JP2023548522A - Composition for use in cancer treatment comprising a combination of an immune checkpoint inhibitor and an antibody-amatoxin conjugate - Google Patents

Abstract

The present application relates to a composition comprising (a) at least one immune checkpoint inhibitor and (b) at least one conjugate, wherein the conjugate comprises (i) a target binding moiety; (ii) at least one and (iii) optionally at least one linker connecting said target binding moiety and said at least one amatoxin. The application further relates to said composition for use in the treatment of patients with cancer, as well as to pharmaceutical formulations comprising said composition and additional excipients, and to methods of manufacturing and uses of said composition (FIG. 1).

Description

This application relates to the field of cancer immunotherapy. In particular, the present application relates to a composition comprising (a) at least one immune checkpoint inhibitor and (b) at least one conjugate, the conjugate comprising: (i) a target binding moiety; (ii) Compositions comprising at least one amatoxin and (iii) optionally at least one linker connecting said target binding moiety and said at least one amatoxin. The application further relates to said compositions and pharmaceutical formulations comprising said compositions and additional excipients for use in the treatment of patients with cancer, as well as methods of making and uses of said compositions.

The control of the immune system is controlled by regulatory cells of the innate and adaptive immune systems such as regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and M2-type macrophages, regulatory cytokines such as IL-10 and TGF-β, This is done through a variety of mechanisms, including immune checkpoints that control T cell activation. Such cells and molecules have the potential to be exploited as mechanisms of immune destruction during the development of cancer and chronic infections. The first is based on immunotherapy. This treatment aims to enhance the host or patient's immune response against different stages of tumor progression, and is an off-target therapy compared to chemotherapy drugs and other treatment methods that directly destroy cancer cells. (Singh et al, 2020).

Immune checkpoints are receptors on the cell membrane of T lymphocytes that regulate the immune reactivity of those cells. Immune checkpoints have anti-inflammatory (suppressive) and inflammatory-inducing (activating) properties, are expressed on the cell membrane of T cells, and interact with their respective soluble or cell-bound ligands. Tumor cells often activate anti-inflammatory immune checkpoint pathways through their respective ligands and suppress anti-tumor immune responses, thereby evading immune surveillance and allowing tumor growth to proceed. Immune checkpoint inhibitors (ICIs) are drugs, especially monoclonal antibodies, that bind to anti-inflammatory immune checkpoints or their ligands and have the ability to disrupt tumor suppression strategies and fight tumors by reactivating the immune system. can be re-established. ICI has been shown to be clinically effective in various tumor types (Dyck and Mills, 2017; Darwin et al, 2018).

Two of the most widely studied and used ICI targets for drug development are the PD-1/PD-L1-PD-L2 and CTLA4/CD80-CD86 receptor ligand signaling pathways. Both pathways result in inhibition of T cell activation, proliferation, and survival. CTLA-4 receptor is mainly expressed on T cells, whereas PD-1 is expressed on activated T cells, B cells, and certain myeloid cells. Furthermore, whereas CTLA-4 acts during the priming phase of T cell activation and limits early T cell activation, PD-1 acts during the later effector phase, primarily in peripheral tissues where T cells 1 ligand (Dyck and Mills, 2017).

Two signals are required for T cell activation: the first signal is recognition of peptide antigens presented by major histocompatibility complexes (MHC), and the second signal is recognition of peptide antigens presented by antigen presenting cells (APCs). Co-stimulation via CD28 is required after binding to expressed CD80 and CD86 (Figure 2). CTLA-4 was one of the first inhibitory receptors identified to be involved in suppressing T cell responses, and is structurally similar to CD28 and binds to CD80 and CD86 with higher affinity than CD28. Join. It has been suggested that CTLA-4 expression prevents T cell activation by reducing costimulatory (secondary) signals through CD28, leading to T cell anergy; anergic T cells have effector functions. (Dyck and Mills, 2017). CTLA-4 expression and function are intrinsically linked to T cell activation. Expression of CTLA-4 increases immediately after binding to the T cell receptor (TCR) (signal 1). Blocking CTLA-4 in vivo has been shown to inhibit CTLA-4 binding to CD80/86 and promote antitumor immunity by suppressing Treg cells and enhancing effector T cell function ( Wei et al., 2018).

The programmed cell death protein 1 (PD-1) molecule is a hydrophobic, intracellular domain with potential phosphorylation sites within an immunotyrosine-based inhibitory motif (ITIM) and an immunoreceptor inhibitory tyrosine-based switch motif (ITSM). It consists of a transmembrane region and an extracellular IgV domain (Li et al, 2016). The suppressive effect of PD-1 on activated T cells requires an activation switch motif (ITSM). Ligand binding of PD-1 leads to recruitment of the tyrosine phosphatase SHP-2 to the ITSM motif and interference with TCR downstream signaling. In addition, binding of PD-1 ligand interferes with signal transduction molecules such as PIP-3 kinase and Ras, which are important for T cell proliferation, cytokine secretion, and metabolism, and induces metabolic changes in T effector cells. Promotes induction of Treg cells. PD-1 ligand binding also leads to T cell exhaustion.

PD-1 is associated with T cells, Tregs, exhausted T cells, B cells, activated monocytes, dendritic cells (DC), natural killer (NK) cells, natural killer T (NKT) cells, epithelial cells, and tumors. It has been detected in cells (Li et al, 2016). PD-1 expression in T cells is induced by antigen stimulation. PD-1 mainly exerts a suppressive effect on peripheral T cells. Two ligands for PD-1 have been identified: PD-L1 (CD274) and PD-L2 (CD273). In cancer, tumor cells and myeloid cells are considered the main cell types that mediate T cell suppression through PD-1 binding (Li et al, 2016; Dyck and Mills, 2017).

In addition to CTLA-4 and PD-1, recent studies have shown that lymphocyte activation gene-3 (LAG-3), T-cell immunoglobulin and mucin domain-containing-3 (TIM-3), T-cell immunoglobulin and Additional immune checkpoint targets have been identified, such as ITIM domain (TIGIT), V domain Ig suppressor of T cell activation (VISTA), CD96, and BTLA (CD272) (Qin et al, 2019).

LAG-3 is normally expressed on activated CD4- and CD8-positive T cells, Tregs, a subpopulation of natural killer (NK) cells, B cells, and plasmacytoid dendritic cells (pDCs). Studies have shown that LAG-3 signaling plays a negative regulatory role in T helper 1 (Th1) cell activation, proliferation, and cytokine secretion. During tumor development and cancer progression, tumor cells use this pathway to evade immune surveillance. It has been reported that MHC-II, galectin-3, LSECtin, a-synuclein, and fibrinogen-like protein 1 (FGL1) interact with LAG-3.

TIM-3, also called hepatitis A virus cellular receptor 2 (HAVCR2), is an N-terminal Ig variable region (IgV)-like domain, a membrane-proximal mucin-like domain containing an O-linked glycosylation site (glycosylation Mucin), belongs to the Ig superfamily with a single transmembrane region and a C-terminal cytoplasmic end. It is known that TIM-3 is expressed not only in T cells but also in various types of immune cells such as B cells, Tregs, NK cells, DCs, monocytes, and macrophages. When four types of ligands bind to the IgV domain of TIM-3: galectin-9, high mobility group protein B1 (HMGB1), carcinoembryonic antigen cell adhesion molecule 1 (Ceacam-1), and phosphatidylserine (PtdSer). Galectin-9 and HMGB1 are known as soluble ligands, and Ceacam-1 and PtdSer are known as surface ligands. It has been reported that the binding of TIM-3 and galectin-9 causes intracellular calcium flux in Th1 cells and induces cell death. Galectin-9 also induced apoptosis of TIM-3 and CD8 positive T cells in colon cancer. The interaction between HMGB1 and TIM-3 primarily affects the innate immune response.

TIGIT proteins contain an extracellular IgV region, a transmembrane domain, an ITIM and a cytoplasmic tail with an immunoglobulin tail tyrosine (ITT)-like phosphorylation motif. It has been demonstrated that TIGIT expression is strongly restricted to lymphocytes, primarily T cell subsets (including Tregs and memory T cells) and NK cells. TIGIT binds two ligands with different affinities: CD155 (PVR or Necl-5) and CD112 (also known as nectin-2, PRR2 or PVRL2). TIGIT exerts its immunosuppressive effects by competing for ligand with other partners such as CD266 (DNAM-1). CD226 delivers positive costimulatory signals and TIGIT delivers inhibitory signals to T cells (Qin et al, 2019).

Currently, there are approximately 4,000 types of immune/oncology-related therapeutic drugs in the drug development pipeline worldwide, and clinical trials for PD-1/PD-L1 blockers alone have already reached 2,250 worldwide. There is. Since the approval of ipilimumab (anti-CTLA-4) for the treatment of metastatic melanoma in the United States in 2011, the number of approved indications for ICIs has increased to well over 20 (Taams and de Gruijl, 2020 ). The U.S. Food and Drug Administration (FDA) has so far approved two classes of ICIs that have shown promising therapeutic efficacy: CTLA-4, PD-1 (CD279) and its ligand PD-L1 (CD274, B7-H1), PD-L2 (CD273, B7-DC), and others are currently in clinical trials (Singh et al, 2020).

Therapeutic monoclonal antibodies that have been approved by regulatory authorities as immune checkpoint inhibitors include ipilimumab for CTLA-4, nivolumab and pembrolizumab for PD-1, and atezolizumab, avelumab, durvalumab and cemiplimab for PD-L1 (Singh et al. al, 2020).

The following tumor types have received regulatory approval for immune checkpoint inhibition therapy: melanoma, squamous and non-squamous non-small cell lung cancer, metastatic small cell lung cancer, renal cell carcinoma, Hodgkin lymphoma, and urinary tract cancer. Road epithelial carcinoma, head and neck squamous cell carcinoma, Merkel cell carcinoma, hepatocellular carcinoma, gastric cancer/gastroesophageal cancer, metastatic colorectal cancer, primary mediastinal B-cell lymphoma, recurrent or metastatic cervical cancer, metastatic cutaneous squamous cell carcinoma (Wei et al., 2018; Singh et al., 2020).

In addition to monotherapy with CTLA-4 or PD-1 inhibitory antibodies, combination therapy including, for example, ipilimumab and nivolumab has been used. Additionally, ICIs have been used in combination with cancer vaccines consisting of cancer antigens and adjuvants that activate innate immune cells such as dendritic cells. Cancer vaccines aim at generating tumor-specific T cells that kill tumor cells through secretion of IFN-γ and lytic granules. In addition, ICI is expected to directly induce tumor cell damage and improve tumor immunity in some cancer types when used in combination with radiotherapy or a histone deacetylase inhibitor.

ICI is considered one of the most important developments in cancer treatment over the past decade. However, a significant number of patients did not respond to monotherapy approaches, particularly with individual ICIs (Dyck and Mills, 2017). Furthermore, the emergence of a new spectrum of immune-related adverse events (irAEs) associated with this approach has also been noted (Martins et al, 2019), highlighting the need for high cancer cell specificity and selectivity of this class of therapeutics. is suggested.

One of the major factors for the clinical success of anticancer drugs is immunogenic cell death (ICD), i.e., immunity to dead cell antigens derived from cancer cells in particular, compared to non-immunogenic cancer cell death such as apoptosis. It has been found that the ability to induce a mode of cell death stimulates the response. ICD involves changes in cell surface composition and release of soluble mediators. These signals act on a series of receptors expressed by dendritic cells, prompting the presentation of tumor antigens to T cells (Kroemer et al, 2013). This cell death was originally observed and studied in the context of anticancer chemotherapy.

ICD has been found to be characterized by a combination of changes in the composition of the cell membrane and the composition of the microenvironment of dying cells (Figure 3). These changes are due to pre-mortem stress and subsequent cell collapse. ICD is always preceded by two types of stress, endoplasmic reticulum (ER) stress and autophagy, and is an adaptive response to stress. As a result of ER stress caused by chemotherapy or the like, ER proteins such as calreticulin (CRT), which is the largest fraction normally secreted into the ER lumen, and heat shock proteins, become exposed on the outer surface of the cell membrane. As a result of and dependent on autophagy, adenosine triphosphate (ATP) is secreted from dying cells. As a result of cell collapse associated with cell death, high mobility group box 1 (HMGB1), a non-histone chromatin protein, is released into the microenvironment.

Exposure of ER proteins including CRT to the cell surface, secretion of ATP, and release of HMGB1 are hallmarks of ICD, as opposed to non-immunogenic cell death. CRT, ATP and HMGB1 interact with CD91 (low-density lipoprotein receptor-related protein 1, LRP1), P2RX7 (purinergic receptor) and TLR4 (Toll-like receptor 4) receptors expressed by dendritic cells, respectively. It promotes engulfment of dying cells, production of cytokines such as IL-1β, and presentation of tumor antigens. Therefore, the patient's dying cancer cells act as a vaccine, recruiting DCs, increasing the number and activity of T lymphocytes, and increasing the number and activity of cytotoxic CD8+ T lymphocytes (CTLs) and regulatory T cells (Tregs) within the tumor. can stimulate a tumor-specific immune response characterized by an increase in the proportion of cancer cells and control residual cancer cells (Kroemer et al, 2013).

Different types of chemotherapeutic agents do not have the same ability to induce ICD. In a study in which 24 cytotoxic chemotherapeutic agents were administered to cancer cells in vivo, only four of them (three anthracyclines and oxaliplatin) were protective, although all drugs induced apoptosis. It has been confirmed that anti-cancer drug induces a strong immune response (Obeid et al, 2007).

Therefore, there is a need for the development of a therapeutic agent or composition that combines the immune checkpoint inhibiting effect of ICI, high cancer cell specificity and selectivity, and ICD-inducing ability.

Amatoxin is a cyclic peptide composed of eight amino acids found in the Amanita phalloides mushroom (see Figure 1). Amatoxin specifically inhibits DNA-dependent RNA polymerase II in mammalian cells, thereby also inhibiting transcription and protein biosynthesis in affected cells. Inhibition of transcription in cells causes arrest of growth and proliferation. The complex between amanitin and RNA polymerase II is not covalently bound, but is extremely tight (KD = 3 nM). Dissociation of amanitin from this enzyme is a very slow process, thus reducing the chance of recovery of affected cells. If inhibition of transcription lasts long enough, cells undergo programmed cell death (apoptosis).

Antibody-drug conjugates (ADCs) comprising amatoxin (antibody-targeted amatoxin conjugate) and tumor antigen-specific antibodies, antibody fragments or derivatives or antibody-like proteins have been described (WO2010/115629A2, WO2016/142049A1, WO2017/ 149077A1).

As shown herein, the inventors unexpectedly discovered that amatoxin-based ADCs containing tumor antigen-specific antibodies as target binding moieties induce immunogenic cell death. Surprisingly, the inventors further observed a synergistic effect of amatoxin-based ADCs and immune checkpoint inhibitors on tumor cell killing activity in vivo. This result was unexpected since neither amatoxin alone nor an amatoxin-based ADC had previously been shown to have such a beneficial effect.

Therefore, in view of the prior art, one object of the present invention was to provide a pharmaceutical composition comprising:
(a) at least one immune checkpoint inhibitor; and (b) at least one conjugate comprising:
(i) a target binding moiety;
(ii) at least one amatoxin; and (iii) optionally at least one linker connecting said target binding moiety and said at least one amatoxin.

One further object of the invention is to provide a composition for use in the treatment of cancer or chronic infections, said composition comprising: (a) at least one immune checkpoint inhibitor; and (b) at least one conjugate, the conjugate comprising:
(i) a target binding moiety;
(ii) at least one amatoxin; and (iii) optionally at least one linker connecting said target binding moiety and said at least one amatoxin.

A further object of the invention is to comprise the composition (for use) as described above, further comprising one or more pharmaceutically acceptable buffers, surfactants, diluents, carriers, excipients, fillers. It is an object of the present invention to provide pharmaceutical formulations containing agents, binders, lubricants, lubricants, disintegrants, adsorbents, and/or preservatives.

These and other objects are achieved by the methods and means described in the independent claims of the invention. The dependent claims relate to specific embodiments.

The general advantages of the invention and its features are described in detail below.

Structural formulas of various amatoxins. The bold numbers (1-8) indicate the standard numbering of the eight amino acids that form amatoxin. The standard designations of atoms in amino acids 1, 3, and 4 are also shown (Greek letters α-γ, Greek letters α-δ, and numbers 1'-7', respectively). Key concepts of immunogenic cell death. It was found that in cancer cells that responded to ICD inducers, as a result of premortem endoplasmic reticulum stress and autophagy, CRT was exposed to the outer leaflet of the cell membrane in the pre-apoptotic stage and secreted ATP during apoptosis. In addition, cells that have undergone ICD have their membranes permeated by secondary necrosis and release nuclear protein HMGB1. CRT, ATP, and HMGB1 bind to receptors CD91, P2RX7, and TLR4, respectively. This promotes recruitment of DCs to the tumor bed (stimulated by ATP), uptake of tumor antigens by DCs (stimulated by CRT), and optimal antigen presentation to T cells (stimulated by HMGB1). Taken together, these processes result in a potent IL-1β- and IL-17-dependent IFN-γ-mediated immune response involving both γδT cells and CTLs. ATP, adenosine triphosphate; CRT, calreticulin; CTL, cytotoxic CD8+ T lymphocytes; DC, dendritic cells; HMGB1, high mobility group box 1; IFN, interferon; IL, interleukin; TLR, Toll -like receptors. (From Kroemer et al. 2013). Key concepts of immune checkpoint inhibition. MHC presentation of peptide antigens to T cell receptors provides the initial signal for T cell activation (1). Through the interaction of CD80 on antigen-presenting cells and CD28 on T cells, T cells receive a second costimulatory activation signal (2). CTLA-4 competes with CD28 to bind to CD80 and sends an inhibitory signal to T cells (3). PD-1 receptors on T cells bind PD-L1 and send inhibitory signals to T cells (4). Tumor cells use this mechanism to prevent T cells from eliminating malignant cells. By using inhibitors that prevent this interaction from occurring (CTLA-4, PD-1, or PD-L1 inhibitors), T cells remain active after identifying tumor cells, allowing the host to remove tumor cells from the host. can be eliminated. CD, cluster of differentiation; CTLA-4, cytotoxic T lymphocyte-associated antigen-4; MHC, major histocompatibility complex; PD-1, programmed cell death protein-1; PD-L1, PD ligand-1; TCR, T cell receptor. (Figure from Sambi et al. 2019). Immunogenic cell death induced by antibody-targeted amatoxin conjugate (ATAC). The Her2 positive cell line BT474 and the CD79b positive cell line BJAB were each exposed to different compounds. Immunogenic cell death (ICD) markers calreticulin (CRT), adenosine triphosphate (ATP), and high mobility group box 1 protein (HMGB1) were measured. Amanitin-binding ADCs induce secretion of ICD markers in a target-dependent manner. (A, B, C) Her2 positive cell line BT474 and (D, E, F) CD79b positive cell line BJAB were treated with no compound (first bar), 100 nM maytansine (second bar), and 100 nM amanitin (third bar), respectively. (bar), 50 nM anti-Her2-amanitin conjugate (4th bar), 50 nM anti-CD79b-amanitin conjugate (5th bar), (A), (D) CRT exposed (positive) cells, (B), (E) ATP secretion, (C), (F) HMGB1 release amount were evaluated. Synergy of avelumab and anti-CD19-amatoxin conjugate in the presence of peripheral blood mononuclear cells (PBMC). Anti-PD-L1 antibody avelumab (20 mg/kg, i.v., days 0, 3, 6, 8, 10, 13) or anti-CD19-amatoxin conjugate (ATAC, 0.1 and 0.3 mg, respectively) /kg, single i.v. on day 0) or a combination of avelumab (20 mg/kg) and ATAC (0.1 and 0.3 mg/kg, respectively) in the Raji xenograft mouse model system. Results of an in vivo antitumor test. Tumor volume was assessed at various time points after tumor cell inoculation. Synergy of avelumab and anti-CD19-amatoxin conjugates in the absence or presence of peripheral blood mononuclear cells (PBMCs). Results of an antitumor test using the anti-PD-L1 antibody avelumab (20 mg/kg, i.v., days 0, 3, 6, 8, 10, 13) or anti-CD19-amatoxin conjugate (ATAC, 0.3 mg/kg, single i.v. on day 0) or a combination of avelumab (20 mg/kg) and ATAC (0.3 mg/kg) without PBMC addition (groups 01-04), respectively. and results of in vivo antitumor test in Raji xenograft mouse model system with addition of PBMC (05-08 groups). Tumor volume was assessed at various time points after tumor cell inoculation. Overview of cytotoxic efficacy of anti-HER2 ATAC in different cell lines. Anti-HER2-LALA-D265C antibodies conjugated to amatoxin containing linkers XIXa, XVIIIa, XIIIa, , XXIIb, XXIb, and the anti-HER2 antibody corresponds to the antibody in the above formula). Anti-Her2 ATAC s. c. effectiveness in. JIMT-1 xenograft model. For the Jimt-1 xenograft model, female NMRI nude mice were injected subcutaneously into the right flank with ^5x10 Jimt-1 breast cancer cells (Mol Cancer Ther. 2004 Dec; 3(12):1585-92) per mouse. Inoculated. Animals were assigned to each experimental group on day 0 when the mean tumor volume was ˜120 mm ³ . On the same day, animals received a single intravenous dose of amanitin-based anti-Her2 antibody drug conjugate (ADC) as directed. Tumor volume and body weight were measured twice weekly. In vivo synergy of pembrolizumab and anti-CD19-amatoxin conjugate treatment in the Raji xenograft mouse model system. Anti-PD-1 antibody pembrolizumab (20 mg/kg, i.v., days 0, 3, 6, 8, 10) or anti-CD19-amatoxin conjugate (anti-CD19 ATAC, 0.1 mg/kg or 0.1 mg/kg). Antitumor study results of pembrolizumab (20 mg/kg) in combination with anti-CD19 ATAC (0.1 mg/kg or 0.3 mg/kg). Tumor volume was assessed at various time points after tumor cell inoculation. Error bars represent SEM. Mouse survival plot showing the synergistic effect of anti-CTLA4 treatment and anti-CD19-amatoxin conjugate combination treatment in the Raji xenograft mouse model. Anti-CD19 ATAC was administered as a single dose of 0.1 mg/kg or 0.3 mg/kg on day 0, and ipilimumab was administered at a dose of 4 mg/kg on days 0, 3, 6, 8, and 10. The combination therapy consisted of anti-CD19 ATAC at 0.1 mg/kg or 0.3 mg/kg (single dose) on day 0, and ipilimumab at 4 mg/kg on days 0, 3, 6, 8, and 10. Administered in doses. Experimental details are described in Example 6.

Before describing the invention in detail, it is important to note that the invention is not limited to particular component parts of the described device, or as such devices and methods may vary, the process of the described method. It should be understood that it is not limited to processes. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms "a," "an," and "the" include singular and/or plural references unless the context clearly dictates otherwise. Please note. Furthermore, it is to be understood that when a parameter range is given that is bounded by numerical values, the range is deemed to include these limits. Additionally, any indication of numerical ranges herein is intended solely to serve as shorthand for individually referring to each individual value within the range. Unless otherwise indicated herein, individual values are incorporated herein as if individually set forth herein. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

The word "substantially" does not exclude "completely". For example, a composition that is "substantially free" of Y may be completely free of Y. If necessary, the word "substantially" can be omitted from the definition of the invention.

Throughout this specification and the claims that follow, unless the context requires otherwise, the term "comprise" and variations such as "comprises" and "comprising" are meant to include the recited member, integer or step. However, it will be understood that this is not meant to exclude other unlisted elements, integers or steps. The term "consist of" is a particular embodiment of the term "comprise" and excludes other non-stated members, integers or steps. In the context of the present invention, the term "comprise" encompasses the term "consist of." Thus, the term "comprising" encompasses not only "including" but also "consisting." For example, a composition "comprising" X may consist of only X, or it may contain something additional, eg, X+Y.

Furthermore, it is to be understood that the embodiments disclosed herein are not meant to be construed as individual embodiments that are unrelated to each other. Features discussed in one embodiment are also meant to be disclosed in connection with other embodiments presented herein. In one case, if a particular feature is not disclosed in one embodiment but is disclosed in another embodiment, a person skilled in the art will understand that this does not mean that said feature is disclosed in said other embodiment. They will understand that this does not necessarily mean what they mean. Those skilled in the art will appreciate that it is within the spirit of this application to disclose the features for other embodiments; however, this is done solely for clarity and to keep the specification manageable. It will be understood that it is not understood.

Furthermore, the contents of the prior art documents referred to herein are incorporated by reference. This refers in particular to prior art documents disclosing standard or conventional methods. In that case, incorporation by reference primarily has the purpose of providing a fully operable disclosure and avoiding lengthy repetition.

According to a first aspect of the invention, there is provided a pharmaceutical composition comprising:
(a) at least one immune checkpoint inhibitor; and (b) at least one conjugate, the conjugate comprising:
(i) a target binding moiety;
(ii) at least one amatoxin; and (iii) optionally at least one linker connecting said target binding moiety and said at least one amatoxin.

According to a second aspect of the invention there is provided a composition for use in the treatment of cancer, said composition comprising:
(a) at least one immune checkpoint inhibitor; and (b) at least one conjugate, the conjugate comprising:
(i) a target binding moiety;
(ii) at least one amatoxin; and (iii) optionally at least one linker connecting said target binding moiety and said at least one amatoxin.

According to a third aspect of the invention there is provided a composition for use in the treatment of chronic infections, said composition comprising:
(a) at least one immune checkpoint inhibitor; and (b) at least one conjugate, the conjugate comprising:
(i) a target binding moiety;
(ii) at least one amatoxin; and (iii) optionally at least one linker connecting said target binding moiety and said at least one amatoxin.

Immune checkpoints, also called immune checkpoint receptors, prevent excessive inflammation and autoimmune diseases by controlling the activation of T cells, while also suppressing anti-tumor immune responses.

In the context of the present invention, the term "immune checkpoint inhibitor" or simply "checkpoint inhibitor" or "ICI" refers directly or indirectly to immune checkpoint receptor proteins found on the surface of immune cells or Refers to any agent or compound that reduces the levels of or inhibits the function of a molecule (e.g., T cells) or, directly or indirectly, on the surface of a soluble compound or immune cell that inhibits said immune checkpoint. Applies to agents or compounds that reduce the level of ligand binding to a receptor protein or molecule or inhibit its function. Such suppressor cells can be, for example, cancer cells, regulatory T cells, tolerogenic antigen presenting cells, myeloid-derived suppressor cells, tumor-associated macrophages, or cancer-associated fibroblasts. The ligand is typically capable of binding immune checkpoint receptor proteins or molecules on immune cells. Non-limiting examples of immune checkpoint receptor protein-ligand pairs include PD-1, PD-L1. PD-1 is an immune checkpoint receptor protein present on T cells. PD-L1, which is overexpressed in cancer cells, binds to PD-1 and helps cancer cells evade attack by the host's immune system. Therefore, immune checkpoint inhibitors either inhibit PD-1 on T cells (i.e., act as PD-1 inhibitors) or inhibit PD-L1 on cancer cells (i.e., PD-L1 inhibition (acting as an agent) to prevent PD-1/PD-L1 interaction and thereby maintain or restore anti-tumor T cell activity or inhibit cancer cell activity.

Thus, immune checkpoint inhibitors are antagonists of immune inhibitory receptors such as PD-1, in which case they inhibit PD-1 or PD-L1 in the PD-1/PD-L1 pathway. Examples of PD-1 or PD-L1 inhibitors include, but are not limited to, humanized or human antibodies that antagonize or block human PD-1 function, such as: pembrolizumab, pidilizumab, cemiplimab, JTX-4014. , spartalizumab, sintilimab (IBI308), dostarlimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-224, PD1-1, PD1-2, PD1-3, PD1-4, PD1-5, BCD-100, AGEN-2034, toripalimab (TAB001, JS001) or AMP-514 (MEDI0680), as well as nivolumab that blocks PD-1, avelumab that blocks PD-L1, durvalumab, cosibelimab (CK-301), WBP- 3155 (CS1001), atezolizumab, a fully human antibody such as recombinant anti-PD-L1 probody CX-072.

For example, Hamid, O. et al. (2013) New England Journal of Medicine 369(2):134-44, pembrolizumab (formerly known as lambrolizumab, trade name Keytruda, also known as MK-3475) is a humanized IgG4 monoclonal antibody that binds to PD-1. Contains a C228P mutation designed to prevent Fc-mediated cytotoxicity. Pembrolizumab is disclosed, for example, in US 8,354,509 and WO2009/114335. It is approved by the FDA for the treatment of patients with unresectable or metastatic melanoma and patients with metastatic NSCLC.

Nivolumab (CAS Registry Number: 946414-94-4; BMS-936558 or MDX1106b) is a fully human IgG4 monoclonal antibody that lacks detectable antibody-dependent cytotoxicity (ADCC) and specifically blocks PD-1. . Nivolumab is disclosed, for example, in US 8,008,449 and WO2006/121168. It is FDA approved for the treatment of patients with unresectable or metastatic melanoma, metastatic NSCLC, and advanced renal cell carcinoma.

Pigilizumab (CT-011, Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD-1. Pigilizumab is disclosed, for example, in WO2009/101611.

PD1-1 to PD1-5 refer to anti-PD-1 antibodies disclosed in WO2018/220169.

Ipilumumab (CAS Registration Number: 477202-00-9, also known as 10D1, MDX010, MDX-101) is a human IgG1 antibody that binds cytotoxic T lymphocyte antigen 4 (CTLA4). CTLA-4 is an inhibitory molecule that competes with stimulatory CD28 for binding to B7 on antigen presenting cells. Both CTLA-4 and CD28 are displayed on the surface of T cells. Ipilimumab is a human IgG1 that binds to CTLA-4 and prevents inhibition of T cell-mediated immune responses against tumors. Ipilimumab is disclosed, for example, in WO 01/14424 as the antibody "10D1".

As used herein, INN is meant to also include all biosimilar antibodies of the corresponding originator antibody disclosed herein, and includes 42 USC § 262 subsection (k) of the United States and equivalents of other jurisdictions. including, but not limited to, biosimilar antibodies approved under the regulations of

Immune checkpoint receptors or molecules include, but are not limited to, PD-1, CTLA-4, LAG-3, TIM-3, TIGIT, VISTA, OX40, GITR, ICOS, CD276 (B7-H3), B7 -H4 (VTCN1), IDO, KIR, CD122, CD137, CD94/NKG2A, CD80, CD86, Galectin-3, LSECtin, CD112, Ceacam-1, Gal-9, PtdSer, HMGB1, HVEM, CD155 and BTLA (CD27 2) including.

Immune checkpoint inhibitors according to the invention may be, for example, small (organic) compounds or large molecules such as peptides or nucleic acids. For example, small molecule immune checkpoint inhibitors according to the invention include CA-170, including its precursor AUNP-12, as disclosed in WO15033301 A1; or, for example, BMS-8 (CAS No. 1675201-90-7 )and so on. In at least one embodiment of the invention, the immune checkpoint inhibitor is an antibody, or an antigen-binding fragment thereof, or an antigen-binding derivative thereof. In a preferred embodiment, the immune checkpoint inhibitor is a monoclonal antibody, or an antigen-binding fragment thereof, or an antigen-binding derivative thereof.

In the context of the present invention, the term "amatoxin" is isolated from the genus Amanita and described by Wieland, T.; and Faulstich H. (Wieland T, Faulstich H., CRC Crit Rev Biochem. 5 (1978) 185-260), as well as all chemical derivatives thereof; and all semisynthetic derivatives thereof. Analogs; In addition, all synthetic analogs constructed from building blocks that follow the master structure of the natural compound (cyclic, 8 amino acids), as well as all synthetic analogs that contain non-hydroxylated amino acids in place of hydroxylated amino acids. Synthetic or semi-synthetic analogs, as well as all synthetic or semi-synthetic analogs, in which the sulfoxide moiety is substituted with a sulfone, thioether, or atom different from sulfur, e.g., a carbon atom, as in the carbanalogues of amanitin. Including those who are present.

As used herein, a "derivative" of a compound has a chemical structure similar to that of the compound, but contains at least one chemical group not present in the compound, and is deficient in at least one chemical group present in the compound. It refers to the species that has The compound to which the derivative is compared is called the "parent" compound. Typically, a "derivative" may be produced from a parent compound in one or more chemical reaction steps.

As used herein, an "analog" of a compound is one that is structurally related to, but not identical to, the compound and exhibits at least one activity of the compound. The compound to which analogs are compared is referred to as the "parent" compound. Said activities include, but are not limited to, binding activities with other compounds; inhibitory activities, such as enzyme inhibiting activities; toxic effects; and activating activities, such as enzyme activating activities. It is noted that there is no requirement that the analog exhibit such activity to the same extent as the parent compound. The compound has at least 1% (more preferably at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 40%) of the activity of the parent compound. An analog is considered an analog in the context of this application if it exhibits the related activity to an extent of at least 50%). Accordingly, as used herein, "analogs of amatoxin" are structurally related to any one of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanulin, and amanulic acid. and at least 1% (more preferably at least 5%, more preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%), Demonstrates inhibitory activity against mammalian RNA polymerase II for at least one of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanulin, and amanuric acid. "Amatoxin analogs" suitable for use in the present invention include mammalian RNA that is less than any one of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanulin, or amanuric acid. It may even exhibit significant inhibitory activity against polymerase II. Inhibitory activity can be measured by determining the concentration at which 50% inhibition occurs (IC ₅₀ value). Inhibitory activity against mammalian RNA polymerase II can be determined indirectly by measuring inhibitory activity against cell proliferation.

A "semi-synthetic analog" is an analog obtained by chemical synthesis using a compound derived from a natural product (plant material, bacterial culture, fungal culture, cell culture, etc.) as a starting material. Typically, the "semi-synthetic analogues" of the invention are those synthesized starting from compounds isolated from mushrooms of the Amanita family. On the other hand, "synthetic analogues" refer to analogues that are synthesized from small (typically petrochemical) building blocks by so-called total synthesis. This total synthesis is usually carried out without the aid of biological processes.

According to some embodiments of the invention, the amatoxin of the conjugate is α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanulin, amanulic acid, and analogs thereof; It can be selected from the group consisting of derivatives, salts.

Functionally, an amatoxin is defined as a peptide or depsipeptide that inhibits mammalian RNA polymerase II. Preferred amatoxins are those that have a functional group (eg, a carboxy group, an amino group, a hydroxy group, a thiol or a thiol-capture group) that can be reacted with a linker molecule or target binding moiety as defined below.

In the context of the present invention, the term "amanitin" includes, in particular, an aspartic acid or asparagine residue in position 1, a proline residue, especially a hydroxyproline residue in position 2, isoleucine, hydroxyisoleucine or dihydroxyisoleucine in position 3, tryptophan or hydroxytryptophan residues, glycine residues at positions 5 and 7, isoleucine residues at position 6, and cysteine residues at position 8, especially derivatives in which cysteine is oxidized to sulfoxide or sulfone derivatives (amanitin). Refers to the two-ring structure based on the master structure of the natural compound (cyclic, 8 all synthetic analogs thereof constructed from building blocks according to amino acids), as well as all synthetic or semi-synthetic analogs containing non-hydroxylated amino acids in place of hydroxylated amino acids; , in each case, derivatives or analogs thereof include those that are functionally active in inhibiting mammalian RNA polymerase II.

As used herein, the term "target binding moiety" refers to any molecule or portion of a molecule that is capable of specifically binding to a target molecule or target epitope. Preferred target binding moieties in the context of this application are (i) antibodies or antigen-binding fragments thereof; (ii) antibody-like proteins; and (iii) nucleic acid aptamers. "Target binding moieties" suitable for use in the present invention typically have a molecular weight of 40,000 Da (40 kDa) or greater.

A "linker" in the context of this application refers to a molecule that increases the distance between two components, e.g. to alleviate steric interference between the target binding moiety and amatoxin, which would otherwise cause amatoxin to It may reduce its ability to interact with polymerase II. The linker can serve another purpose as it can promote release of amatoxin specifically in cells targeted by the target binding moiety. The linker, preferably the bond between the linker and the amatoxin on one side and the bond between the linker and the target binding moiety or antibody on the other side, is stable under physiological conditions extracellularly, e.g. in blood. On the other hand, it is preferred that it can be cleaved intracellularly, particularly within a target cell, such as a cancer cell. To provide this selective stability, the linker may preferably contain functional groups that are pH-sensitive or protease-sensitive. Also, the bond between the linker and the target binding moiety may provide selective stability. Preferably, the linker has a length of at least 1, preferably 1 to 30 atoms (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 atoms), where one side of the linker is reacted with amatoxin and the other side is It had reacted with the target binding moiety. In the context of the present invention, linkers are preferably optionally substituted C _1-30 -alkyl, C _1-30 -heteroalkyl, C _2-30 -alkenyl, C _2-30 -heteroalkenyl, C _{2 -30} -alkynyl, _C2-30 -heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroalkyl group. Linkers can include one or more structural elements such as amides, esters, ethers, thioethers, disulfides, hydrocarbon moieties, and the like. Linkers can also include combinations of two or more of these structural elements. Each one of these structural elements can occur one or more times in the linker, for example two, three, four, five, or six times. In some embodiments, the linker can include a disulfide bond. It is understood that the linker must be attached to the amatoxin and target binding moiety either in a single step or in two or more subsequent steps. For that purpose, the linker preferably carries two groups at the proximal and distal ends, (i) covalently bonding to a group, preferably an activated group, on the amatoxin or target binding moiety; or (ii) activated to form a covalent bond with a group on amatoxin. Therefore, if a linker is present, the chemical groups that are the result of such coupling reactions (e.g., esters, ethers, urethanes, peptide bonds, etc.) are preferably at the distal and proximal ends of the linker. . The presence of a "linker" is optional, ie, the toxin can be linked directly to a residue of the target binding moiety in some embodiments of the target binding site toxin conjugate.

In preferred embodiments of said compositions of the first, second and third aspects, said immune checkpoint inhibitor and/or said conjugate target binding moiety is selected from the group consisting of:
(i) antibodies, preferably monoclonal antibodies;
(ii) an antigen-binding fragment thereof, preferably a variable domain (Fv), Fab fragment or F(ab) ₂ fragment;
(iii) an antigen-binding derivative thereof, preferably a single chain Fv (scFv), and (iv) an antibody-like protein.

The antibody, or antigen-binding fragment thereof, or antigen-binding derivative thereof, can be a murine antibody, chimeric antibody, humanized antibody, or human antibody, or antigen-binding fragment thereof, or antigen-binding derivative thereof, respectively.

As used herein, the term "antibody" refers to a protein consisting of one or more polypeptide chains encoded by an immunoglobulin gene or fragment of an immunoglobulin gene or cDNA derived therefrom. The immunoglobulin genes include the light chain kappa, lambda and heavy chain alpha, delta, epsilon, gamma and mu constant region genes and any of a number of different variable region genes.

The basic immunoglobulin (antibody) structural unit is usually a tetramer consisting of two identical pairs of polypeptide chains: a light chain (L, molecular weight approximately 25 kDa) and a heavy chain (H, molecular weight approximately 50-70 kDa). be. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH or V _H ) and a heavy chain constant region (abbreviated as CH or C _H ). The heavy chain constant region consists of three domains: CH1, CH2 and CH3. Each light chain includes a light chain variable region (abbreviated as VL or _VL ) and a light chain constant region (abbreviated as CL or _CL ). The VH and VL regions can be further subdivided into regions of hypervariability, also called complementarity determining regions (CDRs) inserted between more conserved regions called framework regions (FRs). Each VH and VL region is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains form the binding domain that interacts with the antigen.

CDRs are most important for binding of antibodies or antigen-binding portions thereof. FR can be replaced with other sequences as long as the three-dimensional structure necessary for antigen binding is maintained. Structural changes in the construct most often lead to loss of sufficient binding to the antigen.

The term "antigen-binding portion" of a (monoclonal) antibody refers to one or more fragments of an antibody that, in its native form, retains the ability to specifically bind to the CD20 antigen. Examples of antigen-binding portions of antibodies include Fab fragments (monovalent fragments consisting of VL, VH, CL and CH1 domains), F(ab') ₂ fragments (two Fab fragments linked by a disulfide bridge in the hinge region), Fd fragments consisting of a VH and CH1 domain, Fv fragments consisting of a single-arm VL and VH domain of an antibody, and dAb fragments consisting of a VH domain and an isolated complementarity determining region (CDR).

The antibody or antibody fragment or antibody derivative thereof according to the invention may be a monoclonal antibody. Antibodies may be of the IgA, IgD, IgE, IgG or IgM isotype.

The term "monoclonal antibody" ("mAb") as used herein refers to a preparation of antibody molecules with a single specificity. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to antibodies exhibiting a single binding specificity, with variable and constant regions derived from or based on human germline immunoglobulin sequences, or obtained from completely synthetic sequences. Refers to antibodies that can be used. The method of preparing monoclonal antibodies is independent of binding specificity. Preferably, such antibodies are selected from the group consisting of IgG, IgD, IgE, IgA and/or IgM, or fragments or derivatives thereof, more preferably such antibodies are IgG type antibodies or fragments or derivatives thereof.

As used herein, the term "fragment" refers to fragments of such antibodies that retain target binding ability, such as CDRs (complementarity determining regions), hypervariable regions, variable domains (Fv), IgG chain (consisting of VH, CH1, hinge, CH2 and CH3 regions), IgG light chain (consisting of VL and CL regions), and/or Fab and/or F(ab) ₂ .

As used herein, the term "antigen-binding derivative" or "derivative" refers to a protein construct that differs structurally from the common antibody concept, but nevertheless has some structural relationship, e.g., with a peptide scaffold. A protein consisting of at least one CDR of the antibody from which it is derived. Examples include scFv, Fab and F(ab) ₂ , and dual, triple or more specific antibody constructs. All of these items are explained below.

Other antibody derivatives known to those skilled in the art include diabodies, camel antibodies, domain antibodies, bivalent homodimers consisting of two chains of scFvs, IgAs (two IgG structures linked by a J chain and a secreted component). shark antibodies, antibodies consisting of New World primate frameworks and non-New World primate CDRs, dimeric constructs containing CH3+VL+VH, other scaffold protein formats containing CDRs, and antibody conjugates (e.g., drug , toxins, cytokines, aptamers, nucleic acids such as desoxyribonucleic acid (DNA) or ribonucleic acid (RNA), therapeutic polypeptides, antibodies or fragments or derivatives thereof linked to radioisotopes or labels). The scaffold protein format may include, for example, antibody-like proteins such as ankyrin, affilin proteins, and the like.

As used herein, the term "antibody-like protein" refers to a protein that has been modified (eg, by mutagenesis of an Ig loop) to specifically bind a target molecule. Typically, such antibody-like proteins include at least one variable peptide loop attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of antibody-like proteins to levels comparable to those of antibodies. The length of variable peptide loops usually consists of 10-20 amino acids. The scaffold protein may be any protein that has good solubility. Preferably, the scaffold protein is a globular protein. Antibody-like proteins include, but are not limited to, affibodies, anticalins, and engineered ankyrin repeat proteins (Binz et al., 2005). Antibody-like proteins can be obtained from large libraries of variants, for example by panning from large-scale phage display libraries, and can be isolated in the same way as regular antibodies. Additionally, antibody-like binding proteins can be obtained by combinatorial mutagenesis of surface-exposed residues of globular proteins.

As used herein, the term "Fab" refers to an IgG fragment that includes an antigen binding region, one constant and one variable domain from each heavy and light chain of an antibody. consists of

As used herein, the term "F(ab) ₂ " refers to an IgG fragment consisting of two Fab fragments linked together by disulfide bonds.

As used herein, the term "scFv" refers to an immunoglobulin heavy chain in which a single chain variable region fragment is joined with a short linker, usually containing serine (S) and/or glycine (G) residues. and a fusion of the variable region of a light chain. This chimeric molecule retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker peptide.

Modified antibody formats include, for example, bi- or trispecific antibody constructs, antibody-based fusion proteins, immunoconjugates, and the like.

IgG, scFv, Fab and/or F(ab) ₂ are antibody formats familiar to those skilled in the art. Relevant enabling techniques can be obtained from the respective textbooks.

According to a preferred embodiment of the invention, said antibody, or antigen-binding fragment or derivative thereof, is a murine, chimeric, humanized or human antibody, or an antigen-binding fragment or derivative thereof, respectively. .

Mouse-derived monoclonal antibodies (mAbs) contain proteins from other species that can induce antibodies, which can cause undesirable immunological side effects. To overcome this problem, antibody humanization and maturation methods have been proposed that ideally still retain the specificity and affinity of the non-human parent antibody, but with minimal immunogenicity when applied to humans. (for a review, see Almagro and Fransson 2008). These methods are used, for example, to replace mouse MAb framework regions with the corresponding human framework regions (so-called CDR grafting). WO200907861 discloses the production of humanized forms of murine antibodies by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA technology. US6548640 by Medical Research Council describes CDR grafting techniques and US5859205 by Celltech describes the production of humanized antibodies.

As used herein, the term "humanized antibody" refers to an antibody, fragment or derivative thereof, which includes at least a portion of the constant and/or framework regions of the antibody, and optionally Portions of the CDR regions are derived from or adjusted to human immunoglobulin sequences.

The antibodies, antibody fragments or antibody derivatives thereof disclosed herein may include humanized sequences of preferred VH and VL-based antigen binding regions that maintain particularly appropriate ligand affinity. Amino acid sequence modifications to obtain the humanized sequence may occur in the CDR regions and/or the framework regions of the original antibody and/or the antibody constant region sequences.

The antibody or antibody fragment or derivative thereof can be glycosylated. The glycan can be an N-linked oligosaccharide chain of asparagine 297 of the heavy chain.

Antibodies or fragments or derivatives of the invention can be produced by transfecting host cells with expression vectors containing the coding sequences for antibodies of the invention. Expression vectors or recombinant plasmids are produced by placing the encoded antibody sequences under the control of appropriate regulatory genetic elements, including promoter and enhancer sequences, such as the CMV promoter. The heavy and light chain sequences can be expressed from individual expression vectors that are co-transfected, or from dual expression vectors. The transfection may be a transient transfection or a stable transfection. The transfected cells are then cultured to produce transfected antibody constructs. If stable transfections are performed, stable clones secreting antibodies with appropriately related heavy and light chains can then be grown for e.g. ELISA, subcloning, and future production. selection by screening with appropriate assays.

In some embodiments of compositions according to the invention, the immune checkpoint inhibitor is PD-1, CTLA-4, LAG-3, TIGIT, TIM-3, VISTA, BTLA (CD272), OX40 (CD134). , B7-H4 (VTCN1), CD96, CD278 (ICOS), CD94/NKG2A and CD160, or PD-L1, PD-L2, CD80, CD86, Binds to a ligand of an immune checkpoint receptor selected from the group consisting of galectin-3, LSECtin, CD112, Ceacam-1, Gal-9, PtdSer, HMGB1, HVEM, CD155, OX40L, and CD275 (ICOSLG).

In some embodiments, compositions according to the invention include an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is nivolumab, pembrolizumab, pidilizumab, cemiplimab, JTX-4014, spartalizumab, sintilimab (IBI308) , dostarlimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-224, PD1-1, PD1-2, PD1-3, PD1-4, PD1-5, BCD-100, AGEN-2034, toripalimab (TAB001, JS001), or AMP-514 MEDI0680 Nivolumab, Avelumab, Durvalumab, Cosibelimab (CK-301), WBP-3155 (CS1001), Atezolizumab or CX-072, or antigen-binding fragments thereof, or antigen-binding derivatives thereof ) is an antibody selected from the group consisting of: Preferably, this is the case when the antibody is avelumab, nivolumab, ipilimumab, pembrolizumab, or any of their antigen-binding fragments or antigen-binding derivatives thereof.

In some embodiments of the invention, compositions according to the invention disclosed herein contain two or more immune checkpoint inhibitors, such as 2, 3, 4, 5, The composition includes a combination of six immune checkpoint inhibitors, preferably the composition includes a combination of two immune checkpoint inhibitors. For example, the compositions according to the invention disclosed herein can be used to target different immune checkpoints such as CTLA-4 and PD-1/PD-L1, PD-1/PD-L1 and TIGIT, PD-1/PD- It is preferred to include two or more immune checkpoint inhibitors that target L1 and OX40, PD-1/PD-L1 and VISTA, CTLA4 and TIGIT, CTLA4 and OX40.

Thus, a composition according to the invention may, for example, contain one of the following combinations of immune checkpoint inhibitors:

CTLA4-PD-1/PD-L1:
Ipilimumab in combination with nivolumab, avelumab, pembrolizumab, pidilizumab, PD1-1, PD1-2, PD1-3, PD1-4, PD1-5, durvalumab, atezolizumab

PD-1/PD-L1 and TIGIT:
Tiragolumab in combination with any of nivolumab, avelumab, pembrolizumab, pidilizumab, PD1-1, PD1-2, PD1-3, PD1-4, PD1-5, durvalumab, atezolizumab; or nivolumab, avelumab, pembrolizumab, pidilizumab, PD1- 1, BMS986207 in combination with any of PD1-2, PD1-3, PD1-4, PD1-5, durvalumab, atezolizumab;

PD-1/PD-L1 and OX40:
BMS986178 in combination with any of nivolumab, avelumab, pembrolizumab, pidilizumab, PD1-1, PD1-2, PD1-3, PD1-4, PD1-5, durvalumab, atezolizumab;

PD-1/PD-L1 and VISTA
CI-8993 in combination with any of nivolumab, avelumab, pembrolizumab, pidilizumab, PD1-1, PD1-2, PD1-3, PD1-4, PD1-5, durvalumab, atezolizumab;

PD-1/PD-L1 and OX40:
MEDI0562 in combination with any of nivolumab, avelumab, pembrolizumab, pidilizumab, PD1-1, PD1-2, PD1-3, PD1-4, PD1-5, durvalumab, atezolizumab, or nivolumab, avelumab, pembrolizumab, pidilizumab, PD1-1 PF04518600 in combination with any of , PD1-2, PD1-3, PD1-4, PD1-5, durvalumab, atezolizumab;

TIM-3 and PD-1/PD-L1:
MBG453 in combination with any of nivolumab, avelumab, pembrolizumab, pidilizumab, PD1-1, PD1-2, PD1-3, PD1-4, PD1-5, durvalumab, atezolizumab;

CTLA4 and TIGIT:
Tiragolumab or ipilimumab in combination with one of BMS986207.

CTLA4 and OX40:
MEDI0562, or ipilimumab in combination with one of PF04518600.

In some embodiments, the composition according to the invention comprises a target binding moiety, the target binding moiety of the conjugate binds to a target molecule on the cell surface of a cancer cell, and the target molecule is PSMA, CD19. , CD37, CD269, sialyl Lewis ^a , HER-2/neu, and epithelial cell adhesion molecule (EpCAM). The target molecules PSMA, CD19, CD37, CD269, sialyl Lewis ^a , HER-2/neu, epithelial cell adhesion molecule (EpCAM) disclosed above refer to the following proteins or cell surface antigens: as used herein "PSMA" refers to prostate-specific membrane antigen, which is glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I) or N-acetyl-aspartylglutamate (NAAG) Also known as peptidase, it is an enzyme encoded by the human folate hydrolase (FOLH1) gene. PSMA is a membrane-bound cell surface peptidase that plays different physiological roles and is expressed in various tissues such as the prostate, kidney, small intestine, central and peripheral nervous system. It is highly expressed in malignant prostate epithelial cells and vascular endothelial cells of many solid tumor malignancies including glioblastoma, breast cancer, and bladder cancer.

"CD19" disclosed above refers to B lymphocyte antigen CD19, also known as CD19 molecule (Cluster of Differentiation 19), B lymphocyte surface antigen B4, T cell surface antigen Leu-12, CVID3, and in humans. It is a transmembrane protein encoded by the gene CD19. CD19 is a biomarker for B lymphocyte development and lymphoma diagnosis, and can be used as a target for leukemia immunotherapy.

The term "CD37" as disclosed above refers to the protein encoded by the CD37 gene in humans and which is a member of the "tetraspanin" superfamily or the transmembrane 4 superfamily. Tetraspanins are characterized by the presence of four conserved transmembrane domains and are involved in a wide range of biological phenomena, including cell proliferation, survival, adhesion, cell-to-cell communication, trafficking, cell-to-cell communication via exosomes, tumorigenesis, metastasis, and regulation of immune responses. It is thought to be a "molecular facilitator" of signal transduction. Tetraspanin members have also been reported to have functional roles in a wide range of cellular processes, including cell motility, development and differentiation, activation, proliferation, migration, and tumor invasion (Hemler 2001; Xu-Monette et al. .2016). Increased CD37 expression was observed in B-cell malignancies (Zouet al. 2018). Most B-cell malignancies, such as B-cell non-Hodgkin's lymphoma (NHL) and B-cell chronic lymphocytic leukemia (B-CLL), express CD37.

The target molecule CD269 disclosed above refers to B cell maturation antigen (BCMA or BCM), also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17), a protein encoded by the TNFRSF17 gene in humans. It is. CD269 is involved in leukemia, lymphoma, and multiple myeloma.

The sialyl Lewis ^a (also referred to as CD15) disclosed above is a tetrasaccharide consisting of sialic acid, fucose, and N-acetyllactosamine. Sialyl Lewis ^a mediates phagocytosis and chemotaxis and is found in neutrophils. It is expressed in Hodgkin's disease patients, some B-cell chronic lymphocytic leukemias, acute lymphoblastic leukemias, and most acute nonlymphocytic leukemias. Sialyl Lewis ^a is present in almost all Reed-Sternberg cells, and the presence of such cells can be immunohistochemically confirmed in a biopsy that is diagnostic of Hodgkin's lymphoma.

HER-2/neu, as disclosed above, binds to the receptor tyrosine-protein kinase erbB-2, known as CD340 (cluster of differentiation 340), proto-oncogene Neu, Erbb2 (rodent), or ERBB2 (human). In humans, it is a protein encoded by the ERBB2 gene. ERBB is an abbreviation for erythroblastic oncogene B, a gene isolated from the avian genome. HER2 (from human epidermal growth factor receptor 2), also often called HER2/neu. HER2 is a member of the human epidermal growth factor receptor (HER/EGFR/ERBB) family. Receptor dimerization results in autophosphorylation of tyrosine residues within the cytoplasmic domain of the receptor, initiating various signaling pathways that lead to cell proliferation and tumorigenesis. Amplification or overexpression of HER2 is observed in approximately 15-30% of breast cancers and approximately 10-30% of gastric/esophageal cancers, and plays a role as a prognostic biomarker.

EpCAM stands for "epithelial cell adhesion molecule" and has been disclosed to be a transmembrane glycoprotein that mediates Ca2+-independent homo-type cell-to-cell adhesion in the epithelium. EpCAM is a glycosylated type I membrane protein with a molecular weight of 30-40 kD, and is endocoded by the EPCAM gene in humans. The sequence of the EpCAM molecule predicts the existence of three potential N-linked glycosylation sites. It is composed of 314 types of amino acids. EpCAM is composed of an extracellular domain (242 amino acids) with epidermal growth factor (EGF) and thyroglobulin repeat-like domains, a single transmembrane domain (23 amino acids), and a short intracellular domain (26 amino acids). EpCAM is involved in cell signaling, migration, proliferation, and differentiation. Furthermore, EpCAM has the potential to be cancerous due to its ability to upregulate c-myc, e-fabp, cyclin A and E. EpCAM is sometimes referred to as Ep-CAM, 17-1A, HEA125, MK-1, GA733-2, EGP-2, EGP34, KSA, TROP-1, ESA, and KS1/4. EpCAM is expressed only in epithelia and epithelial-derived neoplasms, and can be used as a diagnostic marker for various cancers.

In the context of this application, the terms "target molecule" and "target epitope" refer to the antigen and epitope of the antigen, respectively, that are specifically bound by a target binding moiety. In particular, the target molecule is a tumor-associated antigen, in particular an antigen or epitope that is present in increased concentration and/or in a different conformation on the surface of one or more tumor cell types compared to the surface of non-tumor cells. be. In particular, said antigen or epitope is present on the surface of one or more tumor cell types, but not on the surface of non-tumor cells. In certain embodiments, the target binding moiety specifically binds to an epitope of an antigen selected from: PSMA, CD19, CD37, CD269, sialyl Lewis ^a , HER-2/neu, epithelial cell adhesion molecule (EpCAM). ). In other embodiments, the antigen or epitope is preferentially expressed on cells involved in autoimmune disease. In certain embodiments, the target binding moiety specifically binds to an epitope of the IL-6 receptor (IL-6R).

In some embodiments, the compositions according to the invention disclosed herein include a conjugate that includes a target binding moiety, and the target binding moiety of the conjugate is D265C, D265A, A118C, L234A, or L235A (according to the EU numbering system). "Kabat-like EU index" or "EU numbering system" refers to the numbering of human IgG1 EU antibodies (e.g., Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, herein (incorporated in its entirety by reference).

In a preferred embodiment, the antibody representing the target binding portion of the conjugate according to the invention disclosed herein comprises an Fc region with the D265C mutation, and said linker (if present) or said amatoxin is One or two of the D265C residues are linked to the antibody, preferably via a disulfide bond.

According to some embodiments of the invention, said antibody representing the target binding portion of said conjugate is heavy chain 118Cys, heavy chain 239Cys, or heavy chain 265Cys according to the EU numbering system, preferably according to the EU numbering system. Genetically engineered to contain heavy chain 265Cys, the linker, or the amatoxin, is connected to the antibody via the heavy chain 118Cys, or heavy chain 239Cys, or heavy chain 265Cys residue, respectively.

According to a preferred embodiment, the antibody representing the target binding portion of the conjugate according to the invention disclosed herein comprises an Fc region comprising the L234 mutation, the L235 mutation and the D265 mutation.

According to a more preferred embodiment, the antibody representing the target binding portion of the conjugate according to the invention disclosed herein comprises an Fc region containing the L234A, L235A and D265C mutations (according to the EU numbering system).

As used herein, the term "genetically engineered" or "genetically modified" means a nucleotide and/or amino acid substitution, insertion, deletion or reversion, or any combination thereof; Biochem. J. (1986) 237, 1-7 or J Biol Chem. 2015 Jan 30; 290(5):2577-2592, for example, by genetic technology methods such as site-directed mutagenesis, to modify the amino acid sequence of a given or naturally occurring polypeptide or protein, or a portion thereof. This relates to changing the division.

As used herein, the term "amino acid substitution" refers to a modification of the amino acid sequence of a protein, in which one or more amino acids are replaced with the same number of different amino acids so that the original protein is Proteins containing different amino acid sequences are produced. Conservative amino acid substitutions are understood to relate to substitutions of similar size, charge, polarity and/or conformation that do not significantly affect the structure and function of the protein. Groups of conservative amino acids in that sense, such as the non-polar amino acids Gly, Ala, Val, He and Leu; the aromatic amino acids Phe, Trp and Tyr; the positively charged amino acids Lys, Arg and His; and the negatively charged amino acids Asp and Glu. Exemplary amino acid substitutions are shown in Table 1 below:

According to some embodiments of the invention, said linker (if present) or said amatoxin is linked to said antibody via any of the natural Cys residues of said antibody, preferably via a disulfide bond. Ru.

^Furthermore , ^{the conjugate according} to ^the invention ^{can be used} for ^-9 M, preferably 10x10 ^-10 M, 9x10 ^-10 M, 8x10 -10 M, 7x10 ^-10 M, 6x10 ^{-10 M} , 5x10 ^-10 M, 4x10 ^-10 M, 3x10 ^-10 M, 2x10 ^-10 M , more preferably 10x10 ^-11 M, 9x10 ^-11 M, 8x10 -11 M, 7x10 ^-11 M, 6x10 ^-11 M, ^{5x10 -11 M} , 4x10 ^-11 M, 3x10 ^-11 M, 2x10 ^-11 M or 1x10 It can have a cytotoxic activity with an IC ₅₀ superior to ^-11 M.

In some embodiments of the invention, the described conjugate comprises an amatoxin comprising (i) amino acid 4 having a 6'-deoxy position and (ii) amino acid 8 having an S-deoxy position.

In some embodiments of the invention, the linker (if present) of the conjugate or the target binding moiety is (i) the γC-atom of amatoxin amino acid 1, or (ii) the δC-atom of amatoxin amino acid 3. or (iii) the 6'-C-atom of the amatoxin amino acid 4.

According to a preferred embodiment of the invention, said conjugate in the described composition comprises a linker.

The linker can be a stable (non-cleavable) linker or a cleavable linker. The cleavable linker can be selected from the group consisting of enzymatically cleavable linkers, preferably protease cleavable linkers, and chemically cleavable linkers, preferably linkers containing disulfide bridges.

A "cleavable linker" is understood as containing at least one cleavage site. As used herein, the term "cleavage site" shall refer to a moiety that is susceptible to a specific cleavage at a defined location under specific conditions. Such conditions are, for example, a particular enzyme or a reductive environment in a particular body or cell compartment.

A "non-cleavable linker" is understood to be resistant to enzymatic cleavage, e.g. by cathepsin B, and upon degradation of the antibody portion of the conjugate of the invention in a target cell (e.g. lysosomal degradation) released from the conjugate.

According to some embodiments of the invention, the cleavage site is an enzymatically cleavable moiety that includes two or more amino acids. Preferably, the enzymatically cleavable moiety is a dipeptide of valine-alanine (Val-Ala), valine-citrulline (Val-Cit), valine-lysine (Val-Lys), valine-arginine (Val-Arg). phenylalanine-lysine-glycine-proline-leucine-glycine (Phe Lys Gly Pro Leu Gly) or alanine-alanine-proline-valine (Ala Ala Pro Val) peptide, or β-glucuronide or β-galactoside. Enzymatically cleavable moieties are sometimes referred to as linkers.

In certain preferred embodiments, the enzymatically cleavable moiety of the invention comprises a dipeptide selected from: Phe-Lys, Val-Lys, Phe-Ala, Val-Ala, Phe-Cit and Val-Cit; In particular, the cleavable linker further comprises a p-aminobenzyl (PAB) spacer between the dipeptide and the amatoxin.

Accordingly, the conjugates of the invention disclosed herein include the dipeptide-PAB moieties Phe-Lys-PAB, Val-LysPAB, Phe-Ala-PAB, Val-Ala-PAB, Phe-Cit as disclosed above. - PAB, or Val-Cit-PAB. Preferably, the cleavable portion of the conjugate of the invention comprises the dipeptide-PAB moiety Val-Ala-PAB.

Here, the PAB moiety is linked to amatoxin.

According to some embodiments, the above-disclosed cleavable moiety or linker containing a thiol-reactive group of the present invention includes bromoacetamide, iodoacetamide, methylsulfonylbenzothiazole, 4,6-dichloro-1,3 , 5-triazin-2-ylamino group methylsulfonylphenyltetrazole or methylsulfonylphenyloxadiazole, pyridine-2-thiol, 5-nitropyridin-2-thiol, methanethiosulfonic acid or maleimide.

According to a preferred embodiment, the thiol-reactive group is a maleimide (maleimidyl moiety) as depicted below:

According to particularly preferred embodiments, the linker of the invention comprises a structure (i) before coupling, or (ii) after coupling to an antibody as disclosed herein.

According to some embodiments, the cleavage site may be cleavable by at least one protease selected from the group consisting of cysteine proteases, metalloproteases, serine proteases, threonine proteases, and aspartate proteases.

Cysteine proteases, also called thiol proteases, have a common catalytic mechanism using nucleophilic cysteine thiol as a catalyst, and are catalytic triads or dyads.

Metalloproteases are proteases in which a metal is involved in the catalytic mechanism. Most metalloproteases require zinc, but some use cobalt. Metal ions are coordinated to proteins via three ligands. There are various ligands that coordinate metal ions, including histidine, glutamic acid, aspartic acid, lysine, and arginine. The fourth coordination is occupied by an unstable water molecule.

Serine protease is an enzyme that cleaves peptide bonds in proteins, and the nucleophilic amino acid in the active site of the enzyme is serine. Serine proteases are broadly classified into two types based on their structure: chymotrypsin-like (trypsin-like) and subtilisin-like.

Threonine proteases are a group of proteolytic enzymes that have a threonine (Thr) residue in their active site. Although the prototype of this class of enzymes is the catalytic subunit of the proteasome, acyltransferases have convergently evolved the same active site geometry and mechanism.

Aspartic proteases are a class of protease enzymes that process peptide substrates catalyzed by activated water molecules bound to one or more aspartic acid residues. Generally, they have two highly conserved aspartates in the active site and are optimally activated at acidic pH. Almost all known aspartyl proteases are inhibited by pepstatin.

In some embodiments of the invention, the cleavable site is cleaved by at least one agent selected from the group consisting of cathepsin A or B, matrix metalloprotease (MMP), elastase, β-glucuronidase, and β-galactosidase. , preferably cathepsin B.

In some embodiments of the invention, the cleavage site is a disulfide bond, and the particular cleavage is performed by a reducing environment, eg, an intracellular environment, eg, acidic pH conditions.

According to some embodiments, the inventive conjugates disclosed herein include a non-cleavable linker. Non-cleavable linkers suitable for use according to the invention include, for example, bonds, -(C=O)-, C ₁ -C ₆ alkylene, C ₁ -C ₆ heteroalkylene, C ₂ -C ₆ alkenylene, C ₂ - One or more groups selected from C ₆ heteroalkenylene, C ₂ -C ₆ alkynylene, C ₂ -C ₆ heteroalkynylene, C ₃ -C ₆ cycloalkylene, heterocycloalkylene, arylene, heteroaryene, and combinations thereof; , each of which may be optionally substituted and/or may contain one or more heteroatoms (eg, S, N, or O) in place of one or more carbon atoms. Non-limiting examples of such groups include ( _CH2 ) _p , (C=O)( _CH2 ) _p , and polyethylene glycol (PEG; ( _CH2CH2O ) _p ) _, where p is unit (which is an integer from 1 to 6 independently selected).

In some embodiments, non-cleavable linkers of the invention include a bond, -(C=O)-, -C(O)NH- group, -OC(O)NH- group, C ₁ -C ₆ alkylene , C ₁ -C ₆ heteroalkylene, C ₂ -C ₆ alkenylene, C 2 -C ₆ heteroalkenylene, C ₂ -C ₆ alkynylene, C _{2 -C 6} heteroalkynylene, C ₃ -C ₆ cycloalkylene, heterocycloalkylene , arylene, heteroarylene, -(CH2CH2O)p- group (p is an integer from 1 to 6), each C ₁ -C ₆ alkylene, C ₁ -C ₆ heteroalkylene, C ₂ - C ₆ alkenylene, C ₂ -C ₆ heteroalkenylene, C ₂ -C ₆ alkynylene, C 2 -C _{6 heteroalkynylene, C 3 -C 6 cycloalkylene ,} heterocycloalkylene _, arylene, or heteroarylene means alkyl, alkenyl, Alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxide, alkoxy, sulfanyl , halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, nitro.

For example, each C ₁ -C ₆ alkylene, C ₁ -C ₆ heteroalkylene _, C 2 -C ₆ alkenylene, C ₂ -C ₆ heteroalkenylene, C ₂ -C ₆ of the non-cleavable linkers disclosed herein Alkynylene, C ₂ -C ₆ heteroalkynylene, C ₃ -C ₆ cycloalkylene, heterocycloalkylene, arylene, or heteroarylene is optionally interrupted with one or more heteroatoms selected from O, S and N. Alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl , hydroxylated, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxylated, mercapto, nitro. Good too. The definitions of chemical groups used in this book are from the "Compendium of Chemical Terminology"("GoldBook") version 2.3.3, goldbook. published by the International Union of Pure and Applied Chemistry (IUPAC). iupac. org, ISBN: 0-9678550-9-8), the contents of which are incorporated herein by reference.

According to a preferred embodiment, the non-cleavable linker of the conjugate of the invention comprises -(CH ₂ ) _n - units, where n is an integer from 2 to 12, such as from 2 to 6, for example n is 1, 2, 3, 4, 5, or 6.

In a preferred embodiment, the non-cleavable linker of the conjugate of the invention comprises -(CH ₂ ) _n -, where n is 6, and the linker is represented by the formula:

In some embodiments, the inventive non-cleavable linkers disclosed herein further include a thiol-reactive group. The thio-reactive groups of the non-cleavable linker as disclosed above include bromoacetamide, iodoacetamide, methylsulfonylbenzothiazole, thiol-reactive groups, 4,6-dichloro-1,3,5-triazine- The 2-ylamino group is selected from methylsulfonylphenyltetrazole or methylsulfonylphenyloxadiazole, pyridine-2-thiol, 5-nitropyridine-2-thiol, methanethiosulfonic acid or maleimide.

According to a preferred embodiment, the thiol-reactive group is a maleimide (maleimidyl moiety) as depicted below. For example, the maleimide-containing non-cleavable linker can have, for example, the following structure, with the wavy line at the end of the linker indicating the point of attachment to amatoxin:

After conjugation of an antibody or target binding moiety, e.g., to a reactive sulfhydryl, as disclosed herein, the meleimidyl moiety of, e.g., a cleavable or non-cleavable linker, as disclosed herein, has the following structure: including:
Here, the wavy line represents the site of attachment of a cleavable or non-cleavable linker as disclosed herein.

According to a preferred embodiment, a conjugate of the invention comprising a cleavable or non-cleavable linker further comprising a thiol-reactive group may be attached to a naturally occurring sulfhydryl moiety in the antibody of the conjugate, or Said cleavable or non-cleavable linker of the conjugate of the invention containing a thiol-reactive group can be described, for example, by Nat Biotechnol. 2008 Aug; 26(8):925-32, may be attached to a sulfhydryl moiety introduced into the antibody by genetic modification. Preferably, a cleavable or non-cleavable linker as disclosed herein containing a thio-reactive group is attached to a sulfhydryl moiety introduced into the Fc region of the antibody of the conjugate of the invention by genetic modification. . A preferred position at which a sulfhydryl moiety may be introduced within the Fc region of said antibody is D265, or A118 (according to EU numbering), more preferably D265.

According to a preferred embodiment of the invention, the conjugate in said composition comprises as linker-amatoxin moiety any of the following compounds of formulas (I) to (XI), respectively:

According to one embodiment, the conjugate of the invention is preferably synthesized by reacting the thiol groups of the antibody with a compound according to any one of formulas XIIa to XXIIa containing a maleimide moiety as a reactive cap. be done. As used herein, the term "reactive cap" refers to a chemical moiety that reacts with an antibody, such as a thiol group, to covalently attach a compound of formulas (XIIa) to (XXIIa) to the antibody.

According to some embodiments, compounds according to formulas (XIIa) to (XXIIa) of the invention are useful for the production of antibody-drug conjugates (ADCs), more specifically antibody-targeted amatoxin conjugates or Used in manufacturing. The inventive ADCs disclosed herein may also be referred to as "ATACs." For example, compounds (XIIa)-(XXIIa) are reacted with a target binding moiety, such as an antibody, such as a human IgG1 antibody, under appropriate conditions to form an ATAC. The compounds of the invention according to formulas (XIIa) to (XXIIa) may be used, for example, as antibodies directed against, for example, PSMA, CD19, CD37, CD269, sialyl Lewis ^a , HER-2/neu, epithelial cell adhesion molecule (EpCAM). can be reacted with target binding moieties such as to obtain the corresponding ATACs. Coupling of compounds (XIIa) to (XXIIa) of the invention to target binding moieties or antibodies is performed, for example, as disclosed in WO2018/115466 A1, and coupling of compounds (XIIb) to (XXIIb) of the invention You may obtain an ATAC containing any of the following.

According to a preferred embodiment of the invention, said conjugate of the invention is a compound according to any one of formulas (XIIb) to (XXIIb) disclosed herein:
Here, n is preferably 1 to 10, preferably 1, 2, 3 to 4, preferably 1, 2 to 5, preferably 4 to 7, preferably 8 to 10.

Furthermore, according to a preferred embodiment of the invention, said conjugate is a compound according to any one of formulas XIII to XXII:

Here, the amatoxin linker moiety is bonded to the ε-amino group of the naturally occurring lysine residue of the antibody, and n is preferably 1 to 8, preferably 1, 2, 3 to 4, preferably is from 2 to 5, preferably from 5 to 7.

Furthermore, according to a preferred embodiment of the invention, said conjugate is a compound according to any one of formulas XXIII, XXIV, XIIIb, XXIIb, XXb and XVIb:
Here, the amatoxin linker moiety is bonded to a thiol group of a cysteine residue of the antibody, and n is preferably 1 to 10, or, for example, n is 2, 4 to 6, more preferably n is 1, 2, 4 or 8.

According to a preferred embodiment according to the invention, said composition comprises an immune checkpoint inhibitor avelumab, nivolumab, pembrolizumab, durvalumab or ipilimumab and an antibody directed against HER2 (αHER2-LALA-D265C) and a formula XXIb, XXIIb, XIIb, XIIIb, XVIIIb, or XIXb, wherein the antibody represented by the respective amatoxin structure disclosed above is αHER2-LALA-D265C. According to a preferred embodiment of the invention, the composition therefore comprises one of the following:

Accordingly, a composition of the invention comprises an antibody as disclosed above coupled to one amatoxin as disclosed above and at least one immune checkpoint inhibitor as disclosed above.

According to a particularly preferred embodiment according to the invention, said composition comprises an immune checkpoint inhibitor avelumab, nivolumab, pembrolizumab, durvalumab or ipilimumab and an antibody directed against CD19 (chiBCE19-D265C) and an antibody of the formula XXIII, XXII , XXb, XXIIb, XXIV, XIIIb, or XVIb, wherein the antibody shown in each amatoxin structure disclosed above is CD19 (chiBCE19-D265C). According to a particularly preferred embodiment of the invention, the composition therefore comprises any of the following:

Accordingly, a composition of the invention comprises an antibody as disclosed above coupled to one amatoxin as disclosed above and at least one immune checkpoint inhibitor as disclosed above.

According to a particularly preferred embodiment, said composition according to the invention comprises an immune checkpoint inhibitor avelumab, nivolumab, pembrolizumab, durvalumab or ipilimumab and an antibody directed against PSMA and a formula XXIII, XXII, XXb, XXIIb, Conjugates comprising an amatoxin according to XXIV, XIIIb, or XVIb, wherein the antibody shown in each amatoxin structure disclosed above is an anti-PSMA antibody. According to a particularly preferred embodiment of the invention, the composition therefore comprises any of the following:

Accordingly, a composition of the invention comprises an antibody as disclosed above coupled to one amatoxin as disclosed above and at least one immune checkpoint inhibitor as disclosed above.

According to a particularly preferred embodiment, the anti-PSMA antibody of the conjugate of the invention disclosed above is an anti-PSMA antibody as disclosed in WO2020/025564. For example, the antibodies of the conjugates of the invention disclosed herein include 3-F11-var1, 3-F11-var2, 3-F11-var3, 3-F11-var4, 3-F11-var5, 3- F11-var6, 3-F11-var7, 3-F11-var8, 3-F11-var9, 3-F11-var10, 3-F11-var11, 3-F11-var12, 3-F11-var13, 3-F11- var14, 3-F11-var115, or 3-F11-var16, preferably the antibody of the conjugate of the invention is 3-F11-var1, 3-F11 as disclosed in WO 2020/025564. -var13 or 3-F11-var16.

According to a particularly preferred embodiment, the anti-PSMA antibody as disclosed above for use in the composition of the invention is selected in its Fc region from L234A, L235A and D265C (numbering according to EU nomenclature). at least one mutation, preferably L234A, L235A and D265C.

According to a particularly preferred embodiment, said composition according to the invention comprises an immune checkpoint inhibitor avelumab, nivolumab, pembrolizumab, durvalumab or ipilimumab and an antibody directed against CD37 and a formula XXIII, XXII, XXb, XXIIb, Conjugates comprising an amatoxin according to XXIV, XIIIb, or XVIb, wherein the antibody shown in each amatoxin structure disclosed above is an anti-CD37 antibody. According to a particularly preferred embodiment of the invention, the composition therefore comprises any of the following:

Accordingly, a composition of the invention comprises an antibody as disclosed above coupled to one amatoxin as disclosed above and at least one immune checkpoint inhibitor as disclosed above.
According to a particularly preferred embodiment, the anti-CD37 antibody disclosed above has L234A, L235A and D265C (numbering according to EU nomenclature) in its Fc region for use in the composition of the invention, preferably Contains at least one mutation selected from L234A, L235A and D265C.

According to a particularly preferred embodiment, said composition according to the invention comprises an immune checkpoint inhibitor avelumab, nivolumab, pembrolizumab, durvalumab or ipilimumab and an antibody directed against CD269 and a formula XXIII, XXII, XXb, XXIIb, Conjugates comprising an amatoxin according to XXIV, XIIIb, or XVIb, wherein the antibody represented by the respective amatoxin structure disclosed above is an anti-CD269 antibody. According to a particularly preferred embodiment of the invention, the composition therefore comprises any of the following:

Accordingly, a composition of the invention comprises an antibody as disclosed above coupled to one amatoxin as disclosed above and at least one immune checkpoint inhibitor as disclosed above.

According to a particularly preferred embodiment, the anti-CD269 antibody of the conjugate of the invention disclosed above is the humanized antibody J22.9-ISY as disclosed in WO2018/115466, and the conjugate is according to formula (I). The amatoxin linker moiety or conjugate is represented by formula (XIIb) disclosed herein.

According to some embodiments, the anti-CD269 antibody as disclosed above for use in the compositions of the invention is selected from, e.g., L234A, L235A and D265C (numbering according to EU nomenclature). It may contain at least one mutation in its Fc region, preferably the Fc region of said antibody contains mutations L234A, L235A and D265C.

According to a particularly preferred embodiment, said composition according to the invention comprises an immune checkpoint inhibitor avelumab, nivolumab, pembrolizumab, durvalumab or ipilimumab and an antibody directed against sialyl Lewis ^a and a formula XXIII, XXII, XXb, Conjugates comprising an amatoxin according to XXIIb, XXIV, XIIIb, or XVIb, wherein the antibody shown in each amatoxin structure disclosed above is an anti-sialyl Lewis ^a antibody. According to a particularly preferred embodiment of the invention, the composition therefore comprises any of the following:

Accordingly, a composition of the invention comprises an antibody as disclosed above coupled to one amatoxin as disclosed above and at least one immune checkpoint inhibitor as disclosed above.

According to some embodiments, the anti-sialyl Lewis ^a antibodies as disclosed above for use in the compositions of the invention include L234A, L235A and D265C (numbering according to EU nomenclature) in their Fc region. ), preferably L234A, L235A and D265C.

According to a particularly preferred embodiment, said composition according to the invention comprises an immune checkpoint inhibitor avelumab, nivolumab, pembrolizumab, durvalumab or ipilimumab and an antibody directed against EpCAM and a formula XXIII, XXII, XXb, XXIIb, Conjugates comprising an amatoxin according to XXIV, XIIIb, or XVIb, wherein the antibody shown in the respective amatoxin structure disclosed above is an anti-EpCAM antibody. According to a particularly preferred embodiment of the invention, the composition therefore comprises any of the following:

Accordingly, a composition of the invention comprises an antibody as disclosed above coupled to one amatoxin as disclosed above and at least one immune checkpoint inhibitor as disclosed above.

According to some embodiments, the anti-EpCAM antibodies as disclosed above for use in the compositions of the invention have in their Fc region L234A, L235A and D265C (numbering according to EU nomenclature). At least one selected mutation, preferably L234A, L235A and D265C.

According to particularly preferred embodiments, the conjugate for use in the compositions of the invention disclosed above comprises formulas XIIb, XIVb, XVIIIb, XXb, or XVIIb, or comprises formulas XIIb, XIVb, XXb or XVIIb. Therefore, according to a particularly preferred embodiment, said composition according to the invention comprises at least one of the immune checkpoint inhibitors avelumab, nivolumab, pembrolizumab, durvalumab or ipilimumab with the formula XXIII, XXII, XXb, XXIIb, XXIV, XIIIb , or the conjugates described in XVIb. According to a particularly preferred embodiment of the invention, the composition therefore comprises at least one of the following:

Accordingly, the composition of the invention at least comprises a conjugate as disclosed above and at least one immune checkpoint inhibitor as disclosed above.

According to one embodiment, the invention provides compositions of the formula (XIIb), (XIIIb), (XIVb), (XVb), (XVIb), (XVIIb), ( XVIIIb), (XIXb), (XXb), (XXIb), (XXIIb).

According to one embodiment, the invention provides an immune checkpoint selected from the group of avelumab, nivolumab, pembrolizumab, ipilimumab or durvalumab for use in the manufacture of the composition according to the invention disclosed herein. This relates to inhibitors.

According to a fourth aspect of the invention, there is provided a pharmaceutical formulation comprising a composition (for use) as described, further comprising one or more pharmaceutically acceptable buffers, surfactants, agents, diluents, carriers, excipients, fillers, binders, lubricants, lubricants, disintegrants, adsorbents, and/or preservatives.

In aqueous form, the pharmaceutical formulation may be ready for administration, whereas in lyophilized form, the formulation may be prepared with preservatives, such as, but not limited to, benzyl alcohol, vitamins, etc., prior to administration. Contains antioxidants such as A, vitamin E, vitamin C, retinyl palmitate, and selenium, the amino acids cysteine and methionine, citric acid and sodium citrate, and synthetic preservatives such as the parabens methylparaben and propylparaben. It can be transferred to liquid form by the addition of water for injection, which may or may not be necessary.

The pharmaceutical formulation may further contain one or more stabilizers which may be, for example, amino acids, sugar polyols, disaccharides and/or polysaccharides. The pharmaceutical formulation may further include one or more surfactants, one or more tonicity agents, and/or one or more metal ion chelators, and/or one or more preservatives.

The pharmaceutical formulations described herein are suitable for at least intravenous, intramuscular or subcutaneous administration. Alternatively, the conjugate according to the invention may be provided in a depot allowing for sustained release of biologically active agent over a period of time.

In yet another aspect of the invention there is provided a primary package, such as a pre-filled syringe or pen, vial or infusion bag, containing the formulation according to the previous aspect of the invention.

Prefilled syringes or pens may contain the formulation either in lyophilized form (which must then be dissolved, eg, in water for injection, before administration) or in aqueous form. The syringe or pen is often a disposable item for single use only, and may have a volume of 0.1 to 20 ml. However, the syringe or pen may also be a multi-use or multi-dose syringe or pen.

The vial may also contain the formulation in lyophilized or aqueous form and may serve as a single or multiple use device. As a multiple use device, the vial can have a larger capacity. The infusion bag usually contains the formulation in aqueous form and may have a capacity of 20 to 5000 ml.

In some embodiments of the invention, compositions for use in the treatment of cancer, or pharmaceutical formulations as described, include melanoma, squamous and non-squamous non-small cell lung cancer, metastatic small cell lung cancer, Renal cell carcinoma, Hodgkin lymphoma, B lymphocyte-related malignant tumor, urothelial carcinoma, head and neck squamous cell carcinoma, Merkel cell carcinoma, hepatocellular carcinoma, gastric cancer/gastroesophageal cancer, metastatic colorectal cancer, multiple myeloma, primary tumor cancer selected from the group consisting of breast cancer, including sexual mediastinal B-cell lymphoma, recurrent or metastatic cervical cancer, metastatic cutaneous squamous cell carcinoma, prostate cancer, and triple negative breast cancer (TNBC).

In a preferred embodiment, the invention is for use in the treatment of B lymphocyte-related malignancies, particularly non-Hodgkin's lymphoma, follicular lymphoma, diffuse large B-cell non-Hodgkin's lymphoma, and chronic lymphocytic leukemia. of the compositions or pharmaceutical formulations disclosed herein.

In some embodiments, the invention relates to immune checkpoint inhibitors as disclosed herein for use in compositions of the invention or in pharmaceutical compositions of the invention. Accordingly, the present invention provides avelumab, nivolumab, pembrolizumab, ipilimumab, PD1-1, PD1-2, PD1-3, PD1 for use in the compositions or pharmaceutical formulations according to the invention disclosed herein. -4, PD1-5, pidilizumab, cemiplimab, JTX-4014, spartalizumab, sintilimab (IBI308), dostarlimab, tripalimab, durvalumab, or atezolizumab.

In some embodiments, the invention relates to the use of avelumab, nivolumab, pembrolizumab, ipilimumab or durvalumab in a composition or pharmaceutical formulation according to the invention as disclosed herein.

The invention further provides a method for the treatment of cancer in a human subject in need thereof, the method comprising: (a) at least one immune checkpoint inhibitor; and (b) at least one conjugate. administering to the subject a composition comprising: (i) a target binding moiety; (ii) at least one amatoxin; and (iii) optionally a combination of the target binding moiety and the at least one amatoxin. at least one linker connecting two amatoxins.

According to one embodiment, the above-disclosed method of treating cancer in a human subject in need thereof includes melanoma, squamous and non-squamous non-small cell lung cancer, metastatic small cell lung cancer, renal cell carcinoma. , Hodgkin lymphoma, urothelial carcinoma, head and neck squamous cell carcinoma, Merkel cell carcinoma, hepatocellular carcinoma, gastric and gastroesophageal cancer, metastatic colorectal cancer, primary mediastinal B-cell lymphoma, recurrent or metastatic cervical cancer, and including administering the compositions or pharmaceutical formulations disclosed above for the treatment of metastatic cutaneous squamous cell carcinoma.

In some embodiments, the methods of treating cancer disclosed above include administering an immune checkpoint inhibitor and a conjugate of the compositions disclosed herein sequentially or simultaneously. include. When administering an immune checkpoint inhibitor and a conjugate of the invention sequentially, it is also possible to administer the checkpoint inhibitor first and then the conjugate, e.g. The gate can also be administered first followed by the immune checkpoint inhibitor. In preferred embodiments, the immune checkpoint inhibitors and conjugates of the invention are administered intravenously (iv), either sequentially or simultaneously, as disclosed above. As used herein, the term "co-administration" refers to administration of an immune checkpoint inhibitor and a conjugate of the invention on the same day, e.g., 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours each other. Refers to administration within an hour, 20 hours, or 23 hours apart.

The present invention further provides a composition for the treatment of cancer comprising (a) at least one immune checkpoint inhibitor and (b) at least one conjugate, the conjugate comprising: (i) a target binding agent; (ii) at least one amatoxin; and (iii) optionally at least one linker connecting said target binding moiety and said at least one amatoxin.

Although the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; It is not limited to the embodiment. Other variations to the disclosed embodiments may be understood and practiced by those skilled in the art from a study of the drawings, the disclosure, and the appended claims in practicing the claimed invention. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" excludes a plural. It's not something you do. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims shall not be construed as limiting the scope.

All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown from 5'->3'.

Example 1 Synthesis of linker toxin

5.15 mg (3.92 μmol) of thioether XIIa (described in WO2018/115466) was dissolved in 1000 μl of acetic acid. At ambient temperature, 2078.99 μl of a solution of m-CPBA (metachloroperbenzoic acid, 69.5%, Aldrich) in acetic acid was added (stock solution: 5.15 mg m-CPBA dissolved in 5000 μl of acetic acid). . After stirring for 3 hours and 15 minutes at ambient temperature, the solution was added dropwise to 15 ml of ice-cold MTBE. The entire mixture was stored at −18° C. for an additional hour and the precipitate was isolated by centrifugation. The solid residue was washed with 15 ml of ice-cold MTBE and dried. The crude product was purified by HPLC to obtain 3.12 mg (59%) of pure product. The product was lyophilized from acetonitrile/water (1/1) to a white amorphous solid.

HPLC purification Prep. HPLC was performed using Phenomenex Luna-C18 (2), 10 μm column (250 x 21.2 mm) (flow 30 ml/min, λ = 290 nm), water, 0.05% TFA (solvent A), pure acetonitrile (solvent B). was performed using the following gradient:

0.0 minutes → 95% A; 14.8 minutes → 50% A; 15.0 minutes → 0% A; 18.0 minutes → 0% A; 18.5 minutes → 95% A, 22.0 minutes → 95%A.

10.09 mg (7.50 μmol) sulfoxide XVa (described in WO2016/142049) was dissolved in 2000 μl acetic acid. At ambient temperature, 1784.28 μl of a solution of m-CPBA (metachloroperbenzoic acid, 69.5%, Aldrich) in acetic acid was added (stock solution): 5.18 mg m-CPBA dissolved in 5000 μl of acetic acid. ). After stirring at ambient temperature for 3.5 hours, the solution was added dropwise to 30 ml of ice-cold MTBE. The entire mixture was stored at −18° C. for an additional 45 minutes and the precipitate was isolated by centrifugation. The solid residue was washed with 30 ml of ice-cold MTBE and dried. The crude product was purified by HPLC to obtain 5.82 mg (57%) of pure product. The product was lyophilized from acetonitrile/water (1/1) to a white amorphous solid.

HPLC purification Prep. For HPLC, use Phenomenex Luna-C18 (2), 10 μm column (250 x 21.2 mm) (flow 30 ml/min, λ = 305 nm), water, 0.05% TFA (solvent A), pure acetonitrile (solvent B). The following gradient was performed using:

0.0 minutes → 95% A; 14.8 minutes → 50% A; 15.0 minutes → 0% A; 18.0 minutes → 0% A; 18.5 minutes → 95% A, 22.0 minutes → 95%A.

20.25 mg (22.01 μmol) of β-amanitin was dissolved in 4000 μl of acetic acid. A solution of m-CPBA (metachloroperbenzoic acid, 69.5%, Aldrich) in 5634.38 μl acetic acid was added at ambient temperature (stock solution): 10.67 mg m-CPBA dissolved in 10 ml acetic acid. ). After stirring for 3.5 hours at ambient temperature, the solution was added dropwise to 60 ml of ice-cold MTBE. The entire mixture was stored at −18° C. for an additional 30 minutes and the precipitate was isolated by centrifugation. The solid residue was washed with 60 ml of ice-cold MTBE and dried. The crude product was purified by HPLC to obtain 10.46 mg (51%) of pure product. The product was lyophilized from acetonitrile/water (1/1) to a white amorphous solid.

HPLC purification Prep.
For HPLC, use Phenomenex Luna-C18 (2), 10 μm column (250 x 21.2 mm) (flow 30 ml/min, λ = 305 nm), water, 0.05% TFA (solvent A), pure acetonitrile (solvent B). The following gradient was performed using:

0.0 minutes → 97% A; 1.0 minutes → 90% A; 23.0 minutes → 86% A; 23.1 minutes → 0% A; 27.0 minutes → 0% A, 27.5 minutes → 97%A; 30.0 minutes → 97%A.

7.99 mg (8.54 μmol) of the product from step 1 was dissolved in 665 μl dry DMF. At ambient temperature, 127.98 μl of a solution of TBTU in dry DMF was added. A solution of 128.04 μl DIPEA in dry DMF was then added (stock solution: 52.25 μl DIPEA in 1500 μl dry DMF). The resulting solution was stirred for 1 minute under an argon atmosphere. Then, 128.05 μl of a solution of BMP-Val-Ala-PAB-NH ₂ (WO2018115466) was added. The resulting reaction mixture was stirred at room temperature for 2 hours. Solvent was removed under reduced pressure. The crude product was purified by HPLC to obtain 7.34 mg (63%) of pure product.

HPLC purification Prep. HPLC was performed using Phenomenex Luna-C18 (2), 10 μm column (250 x 21.2 mm) (flow 15 ml/min, λ = 305 nm) 95% water, 5% methanol, 0.05% TFA (solvent A) and 95% methanol, The following gradient was performed using 5% water, 0.05% TFA (solvent B):

0.0 minutes → 100% A, 5.0 minutes → 50% A, 10.0 minutes → 30% A, 20.0 minutes → 20% A, 45.0 minutes → 0% A, 50.0 minutes → 100A, 55.0 minutes → 100A.

5.07 mg (3.77 μmol) sulfoxide XVIIIa (described in WO2016/142049) was dissolved in 1000 μl acetic acid. At ambient temperature, 1043.57 μl of a solution of m-CPBA (metachloroperbenzoic acid, 69.5%, Aldrich) in acetic acid was added (stock solution: 4.93 mg m-CPBA dissolved in 5000 μl of acetic acid). . After stirring for 3 hours and 15 minutes at ambient temperature, the solution was added dropwise to 15 ml of ice-cold MTBE. The entire mixture was stored at −18° C. for an additional hour and the precipitate was isolated by centrifugation. The solid residue was washed with 15 ml of ice-cold MTBE and dried. The crude product was purified by HPLC to obtain 2.44 mg (48%) of pure product. The product was lyophilized from acetonitrile/water (1/1) to a white amorphous solid.

HPLC purification Prep. For HPLC, use Phenomenex Luna-C18 (2), 10 μm column (250 x 21.2 mm) (flow 30 ml/min, λ = 305 nm), water, 0.05% TFA (solvent A), pure acetonitrile (solvent B). The following gradient was performed using:

0.0 minutes → 95% A; 14.8 minutes → 50% A; 15.0 minutes → 0% A; 18.0 minutes → 0% A; 18.5 minutes → 95% A, 22.0 minutes → 95%A.

5.13 mg (4.67 μmol) sulfoxide XXIa (WO2016142049) was dissolved in 1000 μl acetic acid. At ambient temperature, 1291.27 μl of a solution of m-CPBA (metachloroperbenzoic acid, 69.5%, Aldrich) in acetic acid was added (stock solution: 4.94 mg m-CPBA dissolved in 5000 μl of acetic acid). . After stirring for 3.5 hours at ambient temperature, the solution was added dropwise to 15 ml of ice-cold MTBE. The entire mixture was kept on ice for an additional 30 minutes and the precipitate was isolated by centrifugation. The solid residue was washed with 15 ml of ice-cold MTBE and dried. The crude product was purified by HPLC to obtain 3.6 mg (69%) of pure product. The product was lyophilized from acetonitrile/water (1/1) to a white amorphous solid.

HPLC purification Prep. For HPLC, Phenomenex Luna-C18 (2), 10 μm column (250 x 21.2 mm) (flow 30 ml/min, λ = 305 nm) was used, and water, 0.05% TFA (solvent A), and pure acetonitrile (solvent B) were used. The following gradient was performed using:

0.0 minutes → 95% A; 14.8 minutes → 50% A; 15.0 minutes → 0% A; 18.0 minutes → 0% A; 18.5 minutes → 95% A, 22.0 minutes → 95%A.

Example 2: Antibody-targeted amatoxin conjugates Antibodies were conjugated to amatoxin linker conjugates by the so-called thiomab technology. This approach is carried out by conjugating the maleimide residue of the toxin linker construct to the free SH group of the cysteine residue of the antibody, as shown in the reaction scheme below:

The principles of this conjugation method are disclosed in Junutula et al (2008), the contents of which are incorporated herein by reference.

The antibody used in this experiment contains the D265C substitution in both Fc domains, providing a cysteine residue with such a free SH group. The respective techniques are disclosed in WO2016/142049 A1, the contents of which are incorporated herein by reference, and result in homogeneous products with a fixed drug-to-antibody ratio (“DAR”) of 2 and site-specific Target conjugation is obtained.

Example 3: Induction of immunogenic cell death by antibody-targeted amatoxin conjugates Example 3.1: Treatment of cells for ICD marker measurement The Her2 positive cell line BT474 is a human breast cancer cell line. The CD79b-positive cell line BJAB is a B cell line derived from human Burkitt's lymphoma.

Cells of the Her2 positive cell line BT474 and the CD79b positive cell line BJAB were each plated at 8 x ¹⁰⁴ cells/well in 100 μL of medium. Flat-bottom plates or U-bottom plates were used for BT474 cells and BJAB cells, respectively. The next day, the medium was replaced. This step made it possible to suppress variations between replicate wells and significantly improve the reproducibility of the ATP assay. Cells were then treated with medium alone, maytansine (100 nM), amanitin (100 nM), anti-HER2-amanitin conjugate (50 nM antibody), or anti-CD79b-amanitin conjugate (50 nM antibody) for 16-72 hours.

Example 3.2: Calreticulin Measurement Cells were assayed 24 hours, 48 hours, and 72 hours after treatment. Adherent BT474 cells were lifted with a solution of high purity recombinant cell dissociation enzyme (TrypLE, Thermo-Fisher) at 37°C for 2 minutes. Both BT474 and BJAB cells were washed with PBS/2% FBS and fixed with 0.5% paraformaldehyde for 5 minutes. After washing twice with cold PBS/2% FBS, the cells were incubated with an Fc receptor binding inhibitor (FC block, eBioscience) for 5 minutes to inhibit nonspecific binding through Fc receptors, and then treated with anti-cal reticulum. Incubate for 30 minutes with phospho-FITC (Abcam) or isotype control-FITC (Abcam). Cells were washed twice with PBS/2% FBS. Propidium iodide (PI) was added and samples were analyzed by flow cytometry using FACS Diva software on a FACSCanto instrument (Becton-Dickinson).

Example 3.3: Measurement of extracellular ATP At 16, 40 and 64 hours after treatment, the culture fluid was transferred to a reaction tube (Eppendorf) and centrifuged gently. Extracellular ATP concentration was measured using ENLITEN ATP Assay (Promega). Chemiluminescence was measured with a SpectraMax ME chemiluminescence reader (Molecular Devices).

Example 3.4: Measurement of extracellular HMGB1 Culture fluid was collected 24 hours, 48 hours, and 72 hours after the treatment. Analytes were captured in Nunc Maxisorp 96-well plates coated with 1 μg/mL anti-HMGB1 antibody (clone 1D5, Sigma) at pH 9, and plates were blocked with casein buffer (ThermoFisher). A standard curve was generated using recombinant human HMGB1 protein (R&D Systems) added to the medium incubated at 37° C./5% CO ₂ for the same number of days as the experimental samples. This step has the effect of normalizing the background signal observed in fresh cultures. The experimental sample medium was transferred to a new tube, gently centrifuged to pellet the debris, and added to the prepared plate along with the standard curve. After 1 hour, plates were washed with PBS/0.1% Tween-20 and anti-HMGB1 polyclonal antibody (ab18256, Abcam) was added at 1 μg/mL in PBS for 1 hour. Plates were washed and anti-rabbit peroxidase-conjugated secondary antibody (Jackson Immunoresearch) diluted 1:3000 in PBS was applied. After 30 minutes, plates were washed with PBS/0.1% Tween-20, and bound secondary antibodies were detected using Ultra TMB (Thermo Fisher). Signals were read on a Molecular Devices Spectra Max M5 plate reader. Regarding sample handling, experimental samples could not be frozen for subsequent analysis due to the degradation of the HMGB1 signal. However, it is also possible to store the samples at 4°C and analyze them the next day.

The results of measuring ICD markers from BT474 cells and BJAB cells are shown in FIG. 4.

In contrast to unconjugated maytansine, unconjugated amanitin significantly reduced cell surface exposure of calreticulin (CRT), ATP secretion, and It did not induce HMGB1 release. This finding is consistent with poor cellular uptake of unbound amanitin. However, amanitin-conjugated antibody-drug conjugates (ADCs) induced the exposure and secretion of the ICD markers, respectively, in a target-dependent manner.

In Her2-positive BT474 cells (Figure 4 A-C), anti-HER2-amanitin conjugates induced cell surface exposure of CRT (Figure 4 A), ATP secretion (Figure 4 B), and HMGB1 release (Figure 4 C). but not the anti-CD79b-amanitin conjugate. On the other hand, in CD79b-positive BJAB cells (Fig. 4 D to F), anti-CD79b-amanitin conjugates inhibited cell surface exposure of CRT (Fig. 4 D), ATP secretion (Fig. 4 E), and HMGB1 release (Fig. 4 F). but not the anti-HER2-amanitin conjugate.

Therefore, the exposure of CRT, secretion of ATP, and release of HMGB1 by ATAC in cell lines was found to depend on the specificity of the target binding moiety of ATAC.

Example 4: Synergistic In Vivo Cytotoxic Effects of ATAC and Immune Checkpoint Inhibitors The cytotoxic activity of a combination comprising ICI and ATA has been evaluated using an in vivo tumor mouse model. This study consisted of 6 experimental groups of 12 animals each. CD19-positive Raji cells (human Burkitt's lymphoma, DSMZ) were premixed with human peripheral blood mononuclear cells (PBMC, German Red Cross) and subcutaneously inoculated on test day 0. Treatment consisted of PBS, CD19-specific ATAC chiBCE19-D265C-XIIb alone at a dose of 0.1 mg/kg body weight and 0.3 mg/kg body weight, respectively (single dose i.v.), PD-L1-specific antibody avelumab alone at a dose of 20 mg/kg body weight (i.v. days 0, 3, 6, 8, 10, 13) or the CD19-specific ATAC chiBCE19-D265C-XIIb at a dose of 0.1 mg/kg body weight and a dose of 0.3 mg/kg body weight (single dose i.v.), and the PD-L1 specific antibody avelumab at a dose of 20 mg/kg body weight (iv. days 0, 3, 6, 8, 10, 13). This combination was started on day 0 after cell inoculation. Tumor volume was measured twice a week by caliper measurement, and body weight was measured in parallel. If the tumor volume exceeded ¹⁶⁰⁰ mm or the mouse had to be sacrificed for ethical reasons (according to the German Animal Welfare Act), the animal was sacrificed and a necropsy was performed.

The results are shown in FIG. The reduction in tumor volume obtained when the PD-L1-specific antibody avelumab alone or the CD19-specific ATAC chiBCE19-D265C-XIIb alone was administered at a dose of 0.1 mg/kg body weight compared to the control group (PBS). It was found that they are equivalent. However, the reduction in tumor volume due to the combination of CD19-specific ATAC chiBCE19-D265C-XIIb 0.1 mg/kg body weight and PD-L1-specific antibody avelumab greatly exceeded the sum of the reductions caused by both drugs alone. The same result was observed when ATAC was used at a high concentration of 0.3 mg/kg body weight, confirming the synergistic effect of ATAC and the immune checkpoint inhibitor on tumor cell killing activity in vivo.

Example 4: Dependence of in vivo synergistic cytotoxicity of ATAC and immune checkpoint inhibitors on the presence of PBMCs

The cytotoxic activity of combinations comprising ICI and ATAC has been further evaluated using an in vivo tumor mouse model in the absence and presence of human peripheral blood mononuclear cells (PBMC). The study consisted of 8 experimental groups of 12 animals each. For group 4, CD19-positive Raji cells (human Burkitt's lymphoma, DSMZ) were premixed with human PBMC (German Red Cross) and inoculated subcutaneously on day 0. The remaining four groups were inoculated subcutaneously with Raji cells only. Treatment was with PBS, the CD19-specific ATAC chiBCE19-D265C-XIIb alone (dose 0.3 mg/kg body weight), or the PD-L1-specific antibody avelumab (Bavencio®) alone (20 mg/kg body weight), or Avelumab, a combination of the CD19-specific ATAC chiBCE19-D265C-XIIb and the PD-L1-specific antibody avelumab, starting on day 0 after cell inoculation (see Table 2 for details); , sequential intravenous injections were performed. Tumor volume was measured twice a week by caliper measurement, and body weight was measured in parallel. If the tumor volume exceeded 1600 mm ³ or the mouse had to be sacrificed for ethical reasons (based on German animal welfare legislation), animals were sacrificed and necropsy was performed.

The results are shown in FIG. In the absence of human PBMCs, the combination of CD19-specific ATAC chiBCE19-D265C-XIIb at 0.3 mg/kg body weight and the PD-L1-specific antibody avelumab at 20 mg/kg body weight is different from the combination of CD19-specific ATAC chiBCE19-D265C-XIIb alone The reduction in in vivo tumor volume was not greater than that achieved in the study. On the other hand, in the presence of human PBMC, when the CD19-specific ATAC chiBCE19-D265C-XIIb was used in combination at a dose of 0.3 mg/kg body weight and the PD-L1-specific antibody avelumab at a dose of 20 mg/kg body weight, CD19-specific We found that the reduction in in vivo tumor volume was higher than that with ATAC chiBCE19-D265C-XIIb alone.

In conclusion, the synergistic cytotoxic effect of ATAC and immune checkpoint inhibitors in vivo is dependent on the presence of human PBMC in the mouse model used.

Example 5: Efficacy of amanitin-based anti-CD19 ATAC chiBCE19-D265C-XIIb and pembrolizumab in NOD/SCID mouse Raji tumor xenograft model reconstituted with human PBMC

The study consisted of 6 experimental groups of 9 animals each. For all groups, CD19-positive Raji cells (human Burkitt's lymphoma, DSMZ) were premixed with human PBMC (German Red Cross) and inoculated subcutaneously on day 0. On day 0 after cell inoculation, PBS, CD19-specific ATAC chiBCE19-D265C-XIIb alone at doses of 0.1 mg/kg and 0.3 mg/kg body weight, PD-1-specific antibody pembrolizumab (Keytruda®) alone Administration of the CD19-specific ATAC chiBCE19-D265C-XIIb and the PD-1-specific antibody pembrolizumab was started at a dose of 20 mg/kg body weight (see Table 3 for details). Tumor volume was measured twice a week by caliper measurement, and body weight was measured in parallel. If the tumor volume exceeded 1600 mm ³ or the mouse had to be sacrificed for ethical reasons (based on German animal welfare legislation), animals were sacrificed and necropsy was performed. The results of this study are shown in Figure 9, showing that the synergistic cytotoxic effect of ATAC and anti-PD-1 immune checkpoint inhibitors in vivo was dependent on the presence of human PBMC in the mouse model used and We show that effective amounts of anti-CD19 ATAC are required to induce immunogenic cell death that synergizes with the activity of checkpoint inhibitors.
Example 6: Efficacy of anti-CD19 ATAC chiBCE19-D265C-XIIb and ipilimumab in NOD/SCID mouse Raji tumor xenograft model reconstituted with human PBMC

This study consisted of 6 experimental groups of 12 animals each. For all groups, CD19-positive Raji cells (human Burkitt's lymphoma, DSMZ) were premixed with human PBMC (German Red Cross) and inoculated subcutaneously on day 0. Treatment consisted of PBS, CD19-specific ATAC chiBCE19-D265C-XIIb alone at doses of 0.1 mg/kg and 0.3 mg/kg body weight, and CTLA4-specific antibody ipilimumab (Yervoy®) alone at 4 mg/kg body weight. dose or a combination of the CD19-specific ATAC chiBCE19-D265C-XIIb and the CTLA4-specific antibody ipilimumab starting on day 0 after cell inoculation (see Table 4 for details). If the tumor volume exceeded 1600 mm ³ or the mouse had to be sacrificed for ethical reasons (based on German animal welfare legislation), animals were sacrificed and necropsy was performed. The results of this study are shown in FIG. 10, and show that there was a synergistic effect on survival in animals treated with the combination of CTLA4 immune checkpoint inhibitor and anti-CD19-ATAC (Group 6). This result indicates that an effective dose of anti-CD19 ATAC is required to induce immunogenic cell death, and that its combination with CTLA4 checkpoint inhibitors (e.g., ipilimumab) is evidenced by increased survival in each treatment group. This indicates that they act synergistically.

Claims (19)

A composition for use in the treatment of cancer, the composition comprising: (a) at least one immune checkpoint inhibitor; and (b) at least one conjugate;
The conjugate is
(i) a target binding moiety;
A composition comprising (ii) at least one amatoxin, and (iii) optionally at least one linker connecting said target binding moiety and said at least one amatoxin. 2. A composition for use according to claim 1, wherein the immune checkpoint inhibitor and/or the target binding moiety of the conjugate is selected from the group consisting of:
(i) antibodies, preferably monoclonal antibodies;
(ii) an antigen-binding fragment thereof, preferably a variable domain (Fv), Fab fragment or F(ab) ₂ fragment;
(iii) an antigen-binding derivative thereof, preferably a single chain Fv (scFv), and (iv) an antibody-like protein. The use according to claim 2, wherein the antibody, or antigen-binding fragment or derivative thereof, is a mouse, chimeric, humanized or human antibody, or antigen-binding fragment or derivative thereof, respectively. Composition of. The immune checkpoint inhibitor is directed to an immune checkpoint receptor selected from the group consisting of PD-1, CTLA-4, LAG-3, TIGIT, TIM-3, VISTA, BTLA, CD96 and CD160, or PD- Binds to a ligand of an immune checkpoint receptor selected from the group consisting of L1, PD-L2, CD80, CD86, galectin-3, LSECtin, CD112, Ceacam-1, Gal-9, PtdSer, HMGB1, HVEM, CD155 , a composition for use according to any of claims 1 to 3. The immune checkpoint inhibitor is an antibody selected from the group consisting of nivolumab, pidilizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimumab, and ipilimumab, or an antigen-binding fragment thereof, or an antigen-binding derivative thereof, preferably , the antibody is avelumab, pembrolizumab, nivolumab, or ipilimumab or an antigen-binding fragment thereof or an antigen-binding derivative thereof. For use according to any of claims 1 to 5, wherein the composition comprises a combination of two or more immune checkpoint inhibitors, preferably the composition comprises a combination of two immune checkpoint inhibitors. Composition of. The target binding moiety of the conjugate is a target molecule on the cell surface of cancer cells selected from the group comprising PSMA, CD19, CD37, CD269, sialyl Lewis ^a , HER-2/neu, epithelial cell adhesion molecule (EpCAM). A composition for use according to any of claims 1 to 6, which binds to. 3. The target binding portion of the conjugate is an antibody having an Fc region comprising at least one mutation selected from the group consisting of D265C, D265A, A118C, L234A, and/or L235A (according to the EU numbering system). A composition for use according to any of items 1 to 7. Claim: said antibody comprises an Fc region with a D265C mutation and, if present, said linker or said amatoxin is connected to said antibody via the D265C residue of said antibody, preferably via a thioether bond. Composition for use according to item 8. 10. The amatoxin of the conjugate is selected from α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanulin, amanulic acid, or salts or analogues thereof. A composition for use as described in any of the above. The linker of said conjugate, if present, is a stable linker or a cleavable linker, said cleavable linker being an enzymatically cleavable linker, preferably a protease cleavable linker, and a chemically cleavable linker, Composition for use according to any of claims 1 to 9, selected from the group consisting of linkers, preferably comprising disulfide bridges. The linker or target binding moiety, if present, of the conjugate is (i) the γC-atom of amatoxin amino acid 1, or (ii) the δC-atom of amatoxin amino acid 3, or (iii) the amatoxin amino acid 4. A composition for use according to any of claims 1 to 11, wherein the composition is linked to the amatoxin via the 6'-C-atom of. Composition for use according to any of claims 1 to 12, wherein the conjugate comprises as linker-amatoxin moiety any of the following compounds of formulas (I) to (XII), respectively:
Composition for use according to any of claims 1 to 12, wherein the conjugate is a compound according to any one of formulas XIII to XXII:
Here, the amatoxin linker portion is bonded to the ε-amino group of a naturally occurring lysine residue of the antibody, and n is preferably 1-8. The use according to any of claims 1 to 12, wherein the conjugate is a compound according to any one of formulas XXIII, XXIV, XVIIIb, XVb, XVIb, XVIIb, XVIIIb, XIXb, XXb, XXIIb. A composition for
The amatoxin linker moiety is bonded to the thiol group of the cysteine residue of the antibody, and n is preferably 1 to 10, preferably 1, 2, 3 to 4, preferably 1, 2 to 5, preferably 4-7, preferably 8-10. A composition (for use) further comprising one or more pharmaceutically acceptable buffers, surfactants, diluents, carriers, excipients, fillers, binders, lubricants, lubricants. 16. Pharmaceutical formulation according to any one of claims 1 to 15, comprising , a disintegrant, an adsorbent and/or a preservative. The cancer is melanoma, squamous/non-squamous non-small cell lung cancer, metastatic small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, urothelial carcinoma, head and neck squamous cell carcinoma, Merkel cell carcinoma, hepatocellular carcinoma, gastric cancer. - selected from the group consisting of gastroesophageal cancer, metastatic colon cancer, primary mediastinal B cell lymphoma, recurrent or metastatic cervical cancer, metastatic cutaneous squamous cell carcinoma, according to any one of claims 1 to 15. A composition for use or a pharmaceutical formulation according to claim 16. A method for the treatment of cancer in a human subject in need thereof, the method comprising: a composition comprising: (a) at least one immune checkpoint inhibitor; and (b) at least one conjugate. wherein the conjugate links (i) a target binding moiety, (ii) at least one amatoxin, and (iii) optionally, the target binding moiety and the at least one amatoxin. A method comprising at least one linker. A composition for cancer treatment comprising (a) at least one immune checkpoint inhibitor and (b) at least one conjugate, the conjugate comprising: (i) a target binding moiety; (ii) Use of a composition comprising at least one amatoxin and (iii) optionally at least one linker connecting said target binding moiety and said at least one amatoxin.