CN114369167A - Bifunctional fusion protein composed of anti-PD-L1 antibody and IL-7 - Google Patents

Bifunctional fusion protein composed of anti-PD-L1 antibody and IL-7 Download PDF

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CN114369167A
CN114369167A CN202011102052.1A CN202011102052A CN114369167A CN 114369167 A CN114369167 A CN 114369167A CN 202011102052 A CN202011102052 A CN 202011102052A CN 114369167 A CN114369167 A CN 114369167A
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fusion protein
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刘波
张均利
吴其威
朱冰
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Suzhou Zelgen Biopharmaceutical Co Ltd
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    • C07K14/54—Interleukins [IL]
    • C07K14/5418—IL-7
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Abstract

The invention provides a bifunctional fusion protein consisting of an anti-PD-L1 antibody and IL-7; specifically, the present invention provides a recombinant bifunctional fusion protein comprising: a first structural unit (D1); and a second structural unit (D2); wherein the first building block is an antibody that specifically binds to PD-L1 protein; the second structural unit comprises IL-7, the fusion protein can enrich IL-7 cell factors in a tumor microenvironment, specifically activates an immune system in the tumor microenvironment, and can greatly reduce toxic and side effects.

Description

Bifunctional fusion protein composed of anti-PD-L1 antibody and IL-7
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to a bifunctional fusion protein formed by an anti-PD-L1 antibody and IL-7.
Background
The protein of the immunosuppressive programmed death ligand 1(PD-L1) is generally high in expression in a tumor microenvironment, and PD-L1 can be combined with an immunocyte (mainly T cell) surface receptor PD-1, so that the activation of an immune system is inhibited, and the growth of cancer cells is promoted.
Interleukin 7(IL-7) is a hematopoietic growth factor secreted by stromal cells in the bone marrow and thymus. IL-7 stimulates the differentiation of pluripotent hematopoietic stem cells into lymphoid progenitor cells. It also stimulates the proliferation of all cells of the lymphoid lineage (B cells, T cells and NK cells). IL-7 is a cytokine important for the development of B-cells and T-cells. The cytokine and Hepatocyte Growth Factor (HGF) form a heterodimer, which functions as a pro-progenitor B cell growth stimulating factor. The cytokine can be produced locally by intestinal epithelial cells and epithelial goblet cells, and can act as a regulatory factor for intestinal mucosal lymphocytes.
Although the PD-1/PD-L1 antibody has an unprecedented therapeutic effect on a variety of tumors, it has been effective to date in only about 20% of humans.
Human studies of IL-7 in cancer patients have shown that administration of this cytokine can transiently disrupt the homeostasis of CD8< + > and CD4< + > T cells with a corresponding decrease in the percentage of CD4< + > CD25< + > Foxp3< + > T regulatory cells. However, no actual cancer regression was observed.
Therefore, there is an urgent need in the art to develop a drug that can significantly increase the half-life of IL-7 cytokine, reduce clinical dose, and reduce toxic side effects.
Disclosure of Invention
The invention aims to provide a medicament which can obviously increase the half-life of IL-7 cytokines, reduce clinical dose and reduce toxic and side effects.
In a first aspect of the present invention, a recombinant bifunctional fusion protein is provided, said recombinant bifunctional fusion protein comprising:
a first structural unit (D1); and
a second structural unit (D2);
wherein the first building block is an antibody that specifically binds to PD-L1 protein;
the second building block comprises IL-7,
and the second building block is linked to the end of the heavy chain constant region of the first building block.
In another preferred embodiment, the antibody comprises: an antibody of animal origin (e.g., a murine antibody), a chimeric antibody, a humanized antibody.
In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.
In another preferred embodiment, the antibody is a monoclonal antibody.
In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.
In another preferred embodiment, the D1 and the D2 are connected by a connecting peptide.
In another preferred embodiment, the linker peptide has the structure: - (Gly-Gly-Gly-Ser) n-, wherein n is 1 to 5, preferably 1 to 3.
In another preferred embodiment, the bifunctional fusion protein (monomer) has the structure shown in formula I from N-terminus to C-terminus:
Figure BDA0002725702610000021
wherein the content of the first and second substances,
t1 is IL-7;
l1 is an optional linking element;
VL represents the light chain variable region of an anti-PD-L1 antibody;
CL represents the light chain constant region of the anti-PD-L1 antibody;
VH represents the heavy chain variable region of an anti-PD-L1 antibody;
CH represents the heavy chain constant region of an anti-PD-L1 antibody;
"-" represents a disulfide bond or a covalent bond;
"-" represents a peptide bond.
In another preferred example, the L1 has the following structure: - (Gly-Gly-Gly-Ser) n-, wherein n is 1 to 5, preferably 1 to 3.
In another preferred embodiment, the CH includes CH1, CH2, and CH 3.
In another preferred embodiment, the two monomers of the bifunctional fusion protein form a dimer through disulfide bonds on CH2 and CH 3.
In another preferred embodiment, the IL-7 is derived from a human or non-human mammal, more preferably from a rodent (e.g., mouse, rat), primate, and human.
In another preferred embodiment, the IL-7 includes wild type and mutant.
In another preferred embodiment, the IL-7 includes a full-length, mature form of IL-7, or an active fragment thereof.
In another preferred embodiment, the sequence of the IL-7 is shown in SEQ ID NO. 1.
In another preferred embodiment, the IL-7 also includes derivatives of IL-7.
In another preferred embodiment, the derivatives of IL-7 include modified IL-7, protein molecules having an amino acid sequence homologous to native IL-7 and having native IL-7 activity, dimers or multimers of IL-7, and fusion proteins comprising an IL-7 amino acid sequence.
In another preferred embodiment, the modified IL-7 is PEGylated IL-7.
In another preferred embodiment, the expression "protein molecule having an amino acid sequence homologous to native IL-7 and having native IL-7 activity" means that the amino acid sequence has at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, most preferably at least 98% homology to IL-7; and having IL-7 activity.
In another preferred embodiment, the IL-7 or active fragment thereof comprises an extracellular domain of IL-7.
In another preferred embodiment, the amino acid sequence of VL is shown in SEQ ID No. 2.
In another preferred embodiment, the amino acid sequence of VH is shown in SEQ ID NO. 3.
In another preferred embodiment, the amino acid sequence of the CL is shown in SEQ ID No. 4.
In another preferred embodiment, the amino acid sequence of CH is shown in SEQ ID NO. 5.
In another preferred embodiment, the D1 comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID No. 6.
In another preferred example, the amino acid sequence of the light chain of D1 is shown in SEQ ID No. 7.
In another preferred embodiment, said recombinant bifunctional fusion protein further comprises (preferably coupled to) a detectable label, a targeting label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
In another preferred embodiment, the recombinant bifunctional fusion protein is coupled to a tumor targeting marker conjugate.
In another preferred embodiment, the first building block is an anti-PD-L1 antibody.
In another preferred embodiment, the antibody is of the IgG1 type.
In another preferred embodiment, the antibody has an affinity (nM) for PD-L1 of 0.01-10, preferably 0.08-6, more preferably 0.1-1.
In another preferred embodiment, the recombinant bifunctional fusion protein is a homodimer.
In a second aspect, the present invention provides an isolated polynucleotide encoding a recombinant bifunctional fusion protein according to the first aspect of the present invention.
In another preferred embodiment, said polynucleotide additionally comprises an auxiliary element selected from the group consisting of: a signal peptide, a secretory peptide, a tag sequence (e.g., 6His), or a combination thereof.
In another preferred embodiment, the polynucleotide is selected from the group consisting of: a DNA sequence, an RNA sequence, or a combination thereof.
In a third aspect, the invention provides a vector comprising a polynucleotide according to the second aspect of the invention.
In another preferred embodiment, the vector comprises one or more promoters operably linked to the nucleic acid sequence, enhancer, transcription termination signal, polyadenylation sequence, origin of replication, selectable marker, nucleic acid restriction site, and/or homologous recombination site.
In another preferred embodiment, the vector comprises a plasmid, a viral vector.
In another preferred embodiment, the viral vector is selected from the group consisting of: adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or combinations thereof.
In another preferred embodiment, the vector comprises an expression vector, a shuttle vector and an integration vector.
In a fourth aspect, the present invention provides a host cell comprising a vector according to the third aspect of the present invention, or having a polynucleotide according to the second aspect of the present invention integrated into its genome.
In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell, a plant cell, or a mammalian cell (including human and non-human mammals).
In another preferred embodiment, the host cell is a prokaryotic cell, such as E.coli.
In another preferred embodiment, the yeast cell is a yeast from one or more sources selected from the group consisting of: pichia pastoris, Kluyveromyces, or combinations thereof; preferably, the yeast cell comprises: kluyveromyces, more preferably Kluyveromyces marxianus and/or Kluyveromyces lactis.
In another preferred embodiment, the host cell is selected from the group consisting of: coli, wheat germ cells, insect cells, SF9, Hela, HEK293, 293F cells, CHO cells, yeast cells, or combinations thereof.
In another preferred embodiment, the host cell comprises a CHO cell.
In a fifth aspect, the present invention provides a method of producing a fusion protein according to the first aspect of the invention, said method comprising the steps of:
culturing the host cell of the fourth aspect of the invention under conditions suitable for expression, thereby expressing the fusion protein; and/or isolating the fusion protein.
In a sixth aspect, the invention provides an immunoconjugate comprising:
(a) a recombinant bifunctional fusion protein according to the first aspect of the invention; and
(b) a coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
In another preferred embodiment, the conjugate moiety is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles, and the like.
In a seventh aspect, the present invention provides a pharmaceutical composition comprising:
(i) a recombinant bifunctional fusion protein according to the first aspect of the invention, or an immunoconjugate according to the sixth aspect of the invention; and
(ii) a pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition further comprises other drugs for inhibiting tumor activity.
In another preferred embodiment, the pharmaceutical composition is in the form of injection.
In an eighth aspect, the present invention provides a use of a recombinant bifunctional fusion protein according to the first aspect of the present invention, a polynucleotide according to the second aspect of the present invention, a vector according to the third aspect of the present invention, a host cell according to the fourth aspect of the present invention, or an immunoconjugate according to the sixth aspect of the present invention, for the preparation of a composition or formulation for the treatment or prevention of a tumor.
In another preferred embodiment, the composition or formulation is further used for one or more uses selected from the group consisting of:
1) specifically binds to tumor cells expressing PD-L1;
2) blocking the binding of PD-1 and PD-L1, increasing the activation of T cells (including proliferation and cytokine release);
3) binds to the IL-7 receptor and promotes STAT phosphorylation activation;
4) activation of T cells and NK cells (including proliferation and cytokine release) within human peripheral blood mononuclear cells by IL-7;
(5) increase the number of CD4 and CD 8T cells.
In another preferred embodiment, the composition is a pharmaceutical composition.
In another preferred embodiment, the tumor comprises a solid tumor.
In another preferred embodiment, the solid tumor is selected from the group consisting of: prostate cancer, liver cancer, head and neck cancer, melanoma, non-hodgkin's lymphoma, bladder cancer, glioblastoma, cervical cancer, lung cancer, chondrosarcoma, thyroid cancer, kidney cancer, mesothelioma, osteosarcoma, cholangiocarcinoma, ovarian cancer, gastric cancer, bladder cancer, meningioma, pancreatic cancer, multiple squamous cell tumor, esophageal cancer, lung small cell carcinoma, colorectal cancer, breast cancer, medulloblastoma, breast cancer, or a combination thereof.
In a ninth aspect, the invention provides an in vitro non-therapeutic method of inhibiting tumor growth, comprising the steps of: culturing a tumor cell in the presence of a fusion protein according to the first aspect of the invention or an immunoconjugate according to the sixth aspect of the invention, thereby inhibiting tumor growth.
In another preferred embodiment, the tumor comprises a solid tumor.
In another preferred embodiment, the tumor cells are from one or more tumors selected from the group consisting of: prostate cancer, liver cancer, head and neck cancer, melanoma, non-hodgkin's lymphoma, bladder cancer, glioblastoma, cervical cancer, lung cancer, chondrosarcoma, thyroid cancer, renal cancer, mesothelioma, osteosarcoma, cholangiocarcinoma, ovarian cancer, gastric cancer, bladder cancer, meningioma, pancreatic cancer, multiple squamous cell tumor, esophageal cancer, lung small cell carcinoma, colorectal cancer, breast cancer, medulloblastoma, breast cancer.
In another preferred embodiment, the tumor cells are cells cultured in vitro.
In a tenth aspect, the present invention provides a method for treating tumors, comprising the steps of: administering to a subject in need thereof a fusion protein according to the first aspect of the invention or an immunoconjugate according to the sixth aspect of the invention.
In another preferred embodiment, the tumor comprises a solid tumor.
In another preferred embodiment, the subject is a human.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows a schematic structural diagram of a fusion protein of the present invention.
Detailed Description
The present inventors have surprisingly, through extensive and intensive studies, obtained a recombinant bifunctional fusion protein consisting of a first structural unit (D1) specifically binding to the target molecule PD-L1 protein and a second structural unit (D2) comprising IL-7 in tandem. The fusion protein of the invention is a homodimer. The fusion protein can enrich IL-7 cell factors in a tumor microenvironment, specifically activate an immune system in the tumor microenvironment, and greatly reduce toxic and side effects; in addition, the IL-7 cytokine is fused to the C end of the antibody with longer half-life period, so that the half-life period of the IL-7 cytokine can be obviously increased, the clinical dose can be reduced, and the toxic and side effects can be reduced. On this basis, the present inventors have completed the present invention.
Before the present invention is described in detail, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
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 to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.
As used herein, unless otherwise specified, Fc refers to the Fc fragment of a human immunoglobulin. The term "immunoglobulin Fc region" refers to immunoglobulin chain constant regions, particularly the carboxy-terminal end of or a portion of an immunoglobulin heavy chain constant region, e.g., an immunoglobulin Fc region may comprise two or more domains of heavy chains CH1, CH2, CH3 in combination with an immunoglobulin hinge region, and in preferred embodiments, the immunoglobulin Fc region used comprises at least one immunoglobulin hinge region, one CH2 domain and one CH3 domain, preferably lacking the CH1 domain.
It is known that there are various classes of human immunoglobulins, such as IgA, IgD, IgE, IgM and IgG (including the four subclasses IgG1, IgG2, IgG3 and IgG 4), and it is within the purview of the skilled person to select a particular immunoglobulin Fc region from the particular class and subclass of immunoglobulins, and in a preferred embodiment, the immunoglobulin Fc region is selected from the coding sequence comprising the human immunoglobulin IgG4 subclass Fc region in which one immunoglobulin heavy chain 1 domain (CH1) is deleted, but includes the hinge region and the coding sequences for CH2, CH3 and both domains.
As used herein, the terms "comprising," having, "or" including "include" comprising, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …", and "consisting of … …" are subordinate concepts of "comprising", "having", or "including".
As used herein, unless otherwise indicated, the fusion protein is an isolated protein, unrelated to other proteins, polypeptides or molecules, purified product of recombinant host cell culture or as a purified extract.
Interleukin 7(IL-7)
Interleukin 7(IL-7) is a hematopoietic growth factor secreted by stromal cells in the bone marrow and thymus. IL-7 stimulates the differentiation of pluripotent hematopoietic stem cells into lymphoid progenitor cells. It also stimulates the proliferation of all cells of the lymphoid lineage (B cells, T cells and NK cells). IL-7 is a cytokine important for the development of B-cells and T-cells. The cytokine and Hepatocyte Growth Factor (HGF) form a heterodimer, which functions as a pro-progenitor B cell growth stimulating factor. The cytokine can be produced locally by intestinal epithelial cells and epithelial goblet cells, and can act as a regulatory factor for intestinal mucosal lymphocytes.
Bifunctional fusion proteins
The bifunctional fusion protein constructed by the invention comprises a first structural unit specifically combined with a target molecule PD-L1 and a second structural unit comprising IL-7, can enrich IL-7 cell factors in a tumor microenvironment, specifically activates an immune system in the tumor microenvironment, and can greatly reduce toxic and side effects; in addition, IL-7 can be more accurately enriched in a tumor microenvironment expressing PD-L1 antigen by fusing IL-7 cytokine to the C end of the antibody with longer half-life, and the half-life of the IL-7 cytokine can be obviously increased, the clinical dose is reduced, and the toxic and side effects are reduced.
In a preferred embodiment, the fusion protein of the invention has the structure shown in FIG. 1.
According to the present invention, the recombinant bifunctional fusion protein may be a single multifunctional polypeptide, or it may be a multimeric complex of two or more polypeptides covalently or non-covalently bound to each other.
As used herein, a "fusion protein of the invention", or a "polypeptide", or a "bifunctional fusion protein of the invention" all refer to a fusion protein according to the first aspect of the invention.
The term "fusion protein" as used herein also includes variants of the fusion protein having the above-described activity. These variants include (but are not limited to): deletion, insertion and/or substitution of 1 to 3 (usually 1 to 2, more preferably 1) amino acids, and addition or deletion of one or several (usually up to 3, preferably up to 2, more preferably up to 1) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition or deletion of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the structure and function of the protein. In addition, the term also includes monomeric and multimeric forms of the polypeptides of the invention. The term also includes linear as well as non-linear polypeptides (e.g., cyclic peptides).
The invention also includes active fragments, derivatives and analogs of the above fusion proteins. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains the function or activity of a fusion protein of the invention. The polypeptide fragment, derivative or analogue of the present invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which an antigenic peptide is fused to another compound (such as a compound that increases the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) a polypeptide in which an additional amino acid sequence is fused to the polypeptide sequence (a fusion protein in which a leader sequence, a secretory sequence or a tag sequence such as 6 × His is fused). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.
A preferred class of active derivatives refers to polypeptides formed by the replacement of up to 3, preferably up to 2, more preferably up to 1 amino acid with an amino acid of similar or analogous nature compared to the amino acid sequence of the fusion protein of the invention. These conservative variants are preferably produced by amino acid substitutions according to Table A.
TABLE A
Initial residue(s) Representative substitutions Preferred substitutions
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
The invention also provides analogs of the fusion proteins of the invention. These analogs may differ from the fusion proteins of the invention by amino acid sequence differences, by modifications that do not affect the sequence, or by both. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.
Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.
First structural unit
The recombinant bifunctional fusion protein of the present invention comprises a first structural unit (D1). In the present invention, the first building block specifically binds to the target molecule PD-L1, and in the present invention, the first building block refers to any peptide, polypeptide, nucleic acid molecule, scaffold-type molecule, peptide display molecule or polypeptide-containing construct capable of specifically binding to a specific protein of interest.
As used herein, the term "specifically binds" or the like means that the antigen binding domain forms a complex with a particular antigen characterized by a dissociation constant (KD) of 500pM or less, and does not bind to other unrelated proteins under common test conditions. Preferably, an "unrelated protein" is a protein, peptide or polypeptide that has less than 95% amino acid identity to each other.
Exemplary classifications of antigen-binding domains that can be used in the context of the present invention include antibodies, antigen-binding portions of antibodies, peptides that specifically interact with a particular antigen (e.g., peptibodies), receptor molecules that specifically interact with a particular antigen, proteins that comprise a ligand-binding portion of a receptor that specifically binds a particular antigen, antigen-binding scaffolds (e.g., darpins, HEAT repeat proteins, ARM repeat proteins, triangular tetrapeptide repeat proteins, and other scaffolds based on naturally occurring repeat proteins, etc. [ see, e.g., Boersma and Pluckthun,2011, curr. opin. biotechnol.22:849 and 857, and references cited therein ]) and aptamers or portions thereof.
Methods for determining whether two molecules specifically bind to each other are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, as used in the context of the present invention, an antigen binding domain comprises a peptide sequence as measured in a surface plasmon resonance assay at less than about 500pM, less than about 400pM, less than about 300pM, less than about 200pM, less than about 100pM, less than about 90pM, less than about 80pM, less than about 70pM, less than about 60pM, less than about 50pM, less than about 40pM, less than about 30pM, less than about 20pM, less than about 10pM, as measured as described in the surface plasmon resonance assayA K of less than about 5pM, less than about 4pM, less than about 2pM, less than about 1pM, less than about 0.5pM, less than about 0.2pM, less than about 0.1pM, or less than about 0.05pMDA polypeptide that binds to a particular antigen or portion thereof.
As used herein, the term "surface plasmon resonance" refers to allowing, for example, the use of BIAcoreTMThe system (Biacore Life sciences department of GEHealthcare, Piscataway, NJ) analyzes the optical phenomenon of real-time interactions by detecting changes in protein concentration inside the biosensor matrix.
As used herein, the term "KDBy "is meant the equilibrium dissociation constant for a particular protein-protein interaction (e.g., antibody-antigen interaction). Unless otherwise indicated, K disclosed hereinDThe value is the K determined by surface plasmon resonance measurement at 25 DEG CDThe value is obtained.
Antibodies and antigen-binding fragments thereof
As indicated above, the first building block (D1) may comprise or consist of an antibody or an antigen-binding fragment of an antibody. As used herein, the term "antibody" means any antigen binding molecule or molecular complex comprising at least one Complementarity Determining Region (CDR) that specifically binds to or interacts with a particular antigen (e.g., PD-L1 protein). The term "antibody" includes immunoglobulin molecules comprising 4 polypeptide chains (two heavy (H) and two light (L) chains interconnected by disulfide bonds) as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V)H) And a heavy chain constant region. The heavy chain constant region comprises 3 domains: cH1、CH2 and CH3. Each light chain comprises a light chain variable domain (abbreviated herein as LCVR or V)L) And a light chain constant region. The light chain constant domain comprises a domain (C)L1)。VHRegion and VLThe regions can be further subdivided into hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with more conserved regions, termed Framework Regions (FRs). Each VHAnd VLFrom amino terminus to carboxy terminus in the following order: composition of 3 CDRs and 4 FRs of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 arrangement. In various embodiments of the invention, the FRs of an antibody of the invention (or an antigen-binding portion thereof) may be identical to human germline sequences, or may be modified naturally or artificially. Amino acid consensus sequences can be defined based on side-by-side analysis of two or more CDRs.
The D1 component of the recombinant bifunctional fusion protein of the present invention may comprise or consist of an antigen-binding fragment of an intact antibody molecule. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like include any naturally occurring, enzymatically obtainable, synthetic or genetically modified polypeptide or glycoprotein that specifically binds an antigen to form a complex. Any suitable standard technique such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable domains and optionally antibody constant domains may be used, for example, to derive antigen-binding fragments of antibodies from intact antibody molecules. Such DNA is known and/or readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biological techniques, for example to arrange one or more variable and/or constant domains in a suitable layout, or to introduce codons, generate cysteine residues, modify, add or delete amino acids, and the like.
Non-limiting examples of antigen-binding fragments include: (i) a Fab fragment; (ii) f (ab')2A fragment; (iii) (ii) a fragment of Fd; (iv) (iv) an Fv fragment; (v) single chain fv (scFv) molecules; (vi) a dAb fragment; and (vii) a minimal recognition unit (e.g., an independent Complementarity Determining Region (CDR) such as a CDR3 peptide) consisting of amino acid residues that mimic a hypervariable region of an antibody or a constrained FR3-CDR3-FR4 peptide. As used herein, the expression "antigen-binding fragment" also internally encompasses other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), Small Modular Immunopharmaceuticals (SMIPs), and shark variable IgNAR domains.
Antigen-binding fragments of antibodies will generally compriseAt least one variable field. The variable domain may be of any size or amino acid composition and will typically comprise at least one CDR that is adjacent to or in reading frame with one or more framework sequences. In a region having a sum of VLDomain associated VHIn antigen-binding fragments of domains, VHAnd VLThe domains may be arranged relative to each other in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH、VH-VLOr VL-VLA dimer. Alternatively, the antigen-binding fragment of the antibody may contain monomeric VHOr VLA domain.
In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary layouts of variable and constant domains that may exist within the antigen-binding fragments of antibodies of the invention include: (i) vH-CH1；(ii)VH-CH2；(iii)VH-CH3；(iv)VH-CH1-CH2；(v)VH-CH1-CH2-CH3；(vi)VH-CH2-CH3；(vii)VH-CL；(viii)；)VL-CH1；(ix)VL-CH2；(x)VL-CH3；(xi)VL-CH1-CH2；(xii)VL-CH1-CH2-CH3；(xiii)VL-CH2-CH3; and (xiv) VL-CL. In any layout of variable and constant domains, including any of the exemplary layouts listed above, the variable and constant domains may be directly connected to each other or may be connected by a hinge region or linker region, in whole or in part. The hinge region can be comprised of at least 2 (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that create flexible or semi-flexible linkages between adjacent variable and/or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragment may comprise a sequence of one or more monomeric V having any of the variable and constant domain layouts listed above, with each other and/or with one or more monomeric VHOr VLDomains (e.g., via disulfide bonds)) are in non-covalent association with a homodimer or heterodimer (or other multimer).
The recombinant bifunctional fusion proteins of the present invention may comprise or consist of a human antibody and/or a recombinant human antibody or a fragment thereof. As used herein, the term "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. However, human antibodies can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., in the CDRs and particularly CDR 3) (e.g., mutations introduced by random mutagenesis in vitro or site-specific mutagenesis or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework sequences.
The recombinant bifunctional fusion proteins of the present invention may comprise or consist of a recombinant human antibody or antigen-binding fragment thereof. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (described further below), antibodies isolated from combinatorial libraries of recombinant human antibodies (described further below), antibodies isolated from animals (e.g., mice) that are transgenic for human immunoglobulin genes (see, e.g., Taylor et al, (1992) Nucl. acids sRs.20: 6287-6295), or antibodies prepared, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when using animals transgenic for human Ig sequences, to in vivo endosomal cell mutagenesis) and, thus, the V of the recombinant antibodyHAnd VLThe amino acid sequence of the region is despite being derived from human germline VHAnd VLSequences and related thereto, but may not naturally occur within the human antibody germline repertoire in vivo.
Tumor targeting
In another aspect of the invention, the recombinant bifunctional fusion proteins are useful for targeting tumor cells (e.g., solid tumor cells).
The recombinant bifunctional fusion proteins of the present invention may be conjugated (conjugated) to drugs, toxins, radioisotopes, or other substances that impair cell viability. Alternatively, the drug or toxin may be a substance that does not directly kill the cells, but makes the cells more susceptible to killing by other foreign substances. In yet other embodiments involving tumor targeting, the recombinant bifunctional fusion proteins of the present invention are not themselves conjugated to drugs, toxins, or radioisotopes, but are administered in combination with other antigen binding molecules (referred to herein as "co-ordinating molecules"), such as other anti-tumor antibodies.
According to certain embodiments of the tumor targeting aspect of the invention, the recombinant bifunctional fusion protein (or the cognate antibody) may be conjugated to one or more cytotoxic drugs selected from the group consisting of: calicheamicin, epothilones, methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin C, cisplatin, etoposide, bleomycin, 5-fluorouracil, estramustine, vincristine, etoposide, doxorubicin, paclitaxel, larotaxel, tesetaxel, oteracil (oraxel), docetaxel, dolastatin 10, auristatin E, auristatin PHE, and maytansinoid-based compounds (e.g., DM1, DM4, etc.). The recombinant bifunctional fusion protein (or the synergistic antibody) may also or alternatively be conjugated to a toxin, such as diphtheria toxin, pseudomonas aeruginosa exotoxin a, ricin a chain, abrin a chain, modeccin a chain, alpha-sarcin, aleurites fordii (aleurites fordii) protein, carnation, phytolacca americana (phytolaccaamericina) protein, or the like. The recombinant bifunctional fusion protein (or the cognate antibody) may also or alternatively be conjugated with one or more radioisotopes selected from the group consisting of:225Ac、211At、212Bi、213Bi、186Rh、188Rh、177Lu、90Y、131I、67Cu、125I、123I、77Br、153Sm、166Ho、64Cu、121Pb、224ra and223and Ra. Accordingly, this aspect of the invention includes recombinant bifunctional fusion proteins as antibody-drug conjugates (ADCs) or antibody-radioisotope conjugates (ARCs).
Pharmaceutical compositions and methods of administration
The invention also provides a composition. In a preferred embodiment, said composition is a pharmaceutical composition comprising a recombinant bifunctional fusion protein of the invention, together with a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.
The pharmaceutical composition of the present invention can be used for preventing and treating tumors (e.g., solid tumors). In addition, other therapeutic agents may also be used simultaneously.
The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the specific binding molecule of the present invention (or a conjugate thereof) and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.
In the case of pharmaceutical compositions, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
The main advantages of the invention include:
(a) the fusion protein of the invention can simultaneously target PD-L1 and IL-7 receptors.
(b) The fusion protein can obviously increase the benefit ratio of tumor patients.
(c) By fusing the IL-7 cytokine to the C end of the antibody with longer half-life period, the half-life period of the IL-7 cytokine can be obviously increased, the clinical dose is reduced, and the toxic and side effects are reduced.
(d) By fusing IL-7 cytokines to the C-terminus of an anti-PD-L1 antibody targeted to the tumor microenvironment, IL-7 can be more accurately enriched within the tumor microenvironment expressing the PD-L1 antigen to reduce toxic side effects.
(e) The fusion protein can enrich IL-7 cell factors in a tumor microenvironment, specifically activate an immune system in the tumor microenvironment, and greatly reduce toxic and side effects; in addition, the IL-7 cytokine is fused to the C end of the antibody with longer half-life period, so that the half-life period of the IL-7 cytokine can be obviously increased, the clinical dose can be reduced, and the toxic and side effects can be reduced.
The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specifying the detailed conditions in the following examples, generally followed by conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: ColdSpringHarbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Suzhou JING biopharmaceutical GmbH
<120> bifunctional fusion protein consisting of anti-PD-L1 antibody and IL-7
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Claims (10)

1. A recombinant bi-functional fusion protein, wherein the recombinant bi-functional fusion protein comprises:
a first structural unit (D1); and
a second structural unit (D2);
wherein the first building block is an antibody that specifically binds to PD-L1 protein;
the second building block comprises IL-7,
and the second building block is linked to the end of the heavy chain constant region of the first building block.
2. The fusion protein of claim 1, wherein the bifunctional fusion protein (monomer) has a structure represented by formula I from N-terminus to C-terminus:
Figure FDA0002725702600000011
wherein the content of the first and second substances,
t1 is IL-7;
l1 is an optional linking element;
VL represents the light chain variable region of an anti-PD-L1 antibody;
CL represents the light chain constant region of the anti-PD-L1 antibody;
VH represents the heavy chain variable region of an anti-PD-L1 antibody;
CH represents the heavy chain constant region of an anti-PD-L1 antibody;
"-" represents a disulfide bond or a covalent bond;
"-" represents a peptide bond.
3. An isolated polynucleotide encoding the recombinant bifunctional fusion protein of claim 1.
4. A vector comprising the polynucleotide of claim 3.
5. A host cell comprising the vector of claim 4, or having the polynucleotide of claim 3 integrated into its genome.
6. A method of producing the fusion protein of claim 1, comprising the steps of:
culturing the host cell of claim 5 under conditions suitable for expression, thereby expressing the fusion protein; and/or isolating the fusion protein.
7. An immunoconjugate, comprising:
(a) a recombinant bifunctional fusion protein according to claim 1; and
(b) a coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
8. A pharmaceutical composition comprising:
(i) a recombinant bifunctional fusion protein according to claim 1, or an immunoconjugate according to claim 7; and
(ii) a pharmaceutically acceptable carrier.
9. Use of a recombinant bifunctional fusion protein according to claim 1, a polynucleotide according to claim 3, a vector according to claim 4, a host cell according to claim 5, or an immunoconjugate according to claim 7 for the preparation of a composition or formulation for the treatment or prevention of a tumor.
10. An in vitro non-therapeutic method of inhibiting tumor growth comprising the steps of: culturing tumor cells in the presence of the fusion protein of claim 1 or the immunoconjugate of claim 7, thereby inhibiting tumor growth.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113614109A (en) * 2018-12-21 2021-11-05 Ose免疫疗法公司 Bifunctional anti-PD-1/IL-7 molecules
CN117660358A (en) * 2024-01-31 2024-03-08 青岛华赛伯曼医学细胞生物有限公司 Engineered immune cells expressing secreted fusion proteins and uses thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113614109A (en) * 2018-12-21 2021-11-05 Ose免疫疗法公司 Bifunctional anti-PD-1/IL-7 molecules
CN117660358A (en) * 2024-01-31 2024-03-08 青岛华赛伯曼医学细胞生物有限公司 Engineered immune cells expressing secreted fusion proteins and uses thereof
CN117660358B (en) * 2024-01-31 2024-05-14 青岛华赛伯曼医学细胞生物有限公司 Engineered immune cells expressing secreted fusion proteins and uses thereof

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