Antibodies binding to pd-1 and uses thereof

Abstract

An isolated monoclonal antibody that specifically binds to human PD-1. The present invention also provides nucleic acid molecules encoding the antibody, expression vectors, host cells, and methods for expressing the antibody. The present invention also provides immunoconjugates, bispecific molecules, chimeric antigen receptors, oncolytic viruses, and pharmaceutical compositions comprising the antibody, as well as methods of treatment using the anti-PD-1 antibodies of the present invention.

Description

[0001] This application is a divisional application of the invention patent application entitled "Antibody that binds to PD-1 and its use thereof", which entered the national phase in China on September 24, 2020, with application number 201980021731.5, application date of April 12, 2019. Technical Field

[0002] This invention generally relates to isolated monoclonal antibodies, particularly mouse, chimeric, or humanized monoclonal antibodies, that bind to human PD-1 with high affinity and functional specificity. The invention also provides nucleic acid molecules encoding the antibodies, expression vectors, host cells, and methods for expressing the antibodies. Furthermore, the invention provides immunoconjugates, bispecific molecules, oncolytic viruses, and pharmaceutical compositions comprising the antibodies, as well as diagnostic and therapeutic methods using the anti-PD-1 antibodies of this invention. Background Technology

[0003] Programmed cell death protein 1, also known as PD-1 or CD279, is a member of the CD28 family of T cell regulators and is expressed on activated B cells, T cells, and myeloid cells (Agata et al., (1996) Int Immunol 8:765-72; Okazaki et al., (2002) Curr. Opin. Immunol. 14: 391779-82; Bennett et al., (2003) J Immunol 170:711-8). It comprises a proximal membrane immunoreceptor tyrosine-based inhibitory motif (ITIM) and a distal membrane tyrosine-based switch motif (ITSM) (Thomas, ML (1995) J Exp Med 181:1953-6; Vivier, E and Daeron, M (1997) Immunol Today 18:286-91). Two ligands for PD-1, PD-L1 and PD-L2, have been identified. Both are B7 homologs that bind to PD-1 but not to other CD28 family members.

[0004] Several pieces of evidence suggest that PD-1 and its ligands negatively regulate immune responses. For example, PD-1 has been found to be abundant in various human cancers (Dong et al., (2002) Nat. Med. 8:787-9). Furthermore, the interaction between PD-1 and PD-L1 has been reported to reduce tumor-infiltrating lymphocyte and T-cell receptor-mediated proliferation, as well as immune evasion by cancer cells (Donget et al., (2003) J. Mol. Med. 81:281-7; Blank et al., (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al., (2004) Clin. Cancer Res. 10:5094-100). The study also showed that immunosuppression can be reversed by inhibiting the local interaction between PD-1 and PD-L1, and this effect is additive when the interaction between PD-1 and PD-L2 is also blocked (Iwai et al., (2002) Proc.Nat'l. Acad. Sci. USA 99:12293-7; Brown et al., (2003) J. Immunol. 170:1257-66).

[0005] Animals deficient in PD-1 may develop various autoimmune phenotypes, including autoimmune cardiomyopathy and lupus-like syndrome with arthritis and nephritis (Nishimura et al., (1999) Immunity 11:141-51; Nishimura et al., (2001) Science 291:319-22). Additionally, PD-1 has been found to play a role in autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease (GVHD), type 1 diabetes, and rheumatoid arthritis (Salama et al., (2003) J Exp Med 198:71-78; Prokunina and Alarcon-Riquelme (2004) Hum Mol Genet 13:R143; Nielsen et al., (2004) Lupus 13:510). In mouse B-cell tumor cell lines, the ITSM of PD-1 has been shown to block BCR-mediated calcium channel blockade. 2+ Tyrosine phosphorylation of flux and downstream effector molecules is essential (Okazaki et al., (2001) PNAS 98:13866-71).

[0006] Numerous PD-1-targeting cancer immunotherapies have been developed for disease treatment. One such anti-PD-1 antibody is Nivolumab (marketed by Bristol Myers Squibb under the brand name OPDIVO®), which produced full or partial responses in non-small cell lung cancer, melanoma, and renal cell carcinoma in a clinical trial involving a total of 296 patients (Topalian Slet al., (2012) The New England Journal of Medicine. 366 (26): 2443–54). It was approved in Japan in 2014 and by the US FDA in 2014 for the treatment of metastatic melanoma. Another PD-1-targeting anti-PD-1 antibody is Pembrolizumab (KEYTRUDA). TM MK-3475 (Merck) was also approved by the FDA in 2014 for the treatment of metastatic melanoma. In the United States, it has been used in clinical trials for lung cancer, lymphoma, and mesothelioma.

[0007] Although anti-PD-1 antibodies have been developed and approved, there is still a need for other monoclonal antibodies with enhanced binding affinity for PD-1 and other desired drug properties. Summary of the Invention

[0008] The present invention provides isolated monoclonal antibodies, such as mouse, human, chimeric or humanized monoclonal antibodies, that bind to PD-1 (e.g. human PD-1 and monkey PD-1) and have increased affinity for PD-1, and have comparable (if not better) antitumor effects compared to existing anti-PD-1 antibodies (e.g., Nivolumab).

[0009] The antibodies of this invention can be used in a variety of applications, including the detection of PD-1 protein and the treatment and prevention of PD-1-related diseases, such as cancer, autoimmune cardiomyopathy, autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease (GVHD), type 1 diabetes, and rheumatoid arthritis.

[0010] Accordingly, in one aspect, the present invention relates to isolated monoclonal antibodies (e.g., mouse, chimeric, or humanized antibodies) or their antigen-binding portions that bind to PD-1, having a heavy chain variable region comprising a CDR1 region, a CDR2 region, and a CDR3 region, wherein the CDR1 region, CDR2 region, and CDR3 region comprise, respectively, (1) SEQ ID NO: 1, 2, and 3; (2) SEQ ID NO: 4, 5, and 6; (3) SEQ ID NO: 7, 8, and 9; (4) SEQ ID NO: 10, 11, and 12; (5) SEQ ID NO: 13, 14, and 15; (6) SEQ ID NO: 16, 17, and 18; (7) SEQ ID NO: 19, 20, and 21; (8) SEQ ID NO: 22, 23, and 24; (9) SEQ ID NO: 25, 26, and 27; (10) SEQ ID NO: 28, 29, and 30; or (11) SEQ ID NO: 31, 32 and 33 have amino acid sequences with at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity; wherein the antibody or its antigen-binding fragment binds to PD-1.

[0011] In one aspect, the isolated monoclonal antibody or its antigen-binding portion of the present invention comprises a heavy chain variable region having an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO: 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, or 79, wherein the antibody or its antigen-binding fragment binds to PD-1. SEQ ID NO: 67 and 69-79 may be encoded by the nucleic acid sequences of SEQ ID NO: 102-113, respectively.

[0012] In one aspect, the isolated monoclonal antibody or its antigen-binding portion of the present invention comprises a light chain variable region comprising a CDR1 region, a CDR2 region, and a CDR3 region, wherein the CDR1 region, CDR2 region, and CDR3 region comprise, respectively, (1) SEQ ID NO: 34, 35, and 36; (2) SEQ ID NO: 37, 38, and 39; (3) SEQ ID NO: 40, 41, and 42; (4) SEQ ID NO: 43, 44, and 45; (5) SEQ ID NO: 46, 47, and 48; (6) SEQ ID NO: 49, 50, and 51; (7) SEQ ID NO: 52, 53, and 54; (8) SEQ ID NO: 55, 56, and 57; (9) SEQ ID NO: 58, 59, and 60; (10) SEQ ID NO: 61, 62, and 63; or (1 ... NO: 64, 65 and 66 have amino acid sequences with at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity; wherein the antibody or its antigen-binding fragment binds to PD-1.

[0013] In one aspect, the isolated monoclonal antibody or its antigen-binding portion of the present invention comprises a light chain variable region having an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO: 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96, wherein the antibody or its antigen-binding fragment binds to PD-1. SEQ ID NO: 80 and 86-96 may be encoded by the nucleic acid sequences of SEQ ID NO: 114-125, respectively.

[0014] In one aspect, the isolated monoclonal antibody or its antigen-binding moiety of the present invention comprises a heavy chain variable region and a light chain variable region, each of the heavy chain variable region and the light chain variable region comprising a CDR1 region, a CDR2 region and a CDR3 region, wherein the heavy chain variable regions CDR1, CDR2 and CDR3 and the light chain variable regions CDR1, CDR2 and CDR3 comprise amino acid sequences having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the following sequences respectively: (1) SEQ ID NO: 1, 2, 3, 34, 35 and 36; (2) SEQ ID NO: 4, 5, 6, 37, 38 and 39; (3) SEQ ID NO: 7, 8, 9, 40, 41 and 42; (4) SEQ ID NO: 10, 11, 12, 43, 44 and 45; (5) SEQ ID NO: 13, 14, 15, 46, 47 and 48; (6) SEQ ID NO: 10, 11, 12, 43, 44 and 45; (5) SEQ ID NO: 13, 14, 15, 46, 47 and 48; (6) SEQ ID NO: 10, 11, 12, 43, 44 and 45; (7) SEQ ID NO: 16, 17, 18, 49, 50 and 51; (8) SEQ ID NO: 19, 20, 21, 52, 53 and 54; (9) SEQ ID NO: 22, 23, 24, 55, 56 and 57; (10) SEQ ID NO: 25, 26, 27, 58, 59 and 60; (11) SEQ ID NO: 28, 29, 30, 61, 62 and 63; or (12) SEQ ID NO: 31, 32, 32, 64, 65 and 66, wherein the antibody or its antigen-binding fragment binds to PD-1.

[0015] In one embodiment, the isolated monoclonal antibody or its antigen-binding moiety of the present invention comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region comprising amino acid sequences having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the following sequences: (1) SEQ ID NO: 67 and 80; (2) SEQ ID NO: 68 and 81; (3) SEQ ID NO: 69 and 81; (4) SEQ ID NO: 68 and 82; (5) SEQ ID NO: 68 and 83; (6) SEQ ID NO: 68 and 84; (7) SEQ ID NO: 68 and 85; (8) SEQ ID NO: 68 and 86; (9) SEQ ID NO: 69 and 86; (10) SEQ ID NO: 70 and 87; (11) SEQ ID NO: 71 and 88; (12) SEQ ID NO: 72 and 89; (13) SEQ ID NO: 68 and 84; (14) SEQ ID NO: 68 and 85; (15) SEQ ID NO: 68 and 86; (16) SEQ ID NO: 69 and 86; (17) SEQ ID NO: 70 and 87; (18) SEQ ID NO: 71 and 88; (19) SEQ ID NO: 72 and 89; (10) SEQ ID NO: 70 and 87; (11) SEQ ID NO: 71 and 88; (12) SEQ ID NO: 72 and 89; (13) SEQ ID NO: 68 and 84; (14) SEQ ID NO: 68 and 85; (15) SEQ ID NO: 68 and 86; (16) SEQ (14) SEQ ID NO: 73 and 90; (15) SEQ ID NO: 75 and 92; (16) SEQ ID NO: 76 and 93; (17) SEQ ID NO: 77 and 94; (18) SEQ ID NO: 78 and 95; or (19) SEQ ID NO: 79 and 96, wherein the antibody or its antigen-binding fragment binds to PD-1.

[0016] In one embodiment, the isolated monoclonal antibody or its antigen-binding portion of the present invention comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region and a heavy chain constant region, and the light chain comprising a light chain variable region and a light chain constant region, wherein the heavy chain constant region comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID No: 97, 99, or 129, and the light chain constant region comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID No: 98 or 130, and the heavy chain variable region and light chain variable region comprise the aforementioned amino acid sequences, wherein the antibody or its antigen-binding fragment binds to PD-1. These amino acid sequences of SEQ ID NO: 97 and 99 can be encoded by the nucleic acid sequences of SEQ ID NO: 126 and 128, respectively. SEQ ID NO: 98 can be encoded by SEQ ID NO: 127.

[0017] In some embodiments, the antibody of the present invention comprises two heavy chains and two light chains, or is composed of two heavy chains and two light chains, wherein each heavy chain contains the aforementioned heavy chain constant region, heavy chain variable region, or CDR sequence, and each light chain contains the aforementioned light chain constant region, light chain variable region, or CDR sequence, wherein the antibody binds to PD-1. The antibody of the present invention may be a full-length antibody, such as an IgG1, IgG2, or IgG4 isotype. In other embodiments, the antibody of the present invention may be a single-chain antibody or an antibody fragment, such as a Fab or Fab'2 fragment.

[0018] The antibody or its antigen-binding moiety of the present invention has a higher binding affinity for human PD-1 than prior art anti-PD-1 antibodies (e.g., Nivolumab), with a value of 6.36 × 10⁻⁶. -9 M or less K D It binds to human PD-1 and inhibits the binding of PD-L1 to PD-1. Compared with existing anti-PD-1 antibodies (such as Nivolumab), the antibody of the present invention or its antigen-binding moiety also provides comparable (if not better) anti-tumor effects.

[0019] The present invention also provides immunoconjugates comprising an antibody or antigen-binding portion thereof of the present invention linked to a therapeutic agent (e.g., a cytotoxin). The present invention also provides bispecific molecules comprising an antibody or antigen-binding portion thereof of the present invention linked to a second functional portion (e.g., a second antibody) having a binding specificity different from that of the antibody or antigen-binding portion thereof. Alternatively, the antibody or antigen-binding portion thereof of the present invention can be formulated as part of a chimeric antigen receptor (CAR). The antibody or antigen-binding portion thereof of the present invention can also be encoded by or used in combination with an oncolytic virus.

[0020] The present invention also provides compositions comprising the antibody of the present invention or its antigen-binding moiety, or an immunoconjugate, a bispecific molecule, an oncolytic virus, or a CAR, and a pharmaceutically acceptable carrier.

[0021] The present invention also includes nucleic acid molecules encoding the antibody of the present invention or an antigen-binding portion thereof, as well as expression vectors comprising such nucleic acids and host cells comprising such expression vectors. A method for preparing an anti-PD-1 antibody using host cells comprising an expression vector is also provided, comprising the steps of: (i) expressing the antibody in the host cells, and (ii) isolating the antibody from the host cells or cell cultures thereof.

[0022] In another aspect, the present invention provides a method for modulating an immune response in a subject, comprising administering an antibody of the present invention or its antigen-binding portion to the subject, thereby modulating the immune response in the subject. Preferably, the antibody of the present invention enhances, stimulates, or increases the immune response of the subject. In some embodiments, the method includes administering a composition of the present invention, a bispecific molecule, an immune conjugate, CAR-T cells, or an oncolytic virus encoding or carrying an antibody, or a nucleic acid molecule capable of expressing them in the subject.

[0023] In another aspect, the present invention provides a method for inhibiting tumor growth in a subject, comprising administering to the subject a therapeutically effective amount of an antibody of the present invention or its antigen-binding portion. The tumor may be a solid tumor or a non-solid tumor, including but not limited to lymphoma, leukemia, multiple myeloma, melanoma, colonic adenocarcinoma, pancreatic cancer, colon cancer, gastrointestinal cancer, prostate cancer, bladder cancer, kidney cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, renal cell carcinoma, and nasopharyngeal carcinoma. In some embodiments, the method comprises administering a composition of the present invention, a bispecific molecule, an immunoconjugate, CAR-T cells, or an oncolytic virus encoding or carrying an antibody, or a nucleic acid molecule capable of expressing them in the subject.

[0024] In another aspect, the present invention provides a method for treating an infectious disease in a subject, comprising administering to the subject a therapeutically effective amount of an antibody of the present invention or an antigen-binding portion thereof. In some embodiments, the method comprises administering a composition of the present invention, a bispecific molecule, an immunoconjugate, CAR-T cells, or an oncolytic virus encoding or carrying an antibody, or a nucleic acid molecule capable of expressing them in the subject.

[0025] Furthermore, the present invention provides a method for enhancing an immune response to an antigen in a subject, comprising administering to the subject: (i) an antigen; and (ii) an antibody or an antigen-binding portion thereof, thereby enhancing the subject's immune response to the antigen. The antigen may be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen derived from a pathogen.

[0026] The antibodies of the present invention can be used in combination with at least one other reagent, such as an immunostimulatory antibody (e.g., anti-PD-L1 antibody and / or anti-CTLA-4 antibody), a cytokine (e.g., IL-2 and / or IL-21), or a co-stimulatory antibody (e.g., anti-CD137 and / or anti-GITR antibody).

[0027] Other features and advantages of this disclosure will become apparent from the following detailed description and examples, and should not be construed as limiting. All references, Genbank entries, patents, and published patent applications cited in this application are expressly incorporated herein by reference. Attached Figure Description

[0028] Figure 1 The melting curve of the humanized anti-PD-1 antibody huClEl-V10 is shown.

[0029] Figure 2 The results of size exclusion chromatography for the humanized anti-PD-1 antibody huClE1-V10 are shown. Detailed description

[0030] To ensure easier understanding of this disclosure, certain terms are defined first. Other definitions are set forth throughout the detailed description.

[0031] The term "PD-1" refers to programmed cell death protein 1. The term "PD-1" includes variants, isotypes, homologs, orthologs, and paralogs. For example, in some cases, antibodies specific to human PD-1 protein may cross-react with PD-1 protein from species other than humans (e.g., monkeys). In other embodiments, antibodies specific to human PD-1 protein may be completely specific to human PD-1 protein and not exhibit cross-reactivity with other species or types; or may cross-react with PD-1 from some other species, but not all others.

[0032] The term "human PD-1" refers to a PD-1 protein having an amino acid sequence derived from humans, such as the amino acid sequence of human PD-1 with Genbank accession number NP_005009.2. The terms "monkey or rhesus monkey PD-1" and "mouse PD-1" refer to monkey and mouse PD-1 sequences, respectively, such as those with amino acid sequences having Genbank accession numbers NP_001107830 and CAA48113, respectively.

[0033] The term "immune response" refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by these cells or the liver, which result in selective damage, destruction, or elimination of invading pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues (in cases of autoimmunity or pathological inflammation).

[0034] "Antigen-specific T cell response" refers to a response produced by T cells in response to stimulation of T cells with a T cell-specific antigen. Non-limiting examples of T cell responses to antigen-specific stimulation include proliferation and cytokine production (e.g., IL-2 production).

[0035] As used herein, the term "antibody" includes complete antibodies and any antigen-binding fragments (i.e., "antigen-binding portions") or their single chains. A complete antibody is a glycoprotein consisting of two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated as V in this document). H It consists of a heavy chain constant region and a heavy chain constant region. The heavy chain constant region consists of three structural domains C H1 C H2 and C H3 Composed of. Each light chain consists of a light chain variable region (abbreviated as V in this article). L It consists of a light chain constant region and a light chain constant region. The light chain constant region consists of a structural domain C. L Composition. V H and V L The region can be further subdivided into highly variable regions, called complementary determinant regions (CDRs), interspersed with more conservative regions, called frame regions (FRs). Each V H and V L Composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0036] As used herein, the term "antigen-binding portion" (or simply "antibody portion") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., the PD-1 protein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments covered by the term "antigen-binding portion" of an antibody include: (i) the Fab fragment, which is composed of V... L V H C L and C H1 (ii) a monovalent segment composed of structural domains; (iii) a F(ab')2 segment, which is a divalent segment contained in the hinge region of two Fab segments connected by disulfide bonds; H and C H1 (iv) Fd fragments composed of structural domains; V-shaped segments formed by the antibody single arm L and V H The Fv segment, composed of structural domains, (v) is composed of V HThe dAb fragment consists of the following domains (Ward et al., (1989) Nature 341:544-546); (vi) a separated complementarity-determining region (CDR); and (viii) a nanobody, the heavy chain variable region containing one variable domain and two constant domains. Furthermore, although the two domains V of the Fv fragment... L and V H Encoded by different genes, but they can be linked together using recombination methods via synthetic linkers, thus forming a protein chain, in which V L and V H Regions pair to form monovalent molecules (called single-chain Fvs (scFvs); see, for example, Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be included within the “antigen-binding portion” of the term antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and their utility is screened in the same manner as that of intact antibodies.

[0037] As used herein, “isolated antibody” is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to the PD-1 protein is substantially free of antibodies that specifically bind to antigens other than the PD-1 protein). However, isolated antibodies that specifically bind to the human PD-1 protein may be cross-reactive with other antigens, such as PD-1 proteins from other species. Furthermore, isolated antibodies may be substantially free of other cellular material and / or chemicals.

[0038] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a formulation of an antibody molecule consisting of a single molecule. Monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope.

[0039] As used herein, the term "mouse antibody" is intended to include antibodies having variable regions, wherein both the frame region and the CDR region are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from a mouse germline immunoglobulin sequence. The mouse antibodies of the present invention may include amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "mouse antibody" is not intended to include antibodies in which a germline CDR sequence derived from another mammalian species has been grafted onto a mouse frame sequence.

[0040] The term "chimeric antibody" refers to an antibody prepared by combining genetic material from a non-human source with genetic material from a human. Or more generally, a chimeric antibody is an antibody that has genetic material from one species and genetic material from another species.

[0041] As used in this article, the term "humanized antibody" refers to an antibody derived from a non-human species whose protein sequence has been modified to increase its similarity to naturally occurring human antibody variants.

[0042] The term "isotype" refers to an antibody class (e.g., IgM or IgG1) encoded by a gene in the heavy chain constant region.

[0043] The phrases “antibody that recognizes an antigen” and “antibody that is specific to an antigen” are used interchangeably with the term “antibody that binds specifically to an antigen” in this document.

[0044] As used herein, an antibody that "specifically binds to human PD-1" refers to an antibody that binds to the human PD-1 protein (and possibly PD-1 proteins from one or more non-human species) but substantially does not bind to non-PD-1 proteins. Preferably, the antibody binds to the human PD-1 protein with "high affinity," i.e., K... D 5.0 x 10 -8 M or smaller, more preferably 1.0 x 10 -8 M or smaller, preferably 7.0 x 10 -9 M or smaller.

[0045] As used in this article, the term "substantially non-binding" to proteins or cells refers to K+ that does not bind to proteins or cells or does not bind with high affinity, i.e., K+ that binds to proteins or cells. D 1.0 x 10 -6 M or more, preferably 1.0 x 10 -5 M or larger, more preferably 1.0 x 10 -4 M or above, with 1.0 x 10 being a further preferred size. -3 M or above, with 1.0 x 10 being a further preferred size. -2 M or above.

[0046] The term "high affinity" for IgG antibodies refers to the antibody's affinity for the target antigen (K+). D 1.0 x 10 -6 M or smaller, preferably 5.0 x 10 -8 M or smaller, or even more preferably 1.0 x 10 -8 M or smaller, or even better 7.0 x 10 -9 M or smaller, or even more preferably 1.0 x 10 -9M or smaller. However, "high affinity" binding may differ for other antibody isotypes. For example, "high affinity" binding for IgM isotypes refers to the antibody having a K0... D 10 -6 M or smaller, more preferably 10 -7 M or smaller, or even more preferably 10 -8 M or smaller.

[0047] As used in this article, the term "K" assoc "or "K a "K" refers to the binding rate of a specific antibody-antigen interaction, while, as used herein, the term "K" is used to refer to the binding rate of that interaction. dis "or "K d "K" refers to the dissociation rate of a specific antibody-antigen interaction. As used in this article, the term "K" is... D "" refers to the dissociation constant, which is determined by K d With K a The ratio (i.e., K) d / K a The K of the antibody is obtained and expressed as molar concentration (M). D The value can be determined using methods well-known in the art. Determining antibody K D The preferred method is to use surface plasmon resonance, preferably using a biosensor system such as Biacore. TM system.

[0048] The term "EC" 50 The half-maximum effective concentration (WMC) is the concentration of antibody that induces half the response between baseline and maximum after a specified exposure time.

[0049] The term "IC" 50 The half-maximal inhibitory concentration (WMC), also known as the antibody concentration that inhibits a specific biological or biochemical function by 50% relative to the absence of the antibody, is a specific antibody concentration.

[0050] The term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cattle, horses, chickens, amphibians, and reptiles, but mammals are preferred, such as non-human primates, sheep, dogs, cats, cattle, and horses.

[0051] The term "therapeutic effective amount" refers to the amount of the antibody of the present invention sufficient to prevent or improve symptoms associated with a disease or condition (e.g., cancer) and / or reduce the severity of the disease or condition. Therapeutic effective amount should be understood in relation to the condition being treated, where the actual effective amount can be readily identified by those skilled in the art.

[0052] The various aspects of the invention are further described in detail in the following sections.

[0053] Anti-PD-1 antibodies with increased binding affinity to human PD-1 and better anti-tumor activity

[0054] The antibody or its antigen-binding moiety of the present invention specifically binds to human PD-1 and has improved binding affinity and comparable (if not better) antitumor activity compared to previously described antiPD-1 antibodies (especially compared to Nivolumab).

[0055] The antibody or its antigen-binding moiety of the present invention is preferably 7.0 x 10⁻⁶. -9 M or smaller K D More preferably 5.0 x 10 -10 M or smaller K D Binds to human PD-1 protein. The antibody of this invention also contains approximately 1.0 x 10⁻⁶ units. -7 M to 1.0 x 10 -10 M of K D Combined with cynomolgus monkey PD-1.

[0056] Other functional properties include the ability to block PD-1 / PD-L1 interaction. In one embodiment, the antibody of the present invention can inhibit the binding of PD-1 to PD-L1 at a concentration similar to that of Nivolumab.

[0057] Other functional properties include the antibody's ability to stimulate an immune response, such as an antigen-specific T-cell response. For example, this can be tested by assessing the antibody's ability to stimulate the production of interleukin-2 (IL-2) in an antigen-specific T-cell response. In some embodiments, the antibody binds to human PD-1 and stimulates an antigen-specific T-cell response. In other embodiments, the antibody binds to human PD-1 but does not stimulate an antigen-specific T-cell response. Other methods for assessing the antibody's ability to stimulate an immune response include testing its ability to inhibit tumor growth (e.g., in in vivo tumor transplantation models) or its ability to stimulate an autoimmune response, such as its ability to promote the development of autoimmune diseases in autoimmune models, such as its ability to promote the development of diabetes in NOD mouse models.

[0058] The preferred antibody of the present invention is a human monoclonal antibody. Alternatively, the antibody may be, for example, a chimeric or humanized monoclonal antibody.

[0059] Monoclonal anti-PD-1 antibody

[0060] The preferred antibody of the present invention is a monoclonal antibody with the structure and chemical characteristics described below and in the "Examples" section. V of the anti-PD-1 antibody HThe amino acid sequences are listed in SEQ ID NO: 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, or 79. V of the anti-PD-1 antibody. L The amino acid sequences are shown in SEQ ID NO: 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96. The amino acid sequence ID numbers of the heavy / light chain variable regions of the antibodies are summarized in Table 1 below. Some clones share the same V. H or V L The heavy chain constant region of all clones can be the IgG1 heavy chain constant region, such as the human IgG1 heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 97, or the mouse IgG1 heavy chain constant region having the amino acid sequence listed in, for example, SEQ ID NO: 129, and the light chain constant region of all clones can be the κ constant region, such as the human κ constant region having the amino acid sequence listed in, for example, SEQ ID NO: 98, or the mouse κ constant region having the amino acid sequence listed in, for example, SEQ ID NO: 130. The antibody Fab may contain a heavy chain / light chain variable region, a heavy chain C... H1 Regions (such as one listed in SEQ ID NO: 99) and light chain constant regions.

[0061] Table 1. Amino acid sequence IDs of the heavy / light chain variable regions

[0062]

[0063] The heavy chain variable region CDR and light chain variable region CDR in Table 1 are defined by the IMGT numbering scheme and the Rabat numbering system, respectively. However, as is well known in the art, the CDR region can also be determined by other systems (e.g., the Chothia and CCG systems / methods) based on the heavy chain / light chain variable region sequence.

[0064] V of other anti-PD-1 antibodies that bind to human PD-1 H and V L The sequence (or CDR sequence) is similar to the V of the anti-PD-1 antibody of the present invention. H and V L The sequences (or CDR sequences) are "mixed and matched". Preferably, when V H and V L When chains (or CDRs in such chains) are mixed and matched, the specific V H / V L Yes, V H The sequence is structurally similar to V H Sequence substitution. Similarly, it is preferable to use sequences from a specific V. H / V L Yes, VL Sequence replacement with structurally similar V L sequence.

[0065] Therefore, in one embodiment, the antibody or its antigen-binding portion of the present invention comprises:

[0066] (a) The heavy chain variable region, which contains the amino acid sequences listed in Table 1 above; and

[0067] (b) The light chain variable region, which contains the amino acid sequence listed in Table 1 above, or the V of another anti-PD-1 antibody. L This antibody specifically binds to human PD-1.

[0068] In another embodiment, the antibody or its antigen-binding portion of the present invention comprises:

[0069] (a) The CDR1, CDR2, and CDR3 regions of the heavy chain variable region listed in Table 1 above; and

[0070] (b) The CDR1, CDR2 and CDR3 regions of the light chain variable region listed in Table 1 above, or the CDR of another anti-PD-1 antibody, wherein the antibody specifically binds to human PD-1.

[0071] In yet another embodiment, the antibody or its antigen-binding portion includes the heavy chain variable CDR2 region of an anti-PD-1 antibody binding to the CDRs of other antibodies that bind to human PD-1, such as CDR1 and / or CDR3 from the heavy chain variable regions of different anti-PD-1 antibodies and / or CDR1, CDR2 and / or CDR3 from the light chain variable regions.

[0072] Furthermore, it is well known in the art that the CDR3 domain, independent of the CDR1 and / or CDR2 domains, can be used to determine the binding specificity of an antibody to a homologous antigen, and can predict the generation of multiple antibodies with the same binding specificity based on a common CDR3 sequence. See, for example, Klimka el al., British J. of Cancer 83(2):252-260 (2000); Beiboer el al., J. Mol. Biol. 296:833-849 (2000); Rader et al., Proc. Natl. Acad. Sci. USA 95:8910-8915 (1998); Barbas et al. al.,, J. Am. Chem. Soc. 116:2161-2162 (1994); Barbas et al. Proc. Natl. Acad. Sci. USA. 92:2529-2533 (1995); Ditzel et al.,, J. Immunol. 157:739-749 (1996); Berezovet al.,, BIAjournal 8: Scientific Review 8 (2001); Igarashi et al., J. Biochem (Tokyo) 117:452-7 (1995); Bourgeois et al., J. Virol 72:807-10 (1998); Levi et al. Proc. Natl. Acad. Sci. USA 90:4374-8 (1993); Polymenis and Stoller, J. Immunol. 152:5218-5329 (1994) and Xu and Davis, Immunity 13:37-45 (2000). See also U.S. Patent Nos. 6,951,646; 6,914,128; 6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905 and 5,760,185. The full text of each of these references is incorporated herein by reference.

[0073] Therefore, in another embodiment, the antibody of the present invention comprises CDR2 of the heavy chain variable region of an anti-PD-1 antibody and at least CDR3 of the heavy chain and / or light chain variable region of an anti-PD-1 antibody, or CDR3 of the heavy chain and / or light chain variable region of another anti-PD-1 antibody, wherein the antibody is capable of specifically binding to human PD-1. These antibodies preferably (a) compete with PD-1 for binding; (b) retain functional characteristics; (c) bind the same epitopes; and / or (d) have binding affinity similar to that of the anti-PD-1 antibody of the present invention. In yet another embodiment, the antibody may further comprise CDR2 of the light chain variable region of an anti-PD-1 antibody, or CDR2 of the light chain variable region of another anti-PD-1 antibody, wherein the antibody is capable of specifically binding to human PD-1. In another embodiment, the antibody of the present invention may comprise CDR1 of the heavy and / or light chain variable region of an anti-PD-1 antibody, or CDR1 of the heavy and / or light chain variable region of another anti-PD-1 antibody, wherein the antibody is capable of specifically binding to human PD-1.

[0074] conservative modification

[0075] In another embodiment, the antibody of the present invention comprises heavy chain and / or light chain variable region sequences of CDR1, CDR2, and CDR3 sequences, which differ from the sequence of the anti-PD-1 antibody of the present invention in that they have one or more conserved modifications. It will be understood in the art that certain conserved sequence modifications can be made without removing antigen binding. See, for example, Brummell et al., (1993) Biochem 32: 1180-8; de Wildt et al., (1997) Prot.Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149: 1605-12; Kelley and O'Connell (1993) Biochem.32:6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10:341-6; and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0076] Therefore, in one embodiment, the antibody comprises a heavy chain variable region containing CDR1, CDR2, and CDR3 sequences and / or a light chain variable region containing CDR1, CDR2, and CDR3 sequences, wherein:

[0077] (a) The heavy chain variable region CDR1 sequence contains the sequences listed in Table 1 above, and / or their conservation modifications; and / or

[0078] (b) The heavy chain variable region CDR2 sequence contains the sequences listed in Table 1 above, and / or their conservation modifications; and / or

[0079] (c) The heavy chain variable region CDR3 sequence contains the sequences listed in Table 1 above, and their conservation modifications; and / or

[0080] (d) The light chain variable region CDR1 and / or CDR2 and / or CDR3 sequences contain the sequences listed in Table 1 above; and / or their conservation modifications; and

[0081] (e) Antibody specifically binds to human PD-1.

[0082] The antibody of the present invention has one or more of the functional properties described above, such as high affinity binding to human PD-1 and the ability to induce ADCC or CDC against cells expressing PD-1.

[0083] In various implementation schemes, the antibody may be, for example, a mouse, human, humanized, or chimeric antibody.

[0084] As used herein, the term "conserved sequence modification" is intended to refer to amino acid modifications that do not significantly affect or alter the binding properties of an antibody containing that amino acid sequence. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved amino acid substitution is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are already defined in the art. These families include amino acids with the following side chains: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR region of the antibodies of the present invention can be replaced by other amino acid residues from the same side chain family, and the retained function (i.e., the function described above) of the modified antibody can be tested using the functional analysis methods described herein.

[0085] Engineered and modified antibodies

[0086] One or more V antibodies containing the anti-PD-1 antibody of the present invention can be used. H / V L The antibodies of this invention are prepared by engineering modified antibodies using antibodies with specific sequences as starting materials. Modification can be achieved by modifying one or two variable regions (i.e., V...). H and / or V L Antibodies can be engineered by modifying one or more residues within a region (e.g., within one or more CDR regions and / or one or more frame regions). Alternatively, antibodies can be engineered by modifying residues within a constant region, for example, to alter the effector function of the antibody.

[0087] In some implementations, CDR transplantation can be used for the variable regions of engineered antibodies. Antibodies primarily interact with target antigens through amino acid residues located in the six complementarity-determining regions (CDRs) of the heavy and light chains. Therefore, the amino acid sequences within the CDRs differ more significantly from those outside the CDRs between individual antibodies. Since CDR sequences are responsible for most antibody-antigen interactions, recombinant antibodies that mimic the properties of specific naturally occurring antibodies can be expressed by constructing expression vectors that include CDR sequences from specific naturally occurring antibodies grafted onto the framework sequences of different antibodies with different characteristics (see, for example, Riechmann et al., (1998) Nature 332:323-327; Jones et al., (1986) Nature 321:522-525; Queen et al., (1989) Proc. Natl. Acad. See also USA 86: 10029-10033; U.S. Patent Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0088] Therefore, another embodiment of the present invention relates to an isolated monoclonal antibody or its antigen-binding portion, comprising: a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the sequences of the present invention as described above; and / or a light chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the sequences of the present invention as described above. Although these antibodies comprise the V of the monoclonal antibody of the present invention... H and V L CDR sequences, but they can contain different frame sequences.

[0089] Such framework sequences can be obtained from public DNA databases or publicly available references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in: the “VBase” human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk / vbase), and Kabat et al., (1991); Tomlinson et al., (1992) J. Mol. Biol. 227:776-798; and Cox et al., (1994) Eur. J. Immunol. 24:827-836, as cited above; the contents of each of these are explicitly incorporated herein by reference. As another example, germline DNA sequences of human heavy and light chain variable region genes can be found in the Genbank database. For example, the following heavy chain phylogenetic sequences found in HCo7 HuMAb mice are available from the accompanying Genbank accession numbers: 1-69 (NG--0010109, NT--024637 & BC070333), 3-33 (NG--0010109 & NT--024637), and 3-7 (NG--0010109 & NT--024637). As another example, the following heavy chain phylogenetic sequences found in HCl2 HuMAb mice are available from the accompanying Genbank accession numbers: 1-69 (NG--0010109, NT--024637 & BC070333), 5-51 (NG--0010109 & NT--024637), 4-34 (NG--0010109 & NT--024637), 3-30.3 (CAJ556644) & 3-23 (AJ406678).

[0090] Using one of the sequence similarity search methods known to those skilled in the art, called Gapped BLAST (Altschul et al., (1997), ibid.), the antibody protein sequence was compared with a compiled protein sequence database.

[0091] The preferred framework sequence for the antibody used in this invention is one that is structurally similar to the framework sequence used in the antibody of this invention. V can be... HThe CDR1, CDR2, and CDR3 sequences can be grafted into a frame region having the same sequence as the germline immunoglobulin gene from which the frame sequence originates, or the CDR sequence can be grafted into a frame region containing one or more mutations compared to the germline sequence. For example, it has been found that in certain circumstances, it is beneficial to mutate residues within the frame region to maintain or enhance the antigen-binding capacity of the antibody (see, for example, U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370).

[0092] Another type of variable region modification is to make V H and / or V L Amino acid residue mutations within the CDR1, CDR2, and / or CDR3 regions can improve one or more binding properties (e.g., affinity) of the target antibody. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutations, and the effects on antibody binding or other intended functional properties can be assessed in vitro or in vivo as known in the art. Preferably, conserved modifications (as known in the art) are introduced. Mutations can be amino acid substitutions, additions, or deletions, but substitution is preferred. Furthermore, typically no more than 1, 2, 3, 4, or 5 residues in the CDR region are altered.

[0093] Therefore, in another embodiment, the present invention provides an isolated anti-PD-1 monoclonal antibody or its antigen-binding moiety thereof, comprising a heavy chain variable region comprising: (a) V H CDR1 region, which contains the sequence of the present invention, or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions; (b) V H CDR2 region, which contains the sequence of the present invention, or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions; (c)V H The CDR3 region contains the sequence of the present invention, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; (d)V L CDR1 region, which contains the sequence of the present invention, or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions; (e)V L The CDR2 region contains the sequence of the present invention, or an amino acid sequence having 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions; and (f) V L The CDR3 region contains the sequence of the present invention, or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions.

[0094] The engineered antibodies of the present invention include those that have been tested for V H and / or V LAntibodies that modify framework residues, for example, to improve antibody properties. Typically, such framework modifications are made to reduce antibody immunogenicity. One approach, for example, is to "mutate" one or more framework residues back to their corresponding germline sequences. More specifically, antibodies that have undergone somatic mutations may contain framework residues different from the germline sequence from which the antibody is derived. These residues can be identified by comparing the antibody framework sequence with the germline sequence of the derived antibody.

[0095] Another type of framework modification involves mutating one or more residues within a framework region or even one or more CDR regions to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This method is also known as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043.

[0096] Alternatively, or as an alternative to modifications performed within the frame or CDR region, the antibodies of the present invention can be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cytotoxicity. Furthermore, the antibodies of the present invention can be chemically modified (e.g., one or more chemical moieties can be linked to the antibody), or modified to alter its glycosylation, again altering one or more functional properties of the antibody.

[0097] In one implementation, for C H1 The hinge region is modified to alter the number of cysteine ​​residues in the hinge region, for example, by increasing or decreasing them. This method is described in U.S. Patent No. 5,677,425. Changing C... H1 The number of cysteine ​​residues in the hinge region can, for example, promote the assembly of light and heavy chains or increase or decrease the stability of the antibody.

[0098] In another implementation, the Fc hinge region of the antibody is mutated to reduce the antibody's biological half-life. More specifically, one or more amino acid mutations are introduced into the C10 region of the Fc-hinge segment. H2 -C H3 The domain interface region allows the antibody to bind impaired to staphylococcal protein A (SpA) relative to the natural Fc-hinge domain SpA binding. This method is further described in detail in U.S. Patent No. 6,165,745.

[0099] In another embodiment, the antibody glycosylation is modified. For example, a glycosylated antibody (i.e., an antibody lacking glycosylation) can be prepared. Glycosylation can be altered to, for example, increase the antibody's affinity for the antigen. Such carbohydrate modification can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be performed, resulting in the elimination of one or more variable region framework glycosylation sites, thereby deglycosylating that site. Such deglycosylation can increase the antibody's affinity for the antigen. See, for example, U.S. Patent Nos. 5,714,350 and 6,350,861.

[0100] Alternatively or concurrently, antibodies with altered glycosylation patterns can be prepared, such as low-fucosylated antibodies with reduced fucosylation residues or antibodies with increased bipartite GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC capacity of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing antibodies in host cells with an altered glycosylation mechanism. Cells with altered glycosylation mechanisms have been described in the art and can be used as host cells in which the recombinant antibodies of the present invention are expressed to produce antibodies with altered glycosylation. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosylation gene FUT8 (α(1,6)-fucosylation), resulting in antibodies expressed in Ms704, Ms705, and Ms709 cell lines lacking fucosylation on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines were created by targeted disruption of the FUT8 gene in CHO / DG44 cells using two alternative vectors (see U.S. Patent Publication No. 20040110704 and amane-Ohnuki et al., (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 describes a cell line with a functionally disrupted FUT8 gene encoding fucosyltransferase, resulting in antibodies expressed in such cell lines exhibiting hypofucosylation by reducing or eliminating α-1,6 bond-associated enzymes. EP 1,176,195 also describes cell lines with low enzyme activity to add fucose to N-acetylglucosamine binding to the antibody Fc region or without enzyme activity, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). PCT publication WO 03 / 035835 describes a variant CHO cell line, Led 3, which exhibits reduced ability to link fucose to Asn(297)-linked carbohydrates, resulting in hypofucosylation of antibodies expressed in this host cell (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). Antibodies with modified glycosylation signatures can also be generated in eggs, as described in PCT publication WO 06 / 089231. Alternatively, antibodies with modified glycosylation signatures can be generated in plant cells, such as duckweed. A method for generating antibodies in plant systems is disclosed in U.S. Patent Application No. 040989 / 314911, filed August 11, 2006, corresponding to Alston & Bird LLP Attorney File No. 040989 / 314911.PCT Publication WO 99 / 54342 describes engineered cell lines to express glycoprotein-modified glycosyltransferases (e.g., (l,4)-N-acetylglucosamine aminotransferase III (GnTIII)), resulting in antibodies expressed in engineered cell lines exhibiting an increased bipartite GlcNac structure, thereby leading to increased ADCC activity of the antibodies (see also Umana et al., (1999) Nat. Biotech. 17:176-180). Alternatively, fucosidases can be used to cleave the fucosylate residues of the antibody. For example, fucosidase α-L-fucosidase removes fucosylate residues from antibodies (Tarentino et al., (1975) Biochem. 14:5516-23).

[0101] Another modification of the antibodies contemplated herein is PEGylation. Antibodies can be PEGylated to, for example, increase the antibody's biological (e.g., serum) half-life. To PEGylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG) (e.g., a reactive ester or aldehyde derivative of PEG) under conditions where one or more PEG groups are attached to the antibody or antibody fragment. Preferably, PEGylation is carried out by an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to cover any form of PEG that has been used to derivatize other proteins, such as mono(C1-C2) PEG. 10 Alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In some embodiments, the antibody to be PEGylated is a glycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of this invention. See, for example, EPO 154 316 and EP0 401 384.

[0102] Physical properties of antibodies

[0103] The antibodies of the present invention can be characterized by their various physical properties in order to detect and / or distinguish their different categories.

[0104] For example, antibodies may contain one or more glycosylation sites in the variable region of the light or heavy chain. Such glycosylation sites can lead to increased immunogenicity or altered pK of the antibody due to altered antigen binding (Marshall et al. (1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J Immunol 172:5489-94; Wallick et al. (1988) J Exp Med 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al. (1985) Nature 316:452-7; Mimura et al., (2000) Mol Immunol 37:697-706). Glycosylation is known to occur on motifs containing NXS / T sequences. In some cases, anti-PD-1 antibodies that do not contain glycosylation in the variable region are preferred. This can be achieved by selecting antibodies that do not contain glycosylation motifs in the variable region or by mutating residues in the glycosylation region.

[0105] In a preferred embodiment, the antibody does not contain an asparagine isomer site. Deamidation of asparagine may occur at the NG or DG sequence, resulting in the formation of isoaspartic residues that will be linked into the polypeptide chain and reduce its stability (isoaspartic effect).

[0106] Each antibody will have a unique isoelectric point (pI), which typically falls within the pH range of 6 to 9.5. The pI of IgG1 antibodies typically falls within the pH range of 7–9.5, while the pI of IgG4 antibodies typically falls within the pH range of 6–8. It has been speculated that antibodies with pI values ​​outside the normal range may exhibit certain unfolding and instability under in vivo conditions. Therefore, anti-PD-1 antibodies with pI values ​​falling within the normal range are preferred. This can be achieved by selecting antibodies with pI values ​​within the normal range or by mutating charged surface residues.

[0107] Nucleic acid molecules encoding the antibodies of this invention

[0108] In another aspect, the present invention provides nucleic acid molecules encoding the heavy chain and / or light chain variable regions, or CDRs, of the antibodies of the present invention. The nucleic acids may be present in intact cells, cell lysates, or in partially purified or substantially purified forms. When purified from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) using standard techniques, the nucleic acids are "isolated" or "substantially purified." The nucleic acids of the present invention may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0109] The nucleic acids of this invention can be obtained using standard molecular biology techniques. For antibodies expressed in hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as further described below), cDNA encoding the light and heavy chains of the antibodies prepared from the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acids encoding such antibodies can be recovered from the gene library.

[0110] Preferred nucleic acid molecules of this invention include V encoding a PD-1 monoclonal antibody. H and V L Nucleic acid molecules with sequences or CDRs. Once the encoding V is obtained... H and V L These DNA fragments can be further manipulated using standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, the gene encoding V... L or V H The DNA fragment is operatively linked to another DNA fragment encoding a different protein (such as an antibody constant region or a flexible linker). The term "operatively linked" as used herein is intended to mean that two DNA fragments are linked such that the amino acid sequences encoded by the two DNA fragments remain within the frame.

[0111] By encoding V H DNA and coding heavy chain constant regions (C H1 C H2 and C H3 Another DNA molecule can be operatively linked to, which can encode V HThe isolated DNA from the region is converted into a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art, and DNA fragments covering these regions can be obtained by standard PCR amplification. Heavy chain constant regions can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions, but IgG1 and IgG4 constant regions are most preferred. For Fab fragment heavy chain genes, the DNA encoding V... H The DNA can be operatively linked to only the heavy chain C. H1 Another DNA molecule in the constant region.

[0112] By encoding V L DNA and encoding the light chain constant region C L Another DNA molecule can be operatively linked to encode V. L The isolated DNA from the regions is converted into full-length light chain genes (and Fab light chain genes). The sequences of human light chain constant region genes are known in the art, and DNA fragments covering these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region may be a κ or λ constant region.

[0113] To generate the scFv gene, the gene encoding V will be... H and V L The DNA fragment can be operatively linked to another fragment encoding a flexible linker (e.g., encoding the amino acid sequence (Gly4-Ser)3), enabling V H and V L The sequence can be expressed as a continuous single-chain protein, where V L and V H The zones are connected by flexible joints (see, for example, Bird et al., (1988) Science 242:423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0114] Production of monoclonal antibodies of the present invention

[0115] The monoclonal antibodies (mAbs) of the present invention can be generated using well-known somatic cell hybridization (hybridoma) techniques as described in Kohler and Milstein (1975) Nature 256: 495. Other embodiments for generating monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display techniques. Chimeric or humanized antibodies are also well-known in the art. See, for example, U.S. Patent Nos. 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370, the contents of which are incorporated herein by reference in their entirety.

[0116] Generation of transfected tumors that produce the monoclonal antibodies of the present invention

[0117] The antibodies of the present invention can also be generated in host cells transfected with tumors using combinations of recombinant DNA techniques and gene transfection methods well known in the art (e.g., Morrison, S. (1985) Science 229: 1202). In one embodiment, DNA encoding a partial or full-length light and heavy chain, obtained by standard molecular biology techniques, is inserted into one or more expression vectors, thereby operatively linking the gene to transcriptional and translational regulatory sequences. Herein, the term “operatively linked” refers to linking an antibody gene to a vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene.

[0118] The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody genes. Such regulatory sequences are described, for example, in Goeddel (GeneExpression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenoviruses, such as the adenovirus major late promoter (AdMLP), and polyomaviruses. Alternatively, non-viral regulatory sequences, such as ubiquitin promoters or β-globulin promoters, may be used. Furthermore, regulatory elements consist of sequences from diverse sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and long terminal repeat sequences from human T-cell leukemia virus type 1 (Takebe et al., (1988) Mol. Cell. Biol. 8:466-472). Expression vectors and expression control sequences are selected to ensure compatibility with the host cells used for expression.

[0119] Antibody light chain genes and antibody heavy chain genes can be inserted into the same or separate expression vectors. In a preferred embodiment, the variable region is used to create full-length antibody genes for any antibody isotype, wherein they are inserted into expression vectors that already encode the heavy chain constant region and light chain constant region of the desired isotype, such that V H The segment is operatively connected to C within the carrier. H Section, and V L The segment is operatively connected to C within the carrier. L Segment. Alternatively or concurrently, the recombinant expression vector can encode a signal peptide that promotes the secretion of antibody chains from host cells. The antibody chain gene can be cloned into the vector such that the signal peptide is linked to the N-terminus of the antibody chain gene within the reading frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin).

[0120] In addition to the antibody chain gene and regulatory sequence, the recombinant expression vector of the present invention may carry other sequences, such as sequences regulating vector replication in host cells (e.g., origin of replication) and selectable marker genes. Selectable marker genes facilitate the selection of host cells into which the vector has been introduced (see, for example, U.S. Patent Nos. 4,399,216; 4,634,665 and 5,179,017). For example, typically, selectable marker genes confer resistance to drugs (e.g., G418, hygromycin, or methotrexate) in host cells into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for DHFR host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0121] To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells using standard techniques. Various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-glucan transfection, etc. Although it is theoretically possible to express the antibodies of the present invention in prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells (most preferably mammalian host cells) is most preferred because such eukaryotic cells (especially mammalian cells) are more likely than prokaryotic cells to assemble and secrete appropriately folded and immunologically active antibodies.

[0122] Preferred mammalian host cells for expressing the recombinant antibodies of the present invention include: Chinese hamster ovary (CHO) cells (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used in conjunction with, for example, the DHFR optional marker described in RJ Kaufman and PA Sharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. Particularly for use with NSO myeloma cells, another preferred expression system is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841. When the recombinant expression vector encoding the antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period sufficient to allow expression of the antibody in the host cells or, more preferably, to allow secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0123] Immunoconjugates

[0124] The antibodies of the present invention can be conjugated with therapeutic agents to form immune conjugates, such as antibody-drug conjugates (ADCs). Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binding agents, DNA intercalating agents, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and anti-mitotic agents. In ADCs, the antibody and therapeutic agent are preferably conjugated via a cleavable linker (e.g., a peptide linker, disulfide bond, or hydrazone linker). More preferably, the linker is a peptide linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. ADCs can be prepared as described in the following documents: U.S. Patent Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publications WO 02 / 096910; WO 07 / 038,658; WO 07 / 051,081; WO 07 / 059,404; WO 08 / 083,312; and WO 08 / 103,693; U.S. Patent Publications 20060024317; 20060004081; and 20060247295, the disclosures of which are incorporated herein by reference.

[0125] Bispecific molecules

[0126] On the other hand, this disclosure is characterized by bispecific molecules comprising one or more antibodies of the present invention linked to at least one other functional molecule (e.g., another peptide or protein (e.g., a ligand of another antibody or receptor)) to produce a bispecific molecule that binds to at least two different binding sites or target molecules. Therefore, as used herein, a “bispecific molecule” includes molecules having three or more specificities.

[0127] In one implementation, in addition to anti-Fc binding specificity and anti-PD-1 binding specificity, the bispecific molecule also possesses a third specificity. This third specificity can target anti-enhancing factors (EFs), such as molecules that bind to surface proteins involved in cytotoxic activity to increase the immune response against target cells. For example, anti-enhancing factors can bind to cytotoxic T cells (e.g., via CD2, CD3, CD8, CD28, CD4, PD-1, or ICAM-1) or other immune cells, leading to an increased immune response against target cells.

[0128] Bispecific molecules can take many different forms and sizes. At one end of the size spectrum, bispecific molecules retain the traditional antibody form, except that instead of having two binding arms with the same specificity, they have two binding arms with different specificities. At the other extreme are bispecific molecules composed of two single-chain antibody fragments (scFv) linked by peptide chains, the so-called Bs(scFv)2 construct. Medium-sized bispecific molecules consist of two different F(ab) fragments linked by peptide linkers. These and other forms of bispecific molecules can be prepared by genetic engineering, somatic cell hybridization, or chemical methods. See, for example, Kufer et al., cited above; Cao and Suresh, Bioconjugate Chemistry, 9 (6), 635-644 (1998); and van Spriel et al., Immunology Today, 21 (8), 391-397 (2000), and the references cited therein.

[0129] Oncolytic viruses that encode antibodies or carry antibodies

[0130] Oncolytic viruses preferentially infect and kill cancer cells. The antibodies of the present invention can be used in combination with oncolytic viruses. Alternatively, oncolytic viruses encoding the antibodies of the present invention can be introduced into the human body.

[0131] Pharmaceutical Composition

[0132] In another aspect, this disclosure provides pharmaceutical compositions comprising one or more antibodies of the present invention formulated together with a pharmaceutically acceptable carrier. When the composition comprises more than one antibody, the antibodies can be administered dispersively. The composition may optionally comprise one or more additional pharmaceutically active ingredients, such as another antibody or drug, such as an antitumor drug.

[0133] Pharmaceutical compositions may contain any number of excipients. Excipients that may be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersants or suspending agents, solubilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonants, and combinations thereof. The selection and use of suitable excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0134] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient may be coated in a material to protect it from acids and other natural conditions that may inactivate it. As used herein, the phrase "parenteral administration" refers to administration methods that are typically administered by injection rather than intravenous and local administration, and includes, but is not limited to, intravenous, intramuscular, intraarticular, intrathecal, intracapsular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions. Alternatively, the antibodies of the present invention may be administered via non-parenteral routes, such as local, epidermal, or mucosal administration routes, such as intranasal, oral, vaginal, rectal, sublingual, or local administration.

[0135] Pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated into microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.

[0136] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the target of treatment and the specific route of administration, and is generally the amount of the composition that produces a therapeutic effect. Typically, in one hundred percent, this amount is from about 0.01% to about 99 percent, preferably from about 0.1% to about 70%, and most preferably from about 1% to about 30% of the active ingredient, in combination with a pharmaceutically acceptable carrier.

[0137] Dosing regimens can be adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus injection may be administered, several separate doses may be administered over time, or the dose may be proportionally reduced or increased depending on the urgency of the treatment situation. Formulating parenteral compositions in unit dosage form is particularly advantageous for ease of administration and uniform dosing. As used herein, unit dosage form refers to a physically discrete unit suitable as a single dose to the subject; each unit contains a predetermined amount of the active ingredient, calculated to produce the desired therapeutic effect when used with the desired drug carrier. Alternatively, antibodies can be administered as a sustained-release formulation, in which case less frequent administration is required.

[0138] For antibody administration, the dose can be from about 0.0001 to 100 mg / kg of host body weight, more typically from 0.01 to 5 mg / kg. For example, the dose can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, or 10 mg / kg body weight, or in the range of 1-10 mg / kg. Exemplary treatment regimens require administration once weekly, once every two weeks, once every three weeks, once every four weeks, once monthly, once every three months, or once every three to six months. Preferred dosing regimens for the anti-PD-1 antibody of the present invention include administration of the antibody by intravenous administration of 1 mg / kg body weight or 3 mg / kg body weight using one of the following dosing regimens: (i) six doses once every four weeks, then once every three months; (ii) once every three weeks; (iii) once 3 mg / kg body weight, then 1 mg / kg body weight every three weeks. In some methods, the dose is adjusted to achieve a plasma antibody concentration of about 1-1000 μg / ml (about 25-300 μg / ml in some methods).

[0139] The "therapeuticly effective dose" of the anti-PD-1 antibody of the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of disease, or prevention of impairment or disability caused by disease distress. For example, for treating a subject carrying a tumor, the "therapeuticly effective dose" preferably inhibits tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and more preferably at least about 80% relative to an untreated subject. The therapeutically effective dose of the antibody can reduce the size of the tumor or improve the symptoms of a subject, typically a human or possibly another mammal.

[0140] The pharmaceutical composition may be a controlled-release formulation, including implants, transdermal patches, and microcapsule delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, acid anhydrides, polyethylene glycol, collagen, polyorthoesters, and polylactic acid. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0141] The therapeutic composition can be administered via, for example, medical devices such as: (1) needle-free subcutaneous injection devices (e.g., U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) microinfusion pumps (U.S. Patent No. 4,487,603); (3) percutaneous devices (U.S. Patent No. 4,486,194); (4) infusion devices (U.S. Patent Nos. 4,447,233 and 4,447,224); and (5) permeation devices (U.S. Patent Nos. 4,439,196 and 4,475,196); the disclosures of which are incorporated herein by reference.

[0142] In some embodiments, the monoclonal antibodies of the present invention can be formulated to ensure proper distribution in vivo. For example, to ensure that the therapeutic antibodies of the present invention cross the blood-brain barrier, they can be formulated as liposomes, which may additionally contain a targeting portion to enhance selective transport to specific cells or organs. See, for example, U.S. Patent Nos. 4,522,811; 5,374,548; 5,416,016; and 5,399,331; VV Ranade (1989) J. Clin. Pharmacol. 29:685; Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038; Bloeman et al., (1995) FEBS Lett. 357:140; M. Owais et al., (1995) Antimicrob. AgentsChemother. 39:180; Briscoe et al., (1995) Am. J. Physiol. 1233:134; Schreier et al., (1994) J Biol. Chem. 269:9090; Keinanen and Laukkanen (1994) FEBSLett. 346: 123; and Killion and Fidler (1994) Immunomethods 4:273.

[0143] Uses and methods of the present invention

[0144] The antibodies of the present invention (compositions, bispecific antibodies, immunoconjugates, and other forms, including oncolytic viruses) have a variety of in vitro and in vivo efficacies, including, for example, enhancing immune responses by blocking PD-1. The antibodies can be administered in vitro or ex vivo to cells in cultures, or, for example, in vivo to human subjects, to enhance immunity in various situations. Therefore, in one aspect, the present invention provides a method for modifying an immune response in a subject, the method comprising administering the antibody of the present invention or its antigen-binding portion to the subject, thereby modifying the immune response in the subject. Preferably, the response is enhanced, stimulated, or upregulated.

[0145] Preferred subjects include human patients requiring enhanced immune responses. The method is particularly suitable for treating human patients with diseases that can be treated by enhancing immune responses, such as T-cell-mediated immune responses. In a particular embodiment, the method is particularly suitable for in vivo treatment of cancer. To achieve antigen-specific immune enhancement, anti-PD-1 antibodies can be administered co-administered with the antigen of interest, or the antigen may already be present in the subject to be treated (e.g., a subject with a tumor or a virus). When antibodies against PD-1 are administered with another agent, they can be administered sequentially or simultaneously.

[0146] In view of the ability of the anti-PD-1 antibody of the present invention to inhibit the binding of PD-1 to PD-L1 and / or PD-L2 molecules and stimulate antigen-specific T cell responses, the present invention also provides in vitro and in vivo methods for using antibodies to stimulate, enhance, or upregulate antigen-specific T cell responses. For example, the present invention provides a method for stimulating antigen-specific T cell responses, the method comprising contacting the T cells with the antibody of the present invention to stimulate an antigen-specific T cell response. Any suitable indicator of antigen-specific T cell responses can be used to measure antigen-specific T cell responses.

[0147] Non-limiting examples of such suitable indicators include increased T cell proliferation in the presence of an antibody and / or increased cytokine production in the presence of an antibody. In a preferred embodiment, antigen-specific T cells are stimulated to produce interleukin-2.

[0148] The present invention also provides a method for stimulating an immune response (e.g., an antigen-specific T-cell response) in a subject, comprising administering the antibody of the present invention to the subject, thereby stimulating an immune response (e.g., an antigen-specific T-cell response) in the subject. In a preferred embodiment, the subject is a tumor-carrying subject, and an immune response against the tumor is stimulated. In another preferred embodiment, the subject is a virus-carrying subject, and an immune response against the virus is stimulated.

[0149] In another embodiment, the present invention provides a method for inhibiting tumor cell growth in a subject, comprising administering an antibody of the present invention to the subject thereby inhibiting tumor growth in the subject. In yet another embodiment, the present invention provides a method for treating a viral infection in a subject, comprising administering an antibody of the present invention to the subject thereby treating the viral infection in the subject.

[0150] These and other methods of the present invention will be discussed in further detail below.

[0151] cancer

[0152] Blocking PD-1 with antibodies can enhance the immune response against a patient's cancer cells. In one aspect, this invention relates to treating a subject in vivo with an anti-PD-1 antibody to inhibit the growth of cancerous tumors. The anti-PD-1 antibody can be used alone to inhibit the growth of cancerous tumors. Alternatively, the anti-PD-1 antibody can be used in combination with other immunogenic agents (e.g., oncolytic viruses) or other antibodies used in cancer treatment, as described below.

[0153] Therefore, in one embodiment, the present invention provides a method for inhibiting tumor cell growth in a subject, comprising administering to the subject a therapeutically effective amount of an anti-PD-1 antibody or its antigen-binding portion. Preferably, the antibody is a mouse, chimeric, or humanized anti-PD-1 antibody.

[0154] Preferred cancers for which the growth of the antibodies of the present invention can be inhibited include cancers that are generally responsive to immunotherapy. Non-limiting examples of preferred cancers for treatment include: melanoma (e.g., metastatic malignant melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), breast cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer), whether primary or metastatic. Additionally, the present invention includes refractory or recurrent malignancies for which the growth of the antibodies of the present invention can be inhibited.

[0155] Examples of other cancers that can be treated using the methods of this invention include: bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancers, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia), Chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including asbestos-induced cancers), and combinations of the above cancers. This invention can also be used to treat metastatic cancers, particularly PD-1-expressing metastatic cancers (Iwai et al. (2005) Int. Immunol. 17: 133-144).

[0156] Optionally, antibodies against PD-1 can be combined with immunogenic agents, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), and cells transfected with genes encoding immunostimulatory cytokines (He et al (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as gp100, MAGE antigen, Trp-2, MART1, and / or peptides of tyrosinases, or tumor cells transfected to express the cytokine GM-CSF.

[0157] PD-1 blockade may be more effective when combined with vaccination regimens. Numerous experimental strategies for cancer vaccination have been devised (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition). In one of these strategies, the vaccine is prepared using autologous or allogeneic tumor cells. These cell vaccines have proven to be most effective when tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be an effective antigen-presenting activator for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3539-43).

[0158] Studies of gene expression and large-scale gene expression patterns in various tumors have led to the definition of so-called tumor-specific antigens (Rosenberg, SA (1999) Immunity 10: 281-7). In many cases, these tumor-specific antigens are differentiation antigens expressed in the tumor and the cells from which the tumor originates, such as melanocyte antigen gp100, MAGE antigen, and Trp-2. More importantly, many of these antigens can be demonstrated to be targets of tumor-specific T cells found in the host. PD-1 blockade can be used in combination with a collection of recombinant proteins and / or peptides expressed in the tumor to generate an immune response against these proteins. These proteins are generally recognized as self-antigens by the immune system and are therefore tolerated. Tumor antigens can include the protein telomerase, which is essential for the synthesis of chromosome telomeres and is expressed in more than 85% of human cancers and only in a limited number of somatic tissues (Kim et al. (1994) Science 266: 2011-2013). These somatic tissues can be protected from immune attack in various ways. Tumor antigens can also be "neo-antigens" expressed in cancer cells, because somatic mutations can alter protein sequences or form somatic fusion proteins between two unrelated sequences (i.e., bcr-abl in the Philadelphia chromosome), or they can be idiotypes of B-cell tumors.

[0159] Other tumor vaccines may include proteins derived from viruses associated with human cancer, such as human papillomavirus (HPV), hepatitis viruses (HBV and HCV), and Kaposi's herpes sarcoma virus (KHSV). Another form of tumor-specific antigen that can be used in conjunction with PD-1 blockers is purified heat shock proteins (HSPs) isolated from tumor tissue itself. These heat shock proteins contain protein fragments derived from tumor cells, and these HSPs are highly efficient at delivery to antigen-presenting cells to induce tumor immunity (Suot & Srivastava (1995) Science 269:1585-1588; Tamura et al. (1997) Science 278: 117-120).

[0160] Dendritic cells (DCs) are effective antigen-presenting cells that can be used to elicit antigen-specific responses. DCs can be generated in vitro and loaded with various protein and peptide antigens, as well as tumor cell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs can also be genetically transduced to express these tumor antigens. For immunization purposes, DCs have also been directly fused to tumor cells (Kugler et al. (2000) Nature Medicine 6:332-336). As a method of vaccination, DC immunization can effectively combine with PD-1 blockade to activate a more effective anti-tumor response.

[0161] PD-1 blockers can also be combined with standard cancer treatments. PD-1 blockers can be effectively combined with chemotherapy regimens. In these cases, it is possible to reduce the dose of the administered chemotherapy agent (Mokyr et al. (1998) CancerResearch 58: 5301-5304). An example of such a combination is the use of anti-PD-1 antibodies with decarbazine for the treatment of melanoma. Another example is the use of anti-PD-1 antibodies with interleukin-2 (IL-2) for the treatment of melanoma. The scientific basis for the combined use of PD-1 blockers and chemotherapy is that cell death, a consequence of the cytotoxic effects of most chemotherapy compounds, should lead to elevated levels of tumor antigens in the antigen presentation pathway. Other combination therapies that may synergize with PD-1 blockers through cell death are radiation, surgery, and hormone deprivation. Each of these regimens generates a source of tumor antigens in the host. Angiogenesis inhibitors can also be combined with PD-1 blockers. Inhibition of angiogenesis leads to tumor cell death, which may feed tumor antigens into the host antigen presentation pathway.

[0162] PD-1 blocking antibodies can also be used in combination with bispecific antibodies that target tumor cells with effector cells expressing Fcα or Fcγ receptors (see, for example, U.S. Patent Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. For example, anti-Fc receptor / anti-tumor antigen (e.g., Her-2 / neu) bispecific antibodies have been used to target macrophages to tumor sites. This targeting can more effectively activate tumor-specific responses. The T-cell arms of these responses will be enhanced by using PD-1 blockade. Alternatively, antigens can be delivered directly to dendritic cells (DCs) using bispecific antibodies that bind to tumor antigens and dendritic cell-specific cell surface markers.

[0163] Tumors evade host immune surveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating immunosuppressive proteins expressed by the tumor. These include TGF-β (Kehrl et al. (1986) J.Exp. Med. 163: 1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13:198-200), and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365). Antibodies against each of these entities can be used in combination with anti-PD-1 to counteract the effects of immunosuppressants and promote the host's tumor immune response.

[0164] Other antibodies that activate the host immune response can be used in combination with anti-PD-1. These include molecules on the surface of dendritic cells that activate DC function and antigen presentation. Anti-CD40 antibodies can effectively replace T cell helper activity (Ridge et al. (1998) Nature 393: 474-478) and can be used in combination with PD-1 antibodies (Ito et al. (2000) Immunobiology 201 (5) 527-40). Activating antibodies against T cell costimulatory molecules, such as CTLA-4 (e.g., US Patent No. 5,811,097), OX-40 (Weinberg et al. (2000) Immunol 164: 2160-2169), 4-1BB (Melero et al. (1997) Nature Medicine 3: 682-685 (1997)), and ICOS (Hutloff et al. (1999) Nature 397: 262-266), can also provide increased levels of T cell activation.

[0165] Several experimental treatment options exist involving the in vitro activation and expansion of antigen-specific T cells, and the adoptive transfer of these cells to recipients to stimulate antigen-specific T cells to fight tumors (Greenberg & Riddell (1999) Science 285: 546-51). These methods can also be used to activate T cell responses to infectious agents such as CMV. In vitro activation in the presence of anti-PD-1 antibodies can increase the frequency and activity of adoptively transferred T cells.

[0166] Infectious diseases

[0167] Other methods of the present invention are used to treat patients who have been exposed to specific toxins or pathogens. Therefore, another aspect of the present invention provides a method for treating an infectious disease in a subject, the method comprising administering an anti-PD-1 antibody or its antigen-binding portion to the subject, thereby treating the subject's infectious disease. Preferably, the antibody is a chimeric or humanized antibody.

[0168] Similar to its application in tumors as described above, antibody-mediated PD-1 blockade can be used alone or as adjuvants in combination with vaccines to stimulate immune responses against pathogens, toxins, and self-antigens. Examples of pathogens for which this treatment may be particularly useful include those for which there are currently no effective vaccines or for which conventional vaccines are not entirely effective. These include, but are not limited to, HIV, hepatitis (A, B, and C), influenza, herpes, Giardia, malaria, Leishmania, Staphylococcus aureus, and Pseudomonas aeruginosa. PD-1 blockade is particularly useful for established infections caused by antigens that are altered during infection by agents such as HIV. These novel epitopes are perceived as foreign upon administration of anti-human PD-1, thus evoking a strong T-cell response that is not attenuated by negative signaling through PD-1.

[0169] Examples of pathogenic viruses that cause infections treatable by the methods of the present invention include: HIV, hepatitis (A, B, or C), herpesviruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, and arbovirus encephalitis virus.

[0170] Examples of pathogenic bacteria that cause infections treatable by the methods of this invention include: Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus and Neisseria gonorrhoeae, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacilli, Cholera, Tetanus, Botulinum toxin, Anthrax, Plague, Leptospirosis and Lymes disease bacteria.

[0171] Some examples of pathogenic fungi that cause infections treatable by the methods of the present invention include: Candida (Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, etc.), Cryptococcus neoformans, Aspergillus (Aspergillus fumigatus, Aspergillus niger, etc.), Mucor (Mucor, Absidia, Rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0172] Some examples of pathogenic parasites that cause infections treatable by the methods of this invention include: Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nippostrongylus brasiliensis.

[0173] Of all the methods described above, PD-1 blockers can be used in combination with other forms of immunotherapy, such as cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy, which provides enhanced tumor antigen presentation (see, for example, Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2: 1121-1123).

[0174] Autoimmune response

[0175] Anti-PD-1 antibodies can stimulate and amplify autoimmune responses. In fact, antitumor responses induced by tumor cells and peptide vaccines have revealed that many antitumor responses involve anti-autoreactivity (van Elsas et al. (2001) J.Exp. Med. 194:481-489; Overwijk, et al. (1999) Proc. Natl. Acad. Sci. USA96: 2982-2987; Hurwitz, (2000) ibid.; Rosenberg & White (1996) J. ImmunotherEmphasis Tumor Immunol 19 (1): 81-4). Therefore, it is worth considering combining anti-PD-1 blockers with various autoproteins to design vaccination regimens that can effectively generate immune responses against these autoproteins for disease treatment.

[0176] Other endogenous proteins can also serve as targets, such as IgE for treating allergies and asthma, and TNFα for rheumatoid arthritis. Finally, antibody responses to various hormones can be induced by using anti-PD-1 antibodies. Neutralizing antibody responses to reproductive hormones can be used for contraception. Neutralizing antibody responses to hormones and other soluble factors required for the growth of specific tumors can also be considered potential targets for vaccination.

[0177] Similar approaches to using anti-PD-1 antibodies, as described above, can be used to induce therapeutic autoimmune responses in patients with inappropriate accumulations of other autoantigens, such as cytokines like TNFα and IgE.

[0178] Combination therapy

[0179] In another aspect, the present invention provides a method of combination therapy in which the anti-PD-1 antibody (or its antigen-binding portion) of the present invention is co-administered with one or more other antibodies that effectively stimulate immunity, thereby further enhancing, stimulating, or upregulating the immune response of the subject. In one embodiment, the present invention provides a method of stimulating an immune response in a subject, the method comprising administering an anti-PD-1 antibody and one or more additional immunostimulatory antibodies (e.g., anti-LAG-3 antibody, anti-PD-L1 antibody, and / or anti-CTLA-4 antibody) to the subject, thereby stimulating an immune response in the subject, such as inhibiting tumor growth or stimulating an antiviral response. In another embodiment, an anti-PD-1 antibody and an anti-LAG-3 antibody are administered to the subject. In another embodiment, an anti-PD-1 antibody and an anti-PD-L1 antibody are administered to the subject. In yet another embodiment, an anti-PD-1 antibody and an anti-CTLA-4 antibody are administered to the subject. In yet another embodiment, at least one additional immunostimulatory antibody (e.g., anti-PD-1, anti-PD-L1, and / or anti-CTLA-4 antibody) is a human antibody. Alternatively, at least one additional immunostimulatory antibody may be, for example, a chimeric or humanized antibody (e.g., prepared from mouse anti-LAG-3, anti-PD-L1 and / or anti-CTLA-4 antibodies).

[0180] In another embodiment, the present invention provides a method for treating a hyperproliferative disease (e.g., cancer) comprising administering a PD-1 antibody and a CTLA-4 antibody to a subject. In another embodiment, the anti-PD-1 antibody is administered at a subtherapeutic dose, the anti-CTLA-4 antibody is administered at a subtherapeutic dose, or both are administered at a subtherapeutic dose. In yet another embodiment, the present invention provides a method for altering adverse events associated with treatment of a hyperproliferative disease with an immunostimulant, comprising administering an anti-PD-1 antibody and a subtherapeutic dose of an anti-CTLA-4 antibody to a subject. In some embodiments, the subject is a human. In other embodiments, the anti-CTLA-4 antibody is a human sequence monoclonal antibody 10D1 (described in PCT Publication WO01 / 14424), and the anti-PD-1 antibody is a mouse sequence monoclonal antibody, such as the anti-PD-1 antibody C1H5 described herein. Other anti-CTLA-4 antibodies covered by the methods of this invention include, for example, those disclosed in the following publications: WO 98 / 42752; WO 00 / 37504; US Patent No. 6,207,156; Hurwitz et al. (1998) Proc. Natl. Acad. Sci. USA 95(17): 10067-10071; Camacho et al. (2004) J. Clin. Oncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) CancerRes. 58:5301-5304. In some embodiments, the anti-CTLA-4 antibody is in the form of 5 x 10 -8 M or smaller K D Combined with human CTLA-4, at 1 x 10 -8 M or smaller K D Combined with human CTLA-4, at 5 x 10 -9 M or smaller K D Combined with human CTLA-4, or in 1 x 10 -8 M to 1 x 10 -10 M or smaller K D Combined with human CTLA-4.

[0181] In another embodiment, the present invention provides a method for treating hyperplastic diseases (such as cancer) comprising administering an anti-PD-1 antibody and an anti-LAG-3 antibody to a subject.

[0182] In another embodiment, the present invention provides a method for treating hyperproliferative diseases (such as cancer) comprising administering an anti-PD-1 antibody and an anti-PD-L1 antibody to a subject.

[0183] Blocking PD-1 and one or more second target antigens (e.g., CTLA-4 and / or LAG-3 and / or PD-L1) with antibodies can enhance the immune response against a patient's cancer cells. Cancers in which the growth of the antibodies of this disclosure can be inhibited include those that are typically responsive to immunotherapy. Representative examples of cancers treated with combination therapies of this disclosure include those specifically listed above in the discussion of monotherapy with anti-PD-1 antibodies.

[0184] In some embodiments, the combination of therapeutic antibodies discussed herein may be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions with each antibody administered simultaneously in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic antibodies may be administered sequentially.

[0185] Furthermore, if more than one dose of combination therapy is administered sequentially, the order of administration at each administration point can be reversed or kept in the same order, and sequential administration can be combined with simultaneous administration, or any combination thereof.

[0186] Tumors evade host immune surveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating tumor-expressed, immunosuppressive proteins. These include TGF-β (Kehrl et al. (1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13: 198-200), and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365). In another example, antibodies against each of these entities can be further combined with anti-PD-1 and anti-CTLA-4 and / or anti-LAG-3 and / or anti-PD-L1 antibodies to counteract the immunosuppressive effects and promote the host's tumor immune response.

[0187] Other antibodies that can be used to activate the host immune response can be further combined with anti-PD-1 and anti-CTLA-4 and / or anti-LAG-3 and / or anti-PD-L1 antibodies. These include molecules on the surface of dendritic cells that activate DC function and antigen presentation. Anti-CD40 antibodies (Ridge et al., ibid.) can be combined with anti-PD-1 and anti-CTLA-4 and / or anti-LAG-3 and / or anti-PD-L1 (Ito et al., ibid.). Other activating antibodies targeting T cell co-stimulatory molecules (Weinberg et al., ibid.; Melero et al., ibid.; Hutloff et al., ibid.) can also provide increased levels of T cell activation.

[0188] As mentioned above, bone marrow transplantation is currently used to treat a variety of hematopoietic tumors. Combined PD-1 and CTLA-4 and / or LAG-3 and / or PD-L1 blockers can be used to increase the potency of tumor-specific T cells transplanted from donors.

[0189] Several experimental therapeutic protocols involve the in vitro activation and expansion of antigen-specific T cells, and the adoptive transfer of these cells to receptors for tumor-specific antigen-specific T cells (Greenberg & Riddell, ibid.). These methods can also be used to activate T cell responses to infectious agents such as CMV. It is expected that in vitro activation in the presence of anti-PD-1 and anti-CTLA-4 and / or anti-LAG-3 and / or anti-PD-L1 antibodies will increase the frequency and activity of adoptive T cell transfers.

[0190] In some embodiments, the present invention provides a method for altering adverse events associated with the use of immunostimulants to treat proliferative diseases (e.g., cancer), the method comprising administering an anti-PD-1 antibody and a subtherapeutic dose of anti-CTLA-4 and / or anti-LAG-3 and / or anti-PD-L1 antibodies to a subject. For example, the method of the present invention provides a way to reduce the incidence of colitis or diarrhea induced by immunostimulatory therapeutic antibodies by administering a non-absorbable steroid to a patient. Because any patient who will receive an immunostimulatory therapeutic antibody is at risk of developing colitis or diarrhea caused by such antibody, the entire patient population is suitable for treatment according to the method of the present invention. Although steroids have been used to treat inflammatory bowel disease (IBD) and prevent IBD exacerbations, they have not been used for the prevention of IBD (reducing the incidence of IBD) in patients not diagnosed with IBD. The significant side effects associated with steroids (even non-absorbable steroids) hinder prophylactic use.

[0191] In a further embodiment, the combination of PD-1 with CTLA-4 and / or LAG-3 and / or PD-L1 blockers (i.e., immunostimulatory therapeutic antibodies against PD-1 and against CTLA-4 and / or against LAG-3 and / or against PD-L1) can be further combined with the use of any non-absorbable steroid. As used herein, "non-absorbable steroid" is a glucocorticoid that exhibits extensive first-pass metabolism, resulting in low bioavailability of the steroid after metabolism in the liver, i.e., less than about 20%. In one embodiment of the invention, the non-absorbable steroid is budesonide. Budesonide is a locally acting glucocorticoid that, after oral administration, is primarily metabolized by the liver. ENTOCORT EC TM Astra-Zeneca is a pH- and time-dependent oral formulation of budesonide, designed to optimize drug delivery to the ileum and throughout the colon. ENTOCORT EC TM It is already approved in the United States for the treatment of mild to moderate Crohn's disease involving the ileum and / or ascending colon. ENTOCORT EC TM The standard oral dose for treating Crohn's disease is 6 to 9 mg / day. ENTOCORT EC TM It is released in the intestine, then absorbed and retained in the intestinal mucosa. Once it passes through the target tissues of the intestinal mucosa, ENTOCORT EC... TM It is extensively metabolized by the cytochrome P450 system in the liver into metabolites with negligible glucocorticoid activity. Therefore, its bioavailability is low (approximately 10%). Compared to other glucocorticoids, budesonide's low bioavailability leads to improved treatment rates and poor first-pass metabolism. Compared to systemic corticosteroids, budesonide produces fewer adverse effects, including less hypothalamic-pituitary suppression. However, long-term use of ENTOCORT EC... TM This can lead to systemic glucocorticoid effects, such as excessive cortisol and adrenal suppression. See PDR 58 th ed. 2004; 608-610.

[0192] In another embodiment, the combination of PD-1 with CTLA-4 and / or LAG-3 and / or PD-L1 blockers (i.e., immunostimulatory therapeutic antibodies against PD-1 and anti-CTLA-4 and / or anti-LAG-3 and / or anti-PD-L1 antibodies) bound to a non-absorbable steroid can be further combined with salicylates. Salicylates include 5-ASA agents, such as azulfidine. TM Pharmacia & UpJohn); olsalazine (DIPENTUM) TMPharmacia & UpJohn); Balsalazide (COLAZAL) TM Salix Pharmaceuticals, Inc.); and mesalamine (ASACOL) TM , Procter & Gamble Pharmaceuticals; PENTASA TM ShireUS; CANASA TM , Axcan Scandipharm, Inc.; ROWASA TM Solvay).

[0193] According to the method of the invention, the administration of salicylates in combination with anti-PD-1 and anti-CTLA-4 and / or anti-LAG-3 and / or anti-PD-L1 antibodies and non-absorbable steroids may include any overlapping or sequential administration of salicylates and non-absorbable steroids to reduce the incidence of colitis induced by immunostimulatory antibodies. Thus, for example, a method for reducing the incidence of colitis induced by immunostimulatory antibodies according to the invention includes simultaneous or sequential administration of salicylates and non-absorbable steroids (e.g., administration of salicylates 6 hours after non-absorbable steroids), or any combination thereof. Furthermore, according to the invention, salicylates and non-absorbable steroids may be administered via the same route (e.g., both orally) or via different routes (e.g., salicylates administered orally while non-absorbable steroids are administered rectally), which may differ from the route used for administering the combination of anti-PD-1 and anti-CTLA-4 and / or anti-LAG-3 and / or anti-PD-L1 antibodies.

[0194] The present disclosure is further illustrated by the following examples, which should not be construed as further limitation. All figures and references, Genbank sequences, patents, and published patent applications cited in this application are expressly incorporated herein by reference. Example

[0195] Example 1: Generating mouse anti-PD-1 monoclonal antibodies using hybrid tumor technology

[0196] Immunization

[0197] Mice were immunized according to the method described in E Harlow, D. Lane, Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998. Recombinant human PD-1 protein (Aero biosystems, #PD-l-H5257, containing an extracellular domain, AA Leu 25-Gln 167) with a human IgG1 Fc label at the C-terminus was used as the immunogen. Human PD-1-his protein (Sinobiological, #10377-H08H) was used to determine antiserum titers and screen hybridomas secreting antigen-specific antibodies.

[0198] Specifically, each animal was injected with 25 μg of human PD1 Fc protein in complete Freud adjuvant (Sigma, St. Louis, Mo., USA), followed by 2–3 booster immunizations (depending on antiserum titer) with 25 μg of human PD1 Fc protein in incomplete Freud adjuvant (Sigma, St. Louis, Mo., USA). Antiserum titers were measured using an ELISA-based screening method with recombinant human PD1-his protein. Briefly, diluted serum (60 μl) was added to each well and incubated at 37°C for 40 min. The plates were then washed four times and detected using HRP-goat anti-mouse-IgG (Jackson Immuno research, Cat# 115-036-071), with binding OD observed at 450 nm. A final booster immunization was administered to animals with good titers via intraperitoneal injection prior to hybridoma fusion.

[0199] Hybridoma fusion and screening

[0200] Prior to fusion, cells from the mouse myeloma cell line (SP2 / 0-Agl4, ATCC#CRL-l58l) were cultured to reach the logarithmic growth phase. Spleen cells from immunized mice were aseptically prepared and fused with myeloma cells according to the method described in Kohler G, and Milstein C, "Continuous cultures of fused cells secreting antibody of predefined specificity," Nature, 256: 495-497 (1975). The fused "hybrid cells" were then aliquoted into 96-well plates in DMEM / 20% FCS / HAT medium. Viable hybridoma colonies were observed under a microscope 7–10 days post-fusion. Two weeks later, the supernatant from each well was screened using recombinant human PD1-his protein via an ELISA-based assay. Briefly, ELISA plates were coated overnight at 4°C with 60 μl of human PD1-his (Sino biological, #10377-H08H, 2.0 μl / ml in PBS). The plate was washed four times with PBST and blocked with 200 μl of blocking buffer (5% non-fat emulsion in PBST). Diluted hybridoma supernatant (60 μl) was added to each well and incubated at 37°C for 40 min. The plate was then washed four times and detected using HRP-goat anti-mouse-IgG (Jackson Immuno research, Cat#l 15-036-071), with binding OD observed at 450 nm. Positive hybridomas secreting antibodies binding to human PD1-his were then selected and transferred to 24-well plates. Hybridoma clones producing antibodies with high specific binding and PD1 / PDL1 blocking activity were subcloned, and the antibodies produced by the subclones were purified by protein A affinity chromatography. Briefly, protein A agarose columns (from bestchrom (Shanghai) Biosciences, Cat#AA0273) were washed with PBS buffer at 5 to 10 column volumes. Pass the cell supernatant through the column, then wash the column with PBS buffer until the protein absorbance reaches baseline. Elute the column with elution buffer (0.1 M glycine-HCl, pH 2.7) and immediately collect in a 1.5 ml tube with neutralization buffer (1 M Tris-HCl, pH 9.0). Combine the fractions containing IgG and dialyze overnight in PBS at 4°C.

[0201] Example 2: Affinity of mouse anti-PD-1 monoclonal antibody using BIACORE surface plasmon resonance technology Measurement

[0202] The affinity and binding kinetics of the purified anti-PD-1 mouse monoclonal antibodies (mAbs) generated in Example 1 (i.e., clones C1E1, D1F2, C1F5, D1A1, D1F1, C1E2, C1A1, C1F4, D2C2, 2G2, and C1C5, which were later identified as IgG1 / κ isotypes) were characterized using a Biacore T200 system (GE healthcare, Pittsburgh, PA, USA).

[0203] In short, using a standard amine conjugation kit provided by Biacore (GE Healthcare, Pittsburgh, PA, USA), goat anti-mouse IgG (GE Healthcare, Cat#BR1 00839, Human Antibody Capture Kit) was covalently linked to a CM5 chip (a carboxymethyl dextran-coated chip) via primary amine. Unreacted portions of the biosensor surface were blocked with ethanolamine. Purified anti-PD-1 antibody and 66.7 nM Nivolumab (OPDIVO®) were then flowed onto the chip at a flow rate of 10 μL / min. Recombinant human PD-1-His (Sino biological, #10377-H08H) or cynomolgus monkey PD-1-his protein (Aero biosystems, #PD-l-C5223) in HBS EP buffer (provided by Biacore) were then flowed onto the chip at a flow rate of 30 μL / min. Antigen-antibody binding kinetics were monitored for 2 minutes, and dissociation kinetics for 10 minutes. Using BIA evaluation software, the binding and dissociation curves were fitted to the 1:1 Langmuir binding model.

[0204] Determine k a k d and K D The values ​​are shown in Table 2 below.

[0205] Table 2. Biacore kinetics of mouse anti-PD-1 monoclonal antibodies binding to human or cynomolgus monkey PD-1

[0206]

[0207] The antibody of this invention has a lower K value than nivolumab. D The specific binding to human PD-1 indicates a higher affinity for human PD-1.

[0208] Example 3 Binding activity of mouse anti-PD-1 monoclonal antibody

[0209] 96-well microplates were coated with 2 μg / ml goat anti-mouse IgG Fcγ fragment in PBS (Jackson Immuno Research, #115-006-071, 100 μg / well) and incubated overnight at 4°C. The plates were washed four times with wash buffer (PBS + 0.05% Tween-20, PBST) and then blocked for 2 hours at 37°C with 200 μl / well of blocking buffer (5% w / v non-fat emulsion in PBST). The plates were washed again and incubated with 100 μg / well of the purified anti-PD-1 antibody from Example 1 and Nivolumab (0.004–66.7 nM, serially diluted 5-fold in 2.5% non-fat emulsion in PBST) at 37°C for 40 minutes, followed by four more washes. Plates containing captured anti-PD-1 antibodies were incubated with biotin-labeled human PD-1 Fc protein (SEQ ID NO: 100, 60 nM in 2.5% nonfat emulsion in PBST, 100 μg / well) at 37°C for 40 min. After washing four times, the plates were incubated with streptavidin-conjugated HRP (1:10000 dilution in PBST, Jackson Immuno Research, #016-030-084, 100 μg / well) at 37°C for 40 min. After the final wash, the plates were incubated with 100 μg / well of ELISA substrate TMB (Innoreagents). The reaction was terminated over 15 min at 25°C with 50 μg / well of 1M H₂SO₄, and absorbance was read at 450 nm. Data were analyzed using Graphpad Prism software, and EC50 was reported. 50 value.

[0210] The results are summarized in Table 3 below.

[0211] Table 3. Binding activity of anti-PD-1 antibody to human PD-1

[0212]

[0213] The results showed that the antibody of the present invention specifically binds to human PD-1, and several clones have lower EC50 values ​​than nivolumab. 50 value.

[0214] Example 4: Functional blocking assay and reporting assay using ELISA

[0215] 4.1 Ligand Blocking ELISA

[0216] The ability of the anti-PD-1 antibody of the present invention to block PD-1-PD-L1 interaction was measured using a competitive ELISA assay. Briefly, human PD-L1-Fc protein (SEQ ID NO: 101) was coated onto 96-well microplates at 2 μg / mL PBS and incubated overnight at 4°C. The next day, the plates were washed with wash buffer (PBS + 0.05% Tween-20, PBST) and blocked with 5% non-fat emulsion in PBST at 37°C for 2 hours. The plates were then washed again with wash buffer.

[0217] The anti-PD-1 antibody of the present invention or a dilution of Nivolumab in biotin-labeled human PD-1-Fc (SEQ ID NO: 100, 10 nM in 2.5% non-fat emulsion in PBST) was prepared (starting from 100 nM, serially diluted four times), and incubated at room temperature for 40 min. Then, an antibody / PD-1-Fc-biotin mixture (100 μl / well) was added to the PD-L1-coated plate. After incubation at 37 °C for 40 min, the plate was washed four times with washing buffer. Then, 100 μl / well of streptavidin-conjugated HRP was added, and the plate was incubated at 37 °C for 40 min to detect biotin-labeled human PD-1 binding to PD-L1. The plate was washed again with washing buffer. Finally, TMB was added, and the reaction was terminated with 1 M H2SO4, and the absorbance was read at 450 nm. Data were analyzed using Graphpad Prism software, and IC50 was reported. 50 value.

[0218] 4.2 Benchmark Blocking ELISA

[0219] The binding capacity of the anti-PD-1 antibody blocking benchmark (Nivolumab) of this invention to human PD-1 was measured using a competitive ELISA assay. Briefly, Nivolumab was coated onto 96-well microplates at 2 μl / mL PBS solution and incubated overnight at 4°C. The next day, the plates were washed with wash buffer and blocked with 5% non-fat emulsion in PBST at 37°C for 2 hours. During blocking, biotin-labeled human PD-1 Fc (SEQ ID NO: 100, 10 nM in 2.5% non-fat emulsion in PBST) was mixed with each antibody tested (137 pM–100 nM, 3-fold serial dilutions) and incubated at 25°C for 40 minutes. After washing, the PD-1 / antibody mixture (100 μl / well) was added to the Nivolumab-coated plates and incubated at 37°C for 40 minutes. Wash the plate again with washing buffer, then add 100 μl of SA-HRP per well and incubate at 37°C for 40 minutes to detect the interaction with Opdivo. ®Biotin-labeled human PD-1 was incorporated. The plate was washed with washing buffer. TMB was added, and the reaction was terminated with 1 M H₂SO₄. The absorbance was read at 450 nm. The data were analyzed using Graphpad Prism software, and the IC₂ was reported. 50 value.

[0220] 4.3 Cell-based functional assays

[0221] The activity of antibody blocking cell membrane PD-1 / PD-L1 interaction was assessed using a cell-based reporter assay. The assay consisted of two genetically engineered cell lines: a PD-1 effector cell line stably expressing human PD-1 and an NFAT-responsive element (NFAT-RE)-driven luciferase reporter (Genscript, GS-J2 / PD-1), and a PD-L1 cell line stably expressing human PD-L1 and engineered cell surface protein-antigen peptide / major histocompatibility complex (MHC) (Genscript, GS-C2 / PD-L1, APC cells). When these two cell lines were co-cultured, the PD-1 / PD-L1 interaction inhibited T-cell receptor (TCR)-mediated luciferase expression in PD-1 effector cells (via the NFAT pathway).

[0222] Cell-based functional assays were performed as follows. In short, PD-L1 cells in the logarithmic growth phase were subjected to a assay at a concentration of 5 × 10⁶ cells / mL. 5 The cells were seeded at a density of 1 / ml into 384-well cell culture plates. The next day, a dilution of the anti-PD-1 antibody or Nivolumab of the present invention was prepared in assay buffer (RPMI 1640 + 1% FBS) (starting from 333.3 nM, serially diluted 5-fold). Simultaneously, the culture medium for PD-L1 cells in the 384-well plates was discarded, and then anti-PD-1 antibody (20 μl / well) and PD-1 effector cells (density 6.25 × 10⁶ cells / well) were added. 5 Diluent (20 μl / well) was added to a 384-well cell culture plate. After co-culturing at 37°C for 6 hours, the plate was removed from the incubator, and the luminescence intensity of each well was read using a One-Glo luciferase assay system (Promega, #E6l20) according to the manufacturer's instructions. The dose-response curve was analyzed using Graphpad Prism software, and EC50 was reported. 50 value.

[0223] The results of these three measurements are summarized in Table 4 below.

[0224] Table 4. Ability of anti-PD-1 antibodies to block PD-1 / PD-L1 interaction

[0225]

[0226] It can be seen that the antibodies of the present invention can block the interaction between human PD-1 and human PD-L1, and some of the clones of EC 50 or IC 50 The values ​​are lower than those for Nivolumab. The data also show that the antibodies of this invention can block the interaction between human PD-1 and Nivolumab, with antibodies from clone 2G2 exhibiting partial blocking, indicating that they bind to the same or similar epitopes as Nivolumab.

[0227] Example 5: Generation and Characterization of Chimeric Antibodies

[0228] The variable domains of the heavy and light chains of anti-PD1 mouse mAh C1E1 were cloned into the constant regions of the human IgG1 heavy chain and human κ light chain, respectively. The variable regions of the heavy chain and light chain have the amino acid sequences shown in SEQ ID NO: 67 and 80, respectively, while the amino acid sequences of the constant regions of the human IgG1 heavy chain and human κ light chain are listed in SEQ ID NO: 97 and 98, respectively. Following the protocols described in the foregoing examples, the activity of the resulting chimeric antibody was confirmed in binding capture ELISA, competitive ELISA, and cell-based functional reporter assays. Data showed that the chimeric C1E1 antibody had activity comparable to the benchmark (OPDIVO®), as shown in Table 5 below.

[0229] Table 5. Binding and functional activities of chimeric antibodies

[0230]

[0231] Example 6 Humanization of anti-PD-1 mouse monoclonal antibody C1E1

[0232] The mouse anti-PD1 antibody C1E1 was selected for humanization and further research. Humanization of the mouse antibody was performed using a well-established CDR transplantation method, as described below.

[0233] To select a receptor framework for the humanization of the mouse antibody C1E1, BLAST sequences of the light and heavy chain variable regions of mouse C1E1 were analyzed against the human immunoglobulin gene database. Human germline strains IGVH and IGVK, exhibiting the highest homology with mouse C1E1, were selected as receptor frameworks for humanization. The heavy / light chain variable regions (CDRs) of the mouse antibody were inserted into the selected frameworks, and residues within the frameworks were further mutated to obtain more candidate heavy / light chain variable regions.

[0234] Vectors containing nucleotide sequences encoding the humanized C1E1 heavy / light chain variable region and the human IgG1 heavy chain and human κ light chain constant regions were transiently transfected into 50 ml of 293F suspension cell culture at a 60% to 40% light chain to heavy chain construct ratio, along with 1.2 mg / ml PEI. After six days of incubation in shake flasks, the cell supernatant was collected, centrifuged to separate the cell pellet, and filtered through a 0.22 μm filter before IgG separation. The antibody was purified by protein A affinity chromatography. Briefly, the protein A agarose column (from bestchrom (Shanghai) Biosciences, Cat#AA0273) was washed with 5 to 10 column volumes of PBS buffer. The cell supernatant was then passed through the column, followed by washing with PBS buffer until the protein absorbance reached baseline. The column was eluted with elution buffer (0.1 M glycine-HCl, pH 2.7), and the fractions were immediately collected in 1.5 ml tubes with neutralization buffer (1 M Tris-HCl, pH 9.0). The fractions containing IgG were combined and dialyzed overnight in PBS at 4°C.

[0235] A total of 10 humanized antibodies were obtained, of which 8 were further characterized, namely huClE1-V1 to huClE1-V7 and huClE1-V10. Table 1 above summarizes the amino acid sequences of the heavy / light chain variable regions of the 8 antibodies, and the sequences of the human IgG1 heavy chain and human κ light chain constant regions are listed in SEQ ID NO: 97 and 98, respectively.

[0236] As described in Example 2, the binding affinity of huClE1-V1 to huClE1-V7 to human PD1 was evaluated using BIACORE technology, and the affinity K was... D The values ​​are summarized in Table 6 below. As described in Example 3, the binding activity of antibody huClE1-V10 to human PD1 was assessed by capture binding ELISA, which used goat anti-human IgG Fab capture antibody (Jackson Immuno Research, Cat # 109-005-097) to capture IgG, and the bound EC 50 The values ​​are summarized in Table 7. All eight humanized antibodies have affinity comparable to the chimeric antibody C1E1.

[0237] Table 6. Affinity of humanized C1E1 mAb

[0238]

[0239] Table 7. Binding activity of humanized C1E1 mAb

[0240]

[0241] Then, following the protocols of Examples 2 to 4, the affinity of the humanized antibody huClE1-V10 for human and cynomolgus monkey PD1 was tested by Biacore and by binding capture ELISA, and functional activity was tested by competitive ELISA and by cell-based reporter assay. As shown in Table 8, huClE1-V10 exhibited in vitro activity comparable to that of the chimeric C1E1 antibody.

[0242] Table 8. Binding and functional activity of humanized C1E1 mAh

[0243]

[0244] Example 7 Physicochemical properties of humanized C1E1 antibody

[0245] The physicochemical properties of the anti-PD1 humanized antibody huClE1-V10 were examined using protein thermal displacement assay, cIEF technique, SEC technique, and freeze-thaw method as described below.

[0246] Protein thermal displacement assay for Tm

[0247] The thermal stability of the anti-PD1 humanized antibody huClE1-V10 was measured using the GloMelt™ Thermal Shift Protein Stability Kit (Biotium, Cat#33022-T, lot#:181214). Briefly, the GloMelt™ dye was thawed and brought to room temperature. The vials containing the dye were vortexed and centrifuged. A 10x dye was prepared by adding 5 μL of 200x dye to 95 μL of PBS. 2 μL of 10x dye and 10 μL of antibody were added to a total reaction volume of 20 μL. A melting curve program with the detailed parameters in Table 9 was established. The tubes were briefly spun and then placed in a real-time PCR thermal cycler (Roche, LightCycler 480II). The results were analyzed using Microsoft Excel 2010 software.

[0248] Table 9. Parameters of the melting curve program

[0249]

[0250] cIEF technology determines pI

[0251] The pI and charge heterogeneity of the anti-PD1 humanized antibody huClE1-V10 were determined using capillary isoelectric focusing (cIEF). Briefly, 35 μL of 0.1% MC, 4 μL of pH3-10 Pharmlyte, 2 μL of 0.5 mol / L Arg, 1 μL of Mark-7.05, and 1 μL of Mark-9.99 were added to a tube containing 20 μg of antibody in PBS, followed by the addition of ddH2O to a final volume of 100 μL. The electrophoresis procedure consisted of two phases: initial separation at 1500 V for 1 minute, followed by a 6-minute separation at 3000 V. After a 5-minute exposure for detection, the data were analyzed using Maurice™ Compass™ software (Proteinsimple Inc., USA).

[0252] SEC technology determines clustering

[0253] The percentage of monomers was assessed using size exclusion chromatography (SEC) (Agilent Technologies, 1260 Infinity II). Antibodies were carried in an aqueous mobile phase and passed through a porous stationary phase resin packed in a column. Retention time in the column was a function of the hydrodynamic size of the protein and the pore size in the packed resin bed. Smaller molecules could permeate into the smaller pores of the resin and had longer retention times compared to larger molecules. After elution from the column, the protein was detected by UV absorption. The mobile phase was phosphate-buffered saline, and absorbance was monitored at 280 nm. The flow rate was 0.8 mL / min. The injection volume was 40 μL of 1 mg / mL sample. The column temperature was room temperature. The autosampler temperature was 2–8 °C. The total run time was 25 minutes.

[0254] Determining stability using freeze-thaw method

[0255] The 1 mg / ml antibody solution in 1xPBS formulation (containing 137 mmol / L NaCl, 2.7 mmol / L KCl, 10 mmol / L Na₂HPO₄·12H₂O, and 2 mmol / L KH₂PO₄) was frozen at -80°C for at least 24 hours, and then thawed at room temperature for 30–60 minutes. Five freeze-thaw cycles were repeated for each sample. After certain freeze-thaw cycles, such as the second, third, and fourth cycles, a portion of the solution was removed for SEC analysis before refrozen.

[0256] The instability leading to denaturation was assessed using the GloMelt™ Thermal Shift Protein Stability Kit. Figure 1As shown, the anti-PD1 humanized antibody huC1E1-V10 exhibits a high melting temperature (Tm) typically at 78 °C, and also shows a minor peak at 69.5 °C. cIEF analysis of huClE1-V10 indicated a major isotype percentage of approximately 81.5%, an acidic species percentage of approximately 11.0%, and a basic species percentage of approximately 7.5%. The pI of the huClE1-V10 antibody was calculated using Maurice™ Compass™ software, and the pI value was 8.36. The pI of the huClE1-V10 humanized antibody is significantly higher than 7, therefore a significant positive charge is expected at neutral pH. SEC testing of huClE1-V10 indicated a monomer percentage of 100.0% (see [link to SEC assay]). Figure 2 The stability of the antibody huClE1-V10 during freezing and thawing was assessed by size exclusion chromatography (SEC). No significant increase in aggregation levels was observed over time, and no precipitation or turbidity was observed after five repeated freeze-thaw cycles.

[0257] Example 8: In vivo antitumor efficacy of mouse C1E1 antibody

[0258] The in vivo antitumor activity of mouse C1E1 antibodies was evaluated in NCG mice. Briefly, on day 1, NCG mice were subcutaneously injected with 5 x 10 [units of antibody name missing]. 5 4x10 tumor-activated PBMCs 6 Individual melanoma A375 cells. Tumor volume was measured three times a week using electronic calipers and calculated as (length x width). 2 2. Twenty-four tumor-bearing mice were selected and randomly divided into four groups on day 14. The animals were administered a mediator (5% glucose solution), mouse C1E1, mouse C1E1 Fab, and nivolumab intraperitoneally at doses of 3 mg / kg on days 14, 21, and 28, respectively. Mouse antibody C1E1 contains a mouse heavy chain variable region and a human IgG1 constant region, as well as a mouse light chain variable region and a human Ck1 constant region, with sequences shown in SEQ ID NO: 67, 97, 80, and 98, respectively. Mouse C1E1 Fab contains a mouse heavy chain variable domain and a human IgG1 CH1 constant domain, as well as a light chain variable domain and a human Ck1 constant domain, with sequences shown in SEQ ID NO: 67, 99, 80, and 98, respectively.

[0259] Mice were euthanized on day 35, and tumors were collected and weighed. Tumor growth inhibition rate (%TGI = (1 - mean tumor volume in each treatment group / mean tumor volume in the control group) × 100%) was calculated.

[0260] All treatments were well tolerated in tumor-bearing animals. As shown in Table 10, in the human melanoma A375 xenograft model, treatment with 3 mg / kg of mouse C1E1 and 3 mg / kg of mouse C1E1 Fab were significantly more effective than 3 mg / kg of Nivolumab.

[0261] Table 10. Antitumor efficacy of mouse C1E1 antibody in human melanoma cells A375

[0262]

[0263] a The results were compared with those of the medium control group using the Student's t-test, and *P<0.05.

[0264] Example 9: In vivo antitumor efficacy of herpesvirus T3011 with inserted mouse C1E1 coding sequence

[0265] The antitumor efficacy of herpesvirus T3011 with the mouse C1E1 antibody coding sequence inserted was evaluated in a B16F10-hPD-L1 melanoma xenograft model.

[0266] Except for the insertion of the C1E1 Fab fragment sequence into the genomic region between UL3 and UL4, herpesvirus T3011 is a genetically modified type 1 herpes simplex virus (HSV-1) by removing the inverted repeat region and replacing it with the human IL-12 gene (see, for example, PCT / CN2016 / 080025). The C1E1 Fab sequence contains a mouse heavy chain variable domain and a human IgG1 CH1 constant domain, as well as a mouse light chain variable domain and a human Ckappal constant domain, which have the sequences shown in SEQ ID NO: 67, 99, 80, and 98, respectively.

[0267] In short, female B-hPD-1 humanized mice were subcutaneously injected with 1 x 10⁻⁶ mol / L on the right anterior side. 5 Bl6FlO-hPD-Ll melanoma cells. When the tumor volume reaches approximately 90 mm... 3 Mice were randomly divided into 5 groups of 8 each. These mice were administered a mediator (5% glucose solution) and T3011 (5 × 10⁻⁶) intratumorally, respectively. 6 PFU / mouse), T3011 (1×10) 7 PFU / mouse), T3011 (3×10) 7 PFU / mouse) and NV1020 (3×10) 7PFU / mouse, a recombinant oncolytic HSV-based virus developed by MediGene (formerly NeuroVir) for potential cancer therapy, see, for example, a phase I / II study of oncolytic herpes simplex virus NV1020 in patients with extensively pretreated refractory colorectal cancer that has metastasized to the liver. Hum Gene Ther. 2010 Sep;2l(9): 1119-28). Tumor volume was measured twice weekly.

[0268] Twelve days after drug administration, mice were euthanized and tumors were collected, weighed, and photographed. Relative tumor volume (RTV = TVn / TV0, where TVn refers to the tumor volume on day n and TV0 refers to the tumor volume on day 0), tumor growth inhibition rate (TGI% = 1 - (mean tumor volume of each treatment group / mean tumor volume of the control group) × 100%), and tumor weight inhibition rate (IR) were calculated. TW = (1 - average tumor weight in each treatment group / average tumor weight in the control group) × 100%).

[0269] As shown in Table 11 below, T3011 with the inserted mouse C1E1 antibody coding sequence provided significant antitumor activity in a dose-dependent manner. At 3 x 10⁻⁶... 7 At PFU / mouse dose levels, T3011 administration showed better antitumor efficacy than NV1020 administration.

[0270] Table 11. Antitumor efficacy of herpesvirus T3011 with inserted mouse ClEl coding sequence against B16F10-hPD-Ll melanoma xenograft model

[0271]

[0272] a When compared with the control group using Student's t-test, *P<0.05 and **P<0.01.

[0273] Example 12: In vivo antitumor efficacy of humanized C1E1 antibody

[0274] The effect of a humanized C1E1 antibody on tumor growth was evaluated in an MC38 xenograft model. Briefly, 5 x 10⁻⁶ C1E1 antibodies were subcutaneously injected into the right posterior side of female B-hPD-1 mice. 5 Individual cells. When the tumor volume reaches approximately 100-150 mm... 3Mice were randomly divided into 7 groups of 8 mice each. For three weeks, the mice were administered a carrier (PBS), huClE1-V10, and OPDIVO® intraperitoneally twice weekly at doses of 1 mg / kg, 3 mg / kg, or 10 mg / kg, respectively. The humanized antibody huC11-V10 contains variable and constant regions in the heavy chain and variable and constant regions in the light chain, with sequences shown in SEQ ID NOs: 69, 97, 86, and 98, respectively. Tumor volume was measured during the study.

[0275] Twenty-three days after the start of treatment, mice were euthanized and tumors were collected, weighed, and photographed. Tumor growth inhibition rate (TGI) was calculated. TV (and shown in Table 12.)

[0276] As shown in Table 12 below, tumor-bearing animals tolerated all treatments well. HuClEl-V10 treatment demonstrated dose-dependent antitumor activity in the MC38 xenograft model, comparable to OPDIVO®.

[0277] Table 12. Antitumor efficacy of huClEl-V10 in the MC38 xenograft model

[0278]

[0279] a When compared with the control group using Student's t-test, *P<0.05 and **P<0.01.

[0280] Although the invention has been described above in conjunction with one or more embodiments, it should be understood that the invention is not limited to those embodiments, and the description is intended to cover all possible alternatives, modifications, and equivalents, as may be included within the spirit and scope of the appended claims. All references cited herein are incorporated herein by reference in their entirety.

[0281] The sequences in this application are summarized below.

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[0293] 。

Claims

1. A genetically modified herpes simplex virus type 1 (HSV-1) comprising a polynucleotide encoding a Fab anti-PD-1 antibody, The Fab of the anti-PD-1 antibody comprises a mouse heavy chain variable domain, a human IgG1 CH1 constant domain, a mouse light chain variable domain, and a human κ light chain constant domain, which have sequences of SEQ ID NO: 67, 99, 80, and 98, respectively. The inverted repeat region was replaced by the human IL-12 gene; and The polynucleotide encoding Fab is inserted between UL3 and UL4.

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