RU2802450C2 - New antibodies and nucleotide sequences and their use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to an antibody molecule that specifically binds to CTLA-4, as well as to a pharmaceutical composition containing it. Also a nucleic acid encoding the above molecule, as well as a plasmid, a virus, and a cell containing it are disclosed.

EFFECT: invention is effective for the treatment of cancer.

33 cl, 41 dwg, 8 tbl, 9 ex

Description

Field of technology to which the invention relates

This invention relates to novel anti-CTLA-4 antibody molecules, nucleotide sequences and expression vectors (eg, oncolytic virus) encoding such antibody molecules for use in cancer therapy. The new antibodies improved Treg depletion compared with ipilimumab.

Prerequisites for creating an invention

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 an inhibitory signal to T cells. It is homologous to the T cell co-stimulatory protein CD28, and both CTLA-4 and CD28 bind to CD80 (also designated B7-1) and CD86 (also designated B7-2). CTLA4 is also found in regulatory T cells (Treg) and contributes to its inhibitory function. The CTLA-4 protein contains an extracellular V domain, a transmembrane domain, and a cytoplasmic tail.

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

Promising clinical results with immunomodulatory monoclonal antibodies (mAbs) have renewed belief that the immune system is the key to fighting cancer. The classification of these mAbs into checkpoint blockers (antagonists) or co-stimulatory molecule activators (agonists) has recently been questioned as examples of both types have been found to be able to fight tumors by depleting suppressive regulatory T cells (Tregs).

Immunomodulatory mAbs such as ipilimumab and other anti-CTLA4 antibodies have shown positive results when tested in difficult-to-treat malignancies, although in a minority of patients (Hodi, F. S., et al. 2010, N Engl J Med 363(8): 711-723 ; Beatty, G. L., et al. 2011, Science 331(6024): 1612-1616; Brahmer, J. R., et al. 2012, N Engl J Med 366(26): 2455-2465; Topalian, S. L., et al. 2012 N Engl J Med 366(26): 2443–2454). These promising results have helped bolster belief that the immune system may be the key to fighting cancer. These mAbs were designed to target key molecular regulators of T cells or APC antigen-presenting cells and to enhance anticancer immunity by blocking inhibitory signals (checkpoint blockers) or delivering co-stimulatory signals (agonists). Recently, this binary classification was called into question when it was discovered that the therapeutic activity of anti-CTLA4 antibodies, anti-GITR antibodies and anti-OX40 antibodies, which are all targets of T lymphocytes, involves deletion of suppressive regulatory CD4+ T cells depending on cooperative interaction of activating FcγRs (Bulliard, Jolicoeur et al. 2013, Marabelle, A., et al. 2013, J Clin Invest 123 (6): 2447-2463; Simpson, TR, et al. J Exp Med 210 (9): 1695-1710).

The anti-CTLA-4 monoclonal antibody, ipilimumab (YERVOY®; previously designated 10D1, BMS-734016, MDX 101, MDX-010, MDX-CTLA-4 and MDX-CTLA4), has been approved in several countries for the treatment of melanoma and other diseases and is undergoing clinical trials for other indications (Weber 2008, Overcoming immunologic tolerance to melanoma: targeting CTLA-4 with ipilimumab (MDX-010) Oncologist, 13 (Suppl 4):16-25). Ipilimumab is a fully human monoclonal anti-CTLA-4 antibody (IgG1κ) produced in Chinese hamster ovary cells using recombinant DNA technology. It has 477202-00-9 and 6T8C155666. Ipilimumab is further defined in US Pat. No. 9,789,182, which also specifies the heavy and light chain sequences of ipilimumab (as SEQ ID NOs: 17 and 18, respectively), the VH and/or VL region sequences (as SEQ ID NO: 19 and SEQ ID NO: 20, respectively) and CDR sequences (heavy chain CDR1, CDR2 and CDR3 as indicated in SEQ ID NOs: 21, 22 and 23, and light chain CDR1, CDR2 and CDR3 as indicated in SEQ ID NOs: 24, 25 and 26) .

The 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. 37(5): 473-484.; Blank et al 2015, Therapeutic use of anti-CTLA-4 antibodies. International Immunology, 27(1): 3-10).

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

WO 93/00431 relates to a CTLA4 receptor protein, a CTLA4Ig fusion protein, and a method for regulating cellular interactions using such a fusion protein or monoclonal antibody.

WO 97/20574 relates to a blockade of T cell downregulation associated with CTLA-4 signaling and to a CTLA-4 blocking agent other than an anti-CTLA-4 extracellular domain antibody that enhances the response of mammalian T cells to an antigenic stimulus or reduces the growth of tumor cells in a mammalian host.

WO 00/37504 relates to human anti-CTLA-4 antibodies and the use of such antibodies in the treatment of cancer. WO 00/37504 also relates to the above-mentioned human monoclonal antibody tremelimumab, designated in this patent application 11.2.1.

WO 01/14424 also relates to human antibodies that specifically bind to human CTLA-4, and their use for the treatment of human diseases and infections such as cancer. WO 01/14424 also relates to the human monoclonal antibody ipilimumab mentioned above and further discussed below, designated in this patent application 10D1.

The essence of the invention

This invention provides antibody molecules that specifically bind to CTLA-4 and have an improved depletion effect on CTLA-4 positive cells compared to ipilimumab.

Furthermore, the present invention provides antibody molecules that specifically bind to CTLA-4, wherein the antibody molecules contain 6 CDRs having the sequences of SEQ ID NO: 15, 16, 17, 10, 18 and 19 or 6 CDRs having the sequences of SEQ ID NO: 22, 23, 24, 10, 25 and 26.

In addition, the present invention relates to isolated nucleotide sequences encoding the above antibody molecules.

In addition, the present invention relates to plasmids containing the above nucleotide sequences.

In addition, the present invention relates to viruses, such as oncolytic viruses, containing the above nucleotide sequences or the above plasmids.

In addition, the present invention relates to cells, such as CAR T cells, containing the above nucleotide sequences or the above plasmids.

In addition, the present invention relates to the above antibody molecules, nucleotide sequences, plasmids and/or cells for medical use.

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

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

In addition, this invention relates to pharmaceutical compositions containing at least one of the above antibody molecules, nucleotide sequences, plasmids, viruses and/or cells and, optionally, a pharmaceutically acceptable diluent, carrier or excipient.

In addition, this invention provides methods 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.

In addition, this invention relates to an antibody molecule, an antibody molecule for use, an isolated nucleotide sequence, an isolated nucleotide sequence for use, a plasmid, a plasmid for use, a virus, a virus for use, a cell, a cell for use, a pharmaceutical composition or a method of treatment, such as described herein with reference to the detailed description, examples and/or figures.

Detailed description of this invention

CTLA-4 positive cells include regulatory T cells, Treg cells, Treg or T _reg (formerly known as suppressor T cells, sometimes also called suppressive regulatory T cells) which are a subpopulation of T cells capable of suppressing other immune cells in conditions normal immunity and in conditions of pathological immunity. 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 in that Tregs are also FOXP3-positive (FOXP3+), whereas non-Treg CD4+ are FOXP3-negative (FOXP3-).

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 interactions of CTLA-4 with B7.1 and B7.2. Thus, these antibodies may consequently help overcome CTLA-4-induced suppressive effects on effector T cell proliferation.

By Treg depletion or Treg depletion we mean herein the depletion, removal or elimination of Tregs through physical clearance of cells. Specifically, we refer to the depletion of intratumoral Tregs. Treg depletion can be achieved through ADCC, i.e. antibody-dependent cell-mediated cytotoxicity or antibody-dependent cellular cytotoxicity, and/or ADCP (antibody dependent cellular phagocytosis), i.e. antibody-dependent cellular phagocytosis. This means that when an antibody molecule 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 binding results in Treg depletion. In some embodiments of the present invention, CTLA-4 is preferentially expressed on tumor-infiltrating lymphocytes in the tumor microenvironment or on tumor cells.

ADCC is an immune mechanism by which effector cells bearing the Fc receptor can recognize and destroy antibody-coated target cells expressing tumor-derived antigens on their surface, i.e. in this case CTLA-4. ADCP is a similar mechanism, although it results in the killing of target cells through phagocytosis rather than cytotoxicity.

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

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

The term "antibody molecule" as used herein includes full-length or full-length antibodies, as well as functional fragments of full-length antibodies and derivatives of such antibody molecules.

Functional fragments of a full-length antibody have the same antigen-binding characteristics as the corresponding full-length antibody and include either the same variable domains (ie, VH and VL sequences) and/or the same CDR sequences as the corresponding full-length antibody. That a functional fragment has the same antigen binding characteristics as the corresponding full-length antibody means that it binds to the same epitope on the target as the full-length antibody. Such a functional fragment may correspond to the Fv region of a full-length antibody. Alternatively, such a fragment may be a Fab, also designated F(ab), which is a monovalent antigen-binding fragment containing no Fc regions, or F(ab') ₂ , which is a bivalent antigen-binding fragment containing two antigen-binding Fab sites linked to each other disulfide bonds, or F(ab'), i.e. monovalent variant F(ab') ₂ . Such a fragment may also be a single chain variable fragment (scFv).

A functional fragment does not always contain all six CDRs of the corresponding full-length antibody. It should be noted that molecules containing three or fewer CDR regions (in some cases, even only one CDR region or part thereof) are able to retain the antigen binding activity of the antibody from which the CDRs are derived. For example, Gao et al., 1994, J. Biol. Chem., 269: 32389-93 indicates that the entire VL chain (including all three CDR regions) has high affinity for its substrate.

Molecules containing two CDR regions are described, for example, in Vaughan & Sollazzo 2001, Combinatorial Chemistry & High Proposal Screening, 4: 417-430. Page 418 (right column - 3 "Our Design Strategy") describes a mini-antibody comprising only the H1 and H2 CDR hypervariable regions interspersed within the framework regions. This mini-antibody is described as being able to bind to a target. Based on Pessi et al., 1993, Nature, 362: 367-9, and Bianchi et al., 1994, J. Mol. Biol., 236: 649-59, referenced by Vaughan and Sollazzo and describe the H1 and H2 miniantibodies and their properties in more detail. Qiu et al., 2007, Nature Biotechnology, 25:921-9 showed that a molecule consisting of two linked CDR regions is capable of binding antigen. Quiocho 1993, Nature, 362: 293-4, gives a brief description of the "mini-antibody" technology. Ladner 2007, Nature Biotechnology, 25:875-7, comments that molecules containing two CDR regions are able to retain antigen binding activity.

Antibody molecules containing a single CDR region are described, for example, in Laune et al., 1997, JBC, 272: 30937-44, which shows that a number of hexapeptides derived from the CDR region exhibit antigen binding activity, and notes that synthetic peptides of a complete single CDR region exhibit strong binding activity. Monnet et al., 1999, JBC, 274: 3789-96, show that a number of 12-mer peptides and their associated framework regions have antigen-binding activity, and comment that the CDR3-like peptide alone is capable of binding antigen. In Heap et al., 2005, J. Gen. Virol., 86: 1791-1800, a “microantibody” (a molecule containing a single CDR region) is reported to be able to bind antigen, and a cyclic peptide from an anti-HIV antibody is shown to have antigen-binding activity and function. Nicaise et al., 2004, Protein Science, 13: 1882-91, showed that a single CDR region can confer antigen-binding activity and affinity for its lysozyme antigen.

Thus, antibody molecules having five, four, three, or fewer CDR regions are capable of retaining the antigen-binding properties of the full-length antibodies from which they are derived.

Such an 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 characteristics as the corresponding full-length antibody in the sense that it binds to the same epitope on the target as the full-length antibody.

Thus, the term “antibody molecule”, which is used in this application, refers to all types of antibody molecules and their functional fragments, as well as their derivatives, 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 (Fv), single chain variable fragments (scFv fragments) including divalent single chain variable fragments (di-scFv) and disulfide-linked variable fragments, Fab fragments, F( ab') ₂ , Fab' fragments, antibody heavy chains, antibody light chains, antibody heavy chain homodimers, antibody light chain homodimers, antibody heavy chain heterodimers, antibody light chain heterodimers, antigen-binding functional fragments of such homo- and heterodimers.

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

In some embodiments of the present invention, the antibody is human IgG1. It is known to those in the field of immunology that murine immunoglobulin IgG2a and human immunoglobulin IgG1 interact productively with activating Fc-gamma receptors and demonstrate the ability to activate the deletion of target cells through activation of activating immune cells bearing the Fc-gamma receptor (for example, macrophages and NK- cells), such as ADCP and ADCC. Thus, while murine immunoglobulin IgG2a is the preferred isotype for deletion in mice, human IgG1 is the preferred isotype for deletion in humans. In contrast, it is known that optimal co-stimulation of agonist receptors of the TNFR superfamily, for example 4-1BB, OX40, TNFRII, CD40, depends on the interaction of antibodies with inhibitory FcγRII. In mice, the IgG1 isotype, which binds predominantly to the inhibitory Fc-gamma receptor (FcγRIIB) and only weakly to activating Fc-gamma receptors, is known to be optimal for the co-stimulatory activity of mAbs targeting the TNFR superfamily. Although a direct human equivalent of the mouse IgG1 isotype has not been described, antibodies can be engineered to exhibit similar enhanced binding to inhibitory rather than activating human Fc-gamma receptors. Such engineered TNFR superfamily-targeted antibodies also have improved co-stimulatory activity in vivo in transgenic mice engineered to express activating and inhibitory human Fc-gamma receptors (Dahan et al, 2016, Therapeutic Activity of Agonistic, Human Anti-CD40 Monoclonal Antibodies Requires Selective FcγR Engagement Cancer Cell 29(6):820–31).

As indicated above, the present invention includes various types and forms of antibody molecules, which should be known to one skilled in the field of immunology. It is well known that antibodies used for therapeutic purposes are often modified with additional components that change the properties of the antibody molecule.

Accordingly, this application recognizes that the antibody molecule of the present invention or the antibody molecule used in accordance with the present invention (for example, a monoclonal antibody molecule and/or a polyclonal antibody molecule and/or a bispecific antibody molecule) contains a detectable fragment of the molecule and/ or a cytotoxic fragment of a molecule.

By “detectable molecular fragment” we mean one or more groups consisting of: an enzyme; radioactive atom; fluorescent fragment of a molecule; chemiluminescent fragment of a molecule; bioluminescent fragment of a molecule. The detectable fragment of the molecule allows the antibody molecule to be visualized in vitro and/or in vivo and/or ex vivo.

By "cytotoxic moiety" we mean a radioactive moiety and/or an enzyme, for example, the enzyme being a caspase; and/or a toxin, for example, wherein the toxin is a bacterial toxin or poison; wherein the cytotoxic fragment is capable of inducing cell lysis.

It is also contemplated by this invention that the antibody molecule may be in isolated form and/or purified form, and/or may be PEGylated.

As discussed above, CDR antibodies bind to the target antibody. The assignment of amino acids to each CDR region described in this application corresponds to the definitions according to the work of Kabat EA et al. 1991, in Sequences of Proteins of Immulogical Interest, fifth edition, NIH Publication No. 91-3242, pp xv-xvii.

As may be known to one skilled in the art, there are other methods for assigning amino acids to each CDR region. For example, the International Information System for Immunogenetics (IMGT(R)) (http://www.imgt.org/ and Lefranc and Lefranc “The Immunoglobulin FactsBook” published by Academic Press, 2001).

In a further embodiment of the present invention, the antibody molecule of the present invention or used in accordance with the present invention is an antibody molecule that is capable of competing with the specific antibodies described herein, such as antibody molecules containing the sequences of SEQ ID NO: 15, 16, 17, 10, 18 and 19 or the sequences SEQ ID NO: 22, 23, 24, 10, 25 and 26.

By “capable of competing for” is meant that the competing antibody is capable of inhibiting or otherwise interfering, at least partially, with the binding of an antibody molecule, as defined herein, to a particular target.

For example, such a competitive antibody molecule may be capable of inhibiting the binding of an antibody molecule described herein by at least about 10%; for example, 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 inhibit the ability of an antibody described herein to interfere with or reduce binding to a specific target at least about 10%, for example at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100%.

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

ELISA assays can be used to evaluate epitope-modifying or blocking antibodies. Additional methods useful for detecting a competing antibody are described in Antibodies: A Laboratory Manual, Harlow & Lane, which is incorporated herein by reference (for example, see page 567 - 569, 574 - 576, 583 and 590 - 612, 1988, CSHL, NY, ISBN 0-87969-314-2).

It is well known that an antibody binds specifically to a particular target molecule or antigen, and that this means that the antibody preferentially and selectively binds its target rather than a molecule that is not a target.

The targets of the CTLA-4 antibodies of the present invention or antibodies used in accordance with the present invention are expressed on the surface of cells, i.e. they are a cell surface antigen that may include an epitope (also known in the context of this application as a cell surface epitope) for that antibody. Cell surface antigen and epitope are terms that can be easily understood by one skilled in the field of immunology or cell biology.

By "cell surface antigen" as used herein is meant that a cell surface antigen, or at least an epitope thereof, to which an antibody molecule described herein is exposed on the extracellular side of a cell membrane.

Methods for assessing protein binding are known to those skilled in the field of biochemistry and immunology. It would be desirable for those skilled in the art to be able to use these methods to assess the binding of an antibody to a target and/or the binding of the Fc domain of an antibody to an Fc receptor, and to assess the relative potency or specificity of, or inhibition of, or reduction in, or interference with, these interactions. Examples of methods that can be used to assess protein binding include immunoassays, BIAcore cell-cell interaction assays, Western blotting, radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA) (for a discussion of antibody specificity, see Fundamental Immunology Second Edition, Raven Press, New York, on pages 332-336 (1989) (for discussion of antibody specificity, see Fundamental Immunology, Second Edition, Raven Press, New York) (1989).

Accordingly, as used herein, both “antibody molecule that specifically binds CTLA-4” and “anti-CTLA-4 antibody molecule” refer to an antibody molecule that specifically binds a CTLA-4 target but does not bind a non-target. or binds to the non-target more weakly (eg, with lower affinity) than the target.

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

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

This application also includes the concept that the antibody specifically binds to a CTLA-4 target at least twice as strong, or at least five times as strong, or at least 10 times as strong, or at least 20 times as strong , or at least 50 times stronger, or at least 100 times stronger, or at least 200 times stronger, or at least 500 times stronger, or at least about 1000 times stronger than with non-target.

In addition, this application includes the concept that an antibody specifically binds to a CTLA-4 target if it binds to the target with a K _d of 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 at least about 10 ^-12 K _d , or at least about 10 ^-13 K _d , or at least about 10 ^-14 K _d , or at least about 10 ^-15 K _d .

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

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

In some embodiments of the present invention, the CTLA-4 positive cells are CD4 positive (CD4+) cells, i.e. cells expressing CD4.

In some embodiments of the present invention, the CTLA-4 positive cells are both CD4 positive and FOXP3 positive, i.e. express both CD4 and FOXP3. These cells are Tregs. CD8-positive T cells also express CTLA-4, but Tregs express significantly higher levels of CTLA-4 than CD8-positive T cells. This makes Tregs more susceptible to depletion compared to low-expressing CD8+ cells.

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

Thus, in the tumor microenvironment, Tregs will be the cells that have the highest expression of CTLA-4, causing antibody molecules that specifically bind to CTLA-4 (or anti-CTLA-4 antibody molecules) to have a Treg-depleting effect. This is discussed in more detail below, for example in Example 4 and in connection with FIG. 13.

In some embodiments of the present invention, the CTLA-4 positive cells will be Tregs in the solid tumor. Such Tregs will have very high expression of CTLA-4, and therefore administration of antibody molecules that specifically bind to CTLA-4 will preferentially deplete such Tregs.

As mentioned above, the anti-CTLA-4 antibody molecules described herein are Treg depleting antibody molecules, which means that when administered to a subject such as a human, such antibody molecule specifically binds to CTLA-4 expressed on the surface of Tregs, and this binding leads to Treg depletion.

To decide whether an antibody molecule is an antibody molecule that has an improved depleting effect on CTLA-4-positive cells compared to ipilimumab as stated herein, an in vitro antibody-dependent cellular cytotoxicity (ADCC) assay or an in vivo test in a PBMC model can be used. -NOG/SCID.

An in vitro ADCC test, which is performed using the HK-92 cell line stably transfected to express the CD16-158V allele along with GFP, and the ADCC test includes the following seven sequential steps:

1) CTLA-4-positive cells, CD4-positive cells or Tregs as target cells are isolated from the peripheral blood of healthy donors. This isolation can be performed using a CD4+ T cell isolation kit such as a commercial kit from Miltenyi Biotec.

2) The target cells are then stimulated, eg for 48 hours, with CD3/CD28, eg using CD3/CD28 Dynabeads® and rhIL-2, such as 50 ng/ml rhIL-2. Stimulation can be performed at 37°C.

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

4) The target cells are then incubated for a suitable time, for example 4 hours, in RPMI 1640 + GlutaMAX medium containing HEPES buffer, sodium pyruvate and low IgG FBS. RPMI 1640 + GlutaMAX media can contain 10 mM HEPES buffer, 1 mM sodium pyruvate and 10% FBS low IgG, and the effector cell:target cell ratio can be 2:1.

5) Lysis is determined by flow cytometry.

6) Steps 1-5 are repeated or performed in parallel using ipilimumab instead of the antibody molecule being tested in step 3.

7) The results of lysis of the test antibody molecule are compared with the results of lysis of ipilimumab. The improved lysis of the test antibody molecule compared to ipilimumab demonstrates that the test antibody molecule has an improved depletion effect on CTLA-4 positive cells, CD4 positive cells or Tregs, respectively, depending on which target cells were used.

In some embodiments of the present invention, the improved draining effect in step 7) above is a significantly improved draining effect.

This analysis is demonstrated in more detail below in Example 4 in conjunction with FIG. 12.

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

1) Human PBMC (peripheral blood mononuclear cells) are isolated, washed and resuspended in sterile PBS. In some embodiments of this invention, PBMCs are resuspended in PBS at a concentration of 75 x 10 ⁶ cells/ml.

2) NOG mice are administered IV (intravenously) with an appropriate amount, for example, 200 μl, of the cell suspension from step 1). If 200 μl is administered, this corresponds to 15 x 10 ⁶ cells/mouse.

3) At an appropriate time, eg 2 weeks after injection, spleens from NOG mice are isolated and processed into a single cell suspension. Optionally, a small sample from the single cell suspension is taken to determine CTLA-4 expression on human T cells using FACS to confirm CTLA-4 expression.

4) The cell suspension from step 3) was resuspended in sterile PBS. In some embodiments of this invention, the suspension from the sediment is resuspended in sterile PBS at a concentration of 50 x 10 ⁶ cells/ml. If the optional determination of CTLA-4 expression is included in step 3, the rest of the cell suspension is then resuspended in step 4.

5) SCID mice are injected IP (intraperitoneally) with a suitable amount, eg 200 µl, of the suspension from step 4. If 200 µl is injected, this corresponds to 10×10 ⁶ cells/mouse.

6) A suitable time, such as 1 hour, after injection in step 5) SCID mice are administered an appropriate amount, for example 10 mg/kg, of either the antibody molecule to be tested, ipilimumab, or an isotype control monoclonal antibody.

7) Intraperitoneal fluid of SCID-treated mice is collected at an appropriate time, for example, 24 hours after treatment in step 6).

8) Human T cell subsets are identified and quantified by FACS using the following markers: CD45, CD4, CD8, CD25 and/or CD127.

9) The identification and quantification of T cell subsets from mice treated with the test antibody molecule are compared with the identification and quantification of T cell subsets from mice treated with ipilimumab and the identification and quantification of T cell subsets from mice treated with monoclonal antibody isotype control. The lower number of CTLA-4-positive cells in intraperitoneal fluid from mice treated with the test antibody molecule compared with the number of CTLA-4-positive cells in intraperitoneal fluid from mice treated with ipilimumab demonstrates that the antibody molecule has improved depleting effect on CTLA-4 -positive cells compared to ipilimumab. The lower number of CD4-positive cells in the intraperitoneal fluid of mice treated with the antibody molecule of interest compared with the number of CD4-positive cells in the intraperitoneal fluid of mice treated with ipilimumab demonstrates that the antibody molecule has an improved depletion effect on CD4-positive cells compared with ipilimumab . The lower number of Tregs in the intraperitoneal fluid of mice treated with the antibody molecule of interest compared to the number of Tregs in the intraperitoneal fluid of mice treated with ipilimumab demonstrates that the antibody molecule has an improved depleting effect on Tregs compared with ipilimumab.

In this in vivo test, in some embodiments of the present invention, of greatest interest is the consideration of Treg depletion in step 7.

This analysis is demonstrated in more detail below in Example 4 in conjunction with FIG. 14.

Treg depletion can also be assessed using an antibody-dependent cellular phagocytosis (ADCP) assay, as known to one of ordinary skill in the art.

In some embodiments of the present invention, the antibody molecules have a similar blocking effect on the interactions of CTLA-4 with ligands B7.1 and B7.2 compared to the First. This can be assessed by ELISA (as demonstrated in Fig. 10) or in a more functional assay, where anti-CTLA-4 antibodies enhance IL-2 production by T cells in response to stimulation of PBMCs with SEB.

In some embodiments of the present invention, the anti-CTLA-4 antibody molecule is a human antibody molecule.

In some embodiments of the present invention, the anti-CTLA-4 antibody molecule is a humanized antibody molecule.

In some embodiments of the present invention, the anti-CTLA-4 antibody molecule is a human-derived antibody molecule, meaning that it is derived from a human antibody molecule that has then been modified.

In some embodiments of the present invention, the anti-CTLA-4 antibody molecule is a human IgG1 antibody.

In some embodiments of the present invention, the anti-CTLA-4 antibody is an antibody in the form of a human IgG1 antibody that exhibits improved binding to one or more Fc activating receptors and/or is designed to improve binding to one or more Fc activating receptors; accordingly, in some embodiments of the present invention, the anti-CTLA-4 antibody is an Fc engineered human IgG1 antibody.

In some embodiments of the present invention, the anti-CTLA-4 antibody is a murine or humanized murine IgG2a antibody.

In some embodiments of the present invention, the anti-CTLA-4 antibody is a murine antibody that cross-reacts with human CTLA-4.

In some embodiments of the present invention, the anti-CTLA-4 antibody is a monoclonal antibody.

In some embodiments of the present invention, the anti-CTLA-4 antibody is a polyclonal antibody.

In some embodiments of the present invention, 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 the VL-CDR1 sequence, one of the two VL-CDR2 sequences and/or one of the two alternative VL-CDR3 sequences presented in Table 1 below.

In some embodiments of the present invention, the anti-CTLA-4 antibody molecule is selected from the group consisting of antibody molecules containing 1-6 CDRs selected from the group consisting of the sequences of SEQ ID NOs 3, 6, 8, 10, 12 and 14 .

In some embodiments of the present invention, the anti-CTLA-4 antibody molecule is selected from the group consisting of antibody molecules containing CDRs having the sequences of SEQ ID NOs 3, 6, 8, 10, 12 and 14.

In some embodiments of the present invention, the anti-CTLA-4 antibody molecule is selected from the group consisting of antibody molecules containing 1-6 of the CDRs, VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1 and VL-CDR3,

where VH-CDR1, if present, is selected from the group consisting of the sequences SEQ ID NOs 15, 22, 29 and 35;

where VH-CDR2, if present, is selected from the group consisting of the sequences SEQ ID NO: 16, 23, 30 and 36;

where VH-CDR3, if present, is selected from the group consisting of the sequences SEQ ID NOs 17, 24, 31 and 37;

where VL-CDR1, if present, is selected from the group consisting of the sequences of SEQ ID NOs 10 and 38;

where VL-CDR2, if present, is selected from the group consisting of the sequences SEQ ID NOs 18, 25, 32 and 39;

where VL-CDR3, if present, is selected from the group consisting of the sequences SEQ ID NOs 19, 26 and 40.

In some embodiments of the present invention, the anti-CTLA-4 antibody molecule is an antibody molecule selected from the group consisting of antibody molecules containing 6 CDRs selected from the group consisting of the sequences:

SEQ ID NO: 15, 16, 17, 10, 18 and 19;

SEQ ID NO: 22, 23, 24, 10, 25 and 26;

SEQ ID NO: 29, 30, 31, 10, 32 and 26; And

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

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

In some embodiments of the present invention, the anti-CTLA-4 antibody molecule is an antibody molecule comprising 6 CDRs having the sequences SEQ ID NOs: 22, 23, 24, 10, 25 and 26.

In some embodiments of the present invention, the anti-CTLA-4 antibody molecule is an antibody molecule selected from the group consisting of antibody molecules containing VH selected from the group consisting of the sequences of SEQ ID NOs: 20, 27, 33 and 41.

In some embodiments of the present invention, the anti-CTLA-4 antibody molecule is an antibody molecule selected from the group consisting of antibody molecules containing a VL selected from the group consisting of the sequences of SEQ ID NOs: 21, 28, 34 and 42.

In some embodiments of the present invention, the anti-CTLA-4 antibody molecule is an antibody molecule selected from the group consisting of antibody molecules containing VH and VL selected from the group consisting of the sequences SEQ ID NO: 20-21, 27- 28, 33-34 and 41-42.

In some embodiments of the present invention, 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.

In some embodiments of the present invention, the anti-CTLA-4 antibody molecule comprises a VH having the sequence SEQ ID NO: 27, and a VL having the sequence SEQ ID NO: 28.

Table 1. General CDR sequences of antibodies described in this document

Relevant part of antibody Subsequence Explanation of unidentified amino acid residues SEQ ID NO VH-CDR1 alternative 1 FX ₁ X ₂ YX ₃ MX ₄ WX ₅ R QAPG X ₁ = S or K; X ₂ = D, S or A; X ₃ = Y, S or A; X ₄ = S or N; and X ₅ = V or I 1 VH-CDR1 alternative 2 FSX ₁ YX ₂ MX ₃ WVRQ APG X ₁ = D or S; X ₂ = Y, S or A; and X ₃ = S or N 2 VH-CDR1 alternative 3 FSX ₁ YX ₂ MX ₃ WVRQ APG X ₁ = D or S; X ₂ = Y or S; and X ₃ = S or N 3 VH-CDR2 alternative 1 SX ₁ ISX ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ ADSVKGR X ₁ = G or A; X ₂ = W, G or N; X ₃ = S or T; X ₄ = S or G; X ₅ = R or G; X ₆ = D, S or Y; X ₇ = K, T or I; X ₈ = G, Y, H or D; X ₉ = Y or F 4 VH-CDR2, alternative 2 SX ₁ ISX ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ YADSVKGR X ₁ = G or A; X ₂ = W, G or N; X ₃ = S or T; X ₄ = S or G; X ₅ = R or G; X ₆ = D, S or Y; X ₇ = K, T or I; X ₈ = G, Y, H or D 5 VH-CDR2, alternative 3 SX ₁ ISX ₂ SX ₃ X ₄ X ₅ X ₆ X ₇ YADSVKGR X ₁ = G or A; X ₂ = W or G; X ₃ = S or G; X ₄ = R or G; X ₅ = D or S; X ₆ = K or T; X ₇ = G or Y, H or D 6 VH-CDR3 alternative 1 X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ X ₁₂ X ₁₃ X ₁₄ X ₁₅ X ₁ = T or A; X ₂ = T or R; X ₃ = D, Y or L; X ₄ = L, R, S or G; X ₅ = A, V, S or Y; X ₆ = R, E, G or S; X ₇ = Y, M, L or G; X ₈ = N, H, Y or absent; X ₉ = Q, D or missing; X ₁₀ = W, A, D or none; X ₁₁ = L, F, R or absent; X ₁₂ = A, D, G or missing; X ₁₃ = D, I, M or missing; X ₁₄ = D or missing; and X ₁₅ = V or missing 7 VH-CDR3 alternative 2 X ₁ X ₂ DX ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ X ₁₂ X ₁₃ X ₁ = T or A; X ₂ = T or R; X ₃ = L or R; X ₄ = A or V; X ₅ = R or E; X ₆ = Y or M; X ₇ = N or absent; X ₈ = Q or missing; X ₉ = W or absent; X ₁₀ = L or missing; X ₁₁ = A or absent; X ₁₂ = D or missing 8 VL-CDR1 alternative 1 CX ₁ GSSSNIGX ₂ X ₃ YX ₄ X ₅ X ₆ X ₁ = T or S; X ₂ = A or S; X ₃ = G or N; X ₄ = D or V; X ₅ = V or Y; X ₆ = H or missing 9 VL-CDR1 alternative 2 CTGSSSNIGAGYDVH 10 VL-CDR2 alternative 1 X ₁ NX ₂ X ₃ RPS X ₁ = G, R or D; X ₂ = D, N or S; and X ₃ = N, Q or K eleven VL-CDR2 alternative 2 X ₁ NX ₂ X ₃ RPS X ₁ = G or R; X ₂ = D or N; and X ₃ = N or Q 12 VL-CDR3 alternative 1 CX ₁ X ₂ X ₃ DX ₄ SLX ₅ GX ₆ VX ₇ X ₁ = A or Q; X ₂ = V, A or S; X ₃ = W or Y; X ₄ = D or S; X ₅ = N or S; X ₆ = V, W or P; and X ₇ = V or missing 13 VL-CDR3 alternative 2 CAX ₁ WDDSLNG X ₂ V X ₁ = V or A; and X ₂ = V or W 14

Table 2. Specific anti-CTLA-4 antibody molecules; CDR sequences are in bold in the complete VH and VL sequences

In some embodiments of the present invention, the anti-CTLA-4 antibody molecules described herein may also contain one or both of the constant regions presented in Table 3 below.

Table 3. Sequences of constant regions of antibodies described in this document

Plot Subsequence SEQ ID NO: CH ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 43 C.L. QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 44

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

Table 4. Specific nucleotide sequences encoding anti-CTLA-4 antibody molecules

Clone Coding Subsequence SEQ ID NO: 4-E03 γ1 heavy chain 4-E03 VH GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGGTCCGCCAGGCTCCCGGGAAGGGGCTGGAGTGGGTCTCAGGCATTAGTTGGAGTAGTCGTGACAAAGGCTATGCGGACTCTGTGAAGGGCCGTTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTG CAAATGAACAGCCTGAGAGCCGAGGACACTGCCGTGTATTACTGTACCACAGATCTCGCTAGGTACTGGGGCCAGGGTACACTGGTCACCGTGAGCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA CACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCC AGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA 45 4-E03 λ light chain 4-E03 VL CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTTCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAATGATAACCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCC GGTCCGAGGATGAGGCTGATTATTACTGTGCAGTATGGGATGACAGCCTGAATGGTGTGGTATTCGGCGGAGGAACCAAGCTGACGGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAA GGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATGA 46 5-B07 γ1 heavy chain 5-B07 VH GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTG TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGTGTATTACTGTGCGAGAGATCGGGTAGAGATGAACCAGTGGCTGGCCGACTGGGGCCAGGGTACACTGGTCACCGTGAGCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGA ACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTC CTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCA AGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT CCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA 47 5-B07 λ light chain 5-B07 VL CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTTCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGG TCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGG CGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATGA 48 2-C06 γ1 heavy chain 2-C06 VH GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTAGTGGCAGCGGTGGGTACATACACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATC TGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGTGTATTACTGTGCGACCTATAGCAGTGGCCTGCATGATGCTTTTGATATCTGGGGCCAGGGTACACTGGTCACCGTGAGCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCA GGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCT TCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGG TCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA 49 2-C06 λ light chain 2-C06 VL CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTTCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGACAATAATAAGCGACCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGG TCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGG CGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATGA 50 2-F09 γ1 heavy chain 2-F09 VH GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAAAGCCTATAGCATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATCAGTAACACGGGAGGTAGCACAGACTTCGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTG CAAATGAACAGCCTGAGAGCCGAGGACACTGCCATGTATTACTGTGCGAGATTGGGATATAGTGGCTACGACGACCGTGGTATGGACGTCTGGGGCCAAGGTACACTGGTCACCGTGAGCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTC TTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAG TGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT TCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA 51 2-F09 λ light chain 2-F09 VL CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATTATGTGTACTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACAGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTC CGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGAGTGGTCCTGTGGTATTCGGCGGAGGAACCAAGCTGACGGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAA GGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATGA 52

In some embodiments of the present invention, it is preferred that the antibody molecule binds to both human CTLA-4 (hCTLA-4) and cynomolgus CTLA-4 (cmCTLA-4 or cyno CTLA-4). Cross-reactivity with CTLA-4 expressed on cells of the cynomolgus macaque, also called the crab-eating macaque or Macaca fascicularis, may be advantageous as it allows the antibody molecule to be tested in a monkey without the use of a surrogate antibody, which primarily concerns tolerability.

In some embodiments of the present invention, it is preferred that the antibody molecule binds to both human CTLA-4 (hCTLA-4) and mouse CTLA-4 (mCTLA-4). This can be advantageous as it allows the antibody molecule to be tested in mice, focusing on action and pharmacodynamics, without the need for a surrogate antibody.

In some embodiments of the present invention, the antibody molecule binds to all three of hCTLA-4, cmCTLA-4 and mCTLA-4.

In some embodiments of the present invention, it is necessary to use a surrogate antibody to test the functional activity of the antibody molecule in appropriate in vivo mouse models. To ensure comparability between the effect of an antibody molecule in humans and the in vivo results of a surrogate antibody in mice, it is important to select a functionally equivalent surrogate antibody that has the same in vitro characteristics as the human antibody molecule.

In some embodiments of the present invention, the antibody molecule does not bind human CD28.

One skilled in the medical field will be aware that drugs can be modified by various additives, for example, to change the rate at which the drug is absorbed into the body; and can be modified in various forms, for example, to provide a specific route of administration into the body.

Accordingly, this application includes the antibody molecules, nucleotide sequences, plasmids, viruses and/or cells described herein may be combined with a pharmaceutically acceptable excipient, carrier, diluent, carrier and/or adjuvant into a pharmaceutical composition. In this context, the term pharmaceutical composition may be used interchangeably with the terms pharmaceutical drug, pharmaceutical composition, therapeutic composition, therapeutic drug, therapeutic composition and therapeutic entity.

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

The pharmaceutical compositions described herein may, in some embodiments of the present invention, consist of or contain plasmids containing nucleotide sequences encoding the antibody molecules described above or containing the nucleotide sequences described above.

In some embodiments of the present invention, pharmaceutical compositions may contain nucleotide sequences encoding portions or a complete antibody molecule described herein integrated into a cellular or viral genome or virome. The pharmaceutical composition may then contain a cell or virus as a carrier for delivering the antibody of the invention (or a carrier for delivering the nucleotide sequence encoding the antibody of the invention). For example, in an embodiment of the present invention, the virus may be in the form of a therapeutic oncolytic virus containing nucleotide sequences encoding at least one of the antibody molecules described herein. In some embodiments of the present invention, such an oncolytic virus contains nucleotide sequences encoding a full-length human IgG antibody. In some embodiments of the present invention, such an oncolytic virus contains nucleotide sequences encoding a scFv, Fab, or F(ab') ₂ antibody molecule.

As described in the accompanying claims, in one embodiment, the present invention provides a virus containing 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 “oncolytic” refers to the ability of a virus to selectively replicate in dividing cells (eg, proliferative cells, such as cancer cells) to slow the growth and/or lyse of said dividing cell, either in vitro or in vivo , demonstrating no or minimal replication in nondividing (eg, normal or healthy) cells. "Replication" (or any form of replication such as "replication" and "replication", etc.) means duplication of the virus, which can occur at the nucleic acid level or, preferably, at the level of the infectious virus particle. Such an oncolytic virus can be obtained from any representative virus currently identified. This may be a native virus that is oncolytic in nature, or may be engineered by modifying one or more viral genes so as to enhance tumor selectivity and/or preferential replication in dividing cells, e.g., those involved in DNA replication, nucleic acid metabolism, tropism host, surface attachment, virulence, lysis and spread (see, for example, Wong et al., 2010, Viruses 2: 78-106). It may also be possible to place one or more viral gene(s) under the control of an event or tissue-specific regulatory elements (eg, a promoter). Examples of 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), morbillivirus, adenovirus, poxvirus, retrovirus, measles virus , foam virus, alpha virus, lentivirus, influenza virus, Sinbis virus, myxoma virus, rhabdovirus, picornavirus, coxsackievirus, parvovirus or the like. Such oncolytic viruses are known to those skilled in the field of medicine and virology.

In some embodiments of the present invention, such an oncolytic virus is derived from a herpes virus. Herpesviridae are a large family of DNA viruses that share a common structure and consist of relatively large double-stranded linear DNA genomes encoding 100–200 genes encapsulated in an icosahedral capsid that is enclosed by a lipid bilayer membrane. Although oncolytic herpes virus can be derived from various types of HSV, HSV1 and HSV2 are particularly preferred. The herpes virus can be genetically modified to limit viral replication in tumors or reduce its cytotoxicity in nondividing cells. For example, any viral gene involved in nucleic acid metabolism can be inactivated, either by thymidine kinase (Martuza et al., 1991, Science 252: 854-6), or by ribonucleotide reductase (RR) (Mineta et al., 1994, Cancer Res. 54 : 3363-66) and uracil-N-glycosylase (Pyles et al., 1994, J. Virol. 68: 4963-72). Another aspect involves viral mutants defective in the function of genes encoding virulence factors, such as the ICP34.5 gene (Chambers et al., 1995, Proc. Natl. Acad. Sci. USA 92: 1411-5). Representative examples of oncolytic herpesvirus include NV1020 (eg, 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).

In some embodiments of the present invention, such an oncolytic virus is derived from an adenovirus. Methods for constructing oncolytic adenoviruses are available in the prior art. An advantageous strategy involves replacing viral promoters with tumor-selective promoters or modifying the adenoviral E1 gene product(s) to inactivate its/their binding function to p53 or retinoblastoma protein (Rb), which are altered in tumor cells. In a natural context, the adenovirus gene E1B55kDa interacts with another adenoviral product to inactivate p53 (p53 is often dysregulated in cancer cells), thereby preventing apoptosis. Representative examples of oncolytic adenoviruses include ONYX-015 (eg, Khuri et al., 2000, Nat. Med 6(8):879-85) and H101, also called Oncorine (Xia et al., 2004, Ai Zheng 23(12) :1666-70).

In some embodiments of the present invention, such an oncolytic virus is a poxvirus. In the context of the present invention, the term "poxvirus" refers to a virus belonging to the family Poxviridae, with particular preference to a poxvirus belonging to the subfamily Chordopoxviridae and more preferably to the genus Orthopoxvirus. Vaccinia virus, vaccinia virus, canarypox virus, ectromelia virus, myxoma virus are particularly suitable in the context of the invention. Genomic sequences of such poxviruses are available in the prior art and specialized databases (for example, Genbank under accession number NC_006998, NC_003663 or AF482758.2, NC_005309, NC_004105, NC_001132, respectively).

In specific and preferred embodiments of the present invention, such an oncolytic poxvirus is an oncolytic vaccinia virus. Vaccinia viruses are members of the poxvirus family, characterized by a 200-kb double-stranded DNA genome that encodes numerous viral enzymes and factors that allow the virus to replicate independently of the host cell machinery. Most vaccinia virus particles are intracellular (IMV for intracellular mature virion) with a single lipid envelope and remain in the cytosol of infected cells until lysis. Another infectious form is a double-enveloped virion (EEV for extracellular enveloped virion) that is released from the infected cell without lysing it. Although it can come from any strain of vaccinia virus, Elstree, Wyeth, Copenhagen, Lister, and Western Reserve strains are particularly preferred. In the context of this invention, the gene nomenclature is that of the Copenhagen vaccinia strain unless otherwise indicated. However, correspondence between Copenhagen and other vaccinia strains is generally available in the literature.

Preferably, such oncolytic vaccinia virus is modified by altering one or more viral gene(s). Said modification(s) preferably results in no synthesis or synthesis of a defective viral protein incapable of providing the activity of the protein normally produced by the unmodified gene. Examples of modifications are described in the literature to alter viral genes involved in DNA metabolism, host virulence, the IFN pathway (eg, Guse et al., 2011, Expert Opinion Biol. Ther. 11 (5): 595-608) and the like. Modifications to alter the viral locus include deletion, mutation and/or substitution of one or more nucleotide(s) (contiguous or non-contiguous) in the viral gene or its regulatory elements. The modification(s) can be accomplished in a number of ways known to those skilled in the art using conventional recombinant techniques.

More preferably, such an oncolytic vaccinia virus is modified by changing the gene encoding the thymidine kinase (J2R locus). The enzyme thymidine kinase (TK) (TK - thymidine kinase) is involved in the synthesis of deoxyribonucleotides. TK is required for viral replication in normal cells, since these cells typically have a low concentration of nucleotides, whereas it is required for dividing cells, which contain a high concentration of nucleotides.

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

In some embodiments of the present invention, such an oncolytic virus contains nucleotide sequences encoding an amino acid sequence having at least 80% identity with the sequence listed in Table 2 above. In some embodiments of the present invention, such an oncolytic virus contains an amino acid sequence having at least 85% identity with the sequence listed in Table 2 above. In some embodiments of the present invention, such an oncolytic virus contains an amino acid sequence having at least 90% identity with the sequence listed in Table 2 above. In some embodiments of the present invention, such an oncolytic virus contains an amino acid sequence having at least 95% identity with the sequence listed in Table 2 above.

In some embodiments of the present invention, such an oncolytic virus contains nucleotide sequences encoding the sequences of SEQ ID NO: 20 and ID NO: 21. In some embodiments of the present invention, such an oncolytic virus contains nucleotide sequences encoding the sequences of SEQ ID NO: 27 and ID NO: 28. In some embodiments of the present invention, such an oncolytic virus contains nucleotide sequences encoding the sequences of SEQ ID NO: 33 and ID NO: 34. In some embodiments of the present invention, such an oncolytic virus contains nucleotide sequences encoding the sequences of SEQ ID NO: : 41 and ID NO: 42.

In some embodiments of the present invention, such an oncolytic virus contains nucleotide sequences having at least 80% identity with the sequence listed in Table 4 above. In some embodiments of the present invention, such an oncolytic virus contains nucleotide sequences having at least 85% identity with the sequence listed in Table 4 above. In some embodiments of the present invention, such an oncolytic virus contains nucleotide sequences having at least 90% identity with the sequence listed in Table 4 above. In some embodiments of the present invention, such an oncolytic virus contains nucleotide sequences having at least 95% identity with the sequence listed in Table 4 above.

In some embodiments of the present invention, such an oncolytic virus comprises the sequences SEQ ID NOs: 45 and 46. In some embodiments of the present invention, such an oncolytic virus contains the sequences SEQ ID NOs: 47 and 48. In some embodiments of the present invention, such an oncolytic virus contains the sequences of SEQ ID NOs: 49 and 50. In some embodiments of the present invention, such an oncolytic virus contains the sequences of SEQ ID NOs: 51 and 52.

Some oncolytic viruses are capable of accepting DNA inserts large enough to allow integration of full-length human antibody sequences. Attenuated vaccinia viruses and herpes simplex viruses are examples of therapeutic oncolytic viruses whose genomes are large enough to integrate full-length IgG antibody sequences (Chan, W.M. et al. 2014 Annu Rev Virol 1(1):119-141; Bommareddy, P.K., et al. 2018 Nat Rev Immunol 18(8):498–513). Full-length IgG antibodies are successfully integrated into the oncolytic Vaccinia virus, resulting in the expression and extracellular release (production) of full-length IgG antibodies upon infection of virus-sensitive host cells, such as cancer cells (Kleinpeter, P., et al. 2016, Oncoimmunology 5(10) ): e1220467). Adenoviruses can also be engineered to encode full-length IgG antibodies that are functionally produced and secreted upon cellular infection (Marino, N., et al. 2017 J Clin Invest 123(6):2447-2463).

In a preferred embodiment of the present invention, such an oncolytic virus is a poxvirus (eg, vaccinia virus) defective in TK activity (as a result of a change in the J2R locus) or defective in both TK and RR activity (as a result of a change in both the J2R locus and and at least one of the RR-encoding I4L loci and/or F4L) and containing (a) nucleotide sequences encoding the sequences SEQ ID NO: 20 and ID NO: 21 or (b) nucleotide sequences encoding the sequences SEQ ID NO: 27 and ID NO: 28 or (c) nucleotide sequences encoding the sequences of SEQ ID NO: 33 and ID NO: 34 or (d) nucleotide sequences encoding the sequences SEQ ID NO: 41 and ID NO: 42.

If desired, it may be advantageous for the nucleotide sequence(s) inserted into the oncolytic virus described herein to include additional regulatory elements to facilitate expression, delivery and biological activity. For example, a signal peptide may be included to facilitate secretion outside of a producing cell (eg, an infected cell). The signal peptide is usually inserted at the N-terminus of the encoded polypeptide immediately after the Met initiator. The choice of signal peptides is wide and available to those skilled in the art. For example, signal peptides derived from another immunoglobin (eg, IgG heavy chain) can be used in the context of this invention to ensure secretion of the anti-CTLA4 antibody described herein outside the producing cell. For illustrative purposes, reference may be made to the sequences of SEQ ID NO: 53 and SEQ ID NO: 54 containing the light chain and heavy chain of the antibody 4-E03 described herein, provided with IgG-derived peptide signals.

A particularly preferred oncolytic virus is a vaccinia virus (e.g., Copenhagen strain) defective in both TK and RR activity (as a result of alteration of both the J2R and I4L loci) and containing nucleotide sequences encoding the sequences of SEQ ID NO: 20 and SEQ ID NO: 21 or SEQ ID NO: 53 and SEQ ID NO: 54.

In some embodiments of the present invention, such an oncolytic virus may further comprise additional nucleotide sequence(s) of therapeutic interest, such as nucleotide sequence(s) encoding immunomodulatory polypeptide(s) (i.e., the polypeptide is involved in stimulating an immune response directly or indirectly). Representative examples of suitable immunomodulatory polypeptides include, without limitation, cytokines and chemokines, with particular preference for granulocyte-macrophage colony-stimulating factor (GM-CSF) and, in particular, human, non-human primate, or mouse GM-CSF. Additional nucleotide sequence can be readily obtained by standard molecular biology techniques (eg, PCR amplification, cDNA cloning, chemical synthesis) using sequence data available in the art and the information presented herein. A particularly preferred oncolytic virus is a vaccinia virus (eg, Copenhagen strain) defective in both TK and RR activity (as a result of alteration of both the J2R locus and the I4L loci) and containing nucleotide sequences encoding the sequences of 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, with particular preference for human GM-CSF (for example, having the sequences SEQ ID NO: 55 or SEQ ID NO: 56 ) or mouse GM-CSF (eg, having the sequences SEQ ID NO: 57 or SEQ ID NO: 58).

In addition, the nucleotide sequences to be inserted into such an oncolytic virus can be optimized to ensure high levels of expression in a specific host cell or subject by modifying one or more codon(s). In addition to optimizing codon usage, various modifications may also be provided to prevent clustering of rare, suboptimal codons present in concentrated regions and/or to suppress or modify negative sequence elements that are expected to negatively affect expression levels. Such negative sequence elements include, but are not limited to, regions with very high (>80%) or very low (<30%) GC content; sequence regions rich in AT or GC; unstable straight or inverted repeating sequences; secondary structures R A; and/or internal cryptic regulatory elements, such as internal TATA boxes, chi-sites, ribosome entry sites and/or splice donor/acceptor sites.

In some embodiments of the present invention, the nucleotide sequence(s) are under the control of suitable regulatory elements for their proper expression in the host cell or subject. In the context of this invention, the term “regulatory elements” refers to any element that allows, promotes or modulates the expression of coding nucleotide sequence(s) in a given host cell or subject, including their replication, duplication, transcription, splicing, translation, stability and /or transport inside or outside the expressing cell. Those skilled in the art will appreciate that the choice 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 level of expression, etc. The promoter is of particular importance. In the context of this invention, this may be a constitutive control of the expression of a nucleotide sequence that it controls in many types of host cells, or specific to certain host cells, or regulated in response to certain events or exogenous factors (for example, temperature, nutrient supplementation, hormone, etc.) or according to the phase of the viral cycle (for example, late or early). Promoters adapted for virus-mediated expression are known in the art. Representative examples of oncolytic poxvirus expression include, but are not limited to, p7.5K vaccine, 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 preferred embodiments of the present invention, the nucleotide sequences of the light and heavy chains of an antibody described herein are respectively under the control of promoters having equal transcriptional strength, and preferably under the control of the same promoter (e.g., p7.5K such as described in SEQ ID NO: 59 or pH5.R, such as that described in SEQ ID NO: 60) to obtain a similar level of expression for both chains and therefore optimal assembly of the antibody as a heterotetrameric protein (i.e., avoiding excess unconnected chain). Additional nucleotide sequence (eg, encoding GM-CSF) can be placed under another promoter (eg, pSE/L such as described in SEQ ID NO: 61).

Insertion of nucleotide sequence(s) (possibly equipped with appropriate regulatory elements) into the genome of such an oncolytic virus is accomplished by conventional means, either using appropriate restriction enzymes or, preferably, by homologous recombination. The nucleotide sequence(s) may be independently inserted at any location in the viral genome. Various insertion sites may be considered, for example, in a non-essential viral gene, in an intergenic region, or in a non-coding part of the genome of such an oncolytic virus. The J2R locus and/or I4L locus is particularly suitable for an oncolytic virus that is a poxvirus (eg, oncolytic vaccinia virus). Following insertion of nucleotide sequence(s) into the viral genome, the viral locus at the insertion site may be deleted, at least in part. In one embodiment of the present invention, this deletion or partial deletion may result in the suppression of expression of the viral gene product encoded in whole or in part by the deleted locus, thereby impairing said viral function of the virus. A particularly preferred oncolytic virus is a TK and/or RR defective vaccinia virus containing a heavy chain coding cassette inserted at the J2R locus and a light chain coding cassette inserted at the I4L locus. A cassette encoding an additional nucleotide sequence encoding GM-CSF may be inserted elsewhere in the viral genome or at the J2R or I4L locus, with preference for insertion at the I4L locus.

The present invention also relates to a method for generating such an oncolytic virus as described herein, and in particular an oncolytic poxvirus in a suitable host cell (producer cell). In some embodiments of the present invention, such a method includes one or more homologous recombination step(s) between the viral genome and a carrier plasmid containing the nucleotide sequence(s) to be inserted (possibly with regulatory elements) flanked 5'- end and 3'-end with viral sequences respectively present upstream and downstream of the insertion site. The specified carrier plasmid can be created and introduced into the host cell by routine methods (for example, by transfection). The viral genome can be introduced into a host cell by infection. The size of each flanking viral sequence can vary from at least 100 bp. up to no more than 1500 bp on each side of the nucleotide sequence (preferably from 200 to 550 bp and more preferably from 250 to 500 bp). Homologous recombination to generate such an oncolytic virus is preferably carried out in cultured cell lines (eg HeLa, Vero) or in chick embryonic fibroblast (CEF) cells derived from embryonated eggs.

In some embodiments of the present invention, identification of an oncolytic virus having nucleotide sequences encoding anti-CTLA4, and possibly additional nucleotide sequence (eg, GM-CSF), can be facilitated by the use of a selection and/or detectable gene. In preferred embodiments of the present invention, the transfer plasmid further comprises a selection marker with particular preference for a GPT gene (encoding guanine phosphoribosyltransferase) allowing growth in a selective environment (for example, in the presence of mycophenolic acid, xanthine and hypoxanthine), or a detectable gene encoding a detectable gene product such as GFP, e-GFP or mCherry. In addition, the possibility of using an endonuclease capable of causing a double-strand break in the specified selective or detectable gene may also be considered. Said endonuclease may be in the form of a protein or expressed by an expression vector.

Once generated, such an oncolytic virus can be amplified into a suitable host cell using conventional techniques, including culturing the transfected or infected host cell under suitable conditions to allow production and recovery of infectious particles.

This invention also relates to a method for producing the oncolytic virus described herein. Preferably, the method comprises the steps of a) obtaining a producer cell line, b) transfecting or infecting the resulting producer cell line with an oncolytic virus, c) culturing the transfected or infected producer cell line under suitable conditions to allow virus production, d) reconstituting the produced virus from a culture of said producer cell line and, optionally, e) purifying said recovered virus.

In some embodiments of the present invention, the producer cell is selected from the group consisting of mammalian (e.g., human or non-human) cells, such as HeLa cells (e.g., ATCC-CRM-CCL-2 ^™ or ATCC-CCL-2.2 ^™ ) , HER96, PER-C6 (Fallaux et al., 1998, Human Gene Ther. 9: 1909-17), hamster cell lines such as BHK-21 (ATCC CCL-10), etc., and avian cells, such as those described in WO2005/042728, WO2006/108846, WO2008/129058, WO2010/130756, WO2012/001075, as well as primary chick embryo fibroblast (CEF) derived from chick embryos derived from fertilized eggs. The producer cells are preferably cultured in a suitable medium, which may or may not be supplemented with serum and/or suitable growth factor(s) if necessary (eg, a chemically defined medium, preferably free of animal or human products). The appropriate medium can be easily selected by one skilled in the art depending on the producing cells. Such media are commercially available. Producer cells are preferably cultured at a temperature of +30°C to +38°C (more preferably at about +37°C) for 1 to 8 days before infection. If necessary, several passages of 1 to 8 days can be done to increase the total number of cells.

In step b), producer cells are infected with the oncolytic virus under appropriate conditions using an appropriate multiplicity of infection (MOI) to ensure productive infection of the producer cells. For illustrative purposes, a suitable MOI is in the range of 10 ^-3 to 20, with particular preference for an MOI of 0.01 to 5, and more preferably 0.03 to 1. The infection step is carried out in an environment that may be the same or different from the medium used for culturing producer cells.

In step c), the infected producer cells are then cultured under suitable conditions well known to those skilled in the art until progeny viral particles are formed. Cultivation of infected producer cells is also preferably carried out in a medium, which may be the same or different from the medium/media used for culturing the producer cells and/or during the infection stage, at a temperature between +32°C and +37°C, from 1 to 5 days.

In step d), the viral particles obtained in step c) are collected from the culture supernatant and/or producer cells. Recovery from producer cells may require a step to disrupt the producer cell membrane to allow virus release. Disruption of the producer cell membrane can be caused by a variety of methods well known to those skilled in the art, including, but not limited to, freeze/thaw, hypotonic lysis, sonication, microfluidization, high shear homogenization (also called high speed), or under pressure homogenization. high pressure.

The recovered oncolytic virus may be at least partially purified prior to dosing and use as described herein. A wide variety of purification steps and methods are available in the art, including, for example, clarification, enzymatic treatment (eg, endonuclease, protease, etc.), chromatography and filtration steps. Appropriate methods are described in the art (see, for example, WO2007/147528; WO2008/138533, WO2009/100521, WO2010/130753, WO2013/022764).

In one embodiment of the present invention, the present invention also relates to a cell infected with an oncolytic virus described herein.

The invention also covers pharmaceutical compositions containing a virus, such as an oncolytic virus, as discussed above, and a pharmaceutically acceptable diluent, carrier and/or adjuvant.

The pharmaceutical composition in some embodiments of the present invention may be in the form of a CAR-T cell bearing portions or complete sequences of the antibody described herein as part of the sequence encoding its chimeric T-cell receptor antigen.

The invention also covers pharmaceutical compositions containing a CAR-T cell, as discussed above, and a pharmaceutically acceptable diluent, carrier and/or adjuvant.

The invention also includes other therapeutic modalities or drug "forms" such as antibody-drug conjugates, fusion proteins, etc., and a pharmaceutical composition containing such therapeutic modalities.

The antibody molecules, nucleotide sequences, plasmids, viruses, cells and/or pharmaceutical compositions described herein may be suitable for parenteral administration, including aqueous and/or non-aqueous sterile injectable solutions, which may contain antioxidants, and/or buffers, and/or bacteriostatics and/or solutes that make the intended recipient's blood composition isotonic; and/or aqueous and/or non-aqueous sterile suspensions, which may include suspending agents and/or thickeners. The antibody molecules, nucleotide sequences, plasmids, cells, and/or pharmaceutical compositions described herein may be presented in single or multiple dose containers, such as sealed ampoules and vials, and may be stored in freeze-dried (i.e., lyophilized) state requiring the addition of only a sterile liquid carrier, such as water for injection, immediately before use.

Extemporaneous injection solutions and suspensions can be prepared from sterile powders and/or granules and/or tablets of the type previously described.

When administered parenterally to human patients, the daily dose level of the anti-CTLA-4 antibody molecule typically ranges from 1 mg/kg of the patient's body weight to 20 mg/kg, and in some cases even up to 100 mg/kg is administered in a single or divided dose. Lower doses may be used in special circumstances, such as in combination with prolonged administration. In either case, the physician will determine the actual dosage that will be most appropriate for each individual patient, which will vary depending on the age, weight, and response of the individual patient. The doses given above are typical for the average case. Of course, there may be individual cases where higher or lower dosage ranges are justified, and such cases are within the scope of this invention.

Typically, a pharmaceutical composition (or drug) containing an antibody molecule described herein will contain an anti-CTLA-4 antibody molecule at a concentration of between about 2 mg/ml and 150 mg/ml or between about 2 mg/ml and 200 mg/ml. In some embodiments of the present invention, the pharmaceutical compositions will contain an anti-CTLA-4 antibody molecule at a concentration of 10 mg/ml.

Typically, the pharmaceutical composition (or drug) will contain the oncolytic virus described herein at a concentration of between about 10 ³ to 10 ¹² vp. (viral particles), i.e. (infectious units) or PFU (plaque forming units) depending on the virus and quantitative method. The number of PFU present in a sample can be determined by counting the number of plaques after infection of permissive cells (eg, CEF or Vero cells) to obtain a titer of plaque-forming units (PFU), the number of i.p. by measuring the optical density of A260, and the amount of i.u. using quantitative immunofluorescence, for example using antiviral antibodies. As a general guide, individual dosages that are suitable for a pharmaceutical composition containing a range of oncolytic poxviruses are from about 10 ³ to about 10 ¹⁰ PFU, preferably from about 10 ³ PFU to about 10 ⁹ PFU, preferably from about 10 ⁴ PFU to about 10 ⁸ PFU ; and more preferably from about 10 ⁴ PFU to about 10 ⁷ PFU.

In general, for humans, oral or parenteral administration of the antibody molecules, nucleotide sequences, plasmids, viruses, cells and/or pharmaceutical compositions described herein is the preferred and most convenient route. For veterinary use, the antibody molecules, nucleotide sequences, plasmids, viruses, cells and/or pharmaceutical compositions described herein are administered in a suitable acceptable formulation in accordance with normal veterinary practice, and the veterinary surgeon will determine the dosage regimen and route of administration that will most suitable for a particular animal. Thus, the present invention relates to a pharmaceutical composition containing an amount of an antibody molecule, nucleotide sequences, plasmid, virus and/or cell of the present invention effective for treating various conditions (as described above and further below). It is desirable that the antibody molecules, nucleotide sequences, plasmids, viruses, cells and/or pharmaceutical compositions described herein are adapted for delivery via a route selected from the group consisting of: intravenous; intratumoral; intramuscular; subcutaneous. Administration may be in the form of a single injection or multiple repeated injections (eg, with the same or different doses, by the same or different routes, at the same or different injection sites). For purposes of illustration, individual doses containing approximately ^{104 ,} 5x104, ¹⁰⁵ , ^5x105 , ¹⁰⁶ , 5x106, ¹⁰⁷ , ^{5x107 , 108} , ^5x108 , ¹⁰⁹ , ^5x109 , or ¹⁰¹⁰ PFU of oncolytic poxvirus (eg, the TK- and RR-defective vaccinia virus described herein) are particularly suitable for intratumoral administration.

This invention also includes antibody molecules, nucleotide sequences, plasmids, viruses, cells and/or pharmaceutical compositions described herein, including pharmaceutically acceptable acid addition salts or based addition salts of the polypeptide linking groups of this invention. Acids which are used for the preparation of pharmaceutically acceptable acid addition salts of the above compounds useful in the present invention are acids that form non-toxic acid addition salts, i.e. salts containing pharmacologically acceptable anions, in particular such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate salts , saccharate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate [i.e. 1,1' - methylene bis-(2-hydroxy-3 naphthoate)]. Pharmaceutically acceptable addition salts can also be used to prepare pharmaceutically acceptable salt forms of the agents of the present invention. Chemical bases that can be used as reagents to obtain pharmaceutically acceptable salts of real agents that are acidic in nature are chemical bases that form non-toxic with such compounds salt. So non-toxic salts include, but are not limited to, non-toxic salts derived from pharmacologically acceptable cations such as alkali metal cations (eg potassium and sodium) and alkaline earth metal cations (eg calcium and magnesium). calcium and magnesium), ammonium or water-soluble amine addition salts, such as N-methylglucamine-(meglumine), as well as lower alkanolammonium and others salts of pharmaceutically acceptable organic amines. 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 (eg, spray drying, sludge drying) and/or resuspension methods can be used. Those skilled in the art will appreciate that lyophilization and resuspension may cause the body to lose varying degrees of antibody activity (e.g., with conventional immunoglobulins, IgM antibodies tend to lose more activity than IgG antibodies) and that levels of applications can be adjusted upward. In one embodiment of the present invention, the lyophilized (freeze-dried) polypeptide binding fragment 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.

In some embodiments of the present invention, the viral composition is suitably buffered at a physiological or slightly alkaline pH (eg, from about pH 7 to about pH 9, with particular preference at a pH of from 7 to 8.5 and more particularly close to 8). It may also be useful to include a monovalent salt in the viral composition to ensure appropriate osmotic pressure. Said monovalent salt may in particular be selected from NaCl and KCl, preferably said monovalent salt is NaCl, preferably in a concentration of 10 to 500 mM (eg 50 mM). A suitable viral composition contains 50 g/L sucrose, 50 mM NaCl, 10 mM Tris-HCl and 10 mM MSG, pH 8. The composition can also be formulated to include a cryoprotectant to protect the oncolytic virus at low storage temperatures. Suitable cryoprotectants include, but are not limited to, sucrose (or 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) in the form of high molecular weight polymers such as dextran or polyvinylpyrrolidone (PVP).

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

This application includes that the subject may be a mammal or non-mammalian. Preferably, the mammalian subject is a human or non-mammalian such as a horse, cow, sheep, pig, camel, or dog or cat. Most preferably, the mammalian subject is human.

By “demonstrate” as used herein, it is meant that the subject exhibits a symptom of cancer and/or a diagnostic marker of cancer; and/or a cancer symptom and/or a cancer diagnostic marker can be measured and/or assessed and/or quantified.

A person skilled in the medical field will immediately understand what the symptoms of cancer and diagnostic markers of cancer are, as well as how to measure and/or evaluate and/or quantify whether there is a decrease or increase in the severity of cancer symptoms or a decrease or increase in diagnostic markers of cancer; and how these cancer symptoms and/or cancer diagnostic markers can be used to inform cancer prognosis.

Cancer treatment is often given as a course of treatment, meaning the drug is given over a period of time. The length of treatment will depend on a number of factors, which may include, among other things, the type of drug administered; the type of cancer being treated; the severity of the cancer being treated; age and health status of the subject.

By “during treatment” as used herein, it is meant that the subject is currently undergoing treatment and/or receiving a drug and/or receiving a course of drug.

In some embodiments of the present invention, the cancer that is treated in accordance with the present invention is a solid cancer.

In some embodiments of the present invention, the cancer is selected from the group consisting of advanced solid tumor, 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 cancer, hepatocellular carcinoma, head and neck cancer and colorectal cancer.

Each of the cancers described above is well known, and the symptoms and diagnostic markers of the cancer are well described, as are the medications used to treat these cancers. Accordingly, symptoms, diagnostic markers of cancer and drugs used to treat the above types of cancer are known to those skilled in the medical field.

Clinical determinations of diagnosis, prognosis, and progression of a large number of cancers are based on specific classifications known as cancer staging. These cancer staging systems operate to correlate a number of different cancer diagnostic markers and cancer symptoms to provide a diagnosis and/or prognosis and/or cancer progression. One skilled in the art of oncology will know how to assess the diagnosis and/or prognosis and/or progression of cancer using a cancer staging system, and what diagnostic cancer markers and cancer symptoms should be used to do so.

By “cancer staging” as used herein we mean staging the cancer according to the Rai classification, which includes stage 0, stage I, stage II, stage III and stage IV, and/or the Binet classification, which includes stage A, stage B and stage C, and/or according to the Ann Arbor classification, which includes stage I, stage II, stage III and stage IV.

It is known that cancer can cause abnormalities in cell morphology. These abnormalities often reoccur in certain types of cancer, which means that examination of these changes in morphology (also known as histology) can be used in the diagnosis or prognosis of cancer. Sample imaging techniques for studying cell morphology and preparing samples for imaging are well known in the art; for example, light microscopy or confocal microscopy.

By “histological examination” in this application we mean the presence of small mature lymphocytes and/or the presence of small mature lymphocytes with a narrow border of cytoplasm, the presence of small mature lymphocytes with a dense nucleus devoid of clearly visible nucleoli, and/or the presence of small mature lymphocytes with a narrow border of cytoplasm, and/or with a dense nucleus devoid of clearly visible nucleoli, and/or the presence of atypical cells and/or split cells and/or prolymphocytes.

It is well known that cancer is the result of mutations in a cell's DNA, which can cause the cell to escape cell death or to multiply uncontrollably. Therefore, testing for these mutations (also known as cytogenetic testing) can be a useful tool for assessing the diagnosis and/or prognosis of cancer. An example of this is the deletion of chromosomal location 13q14.1, which is characteristic of chronic lymphocytic leukemia. Methods for studying mutations in cells are well known in the art; for example, fluorescence in situ hybridization (FISH).

By “cytogenetic study” in this application we mean the study of DNA in a cell and, in particular, in a chromosome. Cytogenetic testing can be used to identify changes in DNA that may be associated with the presence of refractory cancer and/or recurrent cancer. Such DNA changes may include: deletions in the long arm of chromosome 13 and/or deletions of the 13q14.1 locus of chromosome, and/or trisomy of chromosome 12, and/or deletions in the long arm of chromosome 12, and/or deletions in the long arm of chromosome 11, and /or deletion 11q, and/or deletion in the long arm of chromosome 6, and/or deletion 6q, and/or deletion in the short arm of chromosome 17, and/or deletion in 17p, and/or translocation t(11:14), and /or translocation (q13:q32), and/or rearrangement of the antigen gene receptor, and/or rearrangement of BCL2, and/or rearrangement of BCL6, and/or translocation t(14:18), and/or translocation t(11:14) , and/or translocation (q13:q32), and/or translocation (3:v), and/or translocation (8:14), and/or translocation (8:v), and/or translocation t(11:14 ) and (q13:q32).

Subjects with cancer are known to exhibit certain physical symptoms, which are often a result of the burden of cancer on the body. These symptoms are often repeated in the same cancer, and therefore may be characteristic of the diagnosis and/or prognosis and/or progression of the disease. The medical professional must understand which physical symptoms are associated with which types of cancer and how assessment of these physical systems may correlate with diagnosis and/or prognosis and/or disease progression. By “physical symptoms” in this application we mean hepatomegaly and/or splenomegaly.

Brief description of graphic materials

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

Fig. 1. Antibodies of this invention specifically bind CTLA-4

Using ELISA, the antibodies were demonstrated to bind to human CTLA-4 and CTLA-4, but not to human CD28 protein. The binding of 2-C06 (Fig. 1A), 4-E-03 (Fig. 1B), 5-B07 (Fig. 1C) was compared with Yerva (Fig. 1D).

Fig. 2. Dose-dependent binding of anti-CTLA-4 mAb to cells transfected with hCTLA-4

Anti-CTLA-4 mAb (Fig. 2A-2D) shows strong binding to CTLA-4-expressing 293T cells similar to Yervoy (Fig. 2E).

Fig. 3. Anti-CTLA-4 mAbs bind to in vitro-activated human CD4+ T cells

CD4+ T cells derived from human peripheral blood were stimulated in vitro. Anti-CTLA-4 mAb binding (solid lines, top row) was analyzed by FACS and compared with Yerva (dashed line, top row) and a commercial FACS antibody (bottom row).

Fig. 4. Blocking binding to in vitro-activated CD4+ T cells

In vitro-activated human CD4+ T cells were stained with Alexa 647-labeled anti-CTLA-4 mAb (black line). Antibody binding was blocked by the rhCTLA-4-Fc protein (gray line).

Fig. 5. Binding to in vitro-activated Cynomolgus CD4+ T cells

CD4+ T cells derived from the peripheral blood of cynomolgus monkeys were stimulated in vitro with CD3/CD28 dynabus. Anti-CTLA-4 mAb binding (solid lines, top row) was analyzed by FACS and compared with Yerva (dashed line, top row) and a commercial FACS antibody (bottom row).

Fig. 6. Blocking cell binding by human and cynomolgus macaque CTLA-4 protein

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

Fig. 7. Binding to 293T cells expressing cynomolgus CTLA-4

293T cells were transiently transfected with cynomolgus CTLA-4, and binding of various concentrations of anti-CTLA-4 mAb was analyzed by FACS.

Fig. 8. Expected lack of binding to resting human/cynomolgus PBMCs

4-E03, as well as 2-C06, 5-B07, and 2-F09 (data not shown), did not exhibit nonspecific binding to different cell subpopulations in human (top row) and cynomolgus monkey (bottom row) PBMCs as analyzed by FACS.

Fig. 9. Expected lack of direct agonist activity

CFSE-labeled CD4+ T cells from healthy donors were stimulated with coated anti-CD3 plus soluble anti-CTLA-4 mAb or anti-CD28.% of dividing cells (CFSElow CD25+ cells) were determined after 3 days by FACS. (A) FACS plots of one representative experiment (B) summarizing the plot of 6 donors.

Fig. 10. CD80/CD86 blocking activity

Anti-CTLA-4 mAbs block the binding of CD80 (Fig. 8A) and CD86 (Fig. 8b) to its ligand CTLA-4, as demonstrated by ELISA.

Fig. 11. Blocking a functional ligand in vitro

PBMCs were stimulated with SEB plus titrated doses of anti-CTLA-4 antibodies. The amount of secreted IL-2 in the supernatant was determined using MSD. This figure shows 1 representative donor out of 6.

Fig. 12. ADCC analysis on in vitro-activated CD4+ T cells

In vitro-activated CD4+ T cells from healthy donors, pre-coated with anti-CTLA-4 mAb at a concentration of 10 μg/ml, were co-cultured with NK cells (NK-92 cell line) in a 2:1 ratio. ADCC activity was assessed by FACS as described below. In fig. shows mean +SD for 4-8 donors.

Fig. 13. CTLA-4 is most highly expressed on tumor-resident Treg cells.

Freshly excised ovarian tumor and blood samples were obtained from patients at the time of surgery. Ascites was collected from patients with different indications of cancer. CTLA expression in this patient material was compared with healthy PBMCs. Tumor samples were crushed and digested. Peripheral blood mononuclear cells were separated by centrifugation. CTLA-4 expression was assessed on CD4+CD25+CD127- Treg cells, CD4+ non-Treg cells and CD8+ effector T cells using flow cytometry. Data represent individual patients/donors with n = 12 for healthy PBMCs, n = 20 for ascites, n = 9 for tumor, and n = 5 for patient blood.

CTLA-4 expression was also analyzed in human T cells that were activated in NOG mice in vivo and then isolated from the spleens of these mice (see FIG. 14).

Fig. 14. Anti-CTLA-4 mAbs mediate Treg depletion in vivo.

Human PBMCs were administered intravenously to NOG mice. Approximately 2 weeks later, spleens were harvested and CTLA-4 expression was analyzed on human Treg cells and CD8+ T cells by FACS. Spleen cells isolated from NOG mice were transferred i.p. to SCID mice. After 1 hour, mice were treated IP with CTLA-4 hIgG1 or control mAb. Intraperitoneal fluid was collected after 24 hours and the frequency of human T cell subsets (14A: Tregs and 14B: CD8+ T cells) was determined by flow cytometry.

Fig. 15. Table summarizing the characteristics of anti-CTLA-4 antibodies.

Fig. 16. Characteristics of surrogate mouse anti-CTLA-4 mAbs.

Fig. 16 AB. Blocking ELISA was performed with m5-B07 to evaluate the blocking characteristics of the ligand. The antibody blocks CD80 binding in FIG. 16A) and CD86 in FIG. 16B) with its ligand CTLA-4 in a dose-dependent manner.

Fig. 16. CD. 5-B07 in mouse IgG2a format mediated Treg deletion in the CT26 tumor model. Balb/c mice were injected subcutaneously with 1 x 10 ⁶ CT26 cells and treatment was initiated when the tumor size was approximately 7 x 7 mm. After 3 injections of 10 mg/kg antibody, single tumor cell suspensions were analyzed for immune cell content by FACS. Fig. 16C: Surrogate ligand blocking antibody 5-B07 causes Treg depletion. This causes a shift in the CD8 ⁺ /Treg T cell ratio, as shown in Fig. 16D.

Fig. 17. Generation COPTG19384 and COPTG19385.

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

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

A) By Western blotting: CEF cells were infected with COPTG19384 at an MOI of 0.05 in triplicate. Cell supernatants were collected after 48 hours and analyzed by WB after electrophoresis under non-reducing conditions and using either anti-Ig (left blot) or anti-light chain (right blot) conjugated to HRP.

B) by ELISA: CEF cells were infected with COPTG19384 at an MOI of 0.05 in triplicate or VVTG17137. Cell supernatants were collected after 48 hours and analyzed by ELISA to detect monoclonal antibody 4-E03.

Fig. 19. Analysis of GM-CSF expression in the supernatant of CEF cells infected with COPTG19384 using ELISA

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

Fig. 20. Replication studies of COP WT, COPTG19384 (two batches) and VVTG17137 (two batches) on normal and tumor hepatocytes

A) Replication rate on normal human hepatocytes.

B) Replication rate on malignant HepG2.

C) Therapeutic indices calculated from replication rates measured on HepG2 and hepatocytes.

Fig. 21. Replication of COPTG19384 and VVTG17137 in reconstructed human skin.

Replication of COPTG19384 and VVTG17137 was assessed after 7 days and varying from 10 to 10 ⁵ PFU. The results represent the means and SEM of three measurements.

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

Fig. 23. Expression levels of both monoclonal antibody 4-E03 and GM-CSF in (A) supernatants of infected HepG2 and LoVo and (B) supernatants of 5 different infected human tumor cell lines

A) Expression levels of 4-E03 and GM-CSF were assessed after 5 days of incubation with an MOI of 10 ^-5 to 10 ^-2 for COPTG19384 and an MOI of 10 ^-2 for VVTG17137 used as a negative control.

B) Expression levels of 4-E03 and GM-CSF were assessed 48 h post-infection with COPTG19384 at an MOI of 0.05.

Fig. 24. Binding of different batches of 4-E03 to the CTLA-4 protein.

Binding of recombinantly produced 4-E03 from CHO (study series 4-E03) or HEK (toxin series 4-E03) cells to (A) human and (B) cynomolgus recombinant protein was compared with the binding of 4-E03 purified from supenatant infected tumor cells MIA PaCa-2 (4-E03 TG) by ELISA.

Fig. 25. Binding of different series of 4-E03 to cells expressing CTLA-4

Binding of recombinantly produced 4-E03 from CHO (study series 4-E03) or HEK (toxin series 4-E03) cells to (A) human and (B) cynomolgus macaque cells expressing CTLA-4 was compared with the binding of supenatant-purified 4-E03 infected tumor cells MIA PaCa-2 (4-E03 TG) by flow cytometry.

Fig. 26. Kinetics of virus accumulation in a xenografted LoVo tumor

Virus accumulation kinetics were assessed in xenografted LoVo tumors following a single IT injection of either COPTG19384 or VVTG17137 at two different doses (10 ⁴ or 10 ⁵ PFU). Solid or dotted lines represent the median of three values determined at each time point.

Fig. 27. Kinetics of accumulation of 4-E03 mAb and GM-CSF in xenografted LoVo tumor.

A) The kinetics of 4-E03 mAb accumulation in xenografted LoVo tumors was assessed after a single it injection of either COPTG19384 or VVTG17137 at two different doses (10 ⁴ or 10 ⁵ PFU) or after a single IP injection of 3 mg/kg monoclonal antibody 4-E03 . Solid or dotted lines represent the median of three values.

B) The kinetics of GM-CSF accumulation in xenografted LoVo tumors was assessed after a single it injection of either COPTG19384 at two different doses (10 ⁴ or 10 ⁵ PFU) or VVTG17137 (10 ⁵ PFU). The solid lines represent the median of the three values determined at each time point.

Fig. 28. Kinetics of concentrations of 4-E03 mAb and GM-CSF in the serum of xenografted LoVo mice

A) The kinetics of 4-E03 mAb serum concentrations were assessed after a single IT injection into a LoVo COPTG19384 or VVTG17137 xenograft tumor at two different doses (10 ⁴ or 10 ⁵ PFU) or after a single IP injection of 3 mg/kg monoclonal antibody 4-E03 . The solid lines represent the median of the three values.

B) Kinetics of GM-CSF concentrations in sera after a single IT injection in a xenografted LoVo tumor COPTG19384 at two different doses (10 ⁴ or 10 ⁵ PFU) or VVTG17137 (10 ⁵ PFU). The dotted lines represent the median of the three values determined at each time point.

Fig. 29. Kinetics of virus accumulation in CT26 tumors.

Kinetics of virus accumulation in CT26 tumors after three it injections (in D0, D2 and D4) of either VVTG18058, COPTG19421, or COPTG19407 at a dose of 10 ⁷ PFU/injection.

Fig. 30. Kinetics of m5-B07 mAb and mGM-CSF accumulation in CT26 tumor

A) Kinetics of m5-B07 mAb concentrations in the CT26 tumor during and after three injections of VVTG18058, COPTG19421 or COPTG19407 (10 ⁷ PFU/injection) or after a single IP injection of 3 mg/kg m5-B07 mAb. The solid lines represent the median of the three values.

B) Kinetics of mGM-CSF concentrations in the CT26 tumor during and after three it injections of VVTG18058, COPTG19421 or COPTG19407 (10 ⁷ PFU/injection) or after a single IP injection of 3 mg/kg m5-B07 monoclonal antibody. The solid lines represent the median of the three values determined at each time point.

Fig. 31: Kinetics of m5-B07 mAb concentrations in CT26 model sera.

Kinetics of m5-B07 mAb concentrations in serum after three injections into the CT26 tumor of either VVTG18058, COPTG19421 or COPTG19407 (10 ⁷ PFU/injection), or after a single IP injection of 3 mg/kg m5-B07 monoclonal antibody. The solid lines represent the median of the three values determined at each time point.

Fig. 32. Antitumor activity in the CT26 model: effect of COPTG19347 +/- anti-PD1 on CT26 tumor growth (A) and mouse survival (B)

CT26 cells were injected subcutaneously into BalB/c mice at D-7. COPTG19347 ( ¹⁰⁷ PFU), VVTG18058 ( ¹⁰⁷ PFU), VVTG18058 or buffer were given by i/t injection at D0, D2 and D4, possibly followed by i/p injection of 250 μg/mouse anti-PD1 RMPI-14 at D7, D10, D14, D17 and D21.

Fig. 33. Dose-response assessment in the CT26 model (compilation of survival data observed after treatment with COPTG19407, COPTG19421 and VVTG18058).

Fig. 34. Antitumor activity of COPTG19407 compared to VVTG18058 plus m5-B07 in the CT26 tumor model.

CT26 cells were injected subcutaneously into Balb/C mice. Treatment of mice began when tumors reached approx. 100 mm ³ . Mice were treated at D0, D2, and D5 with COPTG19407 ( ^8.5x106 PFU it), VVTG18058 ( ^8.5x106 PFU it), m5-B07 (10 mg/kg i.p.), or a combination of VVTG18058 ( ^8.5x106 PFU it ) plus m5-B07 (10 mg/kg i.p.). Fig. 30 AD: Tumor growth and fig. 16 E: survival through time.

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

A20 cells were injected s.c. into Balb/C mice. Treatment of mice began when tumors reached 80-100 mm ³ . Mice were injected at D0, D2 and D4 with vehicle (it), COPTG19407 (4.75× ¹⁰⁶ PFU it), VVTG18058 (4.75× ¹⁰⁶ PFU it), RMP1-14 (anti-mPD-1) (250 μg /mouse i.p.) or a combination of COPTG19407 (4.75×10 ⁶ PFU it) plus RMP1-14 (250 μg/mouse i.p.).

A) Animals of group 1 receiving vehicle

B) Animals of group 2, treated with VVTG18058

C) Animals of group 3 treated with COPTG19407

D) Group 4 animals treated with mouse anti-PD1

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

Fig. 36. Average tumor volume curves of BALB/cN mice bearing subcutaneous A20 tumors.

Each point represents the average of the recorded tumor volume per group. Tumor volumes of all animals were monitored for 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 D7 and treated from D7 to D31. The last mice were sacrificed on D61.

Fig. 37. Antitumor activity in the A20 model: effect of COPTG19407 +/- anti-PD1 on A20 tumor growth (A) and mouse survival (B)

A20 cells were injected s.c. into Balb/C mice. Treatment of mice began when tumors reached 80-100 mm ³ . Mice were treated with vehicle (it) (group 1), anti-PD-1 (group 2), isotype (group 3), VVTG18058 (10 ⁵ PFU it) (group 4), VVTG18058 (10 ⁵ PFU it) + isotype (group 5), VVTG18058 (10 ⁵ PFU it) + anti-PD-1 (group 6), VVTG19407 (10 ⁵ PFU it) (group 7), VVTG19407 (10 ⁵ PFU it) + isotype (group 8) and VVTG19407 (10 ⁵ PFU it) + anti-PD-1 (group 9).

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

C38 cells were injected subcutaneously into C57bl/6 mice. Treatment of mice began when tumors reached 80-100 mm ³ . Mice were injected at D0, D2 and D4 with vehicle (it), COPTG19407 (4.75× ¹⁰⁶ PFU it), VVTG18058 (4.75× ¹⁰⁶ PFU it), RMP1-14 (anti-mPD-1) (250 μg /mouse i.p.) or a combination of COPTG19407 (4.75×10 ⁶ PFU it) plus RMP1-14 (250 μg/mouse i.p.).

A) Animals of group 1 receiving vehicle

B) Animals of group 2, treated with VVTG18058

C) Animals of group 3 treated with COPTG19407

D) Group 4 animals treated with mouse anti-PD1

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

Fig. 39. Average tumor volume curves of C57BL/6 mice bearing subcutaneous C38 tumors.

Each point represents the average of the recorded tumor volume per group. The volume of tumors in all animals was monitored for 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 D7 and treated from D7 to D31. The last mice were sacrificed on D61.

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

EMT6 cells were injected s.c. into Balb/C mice. Treatment of mice began when tumors reached 80-100 mm ³ . Mice were injected at D0, D2 and D4 with vehicle (it), COPTG19407 (4.75× ¹⁰⁶ PFU it), VVTG18058 (4.75× ¹⁰⁶ PFU it), RMP1-14 (anti-mPD-1) (250 μg /mouse i.p.) or a combination of COPTG19407 (4.75×10 ⁶ PFU it) plus RMP1-14 (250 μg/mouse i.p.).

A) Animals of group 1 receiving vehicle

B) Animals of group 2, treated with VVTG18058

C) Animals of group 3 treated with COPTG19407

D) Group 4 animals treated with mouse anti-PD1

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

Fig. 41. Average tumor volume curves of BALB/cN mice bearing subcutaneous EMT6 tumors.

Each point represents the average of the recorded tumor volume per group. The volume of tumors in all animals was monitored for 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 D7 and treated from D7 to D31. The last mice were sacrificed on D56. The curves were stopped after more than 20% of the mice died.

Examples

Specific non-limiting examples will now be described that embody certain aspects of the present invention. In the examples, the rh protein is a human recombinant protein (eg, rhIL-2 is a human recombinant IL-2 protein) and the rcm protein is a cynomolgus recombinant protein (eg, rcmCTLA4 is a cynomolgus recombinant CTLA-4 protein).

In addition to the sequences mentioned above, some additional sequences are used in the examples, which are listed in Table 5 below.

Table 5. Additional sequences used in the examples

Example 1. Preparation of antibodies specific to CTLA-4

Isolation of scFv antibody fragments

n-Coder® scFv library (BioInvent; E, et al. Nat Biotechnol. 2000;18(8):852-6) was used to isolate scFv antibody fragments that recognize human CTLA-4.

The phage library was used in three consecutive pannings against the recombinant human protein. After phage incubation, cells were washed to remove unbound phages. Binding phages were eluted with trypsin and amplified in E. coli. The resulting phage stock solution was converted to scFv format. E. coli was transformed with plasmids carrying scFv, and individual scFv clones were expressed.

Identification of a unique CTLA-4 binding scFv

Converted scFvs from the third panning were analyzed using a homogeneous FMAT assay (Applied Biosystems, Carlsbad, CA, USA) for binding to transfected 293 FT cells expressing human CTLA-4 or a non-target protein.

Briefly, transfected cells were added to clear-bottom plates along with scFv-containing expression plate supernatant (diluted 1:7), mouse anti-His Tag antibody (0.4 μg/ml; R&D Systems), and APC-conjugated goat anti-mouse antibody. (0.2 μg/ml; cat. no. 115-136-146, Jackson Immunoresearch). FMAT plates were incubated at room temperature (approximately 20–25°C) for 9 h before reading. Target specific bacterial clones were classified as active and selectively collected into a 96-well plate.

Binding of IgG to CTLA-4 in ELISA

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

All antibodies were shown to bind to human and cynomolgus CTLA-4 protein, but not to human CD28 protein as determined by ELISA. In addition, 5-B07 was shown to bind to mouse CTLA-4, but not to mouse CD28 (see Fig. 1).

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

Converted IgG clones were assayed for binding to CTLA-4-expressing 293T cells (purchased from Crownbio). Cells were incubated with various concentrations (as shown in Fig. 2) of anti-CTLA-4 mAb at 4°C for 20 min before washing and staining with APC-labeled goat anti-human secondary antibody (cat. no. 109-136-088, Jackson ImmunoResearch). Dead cells were excluded from the analysis using Fixable Viability Dye eFluor780 (eBiosciences). Data acquisition was performed on FACSVerse (BD Biosciences, Franklin Lakes, NJ) and analyzed using FlowJo software (Tree Star, Ashland, OR).

The anti-CTLA-4 mAb was shown to bind CTLA-4-expressing human 293T cells in a dose-dependent manner with a similar EC50 to Yervoy (Figure 2).

293T cells stably transfected with human CTLA-4, 293T cells transiently transfected with cynomolgus CTLA-4, naive human or cynomolgus PBMCs, in vitro-activated human CD4+ T cells, or cyno, were incubated with concentrations of anti-CTLA-4 mAb, indicated at 4°C for 20 minutes before washing and staining with secondary APC-labeled anti-human antibody (Jackson ImmunoResearch).

Example 2 Anti-CTLA-4 mAb specifically binds CTLA-4-expressing (primary) human and cynomolgus macaque cells

CTLA-4-specific mAbs bind primary in vitro-activated human and cynomolgus CD4+ T cells, but not naïve PBMCs isolated from healthy donors

PBMCs were isolated from buffy coat. Briefly, films were diluted 1:3 in PBS and applied to Ficoll-Paque Plus pads (Amersham). Samples were centrifuged at 800 x g for 20 min at 20°C. The upper phase containing plasma was removed and mononuclear cells were isolated from the distinct white band at the plasma/Ficoll interface.

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

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

The antibodies were shown to bind to in vitro-activated human (Fig. 3) and cynomolgus (Fig. 5) CD4+ T cells, but not to resting PBMCs (Fig. 8). Binding to T cells endogenously expressing CTLA-4 is similar to staining with Yerva (Fig. 3, top row, dotted line) and as a positive control with a commercial anti-CTLA-4 FACS antibody from BD Biosciences (clone BNI3; Fig. 3, bottom row).

As shown in FIG. 4, staining of in vitro-activated human CD4+ T cells (black line) can be completely blocked by rhCTLA-4-Fc (gray line), demonstrating the specificity of the antibodies. 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 CTLA-4-expressing cells. IgG binding was detected by FACS.

Transfected 293T cells expressing human and cynomolgus CTLA-4 confirm cynocross-reactivity of the tested antibodies

Antibody cynocross-reactivity was further confirmed in transfected CTLA-4-expressing 293T cells.

As shown in FIG. 6, binding of specific anti-CTLA-4 antibodies to transfected CTLA-4-expressing human cells can be inhibited by human and cynomolgus recombinant protein (both R&D Systems). The antibodies were also shown to bind to transfected cells expressing cynomolgus CTLA-4 (Fig. 7, top row). This binding can again be blocked by the recombinant cynomolgus protein (bottom row, gray line).

Experiments were carried out similarly to the competition assay described above in Example 2 in connection with FIG. 4.

Expected lack of direct agonist activity

In vitro proliferation assays were performed to rule out unanticipated direct agonistic activity (eg, due to nonspecific binding).

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

Fig. 9 demonstrates that none of the anti-CTLA-4 mAbs tested induce T cell proliferation in contrast to anti-CD28 stimulation.

Example 3 Anti-CTLA-4 mAbs Block CD80/CD86 Ligand Binding

Ligand blocking ELISA

The ligand blocking activity of anti-CTLA-4 IgG was assessed by ELISA. For this purpose, recombinant human CTLA-4-Fc protein (R&D Systems) was applied to 96-well plates (Lumitrac 600 LIA plate, Greiner) at a rate of 1 pmol/well. After washing, titrated doses of anti-CTLA-4 mAb were allowed to bind for 1 hour. His-tagged ligands were added at 200 nM and 100 nM, respectively (rhCD80 and rhCD86; R&D Systems), and the plates were incubated for an additional 15 minutes. After washing, bound ligand was detected using an HRP-labeled anti-His antibody (R&D Systems). Super Signal ELISA Pico (Thermo Scientific) was used as substrate and plates were analyzed using a Tecan Ultra Microplate reader.

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

Blocking a functional ligand in vitro

For the SEB PBMC assay, all PBMCs from healthy donors were seeded into 96-well plates (1 × ¹⁰⁵ cells/well) 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 using MSD (Mesoscale) on day 3 according to the manufacturer's instructions.

Antibodies 4-E03 and 2-C06 were shown to enhance IL-2 production, and their efficacy was similar to that of Yervoy. In fig. Figure 11 shows one representative donor out of 6.

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

Antibody-dependent cellular cytotoxicity (ADCC)

ADCC assays were performed using the NK-92 cell line stably transfected to express the CD16-158V allele along with GFP (purchased from Conkwest, San Diego, CA; Binyamin, L., et al., 2008, Blocking NK cell inhibitory self- recognition promotes antibody-dependent cellular cytotoxicity in a model of anti-lymphoma therapy. Journal of immunology 180, 6392-6401). Target CD4+ T cells were isolated from the peripheral blood of healthy donors using a CD4+ T cell isolation kit (Miltenyi Biotec). Cells were stimulated for 48 hours with paramagnetic CD3/CD28 beads (Life Technologies, Thermo Fisher) and 50 ng/ml rhIL-2 (R&D Systems) at 37°C. Target cells were preincubated with mAb at 10 μg/ml for 30 min at 4°C before mixing with NK cells. Cells were incubated for 4 h in RPMI 1640 + GlutaMAX medium (Invitrogen) containing 10 mM HEPES buffer, 1 mM sodium pyruvate, and 10% FBS low IgG at a 2:1 effector cell:target cell ratio. Lysis was determined by flow cytometry. Briefly, at the end of incubation, the cell suspension was stained with BV510-conjugated anti-CD4 (clone RPA-T4, BD Biosciences) along with 10 nM SYTOX Red for dead cells (Invitrogen) or Fixable Viability Dye eFluor780 (eBioscience) for 20 minutes in the dark. at 4°C, and then cells were analyzed using FACSVerse (BD Biosciences).

4-E03 showed significantly improved deletion of CTLA-4+ T cells in vitro compared to Yerva (Fig. 12).

Expression of CTLA-4 on primary patient material

To confirm the translational potential of the above discovery of the depleting activity of anti-CTLA-4 mAbs, CTLA-4 expression was examined in primary patient material.

Ethical approval for the use of clinical samples was obtained from the Ethics Committee of Skåne University Hospital. Informed consent was provided in accordance with the Declaration of Helsinki. Samples were obtained from the Department of Gynecology and Oncology at Skåne University Hospital, Lund. Ascitic fluid was assessed as isolated suspensions of individual cells. Tumor material was cut into small pieces and incubated in R10 with DNase I (Sigma Aldrich) and Liberase ^™ (Roche Diagnostics) for 20 minutes at 37°C. The remaining tissue was mechanically destroyed and, together with the cell suspension, passed through a 70 μm cell filter. Cells isolated from ascitic fluid and tumors were stained. The following antibodies were used to identify different T cell subsets: 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 isotype IgG2a, κ control-PE (G155-178; all from BD Biosciences). Data collection was performed using FACSVerse and data were analyzed using FlowJo.

As shown in FIG. 13, CTLA-4 is most highly expressed on intratumoral Treg cells, making them a good target for depletion of CTLA-4-specific antibodies.

Model PBMC-NOG/SCID

To confirm the in vitro data on the depletion activity of specific anti-CTLA-4 antibodies, we analyzed the depletion ability of anti-CTLA-4 mAbs in the PBMC-NOG/SCID model in vivo. The model is based on the well-known hu-PBMC-NOG model (Sαndergaard H. et al., Clin Exp Immunol. 2013 May;172(2):300-10. doi: 10.1111/cei.12051; Cox J H et al., PLoS One. 2013 Dec 23;8(12):e82944. doi: 10.1371/journal.pone.0082944. eCollection 2013) and was modified in-house as described below.

Mice were bred and maintained at local facilities according to home office guidelines. Female CB 17 scid at eight weeks of age (Bosma GC et al., Nature. 1983 Feb 10;301(5900):527-30) and NOG (NOD/Shi-scid/IL-2Rγ ^null ; Ito M et al, 2002 ,NOD/SCID/γ mouse: an excellent recipient mouse model for engraftment of human cells. Blood 100 (9):3175–3182) mice were supplied by Taconic (Bomholt, Denmark) and maintained in local animal facilities. For the PBMC-NOG/SCID (primary human xenograft) model, human PBMCs were isolated using Ficoll Paque PLUS and, after washing, the cells were resuspended in sterile PBS at 75 × 10 ⁶ cells/ml. NOG mice were injected intravenously with 200 μl of cell suspension, corresponding to 15 × 10 ⁶ cells/mouse. 2 weeks after injection, spleens were isolated and processed into a single cell suspension. A small sample was then taken to determine CTLA-4 expression on human T cells using FACS. As shown in FIG. 13, CTLA-4 is highly expressed on Treg cells compared to other T cells, reflecting the situation in human patients. Most cells were resuspended in sterile PBS at a concentration of 50 × 10 ⁶ cells/ml. SCID mice were injected i.p. with 200 μl of the suspension, which corresponds to 10 × 10 ⁶ cells/mouse. After 1 hour, mice were treated with 10 mg/kg anti-CTLA-4 hIgG1, Yervoy, or isotype control mAb. Intraperitoneal fluid from mice was collected after 24 hours. Human T cell subsets were identified and quantified by FACS using the following markers: CD45, CD4, CD8, CD25, CD127 (all from BD Biosciences).

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).

Example 5 Selected surrogate antibody m5-B07 has the same functional characteristics as 4-E03

In some examples, particularly in vivo examples, the antibody clone 5-B07 in mIgG2a format (also designated m5-B07) was used. This is a murine antibody that is a surrogate antibody for the human antibodies described herein. It was chosen as a surrogate antibody because it binds murine CTLA-4 and thereby blocks ligand binding (Fig. 16 A-B). In addition, it also exhibits Treg depletion activity (Figure 16 CD).

Ligand blocking ELISA

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

As shown in FIG. 16, the antibody blocks the binding of (A) CD80 and (B) CD86 to their ligand CTLA-4.

Treg depletion activity in vivo

The effects of CTLA-4-specific antibodies on tumor T cell subsets in vivo were examined in the CT26 tumor model as described below.

Mice were bred and maintained at local facilities according to home office guidelines. Six- to eight-week-old Balb/C females were provided by Taconic (Bomholt, Denmark) and maintained in local animal facilities. CT26 cells (ATCC) were grown in glutamax-buffered RPMI medium supplemented with 10% FCS. When the cells were semi-confluent, they were detached with trypsin and resuspended in sterile PBS at 10 × 10 ⁶ cells/ml. Mice were injected s.c. with 100 μl of cell suspension, which corresponds to 1 × 10 ⁶ cells/mouse. When the tumors reached approximately 7x7 mm, mice were administered 10 mg/kg of the indicated antibodies twice a week, as shown in the figures. After the third injection, tumors were excised, mechanically divided into small pieces and digested with a mixture of 100 μg/ml Liberase (Roche) and 100 μg/ml DNase (Sigma) at 37°C for 2x5 min with Vortex in between. Then the cell suspension was washed (400 g for 10 min) with PBS containing 10% FBS. Cells were then resuspended in MACS buffer and stained with a panel of antibodies staining CD45, CD3, CD8, CD4, and CD25 (all from BD Biosciences). Before staining, cells were blocked for nonspecific binding using 100μg/ml IVIG. Cells were analyzed using FACS Verse (BD Biosciences). Mouse Treg cells were identified as CD45 ⁺ CD3 ⁺ CD4 ⁺ CD25 ⁺ cells.

As shown in FIG. 16C,5-B07 in mouse IgG2a format mediates tumor Treg deletion associated with D) increased CD8/Treg ratio compared to other CTLA-4-specific n-CoDeR antibodies and the well-characterized commercially available clone 9H10.

Example 6. Production of a virus expressing anti-CTLA4 mAb (COPTG19385) or anti-CTLA4 mAb and GM-CSF (COPTG19384), transgene expression and characterization of genetic stability

COPTG19384 and COPTG19385 are vaccinia viruses (Copenhagen strain) encoding monoclonal anti-CTLA4 antibodies (4-E03). COPTG19384 additionally encodes human GM-CSF. In particular, COPTG19384 and COPTG19385 are defective in thymidine kinase (TK, J2R locus) and ribonucleotide reductase (RR, I4L locus) activity. As shown in FIG. 17, an expression cassette encoding the heavy chain 4-E03 (HC; SEQ ID NO: 54) under the control of the p7.5K promoter (SEQ ID NO: 59) was inserted into the J2R locus, and an expression cassette encoding the light chain (LC, SEQ ID NO: 53) IgG 4-E03 under the control of the p7.5K promoter (SEQ ID NO: 59), was placed in the I4L locus. For COPTG19384, an expression cassette encoding human GM-CSF (SEQ ID NO: 56) under the control of the pSE/L promoter (SEQ ID NO: 61) was also placed at the I4L locus.

The same promoter (p7.5K) was used to control the expression of HC and LC to obtain the same level of expression for both chains and therefore optimal assembly of the antibody as a heterotetrameric protein (i.e. avoiding excess unlinked chain) . However, the same promoter for both antibody chains prevents their integration into the same locus (identical DNA sequences increase the risk of recombination and then deletion of transgenes). Therefore, the cassette encoding 4-E03 HC was inserted into the J2R locus, and the cassette encoding LC 4-E03 into the I4L locus. A cassette encoding the GM-CSF transgene, but under a different promoter (pSE/L), was also inserted into the I4L locus as an antibody light chain.

Receiving COPTG19384

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

A synthetic fragment called the HC Fragment of 1436 bp was obtained containing the HC gene of antibody 4-E03. A fragment “LC fragment” containing the LC gene of antibody 4-E03 and the hGM-CSF gene under the control of pSE/L was obtained synthetically and inserted into a plasmid vector. Coding sequences were optimized to use human codons, a Kozak sequence (ACC) was added before the ATG start codon, and a transcription terminator (TTTTTNT) was added after the stop codon. In addition, some templates were excluded: TTTTTNT, GGGGG, CCCCC, which are detrimental to expression in the poxvirus.

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

COPTG19384 was generated in chick embryo fibroblast (CEF) by two sequential homologous recombinations for sequential insertion into the I4L and J2R loci and using COPTG19156 as the parental virus and two transfer plasmids pTG19367 and pTG19384. CEF was isolated from 12-day embryonated SPF eggs (Charles River). Embryos were mechanically dissected, solubilized in Tryple Select solution (Invitrogen), and cultured in MBE (Eagle-based medium; Gibco) supplemented with 5% FCS (Gibco) and 2 mM L-glutamine.

Homologous recombination between vector plasmids and the parental vaccinia virus produces recombinant vaccinia viruses that have lost the GFP and mCherry expression cassettes and accumulated antibody and GM-CSF expression cassettes. COPTG19156 contains an mCherry gene expression cassette at its I4L locus and a GFP gene expression cassette at its J2R locus. Homologous recombination between the transfer plasmid pTG19367 and the parental COPTG19156 allows the generation of recombinant vaccinia viruses that have lost their GFP expression cassette and acquired the 4-E03 heavy chain expression cassette, and selection was performed by isolating red fluorescent plaques. This intermediate recombinant virus (COPTG19367) was used as the parent virus for a second round of homologous recombination with pTG19384 as a carrier plasmid to generate recombinant vaccinia viruses that had lost the mCherry expression cassette and were given the 4-E03 light chain and GM-CSF expression cassette. Selection COPTG19384 (Fig. 17) was performed by isolating white non-fluorescent plaques.

The COPTG19384 viral stock solution was amplified on CEF in two F175 flasks to create corresponding viral stock solutions that could be aliquoted and stored at -80°C until use. The viral stock solution was titrated on CEF cells and infectious titers were expressed in PFU/ml and calculated using the following formula: number of lytic regions x dilution factor x 4. For illustrative purposes, the resulting viral stock solution was titrated to 6.8 x 10 ⁶ PFU/ml. This stock solution was analyzed by PCR to verify the integrity of the expression cassettes and recombination arms using appropriate primer pairs. The stock solution was also analyzed by sequencing both expression cassettes. Alignment of the sequencing results showed 100% homology with the theoretically expected sequence. If necessary, viral preparations were purified using conventional techniques (eg, as described in WO2007/147528).

Receiving COPTG19385

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

A synthetic fragment called the HC Fragment of 1436 bp was obtained containing the HC gene of antibody 4-E03. Coding sequences were optimized to use human codons, a Kozak sequence (ACC) was added before the ATG start codon, and a transcription terminator (TTTTTNT) was added after the stop codon. In addition, some templates were excluded: TTTTTNT, GGGGG, CCCCC, which are detrimental to expression in the poxvirus.

The HC fragment was inserted into pTG19339 restricted to PvuII by homologous recombination resulting in pTG19367.

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

COPTG19385 was generated in chick embryo fibroblast (CEF) by two sequential homologous recombinations for sequential insertion into the J2R and I4L loci and using COPTG19156 as the parent virus and two transfer plasmids pTG19367 and pTG19385. CEF was isolated from 12-day embryonated SPF eggs (Charles River). Embryos were mechanically dissected, solubilized in Tryple Select solution (Invitrogen), and cultured in MBE (Eagle-based medium; Gibco) supplemented with 5% FCS (Gibco) and 2 mM L-glutamine.

Homologous recombination between vector plasmids and the parental vaccinia virus produces recombinant vaccinia viruses that have lost the GFP and mCherry expression cassettes and accumulated antibody expression cassettes. COPTG19156 contains an mCherry gene expression cassette at its I4L locus and a GFP gene expression cassette at its J2R locus. Homologous recombination between the transfer plasmid pTG19367 and the parental COPTG19156 allows the generation of recombinant vaccinia viruses that have lost their GFP expression cassette and acquired the 4-E03 heavy chain expression cassette, and selection was performed by isolating red fluorescent plaques. This intermediate recombinant virus (COPTG19367) was used as the parent virus for a second round of homologous recombination with pTG19385 as a carrier plasmid to generate recombinant vaccinia viruses that had lost the mCherry expression cassette and the accumulated 4-E03 light chain expression cassette. Selection of COPTG19385 was performed by isolating white non-fluorescent plaques.

The COPTG19385 viral stock solution was amplified on CEF in two F175 flasks to create corresponding viral stock solutions that could be aliquoted and stored at -80°C until use. The viral stock solution was titrated on CEF cells and infectious titers were expressed in PFU/ml and calculated using the following formula: number of lytic regions x dilution factor x 4. For illustrative purposes, the resulting viral stock solution was titrated to 1.04 x 10 ⁷ PFU/ml. This stock solution was analyzed by PCR to verify the integrity of the expression cassettes and recombination arms using appropriate primer pairs. The stock solution was also analyzed by sequencing both expression cassettes. Alignment of the sequencing results showed 100% homology with the theoretically expected sequence. If necessary, viral preparations were purified using conventional techniques (eg, as described in WO2007/147528).

Transgene expression

Virus-mediated expression of monoclonal antibody 4-E03 was assessed in supernatants of CEF cells infected with COPTG19384 by Western blot (WB) and compared with recombinantly produced antibody (40 ng 4-E03). WB allows visualization of the presence of nonfunctional molecules that do not bind CTLA4 (eg, molecules with incomplete chain assembly, aggregates). CEF cells were infected at an MOI of 0.05 with COPTG19384 viral stock in triplicate. Cell supernatants were collected after 48 hours and analyzed by WB after electrophoresis under non-reducing conditions and using either anti-Ig (left blot) or anti-light chain (right blot), HRP-conjugated antibody. The results illustrated in FIG. 18A show that the profile of mAb WB under non-reducing condition produced by infected CEF is close to that of purified 4-E03 with a similar apparent size of 100 to 150 kDa, indicating proper chain folding and assembly.

Quantitative determination of functionally secreted antibodies 4-E03 and GM-CSF in the supernatants was performed using ELISA. ELISA made it possible to quantitatively measure the amount of functional polypeptide produced in cell supernatants. VVTG17137 was used as a negative control. This is a vaccinia virus (Copenhagen strain) with a deletion in the J2R and I4L loci encoding the suicide gene FCU1 (described in WO2009/065546).

To evaluate antibody 4-E03, microplates were coated by overnight incubation at 4°C with 100 μl per well of CTLA4-Fc at 0.25 μg/ml. After incubation, the coating solution was removed, blocking solution was added, and the plates were incubated for 1-2 hours at RT before washing. 4-E03 calibration standards (0.097 to 100 ng/mL) or samples (in triplicate) diluted in blocking solution were added to the wells, and the plates were incubated for 2 hours at 37°C before washing. HRP-conjugated antibodies diluted in blocking solution were added to each well, and the plates were incubated for 1 hour at 37°C before washing. After incubation with TMB solution for 30 min at RT in the dark, 2 M H ₂ SO ₄ (stop solution) was added to stop the enzymatic reaction. Absorbance was read at 450 nm on a microplate reader. Absorbance was plotted against the concentration of antibody in the calibration standard. As illustrated in FIG. 18B, functional mAb4-E03 is produced in cells infected with COPTG19384, reaching concentrations approaching 1 μg/ml.

Virus-mediated expression of GM-CSF in the same supernatants was also assessed using Quantikine® ELISA (R&D Systems Ref SGM00). Briefly, two anti-hGM-CSF antibodies are used in this assay. The first antibody used to capture hGM-CSF in the samples was applied to the surface of the wells of a 96-well plate. The second is conjugated and added to the plate in solution to detect captured hGM-CSF. The concentration of hGM-CSF in the sample is then calculated by interpolation from the calibration curve obtained with some purified hGM-CSF provided in the kit. As illustrated in FIG. 19 the expression level of GM-CSF was approximately 6 μg/ml.

In conclusion, concentrations equal to or greater than 1 μg/ml were detected for both transgenes, indicating satisfactory levels of expression.

Genetic stability:

Genetic stability tests were performed after five passages of the virus on CEF in serum-free medium at a multiple infection (MOI) of 10 ^-4 . Passage P5 was diluted and inoculated onto CEF cells in 60 mm diameter culture dishes to produce 20 to 40 viral plaques per plate. One hundred viral plaques were isolated and subcultured. After one round of amplification, isolated viral plaques were inoculated onto CEF cells and tested by PCR and ELISA. PCR analysis and transgene expression showed that more than 90% of the clones had the correct profile. Since the acceptance criterion for clinical product development is genetic stability greater than or equal to 90%, COPTG19384 was considered genetically stable.

Example 7: In vitro characterization of COPTG19384

Replication studies in hepatocytes: tumor selectivity of virus expressing anti-CTLA4 mAb and GM-CSF

COPTG19384 carries two gene deletions encoding viral enzymes (TK and RR) involved in nucleotide metabolism. When these enzymes function, they allow the virus to replicate in the cytoplasm of most cells, including cells in a quiescent state (i.e., with a low nucleotide pool). Host range studies were performed on primary and malignant cells to ensure that insertion of different transgenes at the J2R and I4L loci did not alter host range selectivity. Reproduction of COPTG19384 was assessed on normal primary human cells (hepatocytes obtained from Biopredic) and on tumor cells from the same organ (HepG2 from hepatocarcinoma, ATCC® HB-8065™). Replication rates and therapeutic indices were calculated and compared with those of wild-type Copenhagen vaccinia virus (COP WT, non-deletion virus) as references for non-selective vaccinia virus and recombinant double-deletion virus VVTG17137 (deleted in the J2R and I4L genes with the suicide gene FCU1, inserted instead of J2R), respectively. Two lots were analyzed: a research lot (Lot 1) and a GMP-manufactured lot (Lot 2). The replication rate was determined as the ratio of the total number of infectious particles at the end of incubation/initial infectious particle (inoculum). The therapeutic index of each virus was determined as the ratio: replication rate on HepG2 cells/replication rate on hepatocytes. The higher the ratio, the higher the selectivity of the virus towards tumor cells.

Primary hepatocytes were grown in liver basal cell medium supplemented with 1.6% hepatocyte culture medium supplement. HepG2 was seeded in a 12-well plate at 4E+05 cells/well and incubated for 24 hours at 37°C with 5% CO ₂ . Before infection, the culture medium was removed and 70 PFU/well of virus was added to each well in either PBS for hepatocytes or PBS plus FCS for HepG2. Infected cells were incubated for 30 min at 37°C with 5% CO ₂ and then 1.5 ml/well of culture medium was added. The plates were incubated at 37°C with 5% CO ₂ for 3 days and then stored at -80°C. The plates were then thawed and the wells sonicated for 30 seconds at 40% amplitude before titration on Vero cells.

Replication rate in normal human hepatocytes:

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 these cells, COP WT spread well with a replication rate of more than 50,000 (Fig. 20A). In other words, each initial viral particle of infection produces about 50,000 new viruses. In the case of two double-deletion recombinant viruses (ie, VVTG17137 and COPTG19384), this replication rate was dramatically reduced to 5-15 depending on the virus or batch of virus (Fig. 20A). This latter result indicates that the attenuated replication relative to normal cells caused by the two deletions was conserved between VVTG17137 and COPTG19384.

Replication rate in HepG2 tumor cells:

HepG2 cells were chosen to monitor the ability of COPTG19384 to replicate in human tumor cells because these cells are the malignant counterpart of normal hepatocytes. In these cells, the five viruses tested had approximately the same replication rate, reaching about 100,000 new viruses, regardless of the original virus tested (Fig. 20B). Therefore, double deletion in both VVTG17137 and COPTG19384 and vectorization of the transgenes did not impair their ability to replicate in malignant cells.

Therapeutic index:

As shown in FIG. 20C, the calculated index for COP WT is only two, which indicates the low selectivity of COP WT for tumor cells compared to normal cells. In contrast, for both VVTG17137 and COPTG19384 (and for both batches of viruses tested), this index ranges from 8.2 E+03 to 1.8 E+04. This confirms that the two recombinant viruses have equally good selectivity for tumor and normal cells.

These results demonstrated that COPTG19384 and VVTG17137 have very similar replication properties on both tumor and healthy cells. Compared to COP WT, their replication on tumor Hep G2 is similar, while on healthy hepatocytes it is severely impaired. Consequently, the deletion of two genes (J2R and I4L) limits the replication of the deleted virus to replicating cells (i.e. with a large pool of nucleotides), including tumor cells. Because transgene expression and replication are closely coupled, COPTG19384 is an effective vector for selective delivery of therapeutic proteins to tumors.

Replication analyzes on CEF and LoVo:

COPTG19384 replication was assessed in CEF (producer cells) isolated from 11 or 12 day specific pathogen-free embryonic eggs (Charles Rivers) and in a human tumor cell line (LoVo; ATCC® CCL-229 ^™ ). CEF and LoVo cells were prepared as a suspension and infected at an MOI of 10 ^-3 for CEF and 10 ^-2 for LoVo (three wells per cell and time point). After incubation at different times, virus titrations were performed on Vero cells (CCL-81™). The replication of COPTG19384 was compared with that of VVTG17137 as a reference. The results show that the replication of COPTG19384 and VVTG17137 was similar in both CEF and LoVo (data not shown).

Replication assays on reconstructed human skin:

Replication of COPTG19384 was also assessed in reconstructed human skin (T-Skin™/Full Thickness Human Skin Model). Thirty six T-Skin™ samples obtained from (EPISKIN SA) were cultured in 6-well plates and maintained in fresh culture medium. VVTG17137 and COPTG19384 were dispensed into each well (in triplicate) to obtain a controlled final concentration (ie, 10 ¹ to 10 ⁵ PFU/well). A negative control corresponding to medium without virus (Mock) was also tested. The plates were incubated at 37°C with 5% CO ₂ for 7 days and T-Skin™ samples were collected and cut into two pieces. The infectious titer was determined on one of two samples using Vero virus titration cells. In fig. 21 shows that COPTG19384 replicates in reconstructed skin to the same extent as the reference sample VVTG17137, confirming that vectorization of either GM-CSF or mAb 4-E03 did not alter the replication behavior of vaccinia virus in reconstructed human skin.

Oncolytic assay

Oncolytic activity is the lytic activity of the tested viral samples against tumor cells. It was assessed by quantifying cell viability after 5 days of incubation on various tumor cell lines: human colorectal adenocarcinoma cell line LoVo (ATCC® CCL-229™), human pancreatic tumor cell line MIA PaCa-2 (ATCC® CCL-1420) and human hepatocarcinoma cell line HepG2 (ATCC® HB-8065™). The oncolytic activity of COPTG19384 was compared with that of VVTG17137 as a reference. A negative control corresponding to uninfected cells (mock infected cells) was also placed on the plates.

Cells were prepared, distributed into Eppendorf tubes (1.2 × 10 ⁶ cells/tube) before virus infection at an MOI of 10 ^-5 to 10 ^-2 and incubated for 30 min at 37°C. The appropriate complete medium was added to an Eppendorf tube and an aliquot of this suspension was added to each well (in triplicate) in a 6-well plate containing 2 ml of the appropriate complete medium. The plates were incubated at 37°C with 5% CO ₂ for 5 days and cell viability was determined in a Vi-Cell counter. The results were expressed as a percentage of the viability of mock-infected cells. Cell supernatants were also collected to determine the concentration of 4-E03mAb and GM-CSF. Fig. 22 demonstrates that the oncolytic activities of COPTG19384 and VVTG17137 are similar in the three tumor cell lines evaluated.

Transgene expression level

Expression levels of both monoclonal antibody 4-E03 and GM-CSF were measured by ELISA (as described in Example 6) in culture supernatants of HepG2 and LoVo cells isolated after oncolysis activity determination (5 days of infection at variable MOI).

Expression levels of 4-E03 and GM-CSF were also measured by ELISA (see Example 6). in the supernatants of 5 cell lines cultured under the following conditions, respectively, human gastric carcinoma cell line Hs-746 T (ATCC® HTB-135™), human ovarian tumor cell line SK-OV-3 (ATCC® HTB-77™), cell human pancreatic tumor line MIA PaCa-2 (ATCC® CCL-1420), human colorectal adenocarcinoma cell line LoVo (ATCC® CCL-229™) and human colorectal carcinoma cell line HCT 116 (ATCC® CCL-247™). Each cell the line was cultured (in triplicate) in 6-well plates (10 ⁶ cells/well) and incubated at 37°C with 5% CO ₂ for 24 hours before infection at an MOI of 0.05. Cell supernatants were then collected 48 h postinfection to determine the concentration of 4-E03mAb and GM-CSF. As expected, MOI, time postinfection, and cell line are important parameters that influence the level of transgene expression in supernatants of infected cells. Fig. 23A demonstrates that a tumor cell line that is quite highly replicative (HepG2) and quite resistant (LoVo) when infected with COPTG19384 is able to produce approximately the same amount of 4-E03 mAb and GM-CSF in its culture supernatants. However, the maximum expression of HepG2 is achieved at an MOI 10 times lower than for LoVo. Moreover, for the 5 tumor cell lines tested, transgene expression was greater than 0.1 and greater than 1 μg/ml for 4-E03 mAb and GM-CSF, respectively. (Fig. 23B). It should be noted that the ELISA assay used to measure the concentration of 4-E03 mAb uses the CTLA4 antigen to capture the antibody. In other words, the antibodies measured by this assay are at least partially functional (i.e. recognize their antigen, other functions of the antibody are performed by the Fc part),

Purification of 4-E03 mAb and glycosylation profile analysis

To obtain fairly large amounts of 4-E03 mAb from infected cells, 15 F175 flasks containing ~4.7 × 10 ⁷ Mia-PACA cells/vial were infected with COPTG19384 at an MOI of 0.01 and incubated for 72 hours. MIA Paca-2 cell culture supernatant (about 450 ml containing 670 μg of 4-E03 mAb as determined by ELISA) was collected, pooled, and clarified by centrifugation to remove most of the cellular debris. Cleared supernatants were filtered through 0.2-μm filters and supplemented with 2 mM EDTA (to inhibit putative metal proteases) and 20 mM Tris pH7.5 (to increase pH). The filtered supernatant was then passed through a protA Hitrap column (GE healthcare, reference 17-5079-01). The column was transferred and connected to an FPLC purifier (GE Healthcare) and the purification program was applied (THM/ProtA 1 mL frac bleu injection loop). The eluted Ab-containing fractions were loaded onto NuPage Bis-Tris 4-12% gels (Thermo NP0323) after adding Laemlly buffer (Biorad) with or without beta-mercaptoethanol to reduce or not the mAb disulfide bonds. The gel was stained with InstantBlue (Expedeon, ISB1L). The three fractions corresponding to the major elution peak were pooled and, after dialysis against drug buffer, the antibody concentration was determined by absorbance at 280 nm. The final concentration of purified mAb was 0.29 mg/ml.

The first characterization was the assessment of chain assembly by electrophoresis under reducing and non-reducing conditions. Under non-reducing conditions, 2 light and 2 heavy chains assemble to form a native and functional antibody. Purified 4-E03 from MIA-infected PaCa-2 and recombinantly produced 4-E03 were found to have indistinguishable electrophoresis profiles under both reducing and non-reducing conditions. In other words, purified 4-E03 from MIA-infected PaCa-2 has the expected ratio of light to heavy chains and is correctly assembled into a heterotetramer with 2 light and 2 heavy chains. The presence of this heterotetramer was also confirmed by mass spectrometry. The purified mAb was subjected to mass spectrometry to analyze glycosylation. Briefly, the mAb was cleaved or not by the IdeS protease, which specifically cleaves IgG at the hinge (resulting in F(ab') ₂ and Fc moieties). The masses of the whole antibody or Fc portions that bear N-glycosylation were determined, and each mass was assigned a theoretical mass calculated based on the primary Fc sequence and glycosylation pattern. The glycosylation profile of 4-E03 mAb purified from MIA-infected PaCa-2 was compared with that of recombinantly produced and purified 4-E03 and MabThera as a reference for human IgG1 used in the clinic. The results show that 4-E03 derived from MIA-infected PaCa2 had a different glycosylation profile from the two reference antibodies with a majority of G0F (88%), while both recombinant 4-E03 and MabThera had a similar glycosylation profile with a typical distribution G0F, G1F and G2F. However, it has been suggested that MIA PaCa-2 is responsible for the low levels of G1F and G2F in purified 4-E03 mAb due to low levels of beta-1,4-galactosyltransferase 1 transcript, which may account for the lack of a galactosyl moiety (and therefore , absence of G1f and G2F) 4-E03 expressed in MIA PaCa-2.

The same type of purification followed by bulk analysis was also performed from the permeate recovered during purification of COPTG19384 obtained at CEF. The results show that the glycosylation profiles of 4-E03 from infected CEF are very similar to those of MabThera or recombinant 4-E03. This latter result suggests that the glycosylation profile of the antibody is more dependent on the cell line used than on the infection itself.

4-E03 purified from the supernatant of infected MIA PaCa-2 cells (4-E03 TG) also exhibited the same binding characteristics as 4-E03 recombinantly produced by CHO (study lot) or HEK (toxin lot) cells (Fig. 24 and 25). This was demonstrated by using an ELISA (as described in Example 1) to test binding to recombinant human CTLA-4 protein (FIG. 24A) and cynomolgus monkey (FIG. 24B). FACS analysis testing binding to human (FIG. 25A) and cynomolgus (FIG. 25B) expressing cells (see Examples 1 and 2) confirmed similar cross-reactivity and binding affinities across batches 4 -E03.

Character of glycosylation and disulfide bonds of GM-CSF

To investigate the glycosylation pattern and the presence of certain disulfide bonds, different human tumor cell lines were infected with COPTG19384 and their supernatants were analyzed by the same WB method. MIA-Paca-2, LoVo, HepG2, and HCT116 cells were infected at an MOI of 0.01 and incubated for 72 h in culture medium without serum. Culture supernatants were collected, clarified by centrifugation, and then filtered on a 0.2-μm filter. Supernatants were stored at -20°C until analysis. They were treated with 8 μl of Rapid PNGase F Buffer 5X followed by incubation at 75°C for 5 minutes. One μl of PNGase F was then added (to remove N-glycans from the glycoprotein) and the mixture was incubated for 30 minutes at 50°C. Twenty-five μl of samples were prepared by adding 5 μl of Laemmli buffer x4 with or without beta-mercaptoethanol (reducing and non-reducing conditions) before submitting for Western blotting. Immune complexes were detected using Amersham ECL Prime Western blotting, and chemiluminescence was detected using a Molecular Imager ChemiDOC XRS (Biorad).

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

Example 8: Pharmacokinetics after intratumoral injection of COPTG19384

Kinetics of tumor and circulatory expression of anti-CTLA4 antibodies, GM-CSF, and viruses following intratumoral (i.t. -intratumoral) administration of vaccinia viruses in the LoVo xenograft model.

Protocol

5 × 10 ⁶ LoVo cells were implanted into the right flank of Swiss nude mice (Charles River, France). After about two weeks, when the tumor volume reached ~120 mm ³ , the mice were randomized and divided into 6 groups of 15 animals.

• Mice from group 1 received it administration of COPTG19384 at a dose of 1 × 10 ⁴ PFU/mouse on D0 (first day of treatment).

• Mice from group 2 received it administration of COPTG19384 at a dose of 1 × 10 ⁵ PFU/mouse at D0.

• Mice from group 3 received it administration of VVTG17137 at a dose of 1 × 10 ⁴ PFU/mouse at D0.

• Mice from group 4 received it administration of VVTG17137 at a dose of 1 × 10 ⁵ PFU/mouse at D0.

• Mice in group 5 received intraperitoneal (IP) administration of 4-E03 at a dose of 3 mg/kg at D0.

• Mice from group 6 received ipilimumab (Yervoy) at a dose of 3 mg/kg at D0.

Tumor and blood from 3 animals were collected on days 1, 3, 6, 10 and 20. Tumors were weighed and homogenized for immediate processing. A quarter of the homogenized tumors were collected for virus titration, the remaining suspension was centrifuged, and supernatants were stored at −20°C until use. The blood was divided into two parts: one was added to a heparin tube (25 IU/100 μl of blood) for titration analysis and frozen at -80°C until analysis. Clarified sera were obtained from another part and stored at -20°C until use. The virus titer was determined in tumor and blood samples by titration on Vero cells.

Virus replication kinetics in the LoVo model:

In the LoVo model, where COPTG19384 was administered once in two doses (1 × 10 ⁴ or 1 × 10 ⁵ PFU), viral replication was monitored and compared with that of VVTG17137 administered under the same conditions. The results shown in FIG. 26 show important variation in the three viral titers measured at each time point. In any case, the results show that both viruses and at both doses replicate in the tumor and maintain a fairly high titer/g tumor from days 3 to 20 after injection. There is no obvious difference in virus titers for a given time point between the two doses of virus or between the two viruses used. Interestingly, all blood samples were negative for virus detection, with the exception of one sample (VVTG17137, dose: 1 x ¹⁰⁷ PFU on day 10), for which only 13 PFU/ml was detected (data not shown).

Together, these results indicate that after a single it injection of either 1x10 ⁴ or 1x10 ⁵ PFU, viral replication was maintained in LoVo tumors for at least 20 days with barely noticeable presence in the bloodstream. It should be noted that the LoVo xenograft model is very favorable for viral replication because it utilizes permissive human and Swiss nude mouse tumor cells, which have a severely compromised immune system with limited antiviral activity.

Kinetics of transgene expression in the LoVo model:

As expected, the kinetics of transgene expression in the tumor followed the kinetics of viral replication with a maximum concentration (Cmax) at day 6 or 10 for both 4-E03 mAb (Fig. 27A) and GM-CSF (Fig. 27B). 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 thereafter the measured mAb concentrations corresponded to the pharmacokinetics of human IgG1 in mice (Fig. 28).

Moreover, tumor concentrations of 4-E03 at Cmax and thereafter (i.e., 6-10 to 20 days post-injection) were approximately 10-fold higher after treatment with COPTG19384 (for both doses) than after a single injection of 4-E03. E03 mAb at a therapeutic dose of 3 mg/kg (Fig. 27A). In contrast, blood mAb concentrations following treatment with COPTG19384 were always lower than concentrations measured after intraperitoneal injection of 3 mg/kg 4-E03 (FIG. 28A). This result demonstrates that mAb vectorization allows high tumor concentrations to be achieved without exceeding or even reaching the blood concentrations obtained with therapeutic mAb dosing.

The kinetics of GM-CSF expression after treatment with COPTG19384 corresponds to the kinetics observed with 4-E03 (Fig. 27B). Interestingly, GM-CSF levels measured in the tumor are lower than 4-E03 levels for the same samples, although in vitro COPTG19384-infected LoVo express about twice as much GM-CSF as 4-E03. GM-CSF blood concentrations were also very low compared to 4-E03 concentrations (Fig. 28B). This result is consistent with the half-life of GM-CSF in vivo, which is very short compared to the half-life of human IgG1.

These results indicate that vectored antibodies and GM-CSF are expressed predominantly in the tumor after i.t. injection of COPTG19384 with minimal systemic exposure. These results confirm that vectorization is particularly suitable for transgenes with toxicological (eg, anti-CTLA4) or pharmacokinetic (eg, GM-CSF) problems.

Kinetics of tumor and bloodstream expression of anti-CTLA4 antibodies, GM-CSF, and viruses after intratumoral administration of vaccinia viruses in the syngeneic CT26 model

Assessing viral activity in an immunocompetent CT26 mouse model requires the generation of several surrogate viruses encoding mouse anti-mCTLA4 with or without mouse GM-CSF:

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

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

• VVTG18058, used as a reference, is a vaccinia virus (Copenhagen strain) deleted in the J2R and I4L genes, without any transgene (“blank” virus).

These vaccinia viruses were generated, like their human counterparts, by two sequential homologous recombinations at the J2R (TK) and then I4L (RR) loci, following the process described in Example 6. ELISA method for quantification of m5-B07 and mGM-antibodies CSFs were similar to those described above (Example 6, “transgene expression” for 4-E03 and GM-CSF), except that the CTLA4-Fc mouse antigen was used to capture the mouse antibody, and the Quantikine ELISA Mouse GM-CSF kit (R&D Systems ) was used to quantify mGM-CSF. The oncolytic activity of these viruses was also assessed in different cell lines (one sarcoma: MCA205 and two colon carcinomas CT26 and MC38) and was similar to that of VVTG18058, showing that vectorization of the mouse antibody with or without mGM-CSF did not affect oncolytic abilities vaccinia virus (data not shown).

Protocol: CT26 cells (2 × ¹⁰⁵ cells) were implanted into the right flank of Balb/c mice (Charles River, France). After about one week, when the tumor volume reached 25-50 mm ³ _, the mice were randomized and divided into 3 groups of 20 animals (groups 1 to 3) and one group of 4 of 10 animals. Tumor and blood were collected and processed as described for the LoVo model, except that they were collected on days 1, 4, 8, and 10 for the first 3 groups and day 1 for group 4.

• Mice from group 1 received it administration of VVTG18058 at a dose of 1 × 10 ⁷ PFU/mouse at D0, D2 and D4.

• Mice from group 2 received it administration of COPTG19407 at a dose of 1 × 10 ⁷ PFU/mouse at D0, D2 and D4.

• Mice from group 3 received it administration of COPTG19421 at a dose of 1 × 10 ⁷ PFU/mouse at D0, D2 and D4.

• Mice from group 4 received i.p. m5-B07 at a dose of 3 mg/kg at D0.

Kinetics of virus replication in the CT26 model:

In the CT26 model, where two surrogate viruses were administered three times (1 × ¹⁰⁷ PFU/injection), viral replication was monitored and compared with that of VVTG18058 administered under the same conditions. The results shown in FIG. 29 show, as for the LoVo model, an important spread of the three virus titers measured at each time point. However, titers for the three viruses were maintained over time and up to 10 days, indicating that the two transgenes did not affect virus clearance or replication at least during this time period. No infectious virus particles were detected in any of the blood samples (data not shown).

Kinetics of transgene expression in the CT26 model

As in the LoVo model, transgene expression in the tumor reflected viral replication. In other words, m5-B07 antibody (FIG. 30A) and mGM-CSF (FIG. 30B) were detected in the tumor at fairly constant levels over the 10 days of monitoring.

In the case of the monoclonal antibody, the Cmax achieved after injections of COPTG19421 or COPTG19407 was approximately 10-fold lower than the Cmax observed with a single IP injection of the m5-B07 antibody at a dose of 3 mg/ml (Fig. 30A). In serum, the difference was even more pronounced with circulating concentrations of m5-B07 being ~100-fold lower following virus treatment compared to injection of 3 mg/kg m5-B07 (FIG. 31).

For GM-CSF, only COPTG19407 treatment resulted in measurable concentrations of mGM-CSF in CT26 tumors, indicating that the measured cytokine was of recombinant rather than endogenous origin. As in the LoVo model, mGM-CSF concentrations measured in the tumor were lower than m5-B07 concentrations (Fig. 30B). Moreover, tumor-produced mGM-CSF was not detected in any serum samples, likely due to the short half-life of the molecule, which precludes any systemic accumulation.

Example 9 Antitumor Activity Studies

COPTG19347 is a vaccinia virus (Copenhagen strain) deleted in the J2R and I4L genes and encoding a whole mouse antibody (namely m5-B07, heavy and light chains) recognizing the mouse antigen CTLA4. COPTG19421 compared to COPTG19347 were both expressed as m5-B07, but under different promoters: namely p7.5K and pH5.R, respectively. Quantification of m5-B07 was assessed in supernatants of infected cells, reaching about 1 μg/ml in infected CT26 at an MOI of 10 ^-1 to about 4 μg/ml in infected MCA205 cells at an MOI of 10 ^-2 . Higher expression of MCA205 compared to CT26 was also observed for mGM-CSF in the culture supernatant of COPTG19407-infected cells (data not shown).

Antitumor activity in mice carrying the CT26 model in combination with anti-PD1

Protocol:

CT26 cells (2 × ¹⁰⁵ cells) were implanted into the right flank of Balb/c mice (Charles River, France). When the tumors reached a volume of 25-50 mm ³ , the mice were randomized into five groups of ten animals. Briefly, mice were treated with three doses of virus administered 2 days apart, followed by intravenous administration of mouse anti-PD1 (RMP1-14 BioXcell) twice weekly for three weeks. In particular,

• Mice in group 1 received vehicle;

• Mice from group 2 received it injection of 1 × 10 ⁷ PFU COPTG19347 in D0, D2 and D4;

• Group 3 mice received 1 x 10 ⁷ PFU COPTG19347 at D0, D2 and D4 and 250 µg/mouse RMP1-14 intraperitoneally at D7, D11, D14, D18 and D22;

• Mice in group 4 were given 250 μg/mouse RMP1-14 IP at D7, D11, D14, D18, and D22;

• Mice in group 5 received 1 x 10 ⁷ PFU of VVTG18058 at D0, D2 and D4.

Tumor sizes were measured twice a week with calipers, and their volume was calculated using the formula (ᴨ/6) (length x width ² ). Animals were sacrificed when the tumor volume reached 2000 mm ³ .

Antitumor activity of COPTG19347 in the CT26 model:

As shown in FIG. 32, treatment with COPTG19347 resulted in not only tumor growth inhibition (Figure 32A) but also tumor regression that eventually transformed into tumor-free mice that survived up to 100 days (Figure 32B). Treatment with COPTG19347 resulted in 60% tumor-free mice at day 100. Co-treatment with an anti-PD-1 antibody did not result in significant improvement in tumor growth inhibition or the percentage of long-lived mice (about 70% tumor-free mice at day 100). In comparison, RPMI-14 treatment had no antitumor effect (same behavior as untreated mice (all died within the first 40 days), whereas VVTG18058 had low activity (about 10% tumor-free mice at day 100).

Dose-response assessment on the CT26 model:

Three surrogate viruses were compared (different promoters to drive m5-B07 and with or without m-GM-CSF) and dose escalation of COPTG19407 was performed compared to VVTG18058 (7.5 x 10 ⁴ , 7.5 x 10 ⁵ or 7. 5 x 10 ⁶ PFU). Experimental conditions were exactly the same as described above, except that co-treatment with anti-PD1 was not applied.

The results of two independent experiments clearly demonstrated that the three viruses tested, COPTG19407, COPTG19421 and COPTG19347, had potent antitumor activity at a dose of 7.5 × 10 ⁶ PFU. This confirms that neither the mGM-CSF encoded in COPTG19407 nor the use of the weakest promoter in COPTG19421 and COPTG19407 impaired the antitumor activity of the armed viruses. At the highest dose tested (i.e. 7.5 x ¹⁰⁶ PFU), the number of tumor-free mice after 80 days was between 5/10 and 7/10 depending on the virus and experiment, compared with 0/10 for treated mice empty virus as shown in the following table.

Table 6. Effect of surrogate viruses COPTG19407, COPTG19421 and COPTG19347 on tumor growth after i.t. injections

Virus name TK locus RR locus Dose (PFU) Number of tumor-free mice in D100 COPTG19407 p7.5K-HC* p7.5K -LC*;
pSE/L-GM-CSF 7.5 x 10 ⁶ 7/10 COPTG19421 p7.5K-HC* p7.5K-LC* 7.5 x 10 ⁶ 5/10 COPTG19347 pH5.R -HC* pH5.R -LC* 7.5 x 10 ⁶ 7/10 VVTG18058 - - 7.5 x 10 ⁶ 0/10 Imitation - 0/10

*HC and LC indicate the heavy and light chains of the mouse m5-B07 anti-mCTLA4 antibody, respectively

Moreover, dose escalation performed with both the empty virus (VVTG18058) and the COPTG19384 surrogate (COPTG19407) demonstrated that even at a relatively low dose (7.5 × ¹⁰⁴ PFU), antibody-expressing virus was still had clear antitumor activity with 4/10 and 2/10 tumor-free mice at 80-98 days compared to 0/10 for VVTG18058 treatment at the same low dose, as shown in the following table.

Table 7. Effect of dose of COPTG19407 or VVTG18058 on tumor growth after i.t. injections

Virus name TK locus RR locus Dose (PFU) Number of tumor-free mice in D100 COPTG19407 p7.5K-HC* p7.5K -LC*;
pSE/L-GM-CSF 7.5 x 10 ⁶ 7/10 7.5 x 10 ⁵ 8/10 7.5 x 10 ⁴ 4/10 VVTG18058 - - 7.5 x 10 ⁶ 0/10 7.5 x 10 ⁵ 0/10 7.5 x 10 ⁴ 0/10

*HC and LC indicate the heavy and light chains of the mouse m5-B07 anti-mCTLA4 antibody, respectively

A compilation of survival data (global survival plot compiled from two independent studies) is presented in Fig. 33 Statistical analysis using the log‐rank test was performed to examine whether there were significant differences between the survival rates of each group.

Antitumor activity of COPTG19407 compared with the combination of VVTG18058 plus m5-B07

CT26 tumor-bearing mice were raised as previously described (Example 5). Briefly, CT26 cells were injected s.c. into Balb/C mice. Treatment of mice began when tumors reached approx. 100 mm ³ . Mice were then treated at D0, D2, and D5 with COPTG19407 (8.5 x ¹⁰⁶ PFU it), VVTG18058 (8.5 x ¹⁰⁶ PFU it), m5-B07 (10 mg/kg i.p.), or a combination of VVTG18058 (8 .5 x 10 ⁶ PFU it) plus m5-B07 (10 mg/kg i.p.). Tumor sizes were then measured twice a week, and mice were sacrificed when tumors reached 2000 mm ³ . As shown in FIG. 34A - 34D, tumor growth was significantly suppressed when mice were treated with COPTG19407 virus expressing anti-CTLA-4 and GM-CSF, whereas the combination of naked virus plus anti-CTLA4 m5-B07 did not result in improved therapy compared with single agent. In groups treated with m5-B07 alone, VVTG18058 virus alone, or a combination of both m5-B07 and VVTG18058, only 20% of mice survived beyond day 70 (Figure 34E). In contrast, 90% of mice survived longer than 100 days after COPTG19407 administration, demonstrating the effectiveness of the vectorization strategy.

Antitumor activity of VVTK-RR-encoding anti-CTLA-4 and GM-CSF in mice bearing subcutaneous murine B-cell lymphoma A20

The A20 cell line is a BALB/c line B lymphoma cell line derived from a spontaneous reticulum cell neoplasm found in an aged BALB/cAnN mouse (ATCC TIB-208™).

Protocol (1):

Tumors were induced by subcutaneous injection of 5 x 10 ⁶ A20 cells into the right flank of a female Balb/cN mouse (Charles River, France). When the tumors reached an average volume of 95 mm ³ , 50 mice were randomized into 5 groups of 10 animals.

• Mice from group 1 received i.t. introduction of the carrier into D0, D2 and D4,

• Mice from group 2 received it injection of VVTG18058 at a dose of 4.75 × 10 ⁶ PFU in D0, D2 and D4,

• Mice from group 3 received it injection of COPTG19407 at a dose of 4.75 × 10 ⁶ PFU in D0, D2 and D4,

• Mice from group 4 received ip injection of anti-PD-1 antibody at a dose of 250 μg on D7, D10, D14, D17, D21 and D24,

• Mice from group 5 received COPTG19407 at a dose of 4.75 × 10 ⁶ PFU in D0, D2 and D4 in combination with i.p. anti-PD-1 antibody at a dose of 250 μg in D7, D10, D14, D17, D21 and D24.

Antitumor effect:

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

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, tumor doubling time could not be accurately calculated because the tumors did not grow exponentially, indicating increased treatment efficacy compared to groups 1, 2, and 4. Similarly, tumor doubling time was calculated using only one animal in group 5. 9 of 10 mice in group 3 had tumors that regressed on D15 and did not increase significantly in volume from D25 until the end of the study at D64. At D64, tumor volumes in these 9 mice ranged from 4 mm³ (technical tumor detection limit) to 59.77 mm³. Similarly, in Group 5, tumors regressed in 9 mice after initiation of treatment, reaching values ranging from 7.24 to 63.21 mm³ at the end of the study in D64. As can be seen in FIG. 35 shows individual tumor volume curves for each treatment group (Groups 1 to 5, corresponding to Fig. 35 A to E), tumors did not grow in animals of groups 3 and 5 treated with COPTG19407, confirming the strong anti-tumor activity of the antibody-expressing virus with or without anti-PD1.

Tumor growth delay was calculated by estimating the time required for tumors to reach an average target volume of 300 mm ³ . The results were similar to those obtained for tumor doubling time, as this parameter could only be calculated for tumors that reached the target volume of 300 mm³, which was not the case for the majority of animals in groups 3 and 5. Groups 1, 2 and 4 had average delays tumor growth by 16, 21 and 17 days, respectively, which did not differ significantly from each other. Additionally, in Groups 3 (n = 2), the average tumor latency was 14 days, indicating that the tumors that did grow in this group grew at the same rate as Groups 1, 2, and 4, however these tumors did regress in both groups of animals. In comparison, the only tumor in group 5 (n = 1) that grew had a significantly (p ≤ 0.0026) longer delay in tumor growth than all other groups, at 43 days.

Tumor growth inhibition (T/G%) was calculated by comparing the mean tumor volume in vehicle-treated group 1 with the other treatment groups. Group 2 had an optimal T/C% of 34% at D22, indicating temporary marginal antitumor activity, but this increased to 71% at D31. Moderate antitumor activity (10-30% T/C%) was observed in group 4. In comparison, both groups 3 and 5 showed significant antitumor activity (T/C% less than 10%) from D27 to D31 (latest calculated T/C value). C%).

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

Protocol (2):

Tumors were induced by subcutaneous injection of 5 × 10 ⁶ A20 cells into the right flank of female BALB/cN mice. When the tumors reached an average volume of 80-100 mm ³ , 90 animals were randomized into 9 groups of 10 animals.

• Mice from group 1 received i.t. introduction of the carrier into D0, D2 and D4.

• Mice in Group 2 received ip anti-PD-1 antibody at a dose of 250 μg/mouse/injection at D0, D4, D7, D10, D14, and D17.

• Mice in group 3 received ip isotype at a dose of 250 μg/mouse/injection at D0, D4, D7, D10, D14 and D17.

• Mice from group 4 received it injection of VVTG18058 at a dose of 1 × 10 ⁵ PFU in D0, D2 and D4.

• Mice from group 5 received it injection of VVTG18058 at a dose of 1 × 10 ⁵ PFU in D0, D2 and D4 and i.p. administration of isotype at a dose of 250 μg/mouse/injection in D0, D4, D7, D10, D14 and D17.

• Mice from group 6 received VVTG18058 at a dose of 1 × 10 ⁵ PFU in D0, D2 and D4 and i.p. anti-PD-1 antibody at a dose of 250 μg/mouse/injection in D0, D4, D7, D10, D14 and D17.

• Mice from group 7 received it injection of COPTG19407 at a dose of 1 x 10 ⁵ PFU in D0, D2 and D4.

• Mice from group 8 received it injection of COPTG19407 at a dose of 1 × 10 ⁵ PFU in D0, D2 and D4 in combination with i.p. administration of isotype at a dose of 250 μg/mouse/injection in D0, D4, D7, D10, D14, D17 and D24.

• Mice from group 9 received COPTG19407 at a dose of 1 × 10 ⁵ PFU in D0, D2 and D4 in combination with i.p. anti-PD-1 antibody at a dose of 250 μg/mouse/injection in D0, D4, D7, D10, D14 and D17.

Antitumor effect:

The dose of COPTG19407 is suboptimal and demonstrates modest antitumor activity similar to that of anti-PD-1 treatment in terms of tumor volume and mouse survival.

In contrast, the combination of COPTG19407 with anti-PD-1 showed strong antitumor activity resulting in lower tumor volume compared to other groups, as shown in FIG. 37A (approximately 290 mm ³ at day 36 compared to approximately 630 mm ³ and 750 mm ³ at day 24 for mice treated with anti-PD-1 alone or COPTG19407 alone, respectively), and much better animal survival as shown in FIG. . 37B (7 animals still alive at day 57 in group 9 compared to only two or one each in groups 2 or 7, respectively).

Study of the antitumor activity of VVTK-RR-encoding anti-CTLA-4 and GM-CSF in mice bearing subcutaneous C38 cell colon tumors

C38 is a murine colon adenocarcinoma derived from the American Type Culture Collection (ATCC CRL-2779™).

Protocol:

Tumor fragments (30-50 mg) were implanted subcutaneously into the right flank of a female C57BL/6J mouse (Janvier, France). When the tumors reached a mean volume of approximately 60 mm ³ , 50 animals were randomized into five groups of ten animals.

• Mice from group 1 received i.t. introduction of the carrier into D0, D2 and D4,

• Mice from group 2 received it injection of VVTG18058 at a dose of 4.75 × 10 ⁶ PFU in D0, D2 and D4,

• Mice from group 3 received it injection of COPTG19407 at a dose of 4.75 × 10 ⁶ PFU in D0, D2 and D4,

• Mice from group 4 received ip injection of mouse anti-PD-1 antibody at a dose of 250 μg on D7, D10, D14, D17, D21 and D24,

• Mice from group 5 received COPTG19407 at a dose of 4.75 × 10 ⁶ PFU in D0, D2 and D4 in combination with i.p. anti-PD-1 antibody at a dose of 250 μg in D7, D10, D14, D17, D21 and D24.

Antitumor effect:

As before, tumor volume in all animals was monitored throughout the study. Tumor doubling time was similar for groups 1 and 2, approximately 6.7 days. Group 4 (n = 5) had a longer tumor doubling time (10.4 days), but there were no significant differences between groups. For groups 3 and 5, tumor doubling time could be calculated, but with fewer animals (n = 2), since most tumors in mice in these groups did not grow exponentially, indicating increased treatment efficacy compared to groups 1, 2, and 4 As illustrated in FIG. 38, 8 of 10 mice in group 3 had tumors that regressed from D15 and did not increase significantly in volume, while five mice had no detectable tumors at the end of the study in D61. Similarly, in Group 5, tumors regressed in 8 mice after initiation of treatment, reaching values ranging from 0 (n = 2) to 47.82 mm³ at the end of the study in D61.

Tumor growth delay was calculated by estimating the time required for tumors to reach an average target volume of 300 mm ³ . The results were similar to those obtained for tumor doubling time, as this parameter could only be calculated for tumors that reached the target volume of 300 mm³, which was not the case for the majority of animals in groups 3 and 5. There were no significant differences between groups. Groups 1, 2, and 4 (n = 5) had a mean tumor growth delay of 23 to 27 days, respectively. In addition, in groups 3 (n = 2) and 5 (n = 3), the mean growth delay was 18 and 24 days, respectively. This indicates that the tumors that did grow in these two groups grew at the same rate as in groups 1, 2, and 4.

Tumor growth inhibition (T/G%) was calculated by comparing the mean tumor volume in vehicle-treated group 1 with the other treatment groups. Group 2 showed no inhibition of tumor growth as T/C% remained above 100% throughout the study. In comparison, groups 3, 4 and 5 all showed significant antitumor activity (T/C% less than 10%) from D31 (group 3 only) to D42 (latest calculated T/C%).

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

Study of the antitumor activity of VVTK-RR-encoding anti-CTLA-4 and GM-CSF in mice bearing subcutaneous EMT6 cell mammary tumors

EMT6 is an ATTC-derived murine mammary carcinoma (ATCC CRL-2755™).

Protocol:

Tumors were induced by subcutaneous injection of 1 × 10 ⁶ EMT6 cells into female BALB/cByJ mice (Charles River, France). When tumors reached a mean volume of approximately 51 mm³, fifty mice were randomized according to individual tumor volume into five groups of ten animals.

• Mice from group 1 received i.t. introduction of the carrier into D0, D2 and D4,

• Mice from group 2 received it injection of VVTG18058 at a dose of 4.75 × 10 ⁶ PFU in D0, D2 and D4,

• Mice from group 3 received it injection of COPTG19407 at a dose of 4.75 × 10 ⁶ PFU in D0, D2 and D4,

• Mice from group 4 received ip injection of anti-PD-1 antibody at a dose of 250 μg on D7, D10, D14, D17, D21 and D24,

• Mice from group 5 received COPTG19407 at a dose of 4.75 × 10 ⁶ PFU in D0, D2 and D4 in combination with i.p. anti-PD-1 antibody at a dose of 250 μg in D7, D10, D14, D17, D21 and D24.

Tumor volumes in all animals were monitored throughout the study by assessing tumor doubling time, tumor growth arrest, and tumor growth inhibition (T/C%) criteria.

Antitumor effect:

Tumor doubling time was similar for groups 1, 2 and 4 and was approximately 5.4 days. For group 3, tumor doubling time could not be calculated because most tumors did not grow exponentially, indicating increased treatment efficacy compared to groups 1, 2, and 4. A similar effect was observed in group 5, where only one animal was used to calculate tumor doubling time . As illustrated in the individual tumor volume plots (FIG. 40), 8 of 10 mice in group 3 had tumors that regressed from D15 and did not grow significantly in volume, and seven mice had no detectable tumors at the end of the study period in D61. Similarly, in Group 5, tumors regressed in 9 mice after initiation of treatment, reaching values ranging from 0 to 13.24 mm³ at the end of the study in D56.

Tumor growth delay was calculated by estimating the time required for tumors to reach an average target volume of 200 mm ³ . The results were similar to those obtained for tumor doubling time, since this parameter could only be calculated for tumors that reached the target volume of 200 mm³, which was not the case for the majority of animals in groups 3 and 5. In groups 1, 2 and 4, the average delay tumor growth was approximately 19 days. Additionally, in groups 3 and 5 (n = 2 for both groups), the mean growth retardation was 24 and 12 days, respectively. This indicates that the tumors that did grow in these two groups grew at the same rate as in groups 1, 2, and 4. There were no significant differences between the groups.

Tumor growth inhibition (T/G%) was calculated by comparing the mean tumor volume in vehicle-treated group 1 with the other treatment groups. Group 2 showed transient suppression of marginal tumor growth at D28, but increased to 79% at D31. Group 4 showed no antitumor activity with T/C% >60% throughout the study. In comparison, both groups 3 and 5 showed significant antitumor activity (T/C% less than 10%) from D24 to D31 (latest calculated T/C%).

Fig. 41 illustrates mean tumor volume curves of BALB/cByJ mice bearing subcutaneous EMT6 tumors, demonstrating the dramatic impact of COPTG19407 virus expressing anti-CTLA4/GM-CSF, with or without anti-PD1, on tumor growth.

CT26 reprocessing

Balb/c mice re-treated with CT26 tumor cells that survived treatment with 10 ⁴ , 10 ⁵ or 10 ⁶ PFU COPTG19421 or 10 ⁶ PFU COPTG19407 were re-treated with CT26 tumor cells or treated with Renca cells (renal adenocarcinoma cells: control) to examine whether the specific antitumor immune response was enhanced.

Table 8. Effect of repeated treatment with CT26 tumor cells or treatment with Renca tumor cells on the number of tumor-free mice

Group No tumors/Total mice CT26 naive control 0/5 VVTG18058 10 ⁵ + CT26 reprocessing 0/1 COPTG19421 10 ⁶ + CT26 reprocessing 3/3 COPTG19421 10 ⁶ + RenCa re-treatment 0/2 COPTG19407 10 ⁴ + CT26 reprocessing 1/1 COPTG19407 10 ⁴ + RenCa re-treatment 0/1 COPTG19407 10 ⁵ + CT26 reprocessing 1/2 COPTG19407 10 ⁵ + RenCa re-treatment 0/2 COPTG19407 10 ⁶ + CT26 reprocessing 2/4 COPTG19407 10 ⁶ + RenCa re-treatment 0/3

The results presented in Table 8 demonstrate that 0/8 mice receiving COPTG19421 or COPTG19407 were tumor-free after re-treatment with RenCa, while 7/10 mice receiving COPTG19421 or COPTG19407 were tumor-free after re-treatment with CT26. This indicates that COPTG19421 and COPTG19407 enhance specific immune memory against CT26 cells.

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List of sequences

<110> BioInvent International AB

Transgene S.A.

<120> NEW ANTIBODIES AND NUCLEOTIDE SEQUENCES AND THEIR APPLICATION

<130>BI86

<150>EP18192311.1

<151> 2018-09-03

<160> 63

<170> Patent In version 3.5

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<220>

<221> MISC_FEATURE

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<221> MISC_FEATURE

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<220>

<221> MISC_FEATURE

<222> (5)..(5)

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<220>

<221> MISC_FEATURE

<222> (7)..(7)

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<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> X at position 9 represents V or I

<400> 1

Phe Xaa Xaa Tyr Xaa Met Xaa Trp Xaa Arg Gln Ala Pro Gly

1 5 10

<210> 2

<211> 14

<212> Protein

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> X in position 3 represents D or S

<220>

<221> MISC_FEATURE

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<223> X in position 3 represents Y, S or A

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> X in position 3 represents S or N

<400> 2

Phe Ser Xaa Tyr Xaa Met Xaa Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 3

<211> 14

<212> Protein

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> X in position 3 represents D or S

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X at position 5 represents Y or S

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> X at position 5 represents S or N

<400> 3

Phe Ser Xaa Tyr Xaa Met Xaa Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 4

<211> 19

<212> Protein

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X in position 2 represents G or A

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X at position 5 represents W, G or N

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> X at position 6 represents S or T

<220>

<221> MISC_FEATURE

<222> (7)..(7)

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<220>

<221> MISC_FEATURE

<222> (8)..(8)

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<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> X at position 9 represents D, S or Y

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> X at position 10 represents K, T or I

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> X at position 11 represents G, Y, H or D

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> X at position 12 represents Y or F

<400> 4

Ser Xaa Ile Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Asp Ser Val

1 5 10 15

Lys Gly Arg

<210> 5

<211> 19

<212> Protein

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X in position 2 represents G or A

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X at position 5 represents W, G or N

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> X at position 6 represents S or T

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> X at position 7 represents S or G

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> X at position 8 represents R or G

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> X at position 9 represents D, S or Y

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> X at position 10 represents K, T or I

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> X at position 11 represents G, Y, H or D

<400> 5

Ser Xaa Ile Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Ala Asp Ser Val

1 5 10 15

Lys Gly Arg

<210> 6

<211> 19

<212> Protein

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X in position 2 represents G or A

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X at position 5 represents W or G

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> X at position 7 represents S or G

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> X at position 8 represents R or G

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> X at position 9 represents D or S

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> X at position 10 represents K or T

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> X at position 11 represents G or Y, H or D

<400> 6

Ser Xaa Ile Ser Xaa Ser Xaa Xaa Xaa Xaa Xaa Tyr Ala Asp Ser Val

1 5 10 15

Lys Gly Arg

<210> 7

<211> 15

<212> Protein

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> X in position 1 represents T or A

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X in position 2 represents T or R

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> X in position 3 represents D, Y or L

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> X in position 4 represents L, R, S or G

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X at position 5 represents A, V, S or Y

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> X at position 6 represents R, E, G or S

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> X at position 6 represents Y, M, L or G

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> X at position 8 is N, H, Y or missing

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> X at position 9 represents Q, D or missing

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> X at position 10 represents W, A, D or none

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> X at position 11 represents L, F, R or none

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> X at position 12 represents A, D, G or none

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> X at position 13 represents D, I, M or none

<220>

<221> MISC_FEATURE

<222> (14)..(14)

<223> X at position 14 represents D or is missing

<220>

<221> MISC_FEATURE

<222> (15)..(15)

<223> X at position 15 represents V or is missing

<400> 7

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10 15

<210> 8

<211> 13

<212> Protein

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> X in position 1 represents T or A

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X in position 2 represents T or R

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> X in position 4 represents L or R

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X at position 5 represents A or V

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> X at position 6 represents R or E

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> X at position 7 represents Y or M

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> X at position 8 is N or missing

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> X at position 9 is Q or missing

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> X at position 10 is W or missing

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> X at position 11 represents L or is missing

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> X at position 12 represents A or is missing

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> X at position 13 represents D or missing

<400> 8

Xaa Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10

<210> 9

<211> 15

<212> Protein

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X in position 2 represents T or S

<220>

<221> MISC_FEATURE

<222> (10)..(10)

<223> X at position 10 represents A or S

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> X at position 11 represents G or N

<220>

<221> MISC_FEATURE

<222> (13)..(13)

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<220>

<221> MISC_FEATURE

<222> (14)..(14)

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<220>

<221> MISC_FEATURE

<222> (15)..(15)

<223> X at position 15 represents H or is missing

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Cys Xaa Gly Ser Ser Ser Asn Ile Gly Xaa Xaa Tyr Xaa Xaa Xaa

1 5 10 15

<210> 10

<211> 15

<212> Protein

<213> Homo sapiens

<400> 10

Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His

1 5 10 15

<210> 11

<211> 7

<212> Protein

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> X in position 1 represents G, R or D

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> X in position 3 represents D, N or S

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> X in position 4 represents N, Q or K

<400> 11

Xaa Asn Xaa Xaa Arg Pro Ser

15

<210> 12

<211> 7

<212> Protein

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> X in position 1 represents G or R

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> X in position 3 represents D or N

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> X in position 3 represents N or Q

<400> 12

Xaa Asn Xaa Xaa Arg Pro Ser

15

<210> 13

<211> 13

<212> Protein

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> X in position 2 represents A or Q

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> X in position 3 represents V, A or S

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> X at position 4 represents W or Y

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> X at position 6 represents D or S

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> X at position 9 represents N or S

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> X at position 11 represents V, W or P

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> The X at position 13 represents a V or is missing

<400> 13

Cys Xaa Xaa Xaa Asp Xaa Ser Leu Xaa Gly Xaa Val Xaa

1 5 10

<210> 14

<211> 12

<212> Protein

<213> Homo sapiens

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> X in position 3 represents V or A

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> X at position 11 represents V or W

<400> 14

Cys Ala Xaa Trp Asp Asp Ser Leu Asn Gly Xaa Val

1 5 10

<210> 15

<211> 14

<212> Protein

<213> Homo sapiens

<400> 15

Phe Ser Asp Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 16

<211> 19

<212> Protein

<213> Homo sapiens

<400> 16

Ser Gly Ile Ser Trp Ser Ser Arg Asp Lys Gly Tyr Ala Asp Ser Val

1 5 10 15

Lys Gly Arg

<210> 17

<211> 7

<212> Protein

<213> Homo sapiens

<400> 17

Thr Thr Asp Leu Ala Arg Tyr

15

<210> 18

<211> 7

<212> Protein

<213> Homo sapiens

<400> 18

Gly Asn Asp Asn Arg Pro Ser

15

<210> 19

<211> 12

<212> Protein

<213> Homo sapiens

<400> 19

Cys Ala Val Trp Asp Asp Ser Leu Asn Gly Val Val

1 5 10

<210> 20

<211> 114

<212> Protein

<213> Homo sapiens

<400> 20

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

1 5 10 15

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

20 25 30

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

35 40 45

Ser Gly Ile Ser Trp Ser Ser Arg Asp Lys Gly Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Thr Thr Asp Leu Ala Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

100 105 110

Ser Ser

<210> 21

<211> 112

<212> Protein

<213> Homo sapiens

<400> 21

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

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gly Asn Asp Asn Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu

65 70 75 80

Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Val Trp Asp Asp Ser

85 90 95

Leu Asn Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 22

<211> 14

<212> Protein

<213> Homo sapiens

<400> 22

Phe Ser Ser Tyr Ser Met Asn Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 23

<211> 19

<212> Protein

<213> Homo sapiens

<400> 23

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

1 5 10 15

Lys Gly Arg

<210> 24

<211> 13

<212> Protein

<213> Homo sapiens

<400> 24

Ala Arg Asp Arg Val Glu Met Asn Gln Trp Leu Ala Asp

1 5 10

<210> 25

<211> 7

<212> Protein

<213> Homo sapiens

<400> 25

Arg Asn Asn Gln Arg Pro Ser

15

<210> 26

<211> 12

<212> Protein

<213> Homo sapiens

<400> 26

Cys Ala Ala Trp Asp Asp Ser Leu Asn Gly Trp Val

1 5 10

<210> 27

<211> 120

<212> Protein

<213> Homo sapiens

<400> 27

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Asp Arg Val Glu Met Asn Gln Trp Leu Ala Asp Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 28

<211> 112

<212> Protein

<213> Homo sapiens

<400> 28

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

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu

65 70 75 80

Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser

85 90 95

Leu Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 29

<211> 14

<212> Protein

<213> Homo sapiens

<400> 29

Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly

1 5 10

<210> 30

<211> 19

<212> Protein

<213> Homo sapiens

<400> 30

Ser Gly Ile Ser Gly Ser Gly Gly Tyr Ile His Tyr Ala Asp Ser Val

1 5 10 15

Lys Gly Arg

<210> 31

<211> 13

<212> Protein

<213> Homo sapiens

<400> 31

Ala Thr Tyr Ser Ser Gly Leu His Asp Ala Phe Asp Ile

1 5 10

<210> 32

<211> 7

<212> Protein

<213> Homo sapiens

<400> 32

Asp Asn Asn Lys Arg Pro Ser

15

<210> 33

<211> 120

<212> Protein

<213> Homo sapiens

<400> 33

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

1 5 10 15

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

20 25 30

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

35 40 45

Ser Gly Ile Ser Gly Ser Gly Gly Tyr Ile His Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Thr Tyr Ser Ser Gly Leu His Asp Ala Phe Asp Ile Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 34

<211> 112

<212> Protein

<213> Homo sapiens

<400> 34

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

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu

65 70 75 80

Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser

85 90 95

Leu Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 35

<211> 14

<212> Protein

<213> Homo sapiens

<400> 35

Phe Lys Ala Tyr Ser Met Ser Trp Ile Arg Gln Ala Pro Gly

1 5 10

<210> 36

<211> 19

<212> Protein

<213> Homo sapiens

<400> 36

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

1 5 10 15

Lys Gly Arg

<210> 37

<211> 15

<212> Protein

<213> Homo sapiens

<400> 37

Ala Arg Leu Gly Tyr Ser Gly Tyr Asp Asp Arg Gly Met Asp Val

1 5 10 15

<210> 38

<211> 14

<212> Protein

<213> Homo sapiens

<400> 38

Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr

1 5 10

<210> 39

<211> 7

<212> Protein

<213> Homo sapiens

<400> 39

Gly Asn Ser Asn Arg Pro Ser

15

<210> 40

<211> 13

<212> Protein

<213> Homo sapiens

<400> 40

Cys Gln Ser Tyr Asp Ser Ser Leu Ser Gly Pro Val Val

1 5 10

<210> 41

<211> 122

<212> Protein

<213> Homo sapiens

<400> 41

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Leu Gly Tyr Ser Gly Tyr Asp Asp Arg Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 42

<211> 112

<212> Protein

<213> Homo sapiens

<400> 42

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

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser Leu

85 90 95

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

100 105 110

<210> 43

<211> 330

<212> Protein

<213> Homo sapiens

<400> 43

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

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

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

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

115 120 125

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

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

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

180 185 190

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

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 44

<211> 105

<212> Protein

<213> Homo sapiens

<400> 44

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

1 5 10 15

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

20 25 30

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

35 40 45

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

50 55 60

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

65 70 75 80

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

85 90 95

Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 45

<211> 1335

<212> DNA

<213> Homo sapiens

<400> 45

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt gactactaca tgagctgggt ccgccaggct 120

cccgggaagg ggctggagtg ggtctcaggc attagttgga gtagtcgtga caaaggctat 180

gcggactctg tgaagggccg tttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac actgccgtgt attactgtac cacagatctc 300

gctaggtact ggggccaggg tacactggtc accgtgagct cagcctccac caagggccca 360

tcggtcttcc ccctggcacc ctcctccaag agcacctctg ggggcacagc ggccctgggc 420

tgcctggtca aggactactt ccccgaaccg gtgacggtgt cgtggaactc aggcgccctg 480

accagcggcg tgcacacctt cccggctgtc ctacagtcct caggactcta ctccctcagc 540

agcgtggtga ccgtgccctc cagcagcttg ggcacccaga cctacatctg caacgtgaat 600

cacaagccca gcaacaccaa ggtggacaag aaagttgagc ccaaatcttg tgacaaaact 660

cacacatgcc caccgtgccc agcacctgaa ctcctggggg gaccgtcagt cttcctcttc 720

cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg 780

gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag 840

gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc 900

agcgtcctca ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc 960

tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaagccaa agggcagccc 1020

cgagaaccac aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc 1080

agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtggggagagc 1140

aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc 1200

ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc 1260

tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg 1320

tctccgggta aatga 1335

<210> 46

<211> 654

<212> DNA

<213> Homo sapiens

<400> 46

cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60

tcctgcactg ggagcagctc caacatcggg gcaggttatg atgtacactg gtatcagcag 120

ctcccaggaa cggcccccaa actcctcatc tatggtaatg ataaccggcc ctcaggggtc 180

cctgaccgat tctctggctc caagtctggc acctcagcct ccctggccat cagtgggctc 240

cggtccgagg atgaggctga ttattactgt gcagtatggg atgacagcct gaatggtgtg 300

gtattcggcg gaggaaccaa gctgacggtc ctaggtcagc ccaaggctgc cccctcggtc 360

actctgttcc cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc 420

ataagtgact tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480

aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540

agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600

acgcatgaag ggagcaccgt ggagaagaca gtggccccta cagaatgttc atga 654

<210> 47

<211> 1353

<212> DNA

<213> Homo sapiens

<400> 47

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt agctatagca tgaactgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac actgccgtgt attactgtgc gagagatcgg 300

gtagagatga accagtggct ggccgactgg ggccagggta cactggtcac cgtgagctca 360

gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1080

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320

cagaagagcc tctccctgtc tccgggtaaa tga 1353

<210> 48

<211> 654

<212> DNA

<213> Homo sapiens

<400> 48

cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60

tcctgcactg ggagcagctc caacatcggg gcaggttatg atgtacactg gtatcagcag 120

ctcccaggaa cggcccccaa actcctcatc tataggaata atcagcggcc ctcaggggtc 180

cctgaccgat tctctggctc caagtctggc acctcagcct ccctggccat cagtgggctc 240

cggtccgagg atgaggctga ttattactgt gcagcatggg atgacagcct gaatggttgg 300

gtgttcggcg gaggaaccaa gctgacggtc ctaggtcagc ccaaggctgc cccctcggtc 360

actctgttcc cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc 420

ataagtgact tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480

aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540

agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600

acgcatgaag ggagcaccgt ggagaagaca gtggccccta cagaatgttc atga 654

<210> 49

<211> 1353

<212> DNA

<213> Homo sapiens

<400> 49

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcaggt attagtggca gcggtgggta catacactat 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac actgccgtgt attactgtgc gacctatagc 300

agtggcctgc atgatgcttt tgatatctgg ggccagggta cactggtcac cgtgagctca 360

gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1080

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320

cagaagagcc tctccctgtc tccgggtaaa tga 1353

<210> 50

<211> 654

<212> DNA

<213> Homo sapiens

<400> 50

cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60

tcctgcactg ggagcagctc caacatcggg gcaggttatg atgtacactg gtatcagcag 120

ctcccaggaa cggcccccaa actcctcatc tatgacaata ataagcgacc ctcaggggtc 180

cctgaccgat tctctggctc caagtctggc acctcagcct ccctggccat cagtgggctc 240

cggtccgagg atgaggctga ttattactgt gcagcatggg atgacagcct gaatggttgg 300

gtgttcggcg gaggaaccaa gctgacggtc ctaggtcagc ccaaggctgc cccctcggtc 360

actctgttcc cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc 420

ataagtgact tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480

aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540

agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600

acgcatgaag ggagcaccgt ggagaagaca gtggccccta cagaatgttc atga 654

<210> 51

<211> 1359

<212> DNA

<213> Homo sapiens

<400> 51

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcaaa gcctatagca tgagctggat ccgccaggct 120

ccagggaagg ggctggagtg ggtctcaggt atcagtaaca cgggaggtag cacagacttc 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac actgccatgt attactgtgc gagattggga 300

tatagtggct acgacgaccg tggtatggac gtctggggcc aaggtacact ggtcaccgtg 360

agctcagcct ccaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 420

tctggggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 480

gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 540

tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 600

cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagaaagtt 660

gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 720

gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 780

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 840

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggagggagcag 900

tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 960

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1020

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1080

gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1140

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1200

cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1260

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1320

tacacgcaga agagcctctc cctgtctccg ggtaaatga 1359

<210> 52

<211> 654

<212> DNA

<213> Homo sapiens

<400> 52

cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60

tcttgttctg gaagcagctc caacatcgga agtaattatg tgtactggta tcagcagctc 120

cccaggaacgg cccccaaact cctcatctat ggtaacagca atcggccctc aggggtccct 180

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240

tccgaggatg aggctgatta ttactgccag tcctatgaca gcagcctgag tggtcctgtg 300

gtattcggcg gaggaaccaa gctgacggtc ctaggtcagc ccaaggctgc cccctcggtc 360

actctgttcc cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc 420

ataagtgact tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480

aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540

agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600

acgcatgaag ggagcaccgt ggagaagaca gtggccccta cagaatgttc atga 654

<210> 53

<211> 236

<212> Protein

<213> Homo sapiens

<400> 53

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

1 5 10 15

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

20 25 30

Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile

35 40 45

Gly Ala Gly Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala

50 55 60

Pro Lys Leu Leu Ile Tyr Gly Asn Asp Asn Arg Pro Ser Gly Val Pro

65 70 75 80

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

85 90 95

Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Val Trp

100 105 110

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

115 120 125

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

130 135 140

Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile

145 150 155 160

Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser

165 170 175

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

180 185 190

Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln

195 200 205

Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser

210 215 220

Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

225 230 235

<210> 54

<211> 463

<212> Protein

<213> Homo sapiens

<400> 54

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

1 5 10 15

Val His Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Tyr Cys Thr Thr Asp Leu Ala Arg Tyr Trp Gly Gln Gly Thr Leu

115 120 125

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

130 135 140

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

210 215 220

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

225 230 235 240

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

245 250 255

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

260 265 270

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

<210> 55

<211> 127

<212> Protein

<213> Homo sapiens

<400> 55

Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His Val

1 5 10 15

Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp Thr

20 25 30

Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe Asp

35 40 45

Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys Gln

50 55 60

Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met Met

65 70 75 80

Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser Cys

85 90 95

Ala Thr Gln Thr Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys Asp

100 105 110

Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu

115 120 125

<210> 56

<211> 144

<212> Protein

<213> Homo sapiens

<400> 56

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

1 5 10 15

Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His

20 25 30

Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp

35 40 45

Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe

50 55 60

Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys

65 70 75 80

Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met

85 90 95

Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser

100 105 110

Cys Ala Thr Gln Thr Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys

115 120 125

Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu

130 135 140

<210> 57

<211> 124

<212> Protein

<213> Mus musculus

<400> 57

Ala Pro Thr Arg Ser Pro Ile Thr Val Thr Arg Pro Trp Lys His Val

1 5 10 15

Glu Ala Ile Lys Glu Ala Leu Asn Leu Leu Asp Asp Met Pro Val Thr

20 25 30

Leu Asn Glu Glu Val Glu Val Val Ser Asn Glu Phe Ser Phe Lys Lys

35 40 45

Leu Thr Cys Val Gln Thr Arg Leu Lys Ile Phe Glu Gln Gly Leu Arg

50 55 60

Gly Asn Phe Thr Lys Leu Lys Gly Ala Leu Asn Met Thr Ala Ser Tyr

65 70 75 80

Tyr Gln Thr Tyr Cys Pro Pro Thr Pro Glu Thr Asp Cys Glu Thr Gln

85 90 95

Val Thr Thr Tyr Ala Asp Phe Ile Asp Ser Leu Lys Thr Phe Leu Thr

100 105 110

Asp Ile Pro Phe Glu Cys Lys Lys Pro Val Gln Lys

115 120

<210> 58

<211> 141

<212> Protein

<213> Mus musculus

<400> 58

Met Trp Leu Gln Asn Leu Leu Phe Leu Gly Ile Val Val Tyr Ser Leu

1 5 10 15

Ser Ala Pro Thr Arg Ser Pro Ile Thr Val Thr Arg Pro Trp Lys His

20 25 30

Val Glu Ala Ile Lys Glu Ala Leu Asn Leu Leu Asp Asp Met Pro Val

35 40 45

Thr Leu Asn Glu Glu Val Glu Val Val Ser Asn Glu Phe Ser Phe Lys

50 55 60

Lys Leu Thr Cys Val Gln Thr Arg Leu Lys Ile Phe Glu Gln Gly Leu

65 70 75 80

Arg Gly Asn Phe Thr Lys Leu Lys Gly Ala Leu Asn Met Thr Ala Ser

85 90 95

Tyr Tyr Gln Thr Tyr Cys Pro Pro Thr Pro Glu Thr Asp Cys Glu Thr

100 105 110

Gln Val Thr Thr Tyr Ala Asp Phe Ile Asp Ser Leu Lys Thr Phe Leu

115 120 125

Thr Asp Ile Pro Phe Glu Cys Lys Lys Pro Val Gln Lys

130 135 140

<210> 59

<211> 124

<212> DNA

<213> Artificial sequence

<220>

<223> p7.5K promoter

<400> 59

ccacccactt tttatagtaa gtttttcacc cataaataat aaatacaata attaatttct 60

cgtaaaagta gaaaatatat tctaatttat tgcacggtaa ggaagtagaa tcataaagaa 120

cagt 124

<210> 60

<211> 114

<212> DNA

<213> Artificial sequence

<220>

<223> pH5.R promoter

<400> 60

tttattctat acttaaaaaa tgaaaataaa tacaaaggtt cttgagggtt gtgttaaatt 60

gaaagcgaga aataatcata aattatttca ttatcgcgat atccgttaag tttg 114

<210> 61

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> pSE/L promoter

<400> 61

aaaaattgaa attttatttt ttttttttgg aatataaata 40

<210> 62

<211> 236

<212> Protein

<213> Mus musculus

<400> 62

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

1 5 10 15

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

20 25 30

Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile

35 40 45

Gly Ala Gly Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala

50 55 60

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

65 70 75 80

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

85 90 95

Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp

100 105 110

Asp Asp Ser Leu Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr

115 120 125

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

130 135 140

Ser Ser Glu Glu Leu Glu Thr Asn Lys Ala Thr Leu Val Cys Thr Ile

145 150 155 160

Thr Asp Phe Tyr Pro Gly Val Val Thr Val Asp Trp Lys Val Asp Gly

165 170 175

Thr Pro Val Thr Gln Gly Met Glu Thr Thr Gln Pro Ser Lys Gln Ser

180 185 190

Asn Asn Lys Tyr Met Ala Ser Ser Tyr Leu Thr Leu Thr Ala Arg Ala

195 200 205

Trp Glu Arg His Ser Ser Tyr Ser Cys Gln Val Thr His Glu Gly His

210 215 220

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

225 230 235

<210> 63

<211> 469

<212> Protein

<213> Mus musculus

<400> 63

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

1 5 10 15

Val His Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Tyr Cys Ala Arg Asp Arg Val Glu Met Asn Gln Trp Leu Ala Asp

115 120 125

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

130 135 140

Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser

145 150 155 160

Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val

165 170 175

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

180 185 190

Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr

195 200 205

Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala

210 215 220

His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly

225 230 235 240

Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu

245 250 255

Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val

260 265 270

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

275 280 285

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

290 295 300

Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser

305 310 315 320

Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met

325 330 335

Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala

340 345 350

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

355 360 365

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

370 375 380

Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr

385 390 395 400

Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr

405 410 415

Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu

420 425 430

Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser

435 440 445

Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser

450 455 460

Arg Thr Pro Gly Lys

465

<---

Claims (51)

1. An antibody molecule that specifically binds to CTLA-4, containing 6 CDRs with the sequences SEQ ID NO: 15, 16, 17, 10, 18 and 19 or 6 CDRs with the sequences SEQ ID NO: 22, 23, 24, 10, 25 and 26. 2. The antibody molecule of claim 1, which, compared to ipilimumab, has an improved depletion effect on CTLA-4 positive cells or CTLA-4 and CD4 positive cells. 3. An antibody molecule according to claim 1 or 2, which has an improved Treg depletion effect compared to ipilimumab. 4. Antibody molecule according to any one of paragraphs. 1-3, which is considered to have an improved depletion effect on CTLA-4-positive cells, CD4-positive cells and/or Tregs compared with ipilimumab if it produces improved depletion in: (i) in vitro ADCC testing, which is carried out using the HK-92 cell line stably transfected to express the CD16-158V allele along with green fluorescent protein (GFP), wherein ADCC testing includes the following sequential steps: 1) CTLA-4-positive cells, CD4-positive cells or Tregs as target cells are isolated from the peripheral blood of healthy donors; 2) then the target cells are stimulated by CD3/CD28 and rhIL-2; 3) then the target cells are pre-incubated with the antibody molecule and then mixed with NK cells; 4) then the target cells are incubated in RPMI 1640 + GlutaMAX medium containing 1 HEPES buffer, sodium pyruvate and low IgG FBS; 5) lysis is determined by flow cytometry; 6) steps 1-5 are repeated or performed in parallel using ipilimumab instead of the antibody molecule in step 3; And 7) the results of lysis of the antibody molecule are compared with the results of lysis of ipilimumab, and the improved lysis of the antibody molecule compared to ipilimumab demonstrates that the antibody molecule has an improved depleting effect on CTLA-4 positive cells, CD4 positive cells and/or Tregs; and/or (ii) in vivo testing on the PBMC-NOG/SCID model, wherein in vivo testing includes the following sequential steps: 1) human PBMCs are isolated, washed and resuspended in sterile PBS; 2) NOG mice are injected intravenously (IV) with the cell suspension from step 1); 3) NOG mouse spleens were isolated and processed into a single cell suspension; 4) the cell suspension from step 3) is resuspended in sterile PBS; 5) SCID mice are injected intraperitoneally (i.p.) with the suspension obtained in step 4; 6) SCID mice are then treated with either the antibody molecule, ipilimumab, or an isotype control monoclonal antibody; 7) collect intraperitoneal fluid from treated SCID mice; 8) human T cell subsets are identified and quantified by FACS using the following markers: CD45, CD4, CD8, CD25, CD127; 9) the identification and quantification of T cell subsets from mice treated with the antibody molecule are compared with the identification and quantification of T cell subsets from mice treated with ipilimumab and the identification and quantification of T cell subsets from mice treated with isotype control monoclonal antibodies, and fewer CTLA-4 positive cells, CD4 positive cells and/or Tregs in intraperitoneal fluid from mice treated with the antibody molecule of interest compared to CTLA-4 positive cells, CD4 positive cells and/or or Tregs in the intraperitoneal fluid of ipilimumab-treated mice demonstrates that the antibody molecule has an improved depletion effect on CTLA-4-positive cells, CD4-positive cells, and/or Tregs compared with ipilimumab. 5. Antibody molecule according to any one of paragraphs. 1-4, characterized in that said antibody molecule contains a variable heavy chain selected from the group consisting of the sequences SEQ ID NO: 20 and 27, and/or a variable light chain selected from the group consisting of the sequences SEQ ID NO: 21 and 28. 6. Antibody molecule according to any one of paragraphs. 1-5, characterized in that said antibody molecule contains a heavy chain constant region of the sequence SEQ ID NO: 43 and/or a light chain constant region of the sequence SEQ ID NO: 44. 7. Antibody molecule according to any one of paragraphs. 1-6, characterized in that said antibody molecule is selected from the group consisting of a full-length antibody, a chimeric antibody, a single chain antibody, Fab, Fv, scFv, Fab' and (Fab') ₂ . 8. Antibody molecule according to any one of paragraphs. 1-7, which binds to human CTLA-4 (hCTLA-4), and/or cynomolgus CTLA-4 (cmCTLA-4), and/or mouse CTLA-4 (mCTLA-4). 9. Antibody molecule according to any one of paragraphs. 1-8, which does not bind to human CD28. 10. Antibody molecule according to any one of paragraphs. 1-9, wherein said antibody molecule is selected from the group consisting of a human IgG antibody, a humanized IgG antibody, and a human-derived IgG antibody. 11. The antibody molecule of claim 10, wherein said antibody molecule is a human IgG1 antibody. 12. Antibody molecule according to any one of paragraphs. 1-11, characterized in that said antibody molecule is a monoclonal antibody. 13. An isolated nucleic acid encoding an antibody molecule as defined in any one of paragraphs. 1-12, containing a nucleotide sequence encoding the amino acid sequence of the specified antibody. 14. The nucleic acid of claim 13, comprising or consisting of a sequence selected from the group consisting of the sequences of SEQ ID 45-52. 15. An expression plasmid containing the nucleic acid of claim 13 or 14, under the control of suitable regulatory elements for proper expression. 16. A virus for expressing the antibody molecule defined in any one of paragraphs. 1-12, containing a nucleic acid as defined in claim 13 or 14, under the control of suitable regulatory elements for proper expression, or a plasmid as defined in claim 15. 17. The virus according to claim 16, which is an oncolytic virus and preferably an oncolytic poxvirus. 18. The virus of claim 17, wherein said oncolytic poxvirus belongs to the subfamily Chordopoxviridae, more preferably to the genus Orthopoxvirus, preferably selected from the group consisting of vaccinia virus, vaccinia virus, canarypox virus, ectromelia virus and myxoma virus. 19. The virus according to claim 18, characterized in that said oncolytic virus is a vaccinia virus defective in both thymidine kinase (TK) and/or ribonucleotide reductase (RR) activity and containing nucleotide sequences encoding the sequences of SEQ ID NO: 20 and ID NO: 21 or the sequences SEQ ID NO: 53 and ID NO: 54. 20. The virus of claim 19, wherein said oncolytic vaccinia virus further comprises a nucleotide sequence encoding GM-CSF, with particular preference for human GM-CSF, such as GM-CSF having the sequence SEQ ID NO: 55 or the sequence SEQ ID NO: 56, or a mouse GM-CSF, such as a GM-CSF having the sequence of SEQ ID NO: 57 or the sequence of SEQ ID NO: 58. 21. Virus according to any one of paragraphs. 16-20, characterized in that it contains a J2R locus and an I4L locus, and a heavy chain encoding cassette is inserted into the J2R locus and a light chain encoding cassette is inserted into the I4L locus. 22. A cell for expressing the antibody molecule defined in any one of paragraphs. 1-12, containing a nucleic acid, as defined in claim 13 or 14, under the control of suitable regulatory elements for proper expression, or a plasmid, as defined in claim 15, or a virus, as defined in any of claims. 16-21. 23. Antibody molecule according to any one of paragraphs. 1-12, nucleic acid according to claim 13 or 14, plasmid according to claim 15, virus according to any one of claims. 16-21 or a cell according to claim 22 for use in medicine. 24. Antibody molecule according to any one of paragraphs. 1-12, nucleic acid according to claim 13 or 14, plasmid according to claim 15, virus according to any one of claims. 16-21 or a cell according to claim 22 for use in the treatment of cancer. 25. Use of an antibody molecule according to any one of paragraphs. 1-12 for the production of a pharmaceutical composition for use in the treatment of cancer. 26. Use of a nucleic acid according to claim 13 or 14 for the production of a pharmaceutical composition for use in the treatment of cancer. 27. Use of the plasmid according to claim 15 for the production of a pharmaceutical composition for use in the treatment of cancer. 28. Use of a virus according to any one of paragraphs. 16-21 for the production of a pharmaceutical composition for use in the treatment of cancer. 29. Use of a cell according to claim 22 for the production of a pharmaceutical composition for use in the treatment of cancer. 30. A pharmaceutical composition for treating cancer in a subject, comprising an effective amount of an antibody molecule as defined in any one of paragraphs. 1-12, nucleic acid according to claim 13 or 14, plasmid according to claim 15, virus according to any one of paragraphs. 16-21 or cells according to paragraph 22. 31. The pharmaceutical composition according to claim 30, further comprising a pharmaceutically acceptable diluent, carrier, excipient and/or excipient. 32. A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of an antibody molecule as defined in any one of claims. 1-12, nucleic acid according to claim 13 or 14, plasmid according to claim 15, virus according to any one of paragraphs. 16-21, cells according to claim 22 or a pharmaceutical composition according to claim 30. 33. Antibody molecule for use according to claim 24, nucleic acid for use according to claim 24, plasmid for use according to claim 24, virus for use according to claim 24, cell for use according to claim 24, use according to any one of claims. 25-29, the pharmaceutical composition of claim 30 or 31, or the method of claim 32, wherein the cancer is a solid cancer.