CN113880955A - Recombinant fusion protein targeting CD47 and CD70, and preparation and application thereof - Google Patents

Abstract

The present application provides a recombinant fusion protein comprising a CD70 antibody or antibody fragment thereof, at least one paratope of the CD70 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, CD70 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 70.

Description

Recombinant fusion protein targeting CD47 and CD70, and preparation and application thereof

Technical Field

The present application relates to a recombinant fusion protein targeting CD47, CD70 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) escape immune surveillance of T lymphocytes by highly expressing membrane proteins PD-L1 and PD-L2, wherein both the membrane proteins PD-L1 and PD-L2 are combined with PD-1 on the surface of the T cells to trigger T cell apoptosis; 2) by expressing CD70, binding to CD27 on the surface of immune cells, T cell apoptosis and T cell depletion are induced, increasing the amount of tregs in large quantities; 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).

CD70

CD70 is a II-form transmembrane protein (b.f. israel et al, 2005) belonging to the tumor necrosis factor superfamily, expressed primarily in highly activated lymphocytes, as well as dendritic cells, and expression of CD70 has also been found in antigen presenting cells. CD70, also known as CD27L, binds to CD27, a receptor expressed on mature T cells, memory B cells, germinal center B cells, and Natural Killer (NK) cells. The interaction of CD70 with CD27 plays an important role in activating lymphocytes and maintaining immune responses. CD70-CD27 signal channel can induce CD4⁺T cells and CD8⁺Proliferation of T cells and secretion of cytokines. CD70-CD27 can also promote B cell activation, proliferation and differentiation, promote the formation of germinal center, and induce the secretion of antibody.

Numerous studies have shown that CD70 is expressed in a variety of tumor tissues, including renal cell carcinoma, nasopharyngeal carcinoma, Epstein-Barr virus-induced cancer, malignant Hodgkin's Lymphoma (HL) (h.j. gruss et al, 1996), non-hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma, follicular lymphoma, B-cell lymphocytic leukemia, Burkitt's lymphoma (s.m.lens et al, 1999), multiple myeloma (j.a. mcearcap et al, 2008), Waldenstrom's macroglobulinemia (a.w.ho et al, 2008), nasopharyngeal carcinoma (a.agaathanggelgro et al, 1995), thymus carcinoma (t.hishima et al, 2000), renal cell carcinoma (c.l.law et al, 2006), glioblastoma (j.schhuh et al, 2002), brain carcinoma (feishid-h et al, hel et al, melanoma (h.2016, h.2016), melanoma (c.l.l.l.ah, 2016), melanoma (hrwal. h.g. h.2016, h.h.2016), melanoma (c.h.g. h.h.h.2016, h.h., 2008). Expression of CD70 is associated with a poor prognosis in B-cell lymphomas, renal cell carcinomas, and breast cancers. Some studies have also found that CD70 is expressed on the surface of primary and metastatic non-small cell lung cancer (NSCLC) tumor cells and in the tumor microenvironment, making CD70 a potential target for the treatment of NSCLC (j.

Tumor cells utilize their expressed CD70 to induce T cell apoptosis and T cell depletion and greatly increase the amount of regulatory T cells (tregs), thereby promoting their own growth (Julie Jacobs et al, 2018). For example, in Acute Myeloid Leukemia (AML), the CD70-CD27 pathway in AML cells can activate stem cell gene expression programs, promoting symmetric cell division and proliferation, while antibody blockade can inhibit cell growth (c.riether et al, 2017). Leukemia Stem Cells (LSCs) can be greatly reduced by blocking the interaction of CD70 with CD27 by the antibody cusatuzumab (c.riether et al, 2020).

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 have limited therapeutic efficacy. For example, the approved PD-L1 antibody, Avelumab

The overall remission rate was only 33%.

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 (Shim h.2020).

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 CD70 antibody and a CD47 binding peptide. The recombinant fusion proteins of the present application are capable of binding to CD70 with comparable or higher activity than the CD70 antibody and CD47 binding protein⁺、CD47⁺And/or CD70⁺CD47⁺Cell, for CD70⁺CD47⁺The cells elicit comparable or higher antibody-dependent cell-mediated cytotoxic effects (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC). The recombinant fusion proteins of the present application also show potent antitumor effects in vivo experiments.

Specifically, the present application discloses a recombinant fusion protein comprising a CD70 antibody or antibody fragment thereof that specifically binds CD70, and a CD47 binding peptide that specifically binds CD47, wherein the CD47 binding peptide is linked to the CD70 antibody or antibody fragment thereof, wherein the CD70 antibody or antibody fragment thereof comprises a heavy chain variable region comprising HV-CDR1, HV-CDR2 and HV-CDR3, HV-CDR1, HV-CDR2 and HV-CDR3 comprising the amino acid sequences shown in SEQ ID NOs:2, 3 and 4, respectively, a light chain variable region comprising FcR 1, LV-CDR2 and LV-CDR3, LV-CDR1, LV-CDR2 and LV-CDR3 comprising the amino acid sequences shown in SEQ ID NOs:5, 6 and 7, respectively, a heavy chain constant region having an LV-binding capacity, a complement protein (e.g., C1 binding complement protein 1q) 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 having at least 95% sequence identity to SEQ ID NO:1, wherein the recombinant fusion protein is capable of simultaneously binding CD47 and CD 70. 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 CD70 antibody or antibody fragment thereof.

The CD70 antibody can be an IgG antibody. The heavy chain variable region of the CD70 antibody or antibody fragment thereof may comprise 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. 8. In one embodiment, the heavy chain variable region may comprise the amino acid sequence set forth in SEQ ID NO 8. The light chain variable region of the CD70 antibody or antibody fragment thereof may comprise 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. 9(X ═ N, or X ═ E). In one embodiment, the light chain variable region may comprise the amino acid sequence shown in SEQ ID No. 9(X ═ N, or X ═ E). In one embodiment, the heavy chain variable region and the light chain variable region of the CD70 antibody or antibody fragment thereof may comprise amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs:8 and 9(X ═ N, or X ═ E), respectively. In some embodiments, the heavy chain variable region and the light chain variable region of the CD70 antibody or antibody fragment thereof may comprise the amino acid sequences set forth in SEQ ID NOs:8 and 9(X ═ N, or X ═ E), respectively.

The heavy chain constant region having FcR binding, and/or binding to a complement protein (e.g., C1q) 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 heavy chain constant region that binds FcR and/or to a complement protein (e.g., C1q) is a human IgG1 heavy chain constant region, or a functional fragment thereof. In some embodiments, the heavy chain constant region that binds FcR and/or binds a complement protein (e.g., C1q) has the amino acid sequence set forth in SEQ ID NO. 10.

The CD70 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 CD70 antibody or antibody fragment thereof can comprise the amino acid sequence set forth in SEQ ID NO. 11.

The heavy chain of the CD70 antibody or antibody fragment thereof can comprise 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. 16. In one embodiment, the heavy chain may comprise the amino acid sequence set forth in SEQ ID NO 16. The light chain of the CD70 antibody or antibody fragment thereof may comprise 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. 14(X ═ N, or X ═ E). In one embodiment, the light chain variable region may comprise the amino acid sequence shown in SEQ ID No. 14(X ═ N, or X ═ E). In one embodiment, the heavy chain variable region and the light chain variable region of the CD70 antibody or antibody fragment thereof may comprise amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs:16 and 14(X ═ N, or X ═ E), respectively. In some embodiments, the heavy chain variable region and the light chain variable region of the CD70 antibody or antibody fragment thereof may comprise the amino acid sequences set forth in SEQ ID NOs:16 and 14(X ═ N, or X ═ E), respectively.

In some embodiments, at least one paratope of the CD70 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 CD70 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 CD70 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 CD70 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 CD70 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:21)、-(Gly-Gly-Gly-Gly-Ser)₃- (SEQ ID NO:20), or- (Gly-Gly-Gly-Gly-Ser)₄- (SEQ ID NO: 22). In some embodiments, the linker is- (Gly-Gly-Gly-Gly-Ser)₃-(SEQ ID NO:20)。

A recombinant fusion protein of the present application can comprise a CD47 binding peptide-linker-CD 70 antibody heavy chain, and a CD70 antibody light chain, wherein the CD47 binding peptide-linker-CD 70 antibody heavy chain comprises 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. 12, and the CD70 antibody light chain comprises 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. 14(X ═ N, or X ═ E). In some embodiments, a recombinant fusion protein of the present application may comprise a CD47 binding peptide-linker-CD 70 antibody heavy chain, wherein the CD47 binding peptide-linker-CD 70 antibody heavy chain comprises the amino acid sequence set forth in SEQ ID No. 12, and a CD70 antibody light chain, wherein the CD70 antibody light chain comprises the amino acid sequence set forth in SEQ ID No. 14(X ═ N, or X ═ E). The amino acid sequences of SEQ ID NOs 12, 14(X ═ N) and 14(X ═ E) can be encoded by the nucleic acid sequences shown in SEQ ID NOs 13, 15(N1 ═ a, N2 ═ C) and 15(N1 ═ G, N2 ═ a), respectively.

A recombinant fusion protein of the present application can comprise a CD70 antibody heavy chain, wherein the CD70 antibody heavy chain comprises 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. 16, and a CD47 binding peptide-linker-CD 70 antibody light chain, wherein the CD47 binding peptide-linker-CD 70 antibody light chain comprises 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. 18. In some embodiments, a recombinant fusion protein of the present application can comprise a CD70 antibody heavy chain, wherein the CD70 antibody heavy chain comprises the amino acid sequence set forth in SEQ ID No. 16, and a CD47 binding peptide-linker-CD 70 antibody light chain, wherein the CD47 binding peptide-linker-CD 70 antibody light chain comprises the amino acid sequence set forth in SEQ ID No. 18. The amino acid sequences shown in SEQ ID NOs:16 and 18 may be encoded by the nucleic acid sequences shown in SEQ ID NOs:17 and 19, respectively.

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 CD70 overexpression or used for preparing medicines for treating diseases related to CD47 and/or CD70 overexpression.

In one aspect, the present application provides a method for treating or alleviating a disease associated with CD47 and/or CD70 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 CD70 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, renal cell carcinoma, nasopharyngeal cancer, Waldenstrom's macroglobulinemia, thymus cancer, melanoma, glioblastoma, and osteosarcoma.

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 IMM4002, IMM4002M, and IMM4003 of the present application. The circular structure represents the mutant SIRP alpha first extracellular domain (SIRP alpha D1) having the amino acid sequence shown in SEQ ID NO. 1. : -represents a glycosylation site. In the recombinant fusion proteins IMM4002 and IMM4002M, the mutant sirpa D1 was linked to the heavy chain N-terminus of the CD70 antibody via a linker peptide. In IMM4003, a mutant sirpa D1 was linked to the N-terminus of the light chain of CD70 antibody via a linker peptide. Wherein the CD70 antibody IMM40H has a heavy and light chain as shown in SEQ ID NOs:16 and 14(X ═ N), respectively, IMM40M has a heavy and light chain as shown in SEQ ID NOs:16 and 14(X ═ E), respectively, and the linker has the amino acid sequence shown in SEQ ID NO: 20. Compared to IMM40H, IMM40M contained an N85E mutation in the light chain variable region that eliminated the original glycosylation site.

FIGS. 2A-2G show IMM4002, IMM4002M, and/or IMM4003 and human CD70 expressing Chinese hamster ovary cells (A), CD47⁺CD70^-Jurkat human T-lymphoid leukemia cell (B), CD47⁺CD70⁺U266 human multiple myeloma cells (C), CD47⁺CD70⁺Raji human B lymphoblastoid (D), CD47⁺CD70⁺U266 human multiple myeloma cells (E), CD47⁺CD70⁺Jeko-1 human mantle cell lymphoma cell (F), and CD47⁺CD70⁺Binding activity of Daudi human Burkitt lymphoma cells (G). The CD70 antibody IMM40H, the CD70 antibody Cusatuzumab (heavy and light chain amino acids are shown in SEQ ID NOs:28 and 29), the CD47 binding protein IMM01 (described in US 2021/0024598A 1, comprising two mutations SIRPa D1(SEQ ID NO:1) linked to an Fc dimer fragment, in which the monomers contain the nucleic acid and amino acid sequences shown in SEQ ID NO:24 and SEQ ID NO:23, respectively), human hIgG1-Fc were used as controls.

FIGS. 3A-3B show that IMM4002 blocks SIRP α -Fc and CD47⁺CD70^-Jurkat human T-lymphoid leukemia cell (A) or CD47⁺CD70⁺Raji human B lymphoblastoma tumor cell lineThe ability of CD47 to bind on cells. IMM01, IMM40H, and hIgG1-Fc were used as controls.

FIG. 4 shows that IMM4002 blocks CD27(ECD) -Fc (SEQ ID NO:30) from CD70⁺The ability of CD70 to bind on chinese hamster ovary cells. Cusatuzumab was a CD70 antibody used as a positive control, and hIgG1-Fc used as a negative control.

FIG. 5 shows IMM4002 blocks CD70⁺The activity of Raji human B lymphoblastoma cells to interact with human T lymphoblastic leukemia cells Jurkat-CAR-CD27, wherein the expression level of CD69 indirectly reflects the binding ability of CD70 to Jurkat-CAR-CD 27. Cusatuzumab was a CD70 antibody used as a positive control, and hIgG1-Fc used as a negative control.

FIGS. 6A-6D illustrate IMM4002 vs. CD47⁺CD70⁺Raji human B lymphoblastoma cell (A), CD47⁺CD70⁺U266 human multiple myeloma cells (B), CD47⁺CD70⁺Daudi human Burkitt lymphoma cell (C), and CD47⁺CD70⁺Jeko-1 human mantle cell lymphoma cell (D) ability to elicit antibody-dependent cellular cytotoxicity (ADCC). IMM01 was used as a positive control and hIgG1-Fc was used as a negative control.

FIGS. 7A-7D illustrate IMM4002 vs. CD47⁺CD70⁺Raji human lymphoblasts (A), U266 human multiple myeloma cells (B), Daudi human Burkitt lymphoma cells (C), and Jeko-1 human mantle cell lymphoma cells (D) ability to elicit antibody-dependent cellular phagocytosis (ADCP). IMM40H was used as a positive control and hIgG1-Fc, which elicited lower ADCP activity, was used as a negative control.

FIG. 8 shows IMM4002 vs. CD47⁺CD70⁺The ability of Raji human lymphoblasts to trigger Complement Dependent Cytotoxicity (CDC). IMM40H was used as a positive control, and hIgG1-Fc and IMM01, which elicited very low CDC, were used as negative controls.

Figure 9 shows the in vivo anti-tumor effect of IMM4002 and IMM4003 in SCID mouse U266 xenograft tumor model.

Figure 10 shows that tumor-bearing mice treated with IMM4002 had higher survival rates.

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.

Through a large number of experiments, the present inventors have invented a novel recombinant multifunctional fusion protein, which can attack tumors through several mechanisms of action, one is to block apoptosis and failure of T cells mediated by CD70 and Treg increase, one is to relieve the check of inhibitory signals mediated by SIRP on immune cells such as macrophages, one is to activate the killing of cancer cells by immune cells such as NK cells and/or macrophages, and the other is to activate the killing of cancer cells by complement system.

A recombinant fusion protein of the present application comprises a CD70 antibody or antibody fragment thereof, at least one paratope of the CD70 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, CD70, and FcR, i) block the interaction between CD70 on cancer cells and CD27 on immune cells, thereby blocking CD 70-mediated T cell apoptosis and failure, and Treg increase; ii) blocking the interaction of CD47 on cancer cells with SIRP on immune cells such as macrophages, relieving the SIRP-mediated inhibitory signal from examination of immune cells such as macrophages; iii) binding of the antibody Fc region to FcR on immune cells such as NK cells or macrophages, activating killing of cancer cells by immune cells NK cells or macrophages, and vi) binding of the antibody Fc region to complement system proteins such as C1q, activating killing of cancer cells by the complement system. In one embodiment, a paratope of a CD70 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 CD70 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 CD70 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 CD70 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 CD70 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, in which there is an N80A mutation at position 80 of SEQ ID NO:1, which mutation may achieve the effect of deglycosylation, compared to the wild-type sirpa D1.

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, the sirpa D1 may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID No. 1, wherein the sirpa D1 is capable of binding to CD47 on the surface of cancer/tumor cells and blocking the interaction of CD47 with SIRP on the surface of immune cells, such as macrophages.

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 CD70 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. The following examples illustrateExemplary suitable linkers of (Gly-Ser), e.g., (Gly-Gly-Gly-Gly-Ser)₃-(SEQ ID NO:20)。

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.

The CD70 antibody can be an IgG antibody. The CD70 antibody may comprise the heavy and light chain variable regions of SEQ ID NOs:8 and 9(X ═ N). The CD70 antibody may contain a N85E mutation in the light chain variable region that eliminates the original glycosylation site. In some embodiments, the CD70 antibody may comprise the heavy chain variable region and the light chain variable region of SEQ ID NOs:8 and 9(X ═ E). In some embodiments, the CD70 antibody is an isolated IgG monoclonal antibody comprising two heavy chains each having the amino acid sequence of SEQ ID NO:16 and two light chains each having the amino acid sequence of SEQ ID NO:14(X ═ N or E), which can be encoded by SEQ ID NO:17 and SEQ ID NOs:15(N1 ═ a, N2 ═ C; or N1 ═ G, N2 ═ a), respectively. The Fab portion (or paratope) of the CD70 antibody may bind to CD70 on the surface of cancer/tumor cells to block the interaction of CD70 with CD27 on the surface of immune cells, thereby blocking the T cell apoptosis and exhaustion mediated by CD70, and Treg increase, while the Fc portion of the CD70 antibody may bind to FcR on the surface of immune cells, such as NK cells and/or macrophages, and/or to complement system proteins, such as C1q, to stimulate the killing of cancer cells by the immune system and/or complement system. In some embodiments, the heavy chain may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID No. 16, wherein the CD70 antibody is capable of binding to CD70 and blocking the interaction between CD70 and CD27 on the surface of immune cells, and is capable of binding to FcR on the surface of immune cells, such as NK cells or macrophages, and/or to a complement system protein to activate the immune system and/or complement system to kill cancer cells. In some embodiments, the light chain may have an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO 14(X ═ N or E), wherein the CD70 antibody is capable of binding to CD70 and blocking the interaction between CD70 and immune cell surface CD 27.

The term "antibody" herein includes whole antibodies, e.g., IgG, IgA, IgD, IgE and IgM, and any antigen-binding fragment (or antigen-binding portion) or single chain thereof. A whole antibody is a glycoprotein comprising at least two heavy chains and two light chains, which are linked via disulfide bonds. Each heavy chain comprises a heavy chain variable region (V)_H) And a heavy chain constant region. The heavy chain constant region comprises three domains, C_H1、C_H2And C_H3. Each light chain comprises a light chain variable region (V)_L) And a light chain constant region. The light chain constant region comprises a domain C_L。V_HAnd V_LRegions may also be subdivided into regions of high degree of variation, i.e., CDR regions, with more conserved Framework Regions (FRs) distributed between the CDR regions. Each V_HAnd V_LConsists of three CDRs and four FR regions, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions of the heavy and light chains comprise binding domains that react with antigen. The constant region of the antibody may mediate the binding of immune proteins to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component of the complement system (C1 q).

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., CD70), and optionally to FcR and/or complement system proteins.

The "paratope", also called antigen binding site, as used herein, refers to a specific part of an antibody that binds to an antigen complementarily, and comprises 6 CDRs, i.e., 3 CDRs from the heavy chain variable region and 3 CDRs from the light chain variable region.

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.

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

The terms "antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refer to the binding of an antibody, such as a CD70 antibody, or an antibody-like protein, such as sirpa-Fc, to the FcR of an immune effector cell, such as a macrophage, such that the immune effector cell actively lyses cell membrane surface antigens to the antibody, such as a CD70 antibody, or target cells, such as cancer cells, that bind to sirpa.

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

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

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

A "CAR" or "chimeric antigen receptor" comprises a) an extracellular antigen-binding domain, b) a transmembrane domain; and c) an intracellular signaling domain. The CAR may further comprise a signal peptide at the N-terminus of the extracellular antigen-binding domain. The CD27-CAR in the present application comprises a signal peptide, a CD27 extracellular segment, a CD8 a hinge region, a CD28 transmembrane and intracellular region, and a CD3 zeta signaling region.

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. The selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy,20th Ed. (Lippincott Williams & Wilkins 2003), 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 CD70, 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, renal cell carcinoma, nasopharyngeal cancer, Waldenstrom macroglobulinemia, thymus cancer, melanoma, glioblastoma, and osteosarcoma.

In one embodiment, diseases associated with overexpression of CD47 and/or CD70 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 IMM40H, IMM40M, IMM4002, IMM4003, and IMM4002M are shown in FIG. 1.

IMM40H and IMM40M are IgG antibodies targeting CD 70. IMM40H has heavy and light chains as shown in SEQ ID NOs:16 and 14(X ═ N), respectively, and IMM40M has heavy and light chains as shown in SEQ ID NOs:16 and 14(X ═ E), respectively. Major represents a glycosylation site, and in contrast to IMM40H, IMM40M contains an N85E mutation in the light chain variable region that eliminates the original glycosylation site.

IMM4002, IMM4002M, and IMM4003 are recombinant fusion proteins targeting CD70 and CD 47. In IMM4002 and IMM4002M, a mutant sirpa D1 was linked to the N-terminus of the antibody heavy chain via a linker peptide. In IMM4003, a mutant sirpa D1 was linked to the N-terminus of the antibody light chain via a linker peptide. Major axis indicates a glycosylation site, the circular structure indicates a first extracellular domain of SIRP α (SIRP α D1) having an amino acid sequence shown in SEQ ID NO:1, and the linker peptide has an amino acid sequence shown in SEQ ID NO: 20.

IMM01 is a CD47 binding Fc fusion protein described in US 2021/0024598A 1 comprising two mutant SIRP α D1(SEQ ID NO:1) linked to an Fc dimer fragment, wherein the monomers comprise the nucleic acid and amino acid sequences shown in SEQ ID NO:24 and SEQ ID NO:23, respectively.

Cusatuzumab is an IgG antibody targeting CD70, with the heavy and light chain amino acids shown in SEQ ID NOs:28 and 29, respectively.

Example 1 construction and protein profiling of IMM40H, IMM40M, IMM4002, IMM4003 and IMM4002M expression vectors To achieve

The coding sequences for IMM40H, IMM40M, IMM4002, IMM4003 and IMM4002M were artificially designed.

Specifically, for the long peptide chain in IMM4002, i.e., the SIRP α D1-linker-CD 70 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 70 antibody heavy chain coding sequence (SEQ ID NO:13), 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 peptide chain in IMM4002, i.e., the CD70 antibody light chain, the same signal peptide sequence as well as the Kozak sequence was added to the 5 ' end of the CD70 antibody light chain coding sequence (SEQ ID NO:15, N1 ═ a, N2 ═ C), and HindIII and XbaI restriction sites were added to the 5 ' and 3 ' ends of the resulting sequence, respectively. The resulting sequences were synthesized by Kinsley science and technology, Inc. and cloned into pMac-H and pMac-L vectors, respectively.

For the long peptide chain in IMM4002M, i.e., the SIRP α D1-linker-CD 70 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 70 antibody heavy chain coding sequence (SEQ ID NO:13) 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 peptide chain in IMM4002, i.e., the CD70 antibody light chain, the same signal peptide sequence as well as the Kozak sequence was added to the 5 ' end of the CD70 antibody light chain coding sequence (SEQ ID NO:15, N1 ═ G, N2 ═ a), and HindIII and XbaI restriction sites were added to the 5 ' and 3 ' ends of the resulting sequence, respectively. The resulting sequences were synthesized by Kinsley science and technology, Inc. and cloned into pMac-H and pMac-L vectors, respectively.

For the long peptide chain in IMM4003, i.e., the CD70 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 CD70 antibody heavy chain coding sequence (SEQ ID NO:17) and a Kozak sequence (SEQ ID NO:26) was added to the 5' end of the signal peptide sequence. For the short peptide chain of IMM4003, i.e., SIRPa D1-linker-CD 70 antibody light chain, the same signal peptide sequence as well as the Kozak sequence was added to the 5 ' end of the SIRPa D1-linker-CD 70 antibody light chain coding sequence (SEQ ID NO:19), and HindIII and XbaI restriction sites were added to the 5 ' and 3 ' ends of the resulting sequence, respectively. The resulting sequences were synthesized by Kinsley science and technology, Inc. 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 1X10 cells the day before transient transformation⁶One/ml Density inoculated to TransFx-C containing 6mM Glutamine^TMCHO transient medium (Cat # SH30942.02, Hyclone); 2) the mass ratio of the heavy-light chain/long-short peptide chain expression vector is 1: 1, preparing the desired DNA at 1. mu.g/ml, adding to 1/20 transient volume of OPTI-MEM medium (Gibco); 3) PEI (MW 40,000, polyethyleneimine hydrochloride, Cat #24765-1, polysciences) was prepared in a mass ratio of PEI: DNA of 1mg/ml, and the desired PEI was added to OPTI-MEM medium (Gibco) in an instantaneous volume of 1/20; 4) slowly adding the PEI diluent into the DNA diluent, uniformly mixing, and incubating for 20 minutes at room temperature; 5) add PEI/DNA mix to cell sap and place cells at 37 degrees 5% CO₂Culturing in an incubator with the rotation speed of 110rpm in an oscillating manner; 6) transfection enhancers (1mM sodium butyrate, 0.25% V/V DMSO) were added every other day while the incubation temperature was lowered to 33 degrees; 7) when the cell viability decreased below 50%, the supernatant was collected by centrifugation at 3000rpm for 5 minutes and affinity purified using Protein A packing.

Example 2 binding of recombinant fusion proteins to cell surface CD47 or/and CD70

Each cell, i.e., Chinese hamster ovary cells expressing human CD70、CD47⁺CD70^-Jurkat human T lymphoid leukemia cell, CD47⁺CD70⁺U266 human multiple myeloma cells, CD47⁺CD70⁺Raji human B lymphoblastoid cell, CD47⁺CD70⁺U266 human multiple myeloma cells, CD47⁺CD70⁺Jeko-1 human mantle cell lymphoma cells, and CD47⁺CD70⁺Daudi human Burkitt's lymphoma cells, 1000rpm centrifugation for 5min, cell collection, PBS washing 1 time, cell density adjustment to 1x10⁶And/ml. Recombinant fusion proteins IMM4002, IMM4003, IMM4002M, and/or control proteins IMM40H, IMM01 (Fc fusion protein that binds CD47, described in US 2021/0024598A 1, comprising two mutations SIRPa D1(SEQ ID NO:1) were linked to an Fc dimer fragment, in which the monomers contained the nucleic acid and amino acid sequences shown in SEQ ID NO:24 and SEQ ID NO:23, respectively), Cusatuzumab (CD70 antibody, heavy and light chain amino acids shown in SEQ ID NOs:28 and 29, respectively), hIgG1-Fc, diluted with a 1% BSA-PBS gradient.

Mu.l of cells were taken, mixed with 100. mu.l of protein solution, and incubated at 4 ℃ for 45 min. After the incubation was completed, 1% BSA-PBS was washed 1 time. Mu.l of anti-human IgG Fc-FITC (Cat # F9512, Sigma) was added, diluted 500-fold and incubated at 4 ℃ for 45 min. After the incubation was completed, 1% BSA-PBS was washed 1 time. Resuspend cells in 200. mu.l of 1% BSA-PBS, flow cytometer using cells (

easyCyte 5HT, Merck Millipore) for FACS analysis.

FIG. 2A shows IMM4002 and IMM4003 with CD70⁺Binding capacity of chinese hamster ovary cells. For the CD70 single positive cell, the order of strong and weak binding capacity is IMM40H>IMM4003>IMM4002, but the difference between the three is small, it is believed that IMM4002 has comparable binding capacity to human CD70 and IMM 40H.

FIG. 2B shows IMM4002 and IMM4003 with CD47⁺CD70^-Binding capacity of Jurkat human T-lymphoid leukemia cells. For the CD47 single positive cell, the order of strong and weak binding capacity is IMM01>IMM4002>And (4) IMM 4003. Wherein the junctions of IMM4002 and IMM4003 with human CD47The combined abilities are equivalent and are 3 to 4 times weaker than IMM01

FIG. 2C shows IMM4002 and IMM4002M with CD47⁺CD70⁺U266 binding ability of human multiple myeloma cells. For the double positive cells, the order of the binding capacity was IMM40H>IMM4002>IMM4002M, wherein IMM4002 and IMM4002M have comparable binding capacity.

FIG. 2D shows IMM4002 and CD47⁺CD70⁺Binding capacity of Raji human B lymphoblasts. For the cells with double positive, the order of strong and weak binding capacity is IMM40H>IMM4002>cusatuzumab>IMM 01. Wherein, the combining ability of IMM4002 and IMM40H is equivalent, and both are obviously stronger than that of cusatuzumab and IMM 01. Since Raji cells express FcR, the negative control hIgG1-Fc also had binding reactions.

FIG. 2E shows IMM4002 and CD47⁺CD70⁺U266 binding ability of human multiple myeloma cells. For the cells with double positive, the order of strong and weak binding capacity is IMM40H>IMM4002>cusatuzumab>IMM 01. Of these, IMM4002 has a slightly weaker binding capacity than IMM40H, but is stronger than cusatuzumab and significantly stronger than IMM 01.

FIG. 2F shows IMM4002 and CD47⁺CD70⁺Binding capacity of Jeko-1 human mantle cell lymphoma cells. For the cells with double positive, the order of strong and weak binding capacity is IMM01>cusatuzuma>IMM4002>IMM40H。

FIG. 2G shows IMM4002 and CD47⁺CD70⁺Binding capacity of Daudi human burkitt lymphoma cells. For the cells with double positive, the sequence of strong and weak binding capacity is IMM4002>IMM01>IMM40H>cusatuzumab。

Example 3 blocking of SIRP α -Fc binding to cell surface CD47 by recombinant fusion proteins

Mu.l of 3. mu.g/ml SIRP α -mFc (wild type human SIRP α + murine IgG1 Fc, SEQ ID NO:27) was mixed with 50. mu.l of IMM4002 and a control protein, each starting at a concentration of 30. mu.g/ml, diluted in a 3-fold gradient. Adding the above mixture to a solution containing 50 μ l of a mixture with a density of 1X10⁶Jurkat or Raji cells in 96-well plates in ml, incubated at 4 ℃ for 45 minutes. Cells were washed with PBS, after which 100. mu.l PE-labeled for murine I was addedA secondary antibody to gG-Fc (Cat #405307, Biolegend) was incubated for 45 min. Cells were washed twice, resuspended in 200 μ l PBS, and analyzed for SIRP α -Fc-CD47 interaction by FACS.

FIG. 3A shows that IMM4002 blocks SIRP α -Fc and CD47⁺CD70^-The ability of Jurkat to bind CD47 on human T-lymphoid leukemia cells, wherein the blocking ability of IMM4002 is comparable to IMM 01.

FIG. 3B shows that IMM4002 blocks SIRP α -Fc and CD47⁺CD70⁺The ability of CD47 binding on Raji human B lymphoblastoma cells was significantly greater for the blockade of IMM4002 than for IMM 01.

Example 4 IMM4002 blocks CD27(ECD) -Fc from CD70 on Chinese hamster ovary cells expressing human CD70 Bonding of

50 μ l of the mixture was mixed at a density of 5X 10⁵The CHO-hCD70 cells/ml were mixed with 50. mu.l of IMM40H, IMM4002, Cusatuzumab, and hIgG-Fc, respectively, starting at 900nM, diluted in a 3-fold gradient. The above mixture was added to a 96-well plate containing 50. mu.l of 2.5. mu.g/ml biotin-CD 27(ECD) -Fc (SEQ ID NO:30, amino acids 1-172 being extracellular domain of CD 27), and the plate was incubated at 4 ℃ for 45 minutes. Cells were washed with PBS, followed by addition of 100. mu.l of PE-labeled streptavidin secondary antibody to biotin (Cat #554061, Biolegend) and incubation for 45 minutes. Cells were washed twice, resuspended in 200. mu.l PBS, and analyzed for CD27-Fc-CD70 interactions by FACS.

As shown in FIG. 4, the activity of IMM4002 in blocking CD70-CD27 was observed in CD70⁺CD47^-On chinese hamster ovary cells, comparable to IMM40H and cusatuzumab.

⁺Example 5 IMM4002 blocks CD70Raji human B lymphoblastoma cells and human T lymphocytic leukemia cells Interaction of cell Jurkat-CAR-CD27

Taking 50 μ l of the mixture with a density of 2X10⁵CD70 of one/ml⁺Raji human B lymphoblastoma cells were incubated with 50. mu.l of IMM4002, IMM40H, Cusatuzumab, IMM01, and hIgG1-Fc at a starting concentration of 16ng/ml, 4-fold dilution gradient, respectively, for 45min at 37 ℃. Then 50. mu.l of a 1X 10-dense solution was added⁶Human T lymphocyte leukemia cell Jurkat-CAR-CD27 (manufactured by the company, the amino acid sequence of CAR-CD27 is shown in SEQ ID NO:31, amino acids 1-172 are extracellular segments of CD 27) at each ml was incubated at 37 ℃ overnight. After adding 0.5% BSA-PBS to the cell mixture, centrifugation was performed, and the cells were resuspended in 50. mu.l 1: a100-diluted dilution of PE-labeled anti-human CD69 antibody (Cat # sc-373799, Santa Cruz) was incubated at 4 ℃ for 45 minutes, and the cells were washed once with 0.5% BSA-PBS and resuspended in 0.5% BSA-PBS. Then, the cells are subjected to flow cytometry (

easyCyte 5HT, Merck Millipore) for FACS analysis. The expression level of CD69 indirectly reflects the degree of binding of CD70 to Jurkat-CAR-CD27 on Raji human B lymphoblastoma cells.

As shown in FIG. 5, IMM4002 and IMM40H showed comparable inhibitory activity against CD70-CD27 binding, slightly stronger than Cusatuzumab, a traditional monospecific CD70 antibody.

Example 6 IMM4002 elicits antibody-dependent cellular cytotoxicity (ADCC) against tumor cells

Each tumor cell was labeled with a 500-fold dilution of 1mM CFSE (Cat #21888-25mg, Sigma).

Taking 50 μ l of the mixture with a density of 6X 10⁵Each tumor cell (used as a targeting cell) was mixed with 100. mu.l of a mixture having a density of 6X 10⁵/ml of NK92MI cells stably expressing Fc γ RIIIa (effector cells), in a ratio of effective targets of 2: 1 mixed and the mixed cells at 5% CO₂Next, 50. mu.l of fusion protein (initial concentration of 1nM each of IMM01 and IMM40H for mixtures of IMM01 and IMM 40H) diluted in a 4-fold gradient at an initial concentration of 1nM, and control protein, respectively, were incubated at 37 ℃ for 4 hours. Thereafter, Propidium Iodide (PI) (Cat # P4170, Sigma) was added to the cell culture broth at a concentration of 5. mu.g/ml, and the cells were analyzed for PI signal by FACS. The percentage of cell lysis due to ADCC was calculated based on the following formula:

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

As shown in FIG. 6AShown for CD47⁺CD70⁺Raji human B lymphoblastoma cells, IMM4002 elicited ADCC effects comparable to the CD70 monospecific antibody IMM40H, and IMM40H + IMM01 mixtures, and much stronger than the monospecific CD47 binding protein IMM 01.

As shown in fig. 6B for CD47⁺CD70⁺U266 human multiple myeloma cells, IMM4002 elicited ADCC effects comparable to CD70 monospecific antibodies IMM40H, and IMM40H + IMM01 mix, and much stronger than the monospecific CD47 binding protein IMM 01.

As shown in fig. 6C for CD47⁺CD70⁺Daudi human burkitt lymphoma cells, IMM4002 elicited ADCC effects that were stronger than the CD70 monospecific antibodies IMM40H, and the IMM40H + IMM01 mixture, and much stronger than the monospecific CD47 binding protein IMM 01.

As shown in fig. 6D for CD47⁺CD70⁺Jeko-1 human mantle cell lymphoma cells, the ADCC effect elicited by IMM4002 was comparable to the CD70 monospecific antibody IMM40H, and IMM40H + IMM01 mixture, and much stronger than the monospecific CD47 binding protein IMM 01.

^{+ +}Example 7 IMM4002 against CD47CD70 human lymphoblast (Raji cells), human multiple myeloma cells (U266 cells), human Burkitt lymphoma cells (Daudi cells) and human mantle cell lymphoma cells (Jeko-1 cells) priming High levels of Antibody Dependent Cellular Phagocytosis (ADCP)

After digesting macrophage THP-1 cells with 0.25% pancreatin at 37 ℃ for 2min, stop with complete medium (1640+ 10% FBS), and blow the cells into suspension, centrifuge at 1000rpm for 5min, collect THP-1 cells and add to 96-well plate, 3 × 10⁵One well containing 100. mu.l of complete medium per well, 200ng/ml PMA stimulated overnight culture.

Tumor cells, i.e., Raji cells, U266 cells, Daudi cells, and Jeko-1 cells, were labeled with CFSE as targeting cells. Specifically, 1mM CFSE was diluted 500 times, added to the cell suspension with adjusted density, mixed well, and stained in a cell incubator for 30 min. After the dyeing is finished, the blank 1640 culture medium is washed for 1 time, andcell density was adjusted to 2x10⁶And/ml. Cells were centrifuged at 1000rpm for 5min, collected, washed 1 time with 1640 medium, and cell density was adjusted to 1 × 10⁶/ml。

Recombinant fusion and control proteins were diluted in a 1640 media fold-gradient, where the initial concentrations of each of IMM01 and IMM40H were consistent with the single substance group for the IMM01 and IMM40H mixtures. Mu.l of the treated tumor cells were mixed with 100. mu.l of the diluted recombinant fusion protein or control protein, 2 more wells per concentration, and incubated in an incubator for 45 min. The supernatant of the well plate inoculated with THP-1 cells was aspirated, 200. mu.l of the above tumor cell-fusion protein mixture was added, and a control group, i.e., THP-1 cells alone, THP-1 and tumor cells were set and incubated in an incubator for 2 hours.

PBS-washed 96-well plates were washed 5 times to remove free tumor-CFSE cells. After washing, 200. mu.l of PBS was added to each well, and the THP-1 cells were suspended by repeated pipetting. THP-1 cells were flow-detected and analyzed for green fluorescence intensity. Phagocytic activity was evaluated by analyzing the green fluorescence intensity of THP-1 cells.

As shown in fig. 7A, for CD47⁺CD70⁺Raji human lymphoblasts, IMM4002, elicited ADCP effects comparable to the CD70 monospecific antibody IMM40H, and much stronger than the Fc fusion protein IMM01 bound to CD47, and at high concentrations than the IMM01+ IMM04H mixture.

As shown in fig. 7B for CD47⁺CD70⁺U266 human multiple myeloma cells, IMM4002 elicited ADCP effects comparable to the CD70 monospecific antibody IMM40H, and much stronger than the Fc fusion protein IMM01 binding to CD47, and at high concentrations than the IMM01+ IMM04H mixture.

As shown in fig. 7C for CD47⁺CD70⁺Daudi human burkitt lymphoma cells, IMM4002 elicited slightly stronger ADCP effects than the CD70 monospecific antibody IMM40H, the Fc fusion protein IMM01 that binds CD47, and the IMM01+ IMM04H mixture.

As shown in fig. 7D for CD47⁺CD70⁺Jeko-1 human mantle cell lymphoma cells, IMM4002 elicited ADCP effects slightly weaker than that of the CD70 monospecific antibody IMM40H, but slightly stronger than that of IMM01+ IMM04H mixed cellsMuch stronger than the Fc fusion protein IMM01 that binds CD 47.

^{+ +}Example 8 IMM4002 elicited high levels of complement fixation against CD47CD70 human lymphoblasts (Raji cells) Dependent Cytotoxicity (CDC)

Serum-free Raji cell suspensions were prepared with cell density adjusted to 3X10⁵Mu.l/ml were inoculated into a 96-well U-bottom cell culture plate and incubated for 30min with 30. mu.l of IMM01, IMM40H, IMM4002, IMM01+ IMM40H mixtures (IMM01 and IMM40H, each starting at 25nM), and hIgG1-Fc, each starting at 25nM, and 4-fold gradient dilutions. To the mixture was added 20. mu.l of 13 fold diluted rabbit serum complement (Cat # CL3111, Accurate Chemical) and incubation was continued for 4 hours. Propidium Iodide (PI) (Cat # P4170, Sigma) was then added to the cell culture medium at a concentration of 5. mu.g/ml and the cells were analyzed by FACS for PI signal to calculate cell lysis.

As shown in figure 8, for Raji human lymphoblasts double positive for human CD47 and CD70, IMM4002 elicited high levels of Complement Dependent Cytotoxicity (CDC) with slightly less activity than the CD70 monospecific antibodies IMM40H, and IMM01+ IMM40H mixture.

Example 9 IMM4002 and IMM4003 show potent antitumor activity in vivo

The right dorsal side of 48 5-6 week-old SCID mice was inoculated with a 5X 10-containing vaccine near the axilla⁶A mix of 100. mu.L of medium and 100. mu.L of Matrigel of human multiple myeloma U266 cells was prepared. When the tumor volume reaches-200 mm³At this time, the mice were randomly divided into 8 groups of 6 mice each, and the divided day was defined as D0. Starting on day D0, groups of mice were injected intraperitoneally with PBS, IMM40H (0.3mg/kg), IMM40H (1.0mg/kg), IMM01(0.5mg/kg), IMM4002(1.2mg/kg), IMM4003(1.2mg/kg), and IMM01+ IMM40H (0.5mg/kg +1.0mg/kg), for 4 weeks, 1 time per week. Dosing was terminated after 4 weeks, with 3.5 weeks of continuous observation at the end of the experiment. Tumor volume and mouse body weight were measured every 3-4 days.

Tumor volume (V) was calculated as (length x width)²)/2. Relative Tumor Volume (RTV) ═ V_t/V₀In which V is_tTumor volume at day t (Dt), V₀Tumor volume at the start of treatment (D0).

Relative tumor inhibition TGI (%) -1-mean relative tumor volume/mean relative tumor volume of negative control group x 100%.

TABLE 1 antitumor Effect of IMM4002, IMM4003 and other therapeutic Experimental endpoints (D52)

As shown in FIG. 9, the mean tumor volume at day 52 (D52) in the PBS control group was 2715.13. + -. 107.27mm during the experiment³Relative Tumor Volume (RTV) mean 14.01. + -. 0.67.

The tumor volume was 61.42. + -. 27.64mm 4 weeks after IMM40H (0.3mg/kg) administration, i.e., at day 28 (D28)³And RTV is 0.30 +/-0.13. Tumor volume and relative tumor volume were significantly reduced during the periods D3-D28 (p) compared to the contemporary vehicle control group<0.05). D52, the tumor volume is 487.78 +/-233.37 mm³And the RTV is 2.43 +/-1.16. Tumor volume and relative tumor volume continued to be significantly reduced (p) within three and half weeks of withdrawal compared to the contemporary vehicle control group<0.01)。

In the IMM40H (1.0mg/kg), IMM01(0.5mg/kg), IMM4002(1.2mg/kg), IMM4003(1.2mg/kg), and IMM01+ IMM40H (0.5mg/kg +1.0mg/kg) groups, the tumor volumes were 0.00. + -. 0.00mm at D28³And RTV is 0.00 +/-0.00. Both tumor volume and relative tumor volume were significantly reduced during periods D3-D28 (p) compared to the contemporary vehicle control group<0.01). D52 shows that the tumor volume is 0.00 +/-0.00 mm³And RTV is 0.00 +/-0.00. Tumor volume and relative tumor volume continued to be significantly reduced (p) within three and half weeks of withdrawal compared to the contemporary vehicle control group<0.01)。

The data show that IMM4002 and IMM4003 show strong anti-tumor effect, and the tumor is completely eliminated at the dosage of 1.2 mg/kg.

Example 10 IMM4002 shows robust in vivo in SCID mouse Raji xenograft in situ tumor model Antitumor activity

Collecting Raji cells in logarithmic growth phase, suspending in RPMI-1640 medium, adjusting cell concentration to 2.5 × 10⁷and/mL. 16 SCID mice, 5-6 weeks old, were inoculated with 0.2mL of cell suspension by rat intravenous injection under sterile conditions. 3 days after inoculation, animals were randomized by body weight so that the difference in body weight among the groups was less than 10% of the mean, daily as D0, and intravenous administration, i.e., IMM4002(3.6mg/kg) or PBS, was started 2 times per week for 3 weeks, according to the animal body weight. The experimental period was 129 days, during which the animal body weight was measured 3 times a week and clinical signs were recorded for 1 observation per day. According to animal welfare related regulations, an individual experimental animal will be removed from the experimental group and euthanized during the experiment if the animal meets any of the following conditions: 1. the weight of the animal is reduced by more than 20 percent (BWL is more than or equal to 20 percent) compared with D0; 2. the animals have serious adverse reactions, such as blindness, paralysis, etc.

As shown in FIG. 10, the PBS control group showed a decrease in the rate of weight change of more than 20% in 1 mouse at D16 and paralysis in 1 mouse at D21, followed by a decrease in the rate of weight change of more than 20% or paralysis in 8 mice by D30, with a mortality of 100% and a median survival time of 25 days. In IMM4002(3.6mg/kg, IV) group, the rate of change in body weight of 1 mouse decreased by more than 20% at D33, 1 mouse died at D94, 1 mouse had hemiplegia at D122, and 3 animals died by the end of the D129 experiment.

Thus, IMM4002 exhibited a strong antitumor effect.

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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Sequence listing

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

<120> recombinant fusion protein targeting CD47 and CD70, and preparation and application thereof

<130> 55525 000XX

<160> 31

<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> 8

<212> PRT

<213> Artificial sequence

<220>

<223> HV-CDR1 of CD70 antibodies IMM40H and IMM40M

<400> 2

Gly Tyr Thr Phe Thr Asp Ser Ala

1 5

<210> 3

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> HV-CDR2 of CD70 antibodies IMM40H and IMM40M

<400> 3

Ile Ser Thr Tyr Asp Gly Asp Thr

1 5

<210> 4

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> HV-CDR3 of CD70 antibodies IMM40H and IMM40M

<400> 4

Ala Arg Arg Gly Tyr Tyr Asp Tyr Asp Trp Phe Pro Tyr

1 5 10

<210> 5

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> LV-CDR1 of CD70 antibodies IMM40H and IMM40M

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Lys Ser Val Ser Ala Ser Gly Tyr Ser Phe

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<210> 6

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> LV-CDR2 of CD70 antibodies IMM40H and IMM40M

<400> 6

Leu Ala Ser

1

<210> 7

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> HV-CDR3 of CD70 antibodies IMM40H and IMM40M

<400> 7

Gln His Ser Arg Glu Leu Pro Pro Thr

1 5

<210> 8

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain variable region of CD70 antibodies IMM40H and IMM40M

<400> 8

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

1 5 10 15

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

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Ser Thr Tyr Asp Gly Asp Thr Asp Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Tyr Tyr Asp Tyr Asp Trp Phe Pro Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 9

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> light chain variable region of CD70 antibodies IMM40H and IMM40M

<220>

<221> other features

<222> (85)..(85)

<223> Xaa can be Asn or Glu

<400> 9

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Ala Ser

20 25 30

Gly Tyr Ser Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Xaa Asp Thr Ala Asn Tyr Tyr Cys Gln His Ser Arg

85 90 95

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

100 105 110

<210> 10

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<223> 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> 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> 590

<212> PRT

<213> Artificial sequence

<220>

<223> Long chain of IMM4002 and IMM4002M (SIRP. alpha. D1-linker-CD 70 antibody IMM40H heavy chain)

<400> 12

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 Lys Lys Pro Gly Ala Ser Val Lys Val

145 150 155 160

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

165 170 175

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

180 185 190

Thr Tyr Asp Gly Asp Thr Asp Tyr Asn Gln Lys Phe Lys Gly Arg Val

195 200 205

Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr Met Glu Leu Ser

210 215 220

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

225 230 235 240

Tyr Tyr Asp Tyr Asp Trp Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val

245 250 255

Thr Val Ser Ala 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> 13

<211> 1830

<212> DNA

<213> Artificial sequence

<220>

<223> Long chain of IMM4002 and IMM4002M (SIRP. alpha. D1-linker-CD 70 antibody IMM40H heavy chain)

<400> 13

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 aagaagcctg gcgcaagcgt gaaggtgagc 540

tgcaaggtga gcggctacac cttcaccgac agcgccctgc actgggtgag acaagcccct 600

ggcaagggcc tggagtggat gggcgtgatc agcacctacg acggcgacac cgactacaat 660

cagaagttca agggcagagt gaccatgacc gaggacacaa gcaccgacac cgcctacatg 720

gagctgagca gcctgagaag cgaggacacc gccgtgtact actgcgctag aagaggctac 780

tacgactacg actggttccc ttactggggc caaggcaccc tggtgaccgt gagcgccgct 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> 14

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> short chain of IMM4002/IMM4002M (light chain of CD70 antibody IMM40H/IMM 40M)

<220>

<221> other features

<222> (85)..(85)

<223> Xaa can be Asn or Glu

<400> 14

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Ala Ser

20 25 30

Gly Tyr Ser Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Xaa Asp Thr Ala Asn Tyr Tyr Cys Gln His Ser Arg

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 15

<211> 853

<212> DNA

<213> Artificial sequence

<220>

<223> short chain of IMM4002/IMM4002M (light chain of CD70 antibody IMM40H/IMM 40M)

<220>

<221> other features

<222> (310)..(310)

<223> n is a or g

<220>

<221> other features

<222> (312)..(312)

<223> n is c or a

<400> 15

atgggatggt catgtatcat cctttttctg gtagcaactg caactggagt acattcagac 60

atcgtgctga cacagagccc tgcaagcctg gccgtgagcc ctggacagag agccaccatc 120

acctgcagag caagcaagag cgtgagcgca agcggctaca gcttcctgca ctggtatcag 180

cagaagcctg gacagcctcc taagctgctg atctacctgg caagcaacct ggagagcggc 240

gtgcctgcta gattcagcgg cagcggcagc ggcaccgact tcaccctgac catcaaccct 300

gtggaggccn angacaccgc caactactac tgtcagcaca gcagagagct gcctcctacc 360

ttcggcggcg gcaccaaggt ggagatcaag cgtgagttct agaggatcca tctgggataa 420

gcatgctgtt ttctgtctgt ccctaacatg ccctgtgatt atccgcaaac aacacaccca 480

agggcagaac tttgttactt aaacaccatc ctgtttgctt ctttcctcag gaactgtggc 540

tgcaccatct gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc 600

tgttgtgtgc ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga 660

taacgccctc caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag 720

cacctacagc ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt 780

ctacgcctgc gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag 840

gggagagtgt tag 853

<210> 16

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<223> Long chain of IMM4003 (heavy chain of CD70 antibody IMM 40M)

<400> 16

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

1 5 10 15

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

20 25 30

Ala Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Ser Thr Tyr Asp Gly Asp Thr Asp Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Tyr Tyr Asp Tyr Asp Trp Phe Pro Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala 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> 17

<211> 1410

<212> DNA

<213> Artificial sequence

<220>

<223> Long chain of IMM4003 (heavy chain of CD70 antibody IMM 40M)

<400> 17

atgggatggt catgtatcat cctttttctg gtagcaactg caactggagt acattcacaa 60

gtgcagctgg tgcagagcgg cgccgaggtg aagaagcctg gcgcaagcgt gaaggtgagc 120

tgcaaggtga gcggctacac cttcaccgac agcgccctgc actgggtgag acaagcccct 180

ggcaagggcc tggagtggat gggcgtgatc agcacctacg acggcgacac cgactacaat 240

cagaagttca agggcagagt gaccatgacc gaggacacaa gcaccgacac cgcctacatg 300

gagctgagca gcctgagaag cgaggacacc gccgtgtact actgcgctag aagaggctac 360

tacgactacg actggttccc ttactggggc caaggcaccc tggtgaccgt gagcgccgct 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> 18

<211> 358

<212> PRT

<213> Artificial sequence

<220>

<223> short chain of IMM4003 (SIRP. alpha. D1-linker-CD 70 antibody IMM40H light chain)

<400> 18

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 Leu

130 135 140

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

145 150 155 160

Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Ala Ser Gly Tyr Ser Phe

165 170 175

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

180 185 190

Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly

195 200 205

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Pro Val Glu Ala

210 215 220

Asn Asp Thr Ala Asn Tyr Tyr Cys Gln His Ser Arg Glu Leu Pro Pro

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Phe Asn Arg Gly Glu Cys

355

<210> 19

<211> 1273

<212> DNA

<213> Artificial sequence

<220>

<223> short chain of IMM4003 (SIRP. alpha. D1-linker-CD 70 antibody IMM40H light chain)

<400> 19

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

atcgtgctga cacagagccc tgcaagcctg gccgtgagcc ctggacagag agccaccatc 540

acctgcagag caagcaagag cgtgagcgca agcggctaca gcttcctgca ctggtatcag 600

cagaagcctg gacagcctcc taagctgctg atctacctgg caagcaacct ggagagcggc 660

gtgcctgcta gattcagcgg cagcggcagc ggcaccgact tcaccctgac catcaaccct 720

gtggaggcca acgacaccgc caactactac tgtcagcaca gcagagagct gcctcctacc 780

ttcggcggcg gcaccaaggt ggagatcaag cgtgagttct agaggatcca tctgggataa 840

gcatgctgtt ttctgtctgt ccctaacatg ccctgtgatt atccgcaaac aacacaccca 900

agggcagaac tttgttactt aaacaccatc ctgtttgctt ctttcctcag gaactgtggc 960

tgcaccatct gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc 1020

tgttgtgtgc ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga 1080

taacgccctc caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag 1140

cacctacagc ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt 1200

ctacgcctgc gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag 1260

gggagagtgt tag 1273

<210> 20

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 20

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

1 5 10 15

<210> 21

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 21

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 22

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> joint

<400> 22

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> 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> nucleic acid sequence of 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

<210> 28

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<223> Cusatuzumab heavy chain

<400> 28

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Val Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asp Ile Asn Asn Glu Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ala Gly Tyr Ser Asn His Val Pro Ile Phe Asp Ser Trp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

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

435 440 445

Ser Pro Gly Lys

450

<210> 29

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> Cusatuzumab light chain

<400> 29

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

1 5 10 15

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

20 25 30

Asn Phe Pro Thr Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Leu

35 40 45

Leu Ile Tyr Asn Thr Asn Thr Arg His Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Asp Asp Glu Ala Glu Tyr Phe Cys Ala Leu Phe Ile Ser Asn

85 90 95

Pro Ser Val Glu Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly Gln

100 105 110

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

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

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

165 170 175

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

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 30

<211> 406

<212> PRT

<213> Artificial sequence

<220>

<223> CD27(ECD)-Fc

<400> 30

Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr Trp Ala Gln

1 5 10 15

Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe Leu Val Lys

20 25 30

Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro Cys Ile Pro

35 40 45

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

50 55 60

Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys Thr Ile Thr

65 70 75 80

Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys Arg Asp Lys

85 90 95

Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu Thr Ala Arg

100 105 110

Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His Leu Pro Tyr

115 120 125

Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met Gln Thr Leu

130 135 140

Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr His Trp Pro

145 150 155 160

Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Glu Phe Glu Pro

165 170 175

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

180 185 190

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

325 330 335

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

340 345 350

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

355 360 365

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

370 375 380

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

385 390 395 400

Ser Leu Ser Pro Gly Lys

405

<210> 31

<211> 397

<212> PRT

<213> Artificial sequence

<220>

<223> CD27-CAR

<400> 31

Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr Trp Ala Gln

1 5 10 15

Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe Leu Val Lys

20 25 30

Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro Cys Ile Pro

35 40 45

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

50 55 60

Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys Thr Ile Thr

65 70 75 80

Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys Arg Asp Lys

85 90 95

Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu Thr Ala Arg

100 105 110

Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His Leu Pro Tyr

115 120 125

Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met Gln Thr Leu

130 135 140

Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr His Trp Pro

145 150 155 160

Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Thr Thr Thr Pro

165 170 175

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

180 185 190

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

195 200 205

Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu Val Val Val

210 215 220

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

225 230 235 240

Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr

245 250 255

Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln

260 265 270

Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys

275 280 285

Phe Ser Arg Ser Ala Glu Pro Pro Ala Tyr Gln Gln Gly Gln Asn Gln

290 295 300

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

305 310 315 320

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

325 330 335

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

340 345 350

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

355 360 365

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

370 375 380

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

385 390 395

Claims (15)

1. A recombinant fusion protein comprising a CD70 antibody or antibody fragment thereof, and a CD47 binding peptide,

the CD70 antibody or antibody fragment thereof comprises a heavy chain variable region comprising amino acid sequences set forth in SEQ ID NOs: 2. 3 and 4, HV-CDR1, HV-CDR2, and HV-CDR3, the light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 5. 6 and 7, a heavy chain constant region having FcR binding ability and 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 CD70 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, CD70, and FcR.

2. The recombinant fusion protein according to claim 1, wherein each paratope of the CD70 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 CD70 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 CD70 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：20)。

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 sequence set forth in i) SEQ ID NOs:8 and 9(X ═ N); or ii) SEQ ID NOs:8 and 9(X ═ E).

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:12, a CD47 binding peptide-linker-CD 70 antibody heavy chain variable region-heavy chain constant region, and a polypeptide having the amino acid sequence set forth in SEQ ID NO: a CD70 antibody light chain variable region-light chain constant region of the amino acid sequence set forth in claim 14(X ═ N);

ii) has the sequence of SEQ ID NO:12, a CD47 binding peptide-linker-CD 70 antibody heavy chain variable region-heavy chain constant region, and a polypeptide having the amino acid sequence set forth in SEQ ID NO: a CD70 antibody light chain variable region-light chain constant region of the amino acid sequence set forth in 14(X ═ E); or

iii) has the sequence of SEQ ID NO:16, and a heavy chain variable region-heavy chain constant region of the CD70 antibody having the amino acid sequence set forth in SEQ ID NO:18, a CD47 binding peptide-linker-CD 70 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 CD70 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, renal cell carcinoma, nasopharyngeal cancer, Waldenstrom's macroglobulinemia, thymus cancer, melanoma, glioblastoma, and osteosarcoma.

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