RU2803460C2 - Humanized antibody against pd-1 and its use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to an isolated anti-PD-1 antibody or its antigen-binding fragment, as well as to a composition, combination and a set containing it. Also an isolated nucleic acid encoding the above antibody or a fragment thereof, as well as a vector and a cell containing it are disclosed. The invention also relates to a method of obtaining an isolated anti-PD-1 antibody or antigen-binding fragment thereof, which involves growing the above cell.

EFFECT: invention is effective for the preparation of a drug for the treatment of a tumor or cancer associated with PD-1, as well as for the treatment of a tumor or cancer associated with PD-1.

22 cl, 25 dwg, 15 tbl, 6 ex

Description

Field of technology

The present invention relates to the field of oncology or cancer immunotherapy. In particular, the present invention provides a recombinant humanized anti-programmed cell death receptor-1 (PD-1) antibody that can be used in immunotherapy of tumors or cancer. The present invention also provides nucleic acid sequences encoding said antibody, vectors containing said nucleic acid sequences, pharmaceutical compositions and kits.

Prior Art Technologies

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 primarily 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 (ie, B7-H1, also known as CD274) and PD-L2 (ie, B7-DC, also known as CD273), both of which are transmembrane protein molecules of the B7 family. In terms of quantity and action, PD-L1 is the main ligand of PD-1. PD-L1 is widely expressed on the surface of various cell types, including hematopoietic cells such as dendritic cells, B cells and T cells, as well as non-hematopoietic cells such as epithelial and endothelial cells. PD-L1 expression on the surface of tumor cells is high, and it can be upregulated by inflammatory cytokines such as interferon-γ and TNF-α. The ligand PD-L2 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, approximately three times greater than PD-L1.

PD-1 plays its biological role by binding to its ligands, inhibiting T cell proliferation, T cell 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, tyrosine residues within the immunoreceptor tyrosine switch motif (ITSM), one domain of the cytoplasmic region of PD-1, are phosphorylated, then the phosphorylated ITSM recruits the phosphatase SHP-2, resulting in inhibition of important downstream pathways through dephosphorylation such as blocking the activation of phosphoinositide 3-kinase (PI3K) and its downstream protein kinase B (PKB or Akt), inhibition of glucose metabolism, production of the cytokine interleukin-2 (IL-2) and expression of the anti-apoptotic protein Bc1-x1; thus, it inhibits the proliferation and activation of T and B cells and the secretion of immunoglobulins, resulting in the suppression of the autoimmune response. Tumor cells use this immunosuppressive mechanism to evade the immune response by binding to PD-L1, highly expressed on PD-1 molecules on the surface of lymphocytes, they evade immune recognition and removal by the body.

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 T cell secretion as well as interferon-γ and IL-2 at tumor sites, reduce the proportion of myeloid-derived suppressor cells (MDSCs), alter the tumor microenvironment, repair and enhance immune killing function of T cells and thus suppress tumor growth. The US Food and Drug Administration (FDA) has now granted marketing authorization for anti-PD-1 antibodies, including Bristol-Myers Squibb's Opdivo (INN nivolumab) and Merck & Co's Keytruda (INN pembrolizumab). Opdivo is approved for the treatment of melanoma, non-small cell lung cancer, squamous cell carcinoma of the head and neck, classical Hodgkin 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, squamous cell carcinoma of the head and neck, classical Hodgkin lymphoma, urothelial carcinoma, high microsatellite instability and gastric cancer. In addition, domestic and international anti-PD-1 antibodies in clinical trials include REGN2810 from Regeneron/Sanofi, JS001 from Top Alliance, SHR-1210 from Hengrui, BGB-A317 from Beigene, IBI308 from Cinda and GLS-010 from Gloria/WuXiPharmaTech and many other anti-PD-1 and anti-PD-L1 antibodies are in preclinical studies.

In conclusion, research on monoclonal antibodies against PD-1 has made some progress. However, there is still a need to improve their efficacy, safety and * create competitive monoclonal biologics in oncology or cancer immunotherapy.

Description of the present invention

The technical terms used in the present invention and their corresponding abbreviations are given in Table 1.

The first aspect of the present invention provides an isolated anti-PD-1 antibody or antigen-binding fragment thereof comprising a light chain variable region or part thereof and/or a heavy chain variable region or part thereof;

wherein said light chain variable region or portion thereof comprises a light chain CDR1 having the amino acid sequence SEQ ID NO: 10, a light chain CDR2 having the amino acid sequence SEQ ID NO: 11, and a light chain CDR3 having the amino acid sequence SEQ ID NO: 12 ; And

said heavy chain variable region or portion thereof comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 13, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 14, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 15.

In a specific embodiment, said antibody or antigen-binding fragment thereof comprises, consists of, or essentially consists of: an amino acid sequence having at least 90%, 92%, 95%, 98%, or 100% identity with a light chain variable region sequence SEQ ID NO: 23 of the specified anti-PD-1 antibodies, and an amino acid sequence having at least 90%, 92%, 95%, 98% or 100% identity with the heavy chain variable region sequence SEQ ID NO: 22 of the specified anti-PD antibodies -1.

In a certain embodiment, said antibody further comprises a light chain constant region and a heavy chain constant region; 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% identity with the kappa constant region sequence light chain with the 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% identity with the sequence of the IgG4 heavy chain constant region with amino acid sequence SEQ ID NO: 24. In a specific embodiment, said antibody is an IgG antibody. In yet another specific embodiment, said antibody is an IgG4 antibody.

In yet another specific embodiment, said antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

In a specific embodiment, said antibody or antigen-binding fragment thereof binds to said recombinant human PD-1 protein with an average affinity K _D of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 . ., or any values thereof, such as about 64.8 pM or about 108 pM, etc. The method for determining the binding affinity K _D is described in the examples of this application.

In yet another specific embodiment, said 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% identity to SEQ ID NO: 1.

A second aspect of the present invention provides an isolated anti-PD-1 antibody or antigen-binding fragment thereof that specifically binds to a peptide sequence selected from at least one of the extracellular regions of the PD-1 protein 60SESFV64, 78KLAAFPEDRSQP89, 128LAPKAQI134.

In a specific embodiment, an isolated antibody or antigen-binding fragment thereof is provided that specifically binds to an amino acid residue selected from at least one of the E61, K78, D85, P130 extracellular region residue of said PD-1 protein.

In yet another specific embodiment, said antigen-binding fragment is present 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 yet another specific embodiment, said single chain antibody molecule is a scFv, di-scFv, tri-scFv, diabody, or scFab.

In a further specific embodiment, said antibody or antigen-binding fragment thereof from the above embodiments forms a covalent or non-covalent conjugate or recombinant multipurpose fusion of another drug molecule, thereby forming a modified drug molecule, wherein said other molecule is selected from a low molecular weight compound and /or biomacromolecules.

In a third aspect, the present invention provides an isolated nucleic acid whose nucleotide sequence encodes said antibody and/or antigen binding fragment of the first and second aspects.

The fourth aspect of the present invention provides a vector containing the specified nucleic acid from the third aspect.

A fifth aspect of the present invention provides an isolated cell expressing said antibody and/or antigen binding fragment from the first and second aspects, and/or containing said nucleic acid from the third aspect or said vector from the fourth aspect.

In yet another specific embodiment, said cells are prokaryotic or eukaryotic cells.

A sixth aspect of the present invention provides a method for producing said antibody and/or antigen binding fragment from the first and second aspects, said method comprising growing cells from the fifth aspect and isolating said antibodies.

A seventh aspect of the present invention provides the use of said antibody and/or antigen binding fragment and/or modified drug molecule of the first and second aspects for the preparation of a drug for treating a tumor or cancer.

In yet another specific embodiment, said tumor or cancer is colon cancer.

An eighth aspect of the present invention provides the use of said antibody and/or antigen binding fragment and/or modified drug molecule of the first aspect for treating a tumor or cancer.

In yet another specific embodiment, said tumor or cancer is colon cancer.

A ninth aspect of the present invention provides a pharmaceutical composition comprising said antibody and/or antigen binding fragment and/or modified drug molecule of the first and second aspects.

A tenth aspect of the present invention provides a pharmaceutical combination comprising said pharmaceutical composition from the ninth aspect and one or more therapeutically active compounds.

An eleventh aspect of the present invention provides a kit comprising said antibody and/or antigen binding fragment and/or modified drug molecule from the first and second aspects, or said pharmaceutical composition from the ninth aspect, or said pharmaceutical combination from the tenth aspect, preferably further comprising a device for introduction.

A twelfth aspect of the present invention provides a method of treating a tumor or cancer, comprising administering to a subject in need thereof a therapeutically effective amount of said isolated anti-PD-1 antibody or antigen-binding fragment thereof or a modified drug molecule of the first and second aspects, or a pharmaceutical composition of a ninth aspect, or a pharmaceutical combination of a tenth aspect, or a set of an eleventh aspect for treating said tumor or cancer, wherein said tumor or cancer is preferably colon cancer.

Brief description of drawings

Fig. 1: Blocking by the mouse PD1-M944 antibody of the binding of PD-L1 (A) and PD-L2 (B) to the PD-1 protein.

Fig. 2: Determination by enzyme-linked immunosorbent assay (ELISA) of binding of the humanized antibody PD1-H944 to recombinant human PD-1 protein (A, B, C, n=3).

Fig. 3: Fluorescence activated cell sorting (FACS) determination of PD1-H944 binding to recombinant Jurkat/PD-1 cells.

Fig. 4: Octet analysis to determine the affinity of PD1-H944 (A1, B1, C1, n=3) and nivolumab (A2, B2, C2, n=3) with recombinant human PD-1 protein.

Fig. 5: ELISA assay for binding of PD1-H944 (A), nivolumab (B) and negative control (C) to recombinant human, monkey and mouse PD-1 protein.

Fig. 6: Binding of PD1-H944 and nivolumab at various concentrations to monkey PD-1 protein (A) and mouse PD-1 protein (B).

Fig. 7: Enzyme-linked immunosorbent assay (ELISA) determination of blocking binding of human PD-L1 (A) and human PD-L2 (B) to recombinant human PD-1 protein by PD1-H944.

Fig. 8: FACS analysis to determine the blocking of human PD-L1 binding to human Jurkat/PD-1 cells by PD1-H944.

Fig. 9: ELISA determination of PD1-H944 binding to recombinant CD 16a protein.

Fig. 10: ELISA determination of PD1-H944 binding to recombinant C1q protein.

Fig. 11: ELISA determination of PD1-H944 binding to human FcRn protein.

Fig. 12: Secretion of IL-2 (top) and interferon-γ (bottom) in a mixed lymph assay (A, B, C are CD4 ⁺ T cells from 3 donors) stimulated by anti-PD-1 antibody.

Fig. 13: Activating function of PD1-H944 in a recombinant human PD-1 reporter gene cell system.

Fig. 14: ADCC function of PD1-H944 determined in a recombinant CD 16a reporter gene cell system (A and B are results from different assay batches; C and D are bioluminescent intensity values at the highest antibody concentration; ** indicates P < 0.01).

Figure 15: Effect of CDC PD1-H944 on CHO-PD-1 cells.

Fig. 16: Curve of changes in body weight of animals after administration.

Fig. 17: Tumor volume growth curve after injection.

Fig. 18: Plot of tumor weight at the end of the analysis (*P<0.05).

Fig. 19: Graph of changes in body weight of animals after administration.

Fig. 20: Graph of tumor volume growth after drug administration.

Fig. 21: Plot of tumor weight at the end of the analysis.

Fig. 22: Homology modeling of PD1-H944 associated with the crystal structure of PD1.

Fig. 23: Model of the PD1 protein showing the mutation site.

Fig. 24: Binding of mutant PD-1 proteins to PD-L1, nivolumab and PD1-H944.

Fig. 25A-B: Amino acid regions where PD1-H944, PD-L1 and nivolumab specifically bind to PD-1.

Detailed Description of the Present Invention

Definitions

Unless otherwise specified, all technical and scientific terms used herein have the meaning commonly understood by one skilled in the art to which the present invention belongs. For the purposes of the present invention, the following terms are defined.

As used in this specification and in the accompanying claims, the singular forms “one,” “other,” and “said” include the plural designation of subject matter 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 (eg, bispecific antibodies) and even antibody fragments. Typically, full-length antibody structures preferably include four polypeptide chains, two heavy (H) chains and two light (L) chains, typically linked 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 heavy or light chain domain of an antibody that is involved in the binding of the antibody to an antigen. The variable regions of the heavy and light chains of natural antibodies ( _VH and _VL , respectively) generally have a similar structure and can be divided into highly variable regions (called complementarity determining regions (CDRs)) interspersed with more conserved regions (called framework regions). FR)).

The term “complementarity determining region” (CDR, eg, CDR1, CDR2 and CDR3) refers to those amino acid residues in the variable region of an antibody whose presence is required for antigen binding. Each variable region typically has three CDR regions, designated CDR1, CDR2 and CDR3. Each complementarity-determining region may contain amino acid residues from the “complementarity-determining region” defined by Kabat (et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. 1991 ) and/or amino acid residues from the "highly variable loop" (Chothia and Lesk; J Mol Biol 196: 901-917 (1987)).

The term “framework” or “FR” region amino acid residues refers to residues in the variable region other than residues in the CDR as defined herein.

Each heavy chain variable region and light chain variable region typically contains 3 CDRs and up to 4 FRs, with said CDRs and FRs arranged from the amino terminus to the carboxyl terminus in the following order, for example: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

The complementarity determining region (CDR) and framework region (FR) of a particular 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 those amino acid sequences in the light and heavy chains of an antibody that are not directly involved in antibody-antigen binding but exhibit various 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 divided into five classes of antibodies: IgA, IgD, IgE, IgG and IgM, of which IgG and IgA can be roughly divided into subclasses (isoforms) such as IgG1, IgG2, IgG3 , IgG4, IgA1 and IgA2. Accordingly, the heavy chains of the five classes of antibodies are divided into α, δ, ε, γ and μ chains, respectively. Antibody light chains are classified into κ and λ, according to the amino acid sequence of their light chain constant region.

An "antigen-binding antibody fragment" contains 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 (eg, one or more CDRs) of the parent antibody. Examples of antigen binding fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd fragments, Fd' fragments, single chain antibody molecules (eg, scFv, di-scFv or tri-scFv, diabody or scFab), single domain antibodies, but are not limited to them.

An "antibody fragment" is a non-intact antibody molecule that at least retains some of the biological properties of the parent antibody, including, but not limited to, the Fc fragment, in addition to the fragments that are described above as "antigen-binding fragments".

The term “modified drug molecule” means a conjugate or recombinant multi-target drug fusion formed by covalently or non-covalently combining an antibody or fragment thereof, such as an antigen-binding fragment, with another molecule, which is a small molecule compound or biomacromolecule.

The term "chimeric" antibodies refers to antibodies in which part of the heavy and/or light chain is derived from a particular source or species and the remainder is derived from another source or species. "Humanized antibodies" are a subset of "chimeric antibodies".

The term “humanized antibody” or “humanized antigen-binding fragment” as used herein means an antibody or antibody fragment that: (i) is derived from a non-human source (eg, a transgenic mouse having a heterologous immune system) and is based on the sequence human germline; or (ii) a chimeric antibody, wherein the variable region is derived from a non-human source and the constant region is derived from a human; or (iii) a CDR graft, wherein the variable region CDR is derived from a non-human source, one or more variable region framework regions are derived from a human, and the constant region, if any, is derived from a human. The goal of “humanization” is to eliminate the immunogenicity of antibodies derived from a non-human source in the human body while maintaining the highest possible affinity. It is advantageous to select a human framework sequence as the template for humanization that is most similar to the framework sequence of the non-human parent antibody. In some cases, it may be necessary to replace one or more amino acids in a human framework sequence with corresponding residues in a construct derived from a non-human source to avoid loss of affinity.

The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e. each individual antibody in a population is identical, except for possible mutations (such as natural mutations) that may be present in very small quantities. Thus, the term "monoclonal" indicates the nature of the antibody in question, that is, not a mixture of unrelated antibodies. Unlike polyclonal antibody preparations, which typically contain different antibodies against different epitopes, each monoclonal antibody in a monoclonal antibody preparation is directed against a single epitope in the antigen. In addition to specificity, monoclonal antibody preparations have the advantage that they usually do not contain other antibodies. The term "monoclonal" should not be understood as requiring the production of the antibody 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 such that said antibody can be used as a therapeutic agent 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 from homologues and variants of the target proteins mentioned above (for example, mutant forms, splice variants or truncated forms of the protein as a result of hydrolysis).

The term "binding affinity" refers to the strength of the sum of non-covalent interactions between the individual binding sites of a molecule and its binding partners. Unless otherwise specified, “binding affinity” as used herein refers to intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (eg, antibody and antigen). "K _D ", "binding rate constant K _on " and "dissociation rate constant K _off " are commonly used to describe the affinity between a molecule (such as an antibody) and its binding partner (such as an antigen). Affinity is the degree of strength to which a ligand binds a particular protein. Binding affinity is influenced by non-covalent intermolecular interactions such as hydrogen bonding, electrostatic interactions, hydrophobic forces and van der Waals forces between two molecules. In addition, the binding affinity between a ligand and its target molecule can be influenced by the presence of other molecules. Affinity can be analyzed by conventional methods known in the art, including the ELISA method described herein.

The term "epitope" includes any determinant protein cluster that specifically binds to an antibody or T-cell receptor. Determinant epitope clusters typically consist of reactive surface groups of the molecule (eg, amino acid or sugar side chains, or combinations thereof) and often have specific three-dimensional structural features as well as specific charge characteristics.

An "isolated" antibody is an identified antibody and an antibody isolated from a cell that naturally expresses the antibody. Isolated antibody includes antibodies in situ in recombinant cells and antibodies that are typically produced in at least one purification step.

"Sequence identity" between two polypeptides or nucleic acid sequences indicates the number of residues that are identical between the specified sequences, as a percentage of the total number of residues. When calculating percent identity, the aligned sequences are aligned to provide the maximum match between sequences, with gaps in the match (if any) eliminated using a specific algorithm. Preferred computer software methods for determining the identity of two sequences include, but are not limited to, GCG software 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 National Center for Biotechnology Information (NCBI) and other sources. The famous 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. Preferred are human FcRs of natural sequence, and preferably are 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 under 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. This neonatal Fc receptor (FcRn) plays an important role in the metabolic fate of IgG-like antibodies in vivo. FcRn protects IgG from the lysosomal degradation pathway, thereby reducing its serum clearance and prolonging its half-life. Therefore, the in vitro binding properties/characteristics of FcRn to IgG indicate its in vivo pharmacokinetic properties in the bloodstream.

The term "effector function" refers to those biological activities attributable to the Fc region of an antibody that 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 antigen uptake by antigen presenters cells, inhibition of cell surface receptors (eg, B cell receptors) and activation of B cells.

The term "effector cell" refers to a leukocyte that expresses one or more FcRs and performs effector functions. In one aspect, said effector cells express FcγRIII to a lesser extent and perform ADCC effector functions. Examples of these human leukocytes are 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, such as blood. Effector cells are typically lymphocytes associated with the 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 are of hematopoietic origin and participate 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 present on certain cytotoxic cells (eg, NK cells, neutrophils, and macrophages), allowing these cytotoxic effector cells to specifically bind to target cells bearing antigens, and subsequently kill said target cells using, for example, a cytotoxin. To evaluate the ADCC activity of a target antibody, in vitro ADCC assays can be performed, such as in vitro ADCC assays described in US Pat. No. 5,500,362 or 5,821,337 or US Pat. No. 6,737,056 (Presta), methods documented in embodiments of the present application. Suitable effector cells for use in such assays include PBMC 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), in which said antibody binds to its corresponding antigen. To assess complement activation, a CDC assay may be performed, such as the CDC assay described in Gazzano-Santoro et al., J. Immunol Methods 202: 163 (1996), methods documented in embodiments of this application, such as those described in the patent US No. 6,194,551 B1 and WO 1999/51642, which describe polypeptide variants having altered Fc region amino acid sequences (polypeptides having a variant Fc region), and polypeptide variants exhibiting increased or decreased C1q binding.

Amino acid sequence and nucleotide sequence of said antibody according to the present invention

The present invention used recombinant human PD-1 protein to immunize a mouse, and then obtained a clone with the M944 scFv antibody, which specifically binds to recombinant human PD-1 with high affinity, by screening a phage antibody library. The nucleotide sequences encoding the heavy chain and light chain variable regions of the indicated M944 scFv antibody were assembled by PCR with the sequences encoding the mouse IgG1 heavy chain constant region and the mouse kappa light chain constant region, respectively, and the resulting assembled sequence was inserted into a vector for transient expression at HEK-293 cultured for expression. High purity mouse PD1-M944 antibody was purified using a protein A purification column. ELISA showed that mouse PD1-M944 antibody was able to block the binding of PD-1 to its ligand.

Then, using the classical humanized CDR transplantation method, the human antibody light chain or heavy chain variable region closest to the mouse antibody light chain or heavy chain variable region was selected as the template. In one embodiment, IGKV3-11*01 is selected as the humanized light chain variable region and IGHV3-21*02 is selected as the heavy chain variable region. The humanized light chain variable region (V _L ) and heavy chain variable region (V _H ) sequences were obtained by inserting each of the three light chain/heavy chain CDRs of a mouse antibody into the corresponding positions of the above human antibody templates. Since the mouse antibody framework key site is required to maintain CDR activity, the key site was reverse mutated into the mouse antibody sequence. The amino acid sequence and nucleotide sequence of the humanized antibody PD1-H944 were obtained by sequentially splicing the light chain/signal peptide sequence of the heavy chain, the variable region light chain/heavy chain sequence of the humanized antibody with a reverse mutation, and the human antibody heavy chain constant region/kappa light chain constant region sequence person, respectively.

Nucleic acids according to the present invention

The present invention also relates to a nucleic acid molecule encoding antibodies or parts thereof according to the present invention. Said sequences of these nucleic acid molecules include, but are not limited to, SEQ ID NOs: 3-7 and 26-33.

These nucleic acid molecules according to the present invention are not limited to the sequences specified in the present description, but also include variants thereof. Embodiments of the present invention may be described with reference to their physical hybridization properties. Those skilled in the art will appreciate that using nucleic acid hybridization techniques, said nucleic acid can be used to identify complementary nucleic acids, as well as equivalents or homologs thereof. It should also be understood that hybridization can occur at less than 100% complementarity. However, with appropriate selection of conditions, hybridization techniques can be used to distinguish between specified DNA sequences based on the structural relevance of the DNA sequence to a particular probe. For information 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)b Current Protocols in Molecular Biology. New York: John Wiley and Sons.

Recombinant and expression vectors

The present invention also provides recombinant constructs containing one or more nucleotide sequences according to the present invention. Said recombinant constructs of the present invention may be used with vectors, wherein said vectors may be, for example, plasmids, phagemids, phages or viral vectors, and said nucleic acid molecules encoding antibodies of the present invention are inserted into said vectors.

Antibodies provided herein can be produced by recombinant expression of nucleotide sequences encoding light and heavy chains or portions thereof in a host cell. To recombinantly express this antibody, a host cell can be transfected with one or more recombinant expression vectors carrying nucleotide sequences encoding light and/or heavy chains or portions thereof, such that said light and heavy chains are expressed in said host cell. To prepare and/or produce nucleic acids encoding heavy and light chains, to incorporate these nucleic acids into recombinant expression vectors, and to introduce said vectors into host cells, standard recombinant DNA techniques are used, such as those described in, for example, 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 procedures documented in US Patent No. 4,816,397 (Boss) et al.

Additionally, nucleotide sequences encoding a variable region of said heavy and/or light chain can be converted into nucleotide sequences encoding, for example, a full-length antibody chain, a Fab fragment, or a ScFv: for example, a DNA fragment encoding a light chain variable portion or a heavy chain variable portion chain can be operably ligated (so that the amino acid sequence encoded by both DNA fragments is in the same reading frame) with another DNA fragment encoding, for example, an antibody constant region or a flexible linker. These human heavy and light chain constant region sequences are known in the art (see, for example, Kabat, E. A., et 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 using standard PCR amplification.

Standard recombinant DNA expression techniques can be used to express antibodies (see, for example, Goeddel; Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). For example, a nucleotide sequence encoding a desired antibody can be inserted into an expression vector, which is subsequently transfected into a suitable host cell. Suitable host cells include 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 choice of 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 expression is constitutive or inducible.

Antibodies of the present invention can be isolated and purified from recombinant cell cultures by well known methods, which well known methods include ammonium sulfate or ethanol precipitation, acid extraction, Protein A affinity chromatography, Protein G affinity chromatography, anion or cation exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and pectin chromatography, but are not limited to. High performance liquid chromatography (HPLC) can also be used for purification. 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, 10, each which are incorporated herein by reference in their entirety.

Antibodies of the present invention include naturally purified products, products of chemical synthesis methods and products obtained by recombinant methods from prokaryotic and eukaryotic hosts, said eukaryotic hosts including, for example, yeast, higher plant cells, insects and mammals. Antibodies of the present invention may be glycosylated or non-glycosylated. Such methods are documented in many standard laboratory manuals, such as Sambrook, cited above, sections 17.37-17.42; Ausubel above, chapters 10, 12, 13, 16, 18 and 20.

Thus, embodiments of the present invention may also be host cells containing said vector or nucleic acid molecules, wherein said host cells may be higher eukaryotic host cells such as mammalian cells, lower eukaryotic host cells such like yeast cells, and can be prokaryotic cells, such as bacterial cells.

Properties and functions of antibodies according to the present invention

The properties and functions of the specified antibody PD1-H944 were analyzed. The analysis showed that the antibody of the present invention has the following advantages: (1) it is able to bind human PD-1 with high affinity and specificity and has a low dissociation rate, which provides good antitumor efficacy; (2) it is able to block PD-L1 binding to PD-1 more potently than nivolumab; (3) compared with nivolumab, binds recombinant human PD-1 with greater sensitivity and specificity; cross-links with recombinant monkey PD-1, but is relatively unbound with mouse PD-1; (4) it is able to effectively block the binding of recombinant human PD-1 to its ligands Pd-L1 and Pd-L2, (5) it is able to effectively reactivate immunosuppressive T cells and activate the recombinant PD-1 reporter system of human cells; (6) demonstrates superior antitumor effect in a humanized PD-1 mouse model bearing MC38 colon cancer tumor, much better than nivolumab (Sino Biological, Inc.) and comparable to the effect of pembrolizumab (Sino Biological, Inc.); (7) It exhibits both low ADCC activity and low CDC activity, and its ADCC activity is lower than that of nivolumab.

Applications

The antibodies of the present invention can be used to treat a variety of tumors or cancers or to formulate drugs to treat a variety of tumors or cancers that specifically target aberrantly expressed PD-1 receptors. Non-limiting examples of these tumors or cancers include colon cancer. Pharmaceutical compositions

The antibodies of the present invention can be formulated with at least one other agent (eg, a stabilizing compound) to form pharmaceutical compositions containing the antibodies of the present invention and one or more pharmaceutically acceptable carriers, diluents or excipients.

Sets

The present invention also relates to pharmaceutical packages and kits containing one or more containers containing the above pharmaceutical compositions according to the present invention. Such containers may be associated with specifications in a form prescribed by the government agency regulating the manufacture, use, or distribution of a drug or biological product that grants approval for its use in humans in the country in which the product is manufactured, used, or distributed.

Production and storage

The pharmaceutical compositions of the present invention can be prepared by methods known in the art, for example, by conventional mixing, dissolving, granulating, lozenge, grinding, emulsifying, encapsulating, pouring or lyophilizing methods.

Already prepared pharmaceutical compositions containing the compounds of the present invention, formulated in a dosage form with a suitable carrier, can be placed in appropriate containers and labeled for the treatment of the specified condition. Such labeling will include information about the quantity, frequency, and route of administration of the drug.

Combinations

Pharmaceutical compositions containing the antibodies of the present invention described above are also combined with one or more other therapeutic agents, such as antineoplastic agents, without the resulting combination causing unacceptable side effects.

The following examples are provided to illustrate the present invention and are not intended to limit the present invention in any way.

Examples

Example 1: Screening for Antibodies Derived from Mouse that Block PD-1 Binding to PD-L1/PD-L2 Using a Phage Antibody Library

1.1. Mouse immunization

Recombinant human PD-1 protein (Sino Biological, Inc, cat. no. 10377-Н08Н) was used to immunize mice. The amino acid sequence of the extracellular region of this human PD-1 protein (UniProtKB Q15116) is Metl-Glnl67 (SEQ ID NO: 1).

This recombinant human PD-1 protein was mixed with Freund's adjuvant, and the mixture, which was administered subcutaneously at intervals of 2 weeks, 3 weeks, 2 weeks and 3 weeks, was used to immunize mice 5 times at a dosage of 20 μg each. Starting with the second immunization, blood was collected seven days after each immunization through the medial canthal plexus of the eye. Mouse anti-PD-1 serum titer was measured by ELISA using recombinant human PD-1 protein coating. The serum titer reached an 8000-fold dilution after the fifth immunization, and mice were intravenously injected with 25 μg of recombinant human PD-1 protein 9 weeks after the fifth immunization. After 4 days, mice were sacrificed and spleen tissue was frozen in liquid nitrogen.

1.2. Phage display library screening

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 corresponding nucleotide sequences encoding the light and heavy chain variable regions of the mouse antibody were amplified and assembled into the nucleotide sequence encoding their scFvs using an overlap extension PCR method in which the corresponding nucleotide sequences encoding the light and heavy chain variable regions were linked via a linker (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 Jones et al., Bio/Technolgy 9(6):579, July 1991), then enzymatically ligated to the pComb3x phage vector (Sino Biological, Inc.) using SƒiI restriction endonuclease (Fermentas), and then electrotransformed X-Blue competent cells to construct a scFv phage display antibody library for immunized mice. By coating ELISA plates with recombinant human PD-1 protein, anti-PD-1 antibody-enriched phage libraries were prepared following a phage antibody coating process (Ref: Antibody Phage Display: Methods and Protocols, Philippa M. O'Brien and Robert Aitken (Eds), Humana Press, ISBN: 9780896037113). Monoclonal phages selected from the enriched library were expressed and then tested for binding to recombinant human PD-1 protein by ELISA. Clones of the strong binding M944 scFv antibody, which specifically binds to recombinant human PD-1, were selected and a sequencing company was commissioned to determine the nucleotide sequences of the M944 scFv antibody (SEQ ID NO:3). 1.3. Generation of mouse PD1-M944 antibody

The nucleotide sequences of the variable regions of the heavy and light chains of the M944 scFv antibody (SEQ ID NO: 4/5) were combined by PCR with the nucleotide sequences of the constant region of the heavy chain of the mouse IgG1 antibody (SEQ ID NO: 6) and the constant region of the mouse kappa light chain (SEQ ID NO: 7), respectively. The resulting nucleotide sequences were then enzymatically digested using HindIII and XbaI (Fermentas) and inserted into the transient expression vector pSTEP2 (Sino Biological, Inc), and the plasmid was extracted and transfected into HEK-293 cells, which were cultured for 7 days. The culture supernatant was purified on a protein A column to obtain a high purity antibody.

1.4 Functional analysis of mouse PD1-M944 antibody

(1) Blocking the binding of recombinant human PD-1 to PD-L1: A solution of 1 μg/ml recombinant human PD-1 protein was applied to 96-well plates in an amount of 100 ml per well overnight at 4°C. The plates were washed the next day and blocked at room temperature for 1 h. 100 μl of a 10 μg/ml solution of biotinylated PD-L1-Fc-biotin protein (SinoBiological, Inc.) was co-incubated with various concentrations (0.4 μg/ml , 2 μg/ml and 10 μg/ml) PD1-M944 or nivolumab (Sino Biological, Inc). These plates were washed to remove unbound antibodies. These plates were incubated with the antibiotic streptavidin/horseradish peroxidase (Beijing Zhong Shan - Golden Bridge Biological Technology Co., Ltd) and then washed several times, and the chromogenic substrate solution was added to obtain color. After stopping the reaction, OD ₄₅₀ was determined. The results demonstrated that the recombinant PD-L1 protein could effectively bind to the PD-1 protein coat, and the binding of the recombinant PD-L1 protein to PD-1 was effectively inhibited by adding different concentrations of PD1-M944 or nivolumab (Figure 1A). These results indicate that the mouse PD1-M944 antibody has good activity in blocking the binding of PD-1 to its ligand PD-L1.

(2) Blocking the binding of recombinant human PD-1 to PD-L2: A solution of recombinant human PD-1 protein at a concentration of 1 μg/ml was applied to a 96-well plate in an amount of 100 ml per well, overnight at 4°C, the next day were washed and blocked at room temperature for 1 h. 100 μl of 0.5 μg/ml biotinylated protein PD-L2-Fc-biotin (Sino Biological, Inc.) were co-incubated with different concentrations (0.5 μg/ml, 2 .5 μg/ml and 12.5 μg/ml) PD1-M944 or nivolumab (Sino Biological, Inc.). These plates were washed to remove unbound antibodies, incubated with the antibiotic streptavidin/horseradish peroxidase (Beijing Zhong Shan - Golden Bridge Biological Technology Co., Ltd), washed several times, and the chromogenic substrate solution was added to obtain color, and after stopping the reaction, the OD was determined ₄₅₀ . The results demonstrated that the recombinant PD-L2 protein could efficiently bind to the PD-1 protein coat, and the binding of the recombinant PD-L2 protein to PD-1 was effectively inhibited by adding different concentrations of PD1-M944 or nivolumab (Figure 1B). These results indicate that the mouse PD1-M944 antibody has good activity in blocking the binding of PD-1 to its ligand PD-L2.

Example 2: Humanization of the Mouse PD1-M944 Antibody Sequence to Produce the Humanized PD1-H944 Antibody Sequence

2.1 Determination of CDR regions for each of the light and heavy chains of the mouse PD1-M944 antibody

The nucleotide sequence of the M944 scFv antibody was determined in Example 1.2, in which the amino acid sequences of the variable regions of the heavy and light chains of the PD1-M944 scFv antibody were determined, see SEQ ID NO: 8 and 9.

The amino acid sequences of the 3 CDR regions of each of the light and heavy chains of the mouse PD1-M944 antibody were determined with reference to the Kabat numbering scheme, see Table 1. The 3 CDR regions of each of the above light and heavy chains were transferred and inserted into the final humanized PD1-M944 antibody. H944 in successive stages, see Examples 2.2 and 2.3.

2.2 Humanization of mouse PD1-M944 antibody by CDR transplantation

Humanization of the mouse antibody was carried out using the classical method of humanization by CDR transplantation. Human Antibody The human antibody light or heavy chain variable regions that are most similar to the mouse light or heavy chain variable regions were selected as a template, and three CDRs (Table 1) from each of the mouse antibody light or heavy chains were inserted into the human antibody variable region to obtain humanized light chain variable region (V _L ) and heavy chain variable region (V _H ) sequences. The specified matrix for each of the variable regions of the light chain of PD1-M944 was the antibody IGKV3-11*01, which is 73.48% homologous to the light chain of PD1-M944, and the specified matrix for each of the variable regions of the heavy chain was the antibody IGHV3-21*02 , which is 81.94% homologous to the heavy chain of PD1-M944.

2.3 Back mutation of the framework region of the humanized variable region sequence

Since key sites in the mouse antibody framework region are required to maintain CDR activity, the corresponding sites were backmutated to those shown in the mouse antibody sequence, where position 89 in the light chain was backmutated to M and position 91 to F in the heavy chain position 44 is on R and position 78 is on N.

The humanized antibody PD1-H944 was produced by humanized CDR transplantation and reverse mutation of the framework region, and the amino acid sequences of its heavy and light chain are shown in SEQ ID NO: 16 and 17, respectively; its heavy and light chain amino acid sequences in signal peptide containing form are shown in SEQ ID NOs: 18 and 19, respectively, containing the sequentially linked heavy/light chain signal peptide sequences (SEQ ID NOs: 20 and 21); humanized antibody heavy chain/light chain variable region sequence (SEQ ID NOs: 22 and 23); and a humanized antibody constant region sequence, which is the human IgG4 antibody heavy chain constant region/human kappa light chain constant region, respectively (SEQ ID NOs: 24 and 25).

Example 3: Preparation of Humanized Antibody PD1-H944

After PCR amplification, the corresponding heavy and light chain nucleotide sequences encoding the PD1-H944 signal peptide-containing antibody (SEQ ID NO:26 and 27) above, which contain nucleotide sequences encoding the heavy/light chain signal peptide concatenated in series ( SEQ ID NO:28/29), humanized antibody heavy/light chain variable region (SEQ ID NO:30/31) and human IgG4 heavy chain constant region/human kappa light chain constant region (SEQ ID NO:32/33), respectively, were digested with two HindIII viXbaI restriction endonucleases (Fermentas) and then inserted into the commercial vector pcDNA3 (Invitrogen). After extraction of the plasmid, a fragment of 1.8 kb was obtained. by triple digestion of the pCDNA3 light chain vector with NruI+NaeI+DraI, then inserted into the pSSE vector of the CHO/dhfr expression system (Sino Biological, Inc), and then a 2.5 fragment was obtained by triple digestion of the pCDNA3 heavy chain vector with using NruI+NaeI+DraI (Fermentas). Then, the light chain vector pSSE (Sino Biological, Inc) obtained in the previous step was also introduced into the vector to obtain the complete vector. Said expression vector is a eukaryotic expression vector containing an amplified DNA element of the dhƒr gene, a NeoR resistance gene, and expression elements of the light and heavy chains of said antibody. The expression vector was transfected into dhfr-DG44 cells, PO1-H944 positive cell lines were obtained through G418 screening, PD1-H944 high expression cell lines were obtained through MTX stepwise screening, and then domesticated in serum-free culture for clonal screening. At each stage, clones with high antibody expression were selected based on ELISA results in combination with cell growth status and key quality attributes of said antibody.

This PD1-H944-producing CHO cell line was grown in serum-free suspension culture to produce the PD1-H944 antibody of high purity and quality.

Example 4: Characterization of Humanized Antibody PD1-H944

4.1 Binding affinity analysis of PD1-H944 to human, mouse and monkey PD-1 antigen

(1) Binding ability of PD1-H944 to recombinant human PD-1 protein Indirect ELISA assay was used to detect specific binding

PD1-H944 with recombinant human PD-1 protein. Various 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) recombinant human PD-1 protein was applied to 96-well plates overnight at 4°C. The next day, the plates were washed and blocked at room temperature for 1 h. After incubation with 100 μl of 2 μg/ml PD1-H944, nivolumab (Bristol-Myers Squibb) and H7N9-R1-IgG4 antibody as a negative control ( Sino Biological, Inc., respectively, the plates were washed to remove unbound antibody, then incubated with goat anti-human IgG F(ab) ₂ /horseradish peroxidase (JACKSON) and washed sequentially, and the chromogenic substrate solution was added to obtain color and detection OD ₄₅₀ . The EC ₅₀ for binding of PD-1-H944 and nivolumab to recombinant human PD-1 protein was 31.5 ng/ml, R ² =0.998 and 179.0 ng/ml, R ² =0.997, respectively. This indicates that PD1-H944 binds to recombinant human PD-1 protein significantly better than nivolumab (Figure 2A-B).

(2) PD1-H944 binding ability to recombinant Jurkat/PD-1 cells

The binding ability of PD1-H944 to recombinant Jurkat/PD-1 cells was measured by cell cytometry using the Jurkat/PD-1 cell line stably expressing recombinant human PD-1 (SinoCellTech Ltd) as the experimental material. Various concentrations of PD1-H944, nivolumab and H7N9-R1-IgG4 antibody as a negative control (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 5x10 ⁵ /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 indicated antibody to the indicated cells was determined by adding goat anti-human IgG antibody with Fc-FITC (Sino Biological, Inc.). The results obtained demonstrated that PD1-H944 specifically bound to Jurkat/PD-1 cells with an EC _{50 of} 9.63 μg/ml, R ² =1.000, nivolumab specifically bound to Jurkat/PD-1 cells with an EC ₅₀ of 10.18 μg/ ml, R ² =0.994, and the H7N9-R1-IgG4 antibody as a negative control did not bind to Jurkat/PD-1 cells (Fig. 3). This indicates that PD1-H944 has a good ability to bind to PD-1 expressed by Jurkat/PD-1 cells and that its binding ability is slightly better than that of nivolumab.

(3) Binding affinity of PD1-H944 to recombinant human PD-1 protein

Binding 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) with PD-1 was detected using the Octet Biomolecular Interaction system along with the H7N9-R1-IgG4 antibody (13.30 nM) as a negative control. The results obtained demonstrated that the average binding affinity of PD1-H944 to recombinant human PD-1 protein was 64.8 pM, the average association rate constant k _on was 3.01E+05 M ^-1 s ^-1 and the average dissociation rate constant _koff was 1.95E-05 s ^-1 ; the average binding affinity _KD of nivolumab to the PD-1 protein was 74.1 mM, the average association rate constant k _on was 6.92E+05 M ^-1 s ^-1 and the average dissociation rate constant k _off was 5.12E-05 s ^-1 (Fig. 4). PD1-H944 has a stronger affinity than nivolumab, which is about 1.14 times that of nivolumab, and PD1-H944 has a lower dissociation rate, so that PD1-H944 has a higher ability to bind to PD-1 protein than nivolumab.

(4) Binding ability of PD1-H944 to recombinant monkey and mouse PD-1 protein

An indirect ELISA assay 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.) at various 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 applied to 96-well plates, 100 µl per well, kept in 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 H7N9-R1-IgG4 antibody as a negative control (all at a concentration of 2 μg/ml ), the plates were washed and a secondary antibody, goat anti-human IgG F(ab) ₂ /horseradish peroxidase, was added to produce color, the OD ₄₅₀ was determined, and this assay was performed in triplicate. The results obtained demonstrated that PD1-H944 bound to recombinant human PD-1 protein and recombinant monkey PD-1 protein with EC _{50 of} 25.8 ng/ml (R ² =0.999) and 32.7 ng/ml (R ² =0.997 ), respectively, but did not bind to recombinant mouse PD-1 protein (Fig. 5A); nivolumab bound to recombinant human PD-1 protein and recombinant monkey PD-1 protein with an EC _{50 of} 113.2 ng/ml (R ² =0.997) and 80.2 ng/ml (R ² =0.997), respectively, and did not bind with mouse PD-1 protein (Fig. 5B); the above antibody, used as a negative control, did not bind to recombinant human PD-1 protein, recombinant monkey PD-1 protein, or recombinant mouse PD-1 protein (Fig. 5). The good binding of PD1-H944 to the monkey PD-1 protein ensures the use of monkeys to evaluate the safety of this drug.

(5) Binding affinity of PD1-H944 to recombinant monkey and mouse PD-1 proteins

The affinities of PD1-H944 and nivolumab for biotinylated monkey and mouse PD-1 proteins (Sino Biological, Inc.) at various concentration gradients (see Fig. 6A-B) were measured using the Octet system and analyzed to obtain K _D values. The results demonstrated that the reported _KD affinity of PD1-H944 for recombinant monkey PD-1 protein was 108 pM, and the reported _KD affinity of nivolumab for recombinant monkey PD-1 protein was 131 pM, both values being comparable (Figure 6A). Both PD1-H944 and nivolumab did not bind to recombinant mouse PD-1 protein ( Fig. 6B ).

4.2. PD1-H944 blocks the binding of PD-1 ligands (PD-L1 and PD-L2) to the human PD-1 protein

Recombinant human PD-1 protein was applied to a 96-well plate in an amount of 100 ml per well and left overnight at 4°C. The plate was washed the next day and blocked at room temperature for 1 hour before adding 1 μg/ml biotinylated human PD-L1 (Sino Biological, Inc.) or 2 μg/ml biotinylated human PD-L2 (Sino Biological, Inc.) . Then, PD1-H944, nivolumab, or H7N9-R1-IgG4 antibody as a negative control was added at various 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), incubated, the antibiotic streptavidin/horseradish peroxidase was added, incubated, and OD ₄₅₀ was determined. For each group, this test was performed in duplicate. The results demonstrated that biotinylated recombinant human PD-L1 and PD-L2 proteins efficiently bound to the human PD-1 protein coating, and the addition of varying concentrations of both PD1-H944 and nivolumab effectively inhibited the binding of recombinant human PD-L1 protein (Fig. 7A) and recombinant human PD-L2 protein (Fig. 7B) with human PD-1. PD1-H944 and nivolumab inhibited recombinant human PD-L1 protein with an IC _{50 of} 0.116 μg/ml (R ² =0.995) and 0.129 μg/ml (R ² =0.997), respectively, and recombinant human PD-L2 protein with an IC _{50 of} 0.446 µg/ml (R ² =0.994) and 0.486 µg/ml (R ² =0.996), respectively. The ability of PD1-H944 to inhibit the binding of human PD-1 to human PD-L1 or human PD-L2 was not significantly different from that of nivolumab. No significant inhibition was observed for the antibody used as a negative control.

4.3. PD1-H944 blocks human PD-1 ligand (PD-L1) binding to cells expressing human PD-1

Jurkat/PD-1 cells stably expressing recombinant human PD-1 at 5 × ¹⁰⁵ cells per tube in logarithmic growth phase were added to PD1-H944, nivolumab and the H7N9-R1-IgG4 antibody as a negative control at various 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. 10 μl of 0.4 μg/ml B7H1-Fc-6hothh (Sino Biological, Inc.) was added, washed with phosphate saline, and unbound antibody was removed by centrifugation. Alexa Fluor® 488 streptavidin conjugate (Life Technologies) was added, incubated at 4°C for 20 minutes, washed and centrifuged sequentially, and 200 μl of phosphate saline was added to resuspend the cells for flow cytometer detection. The results demonstrated that the recombinant human PD-L1 protein could effectively bind to Jurkat/PD-1 cells, and the binding of the recombinant human PD-L1 protein to Jurkat/PD-1 cells was effectively inhibited by the addition of various concentrations of PD1-H944 and nivolumab ( Fig. 8). The IC ₅₀ inhibition concentrations were 1.78 μg/ml (R ² =0.994) and 2.48 μg/ml (R ² =0.989), respectively. Antibody PD1-H944 inhibited the binding of human PD-1 and human PD-L1 slightly better than nivolumab. No significant inhibition was observed when the antibody used as a negative control was added. 4.4. Binding of PD1-H944 to CD16a

CD affinity receptor protein 16a (FcγRIIIa) is the major Fc receptor mediating the effects of ADCC, with CD 16a containing the V158 SNP site having relatively high affinity. The binding of PD1-H944 to the indicated recombinant CD16a protein (V158) was measured by ELISA to evaluate the potential influence of PD1-H944 on the effects of antibody-dependent cell-mediated cytotoxicity (ADCC).

The positive control PD1-H944-1-IgG1(o) used in this assay is the PD1-H944 variable region bound to native IgG1, and the negative control PD1-H944-1-IgG1 is the variable region PD1-H944 bound to the N297A mutant IgG1, whose Fc fragment lacks the ability to bind to CD 16a due to deletion of the N-glycoside. Recombinant human PD-1 protein (10 μg/ml, 100 μl per well) was applied to 96-well plates overnight at 4°C, washed the next day and blocked at room temperature for 1 h. PD1-H944, nivolumab , PD1-H944-1-IgG1(o) antibody (Sino Biological, Inc.) used as a positive control, and PD1-H944-1-IgG1 antibody (Sino Biological, Inc.) used as a negative control were added at various 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) (see Fig. 9), and the plates were washed to remove unbound antibody after incubation. 10 μg/ml recombinant CD16a-AVI-His (V158)+BirA protein (Sino Biological, Inc.) was added to onions after incubation with 1 μg/ml streptavidin/horseradish peroxidase, and the OD ₄₅₀ of the color reaction after incubation was determined.

The results obtained demonstrated that the binding of the antibody used as a positive control for recombinant CD16a protein increased with increasing antibody concentration. PD1-H944, nivolumab, and the negative control antibody did not bind to recombinant CD16a protein at any of the concentrations tested (Figure 9). This suggests that both PD1-H944, an engineered IgG4 subtype antibody, and nivolumab have very low CD16a binding capacity and suggests that PD1-H944 causes very little or no ADCC effect.

4.5. Binding of PD1-H944 to C1q

The binding of PD1-H944 to recombinant C1q protein was measured by ELISA, thereby assessing the potential for PD1-H944 to influence complement-dependent cytotoxicity (CDC) effects.

PD1-H944-1-IgG1(o), obtained by attaching the variable region of PD1-H944 to natural IgG1, was used as a positive control antibody (Sino Biological, Inc.), and H7N9-R1-IgG4 (Sino Biological, Inc. ) was used as an isotype control antibody. Various 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) ml) (see Fig. 10) PD1-H944, nivolumab, positive control antibody, and isotype control antibody were applied to 96-well plates at 100 ml per well overnight at 4°C . The next day, the plates were washed, blocked at room temperature for 1 hour and 100 μl of a 5 μg/ml solution of recombinant C1q protein (Quidel Corporation) was added, respectively, and the plates were incubated for 3 hours. 100 μl of secondary antibody solution against C1q/horseradish peroxidase (Abeam) was added at a dilution of 1:400, incubated for 1 hour, color reaction was carried out and OD ₄₅₀ was detected.

The results obtained demonstrated that the binding of the antibody used as a positive control to the recombinant C1q protein increased with increasing antibody concentration. PD1-H944, nivolumab, and the isotype control antibody had comparable binding ability to recombinant C1q protein, with significantly reduced binding ability of the positive control IgG1 antibody (Figure 10).

4.6. Binding of PD1-H944 to FcRn

The binding of PD1-H944 to recombinant Fc receptor protein (FcRn) was measured by ELISA, thereby assessing the pharmacokinetics of PD1-H944 in humans.

Avidin anti-biotin protein (Thermo Fisher Scientific) at a concentration of 10 μg/ml was applied to 96-well plates at a rate of 100 ml per well, incubated overnight at 4°C and washed the next day, blocked at room temperature for 1 hour and incubated with a solution of 10 μg/ml biotinylated protein FCGRT-His+B2M (Sino Biological, Inc.) for 1 hour, then PD1-H944, nivolumab or isotype control antibody H7N9-R1-IgG4 (Sino Biological, Inc.), used as an isotype control were respectively added at 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) (see Fig. 11), incubated for 1 hour, washed and added 250 ng/ml goat anti-human IgG Fc/horseradish peroxidase (Sino Biological Inc.). The process from antibody dilution to incubation with the secondary antibody was carried out while maintaining the pH at 6.0 and the OD ₄₅₀ was measured.

The results demonstrated that PD1-H944 and nivolumab were able to bind to recombinant human FcRn protein, and the binding ability increased with increasing concentration; at high concentrations (270 μg/ml), PD1-H944 binding to recombinant FcRn protein was approximately 1.62 higher than nivolumab binding (Figure 11). Based on these results, it was suggested that PD1-H944 has good pharmacokinetics in humans.

Example 5: Functional Analysis of Humanized Antibody PD1-H944 5.1. Activation of CD4 ⁺ T cells in mixed lymphoid responses stimulated by PD1-H944

The neutralizing activity of the anti-human PD-1 antibody was determined by analyzing a mixture of CD4 ⁺ T lymphocytes with dendritic cells. Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation, then mononuclear cells were obtained by adherent culture in cell culture medium 1640 (GIBCO) (containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin), containing 160 ng/ml rhIL-4 (Sino Biological, Inc.), then 20 ng/ml rhGM-CSF (R&D Systems) was added to the resulting mononuclear cells and incubated in a CO ₂ incubator for three days, then half of the medium was changed for incubation. After 6 days of incubation, suspended cells known as dendritic cells (DC) were collected. The DC density was adjusted to 2×10 ⁶ cells/ml, mitomycin was added to a final concentration of 50 μg/ml, kept at 37°C for 20 minutes, and washed three times with 1640 medium.

CD4 ⁺ T lymphocytes were sorted from PBMCs isolated from the peripheral blood of another individual using a CD4 ⁺ T lymphocyte sorting kit (MiltenyiBiotec). CD4 ⁺ T cells from each of three blood donors were used in each batch for this analysis.

Sorted CD4 ⁺ T lymphocytes were mixed with DCs pretreated with 50 μg/ml mitomycin in a 10:1 ratio, and 105 cells per well were evenly plated into 96-well plates with PD1-H944, nivolumab, and the H7N9-R1-IgG4 antibody used as a negative control (Sino Biological, Inc.), respectively, and a mixed lymphocyte control group, that is, a group with only CD4 ⁺ T cells and a mixture of DCs without antibody, a DC cell control group, that is, a group with only DC cells without antibody and CD4 ⁺ T lymphocytes, a CD4 ⁺ T cell control group, that is, a group with only CD4 ⁺ T cells without antibody and DC cells, in the same volume of sample dilution medium 1640, the final concentration gradient of each antibody was 1 μg/ ml, 0.1 µg/ml, 0.01 µg/ml, and incubated in an incubator with CO ₂ at 37°C and 5% CO ₂ for 5 days. The culture fluid supernatants were collected, and the expression levels of IL-2 and interferon-γ in the indicated culture fluid supernatants were measured by ELISA.

The results demonstrated that the secretion of interferon-γ and IL-2 was not detected in the supernatants of CD4 ⁺ T cell cultures without DC admixture, while in the control group, mixtures of lymphocytes, a mixture of CD4 ⁺ T cells and DCs significantly increased the secretion of interferon-γ and IL-2 CD4 ⁺ T cells. When anti-PD-1 antibody was added to the lymphocyte mixture system, CD4 ⁺ T cell activation in the mixture of CD4 ⁺ T cell and DC lymphatic responses was significantly enhanced with high secretion of interferon-γ and IL-2. In contrast to both PD1-H944 and nivolumab, which showed a significant effect, the antibody used as a negative control did not show this effect. The results obtained are shown in Fig. 12 indicating that PD1-H944 has better functional activity than nivolumab, can effectively reactivate immunosuppressed T cells.

5.2. PD1-H944 stimulates activation of reporter genes in the IL2 signaling pathway downstream of PD1

In this assay, Jurkat-NFAT-Luc2p-PD-1 effector cells (SinoCellTech Ltd) and CHO-K1-PD-L1-CD3E target cells (SinoCellTech Ltd) were used as experimental materials. PD-1/PD-L1 interaction resulting from co-culture of these two cells inhibits TCR signaling and NFAT-RE-mediated bioluminescence, and addition of anti-PD-1 antibody blocked PD-1/PD-L1 interaction and thus reversed this inhibition. The activation function of PD1-H944 in a recombinant human PD-1 reporter cell system was determined by measuring bioluminescence intensity (R1U) in effector cells.

CHO-K1-PD-L1-CD3E target cells were inoculated at 2×10 ⁴ per well into 96-well plates and cultured overnight in DMEM containing 10% FBS, then the supernatant was removed. PD1-H944, nivolumab, and H7N9-R1-IgG4 antibody (Sino Biological, Inc.) used as a negative control at various 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 in an amount of 40 μl per well (see Fig. 13). 7.5 × 10 ⁴ Jurkat-NFAT-Luc2p-PD-1 effector cells were sequentially added at 40 μl per well and incubated in a CO ₂ incubator for 6 hours. Each assay was performed in triplicate for target cells, effector cells, and negative controls. After 6 hours of incubation, 5× passive lysis buffer (Promega) was added at 20 μl per well and the 96-well plates were then placed in a −80°C freezer for additional detection. For this assay, 96-well plates were placed at -80°C. Allow to thaw at room temperature, stir, and 20 μL of supernatant from the wells was transferred to a 96-well white bottom plate for luminescence detection using the LB960-Microplate Luminol Assay.

The results demonstrated that both PD1-H944 and the control antibody nivolumab caused significant activation of the recombinant human PD-1 cellular reporter gene system, as shown in FIG. 13. In the concentration range of 0.007-150 μg/ml, the EC ₅₀ of PD1-H944 was 0.49 μg/ml, and that of nivolumab was 1.08 μg/ml. PD1-H944 had a significantly lower _EC50 than nivolumab and was 2.21 times more active than nivolumab. The antibody used as a negative control did not have the activating function of the recombinant human PD-1 reporter gene cell system.

5.3. PD1-H944 does not significantly stimulate CD16a pathway Fc receptor ADCC function

In this assay, Jurkat-NFAT-Luc2p-CD16A effector cells (SinoCellTech Ltd) and CHO-PD-1 target cells (SinoCellTech Ltd) were used as assay material to measure the PD1-H944-mediated ADCC effect using a system-based method recombinant highly active reporter gene CD16a (V158). The mechanism is that when two kinds of cells are co-cultured and PD1-H944 is added at the same time, the Fab fragment of PD1-H944 binds to PD-1 overexpressed by the target cells, thus its Fc fragment binds to effector cells bearing Fcγ -receptor CD16A therefore activates Jurkat-NFAT-Luc2p-CD16A effector cells and promotes NFAT-RE-mediated chemiluminescence.

CHO-PD-1 target cells were inoculated at 2 x 10 ⁴ per well into a 96-well plate and cultured overnight in DMEM containing 10% FBS, and the supernatant was removed. These plates were washed twice with RPMI 1640 medium (without phenol red) containing 0.1% BSA, and antibodies PD1-H944-1-IgG 1 (o), PD1-H944, nivolumab and negative control H7N9-R1-IgG4 (Sino Biological , Inc.) were added at various concentrations (see Fig. 14A-B) in an amount of 40 μl per well, 1×10 ⁶ Jurkat-NFAT-Luc2p-CD16A effector cells were added in an amount of 40 μl per well, and placed in an incubator with CO ₂ at 37°C and 5% CO ₂ for 4 hours. Each assay was performed in triplicate for target cells, effector cells, and negative controls. After 4 hours, 20 μl per well of 5× passive lysis buffer (Promega) was added and the 96-well plates were placed in a −80°C freezer for testing. For the assay, 96-well plates were allowed to thaw to room temperature, vortexed, and 20 μL of supernatant from the well was transferred to a 96-well white-bottom plate for luminescence detection using the LB960-Microplate Luminal assay.

The results obtained demonstrated that the antibody PD1-H944-1-IgG1(o) used as a positive control caused a significant ADCC effect, nivolumab caused a weaker ADCC effect, and PD1-H944 caused an even weaker ADCC effect than nivolumab in concentration range 0.018-300 ng/ml (p<0.01). Fig. 14A and FIG. 14B shows results with different assay batches. In FIG. 14B and 14D show the bioluminescence intensity (RLU) corresponding to the highest point of antibody concentration (300 ng/ml) from FIG. 14A and 15B, respectively. In this sensitive ADCC functional assay, cellular activation caused by PD1-H944 binding to CD 16a was significantly lower than that of nivolumab, with repeatable results. The low ADCC activity of PD1-H944 is a good confirmation of the effectiveness and safety of this antibody in the clinic.

5.4. PD1-H944 does not significantly activate CDC function of the complement C1q pathway

After binding to tumor cells overexpressing PD-1, PD1-H944 can activate the classical complement pathway to kill tumor cells, leading to their death. In this assay, the CDC effect of antibody PD1-H944 was examined using the WST-8 method.

CHO-PD-1 cells were resuspended in RPMI 1640 medium containing 0.1% BSA (SinoCellTech Ltd) and uniformly inoculated into 96-night plates at 5 x 10 ⁴ per well, followed by the addition of antibodies at various concentrations (Fig. 15) in an amount of 50 μl per well, and then complement C1q was added at a 1:4 dilution in an amount of 50 μl per well (One lambda). The antibodies were: PD1-H944-1-IgG1(o) antibody (Sino Biological, Inc.) used as a positive control, PD1-H944, nivolumab, and PD1-H944-1-IgG1 antibody (Sino Biological, Inc.) , used as a negative control, with blank well B (no cells) and negative control M (with cells but no antibody) assayed. After incubation for 3 hours at 37°C, 15 μl of WST-8 staining solution was added to each well and absorbance was measured at 450 nM and 630 nM on an ELISA analyzer after color stabilization. Results were calculated using absorbance values OD ₄₅₀ - OD ₆₃₀ , subtracting the value for the empty well. % death = (OD negative control M - OD sample)/OD negative control M × 100%.

The results demonstrated that PD1-H944, nivolumab, and the antibody used as a negative control did not induce a CDC effect on CHO-PD-1 tumor cells overexpressing PD-1, while the antibody used as a positive control did. caused the death effect of CHO-PD-1 cells with a maximum death rate of 82.1%, the results are shown in Fig. 15. The CDC analysis also confirmed that PD1-H944 does not induce a CDC effect on target cells expressing PD-1, demonstrating good safety of the antibody.

5.5 PD1-H944 Effectively Inhibits Tumor Growth in the MC38 Subcutaneous Graft Model of Colon Cancer in Humanized PD-1 Mice in Vivo

(1) Pharmacodynamics study of I antibody PD1-H944 in the MC38 subcutaneous transplant model of colon cancer in mice with humanized PD-1

Logarithmic growth stage MC38 cells (Sun Ran Shanghai Biotechnology Co., Ltd.) were used for tumor inoculation. MC38 cells, resuspended in phosphate saline, were inoculated at 5 x 10 ⁵ cells/0.1 ml into mice with humanized B-hPD-1 (Biocytogen) (derived by replacing part of exon 2 of the PD-1 gene of C57BL/6 mice corresponding part of the human PD-1 genome) subcutaneously on the right side of the chest of mice, for a total of 47 mice. When the tumor volume grew to approximately 100 mm ³ , mice were selected according to their individual tumor volume, randomly assigned into 5 groups using Excel software, with 8 animals in each group. Groups 4 and 5 were administered other anti-PD-1 antibodies not related to this application and therefore their data are not presented in this application. The dosage was started on the day of grouping. Mice were given an intraperitoneal injection (LP.) once every 3 days for 6 consecutive doses, then mice were sacrificed 10 days after the last dose and tumor tissue was removed for weighing. The specific dosage regimen is shown in Table 3 below. The antitumor effect of the drug was assessed by calculating the degree of tumor growth inhibition TGI (%). TGI (%)<60% was considered ineffective; if TGI (%) ≥60% and the tumor volume in the treated group was statistically significantly lower than that of the vehicle control group (P<0.05), this level was considered effective, that is, the antibody had a significant inhibitory effect on tumor growth. TGI (%) was calculated as follows:

TGI (%)=[1-(T _i -T ₀ )/(V _i -V ₀ )] × 100, where

T _i : mean tumor volume in the treatment group on day i,

_T0 : mean tumor volume in the treatment group on day 0,

V _i : average tumor volume in the vehicle control group on day i,

_V0 : mean tumor volume in the vehicle control group on day 0.

Animals were sacrificed at the end of the assay, the tumor was weighed, and the rate of tumor weight inhibition IRTW% was calculated with IRTW%>60% as the efficacy reference calculated as follows. Inhibition rate of tumor weight IRTW (%) = (W vehicle control group - W treatment group) / W vehicle control group × 100, W is tumor weight.

All experimental animals were in good general condition in terms of activity and feeding during the dosing period, and their body weight increased to some extent. After administration of the study drug (group 3) and the control drug (group 2), there was no significant difference (P>0.05) in the body weight of the animals. Body weight changes of all animals are shown in Fig. 16 and Table 4.

The results of tumor volume measurements for each group in the trial are shown in Table 5 and FIG. 17.

Day: number of days after cell inoculation

25 days after the first dose, all animals were sacrificed, tumors were removed, weighed and photographed. Tumor weights were statistically compared between groups, and the results are summarized in Table 6 and FIG. 18.

According to the tumor volume results, nivolumab did not significantly inhibit tumor volume in this model, while the mean tumor weight in the PD1-H944 group was 0.044±0.017 g. Its inhibition rate of tumor weight (IRTW) was as high as 97.4 %. Data analysis showed that tumors in the PD1-H944 group were significantly different from those in the vehicle control group (P<0.05), demonstrating the significant antitumor efficacy of PD1-H944.

At the end of the analysis, the mean tumor volume of the control group with solvent was 2171±430 mm ³ , tumor weight 1.719±0.310 g. The mean tumor volume of the positive control group with nivolumab (20 mg/kg) was 1802±228 mm ³ , TGI% - 17.8%, tumor weight - 1.492 ± 0.274 g. The rate of inhibition of IRTW tumor weight by nivolumab was 13.2%, which was not significantly different from that of the tumor for the vehicle control group (P = 0.480). In contrast, the mean tumor volume in the PD1-H944 group was 171 ± 51 mm ³ , TGI% was 96.7%, tumor weight was 0.044 ± 0.017 g, and tumor weight inhibition rate IRTW was 97.4%, which was significantly different from this tumor value for the vehicle control group (P<0.05), indicating that the indicated antibody PD1-H944 binds to the PD-1 epitope, and demonstrates significant inhibition of colon cancer subcutaneous MC38 transplant tumors at the dosage level of 20 mg/ kg.

(2) Pharmacodynamic study II of PD1-H944 antibody in the MC38 subcutaneous graft model of colon cancer in PD-1 humanized mice

Logarithmic growth stage MC38 cells (Sun Ran Shanghai Biotechnology Co., Ltd.) were used for tumor inoculation. MC38 cells resuspended in phosphate saline were inoculated at 5 x 10 ⁵ cells/0.1 ml into humanized B-hPD-1 mice (Biocytogen) (mice generated by replacing part of exon 2 of the PD-1 gene from C57BL/6 mice corresponding part of the human PD-1 genome). A total of 80 mice were inoculated subcutaneously into the right thorax. When the tumor volume grew to approximately 100 mm ³ , mice were selected 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 treated with other anti-PD-1 antibodies not related to this application and therefore their data are not presented. Dosing began on the day of grouping. Mice were given an intraperitoneal injection (LP.) once every 3 days for 6 consecutive doses, mice were sacrificed 10 days after the last dose, and tumor tissue was removed for weighing. The specific dosage regimen is shown in Table 7 below.

The antitumor effect of the drug was assessed by calculating the degree of tumor growth inhibition TG (%). TGI (%)<60% was considered ineffective; if TGI (%) >60% and the tumor volume in the treated group was statistically significantly lower than that of the vehicle control group (P < 0.05), this level was considered effective, that is, the antibody had a significant inhibitory effect on tumor growth. TGI (%) was calculated as follows.

TGI (%)=[1-(T _i -T ₀ )/(V _i -V ₀ )] × 100, where

T _i : mean tumor volume in the treatment group on day i,

_T0 : mean tumor volume in the treatment group on day 0,

V _i : average tumor volume in the vehicle control group on day i,

_V0 : mean tumor volume in the vehicle control group on day 0.

Animals were sacrificed at the end of the assay, the tumor was weighed, and the rate of tumor weight inhibition IRTW% was calculated with IRTW%>60% as the efficacy reference calculated as follows. Inhibition rate of tumor weight IRTW (%) = (W vehicle control group - W treatment group) / W vehicle control group × 100, W is tumor weight.

All experimental animals were in good general condition in terms of activity and feeding at the time of drug administration, and their body weight increased to some extent. There was no significant difference in the body weight of the animals after the administration of test and control drugs (P>0.05) (Table 8 and Fig. 19).

Tumor volumes for each group in the analysis are shown in Table 9 and FIG. 20.

24 days after the first dose, all animals were sacrificed, tumors were removed, weighed and photographed. Tumor weights were statistically compared between groups and the results are presented in Table 10 and FIG. 21.

For the vehicle control group, tumor volume continued to grow after group formation and the mean tumor volume at the end of the study was 3405 ± 624 mm ³ .

For the positive control group: after administration of pembrolizumab at a dosage of 20 mg/kg, the tumor volume increased slowly compared with the vehicle control group, with a significant difference in tumor volume from the 5th day of administration (P<0.05); The TGI% at the end of the trial was 97.0% and the tumor weight inhibition rate was 93.5%, P<0.05 compared with the vehicle control group. Pembrolizumab demonstrated clear tumor inhibition in this efficacy evaluation model, indicating that this model can be used to evaluate the efficacy of the epitope-binding antibody drug Keytruda.

For the PD1-H944, 8 mg/kg group: after administration of PD1-H944 at a dosage of 8 mg/kg, the tumor volume increased slowly compared with the vehicle control group, the tumor volume was statistically significantly different compared with the 5th day of administration (P< 0.05); The TGI% at the end of the test was 89.7%, and the tumor weight inhibition rate was 87.8%, P<0.05 compared with the vehicle control group.

For the PD1-H944 2 mg/kg group: after administration of PD1-H944 at a dosage of 2 mg/kg, the tumor volume increased slowly compared to the vehicle control group, the tumor volume was statistically significantly different compared to day 5 of administration (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 with vehicle control group. This again demonstrates that the binding of the indicated antibody PD1-H944 to the PD-1 epitope is effective in this model, demonstrating a significant tumor suppression effect on MC38 subcutaneous colon cancer grafts, demonstrating a dose-correlated effect. The antitumor efficacies of PD1-H944 and pembrolizumab in this analysis were also confirmed in terms of tumor weight.

The results of this analysis showed that intraperitoneal administration of PD1-H944 at a dosage of 8 mg/kg or 2 mg/kg every 3 days for 6 consecutive doses significantly suppressed the growth of transplanted tumors in mice with humanized B-hPD-l in the MC38 transplant model (P <0.05), and the characteristics of animals and their body weights in the treatment group did not differ from those of animals from the model group. Given the model design preference for different antibody epitopes, the difference in efficacy between PD1-H944 and pembrolizumab was not assessed in this analysis.

An in vivo pharmacodynamics study examined the antitumor effect of PD1-H944 alone in C57BL/6 mice with humanized PD-1 in the MC38 transplant model of colon cancer. The humanized B-hPD-1 mouse is a C57BL/6 mouse that has a humanized PD-1 gene, making this mouse suitable for assessing in vivo efficacy of an anti-human PD-1 antibody. The model was selective for antibody efficacy against different PD-1 epitopes, and pembrolizumab demonstrated significant tumor inhibition in this model. The results demonstrated that PD1-H944 alone (2, 8, and 20 mg/kg administered every 3 days for 6 consecutive doses) significantly inhibited the growth of transplanted MC38 tumors, with summary data presented in Tables 11 and 12.

Example 6: Identification of amino acid sites specifically binding to PD1-H944

Data from Example 4.2 demonstrate that PD1-H944 effectively competes with PD-L1 (Figure 7A) or PD-L2 (Figure 7B) for binding to the PD-1 protein, demonstrating overlap between epitope binding in PD-1 targeting PD1-H944, and an epitope that is the target of a PD-L1 or PD-L2 ligand.

6.1 Molecular modeling predicting conformational epitopes of PD1-H944

To investigate the interaction between PD1-H944 and PD-1 protein, in this example ZDOCK binding of PD1-H944 and PD-1 protein was performed. Based on the fact that the binding epitope of the PD-1 protein. targeted by PD1-H944 overlaps with the epitope targeted by the PD-L1 ligand. Homology modeling for PD1-H944 was performed using the antibody modeling program in DS 4.0 (Accelrys Software Inc.), and the final validity of the model design was verified using the Ramachandran map. The three-dimensional structure of the PD-1 protein was downloaded from the PDB database (PDB TD: 4ZQK) and initialized by the Protein Preparation program. The binding model of the model PD1-H944 and the PD-1 structure was carried out using the ZDOCK program. The top ten scoring options were optimized using RDOCK. The best model was further analyzed by the protein interface analysis program (Figure 22). The surface interaction docking model demonstrated that the two major peptide sequences binding PD1-H944 to the PD-1 protein were CC and FG folded layers (Table 13).

6.2 Confirmation of the functional epitope binding of the PD1-H944 antibody tested on PD-1 protein mutants

To more accurately confirm the functional epitope of PD1-H944 based on the peptide sequences predicted in Table 13 to be the major binding sites of PD1-H944 to the PD-1 protein, a series of alanine mutation mutants of the PD-1 protein were prepared and analyzed in the assay ELISA. To test the accuracy of the model, several sites outside the model of Example 6.1 were also selected for this study (Table 14), and these mutant sites were grouped into five spatial conformational epitopes (Figure 23).

The binding of each mutant from Table 14 to PD1-H944 was determined by ELISA according to the method of Example 4.2. The mutants have varying degrees of reduced binding compared to non-mutant PD-1 protein. Data were analyzed and plotted using Excel 2007 with the PD-1 protein mutant profile as the horizontal coordinate and the binding rate as

Vertical coordinate quality [binding rate=OD _{(PD-1 mutant protein)} /OD _{(PD-1 protein)} ×100%)]. The results demonstrated that the major binding sites of PD-1 protein to PD-L1 ligand were Site 2, Site 3, Site 4 and Site 5 on the folded layers CC and FG of PD-1 protein, which corresponded to the sites from the crystal complex analysis (PDB ID : 4ZQK). The binding sites for the positive control nivolumab binding to PD-1 protein were mainly located at Site 1 and Site 4 of its N-loop and FG fold layer, and this result is also consistent with the sites from the crystal complex assay (PDB ID: 5WT9); The main binding sites of PD1-H944 to the PD-1 protein were E61, K78, D85 and P130 at Site 3 and Site 4. The results obtained were also consistent with the results of the binding model in the above Example (Figure 24 and Table 15), and the binding accuracy of the model between PD1-H944 and PD-1 protein was confirmed.

The binding model between PD1-H944 and the PD-1 protein shows that the binding epitope on the PD-1 protein targeted by PD1-H944 overlaps with the PD-L1 ligand epitope. This finding supports the idea that PD1-H944 acts as a direct steric hindrance. Meanwhile, the overlap between the epitope targeted by both PD1-H944 and nivolumab suggested that PD1-H944 has a larger region to block PD-L1 binding to the epitopes (Figure 25A-B). Therefore, PD1-H944 may have excellent therapeutic efficacy in the treatment of oncology.

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LIST OF SEQUENCES

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agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg 780

aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct 840

tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc 900

acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac 960

tctcctggta aa 972

<210> 7

<211> 321

<212> DNA

<213> Mouse (Mus musculus)

<400> 7

cgggctgatg ctgcaccaac tgtatccacatc ttcccaccat ccagtgagca gttaacatct 60

ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccaaagacat caatgtcaag 120

tggaagattg atggcagtga acgacaaaat ggcgtcctga acagttggac tgatcaggac 180

agcaaagaca gcacctacag catgagcagc accctcacgt tgaccaagga cgagtatgaa 240

cgacataaca gctatacctg tgaggccact cacaagacat caacttcacc cattgtcaag 300

agcttcaaca ggaatgagtg t 321

<210> 8

<211> 118

<212>PRT

<213> Mouse (Mus musculus)

<400> 8

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Ser Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Asn Leu Phe

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ser Arg Gln Tyr Gly Thr Val Trp Phe Phe Asn Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210> 9

<211> 111

<212>PRT

<213> Mouse (Mus musculus)

<400> 9

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr

20 25 30

Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 10

<211> 12

<212>PRT

<213> Mouse (Mus musculus)

<400> 10

Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe Met His

1 5 10

<210> 11

<211> 11

<212>PRT

<213> Mouse (Mus musculus)

<400> 11

Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ala

1 5 10

<210> 12

<211> 9

<212>PRT

<213> Mouse (Mus musculus)

<400> 12

Gln Gln Ser Lys Glu Val Pro Trp Thr

15

<210> 13

<211> 10

<212>PRT

<213> Mouse (Mus musculus)

<400> 13

Gly Phe Thr Phe Ser Ser Tyr Gly Met Ser

1 5 10

<210> 14

<211> 19

<212>PRT

<213> Mouse (Mus musculus)

<400> 14

Val Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Ser Asp Ser

1 5 10 15

Val Lys Gly

<210> 15

<211> 11

<212>PRT

<213> Mouse (Mus musculus)

<400> 15

Ser Arg Gln Tyr Gly Thr Val Trp Phe Phe Asn

1 5 10

<210> 16

<211> 445

<212>PRT

<213> Man (Homo sapiens)

<400> 16

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Ser Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Gln Tyr Gly Thr Val Trp Phe Phe Asn Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 17

<211> 218

<212>PRT

<213> Man (Homo sapiens)

<400> 17

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr

20 25 30

Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Met Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 18

<211> 464

<212>PRT

<213> Man (Homo sapiens)

<400> 18

Met Gly Trp Ser Leu Ile Leu Leu Phe Leu Val Ala Val Ala Thr Arg

1 5 10 15

Val Leu Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu

50 55 60

Glu Trp Val Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Ser

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

85 90 95

Asn Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ser Arg Gln Tyr Gly Thr Val Trp Phe Phe Asn Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly

225 230 235 240

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

275 280 285

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

355 360 365

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

370 375 380

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

450 455 460

<210> 19

<211> 237

<212>PRT

<213> Man (Homo sapiens)

<400> 19

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu

20 25 30

Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val

35 40 45

Asp Ser Tyr Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly

50 55 60

Gln Pro Pro Arg Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly

65 70 75 80

Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

85 90 95

Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Met Tyr Phe Cys Gln

100 105 110

Gln Ser Lys Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu

115 120 125

Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser

130 135 140

Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn

145 150 155 160

Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala

165 170 175

Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys

180 185 190

Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp

195 200 205

Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu

210 215 220

Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 20

<211> 19

<212>PRT

<213> Man (Homo sapiens)

<400> 20

Met Gly Trp Ser Leu Ile Leu Leu Phe Leu Val Ala Val Ala Thr Arg

1 5 10 15

Val Leu Ser

<210> 21

<211> 19

<212>PRT

<213> Man (Homo sapiens)

<400> 21

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser

<210> 22

<211> 118

<212>PRT

<213> Man (Homo sapiens)

<400> 22

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Ser Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Gln Tyr Gly Thr Val Trp Phe Phe Asn Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 23

<211> 111

<212>PRT

<213> Man (Homo sapiens)

<400> 23

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr

20 25 30

Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Met Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 24

<211> 327

<212>PRT

<213> Man (Homo sapiens)

<400> 24

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 25

<211> 107

<212>PRT

<213> Man (Homo sapiens)

<400> 25

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 26

<211> 1395

<212> DNA

<213> Man (Homo sapiens)

<400> 26

atgggctggt ccctgattct gctgttcctg gtggctgtgg ctaccagggt gctgtctgag 60

gtccaacttg tggagtctgg aggaggactg gtgaagcctg gaggctccct gagactgtcc 120

tgtgctgcct ctggcttcac cttctcctcc tatgggatga gttgggtgag acaggctcct 180

gggaagagat tggagtgggt ggctaccatc tctggaggag gcagggacac ctactactct 240

gactctgtga agggcaggtt cacaatcagc agggacaatg ccaagaacaa cctgtacctc 300

caaatgaact ccctgagggc tgaggacaca gcagtctact actgtagcag acaatatggc 360

acagtgtggt tcttcaactg gggacaaggc accctggtga cagtgtcctc tgctagcacc 420

aagggcccat cggtcttccc gctggcgccc tgctccagga gcacctccga gagcacagcc 480

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacgaagac ctacacctgc 660

aacgtagatc acaagcccag caacaccaag gtggacaaga gagttgagtc caaatatggt 720

cccccatgcc caccctgccc agcacctgag ttcctggggg gaccatcagt cttcctgttc 780

cccccaaaac ccaaggacac tctcatgatc tcccggaccc ctgaggtcac gtgcgtggtg 840

gtggacgtga gccaggaaga ccccgaggtc cagttcaact ggtacgtgga tggcgtggag 900

gtgcataatg ccaagacaaa gccgcgggag gagcagttca acagcacgta ccgtgtggtc 960

agcgtcctca ccgtcctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtc 1020

tccaacaaag gcctcccgtc ctccatcgag aaaaccatct ccaaagccaa agggcagccc 1080

cgagagccac aggtgtacac cctgccccca tcccaggagg agatgaccaa gaaccaggtc 1140

agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggaaagc 1200

aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc 1260

ttcttcctct acagcaggct aaccgtggac aagagcaggt ggcaggaggg gaatgtcttc 1320

tcatgctccg tgatgcatga ggctctgcac aaccactaca cacagaagag cctctccctg 1380

tctctgggta aataa 1395

<210> 27

<211> 714

<212> DNA

<213> Man (Homo sapiens)

<400> 27

atgggctggt cctgtatcat cctgttcctg gtggctacag ccacaggagt gcattctgag 60

attgtgctga cccagagccc tgccaccctg tccctgagcc ctggagagag ggctaccctg 120

tcctgtaggg catctgagtc tgtggactcc tatggcaact cctttatgca ctggtatcaa 180

cagaagcctg gacaaccacc aagactgctg atttatgctg ccagcaacca gggctctgga 240

gtgcctgcca ggttctctgg ctctggctct ggcacagact tcaccctgac catctcctcc 300

ttggaacctg aggactttgc tatgtacttc tgtcaacaga gcaaggaggt gccatggacc 360

tttggacaag gcaccaaggt ggagattaag cgtacggtgg ctgcaccatc tgtcttcatc 420

ttcccgccat ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat 480

aacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt 540

aactcccagg agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc 600

accctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg cgaagtcacc 660

catcagggcc tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg ttag 714

<210> 28

<211> 57

<212> DNA

<213> Man (Homo sapiens)

<400> 28

atgggctggt ccctgattct gctgttcctg gtggctgtgg ctaccagggt gctgtct 57

<210> 29

<211> 57

<212> DNA

<213> Man (Homo sapiens)

<400> 29

atgggctggt cctgtatcat cctgttcctg gtggctacag ccacaggagt gcattct 57

<210> 30

<211> 354

<212> DNA

<213> Man (Homo sapiens)

<400> 30

gaggtccaac ttgtggagtc tggaggagga ctggtgaagc ctggaggctc cctgagactg 60

tcctgtgctg cctctggctt caccttctcc tcctatggga tgagttgggt gagacaggct 120

cctgggaaga gattggagtg ggtggctacc atctctggag gaggcaggga cacctactac 180

tctgactctg tgaagggcag gttcacaatc agcagggaca atgccaagaa caacctgtac 240

ctccaaatga actccctgag ggctgaggac acagcagtct actactgtag cagacaatat 300

ggcacagtgt ggttcttcaa ctggggacaa ggcaccctgg tgacagtgtc ctct 354

<210> 31

<211> 333

<212> DNA

<213> Man (Homo sapiens)

<400> 31

gagattgtgc tgacccagag ccctgccacc ctgtccctga gccctggaga gagggctacc 60

ctgtcctgta gggcatctga gtctgtggac tcctatggca actcctttat gcactggtat 120

caacagaagc ctggacaacc accaagactg ctgatttatg ctgccagcaa ccagggctct 180

ggagtgcctg ccaggttctc tggctctggc tctggcacag acttcaccct gaccatctcc 240

tccttggaac ctgaggactt tgctatgtac ttctgtcaac agagcaagga ggtgccatgg 300

acctttggac aaggcaccaa ggtggagatt aag 333

<210> 32

<211> 984

<212> DNA

<213> Man (Homo sapiens)

<400> 32

gctagcacca agggcccatc ggtcttcccg ctggcgccct gctccaggag cacctccgag 60

agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 240

tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 300

aaatatggtc ccccatgccc accctgccca gcacctgagt tcctgggggg accatcagtc 360

ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 420

tgcgtggtgg tggacgtgag ccadgaagac cccgaggtcc agttcaactg gtacgtggat 480

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 540

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 600

tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 660

gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 720

aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 780

tgggaaagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 840

gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg 900

aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 960

ctctccctgt ctctgggtaa ataa 984

<210> 33

<211> 324

<212> DNA

<213> Man (Homo sapiens)

<400> 33

cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60

ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240

aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300

agcttcaaca ggggagagtg ttag 324

<210> 34

<211> 247

<212>PRT

<213> Mouse (Mus musculus)

<400> 34

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr

20 25 30

Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser Ser Arg Ser

115 120 125

Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly

130 135 140

Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser

145 150 155 160

Tyr Gly Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp

165 170 175

Val Ala Thr Ile Ser Gly Gly Gly Arg Asp Thr Tyr Tyr Ser Asp Ser

180 185 190

Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Asn Leu

195 200 205

Phe Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr

210 215 220

Cys Ser Arg Gln Tyr Gly Thr Val Trp Phe Phe Asn Trp Gly Gln Gly

225 230 235 240

Thr Leu Val Thr Val Ser Ala

245

<210> 35

<211> 18

<212>PRT

<213> Mouse (Mus musculus)

<400> 35

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser Ser Arg

1 5 10 15

Ser Ser

<---

Claims (23)

1. An isolated anti-PD-1 antibody or an antigen-binding fragment thereof containing a light chain variable region or part thereof and a heavy chain variable region or part thereof; wherein said light chain variable region or portion thereof comprises a light chain CDR1 having the amino acid sequence SEQ ID NO: 10, a light chain CDR2 having the amino acid sequence SEQ ID NO: 11, and a light chain CDR3 having the amino acid sequence SEQ ID NO: 12 ; and said heavy chain variable region or portion thereof comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 13, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 14, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 15. 2. An isolated anti-PD-1 antibody or antigen-binding fragment thereof according to claim 1, characterized in that said antibody or antigen-binding fragment thereof contains, consists of, or essentially consists of: an amino acid sequence having at least 90% , 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 23 of the anti-PD-1 antibody light chain variable region, and/or an amino acid sequence having at least 90%, 92%, 95%, 98 % or 100% sequence identity with SEQ ID NO: 22 heavy chain variable region anti-PD-1 antibody. 3. An isolated anti-PD-1 antibody according to claim 1 or 2, characterized in that said antibody contains a light chain constant region and a heavy chain constant region, wherein preferably said light chain constant region contains an amino acid sequence having at least 90% , 92%, 95%, 98%, or 100% identity to the sequence of SEQ ID NO: 25 kappa light chain constant region, and/or said heavy chain constant region contains an amino acid sequence having at least 90%, 92%, 95% , 98% or 100% identity to the sequence of SEQ ID NO: 24 heavy chain constant region of the IgG4 antibody. 4. An isolated anti-PD-1 antibody according to any one of paragraphs. 1-3, which is an IgG antibody, preferably an IgG4 antibody. 5. An isolated anti-PD-1 antibody according to any one of paragraphs. 1-4, which is a monoclonal antibody. 6. An isolated anti-PD-1 antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-5, which binds to recombinant human PD-1 protein with an affinity with an average KD in the range of 20-200 pM, preferably 60-70 pM, more preferably 64.8 pM. 7. An isolated anti-PD-1 antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-6 that specifically binds to a PD-1 protein molecule containing the amino acid sequence of SEQ ID NO: 1 or to a protein molecule having amino acid sequences at least 90%, 92%, 95%, 98% or 100% identical sequence SEQ ID NO: 1. 8. An isolated antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-7, which specifically binds to a peptide sequence selected from at least one of the extracellular regions 60SESFV64, 78KLAAFPEDRSQP89, 128LAPKAQI134 of the PD-1 protein; preferably binds specifically to amino acid residues selected from at least one of the E61, K78, D85 and P130 extracellular regions of the PD-1 protein. 9. An isolated anti-PD-1 antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-8, characterized in that the specified antigen-binding fragment is presented in the form of Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd fragment, Fd fragment, single chain antibody molecule or single antibody domain; wherein said single chain antibody molecule is preferably a scFv, di-scFv, tri-scFv, diabody or scFab. 10. An isolated nucleic acid containing, consisting of, or essentially consisting of a nucleotide sequence encoding an isolated anti-PD-1 antibody or an antigen-binding fragment thereof according to any one of claims. 1-9, preferably an isolated nucleic acid containing, consisting of, or essentially consisting of the nucleotide sequence of SEQ ID NOs: 26 and 27. 11. An expression vector containing the isolated nucleic acid according to claim 10. 12. An isolated host cell for producing an antibody against PD-1 or an antigen-binding fragment thereof according to any one of claims 1 to 9, containing an isolated nucleic acid according to claim 10, and/or containing a vector according to claim 11. 13. A method for producing an isolated anti-PD-1 antibody or an antigen-binding fragment thereof according to any one of claims. 1-9, comprising growing the isolated host cells of claim 12 and purifying an anti-PD-1 antibody or an antigen-binding fragment thereof. 14. The use of an isolated anti-PD-1 antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-9 for the preparation of a medicament for the treatment of a tumor or cancer associated with PD-1. 15. Use according to claim 14, characterized in that said tumor or cancer is colon cancer. 16. The use of an isolated anti-PD-1 antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-9 for the treatment of a tumor or cancer associated with PD-1. 17. Use according to claim 16, characterized in that said tumor or cancer is colon cancer. 18. A pharmaceutical composition for the treatment of a tumor or cancer associated with PD-1, containing an effective amount of an isolated antibody against PD-1 or an antigen-binding fragment thereof according to any one of claims. 1-9. 19. A pharmaceutical combination for treating a tumor or cancer associated with PD-1, comprising an effective amount of the pharmaceutical composition of claim 18 with one or more other therapeutic agents. 20. A kit for the treatment of a tumor or cancer associated with PD-1, containing an isolated antibody against PD-1 or an antigen-binding fragment thereof according to any one of paragraphs. 1-9, a pharmaceutical composition according to claim 18 or a pharmaceutical combination according to claim 19. 21. The kit according to claim 20, further comprising an insertion device. 22. A method of treating a tumor or cancer associated with PD-1, comprising administering to a subject in need thereof a therapeutically effective amount of an isolated anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims. 1-9, or a pharmaceutical composition according to claim 18, or a pharmaceutical combination according to claim 19, or a kit according to claim 20 for treating said tumor or cancer, preferably characterized in that said tumor or cancer is colon cancer.