Patent Application
20240109974
The present application claims the right of priority for Chinese patent application No. 202110140980.5, entitled “HUMANIZED ANTIBODY AGAINST TNFR2 AND USE THEREOF” and submitted to the China National Intellectual Property Administration on Jan. 29, 2021, and for Chinese patent application No. 202111016307.7, entitled “HUMANIZED ANTIBODY AGAINST TNFR2 AND USE THEREOF” and submitted to the China National Intellectual Property Administration on Aug. 31, 2021, the contents of which are all incorporated herein by reference in their entireties.
The present invention relates to the fields of biomedicine and bioengineering, in particular to a humanized antibody against TNFR2 or an antigen-binding fragment thereof a pharmaceutical composition of the humanized antibody against TNFR2 or antigen-binding fragment thereof and the use thereof.
Immunity is a protective response of the body, which is affected by many genes, proteins and cells. Immune abnormalities may cause many diseases, including tumours, immune deficiencies (e.g., acquired immune deficiency syndrome), allergies, rheumatoid arthritis and other diseases. In the past few years, tumour immunotherapy, as a brand-new treatment method, has become a hot spot in the field of tumour treatment research. Antagonistic antibodies targeting immune checkpoint proteins, such as anti-PD-1 and anti-CTLA-4 monoclonal antibodies, have been used to treat many types of cancers, and have achieved revolutionary results, greatly prolonging the life expectancy of patients with malignant tumours. However, there are still many cancer patients who do not respond to treatment with antagonistic antibodies against immune checkpoint proteins or develop resistance or drug resistance after short-term treatment. Therefore, it is necessary to develop new drugs for treating cancers, which can be used alone or in combination with other tumour treatment methods, including in combination with antagonistic antibodies against immune checkpoint proteins, to further improve the efficacy and safety.
The present inventors have found that human TNFR2 is overexpressed on the surface of human Tregs and various human tumour cells, suggesting that human TNFR2 may promote the tumourigenesis in patients and mediate the immunosuppression and immune escape in the tumour microenvironment. It may be a very potential anti-tumour strategy to regulate the function of Treg cells by TNFR2, thus inhibiting the tumourigenesis. The present inventors have prepared an antagonistic antibody against TNFR2, which can: 1) specifically bind to TNFR2 and block the binding of TNFR2 to the ligand TNFα thereof, thus inhibiting the proliferation of Treg cells and as a result the inhibitory function mediated thereby, and promoting the expansion of responder T cells and the anti-tumour function mediated by responder T cells and other immune cells; 2) directly mediate the killing effect on tumour cells with high TNFR2 expression, given that TNFR2 is highly expressed in human tumour cell lines; 3) have a better anti-tumour effect and form prolonged immune memory when used in combination with existing anti-PD-1/L1 antibodies. For the description of the above-mentioned antagonistic antibody against TNFR2, reference can be made to the patent application PCT/CN2020/106057, which is incorporated into the present application by reference in its entirety.
In the present invention, a humanized antibody against TNFR2 or an antigen-binding fragment thereof, and a pharmaceutical composition of the humanized antibody against TNFR2 or antigen-binding fragment thereof are prepared, and the use thereof is verified.
In a first aspect, the present invention provides a humanized antibody that specifically binds to TNFR2, or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region;
In certain embodiments, in the antibody or antigen-binding fragment thereof of the present invention, the heavy chain variable region comprises a VH sequence with a CDR identical to and an FR having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to those of any of SEQ ID NOs: 19-27; the light chain variable region comprises a VL sequence with a CDR identical to and an FR having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to those of any of SEQ ID NOs: 28-37.
In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region selected from the group consisting of
In certain embodiments, in the antibody or antigen-binding fragment thereof of the present invention, the antibody or antigen-binding fragment thereof comprises: CDR1-VH selected from SEQ ID NO: 1, 7, or 13; CDR2-VH selected from SEQ ID NO: 2, 8, or 14; CDR3-VH selected from SEQ ID NO: 3, 9, or 15; CDR1-VL selected from SEQ ID NO: 4, 10, or 16; CDR2-VL selected from SEQ ID NO: 5, 11, or 17; and CDR3-VL selected from SEQ ID NO: 6, 12, or 18.
In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention binds to human TNFR2 with a dissociation constant (KD) no more than 7 nM, and to cynomolgus monkey TNFR2 with a dissociation constant (KD) no more than 5 nM.
In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises or does not comprise a heavy chain constant region and/or a light chain constant region; preferably, the heavy chain constant region comprises a full-length heavy chain constant region or a fragment thereof and the fragment can be selected from a CH1 domain, an Fc domain or a CH3 domain; preferably, the heavy chain constant region and/or the light chain constant region are/is a human heavy chain constant region and/or a human light chain constant region; preferably, the heavy chain constant region can be selected from an IgG heavy chain constant region, such as an IgG1 heavy chain constant region, an IgG2 heavy chain constant region, an IgG3 heavy chain constant region or an IgG4 heavy chain constant region; preferably, the heavy chain constant region is a human IgG1 heavy chain constant region, a human IgG2 heavy chain constant region, a human IgG3 heavy chain constant region or a human IgG4 heavy chain constant region.
In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a full-length antibody, an antibody fragment, Fab, Fab′, F(ab′)2, Fd, Fv, scFv, or a diabody.
In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention is coupled with another molecule via or not via a linker; preferably, the another molecule can be selected from a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a radioisotope, a chemotherapeutic drug or an immunomodulator, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescence label, an ultrasonic contrast agent or a photosensitizer.
In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention has the following properties:
(1) specifically binding to a cell expressing TNFR2 on the cell surface; (2) specifically binding to a Treg cell; (3) inhibiting the binding of TNFα to a TNFR2 protein; (4) inhibiting the binding of TNFα to TNFR2 expressed on the cell surface; (5) inhibiting the Treg proliferation and/or Treg function mediated by TNFα; (6) inhibiting the degradation of IκBα mediated by TNFα; (7) inhibiting the inhibition of Tcon cell proliferation by Treg; (8) mediating the ADCC function against a TNFR2-expressing cell; (9) increasing the ratio of CD8+T/Treg cells in tumour infiltrating lymphocytes, TILs; or/and (10) inhibiting the tumour growth.
In a second aspect, the present invention provides a multispecific antigen-binding molecule, wherein the multispecific antigen-binding molecule comprises at least a first antigen-binding module and a second antigen-binding module, wherein the first antigen-binding module comprises the antibody or antigen-binding fragment thereof of the present invention, and the second antigen-binding module specifically binds to other antigens than TNFR2 or binds to a different TNFR2 antigenic epitope from the first antigen-binding module; preferably, the other antigens are selected from CD3, CD4, CD5, CD8, CD14, CD15, CD16, CD16A, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD70, CD74, CD80, CD86, CD126, CD138, B7, PD-1, PD-L1, PD-2, CTLA4, PRVIG, TIGHT, HAS, CLDN18.2, MSLN, MUC, Ia, HLA-DR, tenascin, EGFR, VEGF, P1GF, ED-B fibronectin, an oncogene product, IL-2, IL-6, IL-15, IL-21, TRAIL-R1 or TRAIL-R2; preferably, the multispecific antibody is “bispecific”, “trispecific” or “teraspecific”.
In a third aspect, the present invention provides a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an extracellular antigen-binding domain, a transmembrane domain and an intracellular signalling domain: the extracellular antigen-binding domain comprises the humanized antibody against TNFR2 or antigen-binding fragment thereof of the first aspect of the present invention or comprises the multispecific antigen-binding molecule of the second aspect of the present invention.
In a fourth aspect, the present invention provides an immune effector cell, wherein the immune effector cell comprises the chimeric antigen receptor of the third aspect of the present invention or a nucleic acid fragment encoding the chimeric antigen receptor of the third aspect of the present invention; preferably, the immune effector cell is selected from a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a monocyte, a macrophage, a dendritic cell or a mast cell; the T cell can be selected from an inflammatory T cell, a cytotoxic T cell, a regulatory T cell (Treg) or a helper T cell; preferably, the immune effector cell is an allogeneic immune effector cell or an autologous immune cell.
In a fifth aspect, the present invention provides an isolated nucleic acid fragment, wherein the nucleic acid fragment encodes the humanized antibody against TNFR2 or antigen-binding fragment thereof of the first aspect, the multispecific antigen-binding molecule of the second aspect or the chimeric antigen receptor of the third aspect of the present invention.
In a sixth aspect, the present invention provides a vector, wherein the vector comprises the nucleic acid fragment of the fifth aspect.
In a seventh aspect, the present invention provides a host cell, wherein the host cell comprises the vector of the sixth aspect; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as a bacterial (Escherchia coli) cell, a fungal (yeast) cell, an insect cell or a mammalian cell (a CHO cell line or a 293T cell line); preferably, the cell lacks a fucosyltransferase, and more preferably, the fucosyltransferase is FUT8.
In an eighth aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the present invention or the multispecific antigen-binding molecule of the present invention, comprising culturing the cell of the seventh aspect, and isolating the antibody or antigen-binding fragment thereof or the multispecific antigen-binding molecule expressed by the cell.
In a ninth aspect, the present invention provides a method for preparing an immune effector cell, comprising introducing a nucleic acid fragment encoding the aforementioned CAR into the immune effector cell, and optionally, further comprising enabling the immune effector cell to express the CAR.
In a tenth aspect, the present invention provides a pharmaceutical composition, wherein the composition comprises the antibody or antigen-binding fragment thereof of the present invention, the multispecific antigen-binding molecule of the present invention, the chimeric antigen receptor of the present invention, the immune effector cell of the present invention, the nucleic acid fragment of the present invention, the vector of the present invention, the cell of the present invention, or a product prepared from the method of the present invention; preferably, the composition further comprises a pharmaceutically acceptable carrier, a diluent or an auxiliary agent; preferably, the composition further comprises an additional anti-tumour agent, an immunotherapeutic agent or an immunosuppressant; preferably, the additional anti-tumour agent is selected from a PD-1 antibody, a PD-L1 antibody or a CTLA-4 antibody.
In an eleventh aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof, the multispecific antigen-binding molecule, the chimeric antigen receptor, the immune effector cell, the nucleic acid fragment, the vector, the cell, a product prepared from the method, and the pharmaceutical composition in the preparation of a drug for treating and/or preventing diseases related to immune abnormalities; preferably, the diseases related to immune abnormalities are those related to Treg cells and/or MDSC functions; preferably, the diseases are cancers or autoimmune diseases; preferably, the cancers are selected from ovarian cancer, advanced epidermal T-cell lymphoma, stage III/IV metastatic colorectal cancer, triple-negative breast cancer, pancreatic cancer, non-small cell lung cancer, and/or advanced solid tumours resistant to CTLA-4 and PD-1 therapies, e.g., metastatic melanoma; preferably, the autoimmune diseases can be selected from rheumatoid arthritis, multiple sclerosis, systemic sclerosis, neuromyelitis optica spectrum disorder, systemic lupus erythematosus, myasthenia gravis and IgG4-related diseases.
In a twelfth aspect, the present invention provides a method for treating and/or preventing diseases related to immune abnormalities, comprising administering to a subject an effective amount of the antibody or antigen-binding fragment thereof, the multispecific antigen-binding molecule, the chimeric antigen receptor, the immune effector cell, the nucleic acid fragment, the vector, the cell, a product prepared from the method, or the pharmaceutical composition of the present invention; preferably, the diseases related to immune abnormalities are those related to Treg cells and/or MDSC functions; preferably, the diseases are cancers or autoimmune diseases; preferably, the cancers are selected from ovarian cancer, advanced epidermal T-cell lymphoma, stage III/IV metastatic colorectal cancer, triple-negative breast cancer, pancreatic cancer, non-small cell lung cancer, and/or advanced solid tumours resistant to CTLA-4 and PD-1 therapies, e.g., metastatic melanoma; preferably, the autoimmune diseases can be selected from rheumatoid arthritis, multiple sclerosis, systemic sclerosis, neuromyelitis optica spectrum disorder, systemic lupus erythematosus, myasthenia gravis and IgG4-related diseases.
In a thirteenth aspect, the present invention further provides an additional anti-tumour therapeutic agent administered to the object, such as a chemotherapeutic agent, a targeted therapeutic agent and an immunotherapeutic agent, including a PD-1/PD-L1 therapeutic agent such as an anti-PD-1/PD-L1 antibody, and an anti-CTLA-4 therapeutic agent such as an anti-CTLA-4 antibody, preferably, the additional anti-tumour therapeutic agent is selected from a PD-1/PD-L1 therapeutic agent; preferably, the PD-1/PD-L1 therapeutic agent is selected from a PD-L1 antibody.
In a fourteenth aspect, the present invention provides a method for detecting TNFR2 in vitro, comprising the step of contacting a sample suspected of containing TNFR2 with the antibody or antigen-binding fragment thereof of the present invention.
Unless defined otherwise, terms used herein have the meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. For a term explicitly defined herein, the meaning of the term shall prevail from the definition.
As used herein, the term “TNFR2” refers to tumour necrosis factor receptor 2, also known as tumour necrosis factor receptor superfamily member 1B (TNFRSF1B) or CD120b, which is a membrane receptor that binds to tumour necrosis factor-α (TNFα). The TNFR2 is preferably human TNFR2.
As used herein, the terms “anti-tumour necrosis factor receptor 2 antibody”, “tumour necrosis factor receptor 2 antibody”, “anti-TNFR2 antibody”, “TNFR2 antibody”. “anti-TNFR2 antibody portion” and/or “anti-TNFR2 antibody fragment” and the like refer to any protein- or peptide-containing molecule that comprises at least a portion of an immunoglobulin molecule capable of specifically binding to TNFR2 (such as but not limited to at least one complementarity determining region (CDR) of a heavy chain or a light chain or a ligand binding portion thereof, a heavy chain variable region or a light chain variable region, a heavy chain constant region or a light chain constant region, and a framework region or any portion thereof). The TNFR2 antibody also includes an antibody-like protein scaffold (such as the tenth fibronectin type III domain (10Fn3)) containing BC, DE and FG structural loops similar in structure and solvent accessibility to CDRs of an antibody. The tertiary structure of the 10Fn3 domain is similar to that of an IgG heavy chain variable region, and those skilled in the art can graft, for example, CDRs of a TNFR2 monoclonal antibody onto a fibronectin scaffold by substituting the residues from BC, DE and FG loops of the 10Fn3 with residues from the CDR-H1, CDR-H2 or CDR-H3 region of the TNFR2 monoclonal antibody.
As used herein, the term “antibody (Ab)” refers to an immunoglobulin molecule that specifically binds to or is immunoreactive with a target antigen, including polyclonal, monoclonal, genetically engineered and other modified forms of an antibody (including but not limited to a chimeric antibody, a humanized antibody, a fully human antibody, a heterologous coupled antibody (such as bispecific, trispecific and tetraspecific antibodies, a diabody, a tribody and a tetrabody), and an antibody conjugate) and an antigen-binding fragment thereof (including, for example, Fab′, F(ab′)2, Fab, Fv, rIgG and scFv fragments). In addition, unless otherwise specified, the term “monoclonal antibody (mAb)” is meant to include an intact antibody molecule and an incomplete antibody fragment (e.g., Fab and F(ab′)2 fragments, which lack the Fc fragment of an intact antibody (cleared from animal circulation more quickly) and therefore lack an Fc-mediated effector function) capable of specifically binding to a target protein (see Wahl et al., J. Nucl. Med. 24:316, 1983; the content of which is incorporated herein by reference).
As used herein, the term “monoclonal antibody” refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic or bacteriophage clone), without limitation by the method by which the antibody is produced.
As used herein, the terms “antigen-binding fragment” and “antibody fragment” are interchangeable and refer to one or more antibody fragments that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. An antibody fragment can be Fab, F(ab′)2, scFv, SMIP, a diabody, a tribody, an affibody, a nanobody, an aptamer or a domain antibody. Examples of binding fragments encompassed by the term “antigen-binding fragment” of an antibody include, but are not limited to: (i) an Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) an F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulphide bond in a hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of VL and VH domains of a single arm of an antibody; (v) dAb comprising VH and VL domains; (vi) a dAb fragment consisting of a VH domain (Ward et al., Nature 341:544-546, 1989); (vii) dAb consisting of a VH or VL domain; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs which may optionally be linked via a synthetic linker. In addition, although two domains VL and VH of an Fv fragment are encoded by independent genes, these two domains can be joined via a linker using a recombination method, wherein the linker enables the preparation of a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (known as single chain Fv (scFv); see, for example, Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same way as an intact antibody. An antigen-binding fragment can be generated by a recombinant DNA technology, by the enzymatic or chemical cleavage of an intact immunoglobulin, or in some embodiments by a chemical peptide synthesis procedure known in the art.
As used herein, the term “complementarity determining region (CDR)” refers to a hypervariable region found in both light chain and heavy chain variable domains. The more conservative portion of a variable domain is called the framework region (FR). As understood in the art, the amino acid positions representing the hypervariable region of an antibody may vary according to the context and various definitions known in the art. Some positions within a variable domain can be regarded as hybrid hypervariable positions, as these positions can be regarded as being within the hypervariable regions under a set of standards (such as IMGT or KABAT) and outside the hypervariable regions under a different set of standards (such as KABAT or IMGT). One or more of these positions may also be found in an extended hypervariable region. The present invention includes an antibody comprising modifications in these hybrid hypervariable positions. The variable domains of a natural heavy chain and light chain each comprise four framework regions predominantly in a sheet configuration, which are linked by three CDRs (CDR1, CDR2 and CDR3) that form a loop linking the sheet structure, and in some cases form part of the sheet structure. The CDRs in each chain are closely held together in order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 by the FR region, and together with the CDRs from other antibody chains, contribute to the formation of an antigen-binding site of an antibody (see Kabat et al., Sequences of Proteins of Immunological Interest. National Institute of Health, Bethesda, Md. 1987; which is incorporated herein by reference). For example, in the present invention, CDR1-VH, CDR2-VH and CDR3-VH refer to the first CDR, the second CDR and the third CDR of a heavy chain variable region (VH), respectively, and these three CDRs constitute the CDR combination of a heavy chain (or a variable region thereof) (the VHCDR combination); CDR1-VL. CDR2-VL and CDR3-VL refer to the first CDR, the second CDR and the third CDR of a light chain variable region (VL), respectively, and these three CDRs constitute the CDR combination of a light chain (or a variable region thereof) (the VLCDR combination).
As used herein, the term “Kabat numbering system” generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service. National Institutes of Health, Bethesda. Md., 1991).
As used herein, the term “VH” refers to a variable region of an antibody immunoglobulin heavy chain (including a heavy chain of Fv, scFv or Fab). The term “VL” refers to a variable region of an immunoglobulin light chain (including a light chain of Fv, scFv, dsFv or Fab).
The term “heavy chain constant region” herein refers to the carboxyl terminus portion of an antibody heavy chain, which is not directly involved in the binding of an antibody to an antigen, but exhibits an effector function, such as an interaction with an Fc receptor, and has a more conservative amino acid sequence relative to the variable domain of an antibody. The “heavy chain constant region” comprises at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof. A “heavy chain constant region” includes a “full-length heavy chain constant region” and a “heavy chain constant region fragment”, the former has a structure substantially similar to that of a natural antibody constant region, while the latter only includes “a portion of the full-length heavy chain constant region”. Exemplarily, a typical “full-length antibody heavy chain constant region” consists of CH1 domain-hinge region-CH2 domain-CH3 domain, which also includes a CH4 domain when an IgE antibody is referred to, and does not include the CH1 domain when a heavy chain antibody is referred to. Exemplarily, a typical “heavy chain constant region fragment” can be selected from CH1, Fc or CH3 domains.
The term “light chain constant region” herein refers to the carboxyl terminus portion of an antibody light chain, which is not directly involved in the binding of an antibody to an antigen, and the light chain constant region can be selected from a constant κ domain or a constant λ domain.
The term “Fc” herein refers to the carboxyl terminus portion of an intact antibody obtained by hydrolysing the antibody with papain, which typically comprises CH3 and CH2 domains of an antibody. The Fc region includes, for example, an Fc region of a natural sequence, a recombinant Fc region and a variant Fc region. Although the boundary of the Fc region of an immunoglobulin heavy chain can be slightly changed, the Fc region of a human IgG heavy chain is usually defined as the region extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU Kabat numbering system) can be removed, for example, during the production or purification of an antibody, or by recombinant engineering of a nucleic acid encoding an antibody heavy chain, so the Fc region may or may not include Lys447.
As used herein, the terms “percent (%) sequence identity” and “percent (%) sequence consistency” are interchangeable, and refer to the percentage of amino acid (or nucleotide) residues of a candidate sequence that are identical to those of a reference sequence upon aligning sequences and introducing gaps (if necessary) in order to achieve maximum percent sequence identity (for example, gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). For the purpose of determining the percent sequence identity, alignment can be achieved in a variety of ways well known to those skilled in the art, for example, using publicly available computer software, such as BLAST, ALIGN or Megalign (DNASTAIi) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full-length range of the sequences aligned. For example, a reference sequence aligned for comparison with a candidate sequence can show that the candidate sequence exhibits a sequence consistency from 50% to 100% over the full length of the candidate sequence or a selected portion of consecutive amino acid (or nucleotide) residues of the candidate sequence. The length of a candidate sequence aligned for comparison purposes may be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of a reference sequence. When a position in a candidate sequence is occupied by the same amino acid (or nucleotide) residue as in the corresponding position in a reference sequence, these molecules are identical at that position.
The term “conservative amino acids” herein generally refers to amino acids that belong to the same class or have similar characteristics (such as charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation and rigidity). Exemplarily, the following amino acids in each group are each other's conservative amino acid residues, and a substitution of amino acid residues in the group is a substitution of conservative amino acids:
As used herein, the term “specific binding” refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of a protein and other biomolecules which are specifically recognized by antibodies or antigen-binding fragments thereof, for example. An antibody or an antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM. For example, an antibody or an antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of up to 100 nM (for example, between 1 pM and 100 nM). An antibody or an antigen-binding fragment thereof that does not show a specific binding to a specific antigen or epitope thereof will show a KD greater than 100 nM (for example, greater than 500 nM, 1 μM, 100 μM, 500 μM or 1 mM) for the specific antigen or epitope thereof. A variety of immunoassays can be used to select antibodies that are specifically immunoreactive with specific proteins or carbohydrates. For example, a solid-phase ELISA immunoassay is conventionally used to select antibodies that are specifically immunoreactive with proteins or carbohydrates. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbour Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbour Press, New York (1999), which describe immunoassays and conditions that can be used to determine the specific immunoreactivity.
As used herein, the term “multispecific antibody” refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. Therefore, terms such as “bispecific”. “trispecific” and “tetraspecific” refer to the number of different epitopes to which an antibody/antigen-binding molecule can bind. The term “bispecific antibody” refers to an antibody with monoclonal binding specificities to at least two different antigens, which is generally a human or humanized antibody. In the present invention, one of the binding specificities can be detected against an antigenic epitope of TNFR2, and the other can be detected against another antigenic epitope of TNFR2 or any other antigen, such as against a cell surface protein, a receptor, a receptor subunit, a tissue-specific antigen, a virus-derived protein, a virus-encoded envelope protein, a bacterium-derived protein or a bacterial surface protein.
As used herein, the term “valence” means the presence of a specified number of binding sites in an antibody/antigen-binding molecule. Therefore, the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” indicate the presence of one binding site, two binding sites, four binding sites and six binding sites in an antibody/antigen-binding molecule, respectively.
As used herein, the term “humanized antibody” refers to a non-human antibody that has been genetically engineered, with amino acid sequences modified to improve the homology with the sequences of a human antibody. Generally speaking, all or part of the CDR regions of a humanized antibody come from a non-human antibody (a donor antibody), and all or part of the non-CDR regions (for example, a variable region FR and/or a constant region) come from a human immunoglobulin (a receptor antibody). A humanized antibody generally retains or partially retains the expected properties of a donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to improve immune cell activities, ability to enhance immune responses, etc.
As used herein, the term “chimeric antibody” refers to an antibody that has a variable sequence of an immunoglobulin from a source organism (such as a rat or a mouse) and a constant region of an immunoglobulin from a different organism (such as human). Methods for producing a chimeric antibody are known in the art. See, for example, Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, Bio Techniques 4:214-221; and Gillies et al., 1985 J Immunol Methods 125:191-202; which are incorporated herein by reference.
As used herein, the term “antibody conjugate” refers to a couplet/conjugate formed by chemical bonding of an antibody molecule to another molecule directly or via a linker. The another molecule can be a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a radioisotope, a chemotherapeutic drug or an immunomodulator, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescence label, an ultrasonic contrast agent or a photosensitizer. An “antibody conjugate” is, for example, an antibody-drug conjugate (ADC), in which the drug molecule is the another molecule.
The term “antigen chimeric receptor (CAR)” herein refers to an artificial immune effector cell surface receptor engineered to express on immune effector cells and specifically bind to antigens, comprising at least (1) an extracellular antigen-binding domain, such as a variable heavy chain or light chain of an antibody, (2) a transmembrane domain anchoring the CAR into an immune effector cell, and (3) an intracellular signalling domain. The CAR can redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC-restricted way by using an extracellular antigen-binding domain.
As used herein, the term “regulatory T cells” or “Tregs”, also formerly known as suppressor T cells, refers to a population of lymphocytes that negatively regulate immune responses of the body, so as to maintain resistance to autoantigens, control excessive immune responses, avoid immune damage to normal cells and prevent the occurrence of autoimmune diseases. Tregs express the following biomarkers: CD4, FOXP3 and CD25, which are considered to be derived from the same lineage as naive CD47 cells. Tregs play an extremely important role in tumourigenesis. A large number of studies have shown that the number of Treg cells in the tumour microenvironment increases significantly, including melanoma, ovarian cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, etc., and the number of Treg cells is closely related to the survival rate of tumour patients. In addition, tumour cells can induce the proliferation of tumour infiltrating Treg cells, and the proliferating Treg cells will secrete a large number of immunosuppressive factors such as TGF-β to inhibit the functions of immune cells such as CD8+ T cells and finally hinder the killing effect of immune cells on tumours, which constitutes an important drug resistance mechanism for the failure of immunotherapies in various solid tumours and haematological tumours. Recent studies have shown that the immunological tolerance of patients treated with immunotherapies such as PD-1/PD-L1 is also closely related to Tregs.
As used herein, the term “vector” includes nucleic acid vectors, such as DNA vectors (e.g., plasmids), RNA vectors, viruses or other suitable replicons (e.g., viral vectors). A variety of vectors have been developed for delivering a polynucleotide encoding an exogenous protein to a prokaryotic or eukaryotic cell. The expression vector of the present invention contains polynucleotide sequences and additional sequence elements, for example, for expressing proteins and/or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express the antibodies and antibody fragments of the present invention include plasmids containing regulatory sequences (such as promoters and enhancer regions) that direct gene transcription. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5′ and 3′ untranslated regions, internal ribosome entry sites (IRESs) and polyadenylation signal sites, so as to direct the effective transcription of genes carried on expression vectors. The expression vector of the present invention may also contain a polynucleotide encoding a marker for selection of a cell containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseothricin.
As used herein, the terms “subject”, “object” and “patient” refer to an organism treated for a specific disease or disorder (such as a cancer or an infectious disease) as described herein. Examples of objects and patients include mammals treated for diseases or disorders (for example, cell proliferative disorders such as cancers or infectious diseases), such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the family Bovidae (such as cattle, bison, buffalo, elk and yak), cows, sheep, horses and bisons.
As used herein, the term “treatment” refers to a surgical or therapeutic treatment with an aim to prevent and slow down (reduce) unwanted physiological changes or lesions in an object to be treated, such as the progression of cell proliferative disorders (such as cancers or infectious diseases). Beneficial or desired clinical outcomes include, but are not limited to, the alleviation of symptoms, the weakening of disease severity, the stabilization of disease state (i.e., no deterioration), the delay or slowing down of disease progression, the amelioration or mitigation of disease state, and remission (whether partial or complete), whether detectable or undetectable. Objects in need of treatment include those who already have a disorder or disease, those who are susceptible to a disorder or disease or those who intend to prevent a disorder or disease. When terms such as slowing down, alleviation, weakening, mitigation, and remission are referred to, their meanings also include elimination, disappearance, non-occurrence and other circumstances.
As used herein, the term “effective amount” refers to an amount of a therapeutic agent that is effective in preventing or relieving a disease symptom or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue or subject. The “effective amount” also refers to an amount of a compound sufficient to relieve symptoms, such as treating, curing, preventing or relieving related medical disorders, or increasing the speed of treating, curing, preventing or relieving these disorders. When an active ingredient is administered alone to an individual, a therapeutically effective dose refers only to the dosage of the ingredient. When a certain combination is applied, a therapeutically effective dose refers to the combined dosage of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially or simultaneously.
The present invention is further described below in conjunction with specific examples. The advantages and features of the present invention will be more clearly described. If no specific conditions are indicated in the examples, conventional conditions or the conditions suggested by the manufacturer shall be followed. Any reagents or instruments used, unless the manufactures stated, are conventional products that are commercially available.
The examples of the present invention are only exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention could be modified or substituted without departing from the spirit and scope of the present invention, but these modifications and substitutions all fall within the scope of protection of the present invention.
1.1 Humanization of an Anti-TNFR2 Antibody
The method of “transplantation of CDRs” was used to humanize an antibody, specifically, a human antibody with the highest homology was selected based on sequences to provide antibody framework regions (FRs), into which complementarity determining regions (CDRs) of an antigen-binding fragment in a target antibody based on the Kabat nomenclature were transplanted to form a humanized antibody variable region sequence in order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Secondly, in order to effectively maintain the activity and affinity of an antibody, based on the antibody structure modelling (MOE software), sites for potential reverse mutation were selected, and the selection criteria were: 1) amino acid residues with antibody framework region located at the VH-VL interface, close to CDRs or directly interacting with CDRs were selected for reverse mutation; such amino acid residues mostly were important for maintaining the conformation of the CDR regions; 2) amino acids embedded in the protein were selected as far as possible for reverse mutation, considering the immunogenicity; 3) mutations with reduced molecular energy were preferred, considering the stability and expression level of an antibody. By testing a humanized antibody with different mutations for its affinity to human TNFR2 and for its binding to a cell expressing TNFR2 on the surface, a humanized antibody with the affinity, antibody characterization and activity function comparable to or better than a murine antibody was screened out.
The complementarity determining regions (CDRs) of an antigen-binding fragment in a TNFR2 antibody based on the Kabat nomenclature are detailed in Table 1. Table 2 shows the VH and VL sequences of a humanized antibody molecule. Table 3 shows the pairing of VH and VL sequences of a humanized antibody against TNFR2.
1.2 Expression of a Humanized Antibody Against TNFR2
The day before transfection, ExpiCHO-S cells were counted, and inoculated into a freshly preheated ExpiCHO expressing medium (Invitrogen, A291002) at a density of 2.5-4×106 cells/mL for overnight culture. On the day of transfection, the ExpiCHO-S cell suspension cultured overnight was taken for counting; as a result, the cell concentration was about 7-10×106 cells/mL, and the viability of cells for transfection was greater than 95%. According to the number of transfected cells, a required number of cells were taken, diluted with the ExpiCHO expressing medium to a final concentration of a density of 6×106 cells/mL, and then placed in a shaker with 8% CO2, at 37° C. and 100 rpm for later use. Transfection preparation: a plasmid was diluted in an OptiPRO™ SFM medium (Invitrogen, 12309019), and the mixture was mixed well by gently shaking a centrifuge tube. A well-mixed ExpiFectamine™ CHO reagent (Invitrogen, A29129) was added to the plasmid diluent, and the mixture was mixed well by gently shaking the centrifuge tube, and then left to stand at room temperature for 2 minutes. The above-mentioned plasmid/ExpiFectamine™ CHO reagent complex was slowly added dropwise into a cell suspension to be transfected, during which a shake flask was shaken. After transfection, the transfected cells were cultured in a shaker with 8% CO2, at a temperature of 37° C. and a speed of 100 rpm. On Day 1 after transfection, the transfected cells were supplemented with 0.6% ExpiFectamine™ CHO Enhancer (Invitrogen, A29129) and 16% ExpiCHO™ Feed (Invitrogen, A29129), during which the shake flask was gently shaken, and then the cells were transferred to a shaker with 5% CO2, at a temperature of 32° C. and a speed of 100 rpm for culture. On Day 5 after transfection, the transfected cells were supplemented with 16% ExpiCHO™ Feed, during which the shake flask was gently shaken. On Day 12 after transfection, a cell supernatant was collected and centrifuged at 4000 g for 10 minutes, and then the supernatant was pipetted for further purification of the antibody.
1.3 Purification of a Humanized Antibody Against TNFR2
The cell culture supernatant collected by high-speed centrifugation was filtered with a 0.45+0.22 μM filter membrane, and the filtrate was subjected to affinity chromatography using Akta Avant 150 (Cytiva) for the first step of purification. The chromatographic medium was Protein A filler Mabselect PrismA (Cytiva, Cat #17549803) that interacted with Fc, and the equilibration buffer was PBS (2.5 g/L Na2HPO4 12H2O, 0.408 g/L NaH2PO4, and 8.76 g/L NaCl, pH 7.2). After the column was equilibrated with 4 column volumes of the equilibration buffer, the cell supernatant was loaded onto the column for binding, with the flow rate controlled at a point where the retention time of the sample on the column is >5 min. After sample loading, the column was washed with PBS (pH 7.2) until the ultraviolet absorption value at A280 dropped to the baseline. Then the column was eluted with 2 column volumes of 20 mM PB+1 M NaCl (3.752 g/L Na2HPO4·12H2O, 1.314 g/L NaH2PO4, and 58.44 g/L NaCl, pH 6.2). Subsequently, the column was washed with PBS (pH 7.2) until the ultraviolet absorption value at A280 and the conductivity reached the baseline. Finally, the chromatographic column was washed with an elution buffer of 20 mM citric acid (2.184 g/L citric acid, and 1.086 g/L sodium citrate, pH 3.4), and the elution peak was collected according to the ultraviolet absorption peak at A280. The collected elution sample was neutralized with 1 M Tris (121.14 g/L Tris) and detected to be neutral using a pH meter. Seven2Go (METTLER TOLEDO). The collected antibody was identified to be more than 95% pure by SEC-HPLC and used for follow-up studies.
0.5 μg/ml human TNFR2 (Human TNFR2-His, Sino 10417-H08H) or cynomolgus monkey TNFR2 (Cynomolgus TNFR2-His, Sino 90102-C08H) was pre-coated with 100 μl/well of the antibody in an ELISA plate. The test humanized antibody against TNFR2 (as described in Example 1, the antibody was comprised of variable regions linked to IgG-Fc; the WT chimeric antibody also adopted the IgG-Fc structure) was subjected to 3.33-fold gradient dilution, loaded with 100 μl/well, and incubated with shaking at room temperature for 1.5 hours. The plate was washed, and then a working solution of murine anti-human IgG Fc-HRP (diluted at 1:10000) was added, loaded with 100 μl/well, and incubated with shaking at room temperature for 1.0 hour. The plate was washed again, and then the substrate TMB of HRP was added to develop colour. A stop solution was added to stop the reaction, and then a microplate reader (MD 13) was used to read the absorbance value. The binding curve of an antibody was drawn with antibody concentration as the abscissa and corresponding OD value as the ordinate, and fitted with four parameters (GraphPad Prism9) to calculate the EC50 value. The smaller the EC50 value, the stronger the binding ability of an antibody to human/cynomolgus monkey TNFR2. The binding effect of a humanized antibody was normalized to its corresponding human-murine chimeric antibody WT (see SEQ ID NOs: 38-43 for the sequence). The higher the percentage value, the better the binding effect of the humanized antibody. The results of binding of an anti-TNFR2 antibody to a human TNFR2 protein are shown in
Biacore was used to detect the specific binding of a test anti-TNFR2 antibody to human and cynomolgus monkey TNFR2 proteins. In this experiment, a Protein A chip was used, the time required for the chip to capture the diluted antibody was determined by manual run, so that the Rmax of the saturated binding to an antigen was 50 RU. Both human (Human TNFR2-His, Sino 10417-H8H) and cynomolgus monkey (Cynomolgus TNFR2-His, Sino 90102-C08H) TNFR2 proteins were subjected to gradient dilution to 32, 16, 8, 4, and 2 nM. The affinity of an antibody for an antigen was determined by using multi-cycle dynamics. In each cycle, an antibody was injected and then a TNFR2 protein at a gradient concentration was injected, so that the antigen and the antibody underwent binding and dissociation processes. After each cycle, the Protein A chip was regenerated with Glycine pH 1.5 (removing the protein on the chip). The affinity KD of an antibody and an antigen was fitted with BIAcore T200 analysis software. The results in Table 5 show that all the test anti-TNFR2 antibodies specifically bind to a human or cynomolgus monkey TNFR2 protein with a high affinity level.
CHO-K1 stable cells transfected with a high-expression plasmid of human TNFR2 (named CHO-TNFR2) were taken. The transfected full-length plasmid of human TNFR2 was purchased from Sino Biological (Cat #HG10417-UT, NCBI Ref Seq: NM_001066.2). The experiment was conducted at a cell density within 80%. The cell medium was discarded; the cells were rinsed with PBS, and digested with 1 ml trypsin for 2 minutes; the digestion was stopped with a Ham's F12 complete medium containing 10% FBS, and then a cell suspension was made. The cell suspension was counted, and then an appropriate amount of cell suspension was centrifuged at 350×g; the supernatant was discarded; a corresponding volume of blocking solution (10% FBS+PBS) at a density of 1×107 cells/ml was added to resuspend the cells, and the mixture was incubated at 4° C. for 30 minutes. After the incubation, the mixture was centrifuged at 350×g to remove the supernatant, and the cells were resuspended in a staining buffer (2% FBS+PBS) to a density of 2×106 cells/ml, and spread onto a 96-well plate, with 50 μl of cell suspension added to each well for later use. The antibody was diluted with PBS for 10-fold gradient dilution from the highest concentration of 20 μg/m (twice the concentration); the diluted antibody was added to the well containing 50 μl of cell suspension, and placed on a microplate shaker for shaking at 500 rpm for 1 minute, so that the antibody was fully mixed with the cells; the mixture was incubated at 4° C. for 1 h. After the incubation, the cells were washed twice with the staining buffer, with 100 μl per well, and centrifuged at 350-g for 5 minutes, and the supernatant was discarded. The PE goat anti-Human IgG Fc antibody (ebioscience, Cat #12-4998-82) was diluted 250-fold with the staining buffer, with a volume of 100 μl per well added to the washed cell wells; the mixture was mixed evenly, and stained at 4° C. for 30 minutes. After the staining, similarly, the cells were washed twice with the staining buffer; finally, the cells were resuspended in 200 μl of staining buffer; signals were detected by a flow cytometer (Thermo Attune NxT). The stronger the signal, the stronger the binding ability of an antibody to TNFR2. The binding curve of an antibody was drawn with antibody concentration as the abscissa and corresponding mean fluorescence intensity (MFI) fold as the ordinate, and fitted with four parameters (GraphPad Prism9) to calculate the EC50 value. The smaller the EC50 value, the stronger the binding ability of an antibody to a CHO-TNFR2 cell. The binding effect of a humanized antibody was normalized to its corresponding human-murine chimeric WT antibody. The higher the percentage value, the better the binding effect of the humanized antibody. As shown in
Human Treg cells were isolated from human PBMCs by using a sorting kit (Stemcell, Cat #18063), stimulated and expanded in vitro by Dynabeads Human Treg Expander (Gibco, Cat #11129D) for 15 days, and then packaged and frozen for storage. The Treg cells isolated and expanded in vitro were recovered overnight, centrifuged at 300×g for 5 minutes the next day, and resuspended in DPBS to obtain a cell suspension; the cell suspension was counted. The cells required for the experiment were added to a centrifuge tube, centrifuged at 300×g for 5 min to remove the supernatant, adjusted to a density of 2×106 cells/ml with a staining buffer, and spread onto a 96-well plate, with 50 μg per well. All the test antibodies and the control antibody anti-Hel isotype (diluted to 200 ng/ml with PBS, respectively, with a total amount of 100 μl) were taken. Each sample was added to each well, with 50 μl per well. The well plate added with the cell suspension and the antibody was placed on a microplate shaker for shaking at a speed of 500 rpm for 1 min, so that the cells were fully mixed with the antibody. After the full mixing, the well plate was placed in a 4° C. freezer, and incubated for 60 min. After the incubation, 100 μl of staining buffer was added to each well; the mixture was centrifuged at 350-×g for 5 min; the supernatant was discarded. An additional 200 μl of staining buffer was added to each well to resuspend the cells; the cell suspension was centrifuged at 350×g for 5 min; the supernatant was discarded. The staining buffer was added to the PE goat anti-Human IgG Fc at a ratio of 250:1 (staining buffer:fluorescently stained antibody) for preparation of a staining solution; the solution was mixed well, and then added to the cell wells, with 100 μl per well. The well plate was placed on a microplate shaker for shaking at a speed of 500 rpm for 1 min, so that the cells were fully mixed with the staining solution. After the full mixing, the well plate was placed in a 4° C. freezer, and incubated for 30 min. The cells were washed twice; finally, 200 μl of PBS was added to each well to resuspend the cells; signals were detected by a flow cytometer (Thermo Attune NxT).
The frozen cynomolgus monkey PMBC cells were recovered, and then resuspended with a complete medium (RPMI640-Glutamax+10% FBS+1×P/S+1×ITS+50 μMPM mercaptoethanol) to obtain a cell suspension; the cell suspension was counted. According to the counting results, the resuspended cells were adjusted to a density of 5×106 cells/ml; 100 μl of cell suspension was spread in each well of a 96-well U-bottom plate. A T cell activating reagent, Immunocult T cell (Stemcell, 1:50) activator+rhIL-2 (R&D, 200 IU), at 20% concentration was formulated; the activating reagent at 20% concentration was added to cells, with 100 μl per well; the mixture was mixed well, then placed in an incubator with 5% CO2 at 37° C., and incubated for three days. The activated cells were collected and counted; the cells were adjusted to a density of 4×105 cells/ml with a staining buffer (a DPBS solution containing 2% FBS), and spread onto a 96-well V-bottom plate, with 50 μl per well. All the test antibodies and the anti-Hel control antibody (diluted to 0.4 μg/ml and 4 μg/md with a staining buffer, respectively) were taken; each sample was added to each well, with 50 μl per well. The well plate added with the cell suspension and the antibody was placed on a microplate shaker for shaking at a speed of 500 rpm for 1 min, so that the cells were fully mixed with the antibody. After the full mixing, the well plate was placed in a 4° C. freezer, and incubated for 30 min. After the incubation, 100 μl of staining buffer was added to each well; the mixture was centrifuged at 350×g for 5 min; the supernatant was discarded. An additional 200 μl of staining buffer was added to each well to resuspend the cells; the cell suspension was centrifuged at 350×g for 5 min; the supernatant was discarded. The antibodies Anti-Human CD8-FTIC (BD-555366, 1 μl/test) and PE goat anti-Human IgG Fc (Invitrogen-124998-82, 0.5 μl/test) were added. The well plate was placed on a microplate shaker for shaking at a speed of 500 rpm for 1 min, so that the cells were fully mixed with the staining solution. After the full mixing, the well plate was placed in a 4° C. freezer, and incubated for 30 min. The cells were washed twice; finally, 200 μl of staining buffer was added to each well to resuspend the cells; signals were detected by a flow cytometer (Thermo Attune NxT).
1 μg/ml human TNFR2 (Novoprotein, Cat #C830) was pre-coated with 100 μl/well of the antibody in an ELISA plate. All the test TNFR2 antibodies were subjected to 2.5-fold gradient dilution from the highest concentration of 30 μg/ml; the diluted antibodies were mixed with 15 ng/ml human TNFα-Biotin (Acro Biosystem, Cat #TNA-H82E3) in equal volume, respectively; the resulting mixture was added to the ELISA plate at 100 μl/well, and incubated with shaking at room temperature for 2.0 hours. The plate was washed, and then a Streptavidin-HRP working solution (diluted at 1:10000) was added at 100 μl/well, and the mixture was incubated with shaking at room temperature for 40 minutes. The plate was washed again, and then the substrate TMB of HRP was added to develop colour. A stop solution was added to stop the reaction, and then a microplate reader (MD 13) was used to read the absorbance value. The inhibitory curve of an antibody was drawn with antibody concentration as the abscissa and corresponding OD value as the ordinate, and fitted with four parameters (GraphPad Prism9) to calculate the IC50 value. The smaller the IC50 value, the stronger the ability of an antibody to inhibit the binding of human TNFα to human TNFR2. The blocking effect of a humanized antibody was normalized to its corresponding human-murine chimeric antibody WT. The higher the percentage value, the better the inhibitory effect of the humanized antibody. The blocking curve of test antibodies is shown in
CHO stable cells highly expressing human TNFR2 were used for the experiment (the same as CHO-TNFR2 in Example 4). The CHO-TNFR2 cells were digested, washed twice with DPBS, then stained with Live/Dead (L/D) (20 minutes at room temperature), and plated at a density of 1×105 cells/50 μl/well. A test anti-TNFR2 antibody was diluted to 6 μg/ml with a staining buffer as the starting concentration: the antibody was subjected to 3.33-fold gradient dilution, with a total of 7 concentration points. The diluted antibody was added to the plated cells at 50 μl/well, and the mixture was mixed well by gentle pipetting, and incubated at 4° C. for 30 minutes. Then the diluted 100 ng/ml of human TNFα-biotin was added at 100 μl/well, and the mixture was mixed well by gentle pipetting, and then incubated at 4° C. for 30 minutes. The mixture was washed twice with a staining buffer, and then 0.12 μg/ml PE-streptavidin (BioLegend, Cat #405204) was added at 100 μl/well; the resulting mixture was incubated at 4° C. for 30 minutes. The mixture was washed twice with a staining buffer, and then 150 μl of staining buffer was added to resuspend the cells; signals were detected by a flow cytometer (Thermo Attune NxT). The inhibitory curve of an antibody was drawn with antibody concentration as the abscissa and corresponding MFI value as the ordinate, and fitted with four parameters (GraphPad Prism9) to calculate the IC50 value. The smaller the IC50 value, the stronger the ability of an antibody to inhibit the binding of human TNFα to human TNFR2. The blocking effect of a humanized antibody was normalized to its corresponding human-murine chimeric WT antibody. The higher the percentage value, the better the inhibitory effect of the humanized antibody. The blocking curve of test antibodies is shown in
Human Treg cells were isolated from human PBMCs by using a sorting kit (Stemcell, Cat #18063), stimulated and expanded in vitro by Dynabeads Human Treg Expander (Gibco, Cat #11129D) for 15 days, and then packaged and frozen for storage. The Treg cells isolated and expanded in vitro were recovered overnight, centrifuged at 300×g for 5 minutes the next day, and resuspended with an AIM-V medium (brand: Gibco, Cat #31035025), the cells were counted, adjusted to a density of 6×106 cells/ml based on the cell counting results, and spread onto a 96-well plate, with 25 μl per well. All the test antibodies and the anti-Hel control antibody (diluted to 40% concentration with the AIM-V medium, 1 μg/ml, 10 μg/ml and 100 μg/ml respectively) were taken. Antibodies at each concentration were added to the corresponding cell well, with 25 μl per well. The mixture was mixed with a multi-channel pipette, and then pre-incubated for 30 min in an incubator with 5% CO2 at 37° C. TNFαα (brand: Novoprotein, Cat #C008) was diluted with the AIM-V medium to 20 ng/ml (twice the working concentration); 50 μl of TNFα was added to the well requiring TNFα activation, and 50 μl of AIM-V medium was added to the control well requiring no activation, so that the final concentration of the antibody was 10%, and the final concentration of TNFαα was 10 ng/ml; the mixture was mixed well by pipetting; the culture plate was incubated for 10 min in an incubator at 37° C. After the incubation, the culture plate was taken out, into which 100 μl of pre-cooled DPBS was added, and the mixture was centrifuged at 4° C. and 300 g for 5 min. Then the cells were treated with 100 μl 1 live/dead violet (diluted 500-fold with pre-cooled DPBS), and reacted on ice for 20 min. 100 μl of staining buffer was added to each well; the mixture was centrifuged at 4° C. and 300 g for 5 mm, 100 μl of 1 fix-perm buffer (brand: BD, Cat #554714) was added to each well, mixed evenly, and fixed at 4° C. for 30 min. After the fixation, 100 μl of 1×perm buffer was added to each well to terminate the fixation; the mixture was centrifuged at 400 g for 5 min; the supernatant was discarded. Then 200 μl of 1×perm buffer was added to each well to wash the cells again. 100 μl of IκBα staining solution (diluted 100-fold with 1×perm buffer) was added to the experimental well, and 1 μl of Rat IgG2b κ isotype control antibody was added to the control well; the mixture was mixed evenly, and incubated at 4° C. for 30 min. After the incubation, 100 μl of 1×perm buffer was added to each well to terminate the fixation; the mixture was centrifuged at 400 g for 5 min; the supernatant was discarded. Then 200 μl of 1×perm buffer was added to each well to wash the cells again. Signals were detected by a flow cytometer (Thermo Attune NxT). Flow cytometry was used to analyse the percentage of IκBa 1low in viable cells or the MFI of IκBα in all viable cells. The lower the proportion of IκBααlow, the stronger the ability of an anti-TNFR2 antibody to inhibit the TNFα-induced degradation of IκBα. The results in
By measuring the proliferation of Treg cells induced by TNFα and IL-2 in the presence of a TNFR2 antibody, the inhibitory effect of this antibody on the proliferation of Treg cells induced by TNFα and IL-2 was determined (Zaragoza B et al., Nat Med. 2016 January; 22(1):16-7). The frozen Treg cells expanded and isolated in vitro were recovered overnight, centrifuged at 400×g for 5 minutes the next day, and resuspended with a medium to obtain a cell suspension; the cell suspension was counted. The Tregs were stained with CellTrace Violet cell proliferation kit (Thermo. Cat #C34557), with 1 μl of storage solution per 1×107 cells used to prepare 1 ml of staining solution. Staining was performed at 37° C. for 20 min; the reaction was neutralized with at least 5 times the volume of complete medium, left to stand for 5 min, and centrifuged at 400×g for 5 min. The cells were resuspended and washed once with a medium, and then the washed cells were centrifuged again to obtain the supernatant. The cells were plated at a density of 1×105 cells per well of a 96-well plate, resuspended with a medium at a density of 1×105 cells per 50 μl, and transferred to a 96-well plate. 50 μl of antibody to be tested was added to each well, with the final concentration of the antibody being 12.5 μg/ml; the mixture was incubated in an incubator for 30 minutes. Then 50 μl of medium containing IL-2 (with a final concentration of 300 IU) and TNFα (with a final concentration of 50 ng/ml) and 50 μl of medium containing anti-CD3/CD28 Dynabeads (Gibco, Cat #11129D, with the ratio of beads to Treg cells of 1:20) were added to each well, with the final volume of each well reaching 200 μl. Two parallel wells were set for each condition; the above-mentioned well plates were mixed evenly, and then incubated in an incubator at 37° C. for three days for FACS detection (Thermo Attune NxT). The inhibitory effect of the antibody on Treg proliferation induced by TNFα and IL-2 was determined by the proportion of Treg proliferation. Results in
By measuring the inhibitory function of Treg cells on responder T cells in the presence of an anti-TNFR2 antibody, the inhibitory effect of the antibody on the inhibitory function of Treg cells was determined. The effector cells, Tregs (CD4+CD25+FoxP3+ T cells) and Conventional T cells (Tcons) (CD4+CD25−T cells), required for the experiment were recovered overnight, centrifuged at 400×g for 5 minutes the next day, and resuspended with a complete medium (RPMI1640-Glutamax+10% FBS+1×P/S+1 ITS+50 μMPM mercaptoethanol) to obtain a cell suspension; the cell suspension was counted. The Tcon cells were stained with CellTrace Violet cell proliferation kit (Thermo, Cat #C34557), with 1 μl of storage solution per 1×107 cells used to prepare 1 ml of staining solution. Staining was performed at 37° C. for 20 min; the reaction was neutralized with at least 5 times the volume of complete medium, left to stand for 5 min, and centrifuged at 400×g for 5 min. The cells were resuspended and washed once with a medium, and then the washed cells were centrifuged again to discard the supernatant. The cells were plated at a density of 1×105 cells per well of a 96-well plate, resuspended with a medium at a density of 1×105 cells per 50 μl, and transferred to a 50 mL centrifuge tube. The Treg cells were prepared at the same time, and the Treg cells and the Tcon cells were co-cultured at a ratio of 1:1. Corresponding anti-CD3/CD28 Dynabeads (Gibco, Cat #11129D) were prepared at a ratio of 1/10 by the number of Tcon cells, and added to a medium containing Treg and Tcon at a volume of 50 μl per well. An antibody to be tested was added to each well, with the final concentration of the antibody being 12.5 μg/ml. Three parallel wells were set for each condition; the above-mentioned well plates were mixed evenly, and then incubated in an incubator at 37° C. for 4 days for flow cytometry. The higher the proliferation proportion of Tcon, the stronger the ability of an antibody to inhibit Treg. The inhibitory results as shown in
PBMCs and the frozen Treg cells were cultured in a complete medium (RPMII640-Glutamax+10% FBS+1×P/S+1×ITS+50 μMPM mercaptoethanol) one day in advance, in which 100 IU/ml of IL-2 was added to the PBMCs. On the day of the experiment, NK cells were isolated from PBMCs according to the requirements of the isolation kit (Stemcell. Cat #17955), and resuspended with a complete medium (without IL-2) at a density of 0.8×106 cells/ml. The target Treg cells or CHO-TNFR2 cells were labelled with a Cell Trace violet reagent; after the labelling, the cells were adjusted to a density of 0.4×106 cells/ml. An antibody to be tested was subjected to gradient dilution with a complete medium to a concentration 4 times the final concentration for later use. According to the distribution map of the experimental plate, the target cells were seeded into a cell plate, with 50 μl per well, and then the diluted drug was spread onto a corresponding well, and co-incubated with the target cells at 37° C. for 30 minutes. After the incubation, 100 μl of effector cell suspension was added to a well requiring effector cells, and 100 μl of medium was added to a well requiring no effector cells; the mixture was incubated at 37° C. for 4 h. After the reaction, 1 μl of PI dye was added to each well for detection on an instrument. The higher the proportion of PI positive in target cells, the more significant the effect of ADCC. The results of ADCC experiment as shown in
By using CRISPR/Cas9 technology and homologous recombination, the hTNFRSF1B-WPRE-PA expression cassette was knocked into the exon2 site of Tnfrsf1b gene in a site-specific manner to obtain a heterozygous BALB/c mouse with the Tnfrsf1b gene knocked into hTNFRSF1B-WPRE-PA in a site-specific manner; then a homozygous transgenic mouse (TNFR2 HuGEMM) expressing human Tnfrsf1b (TNFR2) gene was obtained through breeding. The peripheral blood from a 6-8-week-old female BALB/c mouse (from Beijing Vital River Laboratory Animal Technology Co., Ltd.) and a TNFR2 HuGEMM mouse was treated with ACK (Gibco, Cat #A1049201) to remove red blood cells; the treated peripheral blood was subjected to immunophenotyping by FACS to detect the expression of mTNFR2 and hTNFR2 on CD4+ T cells, CD8+ T cells and CD4+CD25+Foxp3+ Treg cells in the peripheral blood of mice, with all populations of cells gated according to an FMO control (
By measuring the TNFα-induced proliferation of Treg cells in a TNFR2 HuGEMM mouse in the presence of a TNFR2 antibody, TNFR2 signalling pathway and corresponding functions of the TNFR2 HuGEMM mouse genetically engineered were verified to be retained, so that it was ensured that the engineered transgenic mouse could be used to evaluate the in vivo efficacy of a candidate anti-TNFR2 antibody. The spleens from 5 TNFR2 HuGEMM mice and 5 wild-type Balb/c mice were extracted aseptically in a biosafety cabinet for grinding. All the grinded cell suspensions were transferred to a 15 ml centrifuge tube for centrifugation (at 320 g and 4° C., for 7 minutes). The supernatant was poured out. About 2-3 ml of ACK erythrocyte lysate (Gibco, Cat #A1049201) was added, and the mixture was mixed well, and kept at room temperature for 3-5 minutes until erythrocytes were completely lysed; 6-8 ml of RPMI1640 medium was added to stop the lysis, and the mixture was mixed well by inversion. The well-mixed mixture was subjected to centrifugation (at 200 g and 4° C., for 8 minutes). After the centrifugation, the supernatant was poured out, and the cells were resuspended with EasySep buffer, filtered with a 40 μm filter membrane, and counted. Then Treg cells were sorted according to the instruction of Mouse CD4+CD25+ Regulatory T kit (brand: Stemcell, Cat #18783A). The sorted Treg cells were stained with CellTrace Violet cell proliferation kit (Thermo, Cat #C34557), with 1 μl of storage solution per 1×107 cells used to prepare 1 ml of staining solution. Staining was performed at 37° C. for 20 min; the reaction was neutralized with at least 5 times the volume of complete medium, left to stand for 5 min, and centrifuged at 400×g for 5 min. The cells were resuspended and washed once with a medium, and then the washed cells were centrifuged again to obtain the supernatant. The cells were plated at a density of 1×105 cells per well of a 96-well plate, resuspended with a medium at a density of 1×105 cells per 50 μl, and transferred to a 96-well plate. 50 μl of antibodies to be tested (including three humanized antibodies against TNFR2, an anti-murine TNFR2 surrogate antibody Surrogate antibody clone #75-54.7 (BioXcell, BE0247), and an isotype control antibody anti-Hel-mIgG1) were added to each well, with the final concentration of the antibodies being 10 μg/ml; the mixture was incubated in an incubator for 30 minutes. Then 50 μl of medium containing mouse IL-2 (brand: Sino Biological Inc., Cat #51061-MNAE, with a final concentration of 300 IU) and mouse TNFα (brand: Sino Biological Inc. Cat #20180502, with a final concentration of 50 ng/ml) and 50 μl of medium without or with Dynabeads mouse T-activator CD3/CD28 (Gibco, Cat #11452D, with the ratio of beads to Treg cells of 1:20) were added to each well, with the final volume of each well reaching 200 μl. In addition, control wells with Treg cells added with mouse IL-2 only, Treg cells added with mouse TNFα only, and Treg cells added with both mouse IL-2 and human TNFα were set. Two parallel wells were set for each condition; the above-mentioned well plates were mixed evenly, and then incubated in an incubator at 37° C. for three days for FACS detection (Thermo Attune NxT). The inhibitory effect of the antibody on TNFα-induced Treg proliferation was determined by the proportion of Treg proliferation.
An EGE system developed based on CRISPR/Cas9 by Biocytogen Gene Biotechnology Co., Ltd. was used to replace part of Exon2 and Exon3 of mTNFR2 gene in a mouse CT26 cell line with PuroR cassette, so as to achieve the purpose of gene knockout and destroy the expression of endogenous mTNFR2 in the mouse; then the CDS of a chimeric protein from a mouse intracellular region, transmembrane region and a human extracellular region was inserted to prepare the CT26 cell line only expressing hTNFR2 gene (CT26-hTNFR2 KI). A colon cancer animal model in which hTNFR2 gene was knocked into the CT26-hTNFR2 KI cell line was established by using TNFR2 HuGEMM, and an efficacy experiment of an anti-TNFR2 antibody was carried out.
A CT26-hTNFR2 KI cell was taken out from the cell bank, and recovered with a complete 1640 medium (1640 (Gibco, 61870-036)+10% FBS (Gibco. 10099-141C)+1% P/S (Gibco, 15140-122)+1% NEAA (Gibco, 11140-050)+1% Sodium Pyruvate (Gibco, 11360-070)+16 μg/ml puromycin (Gibco, A1 1138-03)); the recovered cell was placed in a cell culture bottle (with cell type, number of generation, date, name of the person performing the culture, etc. marked on the cell bottle wall) and incubated in a CO2 incubator (at a temperature of 37° C. and a CO2 concentration of 5%). Cells were passaged upon covering 80%-90% of the bottom of the culture bottle; after the passage, the cells were continued to be cultured in the CO2 incubator; the process was repeated until the number of cells met the requirements of an in vivo efficacy experiment; subsequently, a cell suspension was prepared, and counted by an automatic cell counter (BECKMAN, Vi-Cell XR); according to the counting results, the cells were resuspended with a PBS solution (HyClone, SH30256.01) to prepare a cell suspension (at a density of 2×106 cells/ml). The above cell suspension was inoculated subcutaneously on the right flank of a mouse at 100 μl/mouse. When the tumour volume reached about 100 mm3, mice with moderate individual tumour volume were selected into groups, and the animals were divided into 8 experimental groups according to the tumour volume using an EXCEL random number based randomization method, see Table 9 for grouping. Dosing was started on the same day (marked as Day 0), with the dosing volume of 10 ml/kg; PBS was used as a vehicle; and the dosing was performed by intraperitoneal injection at a frequency of once every 3 days for a total of 4 doses. The tumour volume and body weight were measured three times a week at a fixed time. The tumour volume is calculated according to the following equation: Tumour volume (mm3)=length (mm)×width (mm)×width (mm)/2, and the tumour inhibition rate is calculated according to the following equation: Equation to calculate the tumour inhibition rate: TGI (%)=[1−T/C] 100%, with a statistical method of Two-way ANOVA used to analyse the experimental data. On Day 11 after the dosing, all dosage groups of the test anti-TNFR2 antibody group had statistical differences compared with the PBS control group, with an obvious efficacy. In addition, there was a dose-dependent relationship between different dosage groups of the same antibody. Moreover, the efficacy of the positive drug anti-mPD-1 (BioXcell, BE0146) at 10 mg/kg was comparable to that of the test anti-TNFR2 antibodies 219-Hu1-mIgG2a and 001-Hu3-mIgG2a at a dose of 3 mg/kg. All these results showed that the test humanized antibodies against TNFR2 had a significant in vivo tumour growth inhibition efficacy and were dose-dependent (A and B m
NOG female mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) aged 7-9 weeks were subcutaneously inoculated with 3.5×106 HT-29 cells (set as Day 0 of the experiment), and 7×106 human peripheral blood mononuclear cells (PBMCs, donor #193 and donor #272, extracted from the screened whole blood of donors by CrownBio (Taicang) Co., Ltd. according to SOP-CP-042) were intraperitoneally injected to the mice on the same day to establish a PBMC humanized colon cancer model. When the tumour volume reached about 95 mm3, mice with moderate individual tumour volume were selected into groups, and the animals were randomized into 5 experimental groups (with G1 as a PBS control group, G2 as a control antibody anti-Hel hIgG1 group, G3 as an anti-TNFR2 antibody 001-Hu3-hIgG1 group, G4 as an anti-TNFR2 antibody 219-Hu1-hIgG1 group, and G5 as a Keytruda group) according to the tumour volume using Study Director™ (version No.: 3.1.399.19, supplied by Studylog System, Inc., S. San Francisco, CA, USA), with 5-7 mice in each group for two donors for in vivo efficacy study. Dosing was started on the day of grouping (Day 13). The dose was 10 mg/kg (with the same meaning as the measuring unit mpk in the figure, the same below), and the drug was injected intraperitoneally twice a week. The tumour volume and body weight were measured twice a week at a fixed time. The tumour volume (TV) is calculated according to the following equation: V=½×a×b2, where a and b represent length and width, respectively. In donor #193, on Day 29 after the tumour inoculation, compared with the control antibody group, the group 219-Hu1 had tumour growth significantly inhibited, with statistical differences (P<0.0001); there was no statistical difference between other treatment groups and the control antibody group. It shows that in donor #193, the anti-TNFR2 antibody 219-Hu1 has a significant efficacy (A in
During the experiment, both the control group and the dosing groups exhibited weight loss and death of mice. According to the results of veterinary autopsy, it was inferred that the death of mice was due to a graft versus host disease (GvHD) reaction caused by injection of PBMCs. At the test dose of 10 mg/kg, the test drugs 001-Hu3 and 219-Hu1 and the positive drug Keytruda produced no obvious drug toxicity as observed in animals, and the treatment was tolerable (C-D in
48 h after the last dosing, mice in the G2 control antibody group, G3 anti-TNFR2 antibody 001-Hu3 group and G4 anti-TNFR2 antibody 219-Hu1 group were selected for tumour infiltrating lymphocyte (TIL) isolation (Miltenyi, Cat #130-095-929), so as to perform immunophenotyping by FACS; 123 count eBeads (eBioscience, Cat #01-1234-42) was added for absolute cell counting, with all populations of cells gated according to an FMO control (A in
An animal model of CT26.1C03 colon cancer cells engineered with subcutaneous inoculation of human TNFR2 gene (hTNFR2 KI-CT26) in genetically engineered BALB/c mice (TNFR2 HuGEMM) expressing human TNFR2 was used to evaluate the combined efficacy experiment of the 219-Hu1-hIgG1 antibody and the anti-murine PD-L1 (anti-mPD-L1, BioXcell, BE0101). Mice were subcutaneously inoculated with 5×105 CT26.1C03 colon cancer cells on the right flank (set as Day 0 of the experiment). When the tumour volume reached about 100 mm3, mice with moderate individual tumour volume were selected into groups, and the animals were divided into 5 experimental groups (with G1 as a control group, G2 as an anti-mPD-L1 10 mg/kg antibody group, G3 as a 219-Hu1-hIgG1 10 mg/kg antibody group, G4 as a 219-Hu1-hIgG1 30 mg/kg antibody group, and G5 as a combination group of 219-Hu1-hIgG1 10 mg/kg+anti-mPD-L1 10 mg/kg antibodies) according to the tumour volume using an EXCEL random number based randomization method, with 10 mice in each group for in vivo efficacy study. Dosing was started on the day of grouping (Day 10). Dosing was performed by intraperitoneal injection twice a week. The tumour volume and body weight were measured three times a week at a fixed time.
On Day 18 after the tumour inoculation (i.e., Day 8 of dosing), compared with the control group (G1), the G2 anti-mPD-L1 10 mg/kg group, the G3 219-Hu1-hIgG1 10 mg/kg group and the G4 219-Hu1-hIgG1 30 mg/kg group had TGITV values of 35.1%, 28.2% and 63.8%, respectively, suggesting that the tumour volume growth was all significantly inhibited in all three treatment groups (G2 vs. G1, P<0.0001; G3 vs. G1, P=0.0002; G4 vs. G1, P<0.0001); the 219-Hu1-hIgG1 10 mg/kg group and the anti-mPD-L1 10 mg/kg group had comparable TGITV values; however, the 219-Hu1-hIgG1 30 mg/kg group had a TGITV value significantly higher than that in the 219-Hu1-hIgG1 10 mg/kg group and the anti-mPD-L1 10 mg/kg group (G2 vs. G4, P=0.0002; G3 vs. G4, P<0.0001). The above results showed that the 219-Hu1-hIgG1 group had a dose-dependent effect within the dose range of 10-30 mg/kg, with tumour volume decreased as the increase of dose. See Table 11 and
On Day 18 after the tumour inoculation, compared with the control group (G1), the combined dosing group of G5 219-Hu1-hIgG1 10 mg/kg+anti-mPD-L1 10 mg/kg also had the tumour volume growth significantly inhibited (TGITV=66.5, vs. G1, P<0.0001); in addition, the combined dosing group of G5 219-Hu1-hIgG1 10 mg/kg+anti-mPD-L1 10 mg/kg also had a mean tumour volume significantly smaller than that in the single-dose groups of G3 219-Hu1-hIgG1 10 mg/kg and G2 anti-mPD-L1 10 mg/kg (TGITV G5 vs. G3 vs. G2=66.5% vs. 28.2% vs. 35.1%: G5 vs. G2, P<0.0001; G5 vs. G3, P<0.0001), indicating that the combined dosing of the 219-Hu1-hIgG1 antibody and the anti-mPD-L1 antibody inhibited the tumour growth more potently compared with two single-dose groups, with statistical differences (****, p<0.0001, single-dose group vs. combined dosing group). See Table 11 and
aMean ± SD.
bThe relative tumour growth rate T/C % = TRTV/CRTV × 100% (TRTV: mean RTV in the treatment groups; CRTV: mean RTV in the negative control group). According to the results of tumour measurement, the relative tumour volume (RTV) is calculated with the calculation equation of RTV = Vt/V0, where V0 is the tumour volume measured at the time of grouping and dosing (i.e., PO-D0) and Vt is the tumour volume measured at a certain time.
cTGITV (%) = [1 − (Ti − T0)/(Vi − V0)] × 100% (Ti: mean tumour volume of treatment groups on Day i of dosing; T0: mean tumour volume of treatment groups on Day 0 of dosing; Vi: mean tumour volume of a solvent control group on Day i of dosing; V0: mean tumour volume of a solvent control group on Day 0 of dosing).
dData are analysed using Two-way ANOVA, in which when compared with G1 Veh, p < 0.05 indicates a significant difference; ***: p < 0.001, ****: p < 0.0001.
eData are analysed using Two-way ANOVA, in which when compared with the combined dosing group of G5, p < 0.05 indicates a significant difference; ****: p < 0.0001.
fData are analysed using Two-way ANOVA, in which when compared with the G4 219-Hu1-hIgG1 30 mpk group, p < 0.05 indicates a significant difference; ***: p < 0.001, ****: p < 0.0001.
During the dosing period, the experimental animals in each group were in good activity and eating state, with a certain degree of increase in body weight. During the observation in the experimental period, no deterioration of animal mental state, activity and body surface observation was found in each group, and all the animals showed no drug toxicity and showed good safety results, indicating that the safety of the 219-Hu1-hIgG1 antibody and the combined dosing group of 219-Hu1-hIgG1+anti-mPD-L1 antibodies was high. See
20 ± 1.4
On Day 18 after the tumour inoculation, the animals in each group were euthanized, and the tumours were isolated and weighed. Compared with the control group (G1), the G2 anti-mPD-L1 10 mg/kg group had the tumour weight significantly reduced (TGITW=37.3%, P-value: 0.0087); the G3 219-Hu1-hIgG1 10 mg/kg group had a tumour weight lower than that of the control group, without a statistical difference (TGITW=25.9%, P-value: 0.0830); the G4 219-Hu1-hIgG1 30 mg/kg group also had a tumour weight significantly lower than that of the control group (TGITW=47.9%, P-value: 0.0013); the combined dosing group of G5 219-Hu1-hIgG1 10 mg/kg+anti-mPD-L1 10 mg/kg also had a tumour weight significantly lower than that of the control group (TGITW=43.0%, P-value: 0.0042). The tumour weight of 219-Hu1-hIgG1 at two doses had a dose-dependent effect, with tumour weight decreased as the increase of dose. The tumour weight of the combined dosing group was lower than that of each single-dose group (G2 and G3), without a statistical difference (
a Mean ± SD.
bTGITw (%) = [1 − Ti/Vi] × 100% (Ti: mean tumour weight of treatment groups on Day i of dosing; Vi: mean tumour weight of a solvent control group on Day i of dosing).
cData are analysed using a One-way ANOVA method, in which when compared with G1 Veh, p < 0.05 indicates a significant difference; **: p < 0.01.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110140980.5 | Jan 2021 | CN | national |
| 202111016307.7 | Aug 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/074228 | 1/27/2022 | WO |
