阅读说明：本技术 靶向cd24的抗体及其制备和用途 (Antibodies targeting CD24 and their preparation and use ) 是由 田文志 李松 陈典泽 郭慧琴 于 2021-10-13 设计创作，主要内容包括：本申请提供一种与CD24特异结合的单克隆抗体或其抗原结合部分。还提供编码该抗体或其抗原结合部分的核酸分子、包含该核酸分子的表达载体和宿主细胞、制备该抗体或其抗原结合部分的方法,以及使用该抗体或其抗原结合部分来治疗与CD24过表达相关的疾病的方法。(The present application provides a monoclonal antibody, or antigen-binding portion thereof, that specifically binds to CD 24. Also provided are nucleic acid molecules encoding the antibodies or antigen-binding portions thereof, expression vectors and host cells comprising the nucleic acid molecules, methods of making the antibodies or antigen-binding portions thereof, and methods of using the antibodies or antigen-binding portions thereof to treat diseases associated with overexpression of CD 24.)

1. An isolated monoclonal antibody, or antigen-binding portion thereof, that binds to CD24, comprising a heavy chain variable region comprising a HV-CDR1 region, a HV-CDR2 region, and a HV-CDR3 region, and a light chain variable region comprising a LV-CDR1 region, a LV-CDR2 region, and a LV-CDR3 region, wherein the amino acid sequences of the HV-CDR1 region, the HV-CDR2 region, the HV-CDR3 region, the LV-CDR1 region, the LV-CDR2 region, and the LV-CDR3 region are set forth in SEQ ID NOs:1, 2,3, 4,5, and 6, respectively.

2. The isolated monoclonal antibody, or antigen-binding portion thereof, of claim 1, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NOs:7 or 10.

3. The isolated monoclonal antibody, or antigen-binding portion thereof, of claim 1, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NOs:8, 9, or 11.

4. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 2, wherein the heavy chain variable region and the light chain variable region comprise i) SEQ ID NOs:7 and 8; ii) SEQ ID NOs:7 and 9; or iii) the amino acid sequences shown in SEQ ID NOs:10 and 11.

5. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 1, which is an IgG1, IgG2, or IgG4 subtype.

6. The isolated monoclonal antibody, or antigen-binding portion thereof, of claim 1, comprising a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region and light chain constant region comprise the amino acid sequences set forth in SEQ ID NOs:12 and 13, respectively.

7. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 1, which is a murine, chimeric, or humanized antibody.

8. A nucleic acid molecule encoding the isolated monoclonal antibody, or antigen binding portion thereof, of any one of claims 1-7.

9. An expression vector comprising the nucleic acid molecule of claim 8.

10. A host cell comprising the expression vector of claim 9.

11. A pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding portion thereof of any one of claims 1-7, the nucleic acid molecule of claim 8, the expression vector of claim 9, or the host cell of claim 10, and at least one pharmaceutically acceptable carrier.

12. Use of the pharmaceutical composition of claim 11 in the manufacture of a medicament for treating a disease associated with CD24 overexpression.

13. The use of claim 12, wherein the disease is selected from ovarian cancer, breast cancer, cervical cancer, endometrial cancer, Acute Lymphocytic Leukemia (ALL), cholangiocarcinoma, bladder cancer, pancreatic cancer, gastric adenocarcinoma, glioblastoma, and colon cancer.

14. The use of claim 12, wherein the disease is selected from acute graft-versus-host disease, anti-aids cocktail therapy induced inflammation, rheumatoid arthritis, systemic lupus erythematosus, metabolism-related fatty liver disease, diabetes, multiple sclerosis, and sepsis.

Technical Field

The present application relates to a monoclonal antibody or antigen-binding portion thereof that specifically binds to CD24, and its preparation and use, particularly its use in the treatment of tumors.

Background

Cancer cells have developed several mechanisms to evade host immune surveillance, including: 1) through high expression of the membrane protein CD24, the membrane protein CD24 is combined with Siglec-10 receptors on the surfaces of immune cells, so that immune activation is inhibited, and immune surveillance of macrophages, T lymphocytes, B lymphocytes and natural killer cells is avoided; 2) by evading immune surveillance by macrophages (M phi) by high expression of CD47, CD47 binds to signal-regulatory protein alpha (sirpa) on the macrophage surface, thereby triggering the generation of inhibitory signals that inhibit phagocytosis of cancer cells by macrophages. It can be seen that cancer cells are quite clever and can proliferate rapidly based on the escape mechanism they develop. Therefore, the development of anticancer drugs that effectively kill cancer cells can be directed to these mechanisms.

CD24 and Siglec-10

Sialic acid binding immunoglobulin-like lectins (Siglecs) are immunoglobulin-like type I transmembrane proteins. In the Siglecs family, Siglec-10 is an inhibitory receptor and is widely expressed on immune cells such as macrophages, B cells, NK cells, and activated T cells. It has five extracellular Ig-like domains, a transmembrane region and a cytoplasmic tail. The IgV domain of Siglec-10 contains a key arginine residue that is involved in sialic acid recognition (Yin, et al, 2020). Siglec-10 expression on T cells is known to interfere with T cell activation by inhibiting the formation of the major histocompatibility complex class I (MHC-I) peptide complex of T cells and the phosphorylation of T cell receptor associated kinases Lck and ZAP-70 (Yin, et al., 2020). Siglec-10 expressed on B cells and NK cells is able to inhibit BCR-mediated and NK cell receptor-mediated signal transduction (Yin, et al, 2020).

CD24 is a glycosyl-phosphatidylinositol anchor protein that is present on the surface of developing T lymphocytes and most B lymphocytes (Yin et al, 2020). Many cancer cells, including ovarian cancer, breast cancer, cervical cancer, endometrial cancer, Acute Lymphocytic Leukemia (ALL), cholangiocarcinoma, bladder cancer, pancreatic cancer, gastric adenocarcinoma, and glioblastoma, also highly express CD24(Barkal et al, 2019; Liu et al, 2013). CD24 on cancer cells interacts with Siglec-10 on immune cells, producing a "do not eat me" signal for immune evasion and protection of tumor cells from immune attack.

Studies have shown that CD24 expression is associated with bladder tumor recurrence (Liu et al, 2013). In ovarian cancer patients, expression of CD24 is an independent predictor of overall survival and is associated with tumor stage, peritoneal and lymph node metastasis; proliferation of CD 24-positive cells is enhanced, a highly aggressive phenotype, and is associated with cisplatin resistance in ovarian cancer cells (Nakamura et al 2017).

Studies have shown that the CD24 monoclonal antibody is able to reduce lung metastasis and prolong overall survival in mouse models of bladder cancer and triple negative breast cancer. The literature also indicates that blocking the CD24-Siglecs 10 interaction by antibodies can cause a reduction in tumor growth via macrophages and prolong the survival of tumor-bearing mice (Barkal, et al, 2019; Chan et al, 2019; overladevest et al, 2011).

Furthermore, previous studies have shown that the CD47/CD24 bispecific antibody can effectively activate myeloid immunity in the brain (WuH, et al, 2021). The combination of the CD24 antibody and CD47 antibody enhanced phagocytosis of human ovarian cancer cells (Barkal et al, 2019). Barakal et al also found that the combination therapy of the CD24 antibody and cetuximab further enhanced phagocytosis of pancreatic cancer cells compared to treatment alone, suggesting that combination therapy comprising neutralization of CD24 may produce synergistic anti-tumor effects.

In addition, studies have shown that CD24 up-regulation is closely related to various diseases such as acute graft-versus-host disease (Toubai, t., et al, 2014), inflammatory response (including hepatic steatosis and fibrosis, low density lipoprotein disorder, etc.) caused by anti-aids cocktail therapy (Tian, r.r., et al, 2018), metabolic-related fatty liver disease (Fairbridge, n.a., et al, 2015), diabetes (E1-Mokhtar, m.a., et al, 2020), multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus (Tan, y., et al, 2016), sepsis (Chen, g.y., et al, 2011). The role of CD24 overexpression in the above-mentioned diseases has also been shown in a number of ongoing clinical trials (see references 8-16).

There is a need in the art for more antibodies to CD24 that have desirable properties.

The citation of any document in this application is not an admission that such document is prior art to the present application.

Disclosure of Invention

The present application provides a novel CD24 antibody, or antigen-binding portion thereof, that can bind to CD24⁺Cells, priming for CD24⁺Antibody-dependent cell-mediated cytotoxicity (ADCC) of cells, and also has a potent antitumor effect in vivo. In particular, tumors in tumor-bearing animals can be completely eliminated by administering a dose of the CD24 antibody of the present application. Furthermore, tumor cells are implanted again after complete tumor elimination, and no tumor formation occurs without further administration.

Thus, in one aspect, the application relates to an isolated monoclonal antibody, or antigen binding portion thereof, which binds to CD24 and may comprise i) a heavy chain variable region comprising a VH-CDR1 region, a VH-CDR2 region, and a VH-CDR3 region, wherein the VH-CDR1 region, VH-CDR2 region, and VH-CDR3 region may comprise a sequence as set forth in SEQ ID NOs: 1. 2 and 3, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the aforementioned sequences; and/or ii) a light chain variable region comprising the VL-CDR1 region, VL-CDR2 region, and VL-CDR3 region, wherein the VL-CDR1, VL-CDR2, and VL-CDR3 regions may comprise the amino acid sequence as set forth in SEQ ID NOs: 4. 5, and 6, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the aforementioned sequences.

The heavy chain variable region of the monoclonal antibody or antigen-binding portion thereof of the present application may comprise a heavy chain variable region as set forth in SEQ id nos:7 or 10, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the above sequence.

The light chain variable region of a monoclonal antibody or antigen binding portion thereof of the present application may comprise a variable region as set forth in SEQ ID NOs: 8. 9 or 11, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the aforementioned sequences.

In some embodiments, a monoclonal antibody or antigen-binding portion thereof of the present application may comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region may comprise a heavy chain variable region and a light chain variable region, respectively, as set forth in (i) SEQ ID NOs:7 and 8; (ii) SEQ ID NOs:7 and 9; or (iii) SEQ ID NOs:10 and 11; or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the above, wherein the antibody or antigen-binding portion thereof binds to CD 24.

The monoclonal antibody or antigen-binding portion thereof of the present application may comprise a heavy chain constant region and/or a light chain constant region. The heavy chain constant region may be an IgG1, IgG2, or IgG4 heavy chain constant region, having or engineered to have FcR binding. In some embodiments, the heavy chain constant region can be a human IgG1 heavy chain constant region having, for example, a sequence identical to SEQ ID NO: 12, or a pharmaceutically acceptable salt thereof, having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The light chain constant region can be a kappa light chain constant region, e.g., a human kappa constant region, having, e.g., a sequence identical to SEQ ID NO: 13, or a variant thereof, and 13 amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. Wherein, the N end of the heavy chain constant region is connected with the C end of the heavy chain variable region, and the N end of the light chain constant region is connected with the C end of the light chain variable region.

The CD24 antibodies of the present application are in some embodiments IgG antibodies, comprising or consisting of two heavy chains and two light chains, wherein each heavy chain comprises a heavy chain constant region sequence, a heavy chain variable region sequence, or a CDR sequence as described above, and each light chain comprises a light chain constant region sequence, a light chain variable region sequence, or a CDR sequence as described above. The antibody of the present application may be a full length antibody such as IgG4, IgG1, or IgG 2. In some embodiments, the antibodies of the present application, or antigen binding portions thereof, can be single chain antibodies, or composed of antibody fragments, such as Fab or F (ab')₂And (3) fragment.

The antibody of the present application may be, for example, a murine, human, chimeric or humanized antibody.

The antibodies or antigen-binding portions thereof of the present application can bind to human CD24 and are capable of blocking CD24-Siglec-10 binding/interaction and have anti-tumor effects in vivo.

The present application also provides immunoconjugates comprising an antibody or antigen-binding portion thereof of the present application linked to a therapeutic agent, such as a cytotoxin or an anti-cancer agent. Also provided are bispecific molecules comprising an antibody or antigen-binding portion thereof of the present application, to which is attached a second functional group, e.g., a second antibody, having a binding specificity that is different from that of the antibody or binding portion thereof of the present application. In another aspect, an antibody or antigen-binding portion thereof of the present application can be part of a Chimeric Antigen Receptor (CAR) or a genetically engineered T Cell Receptor (TCR). The present application also provides immune cells, including T cells, NK cells, and the like, having the above CARs and/or TCRs.

The present application also includes nucleic acid molecules encoding the antibodies or antigen binding portions thereof of the present application, as well as expression vectors comprising the nucleic acids and host cells comprising the expression vectors. The present application also provides a method of producing a CD24 antibody using a host cell containing the above expression vector, comprising: (i) expressing the antibody in a host cell, and (ii) isolating the antibody from the host cell or culture thereof.

The present application also provides pharmaceutical compositions comprising an antibody or antigen-binding portion thereof, an immunoconjugate, a bispecific molecule, an immune cell, a nucleic acid molecule, an expression vector, or a host cell of the present application, and a pharmaceutically acceptable carrier.

In one aspect, the present application provides a method of treating or ameliorating a CD 24-associated disease in a subject comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present application. .

In some embodiments, the CD 24-associated disease is cancer. The cancer may be solid and hematologic tumors, including, but not limited to, ovarian cancer, breast cancer, cervical cancer, endometrial cancer, Acute Lymphocytic Leukemia (ALL), cholangiocarcinoma, bladder cancer, pancreatic cancer, gastric adenocarcinoma, glioblastoma, and colon cancer. In some embodiments, the pharmaceutical compositions of the present application may be administered with at least one other anti-cancer agent, for example a CD47 targeting protein, such as a sirpa D1-Fc protein, which may have an amino acid sequence identical to SEQ ID NO: 19, an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 19 has an N80A mutation at position 80, and a mutation at this position can achieve the effect of sirpa D1 deglycosylation.

In some embodiments, the CD 24-associated disease is an inflammatory disease, including but not limited to acute graft-versus-host disease, inflammatory responses induced by anti-aids cocktail therapy (including hepatic steatosis and fibrosis, low density lipoprotein disorders, etc.), rheumatoid arthritis, and systemic lupus erythematosus.

Other CD 24-related diseases that the pharmaceutical compositions of the present application can treat include, but are not limited to, metabolic-related fatty liver disease, diabetes, multiple sclerosis, and sepsis.

The antibodies of the present application may also be used for in vitro detection of CD24, and the like.

Other features and advantages of the present disclosure will be apparent from the following detailed description and examples, which should not be construed as limiting. All documents, Genbank accession numbers, patents and published patent applications cited in the specification are incorporated herein by reference.

Drawings

The detailed description, which is given below by way of example and is not intended to limit the application to the particular embodiments, can be better understood in conjunction with the accompanying drawings.

Fig. 1 is a schematic structural diagram of the CD24 antibodies IMM47C, IMM47, and IMM47H of the present application. Wherein IMM47C is an IgG antibody comprising a murine heavy chain variable region and a murine light chain variable region, specifically comprising SEQ ID NO: 7, SEQ ID NO: 12, the heavy chain constant region of SEQ ID NO: 8, and SEQ ID NO: 13, a light chain constant region; IMM47 is an IgG antibody comprising a murine heavy chain variable region, and a humanized light chain variable region, specifically comprising SEQ ID NO: 7, SEQ ID NO: 12, the heavy chain constant region of SEQ ID NO: 9, and SEQ ID NO: 13, a light chain constant region; IMM47H is an IgG antibody comprising a humanized heavy chain variable region and a humanized light chain variable region, specifically comprising SEQ ID NO: 10, SEQ ID NO: 12, the heavy chain constant region of SEQ ID NO: 11, and SEQ ID NO: 13, a light chain constant region.

FIG. 2 shows IMM47H and CD24⁺CD47⁺Binding activity of MCF-7 human breast cancer cells, hIgG-Fc was used as a negative control.

FIG. 3 shows IMM47H and CD47⁺CD24⁺Binding activity of REH human acute lymphoma cells. IMM01 (SIRP. alpha. D1-Fc, SEQ ID NO: 19), hIgG-Fc was used as a control.

FIG. 4 shows IMM47C vs. CD24⁺CD47⁺MCF-7 human breast cancer cells ability to elicit antibody-dependent cell-mediated cytotoxic effects (ADCC), and hIgG1-Fc was used as a negative control.

FIG. 5 shows IMM47C vs. CD24⁺CD47⁺The ability of REH human acute lymphoblastic leukemia cells to elicit ADCC, hIgG1-Fc with very low ADCC activity was used as a control with IMM 01.

FIG. 6 shows the ability of IMM47 to elicit ADCC on MC38-hCD24 murine colon cancer cells genetically engineered to express human CD24, with hIgG1-Fc used as a negative control.

FIG. 7 showsIMM47C, and IMM47C in combination with IMM01 in carrying CD24⁺CD47⁺In vivo anti-tumor effects in MCF-7 human breast cancer tumor xenografted CB17-SCID mice.

Figures 8A and 8B show the utility of IMM47 and IMM47C in a B6-Siglec10 transgenic mouse homograft MC38-hCD24 colon cancer model, including the mean tumor volume (a) for each group and the individual tumor volume (B).

Figure 9 shows the anti-tumor effect of IMM47C, IMM47, and IMM47H in a Siglec10 humanized B6 mouse model of a homograft MC38-hCD24KI cell tumor.

Detailed Description

For a better understanding of the present application, certain terms are first defined. Other definitions are listed throughout the detailed description section.

"CD 24" herein refers to differentiation antigen clusterin 24, also known as the thermostable antigen CD 24. The term includes variants, homologues, orthologues and paralogues. For example, an antibody specific for human CD24 may in some cases cross-react with a CD24 protein of another species, such as monkey. In other embodiments, an antibody specific for human CD24 protein may be completely specific for human CD24 protein without cross-reacting with other species or other types of proteins, or may cross-react with CD24 protein of some other species but not all other species.

The term "antibody" herein includes whole antibodies, e.g., IgG, IgA, IgD, IgE and IgM, and any antigen-binding fragment (or antigen-binding portion) or single chain thereof. A whole antibody is a glycoprotein comprising at least two heavy chains and two light chains, which are linked via disulfide bonds. Each heavy chain comprises a heavy chain variable region (V)_H) And a heavy chain constant region. The heavy chain constant region comprises three domains, C_H1、C_H2And C_H3. Each light chain comprises a light chain variable region (V)_L) And a light chain constant region. The light chain constant region comprises a domain C_L。V_HAnd V_LRegions may also be subdivided into regions of high degree of variation, i.e., CDR regions, with more conserved Framework Regions (FRs) distributed between the CDR regions. Each V_HAnd V_LConstructed from three CDR and four FR regionsFrom amino terminus to carboxy terminus, the sequences were FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains comprise binding domains that react with antigen. The heavy chain constant region of an antibody may mediate the binding of immune proteins to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component of the complement system (C1 q). In particular, the heavy chain constant region, comprising an Fc region, is a domain that determines the effector function of an antibody, i.e., how the antibody relates to a particular cellular receptor or other defense protein. Fc receptors (fcrs) are proteins on the surface of certain cells, including B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, mast cells, and the like. The Fc region may interact with Fc receptors and some proteins of the complement system, activating the immune system and/or the complement system.

The term "antigen-binding portion" of an antibody (or simply antibody portion), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., CD24 protein). It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments comprised in the "antigen-binding portion" of an antibody include (i) a Fab fragment consisting of V_L、V_H、C_LAnd C_H1A monovalent fragment of (a); (ii) f (ab')₂A fragment, a bivalent fragment comprising two Fab fragments linked by a hinge region disulfide bridge; (iii) from V_HAnd C_mA constituent Fd fragment; (iv) from antibody single-armed V_LAnd V_H(iii) a Fv fragment of (i); (v) from V_HThe constituted dAb fragment (Ward et al, (1989) Nature 341: 544-546); (vi) an isolated Complementarity Determining Region (CDR); and (vii) a nanobody, a heavy chain variable region comprising a single variable domain and two constant domains. Furthermore, despite the two domains V of the Fv fragment_LAnd V_HEncoded by different genes, which can be joined by recombinant means via a synthetic linker which makes both single protein chains, where V_LAnd V_HThe regions pair to form a monovalent molecule, called single chain fc (scfv). These single chain antibodies are also intended to be encompassed by the termIn the meaning of the text. These antibody fragments can be obtained by conventional techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

The term "isolated antibody" as used herein refers to an antibody that is substantially free of other antibodies having different antigenic specificities. For example, an isolated antibody that specifically binds to CD24 is substantially free of antibodies that specifically bind to antigens other than CD24 protein. However, an isolated antibody that specifically binds to human CD24 protein may have cross-binding properties to other antigens, such as CD24 protein from other species. Furthermore, the isolated antibody is substantially free of other cellular material and/or chemicals.

The term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The term "mouse-derived antibody" refers to an antibody in which the variable region framework and CDR regions are derived from mouse germline immunoglobulin sequences. In addition, if the antibody contains constant regions, it is also derived from mouse germline immunoglobulin sequences. The mouse-derived antibody may comprise amino acid residues not encoded by the mouse germline immunoglobulin sequence, e.g., mutations introduced by random or point mutations in vitro or by somatic mutations in vivo. However, the term "murine antibody" does not include antibodies having CDR sequences from other mammalian species inserted into the mouse framework sequences.

The term "chimeric antibody" refers to an antibody obtained by combining genetic material of non-human origin with genetic material of human origin. Or more generally, a chimeric antibody refers to an antibody that combines genetic material of one species with genetic material of another species.

The term "humanized antibody" refers to an antibody that is derived from a non-human species but whose protein sequence has been modified to increase its similarity to a naturally occurring human antibody.

The heavy chain variable region CDRs and the light chain variable region CDRs of the antibodies or antibody fragments thereof of the present application are determined by the IMGT numbering system. It is well known in the art that the heavy chain variable region and light chain variable region CDRs can be determined by, for example, Chothia, Kabat, AbM, or Contact numbering systems/methods.

The terms "antibody-dependent cellular cytotoxicity", "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refer to a cell-mediated immune defense in which the effector cells of the immune system actively lyse target cells, e.g., cancer cells, to which a cell membrane surface antigen binds an antibody, e.g., a CD24 antibody.

The terms "antibody-dependent cellular phagocytosis", "antibody-dependent cellular phagocytosis" or "ADCP" refer to the binding of an antibody, such as a CD70 antibody, or an antibody-like protein, such as sirpa-Fc, to the FcR of an immune cell, such as a macrophage, causing phagocytosis of a target cell bound to the antibody, such as a CD70 antibody, or to sirpa, causing endocytosis and degradation of the target cell.

The terms "complement dependent cytotoxicity", "complement dependent cytotoxicity" or "CDC" refer to the binding of an antibody, such as a CD70 antibody or an antibody-like protein, such as sirpa-Fc, to a complement system protein, such as C1q, which triggers the complement pathway and lyses the target cells.

The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cows, and horses, are preferred.

The term "EC 50," also called half maximal effect concentration, refers to the concentration of antibody that causes 50% of the maximal effect.

The term "IC 50," also called half maximal inhibitory concentration, refers to the concentration of antibody that causes 50% of the maximal inhibitory effect.

As used herein, "sequence identity" refers to the percentage of nucleotides/amino acids in a sequence that are identical to the nucleotides/amino acid residues in a reference sequence after alignment, if necessary, to introduce gaps in the alignment to achieve the maximum percent sequence identity between the two sequences. Two-by-two or multiple sequence alignments can be performed by one skilled in the art to determine percent sequence identity between two or more nucleic acid or amino acid sequences by a variety of methods, for example, using computer software such as ClustalOmega, T-coffee, Kalign, and MAFFT, among others.

The antibodies of the present application comprise heavy and/or light chain variable region sequences or CDR1, CDR2 and CDR3 sequences that are conservatively modified with one or more of the antibodies of CD24 of the present application. It is known in the art that some conservative sequence modifications do not abolish antigen binding.

The term "conservative sequence modification" as used herein refers to amino acid modifications that do not significantly affect or alter the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the present application by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Groups of amino acid residues having similar side chains are known in the art. These groups of amino acid residues include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in a CDR region of an antibody of the present application can be replaced with other amino acid residues of the same side chain set, and the resulting antibody can be tested for retained function (i.e., the function described above) using the functional assays described herein.

The antibodies of the present application may be provided with one or more of the V's of the CD24 antibodies of the present application_H/V_LAntibodies of sequence were used as starting material to prepare genetically modified antibodies. Antibodies can be made by modifying one or both variable regions (i.e., V)_HAnd/or V_L) In (e.g., in one or more CDR regions and/or one or moreFramework regions) to improve binding affinity and/or increase similarity to antibodies naturally occurring in certain species. For example, the framework regions are modified to provide humanized antibodies. Alternatively, the antibody may be genetically modified by modifying residues in the constant region, for example to alter the effector function of the antibody.

In certain embodiments, CDR region implantation can be used to genetically modify the variable region of an antibody. Antibodies interact with the target antigen primarily through amino acid residues located in the six heavy and light chain Complementarity Determining Regions (CDRs). For this reason, amino acid residues within a CDR are more diverse between individual antibodies than sequences outside the CDR. Because the CDR sequences are responsible for the major antibody-antigen interactions, recombinant antibodies that mimic the properties of a particular native antibody can be expressed by constructing expression vectors that contain CDR sequences of the particular native antibody grafted into the framework sequences of different antibodies of different properties.

Thus, another embodiment of the present application relates to an isolated monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 having the sequences described herein above, and a light chain variable region comprising CDR1, CDR2 and CDR3 having the sequences described herein above, which may comprise different framework sequences. The framework sequences can be obtained from public DNA databases or public references including germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be obtained, for example, at the Vbase human germline sequence database (www.mrc-cpe.cam.ac.uk/Vbase). As another embodiment, germline DNA sequences for human heavy and light chain variable region genes are available in the Genbank database. Preferred framework sequences for use in the antibodies of the present application are those that are structurally similar to the framework sequences used in the antibodies of the present application. The CDR1, CDR2, and CDR3 sequences can be embedded in a framework region having the same sequence as the germline immunoglobulin gene from which the framework sequence was derived, or the CDR sequences can be embedded in a framework region comprising one or more mutations compared to the germline sequence. For example, in some cases, it is beneficial to mutate residues in the framework regions to maintain or enhance the antigen binding properties of the antibody, etc.

Another class of variable region modifications is to modify V_HAnd/or V_LAmino acid residues within the CDR1, CDR2, and/or CDR3 regions are mutated to improve one or more binding properties (e.g., affinity) of the antibody of interest. Point mutations or PCR-mediated mutations can be made to introduce mutations, and their effect on antibody binding or other functional properties can be evaluated in vitro or in vivo assays known in the art. Preferably, conservative modifications known in the art are introduced. The mutation may be an amino acid substitution, addition or deletion, but is preferably a substitution. In addition, typically no more than one, two, three, four, or five residues within a CDR region are altered.

Genetically engineered antibodies of the present application are included at V_HAnd/or V_LFor example, those that make genetic modifications in the framework residues to alter antibody properties. Generally, these backbone modifications are used to reduce the immunogenicity of the antibody. For example, one approach is to "back mutate" one or more backbone residues into the corresponding germline sequence. More specifically, an antibody undergoing somatic mutation may contain framework residues that differ from the germline sequence of the resulting antibody. These residues can be identified by comparing the antibody framework sequences to the germline sequences of the resulting antibody.

Another class of framework modifications comprises mutating one or more residues of the framework region, or even one or more CDR regions, to remove T cell epitopes and thereby reduce the potential immunogenicity of the antibody.

Furthermore, as an alternative to modifications within the framework or CDR regions, the antibodies of the present application may be genetically engineered to include genetic modifications in the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antibody-dependent cellular cytotoxicity. In addition, the antibodies of the present application can be chemically modified (e.g., one or more chemical functional groups can be attached to the antibody), or modified to alter glycosylation thereof, to alter one or more functional properties of the antibody.

In one embodiment of the method of the present invention,C_H1the hinge region of (a) is modified, for example, the number of cysteine residues in the hinge region is increased or decreased. Change C_H1Cysteine residues in the hinge region, for example, to facilitate assembly of the heavy chain light chain or to increase/decrease stability of the antibody.

In another embodiment, the Fc hinge region of an antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the C of the Fc hinge fragment_H2-C_H3A linking region such that the antibody has reduced SpA binding relative to native Fc-hinge domain SpA binding.

In another embodiment, the glycosylation of the antibody is modified. For example, deglycosylated antibodies (i.e., antibodies lacking glycosylation) can be made. Glycosylation can be altered, for example, to increase the affinity of an antibody for an antigen. Such modifications of glycation can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate one or more variable region backbone glycosylation sites, thereby eliminating glycosylation at that location. Such deglycosylation can increase the affinity of the antibody for the antigen. Furthermore, antibodies with altered glycosylation patterns can be prepared, such as low fucosyl antibodies with reduced amounts of fucose residues, or antibodies with increased bisecting GlcNac structures. Altered glycosylation patterns have been shown to increase the ADCC activity of an antibody. Such modifications of glycation can be performed, for example, by expressing the antibody in a host cell with an altered glycosylation system. Cells with altered glycosylation systems are known in the art and can be used as host cells for expression of recombinant antibodies of the present application to produce antibodies with altered glycosylation.

Monoclonal antibodies of the present application may be used in conjunction with the monoclonal antibodies of Kohler and Milstein (1975) Nature 256: 495 was prepared by somatic cell hybridization (hybridoma) technique. Other embodiments for making monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display techniques. Chimeric or humanized antibodies are also well known in the art.

Antibodies of the present application can also be produced in host cell transfectomas using, for example, recombinant DNA technology in conjunction with gene transfection methods (e.g., Morrison, S. (1985) Science 229: 1202). In one embodiment, DNA encoding partial or full length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" refers to the linkage of the antibody genes into a vector such that transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody genes.

The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of an antibody gene. Such regulatory sequences are described, for example, in Goeddel (Gene Expression technology. methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred 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 from Cytomegalovirus (CMV), simian virus 40(SV40), adenoviruses, such as the adenovirus major late promoter (AdMLP), and polyoma virus. Alternatively, non-viral regulatory sequences may be used, such as the ubiquitin promoter or the beta-globin promoter. In addition, the regulatory elements are composed of sequences of different origins, such as the SR α promoter system, which comprises the sequence from the SV40 early promoter and the long terminal repeat of the human T-cell leukemia type I virus (Takebe et al, (1988) mol.cell.biol.8: 466-. The expression vector and expression control sequences are selected to be compatible with the expression host cell used.

The antibody light chain gene and the antibody heavy chain gene may be inserted into the same or different expression vectors. In a preferred embodiment, the variable regions are constructed as full length antibody genes by insertion into expression vectors that already encode the heavy and light chain constant regions of the desired subtype, such that V_HWith C in the carrier_HIs operably connected to, V_LWith C in the carrier_LAre operatively connected. Alternatively, the recombinant expression vector may encode a promoter that facilitates the release of antibody chains from the host cellA secreted signal peptide. The antibody chain gene may be cloned into a vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the present application may carry other sequences, such as sequences that regulate replication of the vector in a host cell (e.g., an origin of replication) and a selectable marker gene. Selectable marker genes can be used to select 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 a selectable marker gene confers drug resistance, e.g., G418, hygromycin, or methotrexate resistance, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for methotrexate selection/amplification of DHFR host cells) and the neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vectors encoding the heavy and light chains are transfected into the host cell by standard techniques. The term "transfection" in its various forms encompasses a variety of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, e.g., electroporation, calcium phosphate precipitation, DEAE-dextrose transfection, and the like. Although it is theoretically possible to express the antibodies of the present application in prokaryotic or eukaryotic host cells, it is preferred that the antibodies are expressed in eukaryotic cells, most preferably mammalian host cells, because eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded and immunologically active antibodies.

Preferred mammalian host cells for expression of recombinant antibodies of the present application include Chinese hamster ovary (CHO cells) (including DHFR-CHO cells administered with a DHFR selectable marker as described in, for example, R.J.Kaufman and P.A.Sharp (1982) J.mol.biol.159: 601-621), as described in Urlaub and Chasin, (1980) Proc.Natl.Acad.Sci.USA 77: 4216-4220), NSO myeloma cells, COS cells and SP2 cells. Another preferred expression system, particularly when using NSO myeloma cells, is the GS gene expression system. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or preferably sufficient to allow secretion of the antibody into the medium in which the host cell is grown. Antibodies can be recovered from the culture medium using protein purification methods.

The antibodies or antigen-binding portions thereof of the present application can be crosslinked to a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binding molecules, DNA intercalators, DNA cross-linkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, inhibitors of topoisomerase I or II, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In an ADC, the antibody and therapeutic agent are preferably cross-linked by a linker that is cleavable, e.g., a peptidic, disulfide, or hydrazone linker. More preferably, the linker is a peptide linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Cit, Ser or Glu.

In another aspect, the present application relates to bispecific molecules comprising one or more antibodies of the present application, or antigen-binding portions thereof, linked to at least one other functional molecule, such as another peptide or protein (e.g., another antibody or receptor ligand), to generate bispecific molecules that bind to at least two different binding sites or targeting molecules. The term "bispecific molecule" includes molecules with three or more specificities. In some embodiments, the bispecific molecule has a third specificity in addition to the Fc binding specificity and the CD24 binding specificity. The third specificity may be, for example, directed to CD47 to better target tumor cells, while sparing other CD24⁺Cells such as immune cells.

Bispecific molecules can occur in a variety of forms and sizes. At one end of the size spectrum, the bispecific molecule remains in the traditional antibody format, except that it has two binding arms, each with a different specificity, instead of having two specificitiesThe same binding arm case. At the other extreme, bispecific molecules are composed of two single-chain antibody fragments (scFv) connected via a peptide chain, called Bs (scFv)₂Constructs. Bispecific molecules of intermediate size comprise two different f (ab) fragments linked by a peptide linker. These and other forms of bispecific molecules can be prepared by genetic engineering, somatic hybridization, or chemical methods.

The present application also provides chimeric antigen receptors comprising a CD24 single chain antibody scFv comprising the heavy and light chain CDRs, or the heavy and light chain variable regions described herein. The CD24 chimeric antigen receptor may comprise (a) an extracellular antigen-binding domain comprising CD24 scFv; (b) a transmembrane domain; and (c) an intracellular signaling domain.

In another aspect, the present application provides nucleic acid molecules encoding the heavy/light chain variable regions or CDRs of the antibodies or antigen-binding portions thereof of the present application. The nucleic acid may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially pure" when purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques. The nucleic acids of the present application may be, for example, DNA or RNA, and may or may not comprise intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

The nucleic acids of the present application can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes), cdnas encoding the light and heavy chains of the antibodies prepared by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding such antibodies can be collected from the gene libraries.

Preferred nucleic acid molecules of the present application include V encoding the CD24 monoclonal antibody_HAnd V_LThose of sequences or CDRs. Once the code V is obtained_HAnd V_LThe DNA fragments of (a), which can be further manipulated by standard recombinant DNA techniques, e.g.will beThe variable region gene is converted into a full-length antibody chain gene, a Fab fragment gene or a scFv gene. In these operations, V is encoded_HOr V_LIs operably linked to another DNA segment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" means that two DNA segments are linked together such that the amino acid sequences encoded by both DNA segments are in reading frame.

Code V_HIsolated DNA of the region may be operably linked to V_HCoding DNA and coding heavy chain constant region (C)_H1、C_H2And C_H3) Into the full-length heavy chain gene. The sequence of the human heavy chain constant region gene is known in the art, and DNA fragments comprising these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is preferably an IgG1 or IgG4 constant region. For the Fab fragment heavy chain gene, V is encoded_HThe DNA of the region may be operably linked to a DNA encoding only heavy chain C_H1Another DNA molecule of the constant region is linked.

Code V_LIsolated DNA of the region may be operably linked to V_LCoding DNA and coding light chain constant region C_LInto a full-length light chain gene. The sequence of the human light chain constant region gene is known in the art, and DNA fragments comprising these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region may be a kappa and lambda constant region.

To create scFv genes, encoding V_HAnd V_LMay be operably linked to another segment encoding a flexible linker, e.g., a 5-20 amino acid peptide, such that V_HAnd V_LThe sequence may be expressed as a continuous single chain protein, wherein V_HAnd V_LThe regions are connected by the flexible joint.

In another aspect, the present application provides a pharmaceutical composition comprising one or more antibodies or antigen-binding portions thereof, immunoconjugates, immune cells, bispecific antibodies, and/or nucleic acid molecules encoding the antibodies or antigen-binding portions thereof, expression vectors, or host cells of the present application formulated together with a pharmaceutically acceptable carrier. The composition may optionally comprise one or more other pharmaceutically active ingredients, for example another anti-tumour protein, such as IMM01, a sirpa D1-Fc fusion protein, for use in combination herein.

The pharmaceutical composition may comprise any number of excipients. Excipients that may be used include carriers, surfactants, thickening or emulsifying agents, solid binders, dispersing or suspending agents, solubilizers, coloring agents, flavoring agents, coatings, disintegrating agents, lubricants, sweetening agents, preservatives, isotonic agents and combinations thereof.

The pharmaceutical composition may be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or bolus injection). Depending on the route of administration, the active ingredient may be encapsulated in a material to protect it from acids and other natural conditions that might inactivate it. "parenteral administration" means a mode other than enteral and topical administration, and is typically performed by injection, including but not limited to intravenous, intramuscular, intraarterial, intramembranous, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, supradural, and intrasternal injection and bolus injection. Alternatively, the antibodies of the present application may be administered by a parenteral route, such as topical, epidermal or mucosal administration, such as intranasal, oral, vaginal, rectal, sublingual or topical.

The pharmaceutical compositions may be in the form of a sterile aqueous solution or dispersion. They may also be formulated in microemulsions, liposomes or other ordered structures suitable for high concentrations of drug.

The amount of active ingredient that is formulated with a carrier material into a single dosage form will vary with the host treated and the particular mode of administration, and is essentially the amount of composition that produces a therapeutic effect. The amount is from about 0.01 to about 99%, by percentage, of the active ingredient in combination with a pharmaceutically acceptable carrier.

The dosage regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a rapid perfusion agent may be administered, multiple divided doses may be administered over time, or the dose may be decreased or increased in proportion to the criticality of the treatment situation. It is particularly advantageous to formulate parenteral compositions in dosage units for convenient administration and uniform dosage. Dosage unit form refers to physically discrete units suitable for single administration to a subject; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the pharmaceutical carrier. Alternatively, the antibody may be administered in a slow release formulation, in which case the frequency of administration required is reduced.

For administration of the antibody, the dosage may be about 0.001-100mg/kg of host body weight.

A "therapeutically effective amount" of the CD24 antibody of the present application causes a reduction in the severity of disease symptoms, an increase in the frequency and duration of the asymptomatic phase. For example, for the treatment of a subject with a tumor, a "therapeutically effective amount" preferably inhibits tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and more preferably by at least about 80% as compared to an untreated subject. A therapeutically effective amount of a therapeutic antibody can reduce tumor size, or alleviate a symptom in a subject, which can be a human or another mammal.

The pharmaceutical composition may be a slow release agent, including implants, and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Pharmaceutical compositions can be administered via medical devices, such as (1) needleless hypodermic injection devices (e.g., U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) micro infusion pumps (us patent 4,487,603); (3) transdermal drug delivery devices (us patent 4,486,194); (4) bolus devices (U.S. Pat. nos. 4,447,233 and 4,447,224); and (5) infiltration equipment (U.S. Pat. Nos. 4,439,196 and 4,475,196).

In certain embodiments, a mab of the present application can be formulated to ensure proper in vivo distribution. For example, to ensure that the therapeutic antibodies of the present application cross the blood brain barrier, the antibodies may be formulated in liposomes, which may additionally contain targeting functional groups to enhance selective delivery to specific cells or organs.

The pharmaceutical compositions of the present application have a variety of in vitro and in vitro applications, relating to, for example, the treatment of cancer, or more generally, for immune enhancement in patients with cancer diseases. The antibodies can be administered to a human subject to inhibit tumor growth, for example, in vivo.

In view of the ability of the pharmaceutical compositions of the present application to inhibit tumor cell proliferation and survival, the present application provides methods of inhibiting tumor cell growth in a subject comprising administering to the subject a pharmaceutical composition of the present application, such that tumor growth is inhibited in the subject. Non-limiting examples of tumors that can be treated by the antibodies of the present application include, but are not limited to, ovarian cancer, breast cancer, cervical cancer, endometrial cancer, Acute Lymphocytic Leukemia (ALL), cholangiocarcinoma, bladder cancer, pancreatic cancer, gastric adenocarcinoma, glioblastoma, and colon cancer, primary or metastatic. The pharmaceutical compositions of the present application can be administered with one or more other antibody or non-antibody based therapeutic agents that are effective in inhibiting tumor growth in a subject. In one embodiment, the application provides a method of inhibiting tumor growth in a subject comprising administering to the subject a CD24 pharmaceutical composition in combination with IMM01, a sirpa D1-Fc fusion protein.

The pharmaceutical compositions of the present application may also be used to treat or alleviate inflammatory diseases such as acute graft versus host disease, inflammatory responses induced by anti-aids cocktail therapy (including hepatic steatosis and fibrosis, low density lipoprotein disorders, etc.), rheumatoid arthritis, and systemic lupus erythematosus.

The pharmaceutical compositions of the present application may also be used to treat or ameliorate CD 24-related diseases including, but not limited to, metabolic-related fatty liver disease, diabetes, multiple sclerosis, and sepsis.

The combination of therapeutic agents discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or simultaneously as separate compositions, wherein each agent is in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents may be administered sequentially.

Furthermore, if multiple combination therapy administrations are performed and the agents are administered sequentially, the order of sequential administration at each time point may be reversed or remain the same, sequential administration may be combined with simultaneous administration or any combination thereof.

The present application will be further illustrated with reference to the following non-limiting examples.

Examples

Figure 1 shows the structures of the CD24 antibodies IMM47C, IMM47, and IMM47H of the present application.

Wherein IMM47C is IgG antibody, and comprises mouse-derived heavy chain variable region and mouse-derived light chain variable region. Specifically, IMM47C comprises SEQ ID NO: 7, SEQ ID NO: 12, the heavy chain constant region of SEQ ID NO: 8, and SEQ ID NO: 13, a light chain constant region.

IMM47 is an IgG antibody comprising a mouse-derived heavy chain variable region, and a humanized light chain variable region. Specifically, IMM47 comprises SEQ ID NO: 7, SEQ ID NO: 12, the heavy chain constant region of SEQ ID NO: 9, and SEQ ID NO: 13, a light chain constant region.

IMM47H is an IgG antibody comprising a humanized heavy chain variable region and a humanized light chain variable region. Specifically, IMM47H comprises SEQ ID NO: 10, SEQ ID NO: 12, the heavy chain constant region of SEQ ID NO: 11, and SEQ ID NO: 13, a light chain constant region.

IMM01 is a sirpa D1-Fc fusion protein that binds CD47, described in US 2021/0024598a1, comprising two mutant sirpa D1, linked to an Fc dimer fragment, whose monomers contain the amino acid sequences as set forth in SEQ ID NOs: 20 and SEQ ID NO: 19 and amino acid sequence shown in the specification. Sirpa D1 in IMM01, compared to wild-type sirpa D1, in SEQ ID NO: 19 at position 80 there is a mutation at N80A, which can achieve the deglycosylation effect.

Example 1 CD24 resistanceGeneration, humanization, expression vector construction and expression of bodies

Mice were inoculated with human CD24 protein, sera thereof were taken, and mice with high titer were selected. A hybridoma cell line producing the CD24 antibody is prepared by taking mouse spleen cells through a hybridoma technology, and cell clones producing the monoclonal antibody with high CD24 binding activity, including cell clones producing IMM47C, are screened through a CD24 binding activity test. Sequencing of the heavy chain variable region and the light chain variable region of IMM47C revealed that the amino acid sequences are shown in SEQ ID NOs:7 and 8.

The IMM47C was humanized by CDR grafting to obtain a partially humanized (light chain variable region only humanized) antibody IMM47 and a fully humanized antibody IMM 47H. Wherein IMM47 comprises SEQ ID NO: 7, and SEQ ID NO: 9, IMM47H comprises SEQ ID NO: 10, and SEQ ID NO: 11, light chain variable region.

The coding sequences for IMM47C, IMM47, and IMM47H were designed artificially.

Specifically, for the heavy chain of IMM47C, 57 nucleotides encoding the mouse IgG1 heavy chain signal peptide (SEQ ID NO: 21) were added to the 5 'end of the IMM47C heavy chain variable region-constant region coding sequence (SEQ ID NO: 14) and a Kozak sequence (SEQ ID NO: 22) was added to the 5' end of the signal peptide sequence. Finally, HindIII and NheI restriction sites were added to the 5 'and 3' ends of the resulting sequence, respectively. For the light chain of IMM47C, the same signal peptide sequence as well as the Kozak sequence was added to the 5 ' end of the IMM47C antibody light chain variable region-constant region coding sequence (SEQ ID NO: 15), and HindIII and XbaI restriction sites were added to the 5 ' and 3 ' ends of the resulting sequence, respectively. The resulting sequences were synthesized by Kinsley Biotechnology Inc. and cloned into pMac-H and pMac-L vectors, respectively.

For the heavy chain of IMM47, 57 nucleotides encoding the mouse IgG1 heavy chain signal peptide (SEQ ID NO: 21) were added to the 5 'end of the IMM47 heavy chain variable region-constant region coding sequence (SEQ ID NO: 14) and a Kozak sequence (SEQ ID NO: 22) was added to the 5' end of the signal peptide sequence. Finally, HindIII and NheI restriction sites were added to the 5 'and 3' ends of the resulting sequence, respectively. For the light chain of IMM47, the same signal peptide sequence as well as the Kozak sequence was added to the 5 ' end of the IMM47 antibody light chain variable-constant region coding sequence (SEQ ID NO: 16), and HindIII and XbaI restriction sites were added to the 5 ' and 3 ' ends of the resulting sequence, respectively. The resulting sequences were synthesized by Kinsley Biotechnology Inc. and cloned into pMac-H and pMac-L vectors, respectively.

For the heavy chain of IMM47H, 57 nucleotides encoding the mouse IgG1 heavy chain signal peptide (SEQ ID NO: 21) were added to the 5 'end of the IMM47H heavy chain variable region-constant region coding sequence (SEQ ID NO: 17) and a Kozak sequence (SEQ ID NO: 22) was added to the 5' end of the signal peptide sequence. Finally, HindIII and NheI restriction sites were added to the 5 'and 3' ends of the resulting sequence, respectively. For the light chain of IMM47H, the same signal peptide sequence as well as the Kozak sequence was added to the 5 ' end of the IMM47H antibody light chain variable region-constant region coding sequence (SEQ ID NO: 18), and HindIII and XbaI restriction sites were added to the 5 ' and 3 ' ends of the resulting sequence, respectively. The resulting sequences were synthesized by Kinsley Biotechnology Inc. and cloned into pMac-H and pMac-L vectors, respectively.

The constructed expression vector is used for transiently expressing the protein by using the CHO-S cell. The general process is as follows: 1) CHO-S cells were plated at 1X10 cells the day before transient transformation⁶One/ml density was inoculated into a transient medium containing 6mM glutamine (TransFx-CTMCHO transient medium, Hyclone); 2) the weight/light chain expression vector was prepared in a weight ratio of 1: 1 from the desired DNA at 1. mu.g/ml, and added to OPTI-MEM medium (Gibco) in transient volume 1/20; 3) PEI (MW 40,000 polyethyleneimine hydrochloride, polysciences) was formulated at 1mg/ml, following the PEI: the desired PEI was prepared in a mass ratio of 4: 1 and added to an instantaneous volume 1/20 of OPTI-MEM medium (Gibco); 4) slowly adding the PEI diluent into the DNA diluent, uniformly mixing, and incubating for 20 minutes at room temperature; 5) add PEI/DNA mix to cell sap and place cells at 37 degrees 5% CO₂Culturing in an incubator with the rotation speed of 110rpm in an oscillating manner; 6) the transfection enhancer (1mM sodium butyrate, 0.25% V/VDMSO) was added every other day while the culture temperature was reduced to 33 degrees; 7) when the cell viability rate is reduced to below 50%, 3000rpm, centrifuging for 5 min, collecting supernatant, and utilizingProtein a packing was affinity purified.

^{+ +}Example 2 binding of CD24 antibody to CD24CD47MCF-7 cells

Will CD24⁺CD47⁺The MCF-7 cell density was adjusted to 1X10⁶Mu.l/ml were taken and incubated in 100. mu.l of IMM47H starting at 30. mu.g/ml and diluted in 3-fold gradients for 1 hour at 4 ℃ with hIgG-Fc used as a negative control. Cells were washed twice with cold PBS, and then incubated for 45 minutes in 100. mu.l FITC-labeled secondary antibody against human IgG-Fc (Cat # F9512, Sigma). Cells were washed twice and resuspended in 200. mu.l PBS. Thereafter, the cells were analyzed by flow cytometry (Merck Millipore,easyCyte 5HT) was performed.

As shown in fig. 2, the CD24 antibodies of the present application, including IMM47H, were capable of specifically binding to CD24⁺CD47⁺MCF-7 cells.

^{+ +}Example 3 binding of CD24 antibodies to CD24CD47REH cells

CD24⁺CD47⁺The REH cell density was adjusted to 1X10⁶Mu.l/ml were taken and incubated in 100. mu.l of IMM47H starting at 30. mu.g/ml and diluted in 3-fold gradients for 1 hour at 4 ℃ using IMM01, hIgG-Fc as controls. Cells were washed twice with cold PBS, and then incubated for 45 minutes in 100. mu.l FITC-labeled secondary antibody against human IgG-Fc (Cat # F9512, Sigma). Cells were washed twice and resuspended in 200. mu.l PBS. Then, the cells are subjected to flow cytometry (easyCyte 5HT, Merck Millipore) for FACS analysis.

As shown in FIG. 3, IMM47H and CD24⁺CD47⁺The specific binding of REH cells is slightly better than that of CD47 binding protein IMM 01.

^{+ +}Example 4 priming of CD24 antibodies against CD24CD47MCF-7 cells to high levels of antibody-dependent cellular mediatorsOf lead Cytotoxicity (ADCC)

Labelled MCF-7 cells were diluted 500-fold at 1mM CFSE (Cat #21888-25mg, Sigma).

Taking 50 μ l of the mixture with a density of 6X 10⁵MCF-7 cells (used as targeting cells) per ml, at a density of 6X 10 to 100. mu.l⁵NK92MI cells (effector cells) stably expressing Fc γ RIIIa (158V) in a 2: 1 effective target ratio mixed at 5% CO₂Next, 50. mu.l of IMM47C diluted in 3-fold gradient at an initial concentration of 1000ng/ml was incubated at 37 ℃ for 4 hours, and hIgG-Fc was used as a negative control. Propidium Iodide (PI) (Cat # P4170, Sigma) was then added to the cell culture media at a concentration of 5. mu.g/ml and the cells were analyzed for PI signal by FACS. The percentage of cell lysis due to ADCC was calculated based on the following formula:

% lysis [% IMM47C treated PI-positive target cells [% negative control protein treated PI-positive target cells ]/[ 100- ] negative control protein treated PI-positive target cells ] × 100

As shown in fig. 4 for CD24⁺CD47⁺MCF-7 human breast cancer cells, IMM47C, elicited significant levels of ADCC.

^{+ +}Example 5 priming of CD24 antibodies against CD24CD47REH cells to high levels of antibody-dependent cell-mediated cells Cytotoxicity (ADCC)

Labeled REH cells were diluted 500-fold in 1mM CFSE (Cat #21888-25mg, Sigma).

Taking 50 μ l of the mixture with a density of 6X 10⁵REH cells (used as targeting cells) per ml, with a density of 6X 10 of 100. mu.l⁵NK92MI cells (effector cells) stably expressing Fc γ RIIIa (158V) in a 2: 1 effective target ratio mixed at 5% CO₂Next, 50. mu.l of IMM47C diluted in 3-fold gradient at an initial concentration of 1000ng/ml was incubated at 37 ℃ for 4 hours, and hIgG-Fc was used as a negative control. Propidium Iodide (PI) (Cat # P4170, Sigma) was then added to the cell culture media at a concentration of 5. mu.g/ml and the cells were analyzed for PI signal by FACS. The percentage of cell lysis due to ADCC was calculated based on the following formula:

% lysis [% IMM47C treated PI-positive target cells [% negative control protein treated PI-positive target cells ]/[ 100- ] negative control protein treated PI-positive target cells ] × 100

As shown in fig. 5 for CD24⁺CD47⁺REH human acute lymphoblastic leukemia cells, IMM47C, elicits significant levels of ADCC.

⁺Example 6 priming of high levels of antibody-dependent cell-mediated targeting of CD24 antibody against CD24MC38-hCD24 cells Cytotoxicity (ADCC)

The labeled MC38-hCD24 mouse colon cancer cells were diluted 500-fold in 1mM CFSE (Cat #21888-25mg, Sigma).

Taking 50 μ l of the mixture with a density of 6X 10⁵MC38-hCD24 cells (used as targeting cells) in ml with 100. mu.l density of 6X 10⁵NK92MI cells (effector cells) stably expressing Fc γ RIIIa in a 2: 1 effective target ratio mixed at 5% CO₂Next, 50. mu.l of IMM47 diluted in a 3-fold gradient at an initial concentration of 1000ng/ml was incubated at 37 ℃ for 4 hours, and hIgG-Fc was used as a negative control. Propidium Iodide (PI) (Cat # P4170, Sigma) was then added to the cell culture media at a concentration of 5. mu.g/ml and the cells were analyzed for PI signal by FACS. The percentage of cell lysis due to ADCC was calculated based on the following formula:

% lysis ═ PI positive target cells treated (% IMM 47-treated PI positive target cells [% negative control protein-treated PI positive target cells)/(100-% negative control protein-treated PI positive target cells) × 100

As shown in figure 6, IMM47 elicited significant levels of ADCC for MC38-hCD24 mouse colon cancer cells expressing human CD 24.

Example 7 CD24 antibody shows potent in vivo anti-tumor Activity in a mouse xenograft model of breast cancer

40 SCID mice, 6-8 weeks old, were inoculated with a beta-estradiol sustained release tablet (0.36mg) 3 days prior to cancer cell inoculation in the left back of each mouse. Right forelimb axilla were injected subcutaneously with MCF-7 breast cancer cells at 1x10 per mouse⁷And (4) cells. When the tumor volume reaches 100-³At the same time, mice were randomly divided into 5 groupsEach group of 8 mice was assigned a day of the group as D0. From this day, groups of mice were injected intraperitoneally with PBS, IMM47C (2.5mg/kg), IMM01(2.5mg/kg), and IMM01+ IMM47C (2.5mg/kg +2.5mg/kg), for 4 weeks, 2 times per week. Dosing was terminated after 4 weeks and the experiment was terminated after 1 week of continuous observation. Tumor volume and mouse body weight were measured every 3-4 days. When the mean tumor volume of the PBS group reaches 3000mm³At this point, all tests were terminated.

Tumor volume (V) was calculated as (length x width)²)/2. TGI (%) ═ 1- (average tumor volume at the end of administration of a treatment group-average tumor volume at the start of administration of the treatment group)/(average tumor volume at the end of treatment of the solvent control group-average tumor volume at the start of treatment of the solvent control group)]×100％。

The protocol and results of the testing are summarized in table 1 below.

TABLE 1 antitumor Effect of IMM47C and other therapeutic Agents

On day 28 after the start of the administration, the tumor volume of the tumor-bearing mice in the solvent control group reached 646.87mm³. Both the IMM 47C-treated group and the IMM 01-treated group delayed tumor growth compared to the solvent control group, and the tumor volumes at day 28 in these two groups were 463.26mm, respectively³(T/C71.60%, TGI 37.30%, p 0.009) and 562.24mm³(T/C: 86.92%, TGI: 17.19%, p: 0.375). The IMM47C + IMM01 combination group had the best tumor suppression effect, and the tumor volume of the group at day 28 was 192.63mm³(T/C＝29.81％，TGI＝92.22％，p＝0.001)。

As can be seen from Table 1 and FIG. 7, the combination of IMM47C and IMM01 produced a synergistic effect with an overall antitumor effect that was much better than IMM47C alone or IMM01 alone.

Example 8 CD24 antibody shows potent in vivo anti-tumor Activity in a Colon cancer mouse Homoplasma model

MC38-hCD24 cells were cultured at 37 ℃ with 5% CO₂In an incubator, a culture mediumThe cells were plated for passage every 3 to 4 days in DMEM medium containing 10% inactivated fetal calf serum. Taking MC38-hCD24 in logarithmic growth phase, suspending in PBS, counting cells, and adjusting cell concentration to 1.0 × 10⁷Per mL, the cell suspension was inoculated subcutaneously into the right flank of 30B 6-Siglec10 male mice (Siglec10 humanized B6 mice) using a 1mL syringe at 100. mu.L/mouse, each inoculated at about 1.0X 10⁶And (4) tumor cells. When the mean tumor volume reached about 100mm³At the time, animals were randomly divided into 3 groups of 10 animals per group by tumor volume. The grouping day was set as D0, and from this day, mice were intraperitoneally injected with IMM47H (10mg/kg), IMM47C (10mg/kg) and PBS, respectively, 2 times per week. After 4 serial administrations, 7 mice per group were re-inoculated with MC38-hCD24 tumor cells at D18, and the cell suspension was inoculated subcutaneously into the left flank of the mice using a 1mL syringe at 100. mu.L/mouse, each at about 1.0X 10⁶And (4) tumor cells. Tumor growth was monitored twice before and after 2 vaccinations a week. Tumors exceed 3000mm according to animal welfare criteria³Tumors need to be euthanized at this time.

Tumor volume (V) was calculated as (length x width)²)/2. Tumor volume inhibition rate TGI (%) [1- (average tumor volume at the end of administration of a certain treatment group-average tumor volume at the start of administration of the treatment group)/(average tumor volume at the end of treatment of the solvent control group-average tumor volume at the start of treatment of the solvent control group)]×100％

The entire experimental protocol is shown in fig. 8B and table 2.

Table 2 tumor size and data analysis after IMM47H and IMM47C treatment.

As shown in Table 2 and FIGS. 8A-8B, tumor-bearing mice were completely cleared after 4 doses of IMM47H and IMM47C drugs, whereas the PBS group had 969.92. + -. 143.93mm tumor sizes³(ii) a After re-inoculation of the tumor cells with D18, 5/7 mice in the PBS group, non-tumorigenic in the IMM47H and MM47C groups, and 5 mice in the blank control group all tumorigenic without further administration.

The results show that IMM47C and IMM47H show strong tumor growth inhibition on a Siglec10 humanized B6 mouse homologous transplantation MC38-hCD24KI cytoma model, and the tumor cells of the secondary inoculation of the administration group have no tumor formation, which indicates that the mice of the administration group may have acquired immune response.

Example 9 CD24 antibody shows potent in vivo anti-tumor Activity in a Colon cancer mouse Homocodel

MC38-hCD24 cells were cultured at 37 ℃ with 5% CO₂The culture medium is DMEM medium containing 10% inactivated fetal calf serum, and the cells are plated for passage after full growth every 3 to 4 days. Taking MC38-hCD24 in logarithmic growth phase, suspending in PBS, counting cells, and adjusting cell concentration to 1.0 × 10⁷Per mL, the cell suspension was inoculated subcutaneously into the right flank of 24B 6-Siglec10 female mice (Siglec10 humanized B6 mice) using a 1mL syringe at 100. mu.L/mouse, each inoculated at about 1.0X 10⁶And (4) tumor cells. When the mean tumor volume reached about 100mm³At the time, animals were randomly divided into 4 groups of 6 animals by tumor volume. The grouping day was designated D0, and from this day, mice in each group were intraperitoneally injected 2 times a week for 4 consecutive weeks with IMM47(3mg/kg), IMM47H (3mg/kg), IMM47C (3mg/kg) and PBS, respectively.

Since the tumor size of the mice at D21 exceeded the preset value, the data from D0-D21 were plotted and analyzed.

Table 3 tumor size and data analysis after IMM47, IMM47H and IMM47C treatment.

As shown in table 2 and figure 9, IMM47, IMM47C, and IMM47H demonstrated strong tumor growth inhibition on Siglec10 humanized B6 mouse xenograft MC38-hCD24KI cell tumor model. Specifically, at a dose of 3mg/kg, tumors were completely removed from 6 mice in the IMM 47C-treated group, 4 mice in the IMM 47-treated group, another 2 mice were slow in tumor growth, 5 mice in the IMM 47H-treated group, and 1 mouse tumor continued to grow.

The sequence information of the present application is summarized below.

While the application has been described in connection with one or more embodiments, it should be understood that the application is not limited to those embodiments. The description herein is intended to cover all modifications and equivalents as may be included within the spirit and scope of the appended claims. All documents cited herein are incorporated by reference in their entirety.

Reference to the literature

1.Toubai，T.，et al.，Siglec-G-CD24 axis controls the severity of graft-versus-host disease in mice.Blood，2014.123(22)：p.3512-23.

2.Shim H.Bispecific Antibodies and Antibody-Drug Conjugates for Cancer Therapy：Technological Considerations.Biomolecules.2020 Feb 26；10(3)：360.

3.Tian，R.R.，et al.，CD24and Fc fusion protein protects SIVmac239-infected Chinese rhesus macaque against progression to AIDS.Antiviral Res，2018.157：p.9-17.

4.Fairbridge，N.A.，et al.，Loss of CD24in Mice Leads to Metabolic Dysfunctions and a Reduction in White Adipocyte Tissue.PLoS One，2015.10(11)：p.e0141966.

5.El-Mokhtar，M.A.，et al.，Altered Regulatory B Cell Subsets in Children with Type 1Diabetes Mellitus.J Immunol Res，2020.2020：p.8935694.

6.Tan，Y.，et al.，CD24：from a Hematopoietic Differentiation Antigen to a Genetic Risk Factor for Multiple Autoimmune Diseases.Clin Rev Allergy Immunol，2016.50(1)：p.70-83.

7.Chen，G.Y.，et al.，Amelioration of sepsis by inhibiting sialidase-mediated disruption of the CD24-SiglecG interaction.Nat Biotechnol，2011.29(5)：p.428-35.

8.Graft Versus Host Disease (GVHD)：https：//clinicaltrials.gov/ct2/show/NCT04095858term＝CD24&draw＝2&rank＝9

9.Antiretroviral therapy induced increase of LDL，HbA1c，hepatic steatosis and fibrosis，and other markers of inflammation：

https：//clinicaltrials.gov/ct2/show/NCT03960541term＝CD24&draw＝2&rank＝2

10.Metabolic Associated Fattv Liver Disease：

https：//clinicaltrials.gov/ct2/show/NCT04720560term＝CD24&draw＝4&rank＝23

11.TyPe 1 Diabetes：

https：//clinicaltrials.gov/ct2/show/NCT02801942term＝CD24&draw＝4&rank＝25

12.Type 2 Diabetes Mellitus and Cardiovascular Diseases：

https：//clinicaltrials.gov/ct2/show/NCT02694575term＝CD24&draw＝5&rank＝34

13.Rheumatoid Arthritis：

https：//clinicaltrials.gov/ct2/show/NCT03793270term＝CD24&draw＝4&rank＝24

14.Systemic Lupus Erythematosus：

https：//clinicaltrials.gov/ct2/show/NCT03178721term＝CD24&draw＝5&rank＝32

15.Multiple Sclerosis

https：//clinicaltrials.gov/ct2/show/NCT03257358term＝CD24&draw＝5&rank＝35

16.Sepsis：

https：//clinicaltrials.gov/ct2/show/NCT01995448term＝CD24&draw＝5&rank＝36

17.Shields RL，Namenuk AK，Hong K，Meng YG，Rae J，Briggs J，Xie D，Lai J，Stadlan A，Li B，Fox JA，Presta LG.High Resolution Mapping of the Binding Site on Human IgG1 for FcγRI，FcγRII，FcγRIII，and FcRn and Design of IgG 1 Variants with Improved Binding to the FcgR.JBC.2001，276：6591-6604.

18.Barkal，A.A.，Brewer，R.E.，Markovic，M.，Kowarsky，M.，Barkal，S.A.，Zaro，B.W.，Krishnan，V.，Hatakeyama，J.，Dorigo，O.，Barkal，L.J.，et al.(2019).CD24 signalling through macrophage Siglec-10 is a targetfor cancer immunotherapy.Nature 572(7769)，392-396.

19.Chan，S.H.，Tsai，K.W.，Chiu，S.Y.，Kuo，W.H.，Chen，H.Y.，Jiang，S.S.，Chang，K.J.，Hung，W.C.，and Wang，L.H.(2019).Identification ofthe Novel Role ofCD24 as an Oncogenesis Regulator and Therapeutic Target for Triple-Negative Breast Cancer.MolCancer Ther 18(1)，147-161.

20.Wang S，Chen K，Lei Q，Ma P，Yuan AQ，Zhao Y，Jiang Y，Fang H，Xing S，Fang Y，Jiang N，Miao H，Zhang M，Sun S，Yu Z，Tao W，Zhu Q，Nie Y，Li N.The state of the art of bispecific antibodies for treating human malignancies.EMBO Mol Med.2021 Aug 24：e14291.doi：10.15252/emmm.202114291.

21.Yin，S.S.，and Gao，F.H.(2020).Molecular Mechanism of Tumor Cell Immune Escape Mediated by CD24/Siglec-10.Front Immunol11，1324.

22.Gardai SJ，McPhillips KA，Frasch SC，Janssen WJ，Starefeldt A，Murphy-Ullrich JE，Bratton DL，Oldenborg PA，Michalak M，Henson PM.Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte.Cell.2005；123：321-334.

23.Gennaro，ed.，Remington：The Science and Practice of Pharmacy，20th Ed.，Lippincott Williams&Wilkins 2003.

24.Liu，C.，Zheng，S.，Shen，H.，Xu，K.，Chen，J.，Li，H.，Xu，Y.，Xu，A.，Chen，B.，Kaku，H.，et al.(2013).Clinical significance of CD24 as a predictor ofbladder cancer recurrence.Oncol Lett 6(1)，96-100.

25.Nakamura et al.CD24 expression is a marker for predicting clinical outcome and regulates the epithelial-mesenchymal transition in ovarian cancer via both the Akt and ERK pathways.Oncol Rep.2017 Jun；37(6)：3189-3200.

26.Obeid M，Panaretakis T，Joza N，Tufi R，Tesniere A，van Endert P，Zitvogel L，Kroemer G.Calreticulin exposure is required for the immunogenicity of gamma-irradiation and UVC lightinduced apoptosis.Cell Death Differ.2007，14∶1848-1850.

27.Orr AW，Pedraza CE，Pallero MA，Elzie CA，Goicoechea S，Strickland DK，Murphy-Ullrich JE.Low density lipoprotein receptor-related protein is a calreticulin coreceptor that signals focal adhesion disassembly.J Cell Biol.2003，161：1179-1189.

28.Overdevest，J.B.，Thomas，S.，Kristiansen，G.，Hansel，D.E.，Smith，S.C.，and Theodorescu，D.(2011).CD24 offers a therapeutic target for control of bladder cancer metastasis based on a requirement for lung colonization.CancerRes 71(11)，3802-11.

Sequence listing

<110> Yimingke biomedical technology (Shanghai) Co., Ltd

<120> CD 24-targeting antibody and preparation and use thereof

<130> 55525 00044

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<170> PatentIn version 3.5

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<223> IMM47C, IMM47 and IMM47H antibody heavy chain HV-CDR-1

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Gly Tyr Ser Ile Thr Ser Gly Tyr Ser

1 5

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<211> 7

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Ile His Tyr Ser Gly Ser Thr

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Ala Arg Gly Ala Asp Tyr Ala Leu Asp Tyr

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Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr

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Trp Ala Ser

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<213> Artificial sequence

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<223> IMM47C, IMM47 and IMM47H antibody light chain LV-CDR-3

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Gln Gln Asn Phe Ile Tyr Pro Leu Thr

1 5

<210> 7

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> IMM47C and IMM47 antibody heavy chain variable region

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Asp Val Gln Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile His Tyr Ser Gly Ser Thr Lys Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Gly Ala Asp Tyr Ala Leu Asp Tyr Trp Gly Gln Arg Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 8

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> IMM47C antibody light chain variable region

<400> 8

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly His

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Asn Phe Ile Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 9

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> IMM47 antibody light chain variable region

<400> 9

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

85 90 95

Asn Phe Ile Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Leu

100 105 110

Lys

<210> 10

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> IMM47H antibody heavy chain variable region

<400> 10

Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile His Tyr Ser Gly Ser Thr Lys Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ala Asp Tyr Ala Leu Asp Tyr Trp Gly Gln Arg Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 11

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> IMM47H antibody light chain variable region

<400> 11

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Asn Phe Ile Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Leu

100 105 110

Lys

<210> 12

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain constant region

<400> 12

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Ala Ala Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 13

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> light chain constant region

<400> 13

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 14

<211> 1398

<212> DNA

<213> Artificial sequence

<220>

<223> nucleic acid sequences of IMM47C and IMM47 antibody heavy chains

<400> 14

atgagagtgc tgattctttt gtgcctgttc acagcctttc ctggtatcct gtctgatgtg 60

cagcttcagg agtcaggacc tgacctggtg aaaccttctc agtcactttc actcacctgc 120

actgtcactg gctactccat caccagtggt tatagctggc actggatccg gcagtttcca 180

ggaaacaaac tggaatggat gggctacata cactatagtg gtagcactaa gtacaaccca 240

tctctcaaaa gtcgaatctc tatcactcga gacacatcca agaaccagtt cttcctgcag 300

ttgaattctg tgactactga ggacacagcc acatatttct gtgcaagagg cgcggactat 360

gctttggact actggggtca acgaacctca gtcaccgtct cctcagctag caccaagggc 420

ccatcggtct tccccctggc accctcctcc aagagcacct ctgggggcac agcggccctg 480

ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 540

ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 600

agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc agacctacat ctgcaacgtg 660

aatcacaagc ccagcaacac caaggtggac aagagagttg agcccaaatc ttgtgacaaa 720

actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 780

ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 840

gtggtggacg tgagccacga agaccctgag gtcaagttca actggtatgt ggacggcgtg 900

gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacgccac gtaccgtgtg 960

gtcagcgtcc tcaccgtcct gcaccaagac tggctgaatg gcaaggagta caagtgcaag 1020

gtctccaaca aagccctccc agcccccatc gccgcaacca tctccaaagc caaagggcag 1080

ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccaa 1140

gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1200

agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 1260

tccttcttcc tctattccaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1320

ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1380

ctgtctccgg gcaaatga 1398

<210> 15

<211> 862

<212> DNA

<213> Artificial sequence

<220>

<223> nucleic acid sequence of IMM47C antibody light chain

<400> 15

atggattcac aggcccaggt tcttatgtta ctgctgctat gggtatctgg tacctgtggg 60

gacattgtga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact 120

atgagctgca agtccagtca gagcctttta tatagtagca atcaaaagaa ctacttggcc 180

tggtaccagc agaaaccagg gcactctcct aaactgctga tttactgggc atccactagg 240

gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 300

atcagcagtg tgaaggctga agacctggca gtttattact gtcagcaaaa ttttatctat 360

ccgctcacgt tcggtgctgg gaccaagctg gagctgaaac gtgagttcta gaggatccat 420

ctgggataag catgctgttt tctgtctgtc cctaacatgc cctgtgatta tccgcaaaca 480

acacacccaa gggcagaact ttgttactta aacaccatcc tgtttgcttc tttcctcagg 540

aactgtggct gcaccatctg tcttcatctt cccgccatct gatgagcagt tgaaatctgg 600

aactgcctct gttgtgtgcc tgctgaataa cttctatccc agagaggcca aagtacagtg 660

gaaggtggat aacgccctcc aatcgggtaa ctcccaggag agtgtcacag agcaggacag 720

caaggacagc acctacagcc tcagcagcac cctgacgctg agcaaagcag actacgagaa 780

acacaaagtc tacgcctgcg aagtcaccca tcagggcctg agctcgcccg tcacaaagag 840

cttcaacagg ggagagtgtt ag 862

<210> 16

<211> 859

<212> DNA

<213> Artificial sequence

<220>

<223> nucleic acid sequence of IMM47 antibody light chain

<400> 16

atgggatggt catgtatcat cctttttctg gtagcaactg caactggagt acattcagac 60

attcagatga cacagagccc tagcagcctg agcgcctccg tgggcgacag agtgaccatc 120

acctgcaaga gcagccaaag cctgctgtac agcagcaatc agaagaacta cctggcctgg 180

tatcagcaga agcctggcaa ggcccctaag ctgctgatct actgggcctc cacaagagag 240

agcggcgtgc ctagcagatt cagcggcagc ggcagcggca ccgacttcac cctgaccatc 300

agcagcctgc agcctgagga cttcgccacc tactactgtc agcagaactt catctaccct 360

ctgaccttcg gcggaggcac caaggtggag ctgaagcgtg agttctagag gatccatctg 420

ggataagcat gctgttttct gtctgtccct aacatgccct gtgattatcc gcaaacaaca 480

cacccaaggg cagaactttg ttacttaaac accatcctgt ttgcttcttt cctcaggaac 540

tgtggctgca ccatctgtct tcatcttccc gccatctgat gagcagttga aatctggaac 600

tgcctctgtt gtgtgcctgc tgaataactt ctatcccaga gaggccaaag tacagtggaa 660

ggtggataac gccctccaat cgggtaactc ccaggagagt gtcacagagc aggacagcaa 720

ggacagcacc tacagcctca gcagcaccct gacgctgagc aaagcagact acgagaaaca 780

caaagtctac gcctgcgaag tcacccatca gggcctgagc tcgcccgtca caaagagctt 840

caacagggga gagtgttag 859

<210> 17

<211> 1401

<212> DNA

<213> Artificial sequence

<220>

<223> nucleic acid sequence of IMM47H antibody heavy chain

<400> 17

atgggatggt catgtatcat cctttttctg gtagcaactg caactggagt acattcagac 60

gtgcagctgc aagagagcgg ccctggcctg gtgaagccta gcgagaccct gagcctgacc 120

tgcaccgtgt ccggctacag catcacaagc ggctacagct ggcactggat cagacagcct 180

cctggcaagg gcctggagtg gatcggctac atccactaca gcggcagcac caagtacaac 240

cctagcctga agagcagagt gaccatcagc gtggacacaa gcaagaatca gttcagcctg 300

aagctgagca gcgtgaccgc cgccgacacc gccgtgtact actgcgctag aggcgccgac 360

tacgccctgg actactgggg acagagaaca agcgtgaccg tgagcagcgc tagcaccaag 420

ggcccatcgg tcttccccct ggcaccctcc tccaagagca cctctggggg cacagcggcc 480

ctgggctgcc tggtcaagga ctacttcccc gaaccggtga cggtgtcgtg gaactcaggc 540

gccctgacca gcggcgtgca caccttcccg gctgtcctac agtcctcagg actctactcc 600

ctcagcagcg tggtgaccgt gccctccagc agcttgggca cccagaccta catctgcaac 660

gtgaatcaca agcccagcaa caccaaggtg gacaagagag ttgagcccaa atcttgtgac 720

aaaactcaca catgcccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc 780

ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 840

gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta tgtggacggc 900

gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacgc cacgtaccgt 960

gtggtcagcg tcctcaccgt cctgcaccaa gactggctga atggcaagga gtacaagtgc 1020

aaggtctcca acaaagccct cccagccccc atcgccgcaa ccatctccaa agccaaaggg 1080

cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 1140

caagtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1200

gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1260

ggctccttct tcctctattc caagctcacc gtggacaaga gcaggtggca gcaggggaac 1320

gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 1380

tccctgtctc cgggcaaatg a 1401

<210> 18

<211> 859

<212> DNA

<213> Artificial sequence

<220>

<223> IMM47H antibody light nucleic acid sequence

<400> 18

atgggatggt catgtatcat cctttttctg gtagcaactg caactggagt acattcagac 60

atcgtgatga cacagagccc tgacagcctg gccgtgagcc tgggcgagag agccaccatc 120

aactgcaaga gctctcagag cctgctgtac agcagcaatc agaagaacta cctggcctgg 180

tatcagcaga agcctggaca gcctcctaag ctgctgatct actgggcaag cacaagagag 240

agcggcgtgc ctgacagatt cagcggcagc ggcagcggca ccgacttcac cctgaccatc 300

agcagcctgc aagccgagga cgtggccgtg tactactgtc agcagaactt catctaccct 360

ctgaccttcg gcggcggcac caaggtggag ctgaagcgtg agttctagag gatccatctg 420

ggataagcat gctgttttct gtctgtccct aacatgccct gtgattatcc gcaaacaaca 480

cacccaaggg cagaactttg ttacttaaac accatcctgt ttgcttcttt cctcaggaac 540

tgtggctgca ccatctgtct tcatcttccc gccatctgat gagcagttga aatctggaac 600

tgcctctgtt gtgtgcctgc tgaataactt ctatcccaga gaggccaaag tacagtggaa 660

ggtggataac gccctccaat cgggtaactc ccaggagagt gtcacagagc aggacagcaa 720

ggacagcacc tacagcctca gcagcaccct gacgctgagc aaagcagact acgagaaaca 780

caaagtctac gcctgcgaag tcacccatca gggcctgagc tcgcccgtca caaagagctt 840

caacagggga gagtgttag 859

<210> 19

<211> 364

<212> PRT

<213> Artificial sequence

<220>

<223> SIRP alpha D1 mutant-Fc (IMM 01)

<400> 19

Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala

1 5 10 15

Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro

20 25 30

Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu

35 40 45

Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser

50 55 60

Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Ala

65 70 75 80

Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys

85 90 95

Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser

100 105 110

Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg

115 120 125

Ala Thr Pro Gln His Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

130 135 140

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

145 150 155 160

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

165 170 175

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

180 185 190

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

195 200 205

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

210 215 220

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

225 230 235 240

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

245 250 255

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

260 265 270

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

275 280 285

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

290 295 300

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

305 310 315 320

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

325 330 335

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

340 345 350

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

355 360

<210> 20

<211> 1095

<212> DNA

<213> Artificial sequence

<220>

<223> SIRP alpha D1 mutant-Fc (IMM 01)

<400> 20

gaggaggagc tgcaggtgat tcagcctgac aagtccgtat cagttgcagc tggagagtcg 60

gccattctgc actgcactgt gacctccctg atccctgtgg ggcccatcca gtggttcaga 120

ggagctggac cagcccggga attaatctac aatcaaaaag aaggccactt cccccgggta 180

acaactgttt cagagtccac aaagagagaa aacatggact tttccatcag catcagtgcc 240

atcaccccag cagatgccgg cacctactac tgtgtgaagt tccggaaagg gagccctgac 300

acggagttta agtctggagc aggcactgag ctgtctgtgc gtgccaaacc ctctgccccc 360

gtggtatcgg gccctgcggc gagggccaca cctcagcacg agcccaaatc ttgtgacaaa 420

actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 480

ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 540

gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 600

gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 660

gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 720

gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 780

ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 840

gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 900

agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 960

tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1020

ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1080

ctgtctccgg gttga 1095

<210> 21

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> nucleic acid sequence of mouse IgG1 heavy chain signal peptide

<400> 21

atgggatggt catgtatcat cctttttctg gtagcaactg caactggagt acattca 57

<210> 22

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Kozak sequence

<400> 22

gccgccacc 9

<210> 23

<211> 597

<212> PRT

<213> Artificial sequence

<220>

<223> human SIRP alpha-mouse IgG1 Fc

<400> 23

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Ser Cys Ala Trp Ser Gly Val Ala Gly Glu Glu Glu Leu

20 25 30

Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Ala Gly Glu Ser

35 40 45

Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro Val Gly Pro Ile

50 55 60

Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu Ile Tyr Asn Gln

65 70 75 80

Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu Ser Thr Lys

85 90 95

Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn Ile Thr Pro Ala

100 105 110

Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro Asp

115 120 125

Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val Arg Ala Lys

130 135 140

Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr Pro Gln

145 150 155 160

His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro Arg Asp

165 170 175

Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp Phe Gln

180 185 190

Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile His Ser

195 200 205

Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser Gln Val Ile

210 215 220

Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg Gly Thr

225 230 235 240

Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu Glu Val Thr

245 250 255

Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr Cys Gln Val

260 265 270

Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu Glu Asn Gly

275 280 285

Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu Asn Lys Asp

290 295 300

Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val Ser Ala His

305 310 315 320

Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp Gly Gln Pro

325 330 335

Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Ala His Pro Lys Glu

340 345 350

Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Ser Asn Glu Arg Asn

355 360 365

Glu Phe Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val

370 375 380

Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val

385 390 395 400

Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile

405 410 415

Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val

420 425 430

Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser

435 440 445

Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu

450 455 460

Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala

465 470 475 480

Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro

485 490 495

Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys

500 505 510

Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr

515 520 525

Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr

530 535 540

Gln Pro Ile Met Asn Thr Asn Gly Ser Tyr Phe Val Tyr Ser Lys Leu

545 550 555 560

Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser

565 570 575

Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser

580 585 590

His Ser Pro Gly Lys

595