Novel combinations of antibodies and uses thereof

Abstract

The present invention relates generally to a combination of an oncolytic virus capable of expressing a first antibody molecule that specifically binds to CTLA-4 and a second antibody molecule that specifically binds to PD-1 and / or PD-L1, and its use in the treatment of cancer.

Description

[Technical field]

[0001] The present invention relates to a combination comprising (i) an oncolytic virus capable of expressing a first antibody molecule that specifically binds to CTLA-4, and (ii) a second antibody molecule that specifically binds to PD-1 and / or PD-L1, the combination being for use in the treatment of a cancer comprising or consisting of a cold tumor in a patient. The present invention also relates to the use of the combination in the manufacture of a medicament for treating a cancer comprising or consisting of a cold tumor in a patient, and to a method for treating a cancer comprising or consisting of a cold tumor in a patient comprising administering the combination.

[0002] Treatment with immune checkpoint blockade antibodies has altered the survival of patients with advanced solid tumors, including metastatic melanoma, non-small cell lung cancer, and mismatch repair-deficient cancers ( Hodi et al., 2010 , Larkin et al., 2015 , Topalian et al., 2012 ).

[0003] However, a large unmet need remains as many patients do not respond or acquire resistance to immune checkpoint blockade (ICB) (Sharma et al., 2017). Reasons for lack of efficacy are believed to include a lack or insufficient tumor infiltrating immune cells (TILs), most notably CD8+ T cells (Chen and Mellman, 2013; Gajewski et al., 2013). Deficiencies in chemotactic and inflammatory signals in the solid tumor tumor microenvironment (TME) are similarly believed to underlie resistance to CAR-T cell therapy (Wagner et al., 2020).

[0004] Therefore, identification of therapeutic agents that induce the recruitment of inflammatory immune cells to "immune desert" or "immune excluded" tumors and convert them into robust systemic adaptive antitumor immunity and CD8+ T cell infiltration accompanied by regression of primary and metastatic tumors is highly desirable.

[0005] Intratumoral oncolytic virus therapy induces T cell infiltration and improves anti-PD-1 immunotherapy (Ribas et al., 2017). Combination therapy with anti-CTLA-4 and anti-PD-1 antibodies enhances efficacy compared to single-agent ICB, possibly due to the increased systemic CD4 + and CD8 + This is due to complementary mechanisms of T cell differentiation and regulation of tumor localization of T effector and regulatory T cells (Arce Vargas et al., 2018; Wei et al., 2019). However, tolerability issues with systemically administered anti-CTLA-4, including the approved ipilimumab, limit clinical use (Postow et al., 2015).

[0006] The efficacy and tolerability of systemic anti-CTLA-4 antibody therapy appear to be related: increasing ipilimumab dose enhanced both efficacy and side effects (Bertrand et al., 2015). Consistent with a central immune checkpoint function of CTLA-4, side effects can be severe and of a systemic autoimmune nature (Tivol et al., 1995).

[0007] Interestingly, CD8 + and CD4 +It was recently reported that depletion of intratumoral ("it") Treg cells overexpressing CTLA-4, compared with effector T cells, contributes to ipilimumab therapeutic activity, and Treg-depleting enhanced anti-CTLA-4 variants showed improved therapeutic activity in tumor-bearing FcγR-humanized mice (Arce Vargas et al., 2018). These findings indicated that tumor-localized therapy with Treg-depleting anti-CTLA-4 antibodies may provide potent therapeutic activity with reduced side effects compared to currently available anti-CTLA-4 therapies, especially when combined with validated and safe immunomodulatory agents, such as blockade of the PD-1 / PD-L1 axis or oncolytic viruses (Marabelle et al., 2013a, Marabelle et al., 2013b).

[0008] Against that background, we herein describe and characterize a Vaccinia virus (VV)-based oncolytic vector incorporating a full-length human recombinant anti-CTLA-4 antibody. We also describe an oncolytic virus encoding a recently discovered full-length human recombinant anti-CTLA-4 antibody. This virus-encoded novel human IgG1 CTLA-4 antibody (designated "4-E03") was identified using a monoclonal antibody ("mAb") and function-first screening for targets associated with superior Treg depletion activity. In a humanized mouse model featuring human intratumor-associated CTLA-4 expression, 4-E03 IgG1 demonstrated enhanced Treg depletion compared to clinically validated ipilimumab. In contrast, 4-E03 shows similar potency in blocking CTLA-4:B7 interactions and overcoming CTLA-4-mediated suppression of effector T cell proliferation compared to ipilimumab. In the present invention, we engineered a tumor-selective oncolytic vaccinia vector to express this novel Treg-depleting, checkpoint-blocking anti-CTLA-4 antibody 4-E03 and GM-CSF (VV GMWe have additionally generated viruses encoding a matched Treg-depleting murine surrogate antibody, enabling proof-of-concept studies in syngeneic immune-competent mouse tumor models representing inflammatory or immune-eliminating tumor microenvironments that are sensitive or resistant to ICB.

[0009] This led to the unexpected discovery that oncolytic viruses expressing anti-CTLA-4 antibodies synergize with anti-PD-1 / PD-L1 antibodies to reject "cold" tumors (also referred to herein as "cold immune tumors"). This was surprising since cold tumors, as well as animals in the "cold tumor" mouse models disclosed herein, are known to be resistant to systemic, intravenous, single agent, or combined ICB with currently available anti-CTLA-4 and / or anti-PD-1.

[0010] As discussed in the Examples and herein, "cold" tumors are poorly infiltrated with T cells. Surprisingly, the inventors found that combined treatment with oncolytic viruses expressing anti-CTLA-4 and anti-PD-1 / PD-L1 antibodies induced a robust influx of T cells into "cold" tumors, which became densely T cell-rich, similar to "hot" tumors.

[0011] The inventors' surprising findings provide a further beneficial therapeutic approach for treating cancers that comprise or consist of "cold" tumors. As described herein, the present invention generally relates to a combination comprising (i) an oncolytic virus capable of expressing a first antibody molecule that specifically binds to CTLA-4, and (ii) a second antibody molecule that specifically binds to PD-1 and / or PD-L1, wherein the combination is for use in treating a cancer that comprises or consists of a cold tumor in a patient.

[0012] In a first aspect, the present invention provides a method for treating cancer in a patient comprising: an oncolytic virus capable of expressing a first antibody molecule that specifically binds to CTLA-4; - a second antibody molecule that specifically binds to PD-1 and / or PD-L1, The cancer comprises or consists of a cold tumor.

[0013] In a second aspect, the present invention provides a method for the manufacture of a medicament for treating cancer in a patient, comprising: an oncolytic virus capable of expressing a nucleotide sequence encoding a first antibody molecule that specifically binds to CTLA-4; and - the use of a second antibody molecule that specifically binds PD-1 and / or PD-L1, The cancer comprises or consists of a cold tumor.

[0014] In a third aspect, the invention provides a method for treating cancer in a patient, wherein the cancer comprises or consists of a cold tumor, the method comprising administering to the patient: an oncolytic virus capable of expressing a first antibody molecule that specifically binds to CTLA-4; - a second antibody molecule that specifically binds PD-1 and / or PD-L1.

[0015] In a fourth aspect, the present invention provides an oncolytic virus capable of expressing a first antibody molecule that specifically binds CTLA-4 for use in combination with a second antibody molecule that specifically binds PD-1 and / or PD-L1 for treating cancer in a patient, wherein the cancer comprises or consists of a cold tumour.

[0016] As discussed above and demonstrated in the accompanying examples, in the above aspects of the invention, treatment with an oncolytic virus capable of expressing a first antibody molecule that specifically binds to CTLA-4 and a second antibody that specifically binds to PD-1 and / or PD-L1 causes an influx of T cells into the patient's cancer cold tumor. This results in an increase in the number and / or density of T cells in the patient's cancer cold tumor, such that the tumor becomes densely T cell rich, similar to a hot tumor. In one embodiment of the invention, the number and / or density of T cells in the patient's cancer cold tumor increases by about 5-25 times or more, for example, about 5 times, or 6 times, or 7 times, or 8 times, or 9 times, or 10 times, or 11 times, or 12 times, or 13 times, or 14 times, or 15 times, or 16 times, or 17 times, or 18 times, or 19 times, or 20 times, or 21 times, or 22 times, or 23 times, or 24 times, or 25 times or more.

[0017] Thus, in a further aspect, the present invention provides a method for increasing the number and / or density of T cells in a cold tumor in a patient's cancer, and / or for mediating the influx of T cells into a cold tumor in a patient's cancer, comprising: an oncolytic virus capable of expressing a first antibody molecule that specifically binds to CTLA-4; - a second antibody molecule that specifically binds to PD-1 and / or PD-L1; and In one embodiment of the present invention, the number and / or density of T cells in the cancer cold tumor of the patient is increased by about 5-25 times or more, for example, about 5 times, or 6 times, or 7 times, or 8 times, or 9 times, or 10 times, or 11 times, or 12 times, or 13 times, or 14 times, or 15 times, or 16 times, or 17 times, or 18 times, or 19 times, or 20 times, or 21 times, or 22 times, or 23 times, or 24 times, or 25 times or more.

[0018] As discussed herein, the oncolytic virus of the present invention can express a first antibody molecule that specifically binds to CTLA-4. Cytotoxic T-lymphocyte-associated antigen (CTLA-4 or CTLA4), also known as CD152, is a member of the B7 / CD28 family that blocks T-cell activation. CTLA-4 is expressed on activated T cells and transmits inhibitory signals to T cells. It is homologous to the T-cell costimulatory protein CD28, and both CTLA-4 and CD28 bind to CD80 (also designated B7-1) and CD86 (also designated B7-2). CTLA4 is also found on regulatory T cells (Tregs), contributing to their inhibitory function. The CTLA-4 protein contains an extracellular V domain, a transmembrane domain, and a cytoplasmic tail.

[0019] Antibodies that bind to CTLA-4 inhibit the immune effector CD4 + and CD8 + It has been proposed that they exert their therapeutic activity through a dual mechanism acting both on T cells and on immunosuppressive regulatory T (T regulatory, Treg) cells. It has been shown that antibodies against CTLA-4, which block the interaction of CTLA-4 with its ligands B7.1 and B7.2 on effector T cells, can enhance immune responses and stimulate potent antitumor immunity (Korman et al 2006, Checkpoint blockade in cancer immunotherapy, Adv Immunol. 90:297-339).

[0020] More recently, Fc effector function and Treg depletion have been shown to contribute to and correlate with the therapeutic activity of anti-CTLA-4 antibodies, including the clinically relevant antibodies ipilimumab and tremelimumab (Arce Vargas, Furness et al. 2018). Efficacy and toxicity (the latter of which can be severe and of an autoimmune nature) are thought to be associated with currently available systemic anti-CTLA-4 regimens. Thus, approaches to deliver highly effective but safe anti-CTLA-4-based ICB are lacking. We have recently demonstrated that intratumorally delivered oncolytic viruses encoding Treg-depleting anti-CTLA-4 antibodies (it-vectored anti-CTLA-4) have broad anti-tumor activity. Herein, we unexpectedly demonstrate that in the context of PD-1 / PD-L1 ICB, it-vectored anti-CTLA-4 has efficacy against poorly immune-infiltrated "cold" tumors that were resistant to systemic antibody-mediated ICB. Furthermore, due to the tumor-restricted anti-CTLA-4 exposure associated with this approach, it-vectored anti-CTLA-4 is shown to be safe and well tolerated compared with approved anti-CTLA-4 regimens.

[0021] As discussed herein, the invention also includes a second antibody molecule that specifically binds PD-1 and / or that specifically binds PD-L1. In some embodiments, the second antibody molecule specifically binds PD-1, in some embodiments, the second antibody molecule specifically binds PD-L1, and in some embodiments, the second antibody molecule specifically binds both PD-1 and PD-L1.

[0022] Programmed cell death protein 1 (PD-1 or PD1), also known as CD279, is found on the surface of T cells and B cells and inhibits T cell activity. PD-1 binds to two ligands, PD-L1 and PD-L2. Programmed death-ligand 1 (PD-L1), also known as CD274, binds to its receptor PD-1 and produces an inhibitory signal that reduces T cell proliferation.

[0023] Antibodies are known to those skilled in the art of immunology and molecular biology. Typically, an antibody comprises two heavy chains (H) and two light chains (L). This complete antibody molecule is sometimes referred to herein as a full-size antibody or full-length antibody. The heavy chain of an antibody comprises one variable region (VH) and three constant regions (CH1, CH2, CH3), and the light chain of an antibody molecule comprises one variable region (VL) and one constant region (CL). The variable regions (sometimes referred to as F) are V The variable regions (collectively called the constant regions) bind to the antibody's target, or antigen. Each variable region contains three loops, called complementarity determining regions (CDRs), which are involved in target binding. The constant regions are not directly involved in binding the antibody to the antigen, but exhibit various effector functions. Depending on the amino acid sequence of the constant regions of their heavy chains, antibodies or immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and in humans, some of these are further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. Another part of an antibody is the Fc domain (also known as the fragment crystallizable domain), which contains the two constant domains of each of the antibody's heavy chains. The Fc domain is involved in the interaction between the antibody and the Fc receptor.

[0024] Fc receptors are membrane proteins that are often found on the cell surface of cells of the immune system (i.e., Fc receptors are found on the target cell membrane, otherwise known as the plasma membrane or cytoplasmic membrane). The role of Fc receptors is to bind antibodies via the Fc domain and internalize the antibody into the cell. In the immune system, this can result in antibody-mediated phagocytosis and antibody-dependent cell-mediated cytotoxicity.

[0025] A subgroup of Fc receptors includes Fcγ receptors (Fc-γ receptors, FcγR) specific for IgG antibodies. There are two types of Fcγ receptors: activating Fcγ receptors (also referred to as activating Fcγ receptors) and inhibitory Fcγ receptors. Activating and inhibitory receptors transmit their signals via immunoreceptor tyrosine-based activation motifs (ITAMs) or immunoreceptor tyrosine-based inhibitory motifs (ITIMs), respectively. In humans, FcγRIIb (CD32b) is an inhibitory Fcγ receptor, while FcγRI (CD64), FcγRIIa (CD32a), FcγRIIc (CD32c), FcγRIIIa (CD16a) and FcγRIV are activating Fcγ receptors. FcγRIIIb is a GPI-linked receptor expressed on neutrophils that lacks ITAM motifs but is also considered activating due to its ability to crosslink lipid rafts and bind other receptors. In mice, the activating receptors are FcγRI, FcγRIII, and FcγRIV.

[0026] It is known that antibodies regulate immune cell activity through interaction with Fcγ receptors. Specifically, how antibody immune complexes regulate immune cell activation is determined by the relative engagement of activating and inhibitory Fcγ receptors. Different antibody isotypes bind to activating and inhibitory Fcγ receptors with different affinities, resulting in different A:I ratios (activating:inhibitory ratios) (Nimmerjahn et al; Science. 2005 Dec 2; 310(5753):1510-2).

[0027] By binding to inhibitory Fcγ receptors, antibodies can inhibit, block, and / or downregulate effector cell function.

[0028] Antibodies can activate effector cell function by binding to activating Fcγ receptors, thereby inducing mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), cytokine release, and / or antibody-dependent endocytosis, and in the case of neutrophils, NETosis (i.e., activation and release of NETs (neutrophil extracellular traps)). Antibodies that bind to activating Fcγ receptors can also cause an increase in certain activation markers, such as CD40, MHCII, CD38, CD80, and / or CD86.

[0029] In some embodiments, an antibody molecule that specifically binds to CTLA-4 is an Fcγ receptor binding antibody. By "Fcγ receptor binding antibody" is meant that the antibody molecule is capable of binding to at least one Fcγ receptor via its Fc region.

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

[0031] A functional fragment of a full-sized antibody has the same antigen-binding characteristics as the corresponding full-sized antibody, and contains either the same variable domains (i.e., VH and VL sequences) and / or the same CDR sequences as the corresponding full-sized antibody. A functional fragment has the same antigen-binding characteristics as the corresponding full-sized antibody, meaning that it binds to the same epitope on the target as the full-sized antibody. Such a functional fragment may correspond to the Fv portion of a full-sized antibody. Alternatively, such a fragment may be Fab, also denoted Fab', which is a monovalent antigen-binding fragment that does not contain the Fc portion, or disulfide-linked or Fab', i.e. (Fab') 2 (Fab') is a bivalent antigen-binding fragment containing two antigen-binding Fab portions linked together by a monovalent variant of2 Such a fragment may also be a single chain variable fragment (scFv).

[0032] In some embodiments, the first antibody molecule and / or the second antibody molecule described herein can be a full size antibody, a chimeric antibody, a single chain antibody, and antigen-binding fragments thereof (e.g., Fab, Fv, scFv, Fab', and (Fab')). 2 ).

[0033] A functional fragment does not always contain all six CDRs of the corresponding full-size antibody. It is understood that a molecule containing three or fewer CDR regions (sometimes only a single CDR or a part thereof) can retain the antigen-binding activity of the antibody from which the CDR is derived. For example, Gao et al., 1994, J.Biol.Chem.,269:32389-93, has described that the entire VL chain (containing all three CDRs) has high affinity for its substrate.

[0034] Molecules containing two CDR regions are described, for example, in Vaughan & Sollazzo 2001, Combinatorial Chemistry & High Throughput Screening, 4:417-430. On page 418 (right column-3 Our Strategy for Design), a minibody is described that contains only H1 and H2 CDR hypervariable regions interspersed within framework regions. The minibody is described as being capable of binding to a target. Pessi et al., 1993, Nature, 362:367-9 and Bianchi et al., 1994, J. Mol. Biol., 236:649-59, referenced by Vaughan & Sollazzo, provide a more detailed description of H1 and H2 minibodies and their properties. Qiu et al., 2007, Nature Biotechnology, 25:921-9 demonstrate that a molecule consisting of two linked CDRs is capable of binding to an antigen. Quiocho 1993, Nature, 362:293-4 provides an overview of "minibody" technology. Ladner 2007, Nature Biotechnology, 25:875-7 observes that molecules containing two CDRs are capable of retaining antigen-binding activity.

[0035] Antibody molecules containing a single CDR region are described, for example, in Laune et al., 1997, JBC, 272:30937-44, where it has been demonstrated that various hexapeptides derived from the CDRs exhibit antigen-binding activity, and it is noted that synthetic peptides of complete single CDRs exhibit strong binding activity. Monnet et al., 1999, JBC, 274:3789-96, have demonstrated that various 12-mer peptides and associated framework regions have antigen-binding activity, and opine that CDR3-like peptides are capable of binding antigens alone. Heap et al., 2005, J. Gen. Virol., 86:1791-1800, report that "microantibodies" (molecules containing a single CDR) are capable of binding antigens, and cyclic peptides from anti-HIV antibodies have been shown to have antigen-binding activity and function. Nicaise et al., 2004, Protein Science, 13:1882-91, show that a single CDR can confer antigen-binding activity and affinity for its lysozyme antigen.

[0036] Thus, antibody molecules having five, four, three or fewer CDRs can retain the antigen-binding properties of the full-length antibody from which they are derived.

[0037] An antibody molecule may be a derivative of a full-length antibody or a fragment of such an antibody. A derivative has the same antigen-binding characteristics as the corresponding full-sized antibody, meaning that it binds to the same epitope on the target as the full-sized antibody.

[0038] Thus, as used herein, the term "antibody molecule" includes monoclonal antibodies, polyclonal antibodies, synthetic antibodies, recombinantly produced antibodies, multispecific antibodies, bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, variable fragments (Fv), single chain variable fragments (scFv fragments), including bivalent single chain variable fragments (di-scFv) and disulfide linked variable fragments, Fab fragments, F(ab')2 The present invention includes all types of antibody molecules, including fragments, Fab' fragments, antibody heavy chains, antibody light chains, antibody heavy chain homodimers, antibody light chain homodimers, antibody heavy chain heterodimers, antibody light chain heterodimers, antigen-binding functional fragments of such homo- and heterodimers, as well as functional fragments and derivatives thereof.

[0039] Furthermore, as used herein, the term "antibody molecule" includes all classes of antibody molecules and functional fragments, including IgG, IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD, and IgE.

[0040] In some embodiments, the antibody is human IgG1. Those skilled in the art recognize that mouse IgG2a and human IgG1 share the ability to productively bind to activating Fc gamma receptors and activate target cell deletion through activation of immune cells (e.g., macrophages and NK cells) bearing activating Fc gamma receptors, for example, by ADCP and ADCC. Thus, mouse IgG2a is the preferred isotype for deletion in mice, while human IgG1 is the preferred isotype for deletion in humans. Conversely, optimal costimulation of agonist receptors of the TNFR superfamily, such as 4-1BB, OX40, TNFRII, CD40, is known to depend on antibody binding of inhibitory FcγRIIB. In mice, the IgG1 isotype, which preferentially binds to inhibitory Fc gamma receptors (FcγRIIB) and only weakly binds to activating Fc gamma receptors, is known to be optimal for mAb-targeted costimulatory activity of the TNFR superfamily. Although a direct equivalent of the mouse IgG1 isotype in humans has not been described, antibodies can be engineered to similarly exhibit improved binding to inhibitory human Fc gamma receptors over activating human Fc gamma receptors. Such engineered TNFR superfamily-targeting antibodies also have improved co-stimulatory activity in vivo in transgenic mice engineered to express human activating and inhibitory Fc gamma receptors (Dahan et al, 2016, Therapeutic Activity of Agonistic, Human Anti-CD40 Monoclonal Antibodies Requires Selective FcγR Engagement. Cancer Cell. 29(6):820-31).

[0041] As outlined above, different types and forms of antibody molecules are encompassed by the present invention and will be known to those skilled in the art of immunology. It is known that antibodies used for therapeutic purposes are often modified with additional moieties that modify the properties of the antibody molecule.

[0042] It is therefore included that the antibody molecules of the invention or used in accordance with the invention (e.g. monoclonal and / or polyclonal and / or bispecific antibody molecules) comprise a detectable moiety and / or a cytotoxic moiety.

[0043] "Detectable moieties" include one or more from the group including: enzymes, radioactive atoms, fluorescent moieties, chemiluminescent moieties, bioluminescent moieties. Detectable moieties allow for visualization of the antibody molecule in vitro, and / or in vivo, and / or ex vivo.

[0044] "Cytotoxic moieties" include radioactive moieties, and / or enzymes (e.g., the enzyme is a caspase), and / or toxins (e.g., the toxin is a bacterial toxin or venom), and cytotoxic moieties are capable of inducing cell lysis.

[0045] It is further included that the antibody molecules may be in isolated and / or purified form and / or may be PEGylated.

[0046] As discussed above, the CDRs of an antibody bind to the antibody target. The amino acid assignments for each CDR described herein are as defined by Kabat EA et al. 1991, "Sequences of Proteins of Immunological Interest" Fifth Edition, NIH Publication No. 91-3242, pp xv-xvii.

[0047] As those skilled in the art will recognize, there are other methods for assigning amino acids to each CDR, such as the International ImMunoGeneTics information system (IMGT®) (http: / / www.imgt.org / and Lefranc and Lefranc, "The Immunoglobulin Facts Book," published by Academic Press, 2001).

[0048] In a further embodiment, the CTLA-4 specific antibody molecule of the invention or for use in accordance with the invention is an antibody molecule capable of competing with specific antibodies described herein, such as antibody molecules comprising SEQ ID NOs: 15, 16, 17, 10, 18, and 19, or SEQ ID NOs: 22, 23, 24, 10, 25, and 26.

[0049] By "capable of competing" it is meant that a competing antibody is capable of at least partially inhibiting or otherwise interfering with the binding of an antibody molecule as defined herein to a particular target.

[0050] For example, such a competing antibody molecule may be capable of inhibiting binding of an antibody molecule described herein by at least about 10%, e.g., at least about 20%, or at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 100% and / or may be capable of inhibiting the ability of an antibody described herein to prevent or reduce binding to a particular target by at least about 10%, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100%.

[0051] Competitive binding can be determined by methods well known to those of skill in the art, such as enzyme-linked immunosorbent assay (ELISA).

[0052] ELISA assays can be used to evaluate epitope-modified or blocking antibodies. Additional methods suitable for identifying competing antibodies are disclosed in Antibodies: A Laboratory Manual, Harlow & Lane, which is incorporated herein by reference (see, e.g., pages 567-569, 574-576, 583, and 590-612, 1988, CSHL, NY, ISBN 0-87969-314-2).

[0053] It is well known that antibodies specifically bind to a defined target molecule or antigen, meaning that the antibody binds preferentially and selectively to that target over non-target molecules.

[0054] The targets of the first and second antibodies according to the invention (CTLA-4, PD1, PD-L1) or used in accordance with the invention are expressed on the surface of cells, i.e. they are cell surface antigens, which will include an epitope for the antibody (otherwise known in this context as a cell surface epitope). Cell surface antigens and epitopes are terms readily understood by those skilled in the art of immunology or cell biology.

[0055] By "cell surface antigen" is meant to include cell surface antigens, or at least epitopes thereof, that are exposed on the extracellular side of the cell membrane to which the antibody molecules described herein are directed.

[0056] Methods for assessing protein binding are known to those skilled in the art of biochemistry and immunology. Those skilled in the art will understand that such methods can be used to assess the relative strength, or specificity, inhibition, or prevention, or reduction, of the binding of an antibody to a target and / or the binding of an antibody's Fc domain to an Fc receptor, and their interactions. Examples of methods that can be used to assess protein binding include, for example, immunoassays, BIAcore, Western blots, radioimmunoassays (RIA), and enzyme-linked immunosorbent assays (ELISA) (for a discussion of antibody specificity, see Fundamental Immunology Second Edition, Raven Press, New York at pages 332-336 (1989)).

[0057] Thus, as used herein, both an "antibody molecule that specifically binds to CTLA-4" and an "anti-CTLA-4 antibody molecule" refer to an antibody molecule that specifically binds to the target CTLA-4, but does not bind to non-targets, or that binds to non-targets weaker (e.g., with lower affinity) than the target.

[0058] Thus, as used herein, both an "antibody molecule that specifically binds to PD-1" and an "anti-PD-1 antibody molecule" refer to an antibody molecule that specifically binds to the target PD-1, but does not bind to a non-target, or that binds to the non-target with less affinity (e.g., lower affinity) than the target.

[0059] Thus, as used herein, both an "antibody molecule that specifically binds to PD-L1" and an "anti-PD-L1 antibody molecule" refer to an antibody molecule that specifically binds to the target PD-L1, but does not bind to a non-target, or binds to the non-target with less affinity (e.g., lower affinity) than the target.

[0060] In some embodiments, an antibody molecule that specifically binds to CTLA-4 (or an anti-CTLA-4 antibody molecule) refers to an antibody molecule that specifically binds to the extracellular domain of CTLA-4. In some embodiments, an antibody molecule that specifically binds to PD-1 (or an anti-PD-1 antibody molecule) refers to an antibody molecule that specifically binds to the extracellular domain of PD-1. In some embodiments, an antibody molecule that specifically binds to PD-L1 (or an anti-PD-L1 antibody molecule) refers to an antibody molecule that specifically binds to the extracellular domain of PD-L1.

[0061] Also included is the meaning that the antibody specifically binds to a target CTLA-4 or PD-1 or PD-L1 at least 2 times stronger, or at least 5 times stronger, or at least 10 times stronger, or at least 20 times stronger, or at least 50 times stronger, or at least 100 times stronger, or at least 200 times stronger, or at least 500 times stronger, or at least about 1000 times stronger, than a non-target.

[0062] Additionally, the antibody is at least about 10 -1 M, or at least about 10 -2 M, or at least about 10 -3 M, or at least about 10 -4 M, or at least about 10 -5 M, or at least about 10 -6 M, or at least about 10 -7 M, or at least about 10 -8 M, or at least about 10 -9 M, or at least about 10 -10 M, or at least about 10 -11 M, or at least about 10 -12 M, or at least about 10 -13 M, or at least about 10 -14 M, or at least about 10 -15 Dissociation constant of M (K D ) it is intended that the antibody specifically binds to the target CTLA-4 or PD-1 or PD-L1.

[0063] As described above, the oncolytic viruses of the present invention express an antibody that specifically binds to CTLA-4.

[0064] In some embodiments, the antibody molecule that specifically binds to CTLA-4 is an Fcγ receptor binding antibody.

[0065] In some embodiments, the first antibody molecule is selected from the group consisting of ipilimumab and tremelimumab.

[0066] In some embodiments, an antibody molecule that specifically binds to CTLA-4 (or an anti-CTLA-4 antibody molecule) does not cross-react with CD28. In some embodiments, an antibody molecule that specifically binds to CTLA-4 (or an anti-CTLA-4 antibody molecule) blocks the binding of CTLA-4 to CD80 and / or CD86, thereby inhibiting CTLA-4 signaling.

[0067] In some embodiments, the antibody molecules that specifically bind to CTLA-4 (or anti-CTLA-4 antibody molecules) described herein have an improved depleting effect on CTLA-4 positive cells compared to ipilimumab.

[0068] That an antibody molecule has a depleting effect on CTLA-4 positive cells means that, when administered to a subject, such as a human, such an antibody specifically binds to CTLA-4 expressed on the surface of CTLA-4 positive cells, and this binding results in the depletion of such cells.

[0069] In some embodiments, the CTLA-4 positive cells are CD4 positive (CD4 + ) cells, i.e., cells that express CD4.

[0070] In some embodiments, the CTLA-4 positive cells are both CD4 positive and FOXP3 positive, i.e., express both CD4 and FOXP3. These cells are Tregs. Although CD8 positive T cells also express CTLA-4, Tregs express significantly higher levels of CTLA-4 than CD8 positive T cells. This allows Tregs to differentiate into CD8 positive T cells, which express less CTLA-4. + are more susceptible to depletion than cells.

[0071] In some situations, CTLA-4 is preferentially expressed on immune cells in the tumor microenvironment (tumor infiltrating cells, TILS).

[0072] Thus, in the tumor microenvironment, Tregs are the cells with the highest expression of CTLA-4, and antibody molecules that specifically bind to CTLA-4 (or anti-CTLA-4 antibody molecules) have a Treg-depleting effect.

[0073] Thus, in some embodiments, the antibody molecule that specifically binds to CTLA-4 is a Treg-depleting antibody.

[0074] As described above, the anti-CTLA-4 antibody molecules described herein may be Treg-depleting antibody molecules, meaning that when administered to a subject, such as a human, such antibody molecules specifically bind to CTLA-4 expressed on the surface of Tregs, which binding results in depletion of Tregs.

[0075] To determine whether an antibody molecule is an antibody molecule having a Treg depleting effect as referred to herein (e.g., which may be an improved depleting effect compared to ipilimumab), an in vitro antibody-dependent cellular cytotoxicity (ADCC) assay or an in vivo test in the PBMC-NOG / SCID model can be used.

[0076] 1. An in vitro ADCC test performed using an NK-92 cell line stably transfected to express the CD16-158V allele together with GFP, the ADCC test comprising the following seven consecutive steps: 1) Isolating CTLA-4 positive cells, CD4 positive cells, or Tregs as target cells from peripheral blood of healthy donors. This isolation can be performed using a CD4 positive cell line, such as a commercially available kit from Miltenyi Biotec. + This can be done using a T cell isolation kit. 2) The target cells are then stimulated with CD3 / CD28, e.g., using CD3 / CD28 Dynabeads® and rhIL-2, e.g., 50 ng / ml rhIL-2, for e.g., 48 hours. Stimulation can be performed at 37°C. 3) The target cells are then pre-incubated with the antibody molecule to be tested, for example at 10 μg / ml, for 30 minutes at 4° C., and then mixed with the NK cells. 4) Next, incubate the target cells in RPMI 1640+GlutaMAX medium containing HEPES buffer, sodium pyruvate, and FBS low IgG for an appropriate time, for example 4 hours. RPMI 1640+GlutaMAX medium may contain 10 mM HEPES buffer, 1 mM sodium pyruvate, and 10% FBS low IgG, and the effector:target cell ratio may be 2:1. 5) Determining lysis by flow cytometry. 6) Repeating or performing steps 1 to 5 in parallel, with ipilimumab (as a control) used in place of the antibody molecule tested in step 3. 7) Comparing the lysis results of the tested antibody molecule with those of ipilimumab. Improved lysis of the tested antibody molecule compared to ipilimumab indicates that the tested antibody molecule has an improved depletion effect on CTLA-4 positive cells, CD4 positive cells or Tregs, respectively, depending on the target cells used.

[0077] In some embodiments, the improved depletion effect in step 7) above is a significantly improved depletion effect.

[0078] The in vivo test is based on the combination of PBMC mice and NOG / SCID mice, which is referred to herein as the PBMC-NOG / SCID model. Both PBMC mice and NOG / SCID mice are known models. The in vivo test in the PBMC-NOG / SCID model includes the following nine consecutive steps: 1) Isolate, wash, and resuspend human PBMCs (peripheral blood mononuclear cells) in sterile PBS. In some embodiments, PBMCs are at a concentration of 75×10 6 Cells / ml are resuspended in PBS. 2) Inject NOG mice iv (intravenously) with an appropriate amount of the cell suspension from step 1), for example 200 μl. If 200 μl is injected, this is equivalent to 15×10 6 Equivalent to cells / mouse. 3) At an appropriate time after injection, for example 2 weeks, isolate the spleen from the NOG mouse and make it into a single cell suspension. Optionally, take a small sample from the single cell suspension to determine the expression of CTLA-4 on human T cells by FACS to confirm the expression of CTLA-4. 4) Resuspending the cell suspension of step 3) in sterile PBS. In some embodiments, the cell suspension is at least 50×10 6 Resuspend in sterile PBS at 1000 cells / ml. If the optional CTLA-4 expression determination is included in step 3, the remaining cell suspension is resuspended in step 4. 5) Inject SCID mice ip with an appropriate amount of the suspension from step 4, for example 200 μl. If 200 μl is injected, this is 10×10 6 Equivalent to cells / mouse. 6) At an appropriate time, e.g., 1 hour, after injection of step 5), treating the SCID mice with an appropriate amount, e.g., 10 mg / kg, of either the antibody molecule to be tested, ipilimumab or an isotype control monoclonal antibody. 7) Collecting the intraperitoneal fluid of the treated SCID mice at an appropriate time, for example 24 hours, after treatment in step 6). 8) Identifying and quantifying human T cell subsets by FACS using the markers CD45, CD4, CD8, CD25 and / or CD127. 9) comparing the results of identification and quantification of T cell subsets from mice treated with the tested antibody molecule with the results of identification and quantification of T cell subsets from mice treated with ipilimumab and with the results of identification and quantification of T cell subsets from mice treated with an isotype control monoclonal antibody. A lower number of CTLA-4 positive cells in the peritoneal fluid of mice treated with the tested antibody molecule compared to the number of CTLA-4 positive cells in the peritoneal fluid of mice treated with ipilimumab indicates that the antibody molecule has an improved depletion effect on CTLA-4 positive cells compared to ipilimumab. A lower number of CD4 positive cells in the peritoneal fluid of mice treated with the tested antibody molecule compared to the number of CD4 positive cells in the peritoneal fluid of mice treated with ipilimumab demonstrates that the antibody molecule has an improved depletion effect on CD4 positive cells compared to ipilimumab. The lower number of Tregs in the peritoneal fluid of mice treated with the tested antibody molecule compared to the number of Tregs in the peritoneal fluid of mice treated with ipilimumab indicates that this antibody molecule has an improved depleting effect on Tregs compared to ipilimumab.

[0079] In this in vivo study, in some embodiments, it is of most interest to examine Treg depletion at step 7.

[0080] Treg depletion can also be assessed in an antibody-dependent cellular phagocytosis (ADCP) assay, as known to those of skill in the art.

[0081] In some embodiments, the antibody molecules have a similar blocking effect on CTLA-4 interactions with B7.1 and B7.2 ligands compared to Yervoy (ipilimumab), which can be assessed by ELISA or in a more functional assay in which anti-CTLA-4 antibodies enhance IL-2 production by T cells in response to stimulation of PBMCs with Staphylococcus Enterotoxin B (SEB).

[0082] In some embodiments, the anti-CTLA-4 antibody molecule is a human antibody molecule. In some embodiments, the anti-CTLA-4 antibody molecule is a humanized antibody molecule. In some embodiments, the anti-CTLA-4 antibody molecule is a human-derived antibody molecule, meaning that it is derived from a human antibody molecule that has been subsequently modified. In some embodiments, the anti-CTLA-4 antibody molecule is a human IgG1 antibody.

[0083] In some embodiments, the first antibody molecule that specifically binds to CTLA-4 is selected from the group consisting of a human IgG antibody, a humanized IgG antibody, and an IgG antibody of human origin.

[0084] In some embodiments, the first antibody molecule that specifically binds to CTLA-4 is a full size antibody, a chimeric antibody, a single chain antibody, and antigen-binding fragments thereof (e.g., Fab, Fv, scFv, Fab', and (Fab') 2 ).

[0085] In some embodiments, the anti-CTLA-4 antibody is an antibody in the form of a human IgG1 antibody that exhibits improved binding to one or several activating Fc receptors and / or has been engineered for improved binding to one or several activating Fc receptors; thus, in some embodiments, the anti-CTLA-4 antibody is an Fc-engineered human IgG1 antibody.

[0086] In some embodiments, the anti-CTLA-4 antibody is a murine or humanized murine IgG2a antibody.

[0087] In some embodiments, the anti-CTLA-4 antibody is a murine antibody that is cross-reactive with human CTLA-4.

[0088] In some embodiments, the CTLA-4 molecule is a monoclonal antibody or an antibody molecule of human origin, hi some embodiments, the anti-CTLA-4 antibody is a polyclonal antibody.

[0089] In some embodiments, the anti-CTLA-4 antibody molecule is an antibody molecule that comprises one of three alternative VH-CDR1 sequences, one of three alternative VH-CDR2 sequences, one of two alternative VH-CDR3 sequences, one of two VL-CDR1 sequences, one of two VL-CDR2 sequences, and / or one of two alternative VL-CDR3 sequences as shown in Table 1 below.

[0090] In some embodiments, the anti-CTLA-4 antibody molecule is selected from the group consisting of antibody molecules comprising 1-6 of the CDRs selected from the group consisting of SEQ ID NOs: 3, 6, 8, 10, 12, and 14.

[0091] In some embodiments, the anti-CTLA-4 antibody molecule is selected from the group consisting of antibody molecules comprising CDRs having SEQ ID NOs: 3, 6, 8, 10, 12, and 14.

[0092] In some embodiments, the anti-CTLA-4 antibody molecule is selected from the group consisting of antibody molecules comprising 1-6 of the CDRs, VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, and VL-CDR3; VH-CDR1, if present, is selected from the group consisting of SEQ ID NOs: 15, 22, 29, and 35; VH-CDR2, if present, is selected from the group consisting of SEQ ID NOs: 16, 23, 30, and 36; VH-CDR3, if present, is selected from the group consisting of SEQ ID NOs: 17, 24, 31, and 37; VL-CDR1, if present, is selected from the group consisting of SEQ ID NOs: 10 and 38; VL-CDR2, if present, is selected from the group consisting of SEQ ID NOs: 18, 25, 32, and 39; The VL-CDR3, if present, is selected from the group consisting of SEQ ID NOs: 19, 26, and 40.

[0093] In some embodiments, the anti-CTLA-4 antibody molecule is selected from the group consisting of antibody molecules comprising six CDRs selected from the group consisting of: SEQ ID NOs: 15, 16, 17, 10, 18, and 19; SEQ ID NOs: 22, 23, 24, 10, 25, and 26; SEQ ID NOs: 29, 30, 31, 10, 32, and 26, and SEQ ID NOs: 35, 36, 37, 38, 39, and 40.

[0094] In some embodiments, the anti-CTLA-4 antibody molecule is an antibody molecule that comprises six CDRs having SEQ ID NOs:15, 16, 17, 10, 18, and 19.

[0095] In some embodiments, the anti-CTLA-4 antibody molecule is an antibody molecule that comprises six CDRs having SEQ ID NOs:22, 23, 24, 10, 25, and 26.

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

[0097] In some embodiments, the anti-CTLA-4 antibody molecule is an antibody molecule selected from the group consisting of antibody molecules comprising a VL selected from the group consisting of SEQ ID NOs: 21, 28, 34, and 42.

[0098] In some embodiments, the anti-CTLA-4 antibody molecule is an antibody molecule selected from the group consisting of antibody molecules comprising a VH and a VL selected from the group consisting of SEQ ID NOs: 20-21, 27-28, 33-34, and 41-42.

[0099] In some embodiments, the anti-CTLA-4 antibody molecule comprises a VH having the sequence of SEQ ID NO:20, and a VL having the sequence of SEQ ID NO:21.

[0100] In some embodiments, the anti-CTLA-4 antibody molecule comprises a VH having the sequence of SEQ ID NO:27, and a VL having the sequence of SEQ ID NO:28.

[0101] In some embodiments, the first antibody molecule comprises a variable heavy chain selected from the group consisting of SEQ ID NOs: 20 and 27. In some further or alternative embodiments, the first antibody molecule comprises a variable light chain selected from the group consisting of SEQ ID NOs: 21 and 28.

[0102] In some embodiments, the anti-CTLA-4 antibody molecule comprises a heavy chain constant region (CH) having the sequence of SEQ ID NO: 43. In some further or alternative embodiments, the anti-CTLA-4 antibody molecule comprises a light chain constant region (CL) having the sequence of SEQ ID NO: 44. In some embodiments, the anti-CTLA-4 antibody molecule comprises the constant regions of SEQ ID NOs: 43 and 44.

[0103] [Table 1]

[0104] [Table 2-1]

[0105] [Table 2-2]

[0106] [Table 2-3]

[0107] In some embodiments, the anti-CTLA-4 antibody molecules described herein may also comprise one or both of the constant regions shown in Table 3 below.

[0108] [Table 3]

[0109] In some embodiments, the anti-CTLA-4 antibody molecule is a molecule encoded by one of the nucleotide sequences shown in Table 4 below.

[0110] [Table 4-1]

[0111] [Table 4-2]

[0112] [Table 4-3]

[0113] [Table 4-4]

[0114] In some embodiments, it is advantageous for the antibody molecule to bind to both human CTLA-4 (hCTLA-4) and cynomolgus monkey CTLA-4 (cmCTLA-4 or cyno CTLA-4). Cross-reactivity with CTLA-4 expressed on cells of cynomolgus monkeys, also called crab-eating macaques or Macaca fascicularis, can be advantageous as it allows for testing of the antibody molecule in monkeys without the use of surrogate antibodies, with particular focus on tolerability.

[0115] In some embodiments, it is advantageous for the antibody molecule to bind to both human CTLA-4 (hCTLA-4) and mouse CTLA-4 (mCTLA-4), which can be advantageous as it allows for testing of the antibody molecule in mice, with a particular focus on efficacy and pharmacodynamics, without the need to use surrogate antibodies.

[0116] In some embodiments, the antibody molecule binds to all three of hCTLA-4, cmCTLA-4 and mCTLA-4.

[0117] In some embodiments, it is necessary to use a surrogate antibody to test the functional activity of the antibody molecule in a relevant in vivo model in mice. To ensure comparability between the effect of the antibody molecule in humans and the in vivo results of the surrogate antibody in mice, it is essential to select a functionally equivalent surrogate antibody that has the same in vitro properties as the human antibody molecule.

[0118] In some embodiments, the first antibody molecule that specifically binds CTLA-4 does not bind human CD28.

[0119] As discussed herein, the second antibody may specifically bind to PD-1. In some embodiments, the antibody molecule that specifically binds to PD-1 is selected from one or more of the following non-limiting examples of anti-PD-1 antibodies: Pembrolizumab (currently approved for use), Nivolumab (currently approved for use), Cemiplimab (currently approved for use), Camrelizumab (currently approved for use), ● Spartalizumab (currently in clinical development), ● Dostarlimab (currently in clinical development), - tislelizumab (currently in clinical development), ● JTX-4014 (currently in clinical development), ● Sintilimab (IBI308) (currently in clinical development), - Toripalimab (JS 001) (currently in clinical development), ●AMP-224 (currently in clinical development), ●AMP-514 (MEDI0680) (currently in clinical development).

[0120] In preferred embodiments, the antibody that specifically binds to PD-1 is pembrolizumab, nivolumab, cemiplimab, or camrelizumab. In some embodiments, the antibody that specifically binds to PD-1 is a combination of two or more of these antibodies. In preferred embodiments, the antibody that specifically binds to PD-1 is pembrolizumab.

[0121] In some embodiments, the anti-PD-1 antibody molecule is a human antibody molecule. In some embodiments, the anti-PD-1 antibody molecule is a humanized antibody molecule.

[0122] In some embodiments, the anti-PD-1 antibody molecule is a human-derived antibody molecule, meaning that it is derived from a human antibody molecule that has been subsequently modified.

[0123] In some embodiments, the anti-PD-1 antibody molecule is a human IgG1 antibody.

[0124] In some embodiments, the anti-PD-1 antibody is an antibody in the form of a human IgG1 antibody that exhibits improved binding to one or several activating Fc receptors and / or has been engineered for improved binding to one or several activating Fc receptors; thus, in some embodiments, the anti-PD-1 antibody is an Fc-engineered human IgG1 antibody.

[0125] In some embodiments, the anti-PD-1 antibody is a murine or humanized murine IgG2a antibody.

[0126] In some embodiments, the second antibody molecule that specifically binds to PD-1 is selected from the group consisting of a human antibody molecule, a humanized antibody molecule, and an antibody molecule of human origin.

[0127] In some embodiments, the second antibody molecule that specifically binds to PD-1 is a full-size antibody, a chimeric antibody, a single chain antibody, and antigen-binding fragments thereof (e.g., Fab, Fv, scFv, Fab', and (Fab') 2 ).

[0128] In some embodiments, the second antibody molecule that specifically binds to PD-1 is selected from the group consisting of a human IgG antibody, a humanized IgG antibody, and an IgG antibody of human origin.

[0129] In some embodiments, the anti-PD-1 antibody is a murine antibody that is cross-reactive with human PD-1.

[0130] In some embodiments, the PD-1 molecule is a monoclonal antibody or an antibody molecule of human origin, hi some embodiments, the anti-PD-1 antibody is a polyclonal antibody.

[0131] In some embodiments, the second antibody molecule is capable of specifically binding to PD-L1. In some embodiments, the antibody molecule that specifically binds to PD-L1 is selected from one or more of the following non-limiting examples of anti-PD-L1 antibodies: Atezolizumab (currently approved for use), Durvalumab (currently approved for use), Avelumab (currently approved for use), ●CS1001 (currently in clinical development), KN035 (envafolimab), a PD-L1 antibody with a subcutaneous formulation currently undergoing clinical evaluation in the United States, China, and Japan; ●CK-301 (currently in clinical development by Checkpoint Therapeutics)

[0132] In preferred embodiments, the antibody that specifically binds to PD-L1 is atezolizumab, durvalumab, or avelumab, hi some embodiments, the antibody that specifically binds to PD-L1 is a combination of two or more of these antibodies.

[0133] In some embodiments, the anti-PD-L1 antibody molecule is a human antibody molecule.

[0134] In some embodiments, the anti-PD-L1 antibody molecule is a humanized antibody molecule.

[0135] In some embodiments, the anti-PD-L1 antibody molecule is a human-derived antibody molecule, meaning that it is derived from a human antibody molecule that has been subsequently modified.

[0136] In some embodiments, the anti-PD-L1 antibody molecule is a human IgG1 antibody.

[0137] In some embodiments, the anti-PD-L1 antibody is an antibody in the form of a human IgG1 antibody that exhibits improved binding to one or several activating Fc receptors and / or has been engineered for improved binding to one or several activating Fc receptors; thus, in some embodiments, the anti-PD-L1 antibody is an Fc-engineered human IgG1 antibody.

[0138] In some embodiments, the anti-PD-L1 antibody is a murine or humanized murine IgG2a antibody.

[0139] In some embodiments, the second antibody molecule that specifically binds to PD-L1 is selected from the group consisting of a human antibody molecule, a humanized antibody molecule, and an antibody molecule of human origin.

[0140] In some embodiments, the second antibody molecule that specifically binds to PD-L1 is a full size antibody, a chimeric antibody, a single chain antibody, and antigen-binding fragments thereof (e.g., Fab, Fv, scFv, Fab', and (Fab') 2 ).

[0141] In some embodiments, the second antibody molecule that specifically binds to PD-L1 is selected from the group consisting of a human IgG antibody, a humanized IgG antibody, and an IgG antibody of human origin.

[0142] In some embodiments, the anti-PD-L1 antibody is a murine antibody that is cross-reactive with human PD-L1.

[0143] In some embodiments, the PD-L1 molecule is a monoclonal antibody or an antibody molecule of human origin, hi some embodiments, the anti-PD-L1 antibody is a polyclonal antibody.

[0144] As described herein, the present invention includes an oncolytic virus capable of expressing a first antibody molecule that specifically binds to CTLA-4.

[0145] As used herein, the term "oncolytic" refers to the ability of a virus to selectively replicate in dividing cells (e.g., proliferating cells such as cancer cells) with the goal of slowing the growth of and / or lysing those dividing cells, either in vitro or in vivo, while exhibiting no or minimal replication in non-dividing (e.g., normal or healthy) cells.

[0146] "Replication" (or any form of replication, such as "replicate" and "replicating") refers to viral duplication that may occur at the level of nucleic acid or, preferably, at the level of infectious viral particles. Such oncolytic viruses can be obtained from any member of the currently identified viruses. It may be a naturally oncolytic virus or may be engineered by modifying one or more viral genes to increase tumor selectivity and / or preferential replication in dividing cells, such as those involved in DNA replication, nucleic acid metabolism, host tropism, surface attachment, pathogenicity, lysis, and spread (see, e.g., Wong et al., 2010, Viruses 2:78-106). It can also be envisioned to place one or more viral genes under the control of an event- or tissue-specific regulatory element (e.g., a promoter).

[0147] Exemplary oncolytic viruses include, but are not limited to, reovirus, Seneca Valley virus (SVV), vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), herpes simplex virus (HSV), morbillivirus, adenovirus, poxvirus, retrovirus, measles virus, foamy virus, alphavirus, lentivirus, influenza virus, sinbis virus, myxoma virus, rhabdovirus, picornavirus, coxsackievirus, parvovirus, etc. Oncolytic viruses are known to those skilled in the art of medicine and virology.

[0148] In some embodiments, such oncolytic viruses are derived from herpes viruses. Herpesviridae is a large family of DNA viruses that all share a common structure and consist of a relatively large double-stranded linear DNA genome encoding 100-200 genes, encapsidated in an icosahedral capsid surrounded by a lipid bilayer membrane. Oncolytic herpes viruses can be derived from various types of HSV, with HSV1 and HSV2 being particularly preferred. Herpes viruses can be genetically modified to limit viral replication in tumors or reduce cytotoxicity in non-dividing cells. For example, any viral gene involved in nucleic acid metabolism can be inactivated, such as thymidine kinase (Martuza et al., 1991, Science 252:854-6), ribonucleotide reductase (RR) (Mineta et al., 1994, Cancer Res. 54:3363-66), or uracil-N-glycosylase (Pyles et al., 1994, J. Virol. 68:4963-72). Another embodiment includes viral mutants defective in the function of genes encoding virulence factors, such as the ICP34.5 gene (Chambers et al., 1995, Proc. Natl. Acad. Sci. USA 92:1411-5). Representative examples of oncolytic herpesviruses include NV1020 (e.g., Geevarghese et al., 2010, Hum. Gene Ther. 21(9):1119-28) and T-VEC (Harrington et al., 2015, Expert Rev. Anticancer Ther. 15(12):1389-1403).

[0149] In some embodiments, such oncolytic viruses are derived from adenoviruses. Methods for engineering oncolytic adenoviruses are available in the art. Advantageous strategies include replacing viral promoters with tumor-selective promoters or modifying E1 adenoviral gene products to inactivate their binding function with p53 or retinoblastoma (Rb) proteins, which are altered in tumor cells. In the natural situation, the adenoviral E1B55kDa gene cooperates with another adenoviral product to inactivate p53 (which is frequently dysregulated in cancer cells) and prevent apoptosis. Representative examples of oncolytic adenoviruses include ONYX-015 (e.g., Khuri et al., 2000, Nat.Med 6(8):879-85) and H101, also called Oncorine (Xia et al., 2004, Ai Zheng 23(12):1666-70).

[0150] In some embodiments, such oncolytic viruses are oncolytic poxviruses. As used herein, the term "poxvirus" refers to viruses belonging to the family Poxviridae, with poxviruses belonging to the subfamily Chordopoxviridae, more preferably the genus Orthopoxvirus, being particularly preferred. Vaccinia virus, cowpox virus, canarypox virus, ectromelia virus, myxoma virus are particularly suitable in the context of the present invention. The genomic sequences of such poxviruses are available in technical and specialized databases (e.g. Genbank, accession numbers NC_006998, NC_003663, or AF482758.2, NC_005309, NC_004105, NC_001132, respectively).

[0151] In a particular preferred embodiment, such an oncolytic poxvirus is an oncolytic vaccinia virus. Vaccinia virus is a member of the Poxviridae family, characterized by a 200 kb double-stranded DNA genome that encodes a number of viral enzymes and factors that allow the virus to replicate independently of the host cell machinery. The majority of vaccinia virus particles are intracellular (IMV for intracellular mature virions), have a single lipid envelope, and remain in the cytosol of infected cells until lysis. Another form of infection is a double-enveloped particle (EEV for extracellular enveloped virions), which buds without lysing the infected cell. Vaccinia virus strains may be derived, with Elstree, Wyeth, Copenhagen, Lister, and Western Reserve strains being particularly preferred. The genetic nomenclature used herein is that of the Copenhagen vaccinia strain, unless otherwise stated. However, the correspondence between Copenhagen and other vaccinia strains is generally available in the literature.

[0152] Preferably, such oncolytic vaccinia viruses are modified by modifying one or more viral genes. The modification preferably results in the absence of synthesis or the synthesis of defective viral proteins that cannot guarantee the activity of the proteins produced under normal conditions by the unmodified genes. Exemplary modifications are disclosed in the literature that aim to modify viral genes involved in DNA metabolism, host virulence, IFN pathway (e.g., Guse et al., 2011, Expert Opinion Ther. 11(5):595-608), etc. Modifications to modify viral loci include deletions, mutations and / or substitutions of one or more nucleotides (whether adjacent or not) within the viral genes or their regulatory elements. Modifications can be carried out by several methods known to the skilled artisan using conventional recombinant techniques.

[0153] More preferably, such oncolytic vaccinia viruses are modified by altering the gene encoding thymidine kinase (locus J2R). The thymidine kinase (TK) enzyme is involved in the synthesis of deoxyribonucleotides. TK is required for viral replication in normal cells, since these cells generally have low nucleotide concentrations, whereas it is not required in dividing cells, which contain high nucleotide concentrations.

[0154] Alternatively, or in combination, such oncolytic vaccinia viruses are modified by altering at least one or both genes encoding ribonucleotide reductase (RR). In the natural context, this enzyme catalyzes the reduction of ribonucleotides to deoxyribonucleotides, which represents a key step in DNA biosynthesis. The viral enzyme is similar in subunit structure to the mammalian enzyme and is composed of two heterologous subunits designated R1 and R2, encoded by the I4L and F4L loci, respectively. In the context of the present invention, either or both of the I4L gene (encoding the R1 large subunit) or the F4L gene (encoding the R2 small subunit) can be inactivated (e.g., as described in WO 2009 / 065546 and Foloppe et al., 2008, Gene Ther., 15:1361-71). The sequences of the J2R, I4L and F4L genes and their locations in the genomes of various poxviruses are available in public databases.

[0155] Thus, in some embodiments, the oncolytic virus is defective in thymidine kinase (TK) and / or ribonucleotide reductase (RR) activity, hi some embodiments, the oncolytic virus is a vaccinia virus defective in thymidine kinase (TK) and / or ribonucleotide reductase (RR) activity.

[0156] In some embodiments, the oncolytic virus comprises a nucleotide sequence encoding a first antibody molecule as defined herein.

[0157] In some embodiments, such oncolytic viruses comprise a nucleotide sequence encoding an amino acid sequence having at least 80% identity to a sequence shown in Table 2 above. In some embodiments, such oncolytic viruses comprise an amino acid sequence having at least 85% identity to a sequence shown in Table 2 above. In some embodiments, such oncolytic viruses comprise an amino acid sequence having at least 90% identity to a sequence shown in Table 2 above. In some embodiments, such oncolytic viruses comprise an amino acid sequence having at least 95% identity to a sequence shown in Table 2 above.

[0158] In some embodiments, such oncolytic viruses comprise a nucleotide sequence encoding SEQ ID NO:20 and SEQ ID NO:21. In some embodiments, such oncolytic viruses comprise a nucleotide sequence encoding SEQ ID NO:27 and SEQ ID NO:28. In some embodiments, such oncolytic viruses comprise a nucleotide sequence encoding SEQ ID NO:33 and SEQ ID NO:34. In some embodiments, such oncolytic viruses comprise a nucleotide sequence encoding SEQ ID NO:41 and SEQ ID NO:42.

[0159] In some embodiments, such oncolytic viruses comprise a nucleotide sequence having at least 80% identity to a sequence shown in Table 4 above. In some embodiments, such oncolytic viruses comprise a nucleotide sequence having at least 85% identity to a sequence shown in Table 4 above. In some embodiments, such oncolytic viruses comprise a nucleotide sequence having at least 90% identity to a sequence shown in Table 4 above. In some embodiments, such oncolytic viruses comprise a nucleotide sequence having at least 95% identity to a sequence shown in Table 4 above.

[0160] In some embodiments, the nucleotide sequence comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 45-52. In some embodiments, such oncolytic viruses comprise SEQ ID NOs: 45 and 46. In some embodiments, such oncolytic viruses comprise SEQ ID NOs: 47 and 48. In some embodiments, such oncolytic viruses comprise SEQ ID NOs: 49 and 50. In some embodiments, such oncolytic viruses comprise SEQ ID NOs: 51 and 52.

[0161] Some oncolytic viruses have the ability to accept DNA insertions large enough to accommodate the integration of full-length human antibody sequences. Attenuated vaccinia virus and herpes simplex virus are examples of therapeutic oncolytic viruses, whose genomes are large enough to allow the integration of full-length IgG antibody sequences (Chan and McFadden 2014, Bommareddy, Shettigar et al. 2018). Full-length IgG antibodies have been successfully integrated into oncolytic vaccinia viruses, resulting in the expression and extracellular release (production) of full-length IgG antibodies upon infection of virus-susceptible host cells, e.g., cancer cells (Kleinpeter, et al. 2016). Adenoviruses can also be engineered to encode full-length IgG antibodies that are functionally produced and secreted upon cell infection (Marino, et al. 2017).

[0162] In a preferred embodiment, such an oncolytic virus is a poxvirus (e.g., a vaccinia virus) that is defective in TK activity (resulting from an alteration of the J2R locus) or in both TK and RR activity (resulting from an alteration of both the J2R locus and at least one of the I4L and / or F4L loci encoding RR), and that comprises (a) a nucleotide sequence encoding SEQ ID NO:20 and No.21, or (b) a nucleotide sequence encoding SEQ ID NO:27 and No.28, or (c) a nucleotide sequence encoding SEQ ID NO:33 and No.34, or (d) a nucleotide sequence encoding SEQ ID NO:41 and No.42.

[0163] In some embodiments, TK and RR activity may be disrupted by introducing a nucleotide sequence encoding a first antibody molecule into the relevant locus (i.e., the J2R, I4L, and / or F4L locus). In some preferred embodiments, the virus comprises a nucleotide sequence encoding a heavy chain of a first antibody molecule inserted into the viral J2R locus and / or comprises a nucleotide sequence encoding a light chain of a first antibody molecule inserted into the viral I4L locus.

[0164] Where appropriate, it may be advantageous for the nucleotide sequence inserted into the oncolytic virus described herein to include additional regulatory elements to facilitate expression, transport and biological activity. For example, a signal peptide may be included to facilitate secretion outside the producer cell (e.g., an infected cell). The signal peptide is usually inserted at the N-terminus of the encoded polypeptide immediately following the Met initiator. The choice of signal peptide is broad and available to the skilled artisan. For example, a signal peptide derived from another immunoglobulin (e.g., heavy chain IgG) can be used in the context of the present invention to secrete the anti-CTLA4 antibody described herein outside the producer cell. For illustration, reference may be made to SEQ ID NO: 53 and SEQ ID NO: 54, which comprise the light and heavy chains of the 4-E03 antibody described herein with an IgG-derived peptide signal.

[0165] A particularly preferred oncolytic virus is a vaccinia virus (e.g., the Copenhagen strain) that is defective in both TK and RR activity (due to alterations in both the J2R and I4L loci) and comprises a nucleotide sequence encoding SEQ ID NO:20 and SEQ ID NO:21 or SEQ ID NO:53 and SEQ ID NO:54.

[0166] In some embodiments, such oncolytic viruses can further comprise additional nucleotide sequences of therapeutic interest, such as nucleotide sequences encoding immunomodulatory polypeptides (i.e., polypeptides involved in stimulating an immune response, either directly or indirectly). Representative examples of suitable immunomodulatory polypeptides include, but are not limited to, cytokines and chemokines, with granulocyte macrophage colony stimulating factor (GM-CSF), particularly human, non-human primate or mouse GM-CSF being specifically preferred.

[0167] Thus, in some embodiments, the oncolytic virus (e.g., vaccinia virus) capable of expressing a first antibody molecule that specifically binds to CTLA-4 further comprises a nucleotide sequence encoding GM-CSF, preferably human GM-CSF (e.g., having SEQ ID NO: 55 or SEQ ID NO: 56) or mouse GM-CSF (e.g., having SEQ ID NO: 57 or SEQ ID NO: 58).

[0168] Additional nucleotide sequences can be readily obtained by standard molecular biology techniques (e.g., PCR amplification, cDNA cloning, chemical synthesis) using sequence data accessible in the art and the information provided herein. Particularly preferred oncolytic viruses are vaccinia viruses (e.g., Copenhagen strains) that are defective in both TK and RR activity (due to alterations in both the J2R and I4L loci) and that include nucleotide sequences encoding SEQ ID NO:20 and SEQ ID NO:21 or SEQ ID NO:53 and SEQ ID NO:54, as well as nucleotide sequences encoding GM-CSF, with human GM-CSF (e.g., having SEQ ID NO:55 or SEQ ID NO:56) or mouse GM-CSF (e.g., having SEQ ID NO:57 or SEQ ID NO:58) being particularly preferred.

[0169] The following table provides the sequences of GM-CSF referred to herein.

[0170] [Table 5]

[0171] Furthermore, the nucleotide sequence inserted into such oncolytic viruses can be optimized to provide high levels of expression in a particular host cell or subject by modifying one or more codons. In addition to optimizing codon usage, various modifications can also be envisaged to prevent clustering of rare non-optimal codons present in concentrated regions and / or to suppress or modify "negative" sequence elements that are expected to adversely affect expression levels. Such negative sequence elements include, but are not limited to, regions with very high (>80%) or very low (<30%) GC content, AT-rich or GC-rich sequence stretches, unstable direct or inverted repeats, RA secondary structures, and / or internal potential regulatory elements such as internal TATA boxes, Kai sites, ribosome entry sites, and / or splicing donor / acceptor sites.

[0172] In some embodiments, the nucleotide sequences are placed under the control of suitable regulatory elements for their proper expression in a host cell or subject. As used herein, the term "regulatory element" refers to any element that allows, contributes to, or regulates the expression of a coding nucleotide sequence in a given host cell or subject, including its replication, duplication, transcription, splicing, translation, stability, and / or transport in and out of the expression cell. It will be understood by those skilled in the art that the choice of regulatory element may depend on factors such as the nucleotide sequence itself, the virus into which it is inserted, the host cell or subject, the desired expression level, etc. The promoter is of special importance. In the context of the present invention, it may be a constitutively directed expression of the nucleotide sequence that is controlled in many types of host cells or is specific for a certain host cell, or is regulated in response to a specific event or exogenous factor (e.g., by temperature, nutrient additives, hormones, etc.) or according to the stage of the viral cycle (e.g., late or early). Promoters adapted for viral-mediated expression are known in the art.

[0173] Representative examples of oncolytic poxvirus expression include, but are not limited to, vaccinia p7.5K, pH5.R, p11K7.5, TK, p28, p11, pB2R, pA35R, K1L, and pSE / L promoters (Erbs et al., 2008, Cancer Gene Ther. 15(1):18-28; Orubu et al. 2012, PloS One 7:e40167), early / late chimeric promoters, and synthetic promoters (Chakrabarti et al., 1997, Biotechniques 23:1094-7; Hammond et al, 1997, J. Virol Methods 66:135-8; and Kumar and Boyle, 1990, Virology 179:151-8). In a preferred embodiment, the nucleotide sequences of the light and heavy chains of the antibodies described herein are each placed under the control of a promoter with the same transcriptional strength, preferably the same promoter (e.g., p7.5K as set forth in SEQ ID NO:59 or pH5.R as set forth in SEQ ID NO:60) to obtain similar levels of expression for both chains and thus optimal assembly of the antibody as a heterotetrameric protein (i.e., avoiding excessive unassociated chains). Additional nucleotide sequences (e.g., encoding GM-CSF) can be placed under a different promoter (e.g., pSE / L as set forth in SEQ ID NO:61).

[0174] The table below provides the sequences of the above promoters.

[0175] [Table 6]

[0176] The insertion of the nucleotide sequence (possibly equipped with suitable regulatory elements) into the genome of such oncolytic viruses is carried out by conventional means, using suitable restriction enzymes or preferably by homologous recombination. The nucleotide sequence can be inserted independently at any position of the viral genome. Various insertion sites can be considered, for example, non-essential viral genes, intergenic regions, or non-coding parts of the genome of such oncolytic viruses. The J2R locus and / or the I4L locus are particularly suitable for oncolytic viruses that are poxviruses (e.g. oncolytic vaccinia viruses). After insertion of the nucleotide sequence into the viral genome, the viral locus at the insertion site can be at least partially deleted. In one embodiment, this deletion or partial deletion can result in suppressed expression of the viral gene product encoded by the completely or partially deleted locus, resulting in a virus deficient in said viral function. A particularly preferred oncolytic virus is a TK and / or RR deficient vaccinia virus, comprising a cassette encoding the heavy chain inserted into the J2R locus and a cassette encoding the light chain inserted into the I4L locus. The cassette encoding the additional GM-CSF-encoding nucleotide sequence can be inserted at another location in the viral genome or at the J2R or I4L locus, with insertion at the I4L locus being preferred.

[0177] The present invention also provides methods for producing such oncolytic viruses, in particular oncolytic poxviruses, as described herein, in suitable host cells (producer cells). In some embodiments, such methods comprise one or more steps of homologous recombination between the viral genome and a transfer plasmid comprising nucleotide sequences (possibly with regulatory elements) to be inserted adjacent to the 5' and 3' viral sequences present upstream and downstream, respectively, of the insertion site. The transfer plasmid can be produced by routine techniques (e.g., by transfection) and introduced into the host cell. The viral genome can be introduced into the host cell by infection. The size of each flanking viral sequence can vary from at least 100 bp up to 1500 bp on each side of the nucleotide sequence (preferably 200-550 bp, more preferably 250-500 bp). The homologous recombination allowing the production of such oncolytic viruses is preferably carried out in cultured cell lines (e.g., HeLa, Vero) or in chicken embryonic fibroblast (CEF) cells obtained from embryonated eggs.

[0178] In some embodiments, identification of oncolytic viruses incorporating anti-CTLA4 encoding nucleotide sequences and possibly additional nucleotide sequences (eg, GM-CSF) can be facilitated by the use of selection and / or detectable genes.

[0179] In a preferred embodiment, the transfer plasmid further comprises a selection marker, of which the GPT gene (encoding guanine phosphoribosyltransferase) that allows growth in selective medium (e.g. in the presence of mycophenolic acid, xanthine and hypoxanthine) is particularly preferred, or a detectable gene that encodes a detectable gene product such as GFP, e-GFP or mCherry. Furthermore, the use of an endonuclease that can provide a double-strand break in the selection or detectable gene may also be considered. The endonuclease may be in the form of a protein or may be expressed by an expression vector.

[0180] Once generated, such oncolytic viruses can be amplified in suitable host cells using conventional techniques, including culturing the transfected or infected host cells under appropriate conditions to allow for the production and recovery of infectious particles.

[0181] The present invention also relates to a method for producing an oncolytic virus as described herein, preferably comprising the steps of a) preparing a producer cell line, b) transfecting or infecting the prepared producer cell line with an oncolytic virus, c) culturing the transfected or infected producer cell line under suitable conditions to allow production of virus, d) recovering the produced virus from the culture of the producer cell line, and optionally e) purifying the recovered virus.

[0182] In some embodiments, producer cells are selected from the group consisting of mammalian (e.g., human or non-human) cells, such as HeLa cells (e.g., ATCC-CRM-CCL-2™ or ATCC-CCL-2.2™), HER96, PER-C6 (Fallaux et al., 1998, Human Gene Ther. 9:1909-17), hamster cell lines such as BHK-21 (ATCC CCL-10), and avian cells such as those described in WO 2005 / 042728, WO 2006 / 108846, WO 2008 / 129058, WO 2010 / 130756, WO 2012 / 001075, and primary chicken embryo fibroblasts (CEF) prepared from chicken embryos obtained from fertilized eggs. The producer cells are preferably cultured in a suitable medium, which may or may not be supplemented with serum and / or suitable growth factors as required (e.g. a chemically defined medium, preferably free of animal or human derived products). A suitable medium may be readily selected by the skilled artisan depending on the producer cells. Such media are commercially available. The producer cells are preferably cultured at a temperature of +30°C to +38°C (more preferably about +37°C) for 1 to 8 days prior to infection. If necessary, several 1 to 8 day passagings can be performed to increase the total number of cells.

[0183] In step b), producer cells are infected with the oncolytic virus under appropriate conditions using an appropriate multiplicity of infection (MOI) to allow productive infection of the producer cells. For illustration purposes, an appropriate MOI is 10 -3 Infection steps are typically performed in a culture medium that may be the same or different from the medium used to culture the producer cells.

[0184] Then, in step c), the infected producer cells are cultured under suitable conditions well known to those skilled in the art until progeny viral particles are produced. The cultivation of the infected producer cells is also preferably carried out for 1 to 5 days at a temperature between +32°C and +37°C in a medium which may be the same or different from the medium / mediums used for the producer cell cultivation and / or infection step.

[0185] In step d), the viral particles produced in step c) are harvested from the culture supernatant and / or from the producer cells. Recovery from the producer cells may require a step that allows disruption of the producer cell membrane to allow release of the virus. Disruption of the producer cell membrane can be induced by various techniques well known to those skilled in the art, including but not limited to freeze / thaw, hypotonic lysis, sonication, microfluidization, high shear (also called high speed) homogenization or high pressure homogenization.

[0186] The oncolytic virus recovered can be at least partially purified before being distributed in doses and used as described herein. A large number of purification steps and methods are available in the art, including clarification, enzyme treatment (e.g., endonuclease, protease, etc.), chromatography, and filtration steps. Suitable methods are described in the art (e.g., WO 2007 / 147528, WO 2008 / 138533, WO 2009 / 100521, WO 2010 / 130753, WO 2013 / 022764).

[0187] In one embodiment, the present invention also provides a cell infected with an oncolytic virus capable of expressing a first antibody molecule as described herein.

[0188] The combination of an oncolytic virus capable of expressing a first antibody molecule that specifically binds to CTLA-4 and a second antibody molecule that specifically binds to PD-1 and / or PD-1 is for use in treating cancer in a patient, wherein the cancer comprises or consists of a cold tumor.

[0189] The subject may be a mammal or a non-mammal. Preferably, the mammalian subject is a human or a non-mammal, such as a horse, cow, sheep, pig, camel, dog, or roe. Most preferably, the mammalian subject is a human.

[0190] Patients may exhibit signs or symptoms that suggest they have cancer. "Exhibiting" includes that the subject exhibits cancer symptoms and / or cancer diagnostic markers and / or that the cancer symptoms and / or cancer diagnostic markers can be measured and / or assessed and / or quantified.

[0191] It will be readily apparent to one skilled in the art of medicine what the cancer symptoms and cancer diagnostic markers are, and how to measure and / or assess and / or quantify whether there is a reduction or increase in the severity of the cancer symptoms or whether there is a reduction or increase in the cancer diagnostic markers, and how those cancer symptoms and / or cancer diagnostic markers can be used to form a prognosis for cancer.

[0192] Cancer treatments are often administered as a course of treatment, i.e., the therapeutic agent is administered over a period of time. The length of time of the course of treatment depends on a number of factors, including, among other things, the type of therapeutic agent administered, the type of cancer being treated, the severity of the cancer being treated, and the age and health of the subject.

[0193] "Currently undergoing treatment" includes a subject currently undergoing a course of treatment and / or receiving a therapeutic agent and / or receiving a series of therapeutic agents.

[0194] As described above, the combination of an oncolytic virus capable of expressing a first antibody molecule that specifically binds to CTLA-4 and a second antibody molecule that specifically binds to PD-1 and / or PD-L1 is particularly for use in treating cancer in a patient, wherein the cancer comprises or consists of a cold tumour.

[0195] Thus, in some embodiments, a cold tumor is treated with a first and a second antibody molecule.

[0196] Those skilled in the art will understand that determining whether a cold tumor has been "treated" involves the same kind of determination that is used for any other type of tumor. For example, those skilled in the art will look for signs such as tumor shrinkage (in both injected and non-injected tumors), which can be measured using CT scans, and / or progression-free survival, and / or overall survival. In other cases, this may be a more subjective effect, such as a reduction in the severity of symptoms reported by the subject. Measuring the therapeutic effect in a subject in response to administration of a therapeutic antibody is well known in the art.

[0197] "Cold tumors" are characterized by a proliferation of inflammatory immune cells, most notably T cells, especially CD8+ +Cold tumors refer to tumors that are poorly infiltrated by T cells. Cold tumors are highly clinically relevant, as tumor immune infiltration, especially CD8+ T cell infiltration, has been widely demonstrated to correlate with longer disease-free survival (DFS) and / or overall survival (OS) in cancers with different histological features and anatomical locations. This has been demonstrated in both primary and metastatic (Bruni et al., 2020) settings, including melanoma, most squamous cell carcinomas (SCC), large cell lung carcinoma, and several types of adenocarcinoma (Galon et al., 2006; Fridman et al., 2012; Fridman et al., 2017; Hu et al., 2018), and has been demonstrated to correlate with and predict responsiveness to ICB (Galon and Bruni, 2019).

[0198] For example, recently, CD8 + T cell density has been shown to correlate with response / progression to ICB with antibodies against CTLA-4 and PD-1 / PD-L1 in melanoma (Tumeh et al. 2014), renal cell carcinoma (McDermott, Huseni et al. 2018) and NSCLC (Thommen, Koelzer et al. 2018) in human solid tumor patients. Similarly, preclinical mouse tumor models differ quantitatively and qualitatively with respect to immune infiltration, with the B16 / C57BL6 model showing a high proportion of CD8 + It is well understood that B16 / C57BL6 tumors are particularly poor with respect to immune cell infiltration, including T (Mosely et al. 2017). Furthermore, similar to human "cold tumors," the B16 / C57BL6 model is particularly resistant to systemic ICB with anti-CTLA-4 and / or anti-PD-1 / L1, making it useful for helping to identify therapies that help overcome "cold tumor" resistance to systemic ICB.

[0199] Clinically relevant assays that quantify tumor cell, immune cell, or combined cellular levels of PD-L1 have been devised and are used in the clinic to help identify patients for treatment with anti-PD-1 / L1 ICB reagents, such as pembrolizumab (see KEYTRUDA® Section 2.1 Prescribing Information, which describes how to select patients for therapy, available at https: / / www.accessdata.fda.gov / drugsatfda_docs / label / 2021 / 125514s096lbl.pdf).

[0200] Recent data based on multicolor immunofluorescence suggest that in melanoma, the invasive margin is CD8 + T cell density as the best complete predictive parameter and tumor CD8 + identified T cell density as the second best predictor of response / progression to PD-1 blockade therapy (Tumeh et al., 2014). Furthermore, principal component analysis demonstrated that CD8 infiltration, PD-1, and PD-L1 significantly correlated with treatment outcome. These data support the conclusion that CD8 + These results support the idea that T cell density may be a useful method for identifying patients with cold tumors. Recently, CD3 + CD8 + An immune-based assay named “Immunoscore” has been developed to quantify T cell infiltration in situ ( Bruni et al., 2020 , Galon et al., 2006 , Lanzi et al., 2020 ).

[0201] Immunoscore is an immunohistochemistry and digital pathology-based scoring system that measures CD3 + and CD8 +The density of T cells is assessed. Briefly, two adjacent slides of formalin-fixed paraffin-embedded tumor blocks are stained with anti-CD3 and anti-CD8 antibodies in an automated stainer. The slides are then scanned and the digital images are used to quantify the density of the cells of interest using digital pathology software. The density is finally converted into an immunoscore ranging from low immunoscore (I0) to high immunoscore (I4).

[0202] An approach linking the Immunoscore to the concept of "hot and cold tumors" has been proposed by Galon and Bruni (Galon et al., 2019; Angell et al., 2020). Using this approach, tumors were classified into four categories based on T cell infiltration: hot immune tumors, altered and immunosuppressed immune tumors, altered and excluded immune tumors, and cold tumors.

[0203] The characteristics of these four types of tumors are detailed in Box 1 of Galon et al., 2019, which describes the tumor types according to the following characteristics: 1. Hot immune tumors (also referred to herein as hot tumors) -High T cell and cytotoxic T cell infiltration (high immunoscore) checkpoint activation (programmed cell death protein 1 (PD-1), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), T cell immunoglobulin mucin receptor 3 (TIM-3), and lymphocyte activation gene 3 (LAG-3)) or otherwise impaired T cell function (e.g., extracellular potassium-driven T cell inhibition) 2. Altered and immunosuppressed immune tumors - Insufficient but not absent T cell and cytotoxic T cell infiltration (intermediate immunoscore) - The presence of soluble inhibitory mediators (transforming growth factor-β (TGFβ), interleukin 10 (IL-10), and vascular endothelial growth factor (VEGF)) -Presence of immune suppressor cells (myeloid-derived suppressor cells and regulatory T cells) -Presence of T cell checkpoints (PD-1, CTLA-4, TIM-3, and LAG-3) 3. Altered and eliminated immune tumors - T cell accumulation at the tumor border (invasive margin) without T cell infiltration within the tumor bed (intermediate immunoscore) -Activation of oncogenic pathways -Epigenetic regulation and reprogramming of the tumor microenvironment - Abnormal tumor vasculature and / or stroma -Hypoxia 4. Cold immune tumors (also referred to herein as cold tumors) - Absence of T cells within the tumor or at the tumor edge (low immunoscore) -Failed T cell priming (low tumor mutational burden, poor antigen presentation, and inherent insusceptibility to T cell killing).

[0204] Thus, in some embodiments, a patient is considered to have a cold tumor if they have a tumor that fits the definition of an altered and immunosuppressed immune tumor, an altered and excluded immune tumor, or a cold immune tumor, as defined above.

[0205] It will be appreciated that each of the above types of tumors will have a different level of response to T cell checkpoint inhibition, with cold immune tumors having the lowest level of response (no response), followed by altered and eliminated immune tumors, and altered and immunosuppressed immune tumors (having suboptimal levels of response), respectively.

[0206] The definition used herein of "cold tumors", i.e. tumors with poor infiltration by immune cells (e.g., CD3+ and CD8+ T cells), encompasses both classical cold immune tumors, altered and excluded tumors, and altered and immunosuppressed tumors, since all of these categories are defined by poor (altered immunity) or absent (excluded and cold immunity) T cell infiltration. This is equivalent to cancer patients with immunoscores I, II, or III, but not IV (the latter being T cell inflammatory tumors). Thus, the present invention is applicable to patients with immunoscores I, II, and III, but not IV.

[0207] Thus, in some embodiments, a patient is considered to have a cold tumor if the tumor has an Immunoscore of I, or II, or III.

[0208] Those skilled in the art will appreciate that other assays and techniques besides immunoscore or T cell density may be useful in identifying particularly resistant "cold type" cancers.

[0209] In some embodiments, cold tumors have anergized (or anergic) lymphocytes, meaning that the lymphocytes are unable to respond to antigens. Methods for determining anergic lymphocytes are well known in the art.

[0210] In some other embodiments, cold tumors have low levels of CD3 positive cells. For example, cold tumors may have less than 10% CD3 positive cells (as a percentage of total cells in the tumor), i.e., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or no CD3 positive cells. Methods for measuring the percentage of CD3 positive cells are known in the art.

[0211] As mentioned above, the cold tumors described herein are typically tumors that are poorly targeted by the immune system. In some alternative or additional embodiments, such cold tumors can also be classified into the following types: - Immune desert tumors, i.e. there is a complete lack of immune response in the tumor due to the lack of tumor-infiltrating T cells. -Immune excluded tumors, i.e., responding T cells are produced but are unable to penetrate the tumor and mount a response against it; T cells may be present in the tumor periphery. - Tumors with poor immune infiltration, i.e. reduced levels of immune cell (T-cell) penetration into the tumor microenvironment.

[0212] As used herein, "cold tumor" also includes all of immune desert tumors, immune excluded tumors, and tumors with poor immune infiltration. These definitions may be used instead of or in addition to the definitions of cold tumors (as altered and immunosuppressed tumors, altered and excluded tumors, or cold immune tumors) above. In some embodiments, an altered and immunosuppressed immune tumor corresponds to a tumor with poor immune infiltration. In some embodiments, an altered and excluded immune tumor corresponds to an immune excluded tumor. In some embodiments, a cold immune tumor corresponds to an immune desert tumor.

[0213] "Comprises or consists of cold tumor" refers to a cancer that can be composed of cold tumors and non-cold tumors. For example, this can occur when an original cancer (which can be a cold tumor) metastasizes and forms a secondary tumor that is not a cold tumor. A patient herein may have multiple tumors, only one of which needs to meet the requirements of a cold tumor for the present invention to be beneficial. In other embodiments, a patient may have a single tumor that is considered a cold tumor.

[0214] Cancers that may be classified into these "cold tumor" subtypes include, but are not limited to, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, hepatocellular carcinoma, lung cancer, bladder cancer, renal cancer, gastric cancer, cervical cancer, Merkel cell carcinoma, ovarian cancer, head and neck cancer, mesothelioma, or breast cancer.

[0215] Any of the above cancers are well known and the symptoms and cancer diagnostic markers are well described, as are the therapeutic agents used to treat those cancers. Thus, the symptoms, cancer diagnostic markers and therapeutic agents used to treat the above types of cancers will be known to those skilled in the art of medicine.

[0216] The clinical definition of the diagnosis, prognosis and progression of most cancers is by a certain classification, known as staging. These staging systems work by collating several different cancer diagnostic markers and cancer symptoms to provide an overview of the diagnosis and / or prognosis and / or progression of cancer. Those skilled in the art of oncology will know how to use the staging systems to assess the diagnosis and / or prognosis and / or progression of cancer, and which cancer diagnostic markers and cancer symptoms to use for this purpose.

[0217] "Cancer staging" includes the Rai staging system, including stage 0, stage I, stage II, stage III, and stage IV, and / or the Binet staging system, including stage A, stage B, and stage C, and / or the Ann Arbour staging system, including stage I, stage II, stage III, and stage IV.

[0218] It is known that cancer can cause abnormalities in cell morphology. These abnormalities often occur in a reproducible manner in a particular cancer, which means that examination of these changes in morphology (otherwise known as histological examination) can be used to diagnose or prognose cancer. Techniques for visualizing and preparing samples for visualization to examine cell morphology are known in the art, for example, optical microscopy or confocal microscopy.

[0219] "Histological examination" includes the presence of small mature lymphocytes, and / or the presence of small mature lymphocytes with narrow cytoplasmic borders, the presence of small mature lymphocytes with dense nuclei lacking discernible nucleoli, and / or the presence of small mature lymphocytes with narrow cytoplasmic borders and dense nuclei lacking discernible nucleoli, and / or the presence of atypical cells, and / or cleaved cells, and / or prolymphocytes.

[0220] It is known that cancer is the result of mutations in the DNA of cells, which can lead to cells evading cell death or uncontrolled proliferation. Therefore, examining these mutations (also known as cytogenetic examination) can be a useful tool for evaluating the diagnosis and / or prognosis of cancer. An example of this is the deletion of chromosomal position 13q14.1, which is characteristic of chronic lymphocytic leukemia. Techniques for testing mutations in cells are well known in the art, such as fluorescence in situ hybridization (FISH).

[0221] "Cytogenetic testing" includes testing of DNA, specifically chromosomes, in cells. Cytogenetic testing can be used to identify DNA alterations that may be associated with the presence of refractory and / or recurrent cancer. Such include deletions in the long arm of chromosome 13, and / or deletions at chromosomal location 13q14.1, 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 deletions of 11q, and / or deletions in the long arm of chromosome 6, and / or deletions of 6q, and / or deletions in the short arm of chromosome 17, and / or deletions of 17p, and / or t(1 1:14) translocation, and / or (q13:q32 translocation, and / or antigen gene receptor rearrangement, and / or BCL2 rearrangement, and / or BCL6 rearrangement, and / or t(14:18) translocation, and / or t(11:14) translocation, and / or (q13:q32 translocation, and / or (3:v) translocation, and / or (8:14) translocation, and / or (8:v) translocation, and / or t(11:14) and (q13:q32) translocations.

[0222] Subjects with cancer are known to exhibit certain physical symptoms, which are often the result of the burden that cancer places on the body. These symptoms often recur with the same cancer, and may be characteristic of the diagnosis and / or prognosis and / or progression of the disease. Those skilled in the art of medicine will understand which physical symptoms are associated with which cancers, and how the evaluation of these body systems can be correlated with the diagnosis and / or prognosis and / or progression of the disease. "Physical symptoms" include hepatomegaly and / or splenomegaly.

[0223] Patients with "cold" tumors are less likely to respond to conventional immune checkpoint blockade therapy (e.g., administration of anti-CTLA-4 or anti-PD-1 antibodies). Thus, in some embodiments, patients with cold tumors are resistant to immune checkpoint blockade therapy.

[0224] Given these observations, resistance to ICB constitutes a significant unmet medical need, and drugs that can help overcome resistance have great therapeutic promise. Thus, an advantage of the present invention is that an oncolytic virus capable of expressing a first antibody that specifically binds to CTLA-4, in combination with an antibody specific for PD-1 and / or PD-L1, provides a synergistic effect that can overcome the resistance, targeting cold tumors that previously could not be treated using immune checkpoint inhibitors.

[0225] The present invention also encompasses pharmaceutical compositions comprising a combination of an oncolytic virus capable of expressing a first antibody molecule that specifically binds to CTLA-4 and a second antibody molecule that specifically binds to PD-1 and / or PD-L1 in combination with a pharma- ceutically acceptable carrier and / or diluent and / or adjuvant. Such pharma- ceutically acceptable carriers, diluents, and adjuvants are known in the art.

[0226] The antibody molecules, nucleotide sequences, plasmids, viruses, cells and / or pharmaceutical compositions described herein may be suitable for parenteral administration, including aqueous and / or non-aqueous sterile injection solutions which may contain antioxidants, and / or buffers, and / or bacteriostats, and / or solutes which render the formulation isotonic with the blood of the intended recipient; and / or aqueous and / or non-aqueous sterile suspensions which may contain suspending agents and / or thickening agents. The antibody molecules, nucleotide sequences, plasmids, cells and / or pharmaceutical compositions described herein may be presented in single-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (i.e. lyophilized) condition requiring only the addition of a sterile liquid carrier, for example water for injections, immediately prior to use.

[0227] Extemporaneous injection solutions and suspensions may be prepared from sterile powders, and / or granules and / or tablets of the kind previously described.

[0228] For parenteral administration to human patients, daily dosage levels of anti-PD-1 and / or anti-PD-L1 antibody molecules will typically be 1 mg / kg to 20 mg / kg of patient body weight, or in some cases up to 100 mg / kg, administered in single or divided doses. In some preferred embodiments, the dosage is 10 mg / kg. Lower doses may be used under special circumstances, for example in combination with chronic administration. In any event, the physician will determine the actual dosage that will be most suitable for any individual patient, which will vary with the age, weight, and response of the particular patient. The dosages described above are exemplary of the average case. There can, of course, be individual cases in which higher or lower dosage ranges are merited, and these are within the scope of the invention.

[0229] Typically, pharmaceutical compositions (or medicaments) described herein comprising the antibody molecules will contain the anti-PD-1 and / or anti-PD-L1 antibody molecules at a concentration of about 2 mg / ml to 150 mg / ml or about 2 mg / ml to 200 mg / ml, hi some embodiments, the pharmaceutical compositions will contain the anti-PD-1 and / or anti-PD-L1 antibody molecules at a concentration of 10 mg / ml or 25 mg / ml.

[0230] In some embodiments, when the anti-PD-1 antibody is pembrolizumab, the antibody is used at a dose of about 25 mg / ml, hi some other embodiments, pembrolizumab is used at a dose of 200 mg (iv) every 3 weeks, or at a dose of 400 mg (iv) every 6 weeks.

[0231] In some embodiments, when the anti-PD-1 antibody is nivolumab, the antibody is used at a dose of about 10 mg / ml. In some embodiments, nivolumab is used at a dose of 240 mg (iv) every 2 weeks, or at a dose of 480 mg (iv) every 4 weeks. In some embodiments, nivolumab may be used in combination with the anti-CTLA-4 antibody ipilimumab, where nivolumab is used at a dose of 1 mg / kg every 3 weeks for up to 4 doses, or at a dose of 3 mg / kg every 2 or 3 weeks.

[0232] In some embodiments, where the anti-PD-L1 antibody is atezolizumab, the antibody is used at a dose of about 60 mg / ml, hi some other embodiments, atezolizumab is used at a dose of 840 mg (iv) every 2 weeks, or at a dose of 1200 mg (iv) every 3 weeks, or at a dose of 1680 mg (iv) every 4 weeks.

[0233] One of skill in the art will appreciate that any of the anti-PD-1 or anti-PD-L1 antibodies described herein may be used in any dose or dosing regimen described in their prescribing information.

[0234] Typically, a pharmaceutical composition (or medicament) is administered in a concentration of about 10% depending on the virus and the quantitative technique. 3 ~10 12 It contains the oncolytic virus described herein at a concentration of vp (viral particle), iu (infectious unit) or pfu (plaque forming unit). The amount of pfu present in a sample can be determined by counting the number of plaques after infection of permissive cells (e.g., CEF or Vero cells) to obtain a titer of plaque forming units (pfu), the amount of vp can be determined by measuring the absorbance at 260 nm, and the amount of iu can be determined by quantitative immunofluorescence, e.g., using an antiviral antibody. As a general guidance, individual doses suitable for pharmaceutical compositions containing oncolytic poxviruses are about 10 3 ~about 10 10 pfu, advantageously about 10 3 pfu~about 10 9 pfu, preferably about 10 4 pfu~about 10 7 pfu, more preferably about 10 6 pfu~about 10 7 pfu range.

[0235] In some embodiments, when the subject is a mouse, the optimal dose of the oncolytic virus is about 10 6 ~10 7In some embodiments, when the subject is a human, the optimal dose of the oncolytic virus is about 10 6 ~10 9 It is pfu.

[0236] Generally, in humans, oral or parenteral administration of the antibody molecules, nucleotide sequences, plasmids, viruses, cells, and / or pharmaceutical compositions described herein is the preferred route and is the most convenient. For veterinary use, the antibody molecules, nucleotide sequences, plasmids, viruses, cells, and / or pharmaceutical compositions described herein are administered as an appropriately acceptable pharmaceutical formulation in accordance with normal veterinary practice, with the veterinarian determining the dosing regime and route of administration that will be most appropriate for a particular animal. Thus, the present invention provides pharmaceutical formulations comprising an amount of the antibody molecules, nucleotide sequences, plasmids, viruses, and / or cells of the present invention effective to treat various conditions (discussed above and further below).

[0237] Preferably, the antibody molecules, nucleotide sequences, plasmids, viruses, cells, and / or pharmaceutical compositions described herein are adapted for delivery by a route selected from intravenous, intratumoral, intramuscular, subcutaneous. Administration can be in the form of a single injection or multiple repeated injections (e.g., at the same or different doses, by the same or different routes, at the same or different administration sites). For illustrative purposes, about 10 4 , 5×10 4 , 10 5 , 5×10 5 , 10 6 , 5×10 6 , 10 7 , 5×10 7 , 10 8 , 5×10 8 , 10 9 , 5×10 9 , or 10 10 Individual doses containing pfu of an oncolytic poxvirus (eg, a TK- and RR-deficient vaccinia virus as described herein) are particularly suitable for intratumoral administration.

[0238] The invention also includes antibody molecules, nucleotide sequences, plasmids, viruses, cells and / or pharmaceutical compositions described herein that comprise pharma- ceutically acceptable acid or base addition salts of the polypeptide binding moieties of the invention. The acids used to prepare pharma- ceutically acceptable acid addition salts of the aforementioned base compounds useful in the invention are, inter alia, those that form non-toxic acid addition salts, i.e., salts containing pharma- ceutically acceptable anions, such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate, [i.e., 1,1'-methylene-bis-(2-hydroxy-3 naphthoate)] salts. Pharmaceutically acceptable base addition salts may also be used to produce pharma-ceutically acceptable salt forms of the agents according to the invention. Chemical bases that may be used as reagents to prepare pharma-ceutically acceptable base salts of the agents which are acidic in nature are those which form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to, those derived from such pharma-ceutically acceptable cations, such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium), ammonium or water-soluble amine addition salts, e.g., N-methylglucamine-(meglumine), and lower alkanolammonium, as well as other pharma-ceutically acceptable organic amine base salts. The antibody molecules, nucleotide sequences, plasmids, viruses and / or cells described herein may be lyophilized for storage and reconstituted in a suitable carrier prior to use. Any suitable lyophilization method (e.g., spray drying, cake drying) and / or reconstitution technique may be used. One of skill in the art will appreciate that lyophilization and reconstitution may lead to varying degrees of antibody activity loss (e.g., in conventional immunoglobulins, IgM antibodies tend to experience greater activity loss than IgG antibodies) and that usage levels may need to be adjusted upward to compensate.In one embodiment, a lyophilized (freeze-dried) polypeptide binding moiety loses about 20% or less, or about 25% or less, or about 30% or less, or about 35% or less, or about 40% or less, or about 45% or less, or about 50% or less of its activity (before lyophilization) when rehydrated.

[0239] In some embodiments, the virus composition is suitably buffered at a physiological or slightly basic pH (e.g., about pH 7 to about pH 9, with a pH between 7 and 8.5, more particularly close to 8, being particularly preferred). It may also be beneficial to include a monovalent salt in the virus composition to ensure appropriate osmolality. The monovalent salt may in particular be selected from NaCl and KCl, preferably the monovalent salt is NaCl, preferably at a concentration of 10 to 500 mM (e.g., 50 mM). A suitable virus composition comprises saccharose 50 g / L, NaCl 50 mM, Tris-HCl 10 mM and sodium glutamate 10 mM, pH 8. The composition may also be formulated to include a cryoprotectant to protect the oncolytic virus at low storage temperatures. Suitable cryoprotectants include, but are not limited to, sucrose (or saccharose), trehalose, maltose, lactose, mannitol, sorbitol, and glycerol, preferably at a concentration of 0.5-20% (also referred to as w / v, weight in g / volume in L), as well as high molecular weight polymers such as dextran or polyvinylpyrrolidone (PVP).

[0240] It will be understood that a composition comprising an oncolytic virus capable of expressing a first antibody molecule, and a composition comprising a second antibody molecule described herein, may be administered simultaneously (i.e., simultaneously) as a single composition.

[0241] Alternatively, these compositions can be administered separately, either at similar times or at different times (e.g., one day or one week apart). For example, the oncolytic virus can be administered before the second antibody molecule. In another example, the second antibody molecule can be administered before the oncolytic virus. Such sequential administration can be achieved by temporal separation of the oncolytic virus and the second antibody molecule. Alternatively, or in combination with the first option, sequential administration can also be achieved by spatial separation of the oncolytic virus and the second antibody molecule, by administering the oncolytic virus capable of expressing an anti-CTLA-4 antibody in a manner such as intratumoral administration, so that it reaches the cancer before the second antibody molecule, and then administering the second antibody molecule in a manner such as systemic administration, so that it reaches the cancer after the oncolytic virus.

[0242] The oncolytic virus of the invention and the second antibody molecule can be administered according to an established therapeutic regimen for each component, as described above, meaning that the administration of each component can be simultaneous or at different times relative to each other.

[0243] In some embodiments, the administration of an oncolytic virus capable of expressing a first antibody molecule and a second antibody molecule described herein may be repeated, for example, the administration may be repeated two, three, four, five times, or as many times as necessary to have a therapeutic effect.

[0244] Preferred, non-limiting examples embodying certain aspects of the present invention will now be described with reference to the following figures and examples. [Brief description of the drawings]

[0245] [Figure 1-1]Biochemical and functional characterization of a novel Treg-depleting αCTLA-4 mAb. (A) Antibody-mediated survival and (B) TIL modulation in CT26 tumor-bearing BALB / c mice. Animals with established tumors received four injections (10 mg / kg) of antibodies with the indicated Treg-associated specificities or a control mIgG2a antibody (n=5-15). [Figure 1-2] Biochemical and functional characterization of novel Treg-depleting αCTLA-4 mAbs. (C) CTLA-4 specific mAbs induce ADCC of in vitro activated CD4+ T cells. Lysed target T cells were identified by FACS. Figures show mean ± SD (n=4-8) **p<0.01 by Student's t-test. (D) Anti-CTLA-4 (IgG1) mAbs mediate in vivo Treg depletion in huPBMC mice. Clone 4-E03 shows enhanced depletion of human Treg cells (upper panel) but not CD8+ T cells (lower panel) compared to ipilimumab. Each dot represents one mouse. Graphs show average data from two experiments. *p<0.05 by one-way ANOVA. [Figure 1-3] Biochemical and functional characterization of novel Treg-depleting αCTLA-4 mAbs. (E) 4-E03 hIgG1 and ipilimumab binding to human, mouse, and cynomolgus CTLA-4 and CD28 by ELISA. (F) 4-E03 IgG1 binding to in vitro activated CTLA-4 expressing human T cells pre-blocked with rhCTLA-4-Fc protein (black line), (G) 4-E03 and 2-C06 block CD80 and CD86 binding to CTLA-4 by ELISA. (H) In vitro functional ligand blocking. Graph shows IL-2 in supernatants after treatment of in vitro activated human PBMCs with αCTLA-4. Representative donors are shown (n=6). [Figure 1-4]Biochemical and functional characterization of novel Treg-depleting αCTLA-4 mAbs. (I) Dose-dependent binding of murine αCTLA-4 to mCTLA-4 transfected cells by flow cytometry. (J) Anti-CTLA-4 mAbs block B7 ligand binding (CD80 and CD86) to recombinant CTLA-4 by ELISA. (K) Treg depletion activity and CD8+ T cell / Treg ratio (n=4-8), and (L) survival induced by α-murine CTLA-4 antibodies in BALB / c mice bearing CT26 tumors. [Figure 2-1] Generation and characterization of oncolytic vaccinia viruses expressing Treg-depleted αCTLA-4 and GM-CSF. (A) Schematic diagram of the vaccinia virus vectors used to encode the heavy (at the J2R locus) and light (at the I4L locus) chains of the αCTLA4 antibody and GM-CSF. [Figure 2-2] Production and characterization of oncolytic vaccinia virus expressing Treg-depleted αCTLA-4 and GM-CSF. (B) Replication kinetics in LoVo cells and (C) oncolytic activity of VVGM-αhCTLA4 (BT-001) against MIA PaCa-2 cells. TG6002 (recombinant J2R and I4L deleted vaccinia virus) was added as a control. (D) Electrophoretic profile after Coomassie blue staining of 4-E03 purified from MIA PaCa-2 BT-001 infected cell cultures. Lanes 2 and 5: recombinant 4-E03, lanes 1 and 4: 4-E03 purified from culture medium of MIA PaCa-2 BT-001 infected cells. Lanes 1 and 2: non-reducing conditions, lanes 3 and 4: reducing conditions. [Figure 2-3] Generation and characterization of oncolytic vaccinia viruses expressing Treg-depleted αCTLA-4 and GM-CSF. (E) Expression levels of 4-E03 and human GM-CSF in the indicated human tumor cells 48 hours after infection with VVGM-αhCTLA4 (BT-001). [Figure 2-4]Production and characterization of oncolytic vaccinia viruses expressing Treg-depleted αCTLA-4 and GM-CSF. (F) Biological activity of GM-CSF produced in E) as determined by TF-1 proliferation assay. Recombinant human GM-CSF (Molgramostim obtained from the European Pharmacopoeia Reference Standard) was included as a positive control. (G and H) Functional evaluation of 4-E03 produced by BT-001-infected MIA PaCa-2 cells. [Figure 2-5] Generation and characterization of oncolytic vaccinia viruses expressing Treg-depleted αCTLA-4 and GM-CSF. (G) In vitro: by binding to immobilized recombinant hCTLA protein as in Fig. 1E, and (H) in vivo: (Treg-depleted) as in Fig. 1D. [Figure 3-1] Intratumoral VVGM-αCTLA4 has in vivo antitumor activity associated with tumor-restricted CTLA-4 receptor saturation and Treg depletion. (A) CT26 tumor-bearing mice were treated with VVGM-αCTLA4 (7.5×106, 7.5×105, or 7.5×104 pfu), VV-αCTLA4 (7.5×106 pfu), or empty control VV (7.5×106 pfu) (n=20 mice / group). [Figure 3-2] Intratumoral VVGM-αCTLA4 has in vivo antitumor activity associated with tumor-restricted CTLA-4 receptor saturation and Treg depletion. (B) Pharmacokinetics of αCTLA-4 in tumor and serum of CT26 tumor-bearing mice after three it injections (days 0, 2, and 4) of VVGM-αCTLA4 at 107 pfu or a single ip 3 mg / kg injection of αCTLA-4 mAb 5-B07 (n=3 mice / time point). The grey area indicates the EC10-EC90 range for CTLA-4 receptor saturation (see Figure 1J). [Figure 3-3] Intratumoral VVGM-αCTLA4 has in vivo antitumor activity associated with tumor-restricted CTLA-4 receptor saturation and Treg depletion. (C) The number of FoxP3+ cells in tumors and spleens 10 days after VVGM-αCTLA4 injection was analyzed by FACS. The graph shows pooled data from three independent experiments (n=13 mice / group). [Figure 4-1a] Intratumoral VVGM-αCTLA4 has broad antitumor activity in syngeneic tumor models across inflammatory and cold tumor microenvironments. (A) BALB / c mice bearing CT26, A20, or EMT6 tumors, or C57BL / 6 mice bearing MC38 or B16 tumors, received three it injections of VVGM-αCTLA4, or control viruses lacking αmCTLA-4 mAb (VV empty or VVGM). Treatments were initiated when tumors had a volume of approximately 50-100 mm3 or 4 days after tumor cell injection (B16 only). Graphs show tumor growth and corresponding survival of individual mice (n=10). [Figure 4-1b] Intratumoral VVGM-αCTLA4 has broad antitumor activity in syngeneic tumor models across inflammatory and cold tumor microenvironments. (A) BALB / c mice bearing CT26, A20, or EMT6 tumors, or C57BL / 6 mice bearing MC38 or B16 tumors, received three it injections of VVGM-αCTLA4, or control viruses lacking αmCTLA-4 mAb (VV empty or VVGM). Treatments were initiated when tumors had a volume of approximately 50-100 mm3 or 4 days after tumor cell injection (B16 only). Graphs show tumor growth and corresponding survival of individual mice (n=10). [Figure 4-2a] Intratumoral VVGM-αCTLA4 has broad antitumor activity in syngeneic tumor models across inflammatory and cold tumor microenvironments. (B) CT26 tumor cells were implanted into the right and left flanks of BALB / c mice. It injections into the right flank tumors (vertical dotted line, same as in A) with VVGM-αCTLA4 were initiated when tumors reached a volume of approximately 100 mm3 (n=9-10). [Figure 4-2b] Intratumoral VVGM-αCTLA4 has broad antitumor activity in syngeneic tumor models across inflammatory and cold tumor microenvironments. (B) CT26 tumor cells were implanted into the right and left flanks of BALB / c mice. It injections into the right flank tumors (vertical dotted line, same as in A) with VVGM-αCTLA4 were initiated when tumors reached a volume of approximately 100 mm3 (n=9-10). [Figure 5-1] Intratumoral VVGM-αCTLA4 induces robust systemic CD8+ T cell-dependent antitumor immunity. (A) BALB / c mice were treated with CD8 (short dashed line) or CD4 (long dashed line) depleting antibodies before and after sc challenge with CT26 tumor cells. Treatment was initiated as in Figure 4A when tumors reached a volume of approximately 20-50 mm3. One representative experiment (out of two) with 10 mice per group is shown. [Figure 5-2] Intratumoral VVGM-αCTLA4 induces robust systemic CD8+ T cell-dependent antitumor immunity. (B-D) CT26 tumor-bearing mice were treated it with VV or ip with αCTLA-4 mAb (clone 5-B07 at 3 mg / kg). Tumor cell suspensions and splenocytes were restimulated ex vivo with VV or CT26 (AH-1)-specific peptides, and the percentage of IFN-γ+ and TNF-α+ CD8+ T cells, or MHC class I-tagged multimer-positive CD8+ T cells, was quantified by FACS. (B) shows flow cytometry dot plots of AH-1 peptide-positive (upper panel) or cytokine-positive (lower panel) splenocytes. [Figure 5-3] Intratumoral VVGM-αCTLA4 induces robust systemic CD8+ T cell-dependent antitumor immunity. (B-D) CT26 tumor-bearing mice were treated it with VV or ip with αCTLA-4 mAb (clone 5-B07 at 3 mg / kg). Tumor cell suspensions and splenocytes were restimulated ex vivo with VV or CT26 (AH-1)-specific peptides, and the percentage of IFN-γ+ and TNF-α+ CD8+ T cells, or MHC class I-tagged multimer-positive CD8+ T cells, was quantified by FACS. Quantification of (C) antigen-specific and (D) IFN-γ+ / TNF-α+ CD8+ T cells in the indicated organs. Each dot represents one mouse. (n=3-6 experiments) *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 by one-way ANOVA. [Figure 5-4]Intratumoral VVGM-αCTLA4 induces robust systemic CD8+ T cell-dependent antitumor immunity. (B-D) CT26 tumor-bearing mice were treated it with VV or ip with αCTLA-4 mAb (clone 5-B07 at 3 mg / kg). Tumor cell suspensions and splenocytes were restimulated ex vivo with VV or CT26 (AH-1)-specific peptides, and the percentage of IFN-γ+ and TNF-α+ CD8+ T cells, or MHC class I-tagged multimer-positive CD8+ T cells, was quantified by FACS. Quantification of (C) antigen-specific and (D) IFN-γ+ / TNF-α+ CD8+ T cells in the indicated organs. Each dot represents one mouse. (n=3-6 experiments) *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 by one-way ANOVA. [Figure 6-1] Intratumor-induced CD8+ T cell antitumor immunity is FcγR- and cDC1-dependent. (A) CT26 tumor-bearing WT and Fcer1g- / - BALB / c mice received it injections of VVGM-αCTLA4 or PBS as in FIG. 4A. Graphs show tumor volume (left and center panels) and mouse survival (right panel). Vertical lines indicate end of treatment. (n=10 mice / group) [Figure 6-2] Intratumor-induced CD8+ T cell antitumor immunity is FcγR- and cDC1-dependent. (B) GO terms enriched in a set of 352 differentially expressed genes that were either up- or downregulated in VVGM-αCTLA4 vs. VVempty-treated CT26 tumors. The 20 enriched terms with the lowest adjusted p-values ​​are shown. [Figure 6-3] Intratumor-induced CD8+ T cell antitumor immunity is FcγR- and cDC1-dependent. (C) Network diagram of differentially expressed genes associated with the five most enriched GO terms from Fig. 6b. Only upregulated genes were found to be associated with these five enriched GO terms. [Figure 6-4]Intratumor-induced CD8+ T cell antitumor immunity is FcγR- and cDC1-dependent. (D) MC38 tumor-bearing WT and Batf3- / - BALB / c mice received it injections of VVGM-αCTLA4 or PBS as in Figures 4A and 6A. Graphs show tumor volume (left and center panels) and mouse survival (right panel). Vertical lines indicate end of treatment. (n=8-10 mice / group). [Figure 7-1] Intratumoral VVGM-αCTLA4 expands peripheral effector CD8+ T cells and reduces Treg and exhausted CD8+ T cells. BALB / c mice bearing CT26 "twin" tumors were treated it with VVGM-αCTLA4 or PBS (right flank tumors only). Injected and contralateral tumors in the spleen were harvested 10 days after treatment and stained with a multi-dimensional panel designed to distinguish T cell populations. (A) itVVGM-αCTLA4 reduced activated CD4+ Treg cells (FoxP3+Klrg1+, "T1"), reduced exhausted CD8+ T cells (PD1+TIM3+, "T2"), and expanded activated effector CD8+ T cells (Klrg1+, "T3") in injected and non-injected tumors (upper panel), as well as expanded activated CD8+ T cells in the spleen (S1, lower panel). [Figure 7-2] Intratumoral VVGM-αCTLA4 expands peripheral effector CD8+ T cells and reduces Treg and exhausted CD8+ T cells. CT26 "twin" tumor-bearing BALB / c mice were treated it with VVGM-αCTLA4 or PBS (right flank tumors only). Splenic injected and contralateral tumors were harvested 10 days after treatment and stained with a multi-dimensional panel designed to identify T cell populations. (B) Quantification of the data shown in A is shown. One representative experiment (out of 3) with 5 mice / group. [Figure 7-3]Intratumoral VVGM-αCTLA4 expands peripheral effector CD8+ T cells and reduces Treg and exhausted CD8+ T cells. CT26 "twin" tumor-bearing BALB / c mice were treated it with VVGM-αCTLA4 or PBS (right flank tumors only). Splenic injected and contralateral tumors were harvested 10 days after treatment and stained with a multi-dimensional panel designed to identify T cell populations. (C) Flow cytometry plots show characteristic markers of selected it T cell clusters. [Figure 8-1] Intratumoral VVGM-αCTLA4 synergizes with αPD-1 to reject "cold" ICB-resistant tumors. (A and B) C57BL / 6 mice bearing two B16 tumors, one large (5×105 cells, treated tumor) and one small (1×105 cells, contralateral), received three it injections of VVGM-αCTLA4 (vertical dotted lines) and ipαPD-1 (29F.1A12, 10 mg / kg, twice weekly for 3 weeks, gray area). (A) Survival (n=10-20), *p<0.05 by log-rank test. [Figure 8-2] Intratumoral VVGM-αCTLA4 synergizes with αPD-1 to reject "cold" ICB-resistant tumors. (A and B) C57BL / 6 mice bearing two B16 tumors, one large (5×105 cells, treated tumor) and one small (1×105 cells, contralateral), received three it injections of VVGM-αCTLA4 (vertical dotted lines) and ipαPD-1 (29F.1A12, 10 mg / kg, twice weekly for 3 weeks, gray area). (B) Tumor growth curves of intratumoral injected tumors and contralateral tumors. [Figure 8-3] Intratumoral VVGM-αCTLA4 synergizes with αPD-1 to reject "cold" ICB-resistant tumors. (C) A20 tumor-bearing BALB / c mice were treated three times with VVGM-αCTLA4 it (at a suboptimal dose of 1×105 pfu), αPD-1 ip (RMP1-14, 10 mg / kg total dose), or a combination of both when tumors reached a volume of approximately 135 mm3. The graph shows the survival of the animals (n=10). [Figure 9-1]Characterization of CTLA4-specific mAbs. (A) Freshly resected ovarian tumor, ascites, and blood samples were compared to healthy PBMCs. CTLA-4 expression was assessed by flow cytometry on CD4+CD25+CD127- Treg cells, CD4+ non-Treg cells, and CD8+ effector T cells and compared to expression on human T cells isolated from NOG spleens 2 weeks after PBMC transfer. Data represent individual patients / donors, n=11 for healthy PBMCs, n=20 for ascites, n=9 for tumors, and n=5 for patient blood. (B) Human PBMCs were injected intravenously into NOG mice. Two to three weeks after transplantation, spleens were excised and cell suspensions were injected ip into SCID recipients followed by treatment with 10 mg / kg of 4-E03 hIgG1, 4-E03 hIgG1 N297Q, or isotype control. Cells were collected by ip wash and quantified 24 hours after treatment. Percent cell depletion was normalized to isotype control. Each dot represents one mouse. [Figure 9-2] Characterization of CTLA4-specific mAbs. (C) The binding kinetics of 4-E03 and ipilimumab to soluble human CTLA-4 was evaluated by Biacore analysis. mAbs were captured on the chip by immobilized anti-human Fc, and different concentrations of CTLA-4 protein were injected in each cycle. The KD values ​​of 4-E03 and ipilimumab were 0.6 nM and 2.7 nM, respectively. [Figure 9-3] Characterization of CTLA4-specific mAbs. (D) Dose-dependent binding of anti-CTLA-4 mAbs to human T cells that endogenously express CTLA-4 by flow cytometry. [Figure 9-4]Characterization of CTLA4-specific mAbs. (E) Binding of titrated doses of mouse surrogate antibody 5-B07 mIgG2a (starting from 10 μg / ml, in 3-fold dilution steps) was tested against mouse CTLA-4 and CD28 by ELISA. (F) 1×106 CT26 cells were injected subcutaneously into BALB / c mice (n=7). When tumors reached a size of approximately 100 mm3, mice received 200 μg (10 mg / kg) of 5-B07 mIgG2a, 5-B07 mIgG1 N297A, or isotype control antibody on days 0, 4, and 7. On day 8, tumors were removed and TILs were analyzed by FACS. Data shown are the mean (+SD) of one representative experiment with n=7 mice per group. [Figure 10-1] Pharmacokinetics of virus and transgene in tumor and blood. (A and B) Using the tumor samples described in Figure 3B, (A) intratumoral concentrations of murine GM-CSF and (B) viral load C-E) pharmacokinetics were measured in LoVo xenograft tumors. LoVo cells were implanted into the right flank of Swiss nude mice. When the tumor volume reached approximately 120 mm3 (defined as D0), mice were treated with a single injection of either 105 pfu of VVGM-hCTLA4 (BT-001) or VV it, or 3 mg / kg of 4-E03 monoclonal antibody ip. Blood and tumors of three mice were collected at each indicated time point. (C) 4-E03, (D) GM-CSF, and (E) virus concentrations were determined by ELISA and titration on Vero cells, respectively. Lines connect the median values ​​at each time point. [Figure 10-2]Pharmacokinetics of virus and transgene in tumor and blood. (A and B) Using the tumor samples described in Figure 3B, (A) intratumoral concentrations of murine GM-CSF and (B) viral load C-E) pharmacokinetics were measured in LoVo xenograft tumors. LoVo cells were implanted into the right flank of Swiss nude mice. When the tumor volume reached approximately 120 mm3 (defined as D0), mice were treated with a single injection of either 105 pfu of VVGM-hCTLA4 (BT-001) or VV it, or 3 mg / kg of 4-E03 monoclonal antibody ip. Blood and tumors of three mice were collected at each indicated time point. (C) 4-E03, (D) GM-CSF, and (E) virus concentrations were determined by ELISA and titration on Vero cells, respectively. Lines connect the median values ​​at each time point. [Figure 10-3] Pharmacokinetics of virus and transgene in tumor and blood. (A and B) Using the tumor samples described in Figure 3B, (A) intratumoral concentrations of murine GM-CSF and (B) viral load C-E) pharmacokinetics were measured in LoVo xenograft tumors. LoVo cells were implanted into the right flank of Swiss nude mice. When the tumor volume reached approximately 120 mm3 (defined as D0), mice were treated with a single injection of either 105 pfu of VVGM-hCTLA4 (BT-001) or VV it, or 3 mg / kg of 4-E03 monoclonal antibody ip. Blood and tumors of three mice were collected at each indicated time point. (C) 4-E03, (D) GM-CSF, and (E) virus concentrations were determined by ELISA and titration on Vero cells, respectively. Lines connect the median values ​​at each time point. [Figure 11-1]Treatment with itVVGM-αCTLA4 induces a sustained antitumor response in treated and untreated tumors. (A) CT26 tumor cells were implanted into the right and left flank of BALB / c mice. It injections (x3, 2 days apart) with VVGM-αCTLA4 into the right flank tumors were started when the tumors reached a volume of approximately 100 mm3. Viral concentrations in treated and contralateral tumors were assessed 1 day after treatment. (n=3 mice per group, mice 1-3 (M1-3), ND=not detected) B-C) ​​CT26 tumor-bearing mice were treated with the indicated doses of VVGM-αCTLA4 as in A). Alternatively, 107 or 105 pfu of VVGM-αCTLA4 were administered iv. Individual tumor growth curves (B) and survival curves (C) are shown. (D) Inhibition of rechallenged tumor growth. CT26 cells were implanted sc into BALB / c mice. It treatment with VVGM-αCTLA4 or blank control VV was scheduled as in A). 100 days after the last VV injection, surviving mice were rechallenged sc with CT26 or Renca cells as indicated in the figure legend of Fig. 4B. [Figure 11-2]Treatment with itVVGM-αCTLA4 induces a sustained antitumor response in treated and untreated tumors. (A) CT26 tumor cells were implanted into the right and left flank of BALB / c mice. It injections (x3, 2 days apart) with VVGM-αCTLA4 into the right flank tumors were started when the tumors reached a volume of approximately 100 mm3. Viral concentrations in treated and contralateral tumors were assessed 1 day after treatment. (n=3 mice per group, mice 1-3 (M1-3), ND=not detected) B-C) ​​CT26 tumor-bearing mice were treated with the indicated doses of VVGM-αCTLA4 as in A). Alternatively, 107 or 105 pfu of VVGM-αCTLA4 were administered iv. Individual tumor growth curves (B) and survival curves (C) are shown. (D) Inhibition of rechallenged tumor growth. CT26 cells were implanted sc into BALB / c mice. It treatment with VVGM-αCTLA4 or blank control VV was scheduled as in A). 100 days after the last VV injection, surviving mice were rechallenged sc with CT26 or Renca cells as indicated in the figure legend of Fig. 4B. [Figure 11-3]Treatment with itVVGM-αCTLA4 induces a sustained antitumor response in treated and untreated tumors. (A) CT26 tumor cells were implanted into the right and left flank of BALB / c mice. It injections (x3, 2 days apart) with VVGM-αCTLA4 into the right flank tumors were started when the tumors reached a volume of approximately 100 mm3. Viral concentrations in treated and contralateral tumors were assessed 1 day after treatment. (n=3 mice per group, mice 1-3 (M1-3), ND=not detected) B-C) ​​CT26 tumor-bearing mice were treated with the indicated doses of VVGM-αCTLA4 as in A). Alternatively, 107 or 105 pfu of VVGM-αCTLA4 were administered iv. Individual tumor growth curves (B) and survival curves (C) are shown. (D) Inhibition of rechallenged tumor growth. CT26 cells were implanted sc into BALB / c mice. It treatment with VVGM-αCTLA4 or blank control VV was scheduled as in A). 100 days after the last VV injection, surviving mice were rechallenged sc with CT26 or Renca cells as indicated in the figure legend of Fig. 4B. [Figure 12] Intratumor-induced CD8 T cell immunity is FcγR dependent. WT and Fcer1g- / - BALB / c mice were challenged sc with 1x106 CT26 cells. When tumors reached approximately 100mm3, mice received three it injections of VVGM-αCTLA4 or PBS control on days 0, 2, and 5 (final dose of 107 pfu). On day 8, tumors and spleens were isolated and FoxP3+CD4+ cells were analyzed by FACS. [Figure 13-1]Treatment with itVVGM-αCTLA4 is dependent on CD8+ T cells. (A) Groups of 10 BALB / c mice were treated or not with 1 mg of CD8- or CD4-depleting antibody (or corresponding isotype control antibody) 3 days before mice were challenged sc with 1×106 CT26 cells. 200 μg of depleting antibody was administered ip 4 days later (day −3 relative to treatment initiation). Mice were then treated it with 1×107 pfu of VVGM-αCTLA4 on days 0, 2, and 4. Percentage of survival to humane endpoint is shown. Data are representative of two independent experiments. [Figure 13-2] Treatment with itVVGM-αCTLA4 is dependent on CD8+ T cells. (B) CT26 tumor-bearing mice were treated it with VV or ip with anti-CTLA-4 mAb (clone 5-B07 at 3 mg / kg). Tumor cell suspensions were restimulated ex vivo with VV or CT26 (AH-1) specific peptide, cultured for 4 h, and the numbers of IFN-γ+ and TNF-α+ CD8+ T cells were quantified by flow cytometry. Each dot represents one mouse. Representative experiment (n=3-6), **p<0.01, ***p<0.005, ****p<0.001 by one-way ANOVA. [Figure 13-3] Processing by itVVGM-αCTLA4 is dependent on CD8+ T cells. (C) and (D) Heatmaps showing median marker expression of (C) 12 intratumoral and (D) 10 splenic CD3+ subpopulations identified by unsupervised clustering. [Figure 13-4] Processing by itVVGM-αCTLA4 is dependent on CD8+ T cells. (C) and (D) Heatmaps showing median marker expression of (C) 12 intratumoral and (D) 10 splenic CD3+ subpopulations identified by unsupervised clustering. [Figure 14-1]B16 tumors are refractory to systemic treatment with anti-CTLA-4 plus anti-PD-1. B16 tumor-bearing C57BL / 6 mice were treated ip with 10 mg / kg anti-PD-1 or a combination of anti-PD-1 and anti-CTLA-4 (10 mg / kg) on ​​days 4, 7, and 11. (A) Data are expressed as mm3 tumor volume on the day after cell inoculation as indicated, and each line represents an individual mouse. (B) The bottom panel shows the percentage of survival to humane endpoints of mice bearing B16 after the indicated treatments. [Figure 14-2] B16 tumors are refractory to systemic treatment with anti-CTLA-4 plus anti-PD-1. B16 tumor-bearing C57BL / 6 mice were treated ip with 10 mg / kg anti-PD-1 or a combination of anti-PD-1 and anti-CTLA-4 (10 mg / kg) on ​​days 4, 7, and 11. (C) B16 tumor-bearing C57BL / 6 mice received three it injections of VVGM-αCTLA4 and / or ip anti-PD-1 as described in FIG. 8. Six days after the last treatment, tumors were harvested and the number of tumor-infiltrating T cells was analyzed by flow cytometry. Each dot represents one mouse. (n=3 experiments). *p<0.05 by one-way ANOVA. The level of T cell infiltration in the highly T cell inflammatory CT26 tumor microenvironment is shown for reference (determined in CT26 tumors approximately 20 days after cell inoculation). EXAMPLES

[0246] Materials and Methods cell line Human embryonic kidney cell line 293T, mouse melanoma B16-F10, mouse colon carcinoma CT26, mouse B-cell lymphoma A20, mouse mammary gland EMT6, and mouse Lewis lung carcinoma cell line (LL / 2) were purchased from American Type Culture Collection (ATCC) and stably transfected with human CTLA-4 (293T-CTLA4) from Crown Bio. Cells were cultured in RPMI+Glutamax (CT26) or DMEM+Glutamax (MC38, B16-F10) supplemented with 10% FCS, 10 mM HEPES, and 1 mM sodium pyruvate. EMT6 cells were maintained in Waymouth's medium supplemented with 15% FCS, 10 mM HEPES, and 1 mM sodium pyruvate. The NK-92 cell line expressing hFcγRIIIA-158V was cultured in supplemented α-MEM medium with GFP (purchased from ATCC) (Binyamin et al., 2008). Primary cells were cultured in R10 medium (RPMI 1640 containing 2 mM glutamine, 1 mM pyruvate, 100 IU / ml penicillin and streptomycin, and 10% FBS, GIBCO by Life Technologies). The human colorectal adenocarcinoma cell line LoVo (ATCC), pancreatic tumor cell line MIA PaCa-2 (ATCC), and human gastric cancer cell line Hs-746 T (ATCC) were grown in DMEM (Gibco) supplemented with 10% FBS and containing 40 mg / L gentamicin. The human ovarian tumor cell line SK-OV-3 (ATCC) and the human colorectal cancer cell line HCT 116 (ATCC) were grown in McCoy's 5A medium (ATCC) supplemented with 10% FBS and containing 40 mg / L gentamicin. The human erythroblast cell line TF-1 (ATCC) was grown in RPMI 1640 (Sigma) supplemented with 10% FBS and containing 40 mg / L gentamicin plus 2 ng / mL GM-CSF.

[0247] mouse Mice were maintained in local pathogen-free facilities. For all experiments, young adult mice were sex- and age-matched and randomly assigned to experimental groups. All procedures were approved by the local ethical committee on experimental animals (Malmo / Lunds djurforsoksetiska namnd) at BioInvent under permit number 17196 / 2018 or 2934 / 2020 or in the transgene APAFIS Nr21622 project 2019072414343465 and were performed in accordance with local ethical guidelines. C57BL / 6 and BALB / c mice were obtained from Taconic, Janvier, or Charles River. The genetically modified strains used were C.129P2(B6)-Fcer1gtm1Rav (Fcer1g-KO on BALB / c background and BALB / cAnNTac WT control) purchased from Taconic, and B6.129S(C)-Batf3tm1Kmm / J (Hildner et al., 2008) (Batf3-KO on C57BL / 6J background and C57BL / 6J WT control) purchased from Jackson Laboratories.

[0248] Human (clinical) samples and ethics Ethics approval was obtained by the ethical committee of Skane University Hospital. Informed consent was provided in accordance with the Declaration of Helsinki. Patient samples were obtained through the Departments of Obstetrics and Gynecology and Oncology at Skane University Hospital, Lund, Sweden. Ascites fluid was evaluated as isolated single cell suspensions.

[0249] Human tissue processing Ovarian tumor samples obtained from patients undergoing surgery were cut into small pieces and incubated in R10 containing DNase I (Sigma) and Liberase™ (Roche Diagnostics) for 20 min at 37°C. The remaining tissue was mechanically dissociated and passed through a 70 μm cell strainer together with the cell suspension. Matched peripheral blood samples were obtained and peripheral blood mononuclear cells were isolated using Ficoll-Paque PLUS (Cytiva) by centrifugation at 800×g for 20 min in Leucosep tubes (Greiner). Human buffy coats were obtained from the Hematology Center of the Hospital of Halmstad (Sweden) and processed according to standard protocols.

[0250] Antibody-dependent cytotoxicity ADCC assays were performed using the NK-92 cell line stably transfected to express the CD16-158V allele along with GFP. + CD4 T cells were isolated from peripheral blood of healthy donors using a T cell isolation kit (Miltenyi Biotec). + Target T cells were isolated. Cells were stimulated with CD3 / CD28 Dynabeads (Life Technologies, Thermo Fisher) to upregulate CTLA-4 and 50 ng / ml recombinant hIL-2 (R&D Systems) for 72 h at 37 °C. Target cells were preincubated with 0.1–10 μg / ml mAb for 30 min at 4 °C and then mixed with NK cells. Cells were incubated for 4 h at a 2:1 effector:target cell ratio. Lysis was determined by flow cytometry. Briefly, at the end of the incubation, cell suspensions were stained with VioGreen-conjugated anti-CD4 (M-T466, Miltenyi Biotec) together with Fixable Viability Dye eFluor780 (eBioscience) for 30 min in the dark at 4 °C, and then cells were analyzed by FACS.

[0251] In vitro functional blockade For the SEB PBMC assay, total PBMCs from healthy donors were plated in 96-well plates (1 × 10 5 Cells were seeded at 1000 x g / well (100 x 100 cells / well) and stimulated with 1 μg / ml Staphylococcus enterotoxin B (SEB, Sigma Aldrich) in the presence of anti-CTLA-4 IgG ranging from 20 to 0.625 μg / ml. After 3 days, supernatants were harvested and IL-2 was quantified by MSD (Meso Scale Discovery, Rockville, USA) according to the manufacturer's instructions.

[0252] In vitro binding assay CTLA-4-expressing transfected cells were incubated with the indicated concentrations of anti-CTLA-4 mAb for 20 min at 4°C, then washed and stained with APC-conjugated goat anti-human secondary antibody (Jackson ImmunoResearch). No binding was observed to cells transfected with empty vector (not shown).

[0253] IgG binding to primary cells was determined using isolated in vitro activated CD4 + Briefly, human peripheral CD4 + T cells were purified from total PBMCs by negative selection using the MACS CD4 T cell isolation kit (Miltenyi Biotec). CD4+ T cells were activated in vitro with CD3 / CD28 dynabeads (Life Technologies) in R10 medium plus 50 ng / ml recombinant hIL-2 (R&D Systems) for 3 days to upregulate CTLA-4 expression. In vitro activated human CD4 + T cells were incubated with the indicated concentrations of anti-CTLA-4 mAb along with anti-CD4. Bound anti-CTLA-4 mAb was detected with APC-labeled goat anti-human IgG. In competitive binding assays, 2 μg / ml of Alexa 647-labeled anti-CTLA-4 mAb was mixed with recombinant human or cynomolgus CTLA-4-Fc protein (50 μg / ml, R&D Systems) and then incubated with CTLA-4-expressing cells. Bound IgG binding was detected by FACS.

[0254] VV production and purification Recombinant viruses were produced by two successive rounds of homologous recombination in chicken embryo fibroblasts (CEFs) using the starting parental Copenhagen vaccinia virus and two transfer plasmids encoding GFP or mCherry at the J2R and I4L loci. The transfer plasmids encoded either the heavy chain of a mAb flanked by J2R recombination arms under the p7.5 promoter, or the light chain of mouse or human GM-CSF flanked by I4L recombination arms under the p7.5 promoter, in addition to or instead of, the mAb under the pSE / L promoter (see FIG. 2A). Recombinant viruses were isolated by several cycles of amplification / isolation of non-fluorescent plaques. Recombinant viruses were then produced on CEFs and purified by 5 μm filtration after cell lysis, followed by purification / concentration using 0.2 μm tangential flow filtration. Finally, the virus was formulated by diafiltration in sucrose 50 g / L, NaCl 50 mM, Tris 10 mM, sodium glutamate 10 mM, pH 8, aliquoted, and stored at -80°C until use.

[0255] All viruses used in this publication were derived from the TK-RR Copenhagen strain. VV: disarmed vaccinia virus or TG6002 (vaccinia virus encoding the FCU1 chimeric enzyme, a benchmark recombinant VV) VV GM : Vaccinia virus encoding mouse GM-CSF VV GM -αCTLA4: vaccinia virus encoding mouse GM-CSF and 5-B07 (anti-mouse CTLA-4, mouse IgG2a) Vaccinia virus encoding VV-αCTLA4:5-B07 VV GM - αhCTLA4 (BT-001): Vaccinia virus encoding human GM-CSF and 4-E03 (anti-human CTLA-4, human IgG1).

[0256] In vitro viral replication, oncolytic activity, and transgene expression The BT-001 replica is available in 10 -3 The viral titers were assessed by measuring the total virus titers at 24, 48, and 72 hours after infection of LoVo cells with BT-001 at a multiplicity of infection (MOI) of 1000 (i.e., 1 virus per 1000 cells). Viral titers were determined by plaque assay in Vero cells.

[0257] After incubating MIA PaCa-2 cells with BT-001 for 5 days at the MOIs indicated in the figure legends, the oncolytic activity of BT-001 was assessed by quantifying cell viability using a cell counter (Vi-Cell). Both the replication and oncolytic activities of BT-001 were benchmarked with those of Copenhagen TK-RR-vaccinia virus TG6002, which is currently undergoing clinical evaluation (Foloppe et al., 2019).

[0258] Transgene expression was assessed after infection of several human tumor cell lines, LoVo, HCT 116 (colon cancer), MIA PaCa-2 (pancreatic cancer), SK-OV3 (ovarian cancer), and Hs176T (gastric cancer), with BT-001 at an MOI of 0.05. Culture supernatants were collected 48 h postinfection, centrifuged, and 0.2 μm filtered, after which 4-E03 and hGM-CSF concentrations were measured by ELISA.

[0259] Antibody purification Approximately 4.7×10 7Fifteen F175 flasks containing 10 MIA PaCa-2 cells / flask were infected with BT-001 at MOI 0.01 in DMEM without bovine serum. 72 hours after infection, cell supernatants were harvested, pooled, centrifuged, and filtered at 0.2 μm before adding EDTA (2 mM final) and Tris pH 7.5 (20 mM final). Pooled supernatants were loaded onto a 1 mL protA Hitrap column (GE healthcare, ref 17-5079-01) pre-equilibrated with PBS at 4 °C. Bound antibodies were eluted with 100 mM glycine HCl pH 2.8 and dialyzed against PBS. Purified 4-E03 was loaded onto SDS-PAGE (NuPage Bis-Tris gels 4-12% Thermo NP0323) under reducing or non-reducing conditions and gels were stained with InstantBlue (Expedeon, ISB1L) Coomassie Blue. This purified antibody (ie, 4-E03 MIA PaCa-2) was further evaluated for CTLA-4 binding and in vivo Treg depletion activity.

[0260] Enzyme-linked immunosorbent assay GMCSF. Human and mouse GM-CSF concentrations were determined using the Quantikine® ELISA GM-CSF immunoassay (R&D Systems).

[0261] Human GM-CSF functionality was assessed using a TF-1 proliferation assay. Cell proliferation of TF-1 cells in the presence of known concentrations of hGM-CSF (standard or from BT-001 infected cells) was measured by a colorimetric assay using the enzymatic conversion of MTS to formazan by dehydrogenases in viable cells (measured by absorbance at 490 nm). The absorbance at 490 nm was plotted against the concentration of GM-CSF and the curves were compared to those obtained with recombinant GM-CSF (i.e., molgramostim).

[0262] Binding to CTLA4 / CD28 proteins. For antibody binding ELISA, purified human CTLA4-Fc, human CD28-Fc (R&D Systems), and mouse CTLA4-Fc (Sino Biologicals) were coated onto assay plates at 1 pmol / well, and mouse CD28-His (R&D Systems) was coated at 5 pmol / well at the same time. Different antibodies were added at 10 μg / ml and allowed to bind for 1 hour at room temperature. Bound n-Coder® mIgG2A or hIgG1 antibodies were detected using either anti-mouse / anti-human H+L-HRP (Jackson Immunoresearch) or anti-mouse / human lambda light chain antibody HRP (Bethyl). Plates were read using a Tecan Ultra with either chromogenic (TMB T0440) or luminogenic (Pierce 37070) substrates.

[0263] Blockade of CD80 / CD86 interactions. For ligand blocking ELISA, purified human CTLA4-Fc (R&D Systems) was coated onto assay plates at 2 pmol / well (for CD80) or 1 pmol / well (for CD86). Antibodies were added at concentrations ranging from 0.4 pM to 67 nM and allowed to bind for 1 h. His-tagged ligands were added at 200 nM and 100 nM, respectively (rhCD80 and rhCD86, R&D Systems), as optimized in pilot experiments by ELISA (data not shown). Plates were incubated for a further 15 min. After washing, bound ligands were detected with an HRP-labeled anti-His antibody (R&D Systems). Plates were analyzed using a Tecan Ultra Microplate reader using Super Signal ELISA Pico (Thermo Scientific) as substrate. Alternatively, mouse CTLA4-Fc (Sino Biological) was coated onto assay plates at 1 pmol / well. Antibodies were added in two-fold dilution series at a starting concentration of 10 μg / ml (67 nM) and allowed to bind for 1 h. His-tagged ligands, CD80 and CD86 (Sino Biological), were added at 50 nM and the plates were incubated for a further 30 min. Detection and reading were performed as above.

[0264] Mouse experiments In vivo tumor experiments. Cultured tumor cells were injected subcutaneously into the left flank only or into both flanks (CT26 1×10 6 Cells, MC38 5×10 5 Cells, A20 5×10 6 cells, EMT6 1 x 10 6 0.5-5×10 cells, B16-F10 5 Unless otherwise stated, mice were cultured at 10 7 VV of pfu GM For tumor growth experiments, tumor sizes of treated and distant tumors were measured twice weekly using calipers and tumor volumes (mm 3) into the formula:(width 2 × length × 0.52) was calculated. If the total tumor burden (treated tumor and contralateral tumor combined) was 2000 mm 3 Animals were euthanized when the tumor reached a volume of 100 μg / ml (experimental endpoint). For functional experiments, tissues were collected and processed at the time points indicated in the figure legends. Mouse tumors were digested in R10 containing DNase I (Sigma) and Liberase™ (Roche Diagnostics) for 15 min at 37° C. Cells were then passed through a 70 μm cell strainer and used directly in the assay. For DC phenotyping, viable leukocytes were enriched after density gradient centrifugation (Cedarline catalogue no. CL5035).

[0265] Primary human xenograft model. PBMC-NOG / SCID mice were cultured with 1–2 × 10 PBMCs isolated from NOG mice (NOD.Cg-PrkdcscidIl2rgtm1Sug / JicTac (Taconic)) using Ficoll-Paque PLUS in 200 μl of PBS. 7 Approximately 2 weeks after injection, SCID mice (CB-Igh-1b / IcrTac-Prkdcscid (Taconic)) were intravenously injected with 10 × 10 PBMCs from reconstituted NOG mice. 6 Splenocytes were injected intraperitoneally. One hour later, mice were treated with 10 mg / kg mAb. Mouse intraperitoneal fluid was collected 24 hours later. Human T cell subsets were identified and quantified by FACS using the following markers: CD45, CD4, CD8, CD25, CD127 (all from BD Biosciences).

[0266] Pharmacokinetics of transgene and virus in tumor and blood. In the CT26 tumor model described above, VV GM VV-αCTLA4 or VV-αCTLA4 were administered under the same conditions as above (i.e., 10 7pfu it injections three times). Tumors and blood of 3 mice / time point were collected on days 1, 4 (before the third injection), 8, and 10. Virus concentrations were measured in whole blood by virus titration on Vero cells and in tumors homogenized in PBS. Concentrations of both 5-B07 and mGM-CSF were measured by ELISA in serum and tumor homogenates. In the xenograft human tumor model, LoVo cells were injected subcutaneously into the left flank of Swiss nude mice. Tumor volumes of approximately 120 mm 3 Approximately 2 weeks after reaching the target concentration, the mice were randomized and divided into two groups (15 mice / group). 5 VV of pfu GM A single it injection of -αhCTLA4 (BT-001) was given to a second group, and an intraperitoneal injection of 3 mg / kg 4-E03 was given to a second group. Tumors and blood / serum from 3 mice / time point were collected on days 1, 3, 6, 10, and 20 after virus injection. Viral titers and concentrations of both 4-E03 and hGM-CSF were measured as described in the previous paragraph.

[0267] Antigen-specific T cell response Antigen-specific T cell responses were analyzed in the spleen, treated tumors, and contralateral tumors. 6 The isolated cells were restimulated with 2 μg / ml of tumor (AH-1, SPSYVYHQF) or virus (S9L8, SPGAAGYDL) specific peptides (BioNordika) (Huang et al., 1996; Russell and Tscharke, 2014). Tumor cells were pulsed for 4 hours in the presence of Brefeldin A (Sigma). Isolated splenocytes were restimulated for 48 hours, the last 4 hours in the presence of Brefeldin A. Cytokine-producing CD8 + T cells were identified by FACS staining for CD45, TCR-β, CD8, TNF-α, IFN-γ, and CD25. In parallel, tumor- and virus-specific CD8 +T cells were identified using MHC class I multimers (pentamer H-2Ld-SPGAAGYDL-R-PE(S9L8) ProImmune, pentamer H-2Ld-TPHPARIGL-R-PE(control) ProImmune, dextramer H-2Ld-SPSYVYHQF-APC(AH-1) Immudex, dextramer H-2Ld-TPHPARIGL-APC(control) Immudex).

[0268] Flow cytometry Dead cells were routinely identified using Fixable Viability Dye eFluor™ 780, Fixable Viability Stain 440 UV, or propidium iodide and excluded from analysis along with doublets. Intracellular staining was performed using FoxP3 Staining Buffer Set (Thermo Fisher Scientific). Sample acquisition was performed on either BD FACS Verse or Fortessa II and data were analyzed using FlowJo 10.7.2. Tumor and splenic CD3 +To generate a UMAP of T cells, data was cleaned using the FlowAI tool (v.2.2), and samples were then barcoded and linked by treatment group and organ. The FlowJo plugin UMAP (v3.1) was run on the resulting flow cytometry standard (FCS) files using default settings (distance function: Euclidean, nearest neighbor: 15, and minimum distance: 0.5), including all correction parameters, as well as forward scatter (FSC) and side scatter (SSC) measurements. For cluster discrimination, the FlowJo plugin xshift (v1.3) was run on the resulting UMAP using default settings (nearest neighbor K=82), including the following parameters: CD4, CD62L, CD25, ICOS, FoxP3, Klrg1, CD44, CTLA-4, PD-1, TIM-3, T-bet, GzmB, Ki-67. The scaled channel values ​​obtained from FlowJo were used to calculate the average expression per cluster for the aforementioned parameters. Average expression heatmaps were generated with the parameter means per cluster, scaled between 0 and 1.

[0269] antibody Monoclonal antibodies for flow cytometry: Anti-human CD4-VioGreen (M-T466) Miltenyi Biotec Catalog No. 130-113-259, Anti-human CD25-BV421 (clone M-A251) BD Biosciences Catalog No. 562442, Anti-human CD127-FITC (clone HIL-7R-M21) BD Biosciences Catalog No. 561697, Anti-human CD8-APC (clone RPA-T8) BD Biosciences Catalog No. 555369, Anti-human CTLA-4-PE (clone BNI3) BD Biosciences Catalog No. 555853, Mouse IgG2a,k Isotype Control-PE BD Biosciences Catalog No. 555574, Anti-mouse CD45.2-PerCP-Cy5.5 (clone 104) BD Biosciences Catalog number 552950, ​​anti-mouse CD45.2-BUV737 (clone 104) BD Biosciences Catalog number 612779, anti-mouse CD25-BV421 (clone 7D4) BD Biosciences Catalog number 564571, anti-mouse CD8-BV786 (clone 53-6.7)BD Biosciences Catalog No. 563332, Anti-Mouse CD4-BV510 (clone RM4-5)BD Biosciences Catalog No. 563106, Anti-Mouse TCRb-Alexa Fluor 488 (clone H57-597)BioLegend Catalog No. 109215, Anti-Mouse PD-1-BB700 (clone RMP1-30)BD Biosciences Catalog No. 748242, Anti-Mouse CTLA-4-PECF594 (clone UC10-4F10-11)BD Biosciences Catalog No. 564332, Anti-Mouse CTLA-4-APC (clone UC10-4B9)BioLegend Catalog No. 106310, Anti-Mouse Klrg1-APC (clone 2F1)BD Biosciences Catalog No. 561620, Anti-Mouse CD62L-BUV395 (clone MEL-14)BD Thermo Fisher Scientific Catalog No. 740218, Anti-Mouse TIM3-PE (clone 5D12)BD Biosciences Catalog No. 566346, Anti-Mouse ICOS-BV605 (clone 7E.17G9)BD Biosciences Catalog No. 745254, Anti-Mouse CD44-APC-Cy7 (clone IM7)BD Biosciences Catalog No. 560568, Anti-Mouse Ki67-Alexa Fluor 700 (clone B56)BD Biosciences Catalog No. 561277, Anti-Human Granzyme B-R718 (clone GB11)BD Biosciences Catalog No. 566964, Anti-Mouse Tbet-BV711 (clone O4-46)BD Biosciences Catalog No. 563320, Anti-Mouse FoxP3-PeCy7 (clone FJK-16s)Thermo Fisher Scientific Catalog number 17-5773-82, anti-mouse IFNg-PeCy7 (clone XMG1.2) BioLegend Catalog No. 505826, Anti-Mouse TNFa-Alexa Fluor 700 (Clone MP6-XT22) BD Biosciences Catalog No. 558000, Pentamer H-2Ld-SPGAAGYDL-R-PE(S9L8) ProImmune, Pentamer H-2Ld-TPHPARIGL-R-PE(Control) ProImmune, Dextramer H-2Ld-SPSYVYHQF-APC(AH-1) Immudex, Dextramer H-2Ld-TPHPARIGL-APC(Control) Immudex.

[0270] Secondary antibodies: Goat anti-mouse IgG (H+L) peroxidase Jackson ImmunoResearch Catalog No. 115-035-003, Goat anti-human IgG (H+L) peroxidase Jackson ImmunoResearch Catalog No. 109-035-003, Goat anti-human IgG, Fc fragment specific APC Jackson ImmunoResearch Catalog No. 109-136-098, Goat anti-human IgG-APC Jackson ImmunoResearch Catalog No. 109-136-088, Goat anti-human kappa light chain HRP Bethyl Catalog No. A80-115P, Goat anti-mouse lambda light chain HRP Bethyl Catalog No. A90-121P, Goat anti-mouse IgG-APC Jackson ImmunoResearch Catalog No. 115-136-146, Anti-His MAb, (clone AD1.1.10) R&D Systems Catalog number MAB050, anti-His-HRP (clone AD1.1.10) R&D Systems Catalog number MAB050H.

[0271] Commercially available antibodies used for in vivo experiments: anti-mouse CD8 (clone 53.6.72) BioXCell Catalog No. BP0004-1, anti-mouse CD4 (clone GK1.5) BioXCell Catalog No. BE0003-1, anti-mouse PD1 (clone 29F.1A12) BioXCell Catalog No. BE0273, anti-trinitrophenol rIgG2a isotype control (clone 2A3) BioXCell Catalog No. BE0089, anti-mouse CTLA-4 (clone 9H10) BioXCell Catalog No. BE0131, anti-mouse PD1 (clone RMP1-14) BioXCell Catalog No. BE0146.

[0272] In-house produced anti-mouse and anti-human antibodies isolated from the n-CoDeR phage display library. Anti-mouse CTLA-4 (clone 5-B07) and anti-human CTLA-4 (clone 4-E03) are described herein.

[0273] RNA sequencing Experimental procedure. CT26 tumor cells were implanted into 10 BALB / c mice per group. Approximately 1 week after implantation, tumor volumes increased to 20–50 mm. 3 When the mice reached 10 mg / kg / day (defined as day 0), they were either not treated or had 10 mg / kg / day in 50 μL of 10 7 Depending on the pfu it, unarmed vaccinia virus (VV empty) or VV GM-αCTLA4. Tumors were harvested on day 4 and RNA was extracted using the Qiagen kitRNeasy plus mini kit. Samples were stored at -80°C until the following day of quality assessment. The quality of the purified RNA was assessed using the Agilent RNA 6000 Nano Kit, Agilent 2100 Bioanalyzer System, and 2100 Expert Software to ensure that at least 25% of the RNA fragments were longer than 200 nt (DV200>25%) required for subsequent 3' mRNA sequencing. Strand-specific libraries were prepared and both ends were sequenced (paired-end sequencing) by IntegraGen (France), resulting in pairs of 100 nt long reads.

[0274] Data analysis. Paired reads were processed by a custom bioinformatics pipeline. Briefly, unique molecular identifier (UMI) sequences were extracted from read 1 and sequences at the 3' ends of captured RNA fragments were extracted from read 2. After quality-based trimming and quality control, read 2 was amplified using STAR (Dobin et al., 2013) to align the Mus musculus complete genome (mm10 assembly) plus VV as extra-artificial chromosomes. GMThe reads were then deduplicated using the program dedup with the method "unique" from the suite of tools UMI tools (Smith et al., 2017). Finally, the deduplicated reads were quantified using HTSeq counts (Anders et al., 2015). The read count data per sample was then normalized using DESeq2 (Love et al., 2014), and genes were considered differentially expressed if the fold change between the two conditions was greater than 2 and the adjusted p-value was less than 0.1 (Benjamini-Hochberg correction for multiple testing). Gene Ontology (GO) enrichment analysis was performed using the set of differentially expressed genes as defined above, either upregulated or downregulated, using the function enrichGO (Yu et al., 2012) from the R package clusterProfiler.

[0275] Isolation of Treg-depleting antibodies specific for tumor Treg-associated receptors Antibodies specific for tumor Treg cell-associated receptors were incubated with CD4 T cells from tumor-bearing mice essentially as previously described. + T cell depleted naive cells and CD11b + Tumor-associated Treg cells (isolated from CT26, 4T1, B16, and Lewis lung tumor-bearing mice) were isolated by subjecting an in vitro CDR-shuffled n-Coder® library to differential biopanning against IL-17 cells (Veitonmaki et al., 2013).

[0276] CTLA-4 mAb production Antibody fragments against human / mouse CTLA-4 were isolated from the n-Coder® scFv phage display library. Enrichment of specific CTLA-4 antibodies was achieved by three successive rounds of panning using biotinylated h / mCTLA-4-His protein (Sino Biological) loaded onto streptavidin Dynabeads or polystyrene balls. The third selection round also included suspension-adapted HEK293-EBNA cells transiently transfected with cDNA encoding the extracellular and transmembrane domains of h / mCTLA-4 or an unrelated non-target protein (Sino Biological). Pre-selection was performed before each selection with biotinylated non-target protein. Bound phages were eluted after each selection round by trypsin digestion and amplified on plates using standard procedures. Phagemids from selection 3 were converted to scFv production format and used in subsequent screening assays evaluating specific binding to soluble (recombinant protein) and cell-bound antigens (transiently transfected cells). Commercially available antibodies were used for evaluation of recombinant and surface-bound human (Yervoy, Bristol Myers Squibb; anti-human APC, Jackson), and anti-mouse CTLA-4 (BioLegend) CTLA-4 by flow cytometry, fluorescence microarray technology (FMAT), and ELISA. Corresponding isotype controls were included as negative controls in all experiments. For primary screening of scFvs, h / mCTLA-4 transfected cells were seeded on FMAT plates. E. coli expressed scFvs were added, followed by deglycosylated mouse anti-His antibody (R&D Systems) and anti-mouse APC (Jackson). Stained cells were detected using an 8200 detection system (Applied Biosystems). Positive clones from the primary screen were re-expressed and re-tested by ELISA for binding to the transfected cells and recombinant proteins. For ELISA, E. coli expressed scFv were added to plates coated with h / mCTLA-4 or a non-target protein.Bound scFv was detected using anti-FLAG-AP (Sigma Aldrich) followed by substrate addition (CDP-star, Life Technologies) and luminescence readout (Tecan Ultra).

[0277] A total of 42 and 31 unique clones were converted to hIgG1 and mIgG2a variants, respectively. VH and VL were PCR amplified and inserted into expression vectors containing the heavy and light chain constant regions of the antibodies, respectively, and transfected into suspension adapted HEK 293EBNA cells (ATCC). Culture medium was harvested 6 days after transfection, and antibodies were purified using a column packed with MabSelect (GE Healthcare) connected to an AKTA Purifier system, following standard procedures. Antibodies were eluted with low pH buffer and then dialyzed into the appropriate formulation buffer using a Spectra / Por Dialysis Membrane 4 (Spectrum Laboratories Inc) before final sterile filtration.

[0278] Antibody purity was assessed by CE-SDS (LabChip XII, Perkin Elmer, Massachusetts, USA) and SE-HPLC (Ultimate 3000, Thermo Fisher Scientific). All preparations were low in endotoxin (<0.1EU / mg protein) as determined using the Chromogenic LAL-Endochrome-K kit (Charles River) adapted from the European Pharmacopoeia 2.6.14, current edition, Bacterial Endotoxins, "Method D. Chromogenic Kinetic Method".

[0279] Purified IgG was then assessed for binding to transfected HEK cells, and primary cells, and to recombinant proteins in both ELISA and Biacore.

[0280] Surface plasmon resonance Binding to recombinant proteins was also tested with surface plasmon resonance (SPR) technology using a Biacore 3000. Anti-human Fc (GE Healthcare) was immobilized on a CM5 sensor chip (GE Healthcare) at a concentration of 330 nM as capture antibody. Optimal concentrations of 4-E03 and ipilimumab, along with the recombinant protein, were evaluated in preliminary studies to obtain good curve fitting and limit mass transfer. Antibody (5 nM in this particular experiment) was added at 10 μl / min for 1 min, followed by titrated concentrations (1.6-50 nM for 4-E03 and 1.6-200 nM for ipilimumab) of human CTLA-4 protein (Sino Biological) at 30 μl / min for 3 min. Between each cycle, the surface was regenerated with 10 mM glycine, pH 1.5.

[0281] Cell transfection cDNAs encoding human and mouse (Sino Biological) CTLA-4 were transfected into suspension-adapted 293FT cells (Life Technologies) using Lipofectamine 2000 (Life Technologies). Transfected cells were cultured in FreeStyle™ 293 Expression Medium (Life Technologies) at 37° C., 5% CO2, 120 rpm for 48 hours. Target expression was analyzed using flow cytometry.

[0282] Data Availability The accession number assigned to the RNAseq data reported in this paper is GEO:GSE176052.

[0283] Quantification and statistical analysis All statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software Inc, La Jolla, CA). p values ​​were calculated using Student's t test or one-way ANOVA. Survival to humane endpoints was plotted using the Kaplan-Meier method with analysis for significance by the log-rank test. Significance was accepted when p<0.05.

[0284] result Identification and characterization of Treg-depleting anti-CTLA-4 antibodies Immune checkpoint blockade and anti-CTLA-4 antibody therapy are clinically validated approaches, but the mechanisms underlying anti-CTLA-4 antibody efficacy have not been fully characterized. The central role of CTLA-4 in maintaining tolerance to self-antigens while allowing effective T cell-mediated recognition and elimination of foreign antigen-expressing cells and tumor cells is well established (Leach et al., 1996, Tivol et al., 1995, Waterhouse et al., 1995). Accumulating data suggest that anti-CTLA-4 antibodies, besides acting to lower the threshold at which T cells recognize tumor antigens and reject tumors, may exert therapeutic activity through the depletion of intratumoral Treg cells following antibody interaction with FcγR-expressing effector cells (Peggs et al., 2009, Simpson et al., 2013) (Ingram et al., 2018). Consistent with this, recent data demonstrated a role for FcγR and Treg depletion in the efficacy of anti-CTLA-4 antibodies, including ipilimumab (Arce Vargas et al., 2018).

[0285] Using the target-agnostic FIRST™ discovery platform (Veitonmaki et al., 2013), we identified a series of antibodies and their associated targets that were able to deplete Treg cells and improve survival in a T cell inflammatory CT26 mouse tumor model (data not shown, Fig. 1A and Fig. 1B). Among these, CTLA-4 and anti-CTLA-4 antibodies were identified, with the anti-CTLA-4 mIgG2a mAb depleting intratumoral Treg cells and conferring survival upon treatment of animals bearing syngeneic CT26 tumors (Fig. 1A). Focused screening of human CTLA-4 specific IgG1 antibodies identified several clones with similar in vitro depletion activity of CTLA-4 expressing human T cells (Fig. 1C). One clone (4-E03) showed consistently stronger CTLA-4 depletion activity compared to other clones when screened across multiple donors. + This clone stood out based on its T cell depletion potency (Figure 1C). To investigate the in vivo relevance of the apparently stronger depletion activity of this clone, we turned to a model in which human PBMCs were engrafted into NOD SCID IL-2R gamma (NSG) mice. Due to graft-versus-host type interactions, human Tregs and CD8+ T cells are highly activated and show similar co-stimulatory and co-inhibitory expression compared to those observed in human tumors (Buchan et al., 2018) (Figure 9A). NOG-hPBMC CD4 + CD25 + CD127 低 Tregs and CD8 + Analysis of T cells indeed revealed CTLA-4 expression levels similar to those observed in T cells from nine ovarian cancer patients (Figure 9A). Furthermore, treatment of NOG-hPBMC mice with 10 mg / kg ipilimumab or additional anti-CTLA-4 antibody clones (2-C06 and 2-F09) upregulated human CD4 + CD25 + CD127 低Similar in vivo depletion activity of Treg cells was demonstrated (Figure 1D). However, 4-E03 induced a significantly greater depletion of human Treg cells. Importantly, compared to Treg cells, 4-E03 induced a significantly greater depletion of human Treg cells in both intratumoral and NOG-hPBMC CD8 and NOG-hPBMC CD8. + Consistent with the lower expression of CTLA-4 on T cells (Figure 9A), 4-E03 inhibited human activating CD8 + 4-E03 antibody preparations did not show T cell depletion (Figure 1D). Biochemical characterization of the 4-E03 antibody preparation, especially HPLC-SEC analysis, showed >95% monomeric IgG (data not shown), ruling out that 4-E03-enhanced Treg depletion resulted from antibody aggregation. Consistent with our and others' observations, anti-CTLA-4 mAb Treg depletion was shown to be dependent on antibody Fc:FcγR interactions. An FcγR binding impaired variant of 4-E03 (IgG1N297Q) showed severe impairment of Treg depletion compared to WT FcγR-proficient IgG1 (Figure 9B).

[0286] These findings indicated that 4-E03 binds to a functionally distinct epitope on CTLA-4. Thus, we characterized the binding and ligand blocking activity of 4-E03 compared to ipilimumab and other anti-CTLA-4 antibodies. All antibodies showed high specificity for the extracellular domain of human CTLA-4, with no observable binding to its closely related human homologue CD28 by ELISA (Figure 1E). 4-E03 and ipilimumab bound to hCTLA-4 with similar efficacy and potency (Figure 1E and Figure 9C), but only 4-E03 showed weak but significant cross-reactivity with mouse CTLA-4 (Figure 1E). These findings are consistent with the two antibodies binding to different epitopes.

[0287] In vitro activated human CD4 +Further comparative analysis of 4-E03 and ipilimumab, assessing binding to endogenously expressed CTLA-4 on T cells (Figure 9D and Figure 1F), blockade of B7:CTLA-4 interactions (Figure 1G), or inhibition of B7:CTLA-4-mediated T cell suppression (Figure 1H), revealed otherwise nearly identical potency and efficacy. In conclusion, the results show that 4-E03 binds to a functionally distinct CTLA-4 epitope, which is associated with stronger Treg depletion, while comparable blockade of B7:CTLA-4 induced T effector cell suppression against the epitope targeted by ipilimumab.

[0288] Since 4-E03 only weakly cross-reacted with mouse CTLA-4 (Figure 1E), we next focused our screen to identify a suitable surrogate for in vivo proof-of-concept studies in immune-competent mouse tumor models. Compared to 4-E03 in the human setting, one clone (5-B07) was identified that showed highly specific binding to mouse CTLA-4 transfected CHO cells (Figure 1I) and mouse CTLA-4 protein (Figure 9E), blocking of B7:CTLA-4 interactions (Figure 1J), and similarly robust depletion of intratumoral mouse Tregs (Figure 1K). Furthermore, anti-mCTLA-4 (5-B07) conferred antitumor activity and improved survival of CT26 tumor-bearing BALB / c mice (Figure 1L). As observed with anti-hCTLA-4 (4-E03) on human cells, anti-mCTLA-4 (5-B07) depletion of mouse Tregs was found to be dependent on Fc:FcγR interactions: Fc:FcγR binding-proficient, but not Fc:FcγR binding-impaired variants of 5-B07 depleted intratumoral Tregs (Figure 9F).

[0289] The similar pronounced Treg depletion activity, along with their similar high specificity for CTLA-4 and blocking activity of CTLA-4:B7 family interactions, indicated the therapeutic potential of anti-hCTLA-4 (4-E03) and anti-mCTLA-4 (5-B07) as suitable MoA-matched surrogates.

[0290] Engineering antibody-encoding oncolytic viruses for tumor-selective CTLA-4 blockade and Treg depletion The frequent side effects of anti-CTLA-4 antibody therapy are consistent with the well-established role of CTLA-4 acting as a central checkpoint to maintain T cell homeostasis and tolerance to self (Tivol et al., 1995, Waterhouse et al., 1995). However, recent studies by Quezada et al. have shown that intratumoral Treg depletion can significantly contribute to ipilimumab clinical activity (Arce Vargas et al., 2018), and that intratumorally delivered Treg-depleting antibodies can result in substantial anticancer activity in mouse tumor models (Fransen et al., 2013, Marabelle et al., 2013b). This dual activity of anti-CTLA-4, acting in the central and peripheral compartments, respectively, suggests that localizing anti-CTLA-4 therapy to the tumor may be an attractive strategy to uncouple anti-CTLA-4 efficacy from toxicity.

[0291] We hypothesized that intratumorally delivered oncolytic viruses (OVs) engineered to express Treg-depleting anti-CTLA-4 represent a particularly attractive means to achieve effective, yet safe, tumor-localized anti-CTLA-4 therapy. Besides enabling local antibody production and CTLA-4 receptor blockade, as well as Treg depletion in the TME, upon infection of tumor cells, OVs are believed to exert both direct and indirect anti-cancer activity and have been approved for cancer immunotherapy (Bommareddy et al., 2018).

[0292] Therefore, we engineered vaccinia virus vectors derived from the attenuated Copenhagen strain (Foloppe et al., 2019) with full-length anti-hCTLA-4 or anti-mCTLA-4 IgG antibody sequences, which have clinically proven safety and potent immunomodulatory effects observed in global smallpox vaccination programs, as well as cytolytic and inflammatory cell infiltration-inducing properties in mouse experimental models of immune desert and immune-exclusion cancers (Fend et al., 2017, Kleinpeter et al., 2016, Liu et al., 2017, Marchand et al., 2018). GM Variant vectors additionally encoding CTLA4 (Figure 2A), a growth factor inducer and enhancer of myelopoiesis and innate immune cell chemotaxis, were also produced and evaluated for therapeutic efficacy.

[0293] After gene reconstitution, the recombinant anti-CTLA-4-encoding virus was confirmed to infect, replicate (Figure 2B), and lyse (Figure 2C) tumor cell lines. Tumor cell lines infected with the engineered oncolytic viruses were further shown to produce full-length IgG antibodies and GM-CSF transgenes (Figures 2D and 2E) with equipotent binding to the CTLA-4 receptor (Figure 2G) and support of GM-CSF-dependent TF-1 cell proliferation (Figure 2F) compared to recombinantly produced proteins. 4-E03 produced by BT-001-infected MIA-PaCa-2 tumor cells was also shown to deplete human Treg cells in vivo (Figure 2H).

[0294] Intratumoral VV GM -αCTLA4 has antitumor activity associated with tumor-selective CTLA-4 receptor saturation and Treg depletion VV GM -αCTLA4 antitumor activity was first evaluated in the CT26 BALB / c model, known to be highly infiltrated by T cells and sensitive to systemic anti-CTLA-4 antibody treatment (Grosso and Jure-Kunkel, 2013). 4 , 7.5×10 5, or 7.5 × 10 6 VV of pfu GM Three intratumoral injections of CT26 tumor-bearing animals with -αCTLA4 demonstrated a dose-dependent antitumor effect, which was observed at 10 6 ~10 7 pfu, with 6-7 of 10 animals cured (Figure 3A). Treatment with anti-CTLA-4 antibody and / or control virus lacking the GM-CSF transgene demonstrated obligate dependence on anti-CTLA-4 antibody (0 / 10 mice survived) and a modest anti-CTLA-4 enhancing effect of GM-CSF on therapeutic efficacy (7 / 10 vs. 5 / 10 mice survived). Thus, based on the established dose-dependent, anti-CTLA-4 antibody-dependent, and GM-CSF enhancing effects, we chose to administer 1x10 7 An intratumorally delivered dose of 100 pfu was used to express the dual transgene-encoding OV (VV GM We investigated the therapeutic and mechanistic evaluation and characterization of CTLA-α (-αCTLA4).

[0295] We next assessed tumor and systemic concentrations of anti-CTLA-4 (Figure 3B), GM-CSF (Figure 10A), and viral particles (Figure 10B) following intratumoral administration of vaccinia OV encoding the transgenes. 7 VV GM Intratumoral injection of VV-αCTLA4 infectious particles into syngeneic murine tumor-bearing immunocompetent mice resulted in intratumoral antibody exposure associated with sustained saturation of CTLA-4 expressing cells in the tumor, but not in the blood (Figure 3B-Figure 1I). GM It administration of VV-αhCTLA4 to immune-deficient mice bearing human tumor xenografts resulted in antibody concentrations several orders of magnitude higher in the tumor compared to blood (Figure S10C-E). Consistent with intratumoral administration, VV achieved receptor saturation concentrations within the tumor but not in the systemic compartment (Figure 3B). GM -αCTLA4 led to almost complete depletion of intratumoral Treg cells in the spleens of CT26 tumor-bearing BALB / c mice, but had no effect on Treg numbers ( Fig. 3C ).

[0296] Collectively, the results demonstrate that intratumoral administration of vaccinia virus encoding anti-CTLA-4 successfully achieves tumor-restricted CTLA-4 receptor saturation and Treg depletion in vivo, supporting the tumor-selective therapeutic properties of anti-CTLA-4 and prompting testing of in vivo efficacy and tolerability in various experimental cancer models.

[0297] VV GM -αCTLA4 has broad antitumor activity We demonstrate the efficacy and safety of itVV in a range of immune-competent mouse cancer models, spanning hematological (A20) and solid tumors of different origins on different genetic mouse backgrounds (CT26 BALB / c colon, EMT6 BALB / c breast, MC38 C57BL / 6 colon, and B16 C57BL / 6 melanoma, Figure 4A), representing highly T cell inflammatory (CT26) to immune exclusionary (B16) tumor microenvironments. GM We then proceeded to evaluate the antitumor activity of -αCTLA4 in models that were either sensitive or resistant to ICB with anti-CTLA-4 or anti-PD-1.

[0298] Surprisingly, VV was administered to C57BL / 6 or BALB / c mice bearing established syngeneic tumors characterized by a diverse immunoinflammatory tumor microenvironment. GM It administration of -αCTLA4 cured the majority of animals (A20=10 / 10, EMT6=8 / 10, MC38=8 / 10, and CT26=10 / 10 surviving mice) (Figure 4A). Equally impressive, in the B16 C57BL / 6 model, characterized by an immune desert TME and resistance to both anti-PD-1 and anti-CTLA-4, itVV GM -αCTLA4 significantly delayed tumor growth and cured 3 / 10 animals. These results support the efficacy of VV in a variety of cancer types, including patients with diverse inflammatory and immune-negative TMEs. GM This demonstrated the broad therapeutic potential of -αCTLA4.

[0299] VV GM Intratumoral treatment with -αCTLA4 induces long-lasting systemic antitumor immunity Preclinical and clinical studies have demonstrated the therapeutic potential of tumor-localized cancer immunotherapy. Intratumoral oncolytic virotherapy alone (Andtbacka et al., 2015) or combined with ICB (Chesney et al., 2018; Ribas et al., 2017) induces durable responses in melanoma cancer patients. Mechanistically, it oncovirus therapy induces or enhances inflammatory cell infiltration into injected tumors, leading to increased tumor antigen presentation, migration to draining lymph nodes, and CD8+ delivery to distant (non-injected) tumor lesions after priming. + It has been proposed to result in T cell trafficking to exert a systemic antitumor "abscopal" effect (Ngwa et al., 2018). In the clinic, such induction of a systemic adaptive antitumor memory response will be important, as cancer patients may present with widespread disease characterized by metastatic, undetectable, or uninjectable tumors.

[0300] We used a multi-pronged approach to identify the itVV GM We assessed whether -αCTLA4 induces abscopal effects and systemic antitumor immunity. First, we used a "twin tumor model" in which tumor cells are implanted subcutaneously into the left and right flanks of each animal, but only one tumor is injected with OV and the other is left untreated, to assess the abscopal effect, which can be shown as a reduction in tumor growth in uninjected tumors.

[0301] Maximally effective VV in CT26 tumor-bearing mice GM Intratumoral injection of a dose of -αCTLA4 resulted in complete rejection of injected tumors (9 / 9) and near complete rejection of uninjected tumors (7 / 9), demonstrating a strong abscopal effect (Figure 4B). The true abscopal nature of itOV administration was confirmed two-fold. First, to exclude a potential therapeutic effect of viral particle diffusion from injected to uninjected tumors, uninjected tumors were analyzed and found to be negative for viral particles (Figure 11A).

[0302] Second, using a twin tumor model, we demonstrated that VV administered locally (it) or systemically (iv) GM -Maximally effective dose of αCTLA4 (10 7 pfu and suboptimal dose (10 5 We compared the antitumor activity of 1000 mg / kg / day (pfu) of 1000 mg / kg / day ... 5 It administration of pfu was at least as effective as a 100-fold higher iv injected dose in rejecting uninjected tumors (Figure 11B and 11C). Finally, consistent with itOV administration inducing immunological memory characteristic of adaptive antigen-specific immune responses, cured animals were protected against rechallenge with the same tumor (CT26), but not with an unrelated tumor (Renca) (Figure 11D).

[0303] VV GM -αCTLA4 inhibits robust systemic CD8 + Eliciting T cell-dependent antitumor immunity The present inventors have + T cell depletion or CD8 + Immunocompromised VV compared with T cell-depleted CT26 tumor-bearing mice GM We investigated the nature of the systemic antitumor immune response by evaluating the therapeutic activity of CD8 + T cell exhaustion is VV GM -αCTLA4 was completely eliminated. CD4 <8172> +<!--8172--> T cell depletion leads to VV GM These data suggest that VV reduced, but did not eliminate, the -αCTLA4 effect. GM -αCTLA4 antitumor activity, but CD8 + In addition to the demonstrated abscopal effect and tumor-specific protection against rechallenge, the results demonstrate that intratumorally delivered VV GM -αCTLA4 inhibits robust systemic CD8 + These results strongly suggest that T cell antitumor immunity was induced.

[0304] Therefore, we next GM -αCTLA4 inhibits tumor-specific and virus-specific CD8 + We evaluated whether VV can induce or expand T cells. CT26 tumor-bearing BALB / c mice were cultured with VV GM Patients were treated intratumorally with -αCTLA4 or systemically (ip) with anti-mCTLA-4 mAb 5-B07 (3 mg / kg) to mimic clinically available anti-CTLA-4 regimens. CT26 tumor-specific and vaccinia-specific CD8 + T cells were quantified by two approaches: direct quantification of tumor-specific CD8+ T cells in harvested spleens using CT26 tumor antigen (AH-1)-specific and vaccinia virus-specific multimers, and IFN-γ following ex vivo stimulation of splenocytes. + TNF-α + CD8 + Evaluation of T cells (Figure 5B) or TILs with the CT26-derived tumor peptide AH-1 and the vaccinia-derived peptide S9L8, respectively. Treatment with PBS or VV encoding only GM-CSF was included as controls. As expected, robust systemic CD8 + Intratumoral VV induces T cell-dependent antitumor immunity GM Consistent with -αCTLA4, itVV GM -αCTLA4 increased tumor-specific CD8 expression in both injected tumor and peripheral (non-injected tumor and splenic) compartments, as assessed by ex vivo stimulation of splenocytes or dextramer staining. + Impressively, itVV induced T cells (Figures 5B to 5D and 13B). GM -αCTLA4 inhibits tumor-specific CD8 + Control treatment with PBS or virus-deficient αCTLA-4 expanded tumor-specific CD8 T cells more effectively by both readouts. + Interestingly, VV GM It treatment with -αCTLA4 also inhibited vaccinia-specific CD8 +T cells were induced.

[0305] Taken together, these data support the notion that intratumoral VV GM -αCTLA4 inhibits robust systemic CD8 + Induced T cell-dependent antitumor immunity.

[0306] Intratumor-induced CD8+ T cell antitumor immunity is FcγR-dependent and correlates with Treg depletion Broad antitumor activity, tumor-specific CD8 in tumors and the periphery + Strong expansion of T cells as well as tumor-restricted depletion of Treg cells were observed in the itVV GM This supported highly effective and safe treatment with -αCTLA4.

[0307] To further evaluate and confirm the role of antibody-mediated Treg depletion underlying antitumor immunity, we investigated the role of antibody-mediated Treg depletion in CT26 tumor-bearing WT and common gamma chain-deficient (Fcer1g - / - ) itVV in BALB / c mice GM The antitumor effects of Fcer1g and Fcer1g-αCTLA4 were compared. - / - Mice lack functional activating Fc gamma receptors and anti-CTLA-4 antibody in vivo Treg depletion and associated anti-cancer activity have previously been shown to be activating FcγR dependent (Arce Vargas et al., 2018; Simpson et al., 2013). GM Consistent with anti-CTLA-4-induced Treg depletion underlying -αCTLA4 antitumor immunity, WT (10 / 10) but not FcγR-deficient animals (3 / 10) were fully protected and cured of their cancers (Figure 6A). The limited but significant antitumor activity observed in FcγR-deficient animals was consistent with our and others' observations that viral vectors (Figure 4A) and CTLA-4:B7 blockade, by themselves, in the absence of Treg depletion (Figures 9F and 12A), delayed tumor growth but conferred only a limited survival advantage.

[0308] In addition to effecting immune effector-mediated ADCC and ADCP of antibody-coated target cells, FcγRs have been shown to promote tumor antigen cross-presentation (DiLillo and Ravetch, 2015), allowing CD8 to mediate tumor antigen cross-presentation to encompass MHCII-restricted extracellular tumor antigens that are normally excluded. + Expanding and enhancing T cell antitumor responses. GM -αCTLA4 antitumor immunity is FcγR dependent and tumor-specific CD8 + Induce more robust expansion of T cells and additionally virus-specific CD8 + The finding that induced T cells in the IL-17 / IL-17 / IL-17 / IL-17 / IL-17 mice showed that it may also promote tumor antigen cross-presentation. This concept was supported by a comparison of VV-injected and untreated CT26 tumor-bearing mice. GM This was reinforced by differential gene expression analysis of tumors taken from mice injected with VV-αCTLA4 (Figures 6B and 6C). In addition to upregulating Batf per se, differential gene analysis demonstrated upregulation of type I IFN response and CD8α markers associated with antigen cross-presenting cDC1 dendritic cells. Additional signatures were identified in VV GM The CD8-αCTLA4-α expression mediated by the CD8-α CTLA4-α β-cells is essential for the induction and activation of antitumor immunity. + supported T cell-dependent (and potentially NK cell-mediated granzyme-dependent) tumor cell lysis.

[0309] VV GM To assess the role of antigen cross-presentation in α-CTLA4-induced antitumor immunity, we generated mice lacking the transcription factor Batf3 (Batf3 - / - Mouse) was used. Batf3 - / - Mice are CD8α + They lack dendritic cells and, as a consequence, have defective antigen cross-presentation and CD8 expression against viruses during infection and against tumor antigens in mouse experimental models of cancer. +These mice show severe impairment of T cell responses (Hildner et al., 2008). Furthermore, cDC1 and antigen cross-presentation are known to mediate the therapeutic activity of immune checkpoint blockade, including αCTLA-4 (Gubin et al., 2014). Therefore, we investigated the effect of cDC1 on the treatment of Batf3+ / - MC38 tumor-bearing mice. + / + and Batf3 - / - itVV in C57BL / 6 mice GM We compared the antitumor activity of Batf3-αCTLA4 and CTLA4-αCTLA4. Strikingly, Batf3 deficiency reduced tumor cell survival in 0 / 8 Batf3 mice compared with 9 / 9 WT mice. - / - As demonstrated by the itVV GM -αCTLA4 abrogated antitumor immunity (Figure 6D).

[0310] Taken together, these results suggest that VV GM -αCTLA4 has both FcγR- and cDC1-dependent antitumor activity, identifying intratumor-induced Treg depletion and tumor antigen cross-presentation as the primary mechanisms, and intratumoral CTLA-4:B7 blockade and tumor lysis as supporting mechanisms, and basal itVV GM -αCTLA4, CD8 + Induced T cell antitumor immunity.

[0311] Intratumoral anti-CTLA-4-VV inhibits peripheral effector CD8 + Expand T cells, Tregs and exhausted CD8 + Reduces T cells The inventors have demonstrated how itVV GM We proceeded to qualitatively characterize how itVV regulates TIL responses in the injected and adjacent tumors, as well as in the periphery. Using multicolor flow cytometry and a high-dimensional antibody panel designed to identify functionally distinct anti- and pro-tumor TIL subsets, we identified 12 T cell clusters across treatment groups (Figures 7 and 13C). Strikingly, itVV GM -αCTLA4 was exhausted (PD-1) in injected tumors compared to mock-treated animals. + TIM-3+ )CD8 + Klrg1 eliminates T cells and prevents exhaustion + Effector CD8 + At the same time, in agreement with the above findings, itVV strongly expanded T cells (Fig. 7). GM -αCTLA4 expresses high levels of CTLA-4 and specifically inhibits Klrg1, which is known to be inhibitory. + CTLA-4, including Tregs + It effectively depleted intratumoral Tregs (Nakagawa et al., 2016) (Figure 7).

[0312] Evaluation of adjacent distant tumors that were not injected with the antibody-encoding virus was similar, but GM Furthermore, intratumoral administration of an oncolytic virus encoding αCTLA4 significantly increased tumor-specific CD8 + Consistent with our observation that itVV expanded T cells (Figures 5B-5D). GM -αCTLA4 inhibits activated granzyme B in the spleen + (Klrg1 + )CD8 + Finally, consistent with the antibody-encoding virus achieving tumor-restricted Treg depletion, the Treg populations depleted in the tumor bed were induced by itVV. GM -αCTLA4 was not significantly altered in the spleen (Fig. 7A).

[0313] Combining intratumoral anti-CTLA-4-VV with anti-PD-1 to reject "cold" distant tumors The inventors' observations indicate that VV GM We hypothesized that α-CTLA4 acts locally in injected tumors, primarily through mechanisms involving anti-CTLA-4 mAb-dependent tumor antigen cross-presentation and Treg depletion, to mediate systemic adaptive antitumor immunity and robust peripheral tumor-specific CD8 + These findings suggest that VV GM -αCTLA4, but CD8 +Anti-PD-1 has shown the potential to synergize with therapeutic agents that help recruit T cells to tumors, primarily through reversal of T cell exhaustion (Hui et al., 2017; Wei et al., 2018), and in some cases stem-like memory CD8 + It is thought to act by recruiting T cells to tumors (Galletti et al., 2020; Simon et al., 2020). Despite evidence that anti-PD-1 can improve survival in multiple solid tumors of different origins, anti-PD-1 does not improve outcomes in patients with poorly immune infiltrated "cold tumors" (Galon and Bruni, 2019), which perhaps represents the greatest unmet medical need in cancer therapy today.

[0314] Therefore, based on their apparently distinct and potentially complementary mechanisms of action, we next investigated the efficacy and safety of anti-PD-1 and VV combination therapy, focusing on ICB-resistant, poorly immune-infiltrated, and poorly immunogenic “cold” cancers, using B16 C57BL / 6 as a model system. GM We investigated the synergistic effects of ICB with anti-PD-1 (10 mg / kg), anti-CTLA-4 (10 mg / kg), or their combination. Previous data demonstrated that B16 tumors are refractory to ICB therapy, including clinically relevant systemic administration of anti-PD-1 (10 mg / kg), anti-CTLA-4 (10 mg / kg), or their combination (Figure 14).

[0315] To mimic the clinical situation in which large palpable tumors are injected with virus encoding an antibody, but small or undetectable metastatic lesions cannot be injected, we performed a randomized controlled trial in which animals had one "large" and one "small" tumor and only the large tumor was injected with virus encoding an antibody. GM We established a twin tumor B16 / C57BL6 model in which PD-1 was injected it with -αCTLA4. Resistance to anti-PD-1 was confirmed by the lack of tumor growth inhibition or survival benefit after systemic treatment with a maximally effective dose of 10 mg / kg (Figure 8A).

[0316] As previously observed, V.V. GMSingle-agent treatment with -αCTLA4 significantly reduced tumor growth of the primary injected tumors (Figures 4 and 8B). GM -αCTLA4 induced only a slight delay in the growth of uninjected tumors (Figure 8B), which did not translate into survival of the animals (Figure 8A). Surprisingly, in contrast to subsequent single-agent or combined anti-PD-1 and anti-CTLA-4 treatments, itVV GM Combined treatment with -αCTLA4 and systemic anti-PD-1 significantly suppressed injected and non-injected tumor growth, and approximately 20% of bearing animals were cured in this ICB treatment-resistant model of "cold" cancer (Figure 8A).

[0317] Furthermore, VV can convert cold, ICB-resistant tumors into a proinflammatory, ICB-responsive phenotype. GM Consistent with -αCTLA4, VV GM Combined treatment with -αCTLA4 (but not anti-PD-1 alone) induced a robust influx of T cells into B16 tumors, which became similarly densely T cell enriched compared to inflamed CT26 tumors (Figure 14C).

[0318] These are the combined itVV GM We show that the synergistic effect of -αCTLA4 and systemic anti-PD-1 was confirmed in BALB / c mice implanted with syngeneic A20 tumors. This model was shown to be hemiresponsive to anti-PD-1, with approximately 20% of the animals cured by full treatment ip dosing (10 mg / kg) (Figure 8C). GM Optimal therapeutic dosing with -αCTLA4 was completely protective (10 / 10 animals were cured, FIG. 4), whereas suboptimal treatment at 1 / 100 of the optimal dose showed non-significant tumor growth inhibition and no survival advantage. GM -αCTLA4 combination cured the majority of animals (7 / 10) (Figure 8C).

[0319] Consideration The inventors have shown that oncovirally encoded Treg-depleting αCTLA-4 has more potent and broader antitumor activity compared to approved systemic αCTLA-4 regimens, yet is safe and well tolerated through the tumor-confined nature of its exposure. GM -αCTLA4 inhibits tumor-specific CD8 + It induced a stronger expansion of T cells and had antitumor activity in a poorly immune-infiltrated "cold" syngeneic mouse tumor model that was resistant to clinically relevant challenge with systemic αCTLA-4 and αPD-1. Notably, our observations support the strong systemic antitumor immunity induced by the oncovirus αCTLA-4, which is strictly derived from an "immune ignition" effect in the injected tumor, itVV. GM These results suggest that -αCTLA4 was not associated with viral spread or antibody exposure to distant uninjected tumors, but rather achieved tumor-restricted CTLA-4 receptor saturation and Treg depletion.

[0320] These observations are GM - has important implications for both the expected clinical efficacy and tolerability of αCTLA-4. From an efficacy perspective, we have demonstrated that oncovirally encoded αCTLA-4 may provide greater therapeutic benefit compared to available (ipilimumab) and Treg-depleted optimized (Arce Vargas, Furness et al. 2018) or "masked" (Gutierrez, Long et al. 2020) systemic αCTLA-4 antibody regimens, as well as compared to previously described oncolytic virus approaches encoding non-Treg-depleted optimized αCTLA-4 (Aroldi, Sacco et al. 2020). At the mechanistic level, VV GM -αCTLA4-induced FcγR-dependent Treg depletion and cDC1 + Antigen cross-presentation was observed with robust CD8 +It underlies T cell proliferation and synergistic therapy with αPD-1 to reject cold tumors. Besides mediating the induction of endogenous antitumor immune responses (Hildner, et al. 2008) and the efficacy of systemic checkpoint blockade therapy (Gubin, Zhang et al. 2014; Salmon, Idoyaga et al. 2016; Garris, Arlauckas et al. 2018), cDC1s mediate intratumoral CD8 + Promotes the proliferation response of TILs and TCF1 + Expanding the pool of stem-like precursors and TIM3 during αPD-1 therapy + Induces the production of terminal effectors (Mao, et al. 2021).

[0321] Similarly, Treg depletion achieved using mAbs against costimulatory or co-inhibitory receptors, such as IL-2R and CTLA-4, was not associated with CD8 + It promotes effector function and may synergize with αPD-1 (Wei, et al. 2019; Solomon, et al. 2020). It reduces Treg and anti-tumor CD8 + Regarding the development of “dual-activity” immunomodulatory antibodies to expand T cells, accumulating data support the understanding of target biology, effector CD8 + We demonstrate both the fine-tuning of T cell enhancing and Treg depleting properties, as well as the importance of the delivery regimen. For example, we recently demonstrated a novel IL-2-mediated CD8+ T cell agonist that depletes Tregs but conserves the critical (IL-2-mediated) growth-survival signaling of CD8+ T cells. + FcγR-competent non-ligand blocking antibodies against IL-2R, which do not starve effector T cells, have been demonstrated to have superior therapeutic potential in cancer therapy compared to ligand blocking antibodies αIL-2R antibodies (Solomon, et al. 2020).

[0322] Similarly, but differently, we recently reported that antibodies against 4-1BB can be engineered to either deplete Tregs or promote effector T cell expansion by antibody isotype switching (altering FcγR binding), but harnessing both mechanisms requires sequential administration or hinge engineering (Buchan et al., 2018). As described herein in the context of oncolytic virus infection, spatial restriction of Treg depletion-enhancing function-blocking anti-CTLA-4 to injected tumors appears to be a particularly promising approach to harness the maximal therapeutic activity of immunomodulatory anti-CTLA-4 antibodies when used alone or in combination with synergistic checkpoint blockade therapies, e.g., anti-PD-1 / L1.

[0323] Several observations support the optimization of Treg depletion in anti-CTLA-4 for tumor-localizing therapy. First, independent studies have established that the therapeutic efficacy of anti-CTLA-4 depends on and correlates with Treg depletion (Arce Vargas et al., 2018; Simpson et al., 2013). The data presented herein on the therapeutic activity of Treg-depleted anti-CTLA-4 clones demonstrates a stronger therapeutic effect in FcγR-competent (Treg-depleted) antibody Fc formats and hosts compared to FcγR-deficient (non-depleted) counterparts, supporting this concept. Second, although clinical outcomes in melanoma patients treated with ipilimumab were recently reported to correlate with FcγR binding and Treg depletion, our data from T cell humanized mouse models suggest that ipilimumab has limited depleting activity against human Treg cells expressing relevant levels of CTLA-4 in tumors, compared to the vectorized anti-CTLA-4 antibody 4-E03 herein. Furthermore, although clinically tolerated doses of ipilimumab (1 mg / kg-3 mg / kg depending on indication and regimen) are associated with only subsaturating CTLA-4 receptor occupancy and submaximal effects (Ribas et al., 2005) (Bertrand et al., 2015), our data demonstrate that oncolytic vectorization and it administration can produce therapeutically optimal exposure (sustained CTLA-4 receptor saturation) in an apparently safe manner, even with Treg depletion-enhanced anti-CTLA-4. Finally, in support of our vectorization of Treg depletion-enhancing and checkpoint blocking “dual activity” αCTLA-4 antibodies, antibody-mediated CTLA-4 blockade has recently been shown to inhibit tumor-specific CD8 + It has been shown to synergize with FcγR-dependent depletion in improving T cell responses. Antibody blockade of CTLA-4 functionally destabilizes intratumoral Tregs, inhibiting B7:CD28 costimulation and antitumor CD8 through a process that includes altered glycolysis and competition for B7 ligands. + Promoted T effector function (Zappasodi, et al. 2021).

[0324] The fact that the full therapeutic dose achieved tumor-restricted anti-CTLA-4 exposure indicates that severe toxicity associated with sustained systemic Treg depletion, such as that observed in the FoxP3-DTR mouse model (Kim et al., 2007), is unlikely to occur. Similarly, since the anti-CTLA-4 checkpoint blockade effect is limited to TILs with tumor antigen specificity, the adverse autoreactivity associated with systemic anti-CTLA-4 should be minimal. In contrast, but consistent with the well-documented central immune checkpoint nature of CTLA-4 (Chambers et al., 1996; Leach et al., 1996), anti-CTLA-4 side effects associated with systemic administration, i.e., body-wide antibody exposure, may be of a severe autoimmune nature and may have fatal consequences (Tivol et al., 1995; Waterhouse et al., 1995).

[0325] Thus, taken together, while anti-CTLA-4 efficacy and tolerability have previously been thought to be dose-dependent, precluding the use of full therapeutic doses of anti-CTLA-4-based regimens, these findings strongly suggest that the spatial restriction of vectored Treg-depleting αCTLA-4 could overcome these current limitations, uncoupling efficacy from tolerability.

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Claims

1. for use in treating cancer in a patient, an oncolytic virus capable of expressing a first antibody molecule that specifically binds to CTLA-4; a second antibody molecule that specifically binds to PD-1 and / or PD-L1, The combination, wherein said cancer comprises or consists of a cold tumor.

2. In the manufacture of a medicament for treating cancer in a patient, an oncolytic virus capable of expressing a nucleotide sequence encoding a first antibody molecule that specifically binds to CTLA-4; - a second antibody molecule that specifically binds to PD-1 and / or PD-L1, The use, wherein the cancer comprises or consists of a cold tumor.

3. an oncolytic virus capable of expressing a first antibody molecule that specifically binds to CTLA-4; a second antibody molecule that specifically binds to PD-1 and / or PD-L1, 1. A pharmaceutical composition for treating cancer in a patient, comprising: The pharmaceutical composition, wherein the cancer comprises or consists of a cold tumor.

4. 1. A pharmaceutical composition for treating cancer in a patient, comprising an oncolytic virus capable of expressing a first antibody molecule that specifically binds to CTLA-4, the pharmaceutical composition is used in combination with a second antibody molecule that specifically binds to PD-1 and / or PD-L1; The pharmaceutical composition, wherein the cancer comprises or consists of a cold tumor.

5. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, wherein said cold tumor is treated by said first and second antibody molecules.

6. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, wherein said antibody molecule that specifically binds to CTLA-4 is an Fcγ receptor binding antibody.

7. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, wherein said antibody molecule that specifically binds to CTLA-4 is a Treg-depleting antibody.

8. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, wherein said oncolytic virus is an oncolytic poxvirus.

9. 9. The combination for use, or use, or pharmaceutical composition according to claim 8, wherein the poxvirus belongs to the subfamily Chordopoxvirinae, more preferably to the genus Orthopoxvirus, preferably selected from the group consisting of vaccinia virus, cowpox virus, canarypox virus, ectromelia virus, and myxoma virus.

10. The combination for use, or use, or pharmaceutical composition according to claim 9, wherein the oncolytic virus is a vaccinia virus that is defective in both thymidine kinase (TK) and / or ribonucleotide reductase (RR) activity and comprises a nucleotide sequence encoding SEQ ID NO: 20 and SEQ ID NO: 21, or SEQ ID NO: 53 and SEQ ID NO:

54.

11. 10. The combination for use, or the use, or the pharmaceutical composition according to claim 9, wherein the vaccinia virus further comprises a nucleotide sequence encoding GM-CSF, with human GM-CSF (e.g. having SEQ ID NO: 55 or SEQ ID NO: 56) or murine GM-CSF (e.g. having SEQ ID NO: 57 or SEQ ID NO: 58) being particularly preferred.

12. 5. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, wherein the virus comprises a nucleotide sequence encoding the heavy chain of said first antibody molecule inserted into the viral J2R locus and / or comprises a nucleotide sequence encoding the light chain of said first antibody molecule inserted into the viral I4L locus.

13. 5. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, wherein the first antibody molecule is selected from the group consisting of antibody molecules comprising 1 to 6 of the CDRs selected from the group consisting of SEQ ID NOs: 3, 6, 8, 10, 12, and 14.

14. the first antibody molecule is selected from the group consisting of antibody molecules comprising 1 to 6 of the CDRs, VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, and VL-CDR3; - VH-CDR1, if present, is selected from the group consisting of SEQ ID NOs: 15, 22, 29 and 35; - VH-CDR2, if present, is selected from the group consisting of SEQ ID NOs: 16, 23, 30 and 36; - VH-CDR3, if present, is selected from the group consisting of SEQ ID NOs: 17, 24, 31 and 37; - VL-CDR1, if present, is selected from the group consisting of SEQ ID NOs: 10 and 38; - VL-CDR2, if present, is selected from the group consisting of SEQ ID NOs: 18, 25, 32 and 39; 5. The combination or use or pharmaceutical composition for use according to any one of claims 1 to 4, wherein VL-CDR3, if present, is selected from the group consisting of SEQ ID NOs: 19, 26 and 40.

15. 5. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, wherein said first antibody molecule comprises six CDRs having SEQ ID NOs: 15, 16, 17, 10, 18, and 19, or said six CDRs having SEQ ID NOs: 22, 23, 24, 10, 25, and 26.

16. 5. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, wherein said first antibody molecule comprises a variable heavy chain selected from the group consisting of SEQ ID NOs: 20 and 27, and / or a variable light chain selected from the group consisting of SEQ ID NOs: 21 and 28.

17. 5. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, wherein said first antibody molecule comprises the heavy chain constant region SEQ ID NO: 43 and / or the light chain constant region SEQ ID NO:

44.

18. 5. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, wherein said first antibody molecule is selected from the group consisting of ipilimumab and tremelimumab.

19. 14. The combination for use, or use, or pharmaceutical composition according to any one of claims 1 to 4, wherein the first antibody molecule is an antibody molecule capable of competing for binding to CTLA-4 with an antibody molecule as defined in claim 13.

20. The first antibody molecule may be a full-size antibody, a chimeric antibody, a single-chain antibody, a Fab, an Fv, an scFv, an Fab', and an (Fab') ２ 5. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, selected from the group consisting of:

21. 5. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, wherein the first antibody molecule is selected from the group consisting of a human IgG antibody, a humanized IgG antibody, and an IgG antibody of human origin.

22. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, wherein said virus comprises a nucleotide sequence encoding a first antibody molecule as defined in claim 13.

23. 23. The combination for use, or the use, or the pharmaceutical composition according to claim 22, wherein said nucleotide sequence comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 45 to 52.

24. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, wherein the second antibody molecule is selected from the group consisting of a human antibody molecule, a humanized antibody molecule, and an antibody molecule of human origin.

25. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, wherein said second antibody molecule is a monoclonal antibody molecule or an antibody molecule of monoclonal origin.

26. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, wherein the second antibody molecule is selected from the group consisting of a full-size antibody, a chimeric antibody, a single-chain antibody, and an antigen-binding fragment thereof.

27. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, wherein the second antibody molecule is a human IgG antibody, a humanized IgG antibody molecule, or an IgG antibody molecule of human origin.

28. 5. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, wherein the second antibody molecule specifically binds to PD1 and is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, camrelizumab, spartalizumab, dostallimab, tislelizumab, JTX-4014, sintilimab (IBI308), toripalimab (JS 001), AMP-224, and AMP-514 (MEDI0680).

29. 5. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, wherein the second antibody molecule specifically binds to PD-L1 and is selected from the group consisting of atezolizumab, durvalumab, avelumab, CS1001, KN035 (embafolimab), and CK-301.

30. The combination for use, or use, or pharmaceutical composition according to any one of claims 1 to 4, wherein the cold tumor is an immune desert tumor, and / or an immune excluded tumor, and / or a tumor with poor immune infiltration.

31. 5. The combination for use, or use, or pharmaceutical composition according to any one of claims 1 to 4, wherein the cold tumor is selected from the group consisting of melanoma, pancreatic cancer, prostate cancer, colorectal cancer, hepatocellular carcinoma, lung cancer, bladder cancer, kidney cancer, gastric cancer, cervical cancer, Merkel cell carcinoma, ovarian cancer, head and neck cancer, mesothelioma, and breast cancer.

32. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, wherein the cancer is resistant to treatment with an antibody molecule that specifically binds to CTLA-4, or is resistant to treatment with an antibody molecule that specifically binds to PD1, or is resistant to treatment with an antibody molecule that specifically binds to PD-L1.

33. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, wherein the cancer is resistant to treatment with an antibody molecule that specifically binds to CTLA-4 and an antibody molecule that specifically binds to PD-L1, or is resistant to treatment with an antibody molecule that specifically binds to CTLA-4 and an antibody molecule that specifically binds to PD-L1, or is resistant to treatment with an antibody molecule that specifically binds to PD-1 and an antibody molecule that specifically binds to PD-L1, or is resistant to treatment with an antibody molecule that specifically binds to CTLA-4, an antibody molecule that specifically binds to PD1 and an antibody molecule that specifically binds to PD-L1.

34. 5. The combination for use, or the use, or the pharmaceutical composition according to any one of claims 1 to 4, wherein the patient has previously been treated with an antibody molecule that specifically binds to CTLA-4, and / or an antibody molecule that specifically binds to PD-1, and / or an antibody molecule that specifically binds to PD-L1, and the patient has proven resistant to said treatment or has become resistant to said treatment after or during said treatment.

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