阅读说明：本技术 靶向cd47和cd38的重组融合蛋白及其制备和用途 (Recombinant fusion protein targeting CD47 and CD38, and preparation and application thereof ) 是由 田文志 李松 陈典泽 于 2021-10-09 设计创作，主要内容包括：本申请提供一种重组融合蛋白,包含CD38抗体或其抗体片段,该CD38抗体或其抗体片段的至少一个互补位在构成该互补位的重链或轻链的N端通过接头与信号调节蛋白(SIRP)的胞外Ig样结构域连接,其中该重组融合蛋白可以同时与CD47、CD38和FcR结合。还提供编码该重组融合蛋白的核酸分子、包含该核酸分子的表达载体、制备该重组融合蛋白的方法、以及使用重组融合蛋白来治疗与CD47和/或CD38过表达相关的疾病的方法。(The present application provides a recombinant fusion protein comprising a CD38 antibody or antibody fragment thereof, at least one paratope of the CD38 antibody or antibody fragment thereof being linked to an extracellular Ig-like domain of a signal-regulatory protein (SIRP) via a linker at the N-terminus of the heavy or light chain constituting the paratope, wherein the recombinant fusion protein can simultaneously bind to CD47, CD38 and FcR. Also provided are nucleic acid molecules encoding the recombinant fusion proteins, expression vectors comprising the nucleic acid molecules, methods of making the recombinant fusion proteins, and methods of using the recombinant fusion proteins to treat diseases associated with overexpression of CD47 and/or CD 38.)

1. A recombinant fusion protein comprising a CD38 antibody or antibody fragment thereof, and a CD47 binding peptide,

the CD38 antibody or antibody fragment thereof comprises a heavy chain variable region comprising amino acid sequences set forth in SEQ ID NOs: 4. 5 and 6, and an HV-CDR1, an HV-CDR2, and an HV-CDR3, the light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 7. LV-CDR1, LV-CDR2, and LV-CDR3 shown in FIGS. 8 and 9, the heavy chain constant region has FcR binding ability and is linked to the C-terminus of the heavy chain variable region,

the CD47 binding peptide comprises an extracellular Ig-like domain of a signal-regulating protein (SIRP) having an amino acid sequence as set forth in SEQ ID NO: as shown in figure 1, the first and second main bodies,

each paratope of the CD38 antibody or antibody fragment thereof is linked to the CD47 binding peptide at the N-terminus of the heavy chain variable region or light chain variable region that constitutes the paratope,

the recombinant fusion protein is capable of simultaneously binding to CD47, CD38, and FcR.

2. The recombinant fusion protein according to claim 1, wherein each paratope of the CD38 antibody or antibody fragment thereof is linked to the CD47 binding peptide at the N-terminus of the heavy chain variable region constituting the paratope.

3. The recombinant fusion protein according to claim 1, wherein each paratope of the CD38 antibody or antibody fragment thereof is linked to the CD47 binding peptide at the N-terminus of the light chain variable region that constitutes the paratope.

4. The recombinant fusion protein of claim 1, wherein the CD38 antibody or antibody fragment thereof is linked to the CD47 binding peptide via a linker.

5. The recombinant fusion protein of claim 4, wherein the linker is- (Gly-Gly-Gly-Gly-Ser)₃-(SEQ ID NO：12)。

6. The recombinant fusion protein of claim 1, wherein the heavy chain variable region and the light chain variable region comprise, respectively, the amino acid sequences set forth in SEQ ID NOs: 2 and 3.

7. The recombinant fusion protein of claim 1, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof.

8. The recombinant fusion protein of claim 1, further comprising a light chain constant region comprising the amino acid sequence of SEQ ID NO: 11, and is linked to the C-terminus of the light chain variable region.

9. The recombinant fusion protein of claim 1, comprising:

i) has the sequence shown in SEQ ID NO: 15, and a CD47 binding peptide-linker-CD 38 antibody heavy chain variable region-heavy chain constant region, and a polypeptide having the amino acid sequence set forth in SEQ ID NO: 17, the variable region-light chain constant region of the light chain of the CD38 antibody; or

ii) has the sequence of SEQ ID NO: 19, and a heavy chain variable region-heavy chain constant region of the CD38 antibody having the amino acid sequence set forth in SEQ ID NO: 21, a CD47 binding peptide-linker-CD 38 antibody light chain variable region-light chain constant region.

10. A nucleic acid molecule encoding the recombinant fusion protein of any one of claims 1-9.

11. An expression vector comprising the nucleic acid molecule of claim 10.

12. A host cell comprising the expression vector of claim 11.

13. A pharmaceutical composition comprising the recombinant fusion protein of any one of claims 1-9, the nucleic acid molecule of claim 10, the expression vector of claim 11, or the host cell of claim 12, and at least one pharmaceutically acceptable excipient.

14. Use of the pharmaceutical composition of claim 13 in the manufacture of a medicament for the treatment of a disease associated with CD47 and/or CD38 overexpression.

15. The use of claim 14, wherein the disease is selected from Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), Acute Lymphocytic Leukemia (ALL), lymphoma, Multiple Myeloma (MM), bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, melanoma, glioma, esophageal cancer, plasma cell myeloma, and prostate cancer.

Technical Field

The present application relates to a recombinant fusion protein targeting CD47, CD38 and/or FcR, and its preparation and use, in particular its use in tumor therapy.

Background

Cancer cells have developed several mechanisms to evade host immune surveillance, including: 1) by high expression of CD38, a large amount of adenosine is produced, so that a large amount of immunosuppressive cells are recruited, and inhibitory signal paths in immune cells such as NK cells, dendritic cells, cytotoxic T cells and the like are activated; 2) escape from immune surveillance by Natural Killer (NK) cells through shedding of MICA/MICB on cancer cell membrane, binding of shed MICA/MICB to NKG2D on NK cell surface, blocking NK cell to MICA/MICB⁺Killing cancer cells; 3) 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 all cancer cells can be directed to these mechanisms.

SIRP and CD47

Signal-regulatory proteins (SIRPs) are transmembrane glycoproteins that include three family members, sirpa (CD172a), SIRP β (CD172b) and SIRP γ (CD172 g). All three proteins contain similar extracellular domains, but have different intracellular domains. The extracellular region comprises three immunoglobulin-like domains, one Ig-V and two Ig-C domains. The intracellular domain of sirpa (CD172a) contains two inhibitory signaling regions that can inhibit signaling and the corresponding cellular functions. The intracellular regions of SIRP β (CD172b) and SIRP γ (CD172g) are very short and do not contain a signaling domain. However, SIRP β (CD172b) is able to function as a signal transduction via adapter proteins such as DAP 12. SIRP is expressed primarily in macrophages (M.phi.), Dendritic Cells (DCs) and neurons.

CD47 is a transmembrane glycoprotein belonging to the immunoglobulin superfamily, expressed on the surface of all cell types including erythrocytes. Ligands for CD47 include integrins, thrombospondin-1, and SIRP. CD47, by interacting with sirpa to signal "do not eat me", can inhibit phagocytosis by macrophages and thereby protect against macrophage attacks such as blood cells.

Many tumors or cancer cells that overexpress CD47 have been shown to inhibit phagocytosis of cancer cells by macrophages. Cancer cells that overexpress CD47 include Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), Acute Lymphocytic Leukemia (ALL), non-hodgkin's lymphoma (NHL), Multiple Myeloma (MM), bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, and pancreatic cancer cells. It has been reported that injection of a CD47 specific antibody that blocks CD47 binding to sirpa into tumor-bearing mice significantly inhibited tumor growth. When the same antibody is injected into mice bearing human leukemia cells, tumor or cancer cells are completely eliminated (Theocharides APA, et al, 2012).

CD38

CD38 is an important metabolic enzyme located on the cell surface, and its active domain faces to the outside of the cell membrane, catalyzing the conversion of NAD + into ADPR, cADPR, etc., which can influence cell growth, T cell activation, etc. by regulating cellular calcium ions. The research shows that CD38 is expressed in high level in plasma cell, T cell, NK cell, dendritic cell and other cells, and is essential for T cell initiation and migration of dendritic cell and neutrophil.

Many solid tumor cells, including hepatocellular carcinoma, non-small cell lung carcinoma, melanoma, pancreatic ductal adenocarcinoma, glioma, breast cancer, gastric cancer, esophageal cancer, are highly expressing CD38(Dwivedi et al, 2021; Wo et al, 2020). For example, 15-23% of lung cancer patients have tumor cells that are CD38 positive (Chen et al, 2018). CD38 can act together with enzymes such as CD203 and CD73 to generate a large amount of immunosuppressive factor, namely adenosine, in the environment of solid tumor tumors. Adenosine can recruit immunosuppressive cells such as Treg cells and MDSC cells to inhibit the activity of the immune system, and can directly bind to A2AR receptor on the surface of immune cells to activate inhibitory signal pathways inside immune cells such as NK cells, dendritic cells and cytotoxic T cells to inhibit the activity of the immune cells. Many non-solid cancer cells also highly express CD38 and levels are inversely correlated with, for example, prognosis for multiple myeloma.

Monoclonal antibodies targeting CD38 showed excellent therapeutic effects in the treatment of hematological malignancies. For example, Daratumumab (Daratumumab) is approved as a single agent or a combination for the treatment of relapsed/refractory multiple myeloma (Usmani et al, 2016). In addition, the CD38 antibodies daratuzumab and Isatuximab are undergoing preclinical and clinical trials for a variety of hematological and non-hematological malignancies, including multiple myeloma, plasma cell myeloma, lymphoma, pancreatic cancer, non-small cell lung cancer, triple negative breast and prostate cancer cancers (Dwivedi et al, 2021).

Research shows that the daratuzumab is combined with CD38 expressed by tumor cells, and induces tumor cell apoptosis through various immune-related mechanisms such as complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP) and the like. The daratuzumab therapy can greatly reduce the number of immunosuppressive cells with high expression of CD38, such as Treg cells, MDSC and the like, and obviously increase CD8 with the function of killing tumors⁺The number of T cells. The stronger CDC activity enables killing of multiple myeloma cells even in the presence of bone marrow stromal cells with immunosuppressive functions.

Fc and FcR

The crystallizable section (Fc region) is the tail region of an antibody and is the domain that determines the effector function of the antibody (i.e., how the antibody is associated with 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. These cells contribute to the protective function of the immune system.

The Fc region can interact with Fc receptors and some proteins of the complement system, activating the immune system.

Therapeutic bispecific or multispecific fusion proteins/antibodies

Antibodies targeting a single antigen may have limited therapeutic efficacy. For example, the approved PD-L1 antibody, AvelumabThe overall remission rate was only 33%. In another example, tumors that highly express CD38 may develop resistance to immunotherapy.

Bispecific or trispecific fusion proteins have been developed in recent years and these fusion proteins have shown rather modest effects in preclinical and clinical trials.

Although conceptually it is not complicated to attach additional binding groups to conventional antibodies, such modifications can significantly alter the structure of the antibody, and the binding force and/or potency of the antibody and additional binding groups can interact (Wang S et al, 2021). To optimize in vivo therapeutic and pharmaceutical properties, careful design and modification in the selection of primary and secondary groups (sequences), balancing of the binding forces of the target, selection of the binding site (N-or C-terminus of the heavy or light chain), structural stability, linker length and sequence are required (shimh.2020).

US 10,800,821B 2 discloses a recombinant bispecific fusion protein of about 90 kdalton, targeting CD47 and FcR, with enhanced antitumor effects observed in treatment of Balb/c nude mice bearing HL cells.

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 recombinant fusion protein comprising a CD38 antibody and a CD47 binding peptide. The recombinant fusion proteins of the present application are capable of binding to CD38 with comparable or higher activity than the CD38 antibody and CD47 binding protein⁺、CD47⁺And/or CD38⁺CD47⁺Cell, for CD38⁺CD47⁺Cell priming is equivalentOr higher antibody-dependent cell-mediated cytotoxic effects (ADCC). The recombinant fusion proteins of the present application also show better anti-tumor effects in vivo experiments than the combination of CD47 binding protein and CD38 antibody.

Specifically, the present application discloses a recombinant fusion protein comprising a CD38 antibody or antibody fragment thereof that specifically binds CD38, and a CD47 binding peptide that specifically binds CD47, wherein the CD47 binding peptide is linked to the CD38 antibody or antibody fragment thereof, wherein the CD38 antibody or antibody fragment thereof comprises a heavy chain variable region comprising HV-CDR1, HV-CDR2 and HV-CDR3, and a light chain variable region comprising HV-CDR1, HV-CDR2 and HV-CDR3, respectively, comprising SEQ ID NOs: 4. 5 and 6, the light chain variable region comprises LV-CDR1, LV-CDR2 and LV-CDR3, LV-CDR1, LV-CDR2 and LV-CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 7. 8 and 9, a heavy chain constant region having FcR binding and linked to the C-terminus of the heavy chain variable region, wherein the CD47 binding peptide comprises a mutant signal-regulatory protein (SIRP) extracellular Ig-like domain comprising an amino acid sequence that is complementary to the amino acid sequence set forth in SEQ ID NO: 1, wherein the recombinant fusion protein is capable of binding to both CD47 and CD 38. The CD47 binding peptide can be bound to the N-terminus of the heavy chain variable region or the light chain variable region of the CD38 antibody or antibody fragment thereof.

The heavy chain variable region of the CD38 antibody or antibody fragment thereof may comprise an amino acid sequence identical to SEQ ID NO: 2, or a variant thereof, 2 has an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In one embodiment, the heavy chain variable region may comprise SE Ω ID NO: 2, or a pharmaceutically acceptable salt thereof. The light chain variable region of the CD38 antibody or antibody fragment thereof may comprise an amino acid sequence identical to SEQ ID NO: 3, having a sequence identity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In one embodiment, the light chain variable region may comprise SEQ ID NO: 3. In one embodiment, the heavy chain variable region and the light chain variable region of the CD38 antibody or antibody fragment thereof may comprise a heavy chain variable region and a light chain variable region that are identical to SEQ ID NOs: 2 and 3 have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the heavy chain variable region and the light chain variable region of the CD38 antibody or antibody fragment thereof may comprise SEQ ID NOs: 2 and 3.

The heavy chain constant region having FcR binding may be a naturally occurring or engineered human IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, or a functional fragment thereof. In some embodiments, the FcR binding heavy chain constant region is a human IgG1 heavy chain constant region, or a functional fragment thereof. In some embodiments, the FcR binding heavy chain constant region has the amino acid sequence of SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof.

The CD38 antibody or antibody fragment thereof can comprise a light chain constant region, such as a human kappa light chain constant region, or a functional fragment thereof, linked to the C-terminus of the light chain variable region. In some embodiments, the CD38 antibody or antibody fragment thereof may comprise the amino acid sequence of SEQ ID NO: 11, or a pharmaceutically acceptable salt thereof.

The heavy chain of the CD38 antibody or antibody fragment thereof may comprise an amino acid sequence identical to SEQ ID NO: 19, or a variant thereof, having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In one embodiment, the heavy chain may comprise SEQ ID NO: 19, or a pharmaceutically acceptable salt thereof. The light chain of the CD38 antibody or antibody fragment thereof may comprise an amino acid sequence identical to SEQ ID NO: 17, or a variant thereof, having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In one embodiment, the light chain variable region may comprise SEQ ID NO: 17. In one embodiment, the heavy chain variable region and the light chain variable region of the CD38 antibody or antibody fragment thereof may comprise a heavy chain variable region and a light chain variable region that are identical to SEQ ID NOs: 19 and 17 have an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the heavy chain variable region and the light chain variable region of the CD38 antibody or antibody fragment thereof may comprise SEQ ID NOs: 19 and 17.

In some embodiments, at least one paratope of the CD38 antibody or antibody fragment thereof is linked to a CD47 binding peptide at the N-terminus of the heavy chain variable region or light chain variable region that makes up the paratope. In some embodiments, each paratope of the CD38 antibody or antibody fragment thereof is linked to a CD47 binding peptide at the N-terminus of the heavy and variable regions or the light chain variable region that make up the paratope. In some embodiments, each paratope of the CD38 antibody or antibody fragment thereof is linked to a CD47 binding peptide at the N-terminus of the heavy chain variable region comprising that paratope. In some embodiments, each paratope of the CD38 antibody or antibody fragment thereof is linked to a CD47 binding peptide at the N-terminus of the light chain variable region that makes up the paratope.

The CD38 antibodies or antibody fragments thereof of the present application can be linked to a CD47 binding peptide via a linker. The linker may be a peptide of 5-30, 10-20, or 15 amino acids in length. The linker may be, for example, - (Gly-Gly-Gly-Gly-Ser)₂-(SEQ ID NO：13)、-(Gly-Gly-Gly-Gly-Ser)₃- (SEQ ID NO: 12), or- (Gly-Gly-Gly-Gly-Ser)₄- (SEQ ID NO: 14). In some embodiments, the linker is- (Gly-Gly-Gly-Gly-Ser)₃-(SEQ ID NO：12)。

The recombinant fusion proteins of the present application can comprise a CD47 binding peptide-linker-CD 38 antibody heavy chain, and a CD38 antibody light chain, wherein the CD47 binding peptide-linker-CD 38 antibody heavy chain comprises an amino acid sequence identical to SEQ ID NO: 15, and a CD38 antibody light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 17, or a variant thereof, having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, a recombinant fusion protein of the present application may comprise a CD47 binding peptide-linker-CD 38 antibody heavy chain, and a CD38 antibody light chain, wherein the CD47 binding peptide-linker-CD 38 antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 15, and the light chain of the CD38 antibody comprises the amino acid sequence set forth in SEQ ID NO: 17. SEQ ID NOs: 15 and 17 may be represented by SEQ ID NOs: 16. and 18.

The recombinant fusion proteins of the present application can comprise a CD38 antibody heavy chain, and a CD47 binding peptide-linker-CD 38 antibody light chain, wherein the CD38 antibody heavy chain comprises an amino acid sequence identical to SEQ ID NO: 19, and a CD 47-binding peptide-linker-CD 38 antibody light chain comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 21 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, a recombinant fusion protein of the present application may comprise a CD38 antibody heavy chain, and a CD47 binding peptide-linker-CD 38 antibody light chain, wherein the CD38 antibody heavy chain comprises the amino acid sequence of SEQ ID NO: 19, CD47 binding peptide-linker-CD 38 antibody light chain comprises the amino acid sequence set forth in SEQ ID NO: 21, or a pharmaceutically acceptable salt thereof. SEQ ID NOs: 19 and 21 may be represented by SEQ ID NOs: 20. and 22.

The present application also provides nucleic acid molecules encoding the recombinant fusion proteins of the present application, as well as expression vectors comprising the nucleic acid molecules, and host cells comprising the expression vectors. Also provided is a method of making a recombinant fusion protein using a host cell of the present application, comprising (i) expressing the recombinant fusion protein in the host cell, and (ii) isolating the recombinant fusion protein from the host cell or cell culture thereof.

The present application also provides a pharmaceutical composition that may comprise a recombinant fusion protein, nucleic acid molecule, expression vector or host cell of the present application, and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises at least one pharmaceutically acceptable adjuvant.

The recombinant fusion protein or the pharmaceutical composition of the application can be used for treating diseases related to CD47 and/or CD38 overexpression or used for preparing medicines for treating diseases related to CD47 and/or CD38 overexpression.

In one aspect, the present application provides a method for treating or alleviating a disease associated with CD47 and/or CD38 overexpression in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present application.

The disease associated with CD47 and/or CD38 overexpression may be Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), Acute Lymphocytic Leukemia (ALL), lymphoma, Multiple Myeloma (MM), bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, melanoma, glioma, esophageal cancer, plasma cell myeloma, and prostate cancer.

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 diagram of the structures of the recombinant fusion proteins IMM5601 and IMM5602 of the present application. The top circular structure represents a mutated sirpa first extracellular domain (sirpa D1) linked via a linker to the N-terminus of the heavy (left panel, IMM5601) or light chain (right panel, IMM5602) of the CD38 antibody IMM 56. The mutant sirpa D1 has the amino acid sequence of SEQ ID NO: 1. The linker has SEQ ID NO: 12. The heavy chain of IMM56 has the amino acid sequence of SEQ ID NO: 19 and the light chain has the amino acid sequence shown in SEQ ID NO: 17, or a pharmaceutically acceptable salt thereof.

FIG. 2 illustrates IMM5601 and IMM5602 with CD38⁺CD47⁺Binding activity of Raji human B lymphoblastoma cells. IMM56 was used as a positive control and IgG was used as a negative control.

FIG. 3 shows IMM5601 and IMM5602 with CD38⁺CD47⁺Binding activity of U266 human multiple myeloma cells. IMM56 was a positive control and IgG was used as a negative control.

FIG. 4 shows IMM5601 and CD38⁺CD47⁺NCI-H9Binding activity of 29 human multiple myeloma cells. IMM56 and IMM01 were used as positive controls, and hIgG was a negative control. IMM01 is described in US 2021/0024598A 1 and comprises two mutations SIRP α D1(SEQ ID NO: 1) linked to an Fc dimer fragment, wherein the monomer comprises the amino acid sequence set forth in SEQ ID NO: 23, or a pharmaceutically acceptable salt thereof.

FIG. 5 illustrates IMM5601 and IMM5602 with CD38^-CD47⁺Binding activity of Jurkat human T cell leukemia cells. IMM01 was used as a positive control, and IMM56 and hIgG were negative controls.

FIG. 6 shows that IMM5601 blocks SIRP α -Fc and CD38⁺CD47⁺Binding capacity of NCI-H929 human multiple myeloma cells. IMM56 and IMM01 were used as positive controls and hIgG was used as negative control.

FIGS. 7A and 7B illustrate IMM5601 and IMM5602 versus CD38⁺CD47⁺Raji human B lymphoblastoma cell (A) and CD38⁺CD47⁺U266 ability of human multiple myeloma cells (B) to elicit antibody-dependent cell-mediated cytotoxicity (ADCC). IMM56 served as a positive control and IgG served as a negative control.

FIG. 8 shows IMM5601 vs. CD38⁺CD47⁺The ability of NCI-H929 cells to elicit antibody-dependent cell-mediated cytotoxicity (ADCC). IMM01 and IMM56 were used as positive controls and hIgG was used as negative control.

FIG. 9 shows the anti-tumor effect of IMM01, IMM56, IMM5601, and IMM56+ IMM01 in CB17 SCID mice xenografted with NCI-H929 human multiple myeloma cells.

Detailed Description

In principle, there are three main different approaches to target two or more pharmacological mechanisms of tumor growth. Most often, a combination of two or more different drugs may be administered to a patient. Although this option allows maximum flexibility for possible drug combinations and different dosages, it faces the problem of: a) patient compliance is poor as there are many medications and different dosing schedules for each medication; b) possible incompatibilities exist due to drug-drug interactions; and c) increased risk of drug side effects. These problems can reduce the effectiveness of the treatment and prevent the achievement of therapeutic goals, particularly in the management of chronic diseases such as cancer.

The second approach is to use a fixed dose combination of multiple drugs in a single dosage form. This approach reduces the burden of drug quantity and improves patient compliance. The disadvantage of fixed-dose combinations is mainly that the choice of the possible dose ratios between the active ingredients is limited, which makes it more difficult to adjust the dose appropriately to the maximum efficacy and minimum adverse effect for the individual patient. Furthermore, the pharmacokinetic profile of the different drugs in the combination may cause complex time-shifts of the drug effect in each target patient, compromising the overall efficacy.

The third approach is to use multifunctional drugs that combine two or more pharmacological mechanisms in a single compound. The design and identification of these multifunctional molecules is more complex and requires extensive research to confirm the optimal ratio of targeted activities in the molecules, whereas the combined pharmacokinetics may result in matching pharmacokinetic activities at the molecular target level. Multifunctional molecules can also be engineered to combine fixed dose combinations with other drugs, thereby combining three, or even four, pharmacological mechanisms in a single tablet to produce further increases in efficacy.

After a large number of experiments, the present inventors have invented a novel recombinant multifunctional fusion protein, which can attack tumors through three mechanisms of action, one is to reduce adenosine catalytically produced by CD38, thereby reducing recruitment of immunosuppressive cells and relieving activation of inhibitory signaling pathways in immune cells such as NK cells, dendritic cells, cytotoxic T cells, etc., one is to relieve macrophage examination by SIRP-mediated inhibitory signals, and the other is to activate NK cells and/or macrophages, etc., to kill cancer cells.

A recombinant fusion protein of the present application comprises a CD38 antibody or antibody fragment thereof, at least one paratope of the CD38 antibody or antibody fragment thereof being linked via a linker to an extracellular Ig-like domain of a signal-regulating protein (SIRP) at the N-terminus of the heavy or light chain constituting the paratope. The recombinant protein can simultaneously bind to CD47, CD38, and FcR, i) reduce adenosine catalytically produced by CD38 on cancer cells, thereby reducing recruitment of immunosuppressive cells and relieving activation of inhibitory signaling pathways in immune cells such as NK cells, dendritic cells, cytotoxic T cells, and the like; ii) blocking the interaction of CD47 on cancer cells with SIRP on macrophages, relieving the macrophages from the detection of SIRP-mediated inhibitory signals; and iii) the antibody Fc region binds to FcR on NK cells or macrophages, activating killing of cancer cells by NK cells or macrophages. In one embodiment, a paratope of a CD38 antibody or antibody fragment thereof is linked to an extracellular Ig-like domain of a signal-regulating protein (SIRP) via a linker at the N-terminus of the heavy or light chain constituting the paratope. In another embodiment, each paratope of the CD38 antibody or antibody fragment thereof is linked to an extracellular Ig-like domain of a signal-regulating protein (SIRP) via a linker at the N-terminus of the heavy or light chain constituting the paratope. In one embodiment, each paratope of the CD38 antibody or antibody fragment thereof is linked to an extracellular Ig-like domain of a signal-regulating protein (SIRP) via a linker at the N-terminus of the heavy chain constituting the paratope. In one embodiment, each paratope of the CD38 antibody or antibody fragment thereof is linked to an extracellular Ig-like domain of a signal-regulating protein (SIRP) via a linker at the N-terminus of the light chain constituting the paratope. The recombinant fusion proteins of the present application are smaller in size (150-180kDa) with a longer half-life of 5-10 days.

The three major components comprised in the fusion protein of the present application are the extracellular Ig-like domain of a signal-regulating protein (SIRP), a linker, and a CD38 antibody or antibody fragment thereof. Those skilled in the art will appreciate that there are many design choices for the three components described above. Preferably, human sequences are used in the treatment of human cancer, since the strong immunogenicity of non-human animal proteins or peptides may cause allergic and other adverse reactions. However, other animal proteins or peptides may be used in the present application and may be humanized for various purposes of use.

Any extracellular Ig-like domain of any SIRP (sirpa, sirpa and SIRP γ) that is capable of binding to CD47 can be selected for the construction of fusion proteins. In one embodiment, the signal-regulating protein in the recombinant fusion protein is sirpa and the extracellular Ig-like domain of the signal-regulating protein is the first extracellular Ig-like domain of sirpa (sirpa D1). In one embodiment, the sirpa D1 is a mutant sirpa D1 that differs from the wild-type sirpa D1 in the amino acid sequence of SEQ ID NO: 1, and the N80A mutation exists at the 80 th position, and the mutation at the position can realize the effect of deglycosylation.

In one embodiment, the recombinant fusion protein comprises an amino acid sequence as set forth in SEQ ID NO: 1, sirpa D1. In another embodiment, sirpa D1 may comprise a sequence identical to SEQ ID NO: 1, wherein sirpa D1 is capable of binding to CD47 on the surface of cancer/tumor cells and blocks the interaction of CD47 with macrophage-surface SIRP.

The linker primarily serves as a spacer between the extracellular Ig-like domain of SIRP and the N-terminus of the heavy or light chain of the CD38 antibody. The linker may be composed of peptide-bonded amino acids, preferably 5-30, 10-20, or 15 amino acids, wherein the amino acids are selected from the 20 naturally occurring amino acids. One or more of these amino acids may be glycosylated or deglycosylated, as is known to those skilled in the art. In one embodiment, 5-30 amino acids, 10-30, 10-20, or 15, may be selected from glycine, alanine, proline, asparagine, glutamine, serine, and lysine. In one embodiment, the linker is composed of a majority of amino acids with steric hindrance, such as glycine and alanine. Exemplary linkers are polyglycine (particularly Gly, poly (Gly-Ala)), and polyalanine. Exemplary suitable linkers shown in the following examples are (Gly-Ser), e.g., (Gly-Gly-Gly-Gly-Ser)₃-(SEQ ID NO：12)。

The linker may also be a non-peptide linker. For example, alkyl linkers such as-NH-, - (CH) may be used₂) s-c (o) -, wherein s-2-20. These alkyl linkers may also be via any non-sterically hindered group such as lower alkyl (e.g. C)_1-4Lower acyl), halogen (e.g. Cl, Br), CN, NH₂And phenyl, etc.

In some embodiments, the CD38 antibody is an isolated monoclonal antibody comprising two heavy chains and two light chains, each heavy chain having the amino acid sequence of SEQ ID NO: 19, each light chain having the amino acid sequence of SEQ ID NO: 17, which can be represented by SEQ ID NO: 20 and SEQ ID NO: 18 encoding. The Fab portion (or paratope) of the CD38 antibody may bind to CD38 on the surface of cancer/tumor cells to reduce adenosine production catalyzed by CD38 on cancer cells, thereby reducing recruitment of immunosuppressive cells and relieving activation of inhibitory signaling pathways in immune cells such as NK cells, dendritic cells, cytotoxic T cells, etc., while the Fc portion of the CD38 antibody may bind to FcR on the surface of NK cells and/or macrophages to stimulate killing of cancer cells by NK cells or macrophages. In some embodiments, the heavy chain may comprise a heavy chain sequence identical to SEQ ID NO: 19, wherein the CD38 antibody is capable of binding to CD38 and blocking the catalytic adenosine-producing effects of CD38, and is capable of binding to an FcR on the surface of an NK cell or macrophage to activate the killing of cancer cells by the NK cell and/or macrophage. In some embodiments, the light chain may have an amino acid sequence identical to SEQ ID NO: 17, wherein the CD38 antibody is capable of binding to CD38 and blocking the catalytic effect of CD38, has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

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_Hl、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_LConsists of three CDRs and four FR regions, from amino terminal to carboxyl terminal, with FR1, CDR1, FR2CDR2, FR3, CDR3 and FR 4. The variable regions of the heavy and light chains comprise binding domains that react with antigen. The constant region of the antibody may be linked to 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).

The term "antibody fragment" herein refers to a portion or fragment of an antibody of the present application that retains the ability to specifically bind to an antigen (e.g., CD38), and optionally to an FcR.

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 immune system effector cells actively lyse cell membrane surface antigens from antibodies, such as the CD38 antibody, or target cells, such as cancer cells, that bind to sirpa.

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.

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.

Also, the present application provides polynucleotides encoding the recombinant fusion proteins and expression vectors for expressing the recombinant fusion proteins. Examples of vectors include, but are not limited to, plasmids, viral vectors, Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), Transformable Artificial Chromosomes (TACs), Mammalian Artificial Chromosomes (MACs), and artificial episomes (HAECs).

The present application provides host cells comprising the above-described expression vectors. Host cells may be transformed or transfected with expression vectors. Suitable host cells include e.coli (e.coli), yeast and other eukaryotes. Preferably, E.coli, yeast or mammalian cell lines (e.g., COS or CHO) are used.

In another aspect, the present application provides a pharmaceutical composition comprising a fusion protein of the present application formulated with a pharmaceutically acceptable adjuvant. The composition may optionally comprise one or more other pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical compositions of the present application may also be administered in combination therapy, e.g., with another immunostimulant, an anti-cancer drug, an anti-viral agent, or a vaccine.

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 aids, stabilizers, colorants, flavorants, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. Selection and use of suitable excipients is described in Gennaro, ed., Remington: the Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins2003), The disclosure of which is incorporated herein by reference.

The primary vehicle or carrier in the pharmaceutical composition may be aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, saline, or artificial cerebrospinal fluid, which may be supplemented with other materials common in injections. For example, the vehicle or carrier may be a neutral buffered saline solution or a saline solution mixed with serum albumin. Other exemplary pharmaceutical compositions comprise Tris buffer, or acetate buffer, which may also comprise sorbitol or a suitable substitute thereof. In one embodiment of the present application, the composition may be prepared for storage by mixing the selected components with the desired purity with any formulation agents (Remington's Pharmaceutical Sciences, supra) in lyophilized or aqueous solution form. In addition, the therapeutic composition may be formulated as a lyophilizate using suitable excipients such as sucrose.

Preferably, the pharmaceutical composition is 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 molecule may be encapsulated in a material to protect it from the action of acids and other natural conditions that may inactivate it. The term "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration typically by injection, including, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, dural, and intrasternal injection and infusion. Alternatively, the antibodies of the present application may be administered by a non-injectable route, such as a topical, epidermal or mucosal mode of administration, e.g., intranasal, oral, vaginal, rectal, sublingual, or topical.

The pharmaceutical compositions may be in the form of sterile aqueous solutions or suspensions. They may also be formulated as microemulsions, liposomes, or other ordered structures suitable for high concentrations of drugs.

The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the subject being treated and the particular route of administration, and is generally the amount of composition that produces a therapeutic effect. Generally, the amount is from about 0.01% to about 99% of the active ingredient, by percentage, in combination with a pharmaceutically acceptable carrier.

The dosage regimen may be adjusted to achieve the optimal desired response (e.g., therapeutic response). For example, multiple divided doses may be administered over time, or the dose may be reduced or increased proportionally to the criticality of the treatment. It is particularly advantageous to formulate parenteral compositions in dosage units for ease of administration and to facilitate uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable for single administration to a subject to be treated; each unit containing a pre-calculated amount of active compound that together with the pharmaceutical carrier produces the desired therapeutic effect. Alternatively, the fusion protein may be administered in a sustained release dosage form, in which case the frequency of administration is reduced.

For administration of the fusion protein, the dosage range is about 0.0001-100mg/kg of recipient body weight. An exemplary treatment regimen is twice weekly.

A "therapeutically effective amount" of a fusion protein of the present application preferably causes a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevents injury or disability caused by the disease. For example, for the treatment of a tumor-bearing subject, a "therapeutically effective amount" means that, relative to an untreated subject, tumor growth is preferably inhibited by at least about 40%, more preferably inhibited by at least about 60%, more preferably inhibited by at least about 80%, more preferably inhibited by at least about 99%. A therapeutically effective amount of a fusion protein of the present application can reduce tumor volume, or alleviate symptoms, in a subject (typically a human, or can be another mammal).

The pharmaceutical compositions may be in controlled release formulations including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

The pharmaceutical compositions can be administered by 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 devices (us patent 4,486,194); (4) infusion devices (U.S. Pat. nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Pat. Nos. 4,439,196 and 4,475,196), the disclosures of which are incorporated herein by reference.

In certain embodiments, the fusion proteins of the present application can be formulated to ensure proper in vivo distribution. For example, to ensure that the therapeutic fusion proteins of the present application cross the blood-brain barrier, the fusion proteins are formulated in liposomes that may additionally contain targeting groups to enhance selective delivery to specific cells or organs. See, for example, U.S. Pat. nos. 4,522,811, 5,374,548, 5,416,016, and 5,399,331.

The present application also relates to in vivo gene therapy, wherein a nucleic acid molecule encoding a fusion protein of the present application or a derivative thereof is introduced directly into a subject. For example, a nucleic acid sequence encoding a recombinant fusion protein of the present application is introduced into a target cell by local injection via a nucleic acid construct with or without a suitable delivery vector, such as an adeno-associated viral vector. Alternative viral vectors include, but are not limited to, retroviral, adenoviral, herpes simplex virus, and papilloma virus vectors. Physical transfer of viral vectors in vivo can be achieved by local injection, liposome-mediated transfer, direct injection (naked DNA), or microprojectile bombardment (gene gun) of the desired nucleic acid construct or other suitable delivery vehicle comprising the desired nucleic acid sequence.

The compositions of the present disclosure may be used alone or in combination with other therapeutic agents for enhancing their therapeutic efficacy or reducing potential side effects.

It is another object of the present application to provide a method for preparing the above recombinant fusion protein and a pharmaceutical composition comprising the same. In one embodiment, the preparation method comprises the following steps: (1) providing a nucleic acid molecule encoding a fusion protein; (2) constructing an expression vector comprising the nucleic acid molecule of (1); (3) transfecting or transforming suitable host cells with the expression vector of (2) and culturing the host cells to express the protein; and (4) purifying the protein. The preparation can be carried out by the skilled worker using techniques known to those skilled in the art.

It is another object of the present application to provide a method for treating cancer using the pharmaceutical composition of the present application, comprising administering to a patient or subject in need thereof an effective amount of the above pharmaceutical composition. In one embodiment, the pharmaceutical composition is used to treat tumors or cancers that overexpress CD47 and/or CD38, including but not limited to Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), Acute Lymphocytic Leukemia (ALL), lymphoma, Multiple Myeloma (MM), bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, melanoma, glioma, esophageal cancer, plasma cell myeloma, and prostate cancer.

In one embodiment, diseases associated with overexpression of CD47 and/or CD38 include, but are not limited to, crohn's disease, allergic asthma, and rheumatoid arthritis.

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

Examples

The structures of exemplary fusion proteins IMM5601 and IMM5602, and control proteins of the present application are briefly described below.

IMM56 is an IgG antibody, two heavy chains and two light chains, wherein the amino acid sequences of the heavy chain variable region, heavy chain constant region, light chain variable region and light chain constant region are set forth in SEQ ID NOs: 2. 10, 3 and 11.

IMM5601 contains the IgG antibody IMM56, two SIRP α D1(SEQ ID NO: 1) linked via linkers (SEQ ID NO: 12) to the N-termini of the two heavy chains of IMM 56. Wherein IMM56 comprises SEQ ID NO: 19, and SEQ ID NO: 17, light chain.

IMM5602 contains the IgG antibody IMM56, two SIRP α D1(SEQ ID NO: 1) linked via linkers (SEQ ID NO: 12) to the N-termini of the two light chains of IMM 56. Wherein IMM56 comprises SEQ ID NO: 19, and SEQ ID NO: 17, light chain.

IMM01 is described in US 2021/0024598 a1 and comprises two mutations sirpa D1(SEQ ID NO: 1) linked to an Fc dimer fragment, the monomers of which contain the amino acid sequences shown in SEQ ID NOs: 24 and SEQ ID NO: 23, and amino acid sequences shown in seq id no.

Example 1 construction of IMM5601 and IMM5602 expression vectors and protein expression

The structures of IMM5601 and IMM5602 are shown in fig. 1. Full-length coding sequences of recombinant fusion proteins IMM5601 and IMM5602 were artificially designed.

Specifically, for the long chain in IMM5601, i.e., SIRP α D1-linker-CD 38 antibody heavy chain, 57 nucleotides encoding the mouse IgG1 heavy chain signal peptide (SEQ ID NO: 25) were added to the 5 'end of the SIRP α D1-linker-CD 38 antibody heavy chain coding sequence (SEQ ID NO: 16) and a Kozak sequence (SEQ ID NO: 26) 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 short chain in IMM5601, i.e., the CD38 antibody light chain, the same signal peptide sequence as well as the Kozak sequence were added to the 5 ' end of the antibody light chain 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 Kirsir and cloned into pMac-H and pMac-L vectors, respectively.

For the long chain in IMM5602, i.e., the CD38 antibody heavy chain, 57 nucleotides encoding the mouse IgG1 heavy chain signal peptide (SEQ ID NO: 25) were added to the 5 'end of the CD38 antibody heavy chain coding sequence (SEQ ID NO: 20) and a Kozak sequence (SEQ ID NO: 26) was added to the 5' end of the signal peptide sequence. For the short chain in IMM5602, i.e., SIRP α D1-linker-CD 38 antibody light chain, the same signal peptide sequence as well as a Kozak sequence was added to the 5' end of the SIRP α D1-linker-CD 38 antibody light chain coding sequence (SEQ ID NO: 22). The resulting sequences were synthesized by Kirsir and cloned into pMac-H and pMac-L vectors, respectively.

The constructed expression vector transiently expresses the protein by using CHO-S cells. The general process is as follows: 1) CHO-S cells were plated at 1X 10 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 ratio of the heavy/light (long/short) chain expression vector was 1: 1, and the desired DNA was prepared at 1. mu.g/ml and added to OPTI-MEM medium (Gibco) in transient volume 1/20; 3) PEI (MW40,000 polyethyleneimine hydrochloride, polysciences) was prepared at 1mg/ml, and the desired PEI was prepared in a PEI: DNA ratio of 4: 1, and added to OPTI-MEM medium (Gibco) in an instantaneous volume of 1/20; 4) slowly adding PEI diluent to the DNA diluent, mixing, and standingIncubation 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 decreased below 50%, 3000rpm, centrifugation for 5 minutes collected supernatant for affinity purification using Protein A packing.

^{+ +}Example 2 binding of IMM5601 and IMM5602 to CD38CD47 human lymphoblastoma cells Raji

100 μ l of 1X 10 was taken⁶CD38 per ml⁺CD47⁺Raji cells, incubated in 100. mu.l gradient dilutions in IMM5601, IMM5602, IMM56 in 1% BSA-PBS and control IgG for 1 hour at 4 ℃. Cells were washed twice with cold 1% BSA-PBS followed by incubation for 45 minutes with a FITC-conjugated secondary antibody against human IgG-Fc (Cat # F9512, Sigma). Cells were washed twice and resuspended in 200. mu.l of 1% BSA-PBS. Thereafter, the cells were analyzed by flow cytometry (Merck Millipore,easyCyte 5HT) was performed.

As shown in FIG. 2, IMM5601 has an EC of 123.0nM₅₀Values were associated with Raji cells, IMM5602 with an EC of 93.54nM₅₀Value for binding to Raji cells, binding activity of which was in comparison with the classical monoclonal antibody CD38 targeting protein IMM56 (EC)₅₀Value 83.43 nM).

^{+ +}Example 3 binding of IMM5601 and IMM5602 to CD38CD47 human multiple myeloma cell U266

100 μ l of 1X 10 was taken⁶/ml CD38⁺CD47⁺U266 cells were incubated in 100. mu.l gradient dilutions in IMM5601, IMM5602, IMM56 in 1% BSA-PBS and control IgG at 4 ℃ for 1 hour. Cells were washed twice with cold 1% BSA-PBS followed by incubation for 45 minutes with a FITC-conjugated 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. 3, IMM5601 has an EC of 20.48nM₅₀Values were associated with U266 cells, IMM5602 with an EC of 18.87nM₅₀The value is combined with U266 cells, and the combination activity is more than that of the traditional single antigen CD38 targeting protein IMM56 (EC)₅₀Value 61.93nM) higher.

^{+ +}Example 4 binding of IMM5601 to CD38CD47 human multiple myeloma cell NCI-H929

100 μ l of 1X 10 was taken⁶/ml CD38⁺CD47⁺NCI-H929 cells, 100 u l gradient dilution in 1% BSA-PBS IMM5601, IMM56, IMM01 and control IgG4 ℃ incubation for 1 hours. Cells were washed twice with cold 1% BSA-PBS followed by incubation for 45 minutes with a FITC-conjugated 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. 4, IMM5601 has an EC of 324.3nM₅₀The value is combined with NCI-H929 cells, and the combination activity is weaker than that of the traditional monoclonal antibody CD38 targeting protein IMM56 (EC)₅₀117.1nM) but much higher than the monospecific CD 47-binding protein IMM01 (EC)₅₀The value was 1718 nM).

^{- +}Example 5 binding of IMM5601 and IMM5602 to CD38CD47 human T lymphoblastic leukemia cells Jurkat

100 μ l of 1X 10 was taken⁶/ml CD38^-CD47⁺Jurkat cells, diluted in 100. mu.l gradient in IMM5601, IMM5602, IMM56, IMM01 and control IgG at 4 ℃ for 1 hour, respectively. Cells were washed twice with cold 1% BSAPBS followed by 45 min incubation with FITC-conjugated secondary antibody against human IgG-Fc (Cat # F9512, Sigma). Cells were washed twice and resuspended in 200. mu.l PBS. After thatThe cells were analyzed by flow cytometry (Merck Millipore,easyCyte 5HT) was performed.

As shown in FIG. 5, IMM5601 has an EC of 14.13nM₅₀Values were associated with Jurkat cells, IMM5602 with an EC of 21.87nM₅₀The value is combined with Jurkat cells, and the combination activity is far higher than that of the traditional monoclonal antibody CD38 targeting protein IMM56 (EC)₅₀Value of 335.8nM), slightly weaker than the monospecific CD47 binding protein IMM01 (EC)₅₀The value was 12.1 nM).

Example 6 IMM5601 blocks the interaction of CD47 with SIRPa

Mu.l of 3. mu.g/ml SIRP α -Fc (wild-type human SIRP α + murine IgG1 Fc, SEQ ID NO: 27) was taken and mixed with 50. mu.l of IMM5601, IMM56, IMM01 and hIgG-Fc, respectively, diluted in a gradient (starting concentration 30. mu.g/ml, 3-fold dilution gradient). Adding the above mixture to a solution containing 50 μ l of a mixture with a density of 1X 10⁶Plates were incubated at 4 ℃ for 45 min in 96-well plates of NCI-H929 cells expressing both CD38 and CD47 at each ml. Cells were washed with PBS and analyzed for SIRPa-Fc-CD 47 interaction by FACS.

As shown in FIG. 6, IMM5601 was able to block SIRPa-Fc from CD38⁺CD47⁺Binding of cells, IC₅₀The value was 30.73nM, which is much higher than the monospecific CD47 binding protein IMM01 (IC)₅₀The value was 2759 nM).

^{+ +}Example 7 IMM5601 and IMM5602 elicit high levels of antibodies against CD38CD47Raji and U266 cells Dependent cell mediated cytotoxicity (ADCC)

The labeled target cells were diluted 500-fold at 1mM CFSE (Cat #21888-25mg, Sigma).

50 μ l of 6X 10⁵/ml CFSE-labeled Raji cells or U266 cells (used as targeting cells) with 100. mu.l 6X 10⁵/ml NK92MI cells (effector cells) stably expressing Fc γ RIIIa were mixed 1: 2 and the mixed cells were in 5% CO₂The samples were then diluted with 50. mu.l gradient (starting concentration 1000ng/ml, 3-fold)Gradient dilution) IMM5601, IMM5602, IMM56 and IgG were incubated at 37 ℃ for 4 hours. Propidium Iodide (PI) (Cat # P4170, Sigma) was then added to the cell culture media at a concentration of 5. mu.g/ml and the cell culture media was analyzed for PI signal by FACS. The percentage of cell lysis due to ADCC was calculated based on the following formula: % lysis ═ PI positive cells treated (% IMM5601, IMM5602, or IMM56 [% PI positive cells treated with negative control protein ]/[ 100 [% PI positive cells treated with negative control protein ] } 100

As shown in figures 7A and 7B, IMM5601 and IMM5602 elicited higher levels of ADCC against Raji cells and U266 cells compared to the monospecific CD38 antibody, IMM 56.

^{+ +}Example 8 IMM5601 elicits high levels of antibody-dependent cell-mediation against CD38CD47NCI-H929 cells Cytotoxicity (ADCC)

The labeled target cells were diluted 500-fold at 1mM CFSE (Cat #21888-25mg, Sigma).

50 μ l of 6X 10⁵/ml CFSE-labeled NCI-H929 cells (used as targeting cells) with 100. mu.l 6X 10⁵/ml NK92MI cells (effector cells) stably expressing Fc γ RIIIa were mixed 1: 2 and the mixed cells were in 5% CO₂Next, the cells were incubated with 50. mu.l of IMM5601, IMM56, IMM01 and hIgG, respectively, at 37 ℃ in a gradient dilution (initial concentration 1000ng/ml, 3-fold gradient dilution). Propidium Iodide (PI) (Cat # P4170, Sigma) was then added to the cell culture media at a concentration of 5. mu.g/ml and the cell culture media was analyzed for PI signal by FACS. The percentage of cell lysis due to ADCC was calculated based on the following formula: % lysis ═ PI positive cells treated (% IMM5601, IMM56, or IMM01 @ PI positive cells treated with negative control protein)/(PI positive cells treated with 100 @ negative control protein × 100

As shown in figure 8, IMM5601 elicited higher levels of ADCC against NCI-H929 cells compared to the monospecific CD38 antibody IMM56 and the CD47 binding protein IMM 01.

Example 9 IMM5601 shows a potent antitumor active

Subcutaneous injection of right anterior axillary fossa in 30 SCID mice aged 6-8 weeksThe beam contains 5 x 10⁶A mixture of 100. mu.L PBS and 100. mu.L Matrigel of NCI-H929 cells. When the tumor volume reaches 100-³At this time, the mice were randomly divided into 5 groups of 6 mice each, and the divided day was defined as D0. Starting on the day, groups of mice were injected intraperitoneally with PBS, IMM01(0.3mg/kg), IMM56(1.0mg/kg), IMM5601(1.2 mg/kg), and IMM01(0.3mg/kg) + IMM56(1.0mg/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.

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 IMM5601 and other therapeutic Agents

The results are shown in FIG. 9 and Table 1. As can be seen, on day 21 after the start of the administration, the tumor volume of the tumor-bearing mice of the solvent control group reached 2697.16mm³. Compared with a solvent control group, the IMM010.3mg/kg group and the IMM 561.0 mg/kg group have obvious tumor inhibition effects, and the average volumes of tumors on the 21 st day are 840.23mm³(T/C31.16%, TGI 72.12%, p 0.012) and 469.63mm³(T/C17.40%, TGI 86.52%, p 0.006%). The combination of IMM56 and IMM01 also has significant tumor inhibiting effect, and the average volume of the tumor on day 21 is 460.16mm³(T/C17.06%, TGI 86.88%, p 0.006%). Compared with the solvent control group, the IMM56011.2mg/kg group has significant tumor inhibiting effect, and the tumor volume is 274.79mm³(T/C10.21%, TGI 94.07%, p 0.004) and complete remission was achieved with 2 animals. As can also be seen in fig. 9, IMM5601 functions faster.

In conclusion, the IMM5601 can play a role more quickly in tumor inhibition, and the overall tumor inhibition effect is better than the combined treatment effect of the traditional monoclonal antibody CD38 targeting protein IMM56 and the monospecific CD47 binding protein IMM 01.

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.

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9.Lee WY，Weber DA，Laur O，Severson EA，McCall I，Jen RP，Chin AC，Wu T，Gemert KM，Parkos CA..Novel Structural Determinants on SIRPa that Mediate Binding to CD47. J Immunol.2007，179:7741-7750

10.Obeid M，Panaretakis T，Joza N，Tuff 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

11.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

12.Shields RL，Namenuk AK，Hong K，Meng YG，Rae J，Briggs J，Xie D，Lai J，Stadlen 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 IgG1 Variants with Improved Binding to the FcgR.JBC.2001，276:6591-6604

13.Suzanne L.Topalian，F.Stephen Hodi，Julie R.Brahmer，Scott N.Gettinger，David C. Smith，David F.McDermott，John D.Powderly，Richard D.Carvajal，Jeffrey A.Sosman， Michael B.Atkins，Philip D.Leming，David R.Spigel，Scott J.Antonia，Leora Horn， Char1es G.Drake，Drew M.Pardoll，Lieping Chen，William H.Sharfman，Robert A. Anders，Janis M.Taube，Tracee L.McMiller，Haiying Xu，Alan J.Korman，Maria Jure- Kunkel，Shruti Agrawal，Daniel McDonald，Georgia D.Kollia，Ashok Gupta，Jon M. Wigginton，and Mario Sznol.Safety，Activity，and Immune Correlates of Anti-PD-1 Antibody in Cancer，N Engl J Med 2012；366:2443-2454

14.Theocharides，A.P.A.；Jin，L.Q.；Cheng，P.Y.；Prasolava，T.K.；Malko，A.V.；Ho， J.M.；Poeppl，A.G.；Rooijen，N.van；Minden，M.D.；Danska，J.S.；Dick，J.；Wang， J.C.Y.J.Exp.Med.2012，Vol.209 No.101883-1899

15.Thompson RH，Gillett MD，Cheville JC，Lohse CM，Dong H，Webster WS，Krejci KG，Lobo JR，Sengupta S，Chen L，Zincke H，Blute ML，Strome SE，Leibovich BC， Kwon ED.Costimulatory B7-H1 in renal cell carcinoma patients:Indicator of tumor aggressiveness and potential therapeutic target.PNAS.2004，101(49):17174-9

16.Tseng D，Volkmer JP，Willingham SB，Contreras-Trujillo H，Fathman JW，Fernhoff NB，Seita J，Inlay MA，Weiskopf K，Miyanishi M，Weissman IL.Anti-CD47 antibody- mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response.PNAS.2013，110:11103-11108

17.Usmani，S.Z.，Weiss，B.M.，Plesner，T.，Bahlis，N.J.，Belch，A.，Lonial，S.，Lokhorst， H.M.，Voorhees，P.M.，Richardson，P.G.，Chari，A.，et al.(2016).Clinical efficacy of daratumumab monotherapy in patients with heavily pretreated relapsed or refractory multiple myeloma.Blood 128，37-44.

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Sequence listing

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

<120> recombinant fusion protein targeting CD47 and CD38, and preparation and application thereof

<130> 55525 00042

<160> 27

<170> PatentIn version 3.5

<210> 1

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> SIRP α first extracellular Ig-like Domain with mutation (SIRP α D1)

<400> 1

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

115 120 125

<210> 2

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> IMM56 antibody heavy chain variable region

<400> 2

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

1 5 10 15

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

20 25 30

Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Thr Ile Tyr Pro Gly Asp Gly Asp Thr Gly Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

Met His Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

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

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 3

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> IMM56 antibody light chain variable region

<400> 3

Asp Ile Val Met Thr Gln Ser His Leu Ser Met Ser Thr Ser Leu Gly

1 5 10 15

Asp Pro Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Arg Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ile Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ala Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Pro Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 4

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> IMM56 antibody heavy chain HV-CDR-1

<400> 4

Gly Tyr Thr Phe Thr Asp Tyr Trp

1 5

<210> 5

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> IMM56 antibody heavy chain HV-CDR-2

<400> 5

Ile Tyr Pro Gly Asp Gly Asp Thr

1 5

<210> 6

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> IMM56 antibody heavy chain HV-CDR-3

<400> 6

Ala Arg Gly Asp Tyr Tyr Gly Ser Asn Ser Leu Asp Tyr

1 5 10

<210> 7

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> IMM56 antibody light chain LV-CDR-1

<400> 7

Gln Asp Val Ser Thr Val

1 5

<210> 8

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> IMM56 antibody light chain LV-CDR-2

<400> 8

Ser Ala Ser

1

<210> 9

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> IMM56 antibody light chain LV-CDR-3

<400> 9

Gln Gln His Tyr Ser Pro Pro Tyr Thr

1 5

<210> 10

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<223> antibody heavy chain constant region

<400> 10

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> 11

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> antibody light chain constant region

<400> 11

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> 12

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 12

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 13

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 13

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 14

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 14

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 15

<211> 590

<212> PRT

<213> Artificial sequence

<220>

<223> Long chain of IMM5601 (SIRP. alpha. D1-linker-IMM 56 antibody heavy chain)

<400> 15

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 Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu

130 135 140

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

145 150 155 160

Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Trp Met Gln Trp

165 170 175

Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Thr Ile Tyr

180 185 190

Pro Gly Asp Gly Asp Thr Gly Tyr Ala Gln Lys Phe Gln Gly Lys Ala

195 200 205

Thr Leu Thr Ala Asp Lys Ser Ser Lys Thr Val Tyr Met His Leu Ser

210 215 220

Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Asp

225 230 235 240

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

245 250 255

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

260 265 270

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

275 280 285

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

290 295 300

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

305 310 315 320

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

325 330 335

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

<210> 16

<211> 1830

<212> DNA

<213> Artificial sequence

<220>

<223> Long chain of IMM5601 (SIRP. alpha. D1-linker-IMM 56 antibody heavy chain)

<400> 16

atgggatggt catgtatcat cctttttctg gtagcaactg caactggagt acattcagag 60

gaggagctgc aggtgattca gcctgacaag tccgtatcag ttgcagctgg agagtcggcc 120

attctgcact gcactgtgac ctccctgatc cctgtggggc ccatccagtg gttcagagga 180

gctggaccag cccgggaatt aatctacaat caaaaagaag gccacttccc ccgggtaaca 240

actgtttcag agtccacaaa gagagaaaac atggactttt ccatcagcat cagtgccatc 300

accccagcag atgccggcac ctactactgt gtgaagttcc ggaaagggag ccctgacacg 360

gagtttaagt ctggagcagg cactgagctg tctgtgcgtg ccaaaccctc tgcccccgtg 420

gtatcgggcc ctggcggcgg tgggagcggc ggcggtggga gcggcggcgg gggctcgcaa 480

gtgcagctgg tgcagagcgg cgccgaggtg gccaagcctg gcacctccgt caagctgagc 540

tgcaaggcct ccggctacac cttcaccgac tactggatgc agtgggtgaa gcagagacct 600

ggccaaggcc tggagtggat cggcaccatc taccctggcg acggcgacac cggctacgct 660

cagaagttcc aaggcaaggc caccctgacc gccgacaaga gcagcaagac cgtgtacatg 720

cacctgagca gcctggcctc cgaggacagc gccgtgtact actgcgctag aggcgactac 780

tacggcagca acagcctgga ctactggggc caaggcacaa gcgtgaccgt gagcagcgct 840

agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 900

acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 960

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 1020

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 1080

atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagagagt tgagcccaaa 1140

tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 1200

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 1260

gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtat 1320

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacgcc 1380

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccaag actggctgaa tggcaaggag 1440

tacaagtgca aggtctccaa caaagccctc ccagccccca tcgccgcaac catctccaaa 1500

gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggaggagatg 1560

accaagaacc aagtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1620

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1680

gactccgacg gctccttctt cctctattcc aagctcaccg tggacaagag caggtggcag 1740

caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1800

aagagcctct ccctgtctcc gggcaaatga 1830

<210> 17

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> short chain of IMM5601 (IMM 56 antibody light chain)

<400> 17

Asp Ile Val Met Thr Gln Ser His Leu Ser Met Ser Thr Ser Leu Gly

1 5 10 15

Asp Pro Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Val

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Arg Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ile Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ala Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Pro Pro Tyr

85 90 95

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

100 105 110

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

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 18

<211> 841

<212> DNA

<213> Artificial sequence

<220>

<223> short chain of IMM5601 (IMM 56 antibody light chain)

<400> 18

atgggatggt catgtatcat cctttttctg gtagcaactg caactggagt acattcagac 60

atcgtgatga cacagagcca cctgagcatg agcacaagcc tgggcgaccc tgtgagcatc 120

acctgcaagg cctcccaaga cgtgagcacc gtggtggcct ggtatcagca gaagcctgga 180

cagagcccta gaagactgat ctacagcgcc tcctacagat acatcggcgt gcctgacaga 240

ttcaccggca gcggcgccgg caccgacttc accttcacca tcagcagcgt gcaagccgag 300

gacctggccg tgtactactg tcagcagcac tacagccctc cttacacctt cggcggaggc 360

accaagctgg agatcaagcg tgagttctag aggatccatc tgggataagc atgctgtttt 420

ctgtctgtcc ctaacatgcc ctgtgattat ccgcaaacaa cacacccaag ggcagaactt 480

tgttacttaa acaccatcct gtttgcttct ttcctcagga actgtggctg caccatctgt 540

cttcatcttc ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct 600

gctgaataac ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca 660

atcgggtaac tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct 720

cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga 780

agtcacccat cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta 840

g 841

<210> 19

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> Long chain of IMM5602 (heavy chain of IMM56 antibody)

<400> 19

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

1 5 10 15

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

20 25 30

Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Thr Ile Tyr Pro Gly Asp Gly Asp Thr Gly Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

Met His Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

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

100 105 110

Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

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

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

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

165 170 175

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

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

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

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

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

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Ala

325 330 335

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

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 20

<211> 1410

<212> DNA

<213> Artificial sequence

<220>

<223> Long chain of IMM5602 (heavy chain of IMM56 antibody)

<400> 20

atgggatggt catgtatcat cctttttctg gtagcaactg caactggagt acattcacaa 60

gtgcagctgg tgcagagcgg cgccgaggtg gccaagcctg gcacctccgt caagctgagc 120

tgcaaggcct ccggctacac cttcaccgac tactggatgc agtgggtgaa gcagagacct 180

ggccaaggcc tggagtggat cggcaccatc taccctggcg acggcgacac cggctacgct 240

cagaagttcc aaggcaaggc caccctgacc gccgacaaga gcagcaagac cgtgtacatg 300

cacctgagca gcctggcctc cgaggacagc gccgtgtact actgcgctag aggcgactac 360

tacggcagca acagcctgga ctactggggc caaggcacaa gcgtgaccgt gagcagcgct 420

agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 480

acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 600

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 660

atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagagagt tgagcccaaa 720

tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 780

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 840

gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtat 900

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacgcc 960

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccaag actggctgaa tggcaaggag 1020

tacaagtgca aggtctccaa caaagccctc ccagccccca tcgccgcaac catctccaaa 1080

gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggaggagatg 1140

accaagaacc aagtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1200

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1260

gactccgacg gctccttctt cctctattcc aagctcaccg tggacaagag caggtggcag 1320

caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1380

aagagcctct ccctgtctcc gggcaaatga 1410

<210> 21

<211> 354

<212> PRT

<213> Artificial sequence

<220>

<223> short chain of IMM5602 (SIRP. alpha. D1-linker-IMM 56 antibody light chain)

<400> 21

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 Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met

130 135 140

Thr Gln Ser His Leu Ser Met Ser Thr Ser Leu Gly Asp Pro Val Ser

145 150 155 160

Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Val Val Ala Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Gln Ser Pro Arg Arg Leu Ile Tyr Ser Ala Ser

180 185 190

Tyr Arg Tyr Ile Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ala Gly

195 200 205

Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala

210 215 220

Val Tyr Tyr Cys Gln Gln His Tyr Ser Pro Pro Tyr Thr Phe Gly Gly

225 230 235 240

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

245 250 255

Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val

260 265 270

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

275 280 285

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

290 295 300

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

305 310 315 320

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

325 330 335

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

340 345 350

Glu Cys

<210> 22

<211> 1261

<212> DNA

<213> Artificial sequence

<220>

<223> short chain of IMM5602 (SIRP. alpha. D1-linker-IMM 56 antibody light chain)

<400> 22

atgggatggt catgtatcat cctttttctg gtagcaactg caactggagt acattcagag 60

gaggagctgc aggtgattca gcctgacaag tccgtatcag ttgcagctgg agagtcggcc 120

attctgcact gcactgtgac ctccctgatc cctgtggggc ccatccagtg gttcagagga 180

gctggaccag cccgggaatt aatctacaat caaaaagaag gccacttccc ccgggtaaca 240

actgtttcag agtccacaaa gagagaaaac atggactttt ccatcagcat cagtgccatc 300

accccagcag atgccggcac ctactactgt gtgaagttcc ggaaagggag ccctgacacg 360

gagtttaagt ctggagcagg cactgagctg tctgtgcgtg ccaaaccctc tgcccccgtg 420

gtatcgggcc ctggcggcgg tgggagcggc ggcggtggga gcggcggcgg gggctcggac 480

atcgtgatga cacagagcca cctgagcatg agcacaagcc tgggcgaccc tgtgagcatc 540

acctgcaagg cctcccaaga cgtgagcacc gtggtggcct ggtatcagca gaagcctgga 600

cagagcccta gaagactgat ctacagcgcc tcctacagat acatcggcgt gcctgacaga 660

ttcaccggca gcggcgccgg caccgacttc accttcacca tcagcagcgt gcaagccgag 720

gacctggccg tgtactactg tcagcagcac tacagccctc cttacacctt cggcggaggc 780

accaagctgg agatcaagcg tgagttctag aggatccatc tgggataagc atgctgtttt 840

ctgtctgtcc ctaacatgcc ctgtgattat ccgcaaacaa cacacccaag ggcagaactt 900

tgttacttaa acaccatcct gtttgcttct ttcctcagga actgtggctg caccatctgt 960

cttcatcttc ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct 1020

gctgaataac ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca 1080

atcgggtaac tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct 1140

cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga 1200

agtcacccat cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta 1260

g 1261

<210> 23

<211> 364

<212> PRT

<213> Artificial sequence

<220>

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

<400> 23

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> 24

<211> 1095

<212> DNA

<213> Artificial sequence

<220>

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

<400> 24

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> 25

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> mouse IgG1 heavy chain signal peptide

<400> 25

atgggatggt catgtatcat cctttttctg gtagcaactg caactggagt acattca 57

<210> 26

<211> 9

<212> DNA

<213> Artificial sequence

<220>

<223> Kozak sequence

<400> 26

gccgccacc 9

<210> 27

<211> 597

<212> PRT

<213> Artificial sequence

<220>

<223> human SIRP alpha-mouse IgG1 Fc

<400> 27

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