Monoclonal antibody against program death-1 protein and its use in medicine

Abstract

The present disclosure relates to an antibody having the ability to bind to an immune checkpoint protein programmed death-1 (PD-1), such as human PD-1, or a nucleic acid encoding such an antibody, wherein the antibody comprises a modification in the Fc region that eliminates or reduces the Fc-mediated effector function of the antibody. The present disclosure also relates to a composition or kit comprising the antibody or nucleic acid, and the use of these antibodies or nucleic acids or compositions in the field of medicine, preferably in the field of immunotherapy, for example in the field of immunotherapy for the treatment of cancer. The present invention further relates to a method for inducing an immune response in a subject, comprising providing to the subject an antibody of the present disclosure or one or more nucleic acids encoding such an antibody, or a composition comprising said antibody or nucleic acid.

Description

[Technical Field]

[0001] The present invention relates to antibodies capable of binding, preferably specifically, to the immune checkpoint protein Programmed Death-1 (PD-1), e.g., human PD-1, or nucleic acids encoding such antibodies, wherein the antibodies comprise modifications in the Fc region that eliminate or reduce Fc-mediated effector functions of the antibody. The present invention also relates to compositions or kits comprising said antibodies or nucleic acids, and to the use of these antibodies or nucleic acids or compositions in the field of medicine, preferably in the field of immunotherapy for the treatment of cancer. The present invention further relates to a method for inducing an immune response in a subject, comprising providing to the subject an antibody capable of binding to the immune checkpoint protein PD-1, e.g., human PD-1, or a nucleic acid encoding such an antibody, or a composition comprising said antibody or nucleic acid. [Background technology]

[0002] Immunotherapy aims to enhance or induce specific immune responses in patients to control infectious or malignant diseases. The identification of an increasing number of pathogen-associated and tumor-associated antigens (TAAs) has broadened the pool of targets suitable for immunotherapy. Cells presenting immunogenic peptides (epitopes) derived from these antigens can be specifically targeted by either active or passive immunization strategies. Active immunization facilitates the induction and expansion of antigen-specific T cells in patients that can specifically recognize and kill diseased cells. In contrast, passive immunization can rely on the adoptive transfer of in vitro expanded and, where appropriate, genetically engineered T cells (adoptive T cell therapy).

[0003] In vertebrates, the evolution of the immune system has resulted in a highly effective network based on two types of defense: innate and adaptive immunity. In contrast to the evolutionarily ancient innate immune system, which relies on invariant receptors that recognize common molecular patterns associated with pathogens, adoptive immunity is based on highly specific antigen receptors and clonal selection on B cells (B lymphocytes) and T cells (T lymphocytes). The immune system plays a key role in the development, progression, and therapy of cancer. CD8 + T cells and NK cells can directly lyse tumor cells, and high tumor infiltration by these cells is generally considered to be advantageous for the outcome of various tumor diseases. + T cells contribute to antitumor immune responses by secreting IFNγ or licensing antigen-presenting dendritic cells (DCs), which then activate CD8 + Primes and activates T cells [Kreiter S. et al. Nature 520, 692-6 (2015)]. CD8 + The recognition and elimination of tumor cells by T cells depends on antigen presentation via major histocompatibility complex (MHC) class I. Antigen-specific T cell responses can be elicited by vaccination. Vaccination can be achieved by administering vaccine RNA, i.e., RNA encoding the antigen or epitope against which an immune response should be induced.

[0004] T cell activation may require not only stimulation via the antigen receptor (TCR) but also additional stimulation via conjugated stimulatory molecules (e.g., CD28). Cancer cells can evade and suppress immune responses by upregulating inhibitory immune checkpoint proteins, such as PD-1 and CTLA-4 on T cells, or PD-L1 on tumor cells, tumor stroma, or other cells within the tumor microenvironment. CTLA4 and PD-1 are known to transmit signals that suppress T cell activation. Blocking the activity of these proteins with monoclonal antibodies has restored T cell function, leading to innovative cancer therapies.

[0005] PD-1 (also known as CD279) is an immunoregulatory receptor expressed on the surface of activated T cells, B cells, and monocytes. The PD-1 protein has two naturally occurring ligands, known as PD-L1 (also known as CD274) and PD-L2 (also known as CD273). A wide variety of cancers, including melanoma, lung cancer, renal cancer, bladder cancer, esophageal cancer, gastric cancer, and other cancers, express PD-L1. Thus, in cancer, the PD-1 / PD-L1 system, through the interaction of PD-L1 with PD-1, can inhibit the proliferation, cytokine release, and cytotoxicity of T lymphocytes, thereby providing cancer cells with an opportunity to evade T cell-mediated immune responses.

[0006] Monoclonal antibodies suitable for regulating the activity of the PD-1 / PD-L1 axis are known. PD-1 / PD-L1 interaction can be inhibited by pembrolizumab (MK-3475, also known as lambrolizumab or Keytruda). Another monoclonal antibody suitable for this purpose is nivolumab (ONO-4538, also known as BMS-936558 or Opdivo).

[0007] Antibody-based cancer therapy has high specificity and a low side effect profile compared to conventional drugs, and therefore may be advantageous over conventional therapies. However, by activating the immune system, immune checkpoint inhibitors can cause autoimmune side effects in some patients. Other patients may fail to respond to treatment.

[0008] Furthermore, anti-PD-1 antibodies may alleviate autoimmune diseases without concomitant suppression of normal immunity. For example, anti-PD-1 binding fragments linked to immunotoxins can delay disease onset in autoimmune diabetes and ameliorate symptoms in a mouse model of autoimmune encephalomyelitis [Zhao P. et al. Nat Biomed Eng. 3(4): 292-305 (2019)].

[0009] It is known in the art that modification of antibody Fc region can result in Fc region that inhibits, for example, the interaction between Fc-gamma receptor and C1q.For example, in IgG1 isotype antibody, examples of amino acid positions that can be modified include L234, L235 and P331.These combinations, for example, L234F / L235E / P331S, can cause significant reduction in binding to human CD64, CD32, CD16 and C1q [Xu et al., 2000, Cell Immunol. 200(1):16-26; Oganesyan et al., 2008, Acta Cryst. (D64):700-4]. In addition, the amino acid substitutions L234F and L235E may result in an Fc region that inhibits interaction with Fc-gamma receptors and C1q [Canfield et al., 1991, J. Exp. Med. (173):1483-91; Duncan et al., 1988, Nature (332):738-40]. The amino acid substitution D265A may reduce binding to all Fcy receptors and prevent ADCC [Shields et al., 2001, J. Biol. Chem. (276):6591-604]. Binding to C1q can be inhibited by mutating positions D270, K322, P329, and P331. Mutation of these positions to either D270A or K322A, or P329A or P331A results in the loss of CDC activity of the antibody (Idusogie EE, et al., 2000, J Immunol. 164: 4178-84).

[0010] Thus, despite the impressive benefits associated with immune checkpoint inhibitor therapy, there remains an unmet need for the development of improved antibodies to target these checkpoints and provide additional benefits to immunotherapy, particularly cancer immunotherapy. Summary of the Invention

[0011] The present disclosure generally provides antibodies useful as therapeutic agents for treating and / or preventing diseases, such as cancer or infectious diseases, where the treatment aims to activate the immune system and / or induce an immune response.

[0012] The antibodies of the present disclosure exhibit binding properties to PD-1, preferably human PD-1, and the ability to block the PD-1 / PD-L1 interaction, thereby inducing an immune response.

[0013] The antibodies of the disclosure may have one or more of the following properties: they (i) bind, preferably specifically bind, to PD-1; (ii) may have binding properties for PD-1 on immune cells; (iii) may have binding properties for PD-1 epitopes; (iv) may have binding properties for non-human PD-1 variants, particularly PD-1 variants from mouse, rat, rabbit, and primates; (v) may prevent or reduce the induction of inhibitory signals by PD-1; (vi) may inhibit the interaction / binding of PD-1 with its ligand, preferably the ligand PD-L1, thereby blocking the inhibitory PD-1 / PD-L1 axis, for example, they may inhibit the binding of human PD-L1 to human PD-1; (vii) may inhibit the immunosuppressive signals of PD-L1 or PD-L2; and (viii) may enhance or initiate immune responses, preferably T cell-mediated, preferably CD8 + (ix) may enhance or initiate immune function by inducing cell proliferation; (x) may inhibit cancer growth; (x) may deplete tumor cells and / or suppress cancer metastasis; and / or (xi) may deplete immune cells and / or ameliorate autoimmune diseases. Thus, the antibodies of the present disclosure capable of binding to PD-1 may ameliorate autoimmune diseases by inhibiting the immunosuppressive signal of PD-1 and / or depleting immune cell activation.

[0014] The anti-PD-1 antibodies of the present disclosure reduce or eliminate Fc-mediated effector functions. Eliminating or reducing binding of the antibody's Fc region to Fc-gamma receptors may be desirable to avoid unwanted inflammatory responses to therapeutic antibodies. For example, reducing or eliminating Fc-mediated effector functions may be useful for avoiding potential toxicity to T cells that normally express PD-1, for example. Even if Fc-gamma receptor binding can be eliminated or reduced by using scFv or Fab fragments of the antibody, full-length antibodies may be preferred when pharmacokinetic activity, in the form of increased antibody half-life, is required for therapeutic purposes. Thus, antibodies containing modified Fc regions that reduce or eliminate Fc-mediated effector functions may be desirable.

[0015] In a first aspect, there is provided an antibody having the ability of binding to PD-1, the antibody comprising a heavy chain having a heavy chain constant region (CH) and a heavy chain variable region (VH), wherein the heavy chain constant region comprises an aromatic amino acid or a nonpolar amino acid at a position corresponding to position 234 of a human IgG1 heavy chain according to EU numbering, and an amino acid other than glycine at a position corresponding to position 236 of a human IgG1 heavy chain according to EU numbering.

[0016] The antibody of the first embodiment comprises at least two modifications in the Fc region. When an antibody comprises such modifications, it may become an inactive or inactivated antibody. The terms "inactive," "inactive," or "inactivated" as used herein refer to an Fc region that is at least unable to bind to any Fc-gamma receptor, unable to induce Fc-mediated cross-linking of FcR, unable to induce FcR-mediated cross-linking of target antigens via the two Fc regions of each antibody, or unable to bind to C1q.

[0017] In one embodiment of the first aspect, there is provided an antibody having the ability of binding to PD-1, the antibody comprising a heavy chain having a heavy chain constant region (CH) and a heavy chain variable region (VH), wherein the heavy chain constant region comprises an aromatic amino acid or a nonpolar amino acid at a position corresponding to position 234 of human IgG1 heavy chain according to EU numbering, and an amino acid other than glycine at a position corresponding to position 236 of human IgG1 heavy chain according to EU numbering, and the heavy chain variable region (VH) comprises HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1 sequence is selected from a sequence having or comprising SYN, SEQ ID NO: 12, or SEQ ID NO: 13, the HCDR2 sequence is selected from a sequence having or comprising SEQ ID NO: 10 or SEQ ID NO: 11, and the HCDR3 sequence is selected from a sequence having or comprising SEQ ID NO: 8 or SEQ ID NO: 9.

[0018] In one embodiment of the first aspect, there is provided an antibody having the ability of binding to PD-1, comprising a heavy chain having a heavy chain constant region (CH) and a heavy chain variable region (VH), wherein the heavy chain constant region comprises an aromatic amino acid or a nonpolar amino acid at a position corresponding to position 234 of the human IgG1 heavy chain according to EU numbering, and an amino acid other than glycine at a position corresponding to position 236 of the human IgG1 heavy chain according to EU numbering; and a light chain having a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1 sequence is selected from a sequence having or comprising SEQ ID NO: 16 or SEQ ID NO: 17, the LCDR2 sequence is selected from a sequence having or comprising QAS or SEQ ID NO: 15, and the LCDR3 sequence is a sequence having or comprising SEQ ID NO: 14.

[0019] In one embodiment of the first aspect, the antibody has the ability of binding to PD-1, comprising a heavy chain having a heavy chain constant region (CH) and a heavy chain variable region (VH), and a light chain having a light chain variable region (VL), wherein the heavy chain constant region comprises an aromatic amino acid or a nonpolar amino acid at a position corresponding to position 234 of the human IgG1 heavy chain according to EU numbering, and an amino acid other than glycine at a position corresponding to position 236 of the human IgG1 heavy chain according to EU numbering, and wherein the heavy chain variable region (VH) comprises HCDR1, HCDR2, and HCDR3 sequences, and wherein the HCDR1 sequence has or is selected from the group consisting of SYN, SEQ ID NO: 12, and SEQ ID NO: 13. the HCDR2 sequence is selected from a sequence having or comprising SEQ ID NO:10 or SEQ ID NO:11, the HCDR3 sequence is selected from a sequence having or comprising SEQ ID NO:8 or SEQ ID NO:9, the light chain variable region (VL) comprises LCDR1, LCDR2 and LCDR3 sequences, the LCDR1 sequence is selected from a sequence having or comprising SEQ ID NO:16 or SEQ ID NO:17, the LCDR2 sequence is selected from a sequence having or comprising QAS or SEQ ID NO:15, and the LCDR3 sequence is a sequence having or comprising SEQ ID NO:14.

[0020] For example, an antibody capable of binding to PD-1 of the first aspect can comprise a heavy chain having a heavy chain constant region (CH) and a heavy chain variable region (VH), and a light chain having a light chain variable region (VH), wherein the heavy chain constant region comprises an aromatic or non-polar amino acid, e.g., phenylalanine (F), at a position corresponding to position 234 of the human IgG1 heavy chain according to EU numbering, optionally an acidic amino acid, e.g., glutamic acid (E), at position 235 of the human IgG1 heavy chain according to EU numbering, and an amino acid other than glycine, e.g., arginine (R), at a position corresponding to position 236 of the human IgG1 heavy chain according to EU numbering, wherein the heavy chain variable region (VH) comprises HCDR1, HCDR2, and HCDR3 sequences, and wherein the HCDR1 sequence is SYN, a sequence having or comprising SEQ ID NO: 12 or SEQ ID NO: 13, the HCDR2 sequence is selected from a sequence having or comprising SEQ ID NO: 10 or SEQ ID NO: 11, and the HCDR3 sequence is selected from a sequence having or comprising SEQ ID NO: 8 or SEQ ID NO: 9; and / or the light chain variable region (VL) comprises LCDR1, LCDR2 and LCDR3 sequences, wherein the LCDR1 sequence is selected from a sequence having or comprising SEQ ID NO: 16 or SEQ ID NO: 17, the LCDR2 sequence is selected from a sequence having or comprising SEQ ID NO: 15, and the LCDR3 sequence is a sequence having or comprising SEQ ID NO: 14.

[0021] In a second aspect, there is provided a hybridoma capable of producing the antibody of the first aspect.

[0022] In a third aspect, there is provided a conjugate comprising the antibody of the first aspect linked to a moiety or agent.

[0023] In a fourth aspect, there is provided a multimer comprising at least two antibodies of the first aspect, or at least two conjugates of the third aspect, or a mixture of one or more antibodies of the first aspect and one or more conjugates of the third aspect.

[0024] In a fifth aspect, - an antibody or fragment thereof according to the first aspect; - an antibody heavy chain or fragment thereof of the antibody of the first aspect; or - an antibody light chain or a fragment thereof of the antibody of the first aspect Nucleic acids are provided that include a gene or nucleic acid sequence encoding The encoded antibody and / or the encoded antibody heavy or light chain, respectively, may be a chain described herein, for example, as described in accordance with an embodiment of the first aspect.

[0025] In a sixth aspect, there is provided a vector comprising one or more of the nucleic acids of the fifth aspect.

[0026] In a seventh aspect, there is provided a host cell comprising the nucleic acid of the fifth aspect or the vector of the sixth aspect.

[0027] In an eighth aspect, there is provided a virus comprising the nucleic acid of the fifth aspect or the vector of the sixth aspect.

[0028] In a ninth aspect, there is provided a composition, preferably a pharmaceutical composition, comprising an active agent and a pharmaceutically acceptable carrier, wherein the active agent is (i) an antibody of the first aspect; (ii) the conjugate of the third aspect; (iii) a multimer of the fourth aspect; (iv) a nucleic acid of the fifth aspect or a combination of nucleic acids of the fifth aspect; (v) a vector of the sixth aspect or a combination of vectors of the sixth aspect; (vi) a host cell of the seventh aspect or a combination of host cells of the seventh aspect; and / or (vii) a virus of the eighth aspect or a combination of viruses of the eighth aspect The composition, preferably a pharmaceutical composition, is provided, which is at least one selected from:

[0029] In a tenth aspect, there is provided a pharmaceutical composition of the ninth aspect for use in the prophylactic and / or therapeutic treatment of disease.

[0030] In an eleventh aspect, there is provided a method of treating or preventing a disease in a subject, comprising administering to the subject at least one active agent, wherein the active agent is: (i) an antibody of the first aspect; (ii) the conjugate of the third aspect; (iii) a multimer of the fourth aspect; (iv) a nucleic acid of the fifth aspect or a combination of nucleic acids of the fifth aspect; (v) a vector of the sixth aspect or a combination of vectors of the sixth aspect; (vi) a host cell of the seventh aspect or a combination of host cells of the seventh aspect; and / or (vii) a virus of the eighth aspect or a combination of viruses of the eighth aspect The method is at least one selected from the following:

[0031] In a twelfth aspect, there is provided a kit for the qualitative or quantitative detection of PD-1 in a sample, the kit comprising an antibody of the first aspect or a conjugate of the third aspect or a multimer of the fourth aspect.

[0032] In a thirteenth aspect, there is provided use of the antibody of the first aspect, or the conjugate of the third aspect, or the multimer of the fourth aspect, or the kit of the twelfth aspect, in a method of determining the presence or amount of PD-1 expressed in a sample, the method comprising:

[0033] (i) contacting a sample with an antibody or conjugate or multimer; (ii) detecting the formation of and / or determining the amount of a complex between the antibody or conjugate or multimer and PD-1.

[0034] Other features and advantages of the above-described embodiments of the present disclosure will be apparent from the following detailed description and claims. Embodiments of one aspect can be combined with any of the other aspects described herein in any manner and in any number. Any permutation and combination of all elements and features described in this application should be considered to be disclosed by the description of this application unless the context indicates otherwise. [Brief explanation of the drawings]

[0035] [Figure 1] Figure 1 shows the binding of IgG1-PD1 to PD-1 from various species. CHO-S cells transiently transfected with various species of PD-1 were incubated with IgG1-PD1, pembrolizumab, or the nonbinding control antibodies IgG1-ctrl-FERR and IgG4-ctrl, and binding was analyzed using flow cytometry. Untransfected CHO-S cells incubated with IgG1-PD1 were included as a negative control. A-B. Data shown are the geometric mean fluorescence intensity (gMFI) ± SD of duplicate wells from one representative experiment out of four. C-D. Data shown are the gMFI ± SD of duplicate wells from one representative experiment out of two. E. Data shown are the geometric mean fluorescence intensity (gMFI) ± SD of duplicate wells from one representative experiment out of four. Abbreviations: gMFI = geometric mean fluorescence intensity; PD-1 = programmed cell death protein 1; PE = R-phycoerythrin. [Figure 2]Figure 2 shows the competitive binding of IgG1-PD1 to PD-L1 and PD-L2 for human PD-1. CHO-S cells transiently transfected with human PD-1 were incubated with 1 μg / mL of biotinylated recombinant human PD-L1 (A) or PD-L2 (B) in the presence of IgG1-PD1 or pembrolizumab. IgG1-ctrl-FERR was included as a negative control. Cells were stained with streptavidin-allophycocyanin, and the percentage of cells binding to biotinylated PD-L1 or PD-L2 was determined by measuring the percentage of streptavidin-allophycocyanin+ cells using flow cytometry. The percentage of streptavidin-allophycocyanin+ cells in the no-antibody control and untransfected samples is indicated by the dashed line. Data shown are from a single replicate of one representative experiment out of three. Abbreviations: Ab = antibody; CHO-S = Chinese hamster ovary, suspension; ctrl = control; FERR = L234F / L235E / G236R-K409R; PD1 = programmed cell death protein 1; PD-L1 = programmed cell death 1 ligand 1; PD-L2 = programmed cell death 1 ligand 2. [Figure 3] Figure 3 shows functional inhibition of the PD-1 / PD-L1 checkpoint by IgG1-PD1. Blockade of the PD-1 / PD-L1 axis was tested using a cell-based bioluminescent PD-1 / PD-L1 blockade reporter assay. Data shown are the mean luminescence ± SD of duplicate wells in one representative experiment of five (pembrolizumab and IgG1-PD1), three (IgG1-ctrl-FERR), or two (nivolumab) experiments. Abbreviations: FERR = L234F / L235E / G236R-K409R; PD1 = programmed cell death protein 1; PD-L1 = programmed cell death 1 ligand 1; RLU = relative light units; SD = standard deviation. [Figure 4]Figure 4 shows the enhancement of CD8+ T cell proliferation by IgG1-PD1 in an antigen-specific T cell proliferation assay. Human CD8+ T cells were electroporated with RNA encoding a CLDN6-specific TCR and RNA encoding PD-1 and labeled with CFSE. T cells were then cocultured with iDCs electroporated with CLDN6-encoding RNA in the presence of IgG1-PD1, pembrolizumab, nivolumab, or IgG1-ctrl-FERR. CFSE dilution in T cells was analyzed by flow cytometry after 4 days and used to calculate the expansion index. Data from one representative donor (26268_B) out of four donors evaluated in three independent experiments is shown. Error bars represent the SD of duplicate wells. Curves were fitted using a four-parameter logarithmic fit using GraphPad Prism. Abbreviations: CFSE = carboxyfluorescein succinimidyl ester; FERR = L234F / L235E / G236R-K409R; PD1 = programmed cell death protein 1; SD = standard deviation. [Figure 5] Figure 5 shows IgG1-PD1-induced IFNγ secretion in an allogeneic MLR assay. Triplicate unique donor pairs of allogeneic human mDCs and CD8+ T cells were cocultured for 5 days in the presence of IgG1-PD1 or pembrolizumab. IgG1-ctrl-FERR and an IgG4 isotype control were included as negative controls. IFNγ secretion in supernatants was analyzed using an IFNγ-specific immunoassay. Data shown are the mean ± standard error of the mean (SEM) concentrations for triplicate unique allogeneic donor pairs. Abbreviations: FERR = L234F / L235E / G236R-K409R; IFN = interferon; IgG = immunoglobulin G; mDC = mature dendritic cells; MLR = mixed lymphocyte reaction; SEM = standard error of the mean. [Figure 6]Figure 6 shows IgG1-PD1-induced cytokine secretion in an allogeneic MLR assay. Triplicate unique donor pairs of allogeneic human mDCs and CD8+ T cells were cocultured for 5 days in the presence of 1 μg / mL of IgG1-PD1 or pembrolizumab. IgG1-ctrl-FERR was included as a negative control. Cytokine secretion was analyzed in the supernatant using Luminex. (A) Cytokine levels are expressed as the mean fold change relative to cytokine levels measured in untreated cocultures. (B) Cytokine production levels for three unique allogeneic donor pairs are shown, with horizontal lines indicating the mean, upper, and lower limits. Abbreviations: FC = fold change; FERR = L234F / L235E / G236R-K409R; GM-CSF = granulocyte-macrophage colony-stimulating factor; IgG = immunoglobulin G; IL = interleukin; MCP-1 = monocyte chemotactic protein 1; mDC = mature dendritic cell; MLR = mixed lymphocyte reaction; TNF = tumor necrosis factor. [Figure 7] Figure 7 shows the binding of C1q to membrane-bound IgG1-PD1. C1q binding to IgG1-PD1 was analyzed using stimulated human CD8+ T cells. After incubation with IgG1-PD1, IgG1-ctrl-FERR, IgG1-ctrl, or the positive control antibody IgG1-CD52-E430G (without inactivating mutations and with hexamerization-enhancing mutations), cells were incubated with human serum as a source of C1q. C1q binding was detected using a FITC-conjugated rabbit anti-C1q antibody. Data shown are the geometric mean fluorescence intensity (gMFI) ± standard deviation (SD) from duplicate wells of one representative donor out of seven across three comparative experiments. Abbreviations: FITC = fluorescein isothiocyanate; gMFI = geometric mean fluorescence intensity; PE = R-phycoerythrocyanin. [Figure 8]Figure 8 shows binding of IgG1-PD1 to FcγR. Binding of IgG1-PD1 to immobilized human recombinant FcγR constructs was analyzed by SPR in a qualified assay (n=1). Binding of IgG1-PD1 to FcγRIa (A), FcγRIIa-H131 (B), FcγRIIa-R131 (C), FcγRIIb (D), FcγRIIIa-F158 (E), and FcγRIIIa-V158 (F). Antibody IgG1-ctrl (without FER-inactivating mutations) was included as a positive control for binding. Abbreviations: ctrl = control; FcγR = Fc gamma receptor; IgG = immunoglobulin G; PD-1 = programmed cell death protein 1; RU = resonance units. [Figure 9] Figure 9 shows the binding of IgG1-PD1 and several other anti-PD-1 antibodies to FcγR. Binding of IgG1-PD1, nivolumab, pembrolizumab, dostallimab, and cemiplimab to immobilized human recombinant FcγR constructs was analyzed by SPR (n=3). Binding of the test antibodies to FcγRIa (A), FcγRIIa-H131 (B), FcγRIIa-R131 (C), FcγRIIb (D), FcγRIIIa-F158 (E), and FcγRIIIa-V158 (F) was shown. IgG1-ctrl and IgG4-ctrl antibodies were included as positive controls for FcγR binding of IgG1 and IgG4 molecules with wild-type Fc regions. Shown are binding responses ± SD from three separate experiments. Abbreviations: ctrl = control; FcγR = Fc gamma receptor; IgG = immunoglobulin G; PD-1 = programmed cell death protein 1; RU = resonance units. [Figure 10]Figure 10 shows the binding of IgG1-PD1 and several other anti-PD-1 antibodies to FcγRIa. Binding of IgG1-PD1, nivolumab, pembrolizumab, dostallimab, and cemiplimab to CHO-S cells transiently expressing human FcγRIa was analyzed by flow cytometry. IgG1-ctrl and IgG1-ctrl-FERR were included as positive and negative controls, respectively. Abbreviations: ctrl = control; FcγR = Fc gamma receptor; FERR = L234F / L235E / G236R-K409R; huIgG = human immunoglobulin G; PD-1 = programmed cell death protein 1; PE = R-phycoerythrin. [Figure 11] Figure 11 shows total human IgG in mouse plasma samples. Mice were intravenously injected with 1 or 10 mg / kg IgG1-PD1 at t=0, and serial plasma samples were collected at 10 minutes, 4 hours, 1 day, 2 days, 8 days, 14 days, and 21 days post-injection. Total huIgG in plasma samples was determined for each mouse by ECLIA. Data are presented as the mean huIgG concentration ± SD for three individual mice. The dashed line indicates the plasma concentration of wild-type (wt) huIgG predicted by a two-compartment model based on IgG clearance in humans [Bleeker et al., 2001, Blood. 98(10):3136-42]. Dotted lines indicate the LLOQ and ULOQ. Abbreviations: huIgG = human IgG; IgG = immunoglobulin G; LLOQ = lower limit of quantification; PD-1 = programmed cell death protein 1; SD = standard deviation; ULOQ = upper limit of quantification. [Figure 12]Figure 12 shows the antitumor activity of IgG1-PD1 in human PD-1 knock-in mice. An MC38 colon cancer syngeneic tumor model was established by SC transplantation in hPD-1 KI mice. Mice were administered 0.5, 2, or 10 mg / kg of IgG1-PD1 or pembrolizumab, or 10 mg / kg of IgG1-ctrl-FERR, 2QW x 3 (9 mice per group). (A) Mean tumor volume ± SEM in each group until the final time point when each group was completed. (B) Tumor volume in the various groups on the final day (day 11) when all groups were completed. Data are tumor volumes of individual mice in each treatment group and mean tumor volume ± SEM for each treatment group. Mann-Whitney analysis was used to compare tumor volumes of treatment groups relative to the IgG1-ctrl-FERR treatment group, with *p<0.05, **p<0.01, and ***p<0.001. C. Kaplan-Meier curves show progression-free survival, defined as the percentage of mice with tumor volumes smaller than 500 mm3. One mouse in the 2 mg / kg IgG1-PD1 group was excluded from the analysis because it died of unknown causes on day 16, before the tumor volume exceeded 500 mm3. Abbreviations: 2QWx3 = twice weekly for 3 weeks; ctrl = control; FERR = L234F / L235E / G236R / K409R mutations; IgG = immunoglobulin G; KI = knock-in; PD-1 = programmed cell death protein 1; SC = subcutaneous; SEM = standard error of the mean. [Figure 13]Figure 13 shows the dynamics of peripheral T cell counts in human PD-1 knock-in mice treated with IgG1-PD1. An MC38 colon cancer syngeneic tumor model was established by SC transplantation in hPD-1 KI mice. Mice were administered 0.5 or 10 mg / kg IgG1-PD1, 10 mg / kg pembrolizumab, or 10 mg / kg IgG1-ctrl-FERR on days 0, 3, and 7 (12 mice per group). Peripheral blood samples were collected from four mice per group after euthanasia on days 2, 4, and 8 and analyzed by flow cytometry. The mean ± SD counts of CD3+ (A), CD4+ (B), and CD8+ (C) T cells per μL of blood within the viable CD45+ leukocyte subpopulation are shown. Mann-Whitney analysis was used to compare treatment groups with the IgG1-ctrl-FERR treatment group, and the 10 mg / kg IgG1-PD1 and 10 mg / kg pembrolizumab groups, *p<0.05. Abbreviations: ctrl = control; FERR = L234F / L235E / G236R / K409R mutations; IgG = immunoglobulin G; KI = knock-in; PD-1 = programmed cell death protein 1; SC = subcutaneous; SD = standard deviation. [Figure 14]Figure 14 shows PD markers for splenic T cell subsets in human PD-1 knock-in mice treated with IgG1-PD1. An MC38 colon cancer syngeneic tumor model was established by SC transplantation in hPD-1 KI mice. Mice were administered 0.5 or 10 mg / kg IgG1-PD1, 10 mg / kg pembrolizumab, or 10 mg / kg IgG1-ctrl-FERR on days 0, 3, and 7 (12 mice per group). Spleens were harvested on days 2, 4, and 8 (n=4 mice per group and time point) and analyzed by flow cytometry. Figures show the mean ± SD of the percentages of effector memory (CD44+ CD62L-), central memory (CD44+ CD62L+), and naive (CD44- CD62L+) CD8+ T cells (A) and the percentage of MHC class II+ cells in the total CD8+ T cell population (B) in the spleen on day 8. Mann-Whitney analysis was used to compare treatment groups with the IgG1-ctrl-FERR treatment group, the 10 mg / kg IgG1-PD1 group, and the 10 mg / kg pembrolizumab group, with *p<0.05. Abbreviations: ctrl = control; FERR = L234F / L235E / G236R / K409R mutations; IgG = immunoglobulin G; KI = knock-in; PD-1 = programmed cell death protein 1; SC = subcutaneous; SD = standard deviation. [Figure 15]Figure 15 shows changes in intratumoral cells in human PD-1 knock-in mice treated with IgG1-PD1. An MC38 colon cancer syngeneic tumor model was established by SC transplantation in hPD-1 KI mice. Mice were administered 0.5 or 10 mg / kg of IgG1-PD1, 10 mg / kg of pembrolizumab, or 10 mg / kg of IgG1-ctrl-FERR on days 0, 3, and 7 (12 mice per group). Xenograft tumors were excised on day 8 (n=4 mice per group) and analyzed by IHC. The mean ± SD percentages of CD3+ T cells (A), CD4+ T cells (B), CD8+ T cells (C), and GZMB+ cells (D) among total nucleated cells on day 8 are shown. Mann-Whitney analysis was used to compare treatment groups with the IgG1-ctrl-FERR treatment group, and the 10 mg / kg IgG1-PD1 and 10 mg / kg pembrolizumab groups, *p<0.05. Abbreviations: ctrl = control; FERR = L234F / L235E / G236R / K409R mutations; GZMB = granzyme B; IgG = immunoglobulin G; KI = knock-in; PD-1 = programmed cell death protein 1; SC = subcutaneous; SD = standard deviation. [Figure 16]Figure 16 shows the binding of IgG1-PD1 and other anti-PD-1 antibodies to human monocyte-derived FcγR+ M2c-like macrophages. (A) Expression of FcγRIa, FcγRII, FcγRIIIa, and PD-1 on relevant isotype controls and unstained M2c-like macrophages from one representative donor out of three tested donors was visualized in an overlay histogram of normalized data. (B) After 24 hours of incubation, binding of IgG1-PD1, pembrolizumab, nivolumab, and control antibodies to human monocyte-derived FcγR+ M2c-like macrophages was analyzed by flow cytometry. Binding is shown relative to that of the background control (binding with secondary antibody only, indicated by the black dotted line). Dots represent three individual donors measured in two independent experiments, and bars and error bars represent the mean ± SD of three donors each. Abbreviations: ctrl = control; FERR = L234F / L235E / G236R / K409R mutations; PD-1 = programmed cell death protein 1; SD = standard deviation. [Figure 17] Figure 17 shows FcγR signaling induced by membrane-bound IgG1-PD1 and other anti-PD-1 antibodies. FcγR signaling induced by membrane-bound IgG1-PD1 and several other anti-PD-1 antibodies was examined using cell-based bioluminescent FcγRI (A), FcγRIIa-R131 (B), FcγRIIa-H131 (C), and FcγRIIb (D) reporter assays. IgG1-CD52-E430G, which contains the E430G mutation that enhances hexamerization, was included as a positive control. Data shown are the mean relative light units ± SD of duplicate wells from one representative experiment out of three. Abbreviations: Ab = antibody; FERR = L234F / L235E / G236R / K409R mutation; PD-1 = programmed cell death protein 1; RLU = relative light units; SD = standard deviation. DETAILED DESCRIPTION OF THE INVENTION

[0036] Reference is made hereinafter to the sequences and SEQ ID NOs specifically set forth in the Sequence Listing. Also, without limiting the disclosure, reference is made to specific examples of antibodies of the first aspect described herein: MAB-19-0202, and MAB-19-0618. These exemplary, but non-limiting, antibodies of the first aspect are designated herein by reference to the antibody designation.

[0037] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0038] Although the present disclosure is described in detail below, it should be understood that the present disclosure is not limited to the particular methodology, protocols, and reagents described herein, as such may vary. It should also be understood that the terms used herein describe particular embodiments only and are not intended to limit the scope of the present disclosure, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0039] The elements of the present disclosure are described below. While these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the disclosure to only the explicitly described embodiments. The specification should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered disclosed by the description of this application unless the context indicates otherwise.

[0040] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., (1995) Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0041] For the practice of the present disclosure, unless otherwise indicated, conventional methods of biochemistry, cell biology, immunology, and recombinant DNA techniques will be described as described in the art (e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0042] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise," and variations such as "comprises" or "comprising," are understood to mean the inclusion of a stated member, integer, or step, or group of members, integers, or steps, but not the exclusion of other members, integers, or steps, or group of members, integers, or steps, although in some embodiments such other members, integers, or steps, or group of members, integers, or steps may be excluded, i.e., the subject matter is the inclusion of a stated member, integer, or step, or group of members, integers, or steps. As used in the context of describing the invention (particularly in the context of the claims), the terms "a," "an," "the," and similar references are intended to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is intended merely to serve as a shorthand method for referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if set forth individually herein.

[0043] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any examples provided herein, or the use of exemplary language (e.g., "such as"), are intended merely to better illustrate the invention and do not pose a limitation on the scope of the invention as set forth in any other claims. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0044] Throughout the text of this specification, several documents are cited. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0045] Terms such as "reducing" or "inhibiting" refer to the ability to cause an overall decrease in levels, preferably by 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more. The term "inhibit" or similar expressions includes complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0046] Terms such as "increasing," "enhancing," "promoting" or "prolonging" preferably relate to an increase, enhancement, promotion or prolongation of approximately at least 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 80%, preferably at least 100%, preferably at least 200%, especially at least 300%. These terms may also relate to an increase, enhancement, promotion or prolongation from zero or an unmeasurable or undetectable level to a level above zero or a measurable or detectable level.

[0047] The term "PD-1" refers to programmed cell death-1 and includes any mutant, conformation, isoform, and species homolog of PD-1 that is naturally expressed by cells or expressed by cells transfected with the PD-1 gene. Preferably, "PD-1" refers to human PD-1, and in particular the protein having the amino acid sequence set forth in SEQ ID NO:22 in the Sequence Listing (NCBI Reference Sequence: NP_005009.2), or preferably the protein encoded by the nucleic acid sequence set forth in SEQ ID NO:24 in the Sequence Listing (NCBI Reference Sequence: NM_005018.2). Alternative names for "PD-1" include CD279 and SLEB2.

[0048] The term "PD-1" includes post-translationally modified variants, isoforms, and species homologs of human PD-1 that are naturally expressed by cells or expressed in / on cells transfected with the PD-1 gene.

[0049] The term "PD-1 variants" is intended to encompass (i) PD-1 splice variants, (ii) PD-1 post-translational modification variants, particularly including variants with different N-glycosylation states, and (iii) PD-1 conformational variants. Such variants may include soluble forms of PD-1.

[0050] PD-1 is a type I membrane protein belonging to the immunoglobulin superfamily (The EMBO Journal (1992), vol. 11, issue 11, pp. 3887-3895). Human PD-1 protein comprises an extracellular domain consisting of amino acids 24-170 of the sequence set forth in SEQ ID NO: 22 in the Sequence Listing, a transmembrane domain (amino acids 171-191 of the sequence set forth in SEQ ID NO: 22), and a cytoplasmic domain (amino acids 192-288 of the sequence set forth in SEQ ID NO: 22). The term "PD-1 fragment," as used herein, encompasses any fragment of the PD-1 protein, preferably an immunogenic fragment. This term also encompasses, for example, the above-mentioned domains of the full-length protein or any fragment of these domains, particularly an immunogenic fragment. A preferred amino acid sequence of the extracellular domain of human PD-1 protein is set forth in SEQ ID NO: 23 in the Sequence Listing.

[0051] The term "extracellular portion" or "extracellular domain" in this context, as used herein, preferably refers to a part of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from the outside of said cell, for example by an extracellularly located binding molecule, such as an antibody. Preferably, the term refers to one or more extracellular loops or domains, or fragments thereof.

[0052] In a first aspect, there is provided an antibody having the ability of binding to PD-1, the antibody comprising a heavy chain constant region, wherein the heavy chain constant region comprises an aromatic amino acid or a nonpolar amino acid at a position corresponding to position 234 of a human IgG1 heavy chain according to EU numbering, and an amino acid other than glycine at a position corresponding to position 236 of a human IgG1 heavy chain according to EU numbering.

[0053] The term "antibody" (Ab), as used herein, refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either, capable of specifically binding to an antigen (particularly an epitope on an antigen) under typical physiological conditions, and preferably has a half-life of at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3 days, 4 days, 5 days, 6 days, 7 days or more, etc., or any other relevant, functionally defined period of time (e.g., a period sufficient to induce, promote, enhance, and / or modulate a physiological response associated with antibody binding to an antigen and / or a period sufficient for the antibody to mobilize effector activity). In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and any combination of the foregoing. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The variable and constant regions are also referred to herein as variable and constant domains, respectively. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) interspersed with highly conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of the VH are referred to as HCDR1, HCDR2, and HCDR3 (or CDR-H1, CDR-H2, and CDR-H3), and the CDRs of the VL are referred to as LCDR1, LCDR2, and LCDR3 (or CDR-L1, CDR-L2, and CDR-L3). The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.The constant region of an antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), and the CH can be further subdivided into a constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged from the amino terminus to the carboxy terminus in the following order: CH1, CH2, CH3). The constant region of an 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 components of the complement system, such as C1q. An antibody can be an intact immunoglobulin derived from natural or recombinant sources, or an immunologically active portion of an intact immunoglobulin. An antibody is typically a tetramer of an immunoglobulin molecule. Antibodies can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, F(ab)2, single-chain antibodies, and humanized antibodies.

[0054] The variable regions of the heavy and light chains of immunoglobulin molecules contain binding domains that interact with antigens. The terms "binding region," "antigen-binding region," and "antigen-binding portion" are used interchangeably herein and refer to the region that interacts with antigens, including both VH and VL regions. As used herein, antibody includes not only monospecific antibodies, but also multispecific antibodies that contain multiple, for example, two or more, for example, three or more different antigen-binding regions.

[0055] In some embodiments, an antibody can be a full-length antibody. When used in the context of an antibody, the term "full-length" indicates that the antibody, rather than a fragment, contains all of the domains of a particular isotype that are normally found in that isotype in nature, such as the VH, CH1, CH2, CH3, hinge, VL, and CL domains of an IgG1 antibody.

[0056] The term "humanized antibody," as used herein, refers to a molecule having an antigen-binding site substantially derived from an immunoglobulin from a non-human species, with the remaining immunoglobulin structure of the molecule being based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise a complete variable domain fused onto a constant domain, or may comprise only the complementarity-determining regions (CDRs) grafted onto appropriate framework regions in the variable domain. The antigen-binding site may be wild-type or modified by one or more amino acid substitutions, e.g., to resemble human immunoglobulins more closely. Some forms of humanized antibodies preserve all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs of a mouse antibody). Other forms have one or more CDRs altered relative to the original antibody.

[0057] The term "chimeric antibody" as used herein refers to an antibody in which a portion of the amino acid sequence of each of the heavy and light chains is homologous to the corresponding sequence of antibodies from a particular species or class, while the remaining segments of the chains are homologous to the corresponding sequence of another antibody. Typically, the variable regions of both the light and heavy chains mimic the variable regions of antibodies from a particular mammal, while the constant regions are homologous to the sequences of antibodies from another species. One distinct advantage of such chimeric forms is that the variable regions can be easily derived from currently known sources, for example, using B cells or hybridomas from readily available non-human host organisms, in combination with constant regions from human cell preparations. While the variable regions have the advantage of being easily prepared and their specificity is not affected by the source, the constant regions are human, and therefore less likely to elicit an immune response from a human subject when injected than constant regions from non-human sources. However, the definition is not limited to this specific example.

[0058] The term "antigen-binding portion" (or simply "binding portion") of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. 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 encompassed by the term "antigen-binding portion" of an antibody include (i) a monovalent fragment consisting of the VL, VH, CL, and CH domains, and (ii) an Fd fragment consisting of the VH and CH domains. Further examples are binding domain immunoglobulin fusion proteins comprising (i) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. Binding domain immunoglobulin fusion proteins are further disclosed in U.S. Patent Application Publication Nos. 2003 / 0118592 and 2003 / 0133939. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0059] The term "epitope" refers to a protein determinant capable of binding to an antibody, where the term "bind" preferably relates to specific binding. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural and charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. The term "epitope" preferably refers to an antigenic determinant in a molecule, i.e., a portion or fragment of a molecule such as an antigen that is recognized by the immune system. For example, an epitope can be recognized by T cells, B cells, or antibodies. An epitope of an antigen may comprise a continuous or discontinuous portion of the antigen and may be about 5 to about 100, e.g., about 5 to about 50, more preferably about 8 to about 30, and most preferably about 10 to about 25 amino acids in length; for example, an epitope may preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In one embodiment, the epitope is about 10 to about 25 amino acids in length. The term "epitope" includes B cell epitopes and T cell epitopes.

[0060] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of an MHC molecule. The terms "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes present in all vertebrates, including MHC class I and MHC class II molecules. MHC proteins or molecules are important in signaling between lymphocytes and antigen-presenting or diseased cells during the immune response; they bind peptide epitopes and present them for recognition by T cell receptors on T cells. MHC-encoded proteins are expressed on the cell surface and present both self-antigens (peptide fragments derived from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. In the case of class I MHC / peptide complexes, the bound peptide is typically about 8 to about 10 amino acids in length, although longer or shorter peptides can also be effective. In the case of class II MHC / peptide complexes, the binding peptides are typically about 10 to about 25 amino acids in length, particularly about 13 to about 18 amino acids in length, although longer or shorter peptides may also be effective.

[0061] The term "amino acid corresponding to position ..." and similar expressions, as used herein, refer to the position number of the amino acid in the human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins can be found by alignment with human IgG1. Thus, an amino acid or segment in one sequence "corresponding" to an amino acid or segment in another sequence means that it has at least 50%, at least 80%, at least 90%, or at least 95% identity to the human IgG1 heavy chain when aligned with the other amino acid or segment using standard sequence alignment programs, e.g., ALIGN, ClustalW, etc., typically with default settings. Methods for aligning sequences or segments in sequences, thereby determining corresponding positions in sequences for amino acid positions according to the present disclosure, are believed to be well known in the art.

[0062] For example, with respect to the amino acid sequence of SEQ ID NO: 2 in the Sequence Listing of the present disclosure, the amino acid positions corresponding to positions 234 to 236 of the human IgG1 heavy chain according to EU numbering are amino acid positions 117 to 119 of SEQ ID NO: 2, with F located at position 117 (corresponding to position 234 of the human IgG1 heavy chain according to EU numbering), E located at position 118 (corresponding to position 235 of the human IgG1 heavy chain according to EU numbering), and R located at position 119 (corresponding to position 236 of the human IgG1 heavy chain according to EU numbering). In the sequences shown below, the FER amino acid sequence is underlined and shown in bold. [ka]

[0063] Unless otherwise indicated herein or otherwise clearly contradicted by context, all references to amino acid positions throughout this disclosure refer to the positions corresponding to the respective positions in the human IgG1 heavy chain according to EU numbering.

[0064] In one embodiment of the first aspect, the antibody comprises a heavy chain constant region that has reduced or depleted Fc-mediated effector function, or induces Fc-mediated effector function to a lesser extent, compared to another antibody comprising the same antigen-binding region and a heavy chain constant region (CH) comprising a human IgG1 hinge, CH2, and CH3 regions.

[0065] In one particular embodiment of the antibody according to the first aspect, the heavy chain constant region (CH) is modified such that the antibody induces Fc-mediated effector function to a lesser extent compared to an identical antibody except for comprising an unmodified heavy chain constant region (CH).

[0066] The term "Fc-mediated effector function", as used herein, particularly refers to such functions selected from the list of IgG Fc receptor (Fc gamma R, FcγR) binding, C1q binding, ADCC, CDC, and combinations thereof.

[0067] In the context of the present disclosure, the term "reduced or depleted Fc-mediated effector function" as used in connection with antibodies, including multispecific antibodies, means that the antibody causes an overall reduction in Fc-mediated effector function (such function being selected from the list of IgG Fc receptor (Fc gamma R, FcγR) binding, C1q binding, ADCC, or CDC) compared to a human IgG1 antibody comprising (i) the same CDR sequences as said antibody, particularly the same CDR sequences comprising the same first and second antigen-binding regions, and (ii) two heavy chains comprising a human IgG1 hinge, CH2, and CH3 regions, preferably at a level of 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more. "Depleted Fc-mediated effector function" or similar expressions includes complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0068] In the context of the present disclosure, the term "induces Fc-mediated effector functions to a lesser extent" when used in relation to antibodies, including multispecific antibodies, means that the antibody induces Fc-mediated effector functions (such functions being in particular selected from the list of IgG Fc receptor (Fc gamma R, FcγR) binding, C1q binding, ADCC or CDC) to a lesser extent compared to a human IgG1 antibody that comprises (i) the same CDR sequences as said antibody, in particular the same CDR sequences comprising the same first and second antigen-binding regions, and (ii) two heavy chains comprising human IgG1 hinge, CH2 and CH3 regions.

[0069] Fc-mediated effector function can be determined by measuring the binding of a binder to Fcγ receptors, binding to C1q, or induction of Fc-mediated cross-linking of Fcγ receptors. In particular, Fc-mediated effector function can be determined by measuring the binding of a binder to C1q and / or IgG Fc-gamma RI.

[0070] In one embodiment of the antibody of the first aspect, the amino acid at the position corresponding to position 236 of a human IgG1 heavy chain according to EU numbering is a basic amino acid.

[0071] The terms "amino acid" and "amino acid residue" may be used interchangeably herein and are not to be understood as limiting. Amino acids are organic compounds containing an amine (-NH2) and a carboxyl (-COOH) functional group, along with a side chain (R group) specific to each amino acid. In the context of the present disclosure, amino acids can be classified based on their structure and chemical properties.

[0072] In this disclosure, amino acid residues are represented using the following abbreviations: Also, unless otherwise specified, amino acid sequences of peptides and proteins are specified from N-terminus to C-terminus (left to right), with the N-terminus being identified as the first residue. Amino acids are designated as three-letter abbreviations, one-letter abbreviations, or full names as follows: Ala: A: alanine; Asp: D: aspartic acid; Glu: E: glutamic acid; Phe: F: phenylalanine; Gly: G: glycine; His: H: histidine; Ile: I: isoleucine; Lys: K: lysine; Leu: L: leucine; Met: M: methionine; Asn: N: asparagine; Pro: P: proline; Gln: Q: glutamine; Arg: R: arginine, Ser: S: serine, Thr: T: threonine, Val: V: valine, Trp: W: tryptophan, Tyr: Y: tyrosine, Cys: C: cysteine.

[0073] Naturally occurring amino acids may also be generally divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.

[0074] In one embodiment of the antibody of the first aspect, the basic amino acid at the position corresponding to position 236 of the human IgG1 heavy chain according to EU numbering is selected from the group consisting of lysine, arginine, and histidine. In one embodiment, the basic amino acid at the position corresponding to position 236 of the human IgG1 heavy chain according to EU numbering is arginine (G236R). Such an amino acid substitution is also referred to herein as G236R. The term "G236R" indicates a substitution of the amino acid glycine (G) with arginine (R) at position 236 of the human IgG1 heavy chain according to EU numbering. Similar terms are used for other amino acid positions and amino acids within the present disclosure. Unless indicated to the contrary, the amino acid positions referred to in these terms are amino acid positions in the human IgG1 heavy chain according to EU numbering.

[0075] In one embodiment of the antibody of the first aspect, the amino acid at the position corresponding to position 234 of the human IgG1 heavy chain according to EU numbering is an aromatic amino acid. In one embodiment, the aromatic amino acid at this position is selected from the group consisting of phenylalanine, tryptophan and tyrosine.

[0076] In one embodiment of the antibody of the first aspect, the amino acid at a position corresponding to position 234 of the human IgG1 heavy chain according to EU numbering is a nonpolar amino acid. In one embodiment, the nonpolar amino acid at this position is selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan. In one embodiment, the nonpolar amino acid at this position is selected from the group consisting of isoleucine, proline, phenylalanine, methionine, and tryptophan.

[0077] In one embodiment of the antibody of the first aspect, the amino acid at the position corresponding to position 234 of a human IgG1 heavy chain according to EU numbering is phenylalanine (L234F).

[0078] Exemplary combinations of possible amino acids at positions corresponding to positions 234 and 236 of the human IgG1 heavy chain according to EU numbering are shown in the table below:

[0079] [Table 2]

[0080] For example, at positions corresponding to positions 234 and 236 in a human IgG1 heavy chain according to EU numbering, the following amino acids may particularly be present in the heavy chain constant region of the antibody of the first aspect: 234F / 236R, 234W / 236R, 234Y / 236R, 234A / 236R, 234L / 236R, 234F / 236K, 234W / 236K, 234Y / 236K, 234A / 236K, 234L / 236K, 234F / 236H, 234W / 236H, 234Y / 236H, 234A / 236H, or 234L / 236H.

[0081] The amino acids or amino acid substitutions at positions 234 and 236 may be present in only one heavy chain of the antibody, or in both heavy chains of the antibody. Each amino acid present in the first and second heavy chains of the antibody may be selected independently of the other.

[0082] For example, at least one heavy chain of the antibody of the first embodiment may comprise the following sequence (SEQ ID NO:2): [ka]

[0083] In one embodiment of the antibody of the first aspect, the heavy chain of a human IgG1 heavy chain has amino acids at positions corresponding to 234 and 236 according to EU numbering as defined above, and further has an acidic amino acid at position corresponding to 235 according to EU numbering. In one embodiment, the acidic amino acid at this position is selected from aspartic acid or glutamic acid. In one embodiment, the amino acid at position corresponding to 235 according to EU numbering is glutamic acid (L235E).

[0084] In one embodiment of the antibody of the first aspect, in the heavy chain constant region, the amino acids at positions corresponding to positions 234, 235, and 236 in a human IgG1 heavy chain according to EU numbering are: 234 is a nonpolar or aromatic amino acid, 235 is an acidic amino acid, and 236 is a basic amino acid.

[0085] Exemplary combinations of possible amino acids at positions corresponding to positions 234, 235 and 236 of the human IgG1 heavy chain according to EU numbering are shown in the table below:

[0086] [Table 3]

[0087] For example, at positions corresponding to positions 234, 235 and 236 in a human IgG1 heavy chain according to EU numbering, in particular the following amino acids may be present in the heavy chain constant region of the antibody of the first aspect: 234F / 235E / 236R, 234W / 235E / 236R, 234Y / 235E / 236R, 234A / 235E / 236R, 234L / 235E / 236R, 234F / 235D / 236R, 234W / 235D / 236R, 234Y / 235D / 236R, 234A / 235D / 236R, 234L / 235D / 236R, 234F / 235L / 236R, 234W / 235L / 236R, 234Y / 235L / 236R, 234A / 235L / 236R, 234L / 235L / 236R, 2 34F / 235A / 236R, 234W / 235A / 236R, 234Y / 235A / 236R, 234A / 235A / 236R, 234 L / 235A / 236R, 234F / 235E / 236K, 234W / 235E / 236K, 234Y / 235E / 236K, 234A / 235E / 236K, 234L / 235E / 236K, 234F / 235D / 236K, 234W / 235D / 236K, 234Y / 2 35D / 236K, 234A / 235D / 236K, 234L / 235D / 236K, 234F / 235L / 236K, 234W / 23 5L / 236K, 234Y / 235L / 236K, 234A / 235L / 236K, 234L / 235L / 236K, 234F / 235A / 236K, 234W / 235A / 236K, 234Y / 235A / 236K, 234A / 235A / 236K, 234L / 235A / 236K, 234F / 235E / 236H, 234W / 235E / 236H, 234Y / 235E / 236H, 234A / 235E / 23 6H, 234L / 235E / 236H, 234F / 235D / 236H, 234W / 235D / 236H, 234Y / 235D / 236 H, 234A / 235D / 236H, 234L / 235D / 236H, 234F / 235L / 236H, 234W / 235L / 236H, 234Y / 235L / 236H, 234A / 235L / 236H, 234L / 235L / 236H, 234F / 235A / 236H, 2 34W / 235A / 236H, 234Y / 235A / 236H, 234A / 235A / 236H, or 234L / 235A / 236H.

[0088] The amino acids or amino acid substitutions at positions 234, 235, and 236 may be present in only one heavy chain of the antibody, or in both heavy chains of the antibody. Each amino acid present in the first and second heavy chains of the antibody may be selected independently of the other.

[0089] For example, at least one heavy chain of the antibody of the first embodiment may comprise the following sequence (SEQ ID NO:2 or SEQ ID NO:55): [ka]

[0090] In the present application, where applicable, any permutation and combination of all described amino acid substitutions at positions 234, 236, and 235, e.g., as shown in Tables 2 and 3, should be considered disclosed by the description of the present application unless the context indicates otherwise. For example, in one embodiment of the antibody, the first heavy chain comprises the amino acids FER at positions corresponding to positions 234-236 in a human IgG1 heavy chain according to EU numbering, or the first heavy chain comprises, essentially consists of, or consists of the amino acid sequence set forth in SEQ ID NO: 2, and the second heavy chain of said antibody comprises other amino acids, e.g., the amino acids AAG or LLG at positions corresponding to positions 234-236 in a human IgG1 heavy chain according to EU numbering, or the second heavy chain of said antibody comprises, essentially consists of, or consists of the amino acid sequence set forth in SEQ ID NO: 1 or 7. In another embodiment of the antibody, the first and second heavy chains comprise the same amino acids at positions corresponding to positions 234-236 of the human IgG1 heavy chain according to EU numbering, i.e., the same aromatic or non-polar amino acid, e.g., F, at the position corresponding to position 234 of the human IgG1 heavy chain according to EU numbering, and the same amino acid other than glycine, e.g., R, such as FER or FLR, in a specific combination, at the position corresponding to position 236 of the human IgG1 heavy chain according to EU numbering.

[0091] In one embodiment of the antibody of the first aspect, the antibody comprises at least one or two heavy chain constant regions, wherein the amino acid corresponding to position 234 is phenylalanine, the amino acid corresponding to position 235 is glutamic acid, and the amino acid corresponding to position 236 is arginine (L234F / L235E / G236R=FER).

[0092] In further embodiments of the antibody of the first aspect, at least one or both heavy chain constant regions may comprise, in addition to the substitution of the amino acid at the position corresponding to position 236 of the human IgG1 heavy chain according to EU numbering, one or more further mutations in the constant region, for example at one or more amino acids at positions corresponding to positions L234, L235, G237, D265, D270, K322, P329, and P331 in the human IgG1 heavy chain according to EU numbering.

[0093] In one embodiment, the amino acid corresponding to position 234 in the human IgG1 heavy chain according to EU numbering is not L and is selected, for example, from F or A, and / or the amino acid corresponding to position 235 in the human IgG1 heavy chain according to EU numbering is not L and is selected, for example, from E or A.

[0094] In one embodiment, the amino acid corresponding to position 237 in the human IgG1 heavy chain according to EU numbering is not G, but is, for example, A, i.e., at least one or both heavy chain constant regions of the antibody may comprise, for example, the amino acid substitution G236R / G237A.

[0095] In one embodiment, the amino acid corresponding to position 265 in the human IgG1 heavy chain according to EU numbering is not D, but is, for example, A, i.e., at least one or both heavy chain constant regions of the antibody may comprise, for example, the amino acid substitution G236R / D265A.

[0096] In one embodiment, the amino acid corresponding to position 270 in the human IgG1 heavy chain according to EU numbering is not D, but is, for example, A, i.e., at least one or both heavy chain constant regions of the antibody may comprise, for example, the amino acid substitution G236R / D270A.

[0097] In one embodiment, the amino acid corresponding to position 322 in the human IgG1 heavy chain according to EU numbering is not K, but is, for example, A, i.e., at least one or both heavy chain constant regions of the antibody may comprise, for example, the amino acid substitution G236R / K322A.

[0098] In one embodiment, the amino acid corresponding to position 329 in the human IgG1 heavy chain according to EU numbering is not P, but is, for example, A, i.e., at least one or both heavy chain constant regions of the antibody may comprise, for example, the amino acid substitution G236R / P329A.

[0099] In one embodiment, the amino acid corresponding to position 331 in the human IgG1 heavy chain according to EU numbering is not P, but is, for example, A or S, i.e., at least one or both heavy chain constant regions of the antibody may comprise, for example, the amino acid substitutions G236R / P331A or G236R / P331S.

[0100] In one embodiment of the antibody of the first aspect, the positions corresponding to positions 234, 236 and G327 in a human IgG1 heavy chain according to EU numbering are substituted, and at least one or both heavy chain constant regions of the antibody may, for example, comprise the amino acid substitutions L234F / G236R / G237A.

[0101] In one embodiment of the antibody of the first aspect, the positions corresponding to positions 234, 236 and 265 in a human IgG1 heavy chain according to EU numbering are substituted, and at least one or both heavy chain constant regions of the antibody may, for example, comprise the amino acid substitutions L234F / G236R / D265A.

[0102] In one embodiment of the antibody of the first aspect, the positions corresponding to positions 234, 236 and 270 in a human IgG1 heavy chain according to EU numbering are substituted, and at least one or both heavy chain constant regions of the antibody may, for example, comprise the amino acid substitutions L234F / G236R / D270A.

[0103] In one embodiment of the antibody of the first aspect, the positions corresponding to positions 234, 236 and 322 in a human IgG1 heavy chain according to EU numbering are substituted, and at least one or both heavy chain constant regions of the antibody may, for example, comprise the amino acid substitutions L234F / G236R / K322A.

[0104] In one embodiment of the antibody of the first aspect, the positions corresponding to positions 234, 236 and 329 in a human IgG1 heavy chain according to EU numbering are substituted, and at least one or both heavy chain constant regions of the antibody may, for example, comprise the amino acid substitutions L234F / G236R / P329A.

[0105] In one embodiment of the antibody of the first aspect, the positions corresponding to positions 234, 236 and 331 in a human IgG1 heavy chain according to EU numbering are substituted, and at least one or both heavy chain constant regions of the antibody may, for example, comprise the amino acid substitutions L234F / G236R / P331A or L234F / G236R / P331S.

[0106] In one embodiment of the antibody of the first aspect, the antibody comprises one or more heavy chain constant regions (CH) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of the CH sequence set forth in SEQ ID NO:2.

[0107] Any teaching given herein regarding particular amino acid sequences, e.g., those shown in the Sequence Listing, is to be interpreted as also relating to sequences that are functionally equivalent to the particular sequence, e.g., variants of the particular sequence that result in amino acid sequences that exhibit the same or similar properties as the particular amino acid sequence.

[0108] For purposes of this disclosure, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those occurring naturally. The term "variant" particularly includes fragments of an amino acid sequence.

[0109] In one embodiment, the amino acid variants of the heavy chain constant region comprise the amino acids at positions 234 and 236, and optionally 235, as defined herein and in the appended claims.

[0110] In one embodiment of the antibody of the first aspect, the antibody comprises one or more, for example two, heavy chain constant regions (CH), wherein the heavy chain constant region comprises the sequence set forth in SEQ ID NO:2 or SEQ ID NO:55.

[0111] The antibody of the first aspect can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

[0112] As used herein, the term "isotype" refers to the immunoglobulin class (e.g., IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgD, IgA (e.g., IgA1, IgA2), IgE, IgM, or IgY) encoded by heavy chain constant region genes. When a particular isotype, e.g., IgG1, is referred to herein, the term is not limited to a particular isotype sequence, e.g., a particular IgG1 sequence, but is used to indicate that the antibody is closer in sequence to that isotype, e.g., IgG1, than to other isotypes. Thus, for example, the IgG1 antibodies disclosed herein may be sequence variants of naturally occurring IgG1 antibodies, including mutations in the constant region.

[0113] IgG1 antibodies can exist in multiple polymorphic variants called allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7), any of which are suitable for use in some embodiments herein. Common allotypic variants in the human population are those designated by the letters a, f, n, z, or combinations thereof. In any of the embodiments herein, the antibody may comprise a heavy chain Fc region comprising a human IgG Fc region. In a further embodiment, the human IgG Fc region comprises human IgG1.

[0114] There are five types of mammalian immunoglobulin heavy chains, namely α, δ, ε, γ, and μ, which constitute the different antibody isotypes, namely IgA, IgD, IgE, IgG, and IgM. In contrast to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at the carboxy terminus.

[0115] Mammals have two types of light chains: lambda and kappa. Immunoglobulin chains contain a variable region and a constant region. The constant region is essentially conserved among different immunoglobulin isotypes, while the variable region is highly diverse and is responsible for antigen recognition.

[0116] For example or in embodiments, the antibody, preferably a monoclonal antibody, of the invention is of the IgG1, Kappa or Lambda isotype, and preferably comprises a human IgG1 / Kappa or human IgG1 / Lambda constant part, or the antibody, preferably a monoclonal antibody, is derived from an IgG1, Lambda (lambda) or IgG1, Kappa (kappa) antibody, preferably a human IgG1, Lambda (lambda) or human IgG1, Kappa (kappa) antibody.

[0117] In one embodiment of the first aspect, the antibody comprises a light chain having a light chain constant region comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of the LC sequence set forth in SEQ ID NO: 6. In one embodiment, the antibody comprises a light chain having a light chain constant region comprising the sequence set forth in SEQ ID NO: 6.

[0118] In one embodiment of the invention, the binding agent is a full length IgG1 antibody, such as IgG1, κ. In one embodiment of the invention, the binding agent is a full length human IgG1 antibody, such as IgG1, κ.

[0119] Other isotypes can be obtained by isotype switching. As used herein, "isotype switching" refers to the phenomenon in which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig classes.

[0120] In one embodiment of the antibody of the first aspect, the antibody of the first aspect may be derivatized, linked, or co-expressed with other binding specificities. In another embodiment, the antibody of the first aspect may be derivatized, linked, or co-expressed with another functional molecule, e.g., another peptide or protein (e.g., a Fab' fragment). For example, the antibody of the first aspect may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent bonding, etc.) to one or more other molecular entities, e.g., another antibody (e.g., to generate a bispecific or multispecific antibody).

[0121] The term "antibody derivative" refers to any modified form of an antibody, such as a conjugate of an antibody with another agent or antibody. As used herein, an antibody is "derived from" a particular germline sequence if the antibody is obtained from a system by immunizing an animal or by screening an immunoglobulin gene library, and the selected antibody is at least 90%, more preferably at least 95%, and even more preferably at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, antibodies derived from a particular germline sequence exhibit no more than 10 amino acid differences, more preferably no more than 5 amino acid differences, and even more preferably no more than 4, 3, 2, or 1 amino acid difference, from the amino acid sequence encoded by the germline immunoglobulin gene.

[0122] The antibody of the first embodiment may be a human antibody. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibody of the first embodiment may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0123] The term "monoclonal antibody," as used herein, refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. In one embodiment, a monoclonal antibody is produced by a hybridoma, where a B cell obtained from a non-human animal, e.g., a mouse, is fused with an immortalized cell.

[0124] The antibody of the first embodiment may be a recombinant antibody. As used herein, the term "recombinant antibody" includes all antibodies prepared, expressed, created, or isolated by recombinant means and / or genetic engineering, including, for example, (a) antibodies isolated from animals (e.g., mice) transgenic or transchromosomal for immunoglobulin genes, or hybridomas prepared therefrom; (b) antibodies isolated from host cells, e.g., transfectomas, transformed to express the antibody; (c) antibodies isolated from recombinant, combinatorial antibody libraries; and (d) antibodies prepared, expressed, created, or isolated by any other means involving splicing immunoglobulin gene sequences into other DNA sequences. Preferably, "recombinant," such as recombinant cells, in the context of this disclosure do not occur in nature. The amino acid variants described herein can be readily prepared by one of skill in the art, for example, by recombinant DNA manipulation. The manipulation of nucleotide sequences to prepare proteins and peptides with substitutions, additions, insertions, or deletions is described in detail, for example, in Sambrook et al. (1989).

[0125] As used herein, a "heterologous antibody" is defined in relation to the transgenic organism producing such an antibody. The term refers to an antibody that has an amino acid sequence or encoding nucleic acid sequence that corresponds to that found in an organism not comprised of the transgenic organism, and generally originates from a species other than the transgenic organism.

[0126] As used herein, a "heterohybrid antibody" refers to an antibody having light and heavy chains of different organismal origins. For example, an antibody having a human heavy chain joined to a murine light chain is a heterohybrid antibody.

[0127] The antibodies of the first aspect are preferably isolated. As used herein, "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to PD-1 is substantially free of antibodies that specifically bind to antigens other than PD-1). However, an isolated antibody that specifically binds to an epitope, isoform, or variant of human PD-1 may have cross-reactivity with other related antigens, such as antigens from other species (e.g., PD-1 species homologs). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals. In one embodiment of the first aspect, the "isolated" monoclonal antibody combination relates to antibodies with different specificities that are combined in a well-defined composition.

[0128] As used herein, the term "naturally occurring" as applied to an entity refers to the fact that the entity is found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a natural source and has not been intentionally modified by man in a laboratory is naturally occurring.

[0129] The term "rearranged," as used herein, refers to the configuration of a heavy or light chain immunoglobulin locus in which a V segment is arranged immediately adjacent to a DJ or J segment in a configuration that encodes essentially a complete VH or VL domain, respectively. Rearranged immunoglobulin (antibody) gene loci can be identified by comparison with germline DNA; rearranged loci will have at least one recombined heptamer / nonamer homology element.

[0130] The term "unrearranged" or "germline configuration" as used herein with respect to a V segment refers to an configuration in which the V segment does not rearrange so that it is immediately adjacent to a D or J segment.

[0131] I. Mechanism of antibody action The following provides a discussion of the mechanisms underlying the therapeutic efficacy of the antibodies of the first aspect, but should not be construed as limiting the invention in any way.

[0132] The antibodies described herein preferably interact with the immune checkpoint PD-1. Binding to PD-1 inhibits the interaction of PD-1 with its ligands (PD-L1 and PD-L2). PD-L1 is expressed, for example, on tumor cells and antigen-presenting cells in the tumor microenvironment. Because the interaction of PD-1 with PD-L1 leads to the suppression of an immune response, preferably a T-cell-mediated immune response, blocking PD-1 with the antibody of the first aspect prevents or at least reduces such suppression of the immune response, or in other words, induces an immune response.

[0133] PD-1 and its ligand interact to prevent or reduce immune responses, but blockade of PD-1 may be more advantageous than blockade of its ligand to achieve this effect, since inhibitory signaling between abnormal cells expressing PD-L2 and lymphocytes expressing PD-1 may help the immune system inhibit an immune response, whereas blockade of PD-L1, for example, still results in a reduced immune response.

[0134] The immune system has the ability to recognize and destroy abnormal cells through two distinct modalities: innate and adaptive immunity. The innate components are macrophages, natural killer (NK) cells, monocytes, and granulocytes. These cells identify molecular patterns involved in cellular transformation and release various cytokines and inflammatory mediators. The innate immune response lacks the memory capacity for foreign antigens that is present in the adaptive immune response. The latter component of the immune system is also characterized by specificity for foreign antigens, conferred by the presence of receptors on lymphocytes. Antigen-presenting cells (APCs) also play a role in the adaptive response by taking up foreign antigens and presenting them to lymphocytes in the context of major histocompatibility complexes. CD4+ T cells have receptors that recognize antigens in the context of MHC class II molecules, release cytokines, and also CD8 + This allows the activation of lymphocytes (CTLs) or B cells. CTLs are part of cell-mediated immunity and, after recognizing antigens presented in the context of MHC class I molecules, can eliminate cells by apoptosis or perforin-mediated cytolysis. It is widely accepted that T cell-mediated immunity plays an important role in antitumor responses. B cells are involved in the release of immunoglobulins and, as such, are part of the humoral immune system.

[0135] The term "immune response" refers to the integrated bodily response to a target, such as an antigen or a cell expressing an antigen, and preferably refers to a cellular immune response, or a humoral immune response as well. The immune response can be protective / preventative / prophylactic and / or therapeutic.

[0136] "Inducing an immune response" can mean that there was no immune response before induction, but it can also mean that there was a certain level of immune response before induction and that the immune response was enhanced after induction. Thus, "inducing an immune response" also includes "enhancing an immune response." Preferably, after inducing an immune response in a subject, the subject is protected from developing a disease such as a cancer disease or the disease state is improved by inducing an immune response. In this case, inducing an immune response can mean that the disease state of the subject is improved, that the subject does not develop metastasis, or that a subject at risk of developing a cancer disease does not develop a cancer disease.

[0137] Terms such as "cellular immune response" and "cellular response" refer to immune responses directed at cells. Innate cellular immune responses are driven by macrophages, natural killer (NK) cells, monocytes, and granulocytes. Adaptive cellular immune responses are characterized by antigen presentation in association with MHC class I or class II involving T cells or T lymphocytes, which act as "helpers" or "killers." Helper T cells (CD4 +T cells (also called T cells) play a central role by regulating the immune response and are responsible for the production of killer cells (cytotoxic T cells, cytolytic T cells, CD8 + T cells (also called CTLs) kill abnormal cells, such as cancer cells, and prevent the production of more abnormal cells. In embodiments, the present disclosure includes stimulating anti-tumor CTL responses against tumor cells that express one or more tumor antigens, and preferably present such tumor antigens on MHC class I.

[0138] "Tumor antigen," as used herein, encompasses any substance, preferably a peptide or protein, that targets and / or induces an immune response, such as a specific reaction with an antibody or T lymphocyte (T cell). Preferably, the antigen comprises at least one epitope, such as a T cell epitope. The tumor antigen or its T cell epitope is preferably presented in the context of an MHC molecule by cells, preferably antigen-presenting cells, including abnormal cells, particularly cancer cells, resulting in an immune response against the antigen (including cells expressing the antigen).

[0139] The antibody of the first embodiment is characterized by its binding property to PD-1, preferably by its ability to inhibit the immunosuppressive signal of PD-1.

[0140] According to the present disclosure, the term "binding" preferably relates to "specific binding." As used herein, the terms "binding" or "capable of binding" or "having the ability to bind" in the context of antibody binding to a given antigen or epitope typically refer to a specific binding of about 10 to 150 ng / mL as determined using Bio-Layer Interferometry (BLI) or, for example, as determined using surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte. -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or less, about 10 -10 M or less or even about 10 -11 K below M DThe antibody binds with an affinity corresponding to the K D at least 10 times lower, such as at least 100 times lower, such as at least 1,000 times lower, such as at least 10,000 times lower, such as at least 100,000 times lower, D The amount at which affinity is increased is the antibody's K D Because it depends on the antibody's K D is very low (i.e., the antibody is highly specific), the affinity for the antigen may be at least 10,000 times lower than the affinity for a nonspecific antigen.

[0141] The term "k" d ”(seconds -1 ) as used herein refers to the dissociation rate constant of a particular antibody-antigen interaction. off Also called value.

[0142] The term “K D " (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0143] If two antibodies bind to the same antigen and the same epitope, the two antibodies have the "same specificity." Whether the antibody being tested recognizes the same epitope as a particular antigen-binding antibody, i.e., whether the antibody binds to the same epitope, can be tested by various methods well known to those skilled in the art.

[0144] Competition between antibodies can be detected by a cross-blocking assay. For example, a competitive ELISA assay can be used as a cross-blocking assay. For example, a target antigen can be coated on the wells of a microtiter plate, and a candidate substance that competes with the antigen-binding antibody and the test antibody can be added. The amount of antigen-binding antibody bound to the antigen in the well is indirectly correlated with the binding ability of the candidate substance that competes with the test antibody for binding to the same epitope. Specifically, the greater the affinity of the candidate substance that competes with the test antibody for the same epitope, the less antigen-binding antibody will bind to the antigen-coated well. The amount of antigen-binding antibody bound to the well can be measured by labeling the antibody with a detectable or measurable label.

[0145] An antibody that competes for binding to an antigen with another antibody, e.g., an antibody comprising the heavy and light chain variable regions described herein, or that has the specificity for the antigen of another antibody, e.g., an antibody comprising the heavy and light chain variable regions described herein, may be a variant of the heavy and / or light chain variable regions described herein, e.g., an antibody that contains modifications and / or a degree of identity in the CDRs described herein.

[0146] In one embodiment of the antibody of the first aspect, an antibody having the ability of binding to PD-1 is provided, wherein the antibody comprises a heavy chain variable region (VH) comprising a complementarity determining region 3 (HCDR3) having or comprising the sequence set forth in any one of SEQ ID NOs: 8. In one embodiment of the antibody of the first aspect, an antibody having the ability of binding to PD-1 is provided, wherein the antibody comprises a heavy chain variable region (VH) comprising a complementarity determining region 3 (HCDR3) having or comprising the sequence set forth in any one of SEQ ID NOs: 9.

[0147] In one embodiment of the antibody of the first aspect, the heavy chain variable region (VH) of said antibody comprises a complementarity determining region 2 (HCDR2) having or comprising the sequence set forth in any one of SEQ ID NOs: 10. In one embodiment, HCDR2 of the heavy chain variable region (VH) has or comprises the sequence set forth in any one of SEQ ID NOs: 11.

[0148] In one embodiment of the antibody of the first aspect, the heavy chain variable region (VH) of said antibody comprises a complementarity determining region 1 (HCDR1) having or comprising a sequence selected from SYN. ​​In one embodiment, HCDR1 of the heavy chain variable region (VH) has or comprises a sequence set forth in any one of SEQ ID NOs: 12. In one embodiment, HCDR1 of the heavy chain variable region (VH) has or comprises a sequence set forth in any one of SEQ ID NOs: 13.

[0149] In one embodiment of the antibody of the first aspect, the antibody comprises a heavy chain variable region (VH) comprising an HCDR3 sequence having or comprising the sequence set forth in SEQ ID NO:8 or SEQ ID NO:9, and preferably further comprising SYN, an HCDR1 sequence having or comprising the sequence set forth in SEQ ID NO:12 or SEQ ID NO:13, and / or an HCDR2 sequence having or comprising the sequence set forth in SEQ ID NO:10 or SEQ ID NO:11.

[0150] In one embodiment of the antibody of the first aspect, the antibody comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2 and HCDR3 sequences, wherein the HCDR1 sequence is selected from a sequence having or comprising SYN, SEQ ID NO: 12 or SEQ ID NO: 13, the HCDR2 sequence is selected from a sequence having or comprising SEQ ID NO: 10 or SEQ ID NO: 11, and the HCDR3 sequence is selected from a sequence having or comprising SEQ ID NO: 8 or SEQ ID NO: 9.

[0151] In one embodiment of the antibody of the first aspect, the antibody comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, which are or comprise SYN, SEQ ID NO: 10, and SEQ ID NO: 8, respectively.

[0152] In one embodiment of the antibody of the first aspect, the antibody comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences are or comprise SEQ ID NO: 12, SEQ ID NO: 11, and SEQ ID NO: 8, respectively.

[0153] In one embodiment of the antibody of the first aspect, the antibody comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences are or comprise SEQ ID NO: 13, SEQ ID NO: 10, and SEQ ID NO: 9, respectively.

[0154] In one embodiment of the first aspect, the antibody comprises a light chain having a light chain variable region (VL).

[0155] The antibodies of the first aspect are preferably characterized in that they comprise a heavy chain variable region (VH) comprising a complementarity determining region 3 (HCDR3) having or comprising a sequence as described herein. In preferred embodiments, complementarity determining regions 1 and 2 of the VH are further defined. In one embodiment, these antibodies of the first aspect further comprise a light chain variable region (VL) comprising at least one of the following:

[0156] - a complementarity determining region 3 (LCDR3) having or comprising the sequence set forth in any one of SEQ ID NOs: 14; - a complementarity determining region 2 (LCDR2) having or comprising the sequence QAS. In one embodiment, the light chain variable region (VL) comprises a complementarity determining region 2 (LCDR2) having or comprising the sequence set forth in any one of SEQ ID NOs: 15, and / or - Complementarity determining region 1 (LCDR1) having or comprising the sequence set forth in any one of SEQ ID NOs: 16. In one embodiment, the light chain variable region (VL) comprises a complementarity determining region 1 (LCDR1) having or comprising the sequence set forth in any one of SEQ ID NOs: 17.

[0157] In one embodiment of the antibody of the first aspect, the antibody comprises a light chain variable region (VL) that comprises an LCDR3 sequence having or comprising SEQ ID NO: 14, and preferably further comprises an LCDR1 sequence having or comprising SEQ ID NO: 16 or SEQ ID NO: 17, and / or an LCDR2 sequence having or comprising SEQ ID NO: 15.

[0158] In one embodiment, the antibody of the first aspect comprises a light chain variable region (VL) comprising LCDR1, LCDR2 and LCDR3 sequences, wherein the LCDR1 sequence is selected from a sequence having or comprising SEQ ID NO: 16 or SEQ ID NO: 17, the LCDR2 sequence is selected from a sequence having or comprising QAS or SEQ ID NO: 15, and the LCDR3 sequence is a sequence having or comprising SEQ ID NO: 14.

[0159] In one embodiment, the antibody of the first aspect comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences are or comprise SEQ ID NO: 16, QAS, and SEQ ID NO: 14, respectively.

[0160] In one embodiment, the antibody of the first aspect comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences are or comprise SEQ ID NO: 17, SEQ ID NO: 15, and SEQ ID NO: 14, respectively.

[0161] In one embodiment of the antibody of the first aspect, the antibody comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise or have the sequences SYN set forth in SEQ ID NO: 10 and SEQ ID NO: 8, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise or have the sequences set forth in SEQ ID NO: 16, QAS, and SEQ ID NO: 14, respectively. Specific examples of such antibodies include, but are not limited to, MAB-19-0202.

[0162] In one embodiment of the antibody of the first aspect, the antibody comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise or have the sequences set forth in SEQ ID NO: 12, SEQ ID NO: 11, and SEQ ID NO: 8, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise or have the sequences set forth in SEQ ID NO: 17, SEQ ID NO: 15, and SEQ ID NO: 14, respectively. Specific examples of such antibodies include, but are not limited to, MAB-19-0202.

[0163] In one embodiment of the antibody of the first aspect, the antibody comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise or have the sequences set forth in SEQ ID NO: 13, SEQ ID NO: 10, and SEQ ID NO: 9, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise or have the sequences set forth in SEQ ID NO: 16, QAS, and SEQ ID NO: 14, respectively. Specific examples of such antibodies include, but are not limited to, MAB-19-0202.

[0164] The terms "heavy chain variable region" (also referred to as "VH") and "light chain variable region" (also referred to as "VL") are used herein in their most general sense and include any sequences that can include complementarity-determining regions (CDRs) interspersed with other regions, also referred to as framework regions (FRs). The framework regions position the CDRs, among other things, so that an antigen-binding site can be formed after folding and pairing of the VH and VL. Preferably, each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. That is, the terms "heavy chain variable region" and "light chain variable region" should not be construed as being limited to sequences that can be found in native antibodies or in the VH and VL sequences exemplified herein (SEQ ID NOS: 18-21 in the Sequence Listing). These terms include any sequence that contains and can appropriately position CDRs, such as sequences derived from the VL and VH regions of a native antibody or sequences set forth in SEQ ID NOS: 18-21 in the Sequence Listing. In particular, those skilled in the art will understand that the sequences of the framework regions can be modified without losing the characteristics of the VH and VL, respectively (including both amino acid substitutions and length-related modifications, i.e., insertion or deletion mutations). In preferred embodiments, any modifications are limited to the framework regions. However, those skilled in the art will be familiar with the fact that CDR regions, hypervariable regions, and variable regions can also be modified without losing the ability to bind to PD-1. For example, the CDR regions can be identical to or highly homologous to the regions specified herein. By "highly homologous," it is intended that one to five, preferably one to four, e.g., one to three, or one to two substitutions can be made in the CDRs. Furthermore, the hypervariable and variable regions can be modified to exhibit substantial homology with the regions specifically disclosed herein.

[0165] The CDRs specified herein are identified using two different CDR identification methods. The first numbering scheme used herein is according to Kabat (Wu and Kabat, 1970; Kabat et al., 1991), and the second scheme is the IMGT numbering scheme (Lefranc, 1997; Lefranc et al., 2005). The third approach uses the intersection of both identification schemes.

[0166] In one embodiment of the antibody of the first aspect, the antibody comprises one or more CDRs, and the set of CDRs or combination of sets of CDRs described herein comprises said CDRs together with their intervening framework regions (also referred to herein as framing regions or FRs), or together with portions of said framework regions. Preferably, the portions comprise at least about 50% of either or both of the first and fourth framework regions, said 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Construction of antibodies of the first aspect produced by recombinant DNA techniques may result in the introduction of N- or C-terminal residues in the variable region encoded by linkers introduced to facilitate cloning or other engineering steps, including the introduction of linkers for joining the variable regions of the present disclosure to additional protein sequences, including immunoglobulin heavy chains, other variable domains (e.g., in the generation of diabodies), or protein tags.

[0167] In one embodiment of the antibody of the first aspect, the antibody comprises a heavy chain variable region (VH) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of the VH sequence set forth in any one of SEQ ID NOs: 18.

[0168] The heavy chain variable region (VH) preferably comprises at least one, more preferably two, and even more preferably all three of the CDR sequences of an antibody heavy chain variable region having or comprising the sequence set forth in any one of SEQ ID NOs: 8 to 13, respectively, or having the sequence SYN.

[0169] In one embodiment of the antibody of the first aspect, the antibody comprises a heavy chain variable region (VH), wherein the VH comprises a sequence set forth in any one of SEQ ID NO:18.

[0170] In one embodiment of the antibody of the first aspect, the antibody comprises a light chain variable region (VL) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of the VL sequence set forth in any one of SEQ ID NOs: 19.

[0171] The light chain variable region (VL) preferably comprises at least one, more preferably two, and even more preferably all three CDR sequences of an antibody light chain variable region having or comprising the sequence set forth in any one of SEQ ID NOs: 14 to 17, respectively, or having the sequence QAS.

[0172] In one embodiment of the antibody of the first aspect, the antibody comprises a light chain variable region (VL), wherein the VL comprises a sequence set forth in any one of SEQ ID NOs:19.

[0173] In one embodiment of the antibody of the first aspect, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises or has the sequence set forth in SEQ ID NO: 18 and the VL comprises or has the sequence set forth in SEQ ID NO: 19. Specific examples of such antibodies include, but are not limited to, MAB-19-0202. Also encompassed by the present disclosure are variants of said heavy chain variable region (VH) and said light chain variable region (VL), as well as respective combinations of these variant VHs and VLs.

[0174] The antibody of the first aspect may be derived from various species, including, but not limited to, rabbit, mouse, rat, guinea pig, and human. The antibody may be polyclonal or monoclonal. In one or preferred embodiments, the antibody of the first aspect is monoclonal. The antibody of the first aspect may comprise a chimeric molecule, in one embodiment, in which an antibody constant region derived from one species, preferably human, is combined with an antigen-binding site derived from another species. In one embodiment, the antibody is a monoclonal chimeric antibody, and the constant region is preferably a human immunoglobin constant portion, e.g., a human IgG1 / κ constant portion. Furthermore, in one embodiment, the antibody of the first aspect comprises a humanized molecule, preferably a monoclonal humanized molecule, in which the antigen-binding site of an antibody derived from a non-human species is combined with constant and framework regions of human origin. In one embodiment, the antibody of the first aspect comprises one or more CDRs, and the set or combination of CDR sets described herein comprises said CDRs in a human antibody framework. In one or preferred embodiment, the antibody of the first aspect is a monoclonal humanized antibody, and the constant region is preferably a human immunoglobin constant part, such as a human IgG1 / κ constant part.

[0175] In one embodiment of the antibody of the first aspect, the antibody comprises a heavy chain variable region (VH) comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of the VH sequence set forth in any one of SEQ ID NOs: 20. In one embodiment, the antibody comprises a heavy chain variable region (VH), wherein the VH comprises a sequence set forth in any one of SEQ ID NOs: 20. In one embodiment, the antibody comprises a light chain variable region (VL), wherein the VL comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of the VL sequence set forth in any one of SEQ ID NOs: 21. In one embodiment, the antibody comprises a light chain variable region (VL), wherein the VL comprises a sequence set forth in any one of SEQ ID NOs: 21.

[0176] In one embodiment of the antibody of the first aspect, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises or has the sequence set forth in SEQ ID NO:20, and the VL comprises or has the sequence set forth in SEQ ID NO:21, or a variant of each of these sequences. Another example of an antibody of the first aspect may comprise a VH comprising or having the sequence set forth in SEQ ID NO:20, or a variant thereof, and a VL comprising or having the sequence set forth in SEQ ID NO:21, or a variant thereof. Specific examples of such antibodies include, but are not limited to, MAB-19-0618. Antibody MAB-19-0618 is derived from MAB-19-0202. Also encompassed by the present disclosure are variants of the heavy chain variable region (VH) and the light chain variable region (VL), as well as respective combinations of these variant VHs and VLs.

[0177] In one embodiment of the antibody of the first aspect, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain constant region that comprises or has the sequence set forth in SEQ ID NO: 2 or 55, and a heavy chain variable region (VH) that comprises or has the sequence set forth in SEQ ID NO: 20, and the light chain comprises a light chain constant region that comprises or has the sequence set forth in SEQ ID NO: 6, and a light chain variable region (VL) that comprises or has the sequence set forth in SEQ ID NO: 21.

[0178] In one embodiment of the antibody of the first aspect, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain constant region that comprises or has the sequence set forth in SEQ ID NO: 2 or 55, and a heavy chain variable region (VH) that comprises the CDR1, CDR2 and CDR3 sequences of the sequence set forth in SEQ ID NO: 20, and the light chain comprises a light chain constant region that comprises or has the sequence set forth in SEQ ID NO: 6, and a light chain variable region that comprises the CDR1, CDR2 and CDR3 sequences of the sequence set forth in SEQ ID NO: 21. For example, the CDR1, CDR2 and CDR3 sequences are as set forth herein.

[0179] Referring to specific examples of the monoclonal chimeric antibody (MAB-19-0202) and the monoclonal humanized antibody of the first aspect, the respective sequences are shown in Tables 3, 4, 6, and 7 in the Examples. The exemplary humanized antibody MAB-19-0618 is a humanized variant of MAB-19-0202.

[0180] In one embodiment of the antibody of the first aspect, the antibody is a monoclonal chimeric antibody, a monoclonal humanized antibody, or a fragment of such an antibody. The antibody may be a whole antibody or an antigen-binding fragment thereof, including, for example, a bispecific antibody. Furthermore, the antigen-binding fragment may include a binding domain immunoglobulin fusion protein comprising (i) a binding domain polypeptide (such as a heavy chain variable region or a light chain variable region) fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. Such binding domain immunoglobulin fusion proteins are further disclosed in U.S. Patent Application Publication Nos. 2003 / 0118592 and 2003 / 0133939.

[0181] In one embodiment of the antibody of the first aspect, the antibody or antigen-binding portion thereof comprises at least two heavy chains and at least two light chains, and the antibody comprises the same heavy chains and / or the same light chains, e.g., the heavy chains and / or light chains set forth herein above or below. For example, the antibody of the first aspect may comprise two heavy chains having the sequences set forth in SEQ ID NO:20 and SEQ ID NO:2 or 55, or respective variants of these sequences, and two light chains having the sequences set forth in SEQ ID NO:21 and SEQ ID NO:6, or respective variants of these sequences.

[0182] In one embodiment of the antibody of the first aspect, one or more, preferably both, heavy chain constant regions have been modified such that C1q binding to the antibody is reduced compared to the wild-type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%. In one embodiment, C1q binding can be determined by ELISA.

[0183] As used herein, "wild-type" or "WT" or "native" refers to an amino acid sequence found in nature, including allelic variants. A wild-type amino acid sequence, peptide, or protein has an amino acid sequence that has not been intentionally modified.

[0184] In one embodiment of the antibody of the first aspect, one or more, preferably both, heavy chain constant regions are modified so that the binding of the antibody to one or more IgG Fc-gamma receptors is reduced compared to the wild-type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%. In one embodiment, the one or more IgG Fc-gamma receptors are selected from at least one of Fc-gamma RI, Fc-gamma RII, and Fc-gamma RIII. In one embodiment, the IgG Fc-gamma receptor is Fc-gamma RI.

[0185] In one embodiment of the antibody of the first aspect, the antibody is unable to induce Fc-gammaRI-mediated effector function or the induced Fc-gammaRI-mediated effector function is reduced compared to the wild-type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%.

[0186] In one embodiment of the antibody of the first aspect, the antibody is unable to induce at least one of complement dependent cytotoxicity (CDC) mediated lysis, antibody dependent cellular lysis (ADCC) mediated lysis, apoptosis, homotypic adhesion and / or phagocytosis, or induces at least one of complement dependent cytotoxicity (CDC) mediated lysis, antibody dependent cellular lysis (ADCC) mediated lysis, apoptosis, homotypic adhesion and / or phagocytosis to a reduced extent, preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%.

[0187] Antibody-dependent cell-mediated cytotoxicity is also referred to herein as "ADCC." ADCC refers to the cell-killing ability of effector cells, particularly lymphocytes, described herein, and preferably requires that target cells be marked by an antibody.

[0188] ADCC occurs when an antibody binds to an antigen on a tumor cell and the Fc domain of the antibody engages with an Fc receptor (FcR) on the surface of an immune effector cell. Several families of Fc receptors have been identified, and specific cell populations are characterized by expressing defined Fc receptors. ADCC can be considered a mechanism that directly induces varying degrees of immediate tumor destruction, leading to antigen presentation and the induction of tumor-directed T cell responses. Preferably, in vivo induction of ADCC results in tumor-directed T cell responses and host-derived antibody responses.

[0189] Complement-dependent cytotoxicity is also referred to herein as "CDC." CDC is another cell killing method induced by antibodies. IgM is the most effective isotype for complement activation. IgG1 and IgG3 are also highly effective in inducing CDC via the classical complement activation pathway. Preferably, in this cascade, the formation of an antigen-antibody complex leads to the CDC of the participating antibody molecules, such as IgG molecules. H This results in the uncloaking of multiple, closely spaced C1q-binding sites on the C1q-2 domain (C1q is one of the three subcomponents of complement C1). Preferably, these uncloaked C1q-binding sites convert the previously low-affinity C1q-IgG interaction to one of high avidity, which triggers a cascade of events involving a series of other complement proteins, resulting in the proteolytic release of the effector cell chemotactic / activating factors C3a and C5a. Preferably, the complement cascade culminates in the formation of a membrane attack complex, which creates pores in the cell membrane, facilitating the free passage of water and solutes into and out of the cell, potentially triggering apoptosis.

[0190] In one embodiment of the antibody of the first aspect, the antibody has reduced or depleted effector function, hi one embodiment, the antibody does not mediate ADCC or CDC, or both.

[0191] In one embodiment of the antibody of the first aspect, one or more, preferably both, heavy chain constant regions of the antibody are modified relative to the wild-type antibody such that binding of the antibody to the neonatal Fc receptor (FcRn) is unaffected.

[0192] In one embodiment of the antibody of the first aspect, the PD-1 that the antibody can bind is human PD-1. In one embodiment, the PD-1 has or comprises the amino acid sequence set forth in SEQ ID NO:22 or SEQ ID NO:23, or the amino acid sequence of PD-1 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO:22 or SEQ ID NO:23, or is an immunogenic fragment thereof. In one embodiment, the antibody is capable of binding to a native epitope of PD-1 present on the surface of a living cell.

[0193] The antibodies of the first aspect can be used synergistically with conventional chemotherapeutic agents or other immunotherapies to attack tumors, for example by using other antibodies that target tumor antigens, thereby inducing an immune response against these tumor cells, or by using other checkpoint inhibitors or activators or angiogenesis inhibitors.

[0194] II. Antibody production Antibodies of the first embodiment can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibodies could be employed, e.g., viral or oncogenic transformation of B lymphocytes or phage display techniques using libraries of antibody genes.

[0195] The preferred animal system for preparing hybridomas secreting monoclonal antibodies is the murine system. Hybridoma production in mice is a well-established procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

[0196] Other preferred animal systems for preparing hybridomas secreting monoclonal antibodies are the rat and rabbit systems (e.g., as described in Spieker-Polet et al., Proc. Natl. Acad. Sci. USA 92:9348 (1995); see also Rossi et al., Am. J. Clin. Pathol. 124: 295 (2005)).

[0197] In yet another preferred embodiment, human monoclonal antibodies against PD-1 can be generated using transgenic or transchromosomal mice that carry parts of the human immune system rather than the mouse system. These transgenic and transchromosomal mice include mice known as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "transgenic mice." Production of human antibodies in such transgenic mice can be carried out as described in detail for CD20 in WO 2004 / 035607.

[0198] Another strategy for generating monoclonal antibodies is to directly isolate the antibody-encoding genes from lymphocytes that produce antibodies of a defined strategy, see, e.g., Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of a defined strategy. For details on recombinant antibody engineering, see also Welschof and Kraus, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8 and Benny KC Lo Antibody Engineering ISBN 1-58829-092-1.

[0199] immunization To generate antibodies against PD-1, animals, such as rabbits or mice, can be immunized as described with carrier-conjugated peptides derived from the PD-1 sequence, enriched preparations of recombinantly expressed PD-1 antigen or fragments thereof, and / or cells expressing PD-1. Alternatively, rabbits or mice can be immunized with DNA encoding full-length human PD-1 or fragments thereof. In the event that immunization with a purified or enriched preparation of PD-1 antigen does not yield antibodies, rabbits or mice can also be immunized with cells, such as a cell line, expressing PD-1 to stimulate an immune response.

[0200] Plasma and serum samples can be collected by tail vein or retroorbital bleeding to monitor the immune response over the course of the immunization protocol. Rabbits or mice with sufficient anti-PD-1 immunoglobulin titers can be used for fusions. Rabbits or mice can be boosted intraperitoneally or intravenously with PD-1-expressing cells 3 days before sacrifice and removal of the spleen to increase the proportion of hybridomas secreting specific antibodies.

[0201] Generation of hybridomas producing monoclonal antibodies To generate hybridomas producing monoclonal antibodies against PD-1, spleen cells and lymph node cells from immunized animals, such as rabbits or mice, can be isolated and fused with a suitable immortalized cell line, such as a mouse or rabbit myeloma cell line. The resulting hybridomas can then be screened for the production of antigen-specific antibodies. Individual wells can then be screened by ELISA for antibody-secreting hybridomas. Antibodies with specificity for PD-1 can be identified by immunofluorescence and FACS analysis using PD-1-expressing cells. Antibody-secreting hybridomas can be replicated and rescreened, and if still positive for anti-PD-1 monoclonal antibodies, they can be subcloned by limiting dilution. Stable subclones can then be cultured in vitro to generate and characterize antibodies in tissue culture medium.

[0202] Generation of monoclonal antibody-producing transfectomas The antibodies of the first embodiment can also be produced in host cell transfectomas, for example, using a combination of recombinant DNA technology and gene transfection methods well known in the art [Morrison, S. (1985) Science 229: 1202].

[0203] The term "transfectoma" as used herein includes recombinant eukaryotic host cells expressing an antibody, such as CHO cells, NS / 0 cells, HEK293 cells, HEK293T cells, HEK293T / 17 plant cells, or fungi, including yeast cells.

[0204] For example, in one embodiment, a gene of interest, e.g., an antibody gene, can be ligated into an expression vector, e.g., a eukaryotic expression plasmid such as those used in the GS gene expression system disclosed in International Publication Nos. 87 / 04462, 89 / 01036, and EP 338841, or other expression systems known in the art. The purified plasmid carrying the cloned antibody gene can be introduced into eukaryotic host cells such as CHO cells, NS / 0 cells, Sp2 / 0 cells, COS cells, Vero cells, HeLa cells, HEK293T cells, HEK293T / 17 cells, or HEK293 cells, or other eukaryotic cells such as plant-derived cells, fungal cells, or yeast cells. The method used to introduce these genes can be a method described in the art, such as electroporation, lipofectin, or lipofectamine. After introducing these antibody genes into host cells, cells expressing the antibody can be identified and selected. These cells are transfectomas, which can be amplified for expression levels and scaled up to produce antibodies. Recombinant antibodies can be isolated and purified from these culture supernatants and / or cells.

[0205] Alternatively, cloned antibody genes can be expressed in other expression systems, including prokaryotic cells such as microorganisms, e.g., E. coli. Furthermore, antibodies can be produced in transgenic non-human animals, such as the milk of sheep and rabbits or in the eggs of hens, or in transgenic plants; see, e.g., Verma, R., et al. (1998) J. Immunol. Meth. 216: 165-181; Pollock, et al. (1999) J. Immunol. Meth. 231: 147-157; and Fischer, R., et al. (1999) Biol. Chem. 380: 825-839.

[0206] Variants of the amino acid sequences described herein can be readily prepared by one skilled in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare proteins and peptides with substitutions, additions, insertions, or deletions is described in detail, for example, in Sambrook et al. (1989).

[0207] The antibody of the first aspect can also be produced in genetically modified viruses, such as RNA viruses, using recombinant DNA techniques well known to those skilled in the art. Recombinant viral genomes that can be used to rescue viral particles expressing antibodies or fragments thereof can be obtained, for example, by a method called "reverse genetics."

[0208] Use of partial antibody sequences to express intact antibodies (i.e., humanized and chimerized). a) Chimerization Although mouse or rabbit monoclonal antibodies can be used as therapeutic antibodies in humans, repeated administration of these antibodies can lead to increased immunogenicity and reduced therapeutic efficacy. The primary immunogenicity is mediated by the heavy chain constant region. The immunogenicity of mouse or rabbit antibodies in humans can be reduced or completely avoided if the respective antibodies are chimerized or humanized. A chimeric antibody is an antibody whose different portions are derived from different animal species, such as an antibody having a variable region derived from a mouse or rabbit antibody and a human immunoglobulin constant region. Antibody chimerization is achieved by combining the variable regions of the heavy and light chains of a mouse or rabbit antibody with the constant regions of the heavy and light chains of a human antibody (e.g., as described in Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy, ISBN-0-89603-918-8). In a preferred embodiment, a chimeric antibody is created by combining a human kappa-light chain constant region with a mouse or rabbit light chain variable region. In a further preferred embodiment, chimeric antibodies can be produced by joining a human lambda-light chain constant region to a mouse or rabbit light chain variable region. Preferred heavy chain constant regions for producing chimeric antibodies are IgG1, IgG3, and IgG4. Other preferred heavy chain constant regions for producing chimeric antibodies are IgG2, IgA, IgD, and IgM.

[0209] b) Humanization Antibodies interact with target antigens primarily 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 are more diverse among individual antibodies than those outside the CDRs. Because the CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody by constructing an expression vector in which the CDR sequences from a particular naturally occurring antibody are grafted onto framework sequences from a different antibody with different properties (see, for example, Riechmann, L. et al. (1998) Nature 332: 323-327; Jones, P. et al. (1986) Nature 321: 522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. USA 86: 10029-10033). Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences. These germline sequences differ from mature antibody gene sequences because they do not contain fully assembled variable genes formed by V(D)J joining during B-cell maturation. Germline gene sequences also differ evenly across the variable regions from sequences of high-affinity secondary repertoire antibodies. For example, somatic mutations are relatively rare in the amino-terminal portion of framework region 1 and the carboxy-terminal portion of framework region 4. Furthermore, many somatic mutations do not significantly alter the binding properties of the antibody. For this reason, it is not necessary to obtain the entire DNA sequence of a particular antibody to reconstruct an intact recombinant antibody with binding properties similar to those of the original antibody (see WO 99 / 45962). Partial sequences of the heavy and light chains spanning the CDR regions are typically sufficient for this purpose. The partial sequences are used to determine which germline variable gene segments and joining gene segments contributed to the recombinant antibody variable genes. The germline sequences are then used to fill in the missing portions of the variable regions. The heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody.To add missing sequences, cloned cDNA sequences can be combined with synthetic oligonucleotides by ligation or PCR amplification. Alternatively, the entire variable region can be synthesized as a set of overlapping short oligonucleotides and combined by PCR amplification to create a completely synthetic variable region clone. This process has certain advantages, such as the elimination or inclusion of specific restriction sites or the optimization of specific codons.

[0210] The nucleotide sequences of heavy and light chain transcripts from hybridomas are used to design overlapping sets of synthetic oligonucleotides to create synthetic V sequences with identical amino acid coding capacities as the native sequences. The synthetic heavy and kappa chain sequences can differ from the native sequences in three ways: strings of repetitive nucleotide bases are interrupted to facilitate oligonucleotide synthesis and PCR amplification; optimal translation start sites are incorporated according to Kozak's rules (Kozak, 1991, J. Biol. Chem. 266: 19867-19870); and a HindIII site is engineered upstream of the translation start site.

[0211] For both the heavy and light chain variable regions, the optimized coding strand sequences and corresponding non-coding sequences are broken down into 30-50 nucleotides approximately at the midpoint of the corresponding non-coding oligonucleotides. Thus, for each strand, the oligonucleotides can be assembled into overlapping double-stranded sets spanning a 150-400 nucleotide segment. This pool is used as a template to generate PCR amplification products of 150-400 nucleotides. Typically, a single set of variable region oligonucleotides is broken down into two pools, which are amplified separately to generate two overlapping PCR products. These overlapping products are then combined by PCR amplification to form the complete variable region. It may also be desirable to include overlapping fragments of the heavy or light chain constant region in the PCR amplification to generate fragments that can be easily cloned into expression vector constructs.

[0212] The reconstructed chimeric or humanized heavy and light chain variable regions are then combined with cloned promoter, leader, translation initiation, constant region, 3' untranslated, polyadenylation, and transcription termination sequences to form an expression vector construct. The heavy and light chain expression constructs can be combined into a single vector and co-transfected, sequentially transfected, or separately transfected into host cells, and then fused to form a host cell expressing both chains. Plasmids for constructing human IgGκ expression vectors are available to those skilled in the art. Plasmids can be constructed to reconstruct complete heavy and light chain minigenes using PCR-amplified V heavy and V kappa light chain cDNA sequences. These plasmids can be used to express fully human antibodies, chimeric IgG1 kappa antibodies, or IgG4 kappa antibodies. Similar plasmids can be constructed for the expression of other heavy chain isotypes or antibodies containing lambda light chains.

[0213] Thus, in accordance with the present disclosure, the structural features of the anti-PD-1 antibodies of the first embodiment can be used to generate structurally related humanized anti-PD-1 antibodies that retain at least one functional property of the antibodies of the first embodiment, such as binding to PD-1. More particularly, one or more of the CDR regions disclosed herein can be recombinantly combined with known human framework regions and CDRs to generate additional recombinantly engineered humanized anti-PD-1 antibodies of the first embodiment.

[0214] The antibody of the first aspect can be obtained by a method comprising the step of immunizing an animal, such as a transgenic mouse, with a protein or peptide having the amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23, or an immunogenic fragment thereof, or with a nucleic acid, host cell, or virus expressing said protein or peptide, or an immunogenic fragment thereof. Preferably, the antibody thus obtained is specific for said protein, peptide, or immunogenic fragment thereof. The nucleic acid, host cell, or virus may be a nucleic acid, host cell, or virus disclosed herein.

[0215] The present disclosure also provides B cells isolated from a non-human animal as described above. The isolated B cells can then be immortalized by fusion with an immortalizing cell to provide a source (e.g., a hybridoma) of the antibody of the first aspect. Such hybridomas (i.e., producing the antibody of the first aspect) are also within the scope of the present disclosure.

[0216] Thus, in a second aspect, a hybridoma capable of producing the antibody of the first aspect is provided. As exemplified herein, the antibody of the first aspect can be obtained directly from the hybridoma that expresses the antibody, or can be cloned and expressed recombinantly in host cells (e.g., CHO cells, or lymphocytic cells). Alternatively, it can be produced recombinantly in transgenic non-human animals or plants.

[0217] III. Antibody Characterization Binding to antigen-expressing cells The ability of the antibodies to bind to PD-1 and / or block PD-1 / ligand interactions can be determined using standard binding assays, such as those described in the Examples, reporter gene blocking assays, T cell proliferation assays, etc.

[0218] Characterization of antibody binding To purify anti-PD-1 antibodies, selected hybridomas can be grown in 2-L spinner flasks for monoclonal antibody purification. Alternatively, anti-PD-1 antibodies can be produced in dialysis-based bioreactors. The supernatant can be filtered and, if necessary, concentrated, and then subjected to affinity chromatography using protein G-Sepharose or protein A-Sepharose. The eluted IgG can be checked by gel electrophoresis and high-performance liquid chromatography to confirm purity. The buffer solution can be exchanged for PBS, and the concentration can be determined by OD280 using an extinction coefficient of 1.43. The monoclonal antibodies can be aliquoted and stored at -80°C. Site-directed or multi-site directed mutagenesis can be used to determine whether the selected anti-PD-1 monoclonal antibodies bind to unique epitopes.

[0219] Determination of PD-1 binding specificity The binding potency of an anti-PD-1 antibody to PD-1 can be determined by ELISA. For example, a PD-1 / Fc chimera can be coated onto a microtiter plate. After blocking, the anti-PD-1 antibody to be tested can be added and incubated. After a subsequent washing step, for example, an anti-human IgG coupled to horseradish peroxidase can be added for detection.

[0220] The binding ability of anti-PD-1 antibodies to cell surface-expressed PD-1 can be analyzed using HEK-293 cells ectopically expressing PD-1. Anti-PD-1 antibodies can be added to these cells at various concentrations and incubated. A fluorescently tagged anti-Ig antibody can then be added, and the cell-associated immunofluorescence signal can be recorded.

[0221] Determining the interrupting capacity The efficacy of anti-PD-1 antibodies in blocking the PD-1 / PD-L1 interaction can be analyzed using a PD-1 / PD-L1 blocking bioassay. PD-L1-expressing cells can be incubated with the antibody to be tested at various concentrations. PD-1-expressing effector cells can be added, and the resulting mixture can be incubated. For example, a luciferase assay reagent can be added, and luminescence can be measured. The PD-1 / PD-L1 blocking bioassay (Promega, catalog number J12150), or an equivalent kit, can be used as described by the manufacturer.

[0222] To characterize the ability of anti-PD1 antibodies to induce T cell proliferation in antigen-specific assays in which the PD-1 / PD-L1 axis is active, dendritic cells (DCs) expressing tumor antigens can be performed. Such assays are described in non-limiting manner in Example 5 below.

[0223] Flow cytometry analysis and immunofluorescence microscopy Flow cytometric or immunofluorescence microscopy analysis can be used by methods well known to those skilled in the art to demonstrate the presence of anti-PD-1 antibodies in the serum of immunized animals or the binding of monoclonal antibodies to live cells expressing PD-1.

[0224] Epitope mapping Mapping of the epitope recognized by the antibody of the first embodiment can be performed as described in detail in "Epitope Mapping Protocols", Methods in Molecular Biology by Glenn E. Morris ISBN-089603-375-9 and "Epitope Mapping: A Practical Approach", Practical Approach Series, 248 by Olwyn M. R. Westwood, Frank C. Hay.

[0225] IV. Bispecific / Multispecific Antibodies that Bind to PD-1 In one embodiment of the antibody of the first aspect, the antibody to PD-1 can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., a Fab' fragment), to generate a bispecific or multispecific molecule that binds to multiple binding sites or target epitopes. For example, the antibody of the first aspect can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association, etc.) to one or more other binding molecules, such as another antibody, peptide, or binding mimetic.

[0226] Accordingly, the present disclosure includes bispecific and multispecific molecules comprising at least one first binding specificity for PD-1 and a second binding specificity (or additional binding specificity) for a second target epitope (or additional target epitope).

[0227] In one embodiment of the antibody of the first aspect, the antibody is a multispecific antibody comprising a first antigen-binding region that binds to PD-1 and at least one additional antigen-binding region that binds to another antigen, hi one embodiment, the antibody is a bispecific antibody comprising a first antigen-binding region that binds to PD-1 and a second antigen-binding region that binds to another antigen.

[0228] The term "bispecific molecule" is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, having two different binding specificities. For example, the molecule may bind to or interact with (a) a cell surface antigen and (b) an Fc receptor on the surface of an effector cell. The term "multispecific molecule" or "heterospecific molecule" is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, having more than two different binding specificities. For example, the molecule may bind to or interact with (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, and (c) at least one other component. Thus, the present disclosure includes, but is not limited to, bispecific, trispecific, tetraspecific, and other multispecific molecules directed against PD-1 and other targets, such as Fc receptors on effector cells. The term "bispecific antibody" also includes diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the domains pair with complementary domains on another chain by using a linker that is too short to allow pairing between the two domains on the same chain, thereby forming two antigen-binding sites (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ, et al. (1994) Structure 2: 1121-1123).

[0229] As used herein, the term "heteroantibody" refers to the linkage of two or more antibodies, derivatives thereof, or antigen-binding regions, at least two of which have different specificities, including binding specificity for an Fc receptor on an effector cell and binding specificity for an antigen or epitope on a target cell, e.g., a tumor cell.

[0230] In one embodiment of the antibody of the first aspect, the first antigen-binding region of the multispecific antibody that binds to PD-1 comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) described herein.

[0231] In one embodiment of the antibody of the first aspect, the first and second binding arms of the multispecific antibody are derived from a full-length antibody, such as a full-length IgG1, λ (lambda) antibody or an IgG1, κ (kappa) antibody as described above. In one embodiment, the first and second binding arms are derived from a monoclonal antibody. For example, or in a preferred embodiment, the first and / or second binding arms are derived from an IgG1, κ or λ isotype, and preferably comprise a human IgG1 / κ or human IgG1 / λ constant portion.

[0232] The first antigen-binding region that binds to PD-1 of a multispecific or bispecific antibody of the invention may comprise the heavy and light chain variable regions of an antibody that competes with PD-L1 and / or PD-L2 for PD-1 binding. In one embodiment of a multispecific or bispecific antibody, the first antigen-binding region that binds to PD-1 comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) described herein.

[0233] In one embodiment of a multispecific or bispecific antibody, the second binding specificity can be directed to another immune checkpoint, thereby inhibiting or activating / stimulating the respective checkpoint. Other checkpoint inhibitors that can be targeted include, but are not limited to, CTLA4, PD-L1, TIM-3, KIR, or LAG-3. Checkpoint activators that can be targeted by the second binding specificity include, but are not limited to, CD27, CD28, CD40, CD122, CD137, OX40, GITR, or ICOS. Thus, bispecific and multispecific molecules are included that can bind to at least one other checkpoint and inhibit PD-1 through their respective binding. The second binding specificity can be antagonistic, such as anti-CTLA4, anti-PD-L1, anti-TIM-3, anti-KIR, or anti-LAG-3, or agonistic, such as anti-CD27, anti-CD28, anti-CD40, anti-CD122, anti-CD137, anti-OX40, anti-GITR, or anti-ICOS. Also included are multispecific molecules that bind to PD-1 and can also bind to at least one other immune checkpoint. Preferred combinations of binding specificities include anti-PD1 and anti-PD-L1, or anti-PD-1 and anti-CTLA4.

[0234] For example, CD28 provides the stimulatory guidance required for T cell activation. Similarly, CD137 (4-1BB, TNFRSF9) is a member of the tumor necrosis factor (TNF) receptor (TNFR) superfamily. CD137 is a costimulatory molecule on CD8+ and CD4+ T cells, regulatory T cells (Tregs), natural killer (NK) and NKT cells, B cells, and neutrophils. On T cells, CD137 is not constitutively expressed but is induced upon T cell receptor (TCR) activation. Stimulation via its natural ligand, 4-1BBL, or agonist antibodies results in signal transduction using TNFR-associated factor (TRAF)-2 and TRAF-1 as adaptors. Initial signaling by CD137 involves K-63 polyubiquitination, ultimately leading to activation of the nuclear factor (NF)-κB and mitogen-activated protein (MAP) kinase pathways. Signaling leads to increased T cell costimulation, proliferation, cytokine production, maturation, and prolonged survival of CD8+ T cells. Agonistic antibodies against CD137 have been shown to promote T cell-mediated antitumor control in various preclinical models [Murillo et al. 2008 Clin. Cancer Res. 14(21): 6895-6906]. Antibodies that stimulate CD137 can induce T cell survival and proliferation, thereby enhancing antitumor immune responses. Antibodies that stimulate CD137 have been disclosed in the prior art, including the human IgG4 antibody urelumab (WO 2005 / 035584) and the human IgG2 antibody utomilumab (Fisher et al. 2012 Cancer Immunol. Immunother. 61: 1721-1733).

[0235] In one embodiment of a multispecific or bispecific antibody, the second binding specificity can confer anti-angiogenic activity. Thus, the second binding specificity can target vascular endothelial growth factor (VEGF) or its receptor VEGFR (e.g., VEGFR1, 2, 3). Alternatively, or in addition, the second binding specificity can target PDGFR, c-Kit, Raf, and / or RET.

[0236] In one embodiment of the multispecific or bispecific antibody, the second or further binding specificity of the bispecific or multispecific molecule of the first aspect is directed against and capable of binding to a tumor antigen, thereby enabling specificity of the antibody for cancer cells. In one embodiment, the cancer cells may be selected from the group consisting of melanoma, lung cancer, renal cell carcinoma, bladder cancer, breast cancer, gastric cancer and gastroesophageal junction cancer, pancreatic adenocarcinoma, ovarian cancer, and lymphoma. The tumor antigen may be a surface antigen or an antigen presented in the context of MHC. The binding specificity may be based, for example, on a B-cell receptor (antibody) or a T-cell receptor.

[0237] The term "tumor antigen," as used herein, refers to a component of a cancer cell that may originate from the cytoplasm, cell surface, and cell nucleus. In particular, it refers to an antigen produced intracellularly or as a surface antigen on a tumor cell. Tumor antigens are typically preferentially expressed by cancer cells (e.g., expressed at higher levels in cancer cells than in non-cancerous cells), and in some cases, are expressed exclusively by cancer cells. Examples of tumor antigens include, but are not limited to, p53, ART-4, BAGE, beta-catenin / m, and Bcr-abL. CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, cell surface proteins of the claudin family, for example CLAUDIΝ-6, CLAUDIN-18.2 and CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9 , MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, Myosin / m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 minor BCR-abL, Pml / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT, and WT-1.

[0238] In one embodiment, the second antigen targeted is NY-ESO-1 (UniProt P78358), tyrosinase (UniProt P14679), MAGE-A3 (UniProt P43357), TPTE (UniProt P56180), KLK2 (UniProt P20151), PSA (KLK3) (UniProt P07288), PAP (ACPP, UniProt P15309), HOXB13 (UniProt Q92826), NKX3-1 (UniProt Q99801), HPV16 E6 / E7 (UniProt P03126 / P03129); HPV18 E6 / E7 (UniProt P06463 / P06788); HPV31 E6 / E7 (UniProt P06463 / P06788); P17386 / P17387); HPV33 E6 / E7 (UniProt P06427 / P06429); HPV45 E6 / E7 (UniProt P21735 / P21736); HPV58 E6 / E7 (UniProt P26555 / P26557), PRAME (UniProt P78395), ACTL8 (UniProt Q9H568), CXorf61 (KKLC1, UniProt Q5H943), MAGE-A9B (UniProt P43362), CLDN6 (UniProt P56747), PLAC1 (UniProt Q9HBJ0), and p53 (UniProt P04637).

[0239] Treatment methods involving these antigens can be aimed at treating cancer, where cancer cells are characterized by the expression of the respective antigens. It is also possible to use combinations of the antigens described herein, particularly NY-ESO-1, tyrosinase, MAGE-A3, TPTE, KLK2, PSA (KLK3), PAP (ACPP), HOXB13, NKX3-1, HPV16 E6 / E7; HPV18 E6 / E7, HPV31 E6 / E7, HPV33 E6 / E7, HPV45 E6 / E7, HPV58 E6 / E7, PRAME, ACTL8, CXorf61 (KKLC1), MAGE-A9B, CLDN6, PLAC1, and p53. Treatment methods involving such antigen combinations may be aimed at treating cancer, where the cancer cells are characterized by expression of two or more antigens from each combination of antigens, or where the cancer cells of a majority (e.g., at least 80%, at least 90%, or even more) of patients with a particular cancer being treated express one or more of each antigen in the combination. Such combinations may include combinations of at least two, at least three, at least four, at least five, or at least six antigens. Thus, combinations may include 3, 4, 5, 6, 7, or 8 antigens.

[0240] For the treatment of cutaneous melanoma, the additional binding specificity(s) may target at least one of the following antigens: NY-ESO-1, tyrosinase, MAGE-A3, and / or TPTE.

[0241] For the treatment of prostate cancer, the additional binding specificity(s) may target at least one of the following antigens: KLK2, PSA (KLK3), PAP (ACPP), HOXB13, and / or NKX3-1.

[0242] For the treatment of breast cancer, the additional binding specificity(s) may target at least one of the following antigens: PRAME, ACTL8, CXorf61 (KKLC1), MAGEA3, MAGE-A9B, CLDN6, NY-ESO-1, and / or PLAC1.

[0243] For the treatment of ovarian cancer, the additional binding specificity(s) may target at least one of the following antigens: CLDN6, p53, and / or PRAME.

[0244] As used herein, the term "effector cell" refers to an immune cell that is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, basophils, and the like.

[0245] "Target cell" is intended to mean any unwanted cell in a subject (e.g., a human or animal) that can be targeted by the antibody of the first aspect. In a preferred embodiment, the target cell is a tumor cell.

[0246] Bispecific and multispecific molecules of the first embodiment can be made using chemical techniques (see, e.g., D. M. Kranz et al. (1981) Proc. Natl. Acad. Sci. USA 78:5807), "polydoma" technology (see, U.S. Pat. No. 4,474,893, Reading), or recombinant DNA techniques.

[0247] In particular, bispecific and multispecific molecules of the first embodiment can be prepared by conjugating the constituent binding specificities, e.g., an anti-CTLA4 binding specificity and an anti-PD-1 binding specificity, using methods known in the art. For example, each binding specificity of the bispecific and multispecific molecules can be generated separately and conjugated to each other. When the binding specificities are proteins or peptides, various coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetylthioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) [see, e.g., Karpovsky et al. (1984) J. Exp. Med. 160: 1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82: 8648]. Other methods include those described by Paulus [Behring Ins. Mitt. (1985) No. 78, 118-132], Brennan et al. [Science (1985) 229: 81-83], and Glennie et al. [J. Immunol. (1987) 139: 2367-2375]. Preferred conjugating agents are SATA and sulfo-SMCC, available from Pierce Chemical Co. (Rockford, IL).

[0248] When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.

[0249] Alternatively, both binding specificities may be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific and multispecific molecules are mAb x mAb, mAb x Fab, Fab x F(ab')2, or ligand x Fab fusion proteins. Bispecific and multispecific molecules of the first embodiment, e.g., bispecific molecules, can be single-chain molecules such as single-chain bispecific antibodies, single-chain bispecific molecules comprising one single-chain antibody and a binding determinant, or single-chain bispecific molecules comprising two binding determinants. Bispecific and multispecific molecules can also be single-chain molecules or comprise at least two single-chain molecules. Methods for preparing bispecific and multispecific molecules are described, for example, in U.S. Patent Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858. Thus, the present disclosure encompasses all of these antibody formats.

[0250] Binding of bispecific and multispecific molecules to specific targets can be confirmed by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assays. Each of these assays generally detects the presence of a protein-antibody complex of interest by using a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, FcR-antibody complexes can be detected using, for example, an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to the antibody-FcR complex. Alternatively, the complexes can be detected using any of a variety of other immunoassays. For example, antibodies can be radiolabeled and used in a radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986). Radioactive isotopes can be detected by means such as the use of a γ-counter or scintillation counter or autoradiography.

[0251] V. Immunoconjugates The anti-PD-1 antibody of the first aspect may be conjugated to a moiety or agent. Such conjugates are also referred to herein as "immunoconjugates."

[0252] In a third aspect, a conjugate is provided comprising the antibody of the first aspect linked to a moiety or agent. In one embodiment of the third aspect, the moiety or agent is selected from the group consisting of a radioisotope, an enzyme, a dye, a drug, a toxin, and a cytotoxic agent. The dye can be, for example, a fluorescent dye or a fluorescent tag. In one embodiment, the moiety or agent can activate immune cells. For example, the moiety or agent can be CD80, which interacts with CD28 on T cells.

[0253] The antibody of the first embodiment may be linked or operably linked (e.g., by chemical coupling, genetic fusion, non-covalent association, etc.) to one or more other molecular entities, such as other antibodies that have binding specificity for PD-1. The one or more other antibodies are preferably antibodies of the first embodiment.

[0254] The moiety or agent can be an enzyme linked to an antibody, which can be used, for example, in an enzyme immunoassay, such as an enzyme-linked immunosorbent assay (ELISA) or enzyme-amplified immunoassay technique (EMIT), or in a Western blot.

[0255] Alternatively or additionally, a radionuclide (radioisotope) can be attached to the antibody as a moiety or drug. Such conjugates can be used for diagnostic purposes (radioimmunoassay, positron emission tomography ("immuno-PET")) as well as for therapeutic purposes. The radionuclide can be conjugated to the antibody via a complexing agent. The antibody of the first embodiment can also be conjugated to a radioisotope, such as iodine-131, yttrium-90, or indium-111, to generate a cytotoxic radiopharmaceutical for treating disorders such as cancer. The antibody of the first embodiment can also be attached to a linker-chelator, such as tiuxetan, that allows the antibody to be conjugated to a radioisotope.

[0256] Alternatively or additionally, the moiety or agent may be a tag, for example a fluorescent tag, also known as a fluorescent label or fluorescent probe. Ethidium bromide, fluorescein and green fluorescent protein are common tags.

[0257] Also included in the third aspect are conjugates comprising a therapeutic moiety or agent. The therapeutic moiety or agent may be a cytokine or CD80, which binds to CD28 to provide a costimulatory signal in T cell responses. The therapeutic moiety or agent may also be a cytotoxin or drug (e.g., an immunosuppressant). Immunoconjugates comprising one or more cytotoxins are referred to as "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is detrimental to, and in particular kills, cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or congeners thereof.

[0258] Therapeutic agents suitable for forming the immunoconjugates of the third embodiment include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). In a preferred embodiment, the therapeutic agent is a cytotoxic agent or a radiotoxic agent. In another embodiment, the therapeutic agent is an immunosuppressant. In yet another embodiment, the therapeutic agent is GM-CSF. In a preferred embodiment, the therapeutic agent is doxorubicin, cisplatin, bleomycin, sulfate, carmustine, chlorambucil, cyclophosphamide, or ricin A.

[0259] The antibody conjugates of the third embodiment can be used to modify a given biological response, and the drug moiety should not be construed as being limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins include, for example, enzymatically active toxins or active fragments thereof, such as abrin, ricin A, pseudomonas exotoxin, diphtheria toxin, etc.; proteins such as tumor necrosis factor or interferon-γ; or biological response modifiers, such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte-macrophage colony-stimulating factor ("GM-CSF"), granulocyte-colony-stimulating factor ("G-CSF"), or other growth factors.

[0260] Techniques for conjugating such therapeutic moieties to antibodies are well known and can be found, for example, in Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), Controlled Drug Delivery (2nd Ed.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Monoclonal Antibodies '84: Biological And Clinical Applications, Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies For Cancer Detection And Therapy, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibodies In Cancer Therapy," Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates," Immunol. Rev., 62: 119-58 (1982). The moiety of the conjugate, e.g., a therapeutic moiety or drug, can be conjugated to the antibody by a linker sequence. Suitable linker sequences are known to those skilled in the art.

[0261] In a fourth aspect, there is provided a multimer comprising at least two antibodies of the first aspect, or at least two conjugates of the third aspect, or a mixture of one or more antibodies of the first aspect and one or more conjugates of the third aspect. In one embodiment, the multimer comprises 4 to 8 antibodies of the first aspect or 4 to 8 conjugates of the third aspect. The antibodies or conjugates of the multimers of these aspects may be linked to each other by peptides. The multimers of the fourth aspect are characterized by an increased number of antigen-binding sites for PD-1.

[0262] Thus, a wide variety of antibody conjugates, bispecific and multispecific molecules, and fusion proteins are encompassed, all of which bind to PD-1-expressing cells and can be used to target other molecules to such cells.

[0263] VI. Nucleic Acids Encoding Antibodies In a fifth aspect, the present disclosure also relates to a nucleic acid or nucleic acid molecule comprising a gene or nucleic acid sequence encoding an antibody or portion thereof described herein, e.g., an antibody chain, e.g., an antibody heavy chain of an antibody having the ability to bind to PD-1 described herein with respect to the first aspect, and / or an antibody light chain of an antibody having the ability to bind to PD-1 described herein with respect to the first aspect.

[0264] The term "nucleic acid molecule" or "nucleic acid," as used herein, is intended to include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules, such as in vitro transcribed RNA (IVT RNA). Nucleic acid, as used herein, includes genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. According to the fourth aspect, the nucleic acid can exist as a single-stranded or double-stranded, linear, or covalently closed circular molecule. For example, the nucleic acid is double-stranded DNA. The nucleic acid can be used for introduction into cells, i.e., transfection, for example, in the form of RNA, which can be prepared by in vitro transcription from a DNA template. The RNA can further be modified before application by adding stabilizing sequences, capping, or polyadenylation.

[0265] As used herein, the term "encoding nucleic acid" means that the nucleic acid, when present in an appropriate environment, preferably a cell, can be expressed to produce the protein or peptide that it encodes.

[0266] The nucleic acid according to the fifth aspect may be isolated. The term "isolated nucleic acid," as used herein, means that the nucleic acid is (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) produced recombinantly by cloning, (iii) purified, for example by cleavage and gel electrophoretic fractionation, or (iv) synthesized, for example by chemical synthesis. An isolated nucleic acid is a nucleic acid that is available for manipulation by recombinant DNA techniques.

[0267] In one embodiment of the fifth aspect, the nucleic acid, e.g., RNA, is associated with at least one agent that has a stabilizing effect on the nucleic acid. The stabilizing effect may include protection from RNA degradation. In one embodiment of this aspect, the at least one agent forms a complex with and / or surrounds the RNA. In one embodiment, the at least one agent comprises at least one agent selected from the group consisting of RNA-complexed lipids, RNA-complexed polymers, and RNA-complexed peptides or proteins. For example, the at least one agent is selected from at least one of the group consisting of polyethyleneimine, protamine, poly-L-lysine, poly-L-arginine, and histones.

[0268] According to the fifth aspect, the nucleic acid may be present alone or in combination with other nucleic acids, which may be homologous or heterologous. In a preferred embodiment, the nucleic acid is operably linked to an expression control sequence, which may be homologous or heterologous with respect to the nucleic acid. The term "homologous" means that the nucleic acid is also naturally operably linked to an expression control sequence, and the term "heterologous" means that the nucleic acid is not naturally operably linked to an expression control sequence.

[0269] A nucleic acid, such as a nucleic acid that expresses an RNA and / or a protein or peptide, and an expression control sequence are "operably" linked to each other when they are covalently linked to each other in such a way that expression or transcription of the nucleic acid is under the control or influence of the expression control sequence. If a nucleic acid is to be translated into a functional protein, an expression control sequence operably linked to a coding sequence will cause transcription of the nucleic acid upon induction of the expression control sequence without frameshifting the coding sequence or rendering the coding sequence intranslatable into the desired protein or peptide.

[0270] The term "expression control sequence" as used herein includes promoters, ribosome binding sites, enhancers, and other control elements that regulate the transcription of genes or the translation of mRNA. In certain embodiments, expression control sequences can be regulated. The exact structure of an expression control sequence can vary as a function of species or cell type, but generally includes 5'-untranscribed sequences, such as the TATA box, capping sequence, and CAAT sequence, involved in the initiation of transcription and translation, respectively, as well as 5'- and 3'-untranslated sequences (5'-UTR; 3'-UTR). More specifically, 5'-untranscribed expression control sequences include promoter regions containing promoter sequences for transcriptional control of operably linked nucleic acids. Expression control sequences can also include enhancer sequences or upstream activator sequences. Expression control sequences or regulatory sequences can be homologous or heterologous to the nucleic acid.

[0271] The term "promoter" or "promoter region," as used herein, refers to a nucleic acid sequence located upstream (5') of a nucleic acid sequence to be expressed, which controls the expression of the sequence by providing a recognition and binding site for RNA polymerase. A "promoter region" may contain additional recognition and binding sites for additional factors involved in regulating gene transcription. A promoter may control the transcription of a gene in a prokaryotic or eukaryotic organism. Furthermore, a promoter may be "inducible" and initiate transcription in response to an inducing agent, or it may be "constitutive" if transcription is not controlled by the inducing agent. A gene under the control of an inducible promoter is not expressed, or is expressed only to a small extent, in the absence of an inducing agent. In the presence of an inducing agent, the gene is switched on or transcription levels increase. This is generally mediated by the binding of specific transcription factors.

[0272] Preferred promoters according to the fifth aspect include the promoters of SP6, T3 and T7 polymerases, the human U6 RNA promoter, the CMV promoter, and artificial hybrid promoters (e.g. CMV) in which part(s) thereof are fused with part(s) of the promoter of the gene of another cellular protein, such as human GAPDH (glyceraldehyde-3-phosphate dehydrogenase), with or without additional intron(s).

[0273] The term "expression" is used herein in its most general sense and includes the production of RNA, or the production of RNA and protein / peptide. It also includes partial expression of a nucleic acid. Furthermore, expression can be transient or stable.

[0274] In one embodiment of the fifth aspect, the nucleic acid is RNA, more preferably in vitro transcribed RNA (IVT RNA) or synthetic RNA.

[0275] Nucleic acids such as RNA can be used for introduction into cells, i.e., transfection, particularly in the form of RNA, which can be prepared by in vitro transcription from a DNA template. RNA can also be further modified prior to application by the addition of stabilizing sequences, capping, or polyadenylation.

[0276] As used herein, the term "RNA" refers to a molecule that contains, and preferably is composed entirely or substantially of, ribonucleotide residues. "Ribonucleotide" refers to a nucleotide that has a hydroxyl group at the 2'-position of a β-D-ribofuranosyl group. The term "RNA" includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially or completely purified RNA, essentially pure RNA, synthetic RNA, and recombinantly produced RNA, such as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may include the addition of non-nucleotide material to one or more nucleotides of the RNA, such as to the end or within the RNA. The nucleotides of an RNA molecule may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0277] The term "RNA" as used herein includes and preferably relates to "mRNA." The term "mRNA" refers to "messenger RNA" and refers to a "transcript" generated using a DNA template and encoding a peptide or polypeptide. The promoter for controlling transcription can be any promoter for any RNA polymerase. A DNA template for in vitro transcription may be obtained by cloning a nucleic acid, particularly a cDNA, and introducing it into an appropriate vector for in vitro transcription. cDNA may also be obtained by reverse transcription of RNA. Typically, mRNA contains a 5'-UTR, a protein-coding region, and a 3'-UTR. mRNA has a limited half-life in cells and in vitro. mRNA can be generated by in vitro transcription from a DNA template. Methods for in vitro transcription are known to those skilled in the art. For example, various in vitro transcription kits are commercially available. As used herein, the term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA.

[0278] The stability and translation efficiency of RNA can be modified as needed. RNA molecules with improved stability and improved translation efficiency can be advantageous, for example, for the RNA-encoded antibodies of the present disclosure. For example, RNA can be stabilized and its translation increased by one or more modifications that have a stabilizing effect on the RNA and / or increase translation efficiency. Such modifications are described, for example, in PCT / EP2006 / 009448, which is incorporated herein by reference. To increase the expression of RNA used in accordance with the present disclosure, the sequence encoding the expressed peptide or protein can be modified within the coding region to enhance translation in cells by increasing GC content and optimizing codons to increase mRNA stability, preferably without changing the sequence of the expressed peptide or protein.

[0279] The term "modification" in the context of RNA, as used herein, includes any modification of RNA that does not occur naturally in said RNA.

[0280] The RNA according to the fifth aspect may have modified ribonucleotides to increase its stability and / or reduce its cytotoxicity. For example, in one embodiment, the RNA comprises 5-methylcytidines partially or fully, preferably fully, substituted for cytidine.

[0281] In some embodiments, one or more uridines in the RNA described herein are replaced with modified nucleosides. In some embodiments, the modified nucleosides are modified uridines. In some embodiments, the modified uridines that replace the uridines are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U). The uridines in the RNA can be partially or completely replaced, preferably completely replaced.

[0282] In some embodiments, at least one RNA contains a modified nucleoside in place of at least one uridine. In some embodiments, at least one RNA contains a modified nucleoside in place of each uridine. In some embodiments, each RNA contains a modified nucleoside in place of at least one uridine. In some embodiments, each RNA contains a modified nucleoside in place of each uridine.

[0283] In one embodiment, the RNA contains other modified nucleosides or further modified nucleosides, such as modified cytidine. For example, in one embodiment, 5-methylcytidine is partially or completely, preferably completely, substituted with cytidine in the RNA. In one embodiment, the RNA contains 5-methylcytidine as well as pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), and 5-methyl-uridine (m 5 In one embodiment, the RNA comprises one or more selected from 5-methylcytidine and N1-methyl-pseudouridine (mU). 1 In some embodiments, the RNA contains 5-methylcytidine in place of each cytidine and N1-methyl-pseudouridine (m ψ) in place of each uridine. 1 ψ).

[0284] In one embodiment, the term "modification" refers to providing an RNA with a 5'-cap or 5'-cap analog. The term "5'-cap" refers to the cap structure found at the 5'-end of an mRNA molecule, generally consisting of a guanosine nucleotide attached to the mRNA via an aberrant 5' to 5' triphosphate linkage.

[0285] Providing RNA with a 5'-cap or 5'-cap analog may be achieved by in vitro transcription of a DNA template in the presence of the 5'-cap or 5'-cap analog, where the 5'-cap is co-transcriptionally incorporated into the generated RNA strand, or the RNA may be generated, for example, by in vitro transcription, and the 5'-cap may be attached to the RNA post-transcriptionally using a capping enzyme, for example, vaccinia virus capping enzyme.

[0286] In some embodiments, the RNA according to the fifth aspect comprises a 5'-UTR and / or a 3'-UTR.

[0287] The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or a corresponding region in an RNA molecule, such as an mRNA molecule. The untranslated region (UTR) can be located 5' (upstream) of the open reading frame (5'-UTR) and / or 3' (downstream) of the open reading frame (3'-UTR). If present, the 5'-UTR is located at the 5'-end upstream of the start codon of the protein-coding region. If present, the 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap. If present, the 3'-UTR is located at the 3'-end downstream of the stop codon of the protein-coding region, although the term "3'-UTR" preferably does not include a polyA sequence. Thus, the 3'-UTR is upstream of the polyA sequence (if present), e.g., directly adjacent to the polyA sequence.

[0288] Incorporating a 3'-UTR into the 3'-untranslated region of an RNA molecule can increase translation efficiency. Incorporating two or more such 3'-untranslated regions can produce a synergistic effect. The 3'-untranslated regions can be autologous or heterologous to the RNA into which they are introduced. In a specific embodiment, the 3'-untranslated region is derived from the human β-globin gene.

[0289] To increase the stability and / or expression of the RNA according to the fifth aspect, it may be modified so that it is present in combination with a polyA sequence having a length of preferably 10 to 500, more preferably 30 to 300, even more preferably 65 to 200, and particularly preferably 100 to 150 adenosine residues. The polyA sequence does not have to be masked.

[0290] The term "poly(A) tail" or "poly(A) sequence," as used herein, relates to an uninterrupted or interrupted sequence of adenyl (A) residues typically located at the 3'-end of an RNA molecule, and an "unmasked poly(A) sequence" means that the poly(A) sequence at the 3'-end of an RNA molecule ends with the A of the poly(A) sequence and is not followed by any nucleotides other than A located at the 3'-end, i.e., downstream, of the poly(A) sequence.

[0291] In some embodiments, the polyA tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template containing repetitive dT nucleotides (deoxythymidylic acid) in the strand complementary to the coding strand. The DNA sequence encoding the polyA tail (coding strand) is called a poly(A) cassette.

[0292] In some embodiments, the poly(A) cassette present in the coding strand of DNA consists essentially of dA nucleotides but is interrupted by random sequences of the four nucleotides (dA, dC, dG, and dT). Such random sequences may be 5-50, 10-30, or 10-20 nucleotides in length. Such cassettes are disclosed in International Publication No. 2016 / 005324, which is incorporated herein by reference. Any poly(A) cassette disclosed in International Publication No. 2016 / 005324 may be used in the present disclosure. Poly(A) cassettes consisting essentially of dA nucleotides but interrupted by random sequences with an even distribution of the four nucleotides (dA, dC, dG, dT), for example, having a length of 5-50 nucleotides, exhibit consistent growth of plasmid DNA in E. coli at the DNA level and are still associated with beneficial properties for supporting RNA stability and translation efficiency at the RNA level, and are therefore encompassed. As a result, in some embodiments, the poly-A tail contained in the RNA molecules described herein consists essentially of A nucleotides, but is interrupted by random sequences of four nucleotides (A, C, G, U). Such random sequences may be 5-50, 10-30, or 10-20 nucleotides in length.

[0293] In this context, "consisting essentially of" means that most nucleotides in the poly-A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the number of nucleotides in the poly-A tail, are A nucleotides, while allowing the remaining nucleotides to be nucleotides other than A nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), or C nucleotides (cytidylic acid). In this context, "consisting of" means that all nucleotides in the poly-A tail, i.e., 100% of the number of nucleotides in the poly-A tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylic acid.

[0294] In some embodiments, nucleotides other than A nucleotides do not flank the polyA tail at its 3'-end, i.e., the polyA tail is not masked or followed at its 3'-end by nucleotides other than A.

[0295] The combination of the above modifications, ie, incorporation of a polyA sequence, unmasking of a polyA sequence, and incorporation of one or more 3'-untranslated regions, has a synergistic effect on improving RNA stability and translation efficiency.

[0296] The term "stability" of RNA relates to the "half-life" of RNA. "Half-life" relates to the period required to eliminate half of the activity, amount, or number of a molecule. In the context used herein, the half-life of RNA refers to the stability of said RNA. The half-life of RNA can affect the "expression period" of RNA. RNA with a long half-life can be expected to be expressed for a long period of time.

[0297] Of course, in accordance with the present disclosure, if it is desired to reduce the stability and / or translation efficiency of an RNA, the RNA can be modified to interfere with the function of such elements that increase RNA stability and / or translation efficiency.

[0298] The term "expression" is used herein in its most general sense and includes the production of RNA and / or the production of a peptide, polypeptide, or protein, for example, by transcription and / or translation. With respect to RNA, the terms "expression" or "translation" particularly relate to the production of a peptide, polypeptide, or protein. It also includes partial expression of a nucleic acid. Furthermore, expression may be transient or stable. According to the present disclosure, an antibody is expressed in a cell if the antibody can be detected in the cell or its lysate by conventional techniques for protein detection, such as techniques using an antibody that specifically binds to the PD-1 antibody.

[0299] As used herein, the term "transcription" refers to the process by which genetic code in a DNA sequence is transcribed into RNA. The RNA can then be translated into protein. As used herein, the term "transcription" includes "in vitro transcription," which refers to the process by which RNA, particularly mRNA, is synthesized in vitro in a cell-free system, preferably using an appropriate cell extract. Preferably, a cloning vector is used to generate the transcript. These cloning vectors are commonly called transcription vectors and are encompassed by the term "vector." According to the present disclosure, the RNA preferably used is in vitro transcribed RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly cDNA, and introducing it into an appropriate vector for in vitro transcription. cDNA can also be obtained by reverse transcription of RNA.

[0300] The term "translation", as used herein, relates to the process in a cell's ribosomes where a chain of messenger RNA directs the assembly of amino acid sequences to make peptides, polypeptides and proteins.

[0301] Terms such as "expressible RNA" and "encoding RNA" are used interchangeably herein and, in relation to a particular peptide or polypeptide, mean that the RNA is capable of being expressed to produce said peptide or polypeptide when present in an appropriate environment, preferably a cell. Preferably, the RNA according to the fifth aspect is capable of interacting with cellular translation machinery to provide an expressible peptide or polypeptide.

[0302] Terms such as "transfecting," "introducing," or "transfecting" are used interchangeably herein and refer to the introduction of nucleic acids, particularly exogenous or heterologous nucleic acids, particularly RNA, into cells. According to the present disclosure, cells may form part of an organ, tissue, and / or organism. According to the present disclosure, administration of nucleic acids is achieved as naked nucleic acids or in combination with an administration reagent. Preferably, administration of nucleic acids is in the form of naked nucleic acids. Preferably, RNA is administered in combination with a stabilizing agent, such as an RNase inhibitor. The present disclosure also contemplates repeated introduction of nucleic acids into cells for long-term sustained expression.

[0303] In a sixth aspect, the present disclosure provides a vector comprising one or more of the nucleic acids of the fifth aspect.

[0304] Where appropriate, the vector of the sixth aspect may further comprise a promoter controlling expression of the nucleic acid. The term "vector" is used herein in its most general sense and includes, for example, any intermediate vehicle for a nucleic acid, allowing the nucleic acid to be introduced into prokaryotic and / or eukaryotic cells and, where appropriate, integrated into the genome. This type of vector is preferably replicated and / or expressed intracellularly. Vectors include plasmids, phagemids, bacteriophages, or viral genomes such as adenovirus or baculovirus vectors, cosmids, or other vectors conventional in, for example, genetic engineering, as well as liposomes. Such vectors include expression vectors and cloning vectors. Expression vectors, including plasmids as well as viral vectors, generally contain a desired coding sequence and appropriate DNA sequences required for expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify a specific desired DNA fragment and may lack functional sequences required for expression of the desired DNA fragment. The term "plasmid", as used herein, generally refers to a construct of extrachromosomal genetic material, usually a circular double-stranded DNA, that is capable of replicating independently of chromosomal DNA.

[0305] The vector of the sixth aspect may further comprise a gene, such as a marker gene, that allows for the selection of the vector in a suitable host cell under appropriate conditions. Furthermore, the vector may comprise expression control elements that allow for the appropriate expression of the coding region in a suitable host. Such control elements are known to those skilled in the art and may include a promoter, a splice cassette, and a translation initiation codon. For example, the nucleic acid of the fifth aspect is operably linked to the above-described expression control sequence that allows expression in eukaryotic or prokaryotic cells. Control elements that ensure expression in eukaryotic or prokaryotic cells are well known to those skilled in the art. Methods for constructing nucleic acid molecules according to the fifth aspect, constructing vectors containing the nucleic acid molecules, introducing the vectors into appropriately selected host cells, and causing or achieving expression are well known in the art.

[0306] Vectors for cloning or expression using recombinant techniques are known in the art and include, for example, plasmid-based expression vectors, adenovirus vectors, retrovirus vectors, or baculovirus vectors. Exemplary vectors include pGEX, pET, pLexA, pBI, pVITRO, pVIVO, and pST such as pST4.

[0307] The type of vector for expressing the antibody of the first aspect may be a vector in which the heavy chain and light chain of the antibody are present in different vectors, or a vector in which the heavy chain and light chain are present in the same vector.

[0308] In one embodiment of the sixth aspect, the vector is a multilamellar vesicle, a unilamellar vesicle, or a mixture thereof.

[0309] In one embodiment of the sixth aspect, the vector is a liposome. In one embodiment, the vector is a cationic liposome. The liposome or cationic liposome may contain a phospholipid, such as phosphatidylcholine, and / or a sterol, such as cholesterol. In one embodiment, the liposome or cationic liposome has a particle size ranging from about 50 nm to about 200 nm.

[0310] In one embodiment of the sixth aspect, the vesicle, liposome, or cationic liposome further comprises a ligand for site-specific targeting. The ligand is, for example, an antibody. In one embodiment, the ligand, for example, an antibody, is capable of binding to cancer cells, particularly cancer cells described herein. In one embodiment, the vector releases RNA in tumor cells and / or enters tumor cells. In one embodiment, the ligand, for example, an antibody, binds to a protein associated with the surface of abnormal cells, such as tumor cells. For example, the ligand or antibody can bind to the extracellular portion of a disease-related antigen.

[0311] In one embodiment of the sixth aspect, the RNA of the fifth aspect can be present in RNA lipoplex particles. The RNA lipoplex particles and compositions comprising the RNA lipoplex particles described herein are useful for delivering RNA to target tissues after parenteral administration, particularly after intravenous administration. The RNA lipoplex particles can be prepared using liposomes and by mixing the liposomes with RNA. The RNA lipoplex particles can be obtained, for example, by mixing the RNA with liposomes or at least one cationic lipid, for example, using an ethanol injection technique.

[0312] The term "ethanol injection technique" refers to a process in which an ethanol solution containing lipids is rapidly injected through a needle into an aqueous solution. This action disperses the lipids throughout the solution and promotes lipid structure formation, e.g., lipid vesicle formation, such as liposome formation. Generally, the RNA lipoplex particles described herein can be obtained by adding RNA to a colloidal liposome dispersion. Using the ethanol injection technique, such a colloidal liposome dispersion is formed, in one embodiment, as follows: an ethanol solution containing lipids, such as a cationic lipid, such as DOTMA, and an additional lipid, is injected into an aqueous solution under stirring. In one embodiment, the RNA lipoplex particles described herein can be obtained without an extrusion process.

[0313] The term "extruding" or "extrusion" refers to the creation of particles having a defined cross-sectional profile, particularly by reducing the particle size, by forcing the particles through a filter with predetermined pores.

[0314] In one embodiment, the RNA lipoplex particles may have an average diameter in the range of about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 nm to about 700 nm, about 400 nm to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In one embodiment, the RNA lipoplex particles have an average diameter in the range of about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter in the range of about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

[0315] The RNA lipoplex particles may exhibit a polydispersity index of less than about 0.5, less than about 0.4, or less than about 0.3. For example, the RNA lipoplex particles may exhibit a polydispersity index in the range of about 0.1 to about 0.3.

[0316] The term "polydispersity index" is used herein as a measure of the particle size distribution of a population of particles, e.g., nanoparticles. The polydispersity index is calculated based on dynamic light scattering measurements by so-called cumulant analysis.

[0317] A composition comprising RNA lipoplex particles may include at least one buffer and / or stabilizer and / or chelating agent. For example, the stabilizer can prevent substantial loss of product quality, particularly substantial loss of RNA activity, during storage, such as freezing, lyophilization, spray drying, or storage of a frozen, lyophilized, or spray-dried composition. A chelating agent refers to a chemical compound capable of forming at least two coordinate covalent bonds with a metal ion, thereby forming a stable, water-soluble complex. Without wishing to be bound by theory, chelating agents may reduce the concentration of free divalent ions, which may otherwise promote RNA degradation. Examples of suitable chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), salts of EDTA, desferrioxamine B, deferoxamine, dithiocarb sodium, penicillamine, calcium pentetate, sodium salt of pentetate, succimer, trientine, nitrilotriacetic acid, trans-diaminocyclohexanetetraacetic acid (DCTA), diethylenetriaminepentaacetic acid (DTPA), bis(aminoethyl)glycolether-N,N,N',N'-tetraacetic acid, iminodiacetic acid, citric acid, tartaric acid, fumaric acid, or salts thereof.

[0318] The freeze-dried or spray-dried composition can be reconstituted before use. In some embodiments, the stabilizer is a carbohydrate. As used herein, the term "carbohydrate" refers to and includes monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides. In embodiments of the present disclosure, the stabilizer is mannose, glucose, sucrose, or trehalose. According to the present disclosure, the RNA lipoplex particle composition may have a stabilizer concentration suitable for the stability of the composition, particularly the stability of the RNA lipoplex particles and the stability of the RNA.

[0319] The term "freezing" generally refers to the solidification of a liquid by the removal of heat.

[0320] The terms "lyophilizing" or "lyophilization" refer to freeze-drying a substance by freezing the substance and then reducing the surrounding pressure to cause the freezing medium in the substance to sublimate directly from the solid phase to the gas phase.

[0321] The term "spray-drying" refers to spray-drying a substance by mixing a (heated) gas with an atomized (atomized) fluid in a vessel (spray dryer), which forms droplets from which the solvent evaporates, resulting in a dry powder.

[0322] The term "reconstitute" refers to the addition of a solvent, such as water, to a dried product to return it to a liquid state, such as its original liquid state.

[0323] Compositions comprising RNA lipoplex particles may be liquid or solid, non-limiting examples of which include frozen or lyophilized forms.

[0324] In the context of the present disclosure, the term "particle" relates to a structured entity formed by a molecule or a molecular complex. In one embodiment, the term "particle" relates to a micro- or nano-sized structure, e.g., a micro- or nano-sized compact structure.

[0325] The term "RNA lipoplex particle," as used herein, refers to a particle containing lipids, particularly cationic lipids, and RNA. The electrostatic interaction between the positively charged liposome and the negatively charged RNA results in the formation of a complex, resulting in the spontaneous formation of the RNA lipoplex particle. Positively charged liposomes can generally be synthesized using a cationic lipid, such as DOTMA, and an additional lipid, such as DOPE. In one embodiment, the RNA lipoplex particle is a nanoparticle.

[0326] As used in this disclosure, "nanoparticle" refers to a particle comprising RNA and at least one cationic lipid and having an average diameter suitable for intravenous administration.

[0327] The term "mean diameter" refers to the average hydrodynamic diameter of particles measured by dynamic light scattering (DLS) by data analysis using the so-called cumulant algorithm, which results in a so-called Z-axis with a length dimension. 平均 , and the dimensionless polydispersity index (PI) (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here, the "average diameter", "diameter" or "size" of a particle is defined as this Z 平均 Used synonymously with the value of

[0328] In one embodiment, the nucleic acid of the fifth aspect or the vector of the sixth aspect comprises a nucleic acid sequence encoding an antibody chain described in the first and sixth aspects. For example, the nucleic acid sequence encoding the antibody or antibody chain may comprise a nucleic acid sequence encoding an antibody described herein (e.g., MAB-19-0202, MAB-19-0618), or the heavy or light chain of one of these antibodies.

[0329] The antibody chain may be a heavy chain (H chain = HC) or a light chain (L chain = LC), each of which is preferably described herein. In one embodiment, the H chain comprises a heavy chain variable region (VH) and a heavy chain constant region, and the heavy chain constant region may comprise a heavy chain CH1 constant region, or a combination of a heavy chain CH1 constant region, a heavy chain CH2 constant region, and a heavy chain CH3 constant region. In one embodiment, the CH1 constant domain and the CH2 constant domain may be connected by a hinge region located between the CH1 constant domain and the CH2 constant domain.

[0330] In one embodiment, the L chain comprises a light chain variable region (VL) and a light chain constant region, which may be a CL kappa constant domain or a CL lambda constant domain. For example, the light chain constant region may have or include the sequence set forth in SEQ ID NO: 6, or a variant thereof.

[0331] In one embodiment, the nucleic acid encoding the antibody or antibody chain of the first aspect comprises a nucleic acid sequence encoding a heavy chain variable region (VH) comprising at least one of the HCDR1, HCDR2, and HCDR3 sequences exemplified herein (SEQ ID NOs: 8-13, SYN in the Sequence Listing). That is, the nucleic acid of the fifth aspect may comprise a nucleic acid sequence encoding an HCDR1, HCDR2, or HCDR3 sequence exemplified herein, or the nucleic acid may comprise a nucleic acid sequence encoding a heavy chain variable region (VH) comprising any combination of the HCDR1, HCDR2, and HCDR3 sequences defined herein. Preferred combinations of individual HCDR1-HCDR3 sequences are as set forth above with respect to each amino acid sequence of the first aspect. This teaching also applies, as appropriate, to the nucleic acid sequence of the fifth aspect.

[0332] In one embodiment, the nucleic acid of the fifth aspect comprises a nucleic acid sequence encoding a light chain variable region (VL) comprising at least one of the LCDR1, LCDR2, and LCDR3 sequences exemplified herein (SEQ ID NOS: 14-17, QAS in the Sequence Listing). That is, the nucleic acid may comprise a nucleic acid sequence encoding an LCDR1, LCDR2, or LCDR3 sequence exemplified herein, or the nucleic acid may comprise a nucleic acid sequence encoding a light chain variable region (VL) comprising any combination of the LCDR1, LCDR2, and LCDR3 sequences defined herein. Preferred combinations of individual LCDR1 to LCDR3 sequences are as set forth above with respect to the respective amino acid sequences of the first aspect. This teaching also applies, as appropriate, to the nucleic acid sequence of the fifth aspect.

[0333] In one embodiment, the nucleic acid of the fifth aspect comprises a nucleic acid sequence encoding the VH and VL sequences exemplified herein (SEQ ID NOs: 18 to 21 in the Sequence Listing).

[0334] In one embodiment, there is provided a nucleic acid, or a vector comprising a nucleic acid, e.g., an RNA or RNA-based vector, or a vector suitable for in vitro transcription, comprising a nucleic acid sequence encoding the heavy chain variable region (VH) and / or light chain variable region (VL) of an antibody that binds PD-1 according to the first aspect.

[0335] In one embodiment, the variant nucleic acid sequence comprises / encodes one or more of the respective CDR1, CDR2, and CDR3 amino acid sequences set forth herein. That is, the variant nucleic acid sequence encoding the heavy chain variable region (VH) can comprise / encode one or more of the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth herein, and for specific combinations of CDR sequences, reference is made to the respective disclosures herein. For example, the variant nucleic acid sequence can comprise / encode the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth herein.

[0336] The variant nucleic acid sequence encoding the light chain variable region (VL) may comprise / encode one or more of the LCDR1, LCDR2, and LCDR3 amino acid sequences set forth herein, and for specific combinations of CDR sequences, reference is made to the respective disclosures herein. For example, the variant nucleic acid sequence may comprise / encode the LCDR1, LCDR2, and LCDR3 amino acid sequences set forth herein.

[0337] The variant nucleic acid sequence may encode a heavy chain variable region (VH) or light chain variable region (VL), respectively, that is capable of providing the same binding specificity and / or functionality as that provided by the heavy chain variable region (VH) or light chain variable region (VL) of the parent sequence.

[0338] The nucleic acid of the fifth aspect, or the vector (eg, RNA or RNA-based vector) of the sixth aspect for producing an anti-PD-1 antibody may be produced by in vitro transcription methods.

[0339] Such methods include inserting the DNA sequence of a heavy chain variable region (VH) or light chain variable region (VL) as defined herein above into an IVT vector (e.g., a pST4 vector) using standard cloning techniques. The vector may include a 5'-UTR as defined herein, a 3'-UTR as defined herein, a poly(A) tail as defined herein above, e.g., a poly(A) tail comprising 30 adenine nucleotides, a linker (L), and further (A30LA70). Furthermore, the IVT vector may optionally include a nucleic acid sequence encoding a secretory signal peptide, e.g., a secretory signal peptide as defined herein.

[0340] To create a template for in vitro transcription, the plasmid DNA can be linearized downstream of the poly(A) tail coding region, e.g., using a restriction endonuclease, thereby creating a template for transcribing mRNA, e.g., by using T7 RNA polymerase.

[0341] Upon in vitro transcription, the RNA may be modified and capped at its 5' end to minimize immunogenicity.

[0342] The thus obtained, appropriately capped RNA is used to transfect host cells, such as NS0 cells, Sp2 / 0 cells, HEK293 cells or derivatives thereof, such as HEK293T, HEK293T / 17 and / or HEK293F, COS cells, Vero cells and / or HeLa cells. In one embodiment, the mammalian host cells are selected from HEK293, HEK293T and / or HEK293T / 17 cells. For transfection, liposomes, such as those described herein above, may be used.

[0343] The transfected cells are used to express the antibody or antibody chain, or fragment thereof. To express both the heavy and light chains of an anti-PD-1 antibody, the host cell is preferably transfected with both types of RNA, i.e., individual RNAs encoding the heavy and light chains of the anti-PD-1 antibody, respectively.

[0344] Anti-PD-1 antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the antibody is produced intracellularly, the cells are then lysed and cellular debris removed, for example, by centrifugation or ultrafiltration. Those skilled in the art are familiar with suitable methods for isolating antibodies produced intracellularly. Methods for isolating antibodies secreted into the periplasmic space are similar. If the antibody is secreted into the culture medium, for example, by using a secretory signal peptide, the supernatant from such an expression system can first be concentrated, for example, by using a commercially available protein concentration filter. A protease inhibitor, such as PMSF, can be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of contaminants. Anti-PD-1 antibodies prepared from transfected host cells can be purified, for example, by using chromatography, such as affinity chromatography, gel electrophoresis, flow cytometry, and / or dialysis.

[0345] The teachings provided herein regarding particular nucleic acid and amino acid sequences, e.g., sequences set forth in the Sequence Listing, are also to be construed as relating to modifications of the particular sequences that result in sequences functionally equivalent to the particular sequences, e.g., amino acid sequences that exhibit the same or similar properties as the particular amino acid sequences, and nucleic acid sequences that encode amino acid sequences that exhibit the same or similar properties as the amino acid sequences encoded by the particular nucleic acid sequences. One important property is to retain binding of the antibody to its target or to maintain the desired effector function of the antibody. Preferably, the modified sequence with respect to the particular sequence, when substituted for the particular sequence in the antibody, retains the binding of the antibody to PD-1 and preferably retains the function of the antibody described herein, e.g., PD-1 inhibition of immunosuppression, CDC-mediated lysis, or ADCC-mediated lysis on cells expressing PD-1.

[0346] For example, nucleic acid and amino acid sequence variants, as described herein, encode or provide antibodies or antigen-binding fragments that provide at least one of the following properties: (i) capable of binding, preferably specifically binding, to PD-1, e.g., human PD-1; (ii) capable of blocking the binding of PD-1 to its ligand; (iii) capable of binding to the same antigen as the parent antibody, preferably with sufficient affinity for diagnostic and / or therapeutic use; and / or (iv) The ability to reduce or deplete effector function.

[0347] Those skilled in the art will recognize that the sequences of the CDRs, hypervariable regions, and variable regions, in particular, can be altered without losing the ability to bind to PD-1. For example, the CDR regions can be identical to or highly homologous to the regions explicitly described herein. By "highly homologous," it is intended that one to five, preferably one to four, e.g., one to three or one to two substitutions can be made in the CDRs. Furthermore, the hypervariable and variable regions can be altered to exhibit substantial homology with the regions specifically disclosed herein.

[0348] The term "variant" as used herein refers to mutants, splice variants, conformations, isoforms, allelic variants, species variants, and species homologs, especially those that occur naturally. Allelic variants refer to changes in the normal sequence of a gene, the significance of which is often unclear. Complete gene sequencing often identifies a large number of allelic variants for a given gene. Species homologs are nucleic acid or amino acid sequences that have a different species origin from the given nucleic acid or amino acid sequence. The term "variant" is intended to encompass any post-translational modification variants and conformational variants.

[0349] For purposes of this disclosure, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those occurring naturally. The term "variant" particularly includes fragments of an amino acid sequence.

[0350] Amino acid insertion variants include the insertion of a single or two or more amino acids into a specific amino acid sequence. In the case of an amino acid sequence variant with an insertion, one or more amino acid residues are inserted into a specific site in the amino acid sequence, but random insertion is also possible with appropriate screening of the resulting product.

[0351] Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, for example 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.

[0352] Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may be at any position in the protein. Amino acid deletion variants, including deletions at the N-terminus and / or C-terminus of the protein, are also referred to as N- and / or C-terminal truncation variants.

[0353] Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another. The replacement of one amino acid with another can be classified as conservative or non-conservative. Preference is given to alterations at positions in the amino acid sequence that are not conserved among homologous proteins or peptides, and / or the replacement of an amino acid with another amino acid with similar properties. Preferably, amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include the substitution of a member of a family of amino acids related by their side chains. In the context of the present disclosure, a "conservative substitution" refers to the substitution of one amino acid with another amino acid with similar structural and / or chemical properties, where the amino acid residue is replaced with another amino acid residue from the same class as defined in either of the two tables above: for example, leucine may be substituted with isoleucine, since both are aliphatic, branched, and hydrophobic. Similarly, aspartic acid may be substituted with glutamic acid, since both are small, negatively charged residues. In one embodiment, conservative amino acid substitutions include substitutions within the following groups: - glycine, alanine; - valine, isoleucine, leucine; - Aspartic acid, glutamic acid; - Asparagine, glutamine; - serine, threonine; - lysine, arginine; and - Phenylalanine, tyrosine.

[0354] Preferably, the degree of similarity, preferably the degree of identity, between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence will be at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given over an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably consecutive amino acids. In a preferred embodiment, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using best sequence alignment, for example, Align, using standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0355] "Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences.

[0356] The term "percentage of identity" is intended to indicate the percentage of amino acid residues that are identical between the two sequences being compared, obtained after the best alignment, and this percentage is purely statistical, with the differences between the two sequences being randomly distributed over their entire length. Sequence comparison between two amino acid sequences is conventionally carried out by comparing these sequences after they have been optimally aligned, said comparison being carried out segment by segment or "window of comparison" in order to identify and compare local regions of sequence similarity. In addition to being generated manually, optimal alignment of sequences for comparison can be generated by the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by the similar search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or by computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0357] The percentage identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions compared, and multiplying the result by 100 to obtain the percentage identity between the two sequences.

[0358] With respect to nucleic acid molecules, the term "variant" includes degenerate nucleic acid sequences, which as used herein are nucleic acids that differ in codon sequence from a reference nucleic acid due to degeneration in the genetic code.

[0359] Furthermore, a "variant" of a given nucleic acid sequence, as used herein, includes a nucleic acid sequence that contains single or multiple, e.g., at least 2, at least 4, or at least 6, preferably up to 3, up to 4, up to 5, up to 6, up to 10, up to 15, or up to 20, nucleotide substitutions, deletions, and / or additions.

[0360] Preferably, the degree of identity between a given nucleic acid sequence and a nucleic acid sequence that is a variant of the given nucleic acid sequence will be at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, or most preferably at least 95%, 96%, 97%, 98%, or 99%. The degree of identity is preferably provided over a region of at least about 30, at least about 50, at least about 70, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, or at least about 400 nucleotides. In a preferred embodiment, the degree of identity is provided over the entire length of the reference nucleic acid sequence.

[0361] "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.

[0362] The term "percentage of identity" is intended to indicate the percentage of nucleotides that are identical between the two sequences being compared, obtained after the best alignment, and this percentage is purely statistical, with the differences between the two sequences being randomly distributed over their entire length. Sequence comparison between two nucleotides is conventionally carried out by comparing these sequences after optimal alignment, said comparison being carried out segment by segment or "window of comparison" to identify and compare local regions of sequence similarity. In addition to being generated manually, optimal alignment of sequences for comparison can be generated by the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by the similar search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or by computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0363] The percentage identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions compared, and multiplying the result by 100 to obtain the percentage identity between the two sequences.

[0364] The terms "part," "fragment," and "portion" are used interchangeably herein and refer to a continuous or discontinuous fraction of a structure. With respect to a particular structure, such as an amino acid sequence or a protein or a nucleic acid sequence, the terms "part," "fragment," and "portion" may designate a continuous or discontinuous fraction of said structure. Preferably, a "part," "a fragment," and "a portion" of a structure, such as an amino acid sequence or a nucleic acid sequence, comprises, and preferably consists of, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% of the entire structure or amino acid sequence or nucleic acid sequence. A portion, part, or fragment of a structure preferably comprises one or more functional properties of said structure. For example, a portion, part, or fragment of an epitope, peptide, or protein is preferably immunologically equivalent to the epitope, peptide, or protein from which it is derived. When a "portion," "part," or "fragment" is a discontinuous fraction, said discontinuous fraction preferably consists of 2, 3, 4, 5, 6, 7, 8, or more portions of the structure, each portion being a contiguous element of the structure. For example, a discontinuous fraction of an amino acid sequence may consist of 2, 3, 4, 5, 6, 7, 8, or more portions, preferably no more than four, of said amino acid sequence, each portion preferably comprising at least 5 consecutive amino acids, at least 10 consecutive amino acids, preferably at least 20 consecutive amino acids, preferably at least 30 consecutive amino acids of the amino acid sequence.

[0365] It should be understood that the nucleic acids described herein also include nucleic acids modified to optimize codon usage in a particular host cell or organism. Differences in codon usage between organisms can cause various problems with heterologous gene expression. Codon optimization by changing one or more nucleotides in the original sequence can optimize the expression of the nucleic acid, particularly translation efficiency, in the same or different host in which the nucleic acid is to be expressed. For example, when a nucleic acid derived from a human encoding an antibody constant region and / or framework region is used to prepare, for example, a chimeric antibody or a humanized antibody, it may be preferable to modify the nucleic acid to optimize codon usage, especially when the nucleic acid may be fused to a heterologous nucleic acid, such as a nucleic acid derived from another organism described herein, and is expressed in cells of an organism other than a human, such as a mouse or hamster. For example, nucleic acid sequences encoding human light and heavy chain constant regions can be modified to contain one or more, preferably at least 1, 2, 3, 4, 5, 10, 15, 20, preferably up to 10, 15, 20, 25, 30, 50, 70, or 100 or more, nucleotide substitutions, resulting in optimized codon usage but no change in amino acid sequence.

[0366] The term "genetic material" includes isolated nucleic acids, DNA or RNA, sections of double helices, sections of chromosomes, or the entire genome of an organism or cell, particularly its exome or transcriptome.

[0367] The term "mutation" refers to a change or difference (nucleotide substitution, addition, or deletion) in a nucleic acid sequence compared to a reference sequence. "Somatic mutations" can occur in any cell of the body except germ cells (sperm and eggs) and are therefore not inherited by children. These changes may (but do not necessarily) cause cancer or other diseases. Preferably, the mutation is a nonsynonymous mutation. The term "nonsynonymous mutation" refers to a mutation, preferably a nucleotide substitution, that results in an amino acid change, such as an amino acid substitution, in the translation product.

[0368] The term "mutation," as used herein, includes point mutations, indels, fusions, chromosolysis, and RNA editing. The term "indel," as used herein, describes a special classification of mutations and is defined as a mutation in which an insertion and deletion co-localize, resulting in a net gain or loss of nucleotides. In the coding region of a genome, an indel results in a frameshift mutation unless its length is a multiple of three. Indels can be contrasted with point mutations, in which nucleotides are inserted and deleted from a sequence; point mutations are a form of substitution in which one nucleotide is replaced. The term "chromosolysis," as used herein, refers to a genetic phenomenon in which a specific region of a genome is disrupted and reconnected by a single disruptive event. The terms "RNA edit" or "RNA editing," as used herein, refer to a molecular process in which the information content of an RNA molecule is altered through chemical changes to the base structure. RNA editing includes nucleoside modifications such as deamination of cytidine (C) to uridine (U) and adenosine (A) to inosine (I), as well as the addition and insertion of non-templated nucleotides. RNA editing in mRNA effectively alters the amino acid sequence of the encoded protein, making it different from that predicted from the genomic DNA sequence.

[0369] According to the present disclosure, a "reference" can be used to correlate and compare results obtained from tumor samples. Typically, the "reference" can be based on one or more normal samples obtained from the patient or one or more different individuals, preferably healthy individuals, particularly individuals of the same species, particularly samples not affected by cancer disease. The "reference" can be empirically determined by testing a sufficiently large number of normal samples.

[0370] In a seventh aspect, there is provided a cell or host cell comprising a nucleic acid of the fifth aspect or a vector of the sixth aspect.

[0371] Cells can be transfected with any carrier that can be associated with the nucleic acid of the fifth aspect, such as RNA, for example, by forming a complex with RNA or by forming a vesicle in which RNA is enclosed or encapsulated, thereby increasing the stability of RNA compared to naked RNA.Useful carriers according to the present disclosure include, for example, cationic lipids, liposomes, particularly cationic liposomes, and lipid-containing carriers such as micelles, and nanoparticles such as lipoplex particles.Cationic lipids can form complexes with negatively charged nucleic acids.Any cationic lipid can be used according to the present disclosure.

[0372] Transfectable cells also include recombinant host cells. The term "recombinant host cell," as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in progeny, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "recombinant host cell" as used herein. Host cells can be prokaryotic and / or eukaryotic host cells. Exogenous nucleic acids and / or vectors can be introduced into these host cells. In one embodiment, the host cell is a eukaryotic host cell, preferably a mammalian host cell. In one embodiment, the mammalian host cell is a CHO (Chinese Hamster Ovary) cell, a derivative of a CHO cell line such as CHO-K1 and CHO pro-3, or a lymphocytic cell. In one embodiment, the mammalian host cell is selected from mouse myeloma cells such as NS0 and Sp2 / 0, HEK293 (human embryonic kidney) cells or derivatives thereof, such as HEK293T, HEK293T / 17, and / or HEK293F, COS cells and Vero cells (all green monkey kidney), and / or HeLa (human cervical carcinoma) cells. In one embodiment, the mammalian host cell is selected from HEK293, HEK293T, and / or HEK293T / 17 cells. Further examples of host cells are microorganisms, such as E. coli, and fungi, such as yeasts, e.g., Saccharomyces cerevisiae, or filamentous fungi, e.g., Neurospora hosts and Aspergillus hosts. Host cells and recombinant host cells include, for example, CHO cells, NS / 0 cells, Sp2 / 0 cells, COS cells, Vero cells, HeLa cells, HEK293 cells, HEK293T cells, HEK293T / 17 cells, and transfectomas such as lymphocytic cells.

[0373] The host cells used to produce the antibodies defined herein can be cultured in a variety of media that are commercially available and familiar to those skilled in the art, any of which may be supplemented with hormones and / or other growth factors as needed.

[0374] In an eighth aspect, there is provided a virus comprising the nucleic acid of the fifth aspect or the vector of the sixth aspect.

[0375] According to the present disclosure, instead of providing / administering nucleic acids by using carriers such as, for example, lipid-containing carriers such as cationic lipids, liposomes, particularly cationic liposomes, micelles, and nanoparticles such as lipoplex particles, the nucleic acid of interest may be provided / administered by using recombinant host cells, preferably cells as defined above, or recombinant viruses encoding antibodies or antibody fragments derived from antibodies.

[0376] These viruses can be DNA or RNA viruses. Several viral vectors have shown promising results regarding their potential to enhance immunotherapy of malignant diseases. Replication-competent and replication-incompetent viruses can be used, with the latter group being preferred. Herpesviruses, adenoviruses, vaccinia viruses, reoviruses, and Newcastle disease viruses are examples of preferred viruses useful in accordance with the present disclosure. In one embodiment, the virus or viral vector is selected from the group consisting of adenoviruses, adeno-associated viruses, vaccinia viruses, and poxviruses, including attenuated poxviruses, Semliki Forest viruses, reoviruses, retroviruses, Newcastle disease viruses, Sindbis viruses, and Ty virus-like particles. Adenoviruses and retroviruses are particularly preferred. Retroviruses are typically replication-deficient (i.e., unable to produce infectious particles).

[0377] Methods for introducing nucleic acids into cells in vitro or in vivo include transfection with nucleic acid calcium phosphate precipitates, transfection with DEAE-associated nucleic acids, transfection or infection with the aforementioned viruses carrying the nucleic acid of interest, liposome-mediated transfection, etc. In certain embodiments, targeting nucleic acids to specific cells is a priority. In such embodiments, the carrier (e.g., retrovirus or liposome) used to administer nucleic acids to cells may have a targeting control molecule attached. For example, molecules such as antibodies specific to surface membrane proteins on target cells or ligands for receptors on target cells may be incorporated into or attached to the nucleic acid carrier. Preferred antibodies include antibodies that selectively bind to tumor antigens. When administration of nucleic acids via liposomes is desired, proteins that bind to surface membrane proteins associated with endocytosis may be incorporated into the liposome formulation to enable targeting control and / or uptake. Such proteins include capsid proteins or fragments thereof specific to specific cell types, antibodies against internalizing proteins, proteins corresponding to intracellular sites, etc.

[0378] Preferably, introduction of RNA encoding a peptide or polypeptide into cells, particularly into cells present in vivo, results in the expression of the peptide or polypeptide in the cells. In certain embodiments, targeting of nucleic acids to specific cells is preferred. In such embodiments, the carrier (e.g., retrovirus or liposome) used for administering nucleic acids to cells exhibits a targeting molecule. For example, a molecule such as an antibody specific to a surface membrane protein on the target cell or a ligand for a receptor on the target cell may be incorporated into or bound to the nucleic acid carrier. When nucleic acids are administered via liposomes, a protein that binds to a surface membrane protein associated with endocytosis may be incorporated into the liposome formulation to enable targeting and / or uptake. Such proteins include capsid proteins, fragments thereof specific to a specific cell type, or antibodies against internalizing proteins, proteins that target intracellular locations, etc.

[0379] Unless otherwise indicated herein or clearly contradicted by context, it will be understood that the teachings given in Section VI of this specification regarding nucleic acids encoding antibodies are also applicable, as appropriate, to nucleic acids / polynucleotides encoding peptides or proteins comprising epitopes of antigens. Spleen-targeted RNA lipoplex particles that can be beneficially used for expressing RNA in antigen-presenting cells are described in WO 2013 / 143683, which is incorporated herein by reference.

[0380] VII. Pharmaceutical Compositions In a ninth aspect, the present disclosure provides compositions, e.g., pharmaceutical compositions, comprising one or a combination of nucleic acids comprising nucleic acid sequences encoding the antibodies, including one or a combination of antibodies comprising the conjugates and / or multimers of the first, third, and / or fourth aspects, and / or host cells or vectors comprising the nucleic acids of the fifth, sixth, and / or seventh aspects. Pharmaceutical compositions can be formulated with pharmaceutically acceptable carriers or diluents, as well as any other known adjuvants and excipients, according to conventional techniques, such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. In one embodiment, the composition comprises a combination of multiple (e.g., two or more) isolated antibodies. In another embodiment, the composition comprises a combination of multiple (e.g., two or more) nucleic acids, vectors, or host cells.

[0381] As used herein, "pharmaceutically acceptable carriers" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for cardiovascular (e.g., intravenous or intraarterial), intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compounds, i.e., antibodies, bispecific and multispecific molecules, nucleic acids, vectors, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0382] A "pharmaceutically acceptable substance" refers to a substance that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects (see, e.g., Berge, SM, et al. (1977) J. Pharm. Sci. 66: 1-19).

[0383] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), saline and buffered aqueous solutions, vegetable oils such as olive oil, injectable organic esters such as ethyl oleate, and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0384] The carrier or composition of the ninth embodiment may also contain a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts that may be included include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphoric acid, as well as those derived from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, and aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, and calcium, and those derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0385] The composition of the ninth embodiment may also contain an antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0386] The composition of the ninth embodiment may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured by both sterilization procedures and the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Furthermore, the inclusion of agents that delay absorption, such as monostearate salts and gelatin, can result in prolonged absorption of injectable pharmaceutical forms.

[0387] Pharmaceutically acceptable carriers include sterile aqueous solution or dispersion and sterile powder for extemporaneously preparing sterile injectable solution or dispersion.The use of such media and agents for pharmaceutically active substances is well known in the art.Except when conventional media or agents are incompatible with active compounds, they are contemplated for use in the pharmaceutical compositions of the ninth aspect.Additional active compounds can also be incorporated into compositions.

[0388] Pharmaceutical compositions are typically sterile and must be stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.

[0389] In one embodiment of the ninth aspect, the pharmaceutical composition is formulated for parenteral administration, preferably for cardiovascular, particularly intravenous or intra-arterial administration.

[0390] The composition of the ninth aspect can be administered by various methods known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. The active compound can be prepared with a carrier that protects the compound from rapid release, such as a controlled-release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable and biocompatible polymers that can be used include ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations are generally known to those skilled in the art. For example, see "Sustained and Controlled Release Drug Delivery Systems," J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0391] In order to administer a compound (for example, the antibody or nucleic acid or vector or a combination of nucleic acids or vectors of the above-mentioned embodiments) by a certain administration route, it may be necessary to coat the compound with or co-administer it with a material to prevent inactivation.For example, the compound may be administered to a subject in a suitable carrier, such as liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.Liposomes include water-in-oil-in-water CGF emulsions, as well as conventional liposomes [Strejan et al. (1984) J. Neuroimmunol. 7: 27].

[0392] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.

[0393] Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization), which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0394] The dosage regimen is adjusted to obtain the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in single dosage form. As used herein, the term "single dosage form" refers to physically discrete units suitable as a unitary dosage for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the necessary pharmaceutical carrier. The specifications of the single dosage form of the present disclosure are determined by and directly depend on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the technology of compounding such active compounds to treat individual hypersensitivity.

[0395] The pharmaceutical formulation or composition of the ninth aspect includes those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration.The formulation may be conveniently presented in a single dosage form and can be prepared by any method known in the art of pharmacy.The amount of active ingredient to produce a single dosage form varies depending on the subject to be treated and the specific mode of administration.The amount of active ingredient to produce a single dosage form is generally the amount of the composition that produces a therapeutic effect.

[0396] Dosage forms for topical or transdermal administration of the composition of the ninth aspect include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and any preservatives, other adjuvants, or excipients that may be required.

[0397] The phrases "parenteral administration" and "parenterally administered," as used herein, mean modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0398] In one embodiment, the anti-PD-1 antibody is administered as a protein, and the antibody can be obtained from a hybridoma, transfectoma, or by in vitro transcription as described herein. In one embodiment, the anti-PD-1 antibody is administered as one or more nucleic acids or one or more vectors as defined herein, e.g., RNA encoding the antibody or chain of the antibody, or a fragment of such an antibody or chain, or RNA containing one or more RNAs, or liposomes.

[0399] In one embodiment, the antibody of the first aspect is administered in crystalline form by subcutaneous injection, see Yang et al. (2003) PNAS, 100 (12): 6934-6939.

[0400] When the compounds of the present disclosure are administered to humans and animals as pharmaceuticals, they can be administered alone or in combination with a pharmaceutically acceptable carrier, preferably a pharmaceutically acceptable carrier as defined above, as a pharmaceutical composition containing, for example, from about 0.01 percent to about 99 percent of the active ingredient, preferably from about 0.1 percent to about 90 percent, and most preferably from about 1 percent to about 50 percent, and may further include adjuvants and / or excipients such as antioxidants or preservatives.

[0401] Regardless of the route of administration selected, the compounds of the present disclosure, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions of the ninth aspect, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0402] Pharmaceutical compositions can be administered using medical equipment known in the art.For example, in preferred embodiments, pharmaceutical compositions of the ninth aspect can be administered using needleless hypodermic injection equipment, such as the equipment disclosed in United States Patent No. 5,399,163; United States Patent No. 5,383,851; United States Patent No. 5,312,335; United States Patent No. 5,064,413; United States Patent No. 4,941,880; United States Patent No. 4,790,824; or United States Patent No. 4,596,556. Examples of well-known implants and modules useful in the present disclosure include those described in the following: U.S. Pat. No. 4,487,603 discloses an implantable microinfusion pump for infusing medication at a controlled rate; U.S. Pat. No. 4,486,194 discloses a therapeutic device for administering medication through the skin; U.S. Pat. No. 4,447,233 discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224 discloses a variable flow rate implantable infusion device for continuous drug delivery; U.S. Pat. No. 4,439,196 discloses an osmotic drug delivery system having a multi-chamber compartment; and U.S. Pat. No. 4,475,196 discloses an osmotic drug delivery system.

[0403] Many other such implants, delivery systems, and modules are known to those skilled in the art. In certain embodiments, the antibodies of the above aspects can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the present disclosure cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patent Nos. 4,522,811; 5,374,548; and 5,399,331. Liposomes may contain one or more moieties that selectively transport to specific cells or organs, thereby enhancing targeted drug delivery [see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29: 685]. Exemplary targeting moieties include folic acid or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al.); mannosides [Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038]; antibodies [PG Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180]; and surfactant protein A receptor [Briscoe et al. (1995) Am. J. Physiol. 1233: 134].

[0404] In one embodiment, the therapeutic compound of the present disclosure is formulated in liposomes. In a more preferred embodiment, the liposomes contain a targeting moiety. In a most preferred embodiment, the therapeutic compound in the liposomes is delivered to the desired area, for example, a site adjacent to the tumor site, by bolus injection. The composition must be fluid enough to be easily squirted. It must also be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0405] In further embodiments, the antibodies of the above aspects can be formulated to prevent or reduce transport across the placenta. This can be accomplished by methods known in the art, such as pegylation of the antibody or the use of F(ab)2' fragments. See also Cunningham-Rundles C, Zhuo Z, Griffith B, Keenan J. (1992), "Biological activities of polyethylene-glycol immunoglobulin conjugates. Resistance to enzymatic degradation." J. Immunol. Methods, 152: 177-190; and Landor M. (1995), "Maternal-fetal transfer of immunoglobulins," Ann. Allergy Asthma Immunol. 74: 279-283.

[0406] The composition must be sterile and fluid enough to be deliverable by syringe.In addition to water, the carrier can be isotonic buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof.Proper fluidity can be maintained, for example, by using a coating such as lecithin, maintaining the required particle size in the case of dispersion, and using surfactants.In many cases, it is preferable to include isotonic agents, such as sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.Prolonged absorption of injectable compositions can be achieved by including an agent that delays absorption, such as aluminum monostearate or gelatin in the composition.

[0407] When the active compound is suitably protected, as described above, the compound may be orally administered, for example, with an inert diluent or an assimilable edible carrier.

[0408] VIII. Uses and Methods of the Invention The antibodies, conjugates, multimers, nucleic acids, vectors, host cells and viruses of the above embodiments have numerous therapeutic utilities relating to the treatment of diseases involving cells expressing PD-1 or its ligands (PD-L1 and / or PD-L2).

[0409] Thus, in a further aspect, the present disclosure relates to the medical use of the antibody, conjugate, multimer, nucleic acid, vector, host cell, virus, or composition of the above-mentioned aspects. In this regard, the present disclosure provides an antibody, conjugate, multimer, nucleic acid, vector, host cell, virus, or composition, preferably a pharmaceutical composition, for use in the treatment of a disease, for example, the treatment of tumors / cancer. The expression "for use in the treatment of a disease, for example, the treatment of tumors / cancer" is used herein interchangeably with "for use in a medicament, in particular in a method for the treatment of cancer"; or the use of the product in the preparation of a pharmaceutical formulation for use in said method of treatment in humans (or more generally in a subject in need thereof).

[0410] Hereinafter, when describing the preferred uses and methods of the present disclosure, reference will be made to the antibody of the first aspect. However, unless otherwise indicated herein or clearly contradicted by the context, it should be understood that this teaching is also applicable to other active agents comprising or encoding antibodies, namely the conjugate of the third aspect, the multimer of the fourth aspect, the nucleic acid of the fifth aspect, the vector of the sixth aspect, the host cell of the seventh aspect, the virus of the eighth aspect, or the composition of the ninth aspect.

[0411] For example, the antibodies or nucleic acids can be administered to cells in culture, e.g., in vitro or ex vivo, or to a subject, preferably a human subject, e.g., in vivo, to treat or prevent various diseases, such as those described herein.

[0412] In a tenth aspect, there is provided a pharmaceutical composition of the ninth aspect for use in the prophylactic and / or therapeutic treatment of disease.

[0413] The term "disease" as used herein refers to any pathological condition, including a cancer or tumor, particularly a tumor or cancer as described herein, or a form of autoimmune disease.

[0414] "Tumor" or "cancer" refers to an abnormal group of cells or tissue that grows by rapid and uncontrolled cell proliferation and continues to grow after the stimulus that initiated the new growth has ceased. Tumors exhibit a partial or complete lack of structural organization and functional coordination with normal tissue, and usually form a distinct mass of tissue that can be either benign or malignant. As used herein, these terms also include metastasis. As used herein, the terms "cancer" and "cancer disease" are used interchangeably with the terms "tumor" and "tumor disease."

[0415] "Metastasis" refers to the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a highly complex process that relies on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane into body cavities and blood vessels, and subsequent infiltration of the target organ after transport by the blood. Finally, the growth of new tumors at the target site relies on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, as tumor cells or components may remain and develop metastatic potential. In one embodiment, the term "metastasis" refers to "distant metastasis," which refers to metastasis away from the primary tumor and the regional lymph node system.

[0416] By "treating" is meant administering a compound or composition to a subject to prevent or eliminate disease, including reducing tumor size or the number of tumors in a subject, halting or slowing disease in a subject, inhibiting or slowing the onset of new disease in a subject, reducing the frequency or severity of symptoms and / or recurrences in a subject currently suffering from or previously suffering from a disease, and / or extending or increasing the longevity of a subject.

[0417] In particular, the term "treating a disease" includes curing, shortening the duration, alleviating, preventing, slowing or inhibiting the progression or worsening of, or preventing or delaying the onset of, a disease or its symptoms.

[0418] " At risk " refers to the subject, i.e., the patient, who is identified as having a higher than normal probability of developing disease, especially cancer, compared with the general population.In addition, the subject who has suffered from or currently suffers from disease, especially cancer, is the subject who has a high risk of developing disease, because such subject may continue to develop disease.The subject who currently suffers from or has suffered from cancer also has a high risk of cancer metastasis.

[0419] In one embodiment of the tenth aspect, the disease is cancer growth and / or cancer metastasis, hi one embodiment, the disease is characterized by comprising abnormal or cancerous cells characterized by expressing PD-L1 and / or characterized by having PD-L1 associated with their surface.

[0420] In one embodiment of the tenth aspect, the pharmaceutical composition of the ninth aspect is for use in a method for preventing or treating cancer or a tumor disease.

[0421] These tumors include solid tumors and / or hematological malignancies. Examples of tumor diseases that can be treated and / or prevented include all cancers and tumor entities, including, but not limited to, carcinomas, lymphomas, germinomas, sarcomas, and leukemias. More specifically, examples of such cancers include bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, cancer of the genital and reproductive organs, Hodgkin's disease (Hodgkin's lymphoma), cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, adrenal cancer, sarcoma of soft tissue, cancer of the bladder, cancer of the kidney, renal cell carcinoma, cancer of the renal pelvis, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal axis tumors, gliomas, meningiomas, and pituitary adenomas. These cancers may be at an early stage, intermediate stage, or advanced stage, e.g., metastatic stage. In one embodiment, the cancer being treated is at an advanced stage.

[0422] Examples of cancers that are particularly susceptible to PD-1 pathway blockade therapy include, but are not limited to, melanoma, including metastatic melanoma, lymphoma, including Hodgkin's lymphoma, non-small cell lung cancer (NSCLC), e.g., advanced NSCLC, and lung cancer, including small cell lung cancer, renal cell carcinoma, bladder cancer, breast cancer, including advanced triple-negative breast cancer, gastric cancer and gastroesophageal junction cancer, pancreatic adenocarcinoma, and ovarian cancer. In one embodiment of the tenth aspect, the cancer is selected from the group consisting of melanoma, lung cancer, renal cell carcinoma, bladder cancer, breast cancer, gastric cancer and gastroesophageal junction cancer, pancreatic adenocarcinoma, ovarian cancer, kidney tumor, glioblastoma, and lymphoma, preferably Hodgkin's lymphoma.

[0423] In one embodiment of the tenth aspect, the pharmaceutical composition of the ninth aspect is specifically delivered to, accumulated in, and / or retained in a target organ or tissue. In one embodiment, the vector or virus releases nucleic acid in the target organ or tissue and / or enters cells of the target organ or tissue. In one embodiment, the antibody is expressed in cells of the target organ or tissue.

[0424] In one embodiment of the tenth aspect, said treatment is monotherapy or combination therapy.Preferably, said combination therapy is at least one treatment selected from the group consisting of chemotherapy, molecular target therapy, radiotherapy and other forms of immunotherapy.The term " immunotherapy " refers to the treatment that involves specific immune response.

[0425] The antibody of the first aspect, the conjugate of the third aspect, the multimer of the fourth aspect, the nucleic acid of the fifth aspect, the vector of the sixth aspect, the host cell of the seventh aspect, the virus of the eighth aspect, or the composition, preferably the pharmaceutical composition, of the ninth aspect, is intended for use in the treatment of a disease in a subject in need thereof.

[0426] As used herein, the term "subject" is intended to include humans and non-human animals that respond to antibodies against PD-1. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals such as non-human primates, domesticated animals such as sheep, dogs, cats, cows, horses, goats, chickens, amphibians, and reptiles, and laboratory animals such as mice, rats, rabbits, and guinea pigs. Preferred subjects include human patients with disorders and / or diseases that can be corrected or ameliorated by killing abnormal cells, preferably those described herein. The terms "subject," "individual," "organism," or "patient" are used interchangeably.

[0427] The term "in vivo" relates to the context of the subject.

[0428] In one embodiment of the tenth aspect, the subject is a human subject.

[0429] In an eleventh aspect, there is provided a method of treating or preventing a disease in a subject, comprising administering to the subject at least one active agent, wherein the active agent is: (i) an antibody of the first aspect; (ii) the conjugate of the third aspect; (iii) a multimer of the fourth aspect; (iv) a nucleic acid of the fifth aspect or a combination of nucleic acids of the fifth aspect; (v) a vector of the sixth aspect or a combination of vectors of the sixth aspect; (vi) a host cell of the seventh aspect or a combination of host cells of the seventh aspect; and / or (vii) a virus of the eighth aspect or a combination of viruses of the eighth aspect The method is at least one selected from the following:

[0430] In one embodiment of the method of the eleventh aspect, a nucleic acid, vector, host cell or virus encoding the heavy chain of the antibody of the first aspect is administered in combination with another nucleic acid, vector host cell or virus encoding the light chain of the antibody of the first aspect.

[0431] In one embodiment of the method of the eleventh aspect, the nucleic acid, vector, host cell or virus administered may encode both the heavy and light chains of the antibody of the first aspect.

[0432] In one embodiment of the method of the eleventh aspect, the pharmaceutical composition of the tenth aspect is administered to the subject.

[0433] In one embodiment of the method of the eleventh aspect, the subject has an affected organ or tissue characterized by cells that express PD-L1 and / or by having PD-L1 associated with their surface.

[0434] In one embodiment of the method of the eleventh aspect, the disease is cancer growth and / or cancer metastasis.

[0435] In one embodiment of the method of the eleventh aspect, the method is for treating or preventing cancer growth and / or cancer metastasis in a subject having or at risk of developing cancer and / or cancer metastasis.

[0436] In one embodiment of the method of the eleventh aspect, an effective amount of an active agent is provided. Preferably, the antibody is provided at a dose in the range of 0.1 to 20 mg / kg, more preferably 0.3 to 10 mg / kg, in one or more doses. The doses may be provided, for example, every 1 to 4 weeks, even more preferably every 2 to 3 weeks, for example, every 2 or 3 weeks.

[0437] In one embodiment of the method of the eleventh aspect, the cancer is selected from the group consisting of melanoma, lung cancer, renal cell carcinoma, bladder cancer, breast cancer, gastric cancer and gastroesophageal junction cancer, pancreatic adenocarcinoma, ovarian cancer, renal tumor, glioblastoma and lymphoma, preferably Hodgkin's lymphoma.

[0438] In one embodiment of the method of the eleventh aspect, the active agent or pharmaceutical composition is administered to the cardiovascular system, preferably by intravenous administration, such as administration into a peripheral vein, or intra-arterial administration. In one embodiment of this method, the active agent or pharmaceutical composition is specifically delivered to, accumulated in, and / or retained in a target organ or tissue. In one embodiment of this method, the target organ or tissue is a cancerous tissue, particularly a cancerous tissue as defined herein. For example, the affected organ or tissue can be characterized by cells expressing a disease-associated antigen and / or by the association of a disease-associated antigen on its surface. The disease-associated antigen can be a tumor-associated antigen. The disease-associated antigen can be associated with the surface of abnormal cells, such as tumor cells. In one embodiment of this method, the vector, host cell, or virus releases nucleic acid in the target organ or tissue and / or enters cells of the target organ or tissue, preferably, an antibody is expressed in the cells of the target organ or tissue.

[0439] In one embodiment of the method of the eleventh aspect, the treatment is a monotherapy or a combination therapy. Preferably, the combination treatment is at least one treatment selected from the group consisting of chemotherapy, molecular targeted therapy, radiation therapy, and other forms of immunotherapy. Other forms of immunotherapy include vaccination, e.g., RNA vaccination, and / or targeting other checkpoint inhibitors, thereby inhibiting (antagonist) or activating / stimulating (agonist) each other checkpoint. Other checkpoint inhibitors that can be targeted include, but are not limited to, CTLA4, PD-L1, TIM-3, KIR, or LAG-3. Checkpoint activators that can be targeted by the second binding specificity include, but are not limited to, CD27, CD28, CD40, CD122, CD137, OX40, GITR, or ICOS. For example, preferred combinations of binding specificities include anti-PD1 and anti-PD-L1, or anti-PD-1 and anti-CTLA4. Alternatively or additionally, the immunotherapy can provide anti-angiogenic activity. For example, by targeting vascular endothelial growth factor (VEGF) or its receptor VEGFR (e.g., VEGFR1, 2, 3). Alternatively or additionally, PDGFR, c-Kit, Raf and / or RET can be targeted.

[0440] In one embodiment of the method of the eleventh aspect, the treatment is a combination therapy, and the treatment comprises administering to the subject: (i) a peptide or protein containing an epitope for inducing an immune response to an antigen in a subject, or a polynucleotide encoding the peptide or protein; and (ii) at least one selected from the antibody of the first aspect, the conjugate of the third aspect, the multimer of the fourth aspect, the nucleic acid of the fifth aspect, the vector of the sixth aspect, the host cell of the seventh aspect, and / or the virus of the seventh aspect.

[0441] In one embodiment, the peptide or protein containing an epitope for inducing an immune response against an antigen in a subject, or the polynucleotide encoding the peptide or protein, and at least one active compound specified in (ii) are administered sequentially. In one embodiment, the at least one active compound specified in (ii) is administered after the administration of the peptide or protein containing an epitope for inducing an immune response against an antigen in a subject, or the polynucleotide encoding the peptide or protein. In one embodiment, the at least one active compound specified in (ii) is administered 6 hours or more, 12 hours or more, or 24 hours or more after the administration of the peptide or protein containing an epitope for inducing an immune response against an antigen in a subject, or the polynucleotide encoding the peptide or protein. In one embodiment, the at least one active compound specified in (ii) is administered 12 to 48 hours after the administration of the peptide or protein containing an epitope for inducing an immune response against an antigen in a subject, or the polynucleotide encoding the peptide or protein.

[0442] In one embodiment of the method of the eleventh aspect, the method comprises administering to the subject RNA encoding a peptide or protein comprising an epitope for inducing an immune response to an antigen in the subject.

[0443] As used herein, a sample may be any sample useful in accordance with the present disclosure, particularly a biological sample such as a tissue sample and / or cell sample containing bodily fluids, and may be obtained by conventional methods such as by taking tissue biopsy, including punch biopsy, blood, bronchial aspirate, sputum, urine, feces or other bodily fluids. The term "biological sample," as used herein, also includes fractions of biological samples.

[0444] The therapeutic effect in the treatments and uses discussed herein is preferably achieved by the functional property of the antibody of the first aspect of mediating cell killing, for example by inhibiting the immunosuppressive signal of PD-1 on cells expressing PD-1, preferably by forming a complex between the antibody and PD-1, and / or by inducing an immune response, more preferably a T cell-mediated immune response.

[0445] In one embodiment, the anti-PD-1 antibody is administered as a protein, and the antibody can be obtained from a hybridoma, transfectoma, or by in vitro transcription as described herein. In one embodiment, the anti-PD-1 antibody is administered as one or more nucleic acids or one or more vectors as defined herein, e.g., as a liposome containing RNA or RNA encoding the antibody or chain of the antibody, or a fragment of such an antibody or chain.

[0446] Antibodies of the first aspect can be initially tested for binding activity relevant to the therapeutic or diagnostic use in vitro, for example, antibodies can be tested using binding assays, reporter gene blocking assays, and / or T cell proliferation assays described herein.

[0447] The antibody of the first aspect can be used to elicit one or more of the following biological activities in vivo or in vitro: binding to PD-1, preferably specifically binding to PD-1; having binding properties for PD-1 on cancer cells or normal cells; having binding properties for a PD-1 epitope; having binding properties for non-human PD-1 variants, particularly PD-1 variants from mouse, rat, rabbit and primate; preventing or reducing the induction of inhibitory signals by PD-1; inhibiting the interaction / binding of PD-1 with a ligand of PD-1, preferably the ligand PD-L1, for example inhibiting the binding of human PD-L1 to human PD-1; inhibiting the immunosuppressive signals of PD-L1 or PD-L2; enhancing or initiating immune function (by this mechanism), preferably enhancing or initiating a T-cell mediated immune response; inhibiting cancer growth; and / or depleting tumour cells and / or suppressing cancer metastasis.

[0448] Combination strategy in cancer treatment is considered desirable, because the resulting synergistic effect may be significantly stronger than the effect of single-agent therapy approach.Therefore, the present disclosure also encompasses that the antibody of the first aspect, the conjugate of the third aspect, the multimer of the fourth aspect, the nucleic acid of the fifth aspect, the vector of the sixth aspect, the host cell of the seventh aspect, the virus of the eighth aspect, and / or the pharmaceutical composition of the ninth aspect can be administered in combination therapy, i.e., in combination with other drugs.

[0449] For example, the anti-PD-1 antibody of the first embodiment can be co-administered with one or more therapeutic agents, such as cytotoxic agents, radiotoxic agents, angiogenic agents, and / or immunosuppressants, to reduce the induction of an immune response against the antibody of the first embodiment. The antibody can be linked to the agent (as an immunoconjugate) or administered separately from the agent. In the latter case (separate administration), the antibody can be administered before, after, or simultaneously with the agent, or can be co-administered with other known therapeutic agents, such as anti-cancer therapeutic agents, such as radiation. Such therapeutic agents include, among others, the anti-neoplastic agents listed above. Co-administration of the anti-PD-1 antibody of the first embodiment with a chemotherapeutic agent provides two anti-cancer agents that act through different mechanisms to produce a cytotoxic effect on tumor cells. Such co-administration can overcome problems due to the development of resistance to the drugs or changes in the antigenicity of tumor cells that make them unresponsive to the antibody.

[0450] The antibody or composition of the above embodiment can be used in conjunction with chemotherapy. Therapeutic agents for chemotherapy include one or more chemotherapeutic drugs, such as taxol derivatives, taxotere, gemcitabine, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepaclorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, and the like). Examples of therapeutic agents include, but are not limited to, cis-dichlorodiamineplatinum(II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin (Adriamycin)), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). In a preferred embodiment, the therapeutic agent is a cytotoxic or radiotoxic agent. In another embodiment, the therapeutic agent is an immunosuppressant. In yet another embodiment, the therapeutic agent is GM-CSF. In a preferred embodiment, the therapeutic agent is doxorubicin, cisplatin (Platinol), bleomycin sulfate, carmustine, chlorambucil, cyclophosphamide (Cytoxan, Procytox, Neosar), or ricin A.

[0451] In another embodiment, the antibody of the first aspect may be administered in combination with a chemotherapeutic agent, and preferably shows therapeutic efficacy in patients suffering from cancers that are particularly amenable to blockade of the PD-1 pathway, such as melanoma, including metastatic melanoma, Hodgkin's lymphoma, non-small cell lung cancer (NSCLC), e.g. advanced NSCLC, and lung cancer, including small cell lung cancer, renal cell carcinoma, bladder cancer, advanced triple-negative breast cancer, including advanced triple-negative breast cancer, gastric cancer and gastroesophageal junction cancer, pancreatic adenocarcinoma, or ovarian cancer.

[0452] In one embodiment, the antibody or pharmaceutical composition of the above aspect is administered together with an immunotherapeutic agent. As used herein, "immunotherapeutic agent" refers to any agent that can be involved in activating a specific immune response and / or immune effector function. The present disclosure contemplates the use of antibodies as immunotherapeutic agents. Without wishing to be bound by theory, antibodies can achieve therapeutic effects on cancer cells through various mechanisms, including inducing apoptosis, blocking components of signaling pathways, or inhibiting tumor cell proliferation. In certain embodiments, the antibody is a monoclonal antibody. Non-limiting examples of anti-cancer antibodies and potential antibody targets (in parentheses) that may be used in combination with the present disclosure include: Abagovomab (CA-125), Abciximab (CD41), Adecatumumab (EpCAM), Afutuzumab (CD20), Alacizumab pegol (VEGFR2), Altumomab pentetate (CEA), Amatuximab (MORAb-009), Anatumomab mafenatox (TAG-72), Apolizumab (HLA-DR), Arcitumomab (CEA), Atezolizumab (PD-L1), Bavituximab (phosphatidylserine), Bectumomab (CD22), Belimumab (BAFF), Bevacizumab (VEGF-A), Bivatuzumab mertansine (CD44 v6), Blinatumomab (CD 19), Brentuximab vedotin (CD30TNFRSF8), Cantuzumab Mertansine (Mucin CanAg), Cantuzumab Ravtansine (MUC1), Capromab Pendetide (Prostate Cancer Cells), Carlumab (CNT0888), Catumaxomab (EpCAM, CD3), Cetuximab (EGFR), Citatuzumab Bogatox (EpCAM), Cixutumumab (IGF-1 Receptor), Claudiximab (Claudin), Civatuzumab Tetraxetan (MUC1), Conatumumab (TRAIL-R2), Dacetuzumab (CD40), Dalotuzumab (Insulin Phospho-like growth factor I receptor), Denosumab (RANKL), Detumomab (B-lymphoma cells), Drozitumab (DR5), Ecromeximab (GD3 ganglioside), Edrecolomab (EpCAM), Elotuzumab (SLAMF7), Enavatuzumab (PDL192), Ensituximab (NPC-1C), Epratuzumab (CD22), Ertumaxomab (HER2 / neu, CD3), Etaracizumab (integrin ανβ3), Farletuzumab (folate receptor 1), FBTA05 (CD20), Ficlatuzumab (SCH 900105), Figitumumab (IGF-1 receptor), Flanvotumab (glycoprotein 75), Fresolimumab (TGF-β), Galiximab (CD80), Ganitumab (IGF-I), Gemtuzumab ozogamicin (CD33), Gevokizumab (ILΙβ), Girentuximab [carbonic anhydrase 9 (CA-IX)], Glembatumumab vedotin (GPNMB), Ibritumomab tiuxetan (CD20), Icrucumab (VEGFR-1), Igovoma (CA-125), Indatuximab ravtansine (SDC1), Intetumumab (CD51), Inotuzumab ozogamicin (CD22), Ipilimumab (CD152), Iratumumab (CD30), Labetuzumab (CEA), Lexatumumab (TRAIL-R2), Libivirumab (hepatitis B surface antigen), Lintuzumab (CD33), Lorvotuzumab mertansine (CD56), Lucatumumab (CD40), Lumiliximab (CD23), Mapatumumab (TRAIL-R1), Matuzumab (EGFR), Mepolizumab (IL5), Milatuzumab (CD74), Mitumomab (GD3 ganglioside), Mogamulizumab (CCR4), Moxetumab Pasdotox (CD22), Nacolomab tafenatox (C242 antigen), Naptumomab Estafenatox (5T4), Namatumab (RON), Necitumumab (EGFR), Nimotuzumab (EGFR), Nivolumab (IgG4), Ofatumumab (CD20), Olaratumab (PDGF-Rα), Onartuzumab (human scatter factor receptor kinase)kinase)], Oportuzumab Monatox (EpCAM), Oregovomab (CA-125), Oxelumab (OX-40), Panitumumab (EGFR), Patritumab (HER3), Pertuzumab (MUC1), Pertuzumab (HER2 / neu), Pintumumab (adenocarcinoma antigen), Pritumumab (vimentin), Racotumomab (N-glycolylneuraminic acid), Radretumab (fibronectin extra domain-B), Rafivirumab (rabies virus glycoprotein), Ramucirumab (VEGFR2), Rilotumumab (HGF), Rituximab (CD20), Robatumumab (IGF-1 receptor), Samalizumab ( CD200), Sibrotuzumab (FAP), Siltuximab (IL6), Tabalumab (BAFF), Tacatuzumab Tetraxetan (alpha-fetoprotein), Taplitumomab Paptox (CD19), Tenatumomab (Tenascin C), Teprotumumab (CD221), Ticilimumab (CTLA4), Tigatuzumab (TRAIL-R2), TNX-650 (IL13), Tositumomab (CD20), Trastuzumab (HER2 / neu), TRBS07 (GD2), Tremelimumab (CTLA4), Tucotuzumab Celmoleukin (EpCAM), Ublituximab (MS4A1), Urelumab (4-1 BB), Volociximab (integrin α5β1), Votumumab (tumor antigen CTAA 16.88), Zalutumumab (EGFR), and Zanolimumab (CD4).

[0453] For example, according to the tenth or eleventh embodiment, a subject receiving the antibody of the first embodiment is further treated with one or more antibodies targeting another immune checkpoint. Immune checkpoint inhibitors that activate tumor defense by disrupting inhibitory interactions between antigen-presenting cells and T lymphocytes include, but are not limited to, anti-PD-L1, anti-CTLA4, anti-TIM-3, anti-KIR, and / or anti-LAG-3. Immunotherapeutic agents that stimulate activation checkpoints such as CD27, CD28, CD40, CD122, CD137, OX40, GITR, or ICOS, i.e., for example, anti-CD27, anti-CD28, anti-CD40, anti-CD122, anti-CD137, anti-OX40, anti-GITR, and / or anti-ICOS, are also encompassed. Particularly preferred combination therapies include, but are not limited to, a combination of anti-PD1 and anti-PD-L1, thereby enhancing the efficiency and blockade of the PD1 pathway by targeting both components, or a combination of anti-PD-1 and anti-CTLA4, preventing blockade of both the PD1 and CTLA4 pathways. For example, combinations of binding specificities include anti-PD1 and anti-PD-L1, or anti-PD-1 and anti-CTLA4. Alternatively or additionally, immunotherapy can provide anti-angiogenic activity, for example, by targeting vascular endothelial growth factor (VEGF) or its receptor VEGFR (e.g., VEGFR1, 2, 3). Alternatively or additionally, PDGFR, c-Kit, Raf, and / or RET can be targeted.

[0454] In another embodiment of the tenth or eleventh aspect, the subject receiving the antibody is further treated with an angiogenesis inhibitor, including an antibody targeting vascular endothelial growth factor (VEGF) or its receptor VEGFR, and one or more chemical compounds that inhibit angiogenesis. Pretreatment or concurrent application of these drugs may improve penetration of the antibody into the tumor mass.

[0455] For example, angiogenesis inhibitors can target VEGF.Suitable VEGF inhibitor is Bevacizumab.Other examples include but are not limited to the multikinase inhibitor (for example, Sunitinib, Sorafenib, Pazopanib) that inhibits VEGFR1, 2, 3, PDGFR, c-Kit, Raf and / or RET.

[0456] In another embodiment of the tenth or eleventh aspect, the subject receiving the antibody is further treated with a compound that inhibits growth factor receptor signaling, including a monoclonal antibody that binds to the EGFR receptor, and a chemical compound that inhibits signaling initiated by the EGFR receptor.

[0457] In another embodiment of the tenth or eleventh aspect, such a therapeutic agent includes an agent that leads to the depletion or functional inactivation of regulatory T cells, such as low-dose cyclophosphamide, and / or an anti-IL2 antibody or an anti-IL2 receptor antibody.

[0458] In another embodiment of the tenth or eleventh aspect, the antibody of the first or second aspect may be administered in combination with one or more antibodies selected from an anti-CD25 antibody, an anti-EPCAM antibody, and an anti-CD40 antibody.

[0459] In yet a further embodiment of the tenth or eleventh aspect, the antibody of the first aspect may be administered in combination with an anti-C3b(i) antibody to enhance complement activation.

[0460] The antibody of the first embodiment can also be used in combination with one or more vaccines to stimulate the immune system against antigens expressed by abnormal cells, such as tumor cells. For example, the antigen can be one or more of the tumor antigens specified herein. Vaccination can be achieved by administering vaccine RNA, i.e., RNA encoding an antigen or epitope against which an immune response is induced. Alternatively, a peptide or protein containing an epitope to induce an immune response against the antigen can be administered.

[0461] Thus, in another embodiment of the tenth or eleventh aspect, the antibody of the first aspect may be administered in combination with a vaccine therapy, i.e. in combination with at least one peptide or protein comprising an epitope for inducing an immune response against an antigen in a subject, or at least one polynucleotide / nucleic acid encoding said peptide or protein.

[0462] Thus, the present disclosure also provides a composition, preferably a pharmaceutical composition, comprising: (i) a peptide or protein comprising an epitope for inducing an immune response against an antigen in a subject, or a polynucleotide encoding the peptide or protein; and (ii) at least one selected from the antibody of the first aspect, the conjugate of the third aspect, the multimer of the fourth aspect, the nucleic acid of the fifth aspect, the vector of the sixth aspect, the host cell of the seventh aspect, and / or the virus of the eighth aspect. In one embodiment, the composition comprises RNA encoding the peptide or protein comprising the epitope for inducing an immune response against the antigen in a subject.

[0463] The term "antigen" relates to an agent that comprises an epitope against which an immune response or immune effector molecule, such as an antibody, is directed and / or should be directed. The term "antigen" includes, inter alia, proteins and peptides. In one embodiment, the antigen is a disease-associated antigen, such as a tumor antigen.

[0464] The term "disease-associated antigen" is used in its broadest sense and preferably refers to any antigen associated with a disease that contains an epitope that stimulates the host's immune system to mount a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Thus, disease-associated antigens, their epitopes, or agents such as peptides or proteins that induce immune responses targeting disease-associated antigens or epitopes can be used for therapeutic purposes, particularly vaccination. Disease-associated antigens may be associated with microbial infections, typically microbial antigens, or with cancer, typically tumors.

[0465] In one embodiment, the antigen against which the immune response is directed (ie, the disease-associated antigen) is a tumor antigen, preferably as specified herein. More preferably, the at least one tumor antigen is selected from the group consisting of NY-ESO-1 (UniProt P78358), Tyrosinase (UniProt P14679), MAGE-A3 (UniProt P43357), TPTE (UniProt P56180), KLK2 (UniProt P20151), PSA (KLK3) (UniProt P07288), PAP (ACPP, UniProt P15309), HOXB13 (UniProt Q92826), NKX3-1 (UniProt Q99801), HPV16 E6 / E7 (UniProt P03126 / P03129); HPV18 E6 / E7 (UniProt P06463 / P06788); HPV31 E6 / E7 (UniProt P06463 / P06788); P17386 / P17387; HPV33 E6 / E7 (UniProt P06427 / P06429); HPV45 E6 / E7 (UniProt P21735 / P21736); HPV58 E6 / E7 (UniProt P26555 / P26557), PRAME (UniProt P78395), ACTL8 (UniProt Q9H568), CXorf61 (KKLC1, UniProt Q5H943), MAGE-A9B (UniProt P43362), CLDN6 (UniProt P56747), PLAC1 (UniProt Q9HBJ0), and p53 (UniProt P04637). The peptide or protein used for vaccination (i.e., vaccine antigen) may comprise the antigen or an epitope thereof. In one embodiment, the vaccine antigens are administered in the form of RNA encoding the vaccine antigens. Treatment methods involving these antigens can be aimed at treating cancer, where cancer cells are characterized by the expression of the respective antigens.It is also possible to use combinations of the antigens described herein, particularly NY-ESO-1, Tyrosinase, MAGE-A3, TPTE, KLK2, PSA (KLK3), PAP (ACPP), HOXB13, NKX3-1, HPV16 E6 / E7; HPV18 E6 / E7, HPV31 E6 / E7, HPV33 E6 / E7, HPV45 E6 / E7, HPV58 E6 / E7, PRAME, ACTL8, CXorf61 (KKLC1), MAGE-A9B, CLDN6, PLAC1, and p53. Treatment methods involving such antigen combinations can be aimed at treating cancers in which the cancer cells are characterized by the expression of each combination of two or more antigens, or in which the cancer cells of a majority (e.g., at least 80%, at least 90%, or even more) of patients with a particular cancer being treated express one or more of the respective antigens of the combination. Such a combination may include a combination of at least two, at least three, at least four, at least five, or at least six antigens. Thus, the combination may include 3, 4, 5, 6, 7, or 8 antigens. In this case, each antigen of the combination can be addressed by administering a peptide or protein (i.e., vaccine antigen) comprising the antigen or an epitope thereof, or RNA encoding the peptide or protein. In a particularly preferred embodiment, each antigen of the combination is addressed by administering RNA encoding a peptide or protein comprising the antigen. Thus, vaccination may involve the administration of different RNA molecules, each encoding a peptide or protein comprising an antigen of the antigen combination. RNA encoding different vaccine antigens or different vaccine antigens of the combination can be administered in a mixture, sequentially, or a combination thereof.

[0466] In one embodiment, the combination of antigens comprises, and preferably consists of, NY-ESO-1, Tyrosinase, MAGE-A3, and TPTE, which may be used to treat cutaneous melanoma.

[0467] In one embodiment, the combination of antigens comprises, and preferably consists of, KLK2, PSA (KLK3), PAP (ACPP), HOXB13, and NKX3-1. This combination can be used to treat prostate cancer.

[0468] In one embodiment, the combination of antigens comprises, and preferably consists of, PRAME, ACTL8, CXorf61 (KKLC1), MAGEA3, MAGE-A9B, CLDN6, NY-ESO-1, and PLAC1. This combination may be used to treat breast cancer, such as triple-negative breast cancer, particularly estrogen receptor-negative, progesterone receptor-negative, and HER2-negative breast cancer.

[0469] In one embodiment, the combination of antigens comprises, and preferably consists of, CLDN6, p53, and PRAME, which may be used to treat ovarian cancer, such as epithelial ovarian cancer.

[0470] The vaccines described herein may consist of one or more RNAs targeting one or more antigens expressed in diseases such as cancer. The active principle may be a single-stranded mRNA that is translated into the respective protein upon entry into antigen-presenting cells (APCs). In addition to a wild-type or codon-optimized sequence encoding the antigen sequence, the RNA may contain one or more structural elements (5'-cap, 5'-UTR, 3'-UTR, poly(A)-tail) optimized to maximize the effectiveness of the RNA in terms of stability and translation efficiency. In one embodiment, the RNA contains all of these elements. In one embodiment, beta-S-ARCA (D1) may be used as a specific capping structure at the 5'-end of the RNA drug substance. The 5'-UTR sequence may be the 5'-UTR sequence of human alpha-globin mRNA, optionally with a "Kozak sequence" optimized for increased translation efficiency. The 3'-UTR sequence may be a combination of two repeat 3'-UTRs from human beta globin mRNA, located between the coding sequence and the poly(A)-tail, to ensure higher maximum protein levels and long-term mRNA persistence. Alternatively, the 3'-UTR may be a combination of two sequence elements (FI elements) derived from the "amino-terminal enhancer of split" (AES) mRNA (termed F) and the mitochondrially encoded 12S ribosomal RNA (termed I). These were identified through an ex vivo selection process for sequences that confer RNA stability and enhance total protein expression (see International Publication No. 2017 / 060314, incorporated herein by reference). Additionally, a 110-nucleotide-long poly(A)-tail may be used, consisting of a stretch of 30 adenosine residues, followed by a 10-nucleotide linker sequence (of random nucleotides), and another 70 adenosine residues. This poly(A)-tail sequence was designed to enhance RNA stability and translation efficiency in dendritic cells.

[0471] Additionally, sec (secretory signal peptide) and / or MITD (MHC class I trafficking domain) may be fused to the antigen coding region so that each element is translated as an N- or C-terminal tag, respectively. Fusion protein tags derived from sequences encoding the human MHC class I complex (HLA-B51, haplotype A2, B27 / B51, Cw2 / Cw3) have been shown to improve antigen processing and presentation. sec is thought to correspond to a 78-bp fragment encoding the secretory signal peptide, which directs translocation of the nascent polypeptide chain into the endoplasmic reticulum. MITD corresponds to the transmembrane and cytoplasmic domains of the MHC class I molecule and is also called the MHC class I trafficking domain. Antigens such as CLDN6, which possess a secretory signal peptide and a transmembrane domain, may not require the addition of a fusion tag. A sequence encoding a short linker peptide consisting primarily of the amino acids glycine (G) and serine (S), as commonly used in fusion proteins, can be used as the GS / Linker.

[0472] The antigen may be administered in combination with a helper epitope to break immune tolerance. The helper epitope may be derived from tetanus toxoid, such as the P2P16 amino acid sequence from Clostridium tetani tetanus toxoid (TT). These sequences may help overcome self-tolerance mechanisms to efficiently induce immune responses against self-antigens by providing help for non-tumor-specific T cells during priming. The tetanus toxoid heavy chain contains an epitope that can indiscriminately bind to MHC class II alleles, resulting in CD4+ T cell proliferation in almost all tetanus-vaccinated individuals. + Furthermore, the combination of TT helper epitopes and tumor-associated antigens can induce memory T cells. + It is known to improve immune stimulation compared to application of tumor-associated antigens alone by providing CD8-mediated T cell help. +To reduce the risk of stimulating T cells, two peptide sequences known to contain promiscuous binding helper epitopes may be used to ensure binding to as many MHC class II alleles as possible, e.g., P2 and P16.

[0473] In one embodiment, the vaccine antigen comprises an amino acid sequence that disrupts immune tolerance. In one embodiment, the amino acid sequence that disrupts immune tolerance comprises a helper epitope, preferably a helper epitope derived from tetanus toxoid. The amino acid sequence that disrupts immune tolerance may be directly fused to the C-terminus of the vaccine sequence, e.g., the antigen sequence, or separated by a linker. Optionally, the amino acid sequence that disrupts immune tolerance may link the vaccine sequence to an MITD. When the vaccine antigen is administered in the form of RNA encoding the vaccine antigen, the amino acid sequence that disrupts immune tolerance may be encoded by the RNA. In one embodiment, the antigen-targeting RNA is administered together with an RNA encoding a helper epitope to enhance the resulting immune response. The RNA encoding this helper epitope may contain structural elements (5'-cap, 5'-UTR, 3'-UTR, poly(A)-tail) optimized to maximize the effectiveness of the RNA in terms of stability and translation efficiency as described above for antigen-encoding RNA. Additionally, a sec (secretory signal peptide) and / or MITD (MHC class I trafficking domain) may be fused to the helper epitope-encoding region, such that each element is translated as an N- or C-terminal tag, respectively, as described above for antigen-encoding RNA. In one embodiment, the RNA is co-administered with additional RNA encoding the helper epitopes P2 and P16 (P2P16) from tetanus toxoid (TT) to enhance the resulting immune response.

[0474] Vaccine RNA was complexed with liposomes to form serum-stable RNA-lipoplexes [RNA (LIP)] may be prepared and administered intravenously (iv). When different combinations of RNA are used, the RNAs may be complexed with liposomes separately to form serum-stable RNA-lipoplexes [RNA (LIP) ] may be prepared and administered intravenously (iv). (LIP) targets antigen-presenting cells (APCs) in lymphoid organs and efficiently stimulates the immune system.

[0475] In one embodiment, the vaccine RNA is co-formulated as a lipoplex particle with RNA encoding an amino acid sequence that disrupts immune tolerance.

[0476] As used herein, "tumor antigen" or "cancer antigen" includes (i) tumor-specific antigens, (ii) tumor-associated antigens, (iii) embryonic antigens on tumors, (iv) tumor-specific membrane antigens, (v) tumor-associated membrane antigens, (vi) growth factor receptors, and (xi) any other type of antigen or substance associated with cancer.

[0477] Any tumor antigen (preferably expressed by tumor cells) can be targeted by the vaccination disclosed herein. In one embodiment, tumor antigens are presented by tumor cells and can be targeted by T cells. The vaccination disclosed herein preferably activates T cells specific for MHC-presented tumor antigens. The tumor antigen may be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSAs are unique to tumor cells and are not present on other cells in the body. TAAs are not unique to tumor cells and are also expressed on normal cells under conditions that do not allow for the induction of immunological tolerance to the antigen. Antigen expression on tumors can occur under conditions that allow the immune system to respond to the antigen. TAAs may be antigens expressed on normal cells during fetal development when the immune system is immature and unable to respond, or they may be antigens that are normally present at very low levels on normal cells but are expressed at much higher levels on tumor cells.

[0478] Peptide and protein antigens can be 2 to 100 amino acids in length, including, for example, at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, or at least 50 amino acids in length. In some embodiments, peptides can be more than 50 amino acids. In some embodiments, peptides can be more than 100 amino acids.

[0479] The peptide or protein antigen can be any peptide or protein that is capable of inducing or increasing the ability of the immune system to develop antibody and T cell responses against a target antigen, for example, a disease-associated antigen.

[0480] In yet another embodiment, the antibody of the first aspect may be administered in combination with radiation therapy and / or autologous peripheral stem cell or bone marrow transplantation.

[0481] Also included in the present disclosure is a combination therapy comprising the composition of the ninth aspect and at least one anti-inflammatory agent or at least one immunosuppressant. In one embodiment, such therapeutic agents include one or more anti-inflammatory agents, such as steroids or NSAIDs (nonsteroidal anti-inflammatory drugs). Preferred agents include, for example, aspirin and other salicylates, Cox-2 inhibitors such as rofecoxib (Vioxx) and celecoxib (Celebrex), and NSAIDs such as ibuprofen (Motrin, Advil), fenoprofen (Nalfon), naproxen (Naprosyn), sulindac (Clinoril), diclofenac (Voltaren), piroxicam (Feldene), ketoprofen (Orudis), diflunisal (Dolobid), nabumetone (Relafen), etodolac (Lodine), oxaprozin (Daypro), and indomethacin (Indocin). Combination therapy according to the present disclosure may also include a combination of (i) an antibody of the first aspect, (ii) a vaccination treatment / therapy as defined above, and (iii) at least one anti-inflammatory agent or at least one immunosuppressant.

[0482] The bispecific and multispecific molecules of the above embodiments can be used to interact with other immune checkpoints, thereby inhibiting or activating / stimulating the other checkpoints, respectively. Other checkpoint inhibitors that can be targeted include, but are not limited to, CTLA4, PD-L1, TIM-3, KIR, or LAG-3. Checkpoint activators that can be targeted by the second binding specificity include, but are not limited to, CD27, CD28, CD40, CD122, CD137, OX40, GITR, or ICOS. Preferred combinations of binding specificities include anti-PD1 and anti-PD-L1, or anti-PD-1 and anti-CTLA4.

[0483] Alternatively, or in addition, the bispecific or multispecific molecules of the above embodiments can be used to confer anti-angiogenic activity, for example, by targeting vascular endothelial growth factor (VEGF) or its receptor VEGFR (e.g., VEGFR1, 2, 3). The second binding specificity may also be capable of targeting PDGFR, c-Kit, Raf, and / or RET.

[0484] Alternatively or additionally, the bispecific or multispecific molecules of the above embodiments can be used to target tumor antigens, preferably those specified above, thereby enabling the specificity of the antibody of the first embodiment for cancer cells.

[0485] For the uses and methods of the tenth and eleventh aspects, the actual dosage level of the active ingredient that may be included in the pharmaceutical composition, preferably such a pharmaceutical composition, may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration and that is not toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the present disclosure employed, the route of administration, the time of administration, the excretion rate of the particular compound employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0486] A physician or veterinarian of ordinary skill in the art can easily determine and prescribe the effective amount of pharmaceutical composition required. For example, the physician or veterinarian can start the dose of the compound of the present disclosure employed in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of the composition of the present disclosure is the amount of the compound that is the lowest effective dose to produce a therapeutic effect. Such an effective amount generally depends on the factors described above. Administration is preferably intravenous, intramuscular, intraperitoneal, or subcutaneous, preferably administered proximal to the target site. If desired, the effective daily dose of the therapeutic composition may be administered as two, three, four, five, six, or more subdoses administered separately at appropriate intervals throughout the day, optionally in unit dosage form. While it is possible to administer the compound of the present disclosure alone, it is preferable to administer the compound as a pharmaceutical composition (formulation).

[0487] In one embodiment, the antibody of the first aspect may be administered by infusion, preferably by slow continuous infusion over an extended period of time, such as greater than 24 hours, to reduce toxic side effects. Administration may also be by continuous infusion over a period of 2 to 24 hours, e.g., 2 to 12 hours. Such a regimen may be repeated one or more times as needed, e.g., after 6 or 12 months. Dosage may be determined or adjusted by measuring the circulating amount of anti-PD-1 antibody in a biological sample at the time of administration using an anti-idiotypic antibody that targets the anti-PD-1 antibody.

[0488] In yet another embodiment, the antibody is administered by maintenance therapy, for example, once a week for a period of six months or more.

[0489] A "therapeutically effective dose" for tumor treatment can be measured by the objective tumor response, which can be a complete response or a partial response. A complete response (CR) is defined as the absence of clinical, radiological, or other disease symptoms. A partial response (PR) is a reduction in collective tumor size of more than 50%. Median time to progression is a measure that characterizes the durability of the objective tumor response.

[0490] The "therapeutically effective dosage" in tumor treatment can also be measured by its ability to stabilize disease progression. The ability of a compound to inhibit cancer can be evaluated in an animal model system that can predict its effectiveness in human tumors. Alternatively, this property of a composition can be evaluated by examining the compound's ability to inhibit cell growth or apoptosis using in vitro assays known to those skilled in the art. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise alleviate symptoms in a subject. Those skilled in the art will be able to determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected. 【0491...

Claims

1. An antibody having the ability to bind to PD-1, comprising a heavy chain having a heavy chain constant region (CH) and a heavy chain variable region (VH), and a light chain having a light chain variable region (VL), The heavy chain constant region contains phenylalanine at the position corresponding to position 234 of the human IgG1 heavy chain according to EU numbering, glutamic acid at the position corresponding to position 235 of the human IgG1 heavy chain according to EU numbering, and arginine at the position corresponding to position 236 of the human IgG1 heavy chain according to EU numbering, (i) The heavy chain variable region (VH) comprises the HCDR1, HCDR2, and HCDR3 sequences, and the light chain variable region (VL) comprises the LCDR1, LCDR2, and HCDR3 sequences, where the HCDR1, HCDR2, and HCDR3 sequences each contain or have the sequences described in SEQ ID NO: 12, SEQ ID NO: 11, and SEQ ID NO: 8, respectively, and the LCDR1, LCDR2, and LCDR3 sequences each contain or have the sequences described in SEQ ID NO: 17, SEQ ID NO: 15, and SEQ ID NO: 14, respectively; or (ii) The heavy chain variable region (VH) comprises the HCDR1, HCDR2, and HCDR3 sequences, and the light chain variable region (VL) comprises the LCDR1, LCDR2, and HCDR3 sequences, where the HCDR1, HCDR2, and HCDR3 sequences each contain or have the sequences described in SEQ ID NO: 13, SEQ ID NO: 10, and SEQ ID NO: 9, respectively, and the LCDR1, LCDR2, and HCDR3 sequences each contain or have the sequences described in SEQ ID NO: 16, QAS, and SEQ ID NO: 14, respectively. antibody.

2. (i) The heavy chain constant region includes a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the amino acid sequence of the heavy chain constant region sequence described in Sequence ID No. 2, or (ii) The isotype of the heavy chain steady region is IgG1. The antibody according to claim 1.

3. The antibody according to claim 1, wherein the heavy chain constant region comprises the sequence described in SEQ ID NO: 2 or 55.

4. (i) The heavy chain variable region (VH) includes a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the amino acid sequence of the heavy chain variable region (VH) sequence described in Sequence ID No. 20, or (ii) The heavy chain variable region (VH) includes the sequence described in Sequence ID No. 20, The antibody according to claim 1.

5. (i) The heavy chain variable region (VH) includes the sequence described in Sequence ID No. 20, and the heavy chain constant region includes the sequence described in Sequence ID No. 2 or Sequence ID No. 55, or (ii) The heavy chain comprises the sequence described in Sequence ID No. 56, The antibody according to claim 4.

6. (i) The light chain variable region (VL) includes a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the amino acid sequence of the light chain variable region (VL) sequence described in Sequence ID No. 21, or (ii) The light chain variable region (VL) includes the sequence described in Sequence ID No. 21, The antibody according to claim 1.

7. (i) The light chain comprises a variable light chain region (VL) containing the sequence described in Sequence ID No. 21, and a constant light chain region containing the sequence described in Sequence ID No. 6, or (ii) The light chain comprises the sequence described in Sequence ID No. 57, The antibody according to claim 6.

8. The antibody according to claim 1, wherein the heavy chain variable region (VH) includes or has the sequence described in SEQ ID NO: 20, and the light chain variable region (VL) includes or has the sequence described in SEQ ID NO:

21.

9. (i) The heavy chain comprises a heavy chain constant region (CH) containing the sequence described in Sequence ID No. 2 or 55, and a heavy chain variable region (VH) containing the sequence described in Sequence ID No. 20, and The light chain comprises a light chain constant region (CL) containing the sequence described in Sequence ID No. 6, and a light chain variable region (VL) containing the sequence described in Sequence ID No.

21. including; or (ii) The heavy chain has the sequence described in Sequence ID 56, and the light chain has the sequence described in Sequence ID 57, The antibody according to claim 1.

10. The antibody according to claim 1, which is a monoclonal antibody, a chimeric antibody, or a humanized antibody, or an antigen-binding fragment of such an antibody.

11. (i) The binding of complement protein C1q to the constant region of the antibody is reduced by at least 70% compared to the corresponding wild-type antibody; (ii) The binding of one or more IgG Fc gamma receptors to the antibody is reduced by at least 70% compared to the corresponding wild-type antibody; (iii) The antibody is unable to induce Fc-gamma RI-mediated effector function, or the induced Fc-gamma RI-mediated effector function is reduced by at least 70% compared to the corresponding wild-type antibody; (iv) The antibody is unable to induce at least one of complement-dependent cell-mediated lysis (CDC), antibody-dependent cell-mediated lysis (ADCC), apoptosis, homoadhesion, and / or phagocytosis, or induces at least one of complement-dependent cell-mediated lysis (CDC), antibody-dependent cell-mediated lysis (ADCC), apoptosis, homoadhesion, and / or phagocytosis by at least 70% reduction; or (v) The binding of the neonatal Fc receptor (FcRn) to the antibody is unaffected compared to the corresponding wild-type antibody. The antibody according to claim 1.

12. (i) PD-1 is human PD-1; or (ii) The antibody binds to the native epitope of PD-1 present on the surface of living cells. The antibody according to claim 1.

13. The antibody according to claim 1, which is a polyspecific antibody comprising a first antigen-binding region that binds to PD-1 and at least one further antigen-binding region that binds to another antigen.

14. (i) a bispecific antibody comprising a first antigen-binding region that binds to PD-1 and a second antigen-binding region that binds to another antigen; or (ii) The first antigen-binding region that binds to PD-1 includes the heavy chain variable region (VH) and / or the light chain variable region (VL), The antibody according to claim 13.

15. The following: (i) The antibody according to any one of claims 1 to 14; (ii) A conjugate comprising an antibody according to any one of claims 1 to 14, which is linked to a portion or a drug, wherein the portion or drug is selected from the group consisting of radioisotopes, enzymes, dyes, drugs, toxins and cytotoxic agents; (iii) A polymer comprising at least two antibodies according to any one of claims 1 to 14, or at least two conjugates according to item (ii), or a mixture of one or more antibodies according to any one of claims 1 to 14 and one or more conjugates according to item (ii); or a polymer comprising 4 to 8 antibodies according to any one of claims 1 to 14 or conjugates according to item (ii); (iv) A nucleic acid comprising an antibody according to any one of claims 1 to 14, an antibody heavy chain as specified in any one of claims 1 to 12, an antibody light chain as specified in any one of claims 1 or 6 to 12, or a nucleic acid sequence encoding an antigen-binding fragment thereof; (v) A vector comprising one or more nucleic acids as described in item (iv); and (vi) Host cells or viruses containing the nucleic acid described in item (iv) or the vector described in item (v). An activator selected from the group consisting of the following.

16. A hybridoma capable of producing the antibody described in any one of claims 1 to 14.

17. A conjugate comprising an antibody according to any one of claims 1 to 14, which is linked to a portion or a drug, wherein the portion or drug is selected from the group consisting of radioisotopes, enzymes, dyes, drugs, toxins and cytotoxic agents.

18. The polymer according to item (iii) of claim 15.

19. (i) The antibody according to any one of claims 1 to 14; (ii) an antibody heavy chain as specified in any one of claims 1 to 12; (iii) an antibody light chain as expressed in any one of claims 1 or 6 to 12; or (iv) Antigen-binding fragments of items (i) to (iii) A nucleic acid containing a nucleic acid sequence that codes for [something].

20. The nucleic acid according to claim 19, wherein the nucleic acid is RNA.

21. The vector according to item (v) of claim 15.

22. A host cell or virus according to item (vi) of claim 15.

23. A pharmaceutical composition comprising the activator described in Claim 15 and a pharmaceutically acceptable carrier.

24. (i) formulated for parenteral administration, or (ii) formulated for cardiovascular, intravenous, or intra-arterial administration; and / or It is intended for use in the preventive and / or therapeutic treatment of disease. The pharmaceutical composition according to claim 23.

25. The pharmaceutical composition according to claim 23, for use in a method for preventing or treating cancer.

26. (i) The cancer is selected from the group consisting of melanoma, lung cancer, renal cell carcinoma, bladder cancer, breast cancer, gastric cancer and gastroesophageal junction cancer, pancreatic adenocarcinoma, ovarian cancer and lymphoma; (ii) The cancer comprises cancer cells characterized by the expression of PD-L1 and / or by the association of PD-L1 on their surface; (iii) Delivered specifically to, accumulated in, and / or retained in a target organ or tissue; (iv) The vector or the virus releases nucleic acid in the target organ or tissue and / or enters the cells of the target organ or tissue; (v) The antibody is expressed in the cells of the target organ or tissue; (vi) The treatment is monotherapy or combination therapy; (vii) The combination therapy is at least one therapy selected from the group consisting of chemotherapy, molecular targeted therapy, radiotherapy, and other forms of immunotherapy; or (viiii) The subject is a human, The pharmaceutical composition according to claim 25.

27. ​​Use of the pharmaceutical composition according to claim 23 in the preparation of a pharmaceutical for preventing or treating cancer.

28. A kit for the qualitative or quantitative detection of PD-1 in a sample, (i) The antibody according to any one of claims 1 to 14; (ii) A conjugate comprising an antibody according to any one of claims 1 to 14, linked to a portion or a drug, wherein the portion or drug is selected from the group consisting of radioisotopes, enzymes, dyes, drugs, toxins and cytotoxic agents; or (iii) A polymer comprising at least two antibodies according to any one of claims 1 to 14, or at least two conjugates according to item (ii), or a mixture of one or more antibodies according to any one of claims 1 to 14 and one or more conjugates according to item (ii); or a polymer comprising 4 to 8 antibodies according to any one of claims 1 to 14 or conjugates according to item (ii). A kit that includes this.

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