Patent Application
20220002412
Sequences are submitted concurrently with this application via EFS-Web in a file named Sequence Listing, created on Dec. 21, 2018, the file having a size of 36,864 bytes. All sequences in the latter file are hereby incorporated by reference.
The present disclosure relates to the field of oncology or cancer immunotherapy. Specifically, the present disclosure provides a recombinant humanized antibody against programmed cell death receptor-1 (PD-1), which can be used in tumor or cancer immunotherapy. The disclosure also provides nucleic acid sequences encoding said antibody, vectors containing said nucleic acid sequences, pharmaceutical compositions and kits.
PD-1 is a type I transmembrane glycoprotein consisting of 288 amino acids with a molecular weight of approximately 50 kDa and is a member of the CD28 family, also known as CD279. It functions as a regulator of programmed cell death and is expressed mainly on the surface of mature CD4+ and CD8+ T cells, but also on natural killer T cells, B cells, monocytes and some dendritic cells.
PD-1 has two ligands, PD-L1 (i.e. B7-H1, also known as CD274) and PD-L2 (i.e. B7-DC, also known as CD273), both of which are transmembrane protein molecules of the B7 family. In terms of amount and action, PD-L1 is the major PD-1 ligand. PD-L1 is widely expressed on the surface of various cell types, including haematopoietic cells such as dendritic cells, B cells and T cells, as well as non-haematopoietic cells such as epithelial and endothelial cells. The expression of PD-L1 on the surface of tumor cells shows high, which can be up-regulated by inflammatory cytokines such as IFN-γ and TNF-α. PD-L2 ligand has high similarity to PD-L1, but its distribution is relatively limited, mainly expressed on the surface of immune cells such as macrophages, dendritic cells and mast cells, and its expression is low. PD-L2 binds PD-1 with higher affinity, about three times that of PD-L1.
PD-1 plays its biological role by binding to its ligands, inhibiting T cell receptor-mediated T cell proliferation, activation and cytokine secretion, thereby suppressing the initial and effector phases of the immune response, maintaining immune stability and preventing the development of autoimmune diseases. When PD-1 binds to its ligand, the tyrosine residues within the immunoreceptor tyrosine-based switch motif (ITSM), one domain of PD-1 cytoplasmic region, is phosphorylated, then the phosphorylated ITSM recruits SHP-2 phosphatase, resulting inhibition of important downstream pathways through dephosphorylation such as the blockage of the activation of phosphoinositide 3-kinase (PI3K) and its downstream protein kinase B (PKB or Akt), the inhibition of glucose metabolism, the production of the cytokine interleukin-2 (IL-2) and the expression of the anti-apoptotic protein Bcl-xl; thus inhibits the proliferation and activation of T and B cells and the secretion of immunoglobulins, as a result, the autoimmune response is suppressed. Tumor cells take advantage of this immunosuppressive mechanism to achieve immune escape, via the binding of PD-L1 highly expressed there into PD-1 molecules on the surface of lymphocytes, they evades from being immune recognized and cleared by organism.
Monoclonal antibodies targeting PD-1 are currently a hot topic in tumor or cancer immunotherapy research. By blocking the binding of PD-1 to its ligand, these monoclonal antibodies can increase secretion of T cells and IFN-γ and IL-2 at tumor sites, reduce the proportion of Myeloid-derived suppressor cells (MDSCs), alter the tumor microenvironment, restore and enhance the immune killing function of T cells, and thus inhibit tumor growth. Currently, the US Food and Drug Administration (FDA) has approved the marketing authorization of PD-1 antibodies, including Bristol-Myers Squibb's Opdivo (generic Nivolumab) and Merck &Co's Keytruda (generic Pembrolizumab). Opdivo is approved for the treatment of melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, classic Hodgkin's lymphoma, urothelial carcinoma, high microsatellite instability carcinoma, advanced renal cell carcinoma and hepatocellular carcinoma; Keytruda is approved for the treatment of melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, classic Hodgkin's lymphoma, urothelial carcinoma, high microsatellite instability and gastric cancer. In addition, domestic and international PD-1 target antibodies in clinical research include Regeneron/Sanofi's REGN2810, Top Alliance's JS001, Hengrui's SHR-1210, Beigene's BGB-A317, Cinda's IBI308 and Gloria/WuXiPharmaTech's GLS-010, and many other anti-PD-1 and anti-PD-L1 antibody drugs in preclinical research.
In conclusion, research on monoclonal antibodies targeting PD-1 has made some progress. However, there is still a need for better efficacy, safety and competitive monoclonal biologics in oncology or cancer immunotherapy.
The technical terms used in the present disclosure and their corresponding abbreviation are shown in Table 1.
A first aspect of the present disclosure provides an isolated PD-1 antibody or antigen-binding fragment thereof comprising a light chain variable region or a portion thereof and/or a heavy chain variable region or a portion thereof;
Wherein the light chain variable region or a portion thereof comprises a light chain CDR1 having an amino acid sequence of SEQ ID NO: 10, a light chain CDR2 having an amino acid sequence of SEQ ID NO: 11 and a light chain CDR3 having an amino acid sequence of SEQ ID NO: 12; and
The heavy chain variable region or portion thereof comprises heavy chain CDR1 having amino acid sequence of SEQ ID NO: 13, heavy chain CDR2 having amino acid sequence of SEQ ID NO: 14 and heavy chain CDR3 having amino acid sequence of SEQ ID NO: 15.
In a specific embodiment, said PD-1 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of: an amino acid sequence having at least 90%, 92%, 95%, 98% or 100% sequence identity to the PD-1 antibody light chain variable region sequence of SEQ ID NO:23 and an amino acid sequence having at least 90%, 92%, 95%, 98% or 100% sequence identity to the PD-1 antibody heavy chain variable region sequence of SEQ ID NO:22.
In a particular embodiment, said antibody further comprises a light chain constant region and a heavy chain constant region; in a particular embodiment, wherein said antibody further comprises a light chain constant region and a heavy chain constant region, preferably said light chain constant region has an amino acid sequence having at least 90%, 92%, 95%, 98% or 100% sequence identity to the kappa light chain constant region of amino acid sequence of SEQ ID NO:25, and/or said heavy chain constant region has an amino acid sequence having at least 90%, 92%, 95%, 98% or 100% sequence identity to the IgG4 heavy chain constant region of amino acid sequence of SEQ ID NO:24.
In a specific embodiment, said antibody is an IgG antibody. In one specific embodiment, said antibody is an IgG4 antibody.
In one specific embodiment, the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.
In a specific embodiment, the antibody or antigen-binding fragment thereof binds to the recombinant human PD-1 protein with an affinity KD average of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 pM or higher, or any ranges with the foregoing values as endpoints, such as about 20-200 pM, or about 60-70 pM, etc., or any values therein, such as about 64.8 pM or about 108 pM, etc. The method for determining the binding affinity KD is as described in the examples of this application.
In one specific embodiment, the antibody or antigen-binding fragment thereof specifically binds to a PD-1 protein molecule comprising the amino acid sequence of SEQ ID NO:1 or to a protein molecule having an amino acid sequence having at least 90%, 92%, 95%, 98% or 100% sequence identity to SEQ ID NO:1.
In one specific embodiment, the antigen-binding fragment is in the form of an Fv, Fab, Fab′, Fab′-SH, F(ab′)2, Fd fragment, Fd′ fragment, single chain antibody molecule, or single domain antibody; in one specific embodiment, the single chain antibody molecule is a scFv, di-scFv, tri-scFv, diabody, or scFab.
In a further specific embodiment, the antibody or antigen-binding fragment thereof in the above embodiments forms a covalent or non-covalent conjugate or a recombinant multi-target fusion drug with another molecule, thereby to form a modified drug molecule, said another molecule being selected from a small molecule compound and/or a biomacromolecule.
In one embodiment, a modified drug molecule may include one of a small molecule and a biomacromolecule; and an isolated PD-1 antibody or antigen-binding fragment. The modified drug molecule may include one of a covalent conjugate, a non-covalent conjugate, and a recombinant multi-target fusion drug.
The present disclosure relates to a pharmaceutical composition including an isolated PD-1 antibody or antigen-binding fragment thereof and a modified drug molecule comprising: one of a small molecule and a biomacromolecule, where the modified drug molecule includes one of a covalent conjugate, a non-covalent conjugate, and a recombinant multi-target fusion drug. The pharmaceutical composition may be a therapeutic agent.
A second aspect of the disclosure provides an isolated nucleic acid whose nucleotide sequence encodes the antibody and/or antigen binding fragment of the first and second aspects.
A third aspect of the present disclosure provides a vector comprising the nucleic acid of the second aspect.
A fourth aspect of the disclosure provides an isolated cell expressing the antibody and/or antigen-binding fragment of the first aspect, and/or comprising the nucleic acid of the second aspect or the vector of the third aspect.
In one specific embodiment, said cells are prokaryotic or eukaryotic cells.
A fifth aspect of the present disclosure provides a method for producing the antibody and/or antigen-binding fragment of the first aspect, said method comprising culturing the cells of the fourth aspect and purifying said antibodies.
A sixth aspect of the present disclosure provides the use of the antibody and/or antigen-binding fragment and/or modified drug molecule of the first aspect for the preparation of medicines for the treatment of tumor or cancer.
In one specific embodiment, said tumor or cancer is colon cancer.
An seventh aspect of the present disclosure provides the use of the antibody and/or antigen-binding fragment and/or modified drug molecule of the first aspect for the treatment of tumor or cancer.
In one specific embodiment, said tumor or cancer is colon cancer.
A eighth aspect of the disclosure provides a pharmaceutical composition comprising the antibody and/or antigen-binding fragment and/or modified drug molecule of the first aspect.
A ninth aspect of the present disclosure provides a pharmaceutical combination comprising the pharmaceutical composition of the eighth aspect and one or more therapeutically active compounds.
An tenth aspect of the present disclosure provides a kit comprising the antibody and/or antigen-binding fragment and/or modified drug molecule of the first aspect, or the pharmaceutical composition of the eighth aspect or the pharmaceutical combination of the ninth aspect, preferably further comprising a device for administration.
A eleventh aspect of the present disclosure provides a method of treating a tumor or cancer comprising administering to a subject in need thereof a therapeutically effective amount of the isolated PD-1 antibody or antigen-binding fragment thereof or modified drug molecule of the first aspect, or the pharmaceutical composition of the eighth aspect, or a pharmaceutical combination of the ninth aspect, or the kit of the tenth aspect, thereby treating said tumor or cancer, preferably wherein said tumor or cancer is colon cancer.
Unless otherwise stated, all technical and scientific terms used herein have the meaning normally understood by a person skilled in the art to which the present disclosure belongs. For the purposes of the present disclosure, the following terms are further defined.
When used herein and in the appended claims, the singular forms “one”, “a/an”, “another” and “said” include the plural designation of the object unless the context clearly indicates otherwise.
The term “antibody” refers to an immunoglobulin molecule and refers to any form of antibody that exhibits the desired biological activity. These include, but are not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and multispecific antibodies (e.g. bispecific antibodies), and even antibody fragments. Typically, full-length antibody structures preferably comprise four polypeptide chains, two heavy (H) chains and two light (L) chains, typically interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region and a heavy chain constant region. Each light chain contains a light chain variable region and a light chain constant region. In addition to this typical full-length antibody structure, the structure also includes other derivative forms.
The term “variable region” refers to the domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to the antigen. Said variable regions of the heavy and light chains can be further subdivided into highly variable regions (called complementary decision regions (CDRs)) interspersed with more conserved regions (called framework regions (FRs)).
The term ‘complementary determining region’ (CDR, e.g. CDR1, CDR2 and CDR3) refers to such amino acid residues in the variable region of an antibody whose presence is necessary for antigen binding. Each variable region typically has three CDR regions identified as CDR1, CDR2 and CDR3. Each complementary determining region may contain amino acid residues from a “complementary determining region” as defined by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. 1991) and/or those residues from the “high-variable loop” (Chothia and Lesk; J MolBiol 196: 901-917 (1987)).
The term “framework” or “FR” residues are those residues within the variable region other than CDR residues as defined herein.
Each heavy chain variable region and light chain variable region typically contains 3 CDRs and up to 4 FRs, said CDRs and FRs being arranged from the amino terminus to the carboxyl terminus in the following order, for example: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
The complementary determining region (CDR) and the framework region (FR) of a given antibody can be identified using the Kabat system (Kabat et al: Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991).
The term “constant region” refers to such amino acid sequences in the light and heavy chains of an antibody that are not directly involved in the binding of the antibody to the antigen but exhibit a variety of effector functions such as antibody-dependent cytotoxicity.
According to the amino acid sequence of the constant region of their heavy chains, intact antibodies can be classified into different “classes”. There are five classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, of which can be further divided into subclasses (isoforms), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The L chains of the antibodies are classified into κ and λ according to the amino acid sequence of constant region. The constant domains of the heavy chains corresponding to different “classes” of antibodies are called α, δ, ε, γ and μ, respectively.
An “antigen-binding fragment of an antibody” comprises a portion of an intact antibody molecule that retains at least some of the binding specificity of the parent antibody and typically includes at least a portion of the antigen-binding region or variable region (e.g. one or more CDRs) of the parent antibody. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab′, Fab′-SH, F(ab′)2, Fd fragments, Fd′ fragments, single chain antibody molecules (e.g. scFv, di-scFv or tri-scFv, diabody or scFab), single domain antibodies.
An “antibody fragment” is a non-intact antibody molecule that retains at least some of the biological properties of the parent antibody, including, but not limited to, an Fc fragment, in addition to those described above as “antigen-binding fragments”.
The term “modified drug molecule” means a conjugate or a recombinant multi-target fusion drug formed by covalent or non-covalent connecting an antibody or fragment thereof, such as an antigen-binding fragment to another molecule which is a small molecule compound or biomacromolecule.
The term “chimeric” antibodies refer to antibodies in which a portion of the heavy and/or light chain is derived from a specific source or species and the remainder is derived from a different source or species. “Humanized antibodies” are a subset of “chimeric antibodies”.
The term “humanized antibody” or “humanized antigen-binding fragment” is defined herein as an antibody or antibody fragment that is: (i) derived from a non-human source (e.g., a transgenic mouse carrying a heterologous immune system) and based on a human germline sequence; or (ii) a chimeric antibody where the variable region is of non-human origin and the constant region is of human origin; or (iii) a CDR transplant where the CDR of the variable region is of non-human origin and one or more frame work regions of the variable region are of human origin and the constant region, if any, is of human origin. The aim of “Humanization” is to eliminate the immunogenicity of antibodies of non-human origin in the human body, while retaining the greatest possible affinity. It is advantageous to select the human framework sequence that is most similar to the framework sequence of the non-human source antibody as the template for humanization. In some cases, it may be necessary to replace one or more amino acids in the human framework sequence with corresponding residues in the non-human construct to avoid loss of affinity.
The term “monoclonal antibody” refers to an antibody derived from a substantially homogeneous population of antibodies, i.e. every single antibody comprised in the population is identical except for possible mutations (e.g. natural mutations) which may be present in very small quantities. The term “monoclonal” therefore indicates the nature of the antibody in question, i.e. not a mixture of unrelated antibodies. In contrast to polyclonal antibody preparations, which usually comprise different antibodies against different epitopes, each monoclonal antibody in a monoclonal antibody preparation is directed against a single epitope on the antigen. In addition to their specificity, monoclonal antibody preparations have the advantage that they are usually not contaminated by other antibodies. The term “monoclonal” should not be understood as requiring the production of said antibodies by any particular method. The term monoclonal antibody specifically includes chimeric antibodies, humanized antibodies and human antibodies.
The antibody “specifically binds” to a target antigen such as a tumor-associated peptide antigen target (in this case, PD-1), i.e. binds said antigen with sufficient affinity to enable said antibody to be used as a therapeutic reagent, targeting a cell or tissue expressing said antigen, and does not significantly cross-react with other proteins, or does not significantly cross-react with proteins other than the homologues and variants of the target proteins mentioned above (e.g. mutant forms, splice variants, or protein hydrolysis truncated forms).
The term “binding affinity” refers to the strength of the sum of the non-covalent interactions between a molecule's individual binding sites and its binding partners. Unless otherwise stated, “binding affinity”, when used herein, refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g. antibody and antigen). “KD”, “binding rate constant kon” and “dissociation rate constant koff” are commonly used to describe the affinity between a molecule (e.g. an antibody) and its binding partner (e.g. an antigen). Affinity, i.e. the tight degree at which a ligand binds a particular protein. Binding affinity is influenced by non-covalent intermolecular interactions such as hydrogen bonding, electrostatic interactions, hydrophobic and van der Waals forces between two molecules. In addition, the binding affinity between a ligand and its target molecule may be influenced by the presence of other molecules. Affinity can be analyzed by conventional methods known in the art, including the ELISA described herein.
The term “epitope” includes any protein determinant cluster that specifically binds to an antibody or T-cell receptor. Epitope determinant clusters typically consist of a molecule's chemically active surface groups (e.g. amino acid or sugar side chains, or a combination thereof) and often have specific three-dimensional structural features as well as specific charge characteristics.
An “isolated” antibody is an antibody that has been identified and isolated from a cell that expresses the antibody. The contaminating components of the cells are substances that interfere with the diagnostic or therapeutic use of said antibodies, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Due to the absence of at least one component of the antibody in the natural environment, isolated natural antibodies include in situ antibodies in recombinant cells. However, isolated antibodies are typically prepared by at least one purification step.
“Sequence identity” between two polypeptide or nucleic acid sequences indicates the number of residues that are identical between said sequences as a percentage of the total number of residues, and is calculated based on the size of the smaller one of the compared molecules. When calculating the percentage identity, the sequences being aligned are matched in such a way as to produce a maximum match between the sequences, with the gaps in the match (if present) being resolved by a specific algorithm. Preferred computer program methods for determining identity between two sequences include, but are not limited to, GCG program packages including GAP, BLASTP, BLASTN and FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410). The above procedures are publicly available from the International Center for Biotechnology Information (NCBI) and other sources. The well-known Smith Waterman algorithm can also be used to determine identity.
The term “Fc receptor” or “FcR” refers to a receptor that binds to the Fc region of an antibody. Human FcRs of natural sequence are preferred, and preferably receptors that bind to IgG antibodies (gamma receptors), which include the FcγRI, FcγRII and FcγRIII isoforms, as well as variants of these receptors. All other FcRs are included in the term “FcR”. The term also includes the neonatal receptor (FcRn), which is responsible for the transport of maternal IgG to the fetus (Guyer et al, Journal of Immunology 117: 587 (1976) and Kim et al, Journal of Immunology 24: 249 (1994)).
The term “neonatal Fc receptor”, abbreviated as “FcRn”, binds to the Fc region of IgG antibodies. The neonatal Fc receptor (FcRn) plays an important role in the metabolic fate of IgG-like antibodies in vivo. FcRn functions to rescue IgG from the lysosomal degradation pathway, thereby reducing its clearance in serum and lengthening its half-life. Therefore, the in vitro FcRn binding properties/characteristics of IgG are indicative of its in vivo pharmacokinetic properties in the circulation.
The term “effector function” refers to those biological activities attributable to the Fc region of an antibody, which vary from isotype to isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated uptake of antigen by antigen-presenting cells, cell surface receptors down-regulation (e.g. B-cell receptors) and B-cell activation.
The term “effector cell” refers to a leukocyte that expresses one or more FcR and performs effector functions. In one aspect, said effector cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils. Effector cells can be isolated from natural sources, for example, blood. Effector cells are usually lymphocytes associated with effector phase and function to produce cytokines (helper T cells), kill cells infected by pathogens (cytotoxic T cells) or secrete antibodies (differentiated B cells).
“Immune cells” include cells that have a haematopoietic origin and play a role in the immune response. Immune cells include: lymphocytes, such as B cells and T cells; natural killer cells; and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils and granulocytes.
“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig binds to Fcγ receptors presented on certain cytotoxic cells (e.g. NK cells, neutrophils and macrophages) allows these cytotoxic effector cells to specifically bind to target cells bearing antigens and subsequently kill said target cells using, for example, a cytotoxin. To assess the ADCC activity of the target antibody, in vitro ADCC assays can be performed, such as the in vitro ADCC assays documented in U.S. Pat. No. 5,500,362 or 5,821,337 or U.S. Pat. No. 6,737,056 (Presta), the methods documented in embodiments of the present application. Useful effector cells for use in such assays include PBMCs and NK cells.
“Complement-dependent cytotoxicity” or “CDC” refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass). To assess complement activation, a CDC assay can be performed, such as the CDC assay recited in Gazzano-Santoro et al., J. Immunol Methods 202: 163 (1996). For example in U.S. Pat. No. 6,194,551 B1 and WO1999/51642, there described polypeptide variants having altered amino acid sequences of the Fc region (polypeptides having a variant Fc region) and polypeptide variants having enhanced or reduced C1q binding.
Amino Acid Sequence and Nucleotide Sequence of the Antibody of the Disclosure
The present disclosure used recombinant human PD-1 protein to immunize mice, and then obtained a M944 scFv antibody clone that specifically binds to recombinant human PD-1 with a high affinity by phage antibody library screening. The nucleotide sequences encoding the heavy and light chain variable regions of the M944 scFv antibody were then assemble by PCR with those encoding the mouse IgG1 heavy chain constant region and the mouse kappa light chain constant region, respectively, and the resulting assembled sequence were inserted into the transient expression vector HEK-293 cultured for expression. The high purity murine antibody PD1-M944 was purified using a protein A purification column. ELISA showed that the murine antibody PD1-M944 was able to block the binding of PD-1 to its ligand.
Then, using the classical method for humanized CDR transplantation, the human antibody light chain or heavy chain variable region closest to the murine light chain or heavy chain variable region was elected as the template. In an embodiment, IGKV3-11*01 is elected to be the humanization temple of light chain variable region, while IGHV3-21*02, heavy chain variable region. The humanized light chain variable region (VL) and heavy chain variable region (VH) sequences were obtained by inserting each of the three CDRs of the murine antibody light chain/heavy chain into the corresponding positions of the above human templates. As the key site of the murine framework region is essential to support the activity of the CDR, the key site was reverse mutated to the sequence of the murine antibody. The amino acid sequence and nucleotide sequence of the humanized antibody PD1-H944 were obtained by sequentially splicing the light chain/heavy chain signal peptide sequence, the sequence of the variable region of the light chain/heavy chain of the reverse mutated humanized antibody, and the sequence of the human IgG4 heavy chain constant region/human kappa light chain constant region, respectively.
Nucleic Acids of the Present Disclosure
The present disclosure also relates to nucleic acid molecules encoding antibodies or portions thereof of the present disclosure. The sequences of these nucleic acid molecules include, but are not limited to, SEQ ID NO: 3-7 and 26-33.
The nucleic acid molecules of the present disclosure are not limited to the sequences disclosed herein, but also include variants thereof. Variants in the present disclosure may be described with reference to their physical properties in hybridization. It will be recognized by those of skill in the art that using nucleic acid hybridization techniques, nucleic acids can be used to identify their complements as well as their equivalents or homologues. It will also be recognized that hybridization can occur at less than 100% complementarity. However, given the appropriate choice of conditions, hybridization techniques can be used to distinguish said DNA sequences based on the structural relevance of the DNA sequence to a particular probe. For guidance on such conditions see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, Cold Spring Harbor, N. Y, 1989 and Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Sedman, J. G., Smith, J. A., &Struhl, K. eds. (1995). Current Protocols in Molecular Biology. New York: John Wiley and Sons.
Recombinant Vectors and Expression
The present disclosure also provides recombinant constructs comprising one or more nucleotide sequences of the present disclosure. The recombinant constructs of the disclosure can be used with vectors, said vectors, for example, being plasmid, phagemid, phage or viral vectors, and the nucleic acid molecules encoding the antibodies of the disclosure are inserted into said vectors.
The antibodies provided herein can be prepared by recombinantly expressing nucleotide sequences encoding light and heavy chains or portions thereof in a host cell. In order to recombinantly express the antibody, the host cell may be transfected with one or more recombinant expression vectors carrying nucleotide sequences encoding the light and/or heavy chains or portions thereof, so that said light and heavy chains are expressed in said host cell. Standard recombinant DNA methodologies are used to prepare and/or obtain nucleic acids encoding heavy and light chains, to incorporate these nucleic acids into recombinant expression vectors and to introduce said vectors into host cells, e.g. Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and those documented in U.S. Pat. No. 4,816,397 by Boss et al.
Furthermore, a nucleotide sequence encoding a variable region of said heavy and/or light chain may be converted into a nucleotide sequence encoding, for example, a full-length antibody chain, a Fab fragment or a ScFv: for example, a DNA fragment encoding a variable region of the light chain or a variable region of the heavy chain may be operably ligated (so that the amino acid sequences encoded by both said DNA fragments are in frame) to another DNA fragment encoding, for example, an antibody constant region or a flexible linker. The sequences of the human heavy and light chain constant regions are known in the art (see, for example, Kabat, E. A., el al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments including these regions can be obtained by standard PCR amplification.
To express the antibodies, standard recombinant DNA expression methods can be used (see, for example, Goeddel; Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). For example, a nucleotide sequence encoding the desired antibody can be inserted into an expression vector, which is subsequently transfected into a suitable host cell. Suitable host cells are prokaryotic and eukaryotic cells. Examples of prokaryotic host cells are bacteria and examples of eukaryotic host cells are yeast, insect or mammalian cells. It should be understood that the design of an expression vector including the selection of a regulatory sequence is determined by a number of factors, such as the choice of host cell, the level of expression of the desired protein and whether the expression is constitutive or inducible.
Bacterial Expression
By inserting a structural DNA sequence encoding the desired antibody together with appropriate translation initiation and termination signals and the functional promoters into an operable reading frame, an available expression vector for use in bacteria is constructed. The vector will contain one or more phenotypic selection markers and an origin of replication to ensure the maintenance of the vector and provide amplification in the host as needed. Suitable prokaryotic hosts for transformation include multiple species of E. coli, Bacillus subtilis, Salmonella typhimurium, as well as Pseudomonas, Streptomyces and Staphylococcus.
The bacterial vector may be, for example, phage-, plasmid- or phagemid-based. These vectors may contain selectable markers and bacterial replication origins, which are derived from commercially available plasmids that usually contain elements of the well-known cloning vector pBR322 (ATCC 37017). After transforming an appropriate host strain and growing the host strain to an appropriate cell density, the selected promoter is de-repressed/induced by an appropriate method (for example, temperature change or chemical induction), and the cells are cultured for an additional time. The cells are usually harvested by centrifugation, disrupted by physical or chemical methods, and the resulting crude extract is retained for further purification.
In a bacterial system, a variety of expression vectors can be advantageously selected according to the intended use of the expressed protein. For example, when a large number of such proteins are to be produced for antibody production or for peptide library screening, for example, a vector that directs high-level expression of a fusion protein product to be easily purified may be required.
Mammalian Expression and Purification
Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high-level protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (e.g., CMV promoter/enhancer), promoters and/or enhancers of simian virus 40 (SV40) (e.g. SV40 promoter/enhancer), promoters and/or enhancers of adenovirus (e.g. adenovirus major late promoter (AdMLP)) and promoters and/or enhancers of polyoma virus. For a further description of viral regulatory elements and their sequences, see, for example, U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al., and U.S. Pat. No. 4,968,615 by Schaffner et al. The recombinant expression vector may also include an origin of replication and a selection marker (see, for example, U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017 by Axel et al). Suitable selection markers include genes that confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced. For example, the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate, while the neo gene confers resistance to G418.
The transfection of the expression vector into the host cell can be performed using standard techniques such as electroporation, calcium phosphate precipitation, and DEAE-dextran transfection.
Suitable mammalian host cells for expressing the antibodies provided herein include Chinese Hamster Ovary (CHO cells) [including dhfr-CHO cells, as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, DHFR selection markers are employed, as described in, for example, R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621], NSO myeloma cells, COS cells, and SP2 cells.
The antibodies of the present disclosure can be recovered and purified from recombinant cell culture by known methods, including but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, protein A affinity chromatography, protein G affinity chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography (“HPLC”) can be used for purification as well. See, for example, Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001), for example, Chapters 1, 4, 6, 8, 9, and 10, each of which is incorporated herein by reference in its entirety.
The antibodies of the present disclosure include natural purified products, products by chemical synthesis methods, and products produced from eukaryotic hosts by recombinant technology, the eukaryotic hosts include, for example, yeast, higher plants, insects, and mammalian cells. The antibodies of the present disclosure can be glycosylated or non-glycosylated. Such methods are described in many standard laboratory manuals, such as Sambrook above, Sections 17.37-17.42; and above described Ausubel, Chapters 10, 12, 13, 16, 18 and 20.
Therefore, embodiments of the present disclosure can be also host cells comprising the vectors or nucleic acid molecules, wherein the host cells may be higher eukaryotic host cells such as mammalian cells, lower eukaryotic host cells such as yeast cells, and can be also prokaryotic cells such as bacterial cells.
Properties and Functions of the Antibodies of the Disclosure
Property and function analysis of said PD1-H944 antibody were performed. The analysis showed that the antibody of the present disclosure has the following advantages: (1) it is able to bind human PD-1 with high affinity and specificity and has a low dissociation rate, thus providing good antitumor efficacy; (2) it is able to block the binding of PD-L1 to PD-1 more completely than Nivolumab; (3) when compared with Nivolumab, it binds recombinant human PD-1 more sensitively and more specifically; comparably cross binds to recombinant monkey PD-1, while neither binds to murine PD-1; (4) it is capable of blocking the binding of recombinant human PD-1 to its ligands Pd-L1 and Pd-L2 effectively, (5) it is capable of effectively re-activating immunosuppressed T cells and activating the recombinant human PD-1 reporter cell system; (6) exhibits an excellent tumor-suppressive effect in MC38 colon cancer tumorbearing humanized PD-1 mouse model, far better than that of Nivolumab (Sino Biological, Inc.) and comparable to that of Pembrolizumab (Sino Biological, Inc.); (7) it exhibits both low ADCC and CDC activities, while its ADCC activity is lower than that of Nivolumab.
Uses
The antibodies of the present disclosure can be used to treat a variety of tumors or cancers or to prepare medicines for the treatment of a variety of tumors or cancers, specifically targeting to aberrantly expressed PD-1 receptors. Non-limiting examples of said tumors or cancers include colon cancer.
Pharmaceutical Compositions
Antibodies of the disclosure may be prepared with at least one other agent (e.g. a stabilizing compound) to form pharmaceutical compositions comprising an antibody of the disclosure and one or more pharmaceutically acceptable carriers, diluents or excipients.
Kits
The present disclosure also relates to pharmaceutical packages and kits comprising one or more containers, said containers containing the foregoing pharmaceutical compositions of the present disclosure. Associated with such containers may be specifications in the form prescribed by the governmental agency governing the manufacture, use or distribution of the drug or biological product, which reflect approval for human administration by the agency in which said product is manufactured, used or distributed.
Preparation and Storage
The pharmaceutical compositions of the present disclosure can be prepared in a manner known in the art, for example by conventional mixing, dissolution, granulation, pastille preparation, grinding, emulsification, encapsulation, embedding or lyophilization methods.
Having already prepared pharmaceutical compositions comprising compounds of the present disclosure formulated in an acceptable carrier, they may be placed in appropriate containers and labeled for the treatment of the condition indicated. Such labeling would include the amount, frequency and administration routes of the drug.
Combinations
The pharmaceutical compositions comprising the antibodies of the present disclosure described above are also combined with one or more other therapeutic agents, such as antineoplastic agents, wherein the resulting combination does not cause unacceptable adverse effects.
The following examples are used to illustrate the disclosure exemplarily and are not intended to limit the disclosure.
Recombinant human PD-1 protein (Sino Biological, Inc, Cat. 10377-H08H) was used to immunize mice. The amino acid sequence of the extracellular region of this human PD-1 protein (UniProtKB Q15116) is Met1-Gln167 (SEQ ID NO: 1).
The recombinant human PD-1 protein was mixed with Freund's adjuvant and the mixture which administered subcutaneously at intervals of 2 weeks, 3 weeks, 2 weeks and 3 weeks was used for immunizing mice for 5 times at a dose of 20 μg each. From the second immunization onwards, blood was collected seven days after each immunization via the medial canthal plexus of the eye. The serum titer of mouse anti-PD-1 was measured by ELISA using coated recombinant human PD-1 protein. Serum titer reached 8000-fold dilution after fifth immunization, and the mice were boosted intravenously with 25 μg recombinant human PD-1 protein 9 weeks after the fifth immunization. 4 days later, the mice were executed and the spleen tissue was frozen in liquid nitrogen.
RNA was extracted from mouse spleen tissue using TriPure Isolation Reagent (Roche), and cDNA was obtained by reverse transcription using a reverse transcription kit (Invitrogen). The respective nucleotide sequences encoding the light and heavy chain variable regions of the murine antibody were amplified and assembled into the nucleotide sequence encoding the scFv thereof via the overlap extension PCR method, wherein the respective nucleotide sequences encoding the light and heavy chain variable regions were linked via a linker, TCTAGTGGTGGCGGTGGTTCGGGCGGTGGTGGAGGTGGTAGTTCTA GATCTTCC(SEQ ID NO:2) (Ref: Rapid PCR-cloning of full-length mouse immunoglobulin variable regions; Cloning immunoglobulin variable domains for expression by the polymerase chain reaction T Joneset.al Bio/Technology 9(6):579, July 1991), then enzymatically ligated into the phage vector pComb3x (Sino Biological, Inc.) by restriction endonuclease Sfi I (Fermentas), and then electro transformed the competent X-Blue to construct a phage display scFv antibody library for immunized mice. By coating recombinant human PD-1 protein on ELISA plates, an anti-PD-1 positive antibody-enriched phage libraries was obtained following the process of phage antibody panning (Ref: Antibody Phage Display: Methods and Protocols, Philippa M. O'Brien and Robert Aitken (Eds), Humana Press, ISBN: 9780896037113).
Monoclonal phages were selected from the enriched library, expressed, and then tested their binding to recombinant human PD-1 protein by ELISA. A clone of the highly binding antibody M944 scFv specifically binding to recombinant human PD-1 was elected and commissioned a sequencing company to sequence the nucleotide sequence of the M944 scFv antibody (SEQ ID NO:3).
The nucleotide sequences of the heavy and light chain variable regions of the M944 scFv antibody (SEQ ID NO: 4/5) were assembled with the nucleotide sequences of the mouse IgG1 heavy chain constant region (SEQ ID NO: 6) and the mouse kappa light chain constant region (SEQ ID NO: 7), respectively, by PCR. The resulting nucleotide sequences were then enzymatically cut by Hind III and Xba I (Fermentas) and inserted into the transient expression vector pSTEP2 (Sino Biological, Inc), and the plasmids were extracted and transfected into HEK-293 cells for 7 days. The culture supernatant was purified with Protein A column for high purity antibody.
(1) Blocking the Binding of Recombinant Human PD-1 to PD-L1:
Recombinant human PD-1 protein at a concentration of 1 μg/mL was coated on a 96-well plate at 100 μL per well overnight at 4° C. The plates were washed the next day and blocked at room temperature for 1 h. 100 μL of 10 μg/mL of biotinylated protein PD-L1-Fc-biotin (SinoBiological, Inc.) was co-incubated with different concentrations (0.4 μg/mL, 2 μg/mL and 10 μg/mL) of PD1-M944 or Nivolumab (Sino Biological, Inc). The plates were washed to remove unbound antibodies. The plates were incubated with antibiotic streptavidin/HRP (Beijing Zhong Shan-Golden Bridge Biological Technology Co., Ltd) and then repeatedly washed, and the substrate chromogenic solution was added for color development. OD450 was detected after termination. The results showed that the recombinant PD-L1 protein could effectively bind to the coated PD-1 protein, and the binding of recombinant PD-L1 protein to PD-1 was effectively inhibited by the addition of different concentrations of PD1-M944 or Nivolumab (
(2) Blocking the Binding of Recombinant Human PD-1 to PD-L2:
Recombinant human PD-1 protein at a concentration of 1 μg/mL was coated on a 96-well plate at 100 μL per well, overnight at 4° C., overnight, washed the next day and blocked at room temperature for 1 h. 100 μL of 0.5 μg/mL of biotinylated protein PD-L2-Fc-biotin (Sino Biological, Inc.) was co-incubated with different concentrations (0.5 μg/mL, 2.5 μg/mL and 12.5 μg/mL) of PD1-M944 or Nivolumab (Sino Biological, Inc.). The plates were washed to remove unbound antibodies, incubated with antibiotic streptavidin/HRP (Beijing Zhong Shan-Golden Bridge Biological Technology Co., Ltd), repeatedly washed, and the substrate chromogenic solution was added for color development, and OD450 was detected after termination. The results showed that the recombinant PD-L2 protein could effectively bind to the coated PD-1 protein, and the binding of recombinant PD-L2 protein to PD-1 was effectively inhibited by the addition of different concentrations of PD1-M944 or Nivolumab (
The nucleotide sequence of the M944 scFv antibody was determined in Example 1.2, from which the amino acid sequences of the heavy and light chain variable regions of the PD1-M944 scFv antibody were deduced, see SEQ ID NOs: 8/9.
The amino acid sequences of the 3 CDRs of each of the light and heavy chains of the murine antibody PD1-M944 were determined reference to the Kabat numbering scheme, see Table 1. The 3 CDRs of each of the aforementioned light and heavy chains were transposed and retained in the final humanized antibody PD1-H944 in the subsequent steps, see Examples 2.2 and 2.3.
The humanization of the murine antibody was performed using the classical humanization method of CDR transplantation. The human antibody light or heavy chain variable region, which is closest to the mouse light or heavy chain variable region, was elected as the template, and three CDRs (Table 1) from each of the mouse light or heavy chain were inserted into the variable region of the human antibody to obtain the humanized light chain variable region (VL) and heavy chain variable region (VH) sequences. The human template for the light chain variable region of PD1-M944 is IGKV3-11*01, which is 73.48% homologous to the light chain of PD1-M944, and the human template for the heavy chain variable region is IGHV3-21*02, which is 81.94% homologous to the heavy chain of PD1-M944.
As key sites in the murine-derived framework region are essential to support CDR activity, the corresponding sites were reversely mutated to those shown in the sequence of the murine antibody, wherein the position 89 in the light chain was reversely mutated to M and position 91 to F, in the heavy chain, the position 44 to R and the position 78 to N.
The humanized antibody PD1-H944 was obtained by CDR humanized transplantation and framework region reverse-mutation, and its heavy and light chain amino acid sequences are shown in SEQ ID NO:16/17, respectively; its heavy and light chain amino acid sequences in the form containing the signal peptide are shown in SEQ ID NO:18/19, respectively, comprising sequentially linked heavy/light chain signal peptide sequences (SEQ ID NO:20/21); the variable region of the heavy chain/light chain of humanized antibody sequence (SEQ ID NO:22/23); and the sequence of the constant region of humanized antibody which is the human IgG4 heavy chain constant region/human kappa light chain constant region respectively (SEQ ID NOs:24/25).
LEPED
AMYFCQQSKEVPWTFGQGT
After PCR amplification, the respective above heavy/light chain nucleotide sequences encoding the signal peptide containing PD1-H944 antibody (SEQ ID NO:26/27), which contains the nucleotide sequence coding for the sequentially linked heavy/light chain signal peptide (SEQ ID NO:28/29), the heavy/light chain variable region of the humanized antibody (SEQ ID NO:30/31) and the human IgG4 heavy chain constant region/human kappa light chain constant region (SEQ ID NO:32/33), respectively, were double digested with restriction endonucleases Hind III and Xba I (Fermentas) then inserted into the commercial vector pcDNA3 (Invitrogen). After plasmid extraction, a 1.8 kb fragment was obtained by triple digestion of pCDNA3 light chain vector with NruI+NaeI+Dra I then inserted into the CHO/dhfr system expression vector pSSE (Sino Biological, Inc), then a 2.5 kb fragment was obtained by triple digestion of the pCDNA3 heavy chain vector with NruI+NaeI+Dra I (Fermentas). Then also inserted to the pSSE (Sino Biological, Inc) light chain vector constructed in the previous step to obtain the complete vector. The expression vector is a eukaryotic expression vector containing the DNA amplification element dhfr gene, the NeoR resistance gene and the expression elements of the antibody light and heavy chains. The expression vector was transfected into dhfr-DG44 cells, PD1-H944 positive cell lines were obtained by G418 screening, PD1-H944 high expression cell lines were obtained by MTX stepwise pressure screening, and then domesticated in serum-free culture for clonal screening. At each step, the clone with high antibody expression were selected on the ground of ELISA assay outcomes, together with cell growth status and the key quality attributes of the antibody drug.
The PD1-H944 producing CHO cell line was cultured in serum-free supplement suspension culture to obtain PD1-H944 antibody of high purity and quality.
An indirect ELISA was used to detect the specific binding of PD1-H944 to recombinant human PD-1 protein. Different concentrations (0.16 ng/mL, 0.49 ng/mL, 1.48 ng/mL, 4.44 ng/mL, 13.33 ng/mL, 40 ng/mL, 120 ng/mL, 360 ng/mL, 1080 ng/mL, 3240 ng/mL and 9720 ng/mL) of recombinant human PD-1 protein was coated on 96-well plates overnight at 4° C. On the next day, the plates were washed and blocked at room temperature for 1 h. After incubation with 100 μL of 2 μg/mL of PD1-H944, Nivolumab (Bristol-Myers Squibb) and negative control antibody H7N9-R1-IgG4 (Sino Biological, Inc.) respectively, the plates were washed to remove unbound antibody, then incubated with goat anti-human IgG F(ab)2/HRP (JACKSON) and washed repeatedly, and the substrate chromogenic solution was added for color development and detection of OD450. EC50 of PD-1-H944 and Nivolumab binding to recombinant human PD-1 protein was 31.5 ng/mL, R2=0.998 and 179.0 ng/mL, R2=0.997, respectively. This indicates that PD1-H944 binds recombinant human PD-1 protein significantly better than Nivolumab (
(2) Binding Ability of PD1-H944 to Recombinant Jurkat/PD-1 Cells
The binding ability of PD1-H944 to recombinant Jurkat/PD-1 cells was measured by flow cytometry using the recombinant human PD-1 stable expression cell line Jurkat/PD-1 (SinoCellTech Ltd) as the experiment material. PD1-H944, Nivolumab and the negative control antibody H7N9-R1-IgG4 in different concentrations (0.195 μg/mL, 0.391 μg/mL, 0.781 μg/mL, 1.562 μg/mL, 3.125 μg/mL, 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL and 200 μg/mL) were added to 5×105/tube recombinant Jurkat/PD-1 cells in logarithmic growth phase, mixed and incubated at 4° C. and then washed and centrifuged to remove unbound antibody. The binding of the antibody to the cells was detected by adding goat anti-human IgG Fc-FITC (Sino Biological, Inc.). The results showed that PD1-H944 specifically bound to Jurkat/PD-1 cells with EC50 of 9.63 μg/mL, R2=1.000, Nivolumab specifically bound to Jurkat/PD-1 cells with EC50 of 10.18 μg/mL, R2=0.994, and the negative control antibody H7N9-R1-IgG4 did not bind to Jurkat/PD-1 cells (
(3) Binding Affinity of PD1-H944 to Recombinant Human PD-1 Protein
The affinity of PD1-H944 (0.90 nM, 1.79 nM, 3.66 nM, 7.24 nM), Nivolumab (0.51 nM, 1.02 nM, 2.05 nM, 4.10 nM) to PD-1 was determined using the Octet Biomolecular Interaction Assay System, with H7N9-R1-IgG4 (13.30 nM) as the negative control antibody. The results showed that the mean KD binding affinity of PD1-H944 to recombinant human PD-1 protein was 64.8 pM, the mean association rate constant kon was 3.01E+05 M−1s−1 and the mean dissociation rate constant koff was 1.95E-05 s−1; the mean KD binding affinity of Nivolumab to PD-1 protein was 74.1 pM, with a mean binding rate constant kon of 6.92E+05 M−1s−1 and a mean dissociation rate constant koff of 5.12E-05 s−1 (
(4) Binding Ability of PD1-H944 to Recombinant Monkey and Mouse PD-1 Protein
An indirect ELISA was used to detect the specific binding of PD1-H944 to recombinant monkey and mouse PD-1 protein. recombinant human, monkey and mouse PD-1 proteins (Sino Biological, Inc.) with different concentrations (0.16 ng/mL, 0.49 ng/mL, 1.48 ng/mL, 4.44 ng/mL, 13.33 ng/mL, 40 ng/mL, 120 ng/mL, 360 ng/mL, 1080 ng/mL, 3240 ng/mL and 9720 ng/mL) were coated on 96-well plates, 100 μL per well, coated overnight at 4° C. —, washed the next day and blocked at room temperature for 1 h. After incubation with 100 μL of PD1-H944, Nivolumab and the negative control antibody H7N9-R1-IgG4 (all in 2 μg/mL), the plates were washed and the secondary antibody, goat anti-human IgG F(ab)2/HRP, was added for color developement, OD450 was detected, the assay was in triplet. The results showed that PD1-H944 could bind recombinant human PD-1 protein and recombinant monkey PD-1 protein with antigenic EC50 of 25.8 ng/mL (R2=0.999) and 32.7 ng/mL (R2=0.997), respectively, while did not cross bind to recombinant mouse PD-1 protein (
(5) Binding Affinity of PD1-H944 to Recombinant Monkey and Mouse PD-1 Protein
The affinity of PD1-H944 and Nivolumab to biotinylated monkey and mouse PD-1 proteins (Sino Biological, Inc.) at different concentration gradients (see
The recombinant human PD-1 protein was coated on a 96-well plate at 100 μL per well and left overnight at 4° C. The plate was washed the next day and blocked at room temperature for 1 hour before 1 μg/mL of human PD-L1-biotin (Sino Biological, Inc.) or 2 μg/mL of human PD-L2-biotin (Sino Biological, Inc.) was added. Then PD1-H944, Nivolumab or negative control antibody H7N9-R1-IgG4 in different concentrations (0.003 μg/mL, 0.008 μg/mL, 0.025 μg/mL, 0.074 μg/mL, 0.222 μg/mL, 0.667 μg/mL, 2 μg/mL, 6 μg/mL, 18 μg/mL) was added, incubated, antibiotic streptavidin/HRP was added, incubated, and OD450 was detected. For each group, the test is performed in doublet. The results showed that biotinylated recombinant human PD-L1 and PD-L2 proteins could effectively bind coated human PD-1 protein, and the addition of different concentrations of PD1-H944 and Nivolumab both effectively inhibited the binding of recombinant human PD-L1 protein (
5×105 cells/tube of recombinant human PD-1 stably expressing Jurkat/PD-1 cells in logarithmic growth phase were added with PD1-H944, Nivolumab and the negative control antibody H7N9-R1-IgG4 in different concentrations (0.260 μg/mL, 0.390 μg/mL, 0.585 μg/mL, 0.878 μg/mL, 1.317 μg/mL, 1.975 μg/mL, 2.963 μg/mL, 4.444 μg/mL, 6.667 μg/mL or 10.000 μg/mL) and incubated at 4° C. Add 10 μL of 0.4 μg/mL of B7H1-Fc-Biotin (Sino Biological, Inc.), wash with PBS and remove unbound antibody by centrifugation. Streptavidin Alexa Fluor® 488 Conjugate (Life Technologies) was added, incubated at 4° C. for 20 minutes, repeated washed and centrifuged, and 200 μL of PBS was added to re-suspend the cells for detection on a flow cytometer. The results showed that the recombinant human PD-L1 protein could effectively bind to Jurkat/PD-1 cells, and the binding of recombinant human PD-L1 protein to Jurkat/PD-1 cells was effectively inhibited when different concentrations of PD1-H944 and Nivolumab were added (
The binding of PD1-H944 to the recombinant CD16a (V158) protein was measured by ELISA to assess the potentiality of PD1-H944 in regard to antibody dependent cell-mediated cytotoxicity (ADCC) effects.
The positive control PD1-H944-1-IgG1 (o) used in this assay is the variable region of PD1-H944 linked to natural IgG1, and the negative control PD1-H944-1-IgG1 is the variable region of PD1-H944 linked to N297A mutant IgG1 whose Fc fragment is deprived of the ability to bind to CD16a due to N-glycoside deletion. Recombinant human PD-1 protein (10 μg/mL, 100 μL/well) was coated on 96-well plates overnight at 4° C., washed the next day and blocked at room temperature for 1 h. PD1-H944, Nivolumab, positive control antibody PD1-H944-1-IgG1 (o)(Sino Biological, Inc.) and the negative control antibody PD1-H944-1-IgG1 (Sino Biological, Inc.) at different concentrations (0.020 μg/mL, 0.078 μg/mL, 0.3125 μg/mL, 1.25 μg/mL, 5 μg/mL, 20 μg/mL and 80 μg/mL) were added (see
The results showed that binding of the positive control antibody to recombinant CD16a protein increased with antibody concentration. PD1-H944, Nivolumab and the negative control antibody did not bind to recombinant CD16a protein at any of the tested concentrations (
The binding of PD1-H944 to recombinant C1q protein was measured by ELISA, thereby assessing the potentiality of PD1-H944 of complement-dependent cytotoxicity (CDC) effects.
PD1-H944-1-IgG1 (o), obtained by linking the variable region of PD1-H944 to natural IgG1, was used as the positive control antibody (Sino Biological, Inc.) and H7N9-R1-IgG4 (Sino Biological, Inc.) was used as the isotype control antibody. Different concentrations (20 μg/mL, 10 μg/mL, 5 μg/mL, 2.5 μg/mL, 1.25 μg/mL, 0.625 μg/mL, 0.313 μg/mL, 0.156 μg/mL and 0.078 μg/mL) (see
The results showed that binding of positive control antibodies to recombinant C1q protein increased with antibody concentration. PD1-H944, Nivolumab and isotype control antibodies had comparable binding capacity to recombinant C1q protein, with significantly lower binding capacity than IgG1-type positive controls (
The binding of PD1-H944 to recombinant human Fc receptor (FcRn) protein was measured by ELISA and thus the pharmacokinetics of PD1-H944 in humans was assessed.
Anti-biotin protein Avidin (Thermo Fisher Scientific) at a concentration of 10 μg/mL was coated onto 96-well plates at 100 μL per well and incubated overnight at 4° C. and washed the next day, blocked at room temperature for 1 hour and incubated with 10 μg/mL of biotinylated FCGRT-His+B2M protein (Sino Biological, Inc.) for 1 hour, then PD1-H944, Nivolumab or isotype control antibody H7N9-R1-IgG4 (Sino Biological, Inc.) in different concentrations (0.123 μg/mL, 0.37 μg/mL, 1.11 μg/mL, 3.33 μg/mL, 10 μg/mL, 30 μg/mL, 90 μg/mL, 270 μg/mL) was added respectively (see
The results showed that PD1-H944 and Nivolumab were able to bind to recombinant human FcRn protein, and the binding capacity increased with concentration; at high concentrations (270 μg/mL), the binding of PD1-H944 to recombinant human FcRn protein was approximately 1.62 times of that of Nivolumab (
The neutralizing activity of anti-human PD-1 antibody was detected by the mixing assay of CD4+ T lymphocytes with DCs. Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation, and then mononuclear cells were obtained by the adhesion culture method, 1640 cell culture medium (GIBCO) (containing 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin) containing 160 ng/mL rhIL-4 (Sino Biological, Inc.) and 20 ng/mL rhGM-CSF (R&D Systems) was added to the resulting mononuclear cells and incubated in a CO2 incubator until the third day, then a half volume of culture medium was changed. After 6 days of incubation, the suspending cells were collected, which are known as DCs. The density of DCs was adjusted to 2×106 cells/mL, mitomycin at a final concentration of 50 μg/mL was added, treated at 37° C. for 20 minutes, washed three times with the 1640 medium for further steps. CD4+ T lymphocytes were sorted from PBMC isolated from the peripheral blood of another person using a CD4+ T lymphocyte sorting kit (MiltenyiBiotec). CD4+ T lymphocytes from each of the three blood donors were used in each of batches of this assay. The sorted CD4+ T lymphocytes were mixed with DCs previously treated with 50 μg/mL mitomycin in a 10:1 ratio, and 105 cell/well were evenly inoculated in 96-well plates with PD1-H944, Nivolumab, and the negative control antibody H7N9-R1-IgG4 (Sino Biological, Inc.), respectively, and another mixed lymphocyte control group, i.e. the group with only CD4+ T lymphocytes and DCs mixture without antibody, DC cells control group, i.e. the group with only DC cells without antibody and CD4+ T lymphocytes, CD4+ T cells control group, i.e. the group with only CD4+ T cells without antibody and DCs cells, with the same volume of sample diluent 1640 medium, the final concentration gradient of each antibody was 1 μg/mL, 0.1 μg/mL, 0.01 μg/mL, and incubated in a CO2 incubator at 37° C. and 5% CO2 for 5 days. The culture supernatants were collected and the expression levels of IL-2 and IFN-γ in the culture supernatants were measured by ELISA.
The results showed that IFN-γ and IL-2 secretion could not be detected in the supernatants of CD4+ T cell cultures without DCs mixing, while in the mixed lymphocyte control group, the mixing of CD4+ T cells and DCs significantly increased the IFN-γ and IL-2 secretion of CD4+ T cells. When anti-PD-1 antibody was added to the mixed lymphocyte system, the activation of CD4+ T cells in the mixed lymphatic response of CD4+ T and DCs significantly promoted with higher IFN-γ and IL-2 secretion. Rather contrary to both PD1-H944 and Nivolumab, which had a significant effect, the negative control antibody did not show this effect. The results are shown in
In this assay, effector cells Jurkat-NFAT-Luc2p-PD-1 (SinoCellTech Ltd) and target cells CHO-K1-PD-L1-CD3E (SinoCellTech Ltd) were used as experiment materials. The PD-1/PD-L1 interaction resulted by co-culture of these two cells inhibits TCR signaling and NFAT-RE-mediated bioluminescence, and the addition of PD-1 antibody blocked the PD-1/PD-L1 interaction and thus released this inhibition. The activation function of PD1-H944 in the recombinant human PD-1 reporter cell system was determined by measuring the intensity of bioluminescence (RLU) in effector cells. Target cells CHO-K1-PD-L1-CD3E cells were inoculated at 2×104/well in 96-well plates and cultured overnight in DMEM medium containing 10% FBS, then the supernatant was removed. PD1-H944, Nivolumab and the negative control antibody H7N9-R1-IgG4 (Sino Biological, Inc.) in different concentrations (0.007 μg/mL, 0.023 μg/mL, 0.082 μg/mL, 0.286 μg/mL, 1.000 μg/mL, 3.499 μg/mL, 12.245 μg/mL, 42.857 μg/mL, 150 μg/mL) were added at 40 μL/well (see
The results showed that both PD1-H944 and the control antibody Nivolumab had a significant activation of the recombinant human PD-1 reporter gene cell system, as shown in
5.3 PD1-H944 does not Significantly Stimulate the ADCC Function of the Fc Receptor CD16a Pathway
In this assay, the effector cell Jurkat-NFAT-Luc2p-CD16A (SinoCellTech Ltd) and the target cell CHO-PD-1 (SinoCellTech Ltd) were used as assay materials to measure the PD1-H944-mediated ADCC effect by the recombinant highly active CD16a (V158) reporter gene system method. Target CHO-PD-1 cells were inoculated at 2×104/well in a 96-well plate and cultured overnight in DMEM medium containing 10% FBS, and the supernatant was removed. The plates were washed twice with RPMI 1640 (phenol red-free) medium containing 0.1% BSA, and then positive control antibody PD1-H944-1-IgG1 (o), PD1-H944, Nivolumab and negative control antibody H7N9-R1-IgG4 (Sino Biological, Inc.) were added at different concentrations (see
The results showed that the positive control antibody PD1-H944-1-IgG1 (o) had a significant ADCC mediating effect, Nivolumab had a weaker ADCC effect while PD1-H944 had an even weaker ADCC effect than Nivolumab in the concentration range of 0.018-300 ng/mL (p<0.01).
5.4 PD1-H944 does not Significantly Activate the CDC Function of the Complement C1q Pathway
After its binding to PD-1 overexpressed tumor cells, PD1-H944 could activate the classical complement pathway to kill tumor cells, leading to the death thereof. In this assay, the CDC effect of PD1-H944 was investigated using the WST-8 method. CHO-PD-1 cells were re-suspended in RPMI 1640 medium containing 0.1% BSA (SinoCellTech Ltd) and inoculated uniformly in 96-well plates at 5×104/well, followed by the addition of antibodies at different concentrations (
The results showed that PD1-H944, Nivolumab and the negative control antibody had no CDC effect on CHO-PD-1, a tumor cell overexpressing PD-1, while the positive control antibody had killing effect on CHO-PD-1 with a maximum killing rate of 82.1%, the results are shown in
(1) Pharmacodynamics Study I of PD1-H944 in a Humanized PD-1 Mouse Model with MC38 Colon Cancer Subcutaneous Transplantation Tumor
MC38 cells at logarithmic growth stage (Sun Ran Shanghai Biotechnology Co., Ltd.) were used for tumor inoculation. MC38 cells re-suspended in PBS were inoculated at 5×105 cells/0.1 mL in B-hPD-1 humanized mice (Biocytogen) (obtained by replacing part of exon 2 of the PD-1 gene of C57BL/6 mice with the corresponding part of the human PD-1 genome) subcutaneously on the right side of the lateral thorax of mice, 47 mice in total. When the tumor volume grew into about 100 mm3, the mice were elected according to their individual tumor volume, randomly assigned to 5 groups using excel software, 8 animals in each group. Groups 4 and 5 were administered with other anti-PD-1 antibodies irrelevant to this application, so their data are not presented herein. Dosing was started on the day of grouping. The mice were administered by intraperitoneal injection (I.P.), once every 3 days for 6 consecutive doses, executed 10 days after the last dose and tumor tissue were removed for weighing. The specific dosing regimen was shown in Table 3 below. Anti-tumor effect of the drug was evaluated by calculating the tumor growth inhibition rate TGI (%). TGI (%)<60% was considered ineffective; TGI (%)≥60% and the tumor volume of the treated group was statistically significantly lower than that of the solvent control group (P<0.05) was considered effective, i.e. it had a significant inhibitory effect on tumor growth. the TGI (%) was calculated as follows.
TGI(%)=[1−(Ti−T0)/(Vi−V0)]×100, where
Ti: the mean tumor volume in the treatment group on day i.
T0: the mean tumor volume in the treatment group on day 0.
Vi: the mean tumor volume of the solvent control group on day i.
V0: mean tumor volume of the solvent control group on day 0.
The animals were executed at the end of the assay, weighed for tumor weight and the tumor weight inhibition rate IRTW % was calculated, with IRTW%>60% as the reference index of effectiveness, calculated as follows.
Inhibition rate of Tumor weight IRTW(%)=(W solvent control−W treatment group)/W solvent control×100, W is tumor weight.
aThe volume of administration is calculated at 10 μL/based on the body weight of the tested animal.
All test animals were in good general condition in terms of activity and feeding during the dosing period, and their body weight increased to some extent. There was no significant difference (P>0.05) in the body weight of the animals after the administration of the test drug (group 3) and the control drug (group 2). The changes in body weight of all animals are shown in
amean ± standard error.
bstatistical comparison of body weight in the treatment group with that of the solvent control group 25 days after administration, t-test.
c Group 2 had 7 mice at end of the experiment.
The tumor volume results for each group in the trial are shown in Table 5 and
25 days after the first dose, all animals were euthanized and the tumors were stripped, weighed and photographed. Tumor weights were statistically compared between groups and the results are summarized in Table 6 and
Consistent with the tumor volume results, Nivolumab did not significantly inhibit tumors in this model, while the mean tumor weight of the PD1-H944 group was 0.044±0.017 g. Its inhibition rate of tumor weight (IRTW) reached 97.4%. Data analysis showed tumors of the PD1-H944 group were significantly different from that of solvent controls (P<0.05), demonstrating the significant anti-tumor efficacy of PD1-H944.
At the end of the assay, the mean tumor volume of the solvent control group was 2171±430 mm3 and the tumor weight was 1.719±0.310 g. The mean tumor volume of the positive control group Nivolumab (20 mg/kg) was 1802±228 mm3, the TGI % was 17.8% and the tumor weight was 1.492±0.274 g. The inhibition rate of tumor weight IRTw was 13.2%, which was not significantly different from the tumor volume of the solvent control group (P=0.480). In contrast, the mean tumor volume of PD1-H944 was 171±51 mm3, TGI % was 96.7%, tumor weight was 0.044±0.017 g and inhibition rate of tumor weight IRTW was 97.4%, which was significantly different compared to the tumor volume of the solvent control (P<0.05), indicating that the donor antibody PD1-H944 bound to PD-1 epitope was effective and showed significant inhibition of MC38 colon cancer subcutaneous graft tumors at a dose level of 20 mg/kg.
(2) Pharmacodynamics Study II of PD1-H944 in a Humanized PD-1 Mouse with MC38 Colon Cancer Subcutaneous Transplantation Tumor
MC38 cells at logarithmic growth stage (Sun Ran Shanghai Biotechnology Co., Ltd.) were used for tumor inoculation. MC38 cells re-suspended in PBS were inoculated at 5×105 cells/0.1 mL in B-hPD-1 humanized mice (Biocytogen) (mice obtained by replacing part of exon 2 of the PD-1 gene of C57BL/6 mice with the corresponding part of the human PD-1 genome). A total of 80 mice were inoculated subcutaneously on the right side of the lateral thorax. When the tumor volume grew into about 100 mm3, the mice were elected according to their individual tumor volume, randomly assigned to 8 groups using excel software, 8 animals in each group. Groups 3, 4, 7 and 8 were administered with other anti-PD-1 antibodies irrelevant to this application, so their data were not presented herein. Dosing was started on the day of grouping. The mice were and administered by intraperitoneal injection (I.P.), once every 3 days for 6 consecutive doses, with mice executed 10 days after the last dose and tumor tissue removed for weighing. The specific dosing regimen is shown in Table 7 below.
aThe volume of administration was calculated based on the body weight of the animal at 10 μL/g;
bPembrolizumab: Sino Biological, Inc.
The anti-tumor effect of the drug was evaluated by calculating the tumor growth inhibition rate TGI (%). TGI (%)<60% was considered ineffective; TGI (%)≥60% and the tumor volume of the treated group statistically lower than that of the solvent control group (p<0.05) was considered effective, i.e. it had a significant inhibitory effect on tumor growth. the TGI (%) was calculated as follows.
TGI(%)[1−(Ti−T0)/(Vi−V0)]×100, where
Ti: the mean tumor volume of the treatment group on day i.
T0: the mean tumor volume of the treatment group on day 0.
Vi: the mean tumor volume of the solvent control group on day i.
V0: mean tumor volume of the solvent control group on day 0.
The animals were executed at the end of the assay, tumor were weighed and the tumor weight inhibition rate IRTW % was calculated, with IRTW %≥60% as the reference index of effectiveness, calculated using the following formula,
Tumor weight inhibition rate IRTW(%)=(W solvent control−W treatment group)/W solvent control×100, W is tumor weight.
All test animals were in good general condition in terms of activity and feeding during the administration of the drug, and to some extent, their body weight increased. There was no significant difference in the body weight of the animals after the administration of the test and control drugs (P>0.05) (Table 8 and
amean ± standard error.
bstatistical comparison of treatment group body weight with solvent control group body weight 23 days after dosing, t-test.
The tumor volumes for each group in the assay were shown in Table 9 and
24 days after the first dose, all animals were euthanized and the tumors were stripped, weighed and photographed. Tumor weights were statistically compared between groups and the results are summarized in Table 10 and
For the solvent control group: tumor volume continued to grow after grouping and the mean tumor volume at the end of the trial was 3405±624 mm3.
For the positive control group: after Pembrolizumab administration at 20 mg/kg, tumor volume increased slowly compared to the solvent control group, with a significant difference in tumor volume from day 5 of administration (P<0.05); the TGI % at the end of the trial was 97.0% and the inhibition rate of tumor weight was 93.5%, P<0.05 compared to the solvent control group. Pembrolizumab showed obvious tumor inhibition on this efficacy model, indicating that this model can be used for the efficacy evaluation of Keytruda epitope binding antibody drug.
For the PD1-H9448 mg/kg group: After PD1-H944 administration at 8 mg/kg, the tumor volume increased slowly compared to the solvent control group, and the tumor volume showed a significant difference from the 5th day of administration (P<0.05); the TGI % at the end of the trial was 89.7% and the inhibition rate of tumor weight was 87.8%, P<0.05 compared to the solvent control group.
For the PD1-H9442 mg/kg group: After PD1-H944 administered at 2 mg/kg, tumor volume increased slowly compared to the solvent control group, with a significant difference in tumor volume from day 5 (p<0.05); the TGI % at the end of the trial was 85.3% and the tumor weight inhibition rate was 84.7%, p<0.05 compared to the solvent control group. This again suggests that the subjected PD1-H944 antibody binding to the PD-1 epitope is effective in this model, displaying a significant tumor-suppressive effect on MC38 colon cancer subcutaneous graft tumors displaying dose correlation. The anti-tumor efficacy of PD1-H944 and Pembrolizumab in this assay was also confirmed in terms of tumor weight.
The results of this assay showed that intraperitoneal administration of PD1-H9448 mg/kg or 2 mg/kg every 3 days for 6 consecutive doses significantly inhibited the growth of MC38 transplanted tumors in PD-1 humanized mice (P<0.05), and the animal characterization and body weights of the administered groups were not different from those of the model group. Considering the preference of the model design for different epitopes of antibodies, this assay did not evaluate the efficacy difference between PD1-H944 and Pembrolizumab.
The in vivo pharmacodynamics study investigated the anti-tumor effect of PD1-H944 alone on subcutaneous transplanted MC38 colon cancer in humanized PD-1 C57BL/6 mouse as the animal model. The B-hPD-1 humanized mouse is a C57BL/6 mouse whose PD-1 gene had been humanized, making the mouse suitable for in vivo evaluation of the efficacy of anti-human PD-1 antibody drugs. The model was selective for the efficacy of different epitopes of PD-1 antibodies, and Pembrolizumab showed significant tumor inhibition in this model. The results showed that PD1-H944 alone (2, 8 and 20 mg/kg administered every 3 days for 6 consecutive doses) significantly inhibited the growth of MC38 transplanted tumors and summary data are presented in Tables 11 and 12.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201811573605.4 | Dec 2018 | CN | national |
This application is a continuation of and claims priority to PCT Application No. PCT/CN2019/126594 filed Dec. 19, 2019, which itself claims priority to Chinese Patent Application No. 201811573605.4 filed Dec. 21, 2018. The contents from all of the above are hereby incorporated in their entirety by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2019/126594 | Dec 2019 | US |
| Child | 17353617 | US |
