CN118255894A

CN118255894A - Bispecific binding molecule and application thereof

Info

Publication number: CN118255894A
Application number: CN202311814093.7A
Authority: CN
Inventors: 张轶博; 芦迪; 霍永庭; 欧颖烨; 丁姣姣
Original assignee: Guangdong Fapon Biopharma Inc
Current assignee: Guangdong Fapon Biopharma Inc
Priority date: 2022-12-27
Filing date: 2023-12-26
Publication date: 2024-06-28
Also published as: WO2024140702A1

Abstract

The present disclosure proposes a bispecific binding molecule comprising: a first antigen binding region comprising a VHH having PD-L1 molecule binding activity, and a second antigen binding region having CD47 molecule binding activity. The bispecific binding molecule has good binding activity with the CD47 and the PD-L1 which are highly expressed on the surface of tumor cells, can effectively block the binding between the CD47 and the PD-L1 on the surface of the cells and corresponding receptors thereof, can effectively relieve immunosuppression, and is used for treating or preventing tumors.

Description

Bispecific binding molecule and application thereof

The present application requests the priority and rights of chinese patent application No. 202211689526.6, entitled "a bispecific binding molecule and its use", filed by the national intellectual property office at 12 and 27 of 2022, and is incorporated herein by reference in its entirety.

Technical Field

The present disclosure relates to the field of biological medicine, in particular, the disclosure relates to a bispecific binding molecule and uses thereof.

Background

Tumor immune monitoring is an important process limiting tumor growth, and macrophages, T cells and natural killer cells (NK) play an important role in the recognition and clearance of tumor cells. Tumor cells escape recognition and clearance of the immune system by modulating the expression of some immune checkpoints.

CD47 is an immune checkpoint of innate immunity and is widely regarded as a "do not eat me" signal that helps to maintain the immune tolerance of non-malignant cells under physiological conditions, but such molecules can help cancer cells of different cancer types survive. Cancer cells utilize the "don't eat me" function of CD47, expressing higher levels of CD47 on the surface than non-malignant cells, initiating inhibitory signaling pathways for binding of CD47 to signal regulatory protein alpha (sirpa), resulting in malignant cells escaping phagocytosis by macrophages. Numerous studies have shown that CD47 is overexpressed in different types of tumors, including myeloma, leiomyosarcoma, acute lymphoblastic leukemia, non-hodgkin's lymphoma, breast cancer, osteosarcoma, head and neck squamous cell carcinoma.

PD-L1 is an immune checkpoint of adaptive immunity, and the immune system normally responds to foreign antigens accumulated in the lymph nodes or spleen, triggering antigen-specific cytotoxic T cells (CD8+T cell proliferation). And the apoptosis receptor-1 (PD-1) is combined with the apoptosis-ligand 1 (PD-L1) to transmit an inhibitory signal and reduce proliferation of lymph node CD8+ T cells, and the PD-1 can control accumulation of antigen-specific T cells in lymph nodes by regulating Bcl-2 genes.

PD-L1 and CD47 both exhibit high expression in tumor cells and can be synchronously regulated by MYC. MYC expressed by tumor cells can regulate tumor microenvironment by acting on congenital and acquired immune cells and cytokines, and activating MYC can up-regulate the expression of CD47 and PD-L1 (MYC can directly act on promoters of CD47 and PD-L1 so as to regulate the expression level of mRNA and protein thereof), thereby leading to immunosuppression and tumor proliferation.

CD47 acts as a natural immune checkpoint, binds to signal-regulating protein α (sirpa) on the surface of macrophages in the tumor microenvironment, and signals "don't eat me" to evade immune surveillance. Thus, CD47 is an extremely attractive target for use in combination with PD-1/PD-L1. However, in 2017, arch Oncology terminated phase I/II clinical trials of anti-CD 47 monoclonal antibody Ti-061; in 2018, celgene terminated the clinical trial of anti-CD 47 monoclonal antibody CC-90002 for treating AmL. In view of the broad expression of CD47, potential problems with using anti-CD 47 antibodies as anti-cancer therapies include possible non-targeted effects, such as anemia. CD47 is also expressed in non-malignant cells of the hematopoietic system, hu47F9-G4, including normal erythrocytes, senescent erythrocytes and platelets Forty Seven, alone or in combination with other antibodies may lead to accidental death of normal erythrocytes, possibly leading to anemia.

The PD-1/PD-L1 antibody has the problems of limited single drug response rate (10% -35%), gradual drug resistance or recurrence in continuous treatment and the like, and the addition of the CD47 not only supplements and upgrades the PD-L1 inhibitor, but also takes congenital and adaptive anti-tumor immune response into consideration, so that the antibody is expected to become a new generation of broad-spectrum anti-tumor therapy.

Although many of the current research enterprises have developed antibodies with weak or little hematological toxicity, how to design CD47 targeted drugs with weak hematological toxicity and good therapeutic effects is still the focus of research and development.

Disclosure of Invention

The present disclosure has been completed based on the following findings by the inventors: the research shows that both PD-L1 and CD47 show high expression in tumor cells, CD47 and PD-L1 increase in tumor microenvironment are related to congenital and acquired immunosuppression of organisms, and tumor proliferation is caused, so the inventor develops PD-L1 and CD47 antibodies, designs various bispecific binding molecules by utilizing the monoclonal antibodies, obtains good binding activity with PD-L1 and CD47 through in-vivo and in-vitro junction detection experiments on the constructed bispecific binding molecules, can effectively block the binding between cell surface PD-L1 and CD47 and corresponding receptors thereof, and promotes T cells to secrete cytokines, and can effectively treat or prevent tumors.

Thus, in a first aspect of the disclosure, the disclosure proposes a bispecific binding molecule. According to an embodiment of the disclosure, the bispecific binding molecule comprises a first antigen binding region having a VHH with PD-L1 molecule binding activity having a complementarity determining region in SEQ ID No. 4; and a second antigen binding region, said second antigen binding region having CD47 molecule binding activity. The bispecific binding molecule according to the embodiment of the disclosure has good binding activity with PD-L1 and CD47, can effectively block the binding between PD-L1 and CD47 on the surface of tumor cells and corresponding receptors thereof, but has no obvious influence on the functions of normal cells expressing CD47 proteins, such as erythrocytes, platelets and the like, so that the bispecific binding molecule can effectively prevent or treat tumors, and has higher safety.

In a second aspect of the disclosure, the disclosure provides a multispecific binding molecule. According to an embodiment of the present disclosure, the multispecific binding molecule comprises a bispecific binding molecule as described previously. The multispecific binding molecules according to the embodiments of the present disclosure have good binding activity with PD-L1 and CD47, and can effectively block the binding between PD-L1 and CD47 on the surface of tumor cells and their corresponding receptors, but have no significant effect on the functions of normal cells expressing CD47 proteins, such as erythrocytes, platelets, etc., and the fusion protein can effectively prevent or treat diseases, and has high safety.

In a third aspect of the disclosure, the disclosure provides an isolated nucleic acid molecule. According to an embodiment of the disclosure, the isolated nucleic acid molecule encodes the bispecific binding molecule of the first aspect or the multispecific binding molecule of the second aspect. The bispecific binding molecules or multispecific binding molecules encoded by the isolated nucleic acid molecules according to the embodiments of the present disclosure have good binding activity with PD-L1 and CD47, can effectively block the binding between PD-L1 and CD47 on the surface of tumor cells and their corresponding receptors, but have no significant effect on the function of normal cells expressing CD47 proteins, such as erythrocytes, platelets, etc., and the bispecific binding molecules can effectively prevent or treat tumors with higher safety.

In a fourth aspect of the disclosure, the disclosure provides an expression vector. According to embodiments of the present disclosure, the expression vector contains the isolated nucleic acid molecules described previously. After the expression vector according to the embodiment of the disclosure is introduced into a suitable receptor cell, the expression of the bispecific binding molecule or the multispecific binding molecule can be effectively realized under the mediation of a regulatory system, so that a large amount of the bispecific binding molecule or the multispecific binding molecule can be obtained.

In a fifth aspect of the disclosure, the disclosure provides a recombinant cell. According to embodiments of the present disclosure, the recombinant cells contain the isolated nucleic acid molecules, expression vectors, or express the bispecific binding molecules or multispecific binding molecules described previously. Recombinant cells according to embodiments of the present disclosure may be used for in vitro expression and in large numbers of the previously described bispecific or multispecific binding molecules capable of binding CD47 and PD-L1 proteins.

In a sixth aspect of the disclosure, the disclosure provides a composition. According to embodiments of the present disclosure, the composition comprises a bispecific binding molecule, a multispecific binding molecule, an isolated nucleic acid molecule, an expression vector, or a recombinant cell as previously described. As described above, the isolated nucleic acid molecule, expression vector or recombinant cell according to the embodiments of the present disclosure can effectively bind to CD47 and PD-L1 proteins, block the binding between PD-L1 and CD47 and its corresponding receptor on the surface of tumor cells, but have no significant effect on the function of normal cells expressing CD47 protein, such as erythrocytes and platelets, and can effectively prevent or treat tumors, and further, the composition comprising the same has the effect of blocking the binding between PD-L1 and CD47 and its corresponding receptor, but does not significantly affect the function of erythrocytes and platelets, and can also effectively prevent or treat tumors.

In a seventh aspect of the disclosure, the disclosure provides the use of a bispecific binding molecule, a multispecific binding molecule, an isolated nucleic acid molecule, an expression vector, a recombinant cell or a composition as described hereinbefore in the manufacture of a medicament. As described above, the isolated nucleic acid molecule, expression vector or recombinant cell can effectively bind to CD47 and PD-L1 proteins, block the binding between PD-L1 and CD47 and its corresponding receptor on the surface of tumor cells, but has no significant effect on the function of normal cells expressing CD47 protein, such as erythrocytes, platelets, etc., and can effectively prevent or treat related diseases (such as tumors), and further, the above bispecific binding molecule or a medicament prepared from a series of substances capable of expressing the bispecific binding molecule or multispecific binding molecule under appropriate conditions can also effectively prevent or treat tumors, with higher safety.

In an eighth aspect of the disclosure, the disclosure provides a medicament. According to an embodiment of the present disclosure, the medicament comprises: the bispecific binding molecules, multispecific binding molecules, isolated nucleic acid molecules, expression vectors, recombinant cells, or compositions described previously. As described above, the bispecific binding molecule or multispecific binding molecule according to some embodiments of the present disclosure, or the bispecific binding molecule or multispecific binding molecule obtained by expressing the nucleic acid molecule, expression vector or recombinant cell under appropriate conditions, and the active ingredient in the composition can effectively bind to CD47 and PD-L1 protein, block the binding between PD-L1 and CD47 on the surface of tumor cells and its corresponding receptor, but have no significant effect on the function of normal cells expressing CD47 protein, such as erythrocytes, platelets, etc., and the bispecific binding molecule can effectively prevent or treat tumors that highly express the CD47 and/or PD-L1, so that the medicament comprising the above-mentioned series of substances also has the effect of effectively preventing or treating tumors that highly express the CD47 and/or PD-L1, and has higher safety.

In a ninth aspect of the disclosure, the disclosure provides the use of a bispecific binding molecule, a multispecific binding molecule, an isolated nucleic acid molecule, an expression vector or a recombinant cell as described previously in the preparation of a kit. According to embodiments of the present disclosure, the kit is used for detecting CD47 and/or PD-L1. As described above, the bispecific binding molecule or multispecific binding molecule can effectively bind to CD47 and/or PD-L1, and thus, the bispecific binding molecule or multispecific binding molecule according to the embodiments of the present disclosure, and a kit prepared by expressing a substance that obtains the bispecific binding molecule or multispecific binding molecule under appropriate conditions can effectively detect whether CD47 and/or PD-L1 is contained in a sample to be tested, and quantitatively analyze the same, wherein the sample to be tested comprises a sample for scientific research, and after detection by the kit, a scientific researcher can obtain a sample that meets expectations. The kit may also diagnose a disease or assess disease prognosis.

In a tenth aspect of the disclosure, the disclosure provides a kit. According to embodiments of the present disclosure, the kit comprises a bispecific binding molecule, a multispecific binding molecule, an isolated nucleic acid molecule, an expression vector, or a recombinant cell as previously described. The bispecific binding molecule or multispecific binding molecule provided according to the embodiments of the present disclosure, or the bispecific binding molecule or multispecific binding molecule obtained by expressing the nucleic acid molecule, expression vector or recombinant cell under suitable conditions can be effectively bound to CD47 and/or PD-L1, so that the kit can be used in scientific research, the kit prepared according to the bispecific binding molecule of the embodiments of the present disclosure can be used for diagnostic or non-diagnostic purposes, for example, in scientific research, can be effectively used for detecting whether the sample to be tested contains CD47 and/or PD-L1, and quantitatively analyzing it to obtain a sample meeting the requirements for subsequent scientific research; in addition, it can be used to determine the status of an individual, such as whether the level of CD47 and/or PD-L1 is above or below normal, after obtaining the level of CD47 and/or PD-L1 in a different tissue of the individual.

In an eleventh aspect of the disclosure, the disclosure provides the use of a bispecific binding molecule, a multispecific binding molecule, an isolated nucleic acid molecule, an expression vector, a recombinant cell, a composition, or a medicament as described previously in the treatment or prevention of a disease. As described above, the bispecific binding molecules have high binding activity to CD47 and/or PD-L1, and can be used for effectively treating or preventing related diseases.

In a twelfth aspect of the disclosure, the disclosure provides the use of a bispecific binding molecule, a multispecific binding molecule, an isolated nucleic acid molecule, an expression vector, a recombinant cell, or a kit as described previously in diagnosing a disease or assessing a prognosis of a disease. As previously described, the bispecific binding molecule or multispecific binding molecule is effective to bind CD47 and/or PD-L1, and thus the kit can be used to detect the level of CD47 and/or PD-L1 in a human or animal, and illustratively, CD47 and/or PD-L1 each exhibit high expression in tumor cells, and the level is altered, so that the disease can be diagnosed or prognostic evaluated by detecting, monitoring, or monitoring the level of CD47 and/or PD-L1.

In a thirteenth aspect of the present disclosure, the present disclosure provides a method of diagnosing a disease. According to an embodiment of the present disclosure, the method comprises: detecting CD47 and/or PD-L1 in a sample to be tested using at least one of the bispecific binding molecules, multispecific binding molecules, isolated nucleic acid molecules, expression vectors, recombinant cells, compositions, and kits described previously; and determining the content of CD47 and/or PD-L1 in the sample to be detected based on the detection result. The increase of CD47 and/or PD-L1 in tumor microenvironment is related to immune escape, the increase of CD47 and/or PD-L1 is effective immunosuppressant, the increase of CD47 and/or PD-L1 expression is usually related to malignant tumors of a plurality of cancers, then the immunosuppression function is dominant to promote tumor proliferation, and the bispecific binding molecule or multispecific binding molecule, or the isolated nucleic acid molecule, expression vector, bispecific binding molecule or multispecific binding molecule expressed by recombinant cells, or the bispecific binding molecule or multispecific binding molecule contained in the composition, kit can be effectively bound with CD47 and/or PD-L1, so the method can be used for effectively detecting the content of CD47 and/or PD-L1 in a sample to be tested from an individual to be tested, and can be used for effectively diagnosing the diseases.

In a fourteenth aspect of the present disclosure, the present disclosure proposes a method of assessing disease prognosis, according to an embodiment of the present disclosure, the method comprising: detecting CD47 and/or PD-L1 in a sample to be tested using at least one of the bispecific binding molecules, multispecific binding molecules, isolated nucleic acid molecules, expression vectors, recombinant cells, compositions, and kits described previously; and determining the content of CD47 and/or PD-L1 in the sample to be detected based on the detection result. As previously mentioned, CD47 and/or PD-L1 increase in tumor microenvironment is associated with immune evasion, CD47 and/or PD-L1 is a potent immunosuppressant, and an increase in CD47 and/or PD-L1 expression is usually associated with a number of malignant tumors of cancer, and then its immunosuppressive function is dominant, contributing to tumor proliferation, whereas the bispecific binding molecules, multispecific binding molecules, or isolated nucleic acid molecules, expression vectors, bispecific binding molecules or multispecific binding molecules expressed by recombinant cells, or bispecific binding molecules contained in a composition, kit, can be effectively bound to CD47 and/or PD-L1, and the method according to the application can be used to effectively detect CD47 and/or PD-L1 content in a test sample derived from a subject, and to evaluate the prognosis of said disease based on said CD47 and/or PD-L1 content.

It is to be understood that within the scope of the present disclosure, the above-described technical features of the present disclosure and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows a block diagram of a bispecific binding molecule in an embodiment of the present disclosure, wherein VH1 represents the heavy chain variable region of a targeted CD47 antibody, VL1 represents the light chain variable region of a targeted CD47 antibody, and VHH represents the VHH sequence of a FPX016N nanobody targeted to PD-L1;

FIG. 2 shows graphs of the results of detection of binding activity of bispecific binding molecules to Raji-PDL1 cells in examples of the present disclosure;

FIG. 3 shows graphs of binding activity assays of bispecific binding molecules to Jurkat cells in examples of the disclosure;

FIG. 4 shows graphs of the results of detection of blocking effect of bispecific binding molecules on cell surface PDL1 binding to PD1 proteins in examples of the present disclosure;

FIG. 5 shows a graph of the detection of the blocking effect of bispecific binding molecules on the binding of cell surface CD47 antigen to SIRPalpha in an embodiment of the present disclosure;

FIG. 6 shows graphs of binding activity assays for bispecific binding molecules to erythrocytes in examples of the disclosure (lower graph is an enlarged partial view of the upper graph);

FIG. 7 shows graphs of binding activity assays for bispecific binding molecules to platelets in examples of the disclosure;

FIG. 8 shows a graph of the results of detection of the effect of bispecific binding molecules on hemagglutination activity in the examples of the present disclosure;

FIG. 9 shows graphs of the results of detection of phagocytosis of tumor cells by macrophages induced by bispecific binding molecules in an embodiment of the present disclosure, wherein the abscissa represents each bispecific binding molecule, control sample, and the ordinate (Phagocytic index) represents the phagocytosis index;

FIG. 10 shows graphs of the results of the effect of bispecific binding molecules on T cell secretion of cytokines IL2, IFNγ in the examples of the present disclosure (left and right graphs are test results for different donors);

FIG. 11 shows a graph of the results of bispecific binding molecules in the examples of the present disclosure on tumor volume and mouse body weight effects, wherein:

Panel A shows a graph of the effect on Tumor volume following administration of bispecific binding molecules in the examples of the present disclosure, wherein the abscissa (Days post engraftment) represents time of administration and the ordinate (Tumor volume) represents Tumor volume;

Panel B shows a graph of the results of the effects of bispecific binding molecules on mouse Body weight following administration in examples of the present disclosure, wherein the abscissa (Days post engraftment) represents time of administration and the ordinate (Body weight) represents mouse Body weight;

figure 12 shows a graph of the results of bispecific binding molecules in the examples of the present disclosure on tumor volume and mouse body weight effects, wherein:

FIG. 13 shows graphs of the results of detection of binding activity of bispecific binding molecules to Raji-PDL1 cells in examples of the present disclosure;

FIG. 14 shows graphs of the results of assays of binding activity of bispecific binding molecules to Jurkat cells in examples of the disclosure.

Detailed Description

Embodiments of the present disclosure, examples of which are illustrated in the accompanying drawings, are described in detail below. The embodiments described below by referring to the drawings are exemplary and intended for the purpose of explaining the present disclosure and are not to be construed as limiting the present disclosure.

Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present disclosure, the meaning of "a plurality" is at least two, such as two, three, etc., unless explicitly specified otherwise.

The terms "comprising," "having," "including," or "containing" are used herein in an open-ended fashion, i.e., including what is indicated in the present disclosure, but not excluding other aspects.

In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

For easier understanding of the present disclosure, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein within this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure belongs. The abbreviations for amino acid residues are standard 3 letter and/or 1 letter codes used in the art to refer to one of the 20 commonly used amino acids.

Bispecific binding molecules or antigen binding fragments described in the present disclosure are generally prepared by biosynthetic methods. The coding nucleic acids of the present disclosure may be conveniently prepared according to the nucleotide sequences described in the present disclosure, or by various known methods by those skilled in the art. Such as, but not limited to: PCR, DNA synthesis, etc., and specific methods can be found in J.Sam Brookfield, guidelines for molecular cloning experiments.

Herein, "full length antibody" refers to a natural traditional antibody or mutant thereof; natural conventional antibodies have a tetrapeptide chain structure formed by joining two identical light chains and two identical heavy chains via inter-chain disulfide bonds, and are generally "Y" type structures, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE). A mutant of a conventional antibody refers to a mutation based on the conventional antibody such that two heavy chains or two light chains are not identical, but the overall structure is still in a "Y" structure, such as KIH mutation in the constant region of the heavy chain, etc.

In this context, the term "antigen-binding fragment" as used without being otherwise specified generally refers to antigen-binding antibody fragments, typically antigen-binding or variable regions, and is exemplary, e.g., including CDR-grafted antibodies, fab ', F (ab') 2, fv or scFv, nanobodies, or the like.

Herein, the term "CDR-grafted antibody" refers to the grafting of CDRs of a species mab to antibody variable regions of another species. For example, the CDRs of a murine mab may be grafted to the variable regions of a human antibody in order to replace the CDRs of a human antibody, allowing the human antibody to acquire the antigen binding specificity of the murine mab while reducing its heterology.

In this context, the term "Fab antibody" or "Fab" generally refers to an antibody comprising only Fab molecules, consisting of VH and CH1 of the heavy chain and the complete light chain, linked by a disulfide bond between the light and heavy chains.

As used herein, the term "Fab '" fragment or "F (ab')" fragment both comprise the VH and CH1 of a heavy chain, the complete light chain, and the hinge region, with a disulfide bond between the light and heavy chains.

As used herein, the term "F (ab ') 2 antibody" or "F (ab ') 2" has two antigen binding F (ab ') moieties linked together by disulfide bonds.

As used herein, the term "Fv antibody" generally refers to an antibody fragment consisting of only the light chain variable region (VL) and the heavy chain variable region (VH) joined by a non-covalent bond.

As used herein, the term "single chain antibody" or "scFv" is an antibody fragment in which the variable regions of the heavy and light chains of the antibody are linked by a short peptide.

The term "nanobody" generally refers to the heavy chain variable region (VHH) portion of a heavy chain antibody that naturally lacks a light chain, which is typically found in camelids, comprising a heavy chain variable region (VHH) and conventional CH2 and CH3 regions, which specifically bind to an antigen through the heavy chain variable region (VHH), which alone may exert an antigen-specific binding effect.

"CDR" or "CDR sequence" as used herein refers to the amino acid sequence responsible for antigen binding in an antibody, also known as the complementarity determining region. For example, it can be defined by Kabat, IMGT, chothia, contact or AbM, etc. commonly used in the art (Kaas,Q et al.IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig super family C-like domains.Dev.Comp.Immunol.29,185-203,(2005);R.M.MacCallum et al.,.Antibody–antigen interactions:contact analysis and binding site topography J.Mol.Biol.(1996);Martin,A.C.R.Protein sequence and structure analysis of antibody variabledomains(Book chapter).In Antibody engineering lab manual Eds.Duebel,S.and Kontermann,R.(2001);Marie-Paule Lefranc et al.IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,Developmental and Comparative Immunology 27(2003)55-77).

As used herein, the term "LCDR" refers to the light chain complementarity determining region and "HCDR" refers to the heavy chain complementarity determining region. The heavy chain variable region (VH) comprises 3 CDR regions: HCDR1, HCDR2 and HCDR3; the light chain variable region (VL) comprises 3 CDR regions: LCDR1, LCDR2, and LCDR3.

The term antibody "variable region" or "variable domain" refers to the domain of an antibody that is involved in binding an antigen by an antibody in the heavy or light chain. Herein, the antibody heavy chain variable region (VH (heavy chain variable region of a conventional antibody) or VHH (heavy chain variable region of a single domain antibody)) and the light chain variable region (VL) each comprise four conserved Framework Regions (FR) and three Complementarity Determining Regions (CDRs). The term "complementarity determining region" or "CDR" refers to a region within the variable region that primarily contributes to specific binding to an antigen; "framework" or "FR" refers to the variable domain residues in the variable region other than the CDR residues. VH comprises 3 CDR regions: HCDR1, HCDR2 and HCDR3; VL comprises 3 CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL is composed of 3 CDRs and 4 FRs arranged from amino-terminus (also called N-terminus) to carboxy-terminus (also called C-terminus) in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

In this context, the term "mutant" may refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.

As used herein, the term "nucleotide" refers to ribonucleotides, deoxynucleotides or modified forms of either type of nucleotide, as well as combinations thereof.

As used herein, the term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector may be introduced.

In this context, the term "transformed" or "transfected" refers to the introduction of a nucleic acid (e.g., vector) into a cell by various techniques known in the art.

In this context, the term "identity" is used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences when compared to the amino acid sequence or nucleic acid sequence of a reference sequence, using conventional methods, e.g., see, ausubel et al, eds. (1995), current Protocols in Molecular Biology, chapter 19 (Greene Publishing and Wiley-Interscience, new York); and ALIGN program (Dayhoff(1978),Atlas of Protein Sequence and Structure 5：Suppl.3(National Biomedical Research Foundation,Washington,D.C.). there are many algorithms for aligning sequences and determining sequence identity, including, needleman et al (1970) J.mol. Biol.48:443 homology comparison algorithm; smith et al (1981) adv.appl.Math.2:482, a local homology algorithm; pearson et al (1988) Proc.Natl. Acad.Sci.85:2444 similarity search method; computer programs utilizing the Smith-Waterman algorithm (Meth. Mol. Biol.70:173-187 (1997)), and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al (1990) J. Mol. Biol. 215:403-410)), are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al, meth. Enzyme, 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al, supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, 8 th edition, madison, wisconsin, USA, and CLUSTAL in the PC/Gene programs provided by Intelligenetics, mountain View, california.

One skilled in the art can replace, add and/or delete one or more (e.g., 1,2, 3,4, 5,6, 7, 8, 9, or 10 or more) amino acids to the sequences of the present disclosure to obtain variants of the sequences of the antibodies or functional fragments thereof without substantially affecting the activity of the antibodies (e.g., retaining at least 95% of the activity). They are all considered to be included within the scope of the present disclosure. Such as substitution of amino acids with similar properties in the variable region. Sequence identity as described in the present disclosure may be measured using sequence analysis software. Such as computer programs BLAST, in particular BLASTP or TBLASTN, using default parameters. The amino acid sequences referred to in this disclosure are all shown in N-terminal to C-terminal fashion.

As used herein, the term "at least 90% identical" refers to a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identical to its corresponding reference sequence, including amino acid sequences, nucleic acid sequences, fusion sequences of DNA and RNA, or fusion sequences of amino acids and nucleic acids.

In the present application, the "antibody igg1.8" is a mutant of the wild-type IgG1 antibody having L234A, L a and K447A mutations compared to the wild-type IgG1 antibody, wherein the amino acid numbering of the wild-type IgG1 antibody is according to the EU numbering system, e.g., the "L234A" means that the leucine numbered at position 234 is replaced with alanine according to the EU numbering system.

Bispecific binding molecules and multispecific binding molecules

In some embodiments, the present disclosure proposes a bispecific binding molecule comprising a first antigen binding region having a VHH of PD-L1 molecule binding activity, the VHH having a complementarity determining region in SEQ ID NO 4; the complementarity determining regions are defined according to Kabat, IMGT, chothia, contact or AbM definitions; and a second antigen binding region, said second antigen binding region having CD47 molecule binding activity. The bispecific binding molecule according to the embodiment of the disclosure has good binding activity with PD-L1 and CD47, can effectively block the binding between PD-L1 and CD47 on the surface of tumor cells and corresponding receptors thereof, but has weaker binding to normal cells expressing CD47 protein, such as erythrocytes, platelets and the like, and has good safety, and can effectively prevent or treat tumors.

The complementarity determining regions of SEQ ID NO. 4 may be obtained according to different definitions, and the specific CDR sequences are shown in the following Table:

According to some embodiments of the present disclosure, the bispecific binding molecules described above may further comprise at least one of the following additional technical features:

according to some embodiments of the disclosure, the VHH has the complementarity determining regions set forth in SEQ ID NOS 1-3.

According to some embodiments of the present disclosure, the VHH has a CDR1 as shown in SEQ ID NO. 1, a CDR2 as shown in SEQ ID NO. 2, and a CDR3 as shown in SEQ ID NO. 3.

According to some embodiments of the present disclosure, the amino acid sequence of the VHH is as shown in SEQ ID NO. 4, or has at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identity to SEQ ID NO. 4 and has the complementarity determining regions shown in SEQ ID NO. 1-3.

According to some embodiments of the disclosure, the second antigen binding region comprises a full length antibody or antigen binding fragment thereof that is anti-CD 47.

According to some embodiments of the present disclosure, the anti-CD 47 full length antibody has LCDR1, LCDR2, and LCDR3 in SEQ ID No. 5, and HCDR1, HCDR2, and HCDR3 in SEQ ID No. 6;

The LCDR1, the LCDR2, the LCDR3, the HCDR1, the HCDR2 and the HCDR3 are defined according to Kabat, IMGT, chothia, contact or AbM definition, and specific sequences are as follows:

According to some embodiments of the present disclosure, the anti-CD 47 full length antibody has LCDR1 of SEQ ID NO. 40, LCDR2 of amino acid sequence WA and LCDR3 of SEQ ID NO. 43, and has HCDR1 of SEQ ID NO. 49, HCDR2 of SEQ ID NO. 54 and HCDR3 of SEQ ID NO. 57; the CDR sequences are defined according to IMGT definition.

According to some embodiments of the present disclosure, the anti-CD 47 full length antibody comprises a light chain variable region as set forth in SEQ ID No. 5 or a light chain variable region having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID No. 5, and a heavy chain variable region as set forth in SEQ ID No. 6 or a heavy chain variable region having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID No. 6.

According to some embodiments of the disclosure, the anti-CD 47 antigen binding fragment comprises at least one of a Fab antibody, a Fab 'antibody, a F (ab') 2 antibody, a Fv antibody, a single chain antibody, and a nanobody.

According to some embodiments of the disclosure, the second antigen binding region comprises a full length antibody to CD 47.

According to some embodiments of the present disclosure, the light chain N-terminus or the light chain C-terminus of the anti-CD 47 full length antibody is linked to one or more of the first antigen binding regions, and/or the heavy chain C-terminus of the anti-CD 47 full length antibody is linked to one or more of the first antigen binding regions.

According to some embodiments of the disclosure, the N-terminus of both light chains of the anti-CD 47 full length antibody is linked to one of the first antigen binding regions.

According to some embodiments of the disclosure, the light chain N-terminus of the full length anti-CD 47 antibody is directly or indirectly linked to the C-terminus of the first antigen binding region.

According to some embodiments of the disclosure, the C-terminal ends of the two heavy chains of the anti-CD 47 full-length antibody are each linked to one of the first antigen-binding regions.

According to some embodiments of the disclosure, the heavy chain C-terminus of the full length anti-CD 47 antibody is directly or indirectly linked to the N-terminus of the first antigen binding region.

According to some embodiments of the disclosure, the C-terminus of both light chains of the anti-CD 47 full length antibody is linked to one of the first antigen binding regions.

According to some embodiments of the disclosure, the light chain C-terminus of the full length anti-CD 47 antibody is directly or indirectly linked to the N-terminus of the first antigen binding region.

According to some embodiments of the disclosure, the C-terminus of both heavy chains of the anti-CD 47 full length antibody is further linked to one of the first antigen binding regions each. According to some embodiments of the disclosure, the N-terminus of the first antigen binding region is directly or indirectly linked to the C-terminus of the first antigen binding region.

In an alternative embodiment of the present disclosure, the C-terminus of each of the two light chains of the anti-CD 47 full length antibody is linked to one of the first antigen binding regions, and the C-terminus of each of the two heavy chains of the anti-CD 47 full length antibody is also linked to one of the first antigen binding regions. In an alternative embodiment of the present disclosure, the light chain C-terminus of the full length anti-CD 47 antibody is directly or indirectly linked to the N-terminus of the first antigen binding region, and/or the N-terminus of the first antigen binding region is directly or indirectly linked to the C-terminus of the first antigen binding region.

According to some embodiments of the disclosure, the C-terminal ends of the two light chains of the anti-CD 47 full-length antibody are each linked to two of the first antigen-binding regions. According to some embodiments of the disclosure, the two first antigen binding regions on the same light chain are present in tandem on the light chain. According to some embodiments of the disclosure, the light chain C-terminus of the full length anti-CD 47 antibody is directly or indirectly linked to the N-terminus of the first antigen binding region.

According to some embodiments of the disclosure, the first antigen binding region is indirectly linked to the full-length anti-CD 47 antibody via a linker peptide;

According to some embodiments of the disclosure, the amino acid sequence of the connecting peptide comprises (GGGGS) n, wherein n is an integer greater than or equal to 1. According to some embodiments of the disclosure, n=1 to 15. According to some embodiments of the disclosure, n has a value of 1,2, 3, 4, 5, 6, 7, 8, 9, or 10.

According to some embodiments of the present disclosure, the amino acid sequence of the linker peptide is shown as SEQ ID NO. 7.

According to some embodiments of the disclosure, at least a portion of the heavy chain constant region or the antibody light chain constant region of the anti-CD 47 full length antibody is derived from at least one of a murine antibody, a human antibody, a primates antibody, or a mutant thereof.

According to some embodiments of the disclosure, at least a portion of the heavy chain constant region or the antibody light chain constant region of the anti-CD 47 full length antibody is derived from a human antibody or mutant thereof.

According to some embodiments of the disclosure, at least a portion of the heavy chain constant region or the antibody light chain constant region of the anti-CD 47 full length antibody is derived from a human antibody IgG or a mutant thereof.

According to some embodiments of the disclosure, at least a portion of the heavy chain constant region or the antibody light chain constant region of the anti-CD 47 full length antibody is derived from a human antibody IgG1 or a mutant thereof.

According to some embodiments of the disclosure, the heavy chain constant region of the full length anti-CD 47 antibody has amino acid mutations at positions 234, 235 and 447 (numbered according to the EU numbering system) as compared to wild-type human IgG 1.

According to some embodiments of the disclosure, the heavy chain constant region of the full length anti-CD 47 antibody has L234A, L a and K447A mutations (numbered according to the EU numbering system) compared to wild-type human IgG 1.

According to some embodiments of the present disclosure, the light chain of the full-length anti-CD 47 antibody comprises the amino acid sequence shown in SEQ ID No. 8 or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity thereto.

According to some embodiments of the present disclosure, the heavy chain of the full length anti-CD 47 antibody comprises the amino acid sequence shown in SEQ ID No. 9 or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identity thereto.

According to some embodiments of the disclosure, the bispecific binding molecule comprises two identical first peptide chains and two identical second peptide chains: the amino acid sequences of the first peptide chain and the second peptide chain are as shown in any one of the following (1) to (5):

(1): the amino acid sequence of the first peptide chain is shown as SEQ ID NO. 9 or has at least 90% of identity with SEQ ID NO. 9; the amino acid sequence of the second peptide chain is shown as SEQ ID NO. 10 or has at least 90 percent of identity with SEQ ID NO. 10;

(2): the first peptide chain amino acid sequence is as shown in SEQ ID NO. 11 or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO. 11; the second peptide chain amino acid sequence is as shown in SEQ ID NO. 8 or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO. 8;

(3): the first peptide chain amino acid sequence is as shown in SEQ ID NO. 9 or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO. 9; the second peptide chain amino acid sequence is as shown in SEQ ID NO. 13 or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO. 13;

(4): the first peptide chain amino acid sequence is as shown in SEQ ID NO. 14 or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO. 14; the second peptide chain amino acid sequence is as shown in SEQ ID NO. 15 or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO. 15;

(5): the first peptide chain amino acid sequence is as shown in SEQ ID NO. 9 or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO. 9; the second peptide chain amino acid sequence is as shown in SEQ ID NO. 16 or has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO. 16.

In other embodiments, the present disclosure provides a multispecific binding molecule comprising a bispecific binding molecule as described above. It will be appreciated by those skilled in the art that the bispecific binding molecule may also be fused to a molecule having other binding activity, the species of which is not particularly limited, as long as both the bispecific binding molecule and the molecule having other binding activity are able to function effectively after fusion, to obtain a molecule capable of specifically binding to three or more different epitopes or different antigens.

As used herein, the term "multispecific binding molecule" refers to a specific binding molecule that can bind to three or more different epitopes or different antigens. In particular, the multispecific binding molecules include trispecific, tetraspecific, penta-specific, or hexa-specific binding molecules, and the like.

Nucleic acid molecules, expression vectors and recombinant cells

In other embodiments, the present disclosure provides an isolated nucleic acid molecule encoding a bispecific binding molecule or a multispecific binding molecule as described above. The bispecific binding molecules or multispecific binding molecules encoded by the isolated nucleic acid molecules according to the embodiments of the present disclosure have good binding activity with PD-L1 and CD47, can effectively block the binding between PD-L1 and CD47 on the surface of tumor cells and their corresponding receptors, but have no significant effect on the function of normal cells expressing CD47 proteins, such as erythrocytes, platelets, etc., and can effectively prevent or treat tumors, and have higher safety.

It is noted that for the isolated nucleic acid molecules mentioned in the present disclosure and claims, one skilled in the art will appreciate that either one or both of the complementary double strands are actually included. For convenience, in the present description and claims, although only one strand is shown in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequences of the present application include DNA forms or RNA forms, one of which is disclosed, meaning the other is also disclosed.

In still other embodiments, the present disclosure provides an expression vector comprising the isolated nucleic acid molecule described previously. In the case of the above-described isolated nucleic acid molecules being attached to a vector, the isolated nucleic acid molecules may be directly or indirectly attached to control elements on the vector, provided that these control elements are capable of controlling translation, expression, etc. of the isolated nucleic acid molecule, i.e., the isolated nucleic acid molecule is operably linked to the control elements. Of course, these control elements may be directly from the carrier itself or may be exogenous, i.e. not from the carrier itself.

"Operably linked" herein refers to the linkage of a foreign gene to a vector such that control elements within the vector, such as transcription control sequences and translation control sequences, and the like, are capable of performing their intended functions of regulating transcription and translation of the foreign gene. The usual vectors may be, for example, plasmids, phages and the like. After the expression vector according to some embodiments of the present disclosure is introduced into a suitable recipient cell, the expression of the bispecific binding molecule or the antibody or antigen binding fragment described above can be effectively achieved under the mediation of a regulatory system, thereby achieving in vitro mass-acquisition of the bispecific binding molecule or the antibody or antigen binding fragment. Of course, the nucleic acid molecules encoding the two identical peptide chains 1 and/or the two identical peptide chains 2 of the bispecific binding molecule may be inserted separately into different vectors, usually into the same vector.

According to some embodiments of the present disclosure, the above expression vector may further include at least one of the following additional technical features:

According to some embodiments of the disclosure, the expression vector is a eukaryotic expression vector, a prokaryotic expression vector, or a virus. The expression of the bispecific binding molecules described above in suitable receptor cells, such as CHO cells, e.coli, etc., is thus achieved.

According to some embodiments of the disclosure, the virus comprises a lentivirus.

In some embodiments, the disclosure provides a recombinant cell comprising an isolated nucleic acid molecule, expression vector, or expressing a bispecific binding molecule or a multispecific binding molecule, as described above. Recombinant cells according to some embodiments of the present disclosure may be used under suitable conditions for in vitro expression and in large amounts of the previously described bispecific or multispecific binding molecules capable of binding CD47 and PD-L1 proteins.

According to some embodiments of the present disclosure, the recombinant cell described above may further comprise at least one of the following additional technical features:

according to some embodiments of the disclosure, the recombinant cell is a eukaryotic cell.

According to some embodiments of the disclosure, the recombinant cell is a mammalian cell.

It should be noted that the recombinant cells described in the present disclosure are not particularly limited, and may be prokaryotic cells, eukaryotic cells, or phage. The prokaryotic cell can be escherichia coli, bacillus subtilis, streptomycete or proteus mirabilis and the like. The eukaryotic cells comprise fungi such as pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces, trichoderma and the like, insect cells such as armyworm and the like, plant cells such as tobacco and the like, and mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells and the like. In some embodiments, the recombinant cells of the present disclosure are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

The term "suitable conditions" as used herein refers to conditions suitable for expression of the bispecific binding molecule or multispecific binding molecule of the present application. Those skilled in the art will readily appreciate that conditions suitable for expression of the bispecific binding molecule or multispecific binding molecule include, but are not limited to, suitable transformation or transfection means, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, suitable cell culture time. The "suitable conditions" are not particularly limited and the person skilled in the art may optimize the conditions for optimal expression of the bispecific binding molecule or multispecific binding molecules according to the specific circumstances of the laboratory.

Composition, pharmaceutical use, medicament and kit

In some embodiments, the present disclosure provides a composition comprising a bispecific binding molecule, a multispecific binding molecule, an isolated nucleic acid molecule, an expression vector, or a recombinant cell as described previously.

The compositions of the present disclosure may also be administered in combination with each other, or with one or more other therapeutic compounds, e.g., with a chemotherapeutic agent. Thus, the composition may also contain a chemotherapeutic agent. The bispecific or multispecific binding molecules of the present disclosure may also be combined with a second therapeutic agent, exemplary agents of which include, but are not limited to, other agents that inhibit PD-L1 and/or CD47 activity (including other antibodies or antigen binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.), and/or agents that interfere with PD-L1 and/or CD47 upstream or downstream signaling.

It is noted that the compositions are food compositions, pharmaceutical compositions, and the like, including combinations that are temporally and/or spatially separated, so long as they are capable of coacting to achieve the objects of the present disclosure. For example, the ingredients contained in the composition may be administered to the subject in whole or separately. When the components contained in the composition are separately administered to a subject, the individual components may be administered to the subject simultaneously or sequentially.

In some specific embodiments, the present disclosure contemplates the use of a bispecific binding molecule, a multispecific binding molecule, an isolated nucleic acid molecule, an expression vector, a recombinant cell, or a composition as described previously in the manufacture of a medicament for the prevention or treatment of a disease.

According to some embodiments of the disclosure, the disease comprises a tumor.

According to some embodiments of the disclosure, the tumor comprises at least one of: ovarian cancer, oral squamous cell carcinoma, head and neck cancer, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, renal cancer, esophageal cancer, melanoma, lymphoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma.

In some specific embodiments, the present disclosure provides a medicament comprising: the bispecific binding molecules, multispecific binding molecules, isolated nucleic acid molecules, expression vectors, recombinant cells, or compositions described previously.

In some embodiments, these medicaments or compositions further comprise a pharmaceutically acceptable carrier, including any solvents, solid excipients, diluents, binders, disintegrants or other liquid excipients, dispersing agents, flavoring or suspending agents, surfactants, isotonic agents, thickening agents, emulsifying agents, preservatives, solid binders, glidants or lubricants, and the like, suitable for the particular target dosage form. In addition to the extent to which any conventional adjuvant is incompatible with the compounds of the present disclosure, such as any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition that occur in a deleterious manner, their use is also contemplated by the present disclosure.

For example, the bispecific binding molecules or multispecific binding molecules of the present disclosure may be incorporated into pharmaceutical compositions suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These pharmaceutical compositions may be prepared in various forms. Such as liquid, semi-solid, and solid dosage forms, and the like, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. Typical pharmaceutical compositions are in the form of injection solutions or infusion solutions.

The effective amount of the bispecific binding molecules or multispecific binding molecules described in the present disclosure can vary depending on the mode of administration, the severity of the disease being treated, and the like. The selection of the preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, depending on the urgent requirements of the treatment situation.

According to some embodiments of the disclosure, the bispecific binding molecule or multispecific binding molecule may be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.

The terms "treat" and "prevent" as used herein and the words derived therefrom do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one of ordinary skill in the art would consider to have a potential benefit or therapeutic effect. In this regard, the presently disclosed methods can provide any amount of any level of treatment or prevention of a neoplasm in a mammal. Moreover, the treatment or prevention provided by the methods of the present disclosure may include the treatment or prevention of a disease being treated or prevented, such as one or more patients or symptoms of a tumor. In addition, for purposes herein, "preventing" may encompass delaying the onset of a disease or symptom thereof or a patient.

In addition, the bispecific binding molecules or multispecific binding molecules according to embodiments of the present disclosure may perform a diagnostic function, rely on the bispecific binding molecules or multispecific binding molecules capable of specifically binding CD47 and/or PD-L1 set forth in the foregoing disclosure, and be combined with diagnostic reagents, so as to perform diagnosis of sites of abnormal expression of CD47 and/or PD-L1 in an organism, such as with diagnostic nuclides, nanomaterials, etc., to achieve visual observation of cells, tissues, organs of abnormal expression of CD47 and/or PD-L1 in an organism, thereby assisting a medical worker or a scientific research worker in more accurate judgment of lesions.

Thus, the bispecific binding molecules or multispecific binding molecules herein may also be made part of a kit or other diagnostic reagent, as desired.

In some embodiments, the disclosure proposes the use of a bispecific binding molecule, a multispecific binding molecule, an isolated nucleic acid molecule, an expression vector, or a recombinant cell as described previously in the preparation of a kit for detecting CD47 and/or PD-L1.

In some embodiments, the disclosure also provides a kit comprising the bispecific binding molecule, multispecific binding molecule, isolated nucleic acid molecule, expression vector, or recombinant cell described above. The kit provided by the disclosure can be used for immunoblotting, immunoprecipitation and the like, and relates to a kit for detection by utilizing the specific binding performance of CD47 and/or PD-L1 proteins and antibodies. These kits may comprise any one or more of the following: antagonists, CD47 and/or PD-L1 antibodies or drug reference materials; a protein purification column; immunoglobulin affinity purification buffers; cell assay diluent; instructions, literature, etc. The bispecific binding molecules can be used in different types of diagnostic tests, for example, to detect the presence of a wide variety of diseases or drugs, toxins or other proteins, etc., in vitro or in vivo. For example, the test may be used to test a disease of interest by testing the serum or blood of a subject, such as: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer, which can be any cell whose growth is unregulated.

Use and method for treating disease, diagnosing disease, prognosis evaluation of disease

In some embodiments, the disclosure contemplates the use of the bispecific binding molecules, multispecific binding molecules, isolated nucleic acid molecules, expression vectors, recombinant cells, compositions, or medicaments described previously in the treatment or prevention of a disease. As described above, the bispecific binding molecule or the multispecific binding molecule has higher binding activity to CD47 and/or PD-L1, and can effectively treat or prevent the disease.

According to some embodiments of the disclosure, the disease comprises a tumor.

In some embodiments, the disease comprises a disease associated with CD47 and/or PD-L1.

In some embodiments, the disclosure provides a bispecific binding molecule, a multispecific binding molecule, an isolated nucleic acid molecule, an expression vector, a recombinant cell, or a composition of any of the foregoing for use as a medicament. In some embodiments, the medicament is for treating a disease.

According to some embodiments of the disclosure, the disease comprises a tumor.

In some embodiments, the present disclosure contemplates the use of a bispecific binding molecule, a multispecific binding molecule, an isolated nucleic acid molecule, an expression vector, a recombinant cell, or a composition as described previously in the manufacture of a medicament for treating or preventing a disease. As described above, the bispecific binding molecule or the multispecific binding molecule has higher binding activity to CD47 and/or PD-L1, and can effectively treat or prevent the disease.

According to some embodiments of the disclosure, the disease comprises a tumor.

In some embodiments, the disclosure proposes the use of the bispecific binding molecules, multispecific binding molecules, isolated nucleic acid molecules, expression vectors, recombinant cells, or kits described previously in diagnosing a disease or assessing a prognosis of a disease. As described above, the bispecific binding molecule or multispecific binding molecule, or the isolated nucleic acid molecule, expression vector or recombinant cell expressed bispecific binding molecule or multispecific binding molecule of the present application can bind to CD47 and/or PD-L1 effectively, so that the method of the present application can be used for detecting and monitoring the content of CD47 and/or PD-L1 in a test sample derived from a subject effectively, and for diagnosing or assessing the prognosis of a disease associated with CD47 and/or PD-L1 effectively.

According to some embodiments of the disclosure, the disease comprises a related disease caused by CD47 and/or PD-L1.

According to some embodiments of the disclosure, the disease comprises a tumor.

In some embodiments, the disclosure proposes the use of the bispecific binding molecules, multispecific binding molecules, isolated nucleic acid molecules, expression vectors, or recombinant cells described previously in the preparation of a kit for diagnosing a disease or assessing a prognosis of a disease. As described above, the bispecific binding molecule or multispecific binding molecule, or the isolated nucleic acid molecule, expression vector or recombinant cell expressed bispecific binding molecule or multispecific binding molecule of the present application can bind to CD47 and/or PD-L1 effectively, so that the method of the present application can be used for detecting and monitoring the content of CD47 and/or PD-L1 in a test sample derived from a subject effectively, and for diagnosing or assessing the prognosis of a disease associated with CD47 and/or PD-L1 effectively.

According to some embodiments of the disclosure, the disease comprises a tumor.

In some embodiments, the present disclosure provides a method of treating or preventing a disease comprising: administering to the subject an effective dose of at least one of the bispecific binding molecule, multispecific binding molecule, isolated nucleic acid molecule, expression vector, recombinant cell, composition, and drug.

In some embodiments, the present disclosure provides a method of treating a disease comprising: administering to a subject in need thereof a therapeutically effective dose of at least one of a bispecific binding molecule, a multispecific binding molecule, an isolated nucleic acid molecule, an expression vector, a recombinant cell, a composition, and a medicament of any of the foregoing. According to some embodiments of the disclosure, the disease comprises a tumor.

In some embodiments, the disclosure provides a method of diagnosing a disease comprising detecting PD-L1 and/or CD47 in a sample to be tested using at least one of: bispecific binding molecules, multispecific binding molecules, nucleic acid molecules, expression vectors, recombinant cells, and kits as described previously; and determining the content of the CD47 and/or the PD-L1 in the sample to be detected based on the detection result of the PD-L1 and/or the CD 47. The increase of CD47 and/or PD-L1 in tumor microenvironment is related to immune escape, the increase of CD47 and/or PD-L1 is effective immunosuppressant, the increase of CD47 and/or PD-L1 expression is usually related to malignant tumors of a plurality of cancers, then the immunosuppression function is dominant to promote tumor proliferation, and the bispecific binding molecule or multispecific binding molecule, or nucleic acid molecule, expression vector, bispecific binding molecule or multispecific binding molecule expressed by recombinant cells, or the bispecific binding molecule or multispecific binding molecule contained in the composition, kit can be effectively bound with CD47 and/or PD-L1, so that the method disclosed by the application can be used for effectively detecting the content of CD47 and/or PD-L1 in a sample to be tested, which is derived from an individual to be tested, and can be used for effectively diagnosing the diseases, such as tumors.

According to some embodiments of the present disclosure, the above method of diagnosing a disease may further include at least one of the following additional technical features:

According to some embodiments of the disclosure, the level of CD47 and/or PD-L1 in the test sample that is not below the minimum criterion for disease is an indication that the test sample is derived from a patient suffering from disease.

According to some embodiments of the disclosure, the sample to be tested comprises at least one of the following: blood, saliva, sweat, tissue, cells, blood, serum, plasma, feces, and urine.

According to some embodiments of the disclosure, the disease comprises a tumor.

In other embodiments, the disclosure proposes a prognostic method for assessing disease, comprising detecting PD-L1 and/or CD47 in a sample to be tested, according to examples of the disclosure, using at least one of the following: bispecific binding molecules, multispecific binding molecules, nucleic acid molecules, expression vectors, recombinant cells, and kits as described previously; and determining the content of the CD47 and/or the PD-L1 in the sample to be detected based on the detection result of the PD-L1 and/or the CD 47. As previously mentioned, CD47 and/or PD-L1 increase in tumor microenvironment is associated with immune evasion, CD47 and/or PD-L1 is a potent immunosuppressant, and increase in CD47 and/or PD-L1 expression is usually associated with malignancy of many cancers, and then its immunosuppressive function is dominant, contributing to tumor proliferation, whereas the bispecific binding molecules or multispecific binding molecules, or nucleic acid molecules, expression vectors, bispecific binding molecules or multispecific binding molecules expressed by recombinant cells, or bispecific binding molecules contained in compositions, kits may be effectively bound to CD47 and/or PD-L1, and the method of the application may be used to effectively detect CD47 and/or PD-L1 content in a test sample derived from a subject, and to evaluate prognosis of a disease based on the CD47 and/or PD-L1 content.

According to some embodiments of the present disclosure, the above-described prognostic method of assessing a disease may further include at least one of the following additional technical features:

According to some embodiments of the disclosure, the sample to be tested is derived from a patient suffering from the disease before or after treatment.

According to some embodiments of the present disclosure, the prognostic effect of the disease is determined based on the content of CD47 and/or PD-L1 in the test sample of a patient suffering from the disease before or after treatment.

According to some embodiments of the present disclosure, a decrease in the content of CD47 and/or PD-L1 in a test sample of a patient suffering from the disease after treatment is an indication that the patient's prognosis is good.

According to some embodiments of the disclosure, the disease comprises a tumor.

Reference in the present disclosure to "patient" or "subject" generally refers to a mammal, such as a primate and/or rodent, particularly a human or mouse.

The amino acid or nucleic acid sequences to which the present disclosure relates are detailed in table 1.

Table 1:

The present disclosure is described below with reference to specific embodiments, it being noted that these embodiments are merely illustrative and do not limit the present disclosure in any way. The specific techniques or conditions are not noted in the examples and are carried out according to the techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, huang Peitang et al, molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

EXAMPLE 1 preparation of bispecific binding molecules

The bispecific binding molecules of this example were fused in different structures with the anti-CD 47 antibody (7A 11H 14) (light chain amino acid sequence: SEQ ID NO:8, heavy chain amino acid sequence: SEQ ID NO: 9) targeting CD47 using the FPX016N nanobody (VHH, amino acid sequence: SEQ ID NO: 4) specifically binding PDL1, and the linker sequence (Gly ₄Ser)₃ (amino acid sequence: SEQ ID NO: 7). The following 5 bispecific binding molecules were designed in this example, and their specific structures are shown in FIG. 1:

1) Bispecific binding molecule 1 (R1281): comprising two identical peptide chains 1 and two identical peptide chains 2, wherein peptide chain 1 is the heavy chain of the CD47 antibody (7A 11H 14) (SEQ ID NO: 9); peptide chain 2 (SEQ ID NO. 10) consists of light chains of PD-L1 nano antibody (SEQ ID NO. 4) and CD47 antibody (7A 11H 14), and the nucleotide sequences for encoding peptide chains 1 and 2 are shown as SEQ ID NO. 17 and 18;

2) Bispecific binding molecule 2 (R1282): comprising two identical peptide chains 3 and 2 identical peptide chains 4, wherein peptide chain 3 (SEQ ID NO: 11) consists of the heavy chain of the CD47 antibody (7A 11H 14) and the PDL1 nanobody (SEQ ID NO: 4); peptide chain 4 (SEQ ID NO: 8) is the light chain of the CD47 antibody (7A 11H 14);

3) Bispecific binding molecule 3 (R1283): comprising two identical peptide chains 5 and two identical peptide chains 6, wherein the peptide chain 5 is the heavy chain (SEQ ID NO: 9) of the CD47 antibody (7A 11H 14); peptide chain 6 (SEQ ID NO: 13) consists of the light chain of the CD47 antibody (7A 11H 14) and the PD-L1 nanobody (SEQ ID NO: 4);

4) Tetravalent bispecific binding molecule 4 (R1332): comprising two identical peptide chains 7 and two identical peptide chains 8, wherein the peptide chain 7 (SEQ ID NO: 14) consists of the heavy chain of the CD47 antibody (7A 11H 14) and the PDL1 nanobody (SEQ ID NO: 4); peptide chain 8 (SEQ ID NO: 15) consisted of the light chain of the CD47 antibody (7A 11H 14) and the PDL1 nanobody (SEQ ID NO: 4), containing four PDL1 binding regions, being tetravalent bispecific binding molecules.

5) Tetravalent bispecific binding molecule 5 (R1335): comprising two identical peptide chains 9 and two identical peptide chains 10, wherein the peptide chains 9 are the heavy chains of the CD47 antibody (7 a11H 14); peptide chain 10 (SEQ ID NO: 16) consisted of two PDL1 nanobodies (SEQ ID NO: 4) serially linked to the light chain of the CD47 antibody (7A 11H 14), containing four PDL1 binding regions, being tetravalent bispecific binding molecules.

In addition, a bispecific binding control molecule expressing CD47 and PDL1 was constructed: SG12473: (having a heavy chain shown as SEQ ID NO:19, a light chain shown as SEQ ID NO: 20); and IBI322 (having heavy chains 1 and 2 shown in SEQ ID NO:21, 22, light chain shown in SEQ ID NO: 23) as a control antibody, wherein the structure of the control bispecific binding molecule antibody is shown in FIG. 1;

control molecule R1297: the fusion protein of PD-L1 nanometer antibody (SEQ ID NO: 4) and Fc contains two identical peptide chains, wherein the amino acid sequence of one peptide chain is shown as (SEQ ID NO: 12);

control molecule TJC4: anti-CD 47 mab comprising 2 identical heavy chains and 2 identical light chains, heavy chains: SEQ ID NO. 24; light chain: SEQ ID NO. 25;

Hu5f9-G4: anti-CD 47 mab comprising 2 identical heavy chains and 2 identical light chains, heavy chains: SEQ ID NO. 26; light chain: SEQ ID NO. 27;

Standard protocols for transient or stable transfection are used for both bispecific binding molecules and control antibodies, and mammalian cells are transiently transfected with the nucleotide encoding the above protein in the expression vector pcDNA3.4.

The expressed plasmid is transiently transfected into human embryo kidney HEK 293 cells, and bispecific binding molecules produced by the cells are isolated and purified, and the band molecular weight of the bispecific binding molecules is about 170kD and 203kD on SDS-PAGE under non-reducing conditions.

Example 2 determination of binding Activity of bispecific binding molecules to tumor cells (FACS)

The bispecific binding molecules constructed in example 1 of the present disclosure bind to target antigens on the corresponding cells. To construct Raji-PDL1 expressing human PDL1 (Raji cells are also called black human Burkitt lymphoma cells, which express CD 47), on the basis of which Raji cells are caused to express human PDL1 simultaneously, thereby obtaining Raji-PDL1 cells) as PDL 1-positive cells, jurkat as CD 47-positive cells, and the cell binding activity thereof was determined with the bispecific binding molecules prepared in example 1 of the present disclosure.

2.1 Detection of binding Activity of bispecific binding molecules with Raji-PDL1 cells Using flow analysis

Enough Raji-PDL1 cells were cultured and collected by centrifugation. Meanwhile, the bispecific binding molecules and the corresponding monoclonal antibody R1297 are diluted by PBS+3% FBS, and the concentration of the bispecific binding molecules is 3-fold gradient dilution from 400nmol, so that 9 concentration gradients are obtained for later use. The collected cells were washed once with PBS+3% FBS, and then the cells were resuspended to 2X 10 ⁶ cells/mL by adding PBS+3% FBS, the cells were plated in 96-well plates, 100. Mu.L per well (2X 10 ⁵ cells), 100. Mu.L of diluted antibody was added, and incubated at 4℃for 30 minutes; the supernatant was removed by centrifugation, the cells were washed twice with PBS, resuspended in diluted PE-labeled anti-human IgG FC antibody (Biolegend, 409304), incubated at 4℃for 30 min in the absence of light, washed twice with PBS, resuspended in 100. Mu.L PBS, and assayed on-press, and the binding affinity EC50 value for the antibody to Raji-PDL1 was calculated by analysis with software GraphPadprism 7.0 at average fluorescence intensity.

As a result, as shown in FIG. 2, bispecific binding molecules such as R1281, R1282, and R1283 were able to bind to Raji-PDL1, similar to R1297.

2.2 Detection of binding Activity of bispecific binding molecules to Jurkat cells Using flow analysis

Enough Jurkat cells were cultured and the cells were collected by centrifugation. Following the same experimental procedure as in the previous examples, cells were resuspended in 100 μl PBS, detected on-press, and the binding affinity EC50 values of antibodies to Jurkat were calculated by analysis with software GraphPadPrism 7.0 at average fluorescence intensity.

As a result, as shown in FIG. 3, each bispecific binding molecule binds to Jurkat cells with similar activity, and IBI322 binds to Jurkat cells much weaker than other molecules.

Example 3 determination of the Activity of bispecific binding molecules for blocking PD1/PDL1 (FACS)

In this example, blocking of PDL1 and PD1 proteins expressed on the cell surface of Raji-PDL1 by the bispecific binding molecule prepared in example 1 was examined.

Enough Raji-PDL1 cells were cultured and collected by centrifugation. Meanwhile, the bispecific binding molecules and the corresponding monoclonal antibodies are diluted by PBS+3% FBS, the concentration is from 800nmol, and the concentration is diluted in a 3-time gradient manner, so that 9 concentration gradients are obtained for standby. The collected cells were washed once with PBS+3% FBS, and then resuspended in PBS+3% FBS to 2X 10 ⁶ cells/mL, plated in 96-well plates at 100. Mu.L per well (2X 10 ⁵ cells), added with 50. Mu.L of diluted antibody, incubated at 4℃for 30min, followed by 50. Mu.L of 1.2ug/mL of PD1-mFc antigen (fusion protein of human PD1 extracellular domain with murine IgG Fc) and incubated at 4℃for 30 min; the supernatant was removed by centrifugation, the cells were washed twice with PBS, resuspended in diluted PE-labeled anti-mouse IgG FC antibody (Biolegend, 405307), incubated at 4℃for 30min in the absence of light, washed twice with PBS, resuspended in 100. Mu.L PBS, and assayed on-press, and the IC50 value for blocking binding of Raji-PDL1 to PD1 was calculated by analysis with software GraphPadprism 7.0 at average fluorescence intensity.

As a result, as shown in FIG. 4, R1281 to R1283 have PD1/PDL1 blocking effect.

EXAMPLE 4 determination of CD 47/SIRPalpha blocking Activity by bispecific binding molecules (FACS)

This example detects the blocking of binding of sirpa to CD47 antigen expressed on the surface of Jurkat cells by the bispecific binding molecule constructed in example 1.

Enough Jurkat cells were cultured and the cells were collected by centrifugation. Meanwhile, the bispecific binding molecules and the corresponding monoclonal antibodies are diluted by PBS+3% FBS, the concentration is from 800nmol, and the concentration is diluted in a 3-time gradient manner, so that 9 concentration gradients are obtained for standby. The collected cells were washed once with PBS+3% FBS, and then resuspended to 2X 10 ⁶ cells/mL with PBS+3% FBS, the cells were plated in 96-well plates at 100. Mu.L per well (2X 10 ⁵ cells), 50. Mu.L of diluted antibody was added, incubated at 4℃for 30 minutes, and then 50. Mu.L of 20ug/mL SIRPalpha-mFc antigen was added, and incubated at 4℃for 30 minutes; the supernatant was centrifuged off, the cells were washed twice with PBS, resuspended in diluted PE-labeled anti-mouse IgG FC antibody (Biolegend, 405307), incubated at 4℃for 30min in the absence of light, washed twice with PBS, resuspended in 100. Mu.L PBS, and assayed on-press, and the IC50 value for blocking binding of CD47 to SIRPalpha was calculated by analysis with software GraphPadprism 7.0 at average fluorescence intensity.

As shown in FIG. 5, R1281-R1283 have CD 47/SIRPalpha blocking effect and are better than the CD47 end blocking activity of IBI 322.

Example 5 determination of bispecific binding molecules to erythrocyte binding Activity (FACS)

This example detects the binding of the bispecific binding molecules constructed in example 1 to erythrocytes.

Human erythrocytes (surface expressed CD 47) were collected. Meanwhile, the bispecific binding molecules and the corresponding monoclonal antibodies are diluted by PBS+3% FBS, the concentration is from 200nmol, and 10 concentration gradients are obtained by 3-time gradient dilution for standby. The collected RBCs were washed once with PBS+3% FBS, and then the cells were resuspended to 2X 10 ⁶ cells/mL with PBS+3% FBS, plated in 96-well plates with 100. Mu.L per well (2X 10 ⁵ cells), added with 100. Mu.L of diluted antibody, and incubated at 4℃for 30 min; the supernatant was removed by centrifugation, the cells were washed twice with PBS, resuspended in diluted PE-labeled anti-human IgG FC antibody (Biolegend, 409304), incubated at 4℃for 30 min in the absence of light, washed twice with PBS, resuspended in 100. Mu.L PBS, and assayed on-machine, and the binding activity of the antibody to RBC was calculated by analysis with software GraphPadprism 7.0 at average fluorescence intensity.

The results are shown in FIG. 6: the red blood cell binding of the bispecific binding molecule is much lower than IBI 322. In R1281-R1283, R1281 red blood cells bound less than monoclonal antibody 7A11H14.

Example 6 determination of bispecific binding molecules to platelet binding Activity (FACS)

This example detects the binding of the bispecific binding molecules constructed in example 1 to platelets.

Human platelets (surface expressed CD 47) were collected. Meanwhile, the bispecific binding molecules and the corresponding monoclonal antibodies are diluted by PBS+3% FBS, the concentration is from 200nmol, and 10 concentration gradients are obtained by 3-time gradient dilution for standby. Collected platelets were washed once with pbs+3% fbs, resuspended in pbs+3% fbs, plated in 96-well plates at 100 μl/well, added with 100 μl diluted antibodies, and incubated at 4 ℃ for 30 min; the supernatant was removed by centrifugation, the cells were washed twice with PBS, resuspended in diluted PE-labeled anti-human IgG FC antibody (Biolegend, 409304), incubated at 4℃for 30 min in the absence of light, washed twice with PBS, resuspended in 100. Mu.L PBS, and assayed on-machine, and the binding activity of the antibody to platelets was calculated by analysis with software GraphPadprism 7.0 at average fluorescence intensity.

As a result, as shown in FIG. 7, the bispecific binding molecules showed much lower platelet binding than Hu5f9-G4, and three of R1281 to R1283 had weaker platelet binding than 7A11H14.

Example 7 bispecific binding molecule pair facilitating hemagglutination Activity assay

This example shows the effect of the bispecific binding molecule prepared in example 1 on hemagglutination activity, and is performed as follows: collecting human whole blood; placing whole blood into a 15mL centrifuge tube, supplementing PBS to 15mL, centrifuging at room temperature, 200 Xg, and discarding the supernatant for 10 mins; RBCs were made up to 15mL with PBS, mixed well, and centrifuged at 1500rpm at 5mins at room temperature. Washing for 3 times; after the last wash, the RBCs concentration was adjusted to 2% using PBS (e.g., 49mL of PBS was added to 1mL of RBCs); carrying out 2-time gradient dilution on the antibody according to 1000nmol, wherein the total concentration gradient is 12; using a 96-well round bottom plate, 50. Mu.L of antibody at the corresponding concentration was added to each well and 50. Mu.L of RBCs were incubated at room temperature for 2 hours, and the reaction results were observed and recorded.

As shown in FIG. 8, hu5F9-G4 in the control antibody caused very significant hemagglutination, and control SG12473 had hemagglutination at high concentrations. R1281 does not cause erythrocyte aggregation.

Example 8 detection of phagocytosis of tumor cells by macrophages induced by bispecific binding molecules

This example was used to detect phagocytosis of tumor cells by macrophages induced by the bispecific binding molecules constructed in example 1, and was performed as follows:

Isolation induction of macrophages (MDM): human venous blood was withdrawn to isolate PBMC, monocytes were isolated with Human CD14 MicroBeads (Miltenyi, 130-050-201), and differentiation was induced by the addition of 50ng/mL GM-CSF, after 1 week to obtain mature macrophages.

Phagocytosis experiment: taking out target cells Raji-PDL1 from an incubator, marking the target cells by CFSE (BD Bioscience, 565082), washing the marked target cells twice by using a complete culture medium, and re-suspending the target cells to 1X 10 ⁶ cells/mL by using the culture medium for later use; taking out the induced macrophages from the incubator, carefully scraping the adherent cells with a cell scraper, and re-suspending the adherent cells to 5X 10 ⁵ cells/mL with a culture medium for later use; the bispecific binding molecules and the corresponding monoclonal antibodies were diluted with medium, starting at 100nM, at 10-fold gradient, yielding 4 concentration gradients for use. Macrophages were added to 96-well flat bottom plates at 100 μl/well (Corning, 3473), labeled target cells, and antibodies were added to 96-well plates at 50 μl/well and incubated for 2h at 37 ℃. After the reaction was completed, the cells were transferred to a 96-well V-plate (Corning, 3894), centrifuged, the detection antibody APC ANTI CD b (Biolegend, 301808) was added, incubated at 4℃for 30min, and after centrifugation, the supernatant was resuspended in 100. Mu.L PBS and detected by flow cytometry. The calculation method of the phagocytic index comprises the following steps: macrophage number of phagocytic target cells/total macrophages x 100%.

The experimental results are shown in fig. 9, where the bispecific binding molecules R1281, R1282 and R1283 mediated phagocytosis significantly stronger than the mab control (7 a11H 14) and stronger than the bispecific binding molecule controls SG12473 and IBI322.

EXAMPLE 9T cell modulating Activity of bispecific binding molecules

The regulatory activity of the bispecific binding molecules constructed in example 1 on T cell immune responses was determined using mixed lymphocyte reactions.

Acquisition of human Dendritic Cells (DCs): human venous blood was withdrawn to isolate PBMC, monocytes were isolated with Human CD14 MicroBeads (Miltenyi, 130-050-201), incubated for 3 days with 50ng/mL GM-CSF and 50ng/mL IL4, changed to continue incubation for 3 days, changed to add 50ng/mL TNFα, and further incubated for 3 days. Thus obtaining DC cells.

Acquisition of human T cells: human venous blood was withdrawn to isolate PBMCs and human CD3T cells were isolated using a T cell isolation kit (Stemcell, 19051).

The collected DC cells and T cells from different people are resuspended in complete medium, inoculated in 96-well plates, and cultured in a mixed manner at 2X 10 ⁴/well and 1X 10 ⁵/well, respectively. And bispecific binding molecules diluted with complete medium and controls were added. The medium was incubated in a carbon dioxide incubator at 37℃for 3 days. After the incubation, the concentration of IL2 in the supernatant was measured using a cytokine detection kit (Invitrogen, 88-7025-88).

The results are shown in figure 10, where the bispecific binding molecule R1281 showed higher release of IL2 than R1297 for secretion of IL2 in the ability of the antibody to promote cytokine secretion by T cells. IL2 release of the remaining molecules was similar to that of the monoclonal antibody control R1297, and superior to SG12473. On ifnγ secretion, R1281 is superior to R1297, SG12473, IBI322.

EXAMPLE 10 evaluation of in vivo anti-tumor efficacy of bispecific binding molecules on human Burkitt lymphoma cells Raji-hPDL1

The present example was used to detect the in vivo activity of the CD47/PD-L1 bispecific antibody constructed in example 1 against human Burkitt lymphoma cell Raji-hPDL1 model, while setting isotype control group, mab control group and combination group.

10.1 Experimental materials

NOD SCID mice, females, for 6-8 weeks (source: beijing Vitre Liwa laboratory animal technologies Co., ltd.); raji-hPDL1 cells (Raji cells provided by Beijing co-operating units, modified in the laboratory, constructed as Raji cells expressing human PDL 1); PBMC (Miaoshun, A10K 971099), RPMI-1640 medium (Gibco), FBS (Gibco, 10091-148), 0.25% trypsin-EDTA (Gibco, 25200056), penicillin-streptomycin (Gibco, 15140122), DMSO (Sigma, D2650), DPBS (Hyclone, SH 30028.02).

10.2 Instrumentation

Electronic balance (Shanghai Shun Hengping scientific instruments Co., ltd., JA 12002), vernier caliper (Shanghai Meinaite practical Co., MNT-150T), microscope (Chongqing Orte optical instruments Co., ltd., BDS 200), medical centrifuge (Hunan Instrument laboratory developing Co., L530R), digital display constant temperature water bath (Pris mechanical Co., ltd., HH-S), carbon dioxide incubator (Japanese Song' S health medical instruments Co., ltd., MCO-18 AC), biosafety cabinet (Guangzhou Qianjiang laboratory science Co., ACZ-451), cell technology device (Shanghai Rui Yu organism, IC 1000)

10.3 Experimental methods

Injecting PBMC cells: PBMC cells were recovered as required by the PBMC cell protocol, cultured for 24h in RPMI-1640 medium containing 20% fetal bovine serum, collected, washed 1 time with pre-chilled DPBS, and cell concentrations adjusted to 1X 10 ⁷/mL with DPBS. Female NOD SCID mice were injected tail-intravenously with PBMC cells at a plating volume of 0.2 mL/mouse, i.e., 2X 10 ⁶/mouse. The day of PBMC injection was recorded as D-7.

Cell culture: human PDL1 was transformed into Raji cells to obtain human Burkitt lymphoma cells (Raji-hPDL) highly expressing human PDL1, and cultured in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (Gibco), 1% glutamine and 1% penicillin-streptomycin (1:1).

Inoculating tumor cells: raji-hPDL1 cells in the logarithmic growth phase were collected, washed twice with pre-chilled DPBS, and cell concentrations were adjusted to 5X 10 ⁷/mL with DPBS. Female NOD SCID mice were inoculated subcutaneously with Raji-hPDL cells at a volume of 0.1 mL/mouse, i.e., 5X 10 ⁶/mouse. Tumor cells were inoculated on the day and recorded as D0.

Administration: when the inoculation is marked as day 0 (D0), when the average tumor volume reaches 60-100 mm ³, the mice can be randomly grouped according to the tumor volume, the average tumor volume of each group of mice is ensured to be the same or similar, the mice are randomly divided into 7 groups according to the tumor volume on day 10 (D10) of the experiment, and the administration is started (the administration scheme is shown in table 1).

Table 1: raji-hPDL1 tumor model dosing, mode and frequency

Note that: n, number of animals; the Dosing Volume was adjusted by animal weight (10L/g); if the weight loss during the administration period exceeds 15%, the administration regimen will be adjusted. Q2D 6 represents once every 2 days for a total of 6 doses.

Recording:

D10 began measuring tumor volume and recording, after which tumor long and short diameters were measured 2 times per week with vernier calipers. The formula is as follows: tumor volumes were calculated as (1/2) ×major diameter× (minor diameter) ², and tumor growth curves are shown (fig. 11). When each mouse reached the end of the experiment (body weight loss of more than 20% or tumor volume of more than 2000mm ³ reached the end of the kernel-day), the CO ₂ asphyxiation method sacrificed the mice.

10.4 Data calculation, statistics and analysis

The comparison between the two groups can be tested with independent samples T. More than 3 groups of comparisons applied One-Way ANOVA. If the F values show significant differences, post hoc analysis between groups can be performed. Data were processed using PRISM GRAPHPAD when p <0.05 indicated a statistically significant difference. Tumor volume v=0.5a×b ², a and b are the major and minor diameters of the tumor, respectively. Tumor growth inhibition TGI (%) = [1- (Ti-T0)/(Vi-V0) ]x100, ti is the average tumor volume of the treatment group on day i, T0 is the average tumor volume of the treatment group at the start of treatment, vi is the average tumor volume of the solvent control group on day i, and V0 is the average tumor volume of the solvent control group at the start of treatment.

10.5 Experimental results

(1) As shown in fig. 11A, R1281, 1282, 1283 (TGI 92.9%,94.7%,96.0%, respectively) had significantly better antitumor effect than control antibody 7a11h14 (tgi=78.9%) and control antibody R1297 (tgi=58.4%) alone, comparable to the combination of 7a11H14 and R1297 (tgi=95.2%).

(2) As shown in FIG. 11B, each of the CD47/PDL1 bispecific antibody administration groups had no effect on the body weight of the mice.

EXAMPLE 11 evaluation of in vivo anti-tumor efficacy of bispecific binding molecules on human melanoma cell A375

This example was used to observe the in vivo activity of the CD47/PD-L1 bispecific antibody constructed in example 1 against the human a375 melanoma model, while isotype control, mab control, and combination were set.

11.1 Experimental materials

NOD SCID mice, females, for 6-8 weeks (source: beijing Vitre Liwa laboratory animal technologies Co., ltd.); a375 cells (Shanghai cell bank, SCSP-533); PBMC (Miaoshun, A10K 971099), RPMI-1640 medium (Gibco), FBS (Gibco, 10091-148), 0.25% trypsin-EDTA (Gibco, 25200056), penicillin-streptomycin (Gibco, 15140122), DMSO (Sigma, D2650), DPBS (Hyclone, SH 30028.02)

11.2 Instrumentation

11.3 Experimental methods

Cell culture: human melanoma cells A375 were cultured in DMEM high-sugar medium (Gibco) containing 10% fetal bovine serum (Gibco), 1% glutamine and 1% penicillin-streptomycin (1:1).

Inoculating tumor cells: a375 cells in the logarithmic growth phase were collected, washed twice with pre-chilled DPBS, and cell concentrations were adjusted to 5X10 ⁷/mL with DPBS. Female NOD SCID mice were subcutaneously inoculated with Raji-hPDL1 cells at a volume of 0.1 mL/mouse, i.e., 5X10 ⁶/mouse. Tumor cells were inoculated on the day and recorded as D0.

Administration: when the inoculation is marked as day 0 (D0), when the average tumor volume reaches 60-100 mm ³, the mice can be randomly grouped according to the tumor volume, the average tumor volume of each group of mice is ensured to be the same or similar, the mice are randomly divided into 3 groups according to the tumor volume on day 10 (D10) of the experiment, and 7 mice in each group are started to be dosed (the dosing scheme is shown in table 2).

Table 2: a375 tumor model dosing, mode and frequency

Note that: n, number of animals; the Dosing Volume was adjusted by body weight (10. Mu.L/g) and Q2D 8 represents one dose every 2 days for a total of 8 doses.

Recording:

D10 began measuring tumor volume and recording, after which tumor long and short diameters were measured 2 times per week with vernier calipers. The formula is as follows: tumor volumes were calculated as (1/2) ×major diameter× (minor diameter) ², and tumor growth curves are shown (fig. 12). When each mouse reached the end of the experiment (body weight loss of more than 20% or tumor volume of more than 2000mm ³ reached the end of the kernel-day), the mice were sacrificed by CO2 asphyxiation.

11.4 Data calculation, statistics and analysis

The comparison analysis was performed between the two groups using independent sample T test, and more than 3 groups of comparisons used One-Way ANOVA. If the F values show significant differences, post hoc analysis between groups can be performed. Data were processed using PRISM GRAPHPAD when p <0.05 indicated a statistically significant difference. Tumor volume v=0.5a×b ², a and b are the major and minor diameters of the tumor, respectively. Tumor growth inhibition TGI (%) = [1- (Ti-T0)/(Vi-V0) ]x100, ti is the average tumor volume of the treatment group on day i, T0 is the average tumor volume of the treatment group at the start of treatment, vi is the average tumor volume of the solvent control group on day i, and V0 is the average tumor volume of the solvent control group at the start of treatment.

11.5 Experimental results

(1) As shown in FIG. 12A, the antitumor effect of R1281 (TGI 28.3%) was significantly better than SG12473 (TGI 6.6%)

(2) As shown in fig. 12B, each dosing group had no effect on the body weight of the mice.

EXAMPLE 12 determination of binding Activity of tetravalent bispecific binding molecules to tumor cells (FACS)

The tetravalent bispecific binding molecules constructed in example 1 of the present disclosure were tested for binding to target antigens on the corresponding cells.

Raji-PDL1 expressing human PDL1 was constructed as PDL 1-positive cells, jurkat as CD 47-positive cells, and its cell binding activity was determined with the bispecific binding molecules prepared in example 1 of the present disclosure.

12.1 Detection of binding Activity of bispecific binding molecules with Raji-PDL1 cells Using flow analysis

The results are shown in FIG. 13, where R1332 and R1335 tetravalent bispecific binding molecules have binding activity to tumor cells.

12.2 Detection of binding Activity of bispecific binding molecules to Jurkat cells Using flow analysis

Enough Jurkat cells were cultured and the cells were collected by centrifugation. The following experimental procedure was identical to that described above, cells were resuspended in 100 μl PBS, detected on-press, and the binding affinity EC50 values for antibodies to Jurkat were calculated by analysis with software GraphPadPrism 7.0 at average fluorescence intensity.

The results are shown in FIG. 14, where tetravalent bispecific binding molecules R1332 and R1335 have binding activity to Jurkat cells.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

Although embodiments of the present disclosure have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the present disclosure, and that variations, modifications, alternatives, and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the present disclosure.

Claims

1. A bispecific binding molecule comprising:

A first antigen binding region comprising a VHH having PD-L1 molecule binding activity, said VHH having a complementarity determining region of SEQ ID No. 4; the complementarity determining regions are defined according to Kabat, IMGT, chothia, contact or AbM definitions; and

A second antigen binding region, said second antigen binding region having CD47 molecule binding activity.

2. The bispecific binding molecule of claim 1, wherein the VHH has the complementarity determining regions set forth in SEQ ID NOs 1 to 3;

Alternatively, the amino acid sequence of the VHH is as shown in SEQ ID NO. 4 or has at least 90% identity with SEQ ID NO. 4.

3. The bispecific binding molecule of claim 1 or 2, wherein the second antigen binding region comprises a full length antibody or antigen binding fragment thereof against CD 47;

Optionally, the full length anti-CD 47 antibody has LCDR1, LCDR2, and LCDR3 of SEQ ID NO. 5, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO. 6;

The LCDR1, the LCDR2, the LCDR3, the HCDR1, the HCDR2, and the HCDR3 are defined according to Kabat, IMGT, chothia, contact or AbM definitions;

Optionally, the anti-CD 47 full length antibody has LCDR1 shown in SEQ ID NO. 40, LCDR2 with amino acid sequence WA and LCDR3 shown in SEQ ID NO. 43, and HCDR1 shown in SEQ ID NO. 49, HCDR2 shown in SEQ ID NO. 54 and HCDR3 shown in SEQ ID NO. 57;

Optionally, the full length anti-CD 47 antibody comprises a light chain variable region shown in SEQ ID No. 5 or having at least 90% identity to SEQ ID No. 5, and a heavy chain variable region shown in SEQ ID No. 6 or having at least 90% identity to SEQ ID No. 6;

Optionally, the antigen binding fragment of anti-CD 47 comprises at least one of a Fab antibody, a Fab 'antibody, a F (ab') ₂ antibody, an Fv antibody, a single chain antibody, and a nanobody.

4. A bispecific binding molecule according to any one of claims 1 to 3, the second antigen-binding region comprising a full-length antibody to CD 47;

Optionally, the light chain N-terminus or light chain C-terminus of the anti-CD 47 full length antibody is linked to one or more of the first antigen binding regions, and/or the heavy chain C-terminus of the anti-CD 47 full length antibody is linked to one or more of the first antigen binding regions;

Optionally, the second antigen binding region comprises a full length antibody to CD47, wherein,

A) The N-terminal of each of the two light chains of the anti-CD 47 full-length antibody is connected with one first antigen binding region; alternatively, the light chain N-terminus of the full-length anti-CD 47 antibody is directly or indirectly linked to the C-terminus of the first antigen binding region;

B) The C-terminal ends of the two heavy chains of the anti-CD 47 full-length antibody are each linked to one of the first antigen-binding regions; alternatively, the heavy chain C-terminus of the full-length anti-CD 47 antibody is directly or indirectly linked to the N-terminus of the first antigen binding region; and/or

C) The C-terminal ends of the two light chains of the anti-CD 47 full-length antibody are each linked to one of the first antigen-binding regions; alternatively, the light chain C-terminus of the full-length anti-CD 47 antibody is directly or indirectly linked to the N-terminus of the first antigen binding region;

Optionally, the C-terminal ends of the two light chains of the full-length anti-CD 47 antibody are each linked to two of the first antigen-binding regions; optionally, two first antigen binding regions on the same light chain are present in tandem on the light chain; alternatively, the light chain C-terminus of the full-length anti-CD 47 antibody is directly or indirectly linked to the N-terminus of the first antigen binding region;

Optionally, the first antigen binding region is indirectly linked to the full-length anti-CD 47 antibody via a linker peptide; optionally, the amino acid sequence of the connecting peptide comprises (GGGGS) n, wherein n is an integer greater than or equal to 1; alternatively, n=1 to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; alternatively, the amino acid sequence of the connecting peptide is shown as SEQ ID NO. 7.

5. The bispecific binding molecule of claim 4, wherein at least a portion of the heavy chain constant region or the light chain constant region of the full length anti-CD 47 antibody is derived from at least one of a murine antibody, a human antibody, and a primates antibody or a mutant thereof;

Optionally, at least a portion of the heavy or light chain constant region of the full length anti-CD 47 antibody is derived from a human antibody or mutant thereof;

optionally, at least a portion of the heavy or light chain constant region of the full length anti-CD 47 antibody is derived from a human antibody IgG or a mutant thereof;

optionally, at least a portion of the heavy or light chain constant region of the full length anti-CD 47 antibody is derived from a human antibody IgG1 or a mutant thereof;

optionally, the heavy chain constant region of the full length anti-CD 47 antibody has amino acid mutations at positions 234, 235 and 447 according to the EU numbering system as compared to wild-type human IgG 1;

Optionally, the heavy chain constant region of the full length anti-CD 47 antibody has L234A, L a and K447A mutations compared to wild-type human IgG 1;

Optionally, the light chain of the full length anti-CD 47 antibody comprises or has at least 90% identity to the amino acid sequence shown in SEQ ID No. 8; and/or the heavy chain of said full length anti-CD 47 antibody comprises or has at least 90% identity to the amino acid sequence shown in SEQ ID No. 9;

Optionally, the bispecific binding molecule comprises two identical first peptide chains and two identical second peptide chains: the amino acid sequences of the first peptide chain and the second peptide chain are as shown in any one of the following (1) to (5):

(2): the amino acid sequence of the first peptide chain is shown as SEQ ID NO. 11 or has at least 90% of identity with SEQ ID NO. 11; the amino acid sequence of the second peptide chain is shown as SEQ ID NO. 8 or has at least 90 percent of identity with SEQ ID NO. 8;

(3): the amino acid sequence of the first peptide chain is shown as SEQ ID NO. 9 or has at least 90% of identity with SEQ ID NO. 9; the amino acid sequence of the second peptide chain is shown as SEQ ID NO. 13 or has at least 90 percent of identity with SEQ ID NO. 13;

(4): the amino acid sequence of the first peptide chain is shown as SEQ ID NO. 14 or has at least 90 percent of identity with SEQ ID NO. 14; the amino acid sequence of the second peptide chain is shown as SEQ ID NO. 15 or has at least 90 percent of identity with SEQ ID NO. 15; and

(5): The amino acid sequence of the first peptide chain is shown as SEQ ID NO. 9 or has at least 90% of identity with SEQ ID NO. 9; the second peptide chain has an amino acid sequence as shown in SEQ ID NO. 16 or has at least 90% identity with SEQ ID NO. 16.

6. A multispecific binding molecule comprising a bispecific binding molecule according to any one of claims 1 to 5.

7. An isolated nucleic acid molecule encoding the bispecific binding molecule of any one of claims 1-5 or the multispecific binding molecule of claim 6; optionally, the nucleic acid molecule has a sequence as shown in SEQ ID NO. 17 or having at least 90% identity to SEQ ID NO. 17 and/or a sequence as shown in SEQ ID NO. 18 or having at least 90% identity to SEQ ID NO. 18.

8. An expression vector comprising the isolated nucleic acid molecule of claim 7.

9. A recombinant cell comprising the isolated nucleic acid molecule of claim 7 or the expression vector of claim 8; or alternatively

Expressing the bispecific binding molecule of any one of claims 1 to 5 or the multispecific binding molecule of claim 6.

10. A composition comprising the bispecific binding molecule of any one of claims 1-5, the multispecific binding molecule of claim 6, the isolated nucleic acid molecule of claim 7, the expression vector of claim 8, or the recombinant cell of claim 9; optionally, the composition further comprises one or more pharmaceutically acceptable carriers, diluents or excipients.

11. A medicament, comprising: the bispecific binding molecule of any one of claims 1 to 5, the multispecific binding molecule of claim 6, the isolated nucleic acid molecule of claim 7, the expression vector of claim 8, the recombinant cell of claim 9, or the composition of claim 10.

12. A kit, comprising: the bispecific binding molecule of any one of claims 1 to 5, the multispecific binding molecule of claim 6, the isolated nucleic acid molecule of claim 7, the expression vector of claim 8, the recombinant cell of claim 9, or the composition of claim 10.

13. Use of a bispecific binding molecule according to any one of claims 1 to 5, a multispecific binding molecule according to claim 6, an isolated nucleic acid molecule according to claim 7, an expression vector according to claim 8, a recombinant cell according to claim 9 or a composition according to claim 10 for the preparation of a medicament for the prevention or treatment of a disease or a kit for diagnosing a disease or assessing prognosis of a disease;

optionally, the disease comprises a tumor;

Optionally, the tumor comprises at least one of: ovarian cancer, oral squamous cell carcinoma, head and neck cancer, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, renal cancer, esophageal cancer, melanoma, lymphoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma.