RU2773655C2 - Combination containing multifunctional antibodies redirecting t-cells and control point modulators, and its applications - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, specifically to a method for initiating, enhancing or prolongating an antitumor response; it can be used in medicine. The method includes the injection of an immune control point modulator after 12-96 hours after the first injection of a multifunctional antibody redirecting T-cells.

EFFECT: method can be used in the effective combination therapy of cancer.

39 cl, 4 dwg, 4 ex

Description

The present invention relates to the field of cancer immunotherapy, in particular to the use of T-cell redirecting multifunctional antibodies and immune checkpoint modulators in the therapeutic/curative treatment of cancer.

Immunotherapy is becoming increasingly important in oncology. This is evidenced in recent years by the approval of Yervoy® (ipilimumab, Bristol Myers Squibb), Opdivo® (nivolumab, Bristol Myers Squibb) and Keytruda® (pembrolizumab, Merck). These monoclonal antibodies (MAbs) are directed against either cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or programmed cell death protein 1 (PD-1). A third cancer target that has been studied in detail is the programmed cell death protein ligand 1 (PD-L1). What these targets have in common is that they are crucial negative regulators of T-lymphocytes, so-called immune checkpoint inhibitory molecules. Immune checkpoints are molecules of the immune system, particularly those found on certain immune cells, that need to be activated (stimulatory or costimulatory checkpoint molecules) or inactivated (inhibitory checkpoint molecules) to initiate an immune response. Many of the immune checkpoints are regulated through interactions between specific receptor-ligand pairs. Often cancers protect themselves from the immune system by using such checkpoints to avoid attack by the immune system.

The PD-1 receptor is expressed on the surface of activated T cells and other immune cells such as B cells. Its ligands (PD-L1 and PD-L2) are expressed on the surface of antigen presenting cells such as dendritic cells or macrophages and other immune cells. Binding of PD-L1 or PD-L2 to PD1 triggers a signal in the T cell that virtually "turns off" the T cell or inhibits it. Under non-pathological conditions, this interaction prevents T cells from attacking other cells in the body. However, cancer cells often take advantage of this system and express high levels of PD-L1 on their surface. Thus, cancer cells are able to “turn off” PD-1-expressing T cells and, therefore, suppress the anti-cancer immune response. Inhibitors of PD1 and/or its ligands, such as inhibitory/antagonistic monoclonal antibodies directed against PD1 or its ligands, can enhance the immune response against cancer cells and, therefore, are promising for the treatment of cancers. Examples of PD1 inhibitory/antagonist monoclonal antibodies currently approved for use include ^Opdivo® (nivolumab; Bristol Myers Squibb) and Keytruda® ( ^{pembrolizumab} ; Merck). Other PD1 pathway inhibitors currently in phase II and/or III clinical trials include pidilizumab (a PD1 inhibitory mAb; CureTech/Medivation), durvalumab (a PD-L1 inhibitory mAb; MedImmune/AstraZeneca), avelumab (PD-L1 inhibitory MAb; Merck Serono/Pfizer); and atezolimab (PD-L1 inhibitory MAb; Roche).

^Yervoy® (ipilimumab; Bristol Myers Squibb), another approved immune checkpoint modulator, is an inhibitory/antagonistic monoclonal antibody against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). CTLA4 is also expressed on the surface of activated T cells and its ligands are expressed on the surface of professional antigen-presenting cells. It is assumed that CTLA-4 regulates the proliferation of T cells in the early phase of the immune response, primarily in the lymph nodes, and affects the function of regulatory T cells. Another CTLA-4 inhibitor currently in phase II clinical trials is, for example, tremelimumab (MedImmune/AstraZeneca).

Binding of naturally occurring ligands B7.1 and B7.2 cCTLA-4 and PD-L1/PD-L2 to PD-1 on activated T cells inhibits positive signals mediated by the T cell receptor (TCR) or the costimulatory receptor CD28, and thereby leads to the suppression of T-cell responses, which is a natural mechanism for overcoming an excessive immunological reaction. Intensive research and clinical studies have shown that blocking immune checkpoint molecules with mAbs leads to sustained T-cell activation that can be used to fight cancer. Thus, antibody-mediated blocking of immune checkpoints is an effective approach to enhance the functions of tumor-reactive T cells.

Because antibodies that inhibit (block) immune checkpoints act in part through non-specific activation of the immune system, there are numerous approaches that involve their use in combination with other anti-cancer regimens. Antibodies that inhibit (block) immune checkpoints are used in combination with chemotherapy to reduce tumor burden. The disadvantage of this approach is that potent chemotherapeutic agents have a negative effect on the function of the immune system, reducing the effectiveness of immunotherapeutic drugs. Alternatively, antibodies that inhibit (block) immune checkpoints are used in combination with other inhibitors of immune checkpoints. An example is the combination therapy using Yervoy® and Opdivo® approved by the FDA in 2015 for the treatment of patients with BRAF V600 wild-type, unresectable or metastatic melanoma. In addition, data have recently been published on the successful results of a phase 1b study of the combination of durvalumab and tremelimumab in the case of non-small cell lung cancer (Antonia, Scott et al., Safety and antitumour activity of Durvalumab plus Trenelimumab in non-small cell lung cancer: a multicentre, phase 1b study, Lancet Oncol., February 5, 2016, pii: S1470-2045(15)00544-6 doi: 10.1016/S1470-2045(15)00544-6 [electronic publication prior to going to press]).

There is, however, the disadvantage of a significant increase in negative side effects (Tsai and Daud, Nivolumab plus Ipilimumab in the treatment of advanced melanoma. Journal of Hematology & Oncology, 8, 2015, p. 123). In addition, the combination of checkpoint modulators only with each other directly affects endogenous tumor-specific immunity, in particular because there is no targeting of tumor-specific antigens.

In view of the foregoing, there is a need for improved immunotherapy for use in the treatment of cancer. Thus, the object of the present invention was to overcome the above disadvantages of current cancer immunotherapy and to develop a new combination for use in the treatment of cancer, which increases the survival of patients suffering from cancer and which reduces the risk of side effects.

This problem is solved using the objects presented below in the present description and the attached claims.

While the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, which may vary. It should also be obvious that the terminology used in the present description does not limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise indicated, all technical and scientific terms used in the context of the present description have the meanings well known to one of ordinary skill in the art.

The elements of the present invention are described below. These elements are listed in specific embodiments of the invention, however, as should be obvious, they can be combined in any way and in any number to obtain additional embodiments of the invention. The various described examples and preferred embodiments of the invention should not be taken as indicating that the present invention is limited only to the described embodiments of the invention. It should be understood that the present description is given as a basis, and it includes embodiments of the invention, in which the embodiments of the invention, given in unambiguous form, are combined with any number of described and/or preferred elements. In addition, it is understood that any permutations and combinations of all elements specified in the present description fall within the scope of the present description, unless the context implies otherwise.

In the context of the present description and in the appended claims, unless the context otherwise requires, the term "comprise" and its variations such as "comprises" and "comprising" should be considered as including the specified member, integer or stage, but this does not exclude any other member, integer, or stage not specified. The term "consists of" is a specific version of the term "comprises" to the exclusion of any other member, integer, or step not specified. In the context of the present invention, the term "comprises" includes the term "consists of". Thus, the concept of “comprising” includes the concept of “including”, as well as “consisting”, for example, the composition “containing” X, may consist only of X, or may include something additional, for example, X + Y.

Reference to a concept in the singular and a similar reference used in the context of the present invention (especially in the context of the claims) implies its mention, both in the singular and in the plural, unless the context implies otherwise or if this does not contradict the context. The listing of ranges of values in the context of the present description is intended only as a shorthand method for individually mentioning each individual value that is in the specified range. Unless otherwise indicated, each individual value is included in the present description, as if it were individually mentioned in it. Nothing in the specification is to be construed as indicating any unclaimed element essential to the practice of the invention.

The term "substantially" does not exclude "completely", for example, a composition that is "substantially free" of Y may be completely free of Y. If necessary, the term "substantially" may be omitted from the definition of the concept in question in the present invention.

The term "about" in relation to the numerical value x means x ± 10%.

Combination for use in the therapeutic treatment of cancer

The first object of the present invention is the combination

(I) an immune checkpoint modulator and

(II) redirecting (isolated) T-cell multifunctional antibody or its antigen-binding fragment, containing:

(a) specificity for T-cell surface antigen,

(b) specificity for a cancer and/or tumor associated antigen, and

(c) a human FcγRI, FcγRIIa and/or FcγRIII binding site, wherein the antibody or antigen-binding fragment thereof binds to human FcγRI, FcγRIIa and/or FcγRIII with higher affinity than to human FcγRIIb,

intended for the therapeutic treatment of cancer.

The combination of T-cell-redirecting multifunctional antibodies (trAt) and immune checkpoint modulators represents a new approach to cancer treatment that integrates several unique and complementary immunotherapies:

First, negative side effects are reduced. Blockade of inhibitory checkpoint molecules leads to strong and non-specific activation of T cells, which can cause serious autoimmune disorders such as colitis, diarrhea, pneumonia, hepatitis, etc. The combination with T-cell redirecting multifunctional antibodies allows activated T-cells to be “guided” away from healthy tissue and “led” to the tumor site. Thus, it is possible to inhibit or reduce autoimmune reactions.

In addition, the Fc receptor binding site of T-cell redirecting multifunctional antibodies recruits and stimulates accessory cells such as dendritic cells (DCs) or macrophages by activating Fc receptors. These cells create additional stimuli for T cells, engulf tumor cell debris, and present tumor-derived peptides to the immune system. Thus, T-cell redirecting multifunctional antibodies not only provide T-cell dependent tumor destruction, but also induce long-term tumor-specific immunological memory.

In addition, as a result of prolonged activation of T cells, therapeutic efficacy is increased. In the present invention, it has been found that T cell activation by T cell redirecting multifunctional antibodies is accompanied by an increase in the expression of immune checkpoint molecules, which in turn downregulate activated T cells. Thus, the use of inhibitory checkpoint blocking antibodies in combination results in sustained and prolonged T cell activation, which is beneficial in terms of direct antitumor activity of multifunctional T cell retargeting antibodies.

Thus, it has been found in the course of the present invention that the combination for use according to the present invention provides, in particular, sustained T cell activation compared to T cell activation induced by (I) immune checkpoint modulator alone and/or (II) a T cell-redirecting multifunctional antibody or an antigen-binding fragment thereof.

In general, the combination of T-cell redirecting multifunctional antibodies with immune checkpoint modulators significantly enhances the therapeutic antitumor efficacy of individual drugs (FIG. 1). In addition, it may even reduce the important negative side effects associated with immune checkpoint modulators.

The components of the combination for use according to the present invention are described in detail below, namely, an immune checkpoint modulator and a T cell redirecting multifunctional antibody comprising specificity against a T cell surface antigen, specificity against a cancer and/or tumor associated antigen, and a binding site. human FcγRI, FcγRIIa and/or FcγRIII, and their preferred embodiments. In addition, the use for therapeutic treatment of cancer and its preferred embodiments are described in detail below. It should be understood that (i) the preferred combination for use in the present invention includes the preferred immune checkpoint modulator, (ii) the preferred combination for use in the present invention includes the preferred multifunctional T-cell redirecting antibody, containing specificity against a T-cell surface antigen, specificity against an antigen associated with cancer and/or tumor, and a binding site for human FcγRI, FcγRIIa and/or FcγRIII; and (III) a preferred combination for use in the present invention includes a preferred use for the therapeutic treatment of cancer. A more preferred combination for use in the present invention comprises (I) a preferred immune checkpoint modulator and a preferred T cell retargeting multifunctional antibody comprising specificity against a T cell surface antigen, specificity against a cancer and/or tumor associated antigen and binding site of human FcγRI, FcγRIIa and/or FcγRIII; (ii) a preferred embodiment of a multifunctional T-cell redirecting antibody comprising specificity against a T cell surface antigen, specificity against a cancer and/or tumor associated antigen, and a human FcγRI, FcγRIIa and/or FcγRIII binding site, and a preferred therapeutic use cancer; or (III) a preferred immune checkpoint modulator and a preferred therapeutic use for cancer. The most preferred combination for use in the present invention comprises (i) a preferred immune checkpoint modulator; (ii) a preferred T-cell redirecting multifunctional antibody comprising specificity for a T cell surface antigen, specificity for a cancer and/or tumor associated antigen, and a human FcγRI, FcγRIIa and/or FcγRIII binding site, and (III) a preferred use for the therapeutic treatment of cancer.

It should be understood that, in general, it is preferable that in the combination intended for use according to the present invention, the immune checkpoint modulator and the T-cell redirecting multifunctional antibody (or antigen-binding fragment thereof) are directed against different targets. In other words, the preferably T-cell-redirecting multifunctional antibody (or antigen-binding fragment thereof) does not contain any specificity or any binding site targeting/targeting the same immune checkpoint molecule (or ligand thereof) as the immune checkpoint modulator included in combination for use in accordance with the present invention. And also, in other words, the immune checkpoint modulator preferably does not modulate the immune checkpoint molecule (or ligand thereof) that is targeted by the T-cell retargeting multifunctional antibody (or antigen-binding fragment thereof) included in the combination for use according to the present invention.

T cell-redirecting multifunctional antibody

In the context of the present description (i.e. in this application), the term "antibody" includes various forms of antibodies, preferably monoclonal antibodies, including (but not limited to) complete antibodies, antibody fragments, human antibodies, chimeric antibodies, recombinant antibodies , humanized antibodies, synthetic antibodies, chemically modified antibodies and genetically engineered antibodies (variant or mutant antibodies), if they retain the characteristic properties proposed in the invention. Preferred examples of antibodies include monoclonal antibodies, bispecific antibodies, mini-antibodies, domain antibodies, synthetic antibodies, antibody mimetics, chimeric antibodies, humanized antibodies, human antibodies, fusion antibodies, antibody conjugates, single chain antibodies, antibody derivatives, antibody analogs, and fragments thereof, respectively. More preferred are recombinant antibodies, especially recombinant monoclonal antibodies. In addition, it is also preferred that the antibody is a multi-chain antibody, i. e. an antibody that contains more than one chain, which distinguishes it from a single-chain antibody. In addition, the antibody or antigen-binding fragment may be wholly or partly derived from a human body or be humanized. Humanization of antibodies is known in the art (see, for example, Shalaby et al., J. Exp. Med. 175, 1992, p. 217; Mocikat et al., Transplantation 57, 1994, p. 405). Preferably, at least (six) CDRs (complementarity determining regions) and/or framework regions, more preferably variable regions, are human-derived and/or humanized. Unless otherwise indicated, the term "antibody" includes, in addition to antibodies containing two full-length heavy chains and two full-length light chains, also their derivatives, variants and fragments. In some cases, an "antibody" may include fewer chains.

Preferably, the antibody or antigen-binding fragment intended/intended for use according to the present invention is a monoclonal antibody or antigen-binding fragment. In the context of the present description, a "monoclonal" antibody (MAb or MoAb) should be understood as an antibody produced by identical immune cells that are clones of a single parent cell, as opposed to polyclonal antibodies, which are produced by several different immune cells. As a rule, it is possible to obtain a monoclonal antibody that specifically binds to a particular substance.

As used herein, the term "human antibody" is intended to include antibodies that have variable and constant regions derived from human immunoglobulin sequences. Human antibodies are well known in the art (van Dijk M. A. and van de Winkel J. G., Curr. Opin. Chem. Biol. 5, 2001, pp. 368-374). Human antibodies can also be produced in transgenic animals (eg mice) which, after immunization, can produce the full range of human antibodies or selected antibodies in the absence of endogenous immunoglobulin production. Transfer of an array of human germline immunoglobulin genes into these germline mutant mice should result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits A. et al., Proc. Natl. Acad. Sci. USA 90, 1993, 2551-2555; Jakobovits A. et al., Nature 362, 1993, pp. 255-258; Bruggemann M. et al., Year Immunol. 7, 1993, p. 3340). Human antibodies can also be generated using phage display libraries (Hoogenboom H. R. and Winter G., J. Mol. Biol. 227, 1992, pp. 381-388; Marks J. D. et al., J. Mol. Biol. 222 , 1991, pp. 581-597). In addition, the techniques described by Cole et al. and Boerner et al. (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 1985, p. 77; and Boerner P. et al., J. Immunol. 147, 1991, pp. 86-95). In the context of the present description, the term "human antibody" also includes antibodies that are modified, for example, in the variable region or in the Fc region, to create the properties required according to the invention.

In the context of the present description, the term "recombinant antibody" is meant to include all antibodies that do not occur in nature, in particular, antibodies obtained, expressed, created or isolated by recombination methods, for example, antibodies isolated from a host cell, such as, for example , as a CHO cell, or from an animal (eg, mouse) cell, or an antibody expressed using a recombinant expression vector that has been transfected into a host cell. These recombinant antibodies have variable and constant regions in a transformed form compared to naturally occurring antibodies.

In the context of the present description, the terms "antigen-binding fragment", "fragment" and "antibody fragment" are used interchangeably to refer to any antibody fragment of the invention that retains the specific binding activity of the antibody intended for use according to the invention, in particular specificity for the T antigen -cell surface and specificity for a cancer and/or tumor associated antigen and has a binding site for human FcγRI, FcγRIIa and/or FcγRIII. Examples of antibody fragments include, but are not limited to, sc (single chain) antibody, scFv-Fc, scFv-CH3, scDiabody-CH3, Diabody-CH3, minibody, scFv-KIH, Fab-scFv-Fc, scDiabody-Fc, Diabody-Fc and scFv-Fc tandem (described, for example, in Spiess C., Zhai Q. and Carter P.J., Molecular Immunology 67, 2015, pp. 95-106). The antibody fragments of the invention can be obtained from antibodies by methods that include cleavage with enzymes such as pepsin or papain and/or by cleavage of disulfide bonds using a chemical reaction. Alternatively, antibody fragments can be obtained by cloning and expression of a portion of the heavy and/or light chain sequences. Also within the scope of the invention are single chain Fv fragments (scFv) comprising a CH3 region derived from the heavy and light chains of the antibody of the invention. For example, the invention includes scFv-CH3 or scFv-Fc containing a CDR from an antibody of the invention. They also include heavy and light chain monomers and dimers, heavy chain single domain antibodies, light chain single domain antibodies, as well as single chain antibodies, such as single chain Fv, in which the heavy and light chain variable domains are joined by a peptidic linker. The antibody fragments of the invention are generally multivalent and may be contained in the various structures described above. For example, scFv molecules can be synthesized to give trivalent "triabody" or tetravalent "tetrabodium". The scFv molecules preferably include an Fc region domain. Although in the specification, including the claims, antigen-binding fragment(s), antibody fragment(s), antibody variant(s) and/or derivative(s) may be unambiguously referred to in some places, it should be understood that the term “antibody or "antibody of the invention" includes all categories of antibodies, namely antigen-binding(s) fragment(s), antibody fragment(s), variant(s) and/or derivative(s) of antibodies.

The antibody or fragment thereof intended/intended for use according to the present invention is a T-cell redirecting multifunctional antibody or fragment thereof.

As used herein, a "T cell redirecting" antibody or fragment thereof is an antibody or fragment thereof that contains both specificity for a T cell surface antigen and specificity for a cancer and/or tumor associated antigen. This allows the antibody or antibody fragment to redirect T cells to cancer cells. The term "specificity for a T-cell surface antigen" means, in particular, that the antibody or antigen-binding fragment intended/intended for use according to the present invention contains a paratope that recognizes an epitope of the T-cell surface antigen. In other words, the expression "specificity for a T-cell surface antigen" means, in particular, that the antibody or antigen-binding fragment intended/intended for use according to the present invention contains a T-cell surface antigen binding site. Accordingly, the expression "specificity for a cancer and/or tumor associated antigen" means, in particular, that the antibody or antigen-binding fragment thereof intended/intended for use according to the present invention contains a paratope that recognizes an epitope of the cancer and/or tumor associated antigen . In other words, the expression "specificity for a cancer and/or tumor associated antigen" means, in particular, that the antibody or antigen-binding fragment thereof intended/intended for use according to the present invention contains a cancer and/or tumor associated antigen binding site.

It is important to note that, unlike canonical ("conventional") antibodies, which have only one specificity, T-cell redirecting antibodies have the ability to bind to at least two different epitopes, namely, one epitope on the cancer/tumor cell and one epitope on the T cell, thereby "redirecting" the T cell to the cancer/tumor cell, resulting in T cell-mediated cell death. Thus, the T cell redirecting antibodies for use in the present invention have the ability to redirect the T cell, i.e. the antibody typically has the ability to reactivate an anergic tumor-specific T cells and/or direct the T cells to the desired antigen (provided by the specificity of the antibody for a cancer and/or tumor associated antigen).

In the context of the present description, a "multifunctional" antibody, or fragment thereof, is an antibody or fragment thereof, which/which has the ability to interact simultaneously with several different binding sites. Because an antibody or fragment thereof for/intended for use in the present invention contains (at least) (a) specificity for a T-cell surface antigen, (b) specificity for a cancer and/or tumor associated antigen, and (c) ) a binding site for human FcγRI, FcγRIIa and/or FcγRIII, then the "multifunctional" antibody or fragment thereof is at least a "trifunctional" antibody or fragment thereof. The concept of "trifunctional" means that the antibody or its fragment have the ability to interact simultaneously with three different binding sites.

As noted above, T-cell redirecting multifunctional antibodies comprise a tumor-associated antigen (TAA) specific arm, a second binding arm specific for a T cell surface antigen such as CD3 expressed on T cells, and a human binding site. FcγRI, FcγRIIa and/or FcγRIII, which most preferentially binds to activating Fcγ receptors such as FcγRI, FcγRIIa and/or FcγRIII present on accessory cells such as macrophages, dendritic cells (DCs), or natural killer cells ( NK). Without going into any theory, when creating the present invention, it was suggested that the mechanism of action of T-cell redirecting multifunctional antibodies in the implementation of antitumor therapy (Lindhofer H., Hess J., Ruf P., Trifunctional Triomab ^® antibodies for cancer therapy, in: Bispecific antibodies, edited by Kontermann RE, Springer, Berlin, 2011, pp. 289-312) is as follows: it is assumed that the first critical step in this mechanism of action is the redirection of T cells on the tumor via bispecific antibody mediated cross-linking of TAA with a T-cell surface antigen such as CD3. Antibody-mediated contact with a T-cell surface antigen such as CD3 as a component of the T-cell receptor complex is the first powerful stimulus for T-cell activation in a major histocompatibility complex (MCHC)-independent manner, which is accompanied by secretion of TNF-α and IFN -γ. However, physiological activation of T cells requires a second signal. Through the FcγRI-, FcγRIIa- and/or FcγRIII-binding site of T cell-redirecting multifunctional antibodies, additional recruitment of opsonized T cells and tumor cells, as well as pro-inflammatory cytokines, FcγRI-, FcγRIIa- and/or FcγRIII-positive immune cells, occurs. A cluster of immune cells of various types is formed on the tumor cell.

It is assumed that the formation of this three-cell complex, consisting of tumor cells, T cells and FcγRI-, FcγRIIa- and / or FcγRIII-positive auxiliary immune cells, leads to several important consequences: firstly, there is a mutual stimulation of auxiliary immune cells and T -cells. T cell-redirecting multifunctional antibody-initiated interaction between T cells and CD14-positive monocytes leads to upregulation of CD83, CD86, and CD40 (Riechelmann H., Wiesneth M., Schauwecker P., Reinhardt P., Gronau S., Schmitt A., Schroen C., Atz J., Schmitt M., Adoptive therapy of head and neck squamous cell carcinoma with antibody coated immune cells: a pilot clinical trial Cancer Immunol Immunother 56, 2007, pp. 1397-1406 Stanglmaier M., Faltin M., Ruf P., Bodenhausen A., Schroder P., Lindhofer H., Bi20 (FBTA05), a novel trifunctional bispecific antibody (anti-CD20_anti-CD3), mediates efficient killing of B-cell lymphoma cells even with very low CD20 expression levels, Int J Cancer 123, 2008, pp. 1181-1189; Zeidler R., Mysliwietz J., Csanady M., Walz A., Ziegler I., Schmitt B., Wollenberg B., Lindhofer H., The Fc-region of a new class of intact bispecific antibody mediates activation of accessory cells and NK cells and induces direct phagocytosis of tumor c ells. Br J Cancer 83, 2000, ss. 261-266). Thus, T cells receive a second costimulatory signal in the form of a CD40/CD40L or CD80-CD86/CD28 interaction. As a result, they are strongly and physiologically significant activated, which is characterized by a high level of IL-2 expression and strong proliferation, detected by detection of the proliferation marker Ki-67. In addition, there is an upregulation of T-cell activation markers CD25 and CD69 (Riechelmann H., Wiesneth M., Schauwecker P., Reinhardt P., Gronau S., Schmitt A., Schroen C., Atz J., Schmitt M ., Adoptive therapy of head and neck squamous cell carcinoma with antibody coated immune cells: a pilot clinical trial Cancer Immunol Immunother 56, 2007, pp. 1397-1406). Conversely, immune helper cells are stimulated by interacting with T cells and cross-linking with FcgR. This stimulation is manifested in high measured levels of pro-inflammatory cytokines such as IL-6 and IL-12, which are mainly secreted by the helper cells. In addition, cross-talk between helper cells and T cells is evidenced by increased release of cytokines characteristic of the Th1 cell response, primarily IL-2 and IFN-γ. As a result, targeted tumor cells are effectively destroyed as a result of a concentrated attack of various types of immune effector cells, which has been demonstrated under allogeneic conditions, as well as in ex vivo autologous human systems (Gronau S.S., Schmitt M., Thess B., Reinhardt P., Wiesneth M. , Schmitt A., Riechelmann H., Trifunctional bispecific antibody-induced tumor cell lysis of squamous cell carcinomas of the upper aerodigestive tract, Head Neck 27, 2005, pp. 376-382). Necrotized and apoptotic tumor cells and particles undergo phagocytosis (Riesenberg R., Buchner A., Pohla H., Lindhofer H., Lysis of prostate carcinoma cells by trifunctional bispecific antibodies (alpha EpCAM_alpha CD3). J Histochem Cytochem 49, 2001, ss. 911-917; Zeidler R., Mysliwietz J., Csanady M., Walz A., Ziegler I., Schmitt B., Wollenberg B., Lindhofer H., The Fc-region of a new class of intact bispecific antibody mediates activation of accessory cells and NK cells and induces direct phagocytosis of tumor cells, Br J Cancer 83, 2000, pp. 261-266) and can be processed and resent by professional antigen presenting cells under stimulated conditions, which is an ideal prerequisite for antitumor immunization.

The present invention has surprisingly found that the T-cell-redirecting multifunctional antibody (or antigen-binding fragment thereof) provided herein induces increased expression of immune checkpoint molecules such as CTLA-4 (cf. Example 2, FIG. 2) . Thus, it is preferred that the T-cell redirecting multifunctional antibody (or antigen-binding fragment thereof) included in the combination for use according to the present invention induces increased expression of immune checkpoint molecules such as CTLA-4.

Preferably, the antibody or antigen-binding fragment thereof for use in accordance with the present invention is a trifunctional antibody or trifunctional antigen-binding fragment thereof, especially a bispecific trifunctional antibody or a bispecific trifunctional antigen-binding fragment thereof.

In the context of the present description, “trifunctional” antibodies (trAt) are understood primarily as a specific class of bispecific antibodies that simultaneously recruit and activate T cells and, above all, auxiliary immune cells such as macrophages, dendritic cells, natural cells - killer (NK), and/or other FcγRI, FcγRIIa and/or FcγRIII expressing cells, in the targeted cancer/targeted tumor through their FcγRI, FcγRIIa and/or FcγRIII binding site. Thus, three-functional bispecific antibodies have two antigen-binding sites (i.e., two paratopes). Typically, these two antigen-binding sites (ie, two paratopes) allow antibodies to bind to cancer/tumor cells (cancer/tumor cell surface antigens) and to T cells (T cell surface antigens). Simultaneously, for example, through their Fc fragment, primarily their Fcγ receptor binding site, positive helper cells such as monocytes/macrophages, natural killer cells, dendritic cells or other Fcγ receptor expressing cells are recruited. Simultaneous activation of these different classes of effector cells leads to the effective destruction of tumor cells through various mechanisms, such as phagocytosis and mediated by perforin cytotoxicity. Typically, the net effect of a trifunctional antibody is to bind T cells, and especially Fcγ receptor-positive helper cells, to tumor cells, resulting in destruction of the tumor cells.

Trifunctional antibodies induce tumor cell clearance, specifically through (i) antibody-mediated cell-dependent cytotoxicity, (ii) T-cell mediated cytolysis, and (iii) induction of anti-tumor immunity. In contrast, canonical (monoclonal and monospecific) antibodies actually act only through the first mechanism of action. In addition, in contrast to canonical antibodies, trifunctional antibodies have a higher cytotoxic potential and can even bind to antigens that are relatively weakly expressed. Thus, trifunctional antibodies, when used in an equivalent dose, are more effective (more than 1000 times) in eliminating tumor cells compared to canonical antibodies.

In general, a T cell redirecting multifunctional antibody for use in the present invention is a multispecific antibody. As used herein, "multispecific" refers to the ability to bind to at least two different epitopes, for example, on different antigens such as T-cell surface antigen and cancer/tumor antigen. Thus, concepts like "bispecific", "trispecific", "tetraspecific", etc. refer to the number of different epitopes that an antibody can bind to. For example, canonical IgG-type monospecific antibodies have two identical epitope-binding sites (paratopes) and therefore can only bind to identical epitopes (not different epitopes). In contrast, a multispecific antibody has at least two different types of paratopes and can therefore bind to at least two different epitopes. In the context of the present description, the term "paratope" refers to the epitope-binding site of an antibody. In addition, individual "specificity" may refer to one, two, three or more identical paratopes in a single antibody (the actual number of paratopes in an individual antibody molecule is referred to as "valence"). For example, an individual native IgG-type antibody is monospecific and bivalent because it has two identical paratopes. Thus, a multispecific antibody contains at least two (different) paratopes. Thus, the term "multispecific antibodies" refers to antibodies that have more than one paratope and have the ability to bind to two or more different epitopes. The term "multispecific antibodies" includes in particular the bispecific antibodies mentioned above, but generally also includes a protein, e.g. an antibody, scaffolds that bind, in particular, three or more different epitopes, e.g. or more paratopes.

In particular, a multispecific antibody or antigen-binding fragment thereof may contain two or more paratopes, some of which may be identical, but as a result, all paratopes of an antibody belong to at least two different types of paratopes, and therefore the antibody has at least two specificities. . For example, a multispecific antibody or antigen-binding fragment thereof provided/provided in the present invention may contain four paratopes, of which every two paratopes are identical (i.e., have the same specificity), and thus, the antibody or fragment thereof is bispecific and tetravalent (two identical paratopes for each of the two specificities). Thus, the term "one specificity" refers in particular to one or more paratopes having the same specificity (this generally means that said one or more paratopes are identical), and thus "two specificities" may be realized by two, three, four, five, six, or more paratopes, as long as they refer to only two specificities. Most preferably, the multispecific antibody contains one single paratope for each (of at least two) specificities, ie. a multispecific antibody contains a total of at least two paratopes. For example, a bispecific antibody contains one single paratope for each of the two specificities, ie. the antibody contains a total of two paratopes. Preferably, the antibody also contains two (identical) paratopes for each of the two specificities, i.e. the antibody contains a total of four paratopes. Preferably, the antibody contains three (identical) paratopes for each of the two specificities, ie. the antibody contains a total of six paratopes.

Preferably, the antibody or antigen-binding fragment thereof to/intended for use in accordance with the present invention is a bispecific antibody or a bispecific antigen-binding fragment thereof.

In the context of the present invention, bispecific antibodies (BiAbs) contain (strictly) two specificities. They are the most preferred type of multispecific antibodies and their antigen-binding fragments. In the context of the present invention, a bispecific antibody may be a bispecific antibody of any format containing an FcγRI, FcγRIIa and/or FcγRIII binding site, for example, the antibody described in Spiess C., Zhai Q. and Carter P.J., Molecular Immunology 67, 2 -15, ss. 95-106. For example, BiAbs may be complete antibodies, such as complete IgG-like molecules, or fragments thereof that are not complete antibodies but retain antibody properties. They can be small recombinant antibodies, for example, in the format of tandem single chain variable fragment molecules (taFv), dimeric antibodies (diabodies) (Db), single chain dimeric antibodies (scDb) and various other derivatives thereof (see, for example, Byrne H et al., Trends Biotech, 31(11), 2013, pp. 621-632, where Fig. 2 shows different formats of bispecific antibodies). Several BiAT formats have the ability to redirect effector cells to target cells that play a key role in the development of diseases. For example, several BiAb formats can redirect effector cells to tumor cells, and various BiAb constructs have been created to redirect cells of the immune system, for example, by binding to Fc receptors on the surface of effector cells and triggering them, or by binding to T-cell receptor complexes ( TCR).

Preferably, the multispecific, in particular bispecific, antibody or antigen-binding fragment thereof is at least divalent, ie. has at least two paratopes. More preferably, the multispecific, in particular bispecific, antibody or antigen-binding fragment thereof is divalent, trivalent, tetravalent or hexavalent. Even more preferably, the multispecific, in particular bispecific, antibody or antigen-binding fragment thereof is divalent or tetravalent. Most preferably, the antibody, or antigen-binding fragment thereof, intended/intended for use in accordance with the present invention is a bispecific bivalent antibody, i.e. an antibody that has two paratopes: one that recognizes an antigen on the surface of a T cell and the other that recognizes an antigen associated with cancer and/or tumor.

Unlike the term "multispecific", e.g., bispecific, trispecific, tetraspecific, etc., the term "multifunctional", e.g., trifunctional, etc., refers to the number of different binding sites in a more general sense, namely, it includes any binding sites (not just those that bind to epitopes). Thus, for example, an FcγRI, FcγRIIa and/or FcγRIII binding site is "classified" as "multifunctional", e.g., trifunctional, etc., but not as "multispecific", e.g., bispecific, trispecific, tetraspecific, etc. P. For example, a trifunctional antibody can be mono-, bi-, or trispecific - but in the context of the present invention (when an antibody has two specificities and a FcγRI, FcγRIIa and/or FcγRIII binding site), a trifunctional antibody is typically bispecific.

In the context of the present description, the term "antigen" refers to a substance of any structure that serves as a target for adaptive immune response receptors, in particular, a target for antibodies, T-cell receptors and/or B-cell receptors. The term "epitope", also known as "antigenic determinant", is a part (or fragment) of an antigen that is recognized by the immune system, in particular by antibodies, T-cell receptors and/or B-cell receptors. Thus, one antigen has at least one epitope, ie. an individual antigen has one or more epitopes. The antigen may be (I) a peptide, polypeptide or protein, (II) a polysaccharide, (III) a lipid, (IV) a lipoprotein or lipopeptide, (V) a glycolipid, (VI) a nucleic acid, or (VII) a small molecule drug or toxin. Thus, an antigen can be a peptide, protein, polysaccharide, lipid, a combination thereof, including lipoproteins and glycolipids, a nucleic acid (e.g., DNA, siRNA, shRNA, antisense oligonucleotides, DNA decoy, plasmid), or a small molecule drug (e.g., ciclosporin A, paclitaxel, doxorubicin, methotrexate, 5-aminolevulinic acid), or any combination thereof. Preferably the antigen is selected from (I) a peptide a, a polypeptide or protein, (II) a polysaccharide, (III) a lipid, (IV) a lipoprotein or lipopeptide and (V) a glycolipid, more preferably the antigen is a peptide, polypeptide or protein.

T cell surface antigen

As used herein, "T cell surface antigen (its epitope)" refers to a T cell surface associated antigen (its epitope) or a T cell surface specific antigen (also referred to as "T cell surface markers"). . They are, in particular, "CD" (cluster of differentiation) molecules specific for T cells. CD molecules are cell surface markers that can be used to identify and characterize leukocytes. The CD nomenclature has been developed and continues to be developed since 1982 based on the study of HLDA (human leukocyte differentiation antigens). Whether or not a certain CD molecule is present on T cells (and therefore represents a T cell surface antigen in the context of the present invention) can be obtained, for example, from various sources known to one of skill in the art, for example, http://www.ebioscience.com/resources/human-cd-chart.htm, BD Bioscience's "Human and Mouse CD Marker Handbook" (available from https://www.bdbiosciences.com/documents/cd_marker_handbook.pdf) or from www.hcdm.org. Thus, examples of T cell surface antigens include, for example, those (human) CD markers listed as present (positive) on T cells in BD Bioscience's "Human and Mouse CD Marker Handbook" (available from https:/ /www.bdbiosciences.com/documents/cd_marker_handbook.pdf) or other CD marker charts sources.

Preferably, the T cell surface antigen is selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD8, CD28, CD40L and CD44. This means that the antibody or antigen-binding fragment intended/intended for use according to the present invention contains a paratope that preferentially recognizes (has the ability to bind to) an epitope of a T cell surface antigen selected from the group consisting of CD3, CD2, CD4 , CD5, CD6, CD8, CD28, CD40L and/or CD44. Said specificity preferably facilitates the recruitment of T cells. In this context, CD is an abbreviation for the "cluster of differentiation" (cluster of nomenclature or classification of determinants) described above. In general, it is known as a protocol used to identify and study cell surface molecules that are targets for cell immunophenotyping. From a physiological point of view, CD molecules act as numerous pathways, often acting through receptors or ligands (the molecule that activates the receptor) that are important to the cell. Typically, a signaling cascade is initiated that changes the behavior of the cell (see the section on cell signaling). Some CD proteins do not play a role in cell signaling but have other functions such as cell adhesion. Currently, human CDs are numbered up to 364. The present invention relates to T-cell-associated CD molecules.

Preferably, the T cell surface antigen for which the T cell retargeting multifunctional antibody or antigen binding fragment thereof is specific is not CD28. More preferably, the T cell redirecting multifunctional antibody or antigen binding fragment thereof does not contain any specificity for CD28/CD28 binding site.

More preferably the T cell surface antigen is CD2 or CD3, most preferably the T cell surface antigen is CD3. This means that the antibody or antigen-binding fragment intended/intended for use according to the present invention contains a paratope that more preferably recognizes a CD2 or CD3 epitope, most preferably the antibody or antigen-binding fragment intended/intended for use according to the present invention contains a paratope , which recognizes the CD3 epitope. CD3 (cluster of differentiation 3) is a T cell co-receptor that promotes the activation of cytotoxic T cells. CD3 is usually a protein complex and consists of four different chains. In mammals, the complex contains a CD3γ chain, a CD3δ chain, and two CD3ε chains. These chains associate with a molecule known as the T cell receptor (TCR) and the ζ chain (zeta chain), resulting in the generation of an activation signal in T lymphocytes. The TCR molecules, ζ chains, and CD3 together form a TCR-containing complex.

Cancer and/or tumor associated antigen

As used herein, "cancer and/or tumor-associated antigen (epitope)" refers to cancer-associated antigen, cancer-specific antigen, tumor-associated antigen and/or tumor-specific antigen (epitope thereof). These epitopes/antigens are typically specific to, or associated with, a particular type of cancer/tumor. Information about acceptable cancer/tumor epitopes and antigens can be obtained, for example, from databases of cancer/tumor epitopes, for example, from the database described in van der Bruggen P., Stroobant V., Vigneron N., Van den Eynde B., Peptide database: T cell-defined tumor antigens. Cancer Immun 2013; URL: http://www.cancerimmunity.org/peptide/, which categorizes human tumor antigens into four major groups based on their expression pattern, or the "Tantigen" database (TANTIGEN, version 1.0, December 1, 2009, developed by Bioinformatics Core at Cancer Vaccine Center, Dana-Farber Cancer Institute; URL: http://cvc.dfci.harvard.edu/tadb/). Specific examples of cancer-related, in particular tumor-associated or tissue-specific antigens that can be used in the context of the present invention include (but are not limited to) the following antigens: Epha2, Epha4, PCDGF, HAAH, mesothelin; ERSAM; NY-ESO-1, MUC1 and NIUC10 p5 mucin glycoprotein (primarily mutant versions), EGFR; cancer antigen 125 (CA 125), epithelial glycoprotein 40 (EGP40) (Kievit et al., Int. 1 Cancer 71, 1997, pp. 237-245), squamous cell carcinoma antigen (SCC) (Lozza et al., Anticancer Res. 17, 1997, pp. 525-529), cathepsin E (Mota et al., Am. J Pathol. 150, 1997, pp. 1223-1229), CDC27 (including mutant protein), triose phosphate isomerase antigens, 707-AP, mycobacterial antigen A60 (Macs et al., J. Cancer Res. Clin. Oncol. 122, 1996, pp. 296-300), AFP, alpha(v)beta(3)-integrin, ART-4, ASC, BAGE, β-catenin/m, BCL-2, bcr-abl, bcr-abl p190, bcr-abl p210, BRCA-1, BRCA-2, CA 19-9 (Tolliver and O'Brien, South Med. J. 90, 1997, pp. 89-90; Tsuruta et al., Urol Int. 58, 1997, pp. 20-24), CA125, CALLA, CAMEL, carbonic anhydrase, CAP-1, CASP-8, CDC27/m, CDK -4/m, CD1, CD2, CD4, CD6, CD7, CD8, CD11, CD13, CD14, CD19, CD20, CD21, CD22, CD23, CD24, CD30, CD33, CD37, CD38, CD40, CD41, CD44v3, CD44v6 , CD47, CD52, CEA (Huang et al., ExperRev. Vaccines 1, 2002, pp. 49-63), c-erb-2, CT9, CT10, Sur-B, Dek- cain, DAM-6 (MAGE-B2), DAM-10 (MAGE-B1), EphA2 (Zantek et al., Cell Growth Differ. 10, 1999, pp. 629-638; Carles-Kinch et al., Cancer Res. 62, 2002, pp. 2840-2847), EphA4 (Cheng et al., Cytokine Growth Factor Rev. 13, 2002, pp. 75-85), tumor-associated Thomsen-Friedenreich antigen (Dahlenborg et al., Int. J Cancer 70, 1997, pp. 63-71), ELF2M, ETV6-AML1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, GD1a, GD1b, GD2, GD3, GnT-V, GM1, GM2, GM3, gp100 (Zajac et al., Int. J Cancer 71, 1997, pp. 491-496), GT1b, GT3, GQ1, HAGE, HER2/neu, HLA, HLA-DR, HLA-A*0201-R170I, HPV-E7, HSP-27, HSP-70, HSP70-2M, HSP-72, HSP-90, HST-2, hTERT, hTRT, iCE, apoptosis inhibitors such as as survivin, adenocarcinoma antigen KH-1 (Deshpande and Danishefsky, Nature 387, 1997, pp. 164-166), KIAA0205, K-ras, LAGE, LAGE-1, LDLR/FUT, Lewis Y antigen, MAGE-1, MAGE -2, MAGE-3, MAGE-6, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MAGE-B5, MAGE-B6, MAGE-C2 , MAGE-C3, MAGE D, MART-1, MART-1/Melan-A (Kawakami and Rosenberg, Int. Rev. Immunol. 14, 1997, pp. 173-192), MC1R, MCSP, MDM-2, MHCII , mTOR, myosin/m, MUC1, MUC2, MUM-1, MU M-2, MUM-3, neo-polyA polymerase, NA88-A, NFX2, NY-ESO-1, NY-ESO-1a (CAG-3), PAGE-4, PAP, proteinase 3 (Molldrem et al. , Blood 88, 1996, pp. 2450-2457; Molldrem et al., Blood 90, 1997, pp. 2529-2534), P15, p53, p97, p190, PD-L1, Pgp, PIK3CA, Pm1/RARα, PRAME, proteoglycan, PSA, PSM, PSMA, RAGE, RAS, RCAS1, RU1, RU2, SAGE, SART-1 , SART-2, SART-3, SP17, SPAS-1, SSX2, SSX4 TEL/AML1, TPI/m, tyrosinase, TARP, telomerase, TRP-1 (gp75), TRP-2, TRP-2/INT2, VEGF , WT-1, Wue antigen, cell surface targets GC182, GT468 or GT512, and alternatively translated NY-ESO-ORF2 and CAMEL proteins derived from the NY-ESO-1 and LAGE-1 genes.

More preferably, the cancer and/or tumor associated antigen is selected from the group consisting of EpCAM, HER2/neu, CEA, MAGE, proteoglycan, VEGF, EGFR, mTOR, PIK3CA, RAS, alpha(v)beta(3)-integrin, HLA , HLA-DR, ASC, carbonic anhydrase, CD1, CD2, CD4, CD6, CD7, CD8, CD11, CD13, CD14, CD19, CD20, CD21, CD22, CD23, CD24, CD30, CD33, CD37, CD38, CD40, CD41 , CD47, CD52, CD138, c-erb-2, CALLA, MHCII, CD44v3, CD44v6, p97, GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1b, GT3, GQ1, NY-ESO-1, NFX2 , SSX2, SSX4, Trp2, gp100, tyrosinase, MUC-1, telomerase, survivin, p53, CA125, Wue antigen, Lewis Y antigen, HSP-27, HSP-70, HSP-72, HSP-90, Pgp, MCSP, EphA2 and cell surface targets GC182, GT468 or GT512. This means that the antibody or antigen-binding fragment intended/intended for use according to the present invention contains a paratope that preferentially recognizes (has the ability to bind to) an epitope of a cancer and/or tumor associated antigen selected from the group consisting of EpCAM, HER2/neu, CEA, MAGE, proteoglycan, VEGF, EGFR, mTOR, PIK3CA, RAS, alpha(v)beta(3)-integrin, HLA, HLA-DR, ASC, carbonic anhydrase, GD1, CD2, CD4, CD6, CD7, CD8, CD11, CD13, CD14, CD19, CD20, CD21, CD22, CD23, CD24, CD30 CD33, CD37, CD38, CD40, CD41, CD47, CD52, CD138, c-erb-2, CALLA, MHCII, CD44v3 , CD44v6, p97, GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1b, GT3, GQ1, NY-ESO-1, NFX2, SSX2, SSX4, Trp2, gp100, tyrosinase, MUC-1, telomerase, survivin , p53, CA125, Wue antigen, Lewis Y antigen, HSP-27, HSP-70, HSP-72, HSP-90, Pgp, MCSP, EphA2 and cell surface targets GC182, GT468 or GT512.

Particularly preferably, the cancer and/or tumor associated antigen is selected from the group consisting of EpCAM, HER2/neu, CEA, MAGE, VEGF, EGFR, mTOR, PIK3CA, RAS, GD2, CD19, CD20, CD30, CD33 and CD38, more preferably the cancer and/or tumor associated antigen is selected from the group consisting of EpCAM, HER2/neu, CEA, GD2, CD19, CD20, CD30, CD33 and CD38, even more preferably the cancer and/or tumor associated antigen is selected from the group consisting of of EpCAM, HER2/neu, GD2 and CD20, and most preferably the cancer and/or tumor associated antigen is EpCAM. This means that the antibody or antigen-binding fragment intended/intended for use according to the present invention contains a paratope that preferentially recognizes the EpCAM, HER2/neu, CEA, MAGE, VEGF, EGFR, mTOR, PIK3CA, RAS, GD2, CD19, CD20, CD30, CD33 and CD38; more preferably, the antibody or antigen-binding fragment thereof for/intended for use in the present invention contains a paratope that preferentially recognizes an EpCAM, HER2/neu, CEA, GD2, CD19, CD20 or CD33 epitope; even more preferably, the antibody or antigen-binding fragment thereof for/intended for use according to the present invention contains a paratope that preferentially recognizes an epitope of EpCAM, HER2/neu, GD2 or CD20; and most preferably, the antibody or antigen-binding fragment thereof for use according to the present invention contains a paratope that preferentially recognizes an EpCAM or GD2 epitope.

Preferably, the cancer and/or tumor associated antigen for which the T cell redirecting multifunctional antibody or antigen binding fragment thereof contains specificity is not PD-L1. More preferably, the T cell redirecting multifunctional antibody or antigen binding fragment thereof does not contain any specificity for PD-L1/PD-L1 binding site. More generally, even more preferably, the cancer and/or tumor associated antigen for which the T cell redirecting multifunctional antibody or antigen binding fragment thereof contains specificity is not an immune checkpoint molecule and/or its ligand. Most preferably, the T cell-redirecting multifunctional antibody or antigen-binding fragment thereof does not contain any specificity for the immune checkpoint molecule and/or its ligand/binding site of the immune checkpoint molecule and/or its ligand.

Preferably, the cancer and/or tumor associated antigen (or epitope thereof, respectively) to be recognized by the antibody or antigen-binding fragment thereof for use in the present invention is EpCAM. Preferably, the cancer and/or tumor associated antigen (or epitope thereof, respectively) to be recognized by the antibody or antigen-binding fragment thereof for use in the present invention is GD2. Preferably, the cancer and/or tumor associated antigen (or epitope thereof, respectively) to be recognized by the antibody or antigen-binding fragment thereof for use in the present invention is Her2/neu. Preferably, the cancer and/or tumor associated antigen (or epitope thereof, respectively) to be recognized by the antibody or antigen-binding fragment thereof for use in the present invention is GD3. Preferably, the cancer and/or tumor associated antigen (or epitope thereof, respectively) to be recognized by the antibody or antigen-binding fragment thereof for use in the present invention is CD20. Preferably, the cancer and/or tumor associated antigen (or epitope thereof, respectively) to be recognized by the antibody or antigen-binding fragment thereof for use in the present invention is CD19. Preferably, the cancer and/or tumor associated antigen (or epitope thereof, respectively) to be recognized by the antibody or antigen-binding fragment thereof for use in the present invention is CD30. Alternatively, the cancer and/or tumor associated antigen (or epitope thereof, respectively) to be recognized by the antibody or antigen-binding fragment thereof for use in the present invention is CEA, MAGE, VEGF, EGFR, mTOR, PIK3CA, or RAS.

Preferably, the antibody or antigen-binding fragment thereof for/intended for use according to the present invention binds (I) by its first specificity, e.g. by its first paratope, to an epitope of a T-cell surface antigen selected from the group consisting of CD2 , CD3, CD4, CD5, CD6, CD8, CD28, CD40L and CD44, preferably CD2 or CD3, more preferably CD3, and (ii) by its second specificity, for example by its second paratope, with associated cancer and/ or a tumor antigen selected from the group consisting of EpCAM, HER2/neu, CEA, MAGE, VEGF, EGFR, mTOR, PD-L1, PIK3CA, RAS, GD2, CD19, CD20, CD30, CD33 and CD38 tumor antigens.

More preferably, the antibody or antigen-binding fragment thereof for/intended for use in the present invention binds (I) by its first specificity, e.g. by its first paratope, to a T-cell surface antigen epitope selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD8, CD28, CD40L and CD44, preferably CD2 or CD3, more preferably CD3, and (ii) by its second specificity, for example by its second paratope, with associated with cancer and /or a tumor antigen selected from the group consisting of EpCAM, HER2/neu, CEA, MAGE, VEGF, EGFR, mTOR, PIK3CA, RAS, GD2, CD19, CD20, CD30, CD33 and CD38 tumor antigens.

An antibody or antigen-binding fragment thereof for/intended for use according to the present invention preferably binds by its first specificity, for example, by a first paratope, to an epitope of a T-cell surface antigen, preferably CD3, and by its second specificity, for example, by means of its second paratope, with a cancer and/or tumor associated antigen, preferably selected from the group consisting of tumor antigens EpCAM, HER2/neu, CEA, MAGE, VEGF, EGFR, mTOR, PIK3CA, RAS, GD2, CD19, CD20, CD30, CD33 and CD38, or with gangliosides GM1, GM2, GM3, GD1a, GD1b, GD3, GT1b, GT3 or GQ1.

Preferably, the antibody or antigen-binding fragment thereof for/intended for use in accordance with the present invention comprises a first specificity for CD3 and a second specificity for a cancer and/or tumor associated antigen selected from the group consisting of EpCAM, HER2/neu, CEA , GD2, CD19, CD20 and CD33.

Preferably also, the cancer and/or tumor associated antigen for which the T-cell redirecting multifunctional antibody or antigen-binding fragment thereof contains specificity is not CD19. More preferably, the T cell redirecting multifunctional antibody or antigen binding fragment thereof does not contain any specificity for CD19/nor any CD19 binding site.

Preferably also, the cancer and/or tumor associated antigen for which the T-cell redirecting multifunctional antibody or antigen-binding fragment thereof contains specificity is not CD20. More preferably, the T cell redirecting multifunctional antibody or antigen binding fragment thereof does not contain any specificity for CD20/nor any CD20 binding site.

Preferably also, the cancer and/or tumor associated antigen for which the T-cell redirecting multifunctional antibody or antigen-binding fragment thereof contains specificity is not a CEA. More preferably, the T cell redirecting multifunctional antibody or antigen binding fragment thereof does not contain any CEA specificity/nor any CEA binding site.

Preferably, an antibody or antigen-binding fragment thereof for use according to the present invention may contain one specificity, preferably one paratope, for CD3 and one specificity, preferably one paratope, for EpCAM (anti-CD3 x anti-EpCAM). Preferably, an antibody or antigen-binding fragment thereof for use according to the present invention may contain one specificity, preferably one paratope, for CD3 and one specificity, preferably one paratope, for GD2 (anti-CD3 × anti-GD2). Preferably, an antibody or antigen-binding fragment thereof for/intended for use in accordance with the present invention may contain one specificity, preferably one paratope, for CD3 and one specificity, preferably one paratope, for Her2/neu (anti-CD3 × anti-Her2/ neu). Preferably, an antibody or antigen-binding fragment thereof for use according to the present invention may contain one specificity, preferably one paratope, for CD3 and one specificity, preferably one paratope, for GD3 (anti-CD3 x anti-GD3). Preferably, an antibody or antigen-binding fragment thereof for use according to the present invention may contain one specificity, preferably one paratope, for CD3 and one specificity, preferably one paratope, for CD20 (anti-CD3 x anti-CD20). Preferably, an antibody or antigen-binding fragment thereof for use according to the present invention may contain one specificity, preferably one paratope, for CD3 and one specificity, preferably one paratope, for CD19 (anti-CD3 × anti-CD19). Preferably, an antibody or antigen-binding fragment thereof for use according to the present invention may contain one specificity, preferably one paratope, for CD3 and one specificity, preferably one paratope, for CEA (anti-CD3 x anti-CEA). Preferably, an antibody or antigen-binding fragment thereof for use according to the present invention may contain one specificity, preferably one paratope, for CD3 and one specificity, preferably one paratope, for MAGE (anti-CD3 x anti-MAGE). Preferably, an antibody or antigen-binding fragment thereof for use according to the present invention may contain one specificity, preferably one paratope, for CD3 and one specificity, preferably one paratope, for VEGF (anti-CD3 x anti-VEGF). Preferably, an antibody or antigen-binding fragment thereof for use according to the present invention may contain one specificity, preferably one paratope, for CD3 and one specificity, preferably one paratope, for EGFR (anti-CD3 x anti-EGFR). Preferably, an antibody or antigen-binding fragment thereof for use according to the present invention may contain one specificity, preferably one paratope, for CD3 and one specificity, preferably one paratope, for mTOR (anti-CD3 x anti-mTOR). Preferably, an antibody or antigen-binding fragment thereof for use according to the present invention may contain one specificity, preferably one paratope, for CD3 and one specificity, preferably one paratope, for PIK3CA (anti-CD3 x anti-PIK3CA). Preferably, an antibody or antigen-binding fragment thereof for use according to the present invention may contain one specificity, preferably one paratope, for CD3 and one specificity, preferably one paratope, for RAS (anti-CD3 x anti-RAS).

Alternatively, an antibody or antigen-binding fragment thereof for use according to the present invention may contain one specificity, preferably one paratope, for CD3 and one specificity, preferably one paratope, for CD30 (anti-CD3 x anti-CD30) . Preferably, an antibody or antigen-binding fragment thereof for use according to the present invention may contain one specificity, preferably one paratope, for CD3 and one specificity, preferably one paratope, for CD33 (anti-CD3 x anti-CD33). Preferably, an antibody or antigen-binding fragment thereof for/intended for use according to the present invention may contain one specificity, preferably one paratope, for CD3 and one specificity, preferably one paratope, for an epitope of the arbovirus E protein (anti-CD3 x anti-protein E arbovirus).

Preferably, the antibody or antigen-binding fragment thereof for/intended for use in the present invention comprises two specificities selected from anti-EpCAM x anti-CD3, anti-CD20 x anti-CD3, anti-HER2/neu x anti-CD3, anti- GD2 × anti-CD3 and anti-CD19 × anti-CD3. More preferably, the antibody or antigen-binding fragment thereof for use according to the present invention contains two specificities selected from anti-EpCAM x anti-CD3, anti-GD2 x anti-CD3 and anti-HER2/neu x anti-CD3. Even more preferably, the antibody or antigen-binding fragment thereof for/intended for use according to the present invention contains two specificities selected from anti-EpCAM x anti-CD3 and anti-GD2 x anti-CD3.

Human FcγRI, FcγRIIa and/or FcγRIII binding site

An antibody or antigen-binding fragment thereof intended/intended for use according to the present invention contains a binding site for human FcγRI, FcγRIIa and/or FcγRIII. More preferably, the antibody or antigen-binding fragment thereof for/intended for use in accordance with the present invention contains a human FcγRIIa binding site. A human FcγRI, FcγRIIa and/or FcγRIII binding site, such as an Fc region, allows the multifunctional antibody to further recruit cells expressing FcγRI, FcγRIIa and/or FcγRIII, such as FcγRI-, FcγRIIa- and/or FcγRIII-positive helper cells, for example, macrophages, dendritic cells, natural killer (NK) cells, and other cells expressing FcγRI, FcγRIIa and/or FcγRIII. Since multifunctional antibodies are at least bispecific (or multispecific) antibodies, they preferably have the ability to simultaneously recruit (I) T cells and (II) FcγRI, FcγRIIa and/or FcγRIII expressing cells, such as immune support cells, for example, monocytes/macrophages, natural killer cells, dendritic cells or other expressing FcγRI, FcγRIIa and/or FcγRIII cells, to (III) targeted cancer/tumor cells and activation of said cells. Simultaneous activation of these different classes of effector cells leads to efficient cytolysis of tumor cells through various mechanisms such as phagocytosis and perforin-mediated cytotoxicity. Typically, the net result of exposure to a preferred multifunctional antibody that contains an FcγRI, FcγRIIa and/or FcγRIII binding site is binding of T cells and Fc receptor positive cells to target cells, e.g., tumor cells, resulting in destruction of the tumor cells. . Multifunctional antibodies initiate the elimination of tumor cells through (I) antibody-mediated cell-dependent cytotoxicity, (II) T-cell mediated cell cytolysis, and (III) induction of antitumor immunity.

In general, Fc-gamma receptors (FcγR) are a family of Fc receptors for IgG. All Fcγ receptors (FcγR) correspond to the immunoglobulin superfamily and are the most important Fc receptors in terms of inducing phagocytosis of opsonized (tagged) microorganisms. This family includes several members: FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIIa (CD16a) and FcγRIIIb (CD16b) in humans and FcγRI, FcγRIIb, FcγRIII and FcγRIV in mice. The complexity of the FcγR family is reflected in the presence of four different IgG subclasses in humans (IgG1-IgG4) and mice (IgG1, IgG2a, IgG2b and IgG3), which bind with different affinity and specificity to different Fcγ receptors (see review by Nimmerjahn F. and Ravetch J.V., Fcγ receptors as regulators of immune responses, Nat Rev Immunol 8, 2008, pp. 34-47). Traditionally, FcγR families have been classified according to their level of receptor affinity for specific IgG subclasses and the type of signaling pathway they trigger, ie. whether it is inhibitory or activating. FcγRIIb is conserved in mice and humans and is the only inhibitory FcγR known, it transmits inhibitory signals via an immunoreceptor tyrosine-based inhibitory motif (ITIM) present in its cytoplasmic region. All other FcγRs, with the exception of the human GPI-anchored FcγRIIIb, activate signaling pathways through ITAM motifs present in their cytoplasmic regions. FcγRIa is the only known high affinity FcγR in mice and humans. All other FcγRs have a 100-1000-fold lower affinity in the range of low to medium micromolar concentrations and are specific for a wider range of IgG subclasses. Inhibitory FcγRIIb is the most widely expressed FcγR and is present on virtually all leukocytes except NK cells and T cells. Finally, IgG1 activity has been shown to be negatively regulated by inhibitory FcγRIIb. Thus, it is assumed that the inhibitory FcγRIIb has a "regulatory" function in relation to IgG responses.

Human FcγRIIa (IgG Fc receptor IIa; CD32a) is a low affinity receptor for IgG and is expressed on macrophages, neutrophils, eosinophils, platelets and dendritic cells. FcγRIIa transmits an activating signal upon binding to a ligand and results in the initiation of inflammatory responses including cytolysis, phagocytosis, degranulation, and cytokine production. Two genetically determined structurally distinct human FcγRIIa allotypes are known: FcγRIIa-R131 products and FcγRIIa-H131 alleles. FcγRIIa responses can be modulated by signals from co-expressed inhibitory receptors such as FcγRIIb (CD32b), with signal strength dependent on the ratio between expression levels of activating and inhibitory receptors.

An antibody or antigen-binding fragment thereof intended/intended for use according to the present invention contains a binding site for human FcγRI, FcγRIIa and/or FcγRIII, wherein the antibody or antigen-binding fragment thereof binds with higher affinity to human FcγRI, FcγRIIa and/or FcγRIII than with human FcγRIIb. This means that if an antibody or antigen-binding fragment thereof binds to only one of FcγRI, FcγRIIa and/or FcγRIII (i.e. (i) the antibody or antigen-binding fragment thereof binds to FcγRI but not to FcγRIIa or FcγRIII; (ii) the antibody or its antigen-binding fragment binds FcγRIIa but not FcγRI or FcγRIII; or (III) the antibody or antigen-binding fragment binds FcγRIII but not FcγRI or FcγRIIa), then the antibody or antigen-binding fragment binds with higher affinity to the specified one from human FcγRI, FcγRIIa and/or FcγRIII than with human FcγRIIb. However, if an antibody or antigen-binding fragment thereof binds to more than one of FcγRI, FcγRIIa and/or FcγRIII (i.e. (i) the antibody or antigen-binding fragment thereof binds to FcγRI and FcγRIIa but not to FcγRIII; (ii) the antibody or its antigen-binding fragment binds FcγRIIa and FcγRIII but not FcγRI; (III) the antibody or its antigen-binding fragment binds FcγRI and FcγRIII but not FcγRIIa; or (IV) the antibody or its antigen-binding fragment binds FcγRI, FcγRIIa and FcγRIII) , it is sufficient if the antibody or antigen-binding fragment thereof binds with higher affinity to one of the human FcγRI, FcγRIIa and/or FcγRIII than to human FcγRIIb. However, more preferably, the antibody, or antigen-binding fragment thereof, binds with higher affinity to two of the human FcγRI, FcγRIIa and/or FcγRIII than to human FcγRIIb. Most preferably, the antibody or antigen-binding fragment thereof binds with higher affinity to all three human FcγRI, FcγRIIa and/or FcγRIII than to human FcγRIIb. In particular, (I) the antibody or antigen-binding fragment most preferably binds with higher affinity to each of the human FcγRI, FcγRIIa and/or FcγRIII than human FcγRIIb - and/or (II) the antibody or antigen-binding fragment most preferably binds to higher affinity for each of said human FcγRI, FcγRIIa and/or FcγRIII for which the antibody or antigen-binding fragment thereof contains a binding site than for human FcγRIIb.

In general, human FcγRI, FcγRIIa and FcγRIII are activating Fc receptors, while human FcγRIIb is an inhibitory Fc receptor. Therefore, an antibody or antigen-binding fragment thereof which/which binds with higher affinity to human FcγRI, FcγRIIa and/or FcγRIII than to human FcγRIIb, as described above, binds to an activating Fc receptor rather than an inhibitory Fc receptor, resulting in a shift in the activation/inhibition ratio towards activation and a decrease in the regulatory effect of the inhibitory FcγRIIb. Consequently, phagocytosis and direct cytolysis are enhanced.

In the context of the present description, "higher affinity for human FcγRIIa than for human FcγRIIb" means, in particular, that the value of the EC ₅₀ (effective concentration at which the level of binding signals is half the maximum) of a particular antibody or fragment, characterizing the binding to FcγRIIa , less than the value of the EU ₅₀ of the same antibody or fragment, characterizing the binding to FcγRIIb. In other words, a lower concentration of the antibody (or fragment thereof) is required to achieve half the maximum FcγRIIa binding level than to achieve half the maximum FcγRIIb binding level. For example, the EC ₅₀ (FcγRIIb)/EC ₅₀ (FcγRIIa) ratio can determine the FcγRIIa/FcγRIIb ratio (which is >1 if the antibody binds with higher affinity to human FcγRIIa than to human FcγRIIb). EC ₅₀ values can be determined, for example, using a standard enzyme-linked immunosorbent assay (ELISA). Alternatively, the binding affinities of an antibody or fragment thereof can also be determined by surface plasma resonance measurement, for example, as described in Richards JO, Karki S., Lazar GA, Chen H., Dang W., Desjarlais JR, Optimization of antibody binding to FcgammaRIIa enhances macrophage phagocytosis of tumor cells. Mol Cancer Ther 7, 2008, pp. 2517-2527). In general, the binding affinities of an antibody or fragment thereof for FcγRIIa and FcγRIIb can be determined using a variety of methods known to those skilled in the art. However, the binding affinities of a particular antibody or fragment thereof to FcγRIIa and FcγRIIb are determined primarily by the same method that determines the binding affinities of FcγRIIa and FcγRIIb.

The above applies also to the expression "higher affinity for human FcγRI than for human FcγRIIb", which means, in particular, that the EC ₅₀ value (effective concentration at which the level of binding signals is half the maximum) of a particular antibody or its fragment, characterizing binding to FcγRI, less than the value of the EU ₅₀ of the same antibody or fragment, characterizing the binding to FcγRIIb. In other words, a lower concentration of antibody (or fragment thereof) is required to achieve half maximum FcγRI binding than half maximum FcγRIIb binding. For example, the EC ₅₀ (FcγRIIb)/EC ₅₀ (FcγRI) ratio can determine the FcγRI/FcγRIIb ratio (which is >1 if the antibody binds with higher affinity to human FcγRI than to human FcγRIIb). EC ₅₀ values can be determined, for example, using a standard enzyme-linked immunosorbent assay (ELISA). Alternatively, the binding affinities of an antibody or fragment thereof can also be determined by measuring surface plasma resonance, for example, as described in Richards JO, Karki S., Lazar GA, Chen H., Dang W., Desjarlais JR, Optimization of antibody binding to FcgammaRIIa enhances macrophage phagocytosis of tumor cells. Mol Cancer Ther 7, 2008, pp. 2517-2527). In general, the binding affinities of an antibody or fragment thereof for FcγRI and FcγRIIb can be determined using a variety of methods known to those skilled in the art. However, the binding affinities of a particular antibody or fragment thereof to FcγRI and FcγRIIb are determined primarily by the same method that determines the binding affinities of FcγRI and FcγRIIb.

The above applies also to the expression "higher affinity for human FcγRIII than for human FcγRIIb", which means, in particular, that the EC ₅₀ value (effective concentration at which the level of binding signals is half the maximum) of a particular antibody or its fragment, characterizing binding to FcγRIII, less than the value of the EU ₅₀ of the same antibody or fragment, characterizing the binding to FcγRIIb. In other words, a lower concentration of antibody (or fragment thereof) is required to achieve half maximum FcγRIII binding than half maximum FcγRIIb binding. For example, the EC ₅₀ (FcγRIIb)/EC ₅₀ (FcγRIII) ratio can determine the FcγRIII/FcγRIIb ratio (which is >1 if the antibody binds with higher affinity to human FcγRIII than to human FcγRIIb). EC ₅₀ values can be determined, for example, using a standard enzyme-linked immunosorbent assay (ELISA). Alternatively, the binding affinities of an antibody or fragment thereof can also be determined by measuring surface plasma resonance, for example, as described in Richards JO, Karki S., Lazar GA, Chen H., Dang W., Desjarlais JR, Optimization of antibody binding to FcgammaRIIa enhances macrophage phagocytosis of tumor cells. Mol Cancer Ther 7, 2008, pp. 2517-2527). In general, the binding affinities of an antibody or fragment thereof for FcγRIII and FcγRIIb can be determined using a variety of methods known to those skilled in the art. However, the binding affinities of a particular antibody or fragment thereof to FcγRIII and FcγRIIb are determined primarily by the same method that determines the binding affinities of FcγRIII and FcγRIIb.

Preferably, an antibody or antigen-binding fragment thereof for/intended for use in accordance with the present invention contains a human FcγRI binding site, wherein the antibody or antigen-binding fragment thereof binds with higher affinity to human FcγRI than to human FcγRIIb. It is also preferred that an antibody or antigen-binding fragment thereof for/intended for use in accordance with the present invention contains a human FcγRIII binding site, wherein the antibody or antigen-binding fragment thereof binds with higher affinity to human FcγRIII than to human FcγRIIb. Most preferably, the antibody or antigen-binding fragment thereof for/intended for use in accordance with the present invention contains a human FcγRIIa binding site, wherein the antibody or antigen-binding fragment thereof binds with higher affinity to human FcγRIIa than to human FcγRIIb.

In particular, a higher ratio of FcγRIIa/FcγRIIb binding levels significantly increases antibody-dependent cell-mediated phagocytosis (ADCP; Richards J.O., Karki S., Lazar G.A., Chen H., Dang W., Desjarlais J.R., Optimization of antibody binding to FcgammaRIIa enhances macrophage phagocytosis of tumor cells. Mol Cancer Ther 7, 2008, pp. 2517-2527) and contributes to the activation and maturation of DC, having a positive effect on the induction of antitumor immunity (Boruchov A.M., Heller G., Veri M.C., Bonvini E., Ravetch J.V., Young J.W., Activating and inhibitory IgG Fc receptors on human DCs mediate opposing functions, J Clin Invest 115, 2005, pp. 2914-2923 Kalergis A.M., Ravetch J.V., Inducing tumor immunity through the selective engagement of activating Fcgamma receptors on dendritic cells. J Exp Med 195, 2002, pp. 1653-1659). In particular, Richards et al., 2008 demonstrated that antibodies binding with higher affinity to human FcγRIIa than to human FcγRIIb mediate stronger phagocytosis by macrophages of target cells carrying the antibody on their surface (Richards J.O., Karki S. , Lazar G.A., Chen H., Dang W., Desjarlais J.R., Optimization of antibody binding to FcgammaRIIa enhances macrophage phagocytosis of tumor cells, Mol Cancer Ther 7, 2008, pp. 2517-2527). Thus, an antibody or antigen-binding fragment thereof for use according to the present invention, which/which binds with a higher affinity to human FcγRIIa than to human FcγRIIb, initiates a stronger macrophage phagocytosis of tumor cells. Preferably, an antibody or antigen-binding fragment thereof for use according to the present invention binds with higher affinity to the R131 form of human FcγRIIa than to human FcγRIIb.

Examples of antibodies having higher affinity for human FcγRIIa than for human FcγRIIb are known in the art. For example, Richards et al., 2008, describe an IgG1 variant that contains the G236A substitution, resulting in a significant increase in the FcγRIIa/FcγRIIb ratio (Richards JO, Karki S., Lazar GA, Chen H., Dang W., Desjarlais JR, Optimization of antibody binding to FcgammaRIIa enhances macrophage phagocytosis of tumor cells, Mol Cancer Ther 7, 2008, pp. 2517-2527). In addition, Lindhofer et al., 2011 described Triomab® antibodies containing the mouse IgG2a/rat IgG2b Fc region, which are also characterized by high FcγRIIa/FcγRIIb ratios (Lindhofer H., Hess J., Ruf P., Trifunctional Triomab ^® antibodies for cancer therapy in: Bispecific antibodies, edited by Kontermann RE, Springer, Berlin, 2011, pp. 289-312), and thus enhance phagocytosis by macrophages of target cells carrying the antibody on their surface, and enhance direct cytolysis of tumor cells.

Although only an FcγRI, FcγRIIa or FcγRIII binding site is sufficient, it is preferred that the antibody or antigen-binding fragment thereof to be/intended for use according to the present invention contains/contains an Fc fragment, in particular an Fc region.

As used herein, "Fc fragment" refers to a sequence from an immunoglobulin heavy chain region that begins at the hinge region located upstream of the papain cleavage site and ends at the C-terminus of an immunoglobulin heavy chain. In particular, an "Fc fragment" contains an FcγRI, FcγRIIa and/or FcγRIII binding site. Preferably, the "Fc fragment" contains the FcγRIIa binding site. However, it is also preferred that the Fc fragment may have a functionality other than binding to the Fc receptor, eg binding to a protein of the complement system. In this case, the FcγRI, FcγRIIa and/or FcγRIII binding site may be present in the antibody separately from the Fc fragment. Therefore, an "Fc fragment" may be the entire Fc region or a portion thereof (eg, a domain). Preferably, the "Fc fragment" has the full functionality of the entire Fc region, for example, including binding to the Fc receptor and optionally binding to a protein of the complement system. Thus, the antibody proposed for use according to the present invention preferably contains a complete Fc region, where the complete Fc region contains at least a hinge domain, a CH2 domain and a CH3 domain. An Fc fragment may also contain one or more amino acid insertions, deletions, or substitutions relative to a naturally occurring Fc region. For example, at least one hinge domain, CH2 domain or CH3 domain (or part thereof) may be deleted as a result of deletions. For example, an Fc fragment may contain or consist of: (i) a hinge domain (or part thereof) fused to a CH2 domain (or part thereof), (ii) a hinge domain (or part thereof) fused to a CH3 domain ( or part thereof), (III) CH2 domain (or part thereof) fused to CH3 domain (or part thereof), (IV) hinge domain (or part thereof), (V) CH2 domain (or part thereof) , or (VI) CH3 domain or part thereof. Preferably the Fc fragment contains a G236A substitution.

Preferably, the Fc fragment/Fc region of an antibody or fragment thereof binds to Fc receptor positive cells that preferably express at least FcγRI, FcγRIIa and/or FcγRIII, more preferably at least FcγRIIa.

Preferably, the binding site for human FcγRI, FcγRIIa and/or FcγRIII (primarily an Fc fragment) in an antibody (or fragment thereof) to be used according to the present invention is mouse IgG2a/rat IgG2b. This means that those parts of the antibody (or its fragment) that are involved in binding to human FcγRI, FcγRIIa and/or FcγRIII, are sequences of mouse IgG2a and/or rat IgG2b. In particular, the antibody (or fragment thereof) contains a binding site for human FcγRI, FcγRIIa and/or FcγRIII (primarily an Fc fragment) of mouse IgG2a and/or rat IgG2b. It is well known to one skilled in the art that mouse IgG2a and rat IgG2b contain a human FcγRI, FcγRIIa and/or FcγRIII binding site (primarily an Fc fragment), since well-known antibodies approved for use in humans, such as catumaxomab, contain Fc- mouse IgG2a/rat IgG2b fragment. Therefore, the combination of both (I) the binding site of human FcγRI, FcγRIIa and/or FcγRIII, especially the Fc fragment, mouse IgG2a and (II) the binding site of human FcγRI, FcγRIIa and/or FcγRIII, especially the Fc fragment, is preferred, rat IgG2b), for example, present in heterologous antibodies presented in the present description. Most preferably, the antibody or fragment thereof comprises a mouse IgG2a/rat IgG2b Fc region. This means in particular that the Fc region of an antibody consists of (I) the Fc region of the mouse IgG2a chain and (II) the Fc region of the rat IgG2b chain. Specifically, one antibody (heavy) chain contains a mouse IgG2a heavy chain Fc region and the other (heavy) chain of an antibody contains a rat IgG2b heavy chain Fc region, and both together form an antibody Fc region as described herein (denoted as "Fc region of mouse IgG2a/rat IgG2b"). Such antibodies are characterized by selectively increased binding to the activating FcγRIIa, but not to its inhibitory partner FcγRIIb (Lindhofer H., Hess J., Ruf P., Trifunctional Triomab ^® antibodies for cancer therapy, in: Bispecific antibodies, edited by Kontermann RE, ed. - in Springer, Berlin, 2011, pp. 289-312).

Antibody Format

An antibody or antigen-binding fragment thereof for/intended for use in accordance with the present invention may be of any format as long as it includes at least the two specificities described above and the FcγRI, FcγRIIa and/or FcγRIII binding site described above. In particular, the term "multifunctional antibodies" preferably includes "whole" antibodies, such as complete IgG molecules or IgG-like molecules, while the term "antigen-binding fragments" preferably refers to small recombinant molecule formats, such as molecules in the form of tandems of single-chain variable fragments (taFv), diabodies (Db), single chain diabodies (scDb) and various other derivatives of these molecules (for example, described in Byrne H. et al., Trends Biotech, 31 (11), 2013, pp. 621-632, where Fig. 2 shows different formats of bispecific antibodies, Weidle U.H. et al., Cancer Genomics and Proteomics 10, 2013, pp. 1-18, see especially Fig. 1, and Chan A.C. and Carter P.J., Nat Rev Immu 10, 2010, pp. 301-316, see especially Fig. 3). Preferred examples include (but are not limited to) Triomab and quadroma antibodies.

Thus, the antibody or framework structure, or antigen-binding fragment thereof, intended/intended for use according to the present invention can be selected from the group including Triomab, hybrid hybridoma (quadroma), anticalin-based multispecific platform (Pieris); diabody; single chain diabodies; tandem single chain Fv fragments, tandem antibodies (TandAb), trispecific Abs (Affimed) (105-110 kDa); Darts (double affinity reorienting antibodies, Macrogenics); derivatives of a multifunctional recombinant antibody (110 kDa), molecules based on the Dock and lock platform (docking and closure), molecules based on the Knob into hole platform (KIH), humanized bispecific antibody in as IgG (REGN1979) (Regeneron); bispecific antibodies Mab ² format (F-Star); DVD-Ig = double variable domain immunoglobulin molecule (Abbvie), kappa-lambda chain antibodies, TBTI = quadrivalent bispecific tandem Ig antibody, and CrossMab.

The antibody, or antigen-binding fragment thereof, intended/intended for use according to the present invention, can be selected from bispecific IgG-like antibodies (BsIgG), including CrossMab; DAF (two-in-one); DAF (four-in-one); DutaMab; DT-IgG; molecules based on the "Knobs-in-holes" platform with a common LC chain; molecules obtained by assembly according to the "knobs-in-holes" type, molecules with a charged pair, molecules with the exchange of Fab-arms; SEEDbody; Triomab; LUZ-Y; fcab; κλ antibodies; and molecules based on an orthogonal Fab fragment (Orthogonal Fab). These bispecific antibody formats are presented and described in, for example, Spiess C., Zhai Q. and Carter P.J. Molecular Immunology 67, 2015, pp. 95-106, see especially FIGS. 1 and the corresponding description, for example, on pp. 95-101.

Preferably, the antibody or antigen-binding fragment thereof for/intended for use according to the present invention can be selected from complemented IgG antibodies with an additional antigen-binding fragment, including DVD-IgG, IgG(H)-scFv; scFv-(H)IgG; IgG(L)-scFv; scFV-(L)IgG; IgG(L,H)-Fv; IgG(H)-V; V(H)-IgG; IgG(L)-V; V(L)-IgG; KIH IgG-scFab; 2scFv-IgG; IgG-2scFv; scFv4-Ig; scFv4-Ig; Zybody; and DVI-IgG (four-in-one). These bispecific antibody formats are presented and described, for example, in Spiess C., Zhai Q. and Carter P.J., Molecular Immunology 67, 2015, ss. 95-106, see especially FIGS. 1 and the corresponding description, for example, on pp. 95-101.

Preferably, the antibody or antigen-binding fragment thereof for/intended for use in the present invention may be selected from bispecific antibody fragments including sc-diabodi-CH3; diabodi-CH3; minibody; TriBi minibody; scFv-CH3 KIH; scFv-KIH; Fab-scFv-Fc; tetravalent HCAb; scDiabody-Fc; diabodi-Fc; scFv-Fc tandem; and intrabody. These bispecific antibody formats are presented and described, for example, in Spiess C., Zhai Q. and Carter P.J., Molecular Immunology 67, 2015, ss. 95-106, see especially FIGS. 1 and the corresponding description, for example, on pp. 95-101.

In particular, the antibody or antigen-binding fragment intended/intended for use according to the present invention can be selected from bispecific antibody conjugates, including IgG-IgG and Cov-X-Body. These bispecific antibody formats are presented and described, for example, in Spiess C., Zhai Q. and Carter P.J., Molecular Immunology 67, 2015, ss. 95-106, see especially FIGS. 1 and the corresponding description, for example, on pp. 95-101.

Preferably, the antibody or antigen-binding fragment thereof for/intended for use in accordance with the present invention is a bispecific, trifunctional antibody.

Preferably, the antibody, or antigen-binding fragment thereof, for/intended for use in the present invention has an IgG-like format (an IgG-based antibody, also referred to as an "IgG-type" antibody), wherein an antibody having an IgG-like format is typically contains two heavy chains and two light chains. In most cases, immunoglobulin G (IgG) is considered as the type of antibody. In the context of the present description, this means a protein complex consisting of four peptide chains, two identical heavy chains and two identical light chains, arranged in a Y-shaped structure typical of antibody monomers. Each IgG typically has two antigen-binding sites (centers), which may be different or identical. IgG, which accounts for approximately 75% of antibodies in human serum, is the most common type of antibody present in the bloodstream. Physiologically, IgG molecules are produced and released into plasma by B cells.

Examples of an antibody having an IgG-like format include quadroma and various IgG-scFv formats (see: Byrne H. et al., Trends Biotech, 31(11), 2013, pp. 621-632, FIGS. 2A-E) , with the preferred being a quadroma, which is preferably created by the fusion of two different hybridomas. In the IgG class, the antibodies may preferably be antibodies based on the IgG1-, IgG2-, IgG3- or IgG4 subclass, with an antibody based on IgG1 (also referred to as "IgG1-type") or IgG2 (also referred to as "IgG2 -type of"). Alternatively, the backbone of multifunctional antibodies or antigen-binding fragments for use in the present invention may be any immunoglobulin class (e.g., IgA, IgG, IgM, etc.) and subclass (e.g., IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, etc.).

Preferred formats for bispecific IgG-like antibodies include, for example, hybridoma (quadroma), common light chain knock-into-holes constructs, various IgG-scFv formats, various scFv-IgG formats, IgG format two-in-one, dual V-domain IgG, IgG-V and V-IgG, which are shown, for example, in FIG. 3 in Chan A.C. and Carter P.J., Nat Rev Immu 10, 2010, pp. 301-316 and described in the referenced article. Other preferred bispecific IgG-like antibody formats include, for example, DAF, CrossMab, IgG-dsscFv, DVD, IgG-dsFV, IgG-scFab, scFab-dsscFv, and Fv2-Fc, which are shown in FIG. 1A in Weidle U.H. et al., Cancer Genomics and Proteomics 10, 2013, pp. 1-18 and described in the referenced article. Other preferred bispecific IgG-like antibody formats include DAF (two-in-one), DAF (four-in-one), DutaMab; DT-IgG; the format created by the "knobs-in-holes" assembly; charged pair format; Fab-arm exchange based format; SEEDbody; Triomab; LUZ-Y; fcab; κλ antibody format; and a format based on an orthogonal Fab fragment; DVD-IgG; IgG(H)-scFv; scFv-(H)IgG; IgG(L)-scFv; scFV-(L)IgG; IgG(L,H)-Fv; IgG(H)-V; V(H)-IgG; IgG(L)-V; V(L)-IgG; KIH IgG-scFab; 2scFv-IgG; IgG-2scFv; scFv4-Ig; scFv4-Ig; zebody; and DVI-IgG (“four-in-one”), which are presented and described in, for example, Spiess C., Zhai Q. and Carter P.J., Molecular Immunology 67, 2015, ss. 95-106, especially in FIG. 1 and in the corresponding section of the description, for example, on cc. 95-101.

и Milstein (Nature 256, 1975, с. 495), у Harlow и Lane (Antibodies, A Laboratory Manual, изд-во Cold Spring Harbor, 1988) или у Galfie (Meth. Enzymol. 73, 1981, с. 3) или в DE 19531346. В частности, многофункциональные антитела или их антигенсвязывающие фрагменты, предназначенные для применения согласно настоящему изобретению, можно получать с помощью трех основных методов: (I) путем химической конъюгации, в которой применяют химическое перекрестное сшивание; (II) путем слияния двух различных линий клеток гибридомы (например, согласно методу, описанному у Milstein и др., Nature 305, 1983, с. 537); или (III) на основе генетических подходов с применением технологии рекомбинантной ДНК (например, согласно методу, описанному у Kurucz и др., J. Immunol. 154, 1995, с. 4576; Holliger и др., Proc. Natl. Acad. Sc. USA 90, 1993, с. 6444).In general, methods for producing antibodies are known in the art. Monoclonal antibodies derived from mammals, such as humans, rats, mice, rabbits, goats or sheep, can be obtained using conventional methods, such as those described in

and Milstein (Nature 256, 1975, p. 495), Harlow and Lane (Antibodies, A Laboratory Manual, Cold Spring Harbor, 1988) or Galfie (Meth. Enzymol. 73, 1981, p. 3) or in DE 19531346. In particular, multifunctional antibodies or antigen-binding fragments thereof for use according to the present invention can be obtained using three main methods: (I) by chemical conjugation, which uses chemical cross-linking; (II) by fusion of two different hybridoma cell lines (eg, according to the method described in Milstein et al., Nature 305, 1983, p. 537); or (iii) based on genetic approaches using recombinant DNA technology (for example, according to the method described in Kurucz et al., J. Immunol. 154, 1995, p. 4576; Holliger et al., Proc. Natl. Acad. Sc USA 90, 1993, p. 6444).

Preferably, antibodies can be obtained by fusion of two different hybridoma cell lines (eg, according to the method described in Milstein et al., Nature 305, 1983, p. 537). At the same time, different lines of hybridoma cells are merged, each of which produces antibodies with one of the required specificities, and among the cell clones (“quadromes”) that produce a heterogeneous population of such antibodies - quadromas (or “hybrid hybridomas”), it is possible to identify and isolate those which secrete the desired multifunctional antibodies.

Alternative approaches include chemical conjugation of two different mAbs and/or smaller antibody fragments. It has been found that oxidative reassociation strategies for binding two different antibodies or antibody fragments are ineffective due to the presence of side reactions during the reoxidation of several native disulfide bonds. Modern methods of chemical conjugation are focused on the use of homo- or hetero-bifunctional cross-linking reagents.

Recombinant DNA technology has produced the widest range of multifunctional antibodies through artificial gene manipulation and represents the most versatile approach to antibody generation (see Byrne, H. et al., Trends Biotech, 31(11), 2013, pp. 621- 632). Accordingly, multifunctional antibodies are produced primarily by recombinant DNA techniques or by (hybrid) hybridoma techniques.

Preferably, the antibody or antigen-binding fragment intended/intended for use according to the present invention is a heterologous antibody or a heterologous antibody fragment. In the context of the present description, the term "heterologous" means that the antibody, or its antigen-negative fragment, contains heavy chains from immunoglobulins of various subclasses (for example, IgG1, IgG2, IgG3 and IgG4 in humans; for example, IgG1, IgG2a, IgG2b and IgG3 in mice and rats ) and/or different origin (species).

Preferably, the antibody or antigen-binding fragment thereof for use in the present invention contains a heavy chain derived from rat and/or mouse. "Derived from" rat and/or mouse means in particular that the amino acid sequence of the antibody heavy chain CH3 region, preferably the amino acid sequence of the antibody heavy chain Fc region, is at least 95%, preferably at least 97% identical, more preferably at least 98%, even more preferably at least 99% and most preferably 100% of the sequence of the CH3 region or Fc region, respectively, of the rat and/or mouse immunoglobulin heavy chain.

Therefore, antibodies "derived" from rats and/or mice also include antibodies containing heavy chains, the sequences of which are at least 95% identical, preferably at least 97%, more preferably at least 98%, still more preferably at least 99% and most preferably 100% of the entire length of the rat and/or mouse immunoglobulin heavy chain CH3 region or Fc region, respectively. In addition, it is also preferred if the antibody or antigen-binding fragment intended/intended for use according to the present invention is a rat and/or mouse antibody or antigen-binding fragment. This means that the sequences of all heavy and light chains contained in an antibody or antigen-binding fragment are at least 95% identical, preferably at least 97%, more preferably at least 98%, even more preferably at least 99% and most preferably 100% rat and/or mouse immunoglobulin heavy or light chain sequences, respectively.

However, since an antibody or antigen-binding fragment thereof for use according to the present invention is preferably for use in humans, it is preferred that at least three CDRs (hypervariable regions) and/or framework regions of the heavy chain (and light) variable region chains) are of human origin or have been humanized to provide specificity for the (human) T cell surface antigen and specificity for the (human) cancer and/or tumor associated antigen. Therefore, a preferred antibody or a preferred antigen binding fragment for use in accordance with the present invention comprises a heavy chain having (I) a CH3 region, preferably an Fc region, whose sequence is/is at least 95% identical, preferably at least at least 97%, more preferably at least 98%, even more preferably at least 99% and most preferably 100% of the rat and/or mouse immunoglobulin heavy chain CH3 or Fc region sequences, respectively; and (II) at least three CDRs (hypervariable regions) and/or framework regions of the heavy chain variable region are of human origin or have been humanized. More preferably, both heavy chains of an antibody or antigen-binding fragment to be used according to the present invention have (I) a CH3 region, preferably an Fc region, whose sequence is/are at least 95%, preferably at least 97% identical, more preferably at least 98%, even more preferably at least 99% and most preferably 100% of the sequence of the CH3 region or Fc region, respectively, of the rat or mouse immunoglobulin heavy chain; and (II) at least three CDRs (hypervariable regions) and/or framework regions of the heavy chain variable region are of human origin or humanized. In addition, preferably also at least three CDRs (hypervariable regions) and/or framework regions of the light chain variable region are of human origin or humanized.

Most preferably, the antibody is a rat/mouse antibody or an antigen-binding fragment thereof. In the context of the present description, the term "rat/mouse antibody" refers to an antibody that contains

(a) a (heavy) chain that differs from the rat (heavy) chain only in that the three CDRs and/or framework regions of the heavy chain variable region are of human origin or humanized (i.e., all sequences other than the CDRs and/or frame regions are rat (heavy) chain sequences); and

(b) a (heavy) chain that differs from the murine (heavy) chain only in that the three CDRs and/or framework regions of the heavy chain variable region are of human origin or humanized (i.e., all sequences other than the CDRs and/or framework regions are mouse (heavy) chain sequences).

Most preferably, the antibody is a mouse IgG2a/rat IgG2b antibody, or an antigen-binding fragment thereof. In the context of the present description, the term "antibody in the form of mouse IgG2a/rat IgG2b" refers to an antibody that contains

(a) a (heavy) chain that differs from the rat IgG2b (heavy) chain only in that the three CDRs and/or framework regions of the heavy chain variable region are of human origin or humanized (i.e., all sequences other than the CDRs and/or or framework regions are rat gG2b (heavy) chain sequences; and

(b) a (heavy) chain that differs from the mouse IgG2a (heavy) chain only in that the three CDRs and/or heavy chain variable region framework regions are of human origin or humanized (i.e., all sequences other than the CDRs and/or or framework regions are mouse IgG2a (heavy) chain sequences.

Preferably, the antibody or antigen-binding fragment thereof for/intended for use in accordance with the present invention is selected from one or more of the following isotype combinations (wherein it is specifically understood that each/each isotype/combination has at least CDRs and/or framework regions, preferably variable regions, preferably of human origin or humanized - even if the isotype is only rat/mouse):

- rat-IgG2b/mouse-IgG2a,

- rat-IgG2b / mouse-IgG2b,

- rat-IgG2b/mouse-IgG3,

- rat-IgG2b/human-IgG1,

- rat-IgG2b/human-IgG2,

- rat-IgG2b/human-IgG3 [eastern allotype G3m (st) = binding to protein A],

- rat-IgG2b/human-IgG4,

- rat-IgG2b/rat-IgG2c,

- murine-IgG2a/human-IgG3 [Caucasian allotypes G3m (b+g) = no protein A binding, marked with * below],

- mouse-IgG2a/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],

- mouse-IgG2a/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],

- mouse-IgG2a/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],

- mouse-[VH-CH1,VL-CL]-human-IgG1/Rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],

- mouse-[VH-CH1,VL-CL]-human-IgG4/Rat-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4[N-terminal region of CH2]-human- IgG3*[CH2 C-terminal region: after amino acid position 251]-human-IgG3*[CH3],

- rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge-CH2-CH3],

- rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG2-[hinge-CH2-CH3],

- rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG3-[hinge-CH2-CH3, eastern allotype],

- rat-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG4-[hinge-CH2-CH3],

- human-IgG1/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*[CH2-CH3],

- human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4[N-terminal CH2]-human-IgG3*[C-terminal CH2: after the amino acid position 251]-human-IgG3*[CH3],

- human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG4[N-terminal CH2]-human-IgG3*[C-terminal CH2: after amino acid position 251]-human-IgG3*[CH3],

- human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG2[N-terminal CH2]-human-IgG3*[C-terminal CH2: after amino acid position 251]-human-IgG3*[CH3],

- human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[Hinge]-human-IgG2[N-terminal CH2]-human-IgG3*[C-terminal CH2: after the amino acid position 251]-human-IgG3*[CH3],

- human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],

- human-IgG1/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],

- human-IgG2/human-[VH-CH1,VL-CL]-human-IgG2-[hinge]-human-IgG3*-[CH2-CH3],

- human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG3*-[CH2-CH3],

- human-IgG4/human-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4[N-terminal CH2]-human-IgG3*[C-terminal CH2: after the amino acid position 251]-human-IgG3*[CH3],

- mouse-IgG2b/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],

- mouse-IgG2b/human-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*[CH2-CH3],

- mouse-IgG2b/mouse-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3],

- mouse-[VH-CH1,VL-CL]-human-IgG4/Rat-[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4-[CH2]-human-IgG3*- [CH3],

- human-IgG1/rat[VH-CH1,VL-CL]-human-IgG1[hinge]-human-IgG4-[CH2]-human-IgG3*[CH3],

- human-IgG1/mouse[VH-CH1,VL-CL]-human-IgG1[hinge]-human-IgG4-[CH2]-human-IgG3*[CH3], and

- human-IgG4/human[VH-CH1,VL-CL]-human-IgG4-[hinge]-human-IgG4-[CH2]-human-IgG3*-[CH3].

Preferably, the antibody or antigen-binding fragment thereof for/intended for use according to the invention is an IgG-type antibody (which is also referred to as an "IgG-like" antibody) containing a binding site for human FcγRI, FcγRIIa and/or FcγRIII, in particular Fc -region. More preferably, the antibody or antigen-binding fragment thereof for/intended for use according to the invention is a trifunctional bispecific antibody which is a heterologous rat/mouse antibody containing a human FcγRI, FcγRIIa and/or FcγRIII binding site, primarily an Fc region. While preferred is an antibody with a combination of subclasses of mouse IgG2a and rat IgG2b. Most preferred is a heterologous rat/mouse antibody containing a binding site for human FcγRI, FcγRIIa and/or FcγRIII, especially the Fc region, with heavy chains derived from the mouse IgG2a and rat IgG2b subclasses, in each case preferably their respective light chains.

For antibodies containing a human FcγRI, FcγRIIa and/or FcγRIII binding site, primarily an Fc region, with heavy chains derived from the mouse IgG2a and rat IgG2b subclasses, primarily for Triomab format antibodies, it has been demonstrated that such antibodies bind to more high affinity with human FcγRIIa than with human FcγRIIb, and are characterized by a significantly higher ratio of FcγRIIa/FcγRIIb binding levels (Lindhofer H., Hess J., Ruf P., Trifunctional Triomab ^® antibodies for cancer therapy. in: Bispecific antibodies, ed. Kontermann RE, Springer, Berlin, 2011, pp. 289-312). Thus, these antibodies lead to increased phagocytosis by macrophages of tumor cells carrying the antibody on their surface, and increased direct cytolysis of tumor cells. Therefore, such antibodies are most preferred.

In general, a multifunctional antibody for use according to the invention preferably has in its Fc region the following combination of isotypes: rat-IgG2b/mouse-IgG2a, rat-IgG2b/mouse-IgG2b, rat-IgG2b/human-IgG1 or mouse-[ VH-CH1,VL-CL]-human-IgG1/rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human IgG3*-[CH2-CH3], where * denotes Caucasian allotypes of G3m( b+g) that do not bind to protein A.

Most preferably, the antibody or antigen-binding fragment intended/intended for use according to the present invention is in Triomab format. Triomab are trifunctional bispecific IgG-like antibodies having specificity for CD3 and specificity for cancer and/or tumor associated antigen. These chimeras consist of two semi-antibodies, each with one light and one heavy chain, derived from the parental mouse IgG2a and rat IgG2b isotypes. Therefore, the Triomab Fc region corresponds to the mouse IgG2a/rat IgG2b isotype.

Preferably, the antibody or antigen-binding fragment thereof for/intended for use according to the invention is selected from the group consisting of catumaxomab (anti-CD3 x anti-EpCAM), FBTA05/lymphomun (anti-CD3 x anti-CD20), ertumaxomab (anti-CD3 × anti-HER2/neu) and/or ectomune (anti-CD3 × anti-GD2), preferably the antibody is atumaxomab and/or ectomune.

The most preferred example of a trifunctional bispecific antibody is catumaxomab ( ^Removab® ) (anti-CD3 x anti-CD3). Removab® was approved by the EMA for the treatment of malignant ascites in 2009 (Linke et al., Catumaxomab - clinical development and future directions., 2010, mAbs 2:2). Other preferred examples of trifunctional bispecific antibodies include (i) FBTA05 (which is also referred to as "lymphomun"), EMA-CD20 trifunctional antibody, (II) ertumaxomab, EMA-HER2 trifunctional antibody, (III) ectomune, EMA-GD2 trifunctional antibody, and (IV) TRBs02, a trifunctional antibody specific for human melanoma (Ruf et al., Int J Cancer, 108, 2004, pp. 725-732).

Immune checkpoint modulator

In the context of the present description (i.e. in this application), the term "immune checkpoint modulator" (which is also called "checkpoint modulator") refers to a molecule or compound that/which modulates (for example, completely or partially reduces, inhibits, interacts with, activates, enhances, enhances or maintains) the function of one or more checkpoint molecules. Thus, the immune checkpoint modulator can be an "immune checkpoint inhibitor" (also referred to as a "checkpoint inhibitor" or "inhibitor") or an "immune checkpoint activator" (also referred to as a "checkpoint activator" or "activator". An "immune checkpoint inhibitor" (also referred to as a "checkpoint inhibitor" or "inhibitor") wholly or partly reduces, inhibits, interacts with, or negatively modulates the function of one or more checkpoint molecules. "checkpoint activator" or "activator") wholly or partially activates, stimulates, enhances, enhances, maintains, or positively modulates the function of one or more checkpoint molecules. Immune checkpoint modulators generally have the ability to modulate (I) self-tolerance and/ or (II) the amplitude and/or duration of the immune response eta. Preferably, the immune checkpoint modulator used according to the present invention modulates the function of one or more human checkpoint molecules and thus is a "human checkpoint modulator". Preferably, the immune checkpoint modulator is an activator or inhibitor of one or more immune checkpoint molecules selected from CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA (CD272), CD40, CTLA-4, IDO, KIR, LAG3, PD-1, PD-L1, PD-L2, TIM-3, VISTA, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, GITR, TNFR and/or FasR/DcR3; or an activator or inhibitor of one or more of their ligands.

Checkpoint molecules (also referred to as "immune checkpoint molecules" or "immune checkpoints") are molecules, such as proteins, that are generally involved in immune response pathways and, for example, regulate T cell activation, proliferation T cells and/or T cell function. Therefore, the function of checkpoint molecules that is modulated (e.g., wholly or partly reduced, inhibited, affected, activated, stimulated, increased, enhanced, or maintained) by checkpoint modulators is typically to (regulate) T cell activation, proliferation T cells and/or T cell function. Thus, immune checkpoint molecules regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses. Many of the immune checkpoint molecules belong to the B7:CD28 family or tumor necrosis factor receptor (TNFR) superfamily and, through binding to specific ligands, activate signaling molecules that are recruited to the cytoplasmic domain (see Susumu Suzuki et al., Current status of immunotherapy Japanese Journal of Clinical Oncology, 2016: doi: 10.1093/jjco/hyv201 [pre-print electronic publication], see first Table 1).

The B7:CD28 family includes the most commonly targeted pathways in immune checkpoint studies, including the CTLA-4 pathway - B7-1/B7-2 and the PD-1 pathway - B7-H1(PDL1)/B7-DC(PD-L2). ). Another member of this family is ICOS-ICOSL/B7-H2. Other members of this family are CD28, B7-H3 and B7-H4.

CD28 is expressed constitutively on nearly all human CD4 ⁺ T cells and on about half of all CD8 ⁺ T cells. Binding to its two ligands, CD80 (B7-1) and CD86 (B7-2), which are expressed on dendritic cells, stimulates T cell expansion. The costimulatory checkpoint molecule CD28 competes with the inhibitory checkpoint molecule CTLA4 for these same ligands, CD80 and CD86 (see Buchbinder E.I. and Desai A., CTLA-4 and PD-1 Pathways - Similarities, Differences and Implications of Their Inhibition, American Journal of Clinical Oncology, 39(1), 2016, pp. 98-106).

Cytotoxic T-lymphocyte-associated protein 4 (CTLA4; also known as CD152) is a CD28 homologue with a much higher binding affinity for B7. The CTLA-4 ligands are CD80 (B7-1) and CD86 (B7-2), similar to CD28. However, unlike CD28, binding of CTLA4 to B7 does not lead to the formation of a stimulatory signal, but interferes with the co-stimulatory signal normally provided by CD28. In addition, it is suggested that the binding of CTLA4 to B7 even results in an inhibitory signal that counteracts the stimulatory signals arising from the binding of CD28:B7 and TCR:MHC. CTLA-4 is considered to be the "leader" among inhibitory immune checkpoints because it delays potentially autoreactive T cells from the initial stage of naïve T cell activation, typically in the lymph nodes (Buchbinder E.I. and Desai A., CTLA -4 and PD-1 Pathways: Similarities, Differences and Implications of Their Inhibition, American Journal of Clinical Oncology, 39(1), 2016, pp. 98-106). Preferred CTLA4 checkpoint inhibitors include the monoclonal antibodies ^Yervoy® (ipilimumab; Bristol Myers Squibb) and tremelimumab (Pfizer/MedImmune). Other preferred CTLA-4 inhibitors are the anti-CTLA4 antibodies described in WO 2001/014424, WO 2004/035607, US 2005/0201994 and EP 1212422 B1. Additional preferred antibodies to CTLA-4 are described in US 5811097, US 5855887, US 6051227, US 6984720, WO 01/14424, WO 00/37504, US 2002/0039581 and US 2002/086014. Other preferred anti-CTLA-4 antibodies that can be used in the context of the present invention include, for example, the antibodies described in WO 98/42752; US 6682736 and US 6207156; in Hurwitz et al., Proc. Natl. Acad. sci. USA, 95(17), 1998, pp. 10067-10071; Camacho et al., J. Clin. Oncology, 22(145): Abstract No. 2505, 2004) (antibody CP-675206); Mokyr et al., Cancer Res., 58, 1998, pp. 5301-5304, in US 5977318, US 6682736, US 7109003 and US 7132281. In the context of the present invention, the most preferred checkpoint molecule is CTLA-4.

The programmed cell death receptor 1 (PD1) has two ligands, PD-L1 (also known as B7-H1 and CD274) and PD-L2 (also known as B7-DC and CD273). The PD1 pathway regulates pre-activated T cells in the later stages of the immune response, mainly in peripheral tissues. Thus, the advantage of PD1 targeting is that it can restore immune function in the tumor microenvironment. Preferred PD1 pathway inhibitors include ^Opdivo® (nivolumab; Bristol Myers Squibb), Keytruda® ( ^{pembrolizumab} ; Merck), durvalumab (MedImmune/AstraZeneca), MEDI4736 (AstraZeneca; see WO 2011/066389 A1), atezolizumab (MPDL3280A). , Roche/Genentech; see US 8217149 B2), pidilizumab (CT-011; CureTech), MEDI0680 (AMP-514; AstraZeneca), avelumab (Merck), MSB-0010718C (Merck), PDR001 (Merck). Novartis), BMS-936559 (Bristol Myers Squibb), REGN2810 (Regeneron Pharmaceuticals), MIH1 (Affymetrix), AMP-224 (Amplimmune, GSK), BGB-A317 (BeiGene), and lambrolizumab (for example, it is under hPD109A and its humanized derivatives h409All, h409A16 and h409A17 are described in WO 2008/156712, Hamid et al., N. Engl. J. Med. 369, 2013, pp. 134-144).

Inducible T cell costimulator (ICOS; also known as CD278) is expressed on activated T cells. Its ligand is ICOSL (B7-H2; CD275), expressed mainly on B cells and dendritic cells. The specified molecule, apparently, is important for the effector function of T cells.

B7-H3 (also known as CD276) was originally considered a co-stimulatory molecule, but is now considered a co-inhibitory molecule. A preferred B7-H3 checkpoint inhibitor is the Fc-optimized monoclonal antibody enoblituzumab (MGA271; MacroGenics; see US 2012/0294796 A1).

B7-H4 (also known as VTCN1) is expressed by tumor cells and tumor-associated macrophages and is involved in tumor escape from immunological surveillance. Preferred inhibitors of B7-H4 are the antibodies described by Dangaj D. et al. in Cancer Research 73(15), 2013, ss. 4820-4829 and in Table 1, a corresponding description is also given in Jenessa B. Smith et al., B7-H4 as a potential target for immunotherapy for gynecologic cancers: A closer look. Gynecol Oncol 134(1), 2014, cc. 181-189. Other preferred examples of B7-H4 inhibitors are antibodies to human B7-H4 as described in, for example, WO 2013/025779 A1 and WO 2013/067492 A1, or soluble recombinant forms of B7-H4 as described in US 2012/0177645 A1.

The TNF superfamily includes, in particular, 19 ligand proteins that bind to 29 cytokine receptors. They are involved in many physiological responses such as apoptosis, inflammation or cell survival (Croft M., C.A. Benedict and C.F. Ware, Clinical targeting of the TNF and TNFR superfamilies. Nat Rev Drug Discov12(2), 2013, cc. 147-68). In the case of cancer, the following checkpoint/pathway molecules are preferred: TNFRSF4 (OX40/0X40L), TNFRSFS (CD40L/CD40), TNFRSF7 (CD27/CD70), TNFRSF8 (CD30/CD30L), TNFRSF9 (4-1BB/4-1BBL) , TNFRSF10 (TRAILR/TRAIL)), TNFRSF12 (FN14/TWEAK), TNFRSF13 (BAFFRTACI/APRIL-BAFF) and TNFRSF18 (GITR/GITRL). Other preferred checkpoint/pathway molecules include Fas ligand and TNFRSF1 (TNFα/TNFR). In addition, the B- and T-lymphocyte attenuator (BTLA)/herpes virus entry mediator (HVEM) pathway are preferred to enhance the immune response, as is CTLA-4 blockade. Therefore, in the context of the present invention, checkpoint modulators are preferred for use in the treatment and/or prevention of cancer which modulate one or more checkpoint molecules selected from TNFRSF4 (OX40/0X40L), TNFRSFS (CD40L/CD40), TNFRSF7 (CD27 /CD70), TNFRSF9 (4-1BB/4-1BBL), TNFRSF18 (GITR/GITRL), FasR/DcR3/Fas-ligand, TNFRSF1 (TNFα/TNFR), BTLA/HVEM and CTLA4.

OX40 (also known as CD134 or TNFRSF4) stimulates the expansion of effector and memory T cells, but it also has the ability to suppress differentiation and activity of regulatory T cells and regulate cytokine production. The OX40 ligand is OX40L (also known as TNFSF4 or CD252). OX40 is transiently expressed after contact with the T cell receptor and is only upregulated on immediately prior antigen-activated T cells in areas of inflammatory lesions. Preferred OX40 checkpoint modulators include MEDI6469 (MedImmune/AstraZeneca), MEDI6383 (MedImmune/AstraZeneca), MEDI0562 (MedImmune/AstraZeneca), MOXR0916 (RG7888; Roche/Genentech) and GSK3174998 (GSK).

, Barbara Nicola Olsen and Anders Elm Pedersen, Anti-CD40-mediated cancer immunotherapy: an update of recent and ongoing clinical trials, Immunopharmacology and Immunotoxicology, 36:2, 2014, cc. 96-104). Предпочтительными примерами модуляторов контрольной точки CD40 являются (I) агонистические антитела к CD, такие, как описанные у Sufia Butt Hassan, Jesper Freddie

, Barbara Nicola Olsen and Anders Elm Pedersen, Anti-CD40-mediated cancer immunotherapy: an update of recent and ongoing clinical trials, Immunopharmacology and Immunotoxicology, 36:2, 2014, cc. 96-104, такие как дацетузумаб (SGN-40), CP-870893, FGK 4.5/FGK 45 и FGK115, предпочтительно дацетузумаб, и (II) антагонистические антитела к CD, такие, как описанные у Sufia Butt Hassan, Jesper Freddie

Barbara Nicola Olsen and Anders Elm Pedersen, Anti-CD40-mediated cancer immunotherapy: an update of recent and ongoing clinical trials, Immunopharmacology and Immunotoxicology, 36:2, 2014, cc. 96-104, такие как лукатумумаб (HCD122, CHIR-12.12). Другие предпочтительные модуляторы иммунной контрольной точки CD40 включают SEA-CD40 (фирма Seattle Genetics), ADC-1013 (фирма Alligator Biosciences), APX005M (фирма Apexigen Inc) и RO7009789 (фирма Roche).CD40 (also known as TNFRSF5) is expressed by various cells of the immune system, including antigen-presenting cells. Its ligand is CD40L, also known as CD154 or TNFSF5, which is transiently expressed on the surface of activated CD4 ⁺ T cells. The signal transmitted by CD40 "allows" the maturation of dendritic cells and thereby triggers the activation and differentiation of T cells. However, CD40 can also be expressed by tumor cells. Thus, stimulation/activation of CD40 in cancer patients can be both beneficial and detrimental. Therefore, stimulatory and inhibitory modulators of this immune checkpoint have been developed (Sufia Butt Hassan, Jesper Freddie

, Barbara Nicola Olsen and Anders Elm Pedersen, Anti-CD40-mediated cancer immunotherapy: an update of recent and ongoing clinical trials, Immunopharmacology and Immunotoxicology, 36:2, 2014, cc. 96-104). Preferred examples of CD40 checkpoint modulators are (I) CD agonist antibodies such as those described in Sufia Butt Hassan, Jesper Freddie

, Barbara Nicola Olsen and Anders Elm Pedersen, Anti-CD40-mediated cancer immunotherapy: an update of recent and ongoing clinical trials, Immunopharmacology and Immunotoxicology, 36:2, 2014, cc. 96-104 such as dacetuzumab (SGN-40), CP-870893, FGK 4.5/FGK 45 and FGK115, preferably dacetuzumab, and (II) CD antagonist antibodies such as those described by Sufia Butt Hassan, Jesper Freddie

Barbara Nicola Olsen and Anders Elm Pedersen, Anti-CD40-mediated cancer immunotherapy: an update of recent and ongoing clinical trials, Immunopharmacology and Immunotoxicology, 36:2, 2014, cc. 96-104 such as lucatumumab (HCD122, CHIR-12.12). Other preferred CD40 immune checkpoint modulators include SEA-CD40 (Seattle Genetics), ADC-1013 (Alligator Biosciences), APX005M (Apexigen Inc) and RO7009789 (Roche).

CD27 (also known as TNFRSF7) supports the antigen-specific expansion of naive T cells and plays an important role in the creation of memory T cells. CD27 is also a marker for memory B cells. The short-term availability of its ligand, CD70 (also known as TNFSF7 or CD27L), on lymphocytes and dendritic cells regulates CD27 activity. In addition, costimulation of CD27 is known to suppress the function of effector Th17 cells. A preferred CD27 immune checkpoint modulator is varlilumab (Celldex). Preferred CD70 immune checkpoint modulators are ARGX-110 (arGEN-X) SGN-CD70A (Seattle Genetics).

CD137 (also known as 4-1BB or TNFRSF9) is a member of the tumor necrosis factor (TNF) receptor family and is strongly associated with co-stimulatory activity on activated T cells. In particular, CD137 signaling (via its ligand CD137L, also known as TNFSF9 or 4-1BBL) results in T cell proliferation and protects T cells, especially CD8 ⁺ T cells, from activation-induced cell death. Preferred CD137 checkpoint modulators are PF-05082566 (Pfizer) and urelumab (BMS).

The glucocorticoid-induced TNFR family-related protein (GITR, also known as TNFRSF18) gene enhances T-cell expansion, including Treg expansion. The GITR ligand (GITRL, TNFSF18) is mainly expressed on antigen-presenting cells. Anti-GITR antibodies have been shown to stimulate an antitumor response through the loss of stability of the Treg lineage. Preferred GITR checkpoint modulators are BMS-986156 (Bristol Myers Squibb), TRX518 (GITR Inc) and MK-4166 (Merck).

Another preferred checkpoint molecule for modulation is BTLA. The B- and T-lymphocyte attenuator (BTLA; also known as CD272) is expressed primarily by CD8 ⁺ -T cells, with surface BTLA expression progressively downregulated as human CD8 ⁺ -T cells differentiate from naive cells. to the effector cell phenotype. However, tumor-specific human CD8 ⁺ T cells express high levels of BTLA. BTLA expression is induced during T cell activation and BTLA expression is retained on Th1 cells but not on Th2 cells. Like PD1 and CTLA4, BTLA interacts with the B7 homologue, B7H4. However, unlike PD-1 and CTLA-4, BTLA mediates T cell inhibition by interacting with the tumor necrosis factor receptor (TNF-R) family of receptors rather than the B7 family of cell surface receptors. BTLA is a ligand for tumor necrosis factor (TNFRSF14) superfamily (receptor) member 14, also known as herpes virus entry mediator (HVEM; herpes virus entry mediator, also known as CD270). BTLA-HVEM complexes negatively regulate T-cell immune responses. Preferred BTLA inhibitors are the antibodies described in Table 1 of Alison Crawford and E. John Wherry, Editorial: Therapeutic potential of targeting BTLA. Journal of Leukocyte Biology 86, 2009, pp. 5-8, first of all, human antibodies presented therein. Other preferred antibodies in this context that block the interaction of human BTLA with its ligand are described in WO 2011/014438, for example "4C7" described in WO 2011/014438.

Another family of checkpoint molecules includes checkpoint molecules associated with two major classes of major histocompatibility complex (MCHC) molecules (MCHC class I and class II). This family includes the Ig-like killer cell receptor (KIR) for class I molecules and the lymphocyte activation gene 3 (LAG-3) for class II molecules.

The killer cell immunoglobulin-like receptor (KIR) is a receptor for class I MHC molecules on natural killer cells. An example of a KIR inhibitor is the monoclonal antibody lirilumab (IPH 2102; Innate Pharma/BMS; see US 8119775 B2 and Benson et al., Blood 120, 2012, pp. 4324-4333).

The signaling of the lymphocyte activation gene 3 (LAG3, also known as CD223) leads to the suppression of the immune response by acting on Treg, as well as direct effects on CD8 ⁺ -T cells. A preferred example of a LAG3 inhibitor is the anti-LAG3 monoclonal antibody BMS-986016 (Bristol-Myers Squibb). Other preferred examples of LAG3 inhibitors are LAG525 (Novartis), IMP321 (Immutep) and LAG3-Ig described in WO 2009/044273 A2 and Brignon et al., Clin. Cancer Res. 15, 2009, pp. 6225-6231, as well as murine or humanized antibodies blocking human LAG3 (eg IMP701 described in WO 2008/132601 A1) or fully human antibodies blocking human LAG3 (such as described in EP 2320940 A2).

Another checkpoint molecule pathway is the TIM-3/GAL9 pathway). T cell immunoglobulin and mucin domain 3 (TIM-3, also known as HAVcr-2) is expressed on activated human CD4 ⁺ T cells and regulates cytokines expressed by Th1 and Th17. TIM-3 acts as a negative regulator of Th1/Tc1 function, triggering cell death after interaction with its ligand, galectin-9 (GAL9). TIM-3 is a cell surface molecule specific for type 1 helper T cells that regulates the induction of peripheral tolerance. Thus, a recent study demonstrated that antibodies to TIM-3 can significantly increase antitumor immunity (Ngiow SF et al., Anti-TIM3 antibody promotes T cell IFN-gammamediated antitumor immunity and suppresses established tumors. Cancer Res, 71( 10), 2011, pp. 3540-3551). Preferred examples of TIM-3 inhibitors are antibodies targeting human TIM3 (eg as described in WO 2013/006490 A2) or, above all, the human TIM3 blocking antibody F38-2E2 described in Jones et al., J Exp Med. 205(12), 2008, pp. 2763-2779.

CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1) is another checkpoint molecule (Huang Y.H. et al., CEACAM1 regulates TIM-3-mediated tolerance and exhaustion. Nature, 517(7534), 2015, pp. 386-390; Gray-Owen S. D. and R. S. Blumberg, CEACAM1: contact-dependent control of immunity Nat Rev Immunol, 6(6), 2006, pp. 433-446). A preferred CEACAM1 modulator is CM-24 (cCAM Biotherapeutics).

Another immune checkpoint molecule is GARP, which plays a role in the ability of tumors to elude a patient's immune system. In ongoing clinical trials, a candidate molecule (ARGX-115) appears to have an effect of interest. Therefore, the ARGX-115 is the preferred GARP setpoint modulator.

In addition, various research groups have demonstrated that another checkpoint molecule, phosphatidylserine (also referred to as "PS"), can be targeted in the treatment of cancer (Creelan B.C., Update on immune checkpoint inhibitors in lung cancer. Cancer Control, 21(1 ), 2014, pp. 80-89; Yin Y. et al., Phosphatidylserine-targeting antibody induces Ml macrophage polarization and promotes myeloid-derived suppressor cell differentiation. Cancer Immunol Res, 1(4), 2013, cc. cc 256-268). A preferred phosphatidylserine (PS) checkpoint modulator is bavatituximab (Peregrine).

Another checkpoint pathway is CSF1/CSF1R (Zhu Y., et al., CSF1/CSF1R Blockade Reprograms Tumor-Infiltrating Macrophages and Improves Response to T-cell Checkpoint Immunotherapy in Pancreatic Cancer Models. Cancer Research, 74(18), 2014, pp. 5057-5069). Preferred CSF1R checkpoint modulators are FPA008 (FivePrime), IMC-CS4 (Eli-Lilly), PLX3397 (Plexxicon), and RO5509554 (Roche).

In addition, the role of the CD94/NKG2A natural killer receptor molecule in cervical carcinoma was evaluated (Sheu B.C. et al., Up-regulation of inhibitory natural killer receptors CD94/NKG2A with suppressed intracellular perforin expression of tumor infiltrating CD8+ Tocyte lymphs in human cervical Cancer Res, 65(7), 2005, pp. 2921-2929) and leukemia (Tanaka J. et al., Cytolytic activity against primary leukemic cells by inhibitory NK cell receptor (CD94/NKG2A)-expressing T cells expanded from various sources of blood mononuclear cells Leukemia, 19(3), 2005, pp. 486-489). A preferred NKG2A checkpoint modulator is IPH2201 (Innate Pharma).

Another preferred checkpoint molecule is IDO, an enzyme indoleamine 2,3-dioxygenase involved in the kynurenine pathway (Ball, H.J. et al., Indoleamine 2,3-dioxygenase-2; a new enzyme in the kynurenine pathway. Int J Biochem Cell Biol, 41(3), 2009, 467-471). Indolamine 2,3-dioxygenase (IDO) is a tryptophan catabolizing enzyme with immune-inhibiting properties. It is known that IDO suppresses T and NK cells, participates in the formation and activation of Treg cells and suppressor cells of myeloid origin, and stimulates tumor angiogenesis. IDO1 is overexpressed in many cancers and has been found to promote escape of tumor cells from the immune system (Liu X. et al., Selective inhibition of ID01 effectively regulates mediators of antitumor immunity. Blood, 115(17), 2010, cc. 3520-3530, Ino K. et al., Inverse correlation between tumoral indoleamine 2,3-dioxygenase expression and tumor-infiltrating lymphocytes in endometrial cancer: its association with disease progression and survival, Clin Cancer Res, 14(8), 2008, cc. 2310-2317) and promotes chronic inflammation when induced by local inflammation (Muller A.J. et al., Chronic inflammation that facilitates tumor progression creates local immune suppression by inducing indoleamine 2,3 dioxygenase. Proc Natl Acad Sci US A, 105 (44), 2008, pp. 17073-17078). Preferred IDO inhibitors are exciguamine A, epadostat (INCB024360; InCyte), indoxymod (NewLink Genetics), NLG919 (NewLink Genetics/Genentech), GDC-0919 (NewLink Genetics/Genentech), F001287 (Flexus Biosciences/BMS) and small molecules such as 1-methyltryptophan, especially 1-methyl-[D]-tryptophan, and IDO inhibitors listed in Table 1 of Sheridan C., IDO inhibitors move center stage in immune-oncology; Nature Biotechnology 33, 2015, pp. 321-322.

Another immune checkpoint molecule suitable for modulation is also a member of the kynurenine metabolic pathway: TDO (tryptophan 2,3-dioxygenase). Several studies have already demonstrated that TDO is of interest in terms of cancer immunity and autoimmunity (Garber K., Evading immunity: new enzyme implicated in cancer. J Natl Cancer Inst, 104(5), 2012, pp. 349-352; Platten M., W. Wick and B. J. Van den Eynde, Tryptophan catabolism in cancer: beyond!DO and tryptophan depletion, Cancer Res, 72(21), 2012, pp. 5435-5440; Platten M. et al., Cancer Immunotherapy by Targeting IDOl/TDO and Their Downstream Effectors, Front Immunol, 5, 2014, p.673).

Another immune checkpoint molecule suitable for modulation is A2AR. The A2A adenosine receptor (A2AR) is regarded as an important checkpoint in cancer therapy because the tumor microenvironment typically contains relatively high concentrations of adenosine, which activates A2AR. This signaling forms a negative immune feedback loop in the immune microenvironment (for a review, see Robert D. Leone et al., A2aR antagonists: Next generation checkpoint blockade for cancer immunotherapy. Computational and Structural Biotechnology Journal 13, 2015, pp. 265-272) . Preferred A2AR inhibitors are istradefillin, PBS-509, ST1535, ST4206, tosadenant, V81444, preladenant, vipadenant, SCH58261, SYN115, ZM241365 and FSPTP.

Another immune checkpoint molecule suitable for modulation is VISTA. The V-domain-containing Ig suppressor of T-cell activation (VISTA; also known as C10orf54) is expressed primarily on hematopoietic cells, so persistent expression of VISTA on leukocytes in tumors may allow VISTA to perform effective blockade in a wide variety of solid tumors. A preferred VISTA inhibitor is JNJ-61610588 (ImmuNext), an anti-VISTA antibody that has recently entered a phase 1 clinical trial.

Another immune checkpoint molecule is CD122. CD122 is the beta subunit of the interleukin-2 receptor. CD122 enhances the proliferation of effector CD8 ⁺ T cells.

The most preferred examples of checkpoint molecules include the "CTLA4 pathway" and "PD1 pathway", which involve CTLA4 and its ligands CD80 and CD86, as well as PD1 with its ligands PD-L1 and PD-L2 (more on the CTLA4 and PD pathways). -1, as well as other molecules involved, are described in Buchbinder, E. I. and Desai, A., CTLA-4 and PD-1 Pathways - Similarities, Differences and Implications of Their Inhibition, American Journal of Clinical Oncology, 39(1) , 2016, pp. 98-106). More broadly, preferred examples of checkpoint molecules include CD27, CD28, CD40, CD 122, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CD40, CTLA-4, IDO, KIR, LAG3 , PD-1, TIM-3, VISTA, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, GITR, TNFR and/or FasR/DcR3, and in particular their ligands.

However, it may also be preferred if the immune checkpoint modulator is not an anti-CD28 antibody. More preferably, the immune checkpoint modulator is not directed against CD28 (ie, CD28 is preferably not targeted by the immune checkpoint modulator described herein).

In addition, it may also be preferred if the immune checkpoint modulator is not a PD-1 inhibitor. More preferably, the immune checkpoint modulator is not an inhibitor/antagonist of the PD-1 pathway (also referred to as the "PD-1 axis", which includes, in addition to PD-1 itself, also its PD-L1 and PD-L2 ligands).

Immune checkpoint molecules are responsible for costimulatory or inhibitory interactions with T cell responses. Therefore, checkpoint molecules can be subdivided into (I) (co-)stimulatory checkpoint molecules and (II) inhibitory checkpoint molecules. Generally, (co-)stimulatory checkpoint molecules positively act in conjunction with antigen stimulation-induced T cell receptor (TCR) signaling, while inhibitory checkpoint molecules negatively regulate TCR signaling. Examples of (co-)stimulatory checkpoint molecules include CD27, CD28, CD40, CD122, CD137, OX40, GITR, and ICOS. Examples of inhibitory checkpoint molecules include CTLA4 as well as PD1 with its PD-L1 and PD-L2 ligands; and A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, TIM-3, VISTA, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR, and FasR/DcR3.

Preferably, the immune checkpoint modulator is an activator of a (co-)stimulatory checkpoint molecule or an inhibitor of an inhibitory checkpoint molecule, or a combination thereof. Thus, more preferably, the immune checkpoint modulator is (i) an activator of CD27, CD28, CD40, CD122, CD137, OX40, GITR and/or ICOS or (ii) an inhibitor of A2AR, B7-H3, B7-H4, BTLA, CD40 , CTLA-4, IDO, KIR, LAG3, PD-1, PDL-1, PD-L2, TIM-3, VISTA, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR and/or FasR/DcR3 .

As mentioned above, a large number of modulators (inhibitors/activators) of CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, CTLA-4, PD1, PDL-1, PD are known. -L2, IDO, LAG-3, BTLA, TIM3, VISTA, KIR, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR and/or FasR/DcR3, some of which are already in clinical trials or even approved for application. Based on these known immune checkpoint modulators, alternative immune checkpoint modulators may be developed in the (near) future. In particular, known preferred checkpoint modulators themselves may be used, or analogues thereof may be used, in particular chimerized, humanized or human forms of antibodies.

More preferably, the immune checkpoint modulator is an inhibitor of an inhibitory checkpoint molecule (but preferably not an inhibitor of a stimulatory checkpoint molecule). Therefore, even more preferably, the immune checkpoint modulator is an inhibitor of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR and/or DcR3 or ligand thereof.

Preferably also, the immune checkpoint modulator is an activator of a stimulatory or costimulatory checkpoint molecule (but preferably not an activator of an inhibitory checkpoint molecule). Therefore, the immune checkpoint modulator is more preferably an activator of CD27, CD28, CD40, CD122, CD137, OX40, GITR and/or ICOS, or a ligand thereof.

More preferably the immune checkpoint modulator is a CTLA-4, PD-1, PD-L1 and/or PD-L2 inhibitor, even more preferably the immune checkpoint modulator is a CTLA-4, PD-1 and/or PD-L1 inhibitor , and most preferably the immune checkpoint modulator is a CTLA-4 and/or PD-1 inhibitor. Most preferred is a CTLA-4 inhibitor.

Therefore, the checkpoint modulator can be selected from known inhibitors of the CTLA-4 pathway and/or the PD-1 pathway. Preferred inhibitors of the CTLA-4 pathway and the PD-1 pathway include the monoclonal antibodies ^Yervoy® (ipilimumab; Bristol Myers Squibb) and tremelimumab (Pfizer/MedImmune), as well as ^Opdivo® (nivolumab; Bristol Myers Squibb), Keytruda® ( ^{pembrolizumab} ; Merck), durvalumab (MedImmune/AstraZeneca), MEDI4736 (AstraZeneca; see WO 2011/066389 A1), MPDL3280A (Roche/Genentech; see US 8217149 B2), pidilizumab (CT-011; CureTech) , MEDI0680 (AMP-514; AstraZeneca), MSB-0010718C (Merck), MIH1 (Affymetrix), and lambrolizumab (e.g., described under the name hPD109A and its humanized derivatives h409All, h409A16 and h409A17 in WO 2008/156712; Hamid et al., N. Engl. J. Med. 369, 2013, pp. 134-144). More preferred checkpoint inhibitors include the CTLA-4 inhibitors ^Yervoy® (ipilimumab; Bristol Myers Squibb) and tremelimumab (Pfizer/MedImmune), and/or the PD-1 inhibitors ^Opdivo® (nivolumab; Bristol Myers Squibb), ^Keytruda® ( pembrolizumab; Merck), pidilizumab (CT-011; CureTech), MEDI0680 (AMP-514; AstraZeneca), AMP-224, and lambrolizumab (e.g., described under the name hPD109A and its humanized derivatives h409All, h409A16 and h409A17 in WO 2008 /156712; in Hamid O. et al., N. Engl. J. Med. 369, 2013, pp. 134-144).

In the context of the present invention, more than one immune checkpoint modulator (e.g. checkpoint inhibitor) is preferably used, in particular at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 different immune checkpoint modulators are used ( eg checkpoint inhibitor), preferably 2, 3, 4 or 5 different immune checkpoint modulators (eg checkpoint inhibitor) are used, more preferably 2, 3 or 4 different immune checkpoint modulators are used (eg checkpoint inhibitor), even more preferably 2 or 3 different immune checkpoint modulators (eg checkpoint inhibitor) are used and most preferably 2 different immune checkpoint modulators (eg checkpoint inhibitor) are used. In this case, the expression "different" immune checkpoint modulators (eg, checkpoint inhibitors) means, in particular, that they modulate (eg, inhibit) different pathways of checkpoint molecules.

Preferably, the PD-1 pathway inhibitor is combined with the CTLA-4 pathway inhibitor. For example, as noted above, combination therapy using nivolumab (an anti-PD1 antibody) and ipilimumab (an anti-CTLA-4 antibody) was approved by the FDA in 2015 for the treatment of patients with BRAF V600 wild-type, unresectable, or metastatic melanoma. In addition, a phase 1b study of the combination of durvalumab (an anti-PD-L1 antibody) and tremelimumab (an anti-CTLA-4 antibody) in non-small cell lung cancer was recently published (Antonia, Scott et al., Safety and antitumour activity of durvalumab plus tremelimumab in non-small cell lung cancer: a multicentre, phase 1b study Lancet Oncol February 5, 2016 pii: S1470-2045(15)00544-6 doi: 10.1016/S1470-2045(15)00544-6. [electronic publication before going to print]). Thus, the preferred combinations of immune checkpoint modulators of the PD-1 pathway and the CTLA-4 pathway are (I) nivolumab (anti-PD1 antibody) and ipilimumab (anti-CTLA-4 antibody) or (II) durvalumab (MEDI4736; anti-PD-L1 antibody) and tremelimumab (anti-CTLA-4 antibody). A combination thereof is also preferred, for example, nivolumab (anti-PD1 antibody) and tremelimumab (anti-CTLA-4 antibody) or durvalumab (MEDI4736; anti-PD-L1 antibody) and ipilimumab (anti-CTLA-4 antibody).

Other preferred combinations of at least two different immune checkpoint modulators in the context of the present invention may comprise a combination selected from (i) a combination of a KIR inhibitor and a CTLA-4 inhibitor such as lirilumab/ipilimumab; (ii) a combination of a KIR inhibitor and a PD-1 pathway inhibitor such as PD-1, eg lirilumab/nivolumab; (iii) a combination of a LAG3 inhibitor and a PD-1 pathway inhibitor, such as a PD-1 inhibitor or a PD-L1 inhibitor, such as those described in Woo et al., Cancer Res. 72, 2012, pp. 917-927 or Butler N.S. et al., Nat Immunol. 13, 2011, pp. 188-195), and preferred examples of such a combination include novilumab/BM8-986016 and PDR001/LAG525; (iv) a combination of checkpoint modulators targeting ICOS and a CTLA-4 inhibitor, such as those described in Fu et al., Cancer Res. 71, 2011, pp. 5445-5454; (V) a combination of checkpoint modulators modulating 4-1BB, CTLA-4 inhibitors such as those described in Curran et al., PLoS One 6(4), 2011, el 9499); (VI) combinations of checkpoint modulators targeting PD1 and CD27, such as novilumab/varlilumab and atozolizumab/varlilumab; (VII) a combination of checkpoint modulators targeting OX40 and CTLA-4, such as MEDI6469/tremelimumab; (VIII) combinations of checkpoint modulators targeting OX40 and PD-1 such as MEDI6469/MEDI4736, MOXR0916/MPDL3280A, MEDI6383/MEDI4736 and GSK3174998/pembrolizumab; (IX) combinations of checkpoint modulators targeting PD-1 and 4-1BB such as novilumab/urelumab, pembrolizumab/PT-05082566 and avelumab/PT-05082566; (X) combinations of checkpoint modulators targeting PD-1 and IDO, such as ipilimumab/indoximod, pembrolizumab/INCB024360, MEDI4736/INCB024360, MPDL3280A/GDC-0919, and atezolizumab/INCB024360; (XI) combinations of checkpoint modulators targeting PD-1 and CSF1R, such as pembrolizumab/PLX3397, novilumab/FPAOOS and MPDL3280A/RO5509554; (XII) combinations of checkpoint modulators targeting PD-1 and GITR, such as novilumab/BMS-986156 and pembrolizumab/MK-4166; (XIII) combinations of checkpoint modulators targeting PD-1 and CD40, such as MPDL3280A/RO7009789; (XIV) combinations of checkpoint modulators targeting PD-1 and B7-H3, such as pembrolizumab/MGA271; (XV) combinations of checkpoint modulators targeting CTLA-4 and B7-H3, such as ipilimumab/MGA271; and (XVI) combinations of checkpoint modulators targeting KIR and 4-1BB, such as lirilumab/urelumab.

Most preferably, the combination of an immune checkpoint modulator and a T cell redirecting multifunctional antibody or fragment thereof for use in the present invention comprises at least (α) a CTLA-4 inhibitor and (β) a PD-1, PD-L1 inhibitor and/ or PD-L2, preferably at least (α) a CTLA-4 inhibitor and (β) a PD-1 inhibitor. Examples of such a preferred combination include ^Yervoy® (ipilimumab; Bristol Myers Squibb) and ^Opdivo® (nivolumab; Bristol Myers Squibb), ^Yervoy® (ipilimumab; Bristol Myers Squibb) and Keytruda® ( ^{pembrolizumab} ; Merck), combination ^Yervoy® (ipilimumab; Bristol Myers Squibb) and durvalumab (MedImmune/AstraZeneca), ^Yervoy® (ipilimumab; Bristol Myers Squibb) and MEDI4736 (AstraZeneca; cf. WO 2011/066389 A1), ^Yervoy® (ipilimumab) combination ; Bristol Myers Squibb) and MPDL3280A (Roche/Genentech; cf. US 8217149 B2), ^Yervoy® (ipilimumab; Bristol Myers Squibb) and pidilizumab (CT-011; CureTech), ^Yervoy® (ipilimumab; Bristol Myers Squibb) and MEDI0680 (AMP-514; AstraZeneca), ^Yervoy® (ipilimumab; Bristol Myers Squibb) and MSB-0010718C (Merck), ^Yervoy® (ipilimumab; Bristol Myers Squibb) and MIH1 (Bristol Myers Squibb) and MIH1 Affymetrix), combination Y ^ervoy® (ipilimumab; Bristol Myers Squibb) and AMP-224, ^Yervoy® (ipilimumab; Bristol Myers Squibb) and lambrolizumab, tremelimumab (Pfizer/MedImmune) and ^Opdivo® (nivolumab; Bristol Myers Squibb), tremelimumab (Pfizer/ MedImmune) and Keytruda® ( ^{pembrolizumab} ; Merck), combination of tremelimumab (Pfizer/MedImmune) and durvalumab (MedImmune/AstraZeneca), combination of tremelimumab (Pfizer/MedImmune) and MEDI4736 (AstraZeneca; cf. WO 2011/066389 A1 ), combination of tremelimumab (Pfizer/MedImmune) and MPDL3280A (Roche/Genentech; cf. US 8217149 B2), combination of tremelimumab (Pfizer/MedImmune) and pidilizumab (CT-011; CureTech), combination of tremelimumab (Pfizer/ MedImmune) and MEDI0680 (AMP-514; AstraZeneca), tremelimumab combination (Pfizer/MedImmune) and MSB-0010718C (Merck), tremelimumab combination (Pfizer/MedImmune) and MIH1 (Affymetrix), tremelimumab combination (Pfizer /Med Immune) and AMP-224 and a combination of tremelimumab (Pfizer/MedImmune) and lambrolizumab.

In the context of the present invention, more than one immune checkpoint modulator (e.g. checkpoint inhibitor) of the same checkpoint pathway is also preferably used, in particular at least 2, 3, 4, 5, 6, 7, 8, 9 are used. or 10 immune checkpoint modulators (e.g. checkpoint inhibitors) of the same checkpoint pathway, preferably 2, 3, 4 or 5 immune checkpoint modulators (e.g. checkpoint inhibitors) of the same checkpoint pathway, more preferably 2, 3 or 4 immune checkpoint modulators (e.g. checkpoint inhibitor) of the same checkpoint pathway are used, even more preferably 2 or 3 immune checkpoint modulators (e.g. checkpoint inhibitor) of the same checkpoint pathway are used points and most preferably 2 immune checkpoint modulators (e.g. checkpoint inhibitor) are used alone same checkpoint path. The preferred checkpoint paths for modulation are the PD-1 path or the CTLA-4 path. For example, a combination of MEDI4736 and MEDI0680 can be used to modulate, primarily to inhibit, the PD-1 pathway.

In the context of the present invention, immune checkpoint modulators may be any type of molecule or agent, as long as they wholly or partly reduce, inhibit, interact with, activate, stimulate, enhance, enhance or maintain the function of one or more of the above described checkpoint molecules. In particular, an immune checkpoint modulator binds to one or more checkpoint molecules, such as checkpoint proteins, or their precursors, e.g., at the DNA or RNA level, thereby modulating (e.g., fully or partially reducing, inhibiting, interacting with activating, stimulating, enhancing, enhancing or maintaining) the function of one or more of the checkpoint molecules described above. Preferred immune checkpoint modulators are oligonucleotides, siRNAs, shRNAs, ribozymes, antisense RNAs, immunotoxins, small molecule inhibitors, and antibodies or antigen-binding fragments thereof (e.g., antibodies or antibody fragments, antagonistic antibodies or antibody fragments, or agonistic antibodies or antibody fragments that block checkpoint molecules).

Preferably, the immune checkpoint modulator is an oligonucleotide. Such an oligonucleotide is preferably used to reduce the expression of a protein, especially to reduce the expression of a checkpoint protein such as the checkpoint receptors or ligands described above. Oligonucleotides are short molecules of DNA or RNA, typically containing 2 to 50 nucleotides, preferably 3 to 40 nucleotides, more preferably 4 to 30 nucleotides, and even more preferably 5 to 25 nucleotides, e.g. 4, 5, 6 , 7, 8, 9 or 10 nucleotides. Oligonucleotides are usually created in the laboratory by solid phase chemical synthesis. Oligonucleotides may be single or double stranded, however, in the context of the present invention, the oligonucleotide is preferably single stranded. More preferably, the checkpoint modulator oligonucleotide is an antisense oligonucleotide. Antisense oligonucleotides are single strand DNA or RNA complementary to a selected sequence, in particular a sequence selected from a DNA or RNA sequence (or fragment thereof) of a checkpoint protein. Antisense RNA is generally used to prevent translation into protein of messenger RNA strands, such as checkpoint protein mRNA, by binding to the mRNA. Antisense DNA is typically used to target a specific complementary (coding or non-coding) RNA. If binding occurs, then such DNA/RNA hybrids can be cleaved by the enzyme RNase H. In addition, antisense morpholino oligonucleotides can be used to knock down genes in vertebrates. For example, Kryczek et al. (Kryczek I., Zou L., Rodriguez P., Zhu G., Wei S., Mottram P. et al., B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma. J Exp Med., 203, 2006, pp. 871-881) constructed a B7-H4-specific morpholino oligonucleotide that specifically blocks the expression of B7-H4 in macrophages, resulting in increased T cell proliferation and reduced tumor volumes in mice with specific in relation to the tumor associated antigen (TAA) T-cells.

Preferably, the immune checkpoint modulator is a siRNA. Small interfering RNA (siRNA), sometimes referred to as short interfering RNA or silence-inducing RNA, is a class of double-stranded RNA molecules typically 20-25 base pairs long. In the RNA interference (RNAi) pathway, siRNA affects the expression of specific genes, such as those encoding checkpoint proteins having complementary nucleotide sequences. The functions of siRNAs, which consist in the destruction of mRNA after transcription, lead to the absence of translation. For gene knockdown, transfection of exogenous siRNA can be used, but the effect can be only short-lived, especially in the case of rapidly dividing cells. This problem can be overcome, for example, by modifying the RNA or by using an expression vector for siRNA. The siRNA sequence can also be modified by introducing a short loop between two strands. The resulting transcript is a short hairpin RNA (shRNA, also referred to as "small hairpin RNA") from which a functional siRNA is formed by normal processing by the Dicer enzyme. shRNA is an advantageous RNAi mediator because it has a relatively slow rate of cleavage and turnover. Therefore, the immune checkpoint modulator is preferably shRNA. For shRNA, as a rule, it is required to use an expression vector, for example, a plasmid or a viral or bacterial vector.

Preferably, the immune checkpoint modulator is an immunotoxin. Immunotoxins are chimeric proteins that contain a targeting moiety (such as an antibody) that typically targets an antigen to a specific cell, such as a cancer cell, linked to the toxin. In the context of the present invention, an immunotoxin containing a targeting fragment that targets the checkpoint molecule is preferred. When an immunotoxin binds to a cell carrying an antigen, such as a checkpoint molecule, it is taken up by endocytosis and the toxin can kill the cell. The immunotoxins preferably contain a (modified) antibody or an antibody fragment linked/linked to a (fragment) of the toxin. Techniques for implementing coupling are well known in the art. The targeting site of an immunotoxin typically contains a Fab fragment of an antibody that targets a specific cell type. The toxin is typically cytotoxic, such as a protein derived from a bacterial or plant protein from which the naturally occurring binding domain has been removed, causing the targeting moiety of the immunotoxin to target the toxin to the antigen on the target cell. However, immunotoxins may also contain a targeting fragment other than an antibody or antibody fragment, such as a growth factor. For example, recombinant fusion proteins containing a toxin and a growth factor are also referred to as recombinant immunotoxins.

Preferably, the immune checkpoint modulator is a small molecule drug (also referred to as a "small molecular weight inhibitor"). A low molecular weight drug is an organic compound with a low molecular weight (up to 900 Da), which, as a rule, affects (regulates) a biological process. In the context of the present invention, a small molecule drug that is an immune checkpoint modulator is an organic compound having a molecular weight of not more than 900 Da that wholly or partially reduces, inhibits, interacts with or negatively modulates the function of one or more of the control molecules described above. points. The upper molecular weight limit of 900 Da allows rapid diffusion across cell membranes and ensures oral bioavailability. More preferably, the molecular weight of the small molecular weight immune checkpoint modulator drug does not exceed 500 Da. For example, various A2AR antagonists known in the art are organic compounds having a molecular weight of less than 500 Da.

Most preferably, the immune checkpoint modulator is an antibody or an antigen-binding fragment thereof. Such immune checkpoint modulator antibodies or antigen-binding fragments thereof include, in particular, antibodies or antigen-binding fragments thereof that bind to immune checkpoint receptors or antibodies that bind to immune checkpoint receptor ligands. Preferably, the immune checkpoint modulator antibodies, or antigen-binding fragments thereof, are immune checkpoint receptor agonists or antagonists or immune checkpoint receptor ligands. Examples of antibody-type checkpoint modulators include the immune checkpoint modulators described above that are currently approved for use, namely ^Yervoy® (ipilimumab; Bristol Myers Squibb), ^Opdivo® (nivolumab; Bristol Myers Squibb) and ^Keytruda® ( pembrolizumab; Merck), and other anti-checkpoint receptor antibodies or anti-checkpoint receptor antibodies described above.

Preferably, the immune checkpoint modulators included in the combination used according to the present invention are antibodies or antigen binding fragments that can partially or completely block the PD-1 pathway (for example, they can be partial or complete antagonists of the PD-1 pathway), in in particular PD-1, PD-L1 or PD-L2, more preferably the antibody may partially or completely block PD-1 (eg, may be partial or complete antagonists of PD-1). Such antibodies or antigen-binding fragments include anti-PD-1 antibodies, human anti-PD-1 antibodies, mouse anti-PD-1 antibodies, mammalian anti-PD-1 antibodies, humanized anti-PD-1 antibodies, anti-PD-1 monoclonal antibodies, anti-PD-1 polyclonal antibodies. PD-1, anti-PD-1 chimeric antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-PD-1 adnectins, anti-PD-1 domain antibodies, anti-PD-1 single chain fragments, antibody heavy chain fragments to PD-1 and fragments of the light chain of antibodies to PD-1. For example, an anti-PD-1 antibody may be an antigen binding fragment. Preferably, the anti-PD-1 antibody has the ability to bind to human PD-1 and partially or completely block the activity of (human) PD-1 (for example, such antibodies can be partial or complete antagonists of PD-1), thereby triggering, in particular, the function immune cells expressing PD-1.

Preferably, the immune checkpoint modulators included in the combination used according to the present invention are antibodies or antigen binding fragments that can partially or completely block the CTLA-4 pathway (for example, they can be partial or complete antagonists of the CTLA-4 pathway). Such antibodies or antigen-binding fragments include anti-CTLA4 antibodies, human anti-CTLA4 antibodies, mouse anti-CTLA4 antibodies, mammalian anti-CTLA4 antibodies, humanized anti-CTLA4 antibodies, monoclonal anti-CTLA4 antibodies, polyclonal anti-CTLA4 antibodies, chimeric anti-CTLA4 antibodies, MDX-010 (ipilimumab ), tremelimumab, anti-CD28 antibodies, anti-CTLA4 adnectins, domain anti-CTLA4 antibodies, single-chain anti-CTLA4 antibody fragments, anti-CTLA4 antibody heavy chain fragments, and anti-CTLA4 antibody light chain fragments. For example, an anti-CTLA4 antibody may be an antigen binding fragment. Preferably, the anti-CTLA4 antibody has the ability to bind to human CTLA4 and partially or completely block the activity of CTLA4 (for example, such antibodies may be partial or complete antagonists of CTLA-4), thereby triggering, in particular, the function of immune cells expressing CTLA4.

Preferred combinations of a preferred immune checkpoint modulator and a T-cell redirecting multifunctional antibody

As indicated above, a preferred combination for use in accordance with the present invention contains the preferred immune checkpoint modulator described above. In addition, a preferred combination for use in accordance with the present invention comprises the preferred T cell retargeting multifunctional antibody described above, or the antigen binding fragment described above, comprising/comprising a (preferred) specificity for a T cell surface antigen, a (preferred) specificity for a cancer and/or tumor associated antigen; and a (preferred) human FcγRI, FcγRIIa and/or FcγRIII binding site.

A more preferred combination for use in the present invention comprises (I) a preferred immune checkpoint modulator as provided herein and (II) a preferred T cell redirecting multifunctional antibody or antigen binding fragment as provided/as provided herein which/ which contains (preferred) specificity for a T cell surface antigen, (preferred) specificity for a cancer and/or tumor associated antigen, and (preferred) human FcγRI, FcγRIIa and/or FcγRIII binding site. The following preferred embodiments of the invention describe a preferred combination for use in accordance with the present invention.

In a preferred combination for use in the present invention, the T cell redirecting multifunctional antibody is a trifunctional IgG-type bispecific antibody in which

a) specificity for T-cell surface antigen is specificity (binding site) for (human) CD3;

b) the specificity for a cancer and/or tumor associated antigen is specificity (binding site) for EpCAM, HER2/neu, GD2 or CD20;

c) the binding site for human FcγRI, FcγRIIa and/or FcγRIII is the Fc region of mouse IgG2a/rat IgG2b.

More preferably, the T cell redirecting multifunctional antibody is catumaxomab and/or ectomab.

It is also preferred that in the combination to be used according to the present invention the immune checkpoint modulator is a CTLA-4, PD-1, PD-L1 and/or PD-L2 inhibitor, and more preferably the immune checkpoint modulator is a CTLA inhibitor -4 and/or PD-1. Most preferably, the immune checkpoint modulator is a CTLA-4 inhibitor.

For example, a preferred combination for use in the present invention contains

- trifunctional bispecific IgG-type antibody having

a) specificity (binding site) for (human) CD3;

b) specificity (binding site) for EpCAM, HER2/neu, GD2 or CD20; and

c) Fc region of mouse IgG2a/rat IgG2b; and

- an inhibitor of CTLA-4, PD-L1, PD-L2 and/or PD-1, preferably an inhibitor of CTLA-4 and/or PD-1.

Another preferred example of a combination for use in the present invention comprises

- trifunctional bispecific IgG-type antibody having

a) specificity (binding site) for (human) CD3;

b) specificity (binding site) for EpCAM, HER2/neu, or GD2; and

c) Fc region of mouse IgG2a/rat IgG2b; and

- an inhibitor of CTLA-4, PD-L1, PD-L2 and/or PD-1, preferably an inhibitor of CTLA-4 and/or PD-1.

Most preferably, the combination for use according to the present invention contains (I) catumaxomab or ectomab and (II) a CTLA-4 inhibitor.

Use in the therapeutic treatment of cancer

The combination of the immune checkpoint modulator provided herein and the T cell redirecting multifunctional antibody or antigen-binding fragment provided herein is for use in the treatment of cancer.

Such a combination of an immune checkpoint modulator as provided herein and a T cell redirecting multifunctional antibody or antigen binding fragment as provided herein has the ability to initiate or enhance the efficacy of checkpoint modulators, particularly under therapeutic conditions, as demonstrated in examples presented in the present description.

In the context of the present description, the term "therapeutic treatment" refers to treatment after the onset of the disease. In particular, "therapeutic treatment" does not include preventive measures taken before the onset of the disease. Because the onset of a disease is often associated with the symptom(s) of the disease, humans or animals are often subjected to "therapeutic" treatment after the diagnosis or at least the (high) likelihood that the individual is suffering from a particular disease is established. The goal of therapeutic treatment is, in particular (I) to alleviate, improve or cure the disease (condition) or (II) to inhibit or slow the progression of the disease (for example, by increasing the average survival time for cancer patients). However, the prevention of the onset of the disease, as a rule, cannot be achieved with the help of therapeutic treatment.

The combination presented in the present description is intended for use (for the preparation of a medicinal product) for the therapeutic treatment of cancer. The concept of "disease" used in the context of the present invention, as a rule, is synonymous with and is used interchangeably with the concepts of "disorder" and "condition" (as in the case of a medical condition), in the sense that they all characterize an abnormal state of the human or animal body. or one of its parts, which impairs the normal functioning, usually manifesting itself in the form of distinctive signs and symptoms, and which leads to a decrease in the duration or quality of life of a person or animal.

Cancers are a group of diseases characterized by abnormal growth of cells, which in particular can invade or spread to other parts of the body. Cancer cells/tissue typically exhibit six hallmarks of cancer, namely (i) cell growth and division in the absence of a corresponding signal; (II) continued growth and division even in the presence of opposing signals; (III) avoidance of programmed cell death; (IV) an unlimited number of cell divisions; (V) stimulating the formation of blood vessels; and (VI) tissue invasion and metastasis formation.

Cancer diseases include diseases caused by impaired apoptosis. The cancer may be a solid tumor, a blood cancer, or a lymphatic cancer. In particular, the cancer may be benign, malignant, or metastatic.

Preferably, in the therapeutic treatment of cancer, the combination for use in accordance with the present invention inhibits/slows ongoing/further tumor (or metastasis) growth, or reduces tumor size (or number of metastases), or prevents tumor and/or metastasis recurrence.

Preferred examples of cancers are preferably selected from the following diseases: acoustic neuroma, anal carcinoma, astrocytoma, basalioma, Behçet's syndrome, bladder cancer, blastoma, bone cancer, brain metastases, brain tumors, brain cancer (glioblastomas), breast cancer glands (breast carcinoma), Burkitt's lymphoma, carcinoids, cervical cancer, colon carcinoma, colorectal cancer, uterine corpus carcinoma, craniopharyngioma, CUP syndrome, endometrial carcinoma, gallbladder cancer, genital tumors including urogenital tract cancers, glioblastoma, gliomas , head/neck tumors, hepatomas, histiocytic lymphoma, Hodgkin's syndromes or lymphomas and non-Hodgkin's lymphomas, pituitary tumor, intestinal cancer, including tumors of the small intestine and gastrointestinal tumors, Kaposi's sarcoma, kidney cancer, renal carcinomas, laryngeal cancer or cancer of the larynx, leukemia, including acute myeloid leukemia (AML), erythroleukemia, acute lymphoid leukemia (AL L), chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL), eyelid tumor, liver cancer, liver metastases, lung carcinomas (=lung cancer=bronchial carcinoma), small cell lung carcinomas and non-small cell lung carcinomas, and adenocarcinoma of the lung, lymphomas, cancer of the lymphatic system, malignant melanomas, carcinomas of the breast (=breast cancer), medulloblastomas, melanomas, meningiomas, mycosis fungoides, neoplastic neurinoma, esophageal cancer, carcinoma of the esophagus (=cancer of the esophagus), oligodendroglioma, ovarian cancer (=carcinoma of the ovary ), ovarian carcinoma, pancreatic carcinoma (=pancreatic cancer), penile carcinoma, penile cancer, larynx cancer, pituitary tumor, plasmacytoma, prostate cancer (=prostate tumors), rectal carcinoma, rectal tumors, kidney cancer, renal carcinomas , retinoblastoma, sarcomas, Schneeberger's disease, skin cancer such as melanoma or non-melanoma skin cancer, including basal cell and squamous cell carcinomas, as well as psoriasis, pemphigus, soft tissue tumors, spinalioma, gastric cancer, testicular cancer, pharyngeal cancer, thymoma, thyroid carcinoma, tongue cancer, urethral cancer, uterine cancer, vaginal cancer, various virus-induced tumors, such as for example, as papilloma-induced carcinomas (eg, cervical carcinoma = cervical cancer), adenocarcinomas, herpesvirus-induced tumors (eg, Burkitt's lymphoma, EBV-induced B-cell lymphoma, cervical carcinoma), hepatitis B virus-induced tumors (hepatocellular carcinomas ), HTLV-1 and HTLV-2 induced lymphomas, vulvar cancer, conditions due to or associated with warts, etc.

Other preferred examples of cancers to be treated with the combination of the immune checkpoint modulator provided herein and the T-cell redirecting multifunctional antibody or fragment thereof provided herein include brain cancer, prostate cancer, breast cancer, cancer ovarian cancer, esophageal cancer, lung cancer, liver cancer, kidney cancer, melanoma, intestinal carcinoma, lung carcinoma, head and neck squamous cell carcinoma, Hjkin's lymphoma, chronic myeloid leukemia, colorectal carcinoma, gastric carcinoma, endometrial carcinoma, myeloid leukemia, squamous cell carcinoma of the lung, acute lymphoblastic leukemia, acute myelogenous leukemia, bladder tumor, promyelocytic leukemia, non-small cell lung carcinoma, plasmacytoma and sarcoma.

More preferably, the cancer is selected from lung cancer, gastric cancer, ovarian cancer, breast cancer, melanoma, prostate cancer, head and neck squamous cell carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphomas, bladder tumor, plasmacytoma and/or sarcoma.

In general, the term “combination” of an immune checkpoint modulator provided herein and a T cell redirecting multifunctional antibody or fragment thereof provided herein means that therapy using an immune checkpoint modulator provided herein is combined with therapy using a redirecting T-cell multifunctional antibody or its fragment presented in the present description. In other words, even if one component (checkpoint modulator or T-cell retargeting multifunctional antibody) is not administered, for example, on the same day as the other component (the other checkpoint modulator or T-cell redirecting multifunctional antibody), then their treatment schedules are intertwined. This means that “combination” in the context of the present invention does not include, in particular, the condition that therapy with one component (checkpoint modulator or T-cell redirecting multifunctional antibody) is started after therapy with the other component (another control point modulator) has ended. dots or a T-cell redirecting multifunctional antibody). More generally, the concept of an "intertwined" treatment schedule with a checkpoint modulator and a T-cell retargeting multifunctional antibody - and therefore a combination of a checkpoint modulator and a T-cell retargeting multifunctional antibody - means that:

(I) not every checkpoint modulator administration (and therefore complete checkpoint modulator therapy) is completed in more than one week (preferably more than 3 days, more preferably more than 2 days, even more preferably more than one day) prior to the first administration of the T-cell-redirecting multifunctional antibody (and therefore, before the start of full therapy with the T-cell-redirecting multifunctional antibody); or

(II) not every administration of the T-cell-targeting multifunctional antibody (and hence the complete therapy using the T-cell-targeting multifunctional antibody) is completed in more than one week (preferably more than 3 days, more preferably more than 2 days, even more preferably more than one day) prior to the first administration of the checkpoint modulator (and hence before the start of full therapy using the checkpoint modulator).

For example, in the case of the combination of the immune checkpoint modulator provided herein and the T cell retargeting multifunctional antibody provided herein to be used according to the present invention, one component (checkpoint modulator or T cell redirecting multifunctional antibody) can be administered once a week, and the second component (another checkpoint modulator or T-cell redirecting multifunctional antibody) can be administered once a month. To implement the "combination" described in this example, as defined in the context of the present invention, the monthly input component should be administered at least once during the same week that the other weekly input component is administered.

As indicated above, the administration of an immune checkpoint modulator and/or a T-cell redirecting multifunctional antibody in a combination for use in accordance with the present invention may require several consecutive administrations, for example, several injections. Thus, the administration can be repeated at least twice, for example once as a primary injection and then as a booster injection.

In particular, the immune checkpoint modulator and/or T-cell redirecting multifunctional antibody in the combination for use in the present invention may be administered repeatedly or continuously. The immune checkpoint modulator and/or T cell redirecting multifunctional antibody in the combination for use according to the present invention can be administered repeatedly or continuously over a period of at least 1, 2, 3 or 4 weeks; 2, 3, 4, 5, 6, 8, 10 or 12 months; or 2, 3, 4 or 5 years. For example, the immune checkpoint modulator included in the combination for use according to the present invention can be administered twice a day, once a day, every two days, every three days, once a week, every two weeks, every three weeks, once a month or every two months. For example, a T-cell redirecting multifunctional antibody in a combination for use in the present invention can be administered twice a day, once a day, every two days, every three days, once a week, every two weeks, every three weeks, once a month or every two months.

Preferably, the T-cell retargeting multifunctional antibody or antigen-binding fragment thereof is administered according to a dose escalation regimen. Generally, "dose escalation regimen" refers to repeated administration of an antibody in which the initial dose (i.e., typically the single dose of the first administration of the antibody, e.g., in total or in relation to one single administration cycle) is less than the final dose. dose (ie, typically a single dose at the final administration of the antibody, eg, in total or in relation to one single administration cycle). In particular, the dose of the antibody is increased with repeated administrations of the antibody according to the dosage escalation regimen. In particular, a dose escalation regimen includes two or more "dose levels" that are administered in ascending order, namely, starting at the lowest dose level, followed by the next higher dose level, followed by the optional next higher dose level. doses, etc. The term "dose level" refers to a specific dose/amount of antibody. For example, a dose level of "10 μg" means that a single dose of 10 μg of the antibody is administered one or more times (e.g., two or three times) before the next higher dose level (e.g., in which a single dose of antibody of 50 μg is administered once or several times). Therefore, at each dose level, single doses may be administered one or more times (eg, two or three times). If more than one single dose is administered at the (same) dose level, this means that the single doses (at the indicated dose level) are the same, ie. the amount of antibody administered with each single dose at the (same) dose level is the same. Therefore, in the dose escalation regimen, with the exception of the initial dose, i.e. at the first administration of the antibody, the single dose administered at each single administration of the antibody is either higher than the dose in the previous administration of the antibody (in order to move to the next higher dose level), or remains the same as in the previous administration of the antibody ( to maintain the "existing" dose level). The term "preceding administration" refers to the administration (antibodies) immediately preceding the administration in question (antibodies). For example, in the case of a third administration (antibody), "prior administration" is the second administration (antibodies) but not the first administration (antibodies); or in the case of a fourth administration (antibody), "previous administration" is the third administration (antibodies) and not the first administration (antibodies) or the second administration (antibodies). For example, in a dose escalation regimen (I), the initial dose may be the lowest dose and each subsequent administration (antibody) single dose may be higher than the corresponding previous administration (antibody) so that only one single dose is administered at each dose level. dose or (II) at one or more (but not all) administrations (antibodies), the single doses may be the same as the corresponding prior administration (antibodies) (so that at one or more dose levels, e.g., at each dose level administered more than one single dose).

Preferably, the combination for use in accordance with the present invention is administered over one or more treatment cycles. In the context of the present invention, a treatment cycle is a course comprising one or more treatments that can be repeated according to a regular schedule with rest periods in between. For example, a combination for use according to the present invention may be administered within one treatment cycle (eg, by single dose or repeated doses) and thereafter monitored for recurrence of the cancer or tumor. In particular, if there is a recurrence of the cancer/tumor, then the next cycle of treatment can be carried out. However, the next treatment cycle can also be carried out as a preventive measure. In particular, the interval between two treatments (e.g., between two single doses of antibody and/or between two single doses of checkpoint modulator) within a single treatment cycle should preferably not exceed one month (31 days), more preferably should not exceed 3 weeks, while the interval between the end of one treatment cycle and the beginning of the next treatment cycle (primarily the administration of the antibody and/or immune checkpoint modulator) should preferably be at least one month, preferably at least two months, more preferably at least 3 months, even more preferably at least 4 months, and most preferably at least 6 months. In other words, the interval between two treatments/administrations (antibody and/or checkpoint modulator) within one treatment cycle is preferably less than one month (for example, no more than two or three weeks), while the interval between two treatment cycles ( regarding administration of the antibody and/or checkpoint modulator) is preferably greater than one month (eg, at least two or three months).

Preferably, one treatment cycle comprises (I) one single administration or (II) administration of one initial dose (first administration) and one or more subsequent administrations of the antibody and/or immune checkpoint modulators. The patient can be subjected to one single or different treatment cycles. Each treatment cycle typically comprises 2 to 28, preferably 2 to 20, more preferably 3 to 10, and even more preferably 5 to 8, such as 6 or 7, single administrations of the antibody and/or immune checkpoint modulator.

Preferably, one treatment cycle includes one or more dose levels. In other words, preferably one treatment cycle comprises (I) repeated administration of the same single doses (one single dose level) or (II) administration of one or more increasing single doses (one or more single doses may be administered at each dose level). , as mentioned above). In the latter case, the dose levels following the initial dose level (administration(s) of the dose intended for the first administration), as a rule, are higher than the initial dose level. In particular, the starting dose level may preferably include only one single administration, i. e. the lowest dose is administered only once at the very beginning of the treatment/administration cycle (first administration). In this case, the single dose(s) in one or more subsequent administrations is(are) higher(s) than the initial dose.

In other words, it is preferable that in the cycle of treatment (starting from the introduction of the initial dose and ending with the administration of the final dose), the single dose at each single administration (except for the initial dose) is not lower than the previous dose administered, i.e. each subsequent dose is equal to or greater than the previous one. More preferably, the treatment cycle follows the dose escalation regimen described above. Even more preferably, when more than one treatment cycle is used, each treatment cycle corresponds to the dosage escalation regimen described above. In this case, the dosing regimen in each treatment cycle can be the same or different. As described above, such a dose escalation regimen may also include doses that are equal to the previous one (ie, administration of more than one single dose at the same (single) dose level). For example, only the initial dose may be lower, and all subsequent single doses administered may be the same (and higher than the initial dose), or the initial dose may be lower, the single dose on the second administration may be higher than the initial dose, but lower than all subsequent single doses administered, with all subsequent single doses being preferably the same (and higher than the initial dose and the second dose). Thus, it is preferred that the one or more single doses administered after the initial dose is greater than the single dose of the previous dose.

The final dose (level) in a treatment cycle generally corresponds to the highest single dose of antibody intended to be administered in one treatment cycle; those. maximum single dose per treatment cycle. In particular, one, two, three, four, five or more single doses corresponding to the maximum single dose may be administered at the end of the treatment cycle.

In general, the basic principle of dose escalation is to avoid exposing the patient to sub-therapeutic doses while maintaining safety and allowing rapid dose escalation. Preferably, during the same treatment cycle, none of the single doses is lower than the previous one.

Typically, one treatment cycle includes at least an initial (first) administration and a second administration of the antibody and/or immune checkpoint inhibitor. In a preferred embodiment of the invention, one treatment cycle may include an initial administration, a second administration, a third administration, a fourth administration, a fifth administration, and preferably a sixth administration of the antibody and/or immune checkpoint inhibitor. In one treatment cycle, subsequent administration of the antibody and/or immune checkpoint inhibitor can preferably be done at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days after the previous administration, preferably the subsequent administration is carried out 2-15 days after the previous administration, more preferably the subsequent administration is carried out 2-10 days after the previous administration, even more preferably the subsequent administration is carried out after 3- 8 days after previous administration.

In the case of a combination comprising the immune checkpoint modulator provided herein and the T cell redirecting multifunctional antibody provided herein, which is intended for use according to the present invention, the immune checkpoint modulator and the T cell redirecting multifunctional antibody are preferably administered at about at the same time.

The term "at about the same time" as used herein means, in particular, simultaneous administration, or, in particular, that immediately after the introduction of the immune checkpoint modulator, a multifunctional T-cell retargeting antibody is administered or immediately after the administration of a retargeting T cells of the multifunctional antibody introduce an immune checkpoint modulator. It should be obvious to a person skilled in the art that the concept of "immediately after" includes the period of time necessary to prepare for the second injection, in particular, the period of time necessary to expose and disinfect the site for the second injection, as well as the corresponding preparation of the "injection device" (eg syringe, pump, etc.). Simultaneous administration also includes administration in which the periods of administration of the checkpoint modulator and the T cell retargeting multifunctional antibody overlap, or in which, for example, one component (checkpoint modulator or T cell retargeting multifunctional antibody) is administered for a longer period of time, such as 30 minutes, 1 hour, 2 hours, or even more, for example, by infusion, and the other component (checkpoint modulator or T-cell redirecting multifunctional antibody) is administered at a certain point in time within such a long period of time. Administration of the immune checkpoint modulator and the T cell redirecting multifunctional antibody at about the same time is particularly preferred if different routes of administration are used and/or administration is at different sites.

Preferably, also in the case of a combination comprising an immune checkpoint modulator provided herein and a T cell redirecting multifunctional antibody provided herein, which is intended for use according to the present invention, the immune checkpoint modulator and the T cell redirecting multifunctional antibody are administered sequentially. For example, the immune checkpoint modulator is preferably administered prior to administration of the T cell redirecting multifunctional antibody. It is also preferred if the immune checkpoint modulator is administered after the T-cell redirecting multifunctional antibody.

When administered sequentially, the time interval between administrations of the first component (checkpoint modulator or T cell retargeting multifunctional antibody) and administration of the second component (other checkpoint modulator and T cell retargeting multifunctional antibody) is preferably no more than one week, more preferably no more than 3 days, even more preferably no more than 2 days, and most preferably no more than 24 hours. T-cell-redirecting multifunctional antibody) and the introduction of the second component (the other of the specified checkpoint modulator and T-cell redirecting multifunctional antibody) is preferably no more than 6 hours, more preferably no more than 3 hours, even more preferably no more than 2 hours and most preferably no more than 1 hour.

However, most preferably, the immune checkpoint modulator is administered after the T-cell redirecting multifunctional antibody or antigen-binding fragment thereof. More preferably, the immune checkpoint modulator is administered at least 6 hours, preferably at least 12 hours, more preferably at least 18 hours, even more preferably at least 24 hours, even more preferably at least 36 hours and most preferably at least 48 hours after the T cell retargeting multifunctional antibody or antigen-binding fragment thereof. In other words, in a preferred embodiment of the invention, the interval between administration of the T cell-redirecting multifunctional antibody and or antigen-binding fragment thereof (given first) and administration of the immune checkpoint modulator (given after the antibody) is at least 6 hours, preferably at least 12 hours. , more preferably at least 18 hours, even more preferably at least 24 hours, even more preferably at least 36 hours, and most preferably at least 48 hours. checkpoint is at least 6 hours, preferably at least 12 hours, more preferably at least 18 hours, even more preferably at least 24 hours, even more preferably at least 36 hours, and most preferably at least 36 hours. at least 48 hours after the first administration of T-cell redirecting polyfu a local antibody or antigen-binding fragment thereof. As another example (in a treatment cycle or in general), the final administration of the immune checkpoint modulator is at least 6 hours, preferably at least 12 hours, more preferably at least 18 hours, even more preferably at least 24 hours, even more preferably at least 36 hours, and most preferably at least 48 hours after the final administration of the T-cell redirecting multifunctional antibody or antigen-binding fragment thereof. Most preferably (in a treatment cycle or in general) each immune checkpoint modulator administration is at least 6 hours, preferably at least 12 hours, more preferably at least 18 hours, even more preferably at least 24 hours. h, even more preferably at least 36 h and most preferably at least 48 h after each administration of a redirecting T-cell multifunctional antibody or antigen-binding fragment. Therefore, most preferably (in a treatment cycle or in general) (I) the first administration of the immune checkpoint modulator is at least 6 hours, preferably at least 12 hours, more preferably at least 18 hours, still more preferably at least 24 hours, even more preferably at least 36 hours, and most preferably at least 48 hours after the first administration of the T cell-redirecting multifunctional antibody or antigen-binding fragment thereof, and (ii) the final administration of the immune control modulator points are carried out after at least 6 hours, preferably at least 12 hours, more preferably at least 18 hours, even more preferably at least 24 hours, even more preferably at least 36 hours and most preferably at least at least 48 hours after the final administration of the T-cell redirecting multifunctional antibody, or its antigen-binding fragment.

Preferably also, the immune checkpoint modulator is administered no more than 96 hours, preferably no more than 84 hours, more preferably no more than 72 hours, and most preferably no more than 60 hours after administration of the T cell-redirecting multifunctional antibody, or its antigen-binding fragment. In other words, in a preferred embodiment of the invention, the interval between administration of the T cell-redirecting multifunctional antibody or antigen-binding fragment thereof (administered first) and administration of the immune checkpoint modulator (administered after the antibody) is no more than 96 hours, preferably no more than 84 hours, more preferably no more than 72 hours and most preferably no more than 60 hours. 72 hours and most preferably no more than 60 hours after the first administration of the T-cell redirecting multifunctional antibody or antigen-binding fragment thereof. In another example (in a treatment cycle or in general), the final administration of the immune checkpoint modulator is at most 96 hours, preferably at most 84 hours, more preferably at most 72 hours, and most preferably at most 60 hours. h after the final administration of a T-cell-redirecting multifunctional antibody or antigen-binding fragment thereof. Most preferably (in a treatment cycle or in general) each immune checkpoint modulator administration is not more than 96 hours, preferably not more than 84 hours, more preferably not more than 72 hours, and most preferably not more than 60 hours. after each administration of a T-cell redirecting multifunctional antibody or antigen-binding fragment thereof. Therefore, most preferably (in a treatment cycle or in general) (I) the first administration of the immune checkpoint modulator is after no more than 96 hours, preferably no more than 84 hours, more preferably no more than 72 hours and most preferably not more than 60 hours after the first administration of the T cell-redirecting multifunctional antibody or antigen-binding fragment thereof, and (ii) the final administration of the immune checkpoint modulator is not more than 96 hours, preferably not more than 84 hours, more preferably not more than 72 hours, and most preferably no more than 60 hours after the final administration of the T-cell redirecting multifunctional antibody or antigen-binding fragment thereof.

Most preferably (in a treatment cycle or in general) (I) the first administration of the immune checkpoint modulator is 12-96 hours, preferably 24-84 hours, more preferably 36-72 hours, and most preferably 48-60 hours after the first administration of the redirector. T cells of the multifunctional antibody or antigen-binding fragment thereof, and/or (II) the final administration of the immune checkpoint modulator is 12-96 hours, preferably 24-84 hours, more preferably 36-72 hours and most preferably 48-60 hours after the final administering a T-cell redirecting multifunctional antibody or antigen-binding fragment thereof.

The present invention has surprisingly found that there is an increase in the expression of immune checkpoint molecules following T cell activation by the T cell redirecting multifunctional antibodies described herein. As demonstrated in Example 2 of this disclosure, activation of T cells by the T cell redirecting multifunctional antibodies provided herein increases the expression of an immune checkpoint molecule, primarily CTLA-4, which is considered to be the "leader" of the inhibitory immune checkpoint molecules described higher, which reaches a peak 48-72 hours after the administration of the antibody. These data suggest that T cell activation by the T cell redirecting multifunctional antibodies provided herein, which is critical in the treatment of cancer and neoplastic diseases, can be prolonged primarily if the immune checkpoint modulator has its effect. when the expression of immune checkpoint molecules increases or even peaks. The preferred order of administration described above (antibody first, then immune checkpoint modulator) and time intervals are based on these data and are administration schedules that are expected to result in prolonged T cell activation and therefore increased efficacy in treatment. cancerous and/or neoplastic diseases.

Preferably, the immune checkpoint modulator included in the combination for use according to the present invention and the T-cell redirecting multifunctional antibody included in the combination intended for use according to the present invention are administered in a therapeutically effective amount. The term "therapeutically effective amount"as used herein means an amount that is sufficient to alleviate the symptoms of the disease or condition being treated, or to inhibit or slow the progression of the disease. In other words, "therapeutically effective amount" means an amount of a multifunctional T-cell redirecting antibody and/or checkpoint modulator that is sufficient to induce a positive change in the disease or disorder, i.e. an amount of a T-cell redirecting multifunctional antibody and/or checkpoint modulator that elicits a desired biological or medical response in a tissue, system, animal or human body. The term also includes an amount of a T-cell-redirecting multifunctional antibody and/or checkpoint modulator sufficient to reduce the progression of the disease, especially to reduce or inhibit tumor growth and, therefore, to elicit a desired (immune) response (i.e. "effective against inhibition amount"). However, at the same time, the "therapeutically effective amount" is preferably small enough to avoid serious side effects, ie to provide a reasonable balance between benefit and risk. The determination of these limits, as a rule, is carried out on the basis of a rational medical assessment. In addition, the "therapeutically effective amount" of the multifunctional T-cell redirecting antibody and/or checkpoint modulator may vary depending on the particular cancer condition being treated, as well as the age and physical condition of the patient being treated, body weight, general health. gender, diet, timing of administration, rate of excretion, combination of drugs, activity of specific components (checkpoint modulator and T-cell redirecting multifunctional antibody), severity of condition, duration of treatment, nature of concomitant therapy, specific pharmaceutically acceptable carrier used, and similar factors, what the treating doctor can do based on his knowledge and experience.

Thus, doses administered to an individual, both single and multiple, must vary depending on many factors, including pharmacokinetic properties, the condition of the individual and his characteristics (gender, age, body weight, health status, constitution), the level of symptoms, simultaneously carried out treatment, frequency of treatments and desired effect.

Preferably, for the treatment of cancer, a therapeutically effective single dose of a T-cell redirecting multifunctional antibody in a combination for use in accordance with the present invention is from about 0.001 to 10 mg, preferably from about 0.01 to 5 mg, more preferably from about 0. 1 to 2 mg per injection, or about 1 nmol to 1 mmol per injection, in particular 10 nmol to 100 µmol per injection, preferably 0.1 to 10 µmol per injection. Also preferably, the therapeutically effective single dose of the T-cell redirecting multifunctional antibody in the combination for use according to the present invention is (per kilogram of body weight), in particular for the treatment of cancer, from about 0.01 to 100 μg/kg, preferably from about 0.1 to 50 µg/kg, more preferably from about 1 to 25 µg/kg, even more preferably from about 2 to 20 µg/kg, and most preferably from about 2.5 to 5 µg/kg.

More preferably, the T cell-redirecting multifunctional antibody or antigen-binding fragment thereof provided herein is administered as a single dose in the range of 0.1 to 5000 μg, preferably as a single dose in the range of 1 to 1000 μg, more preferably as single dose in the range of 2 to 750 μg, even more preferably in a single dose in the range of 3 to 700 μg, even more preferably in a single dose in the range of 5 to 600 μg, and most preferably in a single dose in the range of 10 up to 500 mcg.

In the context of the present invention, "single dose" (or "each dose") is an individual dose that is administered to one patient at one time.

Most preferably, the T-cell-redirecting multifunctional antibody or antigen-binding fragment thereof is administered as a single dose of not more than 1 mg, preferably not more than 0.9 mg, more preferably not more than 0.8 mg, even more preferably not more than 0.75 mg , even more preferably not more than 0.6 mg and most preferably not more than 0.5 mg.

Preferably, the initial dose of the antibody or antigen-binding fragment thereof is in the range of 0.5 to 200 µg, preferably 1 to 150 µg, more preferably 2 to 100 µg, most preferably 5 to 70 µg. The initial dose is a single dose for the first administration and is preferably the lowest dose in a single treatment cycle.

Preferably, the first subsequent dose level of the antibody or antigen-binding fragment thereof is greater than the initial dose level (the amount administered as the initial dose), preferably 1.1-10.0 times, more preferably 1.2-5.0 times, and even more preferably 1.5-3.0 times, and optionally the second subsequent dose level and each subsequent dose level exceeds the initial dose level (the amount administered as the initial dose) 1.1-10.0 times, preferably 1.5- 5.0 times.

The maximum dose (per treatment cycle) of the antibody or antigen-binding fragment thereof is preferably selected from the range of 25-1000 μg, preferably 50-750 μg, more preferably 75-500 μg.

Preferably, the therapeutically effective dose of the immune checkpoint modulator included in the combination for use according to the present invention is (per kilogram of body weight), in particular for the treatment of cancer, from about 0.01 to 100 mg/kg, preferably from about 0. 05 to 50 mg/kg, more preferably from about 0.1 to 25 mg/kg, even more preferably from about 0.5 to 15 mg/kg, and most preferably from about 1 to 10 mg/kg.

The T-cell redirecting multifunctional antibody in the combination for use in the present invention and the immune checkpoint modulator in the combination for use in the present invention may be administered by various routes of administration, for example, systemically or topically. Routes of systemic administration generally include, for example, the transdermal, oral, and parenteral routes, which include subcutaneous, intravenous, intramuscular, intra-arterial, intradermal, and intraperitoneal, and/or intranasal routes of administration. Routes of local administration in general include, for example, routes of local administration, as well as administration directly to the site of the lesion, such as intratumoral administration.

Preferably, the T cell redirecting multifunctional antibody in the combination for use in the present invention and the immune checkpoint modulator in the combination for use in the present invention are administered via the parenteral route. More preferably, the T-cell redirecting multifunctional antibody in the combination for use of the present invention and the immune checkpoint modulator in the combination for use in the present invention are administered via intravenous, intratumoral, intradermal, subcutaneous, intramuscular, intranasal or intranodal route. Even more preferably, the T-cell redirecting multifunctional antibody in the combination for use according to the present invention and the immune checkpoint modulator included in the combination for use according to the present invention are administered intravenously and/or subcutaneously.

Preferably, the T-cell redirecting multifunctional antibody in the combination for use of the present invention and the immune checkpoint modulator in the combination for use in the present invention are administered via the same route of administration, preferably via the parenteral route of administration. , more preferably intravenously and/or subcutaneously.

However, it is also preferred that the T cell-redirecting multifunctional antibody in the combination for use in the present invention and the immune checkpoint modulator in the combination for use in the present invention are administered via different routes of administration, preferably via different routes of administration. parenteral routes, more preferably the immune checkpoint modulator included in the combination for use according to the present invention is administered intravenously, the T-cell redirecting multifunctional antibody included in the combination intended for use according to the present invention is administered via intratumoral, intradermal, subcutaneous , intramuscular or intranodal route, preferably a T-cell redirecting multifunctional antibody, included in the combination intended for use according to the present invention, child subcutaneously.

The T cell redirecting multifunctional antibody in the combination for use in the present invention and the immune checkpoint modulator in the combination for use in the present invention may be in the same or different compositions.

Preferably, the T-cell redirecting multifunctional antibody in the combination for use in the present invention and the immune checkpoint modulator in the combination for use in the present invention are in different compositions. However, various other components, such as various excipients, can be used for the T cell redirecting multifunctional antibody and for the checkpoint modulator. In addition, the T cell redirecting multifunctional antibody and the immune checkpoint modulator can be administered via various routes of administration, and the doses (primarily specific doses) can be adjusted according to actual requirements.

However, it is also preferred that the immune checkpoint modulator and the T cell redirecting multifunctional antibody are in the same composition. Such a composition comprising both an immune checkpoint modulator and a T-cell redirecting multifunctional antibody is described in more detail below ("composition of the present invention").

Whether the composition contains only an immune checkpoint modulator (and does not contain a T-cell redirecting multifunctional antibody), only a T-cell retargeting multifunctional antibody (and does not contain a checkpoint modulator), or contains both components, such a composition can be is a pharmaceutical composition.

In particular, such a composition that contains only an immune checkpoint modulator (and does not contain a T cell retargeting multifunctional antibody), only a T cell retargeting multifunctional antibody (and does not contain a checkpoint modulator), or contains both components, preferably is ( pharmaceutical) composition, which optionally contains a pharmaceutically acceptable carrier and/or excipient, or any excipient, buffer, stabilizer, or other materials well known to those skilled in the art.

In the context of the present invention, a pharmaceutically acceptable carrier typically includes a liquid or non-liquid base of the pharmaceutical composition. The term "compatible" as used herein means that said components of a pharmaceutical composition can be mixed with an antibody or antigen-binding fragment thereof as defined above, such that, under normal conditions of use, there is no interaction that could significantly reduce the pharmaceutical efficacy of the pharmaceutical composition. compositions. Pharmaceutically acceptable carriers and excipients should naturally be of sufficiently high purity and of sufficiently low toxicity to be suitable for administration to the individual to be treated.

Preferably the pharmaceutical composition is in the form of a lyophilized powder or in the form of a liquid composition, preferably in the form of an aqueous solution. Thus, the pharmaceutical composition of the present invention can be supplied as an anhydrous lyophilized powder, or more preferably as a solution (as a solution in a vehicle). If supplied as a lyophilized powder by the manufacturer, it is usually dissolved in an appropriate solution (in an aqueous solution; such as water for injection or saline, optionally buffered, eg PBS) shortly before administration. Liquid drug vials may be designed for single use or for multiple use.

In another preferred embodiment of the invention, the checkpoint modulator, T cell redirecting multifunctional antibody and/or pharmaceutical composition (containing one or both components) are not lyophilized. Thus, it is preferred that the checkpoint modulator, T cell retargeting multifunctional antibody, and/or the pharmaceutical composition (containing one or both of the components) are not lyophilized but are supplied in solution, preferably in an aqueous solution, more preferably in an aqueous buffered solution.

Thus, it is most preferred if the pharmaceutical composition is supplied in liquid form. Thus, a pharmaceutically acceptable carrier will generally contain one or more (compatible) pharmaceutically acceptable liquid carriers. Examples of (compatible) pharmaceutically acceptable liquid carriers include pyrogen-free water, isotonic saline or buffered (aqueous) solutions, for example citrate buffered solutions; polyols such as, for example, polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid, other inorganic or organic polymers such as PLGA, preferably to provide a sustained release effect of the active ingredient present. Preferably, in a liquid pharmaceutical composition, the carrier may be pyrogen-free water; isotonic saline or buffered (aqueous) solutions, e.g. citrate buffered solutions, e.g. buffered with phosphate, citrate, etc. solutions. In particular, water or preferably a buffer, more preferably an aqueous buffer such as citrate buffer, may preferably be used for injection or instillation of the pharmaceutical composition.

Therefore, preferably the pharmaceutical composition contains a buffer, preferably an organic acid buffer (ie an organic acid buffer) such as a citrate buffer, a succinate buffer and a tartrate buffer, more preferably the pharmaceutical composition contains a citrate buffer. Thus, the organic acid buffer is preferably selected from the group consisting of citrate buffer, succinate buffer, tartrate buffer and phosphate-citrate buffer, more preferably selected from the group consisting of citrate buffer, succinate buffer and tartrate buffer. Most preferably the buffer is a citrate buffer. Typically, the buffer may (also) contain a sodium salt, preferably at least 30 mM sodium salt, a calcium salt, preferably at least 0.05 mM calcium salt, and/or optionally a potassium salt, preferably at least 1 mM potassium salt. The sodium, calcium and/or potassium salts may be in the form of their halides, for example chlorides, iodides or bromides, in the form of their hydroxides, carbonates, bicarbonates or sulfates, etc. Examples of sodium salts include (but are not limited to) eg NaCl, NaI, NaBr, Na ₂ CO ₃ , NaHCO ₃ , Na ₂ SO ₄ , examples of optional potassium salts include eg KCl, KI, KBr, K ₂ CO ₃ , KHCO ₃ , K ₂ SO ₄ , and examples of calcium salts include, for example, CaCl ₂ , CaI ₂ , CaBr ₂ , CaCO ₃ , CaSO ₄ , Ca(OH) ₂ . In addition, the buffer may contain organic anions of the above cations.

The pharmaceutical composition may also contain saline (0.9% NaCl), lactated Ringer's solution or PBS (phosphate buffered saline). For example, the pharmaceutical composition may be supplied as a stock solution of the antibody or antigen-binding fragment thereof in an appropriate buffer, such as the above organic acid buffer, preferably a citrate buffer, and only shortly before administration, this stock solution can be diluted with saline (0.9% NaCl), lactated Ringer's solution or PBS until the antibody concentration required for administration is reached.

In addition, one or more compatible solid or liquid excipients or diluents, or encapsulating compounds, which are suitable for administration to the individual to be treated, can also be used for the pharmaceutical composition. Other examples of compounds that may be contained in a pharmaceutical composition include sugars such as, for example, lactose, glucose and sucrose; starches, such as, for example, corn starch or potato starch; cellulose and its derivatives, such as, for example, sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate; powdered tragacanth; malt; gelatin; fat; glidant solids such as, for example, stearic acid, magnesium stearate; calcium sulfate; vegetable oils such as, for example, peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and theobroma oil; polyols such as, for example, polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid. In addition, preservatives, stabilizers, antioxidants and/or other additives may be included as needed. Thus, the pharmaceutical composition may also contain stabilizers such as Tween® 80 or Tween® 20. Optionally, excipients may also be contained in the pharmaceutical composition to impart sustained release capability to the antibody or antigen-binding fragment thereof provided herein.

In a preferred embodiment of the invention, the pharmaceutical composition contains no components other than (i) a T cell redirecting multifunctional antibody or antigen-binding fragment thereof as described herein or an immune checkpoint modulator as described herein; (II) the buffer presented in the present description; and optionally (III) water for injection, saline and/or PBS.

Compositions, especially pharmaceutical compositions, presented in the present description, can be adapted for delivery by repeated administration.

Other materials, as well as methods for preparing formulations and the like, which can be used in the context of compositions, especially pharmaceutical compositions, or in the context of their preparation, are presented in part 5 of Remington's "The Science and Practice of Pharmacy", 22nd edition, 2012, University of the Sciences in Philadelphia, Lippincott Williams & Wilkins.

The subject to be treated is preferably a human or non-human animal, in particular a mammal or a human. More preferably, the subject to be treated is preferably a human. Preferably, the individuals are patients diagnosed with cancer. For example, young (less than 15 years old) or elderly (more than 60 years old) patients can be treated according to the present invention. In the case of elderly patients, it is most preferable to administer the drug in the way prescribed by the attending physician, since this ensures compliance with the regimen and treatment regimen. At the same time, the administration should preferably be painless.

In general, the combination of a T-cell redirecting multifunctional antibody and an immune checkpoint inhibitor of the invention may be most beneficial to age-independent cancer patients who are preferably not undergoing immunosuppressive treatment.

Combination with glucocorticoid

Preferably, the combination for use according to the present invention, which is presented in the present invention, further comprises

(III) glucocorticoid.

In other words, the preferred combination proposed in the present invention contains

(I) immune checkpoint modulator;

(II) an (isolated) multifunctional T-cell-redirecting antibody or antigen-binding fragment thereof, comprising/comprising:

(a) specificity for a T-cell surface antigen;

(b) specificity for a cancer and/or tumor associated antigen; and

(c) a human FcγRI, FcγRIIa and/or FcγRIII binding site, wherein the antibody or antigen-binding fragment thereof binds with higher affinity to human FcγRI, FcγRIIa and/or FcγRIII than to human FcγRIIb;

(III) glucocorticoid,

and it is intended for use in the therapeutic treatment of cancer.

It should be understood that the preferred variants of the immune checkpoint modulator set forth above, the preferred variants of the T cell retargeting multifunctional antibody or antigen-binding fragment thereof set forth above, as well as the preferred combinations or uses (e.g., in relation to the production, treatment of diseases, administration, etc.) d) mentioned above are applicable respectively to the combination according to the present invention, which additionally contains a glucocorticoid.

Glucocorticoids (GC) belong to a class of corticosteroids that bind to the glucocorticoid receptor. GCs are a component of a feedback mechanism in the immune system that reduces certain manifestations of immune function, such as reducing inflammation. Even though GCs are well known to influence several pathological mechanisms in cancer cells and therefore GCs are used at high doses to treat certain types of cancer (e.g. lymphomas and leukemias, in these cases GCs have inhibitory effects on lymphocyte proliferation) , in the context of the present invention, glucocorticoids are administered not for the purpose of treating the cancer or tumor disease itself, but to reduce the side effects of a T-cell redirecting multifunctional antibody or antigen-binding fragment thereof, and/or an immune checkpoint modulator. Therefore, according to the present invention, the glucocorticoid is not administered either as (I) an individual agent for the treatment of cancer, nor (II) as a component of a chemotherapy regimen for cancer treatment, such as C-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone), CHOP ( cyclophosphamide, doxorubicin, vincristine, and prednisone), m-BACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, dexamethasone, and leucovorin), and MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin, and leucovorin). In particular, the combination of the present invention preferably does not contain (another) chemotherapeutic agent such as cyclophosphamide, vincristine, procarbazine, doxorubicin, methotrexate and bleomycin. In addition, the cancer to be treated with the combination of the present invention may not include lymphomas and/or leukemias, especially if the combination of the present invention contains a glucocorticoid. In other words, preferably with the combination of the present invention, in particular if the combination contains a glucocorticoid, a cancer other than lymphomas and/or leukemias can be treated.

Preferably, the glucocorticoid is selected from the group consisting of prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone, dexamethasone, cortisone acetate, prednilidene, deflazacort, cloprednol, fluocortolone and budenoside.

Most preferably, the glucocorticoid is dexamethasone. Dexamethasone has very strong glucocorticoid activity, while it has practically no mineralocorticoid activity. Therefore, dexamethasone is a very strong glucocorticoid. In addition, dexamethasone is a long-acting glucocorticoid (biological half-life of 36-54 hours) and is therefore most suitable for treatments requiring sustained or continuous glucocorticoid activity. Dexamethasone is most suitable, among other uses, also for patients suffering from heart failure or hypertension. In addition, the strong antiphlogistic and immunosuppressive (antiallergic) activity of dexamethasone is therapeutically important. In addition, the advantage is that after i.v. injection of dexamethasone, maximum plasma concentration is reached within a few minutes.

Preferably, the glucocorticoid is administered intravenously (i.v.) or orally (po).

The dose of glucocorticoid is generally selected depending on the type of glucocorticoid used. In the case of dexamethasone, a single dose may preferably be in the range of 1-100 mg, more preferably in the range of 2-80 mg, even more preferably in the range of 5-70 mg and most preferably in the range of 10-50 mg, such as 10, 20 or 40 mg, most preferably 10 or 20 mg. In the case of prednisolone or prednisone, the dose should generally be higher due to its lower efficacy. For example, a single dose of prednisolone or prednisone may preferably be in the range of 50-500 mg, more preferably in the range of 100-400 mg, even more preferably in the range of 150-300 mg, and most preferably in the range of 200-250 mg. Based on the well known glucocorticoid efficacy of various glucocorticoids, those skilled in the art can easily find similar dosage ranges for other glucocorticoids. For example, betamethasone has almost the same glucocorticoid activity as dexamethasone, and thus can be administered at almost the same doses, while, for example, prednilidene has almost the same glucocorticoid activity as prednisone and prednisolone, and thus it can be administered at substantially the same doses.

In general, the glucocorticoid can be administered prior to the T-cell redirecting multifunctional antibody or antigen-binding fragment thereof and/or the immune checkpoint modulator; at about the same time as the T cell-targeting multifunctional antibody, or antigen-binding fragment thereof, and/or the immune checkpoint modulator; or after a T-cell redirecting multifunctional antibody or antigen-binding fragment thereof, and/or an immune checkpoint modulator. Preferably, the glucocorticoid is administered prior to the administration of the T cell-redirecting multifunctional antibody or antigen-binding fragment thereof and/or prior to the administration of the immune checkpoint modulator. Preferably, the glucocorticoid is administered no more than 6 hours, preferably no more than 5 hours, more preferably no more than 4 hours, even more preferably no more than 3 hours, even more preferably no more than 2 hours. and most preferably no more than 1 hour before administration of the T cell-redirecting multifunctional antibody or antigen-binding fragment thereof, and/or no more than 6 hours, preferably no more than 5 hours, more preferably no more than 4 hours, still more preferably no more than 3 hours, even more preferably no more than 2 hours, and most preferably no more than 1 hour prior to administration of the immune checkpoint modulator.

For example, when used in combination with the preferred administration of a T-cell-redirecting multifunctional antibody (or an antigen-binding fragment thereof) and an immune checkpoint modulator described above, in the most preferred embodiment, the glucocorticoid is administered first, followed by the T-cell-redirecting multifunctional antibody ( or an antigen-binding fragment thereof), which can be administered, for example, no more than one or two hours after the administration of the glucocorticoid, and then the immune checkpoint modulator is administered, for example, at least 6 hours, preferably at least 12 hours, more preferably at least 24 hours, even more preferably at least 36 hours, and most preferably at least 48 hours after administration of the T-cell redirecting multifunctional antibody (or antigen-binding fragment thereof). Optionally, the glucocorticoid may also be administered prior to administration of the immune checkpoint modulator, for example, no more than one or two hours prior to administration of the immune checkpoint modulator.

According to treatment schedules requiring repeated administration of the T-cell-redirecting multifunctional antibody (or antigen-binding fragment thereof) and/or immune checkpoint modulator, the glucocorticoid is preferably administered at least until the dose of the antibody/checkpoint modulator is increased (e.g., at the first administration of each dose level). dose escalation regimen). More preferably, the glucocorticoid is administered prior to each administration of the T cell-redirecting multifunctional antibody (or antigen-binding fragment thereof) and/or immune checkpoint modulator. Therefore, the most preferred administration schedule described above is preferably applied for each antibody/checkpoint modulator administration, including, for example, the first and/or final administration of a T-cell retargeting multifunctional antibody (or antigen-binding fragment thereof) and/or an immune checkpoint modulator.

It is also preferred that the glucocorticoid is administered at about the same time as the T-cell redirecting multifunctional antibody or antigen-binding fragment thereof, and/or the glucocorticoid is administered at about the same time as the immune checkpoint modulator. In this case, the expression "about the same time" has the same meaning as indicated above (which is used in the present description).

Kit for use according to the present invention

One of the objects of the present invention is a set, in particular a set of components, which contains

(I) an immune checkpoint modulator and

(II) an (isolated) T-cell-redirecting multifunctional antibody, or antigen-binding fragment thereof, which/which contains:

(a) specificity for a T-cell surface antigen;

(b) specificity for a cancer and/or tumor associated antigen; and

(c) a human FcγRI, FcγRIIa and/or FcγRIII binding site, wherein the antibody or antigen-binding fragment thereof binds with higher affinity to human FcγRI, FcγRIIa and/or FcγRIII than to human FcγRIIb;

intended for use in the therapeutic treatment of cancer, especially in humans.

First of all, such a kit for use according to the present invention contains (I) the immune checkpoint modulator described above (in the context of the combination intended for use according to the present invention), and (II) the T-cell redirecting multifunctional antibody described above. (in the context of the combination intended for use according to the present invention). In addition, such a kit is intended for use in the therapeutic treatment of the cancer mentioned above, primarily in humans, as described above. In other words, the preferred immune checkpoint modulator options described above (in the context of the combination for use in the present invention) are also preferred for the kit of the present invention. Thus, the preferred T cell redirecting multifunctional antibody variants described above (in the context of the combination for use in the present invention) are also preferred for the kit of the present invention. In addition, the preferred uses for the therapeutic treatment of cancer as defined above (in the context of the combination to be used according to the present invention) are also preferred for the kit of the present invention.

For example, the immune checkpoint modulator and/or T cell redirecting multifunctional antibody or antigen-binding fragment thereof may be present in the same composition or in different compositions as described above.

In addition, in view of the preferred single doses of the T cell-targeting multifunctional antibody or antigen-binding fragment thereof described above, the T-cell-targeting multifunctional antibody or antigen-binding fragment thereof is preferably present in the kit of the present invention in single doses, wherein each single dose does not exceed 1 mg, preferably each single dose does not exceed 0.9 mg, more preferably each single dose does not exceed 0.8 mg, even more preferably each single dose does not exceed 0.75 mg, and most preferably each single dose does not exceed 0, 5 mg.

In addition, the kit according to the present invention preferably contains

(III) glucocorticoid.

And in this context, too, the preferred glucocorticoid variants described above (in the context of the combination to be used according to the present invention) are also preferred for the kit of the present invention. For example, the glucocorticoid is preferably selected from the group consisting of prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone, dexamethasone, cortisone acetate, prednilidene, deflazacort, cloprednol, fluocortolone and budenoside, most preferably the glucocorticoid is dexamethasone as described above.

The various components of the kit may be packaged in one or more containers. The above components may be present in lyophilized or anhydrous form, or may be dissolved in a suitable buffer. For example, the kit may comprise a (pharmaceutical) composition comprising the immune checkpoint modulator described above and a (pharmaceutical) composition comprising the T-cell retargeting multifunctional antibody described above, whereby, for example, each composition may be contained in a separate container. The kit may also contain a (pharmaceutical) composition containing both the immune checkpoint modulator and the T-cell redirecting multifunctional antibody mentioned above.

The kit may also contain additional reagents, including, for example, buffers for storing and/or reconstituting the above components, wash solutions, and the like.

Moreover, the kit of the present invention may optionally contain instructions for use. Preferably, the kit further comprises a package insert or label with instructions for treating the cancer described herein using a combination of an immune checkpoint modulator and a T-cell redirecting multifunctional antibody. Optionally, the combination (and hence the indications given in the package insert or label included in the kit) may further include (III) a glucocorticoid as described above. In particular, indications for the use of the combination of the present invention as described above may include the modes of administration described above, especially their preferred variants.

Composition for use according to the present invention

Another object of the present invention is also a composition containing

(I) an immune checkpoint modulator and

(II) an (isolated) T-cell-redirecting multifunctional antibody, or antigen-binding fragment thereof, which/which contains:

(a) specificity for a T-cell surface antigen;

(b) specificity for a cancer and/or tumor associated antigen; and

(c) a human FcγRI, FcγRIIa and/or FcγRIII binding site, wherein the antibody or antigen-binding fragment thereof binds with higher affinity to human FcγRI, FcγRIIa and/or FcγRIII than to human FcγRIIb.

In particular, such a composition of the present invention comprises (I) the immune checkpoint modulator described above (in the context of the combination for use in the present invention) and (II) the T-cell redirecting multifunctional antibody described above (in in the context of the combination to be used according to the present invention). In other words, the preferred variants of the immune checkpoint modulator described above (in the context of the combination for use according to the present invention) are also preferred for the composition of the present invention. Therefore, the preferred variants of the T cell redirecting multifunctional antibody described above (in the context of the combination for use in the present invention) are also preferred for the composition of the present invention.

In addition, a composition comprising an immune checkpoint modulator as described above and a T cell redirecting multifunctional antibody as described above, and preferred embodiments of such a composition as described above (in the context of the combination for use in the present invention). Thus, it should be understood that the same description, first of all, the same preferred embodiments of the invention described above for the composition, are applicable to the composition presented in the present description.

For example, the composition of the present invention preferably contains

(III) glucocorticoid.

And in this context also, the preferred glucocorticoid variants described above (in the context of the combination intended for use according to the present invention) are also preferred for the composition of the present invention. For example, the glucocorticoid is preferably selected from the group consisting of prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone, dexamethasone, cortisone acetate, prednilidene, deflazacort, cloprednol, fluocortolone and budenoside, most preferably the glucocorticoid is dexamethasone as described above.

Preferably the composition is for use in medicine, more preferably the composition is for use in the therapeutic treatment of cancer, especially in humans. Therefore, such a composition is intended for use in the therapeutic treatment of the cancer described above, especially in humans, as described above. In other words, the preferred uses for therapeutic treatment of cancer described above (in the context of the combination intended for use according to the present invention) are also preferred for the composition of the present invention.

Thus, the composition preferably contains a pharmaceutically acceptable carrier. Preferred examples of such a pharmaceutically acceptable carrier are described above.

It is also preferred that the (pharmaceutical) composition is used in a method of treating an individual, preferably a human, who is suffering from a cancer.

Method and Combination Therapy of the Present Invention

Another object of the present invention is a method of therapeutic treatment of cancer or the initiation, enhancement or prolongation of an antitumor response in an individual in need thereof, comprising administering to the individual

(I) an immune checkpoint modulator and

(II) an (isolated) T-cell-redirecting multifunctional antibody, or antigen-binding fragment thereof, which/which contains:

(a) specificity for a T-cell surface antigen;

(b) specificity for a cancer and/or tumor associated antigen; and

(c) a human FcγRI, FcγRIIa and/or FcγRIII binding site, wherein the antibody or antigen-binding fragment thereof binds with higher affinity to human FcγRI, FcγRIIa and/or FcγRIII than to human FcγRIIb.

In particular, such a method of the present invention comprises administering i) the immune checkpoint modulator described above (in the context of the combination for use in the present invention) and (ii) the T-cell redirecting multifunctional antibody described above (in in the context of the combination to be used according to the present invention). In addition, such a method can be used for the therapeutic treatment of the cancer described above, especially in humans, as described above. In other words, the preferred variants of the immune checkpoint modulator described above (in the context of the combination intended for use according to the present invention) are also preferred for the method proposed in the present invention. Thus, the preferred variants of the T-cell redirecting multifunctional antibody described above (in the context of the combination to be used according to the present invention) are also preferred for the method of the present invention. In addition, the preferred uses for therapeutic treatment of cancer described above (in the context of the combination intended for use according to the present invention) are also preferred for the method proposed in the present invention.

For example, the immune checkpoint modulator and/or T cell redirecting multifunctional antibody or antigen-binding fragment thereof may be in the same composition or in different compositions as described above. As another example, the method of the present invention preferably comprises administering to the subject

(III) glucocorticoid.

And in this context also, the preferred glucocorticoid variants described above (in the context of the combination intended for use according to the present invention) are also preferred for the method of the present invention. For example, the glucocorticoid is preferably selected from the group consisting of prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone, dexamethasone, cortisone acetate, prednilidene, deflazacort, cloprednol, fluocortolone and budenoside, most preferably the glucocorticoid is dexamethasone as described above.

Preferably, the subject is a human diagnosed with cancer.

In addition, the preferred modes of administration described above in the context of the combination of the present invention also apply to the method of the present invention. Thus, administration of (I) an immune checkpoint modulator and/or (II) a T-cell redirecting multifunctional antibody or antigen-binding fragment thereof, and/or optionally (III) a glucocorticoid is preferably carried out as described above.

Another aspect of the present invention is also a method for prolonging T cell activation in an individual, comprising administering to the individual a combination of:

(I) an immune checkpoint modulator and

(II) an (isolated) T-cell-redirecting multifunctional antibody, or antigen-binding fragment thereof, which/which contains:

(a) specificity for a T-cell surface antigen;

(b) specificity for a cancer and/or tumor associated antigen; and

(c) a human FcγRI, FcγRIIa and/or FcγRIII binding site, wherein the antibody or antigen-binding fragment thereof binds with higher affinity to human FcγRI, FcγRIIa and/or FcγRIII than to human FcγRIIb.

The present invention surprisingly found that the T-cell redirecting multifunctional antibody (or antigen-binding fragment thereof) provided herein induces an increase in the expression of immune checkpoint molecules such as CTLA-4 (cf. Example 2, Fig. 2) . Namely, as demonstrated in example 2 of the present description, the activation of T cells with T cell redirecting multifunctional antibodies specified in the present description increased the expression of an immune checkpoint molecule, in particular CTLA-4, which is considered the "leader" among the inhibitory immune checkpoints. described above. In general, T cell activation by the T cell retargeting multifunctional antibodies described herein is of critical importance in the treatment of cancer and neoplastic diseases. Immune checkpoint molecules such as CTLA-4 antagonize T cell activation primarily due to their overexpression. Therefore, the administration of an immune checkpoint modulator prolongs the activation of T cells by the T cell-redirecting multifunctional antibody (or antigen-binding fragment thereof) because it counteracts the upregulation of the immune checkpoint molecule, which can - in the absence of the immune checkpoint modulator - stop (or at least lessen) antibody-mediated T-cell activation.

In particular, such a method of the present invention would comprise administering (i) an immune checkpoint modulator as described above (in the context of the combination for use in the present invention), (ii) a T-cell redirecting multifunctional antibody as described above (in in the context of the combination to be used according to the present invention), and optionally (III) the glucocorticoid described above (in the context of the combination to be used according to the present invention). In other words, the preferred variants of the immune checkpoint modulator described above (in the context of the combination for use according to the present invention) are also preferred in the method of the present invention. Thus, the preferred variants of the T cell redirecting multifunctional antibody described above (in the context of the combination for use in the present invention) are also preferred in the method of the present invention. In addition, the preferred uses in the therapeutic treatment of cancer described above (in the context of the combination for use according to the present invention) are also preferred in the method of the present invention (including, for example, the preferred mode of administration).

Another object of the present invention is also a combination therapy for the therapeutic treatment of cancer, where the combination therapy includes the introduction

(I) an immune checkpoint modulator and

(II) an (isolated) T-cell-redirecting multifunctional antibody, or antigen-binding fragment thereof, which/which contains:

(a) specificity for a T-cell surface antigen;

(b) specificity for a cancer and/or tumor associated antigen; and

(c) a human FcγRI, FcγRIIa and/or FcγRIII binding site, wherein the antibody or antigen-binding fragment thereof binds with higher affinity to human FcγRI, FcγRIIa and/or FcγRIII than to human FcγRIIb.

Preferred variants of such a combination therapy include the preferred multifunctional T-cell redirecting antibody variants described above, the checkpoint modulator variants described above, and/or more generally the preferred combination variants for use in the present invention. The kit of the present invention and the (pharmaceutical) composition of the present invention can be used in the method and/or combination therapy of the present invention.

For example, the immune checkpoint modulator and/or T cell redirecting multifunctional antibody or antigen-binding fragment thereof may be in the same composition or in different compositions as described above. As another example, the combination therapy of the present invention preferably comprises administering to an individual

(III) glucocorticoid.

And in this context also, the preferred glucocorticoid variants described above (in the context of the combination intended for use according to the present invention) are also preferred for the combination therapy proposed in the present invention. For example, the glucocorticoid is preferably selected from the group consisting of prednisone, prednisolone, methylprednisolone, triamcinolone, betamethasone, dexamethasone, cortisone acetate, prednilidene, deflazacort, cloprednol, fluocortolone and budenoside, most preferably the glucocorticoid is dexamethasone as described above.

The individuals to be treated with such combination therapy are the same individuals as described in the description of the combination to be used according to the present invention. Preferably, the immune checkpoint modulator and T-cell redirecting multifunctional antibody or antigen-binding fragment thereof is administered to a human.

In addition, the preferred modes of administration described above in the context of the combination of the present invention are also applicable to the combination therapy of the present invention. Thus, the administration of (I) an immune checkpoint modulator and/or (II) a T-cell redirecting multifunctional antibody or antigen-binding fragment thereof, and/or optionally (III) a glucocorticoid is preferably carried out as described above.

Brief description of the drawings

Below is a brief description of the accompanying drawings. The drawings are provided to illustrate the present invention in more detail. But they are in no way intended to limit the essence of the invention.

The drawings show:

in fig. 1 is a schematic illustration of the putative mechanisms underlying the therapeutic combination of T cell redirecting trifunctional antibodies that contain specificity for tumor associated antigen (TAA) and specificity for T cell (e.g. CD3) and antibodies blocking control molecules. points. T cells are activated and redirected to targeted tumor cells by trifunctional antibodies. Thereafter, elimination of the tumor cells occurs via T-cell mediated cytotoxic mechanisms such as induction of apoptosis or perforin-mediated cell lysis. Upregulation of inhibitory immune checkpoint molecules such as CTLA-4 and PD-1 on cytotoxic T cells (1) has a negative effect on T cell-mediated antitumor activity. Blocking inhibitory checkpoint molecules with checkpoint blocking antibodies prevents T cell downregulation and stimulates sustained T cell activation. Consequently, there is an increase in the destruction of tumor cells (2);

in fig. 2 is the induction of CTLA-4 expression on T cells activated with trifunctional antibodies as described in Example 2. T cells isolated from mouse spleen cells were incubated (I) with Surek trifunctional antibody (1 μg/mL), immature dendritic cells (5%), irradiated B78-D14 tumor cells (2.5%; upper panel) or ( II) with BiLu antibody (1 μg/ml), immature dendritic cells (5%) and irradiated B16-EpCAM tumor cells (2.5%; bottom panel) in vitro at 37°C for 3 days. For control, an experiment was performed without the addition of a trifunctional antibody. Every day, the expression of CTLA-4 and CD69 on the cell surface was measured using FACS analysis, which makes it possible to distinguish between CD4 ⁺ - and CD8 ⁺ -T cells. The generalized data obtained in three independent experiments are presented. The "whiskers" (error bars) show the standard deviation;

in fig. 3 - described in example 3 the results of radical combination therapy obtained on the model of melanoma line B78-D14. Monotherapy using HB304 did not give a therapeutic effect (A): Mice (n=5) were infected by intraperitoneal (ip) injection of 5×10 ⁵ live tumor cells of the B78-D14 line and injected (ip) 100 μg of HB304 antibody on days 2 and 5 after tumor cell inoculation (Group B) or subjected to vehicle control (PBS) treatment (Group A). There was no difference in overall survival, all mice died (p=0.4). The addition of HB304 to Surek increased its therapeutic efficacy (B): Mice (n=10) were challenged by intraperitoneal (ip) injection of 1×10 ⁵ live B78-D14 tumor cells and injected with either 50 μg Surek alone (group A) or 50 μg Surek+100 μg HB304 (Group B) on days 2 and 5. No antibody was administered to control mice (Group C). Overall survival with Surek monotherapy increased from 60% to 90% when combined with HB304 antibodies (p=0.08; log-rank test);

in fig. 4 - described in example 4, the results of radical combination therapy obtained on the model of melanoma line B16-EpCAM. Mice (n=10) were challenged by intravenous injection (iv) with 1×10 ⁵ live B16-EpCAM tumor cells and treated with either 10 µg of BiLu trifunctional antibody on days 2 and 5 (Group B) or 100 µg of CTLA-4 blocking antibody HB304 on days 9, 12, 19, 26, 33, 40 (Group D), or mice were subjected to the appropriate combination treatment using BiLu+HB304 (Group C). Control mice were not treated with antibody (group A).

Examples

Below are specific examples illustrating various embodiments of the invention and objects of the invention. However, the scope of the present invention is not limited to the specific embodiments of the invention presented in the present description. The formulations and examples below are provided to enable those skilled in the art to more clearly understand and practice the present invention. The scope of the present invention, however, is not limited to the exemplary embodiments of the invention, which should only be considered as illustrative of particular aspects of the invention, and methods that are functionally equivalent to those presented in the description fall within the scope of the invention. In fact, various modifications of the invention in addition to those presented herein will become apparent to those skilled in the art from the foregoing description and the accompanying drawings and the examples below. All such modifications fall within the scope of the appended claims.

Example 1: Creation of trifunctional antibodies

В.,

N., Mocikat R., Hess J.,

M., Wosch S., Suckstorff I., Zehetmeier C., Lindhofer H., Ganglioside GD2-specific trifunctional surrogate antibody Surek demonstrates therapeutic activity in a mouse melanoma model. Journal of translational medicine. 10, 2012, c. 219). Полученные из квадром супернатанты очищали с помощью хроматографии на белке А, применяя последовательное элюирование с изменением рН, а затем стадию очистки с помощью катионообменной хроматографии. Антитело Surek (

N., Ruf Р., Mysliwietz J., Lindhofer H., Mocikat R., Trifunctional bispecific antibody induce tumor-specific T cells and elicit a vaccination effect. Cancer research.; 72, 2012, cc. 3958-3966; Ruf P.,

В.,

N., Mocikat R., Hess J.,M.,

Wosch S., Suckstorff I., Zehetmeier C, Lindhofer H., Ganglioside GD2-specific trifunctional surrogate antibody Surek demonstrates therapeutic activity in a mouse melanoma model. Journal of translational medicine; 10, 2012, c. 219;

N., Mysliwietz J., Deppisch N., Ruf P., Lindhofer H., Mocikat R., Potential of the trifunctional bispecific antibody surek depends on dendritic cells: rationale for a new approach of tumor immunotherapy. Molecular medicine.; 19, 2013, cc. 54-61; Deppisch N., Ruf P.,

N., Neff F., Buhmann R., Lindhofer H., Mocikat R., Efficacy and tolerability of a GD2-directed trifunctional bispecific antibody in a preclinical model: Subcutaneous administration is superior to intravenous delivery. Molecular cancer therapeutics.; 14, 2015, cc. 1877-1883) представляет собой trAт, которое создавали путем слияния родительских гибридом 17А2 (крысиное антитело в виде IgG2b к мышиному CD3) и Ме361 (мышиное антитело в виде IgG2a к GD2) (Ruf Р.,

М., Ellwart J., Wosch S., Kusterer E., Lindhofer H., Two new trifunctional antibodies for the therapy of human malignant melanoma. International journal of cancer. 2004; 108: 725-732). BiLu (Ruf P, Lindhofer H. Induction of a long-lasting antitumor 10 immunity by a trifunctional bispecific antibody. Blood.; 98, 2001, cc. 2526-2534), содержит такую же анти-СБ3-специфичность и дополнительно включает связывающее плечо мышиного IgG2a, распознающее человеческий ЕрСАМ, полученное из клона С215. Для получения НВ304 (антитело к мышиному CTLA-4) применяли клон гибридомы хомяка UC10-4F10-11 (Walunas T.L., Lenschow 15 D.J., Bakker C.Y., Linsley P.S., Freeman G.J., Green J.M., Thompson C.B., Bluestone J.A., CTLA-4 can function as a negative regulator of T cell activation. Immunity.; 1, 1994, cc. 405-413). Линии клеток культивировали в не содержащей белок среде, имеющей определенный химический состав. Все антитела создавали на фирме Trion Research GmbH. 20Trifunctional antibodies ("TrAb") were generated using the quadroma technology described in Ruf P., Lindhofer H., Induction of a long-lasting antitumor immunity by a trifunctional bispecific antibody. Blood. 98, 2001, pp. 2526-2534; Ruf P.,

AT.,

N., Mocikat R., Hess J.,

M., Wosch S., Suckstorff I., Zehetmeier C., Lindhofer H., Ganglioside GD2-specific trifunctional surrogate antibody Surek demonstrates therapeutic activity in a mouse melanoma model. Journal of translational medicine. 10, 2012, p. 219). The supernatants obtained from the quadromas were purified by protein A chromatography using a sequential pH elution followed by a purification step by cation exchange chromatography. Surek antibody (

N., Ruf R., Mysliwietz J., Lindhofer H., Mocikat R., Trifunctional bispecific antibody induce tumor-specific T cells and elicit a vaccination effect. cancer research.; 72, 2012, pp. 3958-3966; Ruf P.,

AT.,

N., Mocikat R., Hess J., M.,

Wosch S., Suckstorff I., Zehetmeier C, Lindhofer H., Ganglioside GD2-specific trifunctional surrogate antibody Surek demonstrates therapeutic activity in a mouse melanoma model. Journal of translational medicine; 10, 2012, p. 219;

N., Mysliwietz J., Deppisch N., Ruf P., Lindhofer H., Mocikat R., Potential of the trifunctional bispecific antibody surek depends on dendritic cells: rationale for a new approach of tumor immunotherapy. Molecular medicine.; 19, 2013, pp. 54-61; Deppisch N., Ruf P.,

N., Neff F., Buhmann R., Lindhofer H., Mocikat R., Efficacy and tolerability of a GD2-directed trifunctional bispecific antibody in a preclinical model: Subcutaneous administration is superior to intravenous delivery. Molecular cancer therapeutics.; 14, 2015, pp. 1877-1883) is a trAt that was created by fusion of parental hybridomas 17A2 (rat IgG2b to mouse CD3) and Me361 (mouse IgG2a to GD2) (Ruf P.,

M., Ellwart J., Wosch S., Kusterer E., Lindhofer H., Two new trifunctional antibodies for the therapy of human malignant melanoma. International journal of cancer. 2004; 108: 725-732). BiLu (Ruf P, Lindhofer H. Induction of a long-lasting antitumor 10 immunity by a trifunctional bispecific antibody. Blood.; 98, 2001, pp. 2526-2534), contains the same anti-CD3 specificity and additionally includes a binding arm mouse IgG2a recognizing human EpCAM derived from clone C215. To obtain HB304 (antibody to mouse CTLA-4), a clone of hamster hybridoma UC10-4F10-11 (Walunas TL, Lenschow 15 DJ, Bakker CY, Linsley PS, Freeman GJ, Green JM, Thompson CB, Bluestone JA, CTLA-4 can function as a negative regulator of T cell activation, Immunity.; 1, 1994, pp. 405-413). The cell lines were cultured in a protein-free medium having a defined chemical composition. All antibodies were generated by Trion Research GmbH. twenty

Example 2: CTLA-4 is upregulated following trAt-induced T cell activation

To investigate whether the immune checkpoint molecule CTLA-4 is upregulated on the surface of T cells activated with tumor-targeting trifunctional antibodies, 25 enriched T cells from mouse spleen cells were incubated with trifunctional antibodies (Surek or BiLu, cf. example 1; 1 µg/mL), their respective proliferation-incompetent (irradiated) tumor target cells B78-D14 (2.5%; in the case of Surek) or B16-EpCAM (2.5%; in the case of BiLu), and immature dendritic cells 30 (5%) in vitro at 37°C for 3 days.

The overall procedure was as follows: B78-D14 cells (Haraguchi M., Yamashiro S., Yamamoto A., Furukawa K., Takamiya K., Lloyd K.O., Shiku H., Furukawa K., Isolation of GD3 synthase gene by expression cloning of GM3 alpha-2,8-sialyltransferase cDNA using anti-GD2 monoclonal antibody. Proceedings of the national academy of sciences of the United States of America.; 91, 1994, pp. 10455-10459) and B16-EpCAM (Ruf P., Lindhofer H., Induction of a long-lasting antitumor immunity by a trifunctional bispecific antibody. Blood.; 98, 2001, pp. 2526-2534) were grown in RPMI 1640 supplemented with 8.9% and 5% respectively, fetal calf serum, 2 mm L-glutamine, 1 mm sodium pyruvate and 1-fold amount of non-essential amino acids. Then 400 μg/ml G418 and 500 μg/ml hygromycin B were added to B78-D14. Cells were washed extensively in PBS before use. The identity of the cell lines was regularly confirmed based on cell morphology and transgene expression. Immature DCs were obtained by culturing bone marrow progenitors (cells) from C57BL/6 mice in RPMI 1640 medium supplemented with 20% FCS, 2 mM L-glutamine, 100 U/ml penicillin and streptomycin, 50 μM 2-mercaptoethanol, sodium pyruvate and non-essential amino acids in the presence of 100 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF). Medium was changed every second day, cells were frozen at -140° C. on day 7. Frozen DCs were thawed, counted and used directly for T cell stimulation assay. 4×10 ⁶ T cells, which were isolated from the spleens of C57BL/6 naive mice by panning of anti-IgG+M antibody-loaded B lymphocytes (Dianova, Hamburg, Germany), were co-cultured with 2×10 ⁵ DC and 10 ⁵ irradiated (100 Gy) tumor cells in 24-well plates for 3 days. trAb was added at a concentration of 1 μg/ml. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 1x non-essential amino acids, 10 mM HEPES, and 50 μM 2-mercaptoethanol.

No trifunctional antibody was added to the controls. At time points of 0, 24, 48 and 72 hours, CTLA-4 expression on the cell surface was measured using a FACS analysis to distinguish between CD4 ⁺ and CD8 ⁺ T cells using the HB304 antibody. Specifically, T cell analysis was performed by fluorescence activated cell sorting (FACS) using a FACS Calibur flow cytometer and Cell Quest cell analysis software (BD Bioscience, Heidelberg, Germany). T-cell surface markers were stained directly with fluorescently labeled anti-CD4 mAbs (clone RM4-5; BD Biosciences) and CD8 (53-6.7, eBioscience, San Diego, USA). Cell surface expression of CTLA-4 was measured using the fluorescently labeled HB304 antibody. The percentage of positive cells was determined based on comparison with the respective isotype controls. In addition, the activation status of T cells was assessed by measuring the T cell activation marker CD69 (at time points 0, 24, 48 and 72 h) using the FACS analysis described above, while the T cell surface marker CD69 was stained directly with fluorescently labeled Mab (H1.2F3; BD Bioscience).

The results are shown in FIG. 2. Both trifunctional antibodies induced strong activation of CD4 ⁺ - as well as CD8 ⁺ -T cells, followed by upregulation of CTLA-4. Compared to CD69, which peaked as early as 24 hours, CTLA-4 expression was delayed by 1-2 days and peaked at 48-72 hours. No CTLA-4 expression was detected in unactivated T cells. Overall, these results clearly demonstrate that CTLA-4 is upregulated following T cell activation with trifunctional antibodies.

Example 3 Immediate Tumor Killing Enhanced by Combined Treatment with trAt and Anti-CTLA-4 Antibody

В.,

N., Mocikat R., Hess J.,

М., Wosch S., Suckstorff I., Zehetmeier C, Lindhofer H., Ganglioside GD2-specific trifunctional surrogate antibody Surek demonstrates therapeutic activity in a mouse melanoma model, Journal of translational medicine; 10, 2012, c. 219) для осуществления терапевтической/исцеляющей обработки. Эту модель опухоли (выведенная из B16F0 линия меланомы B78-D14) создавали с целью обеспечения экспрессии GD2. Указанный ганглиозид представляет собой перспективный антиген с точки зрения таргетирования мелкоклеточного рака легких и злокачественных заболеваний нейроэктодермального происхождения, таких как нейробластома, глиома, саркома или меланома, у человека.To assess the therapeutic/curative potential of combination therapy using trifunctional T-cell-redirecting antibodies and antibodies blocking inhibitory checkpoint molecules, a widely used B78-D14 mouse model of GD2 ⁺ melanoma was used (Ruf P.,

AT.,

N., Mocikat R., Hess J.,

M., Wosch S., Suckstorff I., Zehetmeier C, Lindhofer H., Ganglioside GD2-specific trifunctional surrogate antibody Surek demonstrates therapeutic activity in a mouse melanoma model, Journal of translational medicine; 10, 2012, p. 219) to carry out a therapeutic/healing treatment. This tumor model (derived from B16F0 melanoma line B78-D14) was created to ensure the expression of GD2. Said ganglioside is a promising antigen for targeting small cell lung cancer and malignant diseases of neuroectodermal origin such as neuroblastoma, glioma, sarcoma or melanoma in humans.

В.,

N., Mocikat R., Hess J., М.,

Wosch S., Suckstorff I., Zehetmeier C., Lindhofer H., Ganglioside GD2-specific trifunctional surrogate antibody Surek demonstrates therapeutic activity in a mouse melanoma model, Journal of translational medicine; 10, 2012, c. 219), служило в качестве суррогатного ггАт, перекрестносвязывающего GD2 с рецептором CD3 на мышиных Т-клетках.trAt Surek, which is specific for GD2 and mouse CD3 (Ruf P.,

AT.,

N., Mocikat R., Hess J., M.,

Wosch S., Suckstorff I., Zehetmeier C., Lindhofer H., Ganglioside GD2-specific trifunctional surrogate antibody Surek demonstrates therapeutic activity in a mouse melanoma model, Journal of translational medicine; 10, 2012, p. 219) served as a surrogate GAT that crosslinked GD2 to the CD3 receptor on mouse T cells.

Ipilimumab, a surrogate antibody HB304 (clone UC10-4F10-11; Walunas T.L., Lenschow D.J., Bakker C.Y., Linsley P.S., Freeman G.J., Green J.M., Thompson C.B., Bluestone J.A., CTLA-4 can function as a negative regulator of T cell activation (Immunity 1, 1994, pp. 405-413) directed against mouse CTLA-4.

C57BL/6 mice were purchased from Taconic (Rie, Denmark). Animal experiments were performed at least in duplicate, including 5 female animals in each group. To test trAt-induced tumor rejection, mice were challenged with 1×10 ⁵ live B78-D14 tumor cells and treated on days 2 and 5 with 50 μg Surek. Simultaneously with Surek, 100 μg HB304 was administered on days 2 and 5. All cells and antibodies were administered ip Control animals were included in each experiment, which were injected with tumor cells and PBS alone. Mice were sacrificed when signs of tumor growth became visible to the naked eye. All animal experiments were carried out in accordance with animal welfare regulations and with permission from the competent authorities.

The results are shown in FIG. 3. After combination therapy with Surek trifunctional antibody and anti-CTLA-4 antibody HB304, which was started 2 days after lethal challenge with B78-D14 melanoma cells, overall survival of B78-D14 infected mice increased. Initial experiments demonstrated that HB304 monotherapy in the B78-D14 tumor model was ineffective: no prolongation of survival was observed compared to the control group, which was not treated with the antibody (FIG. 3A). However, when HB304 antibodies were used in combination with trifunctional antibodies (Surek), the overall survival of mice increased from 60% (Surek monotherapy) to 90% (Surek+HB304 combination). This pronounced increase in overall survival demonstrates that the combination of trifunctional antibodies and anti-CTLA-4 blocking antibodies enhances their therapeutic efficacy (Fig. 3B).

Example 4 Immediate tumor killing is also enhanced by combined treatment with trAt and anti-CTLA-4 antibody in another tumor model

The aim of the study described in this example was to evaluate the therapeutic/curative potential of trifunctional antibodies with the addition of CTLA-4 blocking antibodies obtained in the B78-D14 non-immunogenic tumor model (Example 3) combination therapy on a more immunogenic B16-EpCAM tumor model (Ruf P., Lindhofer H., Induction of a long-lasting antitumor immunity by a trifunctional bispecific antibody, Blood. 98, 2001, cc. 2526-2534), which expresses an antigen, recognizable by clinically significant HAT catumaxomab.

trAt BiLu, which is specific for EpCAM and mouse CD3 (Ruf P., Lindhofer H., Induction of a long-lasting antitumor immunity by a trifunctional bispecific antibody. Blood. 98, 2001, pp. 2526-2534), served in as a surrogate trAt that cross-links EpCAM with the CD3 receptor on mouse T cells.

C57BL/6 mice were purchased from Taconic (Rie, Denmark). Animal experiments were performed at least in duplicate, including 10 female animals in each group. To test trAt-induced tumor rejection, mice were challenged by intravenous (iv) administration of 1×10 ⁵ live B16-EpCAM tumor cells and treated with either 10 μg of BiLu trifunctional antibody on days 2 and 5 (Group B) or 100 μg of blocking CTLA- 4 HB304 antibodies on days 9, 12, 19, 26, 33, 40 (group D), or mice were subjected to combined treatment according to the treatment schedules of both antibodies (group C (combination of treatment schedules of groups B and D)). Control mice were injected with tumor cells and PBS but not treated with antibody (Group A). Mice were sacrificed when signs of tumor growth became visible to the naked eye. All animal experiments were carried out in accordance with animal welfare regulations and with permission from the competent authorities.

The results are shown in FIG. 4. First, maintenance therapy was carried out using HB304, which included six injections on days 9, 12, 19, 26, 33 and 40 after tumor infection. This maintenance therapy significantly prolonged the overall survival of mice and was comparable in efficacy to monotherapy using the BiLu trifunctional antibody, with 20% of animals surviving for an extended period of time in both groups (Fig. 4). It is important to note that the use of a combination of both antibodies led to a further increase in the survival rate up to 40%. Moreover, the longest median survival at 110 days was achieved in the combination-treated group C, compared to 59 days in the BiLu-treated group B and 51 days in the HB304-treated group D. All mice in the control group A died, with median survival was 41 days. Thus, the combination of CTLA-4 blocking antibodies with the trifunctional BiLu antibody had a pronounced positive therapeutic effect.

Claims (58)

1. A method for initiating, enhancing or prolonging an antitumor response in an individual in need thereof, comprising administering to the individual (I) an immune checkpoint modulator which is an inhibitor of an inhibitory checkpoint molecule, (II) a T-cell-redirecting multifunctional antibody that contains: (a) an antigen-binding site specific for CD3, (b) an antigen-binding site specific for a cancer and/or tumor associated antigen, and (c) a human FcγRI, FcγRIIa and/or FcγRIII binding site, wherein the antibody binds with higher affinity to human FcγRI, FcγRIIa and/or FcγRIII than to human FcγRIIb, moreover, the first administration of the immune checkpoint modulator is carried out 12-96 hours after the first administration of the redirecting T-cell multifunctional antibody. 2. The method of claim 1, wherein the immune checkpoint modulator and the T cell redirecting multifunctional antibody are directed to different targets. 3. The method of claim 1 wherein the cancer and/or tumor associated antigen is not an immune checkpoint molecule and/or its ligand, preferably the cancer and/or tumor associated antigen is selected from the group consisting of EpCAM , HER2/neu, CEA, MAGE, proteoglycan, VEGF, EGFR, mTOR, PIK3CA, RAS, alpha (v) beta (3) - integrin, HLA, HLA-DR, ASC, carbonic anhydrase, CD1, CD2, CD4, CD6, CD7, CD8, CD11, CD13, CD14, CD19, CD20, CD21, CD22, CD23, CD24, CD30, CD33, CD37, CD38, CD40, CD41, CD47, CD52, CD138, c-erb-2, CALLA, MHCII, CD44v3, CD44v6, p97, GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1b, GT3, GQ1, NY-ESO-1, NFX2, SSX2, SSX4, Trp2, gp100, tyrosinase, MUC-1, telomerase, survivin, p53, CA125, Wue antigen, Lewis Y antigen, HSP-27, HSP-70, HSP-72, HSP-90, Pgp, MCSP, EphA2 and cell surface target GC182, GT468 or GT512. 4. The method of claim 1 wherein the antibody contains an Fc fragment. 5. The method of claim 4, wherein the antibody comprises an Fc region. 6. The method of claim 5, wherein the antibody comprises an Fc region exhibiting one of the following isotype combinations: rat-IgG2b/mouse-IgG2a, rat-IgG2b/mouse-IgG2b, rat-IgG2b/human-IgG1, or murine-[ VH-CH1, VL-CL]-human-IgG1/rat-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human IgG3 (Caucasian allotype G3m (b+g)) - [CH2-CH3 ]. 7. The method of claim 1 wherein the antibody is selected from the group consisting of catumaxomab, lymphomun, ertumaxomab, ectomab. 8. The method of claim 7 wherein the antibody is catumaxomab or ectomab. 9. The method of claim 1 wherein the immune checkpoint modulator is an inhibitor of A2AR, B7-H3, B7-H4, BTLA, CD40, CTLA-4, IDO, KIR, LAG3, PD-1, PD-L1, PD -L2, TIM-3, VISTA, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR and/or DcR3 or their ligand. 10. The method of claim 9 wherein the immune checkpoint modulator is a CTLA-4, PD-1, PD-L1 and/or PD-L2 inhibitor. 11. The method of claim 10 wherein the immune checkpoint modulator is a CTLA-4 and/or PD-1 inhibitor. 12. The method of claim 1 wherein more than one immune checkpoint modulator is used. 13. The method of claim 12, wherein at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different immune checkpoint modulators are used. 14. The method of claim 13, wherein at least (α) a CTLA-4 inhibitor and (β) a PD-1, PD-L1 and/or PD-L2 inhibitor are used. 15. The method of claim 14, wherein at least (α) a CTLA-4 inhibitor and (β) a PD-1 inhibitor are used. 16. The method of claim 1 wherein the antibody is catumaxomab and/or ectomab and the immune checkpoint modulator is a CTLA-4 inhibitor. 17. The method of claim 1, wherein the immune checkpoint modulator and the T cell redirecting multifunctional antibody are present in the same composition or in different compositions. 18. The method of claim 1, wherein the first administration of the immune checkpoint modulator is 24-84 hours, preferably 36-72 hours, and more preferably 48-60 hours after the first administration of the T-cell redirecting multifunctional antibody. 19. The method of claim 1, wherein the T-cell redirecting multifunctional antibody is administered according to an escalating dosage regimen. 20. A method for prolonging T cell activation in an individual, comprising administering to the individual a combination of (I) an immune checkpoint modulator that is an inhibitor of an inhibitory checkpoint molecule, and (II) a T-cell-redirecting multifunctional antibody that contains: (a) an antigen-binding site specific for CD3, (b) an antigen-binding site specific for a cancer and/or tumor-associated antigen; and (c) a human FcγRI, FcγRIIa and/or FcγRIII binding site, wherein the antibody binds with higher affinity to human FcγRI, FcγRIIa and/or FcγRIII than to human FcγRIIb, moreover, the first administration of the immune checkpoint modulator is carried out 12-96 hours after the first administration of the redirecting T-cell multifunctional antibody. 21. Combination therapy for the therapeutic treatment of cancer, wherein the combination therapy comprises administering (I) an immune checkpoint modulator that is an inhibitor of an inhibitory checkpoint molecule, and (II) a T-cell-redirecting multifunctional antibody that contains: (a) an antigen-binding site specific for CD3; (b) an antigen-binding site specific for a cancer and/or tumor-associated antigen; and (c) a human FcγRI, FcγRIIa and/or FcγRIII binding site, wherein the antibody binds with higher affinity to human FcγRI, FcγRIIa and/or FcγRIII than to human FcγRIIb, moreover, the first administration of the immune checkpoint modulator is carried out 12-96 hours after the first administration of the redirecting T-cell multifunctional antibody. 22. The combination therapy of claim 21, wherein the immune checkpoint modulator and the T-cell redirecting multifunctional antibody are directed to different targets. 23. Combination therapy according to claim 21, in which the antigen, associated with cancer and/or tumor, is not an immune checkpoint molecule and/or its ligand, preferably the antigen associated with cancer and/or tumor is selected from the group consisting of EpCAM, HER2/neu, CEA, MAGE, proteoglycan, VEGF , EGFR, mTOR, PIK3CA, RAS, alpha (v) beta (3)-integrin, HLA, HLA-DR, ASC, carbonic anhydrase, CD1, CD2, CD4, CD6, CD7, CD8, CD11, CD13, CD14, CD19, CD20, CD21, CD22, CD23, CD24, CD30, CD33, CD37, CD38, CD40, CD41, CD47, CD52, CD138, c-erb-2, CALLA, MHCII, CD44v3, CD44v6, p97, GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1b, GT3, GQ1, NY-ESO-1, NFX2, SSX2, SSX4, Trp2, gp100, tyrosinase, MUC-1, telomerase, survivin, p53, CA125, Wue antigen, Lewis Y antigen , HSP-27, HSP-70, HSP-72, HSP-90, Pgp, MCSP, EphA2 and cell surface targets GC182, GT468 or GT512. 24. Combination therapy according to claim 21, in which the antibody contains an Fc fragment. 25. The combination therapy of claim 24, wherein the antibody contains an Fc region. 26. Combination therapy according to claim 25, in which the antibody contains an Fc region exhibiting one of the following combinations of isotypes: rat-IgG2b/mouse-IgG2a, rat-IgG2b/mouse-IgG2b, rat-IgG2b/human-IgG1 or mouse- [VH-CH1, VL-CL]-human-IgG1/rat-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human IgG3 (Caucasian allotype G3m (b+g)) - [CH2- CH3]. 27. The combination therapy of claim 21 wherein the antibody is selected from the group consisting of catumaxomab, lymphomun, ertumaxomab, ectomab. 28. The combination therapy of claim 27 wherein the antibody is catumaxomab or ectomab. 29. Combination therapy according to claim 21, in which the immune checkpoint modulator is an inhibitor of A2AR, B7-H3, B7-H4, BTLA, CD40, CTLA-4, IDO, KIR, LAG3, PD-1, PD-L1, PD-L2, TIM-3, VISTA, CEACAM1, GARP, PS, CSF1R, CD94/NKG2A, TDO, TNFR and/or DcR3 or their ligand. 30. The combination therapy of claim 29 wherein the immune checkpoint modulator is an inhibitor of CTLA-4, PD-1, PD-L1 and/or PD-L2. 31. The combination therapy of claim 30 wherein the immune checkpoint modulator is an inhibitor of CTLA-4 and/or PD-1. 32. The combination therapy of claim 21 wherein more than one immune checkpoint modulator is used. 33. The combination therapy of claim 32 wherein at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different immune checkpoint modulators are used. 34. The combination therapy of claim 33, wherein at least (α) a CTLA-4 inhibitor and (β) an inhibitor of PD-1, PD-L1 and/or PD-L2 are used. 35. Combination therapy according to claim 34, which uses at least (α) CTLA-4 inhibitor and (β) PD-1 inhibitor. 36. The combination therapy of claim 21 wherein the antibody is catumaxomab and/or ectomab and the immune checkpoint modulator is a CTLA-4 inhibitor. 37. The combination therapy of claim 21, wherein the immune checkpoint modulator and the T cell redirecting multifunctional antibody are in the same composition or in different compositions. 38. The combination therapy of claim 21, wherein the first administration of the immune checkpoint modulator is 24-84 hours, preferably 36-72 hours, and most preferably 48-60 hours after the last administration of the T-cell redirecting multifunctional antibody. 39. The combination therapy of claim 21, wherein the T-cell redirecting multifunctional antibody is administered according to an escalating dosage regimen.

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