Novel antibodies and nucleotide sequences and their uses

Abstract

To provide novel anti-CTLA-4 antibody molecules, and nucleotide sequences encoding such antibody molecules and expression vectors such as viruses.SOLUTION: The novel antibody molecules are Treg depleting antibody molecules, and they have an improved depleting effect on CTLA-4 positive cells, such as Tregs, compared with ipilimumab. There are also described, uses of such antibody molecules or nucleotide sequences, or viruses in medicine e.g. in treatment of cancers such as solid tumours.SELECTED DRAWING: Figure 1-1

Description

[Technical field]

[0001] The present invention relates to novel anti-CTLA-4 antibody molecules, nucleotide sequences encoding such antibody molecules and expression vectors (e.g., oncolytic viruses) for their use in cancer therapy. The novel antibodies have improved Treg depletion compared to ipilimumab. [Background technology]

[0002] Cytotoxic T-lymphocyte-associated antigen (CTLA-4 or CTLA4), also known as CD152, is a member of the B7 / CD28 family that blocks T-cell activation. CTLA-4 is expressed on activated T cells and transmits inhibitory signals to T cells. It is homologous to the T-cell costimulatory protein CD28, and both CTLA-4 and CD28 bind to CD80 (also designated B7-1) and CD86 (also designated B7-2). CTLA4 is also found on regulatory T cells (Tregs), contributing to their inhibitory function. The CTLA-4 protein contains an extracellular V domain, a transmembrane domain, and a cytoplasmic tail.

[0003] Antibodies that block the interaction of CTLA-4 with its ligands B7.1 and B7.2 have been shown to be able to enhance the immune response and stimulate potent anti-tumor immunity (Korman et al 2006, Checkpoint blockade in cancer immunotherapy, Adv Immunol. 90:297-339).

[0004] Promising clinical results with immunomodulatory monoclonal antibodies (mAbs) have revived the idea that the immune system holds the key to cancer control. The classification of these mAbs as checkpoint blockers (antagonists) or activators of costimulatory molecules (agonists) has recently been called into question by the discovery that examples of both types can fight tumors through the depletion of suppressive regulatory T cells (Tregs).

[0005] Immunomodulatory mAbs such as ipilimumab and other anti-CTLA4 antibodies have shown positive results when tested in difficult-to-treat malignancies, albeit in small numbers of patients (Hodi, FS, et al. 2010, N Engl J Med 363(8):711-723; Beatty, GL, et al. 2011, Science 331(6024):1612-1616; Brahmer, JR, et al. 2012, N Engl J Med 366(26):2455-2465; Topalian, SL, et al. 2012 N Engl J Med 366(26):2443-2454). These promising results have helped reinvigorate the idea that the immune system may hold the key to cancer control. These mAbs were generated to target key molecular regulators of T cells or antigen-presenting cells (APCs) and enhance anti-cancer immunity through blocking inhibitory signals (checkpoint blockers) or delivering costimulatory signals (agonists). Recently, this binary classification has been questioned when it was revealed that the therapeutic activity of anti-CTLA4, anti-GITR, and anti-OX40 antibodies, all of which target T cells, was accompanied by a loss of inhibitory CD4+ T regulatory cells that depended on the co-engagement of activating FcγRs (Bulliard, Jolicoeur et al. 2013; Marabelle, A., et al. 2013, J Clin Invest 123(6):2447-2463; Simpson, TR, et. J Exp Med 210(9):1695-1710).

[0006] Ipilimumab (YERVOY®, formerly 10D1, BMS-734016, MDX 101, MDX-010, MDX-CTLA-4, MDX-CTLA4), a monoclonal CTLA-4 antibody, is approved in several countries for the treatment of melanoma and is undergoing clinical trials for other indications (Weber 2008, Overcoming immunologic tolerance to melanoma: targeting CTLA-4 with ipilimumab (MDX-010) Oncologist, 13 (Suppl 4): 16-25). Ipilimumab is a fully human anti-CTLA-4 monoclonal antibody (IgG1κ) produced in Chinese hamster ovary cells by recombinant DNA technology. Its variants are 477202-00-9 and 6T8C155666. Ipilimumab is further defined in US9789182, which also provides the sequences of the heavy and light chains of ipilimumab (as SEQ ID NOs: 17 and 18, respectively), the sequences of the VH and / or VL regions (as SEQ ID NOs: 19 and 20, respectively) and the CDR sequences (heavy chain CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 21, 22, and 23, and light chain CDR1, CDR2 and CDR3 as set forth in SEQ ID NOs: 24, 25, and 26).

[0007] A second fully human monoclonal anti-CTLA-4 antibody being tested in several clinical trials is tremelimumab (formerly ticilimumab, CP-675,206) (Ribas 2008, Overcoming immunologic tolerance to melanoma: targeting CTLA-4 with tremelimumab (CP-675,206) Oncologist, 13(Suppl 4): 10-5; Callahan et al 2010, Anti-CTLA-4 Antibody Therapy: Immune Monitoring During Clinical Development of a Novel Immunotherapy. Semin Oncol. 37(5): 473-484.; Blank et al 2015, Therapeutic use of anti-CTLA-4 antibodies.. International Immunology, 27(1): 3-10).

[0008] Anti-CTLA-4 antibodies are described in several patent applications and patents, including the following:

[0009] WO93 / 00431 refers to CTLA4 receptor proteins, CTLA4Ig fusion proteins, and methods for modulating cell interactions using such fusion proteins or monoclonal antibodies.

[0010] WO97 / 20574 refers to blocking of T lymphocyte downregulation associated with CTLA-4 signaling, and to CTLA-4 blocking agents other than antibodies against the extracellular domain of CTLA-4 which increase the response of mammalian T cells to antigenic stimulation or reduce the proliferation of tumor cells in a mammalian host.

[0011] WO00 / 37504 refers to human anti-CTLA-4 antibodies and the use of such antibodies in the treatment of cancer. WO00 / 37504 further refers to the above-mentioned human monoclonal antibody tremelimumab, which is designated as 11.2.1 in its patent application. WO01 / 14424 also refers to human antibodies that specifically bind to human CTLA-4 and their use in the treatment of human diseases and infectious diseases, such as cancer. WO01 / 14424 further refers to the above-mentioned and further below-mentioned human monoclonal antibody ipilimumab, which is designated as 10D1 in its patent application. Summary of the Invention

[0012] The present invention relates to an antibody molecule that specifically binds to CTLA-4 and has an improved depleting effect on CTLA-4 positive cells compared to ipilimumab.

[0013] Furthermore, the present invention relates to an antibody molecule that specifically binds to CTLA-4, which comprises six CDRs having SEQ ID NOs: 15, 16, 17, 10, 18 and 19 or six CDRs having SEQ ID NOs: 22, 23, 24, 10, 25 and 26.

[0014] The present invention further relates to isolated nucleotide sequences encoding the above-described antibody molecules.

[0015] Furthermore, the present invention relates to a plasmid comprising the above-mentioned nucleotide sequence.

[0016] Furthermore, the present invention relates to a virus, such as an oncolytic virus, comprising a nucleotide sequence as defined above or a plasmid as defined above.

[0017] Furthermore, the present invention relates to a cell, such as a CAR T cell, comprising the above-mentioned nucleotide sequence or the above-mentioned plasmid.

[0018] Furthermore, the present invention relates to the antibody molecules, nucleotide sequences, plasmids and / or cells described above for use in medicine.

[0019] Furthermore, the present invention relates to the antibody molecules, nucleotide sequences, plasmids, viruses and / or cells described above for use in the treatment of cancer.

[0020] Furthermore, the present invention relates to the use of the above-mentioned antibody molecules, nucleotide sequences, plasmids, viruses and / or cells for the preparation of a pharmaceutical composition for use in the treatment of cancer.

[0021] Furthermore, the present invention relates to a pharmaceutical composition comprising at least one of the above-mentioned antibody molecules, nucleotide sequences, plasmids, viruses and / or cells, and optionally a pharma- ceutically acceptable diluent, carrier or excipient.

[0022] Furthermore, the present invention relates to a method for the treatment of cancer in a subject, comprising administering to the subject a therapeutically effective amount of at least one of the above-mentioned antibody molecules, nucleotide sequences, plasmids, viruses, cells and / or pharmaceutical compositions.

[0023] Furthermore, the invention relates to an antibody molecule, an antibody molecule for use, an isolated nucleotide sequence, an isolated nucleotide sequence for use, a plasmid, a plasmid for use, a virus, a virus for use, a cell, a cell for use, a use, a pharmaceutical composition or a method of treatment as herein described with reference to the detailed description, examples and / or figures. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] CTLA-4 positive cells are classified as regulatory T cells, Treg cells, Treg or T reg(previously known as suppressor T cells and sometimes referred to as suppressive regulatory T cells), are a subpopulation of T cells capable of suppressing other immune cells under normal and pathological immune environments. Tregs are CD4 positive cells (CD4+ cells). There are other CD4+ T cells that are not Tregs, but they can be separated from non-Treg CD4+ cells in that non-Treg CD4+ cells are FOXP3 negative (FOXP3-), whereas Tregs are also FOXP3 positive (FOXP3+).

[0025] Similar to ipilimumab, the anti-CTLA-4 antibody molecules described herein act at least in part by depleting CTLA-4 positive cells such as Treg.Furthermore, similar to ipilimumab, the anti-CTLA-4 antibody molecules described herein block the interaction of CTLA-4 with B7.1 and B7.2.Therefore, these antibodies are thus useful in overcoming the CTLA-4-induced suppressive effect on effector T cell proliferation.

[0026] As used herein, depletion of Tregs, or Treg depletion, refers to the depletion, deletion, or elimination of Tregs through physical clearance of cells. Specifically, it refers to the depletion of intratumoral Tregs. Depletion of Tregs can be achieved through ADCC, i.e., antibody-dependent cell-mediated cytotoxicity or antibody-dependent cellular cytotoxicity, and / or ADCP, i.e., antibody-dependent cellular phagocytosis. This means that when the antibody molecule described herein is administered to a subject, such as a human, it specifically binds to CTLA-4 expressed on the surface of Tregs, and this binding results in the depletion of Tregs. In some embodiments, CTLA-4 is preferentially expressed on tumor-infiltrating lymphocytes or tumor cells in the tumor microenvironment.

[0027] ADCC is an immune mechanism by which Fc receptor-bearing effector cells can recognize and kill antibody-coated target cells that express a tumor-derived antigen, in this case CTLA-4, on their surface. ADCP is a similar mechanism, but kills target cells by phagocytosis rather than cytotoxicity.

[0028] Antibodies are known to those skilled in the art of immunology and molecular biology. Typically, an antibody comprises two heavy chains (H) and two light chains (L). The complete antibody molecule is sometimes referred to herein as a full-size antibody or full-length antibody. The heavy chain of an antibody comprises one variable region (VH) and three constant regions (CH1, CH2, CH3), and the light chain of an antibody molecule comprises one variable region (VL) and one constant region (CL). The variable regions (sometimes referred to as F) are V The variable regions (collectively called the constant regions) bind to the antibody's target, or antigen. Each variable region contains three loops, called complementarity determining regions (CDRs), which are involved in target binding. The constant regions are not directly involved in binding the antibody to the antigen, but exhibit various effector functions. Depending on the amino acid sequence of the constant regions of their heavy chains, antibodies or immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and in humans, some of these are further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4; IgA1 and IgA2. Another part of an antibody is the Fc domain (also known as the fragment crystallizable domain), which contains the two constant domains of each of the antibody's heavy chains. The Fc domain is involved in the interaction between the antibody and the Fc receptor.

[0029] Fc receptors are membrane proteins that are often found on the cell surface of cells of the immune system (i.e., Fc receptors are found on the target cell membrane - alternatively known as the plasma membrane or cytoplasmic membrane). The role of Fc receptors is to bind antibodies via the Fc domain and internalize the antibody into the cell. In the immune system, this can result in antibody-mediated phagocytosis, and antibody-dependent cell-mediated cytotoxicity.

[0030] As used herein, the term antibody molecule encompasses full-length or full-size antibodies, as well as functional fragments of full-length antibodies and derivatives of such antibody molecules.

[0031] A functional fragment of a full-sized antibody has the same antigen-binding characteristics as the corresponding full-sized antibody, and contains either the same variable region (i.e., VH and VL sequences) and / or the same CDR sequences as the corresponding full-sized antibody. A functional fragment has the same antigen-binding characteristics as the corresponding full-sized antibody, meaning that it binds to the same epitope on the target as the full-sized antibody. Such a functional fragment may correspond to the Fv portion of a full-sized antibody. Alternatively, such a fragment may be designated as Fab, also designated F(ab), which is a monovalent antigen-binding fragment that does not contain an Fc portion, or F(ab'), which is a bivalent antigen-binding fragment that contains two antigen-binding Fab portions linked together by disulfide bonds. 2 , or F(ab'), i.e. F(ab') 2 Such a fragment may also be a single chain variable fragment (scFv).

[0032] Functional fragments do not always contain all six CDRs of the corresponding full-size antibody. It is understood that molecules containing three or less CDR regions (sometimes only a single CDR or a part thereof) can retain the antigen binding activity of the antibody derived from that CDR(s). For example, Gao et al., 1994, J.Biol.Chem.,269:32389-93, has described that the whole VL chain (containing all three CDRs) has high affinity for its substrate.

[0033] Molecules containing two CDR regions are described, for example, in Vaughan & Sollazzo 2001, Combinatorial Chemistry & High Throughput Screening, 4:417-430. On page 418 (right column-3 (Our Strategy for Design)), a minibody is described that contains only H1 and H2 CDR hypervariable regions interspersed within framework regions. The minibody is described as being capable of binding to a target. Pessi et al., 1993, Nature, 362:367-9, and Bianchi et al., 1994, J. Mol. Biol., 236:649-59, referenced by Vaughan & Sollazzo, describe H1 and H2 minibodies and their properties in more detail. Qiu et al., 2007, Nature Biotechnology, 25:921-9, show that a molecule consisting of two combined CDRs is capable of binding to an antigen. Quiocho 1993, Nature, 362:293-4 provides an overview of "minibody" technology. Ladner 2007, Nature Biotechnology, 25:875-7 observes that molecules containing two CDRs can retain antigen-binding activity.

[0034] Antibody molecules containing a single CDR region are described, for example, in Laune et al., 1997, JBC, 272:30937-44, where various hexapeptides derived from CDRs are shown to exhibit antigen-binding activity, and where it is noted that synthetic peptides of complete single CDRs exhibit strong binding activity. Monnet et al., 1999, JBC, 274:3789-96, where various 12-mer peptides and associated framework regions are shown to have antigen-binding activity, and where it is observed that CDR3-like peptides alone are capable of binding to antigens. Heap et al., 2005, J. Gen. Virol., 86:1791-1800, where it is reported that "micro-antibodies" (molecules containing a single CDR) are capable of binding to antigens, and where cyclic peptides from anti-HIV antibodies have been shown to have antigen-binding activity and function. Nicaise et al., 2004, Protein Science, 13:1882-91, show that a single CDR can confer antigen-binding activity and affinity for its lysozyme antigen.

[0035] Thus, antibody molecules having five, four, three or fewer CDRs can retain the antigen-binding properties of the full-length antibody from which they are derived.

[0036] An antibody molecule may be a derivative of a full-length antibody or a fragment of such an antibody. A derivative has the same antigen-binding characteristics as the corresponding full-sized antibody, meaning that it binds to the same epitope on the target as the full-sized antibody.

[0037] Thus, as used herein, the term "antibody molecule" includes monoclonal antibodies, polyclonal antibodies, synthetic antibodies, recombinantly produced antibodies, multispecific antibodies, bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, variable fragments (Fv), single chain variable fragments (scFv fragments), including bivalent single chain variable fragments (di-scFv) and disulfide linked variable fragments, Fab fragments, F(ab') 2 The present invention includes all types of antibody molecules, including fragments, Fab' fragments, antibody heavy chains, antibody light chains, antibody heavy chain homodimers, antibody light chain homodimers, antibody heavy chain heterodimers, antibody light chain heterodimers, antigen-binding functional fragments of such homo- and heterodimers, as well as functional fragments and derivatives thereof.

[0038] Furthermore, as used herein, the term "antibody molecule" includes all classes of antibody molecules and functional fragments, including IgG, IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD, and IgE.

[0039] In some embodiments, the antibody is human IgG1. Those skilled in the art recognize that mouse IgG2a and human IgG1 share the ability to productively bind to activating Fc gamma receptors and activate target cell deletion through activation of immune cells (e.g., macrophages and NK cells) that bear activating Fc gamma receptors, for example, by ADCP and ADCC. Thus, mouse IgG2a is the preferred isotype for deletion in mice, while human IgG1 is the preferred isotype for deletion in humans. Conversely, optimal costimulation of TNFR superfamily agonist receptors, such as 4-1BB, OX40, TNFRII, CD40, is known to depend on antibody binding of inhibitory FcγRII. In mice, the IgG1 isotype, which preferentially binds to inhibitory Fc gamma receptors (FcγRIIB) and only weakly binds to activating Fc gamma receptors, is known to be optimal for TNFR superfamily costimulatory activity targeting mAbs. Although no direct equivalent of the mouse IgG1 isotype in humans has been described, antibodies can be engineered to similarly exhibit improved binding to inhibitory human Fc gamma receptors over activating human Fc gamma receptors. Such engineered TNFR superfamily-targeting antibodies have also improved in vivo costimulatory activity in transgenic mice engineered to express human activating and inhibitory Fc gamma receptors (Dahan et al, 2016, Therapeutic Activity of Agonistic, Human Anti-CD40 Monoclonal Antibodies Requires Selective FcγR Engagement. Cancer Cell. 29(6):820-31).

[0040] As outlined above, different types and forms of antibody molecules are encompassed by the present invention and will be known to those skilled in the art of immunology. It is known that antibodies used for therapeutic purposes are often modified with additional moieties that modify the properties of the antibody molecule.

[0041] Thus, antibody molecules of the invention or used in accordance with the invention (e.g., monoclonal and / or polyclonal and / or bispecific antibody molecules) include those which are provided with a detectable moiety and / or a cytotoxic moiety.

[0042] A "detectable moiety" includes one or more from the group consisting of an enzyme, a radioactive atom, a fluorescent moiety, a chemiluminescent moiety, a bioluminescent moiety. The detectable moiety allows for visualization of the antibody molecule in vitro, and / or in vivo, and / or ex vivo.

[0043] "Cytotoxic moieties" include radioactive moieties and / or enzymes, e.g., enzymes such as caspases and / or toxins, e.g., toxins such as bacterial toxins or venoms, and cytotoxic moieties are capable of inducing cell lysis.

[0044] It is further included that the antibody molecules may be in isolated and / or purified form and / or may be PEGylated.

[0045] As mentioned above, the CDRs of an antibody bind to the antibody target. The amino acid assignments for each CDR described herein are as defined by Kabat EA et al., 1991, "Sequences of Proteins of Immulogical Interest," Fifth Edition, NIH Publication No. 91-3242, pp xv-xvii.

[0046] As those skilled in the art will recognize, there are other methods for assigning amino acids to each CDR, such as the International ImMunoGeneTics information system (IMGT™) (http: / / www.imgt.org / and Lefranc and Lefranc "The Immunoglobulin Facts Book" published by Academic Press, 2001).

[0047] In a further embodiment, the antibody molecule of or for use in accordance with the present invention is an antibody molecule capable of competing with a specific antibody described herein, such as the antibody molecules comprising SEQ ID NOs: 15, 16, 17, 10, 18 and 19 or SEQ ID NOs: 22, 23, 24, 10, 25 and 26.

[0048] By "capable of competing" it is meant that a competing antibody is capable of at least partially inhibiting or otherwise interfering with the binding of an antibody molecule as defined herein to a particular target.

[0049] For example, such a competing antibody molecule may be capable of inhibiting binding of an antibody molecule described herein by at least about 10%, e.g., at least about 20%, or 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%, at least about 95%, at least about 100% and / or may be capable of inhibiting the ability of an antibody described herein to bind to a particular target, and may prevent or reduce by at least about 10%, e.g., 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%, at least about 95%, or at least about 100%.

[0050] Competitive binding can be determined by methods known to those skilled in the art, such as enzyme linked immunosorbent assay (ELISA).

[0051] ELISA assay can be used to evaluate epitope-modifying or blocking antibody.Additional suitable methods for identifying competitive antibody are disclosed in Antibodies: A Laboratory Manual, Harlow & Lane (see, for example, pages 567-569, 574-576, 583, and 590-612, 1988, CSHL, NY, ISBN0-87969-314-2), which is incorporated herein by reference.

[0052] It is well known that antibodies specifically bind to a defined target molecule or antigen, meaning that the antibody binds preferentially and selectively to that target over non-target molecules.

[0053] The target CTLA-4 of the antibodies according to the invention or for use in accordance with the invention is expressed on the surface of a cell, i.e., they are cell surface antigens that comprise an epitope for the antibody (alternatively known in this context as a cell surface epitope). Cell surface antigens and epitopes are terms readily understood by those skilled in the art of immunology or cell biology.

[0054] By "cell surface antigen" we include cell surface antigens, or at least epitopes thereof, that are exposed on the extracellular side of the cell membrane to which the antibody molecules described herein are directed.

[0055] Methods for assessing protein binding are known to those skilled in the art of biochemistry and immunology. Those skilled in the art will understand that these methods can be used to assess the binding of antibody to target and / or the binding of Fc domain of antibody to Fc receptor, and the relative strength or specificity, inhibition, prevention, or reduction of their interactions. Examples of methods that can be used to assess protein binding include, for example, immunoassay, BIAcore, Western blot, radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA) (for a discussion of antibody specificity, see Fundamental Immunology, 2nd Edition, Raven Press, New York, pp. 332-336 (1989)).

[0056] Thus, as used herein, both an "antibody molecule that specifically binds to CTLA-4" and an "anti-CTLA-4 antibody molecule" refer to an antibody molecule that specifically binds to the target CTLA-4 but does not bind to non-targets, or that binds to non-targets weaker (e.g., with lower affinity) than the target.

[0057] In some embodiments, an antibody molecule that specifically binds to CTLA-4 (or an anti-CTLA-4 antibody molecule) refers to an antibody molecule that specifically binds to the extracellular domain of CTLA-4.

[0058] In some embodiments, an antibody molecule that specifically binds to CTLA-4 (or an anti-CTLA-4 antibody molecule) does not cross-react with CD28. In some embodiments, an antibody molecule that specifically binds to CTLA-4 (or an anti-CTLA-4 antibody molecule) blocks the binding of CTLA-4 to CD80 and / or CD86, thereby inhibiting CTLA-4 signaling.

[0059] It also includes the meaning that the antibody specifically binds to the target CTLA-4 at least 2 times stronger, or at least 5 times stronger, or at least 10 times stronger, or at least 20 times stronger, or at least 50 times stronger, or at least 100 times stronger, or at least 200 times stronger, or at least 500 times stronger, or at least about 1000 times stronger than a non-target.

[0060] In addition, the antibody binds to the target CTLA-4 at least about 10 -1 K d , or at least about 10 -2 K d , or at least about 10 -3 K d , or at least about 10 -4 K d , or at least about 10 -5 K d , or at least about 10 -6 K d , or at least about 10 -7 K d , or at least about 10 -8 K d , or at least about 10 -9 K d , or at least about 10 -10 K d , or at least about 10 -11 K d , or at least about 10-12 K d , or at least about 10 -13 K d , or at least about 10 -14 K d , or at least about 10 -15 K d K d By binding, it is meant that the antibody specifically binds to the target CTLA-4.

[0061] As described above, the antibody molecules that specifically bind to CTLA-4 (or anti-CTLA-4 antibody molecules) described herein have an improved depleting effect on CTLA-4 positive cells compared to ipilimumab.

[0062] That an antibody molecule has a depleting effect on CTLA-4 positive cells means that, when administered to a subject, such as a human, such an antibody specifically binds to CTLA-4 expressed on the surface of CTLA-4 positive cells, and this binding results in the depletion of such cells.

[0063] In some embodiments, the CTLA-4 positive cells are CD4 positive (CD4+) cells, ie, cells that express CD4.

[0064] In some embodiments, CTLA-4 positive cells are both CD4 positive and FOXP3 positive, i.e., express both CD4 and FOXP3.These cells are Treg.CD8 positive T cells also express CTLA-4, but Treg expresses significantly higher levels of CTLA-4 than CD8 positive T cells.This makes Treg more likely to be exhausted compared to CD8+ cells that express less.

[0065] In some situations, CTLA-4 is preferentially expressed on immune cells in the tumor microenvironment (tumor infiltrating cells, TILS).

[0066] Thus, in the tumor microenvironment, Tregs are the cells with the highest expression of CTLA-4, and antibody molecules that specifically bind to CTLA-4 (or anti-CTLA-4 antibody molecules) have a Treg-depleting effect, as described in more detail below, e.g., in Example 4 and in relation to FIG.

[0067] In some embodiments, the CTLA-4 positive cells are Tregs in solid tumors. Such Tregs have a very high expression of CTLA-4, so administering an antibody molecule that specifically binds to CTLA-4 preferentially results in the depletion of such Tregs.

[0068] As described above, the anti-CTLA-4 antibody molecules described herein are Treg-depleting antibody molecules, meaning that when administered to a subject, such as a human, such antibody molecules specifically bind to CTLA-4 expressed on the surface of Tregs, and this binding results in the depletion of Tregs.

[0069] To determine whether an antibody molecule has an improved depleting effect on CTLA-4 positive cells compared to ipilimumab as referred to herein, an in vitro antibody-dependent cellular cytotoxicity (ADCC) assay or an in vivo test in the PBMC-NOG / SCID model can be used.

[0070] An in vitro ADCC test performed using an NK-92 cell line stably transfected to express the CD16-158V allele together with GFP, the ADCC test comprising the following seven consecutive steps: 1) Isolating CTLA-4 positive cells, CD4 positive cells, or Tregs as target cells from peripheral blood of healthy donors, which can be done using a CD4+ T cell isolation kit, such as a commercially available kit from Miltenyi Biotec. 2) The target cells are then stimulated with CD3 / CD28, e.g., using CD3 / CD28 Dynabeads® and rhIL-2, e.g., 50 ng / ml rhIL-2, for e.g., 48 hours. Stimulation can be performed at 37°C. 3) The target cells are then pre-incubated with the antibody molecule to be tested, for example at 10 μg / ml, for 30 minutes at 4° C., and then mixed with the NK cells. 4) Next, incubate the target cells in RPMI 1640+GlutaMAX medium containing HEPES buffer, sodium pyruvate, and FBS low IgG for an appropriate time, for example 4 hours. RPMI 1640+GlutaMAX medium may contain 10 mM HEPES buffer, 1 mM sodium pyruvate, and 10% FBS low IgG, and the effector:target cell ratio may be 2:1. 5) Determining lysis by flow cytometry. 6) Repeating or performing steps 1-5 in parallel, with ipilimumab used in place of the antibody molecule tested in step 3. 7) Comparing the lysis results of the tested antibody molecule with those of ipilimumab. Improved lysis of the tested antibody molecule compared to ipilimumab indicates that the tested antibody molecule has an improved depletion effect on CTLA-4 positive cells, CD4 positive cells or Tregs, respectively, depending on the target cells used.

[0071] In some embodiments, the improved depletion effect in step 7) above is a significantly improved depletion effect.

[0072] This assay is presented in more detail below in Example 4 in conjunction with FIG.

[0073] The in vivo test is based on the combination of PBMC mice and NOG / SCID mice, referred to herein as the PBMC-NOG / SCID model. Both PBMC mice and NOG / SCID mice are known models. The in vivo test in the PBMC-NOG / SCID model consists of the following nine consecutive steps: 1) Isolate, wash, and resuspend human PBMCs (peripheral blood mononuclear cells) in sterile PBS. In some embodiments, PBMCs are at a concentration of 75×10 6 Cells / ml are resuspended in PBS. 2) Inject the NOG mice iv (intravenously) with an appropriate amount of the cell suspension from step 1), for example 200 μl. If you are injecting 200 μl, this will give you 15×10 6 Corresponds to cells / mouse. 3) At an appropriate time after injection, for example 2 weeks, isolate the spleen from the NOG mouse and make it into a single cell suspension. Optionally, take a small sample from the single cell suspension to determine the expression of CTLA-4 on human T cells by FACS to confirm the expression of CTLA-4. 4) Resuspending the cell suspension of step 3) in sterile PBS. In some embodiments, the suspension contains 50×10 6 Resuspend in sterile PBS at 1000 cells / ml. If the optional CTLA-4 expression determination is included in step 3, the remaining cell suspension is resuspended in step 4. 5) Inject the SCID mice ip (intraperitoneally) with an appropriate amount of the suspension from step 4, for example 200 μl. If you are injecting 200 μl, this is 10×10 6 Corresponds to cells / mouse. 6) At an appropriate time, e.g., 1 hour, after injection of step 5), treating the SCID mice with an appropriate amount, e.g., 10 mg / kg, of either the antibody molecule to be tested, ipilimumab or an isotype control monoclonal antibody. 7) Collecting the intraperitoneal fluid of the treated SCID mice at an appropriate time, for example 24 hours, after treatment in step 6). 8) Identifying and quantifying human T cell subsets by FACS using the markers CD45, CD4, CD8, CD25 and / or CD127. 9) comparing the results of identification and quantification of T cell subsets from mice treated with the tested antibody molecule with the results of identification and quantification of T cell subsets from mice treated with ipilimumab and with the results of identification and quantification of T cell subsets from mice treated with an isotype control monoclonal antibody. A lower number of CTLA-4 positive cells in the peritoneal fluid of mice treated with the tested antibody molecule compared to the number of CTLA-4 positive cells in the peritoneal fluid of mice treated with ipilimumab indicates that the antibody molecule has an improved depletion effect on CTLA-4 positive cells compared to ipilimumab. A lower number of CD4 positive cells in the peritoneal fluid of mice treated with the tested antibody molecule compared to the number of CD4 positive cells in the peritoneal fluid of mice treated with ipilimumab indicates that the antibody molecule has an improved depletion effect on CD4 positive cells compared to ipilimumab. The lower number of Tregs in the peritoneal fluid of mice treated with the tested antibody molecule compared to the number of Tregs in the peritoneal fluid of mice treated with ipilimumab indicates that this antibody molecule has an improved depleting effect on Tregs compared to ipilimumab.

[0074] In this in vivo study, in some embodiments, it is of most interest to examine Treg depletion at step 7.

[0075] This assay is presented in more detail in Example 4 below, in conjunction with FIG.

[0076] Treg depletion can also be assessed in an antibody-dependent cellular phagocytosis (ADCP) assay, as known to those of skill in the art.

[0077] In some embodiments, the antibody molecules have a similar blocking effect on CTLA-4 interaction with B7.1 and B7.2 ligands compared to Yervoy, which can be assessed by ELISA (shown in FIG. 10) or by a more functional assay in which anti-CTLA-4 antibodies enhance IL-2 production by T cells in response to stimulation of PBMCs with SEB.

[0078] In some embodiments, the anti-CTLA-4 antibody molecule is a human antibody molecule.

[0079] In some embodiments, the anti-CTLA-4 antibody molecule is a humanized antibody molecule.

[0080] In some embodiments, the anti-CTLA-4 antibody molecule is a human-derived antibody molecule, meaning that it is derived from a human antibody molecule that has been subsequently modified.

[0081] In some embodiments, the anti-CTLA-4 antibody molecule is a human IgG1 antibody.

[0082] In some embodiments, the anti-CTLA-4 antibody is also an antibody in the form of a human IgG1 antibody that exhibits improved binding to one or more activating Fc receptors and / or has been engineered for improved binding to one or more activating Fc receptors, and thus in some embodiments the anti-CTLA-4 antibody is an Fc-engineered human IgG1 antibody.

[0083] In some embodiments, the anti-CTLA-4 antibody is a murine or humanized murine IgG2a antibody.

[0084] In some embodiments, the anti-CTLA-4 antibody is a murine antibody that is cross-reactive with human CTLA-4.

[0085] In some embodiments, the anti-CTLA-4 antibody is a monoclonal antibody.

[0086] In some embodiments, the anti-CTLA-4 antibody is a polyclonal antibody.

[0087] In some embodiments, the anti-CTLA-4 antibody molecule is an antibody molecule that comprises one of three alternative VH-CDR1 sequences, one of three alternative VH-CDR2 sequences, one of two alternative VH-CDR3 sequences, one of two VL-CDR1 sequences, one of two VL-CDR2 sequences, and / or one of two alternative VL-CDR3 sequences as shown in Table 1 below.

[0088] In some embodiments, the anti-CTLA-4 antibody molecule is selected from the group consisting of antibody molecules comprising one to six CDRs selected from the group consisting of SEQ ID NOs: 3, 6, 8, 10, 12 and 14.

[0089] In some embodiments, the anti-CTLA-4 antibody molecule is selected from the group consisting of antibody molecules comprising CDRs having SEQ ID NOs: 3, 6, 8, 10, 12 and 14.

[0090] In some embodiments, the anti-CTLA-4 antibody molecule is selected from the group consisting of antibody molecules comprising 1-6 of the CDRs, VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, and VL-CDR3; If present, VH-CDR1 is selected from the group consisting of SEQ ID NOs: 15, 22, 29 and 35; If present, VH-CDR2 is selected from the group consisting of SEQ ID NOs: 16, 23, 30, and 36; If present, the VH-CDR3 is selected from the group consisting of SEQ ID NOs: 17, 24, 31 and 37; If present, VL-CDR1 is selected from the group consisting of SEQ ID NOs: 10 and 38; If present, VL-CDR2 is selected from the group consisting of SEQ ID NOs: 18, 25, 32 and 39; If present, the VL-CDR3 is selected from the group consisting of SEQ ID NOs: 19, 26 and 40.

[0091] In some embodiments, the anti-CTLA-4 antibody molecule is selected from the group consisting of antibody molecules comprising six CDRs selected from the group consisting of: SEQ ID NOs: 15, 16, 17, 10, 18 and 19; SEQ ID NOs: 22, 23, 24, 10, 25 and 26; SEQ ID NOs: 29, 30, 31, 10, 32 and 26; and Sequence numbers 35, 36, 37, 38, 39 and 40.

[0092] In some embodiments, the anti-CTLA-4 antibody molecule is an antibody molecule that comprises six CDRs having SEQ ID NOs:15, 16, 17, 10, 18 and 19.

[0093] In some embodiments, the anti-CTLA-4 antibody molecule is an antibody molecule that comprises six CDRs having SEQ ID NOs:22, 23, 24, 10, 25 and 26.

[0094] In some embodiments, the anti-CTLA-4 antibody molecule is an antibody molecule selected from the group consisting of antibody molecules having a VH selected from the group consisting of SEQ ID NOs: 20, 27, 33 and 41.

[0095] In some embodiments, the anti-CTLA-4 antibody molecule is an antibody molecule selected from the group consisting of antibody molecules having a VL selected from the group consisting of SEQ ID NOs: 21, 28, 34 and 42.

[0096] In some embodiments, the anti-CTLA-4 antibody molecule is an antibody molecule selected from the group consisting of antibody molecules having a VH and VL selected from the group consisting of SEQ ID NOs: 20-21, 27-28, 33-34 and 41-42.

[0097] In some embodiments, the anti-CTLA-4 antibody molecule comprises a VH having SEQ ID NO:20 and a VL having SEQ ID NO:21.

[0098] In some embodiments, the anti-CTLA-4 antibody molecule comprises a VH having SEQ ID NO:27 and a VL having SEQ ID NO:28. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2] [Table 2-3]

[0099] In some embodiments, the anti-CTLA-4 antibody molecules described herein may also comprise one or both of the constant regions shown in Table 3 below. [Table 3]

[0100] In this embodiment, the anti-CTLA-4 antibody molecule is a molecule encoded by one of the nucleotide sequences shown in Table 4 below. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0101] In some embodiments, it is advantageous for the antibody molecule to bind both human CTLA-4 (hCTLA-4) and cynomolgus monkey CTLA-4 (cmCTLA-4 or cynoCTLA-4). Cross-reactivity with CTLA-4 expressed on cells of cynomolgus monkeys, also called crab-eating macaques or Macaca fascicularis, can be advantageous as it allows for testing of the antibody molecule in monkeys without the use of surrogate antibodies, with particular focus on tolerability.

[0102] In some embodiments, it is advantageous for the antibody molecule to bind to both human CTLA-4 (hCTLA-4) and mouse CTLA-4 (mCTLA-4). This can be advantageous as it allows for testing of the antibody molecule in mice, with a particular focus on efficacy and pharmacodynamics, without the need to use surrogate antibodies.

[0103] In some embodiments, the antibody molecule binds to all three of hCTLA-4, cmCTLA-4 and mCTLA-4.

[0104] In some embodiments, testing the functional activity of an antibody molecule in a relevant in vivo model in mice requires the use of a surrogate antibody. To ensure comparability between the effect of the antibody molecule in humans and the in vivo results of the surrogate antibody in mice, it is essential to select a functionally equivalent surrogate antibody that has the same in vitro properties as the human antibody molecule.

[0105] In some embodiments, the antibody molecule does not bind to human CD28.

[0106] For example, it will be known to those skilled in the art of medicine that drugs can be modified with different additives to alter the rate at which they are absorbed by the body, and can be modified in different forms, for example to allow for particular routes of administration to the body.

[0107] Thus, it is included that the antibody molecules, nucleotide sequences, plasmids, viruses and / or cells described herein can be combined with a pharma- ceutically acceptable excipient, carrier, diluent, vehicle and / or adjuvant to form a pharmaceutical composition. In this context, the term pharmaceutical composition can be used interchangeably with the terms pharmaceutical preparation, pharmaceutical formulation, therapeutic composition, therapeutic preparation, therapeutic formulation, and therapeutic entity.

[0108] The pharmaceutical compositions described herein may comprise, or in some embodiments consist of, antibody molecules, nucleotide sequences, plasmids, viruses or cells.

[0109] The pharmaceutical compositions described herein may in some embodiments consist of or comprise a plasmid comprising a nucleotide sequence encoding the above-described antibody molecule or comprising the above-described nucleotide sequence.

[0110] In some embodiments, the pharmaceutical composition may comprise a nucleotide sequence encoding a portion or a complete antibody molecule described herein integrated into a cellular or viral genome or viriome. And, the pharmaceutical composition may comprise a cell or virus as a delivery vehicle for the antibody of the invention (or a delivery vehicle for a nucleotide sequence encoding the antibody of the invention). For example, in some embodiments, the virus may be in the form of a therapeutic oncolytic virus comprising a nucleotide sequence encoding at least one of the antibody molecules described herein. In some embodiments, such an oncolytic virus comprises a nucleotide sequence encoding a full-length human IgG antibody. In some embodiments, such an oncolytic virus comprises a nucleotide sequence encoding a scFv, Fab or F(ab') 2 It comprises a nucleotide sequence that encodes an antibody molecule.

[0111] As described in the appended claims, the present invention relates in one embodiment to a virus comprising the nucleotide sequence of the present invention or the plasmid of the present invention. Preferably, the virus is an oncolytic virus, such as a therapeutic oncolytic virus. As used herein, the term "oncolytic" refers to the ability of a virus to selectively replicate in dividing cells (e.g., proliferating cells, such as cancer cells), either in vitro or in vivo, with the aim of slowing down the growth and / or lysing the dividing cells, while eliminating or minimizing replication in non-dividing (e.g., normal or healthy) cells. "Replication" (or any form of replication, such as "replicate" and "replicating") refers to viral duplication that may occur at the level of nucleic acid or, preferably, at the level of infectious viral particles. Such oncolytic viruses can be obtained from any member of the currently identified viruses. It may be a natural virus that is naturally oncolytic or can be engineered by modifying one or more viral genes to increase tumor selectivity and / or preferential replication in dividing cells, such as those involved in DNA replication, nucleic acid metabolism, host tropism, surface attachment, virulence, lysis and spread (see, for example, Wong et al., 2010, Viruses 2:78-106). It is also possible to envision placing one or more viral gene(s) under the control of an event or tissue-specific regulatory element (e.g., a promoter). Exemplary oncolytic viruses include, but are not limited to, reoviruses, Seneca Valley viruses (SVV), vesicular stomatitis viruses (VSV), Newcastle disease viruses (NDV), herpes simplex viruses (HSV), morbilliviruses, adenoviruses, poxviruses, retroviruses, measles viruses, foamy viruses, alphaviruses, lentiviruses, influenza viruses, Sinbis viruses, myxoma viruses, rhabdoviruses, picornaviruses, coxsackie viruses, parvoviruses, and the like.Oncolytic viruses are known to those skilled in the art of medicine and virology.

[0112] In some embodiments, such oncolytic viruses are derived from herpes viruses. The Herpesviridae family is a large family of DNA viruses that all share a common structure, consisting of a relatively large double-stranded linear DNA genome encoding 100-200 genes, encapsidated within an icosahedral capsid surrounded by a lipid bilayer membrane. Oncolytic herpes viruses can be derived from different types of HSV, with HSV1 and HSV2 being particularly preferred. Herpes viruses can be genetically modified to restrict viral replication in tumors or reduce cytotoxicity in non-dividing cells. For example, any viral gene involved in nucleic acid metabolism can be inactivated, such as thymidine kinase (Martuza et al., 1991, Science 252:854-6), ribonucleotide reductase (RR) (Mineta et al., 1994, Cancer Res. 54:3363-66), or uracil-N-glycosylase (Pyles et al., 1994, J. Virol. 68:4963-72). Another embodiment includes viral mutants defective in the function of genes encoding virulence factors, such as the ICP34.5 gene (Chambers et al., 1995, Proc. Natl. Acad. Sci. USA 92:1411-5). Representative examples of oncolytic herpesviruses include NV1020 (e.g., Geevarghese et al., 2010, Hum. Gene Ther. 21(9):1119-28) and T-VEC (Harrington et al., 2015, Expert Rev. Anticancer Ther. 15(12):1389-1403).

[0113] In some embodiments, such oncolytic viruses are derived from adenoviruses. Methods for engineering oncolytic adenoviruses are available in the art. Advantageous strategies include replacing viral promoters with tumor-selective promoters or modifying E1 adenoviral gene product(s) to inactivate their binding function with p53 or retinoblastoma (Rb) proteins, which are altered in tumor cells. In the natural situation, the adenoviral E1B55kDa gene cooperates with another adenoviral product to inactivate p53 (which is frequently dysregulated in cancer cells) and prevent apoptosis. Representative examples of oncolytic adenoviruses include ONYX-015 (e.g., Khuri et al., 2000, Nat.Med 6(8):879-85) and H101, also called Oncorine (Xia et al., 2004, Ai Zheng 23(12):1666-70).

[0114] In some embodiments, such oncolytic virus is a poxvirus. As used herein, the term "poxvirus" refers to a virus belonging to the family Poxviridae, with poxviruses belonging to the subfamily Chordopoxviridae, more preferably the genus Orthopoxvirus, being particularly preferred. Vaccinia virus, cowpox virus, canarypox virus, ectromelia virus, myxoma virus are particularly suitable in the context of the present invention. The genome sequences of such poxviruses are available in the art and in specialized databases (e.g. Genbank under accession numbers NC_006998, NC_003663 or AF482758.2, NC_005309, NC_004105, NC_001132, respectively).

[0115] In a particular preferred embodiment, such an oncolytic poxvirus is an oncolytic vaccinia virus. Vaccinia virus is a member of the poxvirus family characterized by a 200 kb double-stranded DNA genome that encodes a number of viral enzymes and factors that allow the virus to replicate independently of the host cell machinery. The majority of vaccinia virus particles are intracellular (IMV for intracellular mature virions), have a single lipid envelope, and remain in the cytosol of infected cells until lysis. Another form of infection is a double-enveloped particle (EEV for extracellular enveloped virions) that buds without lysing the infected cell. Vaccinia virus strains may be derived, with Elstree, Wyeth, Copenhagen, Lister, and Western Reserve strains being particularly preferred. The gene nomenclature used herein is that of the Copenhagen vaccinia strain unless otherwise stated. However, the correspondence between Copenhagen and other vaccinia strains is generally available in the literature.

[0116] Preferably, such oncolytic vaccinia viruses are modified by modifying one or more viral gene(s). The modification(s) preferably result in the absence of synthesis or the synthesis of defective viral proteins that cannot guarantee the activity of the proteins produced under normal conditions by the unmodified genes. Exemplary modifications are disclosed in the literature that aim to modify viral genes involved in DNA metabolism, host virulence, IFN pathway (e.g. Guse et al., 2011, Expert Opinion Biol. Ther. 11(5):595-608), etc. Modifications to alter viral loci encompass deletions, mutations and / or substitutions of one or more nucleotide(s) (whether adjacent or not) within the viral gene or its regulatory elements. The modification(s) can be made by several methods known to those skilled in the art using conventional recombinant techniques.

[0117] More preferably, such oncolytic vaccinia viruses are modified by altering the gene encoding thymidine kinase (locus J2R). The thymidine kinase (TK) enzyme is involved in the synthesis of deoxyribonucleotides. TK is required for viral replication in normal cells, since these cells generally have low nucleotide concentrations, whereas it is not required in dividing cells, which contain high nucleotide concentrations.

[0118] Alternatively or in combination, such oncolytic vaccinia viruses are modified by altering at least one or both genes encoding ribonucleotide reductase (RR). In the natural context, this enzyme catalyzes the reduction of ribonucleotides to deoxyribonucleotides, which represents a key step in DNA biosynthesis. The viral enzyme is similar in subunit structure to the mammalian enzyme and is composed of two heterologous subunits designated R1 and R2, encoded by the I4L and F4L loci, respectively. In the context of the present invention, either or both of the I4L gene (encoding the R1 large subunit) or the F4L gene (encoding the R2 small subunit) can be inactivated (e.g., as described in WO2009 / 065546 and Foloppe et al., 2008, Gene Ther., 15:1361-71). The sequences of the J2R, I4L and F4L genes and their locations in the genomes of various poxviruses are available in public databases.

[0119] In some embodiments, such oncolytic viruses comprise a nucleotide sequence encoding an amino acid sequence having at least 80% identity to a sequence shown in Table 2 above. In some embodiments, such oncolytic viruses comprise an amino acid sequence having at least 85% identity to a sequence shown in Table 2 above. In some embodiments, such oncolytic viruses comprise an amino acid sequence having at least 90% identity to a sequence shown in Table 2 above. In some embodiments, such oncolytic viruses comprise an amino acid sequence having at least 95% identity to a sequence shown in Table 2 above.

[0120] In some embodiments, such oncolytic viruses comprise nucleotide sequences encoding SEQ ID NO:20 and SEQ ID NO:21. In some embodiments, such oncolytic viruses comprise nucleotide sequences encoding SEQ ID NO:27 and SEQ ID NO:28. In some embodiments, such oncolytic viruses comprise nucleotide sequences encoding SEQ ID NO:33 and SEQ ID NO:34. In some embodiments, such oncolytic viruses comprise nucleotide sequences encoding SEQ ID NO:41 and SEQ ID NO:42.

[0121] In some embodiments, such oncolytic viruses comprise a nucleotide sequence having at least 80% identity to a sequence shown in Table 4 above. In some embodiments, such oncolytic viruses comprise a nucleotide sequence having at least 85% identity to a sequence shown in Table 4 above. In some embodiments, such oncolytic viruses comprise a nucleotide sequence having at least 90% identity to a sequence shown in Table 4 above. In some embodiments, such oncolytic viruses comprise a nucleotide sequence having at least 95% identity to a sequence shown in Table 4 above.

[0122] In some embodiments, such oncolytic viruses comprise SEQ ID NOs: 45 and 46. In some embodiments, such oncolytic viruses comprise SEQ ID NOs: 47 and 48. In some embodiments, such oncolytic viruses comprise SEQ ID NOs: 49 and 50. In some embodiments, such oncolytic viruses comprise SEQ ID NOs: 51 and 52.

[0123] Some oncolytic viruses have the ability to accept DNA inserts large enough to accommodate the integration of full-length human antibody sequences. Attenuated vaccinia virus and herpes simplex virus are examples of therapeutic oncolytic viruses whose genomes are large enough to allow the integration of full-length IgG antibody sequences (Chan,WMet al 2014 Annu Rev Virol 1(1):119-141, Bommareddy,PK,et al.2018 Nat Rev Immunol 18(8):498-513). Full-length IgG antibodies are successfully integrated into oncolytic vaccinia viruses, resulting in the expression and extracellular release (production) of full-length IgG antibodies upon infection of virus-susceptible host cells, e.g., cancer cells (Kleinpeter,P.,et al.2016,Oncoimmunology 5(10):e1220467). Adenoviruses can also be engineered to encode full-length IgG antibodies that are functionally produced and secreted upon cell infection (Marino, N., et al. 2017 J Clin Invest 123(6):2447-2463).

[0124] In a preferred embodiment, such an oncolytic virus is a poxvirus (e.g., a vaccinia virus) that is defective in TK activity (due to an alteration in the J2R locus) or defective in both TK and RR activity (due to an alteration in both the J2R locus and at least one of the I4L and / or F4L loci encoding RR) and comprises (a) a nucleotide sequence encoding SEQ ID NO:20 and SEQ ID NO:21, or (b) a nucleotide sequence encoding SEQ ID NO:27 and SEQ ID NO:28, or (c) a nucleotide sequence encoding SEQ ID NO:33 and SEQ ID NO:34, or (d) a nucleotide sequence encoding SEQ ID NO:41 and SEQ ID NO:42.

[0125] Where appropriate, it may be advantageous for the nucleotide sequence(s) inserted into the oncolytic virus described herein to include additional regulatory elements to facilitate expression, transport and biological activity. For example, a signal peptide may be included to facilitate secretion outside the producer cell (e.g., infected cell). The signal peptide is usually inserted at the N-terminus of the encoded polypeptide immediately after the Met initiator. The choice of signal peptide is broad and available to those skilled in the art. For example, a signal peptide derived from another immunoglobulin (e.g., heavy chain IgG) can be used in the context of the present invention to secrete the anti-CTLA4 antibody described herein outside the producer cell. For illustration, reference can be made to SEQ ID NO: 53 and SEQ ID NO: 54, which comprise the light and heavy chains of the 4-E03 antibody described herein with an IgG-derived peptide signal.

[0126] A particularly preferred oncolytic virus is a vaccinia virus (e.g., the Copenhagen strain) that is defective in both TK and RR activity (due to alterations in both the J2R and I4L loci) and comprises a nucleotide sequence encoding SEQ ID NO:20 and SEQ ID NO:21 or SEQ ID NO:53 and SEQ ID NO:54.

[0127] In some embodiments, such oncolytic viruses can further comprise additional nucleotide sequence(s) of therapeutic interest, such as nucleotide sequence(s) encoding immunomodulatory polypeptide(s) (i.e., polypeptides involved in stimulating an immune response, directly or indirectly). Representative examples of suitable immunomodulatory polypeptides include, but are not limited to, cytokines and chemokines, with particular preference for granulocyte-macrophage colony-stimulating factor (GM-CSF), in particular human, non-human primate or mouse GM-CSF. Additional nucleotide sequences can be readily obtained by standard molecular biology techniques (e.g., PCR amplification, cDNA cloning, chemical synthesis) using sequence data accessible in the art and the information provided herein. A particularly preferred oncolytic virus is a vaccinia virus (e.g., the Copenhagen strain) that is defective in both TK and RR activity (due to alterations in both the J2R and I4L loci) and contains a nucleotide sequence encoding SEQ ID NO:20 and SEQ ID NO:21 or SEQ ID NO:53 and SEQ ID NO:54, as well as a nucleotide sequence encoding GM-CSF, with human GM-CSF (e.g., having SEQ ID NO:55 or SEQ ID NO:56) or murine GM-CSF (e.g., having SEQ ID NO:57 or SEQ ID NO:58) being particularly preferred.

[0128] Furthermore, the nucleotide sequence inserted into such oncolytic viruses can be optimized to provide high levels of expression in a particular host cell or subject by modifying one or more codon(s). In addition to optimizing codon usage, various modifications can also be envisaged to prevent clustering of rare non-optimal codons present in concentrated regions and / or to suppress or modify "negative" sequence elements that are expected to negatively affect expression levels. Such negative sequence elements include, but are not limited to, regions with very high (>80%) or very low (<30%) GC content; AT-rich or GC-rich sequence stretches; unstable direct or inverted repeats; RA secondary structures; and / or internal potential regulatory elements such as internal TATA boxes, chi sites, ribosome entry sites, and / or splicing donor / acceptor sites.

[0129] In some embodiments, the nucleotide sequence(s) are placed under the control of appropriate regulatory elements for their proper expression in a host cell or subject. As used herein, the term "regulatory element" refers to any element that allows, contributes to, or regulates the expression of the coding nucleotide sequence(s) in a given host cell or subject, including its replication, duplication, transcription, splicing, translation, stability, and / or transport in and out of the expression cell. It will be understood by those skilled in the art that the choice of regulatory element may depend on factors such as the nucleotide sequence itself, the virus into which it is inserted, the host cell or subject, the desired expression level, and the like. The promoter is particularly important. In the context of the present invention, it may be a constitutive directed expression of the nucleotide sequence that is controlled in many types of host cells, or specific for a particular host cell, or regulated in response to a particular event or exogenous factor (e.g., by temperature, nutrient additives, hormones, etc.), or according to the stage of the viral cycle (e.g., late or early). Promoters adapted for viral-mediated expression are known in the art. Representative examples of oncolytic poxvirus expression include, but are not limited to, vaccinia p7.5K, pH5.R, p11K7.5, TK, p28, p11, pB2R, pA35R, K1L and pSE / L promoters (Erbs et al., 2008, Cancer Gene Ther. 15(1):18-28; Orubu et al. 2012, PloS One 7:e40167), early / late chimeric promoters and synthetic promoters (Chakrabarti et al., 1997, Biotechniques 23:1094-7; Hammond et al, 1997, J. Virol Methods 66:135-8; and Kumar and Boyle, 1990, Virology 179:151-8).In a preferred embodiment, the nucleotide sequences of the light and heavy chains of the antibodies described herein are each placed under the control of a promoter with the same transcriptional strength, preferably the same promoter (e.g., p7.5K as set forth in SEQ ID NO:59 or pH5.R as set forth in SEQ ID NO:60) to obtain similar levels of expression for both chains and thus optimal assembly of the antibody as a heterotetrameric protein (i.e., avoiding an excess of unassociated chains). Additional nucleotide sequences (e.g., encoding GM-CSF) can be placed under a different promoter (e.g., pSE / L as set forth in SEQ ID NO:61).

[0130] The insertion of the nucleotide sequence(s) (possibly with suitable regulatory elements) into the genome of such oncolytic viruses is carried out by conventional means, using suitable restriction enzymes or preferably by homologous recombination. The nucleotide sequence(s) can be inserted independently anywhere in the viral genome. Various insertion sites can be considered, for example, non-essential viral genes, intergenic regions, or non-coding parts of the genome of such oncolytic viruses. The J2R locus and / or the I4L locus are particularly suitable for oncolytic viruses that are poxviruses (e.g. oncolytic vaccinia viruses). The viral locus at the insertion site of the insertion of the nucleotide sequence(s) into the viral genome can be at least partially deleted. In one embodiment, this deletion or partial deletion can result in suppressed expression of the viral gene product encoded by the completely or partially deleted locus, resulting in a virus that is deficient in the viral function. A particularly preferred oncolytic virus is a TK and / or RR defective vaccinia virus that comprises a cassette encoding a heavy chain inserted into the J2R locus and a cassette encoding a light chain inserted into the I4L locus. The cassette encoding the additional GM-CSF encoding nucleotide sequence can be inserted elsewhere in the viral genome or into the J2R or I4L locus, with insertion into the I4L locus being preferred.

[0131] The present invention also provides methods for generating such oncolytic viruses, in particular oncolytic poxviruses, as described herein, in suitable host cells (producer cells). In some embodiments, such methods comprise one or more steps of homologous recombination between the viral genome and a transfer plasmid comprising nucleotide sequences (possibly with regulatory elements) to be inserted adjacent to the 5' and 3' viral sequences present upstream and downstream, respectively, of the insertion site. Such transfer plasmids can be generated and introduced into the host cell by routine techniques (e.g. by transfection). The viral genome can be introduced into the host cell by infection. The size of each flanking viral sequence can vary from at least 100 bp up to a maximum of 1500 bp on each side of the nucleotide sequence (preferably 200-550 bp, more preferably 250-500 bp). The homologous recombination allowing the generation of such oncolytic viruses is preferably carried out in cultured cell lines (e.g. HeLa, Vero) or in chicken embryo fibroblast (CEF) cells obtained from embryonated eggs.

[0132] In some embodiments, the identification of oncolytic viruses incorporating anti-CTLA4 coding nucleotide sequences and possibly additional nucleotide sequences (e.g., GM-CSF) can be facilitated by the use of a selection and / or detectable gene. In a preferred embodiment, the transfer plasmid further comprises a selection marker, particularly preferably the GPT gene (encoding guanine phosphoribosyltransferase) or a detectable gene encoding a detectable gene product such as GFP, e-GFP, mCherry, etc., that allows growth in a selection medium (e.g., in the presence of mycophenolic acid, xanthine and hypoxanthine). In addition, the use of an endonuclease capable of providing a double-strand break in the selection or detectable gene can also be considered. The endonuclease can be in the form of a protein or can be expressed by an expression vector.

[0133] Once generated, such oncolytic viruses can be amplified in suitable host cells using conventional techniques, including culturing the transfected or infected host cells under appropriate conditions to allow for the production and recovery of infectious particles.

[0134] The present invention also relates to a method for producing an oncolytic virus as described herein, preferably comprising the steps of a) preparing a producer cell line, b) transfecting or infecting the prepared producer cell line with an oncolytic virus, c) culturing the transfected or infected producer cell line under suitable conditions to allow production of virus, d) recovering the produced virus from the culture of the producer cell line, and optionally e) purifying the recovered virus.

[0135] In some embodiments, producer cells are selected from the group consisting of mammalian (e.g., human or non-human) cells such as HeLa cells (e.g., ATCC-CRM-CCL-2™ or ATCC-CCL-2.2™), HER96, PER-C6 (Fallaux et al., 1998, Human Gene Ther. 9:1909-17), hamster cell lines such as BHK-21 (ATCC CCL-10) and avian cells as described in WO2005 / 042728, WO2006 / 108846, WO2008 / 129058, WO2010 / 130756, WO2012 / 001075, and primary chicken embryo fibroblasts (CEF) prepared from chicken embryos obtained from fertilized eggs. The producer cells are preferably cultured in a suitable medium (e.g., a chemically defined medium, preferably free of animal or human derived products) which may or may not be supplemented with serum and / or appropriate growth factor(s) as required. A suitable medium may be readily selected by the skilled artisan depending on the producer cells. Such media are commercially available. The producer cells are preferably cultured at a temperature of +30 to +38°C (more preferably about +37°C) for 1 to 8 days prior to infection. If necessary, several 1 to 8 day passagings can be performed to increase the total number of cells.

[0136] In step b), producer cells are infected with the oncolytic virus under appropriate conditions using an appropriate multiplicity of infection (MOI) to allow productive infection of the producer cells. For illustration purposes, an appropriate MOI is 10 -3 Infection rates range from 0.01 to 20, with particularly preferred MOIs including 0.01 to 5, more preferably 0.03 to 1. The infection step is carried out in a medium which may be the same or different from the medium used to culture the producer cells.

[0137] Then, in step c), the infected producer cells are cultured under suitable conditions well known to the skilled artisan until progeny viral particles are produced. The cultivation of the infected producer cells is also preferably carried out for 1 to 5 days at a temperature between +32°C and +37°C in a medium which may be the same or different from the medium / mediums used for the producer cell cultivation and / or infection step.

[0138] In step d), the viral particles produced in step c) are collected from the culture supernatant and / or from the producer cells. Recovery from the producer cells may require a step that allows disruption of the producer cell membrane to allow release of the virus. Disruption of the producer cell membrane can be induced by various techniques well known to those skilled in the art, including but not limited to freeze / thaw, hypotonic lysis, sonication, microfluidization, high shear (also called high speed) homogenization or high pressure homogenization.

[0139] The oncolytic virus that is recovered can be at least partially purified before being distributed in doses and used as described herein.A large number of purification steps and methods are available in the art, including clarification, enzyme treatment (e.g., endonuclease, protease, etc.), chromatography and filtration steps.Suitable methods are described in the art (see, for example, WO2007 / 147528, WO2008 / 138533, WO2009 / 100521, WO2010 / 130753, WO2013 / 022764).

[0140] In one embodiment, the present invention also provides a cell infected with an oncolytic virus described herein.

[0141] The present invention also encompasses pharmaceutical compositions comprising a virus, such as an oncolytic virus as described above, and a pharma- ceutically acceptable diluent, vehicle and / or adjuvant.

[0142] The pharmaceutical composition may, in some embodiments, be in the form of a CAR-T cell having a partial or complete antibody sequence described herein as part of its chimeric antigen T cell receptor encoding sequence.

[0143] The present invention also encompasses pharmaceutical compositions comprising the above-described CAR-T cells and a pharma- ceutically acceptable diluent, vehicle and / or adjuvant.

[0144] The invention also includes other therapeutics or drug "forms" such as antibody drug conjugates, fusion proteins, and pharmaceutical compositions comprising such therapeutics.

[0145] The antibody molecules, nucleotide sequences, plasmids, viruses, cells and / or pharmaceutical compositions described herein may be suitable for parenteral administration, including aqueous and / or non-aqueous sterile injection solutions which may contain antioxidants, and / or buffers, and / or bacteriostats, and / or solutes which render the formulation isotonic with the blood of the intended recipient; and / or aqueous and / or non-aqueous sterile suspensions which may contain suspending agents and / or thickening agents. The antibody molecules, nucleotide sequences, plasmids, cells and / or pharmaceutical compositions described herein may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example water for injections, immediately prior to use.

[0146] Extemporaneous injection solutions and suspensions may be prepared from sterile powders, and / or granules and / or tablets of the kind previously described.

[0147] For parenteral administration to human patients, the daily dosage level of the anti-CTLA-4 antibody molecule will usually be 1 mg / kg to 20 mg / kg of patient weight, or in some cases up to 100 mg / kg, administered in single or divided doses. Lower doses may be used under special circumstances, for example in combination with chronic administration. In any event, the physician will determine the actual dosage that will be most suitable for an individual patient, which will vary with the age, weight, and response of the particular patient. The dosages described above are exemplary of the average case. There may, of course, be individual cases in which higher or lower dosage ranges are merited, and these are within the scope of the present invention.

[0148] Typically, pharmaceutical compositions (or medicaments) described herein comprising the antibody molecule will contain the anti-CTLA-4 antibody molecule at a concentration of about 2 mg / ml to 150 mg / ml or about 2 mg / ml to 200 mg / ml, in some embodiments, the pharmaceutical composition will contain the anti-CTLA-4 antibody molecule at a concentration of 10 mg / ml.

[0149] Typically, a pharmaceutical composition (or medicament) contains approximately 10% of the virus and the quantitative technique. 3 ~10 12 The oncolytic virus described herein is contained in a concentration of vp (viral particles), iu (infectious units) or PFU (plaque forming units). The amount of pfu present in a sample can be determined by counting the number of plaques after infection of permissive cells (e.g., CEF or Vero cells) to obtain a plaque forming unit (pfu) titer, the amount of vp can be determined by measuring the A260 absorbance, and the amount of iu can be determined by quantitative immunofluorescence, e.g., using an antiviral antibody. As a general guidance, a suitable individual dose for a pharmaceutical composition containing an oncolytic poxvirus is about 10 3 ~about 10 10 pfu, advantageously about 10 3 pfu~about 10 9 pfu, preferably about 10 4 pfu~about 10 8 pfu, more preferably about 10 4 pfu~about 107 pfu range.

[0150] Generally, in humans, oral or parenteral administration of the antibody molecules, nucleotide sequences, plasmids, viruses, cells, and / or pharmaceutical compositions described herein is the preferred route and is the most convenient. For veterinary use, the antibody molecules, nucleotide sequences, plasmids, viruses, cells, and / or pharmaceutical compositions described herein are administered as an appropriately acceptable pharmaceutical formulation in accordance with normal veterinary practice, and the veterinarian will determine the dosing regime and route of administration that will be most appropriate for a particular animal. Thus, the present invention provides pharmaceutical formulations comprising an amount of the antibody molecules, nucleotide sequences, plasmids, viruses, and / or cells of the present invention effective to treat various conditions (as described above and further below). Preferably, the antibody molecules, nucleotide sequences, plasmids, viruses, cells, and / or pharmaceutical compositions described herein are adapted for delivery by a route selected from the group including intravenous, intratumoral, intramuscular, subcutaneous. Administration can be in the form of a single injection or multiple repeated injections (e.g., at the same or different doses, by the same or different routes, at the same or different administration sites). For purposes of illustration, about 10 of an oncolytic poxvirus (e.g., a TK- and RR-deficient vaccinia virus as described herein) may be used. 4 , 5×10 4 , 10 5 , 5×10 5 , 10 6 , 5×10 6 , 10 7 , 5×10 7 , 10 8 , 5×10 8 , 10 9 , 5×10 9 or 10 10 Individual doses containing pfu are particularly suitable for intratumoral administration.

[0151] The present invention also includes antibody molecules, nucleotide sequences, plasmids, viruses, cells and / or pharmaceutical compositions described herein that contain pharma- ceutically acceptable acid or base addition salts of the polypeptide binding moieties of the present invention. The acids used to prepare pharma- ceutically acceptable acid addition salts of the aforementioned base compounds useful in the present invention are, inter alia, those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate [i.e., 1,1'-methylene-bis-(2-hydroxy-3 naphthoate)] salts. Pharmaceutically acceptable base addition salts may also be used to generate pharma-ceutically acceptable salt forms of the agents according to the invention. Chemical bases that may be used as reagents to prepare pharma-ceutically acceptable base salts of the agents that are acidic in nature are those that form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to, those derived from such pharmacologically acceptable cations, such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium), ammonium or water-soluble amine addition salts, such as N-methylglucamine-(meglumine), and lower alkanolammonium, as well as other pharma-ceutically acceptable organic amine base salts. The antibody molecules, nucleotide sequences, plasmids, viruses and / or cells described herein may be lyophilized for storage and reconstituted in a suitable carrier prior to use. Any suitable lyophilization method (e.g., spray drying, cake drying) and / or reconstitution technique may be used. One of skill in the art will appreciate that lyophilization and reconstitution may lead to varying degrees of loss of antibody activity (e.g., in conventional immunoglobulins, IgM antibodies tend to have greater loss of activity than IgG antibodies), and that usage levels may need to be adjusted upward to compensate.In one embodiment, a lyophilized (freeze-dried) polypeptide binding moiety loses less than about 20%, or less than about 25%, or less than about 30%, or less than about 35%, or less than about 40%, or less than about 45%, or less than about 50% of its activity (before lyophilization) when rehydrated.

[0152] In some embodiments, the virus composition is suitably buffered at a physiological or slightly basic pH (e.g., about pH 7 to about pH 9, with a pH between 7 and 8.5, more particularly close to 8, being particularly preferred). It may also be beneficial to include a monovalent salt in the virus composition to ensure a suitable osmolality. The monovalent salt may be selected, in particular, from NaCl and KCl, and preferably, the monovalent salt is NaCl, preferably at a concentration of 10 to 500 mM (e.g., 50 mM). A suitable virus composition comprises saccharose 50 g / L, NaCl 50 mM, Tris-HCl 10 mM, and sodium glutamate 10 mM, pH 8. The composition may also be formulated to include a cryoprotectant to protect the oncolytic virus at low storage temperatures. Suitable cryoprotectants include, but are not limited to, sucrose (or saccharose), trehalose, maltose, lactose, mannitol, sorbitol, and glycerol, preferably at a concentration of 0.5-20% (also referred to as w / v, weight in g / volume in L), as well as high molecular weight polymers such as dextran or polyvinylpyrrolidone (PVP).

[0153] The anti-CTLA-4 antibody molecules, nucleotide sequences and pharmaceutical compositions described herein can be used for use in the treatment of cancer in a subject.

[0154] The subject may be a mammal or a non-mammal. Preferably, the mammalian subject is a human or a non-mammal, such as a horse, or a cow, or a sheep, or a pig, or a camel, or a dog, or a cat. Most preferably, the mammalian subject is a human.

[0155] "Exhibiting" includes when a subject exhibits cancer symptoms and / or cancer diagnostic markers and / or when the cancer symptoms and / or cancer diagnostic markers can be measured and / or assessed and / or quantified.

[0156] It will be readily apparent to one skilled in the art of medicine what cancer symptoms and cancer diagnostic markers are, and how to measure and / or assess and / or quantify whether there is a reduction or increase in the severity of a cancer symptom or whether there is a reduction or increase in a cancer diagnostic marker, and how the cancer symptoms and / or cancer diagnostic markers can be used to form a prognosis for cancer.

[0157] Cancer treatments are often administered as a course of treatment, i.e., the therapeutic agent is administered over a period of time. The length of time of the course of treatment depends on a number of factors, including, among other things, the type of therapeutic agent administered, the type of cancer being treated, the severity of the cancer being treated, and the age and health of the subject.

[0158] "Currently undergoing treatment" includes that the subject is currently undergoing a course of treatment and / or is currently receiving a therapeutic agent and / or is currently receiving a series of therapeutic agents.

[0159] In some embodiments, the cancer treated according to the present invention is a solid tumor.

[0160] In some embodiments, the cancer is selected from the group consisting of advanced solid tumors, melanoma and other malignant neoplasms of the skin, synovial sarcoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, prostate cancer, mesothelioma, ovarian cancer, breast cancer, renal cell carcinoma, hepatocellular carcinoma, head and neck cancer, and colorectal cancer.

[0161] Any of the above mentioned cancers are known and the symptoms and cancer diagnostic markers are well described, as are the therapeutic agents used to treat those cancers. Thus, the symptoms, cancer diagnostic markers and therapeutic agents used to treat the above types of cancers will be known to those skilled in the art of medicine.

[0162] The clinical definition of the diagnosis, prognosis and progression of most cancers is by a certain classification, known as staging. These staging systems work by collating many different cancer diagnostic markers and cancer symptoms to provide an overview of the diagnosis and / or prognosis and / or progression of cancer. Those skilled in the art of oncology will know how to use staging systems to evaluate the diagnosis and / or prognosis and / or progression of cancer, and which cancer diagnostic markers and cancer symptoms should be used for this purpose.

[0163] "Cancer staging" includes the Rai staging system, including stage 0, stage I, stage II, stage III, and stage IV, and / or the Binet staging system, including stage A, stage B, and stage C, and / or the Ann Arbour staging system, including stage I, stage II, stage III, and stage IV.

[0164] It is known that cancer can cause abnormalities in cell morphology. These abnormalities often occur reproducibly in a particular cancer, which means that the examination of these changes in morphology (also known as histological examination) can be used to diagnose or prognose cancer. Techniques for visualizing and preparing samples for visualizing to examine cell morphology are known in the art, such as, for example, optical microscopy or confocal microscopy.

[0165] "Histological examination" includes the presence of small mature lymphocytes, and / or the presence of small mature lymphocytes with narrow cytoplasmic borders, the presence of small mature lymphocytes with dense nuclei lacking discernible nucleoli, and / or the presence of small mature lymphocytes with narrow cytoplasmic borders and dense nuclei lacking discernible nucleoli, and / or the presence of atypical cells, and / or cleaved cells, and / or prolymphocytes.

[0166] It is known that cancer is the result of mutations in the DNA of cells, which can lead to cell death avoidance or uncontrollable proliferation.Therefore, the examination of these mutations (also known as cytogenetic examination) can be a useful tool for evaluating the diagnosis and / or prognosis of cancer.An example of this is the deletion of chromosomal position 13q14.1, which is characteristic of chronic lymphocytic leukemia.Techniques for examining mutations in cells are known in the art, such as, for example, fluorescent in situ hybridization (FISH).

[0167] "Cytogenetic testing" includes testing of DNA, specifically chromosomes, in cells. Cytogenetic testing can be used to identify DNA alterations that may be associated with the presence of refractory and / or recurrent cancer. These include deletion of the long arm of chromosome 13, and / or deletion at chromosomal location 13q14.1, and / or trisomy of chromosome 12, and / or deletion of the long arm of chromosome 12, and / or deletion of the long arm of chromosome 11, and / or deletion of 11q, and / or deletion of the long arm of chromosome 6, and / or deletion of 6q, and / or deletion of the short arm of chromosome 17, and / or deletion of 17p, and / or t(11:14) translocation, and and / or (q13:q32) translocations, and / or antigen gene receptor rearrangements, and / or BCL2 rearrangements, and / or BCL6 rearrangements, and / or t(14:18) translocations, and / or t(11:14) translocations, and / or (q13:q32) translocations, and / or (3:v) translocations, and / or (8:14) translocations, and / or (8:v) translocations, and / or t(11:14) and (q13:q32) translocations.

[0168] Subjects with cancer are known to exhibit certain physical symptoms, which are often the result of the burden that cancer places on the body. These symptoms often recur with the same cancer, and can be diagnostic and / or prognostic and / or progression features of the disease. Those skilled in the art of medicine will understand which physical symptoms are associated with which cancers, and how the evaluation of these body systems can correlate with the diagnosis and / or prognosis and / or progression of the disease. "Physical symptoms" include hepatomegaly and / or splenomegaly. [Brief description of the drawings]

[0169] In the following examples, reference is made to the following figures:

[0170] [Figure 1-1]The antibodies of the invention specifically bind to CTLA-4. The antibodies were shown by ELISA to bind to human and cynomolgous CTLA-4, but not to human CD28 protein. Binding of 2-C06 (Figure 1A), 4-E-03 (Figure 1B), and 5-B07 (Figure 1C) was compared to Yervoy (Figure 1D). [Figure 1-2] Same as above. [Figure 2-1] Dose-dependent binding of anti-CTLA-4 mAb to hCTLA-4 transfected cells Anti-CTLA-4 mAb (FIGS. 2A-2D) shows strong binding to 293T cells expressing CTLA-4, similar to Yervoy (FIG. 2E). [Figure 2-2] Same as above. [Diagram 3] Anti-CTLA-4 mAb binds to in vitro activated human CD4+ T cells CD4+ T cells obtained from peripheral human blood were stimulated in vitro. Binding of anti-CTLA-4 mAb (solid line, top) was analyzed by FACS and compared to Yervoy (dotted line, top) and a commercial FACS antibody (bottom). [Figure 4] Blockade of binding of in vitro activated CD4+ T cells. In vitro activated human CD4+ T cells were stained with Alexa647-labeled anti-CTLA-4 mAb (black line). Antibody binding was blocked by rhCTLA-4-Fc protein (grey line). [Diagram 5] Binding to in vitro activated cynomolgus monkey CD4+ T cells. CD4+ T cells obtained from peripheral cynomolgus monkey blood were stimulated in vitro with CD3 / CD28 dynabeads. Binding of anti-CTLA-4 mAb (solid line, top) was analyzed by FACS and compared to Yervoy (dotted line, top) and a commercial FACS antibody (bottom). [Figure 6]Blockade of cell binding by human and cynomolgus CTLA-4 proteins. 293T-CTLA-4 cells were stained with Alexa647-labeled anti-CTLA-4 mAb (black line). Antibody binding was blocked by rhCTLA-4-Fc protein (light grey line) and rcmCTLA-4-Fc protein (dark grey line). [Figure 7] Binding to 293T cells expressing cynomolgus CTLA-4. 293T cells were transiently transfected with cynomolgus CTLA-4 and binding of various concentrations of anti-CTLA-4 mAb was analyzed by FACS. [Figure 8] Expected lack of binding to resting human / cynomolgus monkey PBMCs. 4-E03 as well as 2-C06, 5-B07 and 2-F09 (data not shown) show no non-specific binding to different cell subsets of human (top) and cynomolgus monkey (bottom) PBMCs when analyzed by FACS. [Figure 9-1] Expected lack of direct agonist activity CFSE-labeled CD4+ T cells from healthy donors were stimulated with coated anti-CD3 and soluble anti-CTLA-4 mAb or anti-CD28. % dividing cells (CFSElow CD25+ cells) were determined after 3 days by FACS. (A) FACS plot of one representative experiment (B) Summary graphs of 6 donors. [Figure 9-2] Same as above. [Figure 10] CD80 / CD86 Blocking Activity Anti-CTLA-4 mAb blocks the binding of CD80 (FIG. 8A) and CD86 (FIG. 8b) to its ligand CTLA-4 as shown by ELISA. [Figure 11] In vitro functional ligand blockade. PBMCs were stimulated with SEB and titrating doses of anti-CTLA-4 antibody. The amount of secreted IL-2 in the supernatant was determined by MSD. One representative donor out of six is ​​shown in the figure. [Figure 12]ADCC assay of in vitro activated CD4+ T cells. In vitro activated CD4+ T cells from healthy donors pre-opsonized with 10 μg / ml anti-CTLA-4 mAb were co-cultured with NK cells (NK-92 cell line) at a 2:1 ratio. ADCC activity was assessed by FACS as described below. The figure shows the mean + SD of 4-8 donors. [Figure 13] CTLA-4 is most highly expressed on Treg cells present in the tumor. Freshly surgically removed ovarian tumors, blood samples were obtained from patients. Ascites fluid was collected from patients with various cancer indications. CTLA expression in this patient material was compared to healthy PBMCs. Tumor samples were minced and digested. Peripheral blood mononuclear cells were isolated by centrifugation. CTLA-4 expression was assessed on CD4+CD25+CD127- Treg cells, CD4+ non-Treg cells, and CD8+ effector T cells by flow cytometry. Data represent individual patients / donors, n=12 for healthy PBMCs, n=20 for ascites, n=9 for tumors, and n=5 for patient blood. CTLA-4 expression was also analyzed in human T cells activated in NOG mice in vivo and isolated from the spleens of these mice (see Figure 14). [Figure 14] Anti-CTLA-4 mAb mediates Treg depletion in vivo. Human PBMCs were injected intravenously into NOG mice. Approximately 2 weeks later, spleens were harvested and CTLA-4 expression on human Treg cells and CD8+ T cells was analyzed by FACS. Spleen cells isolated from NOG mice were transferred intraperitoneally into SCID mice. One hour later, mice were treated intraperitoneally with CTLA-4hIgG1 or control mAb. Peritoneal fluid was collected 24 hours later and the frequency of human T cell subsets (14A: Treg and 14B: CD8+ T cells) was measured by flow cytometry. [Figure 15] Table summarizing the characteristics of anti-CTLA-4 antibodies [Figure 16-1]Figure 16: Characterization of mouse surrogate anti-CTLA-4 mAbs. Figure 16A-B: To assess ligand blocking properties, a blocking ELISA was performed using m5-B07. The antibody dose-dependently blocks binding of Figure 16A) CD80 and Figure 16B) CD86 to its ligand CTLA-4. Figure 16C-D: Mouse IgG2a format 5-B07 mediated Treg deletion in the CT26 tumor model. Balb / c mice were injected subcutaneously with 1x106 CT26 cells, starting treatment at tumor sizes of approximately 7x7mm. After three injections of 10mg / kg antibody, tumor single cell suspensions were analyzed for immune cell content by FACS. Figure 16C: Ligand blocking surrogate antibody 5-B07 causes depletion of Tregs. This shifts the CD8+ / Treg T cell ratio as shown in Figure 16D. [Figure 16-2] Same as above. [Figure 17] Generation of COPTG19384 and COPTG19385 Schematic diagram of COPTG19384 and COPTG19385 used in this study. COPTG19385 contains a deletion of the J2R gene at the TK locus replaced with an anti-CTLA-4 heavy chain driven by p7.5K and a deletion of the I4L gene at the RR locus replaced with an anti-CTLA-4 light chain driven by p7.5K. COPTG19384 contains a deletion of the J2R gene at the TK locus replaced with an anti-CTLA-4 heavy chain driven by p7.5K and a deletion of the I4L gene at the RR locus replaced with an anti-CTLA-4 light chain driven by p7.5K and GM-CSF driven by pSE / L. [Figure 18]Expression analysis of 4-E03 monoclonal antibody in the supernatant of CEF cells infected with COPTG19384. A) By Western blot: CEF cells were infected in triplicate with COPTG19384 at MOI 0.05. After 48 hours, cell supernatants were collected and analyzed by WB using anti-Ig (left blot) or anti-light chain (right blot) HRP-conjugated antibodies after electrophoresis under non-reducing conditions. B) By ELISA: CEF cells were infected in triplicate with COPTG19384 at MOI 0.05 or with VVTG17137. After 48 hours, cell supernatants were collected and analyzed by ELISA for detection of either 4-E03 monoclonal antibody. [Figure 19] Expression analysis of GM-CSF by ELISA in the supernatant of CEF cells infected with COPTG19384. CEF cells were infected at an MOI of 0.05 with COPTG19384 primary research stock in triplicate or with VVTG17137. Cell supernatants were harvested 48 hours later and analyzed by ELISA for detection of GM-CSF. [Figure 20-1] Replication studies of COP WT, COPTG19384 (2 batches) and VVTG17137 (2 batches) in normal and tumor hepatocytes. A) Replication rates in normal human hepatocytes. B) Replication rates in malignant HepG2. C) Therapeutic index calculated from the replication rates measured in HepG2 and hepatocytes. [Figure 20-2] Same as above. [Figure 21] Replication of COPTG19384 and VVTG17137 in reconstituted human skin. Replication of COPTG19384 and VVTG17137 was assessed after 7 days with inoculum doses varying from 10 to 105 pfu. Results are the mean and SEM of triplicate determinations. [Figure 22-1] Oncolytic activity of COPTG19384 and VVTG17137 in three human tumor cell lines: MIA PaCa-2 (A), LoVo (B), and HepG2 (C). [Figure 22-2] Same as above. [Figure 23-1]Expression levels of both 4-E03 monoclonal antibody and GM-CSF in (A) supernatants of infected HepG2 and LoVo and (B) supernatants of five different infected human tumor cell lines. A) Expression levels of 4-E03 and GM-CSF were assessed after 5 days of incubation at MOIs of 10-5 to 10-2 for COPTG19384 and at MOI of 10-2 for VVTG17137 used as a negative control. B) Expression levels of 4-E03 and GM-CSF were assessed 48 hours post-infection with COPTG19384 at MOI of 0.05. [Figure 23-2] Same as above. [Figure 24] Binding of different batches of 4-E03 to CTLA-4 protein. Binding of 4-E03 recombinantly produced in CHO (4-E03 research batch) or HEK (4-E03 tox batch) cells to (A) human and (B) cynomolgus monkey recombinant proteins was compared to the binding of 4-E03 purified from the supernatant of infected MIA PaCa-2 tumor cells (4-E03TG) by ELISA. [Diagram 25] Binding of different batches of 4-E03 to CTLA-4 expressing cells. Binding of 4-E03 recombinantly produced in CHO (4-E03 research batch) or HEK (4-E03 tox batch) cells to (A) human and (B) cynomolgus monkey CTLA-4 expressing cells was compared to the binding of 4-E03 purified from the supernatant of infected MIA PaCa-2 tumor cells (4-E03TG) by flow cytometry. [Figure 26] Kinetics of viral accumulation in LoVo xenografted tumors. The kinetics of viral accumulation in LoVo xenografted tumors after a single intratumoral injection of either COPTG19384 or VVTG17137 at two different doses (104 or 105 pfu) was evaluated. Solid or dashed lines represent the median of the three values ​​determined at each time point. [Figure 27]Kinetics of 4-E03mAb and GM-CSF accumulation in LoVo xenografted tumors. A) The kinetics of 4-E03mAb accumulation in LoVo xenografted tumors was evaluated after a single intratumoral injection of either COPTG19384 or VVTG17137 at two different doses (104 or 105 pfu) or after a single intraperitoneal injection of 3 mg / kg of 4-E03 monoclonal antibody. Solid or dashed lines represent the median of three values. B) The kinetics of GM-CSF accumulation in LoVo xenografted tumors was evaluated after a single intratumoral injection of either COPTG19384 or VVTG17137 (105 pfu) at two different doses (104 or 105 pfu). Solid lines represent the median of three values ​​determined at each time point. [Figure 28] Kinetics of 4-E03 mAb and GM-CSF concentrations in serum of LoVo xenografted mice. A) Kinetics of 4-E03 mAb concentrations in serum were evaluated after a single intratumoral injection into LoVo xenografted tumors of either COPTG19384 or VVTG17137 at two different doses (104 or 105 pfu) or after a single intraperitoneal injection of 3 mg / kg of 4-E03 monoclonal antibody. The solid line represents the median of three values. B) Kinetics of GM-CSF concentrations in serum after a single intratumoral injection into LoVo xenografted tumors of either COPTG19384 or VVTG17137 (105 PFU) at two different doses (104 or 105 pfu). The dashed line represents the median of three values ​​determined at each time point. [Figure 29] Kinetics of viral accumulation in CT26 tumors Kinetics of viral accumulation in CT26 tumors after three intratumoral injections (D0, D2, and D4) of either VVTG18058, COPTG19421, or COPTG19407 at 107 pfu / injection. [Figure 30A]Kinetics of m5-B07mAb and mGM-CSF accumulation in CT26 tumors. A) Kinetics of m5-B07mAb concentration in CT26 tumors during and after three intratumoral injections of VVTG18058, COPTG19421, or COPTG19407 (107 pfu / injection), or after one intraperitoneal injection of 3 mg / kg of m5-B07 monoclonal antibody. The solid line represents the median of the three values. [Figure 30B] B) Kinetics of mGM-CSF concentrations in CT26 tumors during and after three intratumoral injections of VVTG18058, COPTG19421, or COPTG19407 (107 pfu / injection), or after one intraperitoneal injection of 3 mg / kg of m5-B07 monoclonal antibody. The solid line represents the median of the three values ​​determined at each time point. [Diagram 31] Kinetics of m5-B07mAb concentration in serum in the CT26 model. Kinetics of m5-B07mAb concentration in serum after three intratumoral injections (107 pfu / injection) into CT26 tumors of either VVTG18058, COPTG19421, or COPTG19407, or after a single intraperitoneal injection of 3 mg / kg of m5-B07 monoclonal antibody. The solid line represents the median of the three values ​​determined at each time point. [Diagram 32] Antitumor activity in the CT26 model: effect of COPTG19347+ / -anti-PD1 on CT26 tumor growth (A) and mouse survival (B). CT26 cells were injected subcutaneously into BalB / c mice on D-7. COPTG19347 (107 pfu), VVTG18058 (107 pfu), VVTG18058 or buffer were injected intratumorally on D0, D2, and D4, followed by intraperitoneal injection of 250 μg / mouse anti-PD1 RMPI-14 on D7, D10, D14, D17, and D21. [Diagram 33] Dose-effect assessment in the CT26 model (Compilation of survival data observed following treatment with COPTG19407, COPTG19421 and VVTG18058) [Figure 34-1]Antitumor activity of COPTG19407 compared to VVTG18058 plus m5-B07 in the CT26 tumor model. CT26 cells were injected subcutaneously into BalB / c mice. Treatment of mice began when tumors reached approximately 100 mm3. Mice were injected with COPTG19407 (8.5×106 pfu intratumoral), VVTG18058 (8.5×106 pfu intratumoral), m5-B07 (10 mg / kg intraperitoneal), or a combination of VVTG18058 (8.5×106 pfu intratumoral) and m5-B07 (10 mg / kg intraperitoneal) on D0, D2, and D5. Figure 30A-D: Tumor growth and Figure 16E: survival were followed over time. [Figure 34-2] Same as above. [Figure 34-3] Same as above. [Figure 35-1] Individual tumor volume curves of BALB / c mice bearing subcutaneous A20 tumors. A20 cells were injected subcutaneously into BalB / c mice. Treatment of mice began when tumors reached 80-100 mm3. Mice were injected with vehicle (intratumoral), COPTG19407 (4.75×106 pfu intratumoral), VVTG18058 (4.75×106 pfu intratumoral), RMP1-14 (anti-mPD-1) (250 μg / mouse intraperitoneally) or a combination of COPTG19407 (4.75×106 pfu intratumoral) and RMP1-14 (250 μg / mouse intraperitoneally) on D0, D2, and D4. A) Group 1 animals treated with vehicle. B) Group 2 animals treated with VVTG18058. C) Group 3 animals treated with COPTG19407. D) Group 4 animals treated with mouse anti-PD1. E) Group 5 animals treated with COPTG19407 and mouse anti-PD1. [Figure 35-2] Same as above. [Diagram 36]Mean tumor volume curves of BALB / cN mice bearing subcutaneous A20 tumors. Each point represents the mean of tumor volumes recorded per group. Tumor volumes of all animals were monitored over a period of 64 days. Mice were treated with vehicle (group 1), VVTG18058 (group 2), COPTG19407 (group 3), murine anti-PD1 antibody RMP1-14 (BioXCell) (group 4) and COPTG19407 and RMP1-14 (group 5). Animals were randomized on D7 and treated during the period D7–D31. The last mice were sacrificed on D61. [Figure 37] Antitumor activity in the A20 model: effect of COPTG19407+ / -anti-PD1 on A20 tumor growth (A) and mouse survival (B). A20 cells were injected subcutaneously into BalB / c mice. Treatment of mice began when tumors reached 80-100 mm3. Mice were treated with vehicle (intratumoral) (group 1), anti-PD-1 (group 2), isotype (group 3), VVTG18058 (105 pfu intratumoral) (group 4), VVTG18058 (105 pfu intratumoral) + isotype (group 5), VVTG18058 (105 pfu intratumoral) + anti-PD-1 (group 6), VVTG19407 (105 pfu intratumoral) (group 7), VVTG19407 (105 pfu intratumoral) + isotype (group 8) and VVTG19407 (105 pfu intratumoral) + anti-PD-1 (group 9). [Figure 38-1]Individual tumor volume curves of C57BL / 6 mice bearing subcutaneous C38 tumors. C38 cells were injected subcutaneously into C57BL / 6 mice. Treatment of mice began when tumors reached 80-100 mm3. Mice were injected with vehicle (intratumoral), COPTG19407 (4.75×106 pfu intratumoral), VVTG18058 (4.75×106 pfu intratumoral), RMP1-14 (anti-mPD-1) (250 μg / mouse intraperitoneally) or a combination of COPTG19407 (4.75×106 pfu intratumoral) and RMP1-14 (250 μg / mouse intraperitoneally) on D0, D2, and D4. A) Group 1 animals treated with vehicle. B) Group 2 animals treated with VVTG18058. C) Group 3 animals treated with COPTG19407. D) Group 4 animals treated with mouse anti-PD1. E) Group 5 animals treated with COPTG19407 and mouse anti-PD1. [Figure 38-2] Same as above. [Figure 39] Mean tumor volume curves of C57BL / 6 mice bearing subcutaneous C38 tumors. Each point represents the mean of tumor volumes recorded per group. Tumor volumes of all animals were monitored over a period of 61 days. Mice were treated with vehicle (group 1), VVTG18058 (group 2), COPTG19407 (group 3), murine anti-PD1 antibody RMP1-14 (BioXCell) (group 4), and COPTG19407 and RMP1-14 (group 5). Animals were randomized on D7 and treated during the period D7–D31. The last mice were sacrificed on D61. [Figure 40-1]Individual tumor volume curves of BALB / c mice bearing subcutaneous EMT6 tumors. EMT6 cells were injected subcutaneously into BalB / c mice. Treatment of mice began when tumors reached 80-100 mm3. Mice were injected with vehicle (intratumoral), COPTG19407 (4.75×106 pfu intratumoral), VVTG18058 (4.75×106 pfu intratumoral), RMP1-14 (anti-mPD-1) (250 μg / mouse intraperitoneally) or a combination of COPTG19407 (4.75×106 pfu intratumoral) and RMP1-14 (250 μg / mouse intraperitoneally) on D0, D2, and D4. A) Group 1 animals treated with vehicle. B) Group 2 animals treated with VVTG18058. C) Group 3 animals treated with COPTG19407. D) Group 4 animals treated with mouse anti-PD1. E) Group 5 animals treated with COPTG19407 and mouse anti-PD1. [Figure 40-2] Same as above. [Figure 40-3] Same as above. [Diagram 41] Mean tumor volume curves of BALB / c mice bearing subcutaneous EMT6 tumors. Each point represents the mean of tumor volumes recorded per group. Tumor volumes of all animals were monitored over a period of 61 days. Mice were treated with vehicle (group 1), VVTG18058 (group 2), COPTG19407 (group 3), murine anti-PD1 antibody RMP1-14 (BioXCell) (group 4), and COPTG19407 and RMP1-14 (group 5). Animals were randomized on D7 and treated during the period D7–D31. The last mouse was sacrificed on D56. Curves were stopped after more than 20% of mice had died. EXAMPLES

[0171] Specific non-limiting examples are now described that illustrate certain aspects of the invention, in which rh protein refers to human recombinant protein (e.g., rhIL-2 refers to human recombinant IL-2 protein) and rcm protein refers to cynomolgus recombinant protein (e.g., rcmCTLA4 refers to cynomolgus recombinant CTLA-4 protein).

[0172] In addition to the sequences above, several additional sequences are used in the examples, which are set out in Table 5 below. [Table 5]

[0173] Example 1 - Generation of CTLA-4 specific antibodies Isolation of scFv antibody fragments The n-CoDeR® scFv library (BioInvent; Soderlind E, et al Nat Biotechnol. 2000;18(8):852-6) was used to isolate scFv antibody fragments that recognize human CTLA-4.

[0174] The phage library was used in three successive rounds of panning against recombinant human proteins. After phage incubation, cells were washed to remove unbound phages. Bound phages were eluted with trypsin and amplified in E. coli. The resulting phage stocks were converted to scFv format. E. coli was transformed with the scFv-carrying plasmid to express individual scFv clones.

[0175] Identification of unique CTLA-4 binding scFv The converted scFvs from the third round of panning were assayed for binding to transfected 293FT cells expressing human CTLA-4 or a non-target protein using homogeneous FMAT analysis (Applied Biosystems, Carlsbad, CA, USA).

[0176] Briefly, transfected cells were added to clear-bottom plates along with scFv-containing supernatants from expression plates (diluted 1:7), mouse anti-His Tag antibody (0.4 μg / ml, R&D Systems), and APC-conjugated goat anti-mouse antibody (0.2 μg / ml, catalog number 115-136-146, Jackson Immunoresearch). FMAT plates were incubated for 9 hours at room temperature (approximately 20-25 °C) and then read. Target-specific bacterial clones were classified as active and cherries were harvested into 96-well plates.

[0177] IgG binding to CTLA-4 in ELISA 96-well plates (Lumitrac 600 LIA plates, Greiner) were coated overnight at 4 °C with 1 pmol / well recombinant human CTLA-4-Fc protein (R&D Systems), 1 pmol / well cynomolgus (cm) CTLA-4-Fc protein (R&D Systems), 0.3 pmol / well recombinant mouse CTLA-4-Fc protein (R&D Systems), 1 pmol / well recombinant human CD28-Fc protein (R&D Systems), or 0.3 pmol / well recombinant mouse CD28-Fc protein (R&D Systems). After washing, titrated doses of anti-CTLA-4 mAb ranging from 0 μg / ml to 0.06 ng / ml (66 nM to 0.3 pM) were allowed to bind for 1 h. Plates were then washed again and bound antibodies were detected with anti-human F(ab)-HRP secondary antibody (Jackson ImmunoResearch) diluted to 50ng / ml. Plates were analyzed using a Tecan Ultra Microplate reader with Super Signal ELISA Pico (Thermo Scientific) as substrate.

[0178] All antibodies were shown to bind to human and cynomolgus monkey CTLA-4 proteins but not to human CD28 protein when assayed by ELISA. Additionally, 5-B07 was shown to bind to mouse CTLA-4 but not to mouse CD28 (see Figure 1).

[0179] IgG binding to CTLA-4-expressing 293T cells in flow cytometry The converted IgG clones were analyzed for binding to CTLA-4 expressing 293T cells (purchased from Crownbio). Cells were incubated with different concentrations (shown in Figure 2) of anti-CTLA-4 mAb for 20 min at 4 °C, then washed and stained with APC-labeled goat anti-human secondary antibody (catalog no. 109-°C136-088, Jackson ImmunoResearch). Dead cells were excluded from the analysis using Fixable Viability Dye eFluor780 (eBiosciences). Data collection was performed with FACSVerse (BD Biosciences, Franklin Lakes, NJ) and analyzed with FlowJo software (Tree Star, Ashland, OR).

[0180] Anti-CTLA-4 mAb was shown to bind to human CTLA-4-expressing 293T cells in a dose-dependent manner with an EC50 value similar to that of Yervoy (Figure 2).

[0181] 293T cells stably transfected with human CTLA-4, 293T cells transiently transfected with cynomolgus CTLA-4, naive human or cynomolgus PBMCs, and in vitro activated human or cyno CD4+ T cells were incubated with the indicated concentrations of anti-CTLA-4 mAb for 20 min at 4 °C, then washed and stained with APC-conjugated anti-human secondary antibody (Jackson ImmunoResearch).

[0182] Example 2 - Anti-CTLA-4 mAb specifically binds to human and cynomolgus monkey CTLA-4 expressing (primary) cells CTLA-4-specific mAbs bind to primary human and cynomolgus monkey in vitro activated CD4+ T cells, but not to naive PBMCs isolated from healthy donors. PBMCs were isolated from the buffy coat. Briefly, the buffy was diluted 1:3 with PBS and loaded onto a Ficoll-Paque Plus (Amersham) cushion. Samples were centrifuged at 800xg for 20 minutes at 20°C. The upper plasma-containing phase was removed and mononuclear cells were isolated from the distinct white band at the plasma / ficoll phase.

[0183] Human peripheral CD4 + T cells were purified by negative selection using the MACS CD4 T cell isolation kit (Miltenyi Biotec). CD4+ T cells were activated in vitro for 3 days with CD3 / CD28 Dynabeads (Life Technologies) and 50ng / ml rhIL-2 (R&D Systems) in R10 medium (RPMI containing 2mM glutamine, 1mM pyruvate, 100IU / ml penicillin and streptomycin and 10% FBS (GIBCO from Life Technologies) to upregulate CTLA-4 expression. Cynomolgus CD4+ T cells were isolated using non-human CD4 microbeads (Miltenyi Biotec) and incubated with 50ng / ml PMA (Sigma-Aldrich) and 100ng / ml ionomycin (Sigma-Aldrich) for 3 days.

[0184] Naive human or cynomolgus PBMCs, in vitro activated human or cyno CD4+ T cells were incubated with the indicated concentrations of anti-CTLA-4 mAb for 20 min at 4°C, then washed and stained with APC-labeled anti-human secondary antibody (Jackson ImmunoResearch). Anti-CTLA-4 mAb binding was analyzed by FACS using a BD FACS Verse.

[0185] The antibody was shown to bind to in vitro activated human (Figure 3) and cynomolgus monkey (Figure 5) CD4+ T cells, but not to resting PBMC (Figure 8). Binding to T cells that endogenously express CTLA-4 is similar to staining with Yervoy (Figure 3, top, dotted line) and, as a positive control, a commercially available anti-CTLA-4 FACS antibody from BD Biosciences (clone BNI3, Figure 3, bottom).

[0186] As shown in Figure 4, staining of human in vitro activated CD4+ T cells (black line) was completely blocked by rhCTLA-4-Fc (gray line), indicating the specificity of the antibody. In this competitive binding assay, 2 μg / ml of Alexa647-labeled anti-CTLA-4 mAb was mixed with recombinant human CTLA-4-Fc protein (50 μg / ml) and then incubated with CTLA-4 expressing cells. IgG binding was detected by FACS.

[0187] Transfected 293T cells expressing human and cynomolgus CTLA-4 confirm the cyno cross-reactivity of the tested antibodies The cyno cross-reactivity of the antibody was further confirmed in transfected CTLA-4 expressing 293T cells.

[0188] As shown in Figure 6, binding of CTLA-4 specific antibodies to transfected cells expressing human CTLA-4 is inhibited by human and cynomolgus recombinant proteins (both from R&D Systems). The antibodies were also shown to bind to transfected cells expressing cynomolgus CTLA-4 (Figure 7, top). This binding is again blocked by the cynomolgus recombinant protein (bottom, grey line).

[0189] The experiment was carried out similarly to the competition assay described above in Example 2 in relation to FIG.

[0190] Lack of expected direct agonist activity To exclude unexpected direct agonist activity (eg, due to non-specific binding), an in vitro proliferation assay was performed.

[0191] Human peripheral blood CD4+ T cells were purified from healthy PBMCs by negative selection using the MACS CD4 T cell isolation kit (Miltenyi Biotec) and then labeled with CFSE (2 μM, Molecular Probes). The antibody was incubated at room temperature for 1 h with IgG:F(ab') 2 = 1.5:1, F(ab') 2 Goat anti-human IgG, Fcγ fragment specific or F(ab') 2 Cross-linked with goat anti-mouse IgG, Fcγ fragment specific. 1 × 10 5 Purified human CD4+ T cells were stimulated with plate-bound anti-CD3 (0.5 μg / ml, clone UCHT1, R&D Systems) and 4 μg / ml soluble, cross-linked anti-CTLA-4 or cross-linked anti-CD28 (clone CD28.2, BioLegend) for 72 h at 37° C. Cells were washed and stained with BV421-conjugated anti-CD25 antibody (clone M-A251, BD Biosciences). The percentage of CD25+ / CFSElow dividing cells was analyzed by FACS.

[0192] FIG. 9 shows that none of the anti-CTLA-4 mAbs tested induced T cell proliferation in contrast to anti-CD28 stimulation.

[0193] Example 3 - Anti-CTLA-4 mAb blocks CD80 / CD86 ligand binding Ligand blocking ELISA The ligand blocking activity of anti-CTLA-4 IgG was assessed by ELISA. For this purpose, recombinant human CTLA-4-Fc protein (R&D Systems) was coated at 1 pmol / well onto 96-well plates (Lumitrac 600 LIA plates, Greiner). After washing, increasing or decreasing doses of anti-CTLA-4 mAb were allowed to bind for 1 h. His-tagged ligands were added at 200 nM and 100 nM, respectively (rhCD80 and rhCD86, R&D Systems), and the plates were incubated for a further 15 min. After washing, bound ligands were detected with an HRP-labeled anti-His antibody (R&D Systems). Plates were analyzed using a Tecan Ultra Microplate reader with Super Signal ELISA Pico (Thermo Scientific) as substrate.

[0194] As shown in FIG. 10, the anti-CTLA-4 antibodies tested exhibit ligand blocking activity similar to Yervoy.

[0195] In vitro functional ligand blockade For the SEB PBMC assay, total PBMCs from healthy donors were plated in 96-well plates (1 × 10 5 Cells were seeded onto 1000 wells (1000 x 1000 cells / well) and stimulated with 1 μg / ml Staphylococcal enterotoxin B (SEB, Sigma Aldrich) in the presence of titrated doses of anti-CTLA-4 IgG. IL-2 secretion was measured by MSD (Mesoscale) according to the manufacturer's instructions on day 3.

[0196] Antibodies 4-E03 and 2-C06 were shown to enhance IL-2 production and their potency was shown to be similar to that of Yervoy. Figure 11 shows one representative donor out of six.

[0197] Example 4 - Anti-CTLA-4 mAb depletes CTLA-4 expressing cells in vitro and in vivo Antibody-dependent cytotoxicity (ADCC) ADCC assays were performed using the NK-92 cell line stably transfected to express the CD16-158V allele together with GFP (purchased from Conkwest, San Diego, CA; Binyamin, L., et al., 2008, Blocking NK cell inhibitory self-recognition promotes antibody-dependent cellular cytotoxicity in a model of anti-lymphoma therapy. Journal of immunology 180, 6392-6401). CD4+ target T cells were isolated from peripheral blood of healthy donors using a CD4+ T cell isolation kit (Miltenyi Biotec). Cells were stimulated with CD3 / CD28 Dynabeads (Life Technologies, Thermo Fisher) and 50 ng / ml rhIL-2 (R&D Systems) for 48 h at 37°C. Target cells were preincubated with 0.1–10 μg / ml mAB for 30 min at 4°C and then mixed with NK cells. Cells were cultured for 4 hours in RPMI1640+GlutaMAX medium (Invitrogen) containing 10 mM HEPES buffer, 1 mM sodium pyruvate, and 10% FBS low IgG at an effector:target cell ratio of 2:1. Lysis was determined by flow cytometry. Briefly, at the end of the incubation, cell suspensions were stained with BV510-conjugated anti-CD4 (clone RPA-T4, BD Biosciences) together with 10 nM SYTOX Red dead cell stain (Invitrogen) or Fixable Viability Dye eFluor780 (eBioscience) for 20 minutes in the dark at 4°C, and then cells were analyzed using a FACSVerse (BD Biosciences).

[0198] 4-E03 demonstrated significantly improved depletion of CTLA-4+ T cells in vitro compared to Yervoy (Figure 12).

[0199] CTLA-4 expression in primary patient material To validate the translatability of the above findings regarding the depletion activity of anti-CTLA-4 mAbs, CTLA-4 expression was examined in primary patient material.

[0200] Ethical approval for the use of clinical samples was obtained from the ethical committee of Skane University Hospital. Informed consent was provided in accordance with the Declaration of Helsinki. Samples were obtained through the Department of Gynecology and Oncology at Skane University Hospital, Lund. Ascites was evaluated as an isolated single cell suspension. Tumor material was cut into small pieces and incubated with DNase I (Sigma Aldrich) and Liberase™ (Roche Diagnostics) in R10 for 20 min at 37°C. The remaining tissue was mechanically disrupted and passed through a 70 μm cell strainer together with the cell suspension. Cells isolated from ascites and tumor were stained. To identify the different T cell subsets, the following antibodies were used: CD4-BV510 (RPA-T4), CD25-BV421 (M-A251), anti-CD127-FITC (HIL-7R-M21), CTLA-4-PE (BNI3), CD8-PeCy7 (RPA-T8), CD3-APC (UCHT1), CD45-PercP-Cy5.5 (HI30), mouse IgG2a isotype, kappa control-PE (G155-178, all from BD Biosciences). Data collection was performed using FACSVerse and data were analyzed using FlowJo.

[0201] As shown in FIG. 13, CTLA-4 is most highly expressed on intratumoral Treg cells, making it an excellent target for depleting CTLA-4-specific antibodies.

[0202] PBMC-NOG / SCID model To confirm the in vitro findings regarding the depletion activity of CTLA-4 specific antibodies, we analyzed the depletion capacity of anti-CTLA-4 mAbs in an in vivo PBMC-NOG / SCID model. This model is based on the established hu-PBMC-NOG model (Sondergaard H. et al., Clin Exp Immunol. 2013 May;172(2):300-10. doi:10.1111 / cei.12051; Cox JH et al., PLoS One. 2013 Dec 23;8(12):e82944. doi:10.1371 / journal.pone.0082944.eCollection 2013) and was modified in-house as described below.

[0203] Mice were bred and maintained in a local facility in accordance with Home Office guidelines. Eight-week-old female CB17scid (Bosma GC et al., Nature. 1983 Feb 10;301(5900):527-30) and NOG (NOD / Shi-scid / IL-2Rγ null ;Ito M et al, 2002, NOD / SCID / gamma [ka] Mice were provided by Taconic (Bomholt, Denmark) and maintained at the local animal facility. For the PBMC-NOG / SCID (primary human xenograft) model, human PBMCs were isolated using Ficoll Paque PLUS and washed, after which the cells were cultured at a concentration of 75 × 10 6 The cells were resuspended in sterile PBS at 15 × 10 cells / ml. 6200 μl of cell suspension, equivalent to 100 cells / mouse, was injected intravenously. Two weeks after injection, the spleen was isolated and reduced to a single cell suspension. A small sample was then taken to measure the expression of CTLA-4 on human T cells by FACS. As shown in Figure 13, CTLA-4 is more highly expressed on Treg cells compared to other T cells, reflecting the situation in human patients. The majority of cells were 50 × 10 6 The cells were resuspended in sterile PBS at 10 × 10 cells / ml. 6 200 μl of the suspension, equivalent to cells / mouse, were injected intraperitoneally. After 1 h, mice were treated with either anti-CTLA-4hIgG1, Yervoy, or isotype control mAb at 10 mg / kg. Mouse peritoneal fluid was collected 24 h later. Human T cell subsets were identified and quantified by FACS using the following markers: CD45, CD4, CD8, CD25, CD127 (all from BD Biosciences).

[0204] All antibodies tested demonstrated Treg depletion activity equal to or greater than that of Yervoy. Other T cell populations, such as CD8+ effector T cells, were unaffected (Figure 14).

[0205] Example 5 - Selected surrogate antibody m5-B07 exhibits the same functional characteristics as 4-E03 In some examples, particularly in vivo examples, the antibody clone 5-B07 in mIgG2a format is used (also shown as m5-B07). This is a murine antibody that is a surrogate for the human antibodies disclosed herein. It was selected as a surrogate antibody because it binds to mouse CTLA-4, thereby blocking ligand binding (Figures 16A-B). In addition, it also shows Treg depletion activity (Figures 16C-D).

[0206] Ligand blocking ELISA The ligand blocking activity of 5-B07 was evaluated by ELISA. For this purpose, recombinant mouse CTLA-4-Fc protein (Sino Biological Inc.) was coated at 1 pmol / well onto 96-well plates (Lumitrac 600 LIA plates, Greiner). After washing, titrated doses of anti-CTLA-4 mAb were allowed to bind for 1 h. His-tagged ligands were added at 200 nM and 100 nM, respectively (rmCD80 and rmCD86; Sino Biological Inc.), and the plates were incubated for an additional 15 min. After washing, bound ligands were detected with an HRP-conjugated anti-His antibody (R&D Systems). Plates were analyzed using a Tecan Ultra Microplate reader with Super Signal ELISA Pico (Thermo Scientific) as substrate.

[0207] As shown in FIG. 16, the antibodies block binding of (A) CD80 and (B) CD86 to their ligand CTLA-4.

[0208] In vivo Treg depletion activity The effect of CTLA-4 specific antibodies on tumor T cell subsets in vivo was investigated in the CT26 tumor model as described below.

[0209] Mice were bred and maintained in a local facility in accordance with Home Office guidelines. Female Balb / C mice aged 6–8 weeks were supplied by Taconic (Bomholt, Denmark) and housed in a local animal facility. CT26 cells (ATCC) were grown in Glutamax-buffered RPMI supplemented with 10% FCS. When cells were semi-confluent, they were detached with trypsin and cultured at 10 × 10 6 The mice were resuspended in sterile PBS at 1 × 10 cells / ml. 6100 μl of cell suspension corresponding to cells / mouse were injected subcutaneously. When tumors reached approximately 7 × 7 mm, mice were treated with the indicated antibodies at 10 mg / kg intraperitoneally twice weekly as indicated in the figures. After the third dose, tumors were dissected, mechanically divided into small pieces, and digested using a mixture of 100 μg / ml Liberase (Roche) and 100 μg / ml Dnase (Sigma) for 2 × 5 min at 37 °C with vortexing in between. The cell suspension was then washed (400 g for 10 min) with PBS containing 10% FBS. Cells were then resuspended in MACS buffer and stained with a panel of antibodies staining CD45, CD3, CD8, CD4, and CD25 (all from BD Biosciences). Before staining, cells were blocked for nonspecific binding using 100 μg / ml IVIG. Cells were analyzed using a FACS Verse (BD Biosciences). Mouse Treg cells express CD45 + CD3 + CD4 + CD25 + were identified as cells.

[0210] As shown in FIG. 16C, 5-B07 in mouse IgG2a format mediates Treg depletion of tumors associated with increased CD8 / Treg ratios D) compared to other CTLA-4 specific n-CoDeR antibodies and the well-described commercially available clone 9H10.

[0211] Example 6 - Generation of viruses expressing anti-CTLA4 mAb (COPTG19385) or anti-CTLA4 mAb and GM-CSF (COPTG19384), characterization of transgene expression and genetic stability COPTG19384 and COPTG19385 are vaccinia viruses (Copenhagen strain) encoding the monoclonal antibody anti-CTLA4 (4-E03). COPTG19384 further encodes human GM-CSF. More specifically, both COPTG19384 and COPTG19385 are defective in thymidine kinase (TK, J2R locus) and ribonucleotide reductase (RR, I4L locus) activity. As shown in FIG. 17, an expression cassette encoding the 4-E03 heavy chain (HC, SEQ ID NO: 54) under the control of the p7.5K promoter (SEQ ID NO: 59) was inserted into the J2R locus, and an expression cassette encoding the light chain (LC, SEQ ID NO: 53) of 4-E03 IgG under the control of the p7.5K promoter (SEQ ID NO: 59) was placed into the I4L locus. In the case of COPTG19384, an expression cassette encoding human GM-CSF (SEQ ID NO:56) under the control of the pSE / L promoter (SEQ ID NO:61) was also placed at the I4L locus.

[0212] The same promoter (p7.5K) was used to control the expression of the HC and LC to obtain the same level of expression for both chains and thus obtain optimal assembly of the antibody as a heterotetrameric protein (i.e. to avoid an excess of unassociated chains). However, the presence of the same promoter for both chains of the antibody precludes their insertion in the same locus (identical DNA sequences would increase the risk of recombination and eliminate the transgene). Thus, a cassette encoding 4-E03HC was inserted in the J2R locus and a cassette encoding 4-E03LC was inserted in the I4L locus. A cassette encoding the GM-CSF transgene, but under a different promoter (pSE / L), was also inserted in the I4L locus, like the antibody light chain.

[0213] Generating COPTG19384 Vaccinia virus transfer plasmids pTG19339 and pTG19341 were designed to allow the insertion of nucleotide sequences by homologous recombination at the J2R and I4L loci of the vaccinia virus genome, respectively. They are derived from the plasmid pUC18 into which the flanking sequences (BRG and BRD) surrounding the J2R (pTG19339) or I4L (pTG19341) loci have been cloned. Each plasmid also contains the p7.5K promoter.

[0214] A synthetic fragment of 1436 bp, named "fragment HC", containing the HC gene of 4-E03 antibody was generated. A fragment "LC fragment", containing the LC gene of 4-E03 antibody and the hGM-CSF gene under the control of pSE / L, was generated by synthetic methods and inserted into a plasmid vector. The coding sequence was optimized for human codon usage, and a Kozak sequence (ACC) was added before the ATG start codon and a transcription terminator (TTTTTNT) was added after the stop codon. In addition, some patterns were excluded: TTTTTNT, GGGGG, CCCCC, which are detrimental to expression in poxviruses.

[0215] The HC fragment was inserted by homologous recombination into PvuII-restricted pTG19339, giving rise to pTG19367. The plasmid carrying LC was restricted by SnaB1, and the resulting fragment, "LC-GMCSF," was inserted by homologous recombination into PvuII-restricted pTG19341, giving rise to pTG19384. In this plasmid, the expression cassette was inserted head-to-tail between the recombination arms, allowing homologous recombination at the I4L locus of the vaccinia virus genome.

[0216] COPTG19384 was generated in chicken embryo fibroblasts (CEFs) by two successive homologous recombinations for sequential insertion into the I4L and J2R loci and by using COPTG19156 as the parent virus and the two transfer plasmids pTG19367 and pTG19384. CEFs were isolated from 12-day-old embryonated SPF eggs (Charles River). Embryos were mechanically dissected, solubilized in Tryple Select solution (Invitrogen) and cultured in MBE (Eagle Based Medium, Gibco) supplemented with 5% FCS (Gibco) and 2 mM L-glutamine.

[0217] Homologous recombination between the transfer plasmid and the parental vaccinia virus allows the generation of recombinant vaccinia viruses that have lost the GFP and mCherry expression cassettes and have acquired antibody and GM-CSF expression cassettes. COPTG19156 contains an expression cassette for the mCherry gene at the I4L locus and an expression cassette for the GFP gene at the J2R locus. Homologous recombination between the transfer plasmid pTG19367 and the parental COPTG19156 allows the generation of recombinant vaccinia viruses that have lost the GFP expression cassette and have acquired the 4-E03 heavy chain expression cassette, with selection carried out by isolation of red fluorescent plaques. This intermediate recombinant virus (COPTG19367) was used as the parental virus for a second round of homologous recombination with pTG19384 as the transfer plasmid to generate recombinant vaccinia viruses that have lost the mCherry expression cassette and have acquired the 4-E03 light chain and GM-CSF expression cassettes. Selection of COPTG19384 (Figure 17) was carried out by isolating white non-fluorescent plaques.

[0218] Viral stocks of COPTG19384 were amplified in CEFs in two F175 flasks to generate suitable viral stocks that can be aliquoted and stored at -80°C until use. Viral stocks were scaled in CEF cells and infectious titers expressed in pfu / mL were calculated using the following formula: number of lysed areas x dilution factor x 4. For illustration purposes, the viral stock generated was 6.8x10 6 The stock was adjusted up or down in pfu / mL. The stock was analyzed by PCR to verify the integrity of the expression cassette and the recombination arms using appropriate primer pairs. The stock was also analyzed by sequencing of both expression cassettes. Alignment of the sequencing results showed 100% homology with the theoretically expected sequences. If necessary, virus preparations were purified using conventional techniques (e.g. as described in WO2007 / 147528).

[0219] Generating COPTG19385 Vaccinia virus transfer plasmids pTG19339 and pTG19341 were designed to allow the insertion of nucleotide sequences by homologous recombination at the J2R and I4L loci of the vaccinia virus genome, respectively. They are derived from the plasmid pUC18 into which the flanking sequences (BRG and BRD) surrounding the J2R (pTG19339) or I4L (pTG19341) loci have been cloned. Each plasmid also contains the p7.5K promoter.

[0220] A synthetic fragment of 1436 bp, named "Fragment HC", containing the HC gene of the 4-E03 antibody was generated. The coding sequence was optimized for human codon usage, with a Kozak sequence (ACC) added before the ATG start codon and a transcription terminator (TTTTTNT) added after the stop codon. In addition, some patterns were excluded: TTTTTNT, GGGGG, CCCCC, which are detrimental to expression in poxviruses.

[0221] The HC fragment was inserted into PvuII-restricted pTG19339 by homologous recombination to generate pTG19367.

[0222] A plasmid containing an expression cassette encoding only the 4-E03 light chain was obtained by removing the cassette encoding the hGM-CSF gene under the control of pSE / L in plasmid pTG19384 (described above). pTG19384 was restricted with NheI and XbaI (compatible cohesive ends) and religated to generate pTG19385.

[0223] COPTG19385 was generated in chicken embryo fibroblasts (CEFs) by two successive homologous recombinations for sequential insertion into the J2R and I4L loci, and by using COPTG19156 as the parent virus and the two transfer plasmids pTG19367 and pTG19385. CEFs were isolated from 12-day-old embryonated SPF eggs (Charles River). Embryos were mechanically dissected, solubilized in Tryple Select solution (Invitrogen), and cultured in MBE (Eagle Based Medium, Gibco) supplemented with 5% FCS (Gibco) and 2 mM L-glutamine.

[0224] Homologous recombination between the transfer plasmid and the parental vaccinia virus allows the generation of recombinant vaccinia viruses that have lost the GFP and mCherry expression cassettes and gained an antibody expression cassette. COPTG19156 contains an expression cassette for the mCherry gene at the I4L locus and an expression cassette for the GFP gene at the J2R locus. Homologous recombination between the transfer plasmid pTG19367 and the parental COPTG19156 allows the generation of recombinant vaccinia viruses that have lost the GFP expression cassette and gained the 4-E03 heavy chain expression cassette, with selection carried out by isolation of red fluorescent plaques. This intermediate recombinant virus (COPTG19367) was used as the parental virus for a second round of homologous recombination with pTG19385 as the transfer plasmid to generate recombinant vaccinia viruses that have lost the mCherry expression cassette and gained the 4-E03 light chain expression cassette. Selection of COPTG19385 was carried out by isolation of white non-fluorescent plaques.

[0225] Viral stocks of COPTG19385 were amplified in two F175 flasks of CEFs to generate suitable viral stocks that can be aliquoted and stored at -80°C until use. Viral stocks were scaled in CEF cells and infectious titers expressed in pfu / mL were calculated using the following formula: number of lysed areas x dilution factor x 4. For illustration purposes, the viral stock generated was 1.04 x 10 7 The stock was adjusted up or down in pfu / mL. The stock was analyzed by PCR to verify the integrity of the expression cassette and the recombination arms using appropriate primer pairs. The stock was also analyzed by sequencing of both expression cassettes. Alignment of the sequencing results showed 100% homology with the theoretically expected sequences. If necessary, virus preparations were purified using conventional techniques (e.g. as described in WO2007 / 147528).

[0226] Transgene expression Virus-mediated expression of 4-E03 monoclonal antibody was assessed in supernatants of CEF cells infected with COPTG19384 by Western blot (WB) and compared with recombinantly produced antibody (40 ng of 4-E03). Using WB, the presence of non-functional molecules (e.g., molecules with incomplete chain assembly, aggregates) that do not bind to CTLA4 can be visualized. CEF cells were infected in triplicate with COPTG19384 virus stocks at an MOI of 0.05. Cell supernatants were collected after 48 h and analyzed by WB using anti-Ig (left blot) or anti-light chain (right blot) HRP-conjugated antibodies after electrophoresis in non-reducing conditions. The results shown in Figure 18A show that the WB profile in non-reducing conditions of the mAb produced by infected CEFs is close to that of purified 4-E03, with a similar apparent size of 100-150 kDa, indicating correct chain folding and assembly.

[0227] Quantification of the functionally secreted 4-E03 antibody and GM-CSF in the supernatant was performed by ELISA. ELISA allows the amount of functional polypeptide produced in the cell supernatant to be quantitatively measured. VVTG17137 was used as a negative control. It is a vaccinia virus (Copenhagen strain) deleted at the J2R and I4L loci encoding the suicide gene FCU1 (described in WO2009 / 065546).

[0228] To estimate 4-E03 antibodies, microplates were coated with 100 μL per well of 0.25 μg / mL CTLA4-Fc by overnight incubation at 4°C. After incubation, the coating solution was discarded, blocking solution was added, and the plate was incubated at room temperature for 1-2 h and then washed. 4-E03 calibration standards (0.097-100 ng / mL) or samples diluted in blocking solution (in triplicate) were added to the wells, and the plate was incubated at 37°C for 2 h and then washed. HRP-conjugated antibodies diluted in blocking solution were added to each well, and the plate was incubated at 37°C for 1 h and then washed. After incubation with TMB solution for 30 min at room temperature in the dark, HRP-conjugated antibodies were added to each well. 2 SO4 The enzyme reaction was stopped by adding 2M (stop solution). The absorbance was read at 450 nm on a microplate reader. The absorbance was plotted against the antibody concentration of the calibration standards. As shown in Figure 18B, functional 4-E03 mAb was produced in cells infected with COPTG19384, reaching concentrations approaching 1 μg / mL.

[0229] The virus-mediated expression of GM-CSF was also evaluated in the same supernatants using Quantikine® ELISA (R&D Systems Ref SGM00). Briefly, the assay uses two anti-hGM-CSF antibodies. The first antibody used to capture hGM-CSF in the samples was coated on the well surface of a 96-well plate. The second antibody is conjugated and added to the plate in solution to detect the captured hGM-CSF. The concentration of hGM-CSF in the samples is then calculated by interpolation from a standard curve established with several purified hGM-CSFs provided by the kit. As shown in FIG. 19, the expression level of GM-CSF was approximately 6 μg / mL.

[0230] In conclusion, concentrations above 1 μg / mL were detected for both transgenes, indicating satisfactory levels of expression.

[0231] Genetic Stability: Genetic stability tests were performed using a multiplicity of infection (MOI) of 10 -4 This was performed after five passagings of the virus on CEFs in serum-free medium at 100 ng / mL. Passage P5 was diluted and inoculated into CEF cells in 60 mm culture dishes to obtain 20–40 viral plaques per dish. One hundred viral plaques were isolated and subcultured. After one amplification cycle, the isolated viral plaques were inoculated into CEF cells and tested by PCR and ELISA. PCR analysis and transgene expression showed that more than 90% of the clones had the correct profile. As the acceptance criterion for clinical development of a product is more than 90% genetic stability, COPTG19384 was considered genetically stable.

[0232] Example 7 - In vitro characterization of COPTG19384 Replication studies in hepatocytes: Tumor selectivity of viruses expressing anti-CTLA4 mAb and GM-CSF COPTG19384 has two gene deletions encoding viral enzymes (TK and RR) involved in nucleotide metabolism. If functional, these enzymes allow the virus to replicate in the cytoplasm of most cells, including cells that are dormant (i.e., have low available nucleotide pools). To ensure that the insertion of the different transgenes into the J2R and I4L loci does not alter the host range selectivity, host range studies were performed in primary and malignant cells. Replication of COPTG19384 was evaluated in normal primary human cells (hepatocytes, prepared by Biopredic) and in tumor cells of the same organ (HepG2, ATCC® HB-8065™ derived from hepatocellular carcinoma). Replication rates and therapeutic indices were calculated and compared, respectively, with those of both wild-type Copenhagen vaccinia (COP WT, virus without deletions) as a reference for a nonselective vaccinia virus and recombinant double-deleted VVTG17137 virus (in which the J2R and I4L genes were deleted with the suicide FCU1 gene inserted in place of J2R). Two batches were analyzed: a research batch (batch 1) and a GMP-produced batch (batch 2). Replication rates were determined as the ratio of total infectious particles at the end of incubation / initial infectious particles (inoculum dose). The therapeutic index of each virus was determined as the ratio of replication rate in HepG2 cells / replication rate in hepatocytes. The higher the ratio, the more selective the virus is for tumor cells.

[0233] Primary hepatocytes were grown in Basal Hepatic cell medium supplemented with 1.6% supplement for hepatic cell culture. HepG2 cells were seeded in 12-well plates at 4E+05 cells / well and incubated at 37°C, 5% CO 2The cells were incubated at 37°C for 24 h. Prior to infection, the medium was removed and 70 pfu / well of virus was added to each well in either PBS for hepatocytes or FCS-supplemented PBS for HepG2. Infected cells were incubated at 37°C, 5% CO 2 After incubation at 4°C for 30 min, 1.5 mL / well of medium was added. The plates were incubated at 37°C, 5% CO 2 After incubation at 40° C. for 3 days, the plates were stored at −80° C. The plates were then thawed and the wells were sonicated for 30 seconds at 40% amplitude, followed by titration on Vero cells.

[0234] Replication rate in normal human hepatocytes: We chose normal hepatocytes to monitor the ability of COPTG19384 in normal human cells, since these primary cells are regularly available directly from donors. In these cells, COP WT spread well with a replication rate of over 50,000 (Figure 20A). In other words, for every initial infectious viral particle, approximately 50,000 new viruses were generated. In the case of the two recombinant double-deleted viruses (i.e., VVTG17137 and COPTG19384), this replication rate was dramatically reduced to 5–15 depending on the virus or the batch of virus (Figure 20A). This last result indicates that the attenuated replication in normal cells brought about by the two deletions was conserved between VVTG17137 and COPTG19384.

[0235] Replication rate of tumor cells HepG2: HepG2 cells were chosen to monitor the ability of COPTG19384 to replicate in neoplastic human cells, since they are the malignant counterpart of normal hepatocytes. In these cells, the replication rates of the five viruses tested were very similar, reaching approximately 100,000 new viruses, whatever the virus initially tested (Figure 20B). Thus, the double deletion and transgene vectorization in both VVTG17137 and COPTG19384 did not impair the ability to replicate in malignant cells.

[0236] Therapeutic index: As shown in Figure 20C, the calculated index is only 2 for COP WT, indicating the poor selectivity of COP WT towards tumor and normal cells. Conversely, for both VVTG17137 and COPTG19384 (and both lots of viruses tested), this index varies from 8.2E+03 to 1.8E+04. This confirms that the two recombinant viruses have the same good selectivity towards tumor and normal cells.

[0237] These results showed that COPTG19384 and VVTG17137 have very similar replication properties in both tumor and healthy cells. Compared to COP WT, replication in tumorous Hep G2 is similar but highly impaired in healthy hepatocytes. Thus, deletion of the two genes (J2R and I4L) restricts replication of the deleted virus to proliferating cells (i.e., high nucleotide pools), including tumor cells. Because transgene expression and replication are tightly coupled, COPTG19384 is an efficient vector for selectively delivering therapeutic proteins to tumors.

[0238] Replication assays in CEF and LoVo: Replication of COPTG19384 was assessed in CEFs (producer cells) and tumorigenic human cell lines (LoVo, ATCC® CCL-229™) isolated from 11- or 12-day-old specific pathogen-free eggs (Charles Rivers). CEF and LoVo cells were prepared in suspension and an MOI of 10 for CEFs was used. -3 , and 10 for LoVo -2 (3 wells per cell and time point). After different times of incubation, virus titration was performed on Vero cells (CCL-81™). COPTG19384 replication was compared with VVTG17137 replication as a benchmark. Results show that the replication of COPTG19384 and VVTG17137 was similar in both CEF and LoVo (data not shown).

[0239] Replication assay in reconstituted human skin: Replication of COPTG19384 was also evaluated in reconstituted human skin (T-Skin™ / human full thickness skin model). Thirty-six T-Skin™ samples obtained from EPISKIN SA were cultured in 6-well plates and maintained in fresh medium. The final concentration of COPTG19384 treated (i.e., 10 1 ~10 5 VVTG17137 and COPTG19384 were dispensed into each well (in triplicate) to obtain 100 pfu / well. A negative control corresponding to medium without virus was also tested (Mock). The plates were incubated at 37°C and 5% CO 2 After incubation at RT for 7 days, the T-Skin™ samples were collected and cut into two pieces. The infectious titer was determined in one of the two pieces using Vero cells for virus titration. Figure 21 shows that COPTG19384 replicates in the reconstituted skin to the same extent as the benchmark VVTG17137, supporting the fact that vectorization of both GM-CSF and 4-E03 mAb did not alter the replication behavior of vaccinia virus in human reconstituted skin.

[0240] Tumor lysis assay Oncolytic activity is representative of the lytic activity of the tested virus sample against tumor cells. It was evaluated by quantification of cell viability after 5 days of incubation in different tumor cell lines: human colorectal adenocarcinoma cell line LoVo (ATCC® CCL-229™), human pancreatic tumor cell line MIA PaCa-2 (ATCC® CCL-1420) and human hepatocellular carcinoma cell line HepG2 (ATCC® HB-8065™). Oncolytic activity of COPTG19384 was compared with that of VVTG17137 as a benchmark. A negative control corresponding to uninfected cells was also plated (mock-infected cells).

[0241] Cells were prepared and placed in Eppendorf tubes (1.2 x 10 6 cells / tube), then 10 -5 ~10 -2The cells were infected with virus at an MOI of 100 and incubated at 37°C for 30 min. Appropriate complete medium was added to Eppendorf tubes and aliquots of this suspension were added (in triplicate) to each well of a 6-well plate containing 2 mL of appropriate complete medium. The plates were incubated at 37°C, 5% CO 2 The cells were incubated at 4°C for 5 days and cell viability was measured using a Vi-Cell counter. Results were expressed as a percentage of cell viability of mock-infected cells. Cell supernatants were also collected to measure the concentrations of 4-E03 mAb and GM-CSF. Figure 22 shows that the oncolytic activity of COPTG19384 and VVTG17137 was similar in the three tumor cell lines evaluated.

[0242] Expression level of transgene Expression levels of both 4-E03 monoclonal antibody and GM-CSF were measured by ELISA (described in Example 6) in culture supernatants of HepG2 and LoVo cells harvested after oncolytic activity measurements (5 days after infection at variable MOI).

[0243] The expression levels of 4-E03 and GM-CSF were measured by ELISA (see Example 6) in the supernatants of five cell lines cultured under the following conditions: human gastric cancer cell line Hs-746 T (ATCC® HTB-135™), human ovarian tumor cell line SK-OV-3 (ATCC® HTB-77™), human pancreatic tumor cell line MIA PaCa-2 (ATCC® CCL-1420), human colorectal adenocarcinoma cell line LoVo (ATCC® CCL-229™), and human colorectal cancer cell line HCT116 (ATCC® CCL-247™). Each cell line was cultured in 6-well plates (10 6 Cells / well) (in triplicate) at 37°C and 5% CO 2After incubation with COPTG19384 for 24 h, the cells were infected at an MOI of 0.05. Cell supernatants were then harvested 48 h after infection to determine the concentrations of 4-E03mAb and GM-CSF. As expected, the MOI, time post-infection, and cell line are important parameters that affect the transgene expression levels in the supernatants of infected cells. Figure 23A shows that replication-permissive (HepG2) and resistant (LoVo) tumor cell lines can produce approximately equal amounts of 4-E03mAb and GM-CSF in the culture supernatant when infected with COPTG19384. However, maximum expression in HepG2 is reached at an MOI ten times lower than that of LoVo. Moreover, in the five tumor cell lines tested, transgene expression was above 0.1 and 1 μg / mL for 4-E03mAb and GM-CSF, respectively (Figure 23B). It should be noted that the ELISA assay used to measure the concentration of 4-E03mAb uses the antigen CTLA4 to capture the antibody. In other words, the antibodies measured by this assay are at least partially functional (i.e., they recognize their antigen and other functions of the antibody are carried by the Fc portion),

[0244] Purification and glycosylation profile analysis of 4-E03 mAb To produce a significant amount of 4-E03mAb from infected cells, approximately 4.7 10 7Fifteen F175 flasks containing Mia-PACA cells / flask were infected with COPTG19384 at MOI 0.01 and incubated for 72 h. MIA Paca-2 cell culture supernatants (approximately 450 mL containing 670 μg of mAb 4-E03 as measured by ELISA) were harvested, pooled, and clarified by centrifugation to remove most cell debris. The clarified supernatant was filtered through a 0.2 μm filter and 2 mM EDTA (to inhibit putative metalloproteases) and 20 mM Tris pH 7.5 (to raise the pH) were added. The filtered supernatant was then passed through a protA Hitrap column (GE healthcare, ref. 17-5079-01). The column was transferred and connected to a Purifier FPLC (GE Healthcare) and the purification program (THM / ProtA 1 mL injection loop frac bleu) was applied. Elution fractions containing mAb were loaded onto a 4–12% (Thermo NP0323) NuPage Bis-Tris gel after addition of Laemlli buffer (Biorad) with or without beta-mercaptoethanol to reduce or not the disulfide bonds of the mAb. The gel was stained with InstantBlue (Expedeon, ISB1L). Three fractions corresponding to the main peak of elution were pooled and dialyzed against formulation buffer, after which the antibody concentration was determined by absorbance at 280 nm. The final concentration of purified mAb was 0.29 mg / mL.

[0245] The first characterization was the assessment of chain assembly by electrophoresis under reducing and non-reducing conditions. Under non-reducing conditions, 4-E03 assembles two light chains and two heavy chains to form native functional antibodies. 4-E03 purified from infected MIA PaCa-2 and recombinantly produced 4-E03 appeared to have indistinguishable electrophoretic profiles under both reducing and non-reducing conditions. In other words, 4-E03 purified from infected MIA PaCa-2 has the expected ratio of light and heavy chains and correctly assembles into a heterotetramer of two light and two heavy chains. The presence of this heterotetramer was also confirmed by mass spectrometry. The purified mAb was subjected to mass spectrometry for glycosylation analysis. Briefly, the mAb specifically cleaves IgG at the hinge (F(ab') 2 and Fc part were either digested or not digested with IdeS protease. The mass of whole antibody or Fc part with N-glycosylation was determined and each mass was fitted with the theoretical mass calculated from the primary sequence of Fc and glycosylation pattern. The glycosylation profile of 4-E03 mAb purified from infected MIA PaCa-2 was compared with that of recombinantly produced and purified 4-E03 and MabThera as a benchmark for human IgG1 used in the clinic. The results show that the glycosylation profile of 4-E03 produced from infected MIA PaCa2 had a different glycosylation profile than the two antibody references with a majority of G0F (88%), whereas recombinant 4-E03 and MabThera both have glycosylation profiles similar to the typical G0F, G1F and G2F distribution. However, MIA PaCa-2 has low levels of beta-1,4-galactosyltransferase 1 transcripts, which we suspect may be responsible for the low G1F and G2F species in purified 4-E03mAb, and this may be responsible for the galactosyl moiety (and therefore the lack of G1f and G2F) of 4-E03 expressed in MIA PaCa-2.

[0246] The same type of purification followed by mass spectrometry was also performed from the permeate collected during purification of COPTG19384 produced in CEFs. The results show that the glycosylation profile of 4-E03 from infected CEFs was very similar to that of MabThera or recombinant 4-E03. This latest result suggests that the glycosylation profile of antibodies is more influenced by the cell line used than the infection itself.

[0247] 4-E03 purified from the supernatant of infected MIA PaCa-2 cells (4-E03TG) also exhibits the same binding properties as 4-E03 recombinantly produced by CHO (research batch) or HEK (tox batch) cells (Figures 24 and 25). This was shown by ELISA (described in Example 1) to test binding to recombinant (Figure 24A) human and (Figure 24B) cynomolgus CTLA-4 proteins. FACS analysis (see Examples 1 and 2) testing binding to (Figure 25A) human and (Figure 25B) cynomolgus CTLA-4 expressing cells confirmed similar cross-reactivity and binding affinity for the different 4-E03 batches.

[0248] Glycosylation and disulfide bond patterns of GM-CSF To investigate the glycosylation pattern and the presence of some disulfide bonds, different human tumor cell lines were infected with COPTG19384 and their supernatants were analyzed by the same WB method. MIA-Paca-2, LoVo, HepG2 and HCT116 cells were infected at an MOI of 0.01 and incubated for 72 h in serum-free medium. Culture supernatants were collected, clarified by centrifugation and filtered through a 0.2 μm filter. Supernatants were stored at -20 °C until analysis. They were treated by adding 8 μl of Rapid PNGase F Buffer 5X followed by incubation at 75 °C for 5 min. Then, 1 μL of PNGase F was added (to remove N-glycans from glycoproteins) and the mixture was incubated at 50 °C for 30 min. Before being subjected to Western blotting, 25 μL samples were prepared by adding 5 μL of Laemmli bufferx4 with or without beta-mercaptoethanol (reducing and non-reducing conditions). Immune complexes were detected using Amersham ECL Prime Western blotting, and chemiluminescence was recorded using a Molecular Imager ChemiDOC XRS (Biorad).

[0249] GM-CSF from infected HCT116, LoVo, and MIA PaCa-2 displayed the same pattern of glycosylation, whereas GM-CSF produced by infected HepG2 migrated as a non-N-glycosylated molecule. These results indicate that GM-CSF produced by human tumor cells infected with COPTG19384 has the expected post-translational modifications (i.e., disulfide bonds and N-glycosylation). However, these modifications likely differ depending on the tumor cell line used for infection and their specific metabolic state.

[0250] Example 8: Pharmacokinetics of COPTG19384 after intratumoral injection Kinetics of anti-CTLA4 antibody, GM-CSF and virus expression in tumor and bloodstream following intratumoral (it) injection of vaccinia virus in the LoVo xenograft model. protocol 5×10 6 LoVo cells were transplanted into the right flank of Swiss nude mice (Charles River, France). The tumor volume was approximately 120 mm 3 Approximately 2 weeks after reaching this age, the mice were randomized and divided into 6 groups of 15 animals. Mice in group 1 were treated with 1x10 4 Received intratumoral administration of COPTG19384 at a dose of pfu / mouse. Mice in group 2 were given 1x10 5 Received intratumoral administration of COPTG19384 at a dose of 100 pfu / mouse. Group 3 mice were treated with 1x10 4 received intratumoral administration of VVTG17137 at a dose of pfu / mouse. Group 4 mice were treated with 1x10 5 received intratumoral administration of VVTG17137 at a dose of pfu / mouse. • Mice in group 5 received intraperitoneal (ip) administration of 4-E03 at a dose of 3 mg / kg on D0. • Mice in group 6 received ipilimumab (Yervoy) intraperitoneally at a dose of 3 mg / kg on D0.

[0251] Tumors and blood from three animals were collected on days 1, 3, 6, 10 and 20. Tumors were weighted and homogenized for immediate processing. One quarter of the homogenized tumor was collected for virus titration, the remaining suspension was centrifuged and the supernatant was stored at -20°C until use. Blood was divided into two portions: one was added to heparin tubes (25 IU / 100 μL blood) for titration assays and frozen at -80°C until analysis. Clarified serum was generated from the other portion and stored at -20°C until use. Viral titers were determined in tumor and blood samples by titration on Vero cells.

[0252] Viral replication kinetics in the LoVo model: COPTG19384 was administered in two doses (1 × 104 or 1×10 5 In the LoVo model, where the virus was injected once at 1 × 10 pfu, viral replication was monitored and compared with that of VVTG17137 injected in the same conditions. The results displayed in Figure 26 show an important variance of the three values ​​of viral titer measured at each time point. Anyway, the results also show that with both viruses and both doses, the virus replicates in the tumor and maintains a rather high titer / g of tumor from day 3 up to 20 days after injection. At a given time point, there is no obvious difference in viral titer between the two viral doses or between the two viruses used. Interestingly, in one sample (VVTG17137, dose: 1 × 10 at day 10) where only 13 pfu / mL was detected, 7 All blood samples were negative for virus detection, except for 0.01 pfu (data not shown).

[0253] Together these results are 1×10 4 or 1×10 5 We show that after a single intratumoral injection of either PFU, viral replication was maintained in LoVo tumors for at least 20 days with an almost undetectable presence in the bloodstream. It should be noted that the LoVo xenograft model is highly favorable for viral replication due to the use of permissive human tumor cells and Swiss nude mice, whose immune system is severely compromised and therefore has limited antiviral activity.

[0254] Kinetics of transgene expression in the LoVo model: As expected, the kinetics of transgene expression in tumors followed the kinetics of viral replication, with maximum concentrations (Cmax) for both 4-E03mAb (Figure 27A) and GM-CSF (Figure 27B) at days 6 or 10. In the case of a single injection of 4-E03mAb (or ipilimumab), Cmax in tumors and blood was observed at the first time point (day 1), and the concentrations of mAb measured thereafter were consistent with the pharmacokinetics of human IgG1 in mice (Figure 28).

[0255] Furthermore, the concentration of 4-E03 in the tumor after Cmax (i.e., 6-10 to 20 days after injection) was approximately 10-fold higher after COPTG19384 treatment (both doses) than after a single injection of 4-E03 mAb at a treatment dose of 3 mg / kg (Figure 27A). In contrast, blood concentrations of mAb after COPTG19384 treatment were always inferior to those measured after intraperitoneal injection of 3 mg / kg 4-E03 (Figure 28A). This result indicates that mAb vectorization can reach high concentrations in the tumor without exceeding or reaching the blood concentrations obtained with treatment administration of mAb.

[0256] The expression kinetics of GM-CSF after COPTG19384 treatment follows that observed with 4-E03 (Figure 27B). Interestingly, although the levels of GM-CSF measured in the tumors are below those of 4-E03 in the same samples, in vitro LoVo infected with COPTG19384 express approximately twice as much GM-CSF as 4-E03. The blood levels of GM-CSF were also very low compared to those of 4-E03 (Figure 28B). This result is consistent with the in vivo half-life of GM-CSF, which is very short compared to the half-life of human IgG1.

[0257] These results demonstrate that vectorized antibodies and GM-CSF are expressed primarily in tumors after intratumoral injection of COPTG19384 with minimal systemic exposure. These results confirm that vectorization is particularly suitable for transgenes with toxicological (e.g., anti-CTLA4) or pharmacokinetic (e.g., GM-CSF) issues.

[0258] Kinetics of anti-CTLA4 antibody, GM-CSF, and virus expression in tumors and bloodstream following intratumoral injection of vaccinia virus in the CT26 syngeneic model Evaluation of viral activity in the CT26 immunocompetent mouse model requires the generation of several surrogate viruses encoding murine anti-mCTLA4, with or without murine GM-CSF. ●COPTG19407 is a vaccinia virus (Copenhagen strain) containing an expression cassette encoding the heavy chain of murine m5-B07 IgG2 (SEQ ID NO: 63) under the p7.5 promoter at the J2R locus and an expression cassette encoding the light chain of m5-B07 (SEQ ID NO: 62) under the p7.5 promoter at the I4L locus and murine GM-CSF (SEQ ID NO: 58) under the pSE / L promoter. - COPTG19421 is a vaccinia virus (Copenhagen strain) containing an expression cassette encoding the m5-B07 heavy chain under the p7.5 promoter at the J2R locus and an expression cassette encoding the m5-B07 light chain under the p7.5 promoter at the I4L locus. • VVTG18058, used as the benchmark, is a vaccinia virus (Copenhagen strain) in which the J2R and I4L genes have been deleted and which does not contain any transgene (an "empty" virus).

[0259] These vaccinia viruses were generated with respect to their human counterparts by two successive homologous recombinations at the J2R (TK) and then I4L (RR) loci, following the process described in Example 6. The ELISA method for quantifying m5-B07 antibody and mGM-CSF was similar to that described above, except that mouse CTLA4-Fc antigen was used to capture mouse antibodies and Quantikine ELISA kit mouse GM-CSF (R&D Systems) was used to quantify mGM-CSF (Example 6, "Transgene Expression" of 4-E03 and GM-CSF). The oncolytic activity of these viruses was also evaluated in various cell lines (one sarcoma: MCA205 and two colon carcinomas CT26 and MC38) and was similar to that of VVTG18058, indicating that vectorization of mouse antibodies with or without mGM-CSF did not affect the oncolytic ability of vaccinia viruses (data not shown).

[0260] Protocol: CT26 cells (2 × 10 5The tumor volumes were 25–50 mm. 3 Approximately 1 week after reaching NIH, mice were randomized and divided into three groups of 20 animals (groups 1–3) and one group 4 of 10. Tumors and blood were collected and processed as described for the LoVo model, except that tumors and blood were collected on days 1, 4, 8, and 10 for the first three groups and on day 1 for group 4. Mice in group 1 were treated with 1 × 10 7 received intratumoral administration of VVTG18058 at a dose of pfu / mouse. Mice in group 2 were treated with 1 × 10 7 Received intratumoral administration of COPTG19407 at a dose of 100 pfu / mouse. Mice in group 3 were treated with 1 × 10 7 COPTG19421 was administered intratumorally at a dose of pfu / mouse. • Mice in group 4 received intraperitoneal administration of m5-B07 at a dose of 3 mg / kg on D0.

[0261] Viral replication kinetics in the CT26 model: CT26 model in which two surrogate viruses were injected three times (1 × 10 7 In the 10-mL (pfu / injection) viral replication was monitored and compared to that of VVTG18058 injected in the same conditions. The results displayed in Figure 29 show a significant variance of the three values ​​of viral titers measured at each time point for the LoVo model. However, the titers of the three viruses were maintained over time up to 10 days, indicating that at least in this time frame, the two transgenes did not affect viral clearance or replication. No viral infectious particles were detected in any of the blood samples (data not shown).

[0262] Kinetics of transgene expression in the CT26 model Similar to the LoVo model, transgene expression in tumors reflects viral replication: m5-B07 antibody (Figure 30A) and mGM-CSF (Figure 30B) were detected in tumors at fairly constant levels over the 10 days of monitoring.

[0263] For monoclonal antibodies, the Cmax reached after injection of either COPTG19421 or COPTG19407 was approximately 10-fold lower than that observed after a single intraperitoneal injection of 3 mg / mL m5-B07 antibody (Figure 30A). In serum, the difference was even more pronounced, with circulating concentrations of m5-B07 approximately 100-fold lower after viral treatment compared to 3 mg / kg m5-B07 injection (Figure 31).

[0264] In the case of GM-CSF, only treatment with COPTG19407 resulted in measurable concentrations of mGM-CSF in CT26 tumors, indicating that the measured cytokine was of recombinant origin rather than endogenous. Similar to the LoVo model, mGM-CSF concentrations measured in tumors were lower than those of m5-B07 (Figure 30B). Furthermore, mGM-CSF produced by tumors was not detectable in any serum samples, likely due to the short half-life of the molecule that prevents systemic accumulation.

[0265] Example 9: Antitumor Activity Study COPTG19347 is a vaccinia virus (Copenhagen strain) in which the J2R and I4L genes have been deleted and which encodes the whole mouse antibody (i.e., m5-B07, heavy and light chains) that recognizes the mouse CTLA4 antigen. In COPTG19421 versus COPTG19347, both m5-B07 were expressed but under different promoters, i.e., p7.5K and pH5.R, respectively. Quantification of m5-B07 was approximately 1 μg / mL in infected CT26 at an MOI of 10-1 and 1 μg / mL in infected CT26 at an MOI of 10-2. -2 In infected MCA205 cells, mGM-CSF was assessed in the supernatant of infected cells reaching approximately 4 μg / mL. Higher expression in MCA205 compared to CT26 was also observed in the culture supernatant of cells infected with COPTG19407 (data not shown).

[0266] Antitumor activity in mice bearing the CT26 model in combination with anti-PD1 protocol: CT26 cells (2 x 10 5 The tumors were transplanted into the right flank of Balb / c mice (Charles River, France). 3 When the tumor reached 100%, the mice were randomly divided into 5 groups of 10 animals. Briefly, the mice were treated with intratumoral injections of 100 mg / kg / day, 3 times at 2-day intervals, followed by intraperitoneal treatment with mouse anti-PD1 (RMP1-14 BioXcell) twice a week for 3 weeks. • Mice in group 1 received vehicle. Mice in group 2 were treated with 1 × 10 7 Received intratumoral administration of pfu COPTG19347. Mice in group 3 were treated with 1 × 10 7 They received pfu COPTG19347 intratumorally and 250 μg / mouse RMP1-14 ip intraperitoneally on D7, D11, D14, D18 and D22. • Mice in group 4 received intraperitoneal administration of 250 μg / mouse of RMP1-14 on D7, D11, D14, D18 and D22. Mice in group 5 were treated with 1 × 10 7 Received intratumoral administration of pfu VVTG18058.

[0267] Tumor dimensions were measured twice weekly with calipers and their volumes were calculated according to the formula (С / 6) (length x width). 2 ) was used to calculate the tumor volume. Animals were selected based on their tumor volume. 3 When the rats reached this age, they were euthanized.

[0268] Antitumor activity of COPTG19347 in the CT26 model: As shown in Figure 32, COPTG19347 treatment not only resulted in tumor growth inhibition (Figure 32A), but also tumor regression, ultimately resulting in tumor-free mice surviving up to 100 days (Figure 32B). Treatment with COPTG19347 resulted in 60% tumor-free mice at day 100. Combination treatment with anti-PD-1 antibody did not significantly improve tumor growth inhibition or the percentage of long-term surviving mice (about 70% tumor-free mice at day 100). In comparison, RPMI-14 treatment did not provide an anti-tumor effect (behavior the same as untreated mice (all died within the first 40 days)), while VVTG18058 had low activity (about 10% tumor-free mice at day 100).

[0269] Dose-effect assessment in the CT26 model: Three surrogate viruses were compared (with and without different promoters to drive m5-B07 and m-GM-CSF) and dose escalation of COPTG19407 and VVTG18058 was performed (7.5 × 10 4 , 7.5×10 5 , or 7.5 × 10 6 The experimental conditions were exactly the same as those described above, except that co-treatment with anti-PD1 was omitted.

[0270] Results from two independent experiments showed that of the three viruses tested, COPTG19407, COPTG19421, and COPTG19347, the 7.5 x 10 6 pfu dose. This confirms that neither the mGM-CSF encoded by COPTG19407 nor the use of the weakest promoters in COPTG19421 and COPTG19407 compromised the antitumor activity of the armed virus. As summarized in the following table, the highest dose tested (i.e., 7.5×10 6 pfu), the number of tumor-free mice at 80 days was between 5 / 10 and 7 / 10 depending on the virus and experiment, compared with 0 / 10 in mice treated with empty virus. [Table 6]

[0271] Furthermore, dose escalation performed with both the "empty" virus (VVTG18058) and the COPTG19384 surrogate (COPTG19407) demonstrated a relatively low dose (7.5 × 10) of the virus, compared with 0 / 10 of treatment with VVTG18058 at the same low dose, as shown in the following table: 4 pfu), the antibody-expressing virus showed clear antitumor activity with 4 / 10 and 2 / 10 tumor-free mice at 80–98 days. [Table 7]

[0272] A compilation of survival data (overall survival plots compiled from two independent studies) is shown in Figure 33. Statistical analysis using the log-rank test was performed to determine whether there were significant differences in survival for each group.

[0273] Antitumor activity of COPTG19407 compared with the combination of VVTG18058 and m5-B07 CT26 tumor-bearing mice were established as previously described (Example 5). Briefly, CT26 cells were injected subcutaneously into BalB / c mice. Tumors were approximately 100 mm 3 Treatment of mice began when the mice reached 100%. Mice were then treated with COPTG19407 (8.5 × 10 6 pfu intratumor), VVTG18058 (8.5 × 10 6 pfu intratumorally), m5-B07 (10 mg / kg i.p.), or VVTG18058 (8.5 × 10 6 pfu intratumoral) and m5-B07 (10 mg / kg intraperitoneally) were injected on D0, D2 and D5. Tumor dimensions were measured twice weekly thereafter, and tumors were measured when the tumors reached 2000 mm 3Mice were euthanized when tumor growth reached 100 days. As shown in Figures 34A to 34D, tumor growth was significantly inhibited when mice were treated with the anti-CTLA-4 and GM-CSF expressing virus COPTG19407, but the combination of the unarmed virus and anti-CTLA4 m5-B07 did not result in improved therapy compared to the single agent use. In the groups treated with m5-B07 alone, the virus VVTG18058 alone, or the combination of both m5-B07 and VVTG18058, only 20% of the mice survived after 70 days (Figure 34E). In contrast, 90% of the mice survived for more than 100 days after administration of COPTG19407, demonstrating the efficacy of the vectorization strategy.

[0274] Antitumor activity of VVTK-RR encoding anti-CTLA-4 and GM-CSF in A20 subcutaneous murine B-cell lymphoma-bearing mice The A20 cell line is a BALB / c B cell lymphoma line derived from a spontaneous reticular cell neoplasm found in an aged BALB / cAnN mouse (ATCC TIB-208™).

[0275] Protocol (1): Tumors were grown at 5 × 10 in the right flank of female Balb / cN mice (Charles River, France). 6 Tumors were induced by subcutaneous injection of 100 A20 cells. Tumors had an average volume of 95 mm 3 When the total number of mice reached 10, the 50 mice were randomly divided into 5 groups of 10 animals. Mice in group 1 received intratumoral injections of vehicle on D0, D2, and D4; Mice in group 2 were treated with 4.75 × 10 6 received intratumoral administration of VVTG18058 at a dose of p.f.u. Mice in group 3 were treated with 4.75 × 10 6 received intratumoral administration of COPTG19407 at a dose of p.p.fu. Mice in group 4 received intraperitoneal administration of anti-PD-1 antibody at a dose of 250 μg on D7, D10, D14, D17, D21, and D24. Mice in group 5 were treated with 4.75 × 10 6 They received pfu doses of COPTG19407 administered intratumorally and 250 μg doses of anti-PD-1 antibody administered intraperitoneally on D7, D10, D14, D17, D21 and D24.

[0276] Antitumor activity: Tumor volumes of all animals were monitored throughout the study. Antitumor activity of the treatments was based on evaluation of the following criteria: tumor doubling time, tumor growth delay, and tumor growth inhibition (T / C%).

[0277] Tumor doubling times were similar in groups 1, 2, and 4, ranging from 5.14 days (group 1) to 6.37 days (group 2). For group 3, tumor doubling times could not be calculated accurately because tumors did not grow exponentially, indicating a higher efficacy of treatment compared to groups 1, 2, and 4. Similarly, tumor doubling times were calculated using only one animal in group 5. Nine of the 10 mice in group 3 had tumors that regressed by D15 and did not increase in volume substantially from D25 until the end of the study at D64. At D64, the tumor volume in these nine mice was reduced to 4 mm 3 (Technical limit of tumor detection) ~59.77mm 3 Similarly, in Group 5, tumors regressed in nine mice after treatment initiation, ranging from 7.24 to 63.21 mm at the end of the study at D64. 3 As can be seen in Figure 35, which shows the individual tumor volume curves for each group of treatment (groups 1-5 corresponding to Figures 35A-E), no tumors grew in animals in groups 3 and 5 that received COPTG19407, confirming the strong antitumor activity of the antibody-expressing virus with or without anti-PD1.

[0278] Tumor growth delay was observed when the tumor was 300 mm 3This parameter was calculated by estimating the time it takes to reach the average target volume of 300 mm. 3 This was not the case for the majority of animals in groups 3 and 5, a result similar to that obtained for tumor doubling time, since it could only be calculated for tumors that reached 10 days. The mean tumor growth delays for groups 1, 2, and 4 were 16, 21, and 17 days, respectively, which were not significantly different from each other. Furthermore, the mean tumor delay for group 3 (n = 2) was 14 days, indicating that although tumors grown in this group grew at the same rate as groups 1, 2, and 4, these tumors actually regressed in both animals. In comparison, the single tumor in group 5 (n = 1) that grew showed a tumor growth delay of 43 days, which was significantly (p ≤ 0.0026) longer than all other groups.

[0279] Tumor growth inhibition (T / C%) was calculated by comparing the median tumor volume of vehicle-treated group 1 with the other treatment groups. The optimal T / C% in group 2 was 34% at D22, indicating a transient marginal antitumor activity, but this value increased to 71% by D31. Moderate antitumor activity (10-30% T / C%) was observed in group 4. In comparison, both groups 3 and 5 showed significant antitumor activity (T / C% less than 10%) from D27 to D31 (last calculable value of T / C%).

[0280] FIG. 36 shows the mean tumor volume curves of BALB / cN mice bearing subcutaneous A20 tumors, demonstrating the dramatic effect of anti-CTLA4 and Gm-CSF expressing virus COPTG19407 with or without anti-PD1 on tumor growth.

[0281] Protocol (2): Tumors were grown in the right flank of female BALB / cN mice at 5 × 10 6 Tumors were induced by subcutaneous injection of 100 A20 cells. The mean tumor volume was 80–100 mm. 3 When this was reached, the 90 animals were randomly divided into 9 groups of 10 animals. Mice in group 1 received intratumoral injections of vehicle on D0, D2, and D4. Mice in group 2 received intraperitoneal administration of anti-PD-1 antibody at a dose of 250 μg / mouse / injection on D0, D4, D7, D10, D14 and D17. Mice in group 3 received intraperitoneal injections of isotype at a dose of 250 μg / mouse / injection on D0, D4, D7, D10, D14 and D17. Mice in group 4 were treated with 1 × 10 5 received intratumoral administration of VVTG18058 at a dose of p.f.u. Mice in group 5 were treated with 1 × 10 5 received intratumoral administration of VVTG18058 at a dose of pfu and intraperitoneal administration of isotype at a dose of 250 μg / mouse / injection on D0, D4, D7, D10, D14 and D17. Mice in group 6 were treated with 1 × 10 5 pfu dose of VVTG18058 and intraperitoneal administration of anti-PD-1 antibody at a dose of 250 μg / mouse / injection on D0, D4, D7, D10, D14 and D17. Mice in group 7 were treated with 1 × 10 5 received intratumoral administration of COPTG19407 at a dose of p.f.u. Mice in group 8 were treated with 1 × 10 5 pfu dose of COPTG19407 was administered intratumorally in combination with intraperitoneal administration of isotype at a dose of 250 μg / mouse / injection on DO, D4, D7, D10, D14, D17 and D24. Mice in group 9 were treated with 1 × 10 5 Intratumoral administration of COPTG19407 at a dose of pfu was combined with intraperitoneal administration of anti-PD-1 antibody at a dose of 250 μg / mouse / injection on DO, D4, D7, D10, D14 and D17.

[0282] Antitumor activity: The dose of COPTG19407 was suboptimal and showed modest antitumor activity in terms of tumor volume and mouse survival, similar to anti-PD-1 treatment.

[0283] In contrast, the combination of COPTG19407 and anti-PD-1 demonstrated potent anti-tumor activity, with smaller tumor volumes compared to the other groups as shown in Figure 37A (approximately 630 mm at day 24 in mice treated with anti-PD-1 alone or COPTG19407 alone, respectively). 3 and 750mm 3 On the 36th day, the 3 ), with much better animal survival as shown in FIG. 37B (seven animals still alive on day 57 in group 9 compared to only two or one in groups 2 or 7, respectively).

[0284] Antitumor activity study of VVTK-RR encoding anti-CTLA-4 and GM-CSF in mice bearing C38 subcutaneous colon tumor cells C38 is a murine colon adenocarcinoma derived from the American Type Culture Collection (ATCC CRL-2779™).

[0285] protocol: Tumor fragments (30-50 mg) were implanted subcutaneously into the right flank of female C57BL / 6J mice (Janvier, France). 3 When a mean volume of 1000 mg / kg was reached, the 50 animals were randomly divided into 5 groups of 10 animals. • Mice in group 1 received intratumoral administration of vehicle on D0, D2 and D4. Mice in group 2 were treated with 4.75 × 10 6 received intratumoral administration of VVTG18058 at a dose of p.f.u. Mice in group 3 were treated with 4.75 × 10 6 received intratumoral administration of COPTG19407 at a dose of p.p.fu. Mice in group 4 received intraperitoneal administration of mouse anti-PD-1 antibody at a dose of 250 μg on D7, D10, D14, D17, D21, and D24; Mice in group 5 were treated with 4.75 × 10 6 They received intratumoral administration of COPTG19407 at a dose of pfu and intraperitoneal administration of anti-PD-1 antibody at a dose of 250 μg on D7, D10, D14, D17, D21 and D24.

[0286] Antitumor activity: As before, tumor volumes of all animals were monitored throughout the study. Tumor doubling times were similar in groups 1 and 2 at approximately 6.7 days. Group 4 (n=5) had a longer tumor doubling time (10.4 days), but there was no significant difference between the groups. For groups 3 and 5, tumor doubling times were calculable, but with a smaller number of animals (n=2), as the majority of tumors in mice in these groups did not grow exponentially, indicating a greater efficacy of treatment compared to groups 1, 2, and 4. As can be seen in Figure 38, 8 out of 10 mice in group 3 had tumors that had regressed and not substantially increased in volume since D15, and 5 mice had no detectable tumors at the end of the study on D61. Similarly, in group 5, tumors regressed in 8 mice after the start of treatment, ranging from 0 (n=2) to 47.82 mm at the end of the study on D61. 3 reached a value in the range

[0287] Tumor growth delay was observed when the tumor was 300 mm 3 This parameter was calculated by estimating the time it takes to reach the average target volume of 300 mm. 3The results were similar to those obtained for the tumor doubling time, which was not applicable for the majority of animals in groups 3 and 5, since it could only be calculated for tumors that reached 100% doubling time. There were no significant differences between the groups. The mean tumor growth delay in groups 1, 2, and 4 (n = 5) was 23 to 27 days, respectively. Furthermore, the mean growth delay in groups 3 (n = 2) and 5 (n = 3) was 18 and 24 days, respectively. This indicates that the tumors grown in these two groups grew at a similar rate to groups 1, 2, and 4.

[0288] Tumor growth inhibition (T / C%) was calculated by comparing the median tumor volume of vehicle-treated Group 1 with the other treatment groups. Group 2 did not show tumor growth inhibition as T / C% remained above 100% throughout the study period. In comparison, Groups 3, 4, and 5 all showed significant antitumor activity (T / C% below 10%) from D31 (Group 3 only) to D42 (last calculable value of T / C%).

[0289] FIG. 39 shows the mean tumor volume curves of C57BL / 6 mice bearing subcutaneous C38 tumors, demonstrating the dramatic effect of anti-CTLA4 / GM-CSF expressing virus COPTG19407 with or without anti-PD1 on tumor growth.

[0290] Antitumor activity study of VVTK-RR encoding anti-CTLA-4 and GM-CSF in mice bearing EMT6 subcutaneous mammary tumor cells EMT6 is a murine mammary carcinoma derived from ATTC (ATCC CRL-2755™).

[0291] protocol: Tumors were grown in female BALB / cByJ mice at 1 × 10 6 Tumors were induced by subcutaneous injection of 10 EMT6 cells (Charles River, France). 3 When the mean tumor volume reached 100 μg / kg, the 50 mice were randomly divided into 5 groups of 10 animals according to their individual tumor volumes. Mice in group 1 received intratumoral injections of vehicle on D0, D2, and D4; Mice in group 2 were treated with 4.75 × 10 6 received intratumoral administration of VVTG18058 at a dose of p.f.u. Mice in group 3 were treated with 4.75 × 10 6 received intratumoral administration of COPTG19407 at a dose of p.p.fu. Mice in group 4 received intraperitoneal administration of anti-PD-1 antibody at a dose of 250 μg on D7, D10, D14, D17, D21 and D24; Mice in group 5 were treated with 4.75 × 10 6 They received pfu doses of COPTG19407 administered intratumorally and 250 μg doses of anti-PD-1 antibody administered intraperitoneally on D7, D10, D14, D17, D21 and D24.

[0292] Tumor volumes of all animals were monitored throughout the study by assessing the criteria of tumor doubling time, tumor growth delay, and tumor growth inhibition (T / C%).

[0293] Antitumor activity: Tumor doubling times were similar in groups 1, 2, and 4 at approximately 5.4 days. For group 3, the majority of tumors did not grow exponentially, so tumor doubling times could not be calculated, indicating a higher efficacy of treatment compared to groups 1, 2, and 4. A similar effect was observed in group 5, where only one animal was used for the calculation of tumor doubling times. As can be seen in the graph of individual tumor volumes (Figure 40), 8 out of 10 mice in group 3 had tumors that had regressed and not substantially increased in volume since D15, and 7 mice had no detectable tumors at the end of the study on D61. Similarly, in group 5, tumors regressed in 9 mice after the start of treatment, ranging from 0 (n=8) to 13.24 mm at the end of the study on D56. 3 reached a value in the range

[0294] Tumor growth delay was observed when the tumor was 200 mm 3This parameter was calculated by estimating the time it takes to reach the average target volume of 200 mm. 3 This was not the case for the majority of animals in groups 3 and 5, as it could only be calculated for tumors that reached 100 mg / kg / day, and the results were similar to those obtained for tumor doubling time. The mean tumor growth delay in groups 1, 2, and 4 was approximately 19 days. Furthermore, the mean growth delay in groups 3 and 5 (n=2 in both groups) was 24 and 12 days, respectively. This indicates that tumors growing in these two groups grew at a similar rate to groups 1, 2, and 4. There were no significant differences between the groups.

[0295] Tumor growth inhibition (T / C%) was calculated by comparing the median tumor volume of vehicle-treated group 1 with the other treatment groups. Group 2 showed transient marginal tumor growth inhibition at D28, which increased to 79% at D31. Group 4 showed no antitumor activity, with a T / C%>60% throughout the study period. In comparison, both groups 3 and 5 showed significant antitumor activity (T / C%<10%) from D24 to D31 (last calculable value of T / C%).

[0296] FIG. 41 shows the mean tumor volume curves of BALB / cByJ mice bearing subcutaneous EMT6 tumors, demonstrating the dramatic effect of anti-CTLA4 / GM-CSF expressing virus COPTG19407 with or without anti-PD1 on tumor growth.

[0297] CT26 Re-challenge To study whether a specific antitumor immune response occurred, 4 , 10 5 or 10 6 pfu COPTG1942 or 10 6 CT26 tumor cell-challenged BALB / c mice that survived pfu COPTG19407 treatment were rechallenged with CT26 tumor cells or challenged with Renca cells (renal adenocarcinoma cells: control). [Table 8]

[0298] The results shown in Table 8 indicate that 0 / 8 mice receiving COPTG19421 or COPTG19407 were tumor-free after RenCa challenge, whereas 7 / 10 mice receiving COPTG19421 or COPTG19407 were tumor-free after CT26 rechallenge, indicating that COPTG19421 and COPTG19407 enhanced specific immune memory against CT26 cells.

Claims

1. 1. An antibody molecule that specifically binds to CTLA-4, the antibody molecule comprising: VH-CDR1 of SEQ ID NO: 22, VH-CDR2 of SEQ ID NO: 23, VH-CDR3 of SEQ ID NO: 24, VL-CDR1 of SEQ ID NO: 10, VL-CDR2 of SEQ ID NO: 25, and VL-CDR3 of SEQ ID NO: 26, An antibody molecule having the formula:

2. The antibody molecule of claim 1, which has an improved depletion effect on CTLA-4 positive cells or CTLA-4 positive and CD4 positive cells compared to ipilimumab.

3. The antibody molecule of claim 1 or 2, which has an improved depleting effect on Tregs compared to ipilimumab.

4. The antibody molecule (i) An in vitro ADCC test carried out using the NK-92 cell line stably transfected to express the CD16-158V allele together with GFP, said ADCC test comprising the following successive steps: 1) providing CTLA-4 positive cells, CD4 positive cells, or Tregs as target cells isolated from peripheral blood of a healthy donor; 2) then stimulating the target cells with CD3 / CD28 and rhIL-2; 3) then pre-incubating said target cells with said antibody molecules and then mixing with NK cells; 4) then incubating the target cells in RPMI 1640+GlutaMAX® medium containing one HEPES buffer, sodium pyruvate, and FBS low IgG; 5) determining lysis by flow cytometry; 6) repeating or performing steps 1 to 5 in parallel, with ipilimumab being used in place of said antibody molecule in step 3; 7) comparing the lysis results of the antibody molecule with those of ipilimumab, where improved lysis of the antibody molecule compared to ipilimumab indicates that the antibody molecule has an improved depleting effect on CTLA-4 positive cells, CD4 positive cells and / or Tregs; and / or (ii) An in vivo study in a PBMC-NOG / SCID model, said in vivo study comprising the following sequential steps: 1) washing and resuspending isolated human PBMCs in sterile PBS; 2) intravenously injecting the cell suspension from step 1) into NOG mice; 3) isolating the spleen from said NOG mice and making it into a single cell suspension; 4) resuspending the cell suspension from step 3) in sterile PBS; 5) Injecting SCID mice intraperitoneally with the suspension from step 4; 6) then treating said SCID mice with either said antibody molecule, ipilimumab, or an isotype control monoclonal antibody; 7) collecting the intraperitoneal fluid of the treated SCID mice; 8) Identifying and quantifying human T cell subsets by FACS using the following markers: CD45, CD4, CD8, CD25, CD127; 9) comparing the results of identification and quantification of the T cell subsets from the mice treated with the antibody molecule with the results of identification and quantification of the T cell subsets from the mice treated with ipilimumab and with the results of identification and quantification of the T cell subsets from the mice treated with an isotype control monoclonal antibody, and comparing the number of CTLA-4 positive cells, CD4 positive cells and / or Tregs in the peritoneal fluid of mice treated with the antibody molecule being tested with the number of CTLA-4 positive cells, CD4 positive cells and / or Tregs in the peritoneal fluid of mice treated with ipilimumab; 4. The antibody molecule of any one of claims 1 to 3, which is deemed to have an improved depletion effect on CTLA-4 positive cells, CD4 positive cells and / or Tregs compared to ipilimumab if improved depletion is shown in an in vivo test in a PBMC-NOG / SCID model comprising the step of:

5. The antibody molecule of any one of claims 1 to 4, wherein the antibody molecule comprises a variable heavy chain of SEQ ID NO:27 and / or a variable light chain of SEQ ID NO:

28.

6. The antibody molecule of any one of claims 1 to 5, wherein the antibody molecule comprises a heavy chain constant region of SEQ ID NO: 43 and / or a light chain constant region of SEQ ID NO:

44.

7. The antibody molecule may be a full size antibody, a chimeric antibody, a single chain antibody, a Fab, an Fv, an scFv, an Fab', or an (Fab') ２ The antibody molecule of any one of claims 1 to 6, selected from the group consisting of:

8. 8. The antibody molecule of any one of claims 1 to 7, which binds to human CTLA-4 (hCTLA-4), and / or to cynomolgus monkey CTLA-4 (cmCTLA-4), and / or to mouse CTLA-4 (mCTLA-4).

9. The antibody molecule of any one of claims 1 to 8, which does not bind to human CD28.

10. The antibody molecule of any one of claims 1 to 9, wherein the antibody molecule is selected from the group consisting of a human IgG antibody, a humanized IgG antibody, and an IgG antibody of human origin.

11. The antibody molecule of any one of claims 1 to 10, wherein the antibody molecule is a human IgG1 antibody.

12. The antibody molecule of any one of claims 1 to 11, wherein the antibody molecule is a monoclonal antibody.

13. An isolated polynucleotide encoding an antibody molecule according to any one of claims 1 to 12.

14. 14. The isolated polynucleotide of claim 13, comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 47-48.

15. A plasmid comprising the polynucleotide of claim 13 or 14.

16. 16. A virus comprising a polynucleotide according to claim 13 or 14, or a plasmid according to claim 15.

17. The virus described in claim 16, wherein the virus is an oncolytic virus.

18. The virus of claim 16, wherein the virus is an oncolytic poxvirus.

19. 19. The virus of claim 18, wherein the oncolytic poxvirus belongs to the subfamily Chordopoxviridae.

20. The virus of claim 18, wherein the oncolytic poxvirus belongs to the Orthopoxvirus genus and is selected from the group consisting of vaccinia virus, cowpox virus, canarypox virus, ectromelia virus and myxoma virus.

21. The virus of claim 17, wherein the oncolytic virus is a vaccinia virus defective in both thymidine kinase (TK) and / or ribonucleotide reductase (RR) activity and comprises a nucleotide sequence encoding SEQ ID NO:27 and SEQ ID NO:28, or SEQ ID NO:53 and SEQ ID NO:

54.

22. 22. The virus of claim 21, wherein the oncolytic vaccinia virus further comprises a nucleotide sequence encoding GM-CSF.

23. The virus of claim 21, wherein the oncolytic vaccinia virus further comprises a nucleotide sequence encoding human GM-CSF or mouse GM-CSF.

24. The virus of claim 21, wherein the oncolytic vaccinia virus further comprises a nucleotide sequence encoding a human GM-CSF having SEQ ID NO:55 or SEQ ID NO:56; or a murine GM-CSF having SEQ ID NO:57 or SEQ ID NO:

58.

25. 25. The virus according to any one of claims 16 to 24, wherein the cassette encoding the heavy chain is inserted into the J2R locus and the cassette encoding the light chain is inserted into the I4L locus.

26. A cell comprising a polynucleotide according to claim 13 or 14, or a plasmid according to claim 15, or a virus according to any one of claims 16 to 25.

27. An antibody molecule according to any one of claims 1 to 12, a polynucleotide according to claim 13 or 14, a plasmid according to claim 15, a virus according to any one of claims 16 to 25 or a cell according to claim 26 for use in medicine.

28. An antibody molecule according to any one of claims 1 to 12, a polynucleotide according to claim 13 or 14, a plasmid according to claim 15, a virus according to any one of claims 16 to 25 or a cell according to claim 26 for use in the treatment of cancer.

29. Use of an antibody molecule according to any one of claims 1 to 12, a polynucleotide according to claim 13 or 14, a plasmid according to claim 15, a virus according to any one of claims 16 to 25, or a cell according to claim 26 for the manufacture of a pharmaceutical composition for use in the treatment of cancer.

30. A pharmaceutical composition comprising or consisting of an antibody molecule according to any one of claims 1 to 12, a polynucleotide according to claims 13 or 14, a plasmid according to claim 15, a virus according to any one of claims 16 to 25 or a cell according to claim 26, and optionally a pharma- ceutically acceptable diluent, carrier, vehicle and / or excipient.

31. 31. The pharmaceutical composition of claim 30 for use in the treatment of cancer.

32. A pharmaceutical composition for the treatment of cancer comprising a therapeutically effective amount of an antibody molecule according to any one of claims 1 to 12, a polynucleotide according to claim 13 or 14, a plasmid according to claim 15, a virus according to any one of claims 16 to 25, or a cell according to claim 26.

33. 33. The antibody molecule for use according to claim 28, the polynucleotide for use according to claim 28, the plasmid for use according to claim 28, the virus for use according to claim 28, the cell for use according to claim 28, or the pharmaceutical composition according to claim 31 or 32, wherein the cancer is a solid cancer.

34. The use of claim 29, wherein the cancer is a solid cancer.

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