TW201819413A - CD47 antibody, antigen-binding fragments and pharmaceutical use thereof - Google Patents

Abstract

The present invention relates to a CD47 antibody, antigen-binding fragments, and pharmaceutical use thereof. Furthermore, the present invention relates to chimeric and humanized antibodies comprising the CDR regions of the present CD47 antibody, as well as pharmaceutical compositions comprising the present CD47 antibody and antigen-binding fragments thereof, and their use as anti-cancer drugs. In particular, the present invention relates to a humanized CD47 antibody, and its use in preparation of a medicament for the treatment of CD47 pathway related disease.

Description

   CD47 antibody, its antigen-binding fragment and its medical use   

The present disclosure relates to the fields of biology and medicine; more specifically, the present disclosure relates to a CD47 antibody and an antigen-binding fragment thereof, a chimeric antibody, a humanized antibody thereof, and a pharmaceutical composition including the CD47 antibody or an antigen-binding fragment thereof. And its use as an anticancer drug.

With the deepening of human understanding of the immune system and tumorigenesis mechanisms, tumor immunotherapy has increasingly become a powerful weapon for humans to fight against tumors.

Tumor-targeting monoclonal antibodies are one of the important methods in the field of tumor immunotherapy. Macrophages need two signals to exert phagocytosis effect at the same time: one is the activation of the "eat me" signal on the cell surface, and the other is the inactivation of the "don't eat me" signal on the same cell surface. The absence of any one signal is not enough to trigger phagocytosis. Increasing evidence shows that CD47 is a type of "don't eat me" signal, which inhibits the phagocytosis of macrophages by interacting with SIRP-α on the surface of macrophages.

CD47 is a member of the immunoglobulin Ig superfamily, which is widely expressed on the cell surface of different tissues, such as red blood cells, lymphocytes, platelets, and liver cells. CD47 is highly expressed on various tumor cells. Studies have shown that the expression of CD47 on the surface of many tumor cells is about three times higher than that of normal cells on average. In addition, patients with cancer cells expressing large amounts of CD47 have shorter survival periods than patients with low levels of CD47 expression. Current research has found that blocking anti-CD47 monoclonal antibodies have very good effects in tumor treatment, but the mechanism of action of this process is unclear.

CD47 as a target for cancer treatment has the following incomparable advantages: 1. It is widely expressed on the surface of various cancer cells, so it can be used to treat various types of cancer; 2. Normal cells lack the "eat me" signal, so Blocking CD47 alone does not trigger the phagocytic effect of macrophages on normal cells. Therefore, the side effects of CD47 blockers are very small. Many previous basic experimental data have already supported this view. The researchers demonstrated the effectiveness and safety of CD47 blockade in a mouse allogeneic tumor transplantation model.

There are currently related CD47 patents, such as WO2016065329, WO2016109415, WO2014087248 and WO2014093678. It can effectively block the binding between CD47 and SIRP-α and promote the phagocytosis of tumor cells by macrophages in vivo, which has good clinical prospects.

A first aspect of the present disclosure provides a CD47 antibody or antigen-binding fragment thereof, comprising: a CDR region of an antibody heavy chain variable region (hereinafter referred to as HCDR) and / or a CDR region of an antibody light chain variable region (hereinafter referred to as HCDR) LCDR).

In some embodiments, the HCDR is selected from any one or more of the following CDRs: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and CDRs having at least 85% sequence identity with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 .

In other embodiments, the LCDR is selected from any one or more of the following CDRs: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and at least 85% sequence identity with SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 CDR.

In the context of this disclosure, the expression "at least 85% sequence identity" means at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99%, 100% sequence identity.

In a specific embodiment of the present disclosure, a humanized antibody of CD47 or an antigen-binding fragment thereof is provided, which comprises an antibody heavy chain variable region; wherein the antibody heavy chain variable region comprises SEQ ID NO: 8, SEQ HCDR1, HCDR2 and HCDR3 shown in ID NO: 9 and SEQ ID NO: 10, or CDRs having at least 85% sequence identity with SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.

In another specific embodiment, a CD47 humanized antibody or antigen-binding fragment thereof is provided, which comprises an antibody heavy chain variable region; wherein the antibody heavy chain variable region comprises SEQ ID NO: 14, SEQ ID HCDR1, HCDR2 and HCDR3 shown in NO: 15 and SEQ ID NO: 16, or a CDR having at least 85% sequence identity with SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

In a specific embodiment of the present disclosure, a humanized antibody of CD47 or an antigen-binding fragment thereof is provided, which comprises an antibody light chain variable region; wherein the antibody light chain variable region comprises SEQ ID NO: 11, SEQ LCDR1, LCDR2 and LCDR3 shown in ID NO: 12 and SEQ ID NO: 13, or a CDR having at least 85% sequence identity with SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13.

In another specific embodiment, a humanized antibody of CD47 or an antigen-binding fragment thereof is provided, which comprises an antibody light chain variable region; wherein the antibody light chain variable region comprises SEQ ID NO: 17, SEQ ID LCDR1, LCDR2 and LCDR3 shown in NO: 18 and SEQ ID NO: 19, or a CDR having at least 85% sequence identity with SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19.

In a specific embodiment of the present disclosure, a CD47 antibody or an antigen-binding fragment thereof is provided, which is a mouse-derived antibody or an antigen-binding fragment thereof. In a specific embodiment of the present disclosure, there is provided a CD47 antibody or an antigen-binding fragment thereof, wherein the light chain variable region of the antibody comprises a murine light chain FR region or a mutant sequence thereof; and the heavy chain variable region of the antibody includes Murine heavy chain FR region or its mutated sequence.

In a specific embodiment of the present disclosure, there is provided a CD47 antibody or an antigen-binding fragment thereof, the antibody comprising a heavy chain variable region shown in SEQ ID NO: 4 and a light chain variable shown in SEQ ID NO: 5 Area. In another embodiment, a CD47 antibody or an antigen-binding fragment thereof is provided, the CD47 antibody comprising a heavy chain variable region shown in SEQ ID NO: 6 and a light chain variable region shown in SEQ ID NO: 7.

In a specific embodiment of the present disclosure, a CD47 antibody or an antigen-binding fragment thereof is provided, which is a chimeric antibody or an antigen-binding fragment thereof.

In a specific embodiment of the present disclosure, a CD47 humanized antibody or an antigen-binding fragment thereof is provided. The sequence of the heavy chain FR region on the heavy chain variable region is selected from human germline heavy chain IGHV1-3 * 01 and A combination of hjh6.1 and a mutant sequence thereof, or a combination selected from a human germline heavy chain IGHV1-2 * 02 and hjh6.1 and a mutant sequence thereof.

In a specific embodiment of the present disclosure, a CD47 humanized antibody or an antigen-binding fragment thereof is provided. The sequence of the light chain FR region on the light chain variable region is selected from human germline light chain IGKV4-1 * 01 and hjk2 The combination of .1 and a mutant sequence thereof, or a combination selected from a human germline light chain IGKV1-39 * 01 and hjk4.1 and a mutant sequence thereof.

In a specific embodiment of the present disclosure, a CD47 humanized antibody or antigen-binding fragment thereof is provided. The variable region of the humanized antibody or antigen-binding fragment thereof contains 0 to 10 amino acids. Back mutations include, but are not limited to, back mutations of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

In a specific embodiment of the present disclosure, a CD47 humanized antibody or an antigen-binding fragment thereof is provided, which comprises the heavy chain variable region shown in SEQ ID NO: 20 or a mutant sequence thereof and SEQ ID NO: 21 The light chain variable region or its mutated sequence is shown.

In another specific embodiment of the present disclosure, a CD47 humanized antibody or an antigen-binding fragment thereof is provided, which comprises the heavy chain variable region shown in SEQ ID NO: 22 or a mutant sequence thereof and SEQ ID NO: 23 The light chain variable region or its mutated sequence is shown.

In a specific embodiment of the present disclosure, a CD47 humanized antibody or an antigen-binding fragment thereof is provided, which comprises a light chain variable region mutation sequence, which is described in SEQ ID NO: 21 Based on the light chain variable region shown, the 66th amino acid residue D is replaced with E (denoted by D66E, the same applies hereinafter).

In a specific embodiment of the present disclosure, a CD47 humanized antibody or an antigen-binding fragment thereof is provided, which comprises a heavy chain variable region mutation sequence, and the heavy chain variable region mutation sequence is shown in SEQ ID NO: 20. Based on the shown heavy chain variable region, it contains a mutation selected from the group consisting of R72A, M48I, E46D, V68A, I70L, R38K, R67K, A97S, and combinations thereof. In a specific embodiment, the heavy chain variable region mutation sequence is based on the heavy chain variable region shown in SEQ ID NO: 20 and comprises a mutation selected from the following: R72A, M48I, E46D, V68A, I70L, and Its combination.

In a specific embodiment of the present disclosure, a CD47 humanized antibody or an antigen-binding fragment thereof is provided, which comprises a light chain variable region mutation sequence, which is described in SEQ ID NO: 23. Based on the light chain variable region shown, it contains mutations selected from the group consisting of V58I, I2V, M4I, Q38E, A43T, P44H, and combinations thereof. In a specific embodiment, the light chain variable region mutation sequence is based on the light chain variable region shown in SEQ ID NO: 23, and comprises mutations I2V, M4I, and combinations thereof selected from the following.

In a specific embodiment of the present disclosure, there is provided a CD47 humanized antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region mutation sequence, wherein the heavy chain variable region mutation sequence is shown in SEQ ID NO: 22. Based on the shown heavy chain variable region, it contains a mutation selected from the group consisting of R72V, M48V, V68A, M70L, T74K, A40R, R38K, R67K, and combinations thereof.

In a specific embodiment of the present disclosure, a CD47 antibody or an antigen-binding fragment thereof is provided, wherein the humanized antibody variable region comprises: the heavy chain variable region shown in SEQ ID NO: 26 or a mutant sequence thereof , And the light chain variable region or its mutant sequence shown in SEQ ID NO: 27; or the humanized antibody variable region comprises: the heavy chain variable region or its mutant sequence shown in SEQ ID NO: 28, and The light chain variable region shown in SEQ ID NO: 29 or a mutant sequence thereof.

In a specific embodiment of the present disclosure, there is provided a CD47 chimeric antibody or CD47 humanized antibody whose heavy chain comprises: ‧ a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a mutant sequence thereof; and ‧ Light chain constant region of human κ chain, λ chain or its mutant sequence.

In a specific embodiment of the present disclosure, a CD47 chimeric antibody or a CD47 humanized antibody is provided, whose heavy chain comprises: ‧ a heavy chain constant region of human IgG4 or a mutant sequence thereof, and ‧ of a human κ chain Light chain constant region or its mutated sequence.

In a specific embodiment of the present disclosure, a CD47 chimeric antibody or a CD47 humanized antibody is provided, whose heavy chain comprises: a human IgG4 heavy chain constant region containing F234A and L235A mutations (such as SEQ ID NO: 30) .

The present disclosure further provides a pharmaceutical composition comprising: a therapeutically effective amount of a CD47 antibody or an antigen-binding fragment thereof according to the present disclosure, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

The CD47 antibody or antigen-binding fragment thereof according to the present disclosure may be formulated in a composition as the only pharmaceutically active ingredient in a pharmaceutical composition, or may be formulated in a composition in combination with other active ingredients.

As used herein, "carrier" includes a pharmaceutically acceptable carrier, diluent, or excipient that is not toxic when contacted with a cell or mammal at the dosages and concentrations used. A pharmaceutically acceptable carrier generally refers to a buffered aqueous solution. Examples of pharmaceutically acceptable carriers include, but are not limited to, buffers; antioxidants; polypeptides; proteins; hydrophilic polymers; amino acids; sugars; chelating agents; sugar alcohols; ions; surfactants. As another example, a pharmaceutically acceptable carrier, diluent, or excipient can also be in the form of liposomes, including liposomes that target cancer tissues can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

A "therapeutically effective amount" can be determined by testing the CD47 antibody or antigen-binding fragment thereof of the disclosure in known in vitro and in vivo systems, for example, based on an effective amount that has been demonstrated in an animal body, and then inferred from The amount of people.

For systemic, external or local administration, the CD47 antibody or antigen-binding fragment thereof of the present disclosure is mixed with a suitable pharmaceutical carrier to form a pharmaceutical composition. The concentration of the CD47 antibody or antigen-binding fragment thereof in the composition depends on the absorption, inactivation, metabolic rate, dosing schedule, dosage, dosage form, and other factors known to those skilled in the art of the antibody or antigen-binding fragment thereof.

The pharmaceutical composition of the present disclosure can be administered by any route known to those skilled in the art, such as, but not limited to, oral, topical, intra-brain, intraocular, intracardial, intrathecal, Intravenous, intramuscular, intraperitoneal, intradermal, intratracheal, and combinations thereof.

The most suitable route of administration will vary depending on the intended use. For example, for the treatment of various cancers, such as breast cancer, bladder cancer, and gastrointestinal cancer, topical administration, including administration to the site of tumor growth, is preferred because the advantage lies in the CD47 antibody or its antigen of the present disclosure. Binding fragments can be administered at higher concentrations.

Solutions or suspensions for parenteral, intradermal and subcutaneous applications, including but not limited to: sterile diluents; antibacterials; antioxidants; buffers; and agents for adjusting osmotic pressure. Formulations for parenteral administration can be enclosed in ampoules, syringes or single or multiple dose vials made of glass, plastic or other suitable materials.

The present disclosure further provides a bispecific antibody comprising a CD47 antibody or an antigen-binding fragment thereof according to the present disclosure.

The present disclosure further provides an isolated nucleic acid encoding a CD47 antibody or an antigen-binding fragment thereof according to the present disclosure.

The present disclosure further provides an expression vector expressing a CD47 antibody or an antigen-binding fragment thereof according to the present disclosure.

The present disclosure further provides an expression vector comprising an isolated nucleic acid according to the present disclosure.

The present disclosure further provides a host cell transformed with an expression vector according to the present disclosure.

In a specific embodiment, the host cell is selected from a prokaryotic cell or a eukaryotic cell. In a specific embodiment, the host cell is a eukaryotic cell. In a specific embodiment, the host cell is a mammalian cell.

The present disclosure further provides a method for preparing a CD47 antibody and an antigen-binding fragment thereof, comprising: ‧ culturing a host cell as described above under conditions suitable for expressing the CD47 antibody or an antigen-binding fragment thereof, and The CD47 antibody or antigen-binding fragment thereof of the present disclosure is isolated from a host cell.

The present disclosure further provides a method for inhibiting the growth of tumor cells in an individual, comprising: administering to the individual a CD47 antibody or an antigen-binding fragment thereof of the present disclosure, or a pharmaceutical composition of the present disclosure.

The present disclosure further provides the use of the CD47 antibody or antigen-binding fragment thereof of the present disclosure in the manufacture of a medicament for treating cancer. According to another aspect, there is provided the use of a pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating cancer. According to yet another aspect, the use of a bispecific antibody of the present disclosure in the manufacture of a medicament for treating cancer is provided.

In some embodiments, the CD47 antibody or antigen-binding fragment thereof of the present disclosure is capable of treating cancer, including, but not limited to: ovarian cancer, melanoma, prostate cancer, bowel cancer, gastric cancer, esophageal cancer, breast cancer, lung cancer, Kidney cancer, pancreatic cancer, uterine cancer, liver cancer, bladder cancer, cervical cancer, oral cancer, brain cancer, testicular cancer, skin cancer, thyroid cancer, and hematological malignancies. In some embodiments, the hematological malignancy is selected from the group consisting of myeloma, chronic leukemia, and acute leukemia.

Figure 1 shows that CD47 antibodies of the present disclosure enhance antibody-dependent cell phagocytosis (ADCP) of CCRF-CEM cells in vitro. Means hu055-5; Means hu167-33; Represents 5F9.

FIG. 2 is a erythrocyte agglutination experiment of the CD47 antibody of the present disclosure.

Figure 3 is a erythrocyte lysis experiment of the CD47 antibody of the present disclosure.

Figure 4 shows the pharmacokinetics of CD47 antibody hu055-5 in the present disclosure.

Figure 5 shows the pharmacokinetics of CDhu antibody hu167-33 in the present disclosure in rats.

the term

To make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Except where it is clear that it is otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

The three-letter and one-letter codes for amino acids used in this disclosure are described in J. biol. Chem, 243, p3558 (1968).

CD47 is a widely expressed cell membrane surface immunoglobulin, also known as integrin-related protein. Singal regulatory protein (SIRP) is a member of the inhibitory receptor superfamily and belongs to the immunoglobulin superfamily. It is mainly expressed on the surface of macrophages, dendritic cells and nerve cells, and is regulated by the cell surface. Body contact regulates cell migration and phagocytosis, immune homeostasis, and neuronal networks. CD47 is an extracellular ligand of human signal-regulating protein α (SIRP α). By binding to SIRP α on the surface of macrophages, it conducts inhibitory signals to reduce phagocytic activity, thereby suppressing the innate immune system. This signal is visualized Described as a "don't eat me" signal.

The antibody in the present disclosure refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by an interchain disulfide bond. The composition and arrangement of amino acids in the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. According to this, immunoglobulins can be divided into five categories, or isotypes called immunoglobulins, that is, IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain. The same type of Ig can be divided into different subclasses according to the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into a kappa chain and a lambda chain by different constant regions. Each of the five types of Ig can have a κ chain and a λ chain.

In the present disclosure, the antibody light chain may further include a light chain constant region, which includes a human or murine κ, λ chain constant region, or a mutant sequence thereof.

In the present disclosure, the antibody heavy chain may further include a heavy chain constant region comprising a human or murine IgG1,2,3,4 constant region or a mutant sequence thereof.

The sequence of about 110 amino acids near the N-terminus of the heavy and light chains of the antibody varies greatly and is a variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions (C region). The variable region includes three hypervariable regions (HVR) and four backbone regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of an antibody, also known as complementarity determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) are composed of three CDR regions and four FR regions. The sequence from amine end to carboxyl end is: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

The CDR amino acid residues of the LCVR region and HCVR region of the antibody or its antigen-binding fragment according to the invention conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3) in terms of number and position, or meet the requirements of Kabat and chothia. Numbering rules (HCDR1).

The term "murine antibody" in this disclosure is a monoclonal antibody to human CD47 prepared according to the knowledge and skill in the art. The test article is injected with CD47 antigen during preparation, and then the antibody having the desired sequence or functional characteristics is isolated and expressed. In a preferred embodiment of the present disclosure, the mouse-derived CD47 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a mouse-derived κ, λ chain or a mutant sequence thereof, or further include a mouse-derived IgG1, IgG2, The heavy chain constant region of IgG3 or a mutant sequence thereof.

The term "chimeric antibody" is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, and can reduce the immune response response induced by the murine antibody. To establish a chimeric antibody, a fusion tumor that secretes a mouse-specific monoclonal antibody is first established, and then the variable region gene is cloned from mouse fusion tumor cells, and then the human antibody constant region gene is cloned as needed. The variable region gene and the human constant region gene are linked into a chimeric gene and inserted into a vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system. In a preferred embodiment of the present invention, the antibody light chain of the chimeric antibody further comprises a light chain constant region of a human-derived κ, λ chain, or a mutant sequence thereof. The antibody heavy chain of the chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4, or a mutant sequence thereof, preferably a human IgG2 or IgG4 heavy chain constant region, or is significantly changed by amino acid mutation IgG4 constant region that reduces ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity.

The term "humanized antibody", also known as a CDR-grafted antibody, refers to an antibody produced by transplanting a mouse CDR sequence into a human antibody variable region framework (FR). It can overcome the side effect of the chimeric antibody on the human body induced by the large amount of mouse protein components. Human germline antibody variable region framework (FR) sequences can be obtained from the website of ImMunoGeneTics (IMGT) http://imgt.cines.fr, or from the Journal of Immunoglobulins, 2001ISBN012441351. In order to avoid the decrease in activity caused by the decrease in immunogenicity, reverse mutation (back mutation) of the human antibody variable region may be performed to maintain the activity.

The "antigen-binding fragment" described in the present disclosure refers to a Fab fragment having an antigen-binding activity, a Fab 'fragment, an F (ab') 2 fragment, and an Fv fragment and an scFv fragment that bind to human CD47. The Fv fragment contains the variable region of the antibody heavy and light chains, but has no constant region, and has the smallest antibody fragment with all the antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding. The variable regions of two antibodies can also be linked into a single polypeptide chain with different linkers, called a single chain antibody or a single chain Fv (scFv).

The term "binding to CD47" in the present disclosure refers to the ability to interact with human CD47.

The term "antigen-binding site" in the present disclosure refers to a three-dimensional spatial site on the antigen that is discontinuous and recognized by the antibodies or antigen-binding fragments of the present disclosure.

The "ADCC" in the present disclosure, that is, antibody-dependent cell-mediated cytotoxicity, refers to that an Fc receptor-expressing cell directly kills the antibody package by recognizing the Fc segment of the antibody. Targeted cells.

The fusion protein described in the present disclosure is a protein product in which the two genes obtained are expressed by DNA recombination. Recombinant CD47 extracellular region Fc fusion protein A fusion protein that expresses the extracellular region of CD47 and human antibody Fc fragments by DNA recombination. The CD47 extracellular region refers to a part of the CD47 protein expressed outside the cell membrane, and the sequence is shown in SEQ ID NO: 1.

Methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as Cold Spring Harbor's antibody experimental technical guide, Chapters 5-8 and 15. For example, a mouse can be immunized with human CD47 or a fragment thereof. The obtained antibody can be coated, purified, and can be subjected to amino acid sequencing by a general method. The antigen-binding fragment can also be prepared by a general method. The antibody or antigen-binding fragment according to the invention is genetically engineered to add one or more human FR regions to a CDR region of non-human origin. Human FR germline sequences can be obtained from the website of ImMunoGeneTics (IMGT) http://imgt.cines.fr, or from the Journal of Immunoglobulins, 2001ISBN012441351.

The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using general methods. For example, cDNA sequences encoding the heavy chain CDR and light chain can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more recommended technique, mammalian expression systems cause glycosylation of antibodies, especially the highly conserved N-terminus of the Fe region. Stable pure strains were obtained by expressing antibodies that specifically bind to human CD47. Positive pure strains were expanded in serum-free medium in a bioreactor to produce antibodies. The culture medium in which the antibody is secreted can be purified by a general technique. For example, use a Protein A or G Sepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody was eluted with an acidic buffer, and antibody fragments were detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using a general method. Soluble mixtures and polymers can also be removed by common methods, such as molecular sieves, ion exchange. The resulting product needs to be frozen immediately, such as -80 ° C, or lyophilized.

The antibodies of the present disclosure refer to monoclonal antibodies. The monoclonal antibody or mAb described in the present disclosure refers to an antibody obtained from a single pure strain cell strain, and the cell strain is not limited to a pure strain of eukaryotic, prokaryotic, or phage. Monoclonal antibodies or antigen-binding fragments can be recombined using techniques such as fusion tumor technology, recombinant technology, phage display technology, synthetic technology (such as CDR-grafting), or other existing technologies.

"Administration" and "treatment" when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals, humans, recipients Contact with a subject, cell, tissue, organ, or biological fluid. "Administering" and "treating" may refer to, for example, treatment, pharmacokinetics, diagnostics, research, and experimental methods. The treatment of the cells includes the contact of the reagent with the cells, and the contact of the reagent with the fluid, which is in contact with the cells. "Administering" and "treating" also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnostic, binding composition, or by another cell. "Treatment" when applied to a human, veterinary or research subject means a therapeutic treatment, preventive or preventative measure, research and diagnostic application.

"Treatment" means the administration to a patient of an internal or external therapeutic agent, such as a composition comprising a CD47 antibody or an antigen-binding fragment thereof, that the patient has one or more symptoms of the disease. Generally, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in a treated patient or population, whether by inducing the deterioration of such symptoms or inhibiting the development of such symptoms to any clinically measurable degree. The amount of therapeutic agent (also referred to as a "therapeutically effective amount") that is effective in alleviating the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect on the patient. It can be assessed whether the symptoms of the disease have been alleviated by any clinical testing method commonly used by doctors or other health care professionals to assess the severity or progression of the symptoms. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may not be effective in relieving symptoms of a target disease in a patient, any statistical test method known in the art such as Student's t-test, chi-square test, Mann and Whitney's U-test, Kruskal-Wallis test (H-test), Jonckheere-Terpstra checksum and Wilcoxon test determined that it should reduce symptoms of the target disease in a statistically significant number of patients.

"Mutations" in "mutated sequences" as described in this disclosure include, but are not limited to, "back mutations", "conservative modifications" or "conservative substitutions or substitutions." "Conservative modification" or "conservative substitution or substitution" as described in this disclosure refers to other amino acid replacement proteins with similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, rigidity, etc.) The amino acid in the amino acid allows frequent changes without altering the biological activity of the protein. Those skilled in the art know that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co. ., P. 224, (4th edition)). In addition, structurally or functionally similar amino acid substitutions are unlikely to disrupt biological activity.

The "mutated sequence" in the present invention refers to the nucleotide sequence and the amino group of the present invention obtained by subjecting the nucleotide sequence and the amino acid sequence of the present invention to a suitable modification such as substitution, insertion, or deletion. Acid sequences have nucleotide sequences and amino acid sequences with different percentages of sequence identity. The sequence identity described in the present invention may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , 100%. Sequence comparison and percent identity determination between two sequences can be performed by default settings of the BLASTN / BLASTP algorithms available on the National Center For Biotechnology Institute website.

An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's general health, the route and dosage of the method of administration, and the severity of the side effects. An effective amount may be the maximum dose or dosage regimen to avoid significant side effects or toxic effects.

"Exogenous" refers to a substance that is produced outside the organism, cell, or human depending on the context. "Endogenous" refers to a substance that is produced in a cell, organism, or human body by context.

As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such names include progeny. Thus, the words "transformants" and "transformed cells" include primary test cells and cultures derived therefrom, regardless of the number of metastases. It should also be understood that due to intentional or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Included are mutant offspring that have the same functional or biological activity as those originally screened in the transformed cells.

As used herein, "polymerase chain reaction" or "PCR" refers to a program or technique of amplifying a trace amount of a specific portion of nucleic acid, RNA, and / or DNA as described, for example, in US Patent No. 4,683,195. Generally speaking, it is necessary to obtain sequence information from the end of the target region or beyond, so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified. The 5 'terminal nucleotides of the two primers may coincide with the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage, or plastid sequences transcribed from total cellular RNA. See generally Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51: 263; edited by Erlich, (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). The PCR used herein is considered as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample. The method includes using known nucleic acids and nucleic acid polymerases as primers to amplify or Produce specific parts of nucleic acids.

"As needed" or "as needed" means that the event or environment described later may, but need not, occur, and the description includes the place where the event or environment occurs or does not occur. For example, "including 1-3 antibody heavy chain variable regions as needed" means that an antibody heavy chain variable region of a particular sequence may, but need not, be present.

"Pharmaceutical composition" means a mixture containing one or more CD47 antibodies or antigen-binding fragments thereof described herein with other chemical components, as well as other components such as physiological / pharmaceutically acceptable carriers and excipients. The goal of the pharmaceutical composition is to promote the administration to living organisms, which facilitates the absorption of active ingredients and exerts biological activity.

Examples

The following further describes the present disclosure with reference to the examples, but these examples do not limit the scope of the present disclosure. The experimental methods without specific conditions in the examples of the present disclosure are generally based on general conditions, such as the manual of antibody technology experiments in Cold Spring Harbor and the manual of molecular colonization; or according to the conditions recommended by the raw material or commodity manufacturers. The reagents without specific source are general reagents purchased on the market.

Example 1. Preparation of CD47 antigen and detection protein

Using UniProt Leukocyte surface antigen CD47 (human CD47 protein, Uniprot number: Q08722) as a template for CD47, design the amino acid sequences of the antigens and detection proteins involved in the present disclosure. Optionally, different labels can be fused on the basis of CD47 protein such as his tag or Fc etc.

1. His47 CD47 protein extracellular domain (CD47-ECD-His): (SEQ ID NO: 1). Note: The underlined part is a 6 × his label.

2. CD47 extracellular domain and human IgG1Fc fusion protein (CD47-ECD-Fc) as immunogens and detection reagents: (SEQ ID NO: 2). Note: The underlined portion is the human IgG1-Fc portion.

3. Human SIRP α and human IgG1Fc fusion protein (SIRP α-Fc) as binding and blocking detection reagents: (SEQ ID NO: 3). Note: The underlined part is the huamn-IgG1-Fc part.

Example 2. Purification of CD47, SIRP α Related Recombinant Protein

1. Purification of His-tagged recombinant protein: Centrifuge the cell expression supernatant sample to remove impurities, replace the buffer with PBS, and add imidazole to a final concentration of 5 mM. Equilibrate the nickel column with 5 mM imidazole in PBS and rinse the column volume 2-5 times. The replaced supernatant sample was applied to an IMAC column. The column was rinsed with 5 mM imidazole in PBS until the A280 reading dropped to baseline. The column was then washed with PBS + 10 mM imidazole to remove non-specifically bound foreign proteins, and the effluent was collected. The target protein was further extracted with 300 mM imidazole in PBS, and the eluted peaks were collected. The collected eluate was concentrated and further purified by gel chromatography Superdex200 (GE, 28-9893-35). The mobile phase was PBS. Remove the polymer peaks and collect the eluted peaks. After the obtained protein was identified as correct by electrophoresis, peptide mapping, and LC-MS, it was divided into equipment.

A His-labeled CD47-ECD-His (SEQ ID NO: 1) was obtained for use as an immunogen or detection reagent for the antibodies of the present disclosure. CD47-ECD-His can also be used as an immunogen to stimulate mouse immunity after coupling reaction with KLH by in vitro chemistry.

2. Purification steps of CD47-ECD-Fc and SIRP α-Fc fusion protein: The cell expression supernatant sample was centrifuged at high speed to remove impurities, and the supernatant was subjected to MabSelect Sure (GE, 17-5438-01) affinity chromatography. The MabSelect Sure chromatography column was first regenerated with 0.1M NaOH, washed with pure water and then equilibrated with PBS. After the supernatants were combined, they were washed with PBS until the A280 reading dropped to baseline. The target protein was eluted with 0.1M acetic acid buffer at pH 3.5, and neutralized with 1MTris-HCl. The extracted samples were appropriately concentrated and further purified by Superdex200 (GE, 28-9893-35), which was equilibrated with PBS, and then concentrated to the appropriate concentration in the receiving tube where the target proteins were collected.

This method is used to purify CD47-ECD-Fc (SEQ ID NO: 2) and SIRP α-Fc (SEQ ID NO: 3) fusion proteins. The method can also be used to purify humanized antibody proteins involved in this disclosure.

Example 3. Obtaining and preparing anti-human CD47 fusion tumor monoclonal antibody

Immunity

Anti-human CD47 monoclonal antibodies were produced by immunizing mice. SJL white mice for experiments, female, 6 weeks old (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Jing) 2012-0001).

Rearing environment: SPF level. After the mice were purchased, they were raised in a laboratory environment for 1 week, with a 12 / 12-hour light / dark cycle adjustment, a temperature of 20-25 ° C, and a humidity of 40-60%. Environment-adapted mice were immunized according to different protocols, 6-10 mice per group.

The immune antigens can be CD47-ECD-Fc, CD47-ECD-His6, CD47-ECD-His6-KLH, etc. A single reagent can be used with different immune adjuvants or cross-immunization with different types of immunogens. The immune site can be subcutaneously in the abdominal cavity or the back, or alternately in both locations. An exemplary immunization method is emulsification with Freund's adjuvant (sigma Lot Num: F5881 / F5506): Freund's complete adjuvant (CFA) is first exempted, and Freund's incomplete adjuvant (IFA) is used for the rest of the boost. Antigen and adjuvant ratio of 1: 1,100 μ g / only (first free), 50 μ g / only (booster). Day 0 intraperitoneally (IP) injection of 100 μ g / antigen only after emulsification, once every two weeks after the first immunization, a total of 6-8 weeks. Or cross-immunize with Tiermax (sigma Lot Num: T2684) and Alum (Thremo Lot Num: 77161). Antigen with an adjuvant (TiterMax) ratio of 1: 1, the antigen with an adjuvant (Alum were) ratio of 3: 1,10-20 μ g / only (first immunization), 5 μ g / only (booster immunization). After intraperitoneal injection on Day 0 20/10 μ g / emulsified antigen only, once a week after the first immunization, Titermax used interchangeably and Alum were 6-11 weeks. Four weeks after immunization, antigens were selected for injection on the back or intraperitoneally, depending on back clumps and abdominal swelling.

Cell fusion

Select antibodies with high antibody titers in the serum (see Test Example 1, ELISA method combined with CD47) and titers that approach the plateau for spleen cell fusion. 72 hours before fusion, use CD47-ECD-Fc to intraperitoneally sprint the immunization institute. Select mice. Spleen lymphocytes were fused with myeloma cells Sp2 / 0 cells (ATCC ^® CRL-8287 ^TM ) using an optimized PEG-mediated fusion step to obtain fusion tumor cells. Good fusion hybridoma cells HAT complete medium (containing 20% FBS, 1 × HAT and 1 × OPI of RPMI-1640 medium) were resuspended, aliquoted into 96-well cell culture plates ^{(1 × 10 5/150 μ} l / Well), 37 ° C, 5% CO ₂ incubation. On the fifth day after fusion, HAT complete medium was added, 50 μl / well, and incubated at 37 ° C and 5% CO ₂ . From 7 to 8 days after fusion, according to the cell growth density, the medium was completely changed. The medium was HT complete medium (RPMI-1640 medium containing 20% FBS, 1 × HT and 1 × OPI), 200 μl / well, 37 ℃, 5% CO ₂ incubation.

3. Screening of fusion tumor cells

On days 7-9 after fusion, CD47-binding ELISA was used to detect the cell growth density (see Test Example 1). CD47 / SIRP α binding blocking ELISA test (see Test Example 2) was performed on the positive well cells bound by ELISA, and the positive wells were exchanged with liquid, and expanded to the 24-well plate in time according to the cell density. The cell lines transferred to the 24-well plate were re-tested for seed conservation and first colonization. The first selection (see Test Example 1) is positive for seed conservation, and the second or third selection is performed until a single-cell pure strain is obtained. Multiple fusions resulted in fusion tumor cells that blocked the binding effect of CD47 and SIRP α (see Test Example 2).

Pure fusion tumor strains 055 and 167 were obtained through screening experiments and binding experiments. Antibodies were further prepared by serum-free cell culture, and the antibodies were purified according to the purification examples for use in detection examples.

The sequence of the murine anti-variable region of the pure fusion 055 strain was determined as follows: The murine heavy chain variable region sequence of the pure strain 055: (SEQ ID NO: 4).

Sequence of murine light chain variable region of pure strain 055: (SEQ ID NO: 5). Note: The sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, FR sequences in italics in the sequence, and CDR sequences in the bottom line.

The murine anti-variable region sequence of fusion tumor clone 167 is as follows: The murine heavy chain variable region sequence of pure strain 167: (SEQ ID NO: 6).

Sequence of murine light chain variable region of pure strain 167: (SEQ ID NO: 7). Note: The sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, FR sequences in italics in the sequence, and CDR sequences in the bottom line.

Example 4. Humanization of anti-human CD47 fusion tumor monoclonal antibodies

By comparing the IMGT human antibody heavy and light chain variable region germline gene database and the MOE software, the heavy and light chain variable region germline genes with high homology to 055 and 167 were selected as templates, respectively. The CDRs of the antibodies were transplanted into corresponding human-derived templates to form variable region sequences in the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Amino acid residues are identified and annotated by the Kabat numbering system.

1. Humanized fusion tumor pure strain 055

1.1 Selection of humanized framework of Fusion tumor pure strain 055

The humanized light chain template of the mouse antibody 055 is IGKV4-1 * 01 and hjk2.1, and the humanized heavy chain template is IGHV1-3 * 01 and hjh6.1. The sequence of the humanized variable region is as follows: hu055VH- CDR grafting: (SEQ ID NO: 20).

hu055VL CDR grafting: (SEQ ID NO: 21). Note: The sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, FR sequences in italics in the sequence, and CDR sequences in the bottom line.

1.2 Humanized template selection and reverse mutation design of fusion tumor pure strain 055.

1.3 Humanization of Fusion Tumor Strain 055

The humanized template of the fusion tumor pure strain 055 was designed for back mutation. Different back mutations were combined into different humanized antibodies. See Table 3 for details.

2. 167 humanized fusion tumor pure strains

2.1 The humanized light chain template of the murine antibody 167 is IGKV1-39 * 01 and hjk4.1, and the humanized heavy chain template is IGHV1-2 * 02 and hjh6.1. The sequence of the humanized variable region is as follows: Hu167VH -CDR grafting

(SEQ ID NO: 22).

Hu167VL-CDR grafting

(SEQ ID NO: 23).

2.2 Humanized template selection and reverse mutation design of Fusion tumor pure strain 167, see Table 4 below:     

2.3 Humanization of Fusion Tumor Strain 167

The humanized template of the fusion tumor pure strain 167 was designed for back mutation. Different back mutations were combined into different humanized antibodies. See Table 5 for details.

3. Humanized antibody screening

CD47-binding ELISA method (see Test Example 1), and positive well cells bound by ELISA were tested for CD47 / SIRP α binding blocking ELISA (see Test Example 2, and the results are shown in Table 6). The best combination of back mutations that retains the antibody's ability to bind and block.

Different combinations of 055 humanized antibodies have basically maintained good in vitro blocking activity. The affinity of different humanized antibodies was compared by Biacore. Partial data are shown in Table 8. The results show that the humanized antibody combinations have been maintained. For CD47 good binding ability, especially hu055-3, hu055-4, hu055-5, hu055-6, hu055-7 affinity is basically close to the chimeric antibody ch-055.

Similarly, different combinations of 167 humanized antibodies were tested by CD47-binding ELISA (see Test Example 1), and positive well cells bound by ELISA were tested by CD47 / SIRP α binding blocking ELISA (see Test Example 2) According to the test results, a combination of back mutations that retain the antibody binding ability and blocking ability to the greatest extent is preferred. The results of in vitro blocking experiments showed that most of the antibodies maintained good blocking ability and there was no obvious difference between each other. Some of them were selected for Biacore affinity comparison. The results are shown in Table 9, and most of them remained very good. For the binding capacity of CD47, especially hu167-4, hu167-6, hu-167-9, and hu167-14.

According to the comparison of different humanized combinations according to the affinity and blocking activity, it was found that V58I in the reverse mutation had little effect on the maintenance of affinity, so three light chains were added based on the original design (hu167_VL.1E (I2V), hu167_VL. The hu167-31, hu167-32, and hu167-33 produced by the combination of 1F (M4I), hu167_VL.1G (I2V, M4I)) and hu167_VH.1, and in vitro Biacore detection and blocking activity showed that all three maintained comparable CD47 binding. Ability and blocking activity. The affinities (KD) of hu167-31, hu167-32, hu167-33, and hu167-4 were compared by Biacore test. The results showed that the KD ratios compared to hu167-4 were 1.3, 1.2, and 0.98, suggesting hu167-31, hu167. -32, hu167-33 well maintained the binding ability of the antibody.

Example 5. Construction and expression of IgG4-S228P and IgG4-AA forms of CD47 humanized antibodies

Primer PCR was designed to construct each humanized antibody VH / VK gene fragment, and then homologously recombined with the expression vector pHr (with signal peptide and constant region gene (CH1-FC / CL) fragment) to construct the full-length antibody expression vector VH-CH1 -FC-pHr / VK-CL-pHr. The IgG4-AA antibody form can be obtained by simple point mutation of the IgG4 antibody form. IgG4-AA represents F234A and L235A mutations. The results show that it further reduces the binding capacity of IgG4-Fc to FcyR and further reduces ADCC / CDC. IgG4-S228P represents mutation of amino acid S to P at position 228 of the wild-type IgG4 hinge region. This site mutation can avoid mismatch caused by Fab-exchange of natural IgG4 antibodies in vivo.

The sequence of the heavy chain constant region of IgG4-S228P is as follows: (SEQ ID NO: 24).

IgG4-AA represents the heavy chain constant region sequence resulting from F234A and L235A mutations in the bottom line of the above sequence. Specifically, the heavy chain constant region sequence of IgG4-AA (including S228P) in the present disclosure is as follows: (SEQ ID NO: 30)

The light chain (Kappa chain) constant region sequence of the antibody is as follows: (SEQ ID NO: 25).

The constructed antibody sequence is listed as follows: hu167-33: antibody form IgG4AA

hu167-33 heavy chain sequence: (SEQ ID NO: 26).

hu167-33 light chain sequence: (SEQ ID NO: 27).

hu055-5: antibody form IgG4AA

hu055-5 heavy chain sequence: (SEQ ID NO: 28).

hu055-5 light chain sequence: (SEQ ID NO: 29).

Test case

Test example 1. ELISA experiment of CD47 antibody binding to CD47 protein

The binding capacity of the anti-CD47 antibody was detected by the amount of binding of the antibody to CD47 immobilized on the ELISA plate. It was diluted with PBS CD47-ECD-His (SEQ ID NO: 1) to 1 μ g / ml were coated on 96-well ELISA plates (Costar, CAT # 3590), the plate was washed, blocked, diluted at different concentrations of anti-CD47 antibody After the sample was washed, horseradish peroxidase-sheep anti-human (H + L) antibody (Jackson, CAT # 109-035-088) was added, and the plate was washed with tetramethylbenzidine solution to develop color, and finally added Stop solution, measure OD450 on a microplate reader and calculate its EC50 value.

Test Example 2. Blocking of SIRP α / CD47 binding by CD47 antibody

The ability of an anti-CD47 antibody to block the binding of SIRP α and CD47 was determined by measuring the amount of CD47 binding to SIRP α in the presence of the antibody. Dilution buffer CD47-ECD-Fc (SEQ ID NO.2) to 1 μ g / ml in SIRP coated 96-well ELISA plates (Costar, CAT # 3590), the plate was washed blocking, biotin-labeled with phosphoric acid α-Fc (SEQ ID NO: 3) ( diluted with phosphate buffer to a final concentration of 0.5 μ g / ml) and the antibody sample (diluted with a phosphate buffer) in a mixture of 100 μ l / hole, the plate was washed horseradish Peroxidase-streptavidin (sigma, CAT # S2438), then wash the plate with tetramethylbenzidine solution to develop the color, and finally add the stop solution, measure OD450 on the microplate reader and calculate its IC50 value, see the results Table 6.

Test example 3. Experiment of binding CD47 antibody to CCRF-CEM cells

CCRF-CEM cells (ATCC, CAT # CRM-CCL-119) were cultured in RPMI medium (Hyclone, CAT # SH30809.01B) (containing 10% fetal bovine serum), 1 × 10 ⁶ cells / ml for CCRF-CEM cells after blocking 5% BSA, CD47 antibody sample was added to 1 μ g / ml, after washed twice, and then after the addition of Alexa Fluor 488- sheep (H + L) antibody (Invitrogen, CAT # A11013), washed twice with anti-human The flow cytometer reads the fluorescence signal value.

The FACS test results show that the humanized antibody involved in the present disclosure has strong binding ability to the natural CD47 on the cell surface, which is significantly better than that of the control antibody B6H12 (US9017675B). The results are shown in Table 7.

Test Example 4. CD47 antibody enhances CCRF-CEM cells in vitro antibody-dependent cell-mediated phagocytosis (ADCP)

Monocytes were isolated from human peripheral blood monocytes and cultured in a six-well plate in RPMI medium (Hyclone, CAT # SH30809.01B) (containing 10% fetal bovine serum) for 7 days. 1 × 10 ⁶ cells / ml CCRF-CEM cells were added to CFSE (abcam, ab113853) to a final concentration of 0.02 μ M, incubated for 5-10 min 37 ℃, after washed twice was added six-well plates containing mononuclear cells after differentiation, Add diluted anti-CD47 antibody samples and incubate at 37 ° C for 4 hours. After washing twice, scrape off the differentiated monocytes, add APC-anti-human CD14 antibody (BioLegend, CAT # 325608), and incubate at 4 ° C for 0.5 hours. After washing twice, the fluorescence signal was read by the flow cytometer.

The test results are shown in Figure 1, which shows that the addition of the humanized antibody of the present disclosure can effectively promote ADCP and has a certain dose effect.

Test example 5. CD47 antibody erythrocyte agglutination experiment

Fresh human blood was diluted 1 time with phosphate buffer solution and added to a 96-well plate. Anti-CD47 antibody samples diluted at different concentrations were added and incubated at 37 ° C for 4 hours after shaking. Aspirate the supernatant, observe the cell precipitation in different wells of the 96-well plate, record the red blood cell (RBC) agglutination caused by different concentrations of each antibody in each well, mark it in the manner shown in Figure 2, and count the results Compare.

Agglutination of red blood cells caused by antibodies is a common phenomenon in antibodies in the field of CD47. For example, the patent antibody 5F9 (US9017675B, see patents vh2, SEQ ID NO 37 and v12, SEQ ID NO 42) can be detected in vitro to cause a wide range of concentrations. Red blood cell agglutination occurs, and under the same conditions, hu167-33 of the present disclosure does not cause red blood cell agglutination at different concentrations tested. Another antibody molecule, hu055-5, can cause red blood cell agglutination, but the concentration range of red blood cell agglutination is significantly less than 5F9. The results are shown in Figure 2. The potential advantages of the antibodies of the present disclosure in terms of safety are suggested.

Test Example 6. CD47 Antibody Erythrocyte Lysis Experiment

Fresh human blood was diluted 1 time with phosphate buffer solution and added to a 96-well plate. 0.2mg / ml anti-CD47 antibody samples or Triton X-100 diluted at different concentrations were added. After shaking, incubated at 37 ° C for 4 hours. A 96-well plate with OD540 measured on a microplate reader. The results are shown in Figure 3, showing that the addition of the anti-CD47 antibody shown in the present disclosure does not cause lysis of red blood cells.

Test Example 7. BIAcore Affinity Test for CD47 Antibody

The Biacore instrument was used to test the affinity of the disclosed chimeric antibodies and corresponding humanized antibodies for human CD47-his antigen.

The human Fab capture molecule was covalently coupled to a CM5 biosensor wafer (Cat. # BR-1000-12, according to the method described in the instructions of the human Fab capture kit (Cat. # 28-9583-25, GE). GE), so as to affinity capture the test antibody. Then the human CD47-his (Yiqiao Shenzhou, CAT # 12283-HCCH-50) antigen was flowed on the surface of the wafer, and the reaction signal was detected by the Biacore instrument in real time to obtain the binding and dissociation curves. The affinity value was obtained by fitting, see below Table 8 and Table 9. After the dissociation of each cycle was completed in the experiment, the biochip was washed and regenerated with the regeneration solution provided in the human Fab capture kit.

The corresponding humanized antibody of the disclosed antibody was tested by Biacore and found to have a higher affinity with the antigen, and an optimized humanized antibody with high affinity and few back mutations was further screened.

Test example 8. CD47 antibody in vivo pharmacokinetic experiment

Animals took serum at different time points after administration of different concentrations of anti-CD47 antibody samples. According to the method in the ELISA experiment of anti-CD47 antibody binding to CD47 protein in Test Example 1, the standard curves of different samples were prepared. The serum sample 1: 1000 replaced the anti-CD47 antibody. After adding the reaction system, the concentration of anti-CD47 antibody in serum was converted at different time points according to OD450. The data obtained was analyzed by Phoenix WinNonlin software to calculate pharmacokinetic related parameters.

The drug metabolism parameters of hu055-5 at a dose of 3mpk were measured in rats. The results show that hu055-5 has good drug metabolism performance in rats. The average drug half-life t1 / 2 is about 285 hours. See Figure 4, which indicates that The antibody has good stability in rats. The antibody hu167-33 of the present disclosure was tested for drug metabolism in mice. The drug half-life t1 / 2 was found to be approximately 192 hours at a dose of 10 mpk. See FIG. 5, which shows that the antibody has good stability in mice.

<110> Jiangsu Hengrui Pharmaceutical Co., Ltd., Shanghai Hengrui Pharmaceutical Co., Ltd.

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Claims (26)

    A CD47 antibody or antigen-binding fragment thereof comprising any one or more CDR regions selected from the group consisting of HCDR regions of the variable region of the antibody heavy chain: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 , SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15 SEQ ID NO: 16 a CDR having at least 85% sequence identity; and an LCDR region of an antibody light chain variable region: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17 , SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 CDRs with at least 85% sequence identity.         The CD47 antibody or antigen-binding fragment thereof according to item 1 of the scope of the patent application, wherein the variable region of the heavy chain of the antibody comprises HCDR1 shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 HCDR2 and HCDR3, or CDRs having at least 85% sequence identity with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10; and / or the light chain variable region of the antibody comprises SEQ ID NO: 11, LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 12 and SEQ ID NO: 13, or CDRs having at least 85% sequence identity with SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13.         The CD47 antibody or antigen-binding fragment thereof according to item 1 of the scope of patent application, wherein the variable region of the heavy chain of the antibody comprises HCDR1 shown in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. HCDR2 and HCDR3, or a CDR having at least 85% sequence identity with SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16; and / or the light chain variable region of the antibody comprises SEQ ID NO: 17, LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 18 and SEQ ID NO: 19, or a CDR having at least 85% sequence identity with SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.         The CD47 antibody or antigen-binding fragment thereof according to item 1 of the scope of the patent application, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a murine antibody or antigen-binding fragment thereof, a chimeric antibody or fragment thereof, and / or humanized Antibodies or fragments thereof.         The CD47 antibody or antigen-binding fragment thereof according to item 4 of the patent application scope, wherein the light chain variable region of the murine antibody or antigen-binding fragment thereof comprises a murine-derived light chain FR region or a mutant sequence thereof; The heavy chain variable region of an antibody or antigen-binding fragment thereof comprises a murine heavy chain FR region or a mutant sequence thereof.         The CD47 antibody or antigen-binding fragment thereof according to item 5 of the patent application scope, wherein the CD47 antibody comprises a heavy chain variable region shown in SEQ ID NO: 4 and a light chain variable region shown in SEQ ID NO: 5 Or, the CD47 antibody comprises a heavy chain variable region shown in SEQ ID NO: 6 and a light chain variable region shown in SEQ ID NO: 7.         The CD47 antibody or antigen-binding fragment thereof according to item 4 of the scope of the patent application, wherein the sequence of the heavy chain FR region on the heavy chain variable region of the humanized antibody is selected from human germline heavy chain IGHV1-3 * 01 and The combination of hjh6.1 and its mutant sequence, or a sequence selected from the combination of human germline heavy chain IGHV1-2 * 02 and hjh6.1 and its mutant sequence; the light chain on the variable region of the light chain of the humanized antibody The FR region sequence is selected from the combination of human germline light chain IGKV4-1 * 01 and hjk2.1 and its mutant sequence, or from the combination of human germline light chain IGKV1-39 * 01 and hjk4.1 and its mutant sequence. .         The CD47 antibody or antigen-binding fragment thereof according to item 7 of the scope of the patent application, wherein the FR region on the variable region of the humanized antibody contains 0 to 10 back mutations of amino acids.         The CD47 antibody or antigen-binding fragment thereof according to item 8 of the scope of patent application, wherein the humanized antibody variable region comprises: the heavy chain variable region shown in SEQ ID NO: 20 or a mutant sequence thereof, and SEQ ID The light chain variable region or its mutant sequence shown in NO: 21; or the humanized antibody variable region comprises: the heavy chain variable region or its mutant sequence shown in SEQ ID NO: 22, and SEQ ID NO: The light chain variable region shown in 23 or a mutant sequence thereof.         The CD47 antibody or antigen-binding fragment thereof according to item 9 of the scope of the patent application, wherein: the mutation sequence of the light chain variable region shown in SEQ ID NO: 21 comprises the mutation D66E; the heavy chain shown in SEQ ID NO: 20 The mutated sequence of the variable region comprises a mutation selected from the group consisting of: R72A, M48I, E46D, V68A, I70L, R38K, R67K, A97S, and combinations thereof; the mutated sequence of the light chain variable region shown in SEQ ID NO: 23 Mutations from: V58I, I2V, M4I, Q38E, A43T, P44H, and combinations thereof; the mutation sequence of the heavy chain variable region shown in SEQ ID NO: 22 comprises a mutation selected from the group consisting of: R72V, M48V, V68A, M70L , T74K, A40R, R38K, R67K and their combinations.         The CD47 antibody or antigen-binding fragment thereof according to item 10 of the scope of patent application, wherein: the mutation sequence of the heavy chain variable region shown in SEQ ID NO: 20 comprises a mutation selected from the group consisting of R72A, M48I, E46D, and V68A , I70L and their combinations.         The CD47 antibody or antigen-binding fragment thereof according to item 10 of the scope of the patent application, wherein: the mutation sequence of the light chain variable region shown in SEQ ID NO: 23 comprises a mutation selected from the group consisting of I2V, M4I, and a combination thereof.         The CD47 antibody or antigen-binding fragment thereof according to any one of claims 7 to 9, wherein: the humanized antibody variable region comprises: the heavy chain variable region shown in SEQ ID NO: 26 or A mutated sequence, and the light chain variable region shown in SEQ ID NO: 27 or a mutated sequence thereof; or the humanized antibody variable region comprises: the heavy chain variable region shown in SEQ ID NO: 28 or a mutated sequence thereof And the light chain variable region shown in SEQ ID NO: 29 or a mutant sequence thereof.         The CD47 antibody or antigen-binding fragment thereof according to any one of claims 4, 7 to 12, wherein the heavy chain of the chimeric antibody or humanized antibody comprises: human IgG1, IgG2, IgG3, or IgG4 The heavy chain constant region or its mutant sequence, and the light chain constant region of its κ and λ chains, or its mutant sequence. Constant     Item 14 of the patent range CD47 antibody or antigen-binding fragment thereof, wherein the chimeric antibody or humanized antibody heavy chain comprising: a human heavy chain IgG4 constant region or a mutated sequence, and the human κ Chain light chain constant region or a mutated sequence thereof.     The CD47 antibody or antigen-binding fragment thereof according to item 14 of the application, wherein the heavy chain of the chimeric or humanized antibody comprises: a human-derived IgG4 heavy chain constant region containing the F234A mutation and the L235A mutation SEQ ID NO: 30.         A medicinal composition comprising: a therapeutically effective amount of the CD47 antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 of the patent application scope, and one or more pharmaceutically acceptable carriers, diluents or agents Shape agent.         A bispecific antibody, comprising: the CD47 antibody or the antigen-binding fragment thereof according to any one of claims 1 to 16 of the scope of patent application.         An isolated nucleic acid encoding the CD47 antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 of the patent application scope.         An expression vector containing the isolated nucleic acid according to item 19 of the scope of patent application.         A host cell transformed with the expression vector described in claim 20 of the patent application, the host cell being selected from a prokaryotic cell and a eukaryotic cell.         The host cell according to item 21 of the application, wherein the eukaryotic cell is a mammalian cell.         A method for preparing a CD47 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 16 of the scope of patent application, comprising the steps of: expressing the antibody in a host cell according to the scope of patent application 21; and The antibody is isolated from the cells.         A method for inhibiting the growth of tumor cells in an individual, comprising the steps of: administering to the individual the CD47 antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 of the patent application scope, or the 17th patent application scope Said pharmaceutical composition.         A CD47 antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, a pharmaceutical composition according to claim 17, and a bispecific antibody according to claim 18 For use in preparing a medicament for treating cancer, the cancer is selected from the group consisting of ovarian cancer, melanoma, prostate cancer, bowel cancer, gastric cancer, esophageal cancer, breast cancer, lung cancer, kidney cancer, pancreatic cancer, uterine cancer, liver cancer, Bladder, cervical, oral, brain, testicular, skin, thyroid, and hematological malignancies.         The use according to item 25 of the scope of patent application, wherein the hematological malignancy is selected from the group consisting of myeloma, chronic leukemia, and acute leukemia.