CN116355099A - Recombinant anti-human OX40 and PD-L1 bispecific antibody and application thereof - Google Patents

Recombinant anti-human OX40 and PD-L1 bispecific antibody and application thereof Download PDF

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CN116355099A
CN116355099A CN202211684438.7A CN202211684438A CN116355099A CN 116355099 A CN116355099 A CN 116355099A CN 202211684438 A CN202211684438 A CN 202211684438A CN 116355099 A CN116355099 A CN 116355099A
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焦莎莎
曾大地
王荣娟
张畅
王双
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Beijing Konuoxincheng Technology Co ltd
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Abstract

The invention discloses an OX40/PD-L1 bispecific antibody or antigen binding fragment and application thereof, comprising a polypeptide complex, comprising: a first polypeptide comprising a first heavy chain constant region and a first heavy chain variable region, wherein the first heavy chain constant region comprises a first CH1 domain, a first CH2 domain, and a first CH3 domain; a second polypeptide comprising a second heavy chain constant region and a second heavy chain variable region, wherein the second heavy chain constant region comprises a second CH1 domain, a second CH2 domain, and a second CH3 domain; a third polypeptide comprising a third variable region and linked to the first CH3 domain. Through conjugation and connection of different polypeptide chains of the target spots, the recognition and killing of immune cell T cells on tumor cells in vivo are enhanced, and the antitumor effect is exerted.

Description

Recombinant anti-human OX40 and PD-L1 bispecific antibody and application thereof
Technical Field
The present invention relates to the field of antibody pharmaceuticals. In particular, the invention relates to bispecific antibodies directed against human OX40 and PD-L1, particularly bispecific antibodies or antigen-binding fragments that specifically bind to human OX40 and PD-L1. The invention also relates to therapeutic and diagnostic uses of these bispecific antibodies, particularly in the treatment, prevention and/or diagnosis of human OX40 and PD-L1 related diseases, such as tumor-related diseases.
Background
Bispecific antibodies (bsabs), also known as bifunctional antibodies, recognize and bind two different antigens and epitopes simultaneously and block two different signaling pathways to exert their effects. Bispecific antibody structures can be classified into 2 major classes depending on the structure: bispecific antibodies containing an Fc fragment (IgG-like bispecific antibodies) and bispecific antibodies without an Fc fragment (non-IgG-like bispecific antibodies). IgG-like bsabs of IgG-like bispecific antibodies have an Fc portion with Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP).
At present, cancer antibody therapy has been gradually established, and becomes one of the most successful and important strategies for treating hematological malignancies and solid tumors. Such therapies may function by altering the function of the antigen or receptor (e.g., using agonists or antagonists) or coupling specific drugs to antibodies targeting a particular antigen.
However, the antibody treatment has the problem of lower effective rate in clinical single use, and for most cancer species, if the antibody treatment is not used singly, the effective rate is only 10% -20% on average. There is therefore a need to develop more efficient antibody drug molecules to meet clinical needs.
Disclosure of Invention
The invention aims to solve the problem of insufficient tumor inhibiting effect of tumor drugs in the prior art, specifically, solves the problem of low drug effect of single-target antibody drugs by preparing bispecific or multispecific antibodies, such as the technical problem of low drug effect of OX40 antibodies, and provides OX40/PD-L1 bispecific antibodies or antigen binding fragments.
The present invention has been completed based on the following work of the inventors:
OX40, also known as CD134, ACT45, TNFRSF4, a member of the tumor necrosis factor receptor (tumor necrosis factor receptor, TNFR) superfamily, is an activating receptor expressed on the surface of activated CD4+ T, CD + T cells. OX40 binds to ligand OX40L (CD 252, TNFSF 4) expressed predominantly on the surface of Antigen Presenting Cells (APC) and delivers a co-stimulatory signal. OX40 signal can activate downstream NF- κ B, PI3K and PKB pathways, and continued activation of these pathways eventually can prolong T cell survival and expand T cell memory, promoting cell killing capacity of T cells; in addition, OX40 can further enhance effector T cell function by inhibiting differentiation and activity of regulatory T cells (tregs), improving immunosuppression in tumor microenvironment. anti-OX 40-activated antibodies can exert OX 40L-like functions to activate antigen-dependent T effector cells, and can exert anti-tumor effects by abrogating the inhibitory function of Treg cells. The current clinical anti-OX 40 antibody drugs are generally poor in therapeutic effect.
The receptor for PD-L1 is programmed death protein 1 (PD-1), also known as CD279, a member of the CD28 family of T cell receptors, expressed on the surface of a variety of immune cells, such as activated T cells, B cells, monocytes, and the like. After PD-L1 is combined with its receptor PD-1, it can induce T cell apoptosis, disabling and exhaustion, and can inhibit activation, proliferation and anti-tumor function of tumor antigen specific T cell, so as to implement tumor immune escape. The PD-1/PD-L1 blocking antibody can relieve the immunosuppression effect of PD-L1, enhance the recognition and killing of immune cells such as T cells and the like in vivo on tumor cells, thereby achieving the effect of killing tumors.
Through a molecular cloning technology, the inventor carries out conjugation connection on the PD-L1 nano antibody and the OX40 antibody to obtain an OX40/PD-L1 bispecific antibody, and then examines the affinity, the binding activity, the blocking activity and other biochemical properties of the bispecific antibody, and examines the drug effect of the antibody in a humanized murine colon cancer cell MC38-hPD-L1 model.
Thus, according to one aspect of an embodiment of the invention, there is provided a polypeptide complex comprising:
a first polypeptide comprising a first heavy chain constant region and a first heavy chain variable region, wherein the first heavy chain constant region comprises a first CH1 domain, a first CH2 domain, and a first CH3 domain;
A second polypeptide comprising a second heavy chain constant region and a second heavy chain variable region, wherein the second heavy chain constant region comprises a second CH1 domain, a second CH2 domain, and a second CH3 domain;
a third polypeptide comprising a third variable region and linked to the first CH3 domain;
a fourth polypeptide comprising a fourth heavy chain variable region and linked to the second CH3 domain;
a fifth polypeptide comprising a first light chain constant region and a first light chain variable region, and the first light chain constant region is linked to the first CH1 domain and forms a first antigen binding site;
a sixth polypeptide comprising a second light chain constant region and a second light chain variable region, and wherein the second light chain constant region is linked to the second CH1 domain and forms a second antigen binding site,
wherein the first antigen binding site and the second antigen binding site each specifically bind to human OX40 and the third polypeptide and the fourth polypeptide each specifically bind to human PD-L1.
According to another aspect of an embodiment of the present invention, there is provided a polypeptide complex comprising:
A first polypeptide comprising a first heavy chain constant region and a first heavy chain variable region, wherein the first heavy chain constant region comprises a first CH1 domain, a first CH2 domain, and a first CH3 domain;
a second polypeptide comprising a second heavy chain constant region and a second heavy chain variable region, wherein the second heavy chain constant region comprises a second CH1 domain, a second CH2 domain, and a second CH3 domain;
a third polypeptide comprising a third variable region and linked to the first CH3 domain.
According to the polypeptide complex provided by the embodiment of the invention, different polypeptide chains of the polypeptide complex can simultaneously recognize and bind the same or different antigens and epitopes and block the same or different signal paths to play a role.
In addition, the antibody or antigen-binding fragment according to the above embodiment of the present invention may have the following additional technical features:
according to an embodiment of the invention, the polypeptide complex further comprises:
a fourth polypeptide comprising a fourth heavy chain variable region and linked to the second CH3 domain. The recognition and binding targets of the fourth polypeptide and the third polypeptide may be the same or different, and accordingly, the sequences of the fourth polypeptide and the third polypeptide may be the same or different. The third polypeptide and the fourth polypeptide are nanobodies or VHHs
According to an embodiment of the invention, the polypeptide complex further comprises: a fifth polypeptide comprising a first light chain constant region and a first light chain variable region, and the first light chain constant region is linked to the first CH1 domain and forms a first antigen binding site. Whereby the fifth polypeptide and the first polypeptide together form a first antigen binding site.
According to an embodiment of the invention, the polypeptide complex further comprises: a sixth polypeptide comprising a second light chain constant region and a second light chain variable region, and the second light chain constant region is linked to the second CH1 domain and forms a second antigen binding site. The first antigen binding site and the second antigen binding site may recognize and bind to the same target or different targets, and accordingly, the sixth polypeptide may be identical to the fifth polypeptide sequence or different from the fifth polypeptide sequence according to the recognized target.
Specifically, if the first antigen binding site and the second antigen binding site recognize and bind to the same target, according to an embodiment of the invention, the first light chain variable region is identical in sequence to the second light chain variable region. Also, according to an embodiment of the present invention, the first heavy chain variable region is identical in sequence to the second heavy chain variable region.
According to an embodiment of the invention, the third heavy chain variable region is identical in sequence to the fourth heavy chain variable region. Thus, the targets recognized by the third and fourth polypeptides are identical.
According to an embodiment of the invention, the third polypeptide is linked to the first CH3 domain by a flexible linker peptide.
According to an embodiment of the invention, the flexible connecting peptide comprises consecutive 4 bases G.
According to an embodiment of the invention, the flexible connecting peptide comprises a plurality of GGGGS peptide fragments.
According to an embodiment of the invention, the flexible connecting peptide comprises SEQ ID NO: 4.
According to an embodiment of the invention, the fourth polypeptide is linked to the second CH3 domain via the flexible linker peptide.
According to an embodiment of the invention, the third polypeptide and the fourth polypeptide are nanobodies or VHHs. The third polypeptide and the fourth polypeptide may be nanobody or VHH independently, that is, they may be any one of nanobody and VHH, both may be nanobody, and both may be VHH.
According to an embodiment of the invention, the blocking activity of the third polypeptide and the fourth polypeptide on specific receptors and ligands in the polypeptide complex is not less than 80% of the blocking activity of the isolated third polypeptide and fourth polypeptide. Specifically, according to some embodiments of the present invention, specific receptors and ligands are PD-1 and PD-L1.
According to an embodiment of the invention, the polypeptide complex is an IgG-like antibody.
According to an embodiment of the invention, the first polypeptide and the second polypeptide are Fab, fab ', F (ab') 2, fv, scFv, or dAb.
According to an embodiment of the invention, the complementarity determining regions (VH-CDRs) 1, 2 and 3 of the heavy chain variable regions of the first polypeptide and the second polypeptide, respectively, comprise sequences identical to SEQ ID NOs: 13. SEQ ID NO:14 and SEQ ID NO:15 or an amino acid sequence having 80% identity to SEQ ID NO: 22. SEQ ID NO:23 and SEQ ID NO:24 has an amino acid sequence with 80% identity. The at least 80% identity is any percentage identity of 80% or more, such as at least 82%, preferably at least 85%, more preferably at least 90%, even more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% identity.
Further, the sequence of the antibody or antigen binding fragment may be obtained by inserting, deleting, mutating or modifying one or more nucleotides based on the amino acid sequence shown.
According to an embodiment of the invention, the complementarity determining regions (VL-CDRs) 1, 2 and 3 of the light chain variable region of the fifth polypeptide and the sixth polypeptide, respectively, comprise sequences corresponding to SEQ ID NOs: 19. SEQ ID NO:20 and SEQ ID NO:21 or comprises an amino acid sequence having 80% identity to SEQ ID NO: 25. SEQ ID NO:26 and SEQ ID NO:27 has an amino acid sequence with 80% identity.
In some embodiments of the invention, complementarity determining regions (VH-CDRs) 1, 2 and 3 of the heavy chain variable regions of the first polypeptide and the second polypeptide, respectively, comprise sequences identical to SEQ ID NOs: 13. SEQ ID NO:14 and SEQ ID NO:15 and the complementarity determining regions (VL-CDRs) 1, 2 and 3 of the light chain variable regions of the fifth polypeptide and the sixth polypeptide, respectively, comprise amino acid sequences having 80% identity to SEQ ID NOs: 19. SEQ ID NO:20 and SEQ ID NO:21 has an amino acid sequence with 80% identity.
In some embodiments of the invention, complementarity determining regions (VH-CDRs) 1, 2 and 3 of the heavy chain variable regions of the first polypeptide and the second polypeptide, respectively, comprise sequences identical to SEQ ID NOs: 22. SEQ ID NO:23 and SEQ ID NO:24 and the complementarity determining regions (VL-CDRs) 1, 2 and 3 of the light chain variable regions of the fifth polypeptide and the sixth polypeptide, respectively, comprise amino acid sequences having 80% identity to SEQ ID NOs: 25. SEQ ID NO:26 and SEQ ID NO:27 has an amino acid sequence with 80% identity.
Further, according to some embodiments of the invention, the third polypeptide and the fourth polypeptide are nanobodies having a VHH domain but lacking a VL domain, but still being highly stable. According to an embodiment of the invention, the complementarity determining regions (VHH-CDRs) 1, 2 and 3 of the heavy chain variable region of the third polypeptide and the fourth polypeptide, respectively, comprise sequences corresponding to SEQ ID NOs: 16. SEQ ID NO:17 and SEQ ID NO:18 has an amino acid sequence with 80% identity. The VHH-CDRs sequences of the third and fourth polypeptides remain unchanged for different combinations of the first, second, fifth and sixth polypeptides.
According to an embodiment of the invention, the heavy chain variable region of the first polypeptide and the second polypeptide comprises a sequence identical to SEQ ID NO:2 or SEQ ID NO:10 has an amino acid sequence with 80% identity. Whereby the heavy chain variable region of the first polypeptide and the second polypeptide comprises a sequence identical to SEQ ID NO:2 and the heavy chain variable region comprises an amino acid sequence having 80% identity to SEQ ID NO: 13. SEQ ID NO:14 and SEQ ID NO:15 CDRs with 80% identical amino acid sequence; or, the heavy chain variable region of the first polypeptide and the second polypeptide comprises a sequence identical to SEQ ID NO:10 and the heavy chain variable region comprises an amino acid sequence having 80% identity to SEQ ID NO: 22. SEQ ID NO:23 and SEQ id no:24 CDRs with 80% identical amino acid sequence.
According to an embodiment of the invention, the light chain variable region of the fifth polypeptide and the sixth polypeptide comprises a sequence identical to SEQ ID NO:7 or SEQ ID NO:12 has an amino acid sequence with 80% identity. Whereby the light chain variable region of the fifth polypeptide and the sixth polypeptide comprises a sequence identical to SEQ ID NO:7 and the light chain variable region comprises an amino acid sequence having 80% identity to SEQ ID NO: 19. SEQ ID NO:20 and SEQ ID NO:21 CDRs with 80% identical amino acid sequence; or, the light chain variable region of the fifth polypeptide and the sixth polypeptide comprises a sequence identical to SEQ ID NO:12 and the light chain variable region comprises an amino acid sequence having 80% identity to SEQ ID NO: 25. SEQ ID NO:26 and SEQ id no:27 CDRs with 80% identical amino acid sequence.
According to some embodiments of the invention, the heavy chain variable region of the first polypeptide and the second polypeptide comprises a sequence identical to SEQ ID NO:2 or SEQ ID NO:10 and the light chain variable region of the fifth polypeptide and the sixth polypeptide comprises an amino acid sequence having 80% identity to SEQ ID NO:7 has an amino acid sequence with 80% identity.
According to some embodiments of the invention, the heavy chain variable region of the first polypeptide and the second polypeptide comprises a sequence identical to SEQ ID NO:2 or SEQ ID NO:10 and the light chain variable region of the fifth polypeptide and the sixth polypeptide comprises an amino acid sequence having 80% identity to SEQ ID NO:12 has an amino acid sequence with 80% identity.
Further, based on the above sequences of the first, second, fifth and sixth polypeptides, according to an embodiment of the invention, both the first and the second antigen binding site specifically bind to human OX40. According to an embodiment of the invention, the heavy chain variable region of the third polypeptide and the fourth polypeptide comprises a sequence identical to SEQ ID NO:5 has an amino acid sequence with 80% identity. Whereby the heavy chain variable region of the third polypeptide and the fourth polypeptide comprises a sequence identical to SEQ ID NO:5 and the heavy chain variable region comprises an amino acid sequence having 80% identity to SEQ ID NO: 16. SEQ ID NO:17 and SEQ ID NO:18 CDRs with 80% identical amino acid sequence.
Further, based on the aforementioned sequences of the third polypeptide and the fourth polypeptide, according to an embodiment of the present invention, both the third polypeptide and the fourth polypeptide specifically bind to human PD-L1.
According to an embodiment of the invention, the polypeptide complex is a bispecific antibody. In some embodiments, the first antigen binding site and the second antigen binding site bind to the same target, and the third polypeptide and the fourth polypeptide bind to another target.
According to embodiments of the invention, the polypeptide complexes specifically bind to human OX40 receptor and PD-L1. On the one hand, the OX40/PD-L1 bispecific antibody can enhance the killing effect of the T cells on the tumor cells by blocking the interaction of PD-L1 on the surface of the tumor cells and PD-1 on the surface of immune cells; on the other hand, the antigen-dependent T effector cell can be combined with OX40 so as to activate the antigen-dependent T effector cell and play an anti-tumor role.
In addition, the first antigen binding site and the second antigen binding site may bind to different targets, and the third polypeptide and the fourth polypeptide may bind to different targets, as desired by the antibody design. According to an embodiment of the invention, the polypeptide complex is a trispecific antibody or a tetraspecific antibody.
According to another aspect of the invention, there is provided a nucleic acid. According to an embodiment of the invention, the nucleic acid encodes the aforementioned polypeptide.
According to another aspect of the invention, a carrier is provided. According to an embodiment of the invention, the vector comprises the aforementioned nucleic acid.
According to another aspect of the invention, there is provided a cell. According to an embodiment of the invention, the cell comprises the aforementioned vector.
According to another aspect of the present invention there is provided a method of preparing a polypeptide complex as hereinbefore described. According to an embodiment of the present invention, the method for preparing the aforementioned polypeptide complex comprises:
culturing the aforementioned cells under suitable conditions; and
separating and recovering the polypeptide complex.
According to another aspect of the present invention, there is provided a composition. According to an embodiment of the invention, the composition comprises;
the aforementioned polypeptide complex or the aforementioned nucleic acid; and
pharmaceutically acceptable auxiliary materials.
According to another aspect of the invention, a kit is provided. According to an embodiment of the invention, the kit comprises the aforementioned polypeptide complex or the aforementioned nucleic acid.
According to an embodiment of the invention, the use of the composition for the preparation of a medicament for the prevention and treatment of cancer.
According to embodiments of the present invention, the cancer includes breast cancer, head and neck cancer, ovarian cancer, esophageal cancer, gastric cancer, hodgkin's disease, renal cancer, transitional cell carcinoma, melanoma, skin cancer, anal tumor, squamous cell carcinoma, B-cell lymphoma, metastatic non-small cell lung cancer, advanced solid tumor, and cervical cancer.
According to an embodiment of the invention, the kit is used for detecting cancer.
According to embodiments of the present invention, the cancer includes breast cancer, head and neck cancer, ovarian cancer, esophageal cancer, gastric cancer, hodgkin's disease, renal cancer, transitional cell carcinoma, melanoma, skin cancer, anal tumor, squamous cell carcinoma, B-cell lymphoma, metastatic non-small cell lung cancer, advanced solid tumor, and cervical cancer.
According to the embodiment of the invention, the inventor finds that the anti-OX 40/PD-L1 bispecific antibody can relieve the immunosuppression effect of PD-L1 by blocking the interaction of PD-L1 on the surface of tumor cells and PD-1 on the surface of immune cells, and enhance the recognition and killing of immune cells T cells in vivo on tumor cells, so as to achieve the effect of killing tumors; on the other hand, the anti-tumor effect can be exerted by combining and activating OX40 so as to activate antigen-dependent T effector cells and by eliminating the inhibition function of Treg cells, and a better clinical treatment effect is expected.
For a better understanding of the invention, some terms are first defined. Other definitions are set forth throughout the detailed description.
Unless otherwise indicated, the term "immunoglobulin sequence" is used as a generic term, whether used herein to refer to a heavy chain antibody or to a conventional 4-chain antibody, including full-size antibodies, individual chains thereof, and all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments, such as VHH domains or VH/VL domains, respectively). Furthermore, the term "sequence" (e.g. in terms of "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "VHH sequence" or "protein sequence") as used herein is generally understood to include both the relevant amino acid sequence as well as the nucleic acid or nucleotide sequence encoding it, unless the context requires a more restrictive interpretation.
An immunoglobulin single variable domain may be used as a "binding unit", "binding domain" or "building block" (these terms being used interchangeably) for preparing a polypeptide containing one or more additional immunoglobulin single variable domains that may act as binding units (i.e., for the same or different epitopes of the same target and/or for one or more different targets).
The terms "conjugate," "linked" and "coupled" refer to the association of two or more molecules. Ligation may also be genetic (i.e., recombinant fusion). In a specific context, the term includes reference to linking a ligand (e.g., an antibody moiety) to an effector molecule. The ligation may be accomplished using a variety of art-recognized techniques, such as by chemical or recombinant means. "chemical means a reaction between the antibody moiety and an effector molecule such that a covalent bond is formed between the two molecules to form one molecule.
The terms "vector" and "nucleic acid construct" refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, in which additional DNA segments may be ligated into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and an episomal mammalian vector). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA technology are typically in the form of plasmids. However, other forms of expression vectors are also included, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
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Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to designate like parts throughout the figures. In the drawings:
FIG. 1 shows a schematic diagram of anti-PD-L1/OX 40 bispecific antibody architecture according to one embodiment of the invention;
FIG. 2 shows graphs of affinity assays for hz27G12-hzF2 with human OX40 recombinant proteins according to one embodiment of the invention;
FIG. 3 shows graphs of the results of an affinity assay for hz25A7-hzF2 with human OX40 recombinant protein according to one embodiment of the invention;
FIG. 4 shows graphs of the affinity assay of hz27G12-hzF2 with human PD-L1 recombinant proteins according to one embodiment of the invention;
FIG. 5 shows graphs of the affinity assay results for hz25A7-hzF2 with human PD-L1 recombinant proteins according to one embodiment of the invention;
FIG. 6 shows ELISA assays for binding activity of anti-PD-L1/OX 40 bispecific antibodies to human OX40 extracellular domain recombinant protein according to one embodiment of the invention;
FIG. 7 shows ELISA assays for binding activity of anti-PD-L1/OX 40 bispecific antibodies to human PD-L1 extracellular domain recombinant protein according to one embodiment of the invention;
FIG. 8 shows ELISA assays for blocking activity of anti-PD-L1/OX 40 bispecific antibodies against PD-L1 binding to PD-1 according to one embodiment of the invention;
FIG. 9 shows a graph of the results of a block activity assay of an anti-PD-L1/OX 40 bispecific antibody against a PD-L1/PD-1 signaling pathway according to one embodiment of the invention;
FIG. 10 shows graphs of the results of an analysis of anti-PD-L1/OX 40 bispecific antibodies stimulated HT1080-huOX40 cells to express IL8 according to one embodiment of the invention;
FIG. 11 shows inhibition of growth of colon cancer tumor by anti-PD-L1/OX 40 bispecific antibodies to subcutaneous transplantation of MC38-hPD-L1 murine colon cancer into OX40 humanized transgenic mice according to one embodiment of the invention;
FIG. 12 shows that an OX40 humanized transgenic mouse subcutaneously transplanted MC38-hPD-L1 murine colon carcinoma tumor weight, according to one embodiment of the invention;
FIG. 13 shows a schematic of anti-PD-L1/OX 40 bispecific antibody architecture according to one embodiment of the invention;
FIG. 14 shows mouse weight changes in an OX40 humanized transgenic mouse subcutaneously transplanted with MC38-hPD-L1 murine colon cancer tumor model according to one embodiment of the invention.
Detailed Description
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not noted in the examples and are carried out according to the techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, code Huang Peitang et al, molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used are not manufacturer specific and are conventional products commercially available, for example, from Sigma company.
Example 1: anti-PD-L1/OX 40 bispecific antibody construction
This example utilizes molecular cloning techniques to construct anti-PD-L1/OX 40 bispecific antibodies hz27G12-hzF2 and hz25A7-hzF2. The bispecific antibody format contains four polypeptide chains and can bind to both OX40, PD-L1 antigens. The parent antibodies used to construct the bispecific antibodies were anti-OX 40 humanized antibodies hz27G12, hz25A7 (Chinese patent application number: CN 202010063141.3); anti-PD-L1 humanized nanobody hzF (Chinese patent application No. CN 202010324761.8). The structure of the antibody is shown in FIG. 13, and consists of four polypeptide chains which are bilaterally symmetrical, wherein the left half part consists of a peptide chain 1 and a peptide chain 2, and the right half part consists of the peptide chain 1 and the peptide chain 2. The amino acid sequence of the peptide chain 1 of the bispecific antibody of hz27G12-hzF is shown as SEQ ID NO. 1, and the amino acid sequence of the peptide chain 1 comprises the amino acid sequence of the heavy chain variable region of the anti-OX 40 antibody hz27G12 shown as SEQ ID NO. 2, the amino acid sequence of the heavy chain constant region shown as SEQ ID NO. 3, the amino acid sequence of the (G4S) 4G flexible connecting peptide chain shown as SEQ ID NO. 4 and the amino acid sequence of the anti-PD-L1 nano-antibody shown as SEQ ID NO. 5 from the N end to the C end; the amino acid sequence of the peptide chain 2 of the hz27G12-hzF2 bispecific antibody is shown as SEQ ID NO. 6, and the amino acid sequence of the peptide chain 2 comprises the amino acid sequence of the light chain variable region of the anti-OX 40 antibody hz27G12 shown as SEQ ID NO. 7 and the amino acid sequence of the light chain constant region shown as SEQ ID NO. 8 from the N end to the C end. The amino acid sequence of the peptide chain 1 of the bispecific antibody of hz25A7-hzF is shown as SEQ ID NO 9, and the amino acid sequence of the peptide chain 1 comprises the amino acid sequence of the heavy chain variable region of the anti-OX 40 antibody hz25A7 shown as SEQ ID NO 10, the amino acid sequence of the heavy chain constant region shown as SEQ ID NO 3, the amino acid sequence of the (G4S) 4G flexible connecting peptide chain shown as SEQ ID NO 4 and the amino acid sequence of the anti-PD-L1 nano-antibody shown as SEQ ID NO 5 from the N end to the C end; the amino acid sequences of the second polypeptide and the fourth polypeptide peptide chain 2 of the hz25A7-hzF2 bispecific antibody are shown as SEQ ID NO. 11, and the amino acid sequences of the peptide chain 2 comprise the amino acid sequence of the variable region of the light chain of the anti-OX 40 antibody hz25A7 shown as SEQ ID NO. 12 and the amino acid sequence of the constant region of the light chain shown as SEQ ID NO. 8 from the N end to the C end.
The specific construction and preparation method is as follows: the nucleotide sequences encoding the above-described peptide chains of bispecific antibodies hz27G12-hzF2 and hz25A7-hzF2 were synthesized. The synthesized nucleotide sequences encoding the peptide chains were ligated into the vector pcdna3.1, respectively, using restriction enzymes and ligases, to obtain recombinant vectors each containing the nucleotide sequences encoding the peptide chains. The recombinant vector was verified to be correct by sequencing for subsequent expression. The prepared recombinant plasmid respectively comprising the nucleotide sequences of the coding peptide chain 1 or the peptide chain 2 is simultaneously transferred into HEK293 cells for recombinant expression, and after 5-6 days of cell transfection, the culture supernatant is taken and purified by using a ProteinA affinity chromatography column to obtain the recombinant antibody.
SEQ ID NO:1
EVQLVQSGAEVKKPGASVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSIKYNEKFKDRVTITRDTSTSTVYMELSSLRSEDTAVYYCARHEDPITFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGEVQLVESGGGLVQPGGSLRLSCAASRDSDEGASCMGWFRQAPGKEREGVAIIFNAGERTDYGDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCATVWCGSWVARSWGQGTLVTVSS
SEQ ID NO:2
EVQLVQSGAEVKKPGASVKVSCKASGYTFTEYIIHWVRQAPGQGLEWIGWFYPESGSIKYNEKFKDRVTITRDTSTSTVYMELSSLRSEDTAVYYCARHEDPITFAYWGQGTLVTVSS
SEQ ID NO:3
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
SEQ ID NO:4
GGGGSGGGGSGGGGSGGGGSG
SEQ ID NO:5
EVQLVESGGGLVQPGGSLRLSCAASRDSDEGASCMGWFRQAPGKEREGVAIIFNAGERTDYGDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCATVWCGSWVARSWGQGTLVTVSS
SEQ ID NO:6
EIVMTQSPATLSVSPGERATLSCKASQSVDYEGDSYMNWYQQKPGQAPKLLIYAASNLESGIPARFSGSGSGTDFTLTISSLEPEDFATYYCQQSNEDPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:7
EIVMTQSPATLSVSPGERATLSCKASQSVDYEGDSYMNWYQQKPGQAPKLLIYAASNLESGIPARFSGSGSGTDFTLTISSLEPEDFATYYCQQSNEDPYTFGGGTKVEIK
SEQ ID NO:8
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:9
EVQLVESGGGLVQPGRSLRLSCAASGFTFSSHDMSWVRQAPGKGLELVAAINSEGGRIYYPDTMERRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHYDNYAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGEVQLVESGGGLVQPGGSLRLSCAASRDSDEGASCMGWFRQAPGKEREGVAIIFNAGERTDYGDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCATVWCGSWVARSWGQGTLVTVSS
SEQ ID NO:10
EVQLVESGGGLVQPGRSLRLSCAASGFTFSSHDMSWVRQAPGKGLELVAAINSEGGRIYYPDTMERRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHYDNYAWFAYWGQGTLVTVSS
SEQ ID NO:11
DIQMTQSPSSLSASVGDRVTITCRASKSVSTSGSSYIHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSRELPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:12
DIQMTQSPSSLSASVGDRVTITCRASKSVSTSGSSYIHWYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSRELPLTFGGGTKVEIK
Example 2: anti-PD-L1/OX 40 bispecific antibody affinity assay
anti-PD-L1/OX 40 bispecific antibody affinity was determined using an Octet QKe system instrument from Fortebio, using a method of capturing the Fc fragment of an anti-human antibody with a capture Antibody (AHC) biological probe. The antibodies hz25A7-hzF2 and hz27G12-hzF2 were diluted to 4. Mu.g/ml with PBS buffer and passed over the AHC probe surface for 240s. The human OX40 extracellular domain fusion protein or the human PD-L1 extracellular domain fusion protein was used as mobile phase, binding time 300s, dissociation time 300s. After the experiment was completed, 1 was performed with software: 1Langmuir binding pattern fitting, the kinetic constants of antigen-antibody binding were calculated.
The response curves of hz25A7-hzF2 and hz27G12-hzF2 to human OX40 recombinant protein (10481-H08H, beijing Yiqiao Shenzhou) are shown in FIGS. 2 and 3, and the affinity is calculated by fitting the curves, which shows that the affinities (KD) of hz25A7-hzF2 and hz27G12-hzF2 are respectively: 5nM and 8nM. The response curves of hz25A7-hzF2 and hz27G12-hzF2 to human PD-L1 recombinant protein (10084-H08H, beijing Yiqiao Shenzhou) are shown in FIGS. 4 and 5, the curves are fitted and affinities calculated, and the results show that the affinities (KD) of hz25A7-hzF2 and hz27G12-hzF2 are respectively: 1nM and 1.3nM. Detailed kinetic parameters As shown in Table 1, both hz25A7-hzF2 and hz27G12-hzF2 can bind effectively to OX40 and PD-L1.
TABLE 1 affinity assay results of anti-PD-L1/OX 40 bispecific antibodies with human OX40/PD-L1 recombinant protein
Figure BDA0004019255260000151
Example 3: ELISA detection of anti-PD-L1/OX 40 bispecific antibodies and recombinant protein binding Activity
Human OX40 extracellular domain recombinant protein (OX 40-His) was diluted to 1. Mu.g/mL with PBS, 100. Mu.l/well, coated on an ELISA plate, coated overnight at 4 ℃; blocking 120min with 5% BSA blocking solution at 37deg.C, and washing with PBST plate for 3 times; adding gradient diluted hz25A7-hzF2, hz27G12-hzF2, hz25A7, hz27G12 and NC-huIgG1 samples to be tested (66 nM initial, 3-fold gradient diluted 12 gradients), reacting at 37 ℃ for 60min, and washing the PBST plate for 4 times; adding HRP-anti-human Fc (Cat: 109-035-098,Jackson Immuno Research) diluted by 1:5000, reacting at 37deg.C for 45min, and washing the PBST plate for 4 times; finally, TMB substrate is added for color development, a constant temperature incubator at 37 ℃ is used for reacting for 15min,2M HCl is used for stopping the reaction, and the absorbance of the pore plate at the wavelength of 450nm is read and recorded. As shown in FIG. 6 and Table 2, both hz25A7-hzF2 and hz27G12-hzF2 specifically bind to OX40 recombinant proteins at half-maximal effective binding concentrations (EC 50) of 0.0877nM and 0.0781nM, respectively, consistent with the corresponding mAb.
Human PD-L1 extracellular region recombinant protein (PD-L1-His) was diluted to 1. Mu.g/mL with PBS, 100. Mu.l/well, coated on an ELISA plate, and coated overnight at 4 ℃; blocking 120min with 5% BSA blocking solution at 37deg.C, and washing with PBST plate for 3 times; adding gradient diluted hz25A7-hzF2, hz27G12-hzF2, hzF2 and NC-huIgG1 sample to be tested (66 nM initial, 3-fold gradient diluted 12 gradients), reacting at 37 ℃ for 60min, and washing the PBST plate for 4 times; adding HRP-anti-human Fc (Cat: 109-035-098,Jackson Immuno Research) diluted by 1:5000, reacting at 37deg.C for 45min, and washing the PBST plate for 4 times; finally, TMB substrate is added for color development, a constant temperature incubator at 37 ℃ is used for reacting for 15min,2M HCl is used for stopping the reaction, and the absorbance of the pore plate at the wavelength of 450nm is read and recorded. As a result, as shown in FIG. 7 and Table 2, both hz25A7-hzF2 and hz27G12-hzF2 can specifically bind to PD-L1 recombinant proteins at half-effective binding concentrations (EC 50) of 0.5633nM and 0.6506nM, respectively.
TABLE 2 binding Activity of anti-PD-L1/OX 40 bispecific antibodies to human OX40/PD-L1 extracellular region recombinant protein (EC 50: nM)
Figure BDA0004019255260000161
Example 4: ELISA detection of blocking Activity of anti-PD-L1/OX 40 bispecific antibodies against recombinant PD-L1 and PD-1
Human PD-1 extracellular domain recombinant protein (PD-1-hFc) was diluted to 1. Mu.g/mL with PBS, 100. Mu.l/well, coated on an ELISA plate, coated overnight at 4 ℃; blocking 120min with 5% BSA blocking solution at 37deg.C, and washing with PBST plate for 3 times; 50. Mu.L of hz25A7-hzF, hz27G12-hzF2, hzF2, NC-huIgG1 samples to be tested (66 nM,44nM,29.3nM,19.6nM,13nM,8.7nM,5.8nM,3.9nM,2.6nM,1.7nM,1.1nM,0.8 nM) were added to the ELISA plate, then 50. Mu.L of 1ug/ml PD-L1-mFc was added, multiple wells were set for each concentration point, and after mixing, the reaction was performed at 37℃for 60min, and the PBST plate was washed 4 times; adding HRP-anti-mouse IgG (Cat: 115-035-071,Jackson Immuno Research) diluted by 1:5000 for reaction for 45min, and washing the PBST plate for 4 times; finally, TMB substrate was added for color development, the reaction was stopped by a constant temperature incubator at 37℃for 15min and 2M HCl, and the absorbance of the well plate at a wavelength of 450nm was read and recorded, and the results are shown in FIG. 8 and Table 3.
TABLE 3 blocking Activity of anti-PD-L1/OX 40 bispecific antibodies against PD-L1 binding to PD-1 (IC 50: nM)
Figure BDA0004019255260000171
Both hz25A7-hzF2 and hz27G12-hzF2 can specifically block the binding of PD-L1 and PD-1, the half-effective inhibitory concentrations (IC 50) are 6.840nM and 7.118nM respectively, and the blocking activity is consistent with that of the corresponding monoclonal antibody hzF on the PD-L1 side.
Example 5: reporter gene system to detect blocking activity of anti-PD-L1/OX 40 bispecific antibodies against PD-1/PD-L1
Programmed cell death protein 1 (PD-1) is a receptor expressed on activated T cells and its binding to ligands PD-L1 and PD-L2 has a negative regulatory effect on the immune response. The TCR on the surface of CHO-K1 cells has positive regulation effect and can activate NFAT channel in T cells. When both cells are co-cultured, the PD-1/PD-L1 interaction inhibits T cell activity, allowing cancer cells to evade immune surveillance. When antibodies block this pathway, the NFAT pathway within T cells can mediate substrate luminescence.
CHO-PD-L1-CD3L cells, pancreatin digestion, and complete medium to terminate the reaction. Cell count, cell density was adjusted to 4x10 with complete medium 5 cell/ml, 100. Mu.L per well plating。(4×10 4 cell/well), 37 ℃,5% co 2 Culturing overnight; jurkat-PD1-NFAT cell count, centrifugation at 1000rpm for 5min, working medium resuspension, cell density adjusted to 1.2X10 6 cell/ml; CHO-PD-L1-CD3L cell supernatants were discarded, gradient diluted hz25A7-hzF2 (55 nM start, 2-fold gradient diluted 9 gradients), hz27G12-hzF2 (55 nM start, 2-fold gradient diluted 9 gradients), hzF2 (62.5 nM start, 2-fold gradient diluted 9 gradients), NC-huIgG1 (66.6 nM start, 2-fold gradient diluted 9 gradients) samples were added to 96-well white cell plates, 50 μl/well, and multiplex wells were set for each concentration point. Standing at room temperature for 1h; 50 μl/well of the prepared Jurkat-PD1-NFAT cell suspension was added to the corresponding position in a 96-well plate (6×10) 4 cell/well), after plating of Jurkat-PD1-NFAT cells, the white 96-well plate was placed in a 5% co2 incubator at 37 ℃ for 6h; the Bio-Lite Luciferase Assay System substrate is taken out and melted 1-2 h in advance, and the substrate is placed to room temperature in a dark place at room temperature. The cell plates were removed from the incubator, equilibrated to room temperature (about 10-15 min), and Bio-Lite substrate was added to the cell plates, and incubated for 5min at 50 μl/well in the absence of light. The RLU was read using an microplate reader for the Luminescence mode, intersystem selection 100, and the detection results are shown in fig. 9 and table 4.
TABLE 4 blocking Activity of anti-PD-L1/OX 40 bispecific antibodies against PD-1/PD-L1 Signal pathway (IC 50: nM)
Figure BDA0004019255260000181
The results show that both hz25A7-hzF2 and hz27G12-hzF2 can definitely block the PD-1/PD-L1 signal path, the half-effective inhibition concentrations (IC 50) are respectively 4.75nM and 3.38nM, and the blocking effect (IC 50) is consistent with that of the monoclonal antibody hzF2 corresponding to the PD-L1 side.
Example 6: anti-PD-L1/OX 40 bispecific antibody activation Activity assay
The activation activity of OX 40-side antibodies against PD-L1/OX40 bispecific antibodies was evaluated using a reporter system. HT1080-hOX40 cells were digested the day prior to the experiment, resuspended in complete medium (1640 medium+10% FBS+0.5 μg/mL) and conditionedCell density to 1x10 5 mu.L of cell suspension was added per well to a 96-well cell culture plate at a concentration of 200/mL. The cell culture plates were then placed in a 5% CO2 incubator at 37℃overnight. The next day the antibodies to be tested hz25A7-hzF2, hz27G12-hzF2 were diluted to the appropriate concentration (30. Mu.g/mL start, 3-fold gradient diluted 8 gradients) and added to the cell culture plates and incubated for 6 hours at 37℃in a 5% CO2 incubator. Control groups (hz 25A7, hz27G12, NC-huIgG 1) were also set. Finally, the cell culture supernatant was aspirated, and the content of IL-8 in the supernatant was measured by using an IL-8ELISA kit according to the specification, and the results are shown in FIG. 10 and Table 5.
TABLE 5 anti-PD-L1/OX 40 bispecific antibodies stimulate IL8 activity (EC 50: μg/mL) in HT1080-huOX40 cells
Figure BDA0004019255260000182
Hz25A7-hzF2 and Hz27G12-hzF2 can well activate up-regulation of IL8 expression of HT1080-hOX40 cells, so that the level of the expressed IL8 is increased, and half-effective binding concentrations (EC 50) are 0.247 mug/mL and 2.281 mug/mL respectively. And the activity of the hz25A7-hzF2 bispecific antibody is basically equivalent to that of the corresponding monoclonal antibody hz25A7, and the activity of the hz27G12-hzF2 bispecific antibody is slightly weaker than that of the corresponding monoclonal antibody hz27G12 under the same molar concentration.
Example 7: pharmacodynamics evaluation of OX40 humanized mice and humanized mice colon cancer cells MC38-hPD-L1 model
Murine colon cancer tumor cells MC38-hPD-L1 (1×10) highly expressing human PD-L1 were isolated 6 cell/mouse) was inoculated into right anterior hypochondriac subcutaneous area of female B6-hOX40 humanized mice (C57 BL/6-derived human OX40 transgenic mice) and grown to 100mm in tumor 3 The left and right groups were administered in groups of 3, 6 each, and the modes of administration were as follows in Table 6:
table 6 administration of OX40 humanized transgenic mice humanized murine colon cancer cells MC38-hPD-L1 model
Figure BDA0004019255260000191
Tumor volume and body weight were measured 2 times a week, and tumor-bearing mice body weight and change in tumor volume were recorded as a function of time of administration.
The test results are shown in tables 7, 11, 12 and 14, and on the pharmacodynamic model of the OX40 transgenic mice, the effect of the tested drug hz27G12-hzF is better than that of hz27G12 and NC-huIgG1, and the tested drug shows good tumor inhibition activity, almost all 6 mice are completely inhibited, the weight of the mice is slowly increased, and the drug has no obvious toxic or side effect.
As mentioned finally, the bispecific antibodies of hz25A7-hzF2 and hz27G12-hzF2 of the examples of the invention have high affinity for human PD-L1 and OX40 and an affinity (KD) for human OX40 recombinant protein of 5nM and 8nM, respectively; the affinities (KD) for human PD-L1 recombinant proteins were 1nM and 1.3nM, respectively. Meanwhile, hz25A7-hzF2 and hz27G12-hzF2 can specifically block the combination of PD-1/PD-L1 at the molecular level; at the cellular level, hz27G12-hzF2 can inhibit downstream signaling pathway activity by PD-1/PD-L1 production, and can activate OX40 signaling pathway, exerting immune activating activity; the hz27G12-hzF2 has good tumor inhibiting effect in an in-vivo efficacy experiment.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents.

Claims (25)

1. A polypeptide complex comprising:
a first polypeptide comprising a first heavy chain constant region and a first heavy chain variable region, wherein the first heavy chain constant region comprises a first CH1 domain, a first CH2 domain, and a first CH3 domain;
A second polypeptide comprising a second heavy chain constant region and a second heavy chain variable region, wherein the second heavy chain constant region comprises a second CH1 domain, a second CH2 domain, and a second CH3 domain;
a third polypeptide comprising a third variable region and linked to the first CH3 domain;
a fourth polypeptide comprising a fourth heavy chain variable region and linked to the second CH3 domain;
a fifth polypeptide comprising a first light chain constant region and a first light chain variable region, and the first light chain constant region is linked to the first CH1 domain and forms a first antigen binding site;
a sixth polypeptide comprising a second light chain constant region and a second light chain variable region, and wherein the second light chain constant region is linked to the second CH1 domain and forms a second antigen binding site,
wherein the first antigen binding site and the second antigen binding site each specifically bind to human OX40 and the third polypeptide and the fourth polypeptide each specifically bind to human PD-L1.
2. The polypeptide complex of claim 1, wherein the first light chain variable region is identical in sequence to the second light chain variable region.
3. The polypeptide complex of claim 1, wherein the first heavy chain variable region is identical in sequence to the second heavy chain variable region.
4. The polypeptide complex of claim 1 wherein the third heavy chain variable region is identical in sequence to the fourth heavy chain variable region.
5. The polypeptide complex of claim 1, wherein the third polypeptide is linked to the first CH3 domain by a flexible linker peptide,
optionally, the flexible connecting peptide comprises consecutive 4 bases G, preferably the flexible connecting peptide comprises a plurality of GGGGS peptide fragments, more preferably the flexible connecting peptide comprises the amino acid sequence of SEQ ID NO: 4.
6. The polypeptide complex of claim 5, wherein the fourth polypeptide is linked to the second CH3 domain via the flexible linker peptide.
7. The polypeptide complex of claim 1, wherein the third polypeptide and the fourth polypeptide are nanobodies or VHHs.
8. The polypeptide complex of claim 1, wherein the blocking activity of the third polypeptide and the fourth polypeptide in the polypeptide complex for specific receptors and ligands is not less than 80% of the blocking activity of the isolated third polypeptide and fourth polypeptide.
9. The polypeptide complex of claim 1, wherein the polypeptide complex is an IgG-like antibody.
10. The polypeptide complex of claim 1, wherein the first polypeptide and the second polypeptide are Fab, fab ', F (ab') 2, fv, scFv, or dAb.
11. The polypeptide complex of claim 3, wherein complementarity determining regions (VH-CDRs) 1, 2 and 3 of the heavy chain variable regions of the first polypeptide and the second polypeptide, respectively, comprise sequences identical to SEQ id nos: 13. SEQ ID NO:14 and SEQ ID NO:15 or an amino acid sequence having 80% identity to SEQ ID NO: 22. SEQ ID NO:23 and SEQ ID NO:24 has an amino acid sequence with 80% identity.
12. The polypeptide complex of claim 1, wherein complementarity determining regions (VL-CDRs) 1, 2 and 3 of the light chain variable regions of the fifth polypeptide and the sixth polypeptide, respectively, comprise sequences corresponding to SEQ id nos: 19. SEQ ID NO:20 and SEQ ID NO:21 or comprises an amino acid sequence having 80% identity to SEQ ID NO: 25. SEQ ID NO:26 and SEQ ID NO:27 has an amino acid sequence with 80% identity.
13. The polypeptide complex of claim 4, wherein complementarity determining regions (VHH-CDRs) 1, 2 and 3 of the heavy chain variable regions of the third polypeptide and the fourth polypeptide, respectively, comprise sequences corresponding to SEQ id nos: 16. SEQ ID NO:17 and SEQ ID NO:18 has an amino acid sequence with 80% identity.
14. The polypeptide complex of claim 1, wherein the heavy chain variable regions of the first polypeptide and the second polypeptide comprise a sequence that hybridizes to SEQ ID NO:2 or SEQ ID NO:10 has an amino acid sequence with 80% identity.
15. The polypeptide complex of claim 1, wherein the light chain variable regions of the fifth polypeptide and the sixth polypeptide comprise sequences identical to SEQ ID NO:7 or SEQ ID NO:12 has an amino acid sequence with 80% identity.
16. The polypeptide complex of claim 1, wherein the heavy chain variable regions of the third polypeptide and the fourth polypeptide comprise a sequence that hybridizes to SEQ ID NO:5 has an amino acid sequence with 80% identity.
17. The polypeptide complex of claim 1, wherein the polypeptide complex is a bispecific antibody.
18. The polypeptide complex of claim 1, wherein the polypeptide complex is a trispecific antibody or a tetraspecific antibody.
19. A nucleic acid encoding the polypeptide of any one of claims 1-18.
20. A vector comprising the nucleic acid of claim 19.
21. A cell comprising the vector of claim 20.
22. A method of making the polypeptide complex of claims 1-18, comprising:
(1) Culturing the cell of claim 21 under suitable conditions; and
(2) Isolating and recovering the polypeptide complex of claims 1-18.
23. A composition, comprising;
the polypeptide complex of claims 1-18 or the nucleic acid of claim 19; and
pharmaceutically acceptable auxiliary materials.
24. Use of the composition of claim 23 in the manufacture of a medicament for the prevention and treatment of cancer.
25. The use according to claim 24, wherein the cancer comprises breast cancer, head and neck cancer, ovarian cancer, esophageal cancer, gastric cancer, hodgkin's disease, renal cancer, transitional cell carcinoma, melanoma, skin cancer, anal tumor, squamous cell carcinoma, B-cell lymphoma, metastatic non-small cell lung cancer, advanced solid tumor and cervical cancer.
CN202211684438.7A 2021-12-28 2022-12-27 Recombinant anti-human OX40 and PD-L1 bispecific antibody and application thereof Pending CN116355099A (en)

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