Novel antibodies and nucleotide sequences and uses thereof

Abstract

The present disclosure relates to novel antibodies and nucleotide sequences and uses thereof. In particular, the present invention describes novel anti-CTLA-4 antibody molecules and nucleotide sequences as well as expression vectors encoding such antibody molecules, such as viruses. The novel antibody molecules are Treg depleting antibody molecules, and they have an improved depletion effect on CTLA-4 positive cells, such as Treg, as compared to Ipimumab. The use of such antibody molecules or nucleotide sequences or viruses in medicine, for example in the treatment of cancer, such as solid tumors, is also described.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 2019800559149, application date September 3, 2019, and invention name “Novel antibodies and nucleotide sequences and their uses”. Technical Field

[0002] The present invention relates to novel anti-CTLA-4 antibody molecules, nucleotide sequences and expression vectors (e.g., oncolytic viruses) encoding such antibody molecules for use in cancer treatment. Compared with ipilimumab, the novel antibodies have improved Treg depletion. Background Art

[0003] 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 co-stimulatory protein CD28, and both CTLA-4 and CD28 bind to CD80 (also known as B7-1) and CD86 (also known as B7-2). CTLA4 is also present in regulatory T cells (Tregs) and contributes to their inhibitory function. The CTLA-4 protein contains an extracellular V domain, a transmembrane domain, and a cytoplasmic tail.

[0004] Antibodies that block the interaction of CTLA-4 with its ligands B7.1 and B7.2 can enhance immune responses and have been shown to stimulate potent anti-tumor immunity (Korman et al. 2006, Checkpoint blockade in cancer immunotherapy, Adv Immunol. 90:297-339).

[0005] Promising clinical results with immunomodulatory monoclonal antibodies (mAbs) have revived confidence that the immune system is key to controlling cancer. Recently, the classification of these mAbs as checkpoint blockers (antagonists) or activators of co-stimulatory molecules (agonists) has been questioned, with the discovery that examples of both types can fight tumors through depletion of suppressive regulatory T cells (Tregs).

[0006] Immunomodulatory mAbs, such as ipilimumab and other anti-CTLA4 antibodies, have shown positive results in trials in difficult-to-treat malignancies, albeit in a small number of patients (Hodi, FS et al. 2010, NEJM. N Engl J Med) 363(8):711-723; Beatty, GL et al. 2011, Science ( Science 331(6024):1612-1616; Brahmer, JR et al. 2012, NEJM 366(26):2455-2465; Topalian, SL et al. 2012 NEJM 366(26):2443-2454). These promising results help to reinvigorate the idea that the immune system may be key to controlling cancer. These mAbs are generated to target key molecular regulators on T cells or antigen presenting cells (APCs) and enhance anti-cancer immunity by blocking inhibitory signals (checkpoint blockers) or delivering co-stimulatory signals (agonists). This binary classification was recently questioned when it was discovered that the therapeutic activity of anti-CTLA4, anti-GITR, and anti-OX40 antibodies, all of which target T cells, involves depletion of suppressive CD4+ T regulatory cells that is dependent on the co-engagement of activating FcγRs (Bulliard, Jolicoeur et al. 2013, Marabelle, A. et al. 2013, Journal of Clinical Investigation JClinInvest) 123(6):2447-2463; Simpson, TR, et al. Journal of Experimental Medicine ( J Exp Med)》 210(9):1695-1710).

[0007] Monoclonal CTLA-4 antibody, ipilimumab ( Previously denoted as 10D1, BMS-734016, MDX101, MDX-010, MDX-CTLA-4, and MDX-CTLA4), it has been approved in several countries for the treatment of melanoma and is in 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. It is available in 477202-00-9 and 6T8C155666. Ipilimumab is further defined in US9 789 182, which also lists the sequences of the heavy and light chains of Ipilimumab (as SEQ ID NOs: 17 and 18), the sequences of the VH and / or VL regions (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).

[0008] A second fully human monoclonal anti-CTLA-4 antibody that has been 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. =Oncol., 37(5):473-484.; Blank et al. 2015, Therapeutic use of anti-CTLA-4 antibodies. International Immunology, 27(1):3-10).

[0009] Anti-CTLA-4 antibodies have been described in several patent applications and patents, including the following.

[0010] WO 93 / 00431 relates to CTLA4 receptor proteins, to CTLA4Ig fusion proteins, and to methods of modulating cellular interactions using such fusion proteins or monoclonal antibodies.

[0011] WO 97 / 20574 relates to blocking T lymphocyte downregulation associated with CTLA-4 signaling and to CTLA-4 blockers other than CTLA-4 extracellular domain antibodies that increase mammalian T cell responses to antigenic stimulation or reduce the growth of tumor cells in a mammalian host.

[0012] WO 00 / 37504 relates to human anti-CTLA-4 antibodies and the use of such antibodies in the treatment of cancer. WO 00 / 37504 further relates to the above-mentioned human monoclonal antibody tesilinumab, which is denoted as 11.2.1 in this patent application.

[0013] WO 01 / 14424 also relates to human antibodies that specifically bind to human CTLA-4 and to their use in treating human diseases and infections, such as cancer. WO 01 / 14424 further relates to the human monoclonal antibody ipilimumab, referred to above and discussed further below, which is denoted 10D1 in this patent application. Summary of the invention

[0014] The present invention relates to antibody molecules that specifically bind to CTLA-4 and have an improved depletion effect on CTLA-4 positive cells compared to ipilimumab.

[0015] In addition, the present invention relates to an antibody molecule that specifically binds to CTLA-4, wherein the antibody molecule comprises 6 CDRs having SEQ ID. NO: 15, 16, 17, 10, 18 and 19 or 6 CDRs having SEQ ID. NO: 22, 23, 24, 10, 25 and 26.

[0016] Furthermore, the present invention relates to isolated nucleotide sequences encoding the above antibody molecules.

[0017] Furthermore, the present invention relates to a plasmid comprising the above nucleotide sequence.

[0018] In addition, the present invention relates to a virus comprising the above nucleotide sequence or the above plasmid, such as an oncolytic virus.

[0019] In addition, the present invention relates to a cell comprising the above nucleotide sequence or the above plasmid, such as a CAR T cell.

[0020] Furthermore, the present invention relates to the above antibody molecules, nucleotide sequences, plasmids and / or cells for use in medicine.

[0021] In addition, the present invention relates to the above antibody molecules, nucleotide sequences, plasmids, viruses and / or cells for use in treating cancer.

[0022] In addition, the present invention relates to the use of the above antibody molecules, nucleotide sequences, plasmids, viruses and / or cells for the manufacture of pharmaceutical compositions for the treatment of cancer.

[0023] In addition, the present invention relates to a pharmaceutical composition comprising at least one of the above antibody molecules, nucleotide sequences, plasmids, viruses and / or cells, and optionally a pharmaceutically acceptable diluent, carrier or excipient.

[0024] In addition, the present invention relates to a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of at least one of the above antibody molecules, nucleotide sequences, plasmids, viruses, cells and / or pharmaceutical compositions.

[0025] In addition, the present invention relates to antibody molecules, antibody molecules for use, isolated nucleotide sequences, isolated nucleotide sequences for use, plasmids, plasmids for use, viruses, viruses for use, cells, cells for use, uses, pharmaceutical compositions or methods of treatment as described herein with reference to the detailed description, examples and / or figures. DETAILED DESCRIPTION

[0026] CTLA-4 positive cells include regulatory T cells, Treg cells, Treg or T reg (formerly known as suppressor T cells and sometimes referred to as suppressor regulatory T cells), which are a subset of T cells that are able to suppress other immune cells in normal and pathological immune environments. Tregs are CD4-positive cells (CD4+ cells). There are other CD4+ T cells that are not Tregs; however, Tregs can be separated from non-Treg CD4+ cells because Tregs are also FOXP3-positive (FOXP3+), while non-Treg CD4+ cells are FOXP3-negative (FOXP3-).

[0027] Like ipilimumab, the anti-CTLA-4 antibody molecules described herein act at least in part by depleting CTLA-4 positive cells, such as Tregs. In addition, like ipilimumab, the anti-CTLA-4 antibody molecules described herein block the interaction of CTLA-4 with B7.1 and B7.2. Therefore, these antibodies can therefore help overcome the inhibitory effect of CTLA-4 induced on the proliferation of effector T cells.

[0028] The consumption of Treg or Treg consumption refers to the consumption, deletion or elimination of Treg by physical removal of cells in this article. Specifically, it refers to the consumption of Treg in tumors. The consumption of Treg can be achieved by ADCC, i.e., antibody-dependent cell-mediated cytotoxicity or antibody-dependent cellular toxicity, and / or ADCP, i.e., antibody-dependent cellular phagocytosis. This means that when the antibody molecule as described herein is administered to a subject such as a human, it specifically binds to CTLA-4 expressed on the surface of Treg, and this combination leads to the consumption of Treg. In some embodiments, CTLA-4 is preferentially expressed on tumor infiltrating lymphocytes in the tumor microenvironment or on tumor cells.

[0029] ADCC is an immune mechanism by which effector cells bearing Fc receptors can recognize and kill antibody-coated target cells that express tumor-derived antigens (i.e., in the present case, CTLA-4) on their surface. ADCP is a similar mechanism, although it results in the target cell being killed by phagocytosis rather than cytotoxicity.

[0030] Antibodies are well known to those skilled in the art of immunology and molecular biology. Typically, antibodies include two heavy (H) chains and two light (L) chains. In this article, such complete antibody molecules are sometimes referred to as full-size or full-length antibodies. The heavy chain of an antibody includes a variable domain (VH) and three constant domains (CH1, CH2, and CH3), and the light chain of an antibody molecule includes a variable domain (VL) and a constant domain (CL). The variable domains (sometimes collectively referred to as F V The constant domain is a region that binds to the target or antigen of the antibody. Each variable domain includes three loops, which are called complementary determining regions (CDRs), which are responsible for target binding. The constant domain is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions. According to the amino acid sequence of the heavy chain constant region of the antibody or immunoglobulin, the antibody or immunoglobulin can be divided into different categories. Immunoglobulins are mainly divided into five categories: IgA, IgD, IgE, IgG and IgM, and in humans, several of these categories are further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3 and IgG4; IgA1 and IgA2. Another part of the antibody is the Fc domain (also called the fragment crystallizable domain), which includes two constant domains in the constant domain of each antibody heavy chain. The Fc domain is responsible for the interaction between the antibody and the Fc receptor.

[0031] Fc receptors are membrane proteins that are typically found on the cell surface of cells of the immune system (i.e., Fc receptors are found on the target cell membrane—also known as the plasma membrane or cytoplasmic membrane). The role of Fc receptors is to bind antibodies via the Fc domain and internalize the antibodies into the cell. In the immune system, this can lead to antibody-mediated phagocytosis and antibody-dependent cell-mediated cytotoxicity.

[0032] 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.

[0033] A functional fragment of a full-size antibody has the same antigen binding properties as the corresponding full-size antibody and contains the same variable domains (i.e., VH and VL sequences) and / or the same CDR sequences as the corresponding full-size antibody. The functional fragment having the same antigen binding properties as the corresponding full-size antibody means that the functional fragment binds to the same epitope on the target as the full-size antibody. Such a functional fragment may correspond to the Fv portion of the full-size antibody. Alternatively, such a fragment may be a Fab, also known as a monovalent antigen binding fragment F(ab) or a divalent antigen binding fragment F(ab') 2The monovalent antigen-binding fragment does not contain an Fc portion and the bivalent antigen-binding fragment contains a disulfide bond or F(ab') (i.e., F(ab') 2 Such fragments can also be single chain variable fragments (scFv).

[0034] Functional fragments do not always contain all six CDRs of the corresponding full-size antibody. It should be understood that molecules containing three or fewer CDR regions (in some cases, even just a single CDR or a portion thereof) can retain the antigen-binding activity of the antibody from which one or more CDRs are derived. For example, in Gao et al., 1994, Journal of Biological Chemistry (J. Biol. Chem.), 269: 32389-93, it is described that the entire VL chain (including all three CDRs) has a high affinity for its substrate.

[0035] Molecules containing two CDR regions are described in the following literature: for example, Vaughan and Sollazzo 2001, Combinatorial Chemistry & High Throughput Screening, 4: 417-430. On page 418 (right column - 3 Our design strategy), minibodies containing only H1 and H2 CDR hypervariable regions interspersed within the framework region are described. The minibodies are described as being able to bind to the target. Vaughan and Sollazzo cite the following literature: Pessi et al., 1993, Nature, 362: 367-9 and Bianchi et al., 1994, J. Mol. Biol., 236: 649-59, which describe the H1 and H2 minibodies and their properties in more detail. In Qiu et al., 2007, Nature Biotechnology, 25:921-9, it was shown that molecules consisting of two linked CDRs can bind antigen. Quiocho 1993, Nature, 362:293-4 provides a summary of "miniantibody" technology. Ladner 2007, Nature Biotechnology, 25:875-7 indicates that molecules containing two CDRs can retain antigen binding activity.

[0036] Antibody molecules containing a single CDR region are described in the following literature: for example, Laune et al., 1997, Journal of Biological Chemistry (JBC), 272: 30937-44, where a series of hexapeptides derived from CDRs were shown to exhibit antigen binding activity, and synthetic peptides of intact single CDRs were noted to exhibit strong binding activity. In Monnet et al., 1999, Journal of Biological Chemistry, 274: 3789-96, a series of 12-mer peptides and associated framework regions were shown to have antigen binding activity, and it was noted that only CDR3-like peptides were able to bind antigen. In Heap et al., 2005, Journal of General Virology (J. Gen. Virol.), 86: 1791-1800, it was reported that "miniantibodies" (molecules containing a single CDR) were able to bind antigens, and cyclic peptides from anti-HIV antibodies were shown to have antigen binding activity and function. In Nicaise et al., 2004, Protein Science, 13: 1882-91, it was shown that a single CDR can confer antigen binding activity and affinity for its lysozyme antigen.

[0037] Thus, an antibody molecule with five, four, three or fewer CDRs is able to retain the antigen-binding properties of the full-length antibody from which the CDRs are derived.

[0038] The antibody molecule may also be a derivative of a full-length antibody or a fragment of such an antibody. The derivative has the same antigen binding properties as the corresponding full-size antibody because the derivative binds to the same epitope on the target as the full-size antibody.

[0039] Therefore, as used herein, the term "antibody molecule" includes all types of antibody molecules and functional fragments and derivatives thereof, including: monoclonal antibodies, polyclonal antibodies, synthetic antibodies, recombinantly produced antibodies, multispecific antibodies, bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, variable fragments, single-chain variable fragments including divalent single-chain variable fragments (di-scFv) and disulfide-linked variable fragments (scFv fragments), Fab fragments, F(ab') fragments, 2 Fragments, Fab' fragments, antibody heavy chains, antibody light chains, homodimers of antibody heavy chains, homodimers of antibody light chains, heterodimers of antibody heavy chains, heterodimers of antibody light chains, and antigen-binding functional fragments of such homodimers and heterodimers.

[0040] 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.

[0041] In some embodiments, the antibody is human IgG1. The technician understands that mouse IgG2a and human IgG1 effectively bind to activating Fcγ receptors and can activate the deletion of target cells by activating activating Fcγ receptors with immune cells (e.g., macrophages and NK cells) with, for example, ADCP and ADCC. Therefore, although mouse IgG2a is a preferred isotype for deletions in mice, human IgG1 is a preferred isotype for deletions in humans. On the contrary, it is known that the best costimulation of TNFR superfamily agonist receptors (e.g., 4-1BB, OX40, TNFRII, CD40) depends on the involvement of inhibitory FcγRII antibodies. In mice, it is known that the IgG1 isotype, which preferentially binds to inhibitory Fcγ receptors (FcγRIIB) and only weakly binds to activating Fcγ receptors, is the best choice for the costimulatory activity of TNFR superfamily targeting mAbs. Although no direct equivalent of the mouse IgG1 isotype has been described in humans, antibodies can be engineered to exhibit similar enhanced binding to inhibitory human Fcγ receptors compared to activating human Fcγ receptors. In transgenic mice engineered to express both human activating and inhibitory Fcγ receptors, the costimulatory activity of such engineered TNFR superfamily targeting antibodies in vivo was also improved (1 Dahan et al., 2016, Therapeutic Activity of Agonistic, Human Anti-CD40 Monoclonal Antibodies Requires Selective FcγR Engagement. Cancer Cell. 29(6):820-31).

[0042] As mentioned above, different types and forms of antibody molecules are encompassed by the present invention and are known to those skilled in the art of immunology.It is well known that antibodies used for therapeutic purposes are often modified with additional components that modify the properties of the antibody molecule.

[0043] Thus, it is encompassed that the antibody molecule of the invention or the antibody molecule used according to the invention (eg, a monoclonal antibody molecule and / or a polyclonal antibody molecule and / or a bispecific antibody molecule) comprises a detectable moiety and / or a cytotoxic moiety.

[0044] A "detectable moiety" comprises one or more substances from the group consisting of: an enzyme; a radioactive atom; a fluorescent moiety; a chemiluminescent moiety; a bioluminescent moiety. The detectable moiety allows the antibody molecule to be visualized in vitro and / or in vivo and / or ex vivo.

[0045] A "cytotoxic moiety" comprises a radioactive moiety and / or an enzyme, eg, wherein the enzyme is a caspase, and / or a toxin, eg, wherein the toxin is a bacterial toxin or venom; wherein the cytotoxic moiety is capable of inducing cell lysis.

[0046] It is further comprised that the antibody molecule may be in isolated form and / or purified form, and / or may be pegylated.

[0047] As discussed above, the CDRs of an antibody bind to an antibody target. The amino acid assignments for each CDR described herein conform to the definition according to Kabat EA et al. 1991, "Sequences of Proteins of Immulogical Interest" Fifth Edition, NIH Publication No.: 91-3242, pp. xv-xvii.

[0048] As the skilled person will appreciate, there are other methods for assigning amino acids to each CDR, for example, the International Immunogenetics Information System (IMGT(R)) (http: / / www.imgt.org / , and Lefranc and Lefranc "The Immunoglobulin Facts Book", published by Academic Press, 2001).

[0049] In another embodiment, the antibody molecule of the present invention or used according to the present invention is an antibody molecule capable of competing with the specific antibodies described herein, such as an antibody molecule comprising SEQ ID. NO: 15, 16, 17, 10, 18 and 19 or SEQ ID. NO: 22, 23, 24, 10, 25 and 26.

[0050] "Capable of competing" means that the competing antibody is able to at least partially inhibit or otherwise interfere with the binding of an antibody molecule as defined herein to a specific target.

[0051] For example, such a competing antibody molecule may be capable of inhibiting the 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 inhibiting the ability of an antibody described herein to prevent or reduce binding to a specific target 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%).

[0052] Competitive binding can be determined by methods well known to those skilled in the art, such as enzyme-linked immunosorbent assay (ELISA).

[0053] ELISA assays can be used to assess epitope modifications or blocking antibodies. Additional methods suitable for identifying competitive antibodies are disclosed in the following document: Antibodies: A Laboratory Manual, Harlow and Lane, which is incorporated herein by reference (e.g., see pages 567 to 569, 574 to 576, 583, and 590 to 612, 1988, CSHL, New York, ISBN 0-87969-314-2).

[0054] It is well known that antibodies specifically bind to a defined target molecule or antigen, and this means that the antibody preferentially and selectively binds to its target rather than to non-target molecules.

[0055] The target CTLA-4 of the antibody according to the invention or the target CTLA-4 of the antibody used according to the invention is expressed on the cell surface, i.e. the target is a cell surface antigen, which will comprise the epitope of the antibody (also referred to as cell surface epitope in this context). Cell surface antigens and epitopes are terms that are easily understood by those skilled in the art of immunology or cell biology.

[0056] "Cell surface antigen" comprises a cell surface antigen exposed by an antibody molecule described herein or at least an epitope thereof is on the extracellular side of the cell membrane.

[0057] Methods for assessing protein binding are known to those skilled in the art in the fields of biochemistry and immunology. It will be appreciated by those skilled in the art that those methods can be used to assess the binding of an antibody to a target and / or the binding of an antibody's Fc domain to an Fc receptor; and relative strength or specificity, or the inhibition, prevention or reduction of these interactions. Examples of methods that can be used to assess protein binding are, for example, immunoassays, BIAcore, Western blots, radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA) (for discussion of antibody specificity, see Fundamental Immunology, 2nd edition, Raven Press, New York; pp. 332-336 (1989)).

[0058] Therefore, "antibody molecules that specifically bind to CTLA-4" and "anti-CTLA-4 antibody molecules" herein both refer to antibody molecules that specifically bind to target CTLA-4 but do not bind to non-targets, or bind to non-targets much weaker than the target (e.g., with lower affinity).

[0059] 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.

[0060] In some embodiments, the antibody molecule (or anti-CTLA-4 antibody molecule) that specifically binds to CTLA-4 does not cross-react with CD28. In some embodiments, the antibody molecule (or anti-CTLA-4 antibody molecule) that specifically binds to CTLA-4 blocks the binding of CTLA-4 to CD80 and / or CD86, thereby inhibiting CLTA-4 signaling.

[0061] It also includes the following meanings: the strength of the antibody's specific binding to the target CTLA-4 is at least two times, or at least five times, or at least 10 times, or at least 20 times, or at least 50 times, or at least 100 times, or at least 200 times, or at least 500 times, or at least about 1000 times stronger than the strength of the antibody's specific binding to a non-target.

[0062] In addition, the following meanings are included: if the antibody is 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 Kd, 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 If the antibody specifically binds to the target CTLA-4, then the antibody specifically binds to the target CTLA-4.

[0063] As mentioned above, the antibody molecules that specifically bind to CTLA-4 (or anti-CTLA-4 antibody molecules) described herein have improved depletion of CTLA-4 positive cells compared to ipilimumab.

[0064] That the antibody molecule has a depleting effect on CTLA-4-positive cells means that upon administration 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 such binding results in the depletion of such cells.

[0065] In some embodiments, the CTLA-4 positive cells are CD4 positive (CD4+) cells, ie, cells that express CD4.

[0066] In some embodiments, CTLA-4 positive cells are both CD4 positive and FOXP3 positive, i.e., express CD4 and FOXP3. These cells are Tregs. CD8 positive T cells also express CTLA-4, but the CDLA-4 level expressed by Tregs is significantly higher than that of CD8 positive T cells. Compared with CD8+ cells with lower expression, this makes Tregs more easily consumed.

[0067] In some instances, CTLA-4 is preferentially expressed on immune cells in the tumor microenvironment (tumor infiltrating cells, TILS).

[0068] Therefore, in the tumor microenvironment, Tregs will be the cells with the highest CTLA-4 expression, resulting in an antibody molecule (or anti-CTLA-4 antibody molecule) that specifically binds to CTLA-4 having a Treg depletion effect. This is described below, for example in Example 4 and in combination with Fig.13 Discuss in more detail.

[0069] In some embodiments, CTLA-4 positive cells will be Tregs in solid tumors. Such Tregs will have very high CTLA-4 expression, and therefore administration of an antibody molecule that specifically binds to CTLA-4 will preferentially lead to the depletion of such Tregs.

[0070] As mentioned above, the anti-CTLA-4 antibody molecules described herein are Treg-depleting antibody molecules, which means that upon administration to a subject, such as a human, such antibody molecules specifically bind to CTLA-4 expressed on the surface of Tregs, and such binding results in Treg depletion.

[0071] To determine whether an antibody molecule is one that has improved depletion of 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.

[0072] In vitro ADCC assay was performed using NK-92 cell line stably transfected to express CD16-158V allele and GFP, wherein the ADCC assay included the following seven consecutive steps:

[0073] 1) Isolate CTLA-4 positive cells, CD4 positive cells or Treg as target cells from the peripheral blood of healthy donors. This separation can be performed using a CD4+ T cell isolation kit (such as a commercial kit from Miltenyi Biotec).

[0074] 2) Then use CD3 / CD28, e.g. and rhIL-2, such as 50 ng / ml rhIL-2, to stimulate the target cells, for example, for 48 hours. The stimulation can be performed at 37°C.

[0075] 3) The target cells are then pre-incubated with the antibody molecule to be tested, for example at 10 μg / ml at 4° C. for 30 minutes, and then mixed with the NK cells.

[0076] 4) The target cells are then incubated in RPMI 1640+GlutaMAX medium containing HEPES buffer, sodium pyruvate and FBS low IgG for an appropriate time, such as 4 hours. RPMI 1640+GlutaMAX medium may contain 10 mM HEPES buffer, 1 mM sodium pyruvate and 10% FBS low IgG, and the ratio of effector to target cells may be 2:1.

[0077] 5) Determination of lysis by flow cytometry.

[0078] 6) Repeat or perform steps 1-5 in parallel, wherein ipilimumab is used instead of the antibody molecule tested in step 3.

[0079] 7) Compare 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, depending on which target cells are used.

[0080] In some embodiments, the improved consumption in step 7) above is a significantly improved consumption.

[0081] Combination Fig.12 , which assay is shown in more detail in Example 4 below.

[0082] The in vivo test is based on the combined use of PBMC mice and NOG / SCID mice, which is referred to herein as the PBMC-NOG / SCID model. Both PBMC mice and NOG / SCID mice are well-known models. The in vivo test in the PBMC-NOG / SCID model includes the following nine consecutive steps:

[0083] 1) Human PBMC (peripheral blood mononuclear cells) were isolated, washed and resuspended in sterile PBS. In some embodiments, PBMC were cultured at 75×10 6 Resuspend cells / ml in PBS.

[0084] 2) Inject an appropriate amount (e.g. 200 μl) of the cell suspension from step 1) intravenously (iv) into NOG mice. If 200 μl is injected, this corresponds to 15×10 6 cells / mouse.

[0085] 3) At an appropriate time after injection, such as 2 weeks, spleens from NOG mice are isolated and made into single cell suspensions. Optionally, a small sample is taken from the single cell suspension to determine the expression of CTLA-4 on human T cells by FACS to confirm CTLA-4 expression.

[0086] 4) Resuspend the cell suspension from step 3) in sterile PBS. In some embodiments, the cell suspension is resuspended at 50×10 6 Resuspend at 100 cells / ml in sterile PBS. If the optional CTLA-4 expression determination was included in step 3, the remainder of the cell suspension will be resuspended in step 4.

[0087] 5) Inject an appropriate amount (e.g. 200 μl) of the suspension from step 4 intraperitoneally (IP) into SCID mice. If 200 μl is injected, this corresponds to 10×10 6 cells / mouse.

[0088] 6) At a suitable time, such as 1 hour, after the injection in step 5), the SCID mice are treated with an appropriate amount (such as 10 mg / kg) of the antibody molecule to be tested, ipilimumab or an isotype control monoclonal antibody.

[0089] 7) At an appropriate time, such as 24 hours after the treatment in step 6), collect the intraperitoneal fluid of the treated SCID mice.

[0090] 8) Identify and quantify human T cell subsets by FACS using the following markers: CD45, CD4, CD8, CD25 and / or CD127.

[0091] 9) The identification and quantification results of T cell subsets from mice treated with the tested antibody molecule are compared with the identification and quantification results of T cell subsets from mice treated with ipilimumab and the identification and quantification results of T cell subsets from mice treated with an isotype control monoclonal antibody. Compared with the number of CTLA-4 positive cells in the peritoneal fluid of mice treated with ipilimumab, the number of CTLA-4 positive cells in the peritoneal fluid of mice treated with the antibody molecule to be tested is less, indicating that the antibody molecule has an improved depletion effect on CTLA-4 positive cells compared with ipilimumab. Compared with the number of CD4 positive cells in the peritoneal fluid of mice treated with ipilimumab, the number of CD4 positive cells in the peritoneal fluid of mice treated with the antibody molecule to be tested is less, indicating that the antibody molecule has an improved depletion effect on CD4 positive cells compared with ipilimumab. The number of Tregs in the peritoneal fluid of mice treated with the antibody molecule to be tested was lower than the number of Tregs in the peritoneal fluid of mice treated with ipilimumab, indicating that the antibody molecule has an improved depletion effect on Tregs compared to ipilimumab.

[0092] In this in vivo assay, in some embodiments, it is of greatest interest to observe Treg depletion in step 7.

[0093] Combination Fig.14 , which assay is shown in more detail in Example 4 below.

[0094] As known to the skilled artisan, Treg depletion can also be assessed in an antibody-dependent cellular phagocytosis (ADCP) assay.

[0095] In some embodiments, the antibody molecule has a similar blocking effect on the interaction of CTLA-4 with B7.1 and B7.2 ligands as Yervoy. This can be measured by ELISA (e.g. Fig.10 ) or in 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.

[0096] In some embodiments, the anti-CTLA-4 antibody molecule is a human antibody molecule.

[0097] In some embodiments, the anti-CTLA-4 antibody molecule is a humanized antibody molecule.

[0098] In some embodiments, the anti-CTLA-4 antibody molecule is a human antibody molecule, meaning that it is derived from a human antibody molecule that was subsequently modified.

[0099] In some embodiments, the anti-CTLA-4 antibody molecule is a human IgG1 antibody.

[0100] In some embodiments, the anti-CTLA-4 antibody is an antibody in the form of a human IgG1 antibody that exhibits improved binding to one or several activating Fc receptors and / or is engineered for improved binding to one or several activating Fc receptors; thus, in some embodiments, the anti-CTLA-4 antibody is an Fc-engineered human IgG1 antibody.

[0101] In some embodiments, the anti-CTLA-4 antibody is a murine or humanized murine IgG2a antibody.

[0102] In some embodiments, the anti-CTLA-4 antibody is a murine antibody that cross-reacts with human CTLA-4.

[0103] In some embodiments, the anti-CTLA-4 antibody is a monoclonal antibody.

[0104] In some embodiments, the anti-CTLA-4 antibody is a polyclonal antibody.

[0105] In some embodiments, the anti-CTLA-4 antibody molecule is an antibody molecule comprising 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 presented in Table 1 below.

[0106] In some embodiments, the anti-CTLA-4 antibody molecule is selected from the group consisting of antibody molecules comprising 1 to 6 of the CDRs selected from the group consisting of SEQ ID. Nos: 3, 6, 8, 10, 12 and 14.

[0107] 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.

[0108] In some embodiments, the anti-CTLA-4 antibody molecule is selected from the group consisting of antibody molecules comprising 1 to 6 of the CDRs VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, and VL-CDR3,

[0109] wherein VH-CDR1, if present, is selected from the group consisting of SEQ ID.No: 15, 22, 29 and 35;

[0110] wherein VH-CDR2, if present, is selected from the group consisting of SEQ ID.No: 16, 23, 30 and 36;

[0111] wherein VH-CDR3, if present, is selected from the group consisting of SEQ ID.No: 17, 24, 31 and 37;

[0112] wherein VL-CDR1, if present, is selected from the group consisting of SEQ ID.No: 10 and 38;

[0113] wherein VL-CDR2, if present, is selected from the group consisting of SEQ ID.No: 18, 25, 32 and 39;

[0114] Wherein VL-CDR3, if present, is selected from the group consisting of SEQ ID. No: 19, 26 and 40.

[0115] In some embodiments, the anti-CTLA-4 antibody molecule is an antibody molecule selected from the group consisting of an antibody molecule comprising 6 CDRs selected from the group consisting of:

[0116] SEQ ID. NO: 15, 16, 17, 10, 18 and 19;

[0117] SEQ ID. NO: 22, 23, 24, 10, 25 and 26;

[0118] SEQ ID. NO: 29, 30, 31, 10, 32 and 26; and

[0119] SEQ ID. NO: 35, 36, 37, 38, 39 and 40.

[0120] In some embodiments, the anti-CTLA-4 antibody molecule is an antibody molecule comprising 6 CDRs having SEQ ID. NOs: 15, 16, 17, 10, 18 and 19.

[0121] In some embodiments, the anti-CTLA-4 antibody molecule is an antibody molecule comprising 6 CDRs having SEQ ID. NO: 22, 23, 24, 10, 25 and 26.

[0122] In some embodiments, the anti-CTLA-4 antibody molecule is an antibody molecule selected from the group consisting of an antibody molecule comprising a VH selected from the group consisting of: SEQ ID. NO: 20, 27, 33 and 41.

[0123] In some embodiments, the anti-CTLA-4 antibody molecule is an antibody molecule selected from the group consisting of an antibody molecule comprising a VL selected from the group consisting of SEQ ID. NO: 21, 28, 34 and 42.

[0124] In some embodiments, the anti-CTLA-4 antibody molecule is an antibody molecule selected from the group consisting of an antibody molecule comprising a VH and a VL selected from the group consisting of SEQ ID. NOs: 20-21, 27-28, 33-34 and 41-42.

[0125] In some embodiments, the anti-CTLA-4 antibody molecule comprises a VH having the sequence SEQ ID.No:20 and a VL having the sequence SEQ ID.NO:21.

[0126] In some embodiments, the anti-CTLA-4 antibody molecule comprises a VH having the sequence of SEQ ID.No:27 and a VL having the sequence of SEQ ID.No:28.

[0127] Table 1 : General CDR sequences of antibodies disclosed herein

[0128]

[0129]

[0130] Table 2 : Specific anti-CTLA-4 antibody molecule; CDR sequences are marked in bold in the complete VH and VL sequences

[0131]

[0132]

[0133]

[0134]

[0135] In some embodiments, the anti-CTLA-4 antibody molecules described herein may further include one or two of the constant regions presented in Table 3 below.

[0136] Table 3 : Sequences of the constant regions of the antibodies disclosed herein

[0137]

[0138] In some embodiments, the anti-CTLA-4 antibody molecule is a molecule encoded by one of the nucleotide sequences presented in Table 4 below.

[0139] Table 4 : Specific nucleotide sequence encoding anti-CTLA-4 antibody molecules

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146] In some embodiments, it is advantageous that the antibody molecule binds to both human CTLA-4 (hCTLA-4) and rhesus macaque CTLA-4 (cmCTLA-4 or cyno CTLA-4). Cross-reactivity with CTLA-4 expressed on cells in rhesus macaques (also known as cynomolgus macaques or short-tailed macaques) may be advantageous because it enables the antibody molecule to be tested in monkeys without having to use alternative antibodies, which is of particular concern for tolerability.

[0147] In some embodiments, it is advantageous for the antibody molecule to bind to both human CTLA-4 (hCTLA-4) and murine CTLA-4 (mCTLA-4). This may be advantageous because it enables the antibody molecule to be tested in mice, with particular attention to efficacy and pharmacodynamics, without having to use a surrogate antibody.

[0148] In some embodiments, the antibody molecule binds to all three of hCTLA-4, cmCTLA-4, and mCTLA-4.

[0149] In some embodiments, it is necessary to test the functional activity of the antibody molecule in a relevant in vivo model in mice using a surrogate antibody. In order to ensure the comparability between the effect of the antibody molecule in humans and the in vivo results of the surrogate antibody in mice, a functionally equivalent surrogate antibody with the same in vitro properties as the human antibody molecule must be selected.

[0150] In some embodiments, the antibody molecule does not bind human CD28.

[0151] Those skilled in the medical arts will appreciate that drugs can be modified with various additives, for example to alter the rate at which the drug is absorbed by the body; and can be modified in different forms, for example to allow for specific routes of administration to the body.

[0152] Therefore, comprising antibody molecules as described herein, nucleotide sequences, plasmids, viruses and / or cells can be combined into pharmaceutical compositions with pharmaceutically acceptable excipients, carriers, diluents, vehicles and / or adjuvants. In this context, the term pharmaceutical composition can be used interchangeably with the term pharmaceutical preparation, pharmaceutical formulation, therapeutic composition, therapeutic preparation, therapeutic formulation and therapeutic entity.

[0153] The pharmaceutical compositions described herein may include, or in some embodiments consist of, an antibody molecule, a nucleotide sequence, a plasmid, a virus, or a cell.

[0154] In some embodiments, the pharmaceutical composition described herein may consist of or include a plasmid comprising a nucleotide sequence encoding the above-mentioned antibody molecule or comprising the above-mentioned nucleotide sequence.

[0155] In some embodiments, the pharmaceutical composition may include a nucleotide sequence encoding a portion of an antibody molecule described herein that is integrated into a cell or viral genome or in a virion or a complete antibody molecule. Then, the pharmaceutical composition may include a cell or virus as a delivery vehicle for the antibody of the present invention (or a delivery vehicle for a nucleotide sequence encoding an antibody of the present invention). For example, in an embodiment, the virus may be in the form of a therapeutic oncolytic virus, which includes a nucleotide sequence encoding at least one antibody molecule in an antibody molecule described herein. In some embodiments, such an oncolytic virus includes a nucleotide sequence encoding a full-length human IgG antibody. In some embodiments, such an oncolytic virus includes a nucleotide sequence encoding a scFv, Fab or F (ab') 2 The nucleotide sequence of the antibody molecule.

[0156] As described in the appended claims, in an embodiment, the present invention relates to a virus including a nucleotide sequence of the present invention or a plasmid of the present invention. Preferably, the virus is an oncolytic virus, such as a therapeutic oncolytic virus. As used herein, the term "oncolysis" refers to the ability of a virus to selectively replicate in a dividing cell (e.g., a proliferative cell such as a cancer cell), with the purpose of slowing down the growth and / or lysis of the dividing cell in vitro or in vivo, and not showing or showing very little replication in a non-dividing (e.g., normal or healthy) cell." Replication (Replication)" (or any form of replication, such as "replication (replicate)" and "replication (replicating)", etc.) means the replication of the virus, which can occur at the nucleic acid level or preferably at the infectious virus particle level. Such an oncolytic virus can be obtained from any member of the currently identified virus. It can be a natural oncolytic natural virus, or it can be engineered by modifying one or more viral genes, so as to improve tumor selectivity and / or preferentially replicate in dividing cells, such as those involved in DNA replication, nucleic acid metabolism, host tropism, surface attachment, toxicity, lysis and diffusion (for example, see Wong et al., 2010, "Viruses" 2: 78-106). It is also conceivable that one or more viral genes are placed under the control of an event or tissue-specific regulatory element (such as a promoter). Exemplary oncolytic viruses include but are not limited to reovirus, Seneca Valley virus (SVV), vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), herpes simplex virus (HSV), measles virus, adenovirus, poxvirus, retrovirus, measles virus, foamy virus, alpha virus, slow virus, influenza virus, Simbis virus, myxoma virus, rhabdovirus, picornavirus, coxsackie virus, parvovirus, etc. Such viruses are known to technicians in the field of medicine and virology.

[0157] In certain embodiments, such oncolytic viruses are obtained from herpesviruses. Herpesviridae is a large family of DNA viruses, all of which have the same structure and are composed of a relatively large double-stranded linear DNA genome, which encodes 100-200 genes encapsulated in an icosahedral capsid, which is encapsulated in a lipid bilayer membrane. Although oncolytic herpesviruses can be derived from different types of HSV, HSV1 and HSV2 are particularly preferred. Herpesviruses can be genetically modified to limit viral replication in tumors or to reduce their 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 aspect relates to viral mutants that are 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 herpes viruses 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).

[0158] In some embodiments, such oncolytic viruses are obtained from adenoviruses. The method is that the art can be used for engineering oncolytic adenoviruses. A favorable strategy includes replacing viral promoters with tumor-selective promoters or modifying (one or more) E1 adenovirus gene products to inactivate the binding function of p53 or retinoblastoma (Rb) protein that is changed in tumor cells. In the natural environment, the adenovirus E1B55kDa gene and another adenovirus product synergize to inactivate p53 (p53 is often dysregulated in cancer cells), thereby preventing apoptosis. Representative examples of oncolytic adenoviruses include ONYX-015 (e.g., Khuri et al., 2000, "Natural Medicine (Nat.Med)" 6 (8): 879-85) and H101 (Xia et al., 2004, "Cancer (Ai Zheng)" 23 (12): 1666-70) also known as Oncorine.

[0159] In certain embodiments, such oncolytic viruses are poxviruses. As used herein, the term "poxvirus" refers to a virus belonging to the Poxviridae family, particularly preferably a poxvirus belonging to the Chordopoxviridae subfamily, and more preferably a poxvirus belonging to the Orthopoxvirus genus. Vaccinia virus, cowpox virus, canarypox virus, mousepox virus and myxoma virus are particularly suitable in the context of the present invention. The genomic sequence of this type of poxvirus can be obtained in the art and in a special database (e.g., Genbank, accession numbers are NC_006998, NC_003663 or AF482758.2, NC_005309, NC_004105, NC_001132, respectively).

[0160] In a specific and preferred embodiment, such an oncolytic poxvirus is an oncolytic vaccinia virus. Vaccinia virus is a member of the poxvirus family characterized by a 200kb double-stranded DNA genome, which encodes a variety of viral enzymes and factors that enable the virus to replicate independently of the host cell mechanism. Most vaccinia virus particles are intracellular (for intracellular mature virions, IMV) with a single lipid envelope and are retained in the cytosol of infected cells until lysis. Another infectious form is a double-encapsulated particle (for extracellular enveloped virions, EEV), which sprouts from infected cells without lysing them. Although it can be derived from any vaccinia virus strain, Elstree, Wyeth, Copenhagen, Lister and Western Reserve strains are particularly preferred. Unless otherwise indicated, the gene nomenclature used herein is the nomenclature of the Copenhagen vaccinia strain. However, the correspondence between Copenhagen and other vaccinia strains is usually available in the literature.

[0161] Preferably, such oncolytic vaccinia virus is modified by changing one or more viral genes. The (one or more) modifications preferably result in the lack of synthesis or synthesis of defective viral proteins, and the defective viral proteins cannot ensure the activity of the protein produced by the unmodified gene under normal conditions. Exemplary modifications are disclosed in the literature, and the purpose is to change the viral genes involved in DNA metabolism, host virulence, IFN pathways (for example, Guse et al., 2011, "Expert Opinion Biol. Ther. " 11 (5): 595-608) etc. The modification for changing the viral locus covers the deletion, mutation and / or substitution of one or more nucleotides (continuous or discontinuous) in the viral gene or its regulatory element. Conventional recombinant techniques can be used to carry out (one or more) modifications in a variety of ways known to those skilled in the art.

[0162] More preferably, such oncolytic vaccinia virus is modified by changing the thymidine kinase encoding gene (locus J2R). The thymidine kinase (TK) enzyme is involved in the synthesis of deoxyribonucleotides. TK is required for viral replication in normal cells because these cells usually have low concentrations of nucleotides, while it is dispensable in dividing cells containing high nucleotide concentrations.

[0163] As an alternative or combination of oncolytic vaccinia virus, such oncolytic vaccinia virus is modified by changing at least one or two genes encoding ribonucleotide reductase (RR). In the natural environment, the enzyme catalyzes the reduction of ribonucleotides to deoxyribonucleotides, which is a key step in DNA biosynthesis. The subunit structure of the viral enzyme is similar to that of mammalian enzymes, consisting of two heterologous subunits, and the design of R1 and R2 encoded by I4L and F4L loci, respectively. In the context of the present invention, I4L gene (encoding R1 large subunit) or F4L gene (encoding R2 small subunit) or both can be inactivated (e.g., as described in WO2009 / 065546 and Foloppe et al., 2008, "Gene Ther.)", 15: 1361-71). The sequences of J2R, I4L and F4L genes and their positions in various poxvirus genomes are available in public databases.

[0164] In some embodiments, such oncolytic viruses include nucleotide sequences encoding amino acid sequences having at least 80% identity with the sequences listed in Table 2 above. In some embodiments, such oncolytic viruses include amino acid sequences having at least 85% identity with the sequences listed in Table 2 above. In some embodiments, such oncolytic viruses include amino acid sequences having at least 90% identity with the sequences listed in Table 2 above. In some embodiments, such oncolytic viruses include amino acid sequences having at least 95% identity with the sequences listed in Table 2 above.

[0165] In some embodiments, such oncolytic viruses include nucleotide sequences encoding SEQ.ID.NO:20 and ID.NO:21. In some embodiments, such oncolytic viruses include nucleotide sequences encoding SEQ.ID.NO:27 and ID.NO:28. In some embodiments, such oncolytic viruses include nucleotide sequences encoding SEQ.ID.NO:33 and ID.NO:34. In some embodiments, such oncolytic viruses include nucleotide sequences encoding SEQ.ID.NO:41 and ID.NO:42.

[0166] In some embodiments, such oncolytic viruses include nucleotide sequences having at least 80% identity to the sequences listed in Table 4 above. In some embodiments, such oncolytic viruses include nucleotide sequences having at least 85% identity to the sequences listed in Table 4 above. In some embodiments, such oncolytic viruses include nucleotide sequences having at least 90% identity to the sequences listed in Table 4 above. In some embodiments, such oncolytic viruses include nucleotide sequences having at least 95% identity to the sequences listed in Table 4 above.

[0167] In some embodiments, such oncolytic viruses include SEQ.ID.NO: 45 and 46. In some embodiments, such oncolytic viruses include SEQ.ID.NO: 47 and 48. In some embodiments, such oncolytic viruses include SEQ.ID.NO: 49 and 50. In some embodiments, such oncolytic viruses include SEQ.ID.NO: 51 and 52.

[0168] Some oncolytic viruses have the ability to accommodate large enough DNA insertion to accommodate the integration of full-length human antibody sequences. Attenuated vaccinia virus and herpes simplex virus are examples of therapeutic oncolytic viruses, and their genomes are large enough to allow the integration of full-length IgG antibody sequences (Chan, WM et 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 have been successfully integrated into oncolytic vaccinia viruses, resulting in full-length IgG antibodies being expressed and released (produced) to the extracellular space after infection of host cells susceptible to viruses (e.g., cancer cells) (Kleinpeter, P. et al. 2016, "Tumor Immunology (Oncoimmunology)" 5 (10): e1220467). Adenoviruses can also be engineered to encode full-length IgG antibodies that are functionally produced and secreted following cell infection (Marino, N. et al. 2017 J Clin Invest 123(6):2447-2463).

[0169] In a preferred embodiment, such an oncolytic virus is a poxvirus (e.g., vaccinia virus) that is defective in TK activity (due to changes in the J2R locus) or defective in both TK and RR activity (due to changes in the J2R locus and at least one of the I4L and / or F4L loci encoded by the RR) and comprises (a) a nucleotide sequence encoding SEQ.ID.NO:20 and ID.NO:21 or (b) a nucleotide sequence encoding SEQ.ID.NO:27 and ID.NO:28 or (c) a nucleotide sequence encoding SEQ.ID.NO:33 and ID.NO:34 or (d) a nucleotide sequence encoding SEQ.ID.NO:41 and ID.NO:42.

[0170] When appropriate, it may be advantageous to insert (one or more) nucleotide sequences into the oncolytic virus described herein to include additional regulatory elements that promote expression, transport, and biological activity. For example, a signal peptide may be included to promote secretion outside the producer cell (e.g., infected cell). The signal peptide is usually inserted into the N-terminus of the encoded polypeptide immediately after the Met initiator. The selection of signal peptides is extensive and accessible to those skilled in the art. For example, a signal peptide (e.g., heavy chain IgG) derived from another immunoglobulin can be used in the context of the present invention to allow the anti-CTLA4 antibodies described herein to be secreted outside the producer cell. For the purpose of illustration, SEQ ID NO: 53 and SEQ ID NO: 54 may be referred to, which include the light chain and heavy chain of the 4-E03 antibody described herein, and the light chain and heavy chain both have peptide signals derived from IgG.

[0171] A particularly preferred oncolytic virus is a vaccinia virus (e.g., a Copenhagen strain) that is defective in both TK and RR activity (due to alterations in the J2R locus and the I4L locus) and comprises a nucleotide sequence encoding SEQ.ID.NO:20 and SEQID.NO:21 or SEQ.ID.NO:53 and SEQ ID.NO:54.

[0172] In certain embodiments, such oncolytic viruses may further include additional nucleotide sequences with therapeutic significance (one or more), such as nucleotide sequences encoding (one or more) immunomodulatory polypeptides (i.e., polypeptides directly or indirectly involved in stimulating immune responses). Representative examples of suitable immunomodulatory polypeptides include, but are not limited to, cytokines and chemokines, and particularly preferably granulocyte macrophage colony stimulating factor (GM-CSF) and particularly human, non-human primate or mouse GM-CSF. Additional nucleotide sequences can be easily obtained using sequence data available in the art and information provided herein by standard molecular biology techniques (e.g., PCR amplification, cDNA cloning, chemical synthesis). A particularly preferred oncolytic virus is a vaccinia virus (e.g., the Copenhagen strain) which is defective for both TK and RR activity (due to alterations in the J2R locus and the I4L locus) and comprises a nucleotide sequence encoding SEQ.ID.NO: 20 and ID.NO: 21 or SEQ.ID.NO: 53 and SEQ ID.NO: 54 and a nucleotide sequence encoding GM-CSF, and particularly preferably 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).

[0173] In addition, the nucleotide sequence to be inserted into such an oncolytic virus can be optimized by modifying one or more codons to provide high-level expression in a specific host cell or subject. In addition, in order to optimize codon usage, various modifications can also be envisioned to prevent the presence of rare, non-optimal codons in concentrated areas and / or to suppress or modify "negative" sequence elements that are expected to have a negative impact on 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 forward or reverse repeats; RA secondary structures; and / or internal cryptic regulatory elements, such as internal TATA boxes, chi sites, ribosome entry sites, and / or splicing donor / acceptor sites.

[0174] In certain embodiments, (one or more) nucleotide sequences are placed under the control of suitable regulatory elements so that they are correctly expressed in host cells or subjects. As used herein, the term "regulatory element" refers to any element that allows, contributes or regulates the expression of coding (one or more) nucleotide sequences in a given host cell or subject, including its replication, duplication, transcription, splicing, translation, stability and / or transportation in or outside the expressing cell. It will be appreciated by those skilled in the art that the selection of regulatory elements 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, etc. Promoter is particularly important. In the context of the present invention, it can be a constitutive guide expression of a nucleotide sequence that it controls in many types of host cells or is specific to certain host cells or in response to a specific event or exogenous factor (e.g., by temperature, nutritional additives, hormones, etc.) or regulated according to the stage (e.g., late or early) of the viral cycle. Promoters suitable 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 placed under the control of promoters with the same transcriptional strength, preferably the same promoter (e.g., p7.5K, such as the promoter described in SEQ ID NO: 59, or pH5.R, such as the promoter described in SEQ ID NO: 60), to obtain similar expression levels for both chains, and thus the antibody is optimally assembled as a heterotetrameric protein (i.e., to avoid excessive non-associated chains). Additional nucleotide sequences (e.g., encoding GM-CSF) can be placed under the control of different promoters (e.g., pSE / L, such as the promoter described in SEQ ID NO: 61).

[0175] By conventional methods, using appropriate restriction enzymes or preferably by homologous recombination, (one or more) nucleotide sequences (possibly equipped with appropriate regulatory elements) are inserted into the genome of such an oncolytic virus. (One or more) nucleotide sequences can be independently inserted into any position of the viral genome. Various insertion sites can be considered, such as in non-essential viral genes, intergenic regions or non-coding parts of the genome of such an oncolytic virus. The J2R locus and / or the I4L locus are particularly suitable for oncolytic viruses (e.g., oncolytic vaccinia viruses) as poxviruses. After (one or more) nucleotide sequences are inserted into the viral genome, the viral locus of the insertion site may be at least partially deleted. In one embodiment, this deletion or partial deletion may result in the expression of viral gene products encoded by the locus that is completely or partially deleted being suppressed, thereby resulting in defective viruses for the function of the virus. A particularly preferred oncolytic virus is a TK and / or RR defective vaccinia virus comprising a box encoding a heavy chain inserted into the J2R locus and a box encoding a light chain inserted into the I4L locus. The cassette encoding the additional GM-CSF encoding nucleotide sequence may be inserted into another location in the viral genome or into the J2R or I4L loci, and is preferably inserted into the I4L locus.

[0176] The present invention also provides such oncolytic viruses described herein, and particularly oncolytic poxviruses are generated into a method for a suitable host cell (producer cell). In certain embodiments, such a method includes one or more steps of homologous recombination between a viral genome and a transfer plasmid, wherein the transfer plasmid includes (one or more) nucleotide sequences to be inserted (possibly with regulatory elements), the nucleotide sequence is flanked by 5' and 3', and the viral sequence is present in the upstream and downstream of the insertion site, respectively. The transfer plasmid can be generated and introduced into a host cell by conventional techniques (e.g., by transfection). The viral genome can be introduced into a host cell by infection. The size of each flanking viral sequence can vary between at least 100bp to at most 1500bp on each side of the nucleotide sequence (preferably 200 to 550bp and more preferably 250 to 500bp). Homologous recombination allowing the production of such an oncolytic virus is preferably carried out in a cultured cell line (e.g., HeLa, Vero) or in a chicken embryo fibroblast (CEF) cell obtained from an embryonated egg.

[0177] In some embodiments, the identification of oncolytic viruses incorporating anti-CTLA4 encoding nucleotide sequences and possible additional nucleotide sequences (eg, GM-CSF) can be promoted by using selection and / or detectable genes. In a preferred embodiment, the transfer plasmid further includes a selection marker with a specific preference for the GPT gene (encoding guanine phosphoribosyl transferase), which allows growth in a selective medium (eg, in the presence of mycophenolic acid, xanthine, and hypoxanthine) or encoding a detectable gene product such as GFP, e-GFP, or mCherry. In addition, it is also possible to consider the use of an endonuclease that can provide a double-strand break in the selection or detectable gene. The endonuclease can be in the form of a protein or expressed by an expression vector.

[0178] Once generated, such oncolytic viruses can be amplified into appropriate host cells using conventional techniques, which include culturing the transfected or infected host cells under appropriate conditions to allow for the production and recovery of infectious particles.

[0179] The present invention also relates to a method for producing an oncolytic virus as described herein. Preferably, the method comprises the following steps: 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 produce the virus, d) recovering the produced virus from the culture of the producer cell line, and optionally e) purifying the recovered virus.

[0180] In some embodiments, the producer cell is selected from the group consisting of: mammalian (eg, human or non-human) cells such as HeLa cells (eg, ATCC-CRM-CCL-2 TM or ATCC-CCL-2.2 TM), 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 such as those 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. Producer cells are preferably cultured in an appropriate culture medium, which may be supplemented with serum and / or (one or more) suitable growth factors or not (e.g., a chemically defined culture medium, preferably free of animal or human-derived products) if necessary. Depending on the producer cells, a person skilled in the art can easily select an appropriate culture medium. Such culture media are commercially available. Prior to infection, producer cells are preferably cultured for between 1 and 8 days at a temperature comprised between +30° C. and +38° C., more preferably at about +37° C. If necessary, several passages of 1 to 8 days may be performed to increase the total number of cells.

[0181] In step b), the producer cells are infected with the oncolytic virus under appropriate conditions and using an appropriate multiplicity of infection (MOI) to allow productive infection of the producer cells. For illustration purposes, an appropriate MOI ranges from 10 -3 To 20, and particularly preferably the MOI is comprised between 0.01 and 5, and more preferably between 0.03 and 1. The infection step is carried out in a medium which may be the same as or different from the medium used for the producer cell culture.

[0182] In step c), the infected producer cells are then cultured under appropriate conditions well known to those skilled in the art until progeny virus particles are produced. The cultivation of the infected producer cells is also preferably performed at a temperature between +32°C and +37°C in a culture medium which may be the same or different from the culture medium / culture medium used for the cultivation and / or infection step of the producer cells, for 1 to 5 days.

[0183] In step d), the viral particles produced in step c) are collected from the culture supernatant and / or the producer cells. Recovery from 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 known as high speed) homogenization or high pressure homogenization.

[0184] The recovered oncolytic virus can be at least partially purified before being distributed in dosage and used as described herein. A large number of purification steps and methods are available in this area, including, for example, clarification, enzyme treatment (e.g., endonuclease, protease, etc.), chromatography and filtering steps. Suitable methods are described in the art (see, for example, WO2007 / 147528; WO2008 / 138533, WO2009 / 100521, WO2010 / 130753, WO2013 / 022764).

[0185] In one embodiment, the present invention also provides cells infected with the oncolytic virus described herein.

[0186] The present invention also encompasses pharmaceutical compositions comprising a virus, such as the oncolytic viruses discussed above, and a pharmaceutically acceptable diluent, vehicle, and / or adjuvant.

[0187] In some embodiments, the pharmaceutical composition may be in the form of a CAR-T cell carrying a partial or complete antibody sequence described herein as part of the sequence encoding its chimeric antigen T cell receptor.

[0188] The present invention also encompasses a pharmaceutical composition comprising the CAR-T cells discussed above and a pharmaceutically acceptable diluent, vehicle and / or adjuvant.

[0189] The present invention also encompasses other therapeutic modalities or "shapes" of drugs, such as antibody drug conjugates, fusion proteins, etc., as well as pharmaceutical compositions comprising such therapeutic modalities.

[0190] 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 antibacterial agents and / or solutes that make the formulation isotonic with the blood of the intended recipient; and / or aqueous and / or non-aqueous sterile suspensions, which may include suspending agents and / or thickening agents. Antibody molecules, nucleotide sequences, plasmids, viruses, cells and / or pharmaceutical compositions described herein may be present in unit doses or multi-dose containers (e.g., sealed ampoules and vials), and may be stored under freeze drying (lyophilization) conditions that require only the addition of a sterile liquid carrier (e.g., water for injection) just before use.

[0191] Extemporaneous injection solutions and suspensions may be prepared from sterile powders and / or granules and / or tablets of the kind previously described.

[0192] For parenteral administration to human patients, the daily dosage level of the anti-CTLA-4 antibody molecule administered in single or divided doses is generally 1 mg / kg patient body weight to 20 mg / kg, or even up to 100 mg / kg in some cases. In special cases, for example, in combination with long-term administration, lower doses can be used. In any case, the doctor will determine the actual dose that is most suitable for any individual patient, and the actual dose will vary with the age, weight and response of the particular patient. The above dosages are examples of general situations. Of course, in individual cases, higher or lower dosage ranges should be used, and these dosage ranges are within the scope of the present invention.

[0193] Typically, the pharmaceutical compositions (or medicaments) comprising antibody molecules described herein will contain anti-CTLA-4 antibody molecules at a concentration between about 2 mg / ml and 150 mg / ml or between about 2 mg / ml and 200 mg / ml. In some embodiments, the pharmaceutical composition will contain anti-CTLA-4 antibody molecules at a concentration of 10 mg / ml.

[0194] Typically, a pharmaceutical composition (or drug) will contain a concentration of 10 3 Up to 10 12 The oncolytic viruses described herein can be measured in amounts between vp (viral particles), iu (infectious units) or pfu (plaque forming units), depending on the virus and the quantification technique. The amount of pfu present in a sample can be determined by counting the number of plaques after infecting permissive cells (e.g., CEFs 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 antiviral antibodies). As a general guide, a single dose range suitable for a pharmaceutical composition comprising an oncolytic poxvirus is about 10 3 pfu to about 10 10 pfu, advantageously about 10 3 pfu to about 10 9 pfu, preferably about 10 4 pfu to about 10 8 and more preferably about 10 4 pfu to about 10 7 pfu.

[0195] Typically, 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 in appropriate acceptable formulations according to common veterinary practice, and the veterinarian will determine the dosage regimen and route of administration that will be most suitable for a particular animal. Therefore, the present invention provides pharmaceutical formulations comprising a certain amount of antibody molecules, nucleotide sequences, plasmids, viruses and / or cells of the present invention (as further described above and below) that are effective in treating various conditions. Preferably, the antibody molecules, nucleotide sequences, plasmids, viruses, cells and / or pharmaceutical compositions described herein are suitable for delivery by a route selected from the group comprising the following: 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 administration sites with the same or different doses, using the same or different routes). For illustrative purposes, about 10 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 A single dose of pfu of oncolytic poxvirus (eg, the TK- and RR-deficient vaccinia viruses described herein) is particularly suitable for intratumoral administration.

[0196] The present invention also includes antibody molecules, nucleotide sequences, plasmids, viruses, cells and / or pharmaceutical compositions described herein, which include pharmaceutically acceptable acid or base addition salts of the polypeptide binding portion of the present invention. The acid used to prepare the pharmaceutically acceptable acid addition salts of the above-mentioned base compounds that can be used in the present invention is an acid that forms a non-toxic acid addition salt, i.e., a salt containing a pharmaceutically acceptable anion, such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, sucrose, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate [i.e., 1,1'-methylene-bis-(2-hydroxy-3 naphthoate)] salt, etc. Pharmaceutically acceptable base addition salts can also be used to produce pharmaceutically acceptable salt forms of the medicament according to the present invention. Chemical bases that can be used as reagents to prepare pharmaceutically acceptable base salts of the medicaments of the present invention are chemical bases that form non-toxic base salts with such compounds, and the base salts are acidic in nature. Such non-toxic base salts include, but are not limited to: base salts derived from such pharmaceutically acceptable cations, such as base salts of 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-(glucamine); and other base salts of lower alkanolammonium and pharmaceutically acceptable organic amines, and the like. The antibody molecules, nucleotide sequences, plasmids, viruses and / or cells described herein can be lyophilized for storage and reconstituted in a suitable carrier before use. Any suitable lyophilization method (e.g., spray drying, cake drying) and / or reconstitution technology can be used. Those skilled in the art will understand that lyophilization and reconstitution may result in varying degrees of antibody activity loss (e.g., for conventional immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies), and that the use level may have to be adjusted upward to compensate. In one embodiment, the lyophilized (freeze-dried) polypeptide binding moiety loses no more than about 20%, or no more than about 25%, or no more than about 30%, or no more than about 35%, or no more than about 40%, or no more than about 45%, or no more than about 50% of its activity (before lyophilization) when rehydrated.

[0197] In some embodiments, the viral composition is appropriately buffered at a physiological or slightly alkaline pH (e.g., from about pH 7 to about pH 9, and particularly preferably pH between 7 and 8.5, and more particularly close to 8). It may be beneficial to also include a monovalent salt in the viral composition to ensure appropriate osmotic pressure. The monovalent salt may be particularly selected from NaCl and KCl, preferably, the monovalent salt is NaCl, preferably at a concentration of 10 to 500 mM (e.g., 50 mM). Suitable viral compositions include 50 g / L of sucrose, 50 mM of NaCl, 10 mM of Tris-HCl, and 10 mM of sodium glutamate at pH 8. The composition may also be formulated to include a cryoprotectant for protecting oncolytic viruses at low storage temperatures. Suitable cryoprotectants include, but are not limited to, sucrose (or sucrose), trehalose, maltose, lactose, mannitol, sorbitol and glycerol, preferably at a concentration of 0.5% to 20% (weight in g / volume in L, referred to as w / v), and high molecular weight polymers such as dextran or polyvinylpyrrolidone (PVP).

[0198] The anti-CTLA-4 antibody molecules, nucleotide sequences and pharmaceutical compositions described herein can be used to treat cancer in a subject.

[0199] Including, the subject can be a mammal or a non-mammal. Preferably, the mammalian subject is a human, or a non-mammal, such as a horse, cow, sheep, pig, camel, dog or cat. Most preferably, the mammalian subject is a human.

[0200] “Manifestation” includes: the subject manifests cancer symptoms and / or cancer diagnostic markers, and / or the cancer symptoms and / or cancer diagnostic markers can be measured and / or assessed and / or quantified.

[0201] For those skilled in the medical field, it is easy to understand what the cancer symptoms and cancer diagnostic markers will be and how to measure and / or evaluate and / or quantify whether the severity of the cancer symptoms decreases or increases, or whether the cancer diagnostic markers decrease or increase; and how those cancer symptoms and / or cancer diagnostic markers can be used to form a prognosis for cancer.

[0202] Cancer treatment is usually administered as a course of treatment, that is, the therapeutic agent is administered over a period of time. The length of a course of treatment depends on many factors, which may include 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, among other reasons.

[0203] "During treatment" includes: the subject is currently receiving a course of treatment, and / or is receiving a therapeutic agent, and / or is receiving a course of therapeutic agent.

[0204] In some embodiments, the cancer to be treated according to the present invention is a solid tumor.

[0205] In some embodiments, the cancer is selected from the group consisting of advanced solid tumors, melanoma and other skin malignancies, 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.

[0206] Each of the above cancers is well known, and symptoms and cancer diagnostic markers and therapeutic agents for treating the cancers are also well described. Thus, the symptoms, cancer diagnostic markers and therapeutic agents for treating the above cancer types are known to those skilled in the medical field.

[0207] Clinical definitions of diagnosis, prognosis, and progression of a large number of cancers rely on certain classifications known as staging. Those staging systems are used to collate a large number of 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 understand how to use the staging system to assess the diagnosis and / or prognosis and / or progression of cancer, and which cancer diagnostic markers and cancer symptoms should be used to make the assessment.

[0208] “Cancer stage” includes: Rai staging, which includes stage 0, stage I, stage II, stage III and stage IV; and / or Binet staging, which includes stage A, stage B and stage C; and / or Ann Arbour staging, which includes stage I, stage II, stage III and stage IV.

[0209] It is known that cancer can cause abnormalities in cell morphology. These abnormalities often occur reproducibly in certain cancers, which means that examining these morphological changes (also known as histological examinations) can be used for diagnosis or prognosis of cancer. Techniques for visualizing samples to examine cell morphology and preparing samples for visualization are well known in the art; for example, optical microscopy or confocal microscopy.

[0210] "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 with dense nuclei lacking discernible nucleoli; and / or the presence of atypical cells and / or lysed cells and / or prolymphocytes.

[0211] As is well known, cancer is the result of mutations in the cell's DNA, which may cause the cell to avoid cell death or to proliferate uncontrollably. Therefore, checking these mutations (also referred to as cytogenetic tests) may be a useful tool for assessing cancer diagnosis and / or prognosis. An example of this is the loss of chromosome position 13q14.1, a characteristic of chronic lymphocytic leukemia. The techniques for checking mutations in cells are well known in the art; for example, fluorescence in situ hybridization (FISH).

[0212] "Cytogenetic examination" includes an examination of the DNA in the cells, and specifically the chromosomes. Cytogenetic examination can be used to identify changes in the DNA that may be associated with the presence of refractory cancer and / or recurrent cancer. Such changes may include: a deletion in the long arm of chromosome 13; and / or a deletion in the chromosome locus 13q14.1; and / or a trisomy of chromosome 12; and / or a deletion in the long arm of chromosome 12; and / or a deletion in the long arm of chromosome 11; and / or a deletion in 11q; and / or a deletion in the long arm of chromosome 6; and / or a deletion in 6q; and / or a deletion in the short arm of chromosome 17; and / or a deletion in 17p; and / or a t(11: 14) translocation; and / or (q13:q32) translocation; and / or antigen gene receptor rearrangement; and / or BCL2 rearrangement; and / or BCL6 rearrangement; and / or t(14:18) translocation; and / or t(11:14) translocation; and / or (q13:q32) translocation; and / or (3:v) translocation; and / or (8:14) translocation; and / or (8:v) translocation; and / or t(11:14) and (q13:q32) translocations.

[0213] It is known that subjects with cancer exhibit certain physical symptoms, which are generally due to the burden of the cancer on the body. These symptoms often recur in the same cancer and can therefore serve as a feature of the diagnosis and / or prognosis and / or progression of the disease. Those skilled in the art of medicine will understand which physical symptoms are associated with which cancers, and how to assess those physical symptoms and 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

[0214] In the examples below, reference is made to the following figures:

[0215] Figure 1 : The antibodies of the present invention specifically bind to CTLA-4

[0216] ELISA showed that the antibody bound to human and rhesus monkey CTLA-4, but not to human CD28 protein. Figure 1 A), 4-E-03( Figure 1 B), 5-B07( Figure 1 C) in combination with Yervoy ( Figure 1 D) were compared.

[0217] Figure 2 : Dose-dependent binding of anti-CTLA-4 mAb to hCTLA-4-transfected cells

[0218] Anti-CTLA-4 mAb ( Figure 2 AD) showed strong binding to 293T cells expressing CTLA-4, similar to Yervoy ( Figure 2 E).

[0219] Figure 3 : Anti-CTLA-4 mAb binds to in vitro activated human CD4+ T cells

[0220] CD4+ T cells obtained from peripheral blood were stimulated in vitro. Binding of anti-CTLA-4 mAb was analyzed by FACS (solid line, top row) and compared with Yervoy (dashed line, top row) and commercially available FACS antibodies (bottom row).

[0221] Figure 4 :Binding region on CD4+T cells activated in vitro

[0222] Human in vitro activated CD4+ T cells were stained with anti-CTLA-4 mAb labeled with Alexa 647 (black line). Antibody binding was blocked by rhCTLA-4-Fc protein (grey line).

[0223] Figure 5 :Binding with in vitro activated rhesus monkey CD4+T cells

[0224] CD4+ T cells obtained from peripheral rhesus macaque blood were stimulated in vitro with CD3 / CD28 dynabeads. Binding of anti-CTLA-4 mAb was analyzed by FACS (solid line, top row) and compared with Yervoy (dashed line, top row) and commercial FACS antibodies (bottom row).

[0225] Figure 6 :Blocking of human and rhesus monkey CTLA-4 protein binding to cells

[0226] 293T-CTLA-4 cells were stained with anti-CTLA-4 mAb labeled with Alexa 647 (black line). Antibody binding was blocked by rhCTLA-4-Fc protein (light gray line) and rcmCTLA-4-Fc protein (dark gray line).

[0227] Figure 7 :Binding to 293T cells expressing rhesus macaque CTLA-4

[0228] 293T cells were transiently transfected with rhesus macaque CTLA-4, and the binding of different concentrations of anti-CTLA-4 mAb was analyzed by FACS.

[0229] Figure 8 : Expected lack of binding to quiescent human / rhesus PBMCs

[0230] As analyzed by FACS, 4-E03 as well as 2-C06, 5-B07 and 2-F09 (data not shown) did not show any non-specific binding to different cell subsets in human (top row) and rhesus monkey (bottom row) PBMCs.

[0231] Fig. 9 : Expected to lack direct agonist activity

[0232] CFSE-labeled CD4+ T cells from healthy donors were stimulated with coated anti-CD3 plus soluble anti-CTLA-4 mAb or anti-CD28. % dividing cells (CFSElow CD25+ cells) were determined by FACS 3 days later. (A) FACS graph of a representative experiment, (B) graphs summarizing 6 donors.

[0233] Fig.10 :CD80 / CD86 blocking activity

[0234] As shown by ELISA, anti-CTLA-4 mAb blocked CD80 ( Figure 8 A) and CD86 ( Figure 8 B) Binding to its ligand CTLA-4.

[0235] Fig.11 : In vitro functional ligand blocking

[0236] PBMCs were stimulated with SEB plus titrated doses of anti-CTLA-4 antibodies. The amount of secreted IL-2 in the supernatant was determined by MSD. In this figure, 1 of 6 representative donors is shown.

[0237] Fig.12 :ADCC assay on in vitro activated CD4+ T cells

[0238] In vitro activated CD4+T cells from healthy donors preconditioned with 10 μg / ml anti-CTLA-4 mAb were co-cultured with NK cells (NK-92 cell line) at a ratio of 2: 1. ADCC activity was assessed by FACS as described below. The figure shows the mean + SD of 4-8 donors.

[0239] Fig.13 :CTLA-4 is most highly expressed on tumor-resident Treg cells.

[0240] Freshly resected ovarian tumors and blood samples were obtained from surgical patients. Ascites was collected from patients with different cancer indications. CTLA expression on this patient material was compared with healthy PBMC. Tumor samples were minced and digested. Peripheral blood mononuclear cells were separated by centrifugation. CTLA-4 expression on CD4+CD25+CD127-Treg cells, CD4+ non-Treg cells, and CD8+ effector T cells was assessed by flow cytometry. Data represent individual patients / donors, with n=12 for healthy PBMC, n=20 for ascites, n=9 for tumors, and n=5 for patient blood.

[0241] We also analyzed CTLA-4 expression on human T cells that were activated in NOG mice and then isolated from the spleens of these mice (see Fig.14 ).

[0242] Fig.14 : Anti-CTLA-4 mAb mediates Treg depletion in vivo.

[0243] Human PBMCs were injected intravenously into NOG mice. After approximately 2 weeks, the spleens were removed and the expression of CTLA-4 on human Treg cells and CD8+ T cells was analyzed by FACS. Splenocytes isolated from NOG mice were transferred intraperitoneally into SCID mice. One hour later, mice were treated intraperitoneally with CTLA-4hIgG1 or control mAb. After 24 hours, intraperitoneal fluid was collected and the frequency of human T cell subsets was determined by flow cytometry ( Fig.14 A: Treg and Fig.14 B: CD8+ T cells).

[0244] Fig.15 : Table summarizing the properties of anti-CTLA-4 antibodies

[0245] Fig.16 : Fig.16 : Characterization of mouse alternative anti-CTLA-4 mAb

[0246] Fig.16 AB: Blocking ELISA was performed with m5-B07 to assess the ligand blocking properties. The antibody blocked in a dose-dependent manner. Fig.16 A) CD80 and Fig.16 B) Binding of CD86 to its ligand CTLA-4.

[0247] Fig.16 CD:5-B07 in the form of mouse IgG2a mediated Treg depletion in the CT26 tumor model. Balb / c mice were injected subcutaneously with 1×10 6CT26 cells were used and treatment was started when the tumor size was approximately 7x7 mm. After 3 injections of 10 mg / kg antibody, the immune cell content of tumor single cell suspensions was analyzed by FACS. Fig.16 C: Ligand-blocking surrogate antibody 5-B07 causes Treg depletion. Fig.16 As depicted in D, this leads to CD8 + Shift in the ratio of T cells to Treg cells.

[0248] Fig.17 :Generation of COPTG19384 and COPTG19385

[0249] Schematic representation of COPTG19384 and COPTG19385 used in this study. COPTG19385 contains a J2R gene deletion in the TK locus replaced by the heavy chain of anti-CTLA-4 driven by p7.5K, and an I4L gene deletion in the RR locus replaced by the light chain of anti-CTLA-4 driven by p7.5K. COPTG19384 contains a J2R gene deletion in the TK locus replaced by the heavy chain of anti-CTLA-4 driven by p7.5K, and an I4L gene deletion in the RR locus replaced by the light chain of anti-CTLA-4 driven by p7.5K and GM-CSF driven by pSE / L.

[0250] Fig.18 :Analysis of the expression of 4-E03 monoclonal antibody in the supernatant of CEF cells infected with COPTG19384

[0251] A) By Western blot: CEEF cells were infected with COPTG19384 at an MOI of 0.05 in triplicate. Cell supernatants were harvested 48 hours later and analyzed by WB after electrophoresis under non-reducing conditions using anti-Ig (left blot) or anti-light chain (right blot) HRP-conjugated antibodies.

[0252] B) By ELISA: CEF cells were infected in triplicate with COPTG19384 or VVTG17137 at an MOI of 0.05. Cell supernatants were harvested 48 hours later and analyzed by ELISA to detect 4-E03 monoclonal antibodies.

[0253] Fig.19 : Analysis of GM-CSF expression in supernatants of CEF cells infected with COPTG19384 by ELISA

[0254] CEF cells were infected in triplicate at an MOI of 0.05 with COPTG19384 primary research stock or VVTG17137. Cell supernatants were harvested 48 hours later and analyzed by ELISA to detect GM-CSF.

[0255] Fig. 20 :Replication study of COP WT\COPTG19384 (two batches) and VVTG17137 (two batches) in normal and tumor hepatocytes

[0256] A) Replication rate on normal human hepatocytes.

[0257] B) Replication rate on malignant HepG2 cells.

[0258] C) Therapeutic index calculated from replication rates measured in HepG2 and hepatocytes.

[0259] Fig.21 : Replication of COPTG19384 and VVTG17137 in reconstructed human skin

[0260] After 7 days and when the inoculum size was increased from 10 to 10 5 Replication of COPTG19384 and VVTG17137 was assessed with unequal pfu. Results are means and SEM of triplicate measurements.

[0261] Fig. 22 :Oncolytic activity of COPTG19384 and VVTG17137 in three human tumor cell lines: MIA PaCa-2 (A), LoVo (B) and HepG2 (C)

[0262] Fig.23 : Expression levels of 4-E03 mAb and GM-CSF in (A) supernatants of infected HepG2 and LoVo cells and (B) supernatants of infected 5 different human tumor cell lines

[0263] A) After 5 days of incubation, for COPTG19384, at an MOI of 10 -5 Up to 10 -2 And for VVTG17137 used as negative control, MOI was 10 -2 4-E03 and GM-CSF expression levels were evaluated.

[0264] B) 4-E03 and GM-CSF expression levels were assessed 48 hours after infection with COPTG19384 at an MOI of 0.05.

[0265] Fig.24 :Binding of different batches of 4-E03 and CTLA-4 protein

[0266] Binding of 4-E03 recombinantly produced from CHO (4-E03 research batch) or HEK (4-E03 toxicology batch) cells to (A) human and (B) rhesus macaque recombinant proteins was compared by ELISA to binding of 4-E03 purified from the supernatant of infected MIA PaCa-2 tumor cells (4-E03 TG).

[0267] Fig.25 : Binding of different batches of 4-E03 to cells expressing CTLA-4

[0268] Binding of 4-E03 recombinantly produced by CHO (4-E03 research batch) or HEK (4-E03 toxin batch) cells to (A) human and (B) rhesus macaque CTLA-4 expressing cells was compared to binding of 4-E03 purified from the supernatant of infected MIA PaCa-2 tumor cells (4-E03 TG) by flow cytometry.

[0269] Fig.26 : Kinetics of virus accumulation in LoVo xenograft tumors

[0270] In LoVo xenograft tumors, the expression of 10 4 or 10 5 The kinetics of viral accumulation were assessed after a single intrathoracic injection of COPTG19384 or VVTG17137 (pfu). Solid or dashed lines represent the median of three values ​​determined at each time point.

[0271] Fig. 27 : Kinetics of 4-E03 mAb and GM-CSF accumulation in LoVo xenograft tumors

[0272] A) In two different doses (10 4 or 10 5 The kinetics of 4-E03 mAb accumulation in LoVo xenograft tumors was assessed after a single intrathoracic injection of COPTG19384 or VVTG17137 (pfu) or a single intraperitoneal injection of 3 mg / kg of 4-E03 mAb. Solid or dashed lines represent the median of three values.

[0273] B) After a single intrathoracic injection of COPTG19384 (10 4 or 10 5 pfu) or VVTG17137(10 5 The kinetics of virus accumulation in LoVo xenograft tumors was assessed after 10 min of inoculation with 100 pfu (40 μg / mL). The solid line represents the median of three values ​​determined at each time point.

[0274] Fig.28Kinetics of 4-E03 mAb and GM-CSF concentrations in serum of LoVo xenograft mice

[0275] A) In LoVo xenograft tumors, the expression of 10 4 or 10 5 The kinetics of 4-E03 mAb concentrations in serum were evaluated after a single intrathoracic injection of COPTG19384 or VVTG17137 (pfu) or a single intraperitoneal injection of 3 mg / kg of 4-E03 mAb. The solid line represents the median of three values.

[0276] B) In LoVo xenograft tumors, the expression of 10 4 or 10 5 A single intrathoracic injection of COPTG19384 or VVTG17137 (10 5 The kinetics of GM-CSF concentration in serum was evaluated after 10 min of 4 dHO / mL of GM-CSF. The dashed line represents the median of three values ​​determined at each time point.

[0277] Fig.29 : Kinetics of viral accumulation in CT26 tumors

[0278] In 10 7 The kinetics of viral accumulation in CT26 tumors was assessed after three intrathoracic injections (on days 0, 2, and 4) of VVTG18058, COPTG19421, or COPTG19407.

[0279] Figure 30: Kinetics of m5-B07 mAb and mGM-CSF Accumulation in CT26 Tumors

[0280] A) Three intrathoracic injections of VVTG18058, COPTG19421, or COPTG19407 (10 7 The kinetics of m5-B07 mAb concentration in CT26 tumors was evaluated during or after 10 min (pfu / injection) or after a single intraperitoneal injection of 3 mg / kg of m5-B07 mAb. The solid line represents the median of three values.

[0281] B) Three intrathoracic injections of VVTG18058, COPTG19421, or COPTG19407 (10 7 The kinetics of mGM-CSF concentrations in CT26 tumors were evaluated during or after 10 min of 40 min of 10 min of 2 ...10 min of 20 min of 10 min of 10 min of 20 min of 10 min of 10 min of 10 min of 10 min of 10 min of 10 min of

[0282] Fig.31: Kinetics of m5-B07 mAb concentration in serum of CT26 model

[0283] In CT26 tumors, three intrathoracic injections of VVTG18058, COPTG19421, or COPTG19407 (10 7 The kinetics of m5-B07 mAb concentration in serum was evaluated after 20 min (pfu / injection) or after a single intraperitoneal injection of 3 mg / kg of m5-B07 mAb. The solid line represents the median of three values ​​determined at each time point.

[0284] Figure 32: Antitumor activity in the CT26 model: Effects of COPTG19347 + / - anti-PD1 on CT26 tumor growth (A) and mouse survival (B)

[0285] On day 7, CT26 cells were injected subcutaneously into BalB / c mice. On days 0, 2, and 4, COPTG19347 (10 7 pfu)、VVTG18058(10 7 pfu), VVTG18058, or buffer, and may be followed by intraperitoneal injection of 250 μg / mouse of anti-PD1 RMPI-14 on days 7, 10, 14, 17, and 21.

[0286] Fig.33 : Evaluation of dose effect in the CT26 model (Summary of survival data observed after treatment with COPTG19407, COPTG19421, and VVTG18058)

[0287] Fig.34 : Antitumor activity of COPTG19407 compared with VVTG18058 plus m5-B07 in the CT26 tumor model

[0288] CT26 cells were injected subcutaneously into Balb / C mice. When tumors reached approximately 100 mm 3 At day 0, day 2, and day 5, mice were injected with COPTG19407 (8.5×10 6 pfu, intrathoracic), VVTG18058 (8.5×10 6 pfu, intrathoracic), m5-B07 (10 mg / kg, intraperitoneal), or VVTG18058 (8.5×10 6 pfu, intrathoracic) plus m5-B07 (10 mg / kg, intraperitoneal). Figure 30 AD: tumor growth, and Fig.16 : Survival rate changes over time.

[0289] Fig.35 : Individual tumor volume curves of BALB / c mice bearing subcutaneous A20 tumors.

[0290] A20 cells were injected subcutaneously into Balb / C mice. When the tumors reached 80-100 mm 3 At day 0, day 2, and day 4, mice were injected with vehicle (intraperitoneal), COPTG19407 (4.75×10 6 pfu, intrathoracic), VVTG18058 (4.75×10 6 pfu, intrathoracic), RMP1-14 (anti-mPD-1) (250 μg / mouse, intraperitoneal), or COPTG19407 (4.75×10 6 pfu, intrathoracic) plus RMP1-14 (250 μg / mouse, intraperitoneal).

[0291] A) Group 1 animals treated with vehicle

[0292] B) Group 2 animals treated with VVTG18058

[0293] C) Group 3 animals treated with COPTG19407

[0294] D) Group 4 animals treated with mouse anti-PD1

[0295] E) Group 5 animals treated with COPTG19407 and mouse anti-PD1

[0296] Fig.36 : Mean tumor volume curves of BALB / cN mice bearing subcutaneous A20 tumors.

[0297] Each point represents the mean value of the tumor volume recorded in each group. The tumor volume of all animals was monitored within 64 days. Mice were treated with vehicle (Group 1), VVTG18058 (Group 2), COPTG19407 (Group 3), mouse anti-PD1 antibody RMP1-14 (BioXCell) (Group 4), and COPTG19407 and RMP1-14 (Group 5). Animals were randomized on the 7th day and animals were treated during the 7th to 31st day. Finally, mice were killed on the 61st day.

[0298] Fig.37 :Anti-tumor activity in the A20 model: Effects of COPTG19407+ / - anti-PD1 on A20 tumor growth (A) and mouse survival (B)

[0299] A20 cells were injected subcutaneously into Balb / C mice. When the tumors reached 80-100 mm 3The mice were treated with vehicle (intraperitoneal) (Group 1), anti-PD-1 (Group 2), isotype (Group 3), VVTG18058 (10 5 pfu, intrathoracic) (Group 4), VVTG18058 (10 5 pfu, intrathoracic) + isotype (Group 5), VVTG18058 (10 5 pfu, intrathoracic) + anti-PD-1 (Group 6), VVTG19407 (10 5 pfu, intrathoracic) (Group 7), VVTG19407 (10 5 pfu, intrathoracic) + isotype (Group 8) and VVTG19407 (10 5 pfu, intrathoracic) + anti-PD-1 (Group 9) treatment.

[0300] Fig.38 : Individual tumor volume curves of C57BL / 6 mice bearing subcutaneous C38 tumors.

[0301] C38 cells were injected subcutaneously into C57bl / 6 mice. When tumors reached 80-100 mm 3 At day 0, day 2, and day 4, mice were injected with vehicle (intraperitoneal), COPTG19407 (4.75×10 6 pfu, intrathoracic), VVTG18058 (4.75×10 6 pfu, intrathoracic), RMP1-14 (anti-mPD-1) (250 μg / mouse, intraperitoneal), or COPTG19407 (4.75×10 6 pfu, intrathoracic) plus RMP1-14 (250 μg / mouse, intraperitoneal).

[0302] A) Group 1 animals treated with vehicle

[0303] B) Group 2 animals treated with VVTG18058

[0304] C) Group 3 animals treated with COPTG19407

[0305] D) Group 4 animals treated with mouse anti-PD1

[0306] E) Group 5 animals treated with COPTG19407 and mouse anti-PD1

[0307] Fig.39 : Mean tumor volume curves of C57BL / 6 mice bearing subcutaneous C38 tumors.

[0308] Each point represents the mean value of the tumor volume recorded in each group. The tumor volume of all animals was monitored within 61 days. Mice were treated with vehicle (Group 1), VVTG18058 (Group 2), COPTG19407 (Group 3), mouse anti-PD1 antibody RMP1-14 (BioXCell) (Group 4), and COPTG19407 and RMP1-14 (Group 5). Animals were randomized on the 7th day and treated during the 7th to 31st day. Finally, mice were killed on the 61st day.

[0309] Fig.40 : Individual tumor volume curves of BALB / c mice bearing subcutaneous EMT6 tumors.

[0310] EMT6 cells were injected subcutaneously into Balb / C mice. When tumors reached 80-100 mm 3 At day 0, day 2, and day 4, mice were injected with vehicle (intraperitoneal), COPTG19407 (4.75×10 6 pfu, intrathoracic), VVTG18058 (4.75×10 6 pfu, intrathoracic), RMP1-14 (anti-mPD-1) (250 μg / mouse, intraperitoneal), or COPTG19407 (4.75×10 6 pfu, intrathoracic) plus RMP1-14 (250 μg / mouse, intraperitoneal).

[0311] A) Group 1 animals treated with vehicle

[0312] B) Group 2 animals treated with VVTG18058

[0313] C) Group 3 animals treated with COPTG19407

[0314] D) Group 4 animals treated with mouse anti-PD1

[0315] E) Group 5 animals treated with COPTG19407 and mouse anti-PD1

[0316] Fig.41 : Mean tumor volume curves of BALB / c mice bearing subcutaneous EMT6 tumors.

[0317] Each point represents the mean value of the tumor volume recorded in each group. The tumor volume of all animals was monitored over 61 days. Mice were treated with vehicle (Group 1), VVTG18058 (Group 2), COPTG19407 (Group 3), mouse anti-PD1 antibody RMP1-14 (BioXCell) (Group 4), and COPTG19407 and RMP1-14 (Group 5). Animals were randomized on day 7 and treated from day 7 to day 31. Finally, mice were sacrificed on day 56. After more than 20% of the mice died, the curve stopped. DETAILED DESCRIPTION

[0319] Examples

[0320] Specific non-limiting examples that embody certain aspects of the present invention will now be described. In the examples, rh protein represents human recombinant protein (e.g., rhIL-2 represents human recombinant IL-2 protein), and rcm protein represents rhesus monkey recombinant protein (e.g., rcmCTLA4 represents rhesus monkey recombinant CTLA-4 protein).

[0321] In addition to the sequences mentioned above, some additional sequences were used in the Examples and are listed in Table 5 below.

[0322] Table 5: Additional sequences used in the examples

[0323]

[0324]

[0325] Example 1 - Generation of CTLA-4-specific antibodies

[0326] Isolation of scFv antibody fragments

[0327] scFv library (BioInvent; et al., Nat Biotechnol. 2000; 18(8):852-6) were used to isolate scFv antibody fragments that recognize human CTLA-4.

[0328] Phage pools were used in three consecutive pannings against recombinant human proteins. After phage incubation, cells were washed to remove unbound phage. Bound phage were eluted with trypsin and amplified in E. coli. The resulting phage stock was converted to scFv format. E. coli was transformed with a plasmid carrying scFv and expressed a separate scFv clone.

[0329] Identification of unique CTLA-4 binding scFv

[0330] Transformed scFvs from the third panning were assayed for binding to transfected 293FT cells expressing human CTLA-4 or non-target proteins using a homogenous FMAT assay (Applied Biosystems, Carlsbad, CA, USA).

[0331] Briefly, transfected cells were added to the bottom transparent plate together with the supernatant containing scFv in the expression plate (diluted 1:7), mouse anti-HisTag antibody (0.4 μg / ml; R&D Systems) and APC-coupled goat anti-mouse antibody (0.2 μg / ml; Catalog No. 115-136-146, Jackson Immunoresearch). Before reading, the FMAT plate was incubated at room temperature (about 20-25°C) for 9 hours. Target-specific bacterial clones were classified as active substances, and cherry was selected into 96-well plates.

[0332] Binding of IgG to CTLA-4 in ELISA

[0333] 96-well plates (Lumitrac 600LIA plates, Greiner) were coated with 1 pmol / well of recombinant human CTLA-4-Fc protein (Andy Systems), 1 pmol / well of recombinant rhesus monkey (cm) CTLA-4-Fc protein (Andy Systems), 0.3 pmol / well of recombinant mouse CTLA-4-Fc protein (Andy Systems), 1 pmol / well of recombinant human CD28-Fc protein (Andy Systems) or 0.3 pmol / well of recombinant mouse CD28-Fc protein (Andy Systems) overnight at 4°C. 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 hour. The plates were then washed again, and bound antibodies were detected with anti-human F(ab)-HRP secondary antibodies (Jackson ImmunoResearch) diluted to 50 ng / ml. Super Signal ELISA Pico (Thermo Scientific) was used as substrate and the plates were analyzed using a Tecan Ultra Microplate reader.

[0334] As determined by ELISA, all antibodies were shown to bind to human and rhesus CTLA-4 proteins, but not to human CD28 proteins. In addition, 5-B07 was shown to bind to mouse CTLA-4, but not to mouse CD28 (see Figure 1 ).

[0335] IgG binding to CTLA-4 expressing 293T cells in flow cytometry

[0336] The transformed IgG clones were analyzed for binding to 293T cells expressing CTLA-4 (purchased from Crownbio). The cells were incubated with different concentrations of anti-CTLA-4 mAb (eg Figure 2 The cells were incubated at 4°C for 20 minutes (as shown), then washed and stained with APC-labeled goat anti-human secondary antibody (catalog number 109-136-088, Jackson ImmunoResearch). Dead cells were excluded from the analyte using Fixable Viability Dye eFluor780 (eBiosciences). Data acquisition was performed on a FACSVerse (BD Biosciences, Franklin Lakes, NJ) and analyzed with FlowJo software (Tree Star, Ashland, OR).

[0337] The anti-CTLA-4 mAb was shown to bind to 293T cells expressing human CTLA-4 in a dose-dependent manner, with an EC50 value similar to that of Yervoy ( Figure 2 ).

[0338] 293T cells stably transfected with human CTLA-4, 293T cells transiently transfected with rhesus CTLA-4, native human or rhesus PBMCs, and in vitro activated human or rhesus CD4+ T cells were incubated with a certain concentration of anti-CTLA-4 mAb at 4°C for 20 min, then washed and stained with APC-labeled anti-human secondary antibody (Jackson ImmunoResearch).

[0339] Example 2 - Anti-CTLA-4 mAbs specifically bind to (primary) cells expressing human and rhesus CTLA-4

[0340] CTLA-4-specific mAb binds to primary human and rhesus macaque CD4+ T cells activated in vitro but not to naive PBMCs isolated from healthy donors

[0341] PBMCs were isolated from the buffy coat. Briefly, buffy protein was diluted 1:3 in PBS and loaded onto a Ficoll-Paque Plus (Amersham) cushion. The samples were centrifuged at 800 x g for 20 minutes at 20°C. The upper plasma-containing phase was removed and mononuclear cells were isolated from the distinct white band of the plasma / Ficoll interphase.

[0342] Human peripheral CD4 T-cells were purified by negative selection using the MACS CD4 T-cell isolation kit (Miltenyi Biotec).+ T cells. CD4+ T cells were activated in vitro for 3 days in R10 medium (containing 2 mM glutamine, 1 mM pyruvate, 100 IU / ml penicillin and streptomycin, and 10% FBS (GIBCO from Life Technologies)) with CD3 / CD28 dynabeads (Life Technologies) plus 50 ng / ml rhIL-2 (Andy Systems) to upregulate CTLA-4 expression. Rhesus CD4+ T cells were isolated using non-human CD4 microbeads (Miltenyi Biotech) and incubated for 3 days with 50 ng / ml PMA (Sigma-Aldrich) and 100 ng / ml ionomycin (Sigma-Aldrich).

[0343] Naive human or rhesus PBMC, in vitro activated human or rhesus CD4+ T cells were incubated with the indicated concentrations of anti-CTLA-4 mAb at 4°C for 20 min, then washed and stained with APC-labeled anti-human secondary antibody (Jackson ImmunoResearch). Binding of anti-CTLA-4 mAb was analyzed by FACS using BD FACS Verse.

[0344] Showing antibodies and in vitro activated human ( Figure 3 ) and rhesus monkeys ( Figure 5 )CD4+ T cells, but not resting PBMCs ( Figure 8 ). Binding to T cells endogenously expressing CTLA-4 is comparable to that of Yervoy ( Figure 3 , top row, dashed line) was used as a positive control with a commercial anti-CTLA-4 FACS antibody from BD Biosciences (clone BNI3; Figure 3 , bottom row) were stained similarly.

[0345] like Figure 4 As shown, staining of human in vitro activated CD4+ T cells (black line) can be completely blocked by rhCTLA-4-Fc (grey line), indicating the specificity of the antibody. In this competitive binding assay, 2 μg / ml Alexa 647-labeled anti-CTLA-4 mAb was mixed with recombinant human CTLA-4-Fc protein (50 μg / ml) before incubation with cells expressing CTLA-4. IgG binding was detected by FACS.

[0346] Transfected 293T cells expressing human and rhesus CTLA-4 confirmed the rhesus cross-reactivity of the tested antibodies

[0347] The rhesus cross-reactivity of the antibody was further confirmed on transfected 293T cells expressing CTLA-4.

[0348] like Figure 6 As shown, the binding of CTLA-4 specific antibodies to transfected cells expressing human CTLA-4 can be inhibited by both human and rhesus monkey recombinant proteins (both Andy Systems). It was also shown that the antibodies bind to transfected cells expressing rhesus monkey CTLA-4 ( Figure 7 , upper row). This binding can be blocked again by the rhesus macaque recombinant protein (bottom row, grey line).

[0349] Execute the above example 2 Figure 4 Experiments were similar to those described for the competition assay.

[0350] Expected to lack direct agonist activity

[0351] In vitro proliferation assays were performed to exclude unexpected direct agonist activity (eg due to non-specific binding).

[0352] Human peripheral 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). 2 Goat anti-human IgG (Fcγ fragment specific) or F(ab') 2 Goat anti-mouse IgG (Fcγ fragment specific) with IgG:F(ab') 2 =1.5:1 molar ratio for 1 hour. 1×10 40 cells were stimulated with plate-bound anti-CD3 (0.5 μg / ml; clone UCHT1, Andy Systems) and 4 μg / ml soluble cross-linked anti-CTLA-4 or cross-linked anti-CD28 (clone CD28.2 (BioLegend)) at 37°C. 5 Purified human CD4+ T cells were cultured for 72 hours. The cells were washed and stained with BV421-conjugated anti-CD25 antibody (clone M-A251, BD Biosciences). The percentage of CD25+ / CFSE low dividing cells was analyzed by FACS.

[0353] Fig. 9 showed that, in contrast to anti-CD28 stimulation, none of the anti-CTLA-4 mAbs tested induced T cell proliferation.

[0354] Example 3 - Anti-CTLA-4 mAb Block Ligand Binding of CD80 / CD86

[0355] Ligand blocking ELISA

[0356] The ligand blocking activity of anti-CTLA-4 IgG was evaluated by ELISA. To this end, recombinant human CTLA-4-Fc protein (Andy System) was coated on a 96-well plate (Lumitrac 600LIA plate, Greiner) at 1 pmol / well. After washing, titrated doses of anti-CTLA-4 mAb were allowed to bind for 1 hour. Ligands with His tags were added at 200 nM and 100 nM (rhCD80 and rhCD86; Andy System), respectively, and the plate was further incubated for 15 minutes. After washing, the bound ligand was detected with an HRP-labeled anti-His antibody (Andy System). Super Signal ELISA Pico (Thermo Scientific) was used as a substrate, and the plate was analyzed using a Tecan UltraMicroplate reader.

[0357] like Fig.10 As shown, the anti-CTLA-4 antibodies tested showed similar ligand blocking activity as Yervoy.

[0358] In vitro functional ligand blocking

[0359] For the SEB PBMC assay, whole PBMCs from healthy donors were plated in 96-well plates (1×10 5 Cells / well) were plated and stimulated with 1 μg / ml Staphylococcus enterotoxin B (SEB, Sigma-Aldrich) in the presence of titrated doses of anti-CTLA-4 IgG. IL-2 secretion was measured on day 3 by MSD (Mesoscale) according to the manufacturer's instructions.

[0360] Antibodies 4-E03 and 2-C06 were shown to enhance IL-2 production and were shown to have potency similar to that of Yervoy. Fig.11 In Figure 1, one representative donor out of six is ​​shown.

[0361] Example 4 - Anti-CTLA-4 mAbs deplete CTLA-4 expressing cells in vitro and in vivo

[0362] Antibody-dependent cellular cytotoxicity (ADCC)

[0363] ADCC assays were performed using NK-92 cell lines stably transfected to express the CD16-158V allele and 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 Biotech). Cells were stimulated for 48 hours at 37°C with CD3 / CD28 dynabeads (Life Technologies, Thermo Fisher) and 50ng / ml rhIL-2 (Andy Systems). Target cells were pre-incubated with 10 μg / ml mAB at 4 ° C for 30 minutes and then mixed with NK cells. Cells were incubated for 4 hours with a 2: 1 effector: target cell ratio in RPMI 1640+GlutaMAX medium (Invitrogen) containing 10 mM HEPES buffer, 1 mM sodium pyruvate and 10% FBS low IgG. Lysis was determined by flow cytometry. In short, at the end of the incubation, the cell suspension was stained with BV510-coupled anti-CD4 (clone RPA-T4, BD Biosciences) and 10 nM SYTOX Red dead cell stain (Invitrogen) or repairable vitality dye eFluor780 (eBioscience) in the dark at 4 ° C for 20 minutes, and then FACSVerse (BD Biosciences) was used to analyze the cells.

[0364] Compared with Yervoy, 4-E03 showed significant improvement in the loss of CTLA-4+ T cells in vitro ( Fig.12 ).

[0365] CTLA-4 expression on primary patient material

[0366] To validate the translational potential of the above findings for the depleting activity of anti-CTLA-4 mAbs, CTLA-4 expression was examined on primary patient material.

[0367] The Ethics Committee of Skåne University Hospital Ethical approval for the use of clinical samples was obtained from the University Hospital of Skåne, Lund. Informed consent was provided in accordance with the Declaration of Helsinki. Samples were obtained through the Department of Gynecology and Obstetrics and Oncology at the Skåne University Hospital, Lund. Ascites was evaluated as isolated single-cell suspensions. Tumor material was cut into small pieces and treated with DNase I (Sigma-Aldrich) and Liberase TM (Roche Diagnostics) was incubated at 37 ° C in R10 for 20 minutes. The remaining tissue was mechanically broken and passed through a 70 μm cell strainer together with the cell suspension. The cells separated from ascites and tumors were stained. In order to identify 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, κ control-PE (G155-178; all from BD Biosciences). Data acquisition was performed using FACSVerse and data were analyzed using FlowJo.

[0368] like Fig.13 As shown, CTLA-4 is most highly expressed on intratumoral Treg cells, making it a good target for depletion of CTLA-4-specific antibodies.

[0369] PBMC-NOG / SCID Model

[0370] To confirm the in vitro findings of the depletion activity of CTLA-4-specific antibodies, the depletion capacity of anti-CTLA-4 mAbs was analyzed in the in vivo PBMC-NOG / SCID model. This model is based on the well-established hu-PBMC-NOG model ( 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 modified in-house as described below.

[0371] Mice were bred and maintained in a local facility according to the guidelines of the Ministry of Home Affairs. 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, Mouse: an excellent recipient mouse model for engraftment of human cells (NOD / SCID / γ mouse: an excellent recipient mouse model for engraftment of human cells. Blood 100(9):3175-3182) Mice were provided by Taconic (Bomholt, Denmark) and maintained in the local animal facility. For the PBMC-NOG / SCID (primary human xenograft) model, human PBMCs were isolated using Ficoll Paque PLUS and after washing, the cells were centrifuged at 75×10 6 15 × 10 cells / ml were resuspended in sterile PBS. 200 μl of cell suspension was injected intravenously into NOG mice, corresponding to 15 × 10 6 cells / mouse. Two weeks after injection, spleens were isolated and made into single cell suspensions. Thereafter, a small sample was taken to determine the expression of CTLA-4 on human T cells by FACS. Fig.13 As indicated, CTLA-4 expression was higher on Treg cells compared to other T cells, reflecting the situation in human patients. Most cells were cultured at 50 × 10 6 10 × 10 cells / ml were resuspended in sterile PBS. 200 μl of the suspension was injected intraperitoneally into SCID mice, corresponding to 10 × 10 6 cells / mouse. After 1 hour, mice were treated with 10 mg / kg of anti-CTLA-4hIgG1, Yervoy or isotype control mAb. Intraperitoneal fluid of mice was collected 24 hours later. Human T cell subsets were identified and quantified by FACS using the following markers: CD45, CD4, CD8, CD25, CD127 (all from BD Biosciences).

[0372] All antibodies tested showed similar or better Treg depletion activity than Yervoy. Other T cell populations, such as CD8+ effector T cells, were not affected ( Fig.14 ).

[0373] Example 5 - Selected alternative antibody m5-B07 shows the same functional properties as 4-E03

[0374] In some examples, particularly in vivo examples, the mIgG2a form of antibody clone 5-B07 (also denoted m5-B07) has been used. This is a mouse antibody that is a surrogate antibody to the human antibodies disclosed herein. It has been selected as a surrogate antibody because it binds to murine CTLA-4 and thus blocks ligand binding ( Fig.16 In addition, it also showed Treg depletion activity ( Fig.16 CD).

[0375] Ligand blocking ELISA

[0376] The ligand blocking activity of 5-B07 was evaluated by ELISA. To this end, recombinant mouse CTLA-4-Fc protein (Sino Biological Inc., Beijing) was coated on a 96-well plate (Lumitrac600LIA plate, Greiner) at 1 pmol / well. After washing, titrated doses of anti-CTLA-4 mAb were allowed to bind for 1 hour. Ligands with His tags were added at 200 nM and 100 nM (rmCD80 and rmCD86; Sino Biological Inc., Beijing), and the plate was further incubated for 15 minutes. After washing, the bound ligand was detected with an HRP-labeled anti-His antibody (Andy Systems). SuperSignal ELISA Pico (Thermo Scientific) was used as a substrate, and the plate was analyzed using a Tecan Ultra Microplate reader.

[0377] like Fig.16 As shown, antibodies block the binding of (A) CD80 and (B) CD86 to their ligand CTLA-4.

[0378] In vivo Treg depletion activity

[0379] As described below, the effects of CTLA-4-specific antibodies on T cell subsets in tumors in vivo were investigated in the CT26 tumor model.

[0380] Mice were bred and maintained in a local facility according to the guidelines of the Ministry of Home Affairs. Female Balb / C six to eight weeks old were provided by Taconic (Bonholt, Denmark) and maintained 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 plated at 10 × 10 6 100 μl of cell suspension was subcutaneously injected into mice, corresponding to 1×10 6cells / mouse. As shown in the figure, when the tumor reached approximately 7×7mm, the mice were treated intraperitoneally with 10mg / kg of the indicated antibodies twice a week. After the third administration, the tumor was cut open, mechanically divided into small pieces, and digested with a mixture of 100μg / ml release enzyme (Roche) and 100μg / ml deoxyribonuclease (Sigma) at 37°C for 2×5 minutes, with a Vortex placed in between. The cell suspension was then washed with PBS containing 10% FBS (400g, 10 minutes). Afterwards, the cells were resuspended in MACS buffer and stained with an antibody panel of CD45, CD3, CD8, CD4 and CD25 (all from BD Biosciences). Before staining, 100μg / ml IVIG was used to block nonspecific binding of the cells. The cells were analyzed using FACS Verse (BD Biosciences). Mouse Treg cells were identified as CD45 + CD3 + CD4 + CD25 + cell.

[0381] like Fig.16 As shown in C, 5-B07 in mouse IgG2a format mediates Treg depletion in tumors associated with an increased CD8 / Treg ratio (D) compared to other CTLA-4 specific n-CoDeR antibodies and the well-known commercial clone 9H10.

[0382] Example 6 - Expression of anti-CTLA4 mAb (COPTG19385) or anti-CTLA4 mAb and GM-CSF

[0383] Generation of virus, transgene expression, and characterization of genetic stability of (COPTG19384)

[0384] 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 deficient in thymidine kinase (TK, J2R locus) and ribonucleotide reductase (RR, I4L locus) activities. Fig.17 As shown, 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 at 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 at the I4L locus. For COPTG19384, an expression cassette encoding human GM-CSF (SEQ ID NO: 56) was also placed at the I4L locus under the control of the pSE / L promoter (SEQ ID NO: 61).

[0385] The expression of HC and LC was controlled using the same promoter (p7.5K) in order to achieve the same expression level for both chains and thus optimally assemble the antibody as a heterotetrameric protein (i.e. to avoid excess of non-related chains). However, the same promoter for both chains of the antibody does not allow them to be inserted into the same locus (identical DNA sequences would increase the risk of recombination and then elimination of the transgene). Therefore, the cassette encoding 4-E03 HC was inserted at the J2R locus and the cassette encoding 4-E03 LC was inserted at the I4L locus. But the cassette encoding the GM-CSF transgene under a different promoter (pSE / L) was also inserted into the I4L locus, just like the antibody light chain.

[0386] Generation of COPTG19384

[0387] Vaccinia virus transfer plasmids pTG19339 and pTG19341 are designed to allow insertion of nucleotide sequences into the J2R and I4L loci of the vaccinia virus genome, respectively, by homologous recombination. They are derived from plasmid pUC18, into which the flanking sequences (BRG and BRD) surrounding the J2R (pTG19339) or I4L (pTG19341) loci are cloned. Each plasmid also contains the p7.5K promoter.

[0388] A synthetic fragment of 1436 bp named "Fragment HC" containing the HC gene of the 4-E03 antibody was generated. A fragment "LC fragment" containing the LC gene of the 4-E03 antibody and the hGM-CSF gene was synthetically generated under the control of pSE / L and inserted into a plasmid vector. The coding sequence was optimized for human codon usage, with the addition of a Kozak sequence (ACC) before the ATG start codon and a transcription terminator (TTTTTNT) after the stop codon. In addition, some patterns were excluded: TTTNTT, GGGGG, CCCCC, which are deleterious for expression in poxviruses.

[0389] The HC fragment was inserted into pTG19339 restricted by Pvu II by homologous recombination, resulting in pTG19367. The plasmid carrying the LC was restricted by SnaB1, and the resulting fragment "LC-GMCSF" was inserted into pTG19341 restricted by Pvu II by homologous recombination, resulting in pTG19384. In this plasmid, the expression cassette was inserted from head to tail between the recombination arms, thereby achieving homologous recombination in the I4L locus of the vaccinia virus genome.

[0390] COPTG19384 is generated by two consecutive homologous recombination on chicken embryo fibroblasts (CEF) to continuously insert I4L and J2R loci and by using COPTG19156 as parental virus and two transfer plasmids pTG19367 and pTG19384. CEFs were isolated from 12-day-old SPF embryo eggs (Charles River). Embryos were mechanically torn apart, dissolved in Tryple Select solution (Invitrogen), and cultured in MBE (based on Eagle's culture medium; Gibco) supplemented with 5% FCS (Gibco) and 2mM L-glutamine.

[0391] Homologous recombination between the transfer plasmid and the parental vaccinia virus enables the generation of recombinant vaccinia viruses that have lost the GFP and mCherry expression cassettes and acquired the antibody and GM-CSF expression cassettes. COPTG19156 contains an expression cassette for the mCherry gene in its I4L locus and an expression cassette for the GFP gene in its J2R locus. Homologous recombination between the transfer plasmid pTG19367 and the parental COPTG19156 enables the generation of recombinant vaccinia viruses that have lost their GFP expression cassette and acquired the 4-E03 heavy chain expression cassette, and selection is performed by isolating red fluorescent plaques. This intermediate recombinant virus (COPTG19367) was used as the parental virus for a second round of homologous recombination with pTG19384 as a transfer plasmid to generate a recombinant vaccinia virus that has lost its mCherry expression cassette and acquired the 4-E03 light chain and GM-CSF expression cassettes. COPTG19384 ( Fig.17 ) selection was performed by isolating white non-fluorescent plaques.

[0392] The virus stock of COPTG19384 was amplified on CEF in two F175 flasks to generate the appropriate virus stock, which was aliquoted and stored at -80°C until use. The virus stock was titrated on CEF cells and the infectious titer was expressed in pfu / mL and calculated using the following formula: number of lytic zones × dilution factor × 4. For illustrative purposes, the resulting virus stock titered 6.8 × 10 6 pfu / mL. The stock is analyzed by PCR using appropriate primer pairs to verify the integrity of the expression cassette and the recombination arm. The stock is also analyzed by sequencing the two expression cassettes. The comparison of the sequencing results shows 100% homology with the theoretical expected sequence. If necessary, the virus preparation is purified using conventional techniques (e.g., as described in WO2007 / 147528).

[0393] Generation of COPTG19385

[0394] Vaccinia virus transfer plasmids pTG19339 and pTG19341 are designed to allow insertion of nucleotide sequences into the J2R and I4L loci of the vaccinia virus genome, respectively, by homologous recombination. They are derived from plasmid pUC18, into which the flanking sequences (BRG and BRD) surrounding the J2R (pTG19339) or I4L (pTG19341) loci are cloned. Each plasmid also contains the p7.5K promoter.

[0395] A synthetic fragment named "Fragment HC" of 1436 bp containing the HC gene of the 4-E03 antibody was generated. The coding sequence was optimized for human codon usage, with the addition of a Kozak sequence (ACC) before the ATG start codon and a transcription terminator (TTTTTNT) after the stop codon. In addition, some patterns were excluded: TTTNTT, GGGGG, CCCCC, which are deleterious for expression in poxviruses.

[0396] The HC fragment was inserted into pTG19339 restricted by PvuII by homologous recombination to generate pTG19367.

[0397] A plasmid containing an expression cassette encoding only the 4-E03 light chain was obtained by eliminating 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 give pTG19385.

[0398] COPTG19385 is generated by two consecutive homologous recombination on chicken embryo fibroblasts (CEF) to insert J2R and I4L loci continuously and by using COPTG19156 as parental virus and two transfer plasmids pTG19367 and pTG19385. CEFs were isolated from 12-day-old SPF embryo eggs (Charles River). Embryos were mechanically torn apart, dissolved in Tryple Select solution (Invitrogen), and cultured in MBE (based on Eagle's culture medium; Gibco) supplemented with 5% FCS (Gibco) and 2mM L-glutamine.

[0399] Homologous recombination between the transfer plasmid and the parental vaccinia virus enables the generation of recombinant vaccinia viruses that lose the GFP and mCherry expression cassettes and obtain the antibody expression cassette. COPTG19156 contains an expression cassette for the mCherry gene in its I4L locus and an expression cassette for the GFP gene in its J2R locus. Homologous recombination between the transfer plasmid pTG19367 and the parental COPTG19156 enables the generation of recombinant vaccinia viruses that lose their GFP expression cassette and obtain the 4-E03 heavy chain expression cassette, and selection is performed by isolating red fluorescent plaques. This intermediate recombinant virus (COPTG19367) is used as a parental virus, and a second round of homologous recombination with pTG19385 as a transfer plasmid is performed to generate a recombinant vaccinia virus that loses its mCherry expression cassette and obtains the 4-E03 light chain expression cassette. The selection of COPTG19385 is performed by isolating white non-fluorescent plaques.

[0400] The virus stock of COPTG19385 was amplified on CEF in two F175 flasks to generate the appropriate virus stock, which was aliquoted and stored at -80°C until use. The virus stock was titrated on CEF cells and the infectious titer was expressed in pfu / mL and calculated by the following formula: number of lytic zones × dilution factor × 4. For illustrative purposes, the virus stock generated was titrated to 1.04 × 10 7 pfu / mL. The stock is analyzed by PCR using appropriate primer pairs to verify the integrity of the expression cassette and the recombination arm. The stock is also analyzed by sequencing the two expression cassettes. The comparison of the sequencing results shows 100% homology with the theoretical expected sequence. If necessary, the virus preparation is purified using conventional techniques (e.g., as described in WO2007 / 147528).

[0401] Transgene expression

[0402] The viral-mediated expression of 4-E03 monoclonal antibody was evaluated in the supernatant of CEF cells infected by COPTG19384 by Western blotting (WB), and compared with the antibody (40ng 4-E03) produced by recombinant. WB can allow visualization of the presence of non-functional molecules (e.g., molecules, aggregates with incomplete molecular chain assembly) that do not bind CTLA4. CEF cells were infected by COPTG19384 virus stock in triplicate with MOI 0.05. Cell supernatants were harvested after 48 hours, and analyzed by WB using anti-Ig (left blot) or anti-light chain (right blot) HRP-coupled antibodies after electrophoresis under non-reducing conditions. Fig.18The results shown in A show that under non-reducing conditions, the WB profile of mAbs produced by infected CEFs was close to that of purified 4-E03, with an apparent size between 100 and 150 kDa, indicating correct chain folding and assembly.

[0403] The supernatant of functional secreted 4-E03 antibody and GM-CSF was quantified by ELISA. ELISA allows quantitative measurement of the amount of functional polypeptide produced in the cell supernatant. VVTG17137 was used as a negative control. It is a vaccinia virus (Copenhagen strain) deleted in the J2R and I4L loci, encoding the suicide gene FCU1 (described in WO2009 / 065546).

[0404] To estimate 4-E03 antibodies, the microplate was coated with 100 μL / well of 0.25 μg / mL of CTLA4-Fc by overnight incubation at 4°C. After incubation, the coating solution was discarded, the blocking solution was added, and the plate was incubated for 1 to 2 hours at room temperature and then washed. 4-E03 calibration standards (0.097 to 100 ng / mL) or samples diluted in blocking solution (in triplicate) were added to the wells, and the plates were incubated at 37°C for 2 hours and then washed. HRP-conjugated antibodies diluted in blocking solution were added to each well, and the plates were incubated at 37°C for 1 hour and then washed. After incubation with TMB solution at room temperature in the dark for 30 minutes, H 2 SO 4 2M (stop solution) to stop the enzymatic reaction. Read the absorbance at 450nm on a microplate reader. Plot the absorbance versus the antibody concentration of the calibration standard. Fig.18 As shown in B, functional 4-E03 mAb was produced in cells infected with COPTG19384, reaching a concentration close to 1 μg / mL.

[0405] Also use ELISA (Andy Systems Ref SGM00) assessed viral mediated GM-CSF expression in the same supernatant. Briefly, this assay uses two anti-hGM-CSF antibodies. The first antibody used to capture hGM-CSF in the sample is coated on the well surface of a 96-well plate. The second antibody is coupled and added to the plate in solution to detect the captured hGM-CSF. The concentration of hGM-CSF in the sample is then calculated by interpolation from a calibration curve established with some purified hGM-CSF provided with the kit. Fig.19 As shown, the expression level of GM-CSF was approximately 6 μg / mL.

[0406] In summary, both transgenes were detected at concentrations equal to or higher than 1 μg / mL, indicating satisfactory expression levels.

[0407] Genetic stability:

[0408] The virus was passaged five times on CEF in serum-free medium at a multiplicity of infection (MOI) of 10 -4 After that, a genetic stability test was performed. The P5 generation was diluted and inoculated on CEF cells in 60 mm culture dishes to obtain 20 to 40 virus plaques per dish. One hundred virus plaques were isolated and subcultured. After one amplification cycle, the isolated virus plaques were inoculated on CEF cells and tested by PCR and ELISA. PCR analysis and transgene expression showed that more than 90% of the clones had the correct characteristics. Since the acceptance criteria for clinical development of products is genetic stability better than or equal to 90%, COPTG19384 is considered to be genetically stable.

[0409] Example 7 - In vitro characterization of COPTG19384

[0410] Replication studies in hepatocytes: tumor selectivity of viruses expressing anti-CTLA4 mAb and GM-CSF

[0411] COPTG19384 carries a deletion of two genes encoding viral enzymes (TK and RR) involved in nucleotide metabolism. When functional, these enzymes allow the virus to replicate in the cytoplasm of most cells, including cells in a quiescent state (i.e., with low pools of available nucleotides). Host range studies were performed in primary and malignant cells to verify that the insertion of different transgenes in the J2R and I4L loci did not alter the selectivity of the host range. The expression of the transgenes in normal primary human cells (hepatocytes, prepared by Biopredic) and tumor cells from the same organ (HepG2 from hepatocarcinoma, HBCC-8065 TM ). The replication of COPTG19384 was evaluated on HepG2 cells (100 μg / mL). For non-selective vaccinia viruses and recombinant double-deleted VVTG17137 viruses (deletions in the J2R and I4L genes, and the suicide FCU1 gene inserted instead of J2R), the replication rate and therapeutic index were calculated and compared with those of wild-type Copenhagen vaccinia (COP WT, virus without any deletions) as a reference. Two batches were assayed, a research batch (Batch 1) and a batch produced in a GMP manner (Batch 2). The replication rate was determined as the ratio of total infectious particles / initial infectious particles (inoculum) at the end of incubation. The therapeutic index for each virus was determined as the ratio: replication rate on HepG2 cells / replication rate on hepatocytes. The higher this ratio, the better the selectivity of the virus for tumor cells.

[0412] Primary hepatocytes were grown in basal hepatocyte medium supplemented with 1.6% of supplement for hepatocyte culture medium. HepG2 cells were seeded at 4E+05 cells / well in 12-well plates and incubated with 5% CO 2Incubate at 37°C for 24 hours. Prior to infection, the medium was removed and 70 pfu / well of virus in PBS for hepatocytes or in PBS supplemented with FCS for HepG2 was added to each well. Infected cells were incubated with 5% CO 2 Incubate at 37°C for 30 minutes, then add 1.5 mL / well of culture medium. Incubate the plate at 37°C with 5% CO 2 Incubate for 3 days and then store at -80° C. Plates were then thawed and wells were sonicated at 40% amplitude for 30 seconds prior to titration on Vero cells.

[0413] Replication rate in normal human hepatocytes:

[0414] Normal hepatocytes were chosen to monitor the capacity of COPTG19384 in normal human cells because these primary cells can be routinely obtained directly from donors. In those cells, COP WT propagated well, with a replication rate of more than 50,000 ( Fig. 20 In other words, each initial infectious virus particle produces about 50,000 new viruses. In the case of the two recombinant double-deleted viruses (i.e., VVTG17137 and COPTG19384), this replication rate was significantly reduced to 5 to 15 ( Fig. 20 The final results indicate that the attenuated replication to normal cells caused by the two deletions is retained between VVTG17137 and COPTG19384.

[0415] Replication rate in tumor cells HepG2:

[0416] HepG2 cells were chosen to monitor the ability of COPTG19384 to replicate in tumor human cells because these cells are the malignant counterparts of normal liver cells. In those cells, the replication rates of the five viruses tested were very similar, reaching approximately 100,000 new viruses ( Fig. 20 B). Thus, double deletion in VVTG17137 and COPTG19384 and vectorization of the transgenes do not impair their ability to replicate in malignant cells.

[0417] Therapeutic Index:

[0418] like Fig. 20 As shown in Figure 3, the calculated index of COP WT is only two, indicating that COP WT has poor selectivity for tumor cells and normal cells. In contrast, for VVTG17137 and COPTG19384 (and two copies of the tested viruses), the index ranged from 8.2E+03 to 1.8E+04. This confirms that the two recombinant viruses have the same good selectivity for tumor cells and normal cells.

[0419] These results indicate that COPTG19384 and VVTG17137 have very similar replication properties on both tumor cells and healthy cells. Their replication on tumor Hep G2 was similar compared to COP WT, while it was greatly impaired on healthy hepatocytes. Therefore, the deletion of two genes (J2R and I4L) restricts the replication of the deleted virus to proliferating cells (i.e., with a high nucleotide pool) including tumor cells. Since transgene expression and replication are closely linked, COPTG19384 is an effective vector for selective delivery of therapeutic proteins into tumors.

[0420] Replication assay on CEFs and LoVo:

[0421] In CEFs (producer cells) isolated from 11- or 12-day-old specific pathogen-free embryonic eggs (Charles River) and tumor human cell lines (LoVo; CCL-229 TM ) were used to evaluate the replication of COPTG19384. CEF and LoVo cells were made into suspensions and cultured at an MOI of 10 for CEF. -3 and for LoVo is 10 -2 Infection was performed (three wells per cell and per time point). After different incubation times, Vero cells (CCL-81 TM ). The replication of COPTG19384 was compared with that of VVTG17137 as a benchmark. The results showed that the replication of COPTG19384 and VVTG17137 was similar in both CEF and LoVo (data not shown).

[0422] Replication assay on reconstructed human skin:

[0423] T-Skin TM The replication of COPTG19384 was evaluated on a full-thickness human skin model. Thirty-six T-Skin TM Samples were cultured in 6-well plates and maintained in fresh culture medium. VVTG17137 and COPTG19384 were dispensed into each well (in triplicate) at the final concentration of 10 1 Up to 10 5 pfu / well). A negative control (mock) containing medium without virus was also tested. The plates were incubated at 37°C with 5% CO 2 Incubate for 7 days and collect T-Skin TM The sample was cut into two slices. Virus titration was performed using Vero cells and the infectious titer was determined on one of the two slices. Fig.21COPTG19384 was shown to replicate in reconstructed skin to the same extent as the benchmark VVTG17137, supporting the fact that neither GM-CSF nor vectorization of the 4-E03 mAb altered the replication behavior of vaccinia virus on human reconstructed skin.

[0424] Oncolysis assay

[0425] Oncolytic activity indicates the lytic activity of the tested virus samples on tumor cells. It was assessed by quantifying cell viability after 5 days of incubation on different tumor cell lines: human colorectal adenocarcinoma cell line LoVo ( CCL-229 TM ), human pancreatic tumor cell line MIA PaCa-2 ( CCL-1420) and human hepatoma cell line HepG2 ( HBCC-8065 TM ). The oncolytic activity of COPTG19384 was compared with that of VVTG17137 as a benchmark. A negative control (mock-infected cells) corresponding to uninfected cells was also inoculated.

[0426] Prepare cells and distribute them in Eppendorf tubes (1.2×10 6 cells / tube), and then the MOI was 10 -5 Up to 10 -2 Infect with virus and incubate at 37°C for 30 minutes. Add appropriate complete medium to an eppendorf tube and add aliquots of this suspension to each well of a 6-well plate containing 2 mL of appropriate complete medium (in triplicate). Incubate the plate at 37°C with 5% CO. 2 Incubate for 5 days and determine cell viability on a Vi-Cell counter. Results are expressed as a percentage of cell viability of mock-infected cells. Cell supernatants were also recovered for determination of 4-E03 mAb and GM-CSF concentrations. Fig. 22 The oncolytic activities of COPTG19384 and VVTG17137 were similar in the three tumor cell lines evaluated.

[0427] Transgene expression levels

[0428] The expression levels of 4-E03 mAb and GM-CSF were measured by ELISA (as described in Example 6) in the culture supernatants of HepG2 and LoVo cells recovered after oncolytic activity determination (infection at variable MOI for 5 days).

[0429] The expression levels of 4-E03 and GM-CSF were also measured by ELISA (see Example 6) in the supernatants of five cell lines cultured under the following conditions: HTB-135 TM ), human ovarian tumor cell line SK-OV-3 ( HTB-77 TM ), human pancreatic cancer cell line MIA PaCa-2 ( CCL-1420), human colorectal adenocarcinoma cell line LoVo ( CCL-229 TM ) and human colorectal cancer cell line HCT 116 ( CCL-247 TM Each cell line was plated in a 6-well plate (10 6 Cells / well) were cultured (triplicate) and incubated at 37°C with 5% CO2 for 24 hours before infection at an MOI of 0.05. Cell supernatants were then recovered 48 hours post infection and used to determine the concentrations of 4-E03 mAb and GM-CSF. As expected, MOI, time post infection, and cell line were important parameters affecting the level of transgene expression in the supernatant of infected cells. Fig.23 Figure A shows that when infected with COPTG19384, fairly permissive (HepG2) and fairly resistant (LoVo) tumor cell lines were able to produce approximately the same amounts of 4-E03 mAb and GM-CSF in their culture supernatants. However, the maximum expression in HepG2 was 10-fold lower than in LoVo at an MOI. In addition, for the five tumor cell lines tested, the expression of the transgene was above 0.1 and above 1 μg / mL for 4-E03 mAb and GM-CSF, respectively ( Fig.23 B). It is important to note that the ELISA assay used to measure 4-E03 mAb concentrations uses the antigen CTLA4 to capture the antibody. In other words, the antibodies measured by this assay are at least partially functional (i.e., recognize their antigen, with the other functions of the antibody being assumed by the Fc portion),

[0430] Purification and glycosylation characterization of 4-E03 mAb

[0431] To produce large amounts of 4-E03 mAb from infected cells, a 10 μg / ml 715 F175 flasks of Mia-PACA cells / bottle were infected with COPTG19384 at MOI 0.01 and incubated for 72 hours. Harvest MIA Paca-2 cell culture supernatant (about 450mL, containing 670μg mAb 4-E03, as determined by ELISA), pooled and clarified by centrifugation to remove most of the cell debris. The clarified supernatant was filtered on a 0.2μm filter and 2mM EDTA (to inhibit possible metalloproteinases) and 20mM Tris pH7.5 (to improve pH) were added. The filtered supernatant was then passed through a protA Hitrap chromatographic column (GE Healthcare, No. 17-5079-01). The chromatographic column was transferred and connected to a Purifier FPLC (GE Healthcare) and a purification procedure (THM / ProtA 1mL injection cycle frac bleu) was applied. After adding Laemlli buffer (Biorad) with or without β-mercaptoethanol (for reducing or not reducing the disulfide bonds of mAb), the eluted fractions containing mAb were loaded on NuPage Bis-Tris gel 4-12% (Thermo NP0323). The gel was stained with InstantBlue (Expedeon, ISB1L). The three fractions corresponding to the main peak of elution were pooled and the antibody concentration was determined by absorbance at 280 nm after dialysis against formulation buffer. The final concentration of purified mAb was 0.29 mg / mL.

[0432] The first characterization was to assess chain assembly by electrophoresis under reducing and non-reducing conditions. Under non-reducing conditions, 2 light chains and 2 heavy chains assemble to form a native and functional antibody. It appears that purified 4-E03 from infected MIA PaCa-2 and recombinantly produced 4-E03 have indistinguishable electrophoretic profiles under both reducing and non-reducing conditions. In other words, purified 4-E03 from infected MIA PaCa-2 has the expected light and heavy chain ratio and is correctly assembled as a 2 light chain and 2 heavy chain heterotetramer. Mass spectrometry also confirmed the presence of this heterotetramer. The purified mAbs were subjected to mass spectrometry for glycosylation analysis. Briefly, mAbs were digested or not with IdeS protease, which specifically cleaves IgG at the hinge (generating F(ab') 2and Fc part). The mass of the whole antibody or Fc part carrying N-glycosylation was determined, and each mass was fitted to the theoretical mass calculated based on the primary sequence and glycosylation pattern of Fc. 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, which is a benchmark for human IgG1 used in the clinic. The results showed that the glycosylation profile of 4-E03 produced from infected MIA PaCa2 was different from that of the two antibody references, with the majority of G0F (88%), while recombinant 4-E03 and MabThera had similar glycosylation profiles with typical G0F, G1F, and G2F distribution. However, MIA PaCa-2 was suspected to be responsible for the low G1F and G2F species in the purified 4-E03 mAb, as low levels of Beta-1,4-galactosyltransferase 1 transcripts could be responsible for the lack of galactosyl moieties of 4-E03 expressed in MIA PaCa-2 (thus lacking G1f and G2F).

[0433] The same type of purification was also performed from the permeate recovered during the purification of COPTG19384 produced on CEFs, followed by mass analysis. The results showed 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 cell line used has a greater impact on the glycosylation profile of the antibody than the infection itself.

[0434] 4-E03 purified from the supernatant of infected MIA PaCa-2 cells (4-E03 TG) also exhibited the same binding properties as 4-E03 produced recombinantly by CHO (research batch) or HEK (toxin batch) cells ( Fig.24 and 25 ). The ELISA (described in Example 1) was used to test the recombinant human ( Fig.24 A) people and ( Fig.24 B) The binding of rhesus monkey CTLA-4 protein showed this. Fig.25 A) people and ( Fig.25 B) FACS analysis of binding to rhesus CTLA-4 expressing cells (see Examples 1 and 2) confirmed that different 4-E03 batches have similar cross-reactivity and binding affinity.

[0435] GM-CSF glycosylation and disulfide bond patterns

[0436] To study the glycosylation pattern and the presence of some disulfide bonds, different human tumor cell lines were infected with COPTG19384 and their supernatants were analyzed using the same WB method. MIA-Paca-2, LoVo, HepG2 and HCT116 cells were infected with MOI 0.01 and incubated in serum-free medium for 72 hours. The culture supernatant was harvested, clarified by centrifugation, and then filtered on a 0.2 μm filter. The supernatant was stored at -20 ° C until analysis. It was treated by adding 8 μl Rapid PNGaseF buffer 5X, followed by incubation at 75 ° C for 5 minutes. Then one μL PNGase F (to remove N-glycans from glycoproteins) was added, and the mixture was incubated at 50 ° C for 30 minutes. Before Western blotting, twenty-five μL samples were prepared by adding 5 μL Laemmli buffer × 4 with or without β-mercaptoethanol (reducing and non-reducing conditions). The immune complexes were detected using Amersham ECL Prime Western Blotting, and chemiluminescence was recorded with a Molecular Imager ChemiDOC XRS (Bio-Rad).

[0437] GM-CSF from infected HCT116, LoVo, and MIA PaCa-2 displayed the same glycosylation pattern, while 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 may vary depending on the tumor cell line used for infection and its specific metabolic state.

[0438] Example 8: Pharmacokinetics of COPTG19384 after intratumoral injection

[0439] Anti-CTLA4 antibody, GM-CSF, and Kinetics of viral expression in tumors and bloodstream.

[0440] plan

[0441] 5×10 6 LoVo cells were implanted into the right flank of Swiss nude mice (Charles River, France). About two weeks later, when the tumor volume reached about 120 mm 3 The mice were randomly divided into 6 groups, with 15 animals in each group.

[0442] Mice from Group 1 received an intrathoracic dose of 1 × 10 4 pfu / mouse of COPTG19384.

[0443] Mice from group 2 received an intrathoracic dose of 1 × 10 5 pfu / mouse of COPTG19384.

[0444] Mice from Group 3 received an intrathoracic dose of 1 × 10 4 pfu / mouse of VVTG17137.

[0445] Mice from Group 4 received an intrathoracic dose of 1 × 10 5 pfu / mouse of VVTG17137.

[0446] - Mice from Group 5 received intraperitoneal (ip) administration of 4-E03 on day 0 at a dose of 3 mg / kg.

[0447] - Mice from Group 6 received ipilimumab (Yervoy) at a dose of 3 mg / kg intraperitoneally on day 0.

[0448] Tumors and blood were collected from 3 animals on days 1, 3, 6, 10, and 20. Tumors were weighed and homogenized for immediate processing. One quarter of the homogenized tumor was collected for viral titration, and the remaining suspension was centrifuged and the supernatant was stored at -20°C until use. The blood was divided into two parts: one part was added to a heparin tube (25 IU / 100 μL of blood) for titration assay and frozen at -80°C until analysis. Clarified serum was produced from the other part and stored at -20°C until use. Viral titers in tumor and blood samples were determined by titration on Vero cells.

[0449] Kinetics of viral replication in the LoVo model:

[0450] In the LoVo model, two doses (1×10 4 or 1×10 5 pfu) were injected once with COPTG19384, and viral replication was monitored and compared with that of VVTG17137 injected under the same conditions. Fig.26 The results presented in show a significant dispersion of the three values ​​of viral titer measured at each time point. In any case, the results also show that both viruses and both doses of virus replicated in the tumor and maintained relatively high titers / g tumor from day 3 to up to day 20 after injection. For a given time point, there were no significant differences in viral titers between the two doses of virus or between the two viruses used. Interestingly, except for one sample, in which only 13 pfu / mL was detected (data not shown) (VVTG17137, dose at day 10: 1×10 7 All blood samples tested negative for the virus except for 1.

[0451] These results together suggest that a single intrathoracic injection of 1 × 10 4 or 1×10 5 pfu, viral replication was sustained in LoVo tumors for at least 20 days and was barely detectable in the bloodstream. It must be noted that the LoVo xenograft model is highly favorable for viral replication because it uses human tumor cells and Swiss nude mice whose immune systems are severely compromised and therefore have limited antiviral activity.

[0452] Kinetics of transgene expression in the LoVo model:

[0453] As expected, for 4-E03 mAb ( Fig. 27 A) and GM-CSF ( Fig. 27 B), the kinetics of transgene expression in tumors closely followed those of viral replication, reaching maximum concentrations (Cmax) on day 6 or 10. In the case of a single injection of 4-E03 mAb (or ipilimumab), Cmax in tumor and blood was observed at the first time point (day 1), and mAb concentrations measured thereafter were consistent with the pharmacokinetics of human IgG1 in mice ( Fig.28 ).

[0454] Furthermore, after COPTG19384 treatment (for both doses), the concentration of 4-E03 entering the tumor at Cmax and thereafter (i.e., 6-10 to 20 days after injection) was approximately 10-fold higher than after a single injection of 4-E03 at a therapeutic dose of 3 mg / kg ( Fig. 27 In contrast, after COPTG19384 treatment, blood concentrations of mAb were consistently lower than those measured after intraperitoneal injection of 3 mg / kg 4-E03 ( Fig.28 A). This result indicates that vectorization of the mAb allows achieving high concentrations in the tumor without exceeding or even reaching the blood concentrations obtained at therapeutic doses of the mAb.

[0455] After COPTG19384 treatment, the expression kinetics of GM-CSF followed the kinetics observed with 4-E03 ( Fig. 27 B). Interestingly, for the same samples, the levels of GM-CSF measured in tumors were lower than those of 4-E03, but LoVo infected in vitro with COPTG19384 expressed GM-CSF two-fold higher than 4-E03. The blood concentration of GM-CSF was also very low compared to 4-E03 ( Fig.28 B). This result is consistent with the in vivo half-life of GM-CSF, which is very short compared to the in vivo half-life of human IgG1.

[0456] These results show that vectored antibodies and GM-CSF are expressed primarily in tumors following intrathoracic 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) concerns.

[0457] Anti-CTLA4 antibody, GM-CSF, and virus expression in tumors after intratumoral injection of vaccinia virus in the CT26 syngeneic model and the dynamics of expression in the bloodstream

[0458] Evaluation of viral activity in the CT26 immunocompetent murine model requires the generation of several alternative viruses encoding murine anti-mCTLA4 with or without murine GM-CSF:

[0459] COPTG19407 is a vaccinia virus (Copenhagen strain) containing an expression cassette encoding the heavy chain of murine m5-B07 IgG2 (SEQ ID NO:63) at the J2R locus under the p7.5 promoter, as well as an expression cassette encoding the light chain of m5-B07 (SEQ ID NO:62) at the I4L locus under the p7.5 promoter and murine GM-CSF (SEQ ID NO:58) under the pSE / L promoter.

[0460] COPTG19421 is a vaccinia virus (Copenhagen strain) containing an expression cassette encoding the heavy chain of m5-B07 under the p7.5 promoter at the J2R locus, and an expression cassette encoding the light chain of m5-B07 under the p7.5 promoter at the I4L locus.

[0461] • VVTG18058 used as a benchmark is a vaccinia virus (Copenhagen strain) deleted in the J2R and I4L genes without any transgenes ("empty" virus).

[0462] These vaccinia viruses were generated by two consecutive homologous recombination at the J2R (TK) and then I4L (RR) loci, as described in Example 6, for the human counterpart. The ELISA method for quantifying the m5-B07 antibody and mGM-CSF was similar to that described above (Example 6, "Transgenic Expression" of 4-E03 and GM-CSF), except that the mouse antibody was captured using the mouse CTLA4-Fc antigen, and the mGM-CSF was quantified using the Quantikine ELISA kit mouse GM-CSF (Andy Systems). The oncolytic activity of these viruses was also evaluated in various cell lines (a sarcoma: MCA205 and two colon cancers CT26 and MC38), and it was found that similar to VVTG18058, the vectorization of the mouse antibody with or without mGM-CSF did not affect the oncolytic ability of the vaccinia virus (data not shown).

[0463] Protocol: CT26 cells (2×10 5 cells) were implanted into the right flank of Balb / c mice (Charles River, France). About a week later, when the tumor volume reached 25-50 mm 3 At 4 hr, mice were randomized into 3 groups of 20 animals each (Groups 1 to 3) and a Group 4 of 10 animals. Tumors and blood were collected and processed as described for the LoVo model, except that the first 3 groups were collected on days 1, 4, 8, and 10, while Group 4 was collected on day 1.

[0464] Mice from Group 1 received intrathoracic administration of 1 × 10 7 pfu / mouse of VVTG18058.

[0465] Mice from group 2 received intrathoracic administration of 1 × 10 7 pfu / mouse of COPTG19407.

[0466] Mice from Group 3 received intrathoracic administration of 1 × 10 7 pfu / mouse of COPTG19421.

[0467] • Mice from Group 4 received m5-B07 at a dose of 3 mg / kg intraperitoneally on day 0.

[0468] Kinetics of viral replication in the CT26 model:

[0469] In the CT26 model, two alternative viruses (1×10 7 pfu / injection), and virus replication was monitored and compared with the replication of VVTG18058 virus injected under the same conditions. Fig.29 The results presented in show a significant dispersion of the three values ​​of viral titers measured at each time point. However, the titers of the three viruses persisted until day 10, suggesting that the two transgenes did not affect viral clearance or replication, at least within this time window. No viral infectious particles were detected in any blood sample (data not shown).

[0470] Kinetics of transgene expression in the CT26 model

[0471] Like the LoVo model, transgene expression in tumors reflects viral replication. In other words, constant levels of m5-B07 antibody (Figure 30A) and mGM-CSF (Figure 30B) were detected in tumors over the 10 days of monitoring.

[0472] In the case of monoclonal antibodies, the Cmax achieved after COPTG19421 or COPTG19407 injection was approximately 10-fold lower than the Cmax observed after a single intraperitoneal injection of 3 mg / mL of the m5-B07 antibody (Figure 30A). In serum, the circulating concentration of m5-B07 after virus treatment was approximately 100-fold lower than that of m5-B07 injected at 3 mg / kg, a more pronounced difference ( Fig.31 ).

[0473] For GM-CSF, only treatment with COPTG19407 produced measurable mGM-CSF concentrations in CT26 tumors, indicating that the measured cytokines had a recombinant, rather than an endogenous, origin. As in the LoVo model, mGM-CSF concentrations measured in tumors were lower than those of m5-B07 (Figure 30B). In addition, mGM-CSF produced by tumors was not detectable in any serum sample, likely due to the short half-life of the molecule, which prevents any systemic accumulation.

[0474] Example 9: Antitumor activity study

[0475] COPTG19347 is a vaccinia virus (Copenhagen strain) deleted in the J2R and I4L genes and encoding a complete murine antibody (i.e., m5-B07 (heavy and light chains) that recognizes the murine CTLA4 antigen. Both COPTG19421 and COPTG19347 express m5-B07, but under different promoters: p7.5K and pH5.R, respectively. Quantification of m5-B07 was assessed in infected cell supernatants, reaching 10 μg / mL at an MOI of 10 μg / mL. -1 About 1 μg / mL in infected CT26 cells at an MOI of 10 -2 Regarding mGM-CSF, higher expression of MCA205 relative to CT26 was also observed in the culture supernatant of cells infected with COPTG19407 (data not shown).

[0476] Antitumor activity in the mouse-bearing CT26 model in combination with anti-PD1

[0477] plan:

[0478] CT26 cells (2×10 5 cells) were implanted into the right flank of Balb / c mice (Charles River, France). 3 When the volume was 100 mg / kg, the mice were randomly divided into five groups of ten. Briefly, the mice were treated with three intrathoracic administrations of virus (2 days apart) followed by intraperitoneal treatment of mouse anti-PD1 (RMP1-14 BioXcell) twice a week for three weeks. More specifically,

[0479] Mice from Group 1 received vehicle;

[0480] Mice from group 2 received intrathoracic administration of 1×10 7 pfu of COPTG19347;

[0481] Mice from Group 3 received intrathoracic administration of 1×10 7 pfu of COPTG19347 and received 250 μg / mouse of RMP1-14 i.p. on days 7, 11, 14, 18, and 22;

[0482] Mice from Group 4 received intraperitoneal administration of 250 μg / mouse of RMP1-14 on days 7, 11, 14, 18, and 22;

[0483] Mice from Group 5 received intrathoracic administration of 1×10 7 pfu of VVTG18058.

[0484] Tumor size was measured twice a week with calipers and the formula (Π / 6) (length × width 2 ) to calculate its volume. When the tumor volume reaches 2000mm 3 When , the animals were euthanized.

[0485] Antitumor Activity of COPTG19347 in the CT26 Model:

[0486] As shown in Figure 32, COPTG19347 treatment not only resulted in tumor growth inhibition ( Fig.32A ), and also produced tumor regression, which ultimately translated into tumor-free mice with survival times of up to 100 days ( Fig.32B ). Treatment with COPTG19347 resulted in 60% tumor-free mice at day 100. Co-treatment with anti-PD-1 antibodies did not significantly improve tumor growth inhibition or the percentage of mice surviving for a long time (approximately 70% tumor-free mice at day 100). In contrast, RPMI-14 treatment did not provide any anti-tumor effect (behavior was identical to untreated mice (all died within the first 40 days), while VVTG18058 was less active (approximately 10% tumor-free mice at day 100).

[0487] Dose-effect evaluation in the CT26 model:

[0488] Three alternative viruses (different promoters driving m5-B07 and with or without m-GM-CSF) were compared 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 above except that the co-treatment with anti-PD1 was omitted.

[0489] The results of two independent experiments clearly showed that the three viruses tested, COPTG19407, COPTG19421 and COPTG19347, had a 6 pfu. This confirms that the mGM-CSF encoded in COPTG19407, or the use of the weakest promoter in COPTG19421 and COPTG19407, does not impair the antitumor activity of the armed virus. 6 pfu), the number of tumor-free mice at day 80 ranged between 5 / 10 and 7 / 10, depending on the virus and the experiment, compared with 0 / 10 for mice treated with empty virus, as summarized in the table below.

[0490] Table 6 : Effects of the alternative viruses COPTG19407, COPTG19421, and COPTG19347 on tumor growth after intrathoracic injection

[0491]

[0492]

[0493] *HC and LC represent the heavy chain and light chain of m5-B07 mouse anti-mCTLA4 antibody, respectively

[0494] Furthermore, dose escalation of the “empty” virus (VVTG18058) and the COPTG19384 surrogate (COPTG19407) showed that even at relatively low doses (7.5 × 10 4 pfu), the virus-expressing antibody still had significant antitumor activity, with 4 / 10 and 2 / 10 tumor-free mice on days 80-98, while 0 / 10 were treated with VVTG18058 at the same low dose, as shown in the table below.

[0495] Table 7: Effect of the dose of COPTG19407 or VVTG18058 on tumor growth after intrathoracic injection

[0496]

[0497] *HC and LC represent the heavy chain and light chain of m5-B07 mouse anti-mCTLA4 antibody, respectively

[0498] A compilation of survival data (global survival maps compiled from two independent studies) is presented in Fig.33 Statistical analysis was performed using the log-rank test to find out if there was a significant difference in survival rate between the groups.

[0499] Antitumor activity of COPTG19407 compared with the combination of VVTG18058 and m5-B07

[0500] Mice bearing CT26 tumors were established as previously described (Example 5). Briefly, CT26 cells were injected subcutaneously into Balb / C mice. When tumors reached approximately 100 mm 3 The mice were then injected with COPTG19407 (8.5×10 6 pfu, intrathoracic), VVTG18058 (8.5×10 6 pfu, intrathoracic), m5-B07 (10 mg / kg, intraperitoneal), or VVTG18058 (8.5×10 6 pfu, intrathoracic) plus m5-B07 (10 mg / kg, intraperitoneal). Tumor size was then measured twice a week, and when the tumor reached 2000 mm 3 The mice were euthanized at the time of Fig.34 As shown in Figures A to D of Figures 34, when mice were treated with the virus COPTG19407 expressing anti-CTLA-4 and GM-CSF, tumor growth was significantly inhibited, while the combination of unarmed virus plus anti-CTLA4 m5-B07 did not result in improved treatment compared to single agent use. In the groups treated with m5-B07 alone, virus VVTG18058 alone, or a combination of m5-B07 and VVTG18058, only 20% of the mice survived after day 70 ( Fig.34 In contrast, 90% of mice survived for more than 100 days after COPTG19407 administration, demonstrating the efficacy of the vectorization strategy.

[0501] VVTK-RR-encoded anti-CTLA-4 and GM-CSF anti-tumor activity in mice bearing A20 subcutaneous murine B-cell lymphoma Tumor activity

[0502] The A20 cell line is a BALB / c B-cell lymphoma (ATCC TIB-208) derived from a spontaneous reticulocyte neoplasm found in old BALB / cAnN mice. TM ).

[0503] Solution (1):

[0504] By dividing 5×10 6Tumors were induced by subcutaneous injection of A20 cells into the right flank of female Balb / cN mice (Charles River, France). When the average tumor volume reached 95 mm 3 At 4 pm, 50 mice were randomly divided into 5 groups, with 10 animals in each group.

[0505] Mice from Group 1 received intrathoracic administration of vehicle on days 0, 2, and 4,

[0506] Mice from group 2 received intrathoracic administration of 4.75 × 10 6 pfu's VVTG18058,

[0507] Mice from Group 3 received intrathoracic administration of 4.75 × 10 6 pfu of COPTG19407,

[0508] Mice from Group 4 received intraperitoneal administration of anti-PD-1 antibody at a dose of 250 μg on days 7, 10, 14, 17, 21, and 24,

[0509] Mice from Group 5 received intrathoracic administration of 4.75 × 10 6 pfu of COPTG19407, together with anti-PD-1 antibody at a dose of 250 μg administered intraperitoneally on days 7, 10, 14, 17, 21, and 24.

[0510] Antitumor Activity:

[0511] Tumor volume was monitored in all animals throughout the study.The antitumor activity of treatment was based on the evaluation of criteria for tumor doubling time, tumor growth delay, and tumor growth inhibition (T / C %).

[0512] Tumor doubling times were similar for Groups 1, 2, and 4, ranging from 5.14 days (Group 1) to 6.37 days (Group 2). For Group 3, the tumor doubling time could not be accurately calculated as the tumors did not grow exponentially compared to Groups 1, 2, and 4, indicating an increased therapeutic effect. Similarly, only one animal was used in Group 5 to calculate the tumor doubling time. Of the 10 mice in Group 3, 9 tumors regressed by Day 15 and did not grow substantially in size from Day 25 until the end of the study on Day 64. On Day 64, the tumor volume of these 9 mice ranged from 4 mm to 6 mm. 3 (Technical limit of tumor detection) to 59.77mm 3Similarly, in Group 5, 9 mice experienced tumor regression after initiation of treatment, reaching a range of 7.24 to 63.21 mm at the end of the study on day 64. 3 The values ​​of . Fig.35 It can be seen that (groups 1 to 5 correspond to Fig.35 A to E ), tumors did not grow in animals of groups 3 and 5 that received COPTG19407, confirming the strong antitumor activity of the virus expressing antibodies with or without anti-PD1.

[0513] The average target volume of the tumor was estimated to be 300 mm 3 The time taken to reach the target volume of 300 mm was used to calculate tumor growth delay. The results are similar to those obtained by tumor doubling time, as only tumors that reach the target volume of 300 mm can be targeted. 3 This parameter was calculated for tumors in Group 1, 2, and 4, which was not the case for most animals in Group 3 and Group 5. The average tumor growth delays for Groups 1, 2, and 4 were 16, 21, and 17 days, respectively, which were not significantly different from each other. In addition, the average tumor delay for Group 3 (n=2) was 14 days, indicating that the tumors that did grow in this group grew at the same rate as Groups 1, 2, and 4, but these tumors actually regressed in the animals. In contrast, the tumor growth delay of a single tumor grown in Group 5 (n=1) was significantly longer (p≤0.0026) than all other groups, at 43 days.

[0514] 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 had the best T / C% at day 22, which was 34%, indicating a transient marginal anti-tumor activity, but by day 31, this value increased to 71%. Moderate anti-tumor activity was observed in Group 4 (10-30% T / C%). In contrast, Groups 3 and 5 showed significant anti-tumor activity (T / C% less than 10%) from day 27 to day 31 (the last calculable T / C% value).

[0515] Fig.36 Shown are mean tumor volume curves of BALB / cN mice bearing subcutaneous A20 tumors, demonstrating the significant effect of the anti-CTLA4 and Gm-CSF expressing virus COPTG19407 on tumor growth with or without anti-PD1.

[0516] Solution (2):

[0517] By dividing 5×10 6 Tumors were induced by subcutaneous injection of A20 cells into the right flank of female BALB / cN mice. 3When the average volume of 100 μg / mL was measured, 90 animals were randomly divided into 9 groups, with 10 animals in each group.

[0518] Mice from Group 1 received intrathoracic administration of vehicle on days 0, 2, and 4

[0519] Mice from Group 2 received intraperitoneal administration of anti-PD-1 antibody at a dose of 250 μg / mouse / injection on days 0, 4, 7, 10, 14, and 17

[0520] Mice from Group 3 received intraperitoneal administration of 250 μg / mouse / injection of the isotype on days 0, 4, 7, 10, 14, and 17.

[0521] Mice from Group 4 received intrathoracic administration of 1 × 10 5 pfu's VVTG18058

[0522] Mice from Group 5 received intrathoracic administration of 1 × 10 5 pfu of VVTG18058, and received intraperitoneal administration of 250 μg / mouse / injection of the isotype on days 0, 4, 7, 10, 14, and 17.

[0523] Mice from Group 6 received intrathoracic administration of 1 × 10 5 pfu of VVTG18058, and received intraperitoneal administration of anti-PD-1 antibody at a dose of 250 μg / mouse / injection on days 0, 4, 7, 10, 14, and 17

[0524] Mice from group 7 received intrathoracic administration of 1 × 10 5 pfu of COPTG19407

[0525] Mice from Group 8 received intrathoracic administration of 1 × 10 5 pfu of COPTG19407, along with the isotype, received intraperitoneal administration at a dose of 250 μg / mouse / injection on days 0, 4, 7, 10, 14, 17, and 24.

[0526] Mice from group 9 received intrathoracic administration of 1 × 10 5pfu of COPTG19407, together with anti-PD-1 antibody at a dose of 250 μg / mouse / injection administered intraperitoneally on days 0, 4, 7, 10, 14, and 17.

[0527] Antitumor Activity:

[0528] The COPTG19407 dose was suboptimal and exhibited mild antitumor activity similar to anti-PD-1 treatment in terms of tumor volume and mouse survival.

[0529] In contrast, the combination of COPTG19407 and anti-PD-1 showed strong antitumor activity, with smaller tumor volumes compared with the other groups, e.g. Fig.37 As shown in A (compared to mice receiving anti-PD-1 alone or COPTG19407 alone, the mean value was about 630 mm on day 24). 3 and 750mm 3 In comparison, on day 36 it was approximately 290 mm 3 ), and the survival rate of animals is much better, e.g. Fig.37 (In Group 9, 7 animals were alive on Day 57, compared to only two or one in Group 2 or 7, respectively).

[0530] VVTK-RR-encoded anti-CTLA-4 and GM-CSF anti-tumor effects in mice bearing C38 subcutaneous colon tumor cells Tumor activity study

[0531] C38 is from the American Type Culture Collection (ATCC CRL-2779 TM ) of rat colon adenocarcinoma.

[0532] plan:

[0533] Tumor fragments (30-50 mg) were implanted subcutaneously into the right flank of female C57BL / 6J mice (Janvier, France). When the average tumor volume reached approximately 60 mm3, 50 animals were randomly divided into five groups of ten.

[0534] Mice from Group 1 received intrathoracic administration of vehicle on days 0, 2, and 4,

[0535] Mice from group 2 received intrathoracic administration of 4.75 × 10 6 pfu's VVTG18058,

[0536] Mice from Group 3 received intrathoracic administration of 4.75 × 10 6 pfu of COPTG19407,

[0537] Mice from Group 4 received intraperitoneal administration of 250 μg of murine anti-PD-1 antibody on days 7, 10, 14, 17, 21, and 24,

[0538] Mice from Group 5 received intrathoracic administration of 4.75 × 10 6 pfu of COPTG19407, together with anti-PD-1 antibody at a dose of 250 μg administered intraperitoneally on days 7, 10, 14, 17, 21, and 24.

[0539] Antitumor Activity:

[0540] As before, tumor volume was monitored in all animals throughout the study. Tumor doubling times were similar in Groups 1 and 2, approximately 6.7 days. Tumor doubling times were longer in Group 4 (n=5) (10.4 days), but not significantly different between groups. For Groups 3 and 5, tumor doubling times were calculable, but with fewer animals (n=2), as the majority of tumors in mice in these groups did not grow exponentially compared to Groups 1, 2, and 4, indicating increased efficacy of the treatment. Fig.38 It can be seen that in Group 3, 8 of the 10 mice had tumors that regressed from Day 15 with no substantial growth in size, and five mice had no detectable tumors at the end of the study on Day 61. Similarly, in Group 5, 8 mice had tumors that regressed after the start of treatment, reaching a range of 0 (n=2) to 47.82 mm at the end of the study on Day 61. 3 The value of .

[0541] The average target volume of the tumor was estimated to be 300 mm 3 The time taken to reach the target volume of 300 mm was used to calculate tumor growth delay. The results are similar to those obtained by tumor doubling time, as only tumors that reach the target volume of 300 mm can be targeted. 3 This parameter was calculated for tumors in Groups 1, 2, and 4, which was not the case for most animals in Groups 3 and 5. There were no significant differences between the groups. The mean tumor growth delays for Groups 1, 2, and 4 (n=5) were 23 to 27 days, respectively. In addition, the mean growth delays for Groups 3 (n=2) and 5 (n=3) were 18 and 24 days, respectively. This suggests that the tumors that did grow in these two groups grew at a similar rate to those in Groups 1, 2, and 4.

[0542] 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 any tumor growth inhibition, as T / C% remained above 100% for the duration of the study. In contrast, groups 3, 4, and 5 all showed significant antitumor activity (T / C% less than 10%) from day 31 (group 3 only) to day 42 (the last calculable T / C% value).

[0543] Fig.39 Mean tumor volume curves of C57BL / 6 mice bearing subcutaneous C38 tumors are shown, demonstrating the significant effect of the anti-CTLA4 / GM-CSF expressing virus COPTG19407 on tumor growth with or without anti-PD1.

[0544] VVTK-RR-encoded anti-CTLA-4 and GM-CSF antibodies in mice bearing EMT6 subcutaneous breast tumor cells Tumor activity studies

[0545] EMT6 is derived from ATTC (ATCC CRL-2755 TM ) of rat breast cancer.

[0546] plan:

[0547] Female BALB / cByJ mice (Charles River, France) were injected subcutaneously with 1 × 10 6 EMT6 cells were used to induce tumors. When the average tumor volume reached about 51 mm 3 At the time of the experiment, fifty mice were randomly divided into five groups according to the individual tumor volume, with ten mice in each group.

[0548] Mice from Group 1 received intrathoracic administration of vehicle on days 0, 2, and 4,

[0549] Mice from group 2 received intrathoracic administration of 4.75 × 10 6 pfu's VVTG18058,

[0550] Mice from Group 3 received intrathoracic administration of 4.75 × 10 6 pfu of COPTG19407,

[0551] Mice from Group 4 received intraperitoneal administration of anti-PD-1 antibody at a dose of 250 μg on days 7, 10, 14, 17, 21, and 24,

[0552] Mice from Group 5 received intrathoracic administration of 4.75 × 10 6pfu of COPTG19407, together with anti-PD-1 antibody at a dose of 250 μg administered intraperitoneally on days 7, 10, 14, 17, 21, and 24.

[0553] Tumor volume was monitored in all animals throughout the study by evaluating the criteria of tumor doubling time, tumor growth delay, and tumor growth inhibition (T / C %).

[0554] Antitumor Activity:

[0555] The tumor doubling time for groups 1, 2, and 4 was similar at approximately 5.4 days. For group 3, the tumor doubling time could not be calculated because most tumors did not grow exponentially compared to groups 1, 2, and 4, indicating increased efficacy of the treatment. A similar effect was observed in group 5, where only one animal was used to calculate the tumor doubling time. As can be seen from the graph of individual tumor volumes ( Fig.40 ), in Group 3, 8 of the 10 mice had tumors that regressed from day 15 without substantial growth in size, and seven mice had no detectable tumors at the end of the study on day 61. Similarly, in Group 5, 9 mice had tumors that regressed after the start of treatment, reaching a range of 0 (n=8) to 13.24 mm at the end of the study on day 56. 3 The value of .

[0556] The average target volume of the tumor was estimated to be 200 mm 3 The time taken to reach the target volume of 200 mm was used to calculate tumor growth delay. The results are similar to those obtained by tumor doubling time, as only tumors that reach the target volume of 200 mm can be targeted. 3 This parameter was calculated for tumors in groups 1, 2, and 4, which was not the case for most animals in groups 3 and 5. The average tumor growth delay for groups 1, 2, and 4 was approximately 19 days. In addition, the average growth delay for groups 3 and 5 (n=2 for both groups) was 24 days and 12 days, respectively. This suggests that the tumors that did grow in these two groups grew at a similar rate to those in groups 1, 2, and 4. There were no significant differences between the groups.

[0557] 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 on day 28, but increased to 79% on day 31. Group 4 showed no anti-tumor activity, with T / C%>60% for the duration of the study. In contrast, groups 3 and 5 showed significant anti-tumor activity (T / C% less than 10%) from day 24 to day 31 (the last calculable T / C% value).

[0558] Fig.41Shown are mean tumor volume curves of BALB / cByJ mice bearing subcutaneous EMT6 tumors, demonstrating a significant effect of the anti-CTLA4 / GM-CSF expressing virus COPTG19407 on tumor growth with or without anti-PD1.

[0559] CT26 attacks again

[0560] In use 10 4、 10 5 or 10 6 pfu of COPTG19421 or 10 6 Surviving Balb / c mice challenged with CT26 tumor cells were treated with pfu of COPTG19407 and then re-challenged with CT26 tumor cells or challenged with RENCA cells (renal adenocarcinoma cells: control) to investigate whether specific anti-tumor immune responses were enhanced.

[0561] Table 8: Effect of Rechallenge with CT26 Tumor Cells or Challenge with Renca Tumor Cells on the Number of Tumor-free Mice

[0562] Group No tumor / total number of mice CT26 control natural 0 / 5 <![CDATA[VVTG18058 10 5 +CT26 attacks again]]> 0 / 1 <![CDATA[COPTG19421 10 6 +CT26 attacks again]]> 3 / 3 <![CDATA[COPTG19421 10 6 +RenCa Attack]]> 0 / 2 <![CDATA[COPTG19407 10 4 +CT26 attacks again]]> 1 / 1 <![CDATA[COPTG19407 10 4 +RenCa Attack]]> 0 / 1 <![CDATA[COPTG19407 10 5 +CT26 attacks again]]> 1 / 2 <![CDATA[COPTG19407 10 5 +RenCa Attack]]> 0 / 2 <![CDATA[COPTG19407 10 6 +CT26 attacks again]]> 2 / 4 <![CDATA[COPTG19407 10 6 +RenCa Attack]]> 0 / 3

[0563] The results presented in Table 8 show that 0 / 8 mice receiving COPTG19421 or COPTG19407 after RenCa challenge were tumor-free, while 7 / 10 mice receiving COPTG19421 or COPTG19407 after CT26 re-challenge were tumor-free. This suggests that COPTG19421 and COPTG19407 enhance specific immune memory against CT26 cells.

Claims

1. An antibody molecule that specifically binds to CTLA-4 and has an improved depletion effect on CTLA-4 positive cells compared to ipilimumab.

2. The antibody molecule according to claim 1, which has an improved depletion effect on CD4-positive cells compared to ipilimumab. 3 . The antibody molecule according to claim 1 , which has an improved depletion effect on Tregs compared to ipilimumab.

4. The antibody molecule according to any one of claims 1 to 3, which is considered to have an improved depletion effect on CTLA-4 positive cells, CD4 positive cells and / or Tregs compared to ipilimumab if the antibody molecule gives improved depletion in: (i) an in vitro ADCC test performed using a NK-92 cell line stably transfected to express the CD16-158V allele and GFP, wherein the ADCC test comprises the following consecutive steps: 1) Isolate CTLA-4 positive cells, CD4 positive cells or Treg as target cells from the peripheral blood of healthy donors; 2) then stimulating the target cells with CD3 / CD28 and rhIL-2; 3) The target cells are then pre-incubated with the antibody molecule and then mixed with NK cells; 4) The target cells were then incubated in RPMI 1640+GlutaMAX medium containing 1HEPES buffer, sodium pyruvate and FBS low IgG; 5) determination of lysis by flow cytometry; 6) repeating or performing steps 1-5 in parallel, wherein ipilimumab is used instead of the antibody molecule in step 3; and 7) comparing the result of the lysis of the antibody molecule with the result of the lysis of ipilimumab, and the improved lysis of the antibody molecule compared to ipilimumab indicates that the antibody molecule has an improved depletion effect on CTLA-4 positive cells, CD4 positive cells and / or Tregs; and / or (ii) in vivo testing in a PBMC-NOG / SCID model, wherein the in vivo testing comprises the following consecutive steps: 1) Isolate, wash and resuspend human PBMCs in sterile PBS; 2) Injecting the cell suspension from step 1) intravenously into NOG mice; 3) isolating the spleen from the NOG mouse and preparing a single cell suspension; 4) resuspending the cell suspension from step 3) in sterile PBS; 5) Inject the suspension from step 4 intraperitoneally into SCID mice; 6) then treating the SCID mice with the antibody molecule, ipilimumab or an isotype control monoclonal antibody; 7) collecting the intraperitoneal fluid of the treated SCID mice; 8) Identify and quantify 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 the lower number of CTLA-4-positive cells, CD4-positive cells and / or Tregs in the intraperitoneal fluid from the mice treated with the antibody molecule to be tested compared with the number of CTLA-4-positive cells, CD4-positive cells and / or Tregs in the intraperitoneal fluid from the mice treated with ipilimumab indicates that the antibody molecule has an improved depletion effect on CTLA-4-positive cells, CD4-positive cells and / or Tregs compared with ipilimumab.

5. The antibody molecule according to any one of claims 1 to 4, wherein the antibody molecule is selected from the group consisting of antibody molecules comprising 1 to 6 of the CDRs, wherein the CDRs are selected from the group consisting of SEQ ID. No: 3, 6, 8, 10, 12 and 14.

6. The antibody molecule according to any one of claims 1 to 5, wherein the antibody molecule is selected from the group consisting of antibody molecules comprising 1 to 6 of CDRV, VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, and VL-CDR3, wherein VH-CDR1, if present, is selected from the group consisting of SEQ ID.No: 15, 22, 29 and 35; wherein VH-CDR2, if present, is selected from the group consisting of SEQ ID.No: 16, 23, 30 and 36; wherein VH-CDR3, if present, is selected from the group consisting of SEQ ID.No: 17, 24, 31 and 37; wherein VL-CDR1, if present, is selected from the group consisting of SEQ ID.No: 10 and 38; wherein VL-CDR2, if present, is selected from the group consisting of SEQ ID.No: 18, 25, 32 and 39; Wherein VL-CDR3, if present, is selected from the group consisting of SEQ ID. No: 19, 26 and 40.

7. The antibody molecule according to any one of claims 1 to 6, wherein the antibody molecule comprises 6 CDRs having SEQ ID. NO: 15, 16, 17, 10, 18 and 19 or 6 CDRs having SEQ ID. NO: 22, 23, 24, 10, 25 and 26.

8. An antibody molecule that specifically binds to CTLA-4, wherein the antibody molecule comprises 6 CDRs having SEQ ID. NO: 15, 16, 17, 10, 18 and 19, or 6 CDRs having SEQ ID. NO: 22, 23, 24, 10, 25 and 26.

9. The antibody molecule according to any one of claims 1 to 8, wherein the antibody molecule comprises a variable heavy chain selected from the group consisting of SEQ.ID.NO: 20 and 27 and / or a variable light chain selected from the group consisting of SEQ.ID.NO: 21 and 28.

10. The antibody molecule according to any one of claims 1 to 9, 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.

11. The antibody molecule according to any one of claims 1 to 6, wherein the antibody molecule is an antibody molecule capable of competing with the antibody molecule according to any one of claims 7 to 10 for binding to CTLA-4.

12. The antibody molecule according to any one of claims 1 to 11, wherein the antibody molecule is selected from the group consisting of full-length IgG antibody, chimeric antibody, single-chain antibody, Fab, Fv, scFv, Fab' and (Fab')2.

13. The antibody molecule according to any one of claims 1 to 13, which binds to human CTLA-4 (hCTLA-4) and / or rhesus monkey CTLA-4 (cmCTLA-4) and / or mouse CTLA-4 (mCTLA-4).

14. The antibody molecule according to any one of claims 1 to 13, which does not bind to human CD28. 15 . The antibody molecule according to claim 1 , wherein the antibody molecule is selected from the group consisting of human IgG antibody, humanized IgG antibody and human IgG antibody. The antibody molecule according to claim 15 , wherein the antibody molecule is a human IgG1 antibody.

17. The antibody molecule according to any one of claims 1 to 16, wherein the antibody molecule is a monoclonal antibody.

18. An isolated nucleotide sequence encoding the antibody molecule according to any one of claims 1 to 17.

19. The isolated nucleotide sequence according to claim 18, comprising or consisting of: A sequence selected from the group consisting of SEQ ID 45-52.

20. A plasmid comprising the nucleotide sequence according to claim 18 or 19.

21. A virus comprising the nucleotide sequence according to claim 18 or 19 or the plasmid according to claim 20.

22. The virus according to claim 21, which is an oncolytic virus, and preferably an oncolytic poxvirus.

23. The virus according to claim 22, wherein the poxvirus belongs to the subfamily Chordopoxvirinae, more preferably to the genus Orthopoxvirus, preferably selected from the group consisting of vaccinia virus, cowpox virus, canarypox virus, mousepox virus and myxoma virus.

24. The virus according to claim 23, wherein the oncolytic virus is a vaccinia virus that is defective in both thymidine kinase (TK) and / or ribonucleotide reductase (RR) activity and comprises a nucleotide sequence encoding SEQ.ID.NO:20 and ID.NO:21 or SEQ.ID.NO:53 and ID.NO:

54.

25. The virus of claim 24, wherein the oncolytic vaccinia virus further comprises a nucleotide sequence encoding GM-CSF, with a particular preference for 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).

26. The virus according to any one of claims 21 to 25, wherein the cassette encoding the heavy chain is inserted at the J2R locus, and the cassette encoding the light chain is inserted at the I4L locus.

27. A cell comprising a nucleotide sequence according to claim 18 or 19, or a plasmid according to claim 20, or a virus according to any one of claims 21 to 26.

28. An antibody molecule according to any one of claims 1 to 17, a nucleotide sequence according to claim 18 or 19, a plasmid according to claim 20, a virus according to any one of claims 21 to 26 or a cell according to claim 27 for use in medicine.

29. The antibody molecule according to any one of claims 1 to 17, the nucleotide sequence according to claim 18 or 19, the plasmid according to claim 20, the virus according to any one of claims 21 to 26 or the cell according to claim 27 for use in treating cancer.

30. Use of an antibody molecule according to any one of claims 1 to 17, a nucleotide sequence according to claim 18 or 19, a plasmid according to claim 20, a virus according to any one of claims 21 to 26 or a cell according to claim 27 for the manufacture of a pharmaceutical composition for the treatment of cancer.

31. A pharmaceutical composition comprising or consisting of: An antibody molecule according to any one of claims 1 to 17, a nucleotide sequence according to claim 18 or 19, a plasmid according to claim 20, a virus according to any one of claims 21 to 26 or a cell according to claim 27, and optionally a pharmaceutically acceptable diluent, carrier, vehicle and / or excipient.

32. The pharmaceutical composition according to claim 31 , for use in the treatment of cancer.

33. A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of an antibody molecule according to any one of claims 1 to 17, a nucleotide sequence according to claim 18 or 19, a plasmid according to claim 20, a virus according to any one of claims 21 to 26, a cell according to claim 27, or a pharmaceutical composition according to claim 31.

34. An antibody molecule for use according to claim 24, a nucleotide sequence for use according to claim 29, a plasmid for use according to claim 29, a virus for use according to claim 29, a cell for use according to claim 29, a use according to claim 30, a pharmaceutical composition according to claim 32, or a method according to claim 33, wherein the cancer is a solid cancer.

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