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CN118546255A - Difunctional fusion protein targeting alpha v beta 3 and CD47 and application thereof - Google Patents

Difunctional fusion protein targeting alpha v beta 3 and CD47 and application thereof Download PDF

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CN118546255A
CN118546255A CN202310167358.2A CN202310167358A CN118546255A CN 118546255 A CN118546255 A CN 118546255A CN 202310167358 A CN202310167358 A CN 202310167358A CN 118546255 A CN118546255 A CN 118546255A
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王文义
尹晴晴
李元念
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Hengyi Biomedicine Shanghai Co ltd
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Abstract

The present invention provides a novel fusion protein which is a bifunctional fusion protein targeting both αvβ3 and CD47, said fusion protein comprising the following parts: (a) an anti- αvβ3 antibody or antibody fragment; and (b) a domain1 fragment of sirpa linked to the N-terminus of the heavy and/or light chain of an anti- αvβ3 antibody or antibody fragment.

Description

Difunctional fusion protein targeting alpha v beta 3 and CD47 and application thereof
Technical Field
The invention belongs to the technical field of fusion proteins, and particularly relates to a difunctional fusion protein for simultaneously targeting alpha v beta 3 and CD47 and application thereof.
Background
Integrins are a class of transmembrane heterodimeric glycoproteins formed by non-covalent binding of two polypeptide chains, α and β, which are important members of the cell adhesion molecule family. It has now been found that 18 different alpha subunits and 8 beta subunits can be combined into at least 24 integrins. Integrins mediate cell-to-cell and cell-to-extracellular matrix adhesion primarily, while mediating intracellular and extracellular signal transduction. The integrin and its ligand combination can induce extracellular signal transmission, so that the cell can make specific reaction to make the physiological function of integrin be implemented; while extracellular signaling can modulate the ability of integrins to bind to their ligands and regulate the expression of cell surface integrins. Integrin αvβ3 consists of the αv subunit (CD 51, 150000) and the β3 subunit (CD 61, 105000), wherein the extracellular domain of the α chain specifically recognizes polypeptides containing the RGD sequence, mediating the adhesion of integrins to the extracellular matrix. Integrin αvβ3 is not expressed or expressed in normal tissue organs and mature vascular endothelial cells, but is highly expressed on the cell surface of various tumors (including lung cancer, glioblastoma, breast cancer and osteosarcoma) and in neovascular endothelial cells, and plays an important role in the processes of angiogenesis, invasion and metastasis of tumors, so that it is a target for treating cancers. Integrin αvβ3 domains are numerous and the activation process is very complex, with the key domains for activation being the β -propeller domain, βi domain and thigh domain. In addition, many tumor-associated targeted drugs have been developed for αvβ3, such as tumor imaging agents, targeted delivery therapeutic agents, and direct antitumor agents. Recent studies have found that integrin avβ3 may be a candidate for a more selective antigen for mesenchymal tumor cells because it is less expressed in normal adult tissues and integrin avβ3 is enriched on epithelial tumors as they become more invasive, advanced and resistant (e.g., transformed into mesenchymal cells by epithelial-mesenchymal transformation, i.e., EMT processes).
CD47, on the other hand, is also known as integrin-associated protein (integrin associated protein, IAP). CD47 is 5 transmembrane protein with molecular weight of about 50kDa, belonging to immunoglobulin superfamily. The extracellular N-terminal is Igv domain, which is connected with alpha v beta 3 (CD 51/CD 61) and alpha IIb beta 3 (CD 41/CD 61) integrins. CD47 is involved in a variety of physiological functions, such as cell transfer, T-cell and dendritic cell (DENDRITIC CELL, DC) activation, axon development, and the like.
CD47 is expressed on all cell types including erythrocytes, while CD47 is highly expressed on tumor cells. There are two ligands for CD47, signaling regulatory protein α (Signal Regulatory Protein α, sirpa) and thrombin-sensitive protein-1 (Thrombospondin-1, tsp 1), respectively. Sirpa is a receptor type transmembrane glycoprotein containing immunoglobulin structural domain, belongs to SIRP family, and is mainly expressed on macrophages and nerve cells. In the CD 47-sirpa pathway, CD47 protein binds sirpa to phosphorylate its immunoreceptor tyrosine inhibitory motif ITIM, followed by intracellular recruitment of SHP-1 protein, producing a cascade of reactions that inhibits phagocytosis by macrophages. Whereas normal erythrocytes are not phagocytosed by the inhibitory signal generated by the binding of CD47 on the cell membrane surface to sipra of macrophages. TSP1 is a homotrimer consisting of 3 peptide chains that interacts with other cell surface receptors, matrix components, and growth factors to participate in processes such as cell proliferation, apoptosis, adhesion, migration, angiogenesis, and the like.
Studies have shown that CD47 is highly expressed in a variety of tumors such as leukemia and lymphoma. By highly expressing CD47, cancer cells can camouflage as self cells to release anti-phagocytic signals, inhibit macrophage-mediated phagocytosis, and generate immune escape, thereby promoting the progression and diffusion of tumors, and being related to poor prognosis of tumor patients. Furthermore, expression of CD47 correlated positively with expression of PD-1, treg marker Foxp3, MDSC marker CD11b and CD 33. The anti-CD 47 treatment can promote macrophage in the tumor mouse model to polarize to M1 subtype, reduce the expression of PD-1 in effector T cells of the tumor mouse model, increase the secretion of IFN-gamma, reduce the number of immunosuppressive cells Tregs and MDSCs, improve tumor microenvironment and delay the growth of tumor.
Disclosure of Invention
Currently, in the study of the anti-tumor mechanism of action of antibodies targeting αvβ3, it has been found that it kills αvβ3 expressing tumor cells mainly by inducing ADCC function of macrophages (PCT/US 2021/028775), which was also confirmed in vitro studies, while in vitro studies found that the antibodies alone failed to elicit ADCP function of macrophages on tumor cells, presumably related to CD47 expressed on the surface of tumor cells.
In order to further utilize macrophages to kill cancer cells, the inventor newly designs a fusion protein of an antibody targeting the alpha v beta 3 and SIRP alpha, so that the antibody fusion protein can be aggregated in tumors through the targeting of the alpha v beta 3 to avoid the systemic toxicity of targeting CD47, and on the other hand, the macrophages can play the functions of ADCC and ADCP at the same time, thereby further improving the killing of the tumors.
Namely, the invention designs a novel recombinant bifunctional fusion protein which can simultaneously target the alpha v beta 3 and the CD47, can simultaneously play the roles of macrophage-mediated antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), and has better anti-tumor effect. For example, the invention encompasses the following aspects:
a fusion protein comprising the following portions:
(a) An anti- αvβ3 antibody or antibody fragment; and
(B) The domain 1 fragment of sirpa,
The domain1 fragment of sirpa is linked to the N-terminus of the heavy and/or light chain of an anti- αvβ3 antibody or antibody fragment.
2, The fusion protein of claim 1, wherein the domain 1 of sirpa has the sequence shown in SEQ ID No. 8 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence.
The fusion protein of claim 1 or 2, wherein the anti- αvβ3 antibody or antibody fragment is a humanized anti- αvβ3 antibody or antibody fragment.
The fusion protein according to any one of claims 1 to 3, wherein the anti- αvβ3 antibody or antibody fragment is an lgG4 subtype antibody or an antibody fragment derived from an lgG4 subtype antibody.
The fusion protein of any one of claims 1-4, wherein the heavy chain variable region of the anti- αvβ3 antibody or antibody fragment comprises a sequence as set forth in SEQ ID No. 9 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the light chain variable region of the anti- αvβ3 antibody or antibody fragment comprises a sequence as set forth in SEQ ID No. 10 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.
The fusion protein of any one of claims 1-5, wherein the heavy chain of the anti- αvβ3 antibody or antibody fragment comprises the sequence set forth in SEQ ID No. 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the light chain of the anti- αvβ3 antibody or antibody fragment comprises the sequence set forth in SEQ ID No. 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.
The fusion protein of any one of claims 1-6, wherein the domain1 fragment of sirpa is linked to the N-terminus of the heavy and/or light chain of an anti- αvβ3 antibody or antibody fragment by a linker.
The fusion protein according to any one of claims 1 to 7, wherein the linker is (G4S) 3 linker.
The fusion protein according to any one of claim 1 to 8, which comprises 2 first peptide chains and 2 second peptide chains, wherein,
The first peptide chain comprises, in order from the N-terminus: a domain1 fragment of sirpa, an optional linker, a heavy chain variable region, a heavy chain constant region, and an Fc region of an anti- αvβ3 antibody or antibody fragment;
the second peptide chain comprises: the light chain variable region and the light chain constant region of an anti- αvβ3 antibody or antibody fragment.
The fusion protein according to any one of claim 1 to 9, which comprises 2 first peptide chains and 2 second peptide chains, wherein,
The first peptide chain comprises, in order from the N-terminus: a heavy chain variable region, a heavy chain constant region, and an Fc region of an anti- αvβ3 antibody or antibody fragment;
The second peptide chain comprises: domain1 of sirpa, an optional linker, a light chain variable region of an anti- αvβ3 antibody or antibody fragment, and a light chain constant region.
The fusion protein of any one of claims 1-10, wherein the first peptide chain comprises the sequence set forth in SEQ ID No. 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence and the second peptide chain comprises the sequence set forth in SEQ ID No. 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence; or the first peptide chain comprises the sequence as set forth in SEQ ID NO. 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence and the second peptide chain comprises the sequence as set forth in SEQ ID NO. 6 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence.
An isolated nucleic acid molecule encoding a fusion protein as described.
The nucleic acid molecule of claim 12 which is a vector.
14 The nucleic acid molecule of item 12 or 13 comprising (a) and (b) below; or (c) and (d):
(e) A sequence as set forth in SEQ ID NO. 11 or at least 90% thereto,
91％、92％、93％、94％、95％、96％、97％、98％、99％、
A nucleic acid sequence of 100% identity;
(f) The sequence shown in SEQ ID NO. 14 or at least 90% of the sequence,
91％、92％、93％、94％、95％、96％、97％、98％、99％、
A nucleic acid sequence of 100% identity;
(g) A sequence as set forth in SEQ ID NO 12 or having at least 90% of said sequence,
91％、92％、93％、94％、95％、96％、97％、98％、99％、
A nucleic acid sequence of 100% identity;
(h) The sequence shown in SEQ ID NO. 13 or at least 90% of the sequence,
91％、92％、93％、94％、95％、96％、97％、98％、99％、
A nucleic acid sequence of 100% identity.
A pharmaceutical composition comprising the fusion protein of any one of claims 1-11 or the nucleic acid molecule of any one of claims 12-14.
A recombinant cell expressing the fusion protein of any one of claims 1-11 or comprising the nucleic acid molecule of any one of claims 12-14.
Use of the fusion protein of any one of claims 1 to 11 or the nucleic acid molecule of any one of claims 12 to 14 for the treatment of a disease.
The use of claim 17, wherein the disease is a tumor.
The use of claim 17 or 18, wherein the disease is an epithelial tumor.
The use of claim 19, wherein one or more of the epithelial cells in the epithelial tumor have been at least partially transformed into mesenchymal cells.
The use according to any one of claims 17 to 20, wherein the disease is non-small cell lung cancer.
Drawings
FIG. 1 schematic structural diagram of a recombinant bifunctional fusion protein targeting αvβ3 and CD 47.
FIG. 2 FACS binding experiments of bifunctional fusion proteins to H1975 tumor cells.
FIG. 3 experimental verification that the bifunctional fusion protein was capable of inducing macrophage-mediated ADCP.
FIG. 4 determination of macrophage mediated tumor killing by bifunctional fusion proteins.
Detailed Description
Various aspects of the application are described in detail below. The following description is not intended to be limiting, and those skilled in the art can make any modifications and substitutions based on the technical knowledge in the art without significantly impeding the technical effects of the present application.
The practice of the present application will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology and cell biology, which are within the skill of the art. A description of these methods can be found, for example, in Sambrook et al, molecular Cloning: A Laboratory Manual (3 rd edition, 2001); sambrook et al, molecular Cloning: A Laboratory Manual (2 nd edition, 1989); maniatis et al, molecular Cloning: A Laboratory Manual (1982); ausubel et al Current Protocols in Molecular Biology (John Wiley and Sons, 7 th of 2008, updates );Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub.Associates and Wiley-Interscience;Glover,DNA Cloning:A Practical Approach,vol.I&II(IRL Press,Oxford,1985);Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992);Transcription and Translation(B.Hames&S.Higgins,Eds.,1984);Perbal,A Practical Guide to Molecular Cloning(1984);Harlow and Lane,Antibodies,(Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1998)Current Protocols in Immunology Q.E.Coligan,A.M.Kruisbeek,D.H.Margulies,E.M.Shevach and w.strober, eds., 1991); annual Review of Immunology; journal monographs such as ADVANCES IN Immunology.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present application, the following terms are defined below.
In the present application, the term "antibody" generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a particular antigen. For example, an antibody may comprise an immunoglobulin of at least two heavy (H) chains and two light (L) chains interconnected (complexed) by disulfide bonds, and include any molecule comprising an antigen binding portion thereof. The term "antibody" includes monoclonal antibodies, polyclonal antibodies, and also includes antibody fragments or antibody derivatives. In addition, the term "antibody" includes human antibodies, humanized antibodies, and also chimeric antibodies, single domain antibodies (e.g., dAb or V H H), single chain antibodies (e.g., scFv), and antibody fragments that bind to an antigen (e.g., fab ', and F (ab') 2 fragments). The term "antibody" also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, non-glycosylated antibodies, as well as any of the antigen-binding antibody fragments and derivatives thereof described. Each heavy chain may be composed of a heavy chain variable region (V H) and a heavy chain constant region C H. Each light chain may be composed of a light chain variable region (V L) and a light chain constant region C L. The V H and V L regions can be further distinguished as hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR), and it is noted that in this specification, the Complementarity Determining Regions (CDRs) in the heavy chain variable region are termed heavy chain complementarity determining regions (HCDR) and the Complementarity Determining Regions (CDRs) in the light chain variable region are termed light chain complementarity determining regions (LCDR). Each V H and V L may consist of three CDRs and four FR regions, which may be arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The heavy chain constant region typically comprises at least three heavy chain constant region domains, namely the C H domain, the C H domain and the C H domain, the heavy chain constant regions of IgG, igA and IgD comprise the three domains C H1、CH and C H, The heavy chain constant regions of IgM and IgE include four domains of C Hl、CH2、CH and C H. the variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate binding of the immunoglobulin to host tissues or factors including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).
The term "antibody fragment" or "antigen-binding fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof, and may refer to an antigen-binding domain of an intact antibody, e.g., an antigenically determined variable region, sufficient to confer recognition and specific binding of the antibody fragment to a target, e.g., an antigen. Examples of antibody fragments include, but are not limited to: fab, fab ', F (ab') 2, and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdabs (V H or V L), camelid (camelid) V H H domains, and multispecific antibodies formed from antibody fragments. The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are linked consecutively by a short flexible peptide linker, capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specifically indicated, herein an scFv may have V L and V H variable regions in either order, e.g., an scFv may comprise V L -linker-V H or may comprise V H -linker-V L relative to the N-and C-termini of the polypeptide.
The term "functional variant" of a protein or polypeptide sequence in the present application refers to an amino acid sequence having a sequence modification compared to the parent by 1 or more, e.g. 1-30, or 1-20 or 1-10, e.g. 1 or 2 or 3 or 4 or 5 amino acid substitutions, deletions, additions and/or insertions, the functional variant substantially retaining the biological properties of the protein or polypeptide sequence prior to modification. In one embodiment, the application encompasses variants of any of the protein or polypeptide sequences described in the present application. In certain embodiments, a functional variant of a protein or polypeptide sequence retains at least 60%,70%,80%,90%, or 100% of the biological activity of the parent prior to modification. The functional variants of the application may be antibodies, and specifically include antibody variable regions (e.g., V H or V L), antibody constant regions (e.g., C H or C L), A heavy chain CDR region (HCDR 1, HCDR2 or HCDR 3), a light chain CDR region (LCDR 1, LCDR2 or LCDR 3), a C H1、CH or C H domain of a heavy chain, and the like. For antibodies, the biological activity thereof includes, for example, antigen binding capacity. In certain embodiments, the functional variant of the antibody comprises amino acid modifications that do not result in the antibody variant losing binding to the antigen, but optionally may confer properties such as increased antigen affinity and different effector functions. It will be appreciated that the heavy chain variable region or the light chain variable region, or each CDR region, of an antibody may be modified individually or in combination. In certain embodiments, the amino acid modifications in one or more or all three HCDRs do not exceed 1,2, 3, 4,5, 6, 7, 8, 9, or 10. In certain embodiments, the amino acid modifications in one or more or all three LCDRs do not exceed 1,2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the amino acid modification may be a conservative sequence modification, such as a conservative amino acid substitution. In certain embodiments, the functional variant has at least 70%, 80%, 85%, 90% or 95% or 99% or more sequence identity to the parent. Similarly, a "functional variant" of a nucleic acid molecule refers in the present application to a nucleic acid molecule which encodes the same amino acid as the parent nucleic acid molecule or a nucleic acid molecule which encodes a functional variant of said protein.
The term "conservative sequence modification" refers to the amino acid modification that does not significantly affect or modify the binding characteristics of an antibody or antibody fragment comprising the amino acid sequence. Conservative modifications include amino acid substitutions, additions and deletions. Modifications the antibodies or antibody fragments of the application may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions of an amino acid residue with another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the fusion proteins of the application may be replaced with other amino acid residues from the same side chain family, and modified fusion proteins may be tested using the functional assays described herein.
In the present application, the term "isolated" generally refers to an antibody that has been separated from components in its natural environment. In certain embodiments, the antibodies are purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse phase HPLC). For reviews of methods for evaluating antibody purity see Flatman, s.et al, j. Chrom.b848 (2007) 79-87.
In the present application, the term "nucleic acid molecule" generally refers to any length of isolated form of nucleotide, deoxyribonucleotide or ribonucleotide or analog thereof, either isolated from the natural environment or synthesized synthetically. The nucleic acid molecules of the application may be isolated. For example, it may be produced or synthesized by: (i) amplified in vitro, e.g. by Polymerase Chain Reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified, e.g. fractionated by cleavage and gel electrophoresis, or (iv) synthesized, e.g. by chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA techniques. In the present application, nucleic acids encoding the antibodies or antigen binding fragments thereof may be prepared by a variety of methods known in the art, including, but not limited to, overlap extension PCR using restriction fragment procedures or using synthetic oligonucleotides, see Sambrook et al ,Molecular Cloning,ALaboratory Manual,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1989; and Ausube et al Current Protocols in Molecular Biology, greene Publishing and Wiley-Interscience, new York N.Y.,1993.
In the present application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host for transferring an inserted nucleic acid molecule into and/or between host cells. The vector may include a vector mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The carrier also includes a carrier having a plurality of functions as described above. The vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable host cell. Typically, the vector will produce the desired expression product by culturing a suitable host cell comprising the vector. In the present application, one or more of the nucleic acid molecules may be contained in the vector. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in an appropriate host cell and under appropriate conditions. In addition, the vector may also contain expression control elements that allow for proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers and other control elements which regulate gene transcription or mRNA translation, and the like. In certain embodiments, the expression control sequence is a tunable element. The specific structure of the expression control sequences may vary depending on the species or cell type function, but typically comprises 5' non-transcribed and 5' and 3' non-translated sequences involved in transcription and translation initiation, respectively, such as TATA boxes, capping sequences, CAAT sequences, and the like. For example, a 5' non-transcriptional expression control sequence may comprise a promoter region that may comprise a promoter sequence for a transcriptional control functional attachment nucleic acid. The vectors of the present application may be selected from the group consisting of plasmids, retroviral vectors and lentiviral vectors.
In the present application, the term "plasmid" generally refers to a DNA molecule other than a chromosome or pseudonucleus in an organism such as bacteria, yeast, etc., which exists in cytoplasm, has autonomous replication ability, enables it to maintain a constant copy number in daughter cells, and expresses carried genetic information. Plasmids are used as vectors for genes in genetic engineering studies.
In the present application, "sequence identity" generally refers to the degree of sequence identity on a nucleotide-by-nucleotide or amino acid-by-amino acid basis over a comparison window. The "percent sequence identity" may be calculated by: the two optimally aligned sequences are compared in a comparison window, the number of positions in the two sequences where the same nucleobase (e.g., A, T, C, G, I) or the same amino acid residue (e.g., ala, pro, ser, thr, gly, val, leu, ile, phe, tyr, trp, lys, arg, his, asp, glu, asn, gln, cys and Met) is present is determined to yield the number of matched positions, the number of matched positions is divided by the total number of positions in the comparison window (i.e., window size), and the result is multiplied by 100 to yield the percent sequence identity. The optimal alignment for determining percent sequence identity can be accomplished in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length sequence being compared or over the region of the target sequence.
In the present application, the term "pharmaceutically acceptable adjuvant" generally refers to a pharmaceutically acceptable formulation carrier, solution or additive that enhances the properties of the formulation. Such additives are well known to those skilled in the art.
In the present application, the term "cancer" generally refers to or describes a physiological condition of a mammal, which is typically characterized by a deregulation of cell proliferation or survival. In the present application, hyperproliferative diseases, known as cancers, include, but are not limited to, solid tumors, such as those occurring in the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid, and their distal metastases. Such diseases also include lymphomas, sarcomas, and leukemias. Examples of breast cancer include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ. Examples of respiratory cancers include, but are not limited to, small cell lung cancer and non-small cell lung cancer, as well as bronchial adenomas and pleural pneumoblastomas. Examples of brain cancers include, but are not limited to, brain stem and hypothalamic keratomas, cerebellum and brain astrocytomas, medulloblastomas, ependymomas, and neuroectodermal and pineal tumors. Male genital tumors include, but are not limited to, prostate and testicular cancers. Female genital tumors include, but are not limited to, endometrial, cervical, ovarian, vaginal, and vulvar cancers, as well as uterine tumors. Digestive tract tumors include, but are not limited to, anal, colon, colorectal, esophageal, gallbladder, stomach, pancreas, rectum, small intestine, and salivary gland cancers. Urinary tract tumors include, but are not limited to bladder, penile, kidney, renal pelvis, ureter, and urinary tract cancers. Eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma. Examples of liver cancer include, but are not limited to, hepatocellular carcinoma (hepatoma with or without fibrolamellar variation), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular cholangiocarcinoma. Skin cancers include, but are not limited to, squamous cell carcinoma, kaposi's sarcoma, malignant melanoma, merkel cell skin cancer, and non-melanoma skin cancers. Head and neck cancers include, but are not limited to, laryngeal, hypopharyngeal, nasopharyngeal, oropharyngeal cancers, and lip and oral cancers. Lymphomas include, but are not limited to, AIDS-related lymphomas, non-hodgkin's lymphomas, cutaneous T-cell lymphomas, hodgkin's disease, and central nervous system lymphomas. Sarcomas include, but are not limited to, soft tissue sarcomas, osteosarcomas, malignant fibrous histiocytomas, lymphosarcomas, and rhabdomyosarcomas. Leukemia includes, but is not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.
The term "therapeutically effective amount" refers to an amount of an active compound that is sufficient to elicit the biological or medical response desired by the clinician in the subject. The "therapeutically effective amount" of an antibody of the application may be determined by one skilled in the art depending on the route of administration, the weight, age, condition of the subject, and the like. For example, a typical daily dosage range may be 0.01mg to 100mg of active ingredient per kg body weight. Administration of the antibodies of the application includes, but is not limited to, injection, e.g., by intravenous, intramuscular, intraarterial, subcutaneous, intraperitoneal, and the like.
The term "and/or" is understood to mean either one of the selectable items or both of the selectable items.
As used in this disclosure, the terms "comprises" and "comprising" are intended to include the stated element, integer or step, but not to exclude any other element, integer or step. In the present application, when the terms "comprises" or "comprising" are used, they also encompass the circumstance that the recited elements, integers or steps are included unless indicated otherwise. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass antibody variable regions consisting of that particular sequence.
In the present application, the term "about" generally means ranging from 0.5% to 10% above or below the specified value, e.g., ranging from 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.
The invention provides a novel recombinant bifunctional fusion protein, which can simultaneously target alpha v beta 3 and CD47, can simultaneously play the roles of ADCC and ADCP mediated by macrophages, and has better anti-tumor effect. In one aspect, the invention also provides nucleic acid molecules encoding the fusion proteins. In another aspect, the invention also provides a pharmaceutical composition comprising said fusion protein and/or said nucleic acid molecule. In another aspect, the invention also provides a use of said fusion protein and/or said nucleic acid molecule in the treatment of a disease. In another aspect, the invention also provides a method of preparing the fusion protein. The following description will be made with respect to specific embodiments of various aspects of the present invention.
Fusion proteins
The inventor of the invention designs a novel recombinant difunctional fusion protein by fusing fragments of an anti-alpha v beta 3 antibody and SIRP alpha protein, and the novel fusion protein can simultaneously target alpha v beta 3 and CD47 with high affinity, can simultaneously play roles of macrophage-mediated ADCC and ADCP, and has better anti-tumor effect.
In one embodiment of the fusion protein, the fusion protein is a fusion protein comprising an anti- αvβ3 antibody or antibody fragment, and sirpa or sirpa fragment. In one embodiment, the SIRPalpha fragment is a SIRPalpha domain1 fragment, SIRPalpha domain1 refers to the amino acid sequence corresponding to E31-S149 of SIRPalpha protein (SEQ ID NO: 3), and in one embodiment, the fusion protein may further comprise other functional fragments. In one embodiment, the fusion protein is a recombinant protein of an anti- αvβ3 antibody or antibody fragment and a domain1 fragment of sirpa. In one embodiment, the domain1 fragment of sirpa may be linked to the N-and/or C-terminus of an anti- αvβ3 antibody or antibody fragment, preferably to the N-terminus of the heavy or light chain of an anti- αvβ3 antibody or antibody fragment. In a most preferred embodiment, the fusion protein is a fusion protein of a domain1 fragment of sirpa with an anti- αvβ3 antibody, and the domain1 fragment of sirpa is linked to the N-terminus of the heavy or light chain of the anti- αvβ3 antibody. In one embodiment, "heavy chain" refers to a peptide chain comprising the V H fragment of an anti- αvβ3 antibody and "light chain" refers to a peptide chain comprising the V L fragment of an anti- αvβ3 antibody.
In one embodiment, the domain 1 of SIRPalpha has the sequence shown in SEQ ID NO. 8 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to the sequence.
In one embodiment, the anti- αvβ3 antibody or antibody fragment may be derived from a variety of animals, such as antibodies or antibody fragments of human, sheep, mouse, rabbit, etc., and in a preferred embodiment, the antibody or antibody fragment is a human or humanized antibody or antibody fragment thereof. In one embodiment, the antibody or antibody fragment is an antibody of the IgG4 subtype or a fragment derived from an antibody of the IgG4 subtype. In one embodiment, the antibody is HY1272 (ABT 101), a novel IgG 4-type antibody that is humanized on the basis of the reported anti- αvβ3 antibody LM609 (see patent PCT/US 2021/028775).
In one embodiment, the heavy chain variable region of the anti- αvβ3 antibody or antibody fragment comprises a sequence as set forth in SEQ ID No. 9 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence, and the light chain variable region of the anti- αvβ3 antibody or antibody fragment comprises a sequence as set forth in SEQ ID No. 10 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence.
In one embodiment, the heavy chain of the anti- αvβ3 antibody or antibody fragment comprises a sequence as set forth in SEQ ID No. 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence, and the light chain of the anti- αvβ3 antibody or antibody fragment comprises a sequence as set forth in SEQ ID No.2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence.
In one embodiment, the anti- αvβ3 antibody or antibody fragment is an anti- αvβ3 antibody LM609 or fragment thereof, or an antibody Abegrin or fragment thereof after humanization and affinity maturation of LM609, or an HY1272 or fragment thereof, preferably an anti- αvβ3 antibody HY1272, of subtype from IgG1 engineered to IgG4 (to enhance affinity for CD64 on macrophage surface). In one embodiment, the domain1 fragment of sirpa is linked to an anti- αvβ3 antibody or antibody fragment by a linker, which may be arbitrarily selected by those skilled in the art, for example using the sequences AAA, EPKSA, G 4S,(G4S)2,(G4S)3, etc., preferably the linker is (G 4S)3 linker.
In one embodiment, the fusion protein comprises 2 first peptide chains and 2 second peptide chains, wherein the first peptide chains comprise, in order from the N-terminus: a domain1 fragment of sirpa, an optional linker, a heavy chain variable region, a heavy chain constant region, and an Fc region of an anti- αvβ3 antibody or antibody fragment; the second peptide chain comprises: the light chain variable region and the light chain constant region of an anti- αvβ3 antibody or antibody fragment, in one embodiment, the Fc region is preferably an Fc region derived from IgG4, in one embodiment, 2 Fc regions are linked by disulfide bonds.
In one embodiment, the fusion protein comprises 2 first peptide chains and 2 second peptide chains, wherein the first peptide chains comprise, in order from the N-terminus: a heavy chain variable region, a heavy chain constant region, and an Fc region of an anti- αvβ3 antibody or antibody fragment; the second peptide chain comprises: domain1 of sirpa, an optional linker, a light chain variable region of an anti- αvβ3 antibody or antibody fragment, and a light chain constant region. In one embodiment, the fusion protein has a structure as shown in FIG. 1. In one embodiment, the first peptide chain of the fusion protein comprises the amino acid sequence as set forth in SEQ ID NO:5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence and said second peptide chain comprises the amino acid sequence as set forth in SEQ ID NO:2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence; or the first peptide chain comprises the amino acid sequence as set forth in SEQ ID NO:1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence and said second peptide chain comprises the amino acid sequence as set forth in SEQ ID NO:6 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence.
Nucleic acids, vectors, cells, methods of preparation and compositions
In one embodiment, the application provides an isolated nucleic acid molecule that encodes a recombinant protein of the application. For example, the isolated nucleic acid molecule may comprise SEQ ID No: 11-14 or a functional variant thereof.
The nucleic acid molecules of the application may be isolated. For example, it may be produced or synthesized by: (i) amplified in vitro, e.g. by Polymerase Chain Reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified, e.g. fractionated by cleavage and gel electrophoresis, or (iv) synthesized, e.g. by chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA techniques.
In one embodiment, the application provides a vector which may comprise the nucleic acid molecule. In one embodiment of the present application, the vector may be selected from one or more of a plasmid, a retrovirus vector, and a lentiviral vector. The lentiviral vectors of the application may comprise a nucleic acid sequence encoding a recombinant receptor of the application. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in an appropriate host cell and under appropriate conditions. In addition, the vector may also contain expression control elements that allow for proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers and other control elements which regulate gene transcription or mRNA translation, and the like. In certain embodiments, the expression control sequence is a tunable element. The specific structure of the expression control sequences may vary depending on the species or cell type function, but typically comprises 5' non-transcribed and 5' and 3' non-translated sequences involved in transcription and translation initiation, respectively, such as TATA boxes, capping sequences, CAAT sequences, and the like. For example, a 5' non-transcriptional expression control sequence may comprise a promoter region that may comprise a promoter sequence for a transcriptional control functional attachment nucleic acid. One or more nucleic acid molecules of the application may be operably linked to the expression control element. The vector may include, for example, a plasmid, cosmid, virus, phage, or other vector commonly used in, for example, genetic engineering.
In one embodiment, the application provides a cell expressing said fusion protein or comprising said nucleic acid molecule or said vector. In one embodiment of the application, the cell may be a HEK293 cell.
In one embodiment, the application provides a method of preparing the cell comprising introducing into a host cell a vector of the application. For example, the vectors of the application may be introduced into such host cells, e.g., HEK293 cells. In certain embodiments, each or each cell may comprise one or more vectors of the application. In certain embodiments, each or each cell may comprise a plurality (e.g., 2 or more) or a plurality (e.g., 2 or more) of vectors described herein. In one embodiment of the application, the vector of the application may be introduced into the cell by methods known in the art. For example, the CAR molecule can be added to the genome by way of gene editing (e.g., CRISPR, cas 9). In one embodiment of the present application, the vector of the present application with the sequence for expressing the fusion protein may be introduced into the host cell by methods known in the art, such as electroporation, lipofectamine 2000, invitrogen, etc.
In one embodiment, the application provides a pharmaceutical composition that may comprise the fusion protein and a pharmaceutically acceptable adjuvant. The pharmaceutically acceptable adjuvants may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counter ions, metal complexes and, or nonionic surfactants, and the like. In one embodiment of the application, the pharmaceutical composition may be formulated for oral administration, intravenous administration (e.g., intravenous injection, i.v.), intramuscular administration (e.g., intramuscular injection, i.m.), in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration through a subcutaneous depot.
Pharmaceutical use
In one embodiment, the application provides the use of said fusion protein, said nucleic acid molecule or said vector for the preparation of a medicament for the treatment of a disease or disorder. In one embodiment of the application, the disease or disorder may be cancer or malignancy. In one embodiment of the application, the disease or disorder is an epithelial tumor. In a preferred embodiment, the disease or condition is refractory, recurrent, advanced solid tumors following epithelial-mesenchymal transition of the epithelial tumor. In a preferred embodiment, the disease or disorder is, for example, pancreatic cancer, non-small cell lung cancer, colon cancer, prostate cancer, and the like.
In one embodiment, the application provides said fusion protein, said nucleic acid molecule or said vector, which treats a disease or disorder associated with EBv.
In one embodiment, the application provides a method of treating a disease or disorder comprising administering to a subject said fusion protein, said nucleic acid molecule or said vector.
Without intending to be limited by any theory, the following examples are merely illustrative of the manner in which the chimeric antigen receptor, vector, cell, composition of the application works and are not intended to limit the scope of the application.
Examples
Example 1: construction and expression of HY1272-SIRP alpha fusion protein
Two HY 1272-SIRPalpha fusion proteins are designed, and the structural schematic diagram is shown in figure 1. The construction method is that the sequence of human SIRP alpha domain1 (the amino acid sequence corresponding to SEQ ID NO:3E 31-S149) is respectively constructed at the N end (nitrogen end) of a heavy chain sequence (shown as SEQ ID NO: 1) and a light chain sequence (shown as SEQ ID NO: 2) of an anti-alpha v beta 3 antibody HY1272, the middle is connected through a (G4S) 3 linker, and the obtained bifunctional fusion proteins are respectively named HY1272-SIRP alpha-1 and HY1272-SIRP alpha-2, and the heavy chain sequences of HY1272-SIRP alpha-1 are shown as SEQ ID NO:5, the light chain sequence of HY1272-SIRP alpha-2 is shown as SEQ ID NO: shown at 6.
The designed sequences were subjected to gene synthesis, expression vector construction, transient transfection of HEK293 cells and protein a purification, and the expression results are shown in table 1 below:
TABLE 1 HEK293 transient transfection expression purification data for bifunctional fusion proteins
Meanwhile, as a control, SIRPalpha domain1 (an amino acid sequence corresponding to SEQ ID NO:3E 31-S149) is constructed at the N-terminal of human IgG4 Fc, and the constructed molecule is named SIRPalpha-Fc (shown as SEQ ID NO: 7), and transient transfection and expression purification in HEK293 are performed.
Example 2: FACS binding efficacy detection on tumor cells
H1975 cells (ATCC, CRL-5908) are human non-small cell lung cancers that highly express αvβ3. To examine the binding capacity of the constructed fusion proteins to tumor cells, the binding efficacy of the individual molecules to H1975 cells was examined using FACS. The antibody was incubated with H1975 cells for 1 hour using gradient dilution with an initial working concentration of 200nM,1:4 gradient dilutions were performed for a total of 8 concentration points. The cells were then washed 3 times using FACS buffer, incubated with goat anti-human Fc-PE secondary antibody (abcam, ab98596, 1:300) for 30min, and detected by flow cytometry after washing.
As shown in fig. 2, each molecule has clear binding to H1975 cells compared to isotype control IgG, wherein the two bifunctional proteins have optimal binding efficacy, which is significantly better than monoclonal antibody HY1272, and significantly better than sirpa-Fc control, and is presumably due to the ability of the bifunctional proteins to bind simultaneously to both the αvβ3 and CD47 targets that are highly expressed on the tumor cell surface.
Example 3: antibody dependent cellular phagocytosis (Antibody Dependent Cellular Phagocytosis, ADCP) assay
Activation of the CD 47-sirpa signaling pathway can inhibit phagocytosis by macrophages. To verify whether the HY 1272-SIRPalpha bifunctional fusion protein can kill tumors through ADCP, we examined the effect of the fusion protein on phagocytosis of tumor cells by human macrophages.
Human Peripheral Blood Mononuclear Cells (PBMC) were placed in macrophage differentiation medium (RPMI 1640, 10%FB S,8%human serum,20mM HEPES,1%Glutamax,1%penicillin/streptomycin, and 50ng/mL M-CSF) and cultured for 5-6 days in 37% CO 2 to obtain macrophages as effector cells of ADCP (Effector cells). Macrophages were labeled with CELL TRACE violet (Thermo FISHER SCIENTIFIC) dye; h1975 non-small cell lung cancer cell line was labeled with pHrodo (Thermo FISHER SCIENTIFIC) as target cell (TARGET CELLS). Both cells were then separately resuspended in ADCP assay buffer (RPMI-1640, 10%FBS,25mM HEPES,1%Glutamax,1%penicillin/streptomycin) and finally treated with effector cells (E): target cells (T) were expressed as 2:1, and incubating for 4.5h at 37 ℃ under the condition of 5% CO 2, and adding 10 mug/mL of a test substance (HY 1272, HY1272-SIRP alpha-1, HY1272-SIRP alpha-2, SIRP alpha-Fc or IgG4 isotype control) into the incubation system. Finally phagocytosis was analyzed by flow cytometry.
As a result, as shown in FIG. 3, each of HY 1272-SIRPalpha-1, HY 1272-SIRPalpha-2 or SIRPalpha-Fc showed a significant ADCP effect, whereas HY1272 did not, and the ADCP effect of HY 1272-SIRPalpha-1 and HY 1272-SIRPalpha-2 was significantly stronger than that of SIRPalpha-Fc. This result suggests that the HY 1272-SIRPalpha bifunctional protein can kill tumors through macrophage-mediated ADCP.
Example 4: tumor cell killing experiment
Since HY 1272-SIRPalpha can kill tumors not only through macrophage-mediated ADCC, but also through macrophage-mediated ADCP, we evaluate the overall killing effect of the fusion protein on tumor cells.
Human Peripheral Blood Mononuclear Cells (PBMC) were placed in macrophage differentiation medium (RPMI 1640, 10%FBS,8%human serum,20mM HEPES,1%Glutamax,1%penicillin/streptomycin, and 50ng/mL M-CSF) and cultured at 37℃for 5-6 days with 5% CO 2 to obtain macrophages as effector cells (Effector cells). H1975 non-small cell lung cancer cell line as target cell (TARGET CELLS) usingCell cytotoxicity assay kit (Perkinelmer) tested for human macrophage killing H1975 non-small cell lung cancer cells according to the protocol. BATDA labeling was performed using H1975 cells as target cells. Effector cells (E): target cells (T) were each expressed as 0:1 or 10:1, and incubating for 24 hours at 37 ℃ under the condition of 5% CO 2, wherein 10 mug/mL of a test substance (HY 1272, HY 1272-SIRPalpha-1, HY 1272-SIRPalpha-2 or SIRPalpha-Fc) or isotype control is added into the incubation system. Transferring the cell culture supernatant into a new pore plate, adding Europium solution, mixing, measuring time-resolved fluorescence (time-resolved fluorescence), and calculating cell killing rate according to the formula in the kit specification.
As shown in fig. 4, each of the 4 test substances was able to promote the killing of H1975 tumor cells by macrophages, but HY 1272-sirpa-1 and HY 1272-sirpa-2 exhibited significantly better tumor killing activity than HY1272 or sirpa-Fc. The result shows that the HY 1272-SIRPalpha bifunctional protein has better anti-tumor activity.
The various sequences used in this specification will be given below
Heavy chain sequence of SEQ ID NO. 1HY1272
Light chain sequence of SEQ ID NO. 2HY1272
SEQ ID NO:3 human SIRP alpha sequence
SEQ ID NO. 4 human IgG4 Fc region (S228P) sequence
Heavy chain sequence of SEQ ID NO 5HY1272-SIRP alpha-1
Light chain sequence of SEQ ID NO. 6HY1272-SIRP alpha-2
SEQ ID NO. 7SIRP alpha-Fc sequence
Domain 1 sequence of 8 human SIRPalpha with SEQ ID NO
Heavy chain variable region sequence of SEQ ID NO 9HY1272
Light chain variable region sequence of SEQ ID NO. 10HY1272
Heavy chain nucleic acid sequence of SEQ ID NO. 11HY1272
Light chain nucleic acid sequence of SEQ ID NO. 12HY1272
Heavy chain nucleic acid sequence of 13HY1272-SIRP alpha-1
Light chain nucleic acid sequence of SEQ ID NO. 14HY1272-SIRP alpha-2

Claims (21)

1. A fusion protein comprising the following portions:
(a) An anti- αvβ3 antibody or antibody fragment; and
(B) The domain 1 fragment of sirpa,
The domain1 fragment of sirpa is linked to the N-terminus of the heavy and/or light chain of an anti- αvβ3 antibody or antibody fragment.
2. The fusion protein of claim 1, wherein the domain1 of sirpa has the sequence set forth in SEQ ID No. 8 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto.
3. The fusion protein of claim 1 or 2, wherein the anti- αvβ3 antibody or antibody fragment is a humanized anti- αvβ3 antibody or antibody fragment.
4. A fusion protein according to any one of claims 1 to 3, wherein the anti- αvβ3 antibody or antibody fragment is an lgG4 subtype antibody or an antibody fragment derived from an lgG4 subtype antibody.
5. The fusion protein of any one of claims 1-4, wherein the heavy chain variable region of the anti- αvβ3 antibody or antibody fragment comprises a sequence as set forth in SEQ ID No. 9 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence, and the light chain variable region of the anti- αvβ3 antibody or antibody fragment comprises a sequence as set forth in SEQ ID No. 10 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence.
6. The fusion protein of any one of claims 1-5, wherein the heavy chain of the anti- αvβ3 antibody or antibody fragment comprises the sequence set forth in SEQ ID No. 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity thereto, and the light chain of the anti- αvβ3 antibody or antibody fragment comprises the sequence set forth in SEQ ID No. 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, 100% identity thereto.
7. The fusion protein of any one of claims 1-6, wherein the domain1 fragment of sirpa is linked to the N-terminus of the heavy and/or light chain of an anti- αvβ3 antibody or antibody fragment by a linker.
8. The fusion protein according to any one of claims 1 to 7, wherein the linker is (G4S) 3 linker.
9. The fusion protein according to claim 1 to 8, which comprises 2 first peptide chains and 2 second peptide chains, wherein,
The first peptide chain comprises, in order from the N-terminus: a domain1 fragment of sirpa, an optional linker, a heavy chain variable region, a heavy chain constant region, and an Fc region of an anti- αvβ3 antibody or antibody fragment;
the second peptide chain comprises: the light chain variable region and the light chain constant region of an anti- αvβ3 antibody or antibody fragment.
10. The fusion protein according to any one of claim 1 to 9, comprising 2 first peptide chains and 2 second peptide chains, wherein,
The first peptide chain comprises, in order from the N-terminus: a heavy chain variable region, a heavy chain constant region, and an Fc region of an anti- αvβ3 antibody or antibody fragment;
The second peptide chain comprises: domain1 of sirpa, an optional linker, a light chain variable region of an anti- αvβ3 antibody or antibody fragment, and a light chain constant region.
11. The fusion protein of any one of claims 1-10, wherein the first peptide chain comprises the sequence as set forth in SEQ ID No. 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence and the second peptide chain comprises the sequence as set forth in SEQ ID No. 2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence; or the first peptide chain comprises the sequence as set forth in SEQ ID NO. 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence and the second peptide chain comprises the sequence as set forth in SEQ ID NO. 6 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence.
12. An isolated nucleic acid molecule encoding a fusion protein as described.
13. The nucleic acid molecule of claim 12 which is a vector.
14. The nucleic acid molecule of claim 12 or 13, comprising (a) and (b) below; or (c) and (d):
(a) A sequence as set forth in SEQ ID NO. 11 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence;
(b) A sequence as set forth in SEQ ID NO. 14 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence;
(c) A sequence as set forth in SEQ ID NO. 12 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence;
(d) The sequence as set forth in SEQ ID NO. 13 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to said sequence.
15. A pharmaceutical composition comprising the fusion protein of any one of claims 1-11 or the nucleic acid molecule of any one of claims 12-14.
16. A recombinant cell expressing the fusion protein of any one of claims 1-11 or comprising the nucleic acid molecule of any one of claims 12-14.
17. Use of a fusion protein according to any one of claims 1 to 11 or a nucleic acid molecule according to any one of claims 12 to 14 for the treatment of a disease.
18. The use according to claim 17, wherein the disease is a tumor.
19. The use according to claim 17 or 18, wherein the disease is an epithelial tumour.
20. The use of claim 19, wherein one or more of the epithelial cells in the epithelial tumor have been at least partially transformed into mesenchymal cells.
21. The use according to any one of claims 17 to 20, wherein the disease is non-small cell lung cancer.
CN202310167358.2A 2023-02-24 2023-02-24 Difunctional fusion protein targeting alpha v beta 3 and CD47 and application thereof Pending CN118546255A (en)

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JP2008510007A (en) * 2004-08-16 2008-04-03 メディミューン，インコーポレーテッド Eph receptor Fc variants with enhanced antibody-dependent cellular cytotoxic activity
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WO2016024021A1 (en) * 2014-08-15 2016-02-18 Merck Patent Gmbh Sirp-alpha immunoglobulin fusion proteins
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