CN116744969A

CN116744969A - Composition comprising a combination of an immune checkpoint inhibitor and an antibody-amatoxin conjugate for use in cancer therapy

Info

Publication number: CN116744969A
Application number: CN202180088954.0A
Authority: CN
Inventors: M·库尔克; T·黑希勒; A·帕尔; C·米勒; S·维尔纳-西蒙
Original assignee: Heidelberg Pharma Research GmbH
Current assignee: Heidelberg Pharma Research GmbH
Priority date: 2020-11-04
Filing date: 2021-11-04
Publication date: 2023-09-12
Also published as: CL2023001266A1; WO2022096604A1; IL302034A; EP4240424A1; CA3195572A1; AU2021373298A1; MX2023005192A; JP2023548522A; KR20230104653A

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 amatoxin, and (iii) optionally at least one linker linking the target binding moiety to the at least one amatoxin. The application also relates to the compositions for treating patients suffering from cancer, as well as pharmaceutical formulations comprising the compositions and additional excipients, and methods of making and using the compositions.

Description

Composition comprising a combination of an immune checkpoint inhibitor and an antibody-amatoxin conjugate for use in cancer therapy

Technical Field

The present application relates to the field of cancer immunotherapy. In particular, the present application relates to compositions 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 amatoxin, and (iii) optionally at least one linker linking the target binding moiety to the at least one amatoxin. The application also relates to the compositions for treating patients suffering from cancer, as well as pharmaceutical formulations comprising the compositions and additional excipients, and methods of making and using the compositions.

Background

Modulation of the immune system is mediated by a variety of mechanisms, including regulatory cells of the innate and adaptive immune system, such as regulatory T cells (tregs), myeloid-derived suppressor cells (MDSCs), and M2-type macrophages; regulatory cytokines such as IL-10 and TGF-beta; and immune checkpoints that control T cell activation. Such cells and molecules are useful as immune-subversion mechanisms during the development of cancer and chronic infectious diseases. On the other hand, however, one of the most successful cancer treatment strategies developed so far is based on immunotherapy. This treatment is intended to enhance the immune response of the host or patient to the various stages of tumor progression and to reduce off-target outcome compared to chemotherapeutic drugs or other modes of treatment that directly destroy cancer cells (Singh et al 2020).

An immune checkpoint is a receptor on the cell membrane of T lymphocytes that modulates the immunoreactivity of the cells. Anti-inflammatory (inhibitory) and pro-inflammatory (activating) immune checkpoints exist that are expressed on the cell membrane of T cells and interact with the respective ligand (soluble or cell-binding ligand). Tumor cells typically activate the anti-inflammatory immune checkpoint pathways via respective ligands that inhibit the anti-tumor immune response, thereby evading immune surveillance and allowing tumor growth to progress. An Immune Checkpoint Inhibitor (ICI) is an agent, in particular a monoclonal antibody, which binds to an anti-inflammatory immune checkpoint or its ligand and can interrupt this tumor suppression strategy by reactivating the immune system, thus restoring its anti-tumor capacity. ICI has been shown to be clinically effective against a variety of tumor types (Dyck and Mills,2017; darwin et al, 2018).

The two ICI targets most widely studied and used in drug development are PD-1/PD-L1-PD-L2 and CTLA4/CD80-CD86 receptor-ligand signaling pathways. Both of these pathways lead to inhibition of T cell activation, proliferation and survival. Whereas CTLA-4 receptor is expressed predominantly in T cells, PD-1 is expressed in activated T cells, B cells and certain bone marrow cells. Furthermore, while CTLA-4 functions in the priming phase of T cell activation and limits early T cell activation, PD-1 functions in the late phase (during effector phase) mainly in peripheral tissues where T cells encounter PD-1 ligands (Dyck and Mills, 2017).

T cell activation requires two signals: peptide antigens presented by Major Histocompatibility Complex (MHC) were identified as a first signal and co-stimulated by CD28 as a second signal after binding to CD80 or CD86 expressed by Antigen Presenting Cells (APC) (fig. 2). CTLA-4 is one of the inhibitory receptors originally identified as playing a role in inhibiting T cell responses, is similar in structure to CD28, and binds CD80 and CD86 with higher affinity than CD 28. CTLA-4 expression has been proposed to interfere with T cell activation by reducing CD 28-mediated co-stimulatory (second) signaling, resulting in T cell anergy; anergic T cells have limited effector functions (Dyck and Mills, 2017). CTLA-4 expression and function are intrinsically associated with T cell activation; CTLA-4 is upregulated immediately after T Cell Receptor (TCR) engagement (signal 1). Blocking CTLA-4 in vivo has been shown to inhibit binding of CTLA-4 to CD80/86 and promote anti-tumor immunity by inhibiting Treg cells and enhancing effector T cell function (Wei et al, 2018).

The programmed cell death protein 1 (PD-1) molecule consists of an intracellular domain with potential phosphorylation sites within the immune tyrosine-based inhibitory motif (ITIM) and the immune receptor-based inhibitory tyrosine-based opening Guan Jixu (ITSM), a hydrophobic transmembrane region, and an extracellular IgV domain (Li et al 2016). Inhibition of active T cells by PD-1 requires activated switch Guan Jixu (ITSM); PD-1 ligand binding results in the recruitment of tyrosine phosphatase SHP-2 to the ITSM motif, thereby interfering with TCR downstream signaling. In addition, PD-1 ligand binding results in interference of signaling molecules important for T cell proliferation, cytokine secretion and metabolism (such as PIP-3 kinase and Ras), in addition to inducing metabolic changes in T effector cells and promoting induction of Treg cells. PD-1 ligand binding can also lead to T cell depletion.

PD-1 (Li et al 2016) has been detected on T cells, tregs, depleting T cells, B cells, activated monocytes, dendritic Cells (DCs), natural Killer (NK) cells, natural Killer T (NKT) cells, epithelial cells and tumor cells. PD-1 expression on T cells is induced by antigen stimulation. PD-1 exerts its inhibitory effect mainly on peripheral T cells. Two ligands for PD-1, PD-L1 (CD 274) and PD-L2 (CD 273), have been identified. In cancer, tumor cells and bone marrow cells are considered to be the major cell types that mediate T cell inhibition by PD-1 binding (Li et al 2016; dyck and Mills, 2017).

In addition to CTLA-4 and PD-1, recent studies have identified additional immune checkpoint targets such as lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin-containing domain 3 (TIM-3), T cell immunoglobulin and ITIM domains (TIGIT), T cell activation V domain Ig inhibitor (VISTA), CD96, and BTLA (CD 272) (Qin et al, 2019).

LAG-3 is typically expressed on activated CD 4-positive and CD 8-positive T cells, tregs, natural Killer (NK) cell subsets, B cells, and plasmacytoid dendritic cells (pdcs). Studies have shown that LAG-3 signaling plays a negative regulatory role in T helper 1 (Th 1) cell activation, proliferation, and cytokine secretion. During tumorigenesis and cancer progression, tumor cells utilize this pathway to evade immune surveillance. MHC-II, galectin-3, LSECtin, alpha-synuclein and fibrinogen-like protein 1 (FGL 1) have been described to interact with LAG-3.

TIM-3, also known as hepatitis a virus cell receptor 2 (HAVCR 2), belongs to the Ig superfamily, with an N-terminal Ig variable region (IgV) like domain, a near-membrane mucin-like domain containing an O-linked glycosylation site (glycosylated mucin domain), a single transmembrane region, and a C-terminal cytoplasmic tail. TIM-3 expression is not limited to T cells, but is known to be expressed on different types of immune cells, including B cells, tregs, NK cells, DCs, monocytes, and macrophages. Four different ligands were reported to bind to the IgV domain of TIM-3, including galectin 9, high mobility group B1 protein (HMGB 1), carcinoembryonic antigen cell adhesion molecule 1 (Ceacam-1) and phosphatidylserine (PtdSer), with galectin-9 and HMGB1 being soluble ligands and Ceacam-1 and PtdSer being surface ligands. It has been described that the engagement of TIM-3 with galectin 9 triggers intracellular calcium flow in Th1 cells, thereby inducing 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 has a major impact on the innate immune response.

TIGIT proteins comprise an extracellular IgV region, a transmembrane domain, and a cytoplasmic tail with ITIM and an Immunoglobulin Tail Tyrosine (ITT) -like phosphorylation motif. TIGIT expression has been shown to be strictly limited by lymphocytes, mainly T cell subsets (including tregs and memory T cells) and NK cells. TIGIT binds with different affinities to two ligands, CD155 (PVR or Necl-5) and CD112 (connector-2, also known as PRR2 or PVRL 2). TIGIT exerts its immunosuppressive effect by competing with other counterparts, such as CD266 (DNAM-1), for the ligand. CD226 delivers a positive co-stimulatory signal, while TIGIT delivers an inhibitory signal into T cells (Qin et al, 2019).

There are currently approximately 4,000 active immunooncology-related therapeutic agents in the global drug development product line, and the number of clinical trials exploring the blockade of PD-1/PD-L1 alone has reached 2,250. Since 2011 US approved ipilimumab (anti-CTLA-4) for the treatment of metastatic melanoma, the number of ICI approved indications has increased to well over 20 (Taams and de Gruijl, 2020). To date, two classes of ICI that show promising therapeutic results have been approved by the U.S. food and drug administration (US Food and Drug Administration, FDA): CTLA-4, as well as PD-1 (CD 279) and its ligands PD-L1 (CD 274, B7-H1) and PD-L2 (CD 273, B7-DC), 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 against CTLA-4; sodium Wu Shankang for PD-1 and palbociclizumab; and atilizumab, avilamab, dewaruzumab, and cimetidine Li Shan antibodies against PD-L1 (Singh et al 2020).

Tumor types for which immune checkpoint blocking therapies have been approved by regulatory authorities include melanoma, squamous and 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 and gastroesophageal carcinoma, metastatic colorectal cancer, primary mediastinal B-cell lymphoma, recurrent or metastatic cervical cancer, and metastatic skin squamous cell carcinoma (Wei et al, 2018; singh et al, 2020).

In addition to monotherapy with CTLA-4 or PD-1 blocking antibodies, combination therapies comprising, for example, ipilimumab and nivolumab are used. Furthermore, ICI is used in combination with cancer vaccines, typically consisting of cancer antigens and an adjuvant source that activates innate immune cells (such as dendritic cells); cancer vaccines aim to generate tumor-specific T cells that kill tumor cells via secretion of IFN- γ or lytic particles. ICI is also used in combination with radiotherapy, and with histone deacetylase inhibitors, which can induce direct tumor cytotoxicity and improve tumor immunogenicity for some cancer types.

ICI is considered to be one of the most important developments in cancer therapy in the past decade. However, a significant fraction of patients do not respond, particularly in monotherapy with ICI alone (Dyck and Mills, 2017). In addition, the occurrence of a series of new immune related adverse events (irAE) associated with this approach was noted (Martins et al, 2019), indicating the need for higher cancer cell specificity and selectivity for such therapeutic agents.

One of the major aspects of the clinical success of anticancer agents has been shown to be their ability to induce Immunogenic Cell Death (ICD), i.e. a pattern of cell death that stimulates an immune response against dead cell antigens, in particular derived from cancer cells, compared to non-immunogenic cancer cell death, e.g. by apoptosis. ICD involves a change in cell surface composition and release of a soluble medium; these signals act on a range of receptors expressed by dendritic cells, thereby stimulating them to present tumor antigens to T cells (Kroemer et al, 2013). This pattern of cell death was initially observed and studied in the context of anticancer chemotherapy.

ICDs have been found to be characterized by a combination of alterations in plasma membrane composition and microenvironment composition of dead cells (fig. 3). These changes are caused by pre-death stress and subsequent cell disintegration. ICDs must be preceded by two types of stress, endoplasmic Reticulum (ER) stress and autophagy as an adaptive response to stress. The largest part of Calreticulin (CRT) is usually hidden in the ER lumen, and heat shock proteins and other ER proteins are exposed at the outer surface of the plasma membrane due to ER stress caused by, for example, chemotherapy. Adenosine Triphosphate (ATP) is secreted by dead cells as a result of and depending on autophagy. Non-histone chromatin protein high mobility group box 1 (HMGB 1) is released into the microenvironment due to cell disintegration associated with cell death.

Exposure of CRT and other ER proteins at the cell surface, secretion of ATP, and release of HMGB1 are markers of ICD, not non-immunogenic cell death. CRT, ATP and HMGB1 interact with CD91 (low density lipoprotein receptor-related protein 1, lrp 1), P2RX7 (purinergic receptor) and TLR4 (Toll-like receptor 4) receptors, respectively, which are expressed by dendritic cells and promote endocytosis of dead cells, production of cytokines such as IL-1 β, and presentation of tumor antigens. Thus, the patient's dead cancer cells are used as vaccines to stimulate tumor-specific immune responses, characterized by increased DC recruitment, number and activity of T lymphocytes, and increased ratio of cytotoxic CD8 positive T lymphocytes (CTLs) relative to regulatory T cells (tregs) within the tumor, which in turn can control residual cancer cells (Kroemer et al, 2013).

Different chemotherapeutic agents are not equivalent in their ability to induce ICD. In one study, where 24 different cytotoxic chemotherapeutic agents were tested against cancer cells in vivo, it was observed that only four of them (three anthracyclines and oxaliplatin) did elicit a protective anti-cancer immune response, although all agents induced apoptosis (Obeid et al, 2007).

Thus, there is a need to develop therapeutic agents or compositions that bind to the immune checkpoint inhibitory effects of ICI with higher cancer cell specificity and selectivity and a higher ability to induce ICD.

Amatoxins are cyclic peptides composed of 8 amino acids, which are found in amanita (Amanita phalloides) mushrooms (see fig. 1). Amatoxins specifically inhibit DNA-dependent RNA polymerase II of mammalian cells, thereby also inhibiting transcription and protein biosynthesis of the affected cells. Inhibition of transcription in cells causes growth and proliferation to cease. Although not covalently bound, the complex between amanitine and RNA polymerase II is very tight (kd=3 nM). Dissociation of amanitine from enzymes is a very slow process, thus making recovery of the affected cells less likely. When transcriptional inhibition continues long enough, the cell will undergo programmed cell death (apoptosis).

Antibody Drug Conjugates (ADCs) comprising amatoxins (antibody-targeted amatoxin conjugates) and tumor antigen specific antibodies, antibody fragments or derivatives or antibody-like proteins have been described (WO 2010/115629A2, WO2016/142049A1, WO 2017/149977 A1).

As shown by the present application, the inventors unexpectedly found that amatoxin-based ADCs comprising tumor antigen specific antibodies as target binding moieties induced 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. These results are unexpected because no amatoxins alone or amatoxin-based ADCs have been previously shown to have this benefit.

Summary of The Invention

Accordingly, in view of the prior art, it is an object of the present invention to provide a pharmaceutical composition comprising:

(a) At least one immune checkpoint inhibitor, and

(b) At least one conjugate, wherein the conjugate comprises

(i) A target-binding moiety that is capable of binding to a target,

(ii) At least one amatoxin, and

(iii) Optionally at least one linker linking the target binding moiety to the at least one amatoxin.

It is another object of the present invention to provide a composition for treating cancer or chronic infectious diseases, the composition comprising:

(a) At least one immune checkpoint inhibitor, and

(b) At least one conjugate, wherein the conjugate comprises

(i) A target-binding moiety that is capable of binding to a target,

(ii) At least one amatoxin, and

It is another object of the present invention to provide a pharmaceutical formulation comprising the composition (for use) and further comprising one or more pharmaceutically acceptable buffers, surfactants, diluents, carriers, excipients, fillers, binders, lubricants, glidants, disintegrants, adsorbents and/or preservatives.

These and other objects are achieved by the method and means according to the independent claims of the present invention. The dependent claims relate to specific embodiments.

The general benefits of the invention and its features are discussed in detail below.

Drawings

FIG. 1 shows the structural formulae of various amatoxins. The numbers (1 to 8) in bold type designate the standard numbering of the eight amino acids forming the amatoxins. Also shown are the standard designations of atoms in amino acids 1, 3 and 4 (greek letters alpha to gamma, greek letters alpha to delta, and numbers 1 'to 7', respectively).

FIG. 2. Principle concept of immunogenic cell death. As a result of pre-mortem endoplasmic reticulum stress and autophagy, cancer cells that respond to ICD inducers expose CRT to the outer leaflet of their plasma membrane during the pre-apoptotic phase and secrete ATP during apoptosis. Furthermore, cells undergoing ICD release the nucleoprotein HMGB1, as their membranes become permeabilized during secondary necrosis. CRT, ATP and HMGB1 bind to the receptors CD91, P2RX7 and TLR4, respectively. This favors recruitment of DCs to the tumor bed (stimulated by ATP), endocytic tumor antigens of DCs (stimulated by CRT), and optimal antigen presentation to T cells (stimulated by HMGB 1). Together, these processes lead to potent IL-1 β and IL-17 dependent, IFN- γ mediated immune responses (involving both γδ T cells and CTLs). ATP, adenosine triphosphate; CRT, calreticulin; CTL, cytotoxic cd8+ T lymphocytes; DC, dendritic cells; HMGB1, high mobility group protein 1; IFN, interferon; IL, interleukin; TLR, toll-like receptor (from Kroemer et al, 2013).

Fig. 3. Principle concept of immune checkpoint inhibition. MHC presents peptide antigens to T cell receptors, providing a first signal for T cell activation (1). The T cells received a second costimulatory activation signal through interaction between CD80 on the antigen-presenting cells and CD28 on the T cells (2). CTLA-4 competes with CD28 for binding to CD80 and delivers an inhibitory signal to T cells (3). The PD-1 receptor on T cells binds to PD-L1, thereby delivering an inhibitory signal to T cells (4). The use of these mechanisms by tumor cells prevents T cells from clearing malignant cells. By using inhibitors (CTLA-4, PD-1 or PD-L1 inhibitors) that prevent this interaction, T cells remain active after identifying tumor cells and can clear them from the host. 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 (panels from Sambi et al, 2019).

Figure 4 antibody-targeted amatoxin conjugate (ATAC) -induced immunogenic cell death. The Her2 positive cell line BT474 and the CD79b positive cell line BJAB were exposed to different compounds, respectively. Immunogenic Cell Death (ICD) marker Calreticulin (CRT), adenosine Triphosphate (ATP), and high mobility group protein 1 (HMGB 1) were assayed. Amanitine conjugated ADCs induce secretion of ICD markers in a target-dependent manner. Exposing (A, B, C) Her2 positive cell line BT474 and (D, E, F) CD79b positive cell line BJAB to no compound (first bar), 100nM maytansine (second bar), 100nM amanitine (third bar), 50nM anti-Her 2-amanitine conjugate (fourth bar) and 50nM anti-CD 79 b-amanitine conjugate (fifth bar), respectively; and (a), (D) CRT exposed (positive) cells were evaluated; (B), (E) ATP secretion; and (C), (F) HMGB1 release.

Figure 5 synergistic effect of avermectin and anti-CD 19-amatoxin conjugates in the presence of Peripheral Blood Mononuclear Cells (PBMC). Anti-tumor studies in vivo in Raji xenograft mouse model systems using anti-PD-L1 antibody avermectin (20 mg/kg, intravenously, days 0, 3, 6, 8, 10, 13), or anti-CD 19-amatoxin conjugate (ATAC, 0.1 and 0.3mg/kg, respectively; single dose intravenously, day 0), or a combination of avermectin (20 mg/kg) and ATAC (0.1 and 0.3mg/kg, respectively). Tumor volumes were assessed at various time points after tumor cell inoculation.

Figure 6 synergistic effect of avermectin and anti-CD 19-amatoxin conjugates in the absence or presence of Peripheral Blood Mononuclear Cells (PBMC). Anti-tumor studies in vivo in Raji xenograft mouse model systems using the anti-PD-L1 antibody avermectin (20 mg/kg, intravenously, days 0, 3, 6, 8, 10, 13), or anti-CD 19-amatoxin conjugate (ATAC, 0.3mg/kg; single dose intravenously, day 0), or a combination of avermectin (20 mg/kg) and ATAC (0.3 mg/kg) in the absence of PBMC (group 01-04) and in the presence of PBMC (group 05-08), respectively. Tumor volumes were assessed at various time points after tumor cell inoculation.

Figure 7 is an overview of the cytotoxic efficacy of anti-HER 2 ATAC in different cell lines. An anti-HER 2-LALA-D265C antibody conjugated to an amatoxin XIXa, XVIIIa, XIIIa, XIIa, XXIIa, XXIa comprising a linker (the corresponding conjugated amatoxin conjugated to each anti-HER 2-LALA-D265C antibody was named XIXb, XVIIIb, XIIIb, XIIb, XXIIb, XXIb, wherein the anti-HER 2 antibody corresponds to the antibody in the formula).

FIG. 8. Efficacy of anti-Her 2 ATAC in a subcutaneous JIMT-1 xenograft model. For Jimt-1 xenograft model, female NMRI nude mice were inoculated subcutaneously 5×10 in the right flank ⁶ Fine Jimt-1 breast cancerCells (Mol Cancer Ther.2004, 12 months; 3 (12): 1585-92)/mouse. At about 120mm ³ Animals were assigned to the corresponding experimental group on day 0 at the mean tumor volume. On the same day, animals received a single intravenous dose of amanitine-based anti-Her 2 Antibody Drug Conjugate (ADC) as indicated. Tumor volumes and body weights were measured twice weekly.

FIG. 9 synergistic effect of treatment with pambo Li Zhushan anti-CD 19-amatoxin conjugates in vivo in Raji xenograft mouse model systems. Antitumor study results using anti-PD-1 antibody palbociclib (20 mg/kg intravenously, day 0, 3, 6, 8, 10), or anti-CD 19-amatoxin conjugate (anti-CD 19 ATAC,0.1mg/kg or 0.3mg/kg; single dose intravenously, day 0), or a combination of palbociclib (20 mg/kg) and anti-CD 19 ATAC (0.1 mg/kg or 0.3 mg/kg). Tumor volumes were assessed at various time points after tumor cell inoculation. Error bars represent SEM.

Figure 10 depicts a graph of mouse survival for the synergistic effect of combination therapy with anti-CTLA 4 therapy and anti-CD 19-amatoxin conjugate in Raji xenograft mouse model. anti-CD 19 ATAC was administered at a single dose of 0.1mg/kg or 0.3mg/kg on day 0 and ipilimumab at a dose of 4mg/kg on days 0, 3, 6, 8 and 10. anti-CD 19 ATAC at a dose of 0.1mg/kg or 0.3mg/kg (all single doses) on day 0 and ipilimumab at a dose of 4mg/kg on days 0, 3, 6, 8, 10. Experimental details are provided in example 6.

Detailed Description

Before describing the present invention in detail, it is to be understood that this invention is not limited to the particular constituent elements of the described apparatus or process steps of the described method as such apparatus and method may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include singular and/or plural referents unless the context clearly dictates otherwise. Furthermore, it should be understood that where a range of parameters defined by numerical values is given, that range is deemed to include such limiting values. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The word "substantially" does not exclude "complete", e.g., a composition that is "substantially free" of Y may be completely free of Y. The word "substantially" may be omitted from the definition of the application, where necessary.

Throughout the specification and the claims which follow, unless the context requires otherwise, the term "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated member, integer or step but not the exclusion of any other non-stated member, integer or step. The term "consisting of …" is a specific embodiment of the term "comprising" wherein any other unrecited member, integer or step is excluded. In the context of the present application, the term "comprising" encompasses the term "consisting of …". Thus, the term "comprising" encompasses "comprising" as well as "consisting of …", e.g., a composition "comprising" X may consist of X alone or may include additional components, such as x+y.

It should also be understood that the embodiments disclosed herein are not intended to be construed as separate embodiments that are not related to each other. Features discussed with respect to one embodiment are also intended to be disclosed in connection with other embodiments shown herein. If a particular feature is not disclosed with respect to one embodiment, but with respect to another embodiment, then the skilled artisan will appreciate that such feature is not necessarily intended to be disclosed with respect to such other embodiment. The skilled person will appreciate that the gist of the application is to disclose the features also for other embodiments, but only for the sake of clarity and to keep the description in a controllable number, which has not been done.

Furthermore, the content of the prior art documents cited herein is incorporated by reference. This refers in particular to prior art documents disclosing standard or conventional methods. In this case, the primary purpose of the incorporation by reference is to provide a sufficient disclosure that can be achieved and avoid lengthy repetition.

According to a first aspect of the present invention there is provided a pharmaceutical composition comprising:

(a) At least one immune checkpoint inhibitor, and

(b) At least one conjugate, wherein the conjugate comprises

(i) A target-binding moiety that is capable of binding to a target,

(ii) At least one amatoxin, and

According to a second aspect of the present invention there is provided 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, wherein the conjugate comprises

(i) A target-binding moiety that is capable of binding to a target,

(ii) At least one amatoxin, and

According to a third aspect of the present invention there is provided a composition for use in the treatment of a chronic infectious disease, the composition comprising:

(a) At least one immune checkpoint inhibitor, and

(b) At least one conjugate, wherein the conjugate comprises

(i) A target-binding moiety that is capable of binding to a target,

(ii) At least one amatoxin, and

Immune checkpoints (also known as immune checkpoint receptors) control T cell activation, preventing overshoot (overswing) inflammation and autoimmune diseases, but also inhibiting anti-tumor immune responses.

In the context of the present invention, the term "immune checkpoint inhibitor" or simply "checkpoint inhibitor" or "ICI" refers to any agent or compound that directly or indirectly reduces the level of or inhibits the function of an immune checkpoint receptor protein or molecule present on the surface of an immune cell (e.g. a T cell) as a soluble compound or on the surface of an immune cell-inhibiting cell, or that directly or indirectly reduces the level of or inhibits the function of a ligand that binds to said immune checkpoint receptor protein or molecule. Such inhibitory cells may 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 an immune checkpoint receptor protein or molecule to an immune cell. Non-limiting examples of immune checkpoint receptor protein-ligand pairs are PD-1, PD-L1.PD-1 is an immune checkpoint receptor protein present on T cells. PD-L1, which can be overexpressed by cancer cells, binds to PD-1 and helps the cancer cells evade attack by the host immune system. Thus, immune checkpoint inhibitors prevent PD-1/PD-L1 interactions by blocking PD-1 on T cells (i.e., acting as a PD-1 inhibitor) or PD-L1 on cancer cells (i.e., acting as a PD-L1 inhibitor), thereby maintaining or restoring anti-tumor T cell activity or blocking inhibitory cancer cell activity.

Thus, an immune checkpoint inhibitor is an antagonist of an immunosuppressive receptor (e.g., PD-1), in which case it inhibits 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 palbociclizumab, pilidab, cimetidine Li Shan antibody, JTX-4014, sabdariffa-zumab, signal di Li Shan antibody (IBI 308), multi-talizumab (TSR-042, WBP-285), incmcga 00012 (MGA 012), AMP-224, PD1-1, PD1-2, PD1-3, PD1-4, PD1-5, BCD-100, AGEN-2034, terlipressin Li Shan antibody (TAB 001, JS 001), or AMP-514 (MEDI 0680), and fully human antibodies, such as PD-1 blocking marumumab or blocking PD-L1 such as avermectin, de tile Lu Shankang, ke Xili monoclonal antibody (CK-301), WBP-3155 (CS 1001), and atide monoclonal or recombinant pre-PD-L1 CX-antibodies.

Palbociclib (formerly also known as landolizumab; trade name Keytruda; also known as MK-3475) is disclosed, for example, in Hamid, o.et al (2013) New England Journal of Medicine 369 (2): 134-44, is a humanized IgG4 monoclonal antibody that binds to PD-1; it contains mutations at C228P designed to prevent Fc-mediated cytotoxicity. Palbociclib is disclosed in, for example, US 8,354,509 and WO 2009/114335. It is approved by the FDA for the treatment of patients with unresectable or metastatic melanoma and patients with metastatic NSCLC.

Nawuzumab (CAS registry number 946414-94-4; BMS-936558 or MDX1106 b) is a fully human IgG4 monoclonal antibody that specifically blocks PD-1, which lacks detectable antibody-dependent cellular cytotoxicity (ADCC). Nivolumab is disclosed in, for example, US 8,008,449 and WO 2006/121168. It has been approved by the FDA for the treatment of patients with unresectable or metastatic melanoma, metastatic NSCLC, and advanced renal cell carcinoma.

Pittuzumab (CT-011; cure Tech) is a humanized IgG1k monoclonal antibody that binds PD-1. Pittuzumab is disclosed in, for example, WO 2009/101611.

PD1-1 to PD1-5 refer to anti-PD-1 antibodies as disclosed in WO 2018/220169.

Ipilimumab (Ipilumumab) (CAS accession number 477202-00-9, which may also be referred to as 10D1, or MDX010, MDX-101) is a human IgG1 antibody that binds to cytotoxic T lymphocyte antigen 4 (CTLA 4). CTLA-4 is an inhibitory molecule that competes with stimulatory CD28 for binding to B7 on antigen presenting cells. CTLA-4 and CD28 are both present on the surface of T cells. Ipilimumab is a human IgG1 that binds CTLA-4, preventing T cell mediated inhibition of immune responses to tumors. Ipilimumab is disclosed as antibody "10D1", for example, in WO 01/14424.

The INN as used herein is also intended to encompass all biosimilar antibodies to the corresponding original antibodies as disclosed herein, including but not limited to those biosimilar antibodies authorized in accordance with the equivalent regulations of the 42USC ≡262 division (k) of US and other jurisdictions.

Immune checkpoint receptors or molecules include, but are not limited to, for example, PD-1, CTLA-4, LAG-3, TIM-3, TIGIT, VISTA, OX, GITR, ICOS, CD (B7-H3), B7-H4 (VTCN 1), IDO, KIR, CD122, CD137, CD94/NKG2A, CD80, CD86, galectin-3, LSECtin, CD112, ceacam-1, gal-9, ptdSer, HMGB1, HVEM, CD155, and BTLA (CD 272).

An immune checkpoint inhibitor according to the invention may for example be a small molecule (organic) compound or a large molecule such as a peptide or a nucleic acid. For example, small molecule immune checkpoint inhibitors according to the invention include CA-170, including its precursor AUNP-12, as disclosed in WO 15033301A 1; or for example BMS-8 (CAS number 1675201-90-7). In at least one embodiment of the invention, the immune checkpoint inhibitor is an antibody, or antigen-binding fragment thereof, or 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 "amatoxins" includes all cyclic peptides consisting of 8 amino acids as isolated from Amanita (Amanita) and described in Wieland, t. and Faulstich h. (Wieland T, faulstich h.), CRC Crit Rev biochem.5 (1978) 185-260); further all chemical derivatives thereof; further all semisynthetic analogs thereof; all synthetic analogues thereof further built up from synthetic blocks according to the main structure of the natural compound (cyclic, 8 amino acids), all synthetic or semisynthetic analogues further containing non-hydroxylated amino acids instead of hydroxylated amino acids, further all synthetic or semisynthetic analogues in which the sulfoxide moiety is replaced by a sulfone, a thioether, or by an atom other than sulfur, for example a carbon atom, as in amanitine carbon-based analogues (carbanalog).

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

As used herein, an "analog" of a compound is structurally related to, but not identical to, the compound and exhibits at least one activity of the compound. The compound to which the analog is compared is referred to as the "parent" compound. The aforementioned activities include, but are not limited to: binding activity to another compound; inhibitory activity, e.g., enzyme inhibitory activity; toxic effects; activation activity, such as enzyme activation activity. It is not required that the analogs exhibit such activity to the same extent as the parent compound. A compound is considered an analogue in the context of the present application if it exhibits a relevant activity to an extent of 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%, and more preferably at least 50%) of the activity of the parent compound. Thus, as used herein, "analogs of amatoxins" refers to the following compounds: is structurally related to any of alpha-amanitine, beta-amanitine, gamma-amanitine, epsilon-amanitine, tripelen-amanitine (amanin), amanita-amide, amanita-non-toxic cyclic peptide, and a monohydroxy-amanita-carboxylic acid, and exhibits an inhibitory activity against mammalian RNA polymerase II of 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%, and more preferably at least 50%) as compared to at least one of alpha-amanitine, beta-amanitine, gamma-amanita-e, epsilon-amanita-amide, amanita-non-toxic cyclic peptide, and a monohydroxy-amanita-carboxylic acid. "analogs of amatoxins" suitable for use in the present application may even exhibit a higher level than any of alpha-amanitine, beta-amanitine, gamma-amanitine, epsilon-amanitine, tripeptide, amanitamide, amanita non-toxic cyclic peptide or monohydroxy amanita carboxylic acid Inhibitory activity against mammalian RNA polymerase II. By determining the concentration at which 50% inhibition occurs (IC ₅₀ Value) to measure inhibitory activity. The inhibitory activity against mammalian RNA polymerase II can be determined indirectly by measuring the inhibitory activity against cell proliferation.

"semisynthetic analog" refers to an analog obtained by chemical synthesis using a compound from a natural source (e.g., plant material, bacterial culture, fungal culture, or cell culture) as a starting material. Typically, the "semisynthetic analogs" of the invention are synthesized starting from compounds isolated from a fungus of the Amanitaceae family (Amanitaceae). In contrast, "synthetic analogs" refer to analogs synthesized from smaller (typically petrochemical) synthetic blocks by so-called total synthesis. Typically, such total synthesis is performed without the aid of biological processes.

According to some embodiments of the invention, the amatoxins of the conjugates may be selected from the group consisting of α -amanitine, β -amanitine, γ -amanitine, ε -amanitine, tri-hydroxy amanitine, amanitamide, amanita non-toxic cyclic peptide, mono-hydroxy amanita carboxylic acid, and analogs, derivatives, and salts thereof.

Functionally, amatoxins are defined as peptides or depsipeptides that inhibit mammalian RNA polymerase II. Preferred amatoxins are those having a functional group (e.g., a carboxyl, amino, hydroxyl, sulfhydryl or sulfhydryl capturing group) capable of reacting with a linker molecule or target binding moiety as defined below.

In the context of the present invention, the term "amanitine" particularly refers to a bicyclic structure based on: aspartic acid or asparagine residues at position 1, proline residues at position 2 (in particular hydroxyproline residues), isoleucine at position 3, hydroxyisoleucine or dihydroxyisoleucine, tryptophan or hydroxytryptophan residues at position 4, glycine residues at positions 5 and 7, isoleucine residues at position 6, and cysteine residues at position 8, in particular derivatives of cysteine oxidized to sulfoxide or sulfone derivatives (for numbering and representative examples of amanitine, see fig. 1), and furthermore all chemical derivatives thereof; further all semisynthetic analogs thereof; all synthetic analogues thereof, which are further built up from synthetic building blocks according to the main structure of the natural compound (cyclic, 8 amino acids), further contain all synthetic or semisynthetic analogues of non-hydroxylated amino acids instead of hydroxylated amino acids, further all synthetic or semisynthetic analogues, in each case wherein any such derivatives or analogues are functionally active by inhibiting mammalian RNA polymerase II.

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

In the context of the present application, a "linker" refers to a molecule that increases the distance between the two components, e.g., to mitigate steric interference between the target binding moiety and the amatoxin, which might otherwise reduce the ability of the amatoxin to interact with RNA polymerase II. The linker may be used for another purpose, e.g. it may facilitate specific release of amatoxins in cells targeted by the target binding moiety. Preferably, the linker, and 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 extracellular physiological conditions (e.g. blood) while it can be cleaved within the cell, in particular within the target cell (e.g. cancer cell). To provide such selective stability, the linker may comprise a functional group that is preferably pH sensitive or protease sensitive. Alternatively, the bond linking the linker to the target binding moiety may provide selective stability. Preferably, the length of the linker is at least 1, preferably 1-30 atoms long (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), wherein one side of the linker has been reacted with the amatoxin and the other side has been reacted with the target binding moiety. In the context of the present application, a linker Preferably optionally substituted C _1-30 -alkyl, C _1-30 -heteroalkyl, C _2-30 -alkenyl, C _2-30 -heteroalkenyl, C _2-30 Alkynyl, C _2-30 -heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl. The linking group may contain one or more structural elements such as amides, esters, ethers, thioethers, disulfides, hydrocarbon moieties, and the like. The linker may also contain a combination of two or more of these structural elements. Each of these structural elements may be present in the linker more than once, for example, two, three, four, five or six times. In some embodiments, the linker may comprise a disulfide bond. It will be appreciated that the linker must be attached to the amatoxin and the target binding moiety in a single step or in two or more subsequent steps. To this end, the linker carries two groups, preferably at the proximal and distal ends, which may (i) form a covalent bond with a group (preferably an activating group) on the amatoxin or target binding peptide, or (ii) be or may be activated to form a covalent bond with a group on the amatoxin. Thus, if a linker is present, it is preferred that chemical groups are located distal and proximal to the linker, which are the result of such coupling reactions, e.g., esters, ethers, carbamates (urethanes), peptide bonds, etc. The presence of a "linker" is optional, i.e., in some embodiments of the target-binding moiety toxin conjugate, the toxin may be directly linked to a residue of the target-binding moiety.

In a preferred embodiment of the composition of the first, second and third aspects, the immune checkpoint inhibitor and/or target binding moiety of the conjugate is selected from the group consisting of

(i) Antibodies, preferably monoclonal antibodies,

(ii) Antigen binding fragments thereof, preferably variable domains (Fv), fab fragments or F (ab) ₂ The length of the segment is defined by,

(iii) Antigen binding derivatives thereof, preferably single chain Fv (scFv), and

(iv) An antibody-like protein.

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

As used herein, the term "antibody" shall refer to a protein consisting of one or more polypeptide chains encoded by immunoglobulin genes or immunoglobulin gene fragments or cdnas derived therefrom. The immunoglobulin genes include light chain kappa, lambda and heavy chain alpha, delta, epsilon, gamma and mu constant region genes, as well as any of a variety of different variable region genes.

The basic immunoglobulin (antibody) structural units are typically tetramers composed of two identical pairs of polypeptide chains (light chain (L, molecular weight of about 25 kDa) and heavy chain (H, molecular weight of about 50-70 kDa). Each heavy chain consists of a heavy chain variable region (abbreviated as VH or V _H ) And a heavy chain constant region (abbreviated as CH or C _H ) Composition is prepared. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain contains a light chain variable region (abbreviated as VL or V _L ) And a light chain constant region (abbreviated as CL or C _L ). VH and VL regions can be further subdivided into regions of hypervariability, also known as Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, known as Framework Regions (FR). Each VH and VL region is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains form binding domains that interact with antigens.

CDRs are most important for binding of antibodies or antigen binding portions thereof. If the three-dimensional structure required for antigen binding is preserved, the FR may be replaced by other sequences. Structural changes in the construct most often result in a loss of adequate binding to the antigen.

The term "antigen binding portion" of a (monoclonal) antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to the CD20 antigen in its native form. Examples of antigen-binding portions of antibodies include Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains, F (ab') ₂ Fragments, bivalent fragments comprising two Fab fragments linked via a disulfide bridge at the hinge region, fd fragments consisting of VH and CH1 domains, fv fragments consisting of VL and VH domains of a single arm of an antibody, and VH domains and fragments An isolated Complementarity Determining Region (CDR) comprising a dAb fragment.

The antibody or antibody fragment or antibody derivative thereof according to the present invention may be a monoclonal antibody. The antibody may be of the IgA, igD, igE, igG or IgM isotype.

As used herein, the term "monoclonal antibody" ("mAb") refers to a preparation of monospecific antibody molecules. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity, having variable and constant regions derived from or based on human germline immunoglobulin sequences or derived from fully synthetic sequences. The method of preparing monoclonal antibodies is independent of binding specificity. Preferably, such antibodies are selected from 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.

The term "fragment" as used herein shall refer to fragments of such antibodies that retain target binding capacity, e.g., CDRs (complementarity determining regions), hypervariable regions, variable domains (Fv), igG heavy chains (consisting of VH, CH1, hinge regions, CH2 and CH3 regions), igG light chains (consisting of VL and CL regions), and/or Fab and/or F (ab) ₂ 。

As used herein, the term "antigen binding derivative" or "derivative" shall refer to a protein construct that differs in structure from, but still has some structural relationship to, the common antibody concept, e.g., a protein consisting of the peptide scaffold and at least one CDR of the derived original antibody. Examples include, for example, scFv, fab and/or F (ab) ₂ And bi-, tri-or higher specific antibody constructs. All of these items are explained below.

Other antibody derivatives known to the skilled person are diabodies, camelid antibodies, domain antibodies, bivalent homodimers with two chains consisting of scFv, igA (two IgG structures linked by a J chain and a secretory component), shark antibodies, antibodies consisting of a new world primate framework plus non-new world primate CDRs, dimerization constructs comprising ch3+vl+vh, other scaffold protein forms comprising CDRs, and antibody conjugates (e.g. antibodies or fragments or derivatives thereof linked to a drug, a toxin, a cytokine, an aptamer, a nucleic acid such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), a therapeutic polypeptide, a radioisotope or a label). The scaffold protein forms may comprise, for example, antibody-like proteins, such as ankyrin and affilin proteins, and the like.

As used herein, the term "antibody-like protein" refers to a protein that is engineered (e.g., by mutagenesis of the Ig ring) to specifically bind to a target molecule. Typically, such antibody-like proteins comprise at least one variable peptide loop linked at both ends to a protein scaffold. This dual structural limitation greatly increases the binding affinity of the antibody-like protein to a level comparable to that of antibodies. The variable peptide loop typically consists of 10 to 20 amino acids in length. The scaffold protein may be any protein with good solubility properties. Preferably, the scaffold protein is a globular protein. Antibody-like proteins include, but are not limited to, affibodies, anticalin, and engineered ankyrin repeat proteins (Binz et al 2005). The antibody-like proteins may be derived from large libraries of mutants, for example by panning from a large phage display library, and may be isolated similarly to conventional antibodies. Furthermore, antibody-like binding proteins can be obtained by combinatorial mutagenesis of surface exposed residues in globular proteins.

As used herein, the term "Fab" refers to IgG fragments comprising antigen binding regions, which fragments consist of one constant and one variable region from each of the heavy and light chains of an antibody.

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

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

Modified antibody forms are, for example, bispecific or trispecific antibody constructs, antibody-based fusion proteins, immunoconjugates, etc.

IgG, scFv, fab and/or F (ab) ₂ Are well known to the skilled artisan. Related implementation techniques are available from respective textbooks.

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

Monoclonal antibodies (mabs) derived from mice may cause undesirable immunological side effects due to the fact that they contain proteins from another species that may elicit antibodies. To overcome this problem, antibody humanization and maturation methods were designed to generate antibody molecules with minimal immunogenicity when applied to humans, while still ideally preserving the specificity and affinity of the non-human parent antibodies (for reviews see Almagro and frankson 2008). Using these methods, for example, the framework regions of the mouse mAb are replaced by corresponding human framework regions (so-called CDR grafting). WO200907861 discloses the generation of humanized forms of mouse antibodies by joining CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques. US6548640 of the medical research committee (Medical Research Council) describes CDR grafting techniques and US5859205 of Celltech describes the production of humanized antibodies.

As used herein, the term "humanized antibody" relates to an antibody, fragment or derivative thereof, wherein at least a portion of the constant and/or framework regions, and optionally a portion of the CDR regions, of the antibody are derived from or modulated into a human immunoglobulin sequence.

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

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

The antibodies or fragments or derivatives of the invention may be produced by transfecting a host cell with an expression vector comprising the coding sequence of the antibody according to the invention. Expression vectors or recombinant plasmids are produced by placing the coding 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 may be expressed by co-transfected separate expression vectors or by dual expression vectors. The transfection may be transient or stable. The transfected cells are then cultured to produce the transfected antibody construct. When stable transfection is performed, stable clones secreting antibodies with properly associated heavy and light chains are then selected by screening with a suitable assay (e.g., ELISA), subcloned and propagated for future production.

In some embodiments of compositions according to the invention, the immune checkpoint inhibitor binds to an immune checkpoint receptor selected from the group consisting of PD-1, CTLA-4, LAG-3, TIGIT, TIM-3, VISTA, BTLA (CD 272), OX40 (CD 134), B7-H4 (VTCN 1), CD96, CD278 (ICOS), CD94/NKG2A and CD160, or a ligand of an immune checkpoint receptor selected from the group consisting of PD-L1, PD-L2, CD80, CD86, galectin-3, LSECtin, CD112, ceacam-1, gal-9, ptdSer, HMGB1, HVEM, CD155, OX40L, CD275 (ICOSLG).

In some embodiments, the composition according to the invention comprises an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an antibody selected from the group consisting of: na Wu Shankang, pabolizumab, pilizumab, simip Li Shan antibody, JTX-4014, stbadizumab, xindi Li Shan antibody (IBI 308), duodalizumab (TSR-042, WBP-285), INCMGA00012 (MGA 012), AMP-224, PD1-1, PD1-2, PD1-3, PD1-4, PD1-5, BCD-100, AGEN-2034, terlipp Li Shan antibody (TAB 001, JS 001), or AMP-514MEDI0680 Nawuzumab, avizumab, dewar Lu Shankang, ke Xili mab (CK-301), WBP-3155 (CS 1001), aprilizumab or CX-072, or antigen binding fragments thereof, or antigen binding derivatives thereof. Preferably, wherein the antibody is one of avilamab, nivolumab, ipilimumab, palbociclizumab, or antigen binding fragments thereof, or antigen binding derivatives thereof.

In some embodiments of the invention, a composition according to the invention as disclosed herein comprises a combination of two or more immune checkpoint inhibitors, e.g. two, three, four, five, six immune checkpoint inhibitors as disclosed herein, preferably the composition comprises a combination of two immune checkpoint inhibitors. For example, it is preferred that a composition according to the invention as disclosed herein comprises two or more immune checkpoint inhibitors that target different immune checkpoints, such as CTLA-4 and PD-1/PD-L1, PD-1/PD-L1 and TIGIT, PD-1/PD-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 comprise one of the following combinations of immune checkpoint inhibitors:

CTLA4—PD-1/PD-L1：

the combination of the ipilimumab and one of the nivolumab, the avermectin, the palbociclib, the pilidazumab, the PD1-1, the PD1-2, the PD1-3, the PD1-4, the PD1-5, the Dewa Lu Shankang and the actigb;

PD-1/PD-L1 and TIGIT:

a combination of tirizumab Li Youshan antibody and one of nivolumab, avermectin, palbociclib, pilidazumab, PD1-1, PD1-2, PD1-3, PD1-4, PD1-5, dewa Lu Shankang, actigizumab; or BMS986207 is combined with one of Nawuzumab, avermeizumab, palbociclizumab, pituzumab, PD1-1, PD1-2, PD1-3, PD1-4, PD1-5, dewa Lu Shankang, alteizumab;

PD-1/PD-L1 and OX40:

BMS986178 in combination with one of nivolumab, avilamab, palbociclib, pilidab, PD1-1, PD1-2, PD1-3, PD1-4, PD1-5, dewa Lu Shankang, actigb;

PD-1/PD-L1 and VISTA

CI-8993 is combined with one of nivolumab, avilamab, palbociclib, pilidazoab, PD1-1, PD1-2, PD1-3, PD1-4, PD1-5, dewar Lu Shankang, actigb;

PD-1/PD-L1 and OX40:

MEDI0562 in combination with one of nivolumab, avilamab, palbociclib, pilidamide, PD1-1, PD1-2, PD1-3, PD1-4, PD1-5, dewa Lu Shankang, actigb, or PF04518600 in combination with one of nivolumab, avilamab, palbociclib, pilidamide, PD1-1, PD1-2, PD1-3, PD1-4, PD1-5, dewa Lu Shankang, actigb;

TIM-3 and PD-1/PD-L1:

MBG453 is combined with one of Nawuzumab, averment monoclonal antibody, pabrizumab, pierizumab, PD1-1, PD1-2, PD1-3, PD1-4, PD1-5, dewa Lu Shankang, abilizumab;

CTLA4 and TIGIT:

a combination of ipilimumab with one of tirelin Li Youshan antibody or BMS 986207.

CTLA4 and OX40:

a combination of ipilimumab with one of MEDI0562 or PF 04518600.

In some embodiments, a composition according to the invention comprises a target binding moiety, wherein the target binding moiety of the conjugate binds to a target molecule on the cell surface of a cancer cell, wherein the target molecule is PSMA, CD19, CD37, CD269, sialyl Lewis oligosaccharides (sialyl Lewis) ^a One of HER-2/neu, epithelial cell adhesion molecule (EpCAM). The target molecules PSMA, CD19, CD37, CD269, sialyl Lewis oligosaccharides as disclosed above ^a HER-2/neu, epithelial cell adhesion molecule (EpCAM) refers to the following proteins or cell surface antigens: as used herein, "PSMA" refers to a prostate specific membrane antigen, also known as glutamate carboxypeptidaseII (GCPII), N-acetyl-L-aspartyl-L-glutamic acid peptidase I (NAALADase I) or N-acetyl-aspartyl glutamic acid (NAAG) peptidase is an enzyme encoded by the human folate hydrolase (FOLH 1) gene. PSMA is a membrane-bound cell surface peptidase that plays different physiological roles and is expressed in various tissues (e.g., prostate, kidney, small intestine, central and peripheral nervous system). It is highly expressed by malignant prostate epithelial cells and vascular endothelial cells of many solid tumor malignancies, including glioblastoma, breast and bladder cancers.

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

The term "CD37" as disclosed above refers to a protein encoded by the CD37 gene in humans and which is a member of the "four-way transmembrane protein" superfamily or the transmembrane 4 superfamily. Four transmembrane proteins are characterized by the presence of four conserved transmembrane domains, which are thought to be "molecular promoters" of signal transduction, involved in a variety of biological processes including cell growth, survival, adhesion, intercellular communication and trafficking, intercellular communication via exosomes, tumorigenesis, metastasis, and modulation of immune responses. Four-way transmembrane protein members have also been described as having functional roles in a wide variety of cellular processes including cell motility, development and differentiation, activation, proliferation, migration and tumor invasion (Hemler 2001; xu-Monete et al, 2016). Increased CD37 expression was found in B cell malignancies (Zou et al, 2018). Most B cell malignancies express CD37, including B cell non-hodgkin's lymphoma (NHL) and B cell chronic lymphocytic leukemia (B-CLL).

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

Sialyl lewis oligosaccharides as disclosed above ^a (also referred to as CD 15) refers to a tetrasaccharide consisting of sialic acid, fucose and N-acetylgalactosamine. Sialylated lewis oligosaccharides ^a Mediate phagocytosis and chemotaxis present on neutrophils; expressed in hodgkin's disease and in some B-cell chronic lymphocytic leukemia, acute lymphoblastic leukemia, and in most acute non-lymphoblastic leukemia patients. Sialylated lewis oligosaccharides ^a Is present on almost all li-schoendose (Reed-Sternberg) cells and can be used in immunohistochemistry to identify the presence of such cells in a biopsy, thus diagnosing hodgkin's lymphoma.

As disclosed above HER-2/Neu refers to the receptor tyrosine protein kinase erbB-2, also known as CD340 (cluster of differentiation 340), proto-oncogene Neu, erbb2 (rodent) or ERBB2 (human), which is a protein encoded by the ERBB2 gene in humans. ERBB is abbreviated from erythroblast oncogene B (a gene isolated from the avian genome). It is also often referred to as HER2 (from HER 2) or HER2/neu. HER2 is a member of the human epidermal growth factor receptor (HER/EGFR/ERBB) family. Dimerization of this receptor results in autophosphorylation of tyrosine residues within the cytoplasmic domain of the receptor and initiates a variety of signaling pathways leading to cell proliferation and tumorigenesis. Amplification or overexpression of HER2 occurs in approximately 15-30% of breast cancers and 10-30% of stomach/gastroesophageal cancers and serves as a prognostic and predictive biomarker.

EpCAM as disclosed above refers to an "epithelial cell adhesion molecule," which is a transmembrane glycoprotein that mediates ca2+ -independent homotypic cell-cell adhesion in the epithelium. EpCAM is a glycosylated type I membrane protein with a molecular weight of 30-40kD and is encoded by EpCAM genes in humans. The sequence of EpCAM molecules predicts the presence of three potential N-linked glycosylation sites. It consists of 314 amino acids. EpCAM consists of an extracellular domain (242 amino acids), a single transmembrane domain (23 amino acids) and a short intracellular domain (26 amino acids) with an Epidermal Growth Factor (EGF) -and thyroglobulin-like repeat domain. EpCAM is involved in cell signaling, migration, proliferation and differentiation. Furthermore, epCAM has oncogenic potential via its ability to up-regulate c-myc, E-fabp, and cyclin a & E. EpCAM may also be referred to as Ep-CAM, 17-1A, HEA, MK-1, GA733-2, EGP34, KSA, TROP-1, ESA or KS1/4.EpCAM is expressed only in epithelium and epithelium-derived neoplasms and can be used as a diagnostic marker for a variety of cancers.

In the context of the present application, the terms "target molecule" and "target epitope" refer to an antigen and an epitope of an antigen, respectively, to which a target binding moiety specifically binds. In particular, the target molecule is a tumor-associated antigen, in particular an antigen or epitope present on the surface of one or more tumor cell types at an increased concentration and/or in a different spatial configuration compared to the non-tumor cell surface. In particular, the 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 the group consisting of: PSMA, CD19, CD37, CD269, sialyl Lewis oligosaccharides ^a HER-2/neu, epithelial cell adhesion molecule (EpCAM). In other embodiments, the antigen or epitope is preferentially expressed on cells involved in autoimmune diseases. In certain embodiments, the target binding moiety specifically binds to an epitope of the IL-6 receptor (IL-6R).

In some embodiments, a composition according to the invention as disclosed herein comprises a conjugate comprising a target binding moiety, wherein the target binding moiety of the conjugate is an antibody having an Fc region comprising at least one mutation selected from D265C, D265A, A118C, L234A or L235A (according to the EU numbering system). "EU index as in Kabat" or "EU numbering system" refers to the numbering of the human IgG1 EU antibodies (see, e.g., edelman et al, 1969,Proc Natl Acad Sci USA 63:78-85, incorporated herein by reference in its entirety).

In a preferred embodiment, an antibody representing the target binding moiety of a conjugate according to the invention as disclosed herein comprises an Fc region carrying the D265C mutation, and the linker (if present) or the amatoxin is linked to the antibody via one or both of the D265C residues of the antibody, preferably via a disulfide bond.

According to some embodiments of the invention, the antibody representing the target binding portion of the conjugate is genetically engineered to comprise a heavy chain 118Cys, a heavy chain 239Cys, or a heavy chain 265Cys (according to the EU numbering system), preferably a heavy chain 265Cys (according to the EU numbering system), and wherein the linker (if present) or the amatoxin is linked to the antibody via the heavy chain 118Cys, or the heavy chain 239Cys, or a heavy chain 265Cys residue, respectively.

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

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

As used herein, the term "genetically engineered" or "genetically engineered" refers to the modification of an amino acid sequence or portion thereof of a given or native polypeptide or protein by genetic techniques (e.g., site-directed mutagenesis as described in biochem.j. (1986) 237,1-7 or J Biol chem.2015, 30, 1, 5; 290 (5): 2577-2592) in the sense of nucleotide and/or amino acid substitutions, insertions, deletions or inversions, or any combination thereof.

As used herein, the term "amino acid substitution" refers to 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, thereby producing a protein containing an amino acid sequence that differs from the original protein. Conservative amino acid substitutions are understood to relate to substitutions that do not significantly affect the structure and function of the protein due to similar size, charge, polarity, and/or conformation. A conserved amino acid group in this sense represents, for example, the nonpolar amino acids Gly, ala, val, ile and Leu; aromatic amino acids Phe, trp and Tyr; positively charged amino acids Lys, arg and His; and the negatively charged amino acids Asp and Glu. Exemplary amino acid substitutions are presented in table 1 below:

original residue	Examples of substitutions
		Ala(A)	Val、Leu、Ile、Gly
Arg(R)	His、Lys
		Asn(N)	Gln
Asp(D)	Glu
		Cys(C)	Ser
Gln(Q)	Asn
		Glu(E)	Asp
Gly(G)	Pro、Ala
		His(H)	Lys、Arg
Ile(I)	Leu、Val、Met、Ala、Phe
		Leu(L)	Ile、Val、Met、Ala、Phe
Lys(K)	Arg、His
		Met(M)	Leu、Ile、Phe
Phe(F)	Leu、Val、Ile、Tyr、Trp、Met
		Pro(P)	Ala、Gly
Ser(S)	Thr
		Thr(T)	Ser
Trp(W)	Tyr、Phe
		Tyr(Y)	Trp、Phe
Val(V)	Ile、Met、Leu、Phe、Ala

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

Furthermore, conjugates according to the invention may have a conjugate of better than 10X 10 ^-9 M、9×10 ^-9 M、8×10 ^-9 M、7×10 ^-9 M、6×10 ^-9 M、5×10 ^-9 M、4×10 ^-9 M、3×10 ^-9 M、2×10 ^-9 M, preferably better than 10X 10 ^-10 M、9×10 ^-10 M、8×10 ^- ¹⁰ M、7×10 ^-10 M、6×10 ^-10 M、5×10 ^-10 M、4×10 ^-10 M、3×10 ^-10 M、2×10 ^-10 M, and more preferably better than 10X 10 ^-11 M、9×10 ^-11 M、8×10 ^-11 M、7×10 ^-11 M、6×10 ^-11 M、5×10 ^-11 M、4×10 ^-11 M、3×10 ^-11 M、2×10 ^-11 M or 1X 10 ^-11 IC of M ₅₀ Is a cytotoxic activity of (a) a cell.

In some embodiments of the invention, the conjugate as described 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 of the conjugate (if present) or the target binding moiety is linked to the amatoxin via (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 amatoxin amino acid 4.

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

The linker may be a stable (non-cleavable) or cleavable linker. The cleavable linker may be selected from the group consisting of an enzymatically cleavable linker, preferably a protease cleavable linker, and a chemically cleavable linker, preferably a linker comprising a disulfide bridge.

"cleavable linker" is understood to comprise 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 position under specific conditions. The conditions are, for example, a reducing environment in a specific enzyme or a specific body or cellular compartment.

"non-cleavable linker" is understood to mean that it does not undergo enzymatic cleavage by, for example, cathepsin B and is released from the conjugate of the invention during degradation (e.g., lysosomal degradation) of the antibody moiety of the conjugate of the invention within the target cell.

According to some embodiments of the invention, the cleavage site is an enzymatically cleavable moiety comprising two or more amino acids. Preferably, the enzymatically cleavable moiety comprises valine-alanine (Val-Ala), valine-citrulline (Val-Cit), valine-lysine (Val-Lys), valine-arginine (Val-Arg) dipeptide, phenylalanine-lysine-glycine-proline-leucine-glycine (Phe Lys Gly Pro Leu Gly) or alanine-proline-valine (Ala Ala Pro Val) peptide or β -glucuronide or β -galactoside. The enzymatically cleavable moiety may also be referred to as a linker.

In a particularly preferred embodiment, the enzymatically cleavable moiety according to the present invention comprises a dipeptide selected from the group consisting of Phe-Lys, val-Lys, phe-Ala, val-Ala, phe-Cit and Val-Cit, in particular wherein the cleavable linker further comprises a p-aminobenzyl (PAB) spacer between the dipeptide and the amatoxin:

thus, a conjugate of the invention as disclosed herein may comprise an enzymatically cleavable moiety comprising any one of the dipeptide-PAB moieties Phe-Lys-PAB, val-LysPAB, phe-Ala-PAB, val-Ala-PAB, phe-Cit-PAB or Val-Cit-PAB as disclosed above.

Preferably, the cleavable moiety of the conjugate of the present invention comprises the dipeptide-PAB moiety Val-Ala-PAB.

Whereby the PAB moiety is linked to the amatoxins.

According to some embodiments, the cleavable moiety or linker of the present invention as disclosed above comprises a thiol-reactive group selected from bromoacetamide, iodoacetamide, methylsulfonylbenzothiazole, 4, 6-dichloro-1, 3, 5-triazin-2-ylamino group methyl-sulfonylphenyl tetrazole or methylsulfonylphenyl oxadiazole, pyridine-2-thiol, 5-nitropyridine-2-thiol, methane thiosulfonate or maleimide.

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

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

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

Cysteine proteases (also known as thiol proteases) are proteases that share a common catalytic mechanism involving nucleophilic cysteine thiols in catalytic triplets or diads.

Metalloproteinases are proteases whose catalytic mechanism involves metals. Most metalloproteases require zinc, but some use cobalt. The metal ion coordinates to the protein via three ligands. The ligands that coordinate the metal ions may vary with histidine, glutamic acid, aspartic acid, lysine and arginine. The fourth coordinated position is occupied by an unstable water molecule.

Serine proteases are enzymes that cleave peptide bonds in proteins; serine serves as a nucleophilic amino acid at the active site of the enzyme. Serine proteases fall into two broad categories based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like.

Threonine proteases are a family of proteolytic enzymes containing a threonine (Thr) residue in the active site. The prototype members of this class of enzymes are catalytic subunits of the proteasome, however, acyltransferases tend to evolve the same active site geometry and mechanism.

Aspartic proteases are a catalytic type of protease that use activated water molecules bound to one or more aspartic acid residues to catalyze their peptide substrates. In general, they have two highly conserved aspartic acids at the active site and are optimally active at acidic pH. Almost all known aspartyl proteases are inhibited by pepstatin.

In some embodiments of the invention, the cleavable site may be cleaved by at least one agent selected from the group consisting of cathepsin a or B, matrix Metalloproteinase (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 specific cleavage is performed by a reducing environment, such as an intracellular reducing environment, e.g., acidic pH conditions.

According to some embodiments, the conjugates of the invention as disclosed herein comprise a non-cleavable linker. Non-cleavable linkers suitable for use in accordance with the present invention may, for example, comprise one or more groups selected from the group consisting of: bond, - (c=o) -, C ₁ -C ₆ Alkylene, C ₁ -C ₆ Alkylene, C ₂ -C ₆ Alkenylene, C ₂ -C ₆ Alkenylene, C ₂ -C ₆ Alkynylene, C ₂ -C ₆ Alkynylene, C ₃ -C ₆ Cycloalkylene, heterocycloalkylene, arylene, heteroarylene, and combinations thereof, each of which may be optionally substituted, and/or may include one or more heteroatoms (e.g., S, N or O) in place of one or more carbon atoms. Non-limiting examples of such groups include (CH ₂ ) _p 、(C＝O)(CH ₂ ) _p Polyethylene glycol (PEG), (CH) ₂ CH ₂ O) _p ) Units, where p is an integer from 1 to 6, which are in each case independently selected.

In some embodiments, the non-cleavable linkers of the present invention comprise one or more of the following: bond, - (C=O) -, -C (O) NH-group, -OC (O) NH-group, C ₁ -C ₆ Alkylene, C ₁ -C ₆ Alkylene, C ₂ -C ₆ Alkenylene, C ₂ -C ₆ Alkenylene, C ₂ -C ₆ Alkynylene, C ₂ -C ₆ Alkynylene, C ₃ -C ₆ Cycloalkylene, heterocycloalkylene, arylene, heteroarylene, - (CH) ₂ CH ₂ O) _p -a group wherein p is an integer from 1 to 6, wherein each C ₁ -C ₆ Alkylene, C ₁ -C ₆ Alkylene, C ₂ -C ₆ Alkenylene, C ₂ -C ₆ Alkenylene, C ₂ -C ₆ Alkynylene, C ₂ -C ₆ Alkynylene, C ₃ -C ₆ The cycloalkylene, heterocycloalkylene, arylene or heteroarylene may be optionally substituted in each case with 1 to 5 substituents independently selected from the group consisting of: alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, amido, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl, halogen, carboxyl, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

For example, each C of the non-cleavable linkers as disclosed herein ₁ -C ₆ Alkylene, C ₁ -C ₆ Alkylene, C ₂ -C ₆ Alkenylene, C ₂ -C ₆ Alkenylene, C ₂ -C ₆ Alkynylene, C ₂ -C ₆ Alkynylene, C ₃ -C ₆ Cycloalkylene, heterocycloalkylene, arylene or heteroarylene groups may optionally be interrupted by one or more heteroatoms selected from O, S and N, and may be present in each case, for exampleOptionally substituted with 1 to 5 substituents independently selected from the group consisting of: alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, amido, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl, halogen, carboxyl, trihalomethyl, cyano, hydroxy, mercapto, and nitro. As used herein, the definition of chemical groups shall have the meaning and be defined as provided in "Compendium of Chemical Terminology" ("Gold Book") published by the international union of pure and applied chemistry (International Union of Pure and Applied Chemistry, IUPAC) (version 2.3.3, gold Book, 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, wherein n is an integer from 2 to 12, such as 2 to 6, e.g. 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 -, wherein n is 6 and the linker is represented by the formula:

in some embodiments, the non-cleavable linkers of the invention as disclosed herein further comprise a thiol reactive group. The thiol-reactive group of the non-cleavable linker as disclosed above may be selected, for example, from bromoacetamide, iodoacetamide, methylsulfonylbenzothiazole, 4, 6-dichloro-1, 3, 5-triazin-2-ylamino group methyl-sulfonylphenyl tetrazole or methylsulfonylphenyl oxadiazole, pyridine-2-thiol, 5-nitropyridine-2-thiol, methane thiosulfonate or maleimide.

According to a preferred embodiment, the thiol-reactive group is a maleimide (maleimide based moiety) as disclosed above. For example, the non-cleavable linker comprising the maleimide may, for example, have the structure where the wavy line at the end of the linker indicates the point of attachment to the amatoxin:

upon conjugation with a reactive thiol group on an antibody or target binding moiety, e.g., as disclosed herein, a maleimide moiety, e.g., a cleavable or non-cleavable linker, as disclosed herein, comprises the following structure:

The wavy line herein represents the attachment site of a cleavable or non-cleavable linker as disclosed herein.

According to a preferred embodiment, the conjugates of the invention comprising a cleavable or non-cleavable linker further comprising a thiol reactive group may be coupled to a thiol moiety naturally occurring in the antibody of the conjugate, or said cleavable or non-cleavable linker of the conjugates of the invention comprising a thiol reactive group may be coupled to a thiol moiety introduced into the antibody by genetic engineering, as described for example in Nat biotechnol.2008, 8; 26 925-32. Preferably, a cleavable or non-cleavable linker as disclosed herein comprising a thiol reactive group is coupled to a thiol moiety in the Fc region of an antibody that is introduced into the conjugates of the invention by genetic engineering. Preferred positions within the Fc region of the antibody at which a sulfhydryl moiety may be introduced comprise D265 or a118 (numbering according to EU), more preferably D265.

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

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according to one embodiment, the conjugates of the invention are preferably synthesized by reacting the thiol groups of the antibodies with a compound according to any one of formulas XIIa to XXIIa containing a maleimide moiety as a reactive cap. As used herein, the term "reactive cap" refers to a chemical moiety that reacts with, for example, a thiol group of an antibody to covalently attach compounds of formulas (XIIa) to (XXIIa) to the antibody.

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According to some embodiments, the compounds according to formulas (XIIa) - (XXIIa) of the invention are used to generate or prepare antibody-drug conjugates (ADCs), more particularly antibodiesTargeted amatoxin conjugates. The ADC of the present invention as disclosed herein may also be referred to as an "ATAC". For example, compounds (XIIa) to (XXIIa) react with a target binding moiety (such as, for example, an antibody, e.g., a human IgG1 antibody) under suitable conditions to form ATAC. The compounds according to the invention of the formulae (XIIa) - (XXIIa) can be used, for example, in combination with a target binding moiety (e.g.for PSMA, CD19, CD37, CD269, sialyl Lewis oligosaccharides) ^a Antibodies to HER-2/neu, epithelial cell adhesion molecule (EpCAM) to obtain the corresponding ATAC. The coupling of the compounds (XIIa) - (XXIIa) of the invention to a target binding moiety or antibody may be carried out as disclosed, for example, in WO2018/115466A1 to give an ATAC comprising one of the compounds (XIIb) - (XXIIb) of the invention.

According to a preferred embodiment of the present invention, the conjugate of the present invention is a compound according to any of formulae (XIIb) to (XXIIb) as disclosed below:

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wherein 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 present invention, the conjugate is a compound according to any one of formulas XIII to XXII:

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wherein the amatoxin linker moiety is coupled to the epsilon-amino group of a naturally occurring lysine residue of said antibody, and wherein n is preferably 1 to 8, preferably 1, 2, 3 to 4, preferably 2 to 5, preferably 5 to 7.

Furthermore, according to a preferred embodiment of the invention, the conjugate is a compound according to any one of formulas XXIII, XXIV, XIIIb, XXIIb, XXb and XVIb:

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wherein the amatoxin linker moiety is coupled to a thiol group of a cysteine residue of the antibody, and wherein n is preferably 1 to 10, or for example wherein n is 2, 4 to 6, more preferably wherein n is 1, 2, 4 or 8.

According to a preferred embodiment of the invention, the composition comprises at least one of the immune checkpoint inhibitors avermectin, naloxone Wu Shankang, palbociclizumab, dewaruzumab or ipilimumab, and a conjugate comprising an antibody against HER2 (αher 2-LALA-D265C) and an amatoxin according to formula XXIb, XXIIb, XIIb, XIIIb, XVIIIb or XIXb, wherein the antibody represented in the respective amatoxin structure as disclosed above is αher2-LALA-D265C as disclosed herein. Thus, according to a preferred embodiment of the present invention, the composition comprises one of the following:

Thus, the compositions of the invention comprise an antibody as disclosed above coupled to an amatoxin as disclosed above, and at least one immune checkpoint inhibitor as disclosed above.

According to a particularly preferred embodiment of the invention, the composition comprises at least one of the immune checkpoint inhibitors avermectin, nal Wu Shankang, palbociclizumab, dewaruzumab or ipilimumab, and a conjugate comprising an antibody against CD19 (chiBCE 19-D265C) and amatoxins according to formula XXIII, XXII, XXb, XXIIb, XXIV, XIIIb or XVIb, wherein the antibody represented in the respective amatoxin structure as disclosed above is CD19 (chiBCE 19-D265C). Thus, according to a particularly preferred embodiment of the invention, the composition comprises one of the following:

According to a particularly preferred embodiment, the composition according to the invention comprises at least one of the immune checkpoint inhibitors avermectin, nal Wu Shankang, palbociclizumab, dewaruzumab or ipilimumab, and a conjugate comprising an antibody against PSMA and an amatoxin according to formula XXIII, XXII, XXb, XXIIb, XXIV, XIIIb or XVIb, wherein the antibody represented in the respective amatoxin structure as disclosed above is an anti-PSMA antibody. Thus, according to a particularly preferred embodiment of the invention, the composition comprises one of the following:

According to a particularly preferred embodiment, the anti-PSMA antibody of the conjugate of the invention as disclosed above is an anti-PSMA antibody as disclosed in WO 2020/025564. For example, the antibody of the conjugate of the invention as disclosed herein is one of 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 one of 3-F11-var1, 3-F11-var13 or 3-F11-var16, as disclosed in WO 2020/025564.

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

According to a particularly preferred embodiment, the composition according to the invention comprises at least one of the immune checkpoint inhibitors avermectin, nal Wu Shankang, palbociclizumab, dewaruzumab or ipilimumab, and a conjugate comprising an antibody against CD37 and an amatoxin according to formula XXIII, XXII, XXb, XXIIb, XXIV, XIIIb or XVIb, wherein the antibody represented in the respective amatoxin structure as disclosed above is an anti-CD 37 antibody. Thus, according to a particularly preferred embodiment of the invention, the composition comprises one of the following:

According to a particularly preferred embodiment, the anti-CD 37 antibody as disclosed above for use in the composition of the invention comprises at least one mutation in its Fc region selected from L234A, L235A and D265C (numbered according to EU nomenclature), preferably L234A, L a and D265C.

According to a particularly preferred embodiment, the composition according to the invention comprises at least one of the immune checkpoint inhibitors avermectin, nal Wu Shankang, palbociclizumab, dewaruzumab or ipilimumab, and a conjugate comprising an antibody against CD269 and an amatoxin according to formula XXIII, XXII, XXb, XXIIb, XXIV, XIIIb or XVIb, wherein the antibody represented in the respective amatoxin structure as disclosed above is an anti-CD 269 antibody. Thus, according to a particularly preferred embodiment of the invention, the composition comprises one of the following:

According to a particularly preferred embodiment, the anti-CD 269 antibody of the conjugate of the invention as disclosed above is a humanized antibody J22.9-ISY as disclosed in WO2018/115466, and wherein the conjugate comprises an amatoxin linker-moiety according to formula (I), or wherein the conjugate is represented by formula (XIIb) as disclosed herein.

According to some embodiments, an anti-CD 269 antibody as disclosed above for use in a composition of the invention may for example comprise at least one mutation selected from L234A, L a and D265C (numbered according to EU nomenclature) in its Fc region, preferably the Fc region of the antibody comprises the mutations L234A, L a and D265C.

According to a particularly preferred embodiment, the composition according to the invention comprises at least one of the immune checkpoint inhibitors avermectin, na Wu Shankang, palbociclizumab, dewaruzumab or ipilimumab, and comprises a drug against sialyl lewis oligosaccharides ^a Conjugate of amatoxins according to formula XXIII, XXII, XXb, XXIIb, XXIV, XIIIb or XVIb, wherein the antibody represented in each amatoxin structure as disclosed above is an anti-alpha sialyl lewis oligosaccharide ^a An antibody. Thus, according to a particularly preferred embodiment of the invention, the composition comprises one of the following:

According to some embodiments, an anti-sialyl lewis oligosaccharide as disclosed above for use in the compositions of the present invention ^a The antibody comprises at least one mutation in its Fc region selected from L234A, L235A and D265C (numbered according to EU nomenclature), preferably L234A, L a and D265C.

According to a particularly preferred embodiment, the composition according to the invention comprises at least one of the immune checkpoint inhibitors avermectin, nal Wu Shankang, palbociclizumab, dewaruzumab or ipilimumab, and a conjugate comprising an antibody against EpCAM and an amatoxin according to formula XXIII, XXII, XXb, XXIIb, XXIV, XIIIb or XVIb, wherein the antibody represented in the respective amatoxin structure as disclosed above is an anti-EpCAM antibody. Thus, according to a particularly preferred embodiment of the invention, the composition comprises one of the following:

According to some embodiments, the anti-EpCAM antibody as disclosed above for use in the compositions of the invention comprises at least one mutation in its Fc region selected from L234A, L235A and D265C (numbered according to EU nomenclature), preferably L234A, L235A and D265C.

According to a particularly preferred embodiment, the conjugate for use in the composition of the invention as disclosed above comprises or is according to formula XIIb, XIVb, XVIIIb, XXb or XVIIb. Thus, according to a particularly preferred embodiment, the composition according to the invention comprises at least one of the immune checkpoint inhibitors avermectin, naloxone Wu Shankang, palbociclizumab, dewaruzumab or ipilimumab, and a conjugate according to formula XXIII, XXII, XXb, XXIIb, XXIV, XIIIb or XVIb. Thus, according to a particularly preferred embodiment of the present invention, the composition comprises at least one of the following:

thus, the compositions of the invention comprise at least a conjugate as disclosed above and at least one immune checkpoint inhibitor as disclosed above.

According to one embodiment, the present invention relates to conjugates according to formula (XIIb), (XIIIb), (XVb), (XXVIb), (XVIb), (XVIIb), (XIXVIb), (XXb), (XXIb), (XXIIb) for the preparation of the composition according to the invention.

According to one embodiment, the present invention relates to an immune checkpoint inhibitor selected from the group consisting of avermectin, nal Wu Shankang, palbociclizumab, ipilimumab or Dewaruzumab for the preparation of a composition according to the present invention as disclosed herein.

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

In the aqueous form, the pharmaceutical formulation may be ready for administration, whereas in the lyophilized form, the formulation may be converted to a liquid form prior to administration, for example by the addition of water for injection, which may or may not contain a preservative, such as, but not limited to, benzyl alcohol; antioxidants such as vitamin a, vitamin E, vitamin C, retinyl palmitate and selenium; amino acids cysteine and methionine; citric acid and sodium citrate; synthetic preservatives such as parabens (methyl and propyl).

The pharmaceutical formulation may further comprise one or more stabilizing agents, which may be, for example, amino acids, sugar alcohols, disaccharides and/or polysaccharides. The pharmaceutical formulation may further comprise one or more surfactants, one or more isotonic agents, and/or one or more metal ion chelating agents, and/or one or more preservatives.

The pharmaceutical formulations as described herein may be suitable for at least intravenous, intramuscular or subcutaneous administration. Alternatively, the conjugates according to the invention can be provided in a depot formulation which allows for sustained release of the bioactive agent over a specific period of time.

In a further aspect of the invention there is provided a primary package, such as a prefilled syringe or pen, vial or infusion bag, containing the formulation according to the previous aspect of the invention.

The prefilled syringe or pen may contain a formulation in lyophilized form (which must then be dissolved, for example, with water for injection prior to administration) or in aqueous form. The syringe or pen is typically a disposable article for single use only and may have a volume of 0.1 to 20 ml. However, the injector or pen may also be a multi-use or multi-dose injector or pen.

The vials may also contain formulations in lyophilized or aqueous form and may serve as single or multiple use devices. As a multiple use device, the vial may have a relatively large volume. The infusion bag typically contains the formulation in aqueous form and may have a volume of 20 to 5000 ml.

In some embodiments of the invention, the composition for treating cancer or the pharmaceutical formulation as described relates to a cancer selected from the group consisting of: melanoma, squamous and non-squamous non-small cell lung cancer, metastatic small cell lung cancer, renal cell carcinoma, hodgkin lymphoma, B-lymphocyte-associated malignancy, urothelial cancer, head and neck squamous cell carcinoma, merkel cell carcinoma, hepatocellular carcinoma, gastric and gastroesophageal cancer, metastatic colorectal cancer, multiple myeloma, primary mediastinal B-cell lymphoma, recurrent or metastatic cervical cancer, and metastatic skin squamous cell carcinoma, prostate cancer, breast cancer including Triple Negative Breast Cancer (TNBC).

In a preferred embodiment, the present invention relates to said composition or pharmaceutical formulation as disclosed herein for the treatment of B-lymphocyte-associated malignancies, in particular for the treatment of non-hodgkin's lymphoma, follicular lymphoma, diffuse large B-cell non-hodgkin's lymphoma and chronic lymphocytic leukemia.

In some embodiments, the invention relates to an immune checkpoint inhibitor as disclosed herein for use in the composition of the invention or the pharmaceutical composition of the invention. Accordingly, the present invention relates to an avermectin, a nal Wu Shankang, a palbociclizumab, an ipilimumab, a PD1-1, a PD1-2, a PD1-3, a PD1-4, a PD1-5, a pidotizumab, a cimip Li Shan antibody, a JTX-4014, a swabber antibody, a singdi Li Shan antibody (IBI 308), a rituximab, a terlipressin Li Shan antibody, a de valuzumab, or an atiturn Li Zhushan antibody for use in a composition or a pharmaceutical formulation according to the present invention as disclosed herein.

In some embodiments, the invention relates to the use of avermectin, nal Wu Shankang, palbociclizumab, ipilimumab or Devaluzumab in a composition or pharmaceutical formulation according to the invention as disclosed herein.

The invention further relates to a method for treating cancer in a human individual in need thereof, wherein the method comprises administering to the individual 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 amatoxin, and (iii) optionally at least one linker linking the target binding moiety to the at least one amatoxin.

According to one embodiment, a method of treating cancer in a human individual in need thereof as disclosed above comprises administering a composition or pharmaceutical formulation as disclosed above for the treatment of melanoma, squamous and 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 and gastroesophageal carcinoma, metastatic colorectal cancer, primary mediastinal B-cell lymphoma, recurrent or metastatic cervical cancer, and metastatic skin squamous cell carcinoma.

In some embodiments, the methods of treating cancer as disclosed above comprise sequentially or simultaneously administering an immune checkpoint inhibitor and a conjugate of a composition as disclosed herein. In the case of sequentially administering the immune checkpoint inhibitor and the conjugate of the present invention, the checkpoint inhibitor may be administered first, followed by administration of the conjugate, or, for example, the conjugate of the present invention may be administered first, followed by administration of the immune checkpoint inhibitor. In a preferred embodiment, the immune checkpoint inhibitor and the conjugate of the invention are administered intravenously (i.v.) 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 within the same day, e.g., 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 20 hours or 23 hours of each other.

The invention also relates to the use of 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 amatoxin, and (iii) optionally at least one linker linking the target binding moiety to the at least one amatoxin, for the treatment of cancer.

Examples

While 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; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. 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 as 5'- >3'.

Example 1: synthesis of linker toxins

Synthesis of linker toxin XIIIa

5.15mg (3.92. Mu. Mol) of thioether XIIa (as described in WO 2018/115466) are dissolved in 1000. Mu.l of acetic acid. At ambient temperature, 2078.99. Mu.l of m-CPBA (m-chloroperoxybenzoic acid, 69.5%, aldrich) in acetic acid (stock solution: 5.15mg of m-CPBA in 5000. Mu.l of acetic acid) was added. After stirring for 3 hours and 15 minutes at ambient temperature, the solution was added dropwise to 15ml of ice-cold MTBE. The whole mixture was stored at-18 ℃ for an additional 1 hour and the precipitate was separated by centrifugation. The solid residue was washed with 15ml ice-cold MTBE and dried. The crude product was purified by HPLC to give 3.12mg (59%) of pure product. The product was freeze-dried from acetonitrile/water (1/1) to a white amorphous solid.

And (3) HPLC purification:

preparative HPLC was performed using a Phenomenex Luna-C18 (2), 10 μm column (250×21.2 mm) (flow rate 30 ml/min, λ=290 nm), with water, 0.05% TFA (solvent a) and pure acetonitrile (solvent B) as gradient:

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

MS(ESI+)[MH] ⁺ Measured value 1346.7 calculated value 1346.47 (C ₆₁ H ₈₁ N ₁₄ O ₁₉ S)

[M+Na] ⁺ Measured value 1368.5 calculated value 1368.45 (C ₆₁ H ₈₀ N ₁₄ NaO ₁₉ S)

Synthesis of linker toxin XVIa

Variant a)

10.09mg (7.50. Mu. Mol) of sulfoxide XVa (as described in WO 2016/142049) was dissolved in 2000. Mu.l of acetic acid. At ambient temperature, 1784.28. Mu.l of m-CPBA (m-chloroperoxybenzoic acid, 69.5%, aldrich) in acetic acid (stock solution: 5.18mg of m-CPBA in 5000. Mu.l of acetic acid) was added. After stirring at ambient temperature for 3.5 hours, the solution was added dropwise to 30ml of ice-cold MTBE. The whole mixture was stored for an additional 45 minutes at-18 ℃ and the precipitate was isolated by centrifugation. The solid residue was washed with 30ml ice-cold MTBE and dried. The crude product was purified by HPLC to give 5.82mg (57%) of pure product. The product was freeze-dried from acetonitrile/water (1/1) to a white amorphous solid.

And (3) HPLC purification:

Preparative HPLC was performed using a Phenomenex Luna-C18 (2), 10 μm column (250×21.2 mm) (flow rate 30 ml/min, λ=305 nm), with water, 0.05% tfa (solvent a) and pure acetonitrile (solvent B) as gradient:

MS(ESI+)[MH] ⁺ Measured value 1362.4 calculated value 1362.47 (C ₆₁ H ₈₁ N ₁₄ O ₂₀ S)

[M+Na] ⁺ Measured value 1384.6 calculated value 1384.45 (C ₆₁ H ₈₀ N ₁₄ NaO ₂₀ S)

Variant b)

Step 1

20.25mg (22.01. Mu. Mol) of β -amanitine was dissolved in 4000. Mu.l of acetic acid. At ambient temperature, 5634.38. Mu.l of m-CPBA (m-chloroperoxybenzoic acid, 69.5%, aldrich) in acetic acid (stock solution: 10.67mg m-CPBA in 10ml acetic acid) was added. After stirring at ambient temperature for 3.5 hours, the solution was added dropwise to 60ml ice-cold MTBE. The whole mixture was stored at-18 ℃ for an additional 30 minutes and the precipitate was isolated by centrifugation. The solid residue was washed with 60ml ice-cold MTBE and dried. The crude product was purified by HPLC to give 10.46mg (51%) of pure product. The product was freeze-dried from acetonitrile/water (1/1) to a white amorphous solid.

And (3) HPLC purification:

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

MS(ESI+)[MH] ⁺ Measured value 936.9; calculated 936.98 (C) ₃₉ H ₅₄ N ₉ O ₁₆ S)

[M+Na] ⁺ Found 958.9; calculated 958.96 (C) ₃₉ H ₅₃ N ₉ NaO ₁₆ S)

Step 2

7.99mg (8.54. Mu. Mol) of the product of step 1 are dissolved in 665. Mu.l of anhydrous DMF. 127.98 μl of dry DMF solution of TBTU was added at ambient temperature. Thereafter, 128.04. Mu.l of a dry DMF solution of DIPEA (stock solution: 52.25. Mu.l of DIPEA in 1500. Mu.l of dry DMF) was added. The resulting solution was stirred for 1 minute at ambient temperature under argon. Then, 128.05. Mu.l of BMP-Val-Ala-PAB-NH was added ₂ (WO 2018115466) solution. The resulting reaction mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by HPLC to give 7.34mg (63%) of pure product.

And (3) HPLC purification:

preparative HPLC was performed using a Phenomenex Luna-C18 (2), 10 μm column (250×21.2 mm) (flow rate 15 ml/min, λ=305 nm), 95% water, 5% methanol, 0.05% TFA (solvent a) and 95% methanol, 5% water, 0.05% TFA (solvent B) as the following gradient:

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

Synthesis of linker toxin XIXa

5.07mg (3.77. Mu. Mol) of sulfoxide XVIIIa (as described in WO 2016/142049) was dissolved in 1000. Mu.l of acetic acid. At ambient temperature, 1043.57. Mu.l of m-CPBA (m-chloroperoxybenzoic acid, 69.5%, aldrich) in acetic acid (stock solution: 4.93mg m-CPBA in 5000. Mu.l acetic acid) was added. After stirring for 3 hours and 15 minutes at ambient temperature, the solution was added dropwise to 15ml of ice-cold MTBE. The whole mixture was stored at-18 ℃ for an additional 1 hour and the precipitate was separated by centrifugation. The solid residue was washed with 15ml ice-cold MTBE and dried. The crude product was purified by HPLC to give 2.44mg (48%) of pure product. The product was freeze-dried from acetonitrile/water (1/1) to a white amorphous solid.

And (3) HPLC purification:

MS(ESI+)[MH] ⁺ Measured value 1362.4; calculated 1362.47 (C) ₆₁ H ₈₁ N ₁₄ O ₂₀ S)

[M+Na] ⁺ Measured value 1384.6; calculated 1384.45 (C) ₆₁ H ₈₀ N ₁₄ NaO ₂₀ S)

Synthesis of linker toxin XXIIa

5.13mg (4.67. Mu. Mol) of sulfoxide XXIa (WO 2016142049) was dissolved in 1000. Mu.l of acetic acid. At ambient temperature, 1291.27. Mu.l of m-CPBA (m-chloroperoxybenzoic acid, 69.5%, aldrich) in acetic acid (stock solution: 4.94mg m-CPBA in 5000. Mu.l acetic acid) was added. After stirring at ambient temperature for 3.5 hours, the solution was added dropwise to 15ml of ice-cold MTBE. The whole mixture was stored on ice for an additional 30 minutes and the precipitate was separated by centrifugation. The solid residue was washed with 15ml ice-cold MTBE and dried. The crude product was purified by HPLC to give 3.6mg (69%) of pure product. The product was freeze-dried from acetonitrile/water (1/1) to a white amorphous solid.

And (3) HPLC purification:

MS(ESI+)[MH] ⁺ Measured value 1115.4 calculated value 1115.22 (C ₄₉ H ₆₈ N ₁₁ O ₁₇ S)

[M+Na] ⁺ Measured value 1137.2 calculated value 1137.20 (C ₄₉ H ₆₇ N ₁₁ NaO ₁₇ S)

Example 2: antibody-targeted amatoxin conjugates

The antibodies were conjugated to the amatoxin linker conjugates by the so-called Thiomab technique. In this method, conjugation is performed by conjugating the maleimide residue of the toxin linker construct to the free SH group of the cysteine residue in the antibody, as shown in the following reaction scheme:

/>

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

The antibodies used in this experiment contained D265C substitutions in both Fc domains in order to provide cysteine residues with such free SH groups. The corresponding technology is disclosed in WO2016/142049A1, the disclosure of which is incorporated herein by reference, and results in a homogeneous product with an immobilized drug-to-antibody ratio ("DAR") of 2 and site-specific conjugation.

Example 3: antibody-targeted amatoxin conjugates induce immunogenic cell death

Example 3.1: cell processing for ICD marker measurement

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 Her2 positive cell line BT474 and CD79b positive cell line BJAB were treated at 8X 10, respectively ⁴ Individual cells/well were plated into 100 μl of medium. For BT474 or BJAB cells, flat bottom or U-shaped bottom plates were used, respectively. The next day, the medium was changed. This step significantly improves ATP assay reproducibility by reducing variation between duplicate wells. The cells were then treated with medium alone, maytansine (100 nM), amanitine (100 nM), anti-HER 2-amanitine conjugate (50 nM antibody) or anti-CD 79 b-amanitine conjugate (50 nM antibody) for 16-72 hours.

Example 3.2: calreticulin measurement

Cells were assayed 24, 48 and 72 hours after treatment. The adherent BT474 cells were lifted with highly purified recombinant cell dissociation enzyme (TrypLE, thermo-Fisher) solution for 2 min at 37 ℃. BT474 and BJAB cells were both washed with PBS/2% FBS and then fixed in 0.5% paraformaldehyde for 5 min. After washing twice with cold PBS/2% FBS, cells were incubated for 5 min with Fc receptor binding inhibitors (Fc blockers, eBioscience) that block Fc receptor-mediated non-specific binding, then with anti-calreticulin-FITC (Abcam) or isotype control-FITC (Abcam) for 30 min. Cells were washed twice with PBS/2% FBS. Propidium Iodide (PI) was added and samples were analyzed by flow cytometry on a FACSCanto instrument (Becton-Dickinson) using FACS Diva software.

Example 3.3: extracellular ATP measurement

At 16, 40 and 64 hours after treatment, the medium was transferred to a reaction tube (Eppendorf) and gently centrifuged. Extracellular ATP concentration was determined by using the ENLITEN ATP assay (Promega). Chemiluminescence was measured on a SpectraMax ME chemiluminescent reader (Molecular Devices).

Example 3.4: extracellular HMGB1 measurement

Media was collected 24, 48 and 72 hours after treatment. Analytes were captured on Nunc Maxisorp 96-well plates coated with 1 μg/mL anti-HMGB 1 antibody (clone 1d5, sigma) at pH 9 and plates were blocked with casein buffer (ThermoFisher). Standard curves recombinant human HMGB1 protein (R&D Systems) which have been generated at 37 ℃/5% CO ₂ Incubate for the same days as the experimental samples. This step has the effect of normalizing the background signal observed in fresh medium. The medium for the experimental samples was transferred to a fresh tube, gently centrifuged to pellet the debris, and then added to the prepared plate with a standard curve. After 1 hour, the plates were washed with PBS/0.1% Tween-20 and 1. Mu.g/mL of anti-HMGB 1 polyclonal antibody (ab 18256, abcam) was added to the PBS for 1 hour. Plates were washed and anti-rabbit peroxidase conjugated secondary antibody diluted 1:3000 with PBS was applied (Jackson Immunoresearch). After 30 minutes, the plates were washed with PBS/0.1% Tween-20 and bound secondary antibodies were detected using Ultra TMB (Thermo Fisher); the signal is read on a Molecular Devices Spectra Max M board reader. With respect to sample processing, the experimental samples were not frozen for subsequent analysis due to HMGB1 signal degradation. However, the samples can be stored at 4 ℃ for analysis the next day.

ICD marker measurements of BT474 and BJAB cells, respectively, are shown in fig. 4.

In contrast to unconjugated maytansine, unconjugated amanitine did not induce cell surface exposure, ATP secretion, or HMGB1 release of Calreticulin (CRT) of either cell line, as compared to control cells treated with medium alone. This finding is consistent with poor cellular uptake of unconjugated amanitin. However, amanitine-conjugated antibody-drug conjugates (ADCs) induced exposure and secretion of the ICD markers, respectively, in a target-dependent manner.

In Her 2-positive BT474 cells (fig. 4A-C), anti-Her 2-amanitine conjugates, but not anti-CD 79B-amanitine conjugates induced cell surface exposure of CRT (fig. 4A), ATP secretion (fig. 4B) and HMGB1 release (fig. 4C). In contrast, in CD79 b-positive BJAB cells (FIGS. 4D-F), anti-CD 79 b-amanitine conjugates, but not anti-HER 2-amanitine conjugates, induced cell surface exposure of CRT (FIG. 4D), ATP secretion (FIG. 4E) and HMGB1 release (FIG. 4F).

Thus, ATAC-induced CRT exposure, ATP secretion and HMGB1 release in the cell line depend on the specificity of the target binding moiety of ATAC.

Example 4: synergistic cytotoxic effects of ATAC and immune checkpoint inhibitors in vivo

Cytotoxic activity comprising a combination of ICI and ATAC has been evaluated in vivo by using a tumor mouse model. The study consisted of 6 experimental groups of 12 animals each. CD 19-positive Raji cells (human burkitt lymphoma, DSMZ) were premixed with human peripheral blood mononuclear cells (PBMC, german Red Cross) and inoculated subcutaneously on study day 0. Treatment with PBS, a dose of 0.1mg/kg body weight and a dose of 0.3mg/kg body weight, respectively (single dose, intravenous) of CD 19-specific ATAC chiBCE19-D265C-XIIB, a dose of 20mg/kg body weight (intravenous, day 0, 3, 6, 8, 10, 13) of PD-L1-specific antibody avermectin, or a combination of a dose of 0.1mg/kg body weight and a dose of 0.3mg/kg body weight, respectively (single dose, intravenous), of CD 19-specific ATAC chiBCE19-D265C-XIIB and a dose of 20mg/kg body weight (intravenous, day 0, 3, 6, 8, 10, 13) of PD-L1-specific antibody avermectin was initiated at day 0 post cell inoculation. Tumor volumes were measured twice weekly by caliper measurement and body weights were measured in parallel. When the tumor volume is>1600mm ³ At the time or when it is desired to kill the mice for ethical reasons (according to German animal welfare (German animal welfare legislation)), the animals are sacrificed and necropsies are performed.

The results of the study are shown in fig. 5. The tumor volume reduction achieved by either PD-L1-specific antibody avermectin alone or CD 19-specific ATAC chiBCE19-D265C-XIIB alone at a dose of 0.1mg/kg body weight was comparable to that of the control (PBS). However, the tumor volume reduction achieved with the combination of CD 19-specific ATAC chiBCE19-D265C-XIIb and the PD-L1-specific antibody avermectin at a dose of 0.1mg/kg body weight was significantly higher than the sum of the reductions achieved with the two individual agents. The same was observed when ATAC was used at a higher concentration of 0.3mg/kg body weight, indicating that ATAC and immune checkpoint inhibitors have a synergistic effect in tumor cell killing activity in vivo.

Table 1: average tumor volume at day 31 after tumor cell inoculation. TV: tumor volume; SEM: standard error of average value

	Average TV [ mm ³ ]	SEM
			PBS 10mL/kg	493.6	106.1
Abamectin 20mg/kg	353.1	76.3
			ATAC 0.1mg/kg	349.9	87.7
ATAC 0.1mg/kg and Avermectin 20mg/kg	165.5	26.9
			ATAC 0.3mg/kg	176.0	87.5
ATAC 0.3mg/kg and Avermectin 20mg/kg	27.4	22.1

Example 4: dependence of synergistic cytotoxic effects of ATAC and immune checkpoint inhibitors on the presence of PBMCs in vivo

Cytotoxic activity of the combination comprising ICI and ATAC was further assessed in vivo by using a tumor mouse model in the presence and absence of human Peripheral Blood Mononuclear Cells (PBMC). The study consisted of 8 experimental groups of 12 animals each. For the 4 groups, CD 19-positive Raji cells (human burkitt lymphoma, DSMZ) were premixed with human PBMC (German Red Cross) and inoculated subcutaneously on day 0. The remaining 4 groups were inoculated subcutaneously with Raji cells only. Ab-antibody specific for PD-L1-antibody alone at a dose of PBS, 0.3mg/kg body weight, or 20mg/kg body weight, starting at day 0 after cell inoculation with CD 19-specific ATAC chiBCE19-D265C-XIIB alone Or a combination of CD 19-specific ATAC chiBCE19-D265C-XIIb and the PD-L1-specific antibody Avermectin (see Table 2 for details), whereby Avermectin and CD 19-specific ATAC chiBCE19-D265C-XIIb are administered intravenously sequentially. Tumor volumes were measured twice weekly by caliper measurement and body weights were measured in parallel. When the tumor volume is>1600mm ³ At the time or when it is desired to kill the mice for ethical reasons (according to the German animal welfare method), the animals are sacrificed and necropsies are performed.

Table 2: experimental details studied in example 4

The results of the study are shown in fig. 6. In the absence of human PBMC, the combination of a dose of 0.3mg/kg body weight of CD 19-specific ATAC chiBCE19-D265C-XIIb with a dose of 20mg/kg body weight of PD-L1-specific antibody avermectin did not result in a reduction in tumor volume in vivo that was higher than that achieved by CD 19-specific ATAC chiBCE19-D265C-XIIb alone. In contrast, in the presence of human PBMC, a dose of 0.3mg/kg body weight of CD 19-specific ATAC chiBCE19-D265C-XIIb combined with a dose of 20mg/kg body weight of PD-L1-specific antibody avermectin resulted in a higher reduction in tumor volume in vivo than was achieved by CD 19-specific ATAC chiBCE19-D265C-XIIb alone.

In summary, the synergistic cytotoxic effects of ATAC and immune checkpoint inhibitors in vivo depend on the presence of human PBMCs in the mouse model used.

Example 5: efficacy of amanitine-based anti-CD 19 ATAC chiBCE19-D265C-XIIB and palbociclizumab in Raji tumor xenograft models of NOD/SCID mice reconstituted with human PBMC

Table 3: experimental details of the study of example 5

* The administration of chiBCE19-D265C-XIIb and palbociclib was performed sequentially on day 0

The study consisted of 6 experimental groups of 9 animals each. For all groups, CD19 positive Raji cells (human burkitt lymphoma, DSMZ) were premixed with human PBMC (German Red Cross) and inoculated subcutaneously on day 0. The individual CD 19-specific ATAC chiBCE19-D265C-XIIB, or the individual PD-1-specific antibody palbociclizumab, at a dose of 20mg/kg body weight, at day 0 post cell inoculation with PBS, at a dose of 0.1mg/kg and 0.3mg/kg body weight, respectively, was initiatedOr CD 19-specific ATAC chiBCE19-D265C-XIIb in combination with the PD-1-specific antibody palbociclizumab (see Table 3 for details). Tumor volumes were measured twice weekly by caliper measurement and body weights were measured in parallel. When the tumor volume is >1600mm ³ At the time or when it is desired to kill the mice for ethical reasons (according to the German animal welfare method), the animals are sacrificed and necropsies are performed. The results of the study are shown in fig. 9, which shows that the synergistic cytotoxic effect of ATAC and anti-PD-1 immune checkpoint inhibitor in vivo depends on the presence of human PBMC in the mouse model used, and that an effective dose of anti-CD 19 ATAC is required in order to induce immunogenic cell death, which is synergistic with the activity of the immune checkpoint inhibitor.

Example 6: efficacy of anti-CD 19 ATAC chiBCE19-D265C-XIIb and ipilimumab in Raji tumor xenograft model of NOD/SCID mice reconstituted with human PBMC

Table 4: experimental details of the study of example 6, all animals were vaccinated (subcutaneously) with Raji+ PBMC cells on day 0

* The administration of chiBCE19-D265C-XIIB and ipilimumab was performed sequentially on day 0

The study consisted of 6 experimental groups of 12 animals each. For all groups, CD19 positive Raji cells (human burkitt lymphoma, DSMZ) were premixed with human PBMC (German Red Cross) and inoculated subcutaneously on day 0. The CD 19-specific ATAC chiBCE19-D265C-XIIB alone or the CTLA 4-specific antibody ipilimumab alone at a dose of 4mg/kg body weight at day 0 after cell inoculation with PBS, at a dose of 0.1mg/kg and 0.3mg/kg body weight was started Or CD 19-specific ATAC chiBCE19-D265C-XIIb in combination with the CTLA 4-specific antibody ipilimumab (see Table 4 for details). When the tumor volume is>1600mm ³ Time or for ethical reasons (according to German animal welfareMethod) and the need to kill the mice, the animals are sacrificed and necropsies are performed. The results of the study are shown in figure 10, which shows the synergistic effect of survival in animals receiving CTLA4 immune checkpoint inhibitor and anti-CD 19-ATAC combination therapy (group 6). The results indicate that an effective amount of anti-CD 19 ATAC is required to induce immunogenic cell death, which works synergistically in combination with CTLA4 checkpoint inhibitors (e.g., ipilimumab), as demonstrated by the prolongation of survival of the corresponding treatment group.

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Claims

1. A composition for treating cancer comprising:

(a) At least one immune checkpoint inhibitor, and

(b) At least one conjugate, wherein the conjugate comprises

(i) A target-binding moiety that is capable of binding to a target,

(ii) At least one amatoxin, and

2. The composition for use according to claim 1, wherein the immune checkpoint inhibitor and/or the target binding moiety of the conjugate is selected from

(i) Antibodies, preferably monoclonal antibodies,

(iv) An antibody-like protein.

3. The composition for use according to claim 2, wherein the antibody or antigen-binding fragment or antigen-binding derivative thereof is a murine, chimeric, humanized or human antibody, or an antigen-binding fragment or antigen-binding derivative thereof, respectively.

4. A composition for use according to any one of claims 1 to 3, wherein the immune checkpoint inhibitor binds to an immune checkpoint receptor selected from PD-1, CTLA-4, LAG-3, TIGIT, TIM-3, VISTA, BTLA, CD and CD160, or to a ligand of an immune checkpoint receptor selected from PD-L1, PD-L2, CD80, CD86, galectin-3, lseclin, CD112, caracam-1, gal-9, ptdSer, HMGB1, HVEM, CD 155.

5. The composition for use according to any one of claims 2 to 4, wherein the immune checkpoint inhibitor is an antibody selected from the group consisting of: nivolumab, pizetimibe mab, palbociclizumab, atilizumab, avilamab, devaluzumab, cimaprevin Li Shan, and ipilimumab, or antigen-binding fragments thereof, or antigen-binding derivatives thereof, preferably wherein the antibody is avilamab, palbociclizumab, nivolumab, or ipilimumab, or antigen-binding fragments thereof, or antigen-binding derivatives thereof.

6. Composition for use according to any one of claims 1 to 5, wherein the composition comprises a combination of two or more immune checkpoint inhibitors, preferably wherein the composition comprises a combination of two immune checkpoint inhibitors.

7. The composition for use according to any one of claims 1 to 6, wherein the target binding moiety of the conjugate binds to a target molecule on the cell surface of a cancer cell selected from the group consisting of: PSMA, CD19, CD37, CD269, sialyl Lewis oligosaccharides ^a HER-2/neu, epithelial cell adhesion molecule (EpCAM).

8. The composition for use according to any one of claims 1 to 7, wherein the target-binding moiety of the conjugate is an antibody having an Fc region comprising at least one mutation selected from D265C, D265A, A C, L234A and/or L235A (according to the EU numbering system).

9. The composition for use according to claim 8, wherein the antibody comprises an Fc region carrying a D265C mutation, and wherein the linker, if present, or the amatoxin is linked to the antibody via the D265C residue of the antibody, preferably via a thioether linkage.

10. The composition for use according to any one of claims 1 to 9, wherein the amatoxin of the conjugate is selected from the group consisting of a-amanitine, β -amanitine, γ -amanitine, epsilon-amanitine, tripelen, tripelennamide, monohydroxy amanitamide, monohydroxy amanitecarboxylic acid, or salts or analogues thereof.

11. The composition for use according to any one of claims 1 to 9, wherein the linker of the conjugate, if present, is a stable or cleavable linker, and wherein the cleavable linker is selected from the group consisting of an enzymatically cleavable linker, preferably a protease cleavable linker, and a chemically cleavable linker, preferably a linker comprising a disulfide bridge.

12. The composition for use according to any one of claims 1 to 11, wherein the linker of the conjugate, if present, or the target binding moiety is linked to the amatoxin via (i) the yc atom of amatoxin amino acid 1, or (ii) the δc atom of amatoxin amino acid 3, or (iii) the 6' -C atom of amatoxin amino acid 4.

13. The composition for use according to any one of claims 1 to 12, wherein the conjugate comprises as linker-amatoxin moiety any one of the following compounds of formula (I) to (XII), respectively:

14. the composition for use according to any one of claims 1 to 12, wherein the conjugate is a compound according to any one of formulas XIII to XXII:

wherein the amatoxin-binding moiety is coupled to an epsilon-amino group of a naturally occurring lysine residue of the antibody, and wherein n is preferably 1 to 8.

15. The composition for use according to any one of claims 1 to 12, wherein the conjugate is a compound according to any one of formula XXIII, XXIV, XVIIIb, XVb, XVIb, XVIIb, XVIIIb, XIXb, XXb, XXIIb:

/>

wherein the amatoxin linking moiety is coupled to a thiol group of a cysteine residue of the antibody, and wherein 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.

16. Pharmaceutical formulation comprising a composition for use according to any one of claims 1 to 15, and further comprising one or more pharmaceutically acceptable buffers, surfactants, diluents, carriers, excipients, fillers, binders, lubricants, glidants, disintegrants, adsorbents and/or preservatives.

17. The composition for use according to any one of claims 1 to 15 or the pharmaceutical formulation according to claim 16, wherein the cancer is selected from melanoma, squamous and non-squamous non-small cell lung cancer, metastatic small cell lung cancer, renal cell carcinoma, hodgkin's lymphoma, urothelial cancer, 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 metastatic skin squamous cell carcinoma.

18. A method of treating cancer in a human subject in need thereof, wherein the method comprises administering to the subject 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 amatoxin, and (iii) optionally at least one linker linking the target binding moiety to the at least one amatoxin.

19. Use of 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 amatoxin, and (iii) optionally at least one linker linking the target binding moiety to the at least one amatoxin, for the treatment of cancer.