μg/mL Carb and 1% (w/v) glucose, and cultured at 37℃ overnight. The next day, the colonies were scraped into liquid 2YT medium supplemented with 1/3 (v/v) of 80%glycerol and were stored at -80℃.

The direct PCR amplified the repertoire of VHH genes with the use of FR1 primer set and camelid IgG2 and IgG3 hinge region specific primers. The PCR products with corresponding restriction enzyme site were used for phage library construction as descripted above.

Both libraries were used for phage display selection.

1.3 Cell and protein panning and hits selection

Phage display selection of anti-CD47 specific VHH fragments

To select VHH fragments that would effectively bind to CD47, the methods of cell panning and protein panning were employed.

For panning against protein, 20 μg of recombinant human or mouse CD47 ECD protein were immobilized in 5 ml immune tube (Nunc, Rochester, MN, USA) overnight at 4℃ with shaking at 400 rpm. The next day, after washing away unbound protein, the tube was blocked with 10%skim milk for 1 h at 25℃. Approximately 10¹² cfu phages from the immune phage libraries added into non-coated immune tube blocked with 10%skim milk and 100 μg Fc proteins to deplete the non-specifically bound phage, then treated phage described above were added into the tube and incubated at 25℃ for 2 h. After extensive washing with PBST, the nonspecifically adsorbed phages were discarded, and the target specifically bound phages were eluted by Glycine-HCl (pH2.2) and then neutralized by 1 M Tris-HCl (pH8.0) for infection of exponentially growing TG1 cells. The infected TG1 cells were plated on 2YT agar plates containing 2% (w/v) glucose and 100 μg/ml ampicillin and cultured overnight at 37℃. On the next day, the colonies were scraped off the plate with 3 ml 2YT and frozen at -80℃ by adding in 1/3 (v/v) 80%glycerol. The scraped bacteria libraries were inoculated into 2YT-Carb contain 100 μg/ml ampicillin, infected with helper phage M13Ko7 in 2YT medium with 50 μg/ml kanamycin and 1 mM IPTG for phage rescue and used as input for the next round of panning.

For cell-based panning, 10¹² cfu phages were blocked with 5%FBS-PBS. Then incubated with 1×10⁷ A20 cells at 4℃ for 1 h with tumbling at 12 rpm. The cells were washed with ice cold 5%FBS-PBS and the cell bound phage particles were eluted as described above.

Results

Blood samples were collected from immunized alpaca and peripheral blood mononuclear cells (PBMCs) were purified for RNA extraction. After library construction, panning and a serial of screenings, top hit W3456-P5R1-1C1 showed potent binding to human and cynomolgus monkey CD47-expresssing cells, while keep negligible binding to human RBCs. The amino acid sequence of W3456-P5R1-1C1 was listed in Table 4.

Table 4. Variable region amino acid sequence of W3456-P5R1-1C1

VHH protein expression and screening

After desired panning steps, phage infected TG1 cell colonies grown on the plates were scraped and pFL249 phagemid containing VHH fragments were extracted. The VHH fragments were cloned by digestion of pFL249 plasmids with Sfi I and Not I and then ligated into expression vector pETbac containing genes of hexa-histidine-and c-Myc-tag. The ligation products were transformed into E. coli BL21 (DE3) competent cells and then cultured in ZYM-5052 medium at 25℃ for 48 h with shaking at 230 rpm. Then the bacterial culture supernatants were collected for ELISA, competition ELISA and FACS tests.

ELISA was used as the first screen method to test the binding of VHH to human and cynomolgus CD47 ECD protein. Briefly, 96-well plates (Nunc, Rochester, MN, USA) were coated with recombinant human CD47 ECD protein and recombinant mFc-tagged cynomolgus CD47 ECD protein overnight at 4℃. After blocking and washing, the BL21 E. coli supernatants were transferred to the coated plates and incubated at room temperature for 1 h. The plates were then washed and subsequently incubated with secondary antibody Goat Anti-c-Myc-HRP (Bethyl, Montgomery, TX, USA) for 1 h. After washing, TMB substrate was added, and the reaction was stopped by 2 M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Device, Sunnyvale, CA, USA) . Subsequently, competition ELISA was used as the second screen method to further test whether the VHHs binding to human and cynomolgus CD47 ECD protein were compete with the ligand of human CD47 ECD protein. Briefly, 96-well plates (Nunc, Rochester, MN, USA) were coated with recombinant human CD47 ECD protein overnight at 4℃. After blocking and washing, the BL21 E. coli supernatants with recombinant mFc-tagged ligand of human CD47 ECD protein were transferred to the coated plates and incubated at room temperature for 1 h. The plates were then washed and subsequently incubated with secondary antibody Goat Anti-Mouse IgG-Fc-HRP (Bethyl, Montgomery, TX, USA) for 1 h. Remaining procedures were same as ELISA method. In order to confirm the native binding of anti-CD47 VHH on conformational CD47 molecules expressed on cell membrane, flow cytometry analysis was performed with human CD47 transfected CHO-K1 cells and hRBCs. The cells were firstly incubated with the E. coli culture supernatant samples in 96-well U-bottom plates (BD, Franklin Lakes, NJ, USA) at a density of 1×10⁵ cells/well at 4℃ for 1 h, then with a secondary antibody Goat Anti-c-Myc-PE (Bethyl, Montgomery, TX, USA) at 4 ℃ for 30 min. 2 times of washings were applied between each steps and the cells were resuspended in 1X PBS/1%BSA for flow cytometry analysis (IntelliCyt, Albuquerque, NM, USA) .

1.4 Sequencing

The positive E. coli clones selected by ELISA, competition ELISA and FACS screening were sent to Biosune (Shanghai, China) for nucleotide sequencing of VHH gene. The sequencing results were analyzed using CLC Main Workbench (Qiagen, Hilden, Germany) .

1.5 Recombinant VHHs production

The BL21 E. coli clones harboring VHH gene were cultured in 40 ml of ZYM-5052 medium at 25℃ for 48 h with shaking at 230 rpm. The expression of his-and c-Myc-tag fused VHH protein in BL21 supernatant was confirmed by SDS-PAGE, and then purified using Ni-NTA column. The purity of VHH was determined by SEC-HPLC. For low supernatant expression clones, ultrasonic (Scientz, Ningbo, China) breaking E. coli cells was used to release soluble VHH proteins.

1.6 Humanization and IgG generation

“Best Fit” approach was used to humanize the selected lead W3456-P5R1-1C1. For heavy chain amino acid sequences of corresponding V-genes were blasted against in-house human germline V-gene database. The sequence of humanized VH-gene was derived by replacing human CDR sequences in the top hit with Alpaca CDR sequence using Kabat CDR definition. First sequence was derived as for heavy chain. Additional sequences were created by blasting Alpaca frameworks against human germline V-gene database. Frameworks were defined using extended CDR definition where Kabat CDR1 was extended by 5 amino acids at N-terminus. Top three hits were used to derive sequences of humanized VH-genes. Humanized genes were back-translated, codon-optimized for mammalian expression, and synthesized by Sangon Biotech. Synthetic genes were re-cloned into a pcDNA3.4 plasmid (IgG-expressing) , expressed, and purified.

After humanized variants were screened, top lead W3456-P5R1-1C1-z19 was selected, the sequence was separately cloned into expression vectors with the constant region of human IgG1 or IgG4 (with S228P mutation) . Then, the plasmid was transfected into EXpi293 cells according to manufacturer’s instructions (Expi293F Transfection Kit, Invitrogen) . The cells were cultured in an incubator at 37℃, 8%CO2 and then the supernatant was collected after 5 days of culturing. The proteins were purified using Protein A column and SEC column. Final leads were named W3456-P5R1-1C1-z19-uIgG4V1 and W3456-P5R1-1C1-z19-uIgG1, which will be further characterized. The antibodies, W3456-P5R1-1C1-z19-uIgG4V1 and W3456-P5R1-1C1-z19-uIgG1, were also referred to as “lead antibodies” , “lead mAbs” or “leads” hereafter.

Results

Sequence of top lead W3456-P5R1-1C1 was humanized according to humanization strategy. After humanized variants screening and sequence analysis, final lead was selected and named as W3456-P5R1-1C1-z19. The heavy chain variable region sequence was showed in Table 5 and was converted into human IgG1 or IgG4 backbone format with S228P mutation, which were named as W3456-P5R1-1C1-z19-uIgG1 and W3456-P5R1-1C1-z19-uIgG4V1. The molecular information of the final leads was listed in Table 6.

Table 5. Variable region amino acid sequence of W3456-P5R1-1C1-z19

Table 6. Information summary of final lead molecules

Example 2. In-vitro Characterization

2.1 Antibody binding to human and cynomolgus monkey CD47-expressing cells (FACS)

Binding of lead antibody to human and cynomolgus monkey CD47-expressing cells was determined by FACS. Briefly, the engineering CD47-expressing cells were coated into 96-well U-bottom plates at a density of 1x10⁵ cells/well and centrifuged at 1500 rpm at 4℃ for 4 min before removing the supernatant. Then the lead antibody at various concentrations were added to re-suspend cells and incubated at 4 ℃ for 1 hr. The cells were washed twice with 180 μL 1%BSA-PBS. The secondary antibody, goat anti-human IgG-Fc PE was added to re-suspend cells and incubated at 4 ℃ in the dark for 30 min followed by washing with 180 μL 1%BSA-PBS. At the end, the cells were re-suspended in 100 μL 1%BSA-PBS, and the fluorescence intensity was measured by FACS (BD Canto II) and analyzed by FlowJo Version software. The binding EC50s were calculated by using GraphPad Prism software equation: Nonlinear regression (curve fit) –log (agonist) vs. response–Variable slope.

Results

Binding activity of final leads to human and cynomolgus monkey CD47 were performed by FACS using engineered CD47-expression stable cell lines. Results showed both leads could bind to human and cynomolgus CD47-expression cells in a dose dependent manner (Figure 1) and binding EC₅₀ was shown in Table 7.

Table 7. EC₅₀ of the antibodies

NA: Not applicable.

2.2 Antibody binding to tumor cells (FACS)

Binding of lead antibody to tumor cell was also determined by FACS. Briefly, tumor cells were coated into 96-well U-bottom plates at a density of 1x10⁵ cells/well and centrifuged at 1500 rpm at 4℃ for 4 min before removing the supernatant. The lead antibody at various concentrations were added and incubated at 4 ℃ for 1 h. The cells were washed twice with 180 μL 1%BSA-PBS. The secondary antibody, goat Anti-human IgG-Fc PE was added to re-suspend cells and incubated at 4 ℃ in the dark for 30 min followed by washing with 180 μL 1%BSA-PBS. At the end, the cells were re-suspended in 100 μL 1%BSA-PBS, and the fluorescence intensity was measured by FACS (BD Canto II) and analyzed by FlowJo Version software. The binding EC50s were calculated by using GraphPad Prism software equation: Nonlinear regression (curve fit) –log (agonist) vs response–Variable slope.

Results

Both blood tumor and solid tumor cells could escape macrophage-mediated phagocytosis by overexpressing CD47. Binding activity of final lead antibodies (i.e., W3456-P5R1-1C1-z19-uIgG4V1, and W3456-P5R1-1C1-z19-uIgG1) to tumor cells was performed by FACS. The data as shown in Figure 2 and Table 8 indicated that the lead antibodies could specifically bind to Raji and MDA-MB-231 tumor cells in a dose-dependent manner, comparable to 5F9, much potent than TJ-C4.

Table 8. EC₅₀ of the antibodies

NA: Not applicable.

2.3 Antibody binding to human RBC (FACS)

Since CD47 was expressed on human red blood cells (RBCs) , the binding activity of lead antibody on human RBCs was evaluated by FACS. Human red blood cells were isolated from trisodium citrate-treated fresh human blood by centrifuging at 2000 rpm for 10 min and discarding the supernatant serum. Human RBC cells were coated into 96-well U-bottom plates at a density of 1x10⁵ cells/well and centrifuged at 1500 rpm at 4℃ for 4 min before removing the supernatant. Then the lead antibody at various concentrations were added to re-suspend cells and incubated at 4 ℃ for 1 hr. The cells were washed twice with 180 μL 1%BSA-PBS. The secondary antibody, goat Anti-human IgG-Fc PE was added to re-suspend cells and incubated at 4 ℃ in the dark for 30 min followed by washing with 180 μL 1%BSA-PBS. At the end, the cells were re-suspended in 100 μL 1%BSA-PBS, and the fluorescence intensity was measured by FACS (BD Canto II) and analyzed by FlowJo Version software. The binding EC50s were calculated by using GraphPad Prism software equation: Nonlinear regression (curve fit) –log (agonist) vs. response–Variable slope.

Results

Since CD47 was also expressed on human red blood cells (RBCs) , the binding activity of leads on human RBCs was evaluated by FACS for the purpose of testing safety of the anti-CD47 antibodies. As shown in Figure 3 and Table 9, 5F9 showed potent binding to human RBCs, while lead mAbs showed negligible binding to human RBCs, which means that the lead mAbs may have significant benefits by reducing anemia in clinical trials or conventional treatment of CD47-related diseases.

Table 9. EC₅₀ of the antibodies to human RBCs

NA: Not applicable.

2.4 Hemagglutination assay (HA) on Human RBCs

To evaluate hemagglutination activity (HA) of W3456-P5R1-1C1-z19-uIgG4V1 on RBCs, HA testing was performed using human red blood cells (hRBCs) . hRBCs were isolated from trisodium citrate-treated fresh human blood by centrifuging at 2000 rpm for 10 min and discarding the supernatant serum. 25 μL of hRBCs suspension diluted with DPBS were added to one well of U-bottom 96-well plate (approximately ～4x10⁶ RBC/well) , followed by addition of 25 μL of lead antibodies (dilution range from 667 nM to 0.667 nM, equal to 100 μg/ml to 0.1 μg/ml) , then mixed well gently and incubated at 37℃ for 1 h. The RBC were re-suspended in DPBS and examined under microscope. The formation of hRBC clusters were defined as HA positive, whereas the hRBCs remained in the intact and dissociative condition were defined as HA negative.

Results

To evaluate hemagglutination activity (HA) of the lead mAbs on RBCs, HA testing was performed using human RBCs. The RBCs were examined under microscope, as shown in Figure 4. The formation of RBC clusters was defined as HA positive, which was shown in 5F9, whereas RBCs remained in the intact and dissociative condition were defined as HA negative, as shown as isotype control. Figure 4 showed both lead mAbs were completely HA negative.

The results that the lead mAbs had reduced or no RBCs binding and no hemagglutination activity indicated that the lead mAbs had minimized potential side effect on RBC system.

2.5 Human ligand SIRPα competition assay (FACS)

To assess ligand (SIRPα) blocking activity of W3456-P5R1-1C1-z19-uIgG4V1, the cell-based competition assay was performed using human CD47-expressing stable cells and Jurkat tumor cells. Briefly, taking the human CD47-expressing stable cells as an example, the engineered cells expressing human CD47 were coated onto 96-well U-bottom plates with 1x10⁵ cells/well and centrifuged at 1500 rpm at 4℃ for 4 min before removing the supernatant. The mixture of lead antibody at various concentrations and human SIRPα protein (1 μg/ml) was added and incubated for 2 h. The cells were washed twice with 200 μL 1%BSA-PBS. The secondary antibody, mouse anti-His tag-Biotin was added to re-suspended cells and incubated at 4 ℃ in the dark for 1 h followed by washing with 200 μL 1%BSA-PBS. The third antibody, anti-Streptadvitin-PE was added to re-suspended cells and incubated at 4 ℃ in the dark for 30 min followed by washing with 200 μL 1%BSA-PBS. At the end, the cells were re-suspended in 100 μL 1%BSA-PBS, and the fluorescence intensity was measured by FACS (BD Canto II) and analyzed by FlowJo Version software. The competitive binding IC50s were calculated by using GraphPad Prism software equation: Nonlinear regression (curve fit) –log (antagonist) vs. response–Variable slope. The inhibition rate was calculated using the equation: [ (MFI _ligand only -MFI _{blocking samples}) /MFI _ligand only x 100%] .

Results

To assess ligand (SIRPα) blocking activity of the lead mAbs, the cell-based competition assay was performed using human CD47-expressing stable cells and Jurkat tumor cells. Results were shown in Figure 5 and Table 10, demonstrating that the lead mAbs can competitively block the binding of human CD47 to its ligand (SIRPα) , as potent as 5F9.

Table 10. IC₅₀ of the antibodies

NA: Not applicable.

2.6 Human CD47 affinity (SPR)

Human CD47 binding affinity of W3456-P5R1-1C1-z19-uIgG4V1 was performed by SPR assay using Biacore T200. The lead antibody was captured on an anti-human IgG Fc antibody immobilized CM5 sensor chip (GE) . Human CD47 protein at different concentrations were injected over the sensor chip at a flow rate of 30 uL/min for an association phase of 180 s, followed by 3600 s dissociation. The chip was regenerated by 10 mM glycine (pH 1.5) after each binding cycle. The sensorgrams of blank surface and buffer channel were subtracted from the test sensorgrams. The experimental data was fitted by 1: 1 model using Langmiur analysis. Molecular weight of 55 kDa was used to calculate the molar concentration of analyte antigen.

Results

Human CD47 binding affinity of leads was performed by SPR. The affinity KD value was shown in Table 11 and binding kinetics curve were shown in Figure 6.

Table 11. Lead mAb human CD47 binding affinity result, measured by SPR

2.7 Thermal stability

Differential scanning fluorometry (DSF) was used to evaluate lead mAb thermal stability. Briefly, the T_m of antibodies was investigated using QuantStudio^TM 7 Flex Real-Time PCR system (Applied Biosystems) . 19 μL of antibody solution was mixed with 1 μL of 62.5 X SYPRO Orange solution (Invitrogen) and transferred to a 96 well plate (Biosystems) . The plate was heated from 26 ℃ to 95 ℃ at a rate of 0.9 ℃/min, and the resulting fluorescence data was collected. The negative derivatives of the fluorescence changes with respect to different temperatures were calculated, and the maximal value was defined as melting temperature T_m. If a protein has multiple unfolding transitions, the first two T_m were reported, named as T_m1 and T_m2, respectively. Data collection and T_m calculation were conducted automatically by the operation software.

Results

Differential scanning fluorometry (DSF) was used to evaluate lead mAb thermal stability. The data indicated lead mAb displayed good thermal stability in two different buffers as shown in Table 12 and Figure 7.

Table 12. Lead mAb thermal stability testing by DSF

2.8 Serum stability

Serum stability assay of the lead mAb was performed in human serum. Freshly collected human blood was statically incubated in polystyrene tubes without anticoagulant for 30 min at room temperature. Serum was collected after centrifugation the blood at 4000 rpm for 10 min. Gently mixing antibodies with serum and the serum-antibody mixture was incubated at 37℃. The samples were collected on day 0, day 1, day 4, day 7 and day14, respectively and were quickly-frozen down at the indicated time at -80℃ until use. The samples were used to assess their binding ability to human CD47-expressing cells. Briefly, serial dilutions of antibodies were added to CD47-expressing cells and incubated for 1 h at 4℃. The cells were washed two times with 200 μL PBS with 1%BSA. PE conjugated goat anti-human IgG Fc diluted 1: 150 with FACS buffer were added to the cells and incubated at 4℃ for 30 minutes. Additional washing steps were performed twice with 200 μL FACS buffer followed by centrifugation at 1500rpm for 4 min at 4℃. Finally, the cells were re-suspended in 100 μl FACS buffer and fluorescence values were measured by FACS and analyzed by FlowJo.

Results

Serum stability of lead mAb was performed in human serum. Lead mAb was co-cultured with human serum at 37℃ for 0, 1, 4, 7 and 14 days, respectively, and the binding activity was tested by FACS. The data showed serum culturing had no adverse effect on human CD47 binding ability of the lead mAb, as shown in Figure 8.

2.9 Non-specific protein binding (ELISA)

The non-specific binding against 16 different proteins was performed by ELISA. The 96-well high binding plates were coated with 16 different proteins (as indicated in Table 13) at 1 μg/mL at 4℃ overnight and blocked with 2%BSA-PBS for 1 h. The lead antibody at 10 μg/ml were added and incubated for 2 h. Then the Goat anti-human IgG-Fc-HRP secondary antibody was added and incubated for 1 h. The TMB peroxidase substrate solution was added and then reaction was stopped after 12 minutes using 2M HCl. All incubation steps were performed at room temperature, and the plates were washed 5 times with PBST at pH 7.4 between steps. The absorbance of testing samples was measured at 450 nm with a multiwall plate reader (M5e) .

Results

The non-specific binding testing of the lead mAb against 16 different proteins was performed by ELISA. The data indicated that the lead mAb showed no non-specific binding to the tested 12 proteins as shown in Table 13.

2.10 Non-specific cell binding (FACS)

The non-specific binding against 17 different cells (as indicated in Table 14, except CHO-K1 is from rodent animal, other 16 kinds of cells were human-derived. ) was performed by FACS. The 17 different cells were transferred into 96-well U-bottom plates with 1x10⁵ cells/well and centrifuged at 1500 rpm for 4 min at 4℃ before removing supernatant. The lead antibody at 10 μg/ml were added to the cells and incubated at 4℃ for 1 h. The cells are washed twice with 180 μL 1%BSA-PBS. Goat Anti-Human IgG Fc PE secondary antibody (Jackson, Catalog number 109-115-098) was added to re-suspended cells and incubated at 4 ℃ in the dark for 30 min. Additional washing steps are performed twice with 180 μL 1%BSA/1XPBS followed by centrifugation at 1500 rpm for 4 min at 4℃. Finally, the cells are re-suspended in 100 μL 1%BSA-PBS, then fluorescence intensity was measured by flow cytometry (BD Canto II) and analyzed by FlowJo.

Results

The non-specific binding testing of the lead mAb against 16 different human origin tumor cells and rat-derived CHO-K1 cells was conducted by FACS. The data showed lead mAb bind to all 16 human origin tumor cells tested, but did not bind with rat origin CHO-K1 cell, indicating CD47 is widely expressed on human tumor cells as shown in Table 14.

2.11 Phagocytosis assay (FACS)

Phagocytic activity of lead mAb was evaluated using human PBMC-derived macrophages and Raji cells, A375 tumor cells and human RBC as target cells. Human PBMCs were isolated from fresh human blood, and the CD14 positive monocytes were isolated from PBMC by hCD14 Microbeads. The CD14 positive monocytes were differentiated into macrophages by incubating them in the 10%FBS RPIM1640 medium with 100 ng/ml rhM-CSF for 7 days. These monocytes derived macrophages (MDMs) became adherent, allowing other cells to be washed away. MDMs were scraped and seeded into 96-well plates. Several human tumor cell lines or human RBCs were chosen as a target cell type because of their high CD47 expression. Target cells were labeled with 1μM CFSE at 37℃ for 30 min, then washed and added to MDMs at a ratio of 1: 1 tumor cells per phagocyte, and the anti-CD47 antibody was added at various concentrations. Phagocytosis of target cells was allowed for 2 hours. Subsequently, the cells were stained with an antibody to the macrophage marker CD14 conjugated to APC, and analyzed by flow cytometry. Phagocytosis was measured by gating on live cells which were FL4 positive (CD14+) , and then the percent of FL1 (CFSE+) positive cells was assessed. The macrophages that ingested tumor cells were counted and calculated as an index: phagocytosis Index %=Percentage _{CFSE+/CD14-APC+} / (Percentage _CFSE+/CD14- _APC+ + Percentage _{CFSE-/CD14-APC+}) x 100%.

Results

Phagocytic activity of lead mAb was evaluated using human PBMC-derived macrophages and tumor cells of Raji cells, A375 cells, and human red blood cells, respectively. The macrophages that ingested tumor cells were counted and calculated as an index: Phagocytosis Index %= Percentage _{CFSE+/CD14-APC+} / (Percentage _{CFSE+/CD14-APC+} + Percentage _{CFSE-/CD14-APC+}) x 100%. Lead mAb induced potent phagocytosis against tumor cells, while induced much low phagocytosis against human red blood cells, which were shown in Figure 9 and Table 15.

Table 15. Macrophage-mediated phagocytosis assay

(NA: Not applicable)

2.12 ADCC assay

Human PBMCs were used as effector cells and Raji cells were used as target cells. Human PBMCs were isolated from fresh human blood. 2x10⁴ target cells in 40 μL RPMI1640 (no phenol) medium containing 1%FBS were added per well in a 96-well U-bottom plate. Then, serial-diluted antibodies in 20 μL RPMI1640 (no phenol) medium containing 1%FBS were added to each well. After 15 min incubation at 37℃, 4 x10⁵ PBMC in 40 μL RPMI1640 (no phenol) medium containing 1%FBS were added to each well to give a 20: 1 E/T ratio. After incubation at 37℃ for 4 h, mixtures were centrifuged at 1500 rpm for 5 min and 70 μL of supernatant was transferred for detection. Cell death was evaluated using LDH Cytotoxicity Detection Kit (Roche) according to manufacturer’s instructions.

2.13 CDC assay

Raji cells were used as target cells. 2x10⁴ target cells in 40 μL RPMI1640 (no phenol) medium containing 1%FBS were added per well in a 96-well U-bottom plate. Then, serial-diluted antibodies in 20 μL RPMI1640 (no phenol) medium containing 1%FBS were added to each well. After 15 min incubation at 37℃, normal human complement in 40 μL RPMI1640 (no phenol) medium containing 1%FBS were added to each well. After incubation at 37℃ for 4 h, mixtures were centrifuged at 1500 rpm for 5 min and 70 μL of supernatant was transferred for detection. Cell death was evaluated using LDH Cytotoxicity Detection Kit (Roche) according to manufacturer’s instructions.

Results

The lead mAbs were evaluated for their ADCC and CDC ability against Raji tumor cells. The lead mAb with IgG1 format (i.e., W3456-P5R1-1C1-z19-uIgG1) showed strong ADCC against Raji cells, while IgG4 format-based 5F9 and TJ-C4 showed much weaker ADCC activity (Figure 10A) ; No CDC activity against Raji tumor cells was observed for both lead mAbs, as shown in Figure 10B.

Example 3. In vivo characterization

3.1 Anti-tumor efficacy in Raji/CB-17 SCID mice model

Lead mAb efficacy study was tested on Raji B lymphatic cancer model in CB-17 SCID mice. Female CB-17 SCID mice (Shanghai Lingchang Biotech Co., LTD) of 7-8 weeks old were used in the study. The parental Raji cell line was from ATCC. The cells were cultured in RPMI1640 medium supplemented with 10%heat inactivated fetal bovine serum at 37℃ in an atmosphere of 5%CO2 in air. The tumor cells were routinely sub-cultured 3 times a week. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

For the therapeutic model, the Raji cells (1.0×10⁶ cells/200μL Matrigel/PBS mixture) were inoculated subcutaneously into a CB-17 SCID mouse. Body weight was weighed and tumor growth was measured using calipers. When the tumor volume reached approximately 105 mm³, animals were randomly grouped into 6 groups (n=8/group) : G1 (IgG4 isotype control, 1 mg/kg) group, G2 (5F9, 1 mg/kg) group, G3 (TJ-C4, 1 mg/kg) group, G4 (W3456-P5R1-1C1-z19-uIgG4V1, 2 mg/kg) group, G5 (W3456-P5R1-1C1-z19-uIgG4V1, 0.5 mg/kg) group and G6 (W3456-P5R1-1C1-z19-uIgG4V1, 0.1 mg/kg) group, as shown in Table 16, and the day of grouping was considered as day 0. Then they were injected intraperitoneally twice per week for a total of 6 times at day 0, day 4, day 7, day 11, day 14 and day 18 post grouping, respectively. For all tumor-bearing mice, body weight was weighed and tumor growth was measured twice a week using calipers. All the procedures related to animal handling, care and the treatment in the study were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Model Organisms Animal Co., Ltd following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) .

Tumor volume was calculated with the formula 1/2 (length × width²) . The results were represented by mean and the standard error (Mean ± SEM) . Data were analyzed using Two way RM ANOVA Tukey’s multiple comparisons test with Graphpad Prism and p<0.05 was considered to be statistically significant. TGI (Tumor growth inhibition, %) = [1- (Ti-T0) / (Vi-V0) ] ×100%, wherein Ti and Vi refer to the mean tumor volume size in the treated and vehicle groups, respectively, on a given day. T0 and V0 refer to the mean tumor volume size in the treated and vehicle groups, respectively, on grouping day (i.e., day 0) .

Table 16. Anti-tumor efficacy study plan

Results

Lead mAb efficacy study was tested on Raji B lymphatic cancer model in CB-17 SCID mice. As shown in Figure 11A and Table 17a, at day 28 post grouping, mean body weight of the animals in G1 group to G6 group showed no significant reduction, which indicated W3456-P5R1-1C1-z19-uIgG4V1, 5F9 and TJ-C4 were not toxic.

As shown in Figure 11B and Table 17b, at day 28 post grouping, compared with the G1 (IgG4 isotype control, 1 mg/kg) vehicle group, the tumor growth inhibition (TGI %) of G2 group to G6 group were 89.58%, 77.85%, 99.59%, 86.75%and 76.68%, respectively. In addition, the data showed statistically significant difference (P<0.05) , which indicated the obvious anti-tumor efficacy of W3456-P5R1-1C1-z19-uIgG4V1, 5F9 and TJ-C4. Furthermore, W3456-P5R1-1C1-z19-uIgG4V1 showed similar efficacy to 5F9 and much better efficacy than TJ-C4.

Table 17a. Body weight before grouping and day 28 post grouping

Notes:

a. Mean ± SEM;

b. Statistical analysis of mean body weight at day 28 post grouping using T-test between G1 group and the treatment group.

Table 17b. Tumor growth inhibition (TGI) at day 28 post grouping

Note:

a. Mean ± SEM;

3.2 Anti-tumor efficacy in MDA-MB-231/CB-17 SCID mice model

As for the efficacy of the Lead mAb, a study was tested on MDA-MB-231 human triple negative breast cancer model in CB-17 SCID mice. Female CB-17 SCID mice (Shanghai Lingchang Biotech Co., LTD) of 6-8 weeks old were used in the study. The parental MDA-MB-231 cell line was from Creative Bioarray Co., LTD. The cells were cultured in L-15 medium (ATCC) supplemented with 10%heat inactivated fetal bovine serum and 1%Penicillin Streptomycin at 37℃ in an atmosphere without CO₂ in air. The tumor cells were routinely sub-cultured 3 times a week. The tumor cells growing in an exponential growth phase were harvested and counted for tumor inoculation.

For the therapeutic model, the MDA-MB-231 cells (4.5×10⁶ cells/200 μL in Matrigel/PBS mixture) were inoculated subcutaneously into CB-17 SCID mouse. Body weight was weighed and tumor growth was measured using calipers. When the tumor volume reached approximately 150 mm³, animals were randomly grouped into 10 groups (n=7/group) : G1 (IgG4 isotype control, 1 mg/kg) group, G2 (IgG1 isotype control, 1 mg/kg) group, G3 (5F9, 1 mg/kg) group, G4 (TJ-C4, 1 mg/kg) group, G5 (W3456-P5R1-1C1-z19-uIgG4V1, 2 mg/kg) group, G6 (W3456-P5R1-1C1-z19-uIgG4V1, 0.5 mg/kg) group, G7 (W3456-P5R1-1C1-z19-uIgG4V1, 0.1 mg/kg) group, G8 (W3456-P5R1-1C1-z19-uIgG1, 2 mg/kg) group, G9 (W3456-P5R1-1C1-z19-uIgG1, 0.5 mg/kg) group, and G10 (W3456-P5R1-1C1-z19-uIgG1, 0.1 mg/kg) group, as shown in Table 18, and the day of grouping was considered as day 0. Then they were injected intraperitoneally twice per week for a total of 6 times at day 0, day 3, day 7, day 10, day 14 and day 17 post grouping, respectively. For all tumor-bearing mice, body weight was weighed and tumor growth was measured twice a week using calipers. All the procedures related to animal handling, care and the treatment in the study were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Model Organisms Co., LTD following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) .

Tumor volume was calculated with the formula 1/2 (length × width²) . The results were represented by mean and the standard error (Mean ± SEM) . Data were analyzed using Two way RM ANOVA Tukey’s multiple comparisons test with Graphpad Prism and p<0.05 was considered to be statistically significant.

TGI (%) = [1- (Ti-T0) / (Vi-V0) ] ×100%, wherein Ti and Vi refer to the mean tumor volume size in the treated and vehicle groups, respectively, on a given day. T0 and V0 refer to the mean tumor volume size in the treated and vehicle groups, respectively, on grouping day (i.e., day 0) .

Table 18. Anti-tumor efficacy study plan

Results

As shown in Figure 12A and Table 19a, at day 32 post grouping, mean body weight of the animals in G1 group to G10 group showed no significant reduction, which indicated W3456-P5R1-1C1-z19-uIgG4V1, W3456-P5R1-1C1-z19-uIgG1, W345-BMK2 and W345-BMK8 were not significantly toxic.

As shown in Figure 12B and Table 19b, at day 32 post grouping, compared with the G1 (W332-1.80.12. xAb. hIgG4, 1 mg/kg) vehicle group, the tumor growth inhibition (TGI %) of G2 group to G10 group were 23.58%, 77.31%, 40.19%, 87.17%, 86.78%, 38.70%, 106.93%, 109.61%and 73.36%, respectively. In addition, the data showed statistically significant difference (P<0.05) , which indicated the obvious anti-tumor efficacy of W3456-P5R1-1C1-z19-uIgG4V1, W3456-P5R1-1C1-z19-uIgG1, W345-BMK2 and W345-BMK8. Furthermore, W3456-P5R1-1C1-z19-uIgG4V1 and W3456-P5R1-1C1-z19-uIgG1 at the dosage of 2 mg/kg and 0.5 mg/kg showed better efficacy than W345-BMK2 at the dosage of 1 mg/kg, and much better efficacy than W345-BMK8 at the dosage of 1 mg/kg. W3456-P5R1-1C1-z19-uIgG4V1 at the dosage of 0.1 mg/kg showed similar efficacy to W345-BMK8 at the dosage of 1 mg/kg. W3456-P5R1-1C1-z19-uIgG1 at the dosage of 0.1 mg/kg showed similar efficacy to W345-BMK2 at the dosage of 1 mg/kg.

Therefore, W3456-P5R1-1C1-z19-uIgG4V1 and W3456-P5R1-1C1-z19-uIgG1 mAbs displayed significant anti-tumor activity. Furthermore W3456-P5R1-1C1-z19-uIgG4V1 and W3456-P5R1-1C1-z19-uIgG1 mAbs showed better efficacy than W345-BMK2, and much better efficacy than W345-BMK8.

Table 19a. Body weight before grouping at day 32 post grouping

Notes: a. Mean ± SEM;

b. Statistical analysis of mean body weight at day 32 post grouping using T-test between G1 group and the treatment group.

Table 19b. Tumor growth inhibition (TGI) at day 32 post grouping

Note: a. Mean ± SEM.

Example 4. Pharmacokinetics and toxicology studies in cynomolgus monkeys

To determine the pharmacokinetics and preliminary toxic effects of the lead antibody, four male and four female monkeys (3.0 to 5.0 years old and body weight range was from 2.620 kg to 5.256 kg at initiation of dosing) were randomly assigned to four dosing groups (Group 1～Group 4, 1 male + 1 female, i.e., 1 M+1 F) , and injected intravenously (Group 1 and 2: W3456-P5R1-1C1-z19-uIgG1; Group 3 and 4: W3456-P5R1-1C1-z19-uIgG4V1) at different dose level (Group 1 and 3: 10 mg/kg; Group 2 and 4: 30 mg/kg) on Day 0, as shown in Table 20. Assessments included mortality, clinical observations, body weight, food consumption, body temperature and clinical pathology. Immunological and pharmacokinetics analysis was also performed.

Table 20. Grouping and testing antibody dosing information

Results

The summary for PK parameters was listed in Table 21.

Table 21. PK parameters

In summary, for W3456-P5R1-1C1-z19. uIgG1, the systemic exposure for Cmax increased 2.7-fold, while 4.6-fold for AUC0-t as the dosage increased from 10 to 30 mg/kg. For W3456-P5R1-1C1-z19. uIgG4V1, the systemic exposure for Cmax increased 3.2-fold, while 5.8-fold for AUC0-t as the dosage increased from 10 to 30 mg/kg. The clearance of W3456-P5R1-1C1-z19. uIgG1 at the dosage of 10 mg/kg and 30 mg/kg was 57.8 mL/day/kg, 37.2 mL/day/kg, and W3456-P5R1-1C1-z19. uIgG4V1 was 36.4 mL/day/kg, 19.1 mL/day/kg, respectively. For the two Abs, the pharmacokinetic parameter results showed a dose-dependent situation.

All animals were not ADA (anti-drug antibody) positives before 7 days, 2501 (female) and 3501 (female) were ADA negative at all time points. 1001 (male) was positive for ADA on the day 10, day 14 and day 28 after injection, and 2001 (male) , 3001 (male) , 4001 (male) were positive at day 14 and day 28.1501 (female) and 3501 (female) were positive for ADA on the day 28 after injection.

The concentrations of IL-6 in serum changed significantly at 1h after W3456-P5R1-1C1-z19-IgG1 injection at 30 mg/kg. No significant change of IL-2, TNF, IFN-γ, IL-4 and IL-5.

As shown in Figure 13 and Table 21, W3456-P5R1-1C1-z19-uIgG4V1 and W3456-P5R1-1C1-z19-uIgG1 mAbs had good stability in serum.

As shown in Figure 15, no change in T cells of all animals after administration. Animals of 1001 and 1501 slightly increased in B cells on day 1 after administration, then decreased on day 3 to day 7, and returned to normal levels on day 28.

As shown in Figure 14, referred to reference range, during the period from Day 1 to Day 21 after dosing, RBC, HGB, HCT and PLT values were decreased, and RT value was increased in the monkeys of Group 1 and 2, meanwhile, decreased PLT and increased RT values were observed in group 3 and 4, but all the changes could be restored on Day 28.

The results shown in Figures 14 and 15 were obtained from monkeys, and could demonstrate that the antibodies of the present application were safe enough for use in mammal subjects, e.g., human subjects.

Referred to reference range, during the period from Day 1 to Day 7 after dosing, the clinical chemistry parameters of each group have a transient increase mainly including the following: TBIL, DBIL and IBIL values in the monkeys of Group 2 and 3, female monkey of Group 1 and male monkey of Group 4; CK and LDH values in the monkeys of Group 2 and 3 and female monkey of Group 1; AST value in the monkeys of Group 2 and 3; ALT value in the monkeys of Group 2. But all the increased parameters above were returned to normal.

Those skilled in the art will further appreciate that the present invention may be embodied in other specific forms without departing from the spirit or central attributes thereof. In that the foregoing description of the present invention discloses only exemplary embodiments thereof, it is to be understood that other variations are contemplated as being within the scope of the present invention. Accordingly, the present invention is not limited to the particular embodiments that have been described in detail herein. Rather, reference should be made to the appended claims as indicative of the scope and content of the invention.

References

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Claims

An isolated anti-CD47 antibody or the antigen-binding portion thereof, wherein the isolated antibody or the antigen-binding portion thereof comprises a heavy chain variable region comprising:

(a) CDR1 comprising SEQ ID NO: 1, or an amino acid sequence having at least 90%sequence identity with SEQ ID NO: 1, or an amino acid sequence differing from SEQ ID NO: 1 by an amino acid addition, deletion or substitution of not more than 1 amino acid;

(b) CDR2 comprising SEQ ID NO: 2, or an amino acid sequence having at least 85%or 90%sequence identity with SEQ ID NO: 2, or an amino acid sequence differing from SEQ ID NO: 2 by amino acid addition, deletion or substitution of not more than 2 amino acids, and

(c) CDR3 comprising SEQ ID NO: 3.
The isolated anti-CD47 antibody or the antigen-binding portion thereof according to claim 1, wherein the anti-CD47 antibody is a single domain antibody, preferably a humanized single domain antibody.
The isolated anti-CD47 antibody or the antigen-binding portion thereof according to claim 1, wherein the anti-CD47 antibody or the antigen-binding portion thereof comprises a heavy chain variable region comprising:

an amino acid sequence represented by SEQ ID NO: 4 or 5, or

an amino acid sequence having at least 80%, 85%, 90%, 95%or 99%sequence identity with SEQ ID NO: 4 or 5 while maintaining similar binding specificity as SEQ ID NO: 4 or 5, or

an amino acid sequence differing from SEQ ID NO: 4 or 5 by amino acid addition, deletion or substitution of one or more amino acids while maintaining similar binding specificity as SEQ ID NO: 4 or 5.
The isolated anti-CD47 antibody or the antigen-binding portion thereof according to any one of the preceding claims, further comprising a Fc region, preferably human IgG1 Fc region or human IgG4 Fc region, more preferably human IgG4 Fc region with mutation (s) .
The isolated anti-CD47 antibody or the antigen-binding portion thereof according to any one of the preceding claims, wherein the anti-CD47 antibody or the antigen-binding portion thereof comprises or consists of:

an amino acid sequence represented by SEQ ID NO: 7 or 9, or

an amino acid sequence having at least 80%, 85%, 90%, 95%or 99%sequence identity with SEQ ID NO: 7 or 9 while maintaining similar binding specificity as SEQ ID NO: 7 or 9, or

an amino acid sequence differing from SEQ ID NO: 7 or 9 by amino acid addition, deletion or substitution of one or more amino acids while maintaining similar binding specificity as SEQ ID NO: 7 or 9.
A conjugate comprising the anti-CD47 antibody or the antigen-binding portion thereof according to any one of the preceding claims and one or more moieties conjugated to the antibody or the antigen-binding portion thereof, wherein the conjugated moieties are selected from detectable labels, pharmacokinetic modifying moieties, purification moieties, moieties for targeted delivery, half-life improving moieties, or cytotoxic moieties.
A pharmaceutical composition, comprising at least one anti-CD47 antibody or antigen-binding portion thereof according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.
An isolated nucleic acid molecule, comprising a nucleic acid sequence encoding the heavy chain variable region or the heavy chain of the anti-CD47 antibody or antigen-binding portion thereof according to any one of claims 1 to 5.
A vector, comprising the isolated nucleic acid molecule according to claim 8.
A host cell, comprising the isolated nucleic acid molecule according to claim 8 or the vector according to claim 9.
A method for preparing an anti-CD47 antibody or antigen-binding portion thereof according to any one of claims 1 to 5, comprising expressing the anti-CD47 antibody or antigen-binding portion thereof in the host cell according to claim 10 and isolating the anti-CD47 antibody or antigen-binding portion thereof from the host cell.
A method for preventing or treating CD47-related diseases in a subject, comprising administering the anti-47 antibody or antigen-binding portion thereof according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 7 to the subject.
The method according to claim 12, wherein the CD47-related diseases are hematologic diseases or solid tumors, preferably acute myelocytic leukemia (AML) , non-Hodgkin’s lymphoma (NHL) , breast cancer, non-small-cell lung cancer (NSCC) , ovarian cancer, myelodysplastic syndrome, brain cancer, squamous cell carcinoma of the head and neck, and advanced malignant tumors.
The method according to claim 12, further comprising administrating other pharmaceutical formulations or other therapies for combined therapy.
A method for improving clinical prognosis in a subject in need thereof, comprising administering the anti-47 antibody or antigen-binding portion thereof according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 7 to the subject.
A method for increasing tumor cells phagocytosis in a subject, comprising administering the anti-47 antibody or antigen-binding portion thereof according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 7 to the subject.
A method for increasing tumor cells phagocytosis in vitro, comprising contacting the tumor cells with the anti-47 antibody or antigen-binding portion thereof according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 7.
A method for inhibiting growth of tumor cells or reducing metastasis of tumor cells in a subject, comprising administering an effective amount of the anti-47 antibody or antigen-binding portion thereof according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 7.
The method according to any one of claims 12 to 18, wherein the subject is a mammal, preferably a human.
A kit, comprising a container which comprises at least one anti-CD47 antibody or antigen-binding portion thereof according to any one of claims 1 to 5, and an instruction.