Amputated iga antibodies for the treatment of disease

Abstract

The present invention relates to a construct, which comprises one or more domain, preferably at most one domain that binds one or more epitope of a single antigen; and a domain that binds FcαRI, in particular for use in the treatment of diseases with low disease-associated antigen (over)expression, e.g. wherein the disease is characterized by presence of diseased cells expressing per cell between 50.000-200.000 antigen moieties. The construct may bind at most one specific antigen moiety. The present disclosure also relates to a pharmaceutical composition comprising the construct.

Description

[0001] Amputated IgA antibodies for the treatment of disease

[0002] Technical field

[0003] The present invention particularly relates to the field of constructs such as (IgA) antibodies that bind one antigen moiety, in particular for use in the treatment of diseases with low disease-associated antigen (over)expression. These antibodies find applications in research as well as in diagnostic and / or therapeutic methods e.g. immunotherapy, such as in treatment of a cancer.

[0004] Background of the invention

[0005] Monoclonal antibodies (mAb) have become the most successful class of biologies in history to fight cancer. This is due to the highly specific nature of antibodies, whereby the Fab-region specifically binds part of a novel or overexpressed epitope on tumor cells and / or other cells in the tumor microenvironment such as immune cells. Recognition of the antibody by Fc-receptor expressing cells thereby activates the immune system in a controlled fashion.

[0006] All clinically-approved antibodies are of the IgG isotype. This is partially explained by a collection of clinically attractive characteristics unavailable to other antibody isotypes, such as a long biological half-life, FcyR binding and complement binding.

[0007] One example in which IgG antibody therapy is used, is for the treatment of Hodgkin Lymphoma. Here, the malignant Hodgkin-Reed-Sternberg (HRS) cells express CD30, a suitable tumor antigen due to its almost exclusive expression on lymphoma cells. Brentuximab-vedotin, an anti-CD30 IgG antibody-drug conjugate, is an efficient immunotherapeutic against Hodgkin lymphoma. However, it can have significant side-effects, such as neurotoxicity and cytopenia due to the conjugated drug Mono-Methyl Auristatin E.

[0008] Still many types of cancer do not respond well to antibody therapy or relapses occur. This resistance can be due to:

[0009] 1) low expression of the target antigen (naturally or induced by the antibody therapy and / or other cancer therapy such as chemotherapy and / or radiotherapy);

[0010] 2) exhaustion of the immune cells;

[0011] 3) induction of anti-apoptotic proteins;

[0012] 4) upregulation of complement regulatory proteins; 5) induction of pro-tumorigenic immune cells.

[0013] Therefore, a novel immunotherapeutic intervention strategy is required to overcome the current issues with IgG-based therapeutics.

[0014] It is an objective of the current invention to provide new and / or improved immunotherapeutic intervention strategies, preferably with improved therapeutic potential for cancer therapy.

[0015] Summary of the invention

[0016] The current inventors surprisingly found that IgA antibodies with monovalent binding, particularly amputated IgA (i.e. an IgA antibody comprising only one Fab arm) may result in improved cancer treatment, in particular for tumors with low tumor-associated antigen expression, particularly with tumor-associated expression of one of the following antigens: CD30, B7H3, fibronectin, CD19, CD21, CD22, mesothelin, CD123, PDGFRb, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PDL1, PD1, CTLA4 and Her3.

[0017] Compared to the clinically approved antibodies that have been disclosed previously (for example anti-CD30 IgG antibody-drug conjugate), the current inventors surprisingly found that IgA, particularly amputated IgA (i.e. an IgA antibody comprising only one Fab arm) may result in surprisingly enhanced activation of FcaRI expressing leukocytes, such as neutrophils, monocytes and macrophages, while avoiding significant side-effects, such as neurotoxicity and cytopenia.

[0018] IgA triggers a vastly different immune response against cancer, when compared to IgG. Especially neutrophils, which are efficient in killing cancer cells, are very sensitive to IgA activation. However, treatment with regular bivalent IgA is limited by its requirement for higher target density, when compared to IgG.

[0019] To overcome this problem, the present disclosure provides amputated IgA antibodies (i.e. IgA antibodies comprising only one Fab arm) that bind only one moiety of its target, i.e. chosen from CD30, B7H3, fibronectin, CD19, CD21 , CD22, mesothelin, CD123, PDGFRb, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PDL1, PD1 , CTLA4, and Her3.

[0020] The IgA antibodies are amputated in the sense that they only have one Fab arm, i.e. the other Fab arm is amputated (i.e. an IgA antibody comprising only one Fab arm). The invention builds on the concept that one-armed IgA (i.e. IgA antibody comprising only one Fab arm) binds a single antigen, thereby effectively doubling the number of Fc-domains available for leukocyte activation, as compared to double armed IgA which binds two antigens and has only one Fc domain.

[0021] Detailed description of the invention

[0022] The present disclosure relates to a construct comprising:

[0023] - one or more domain(s) that bind(s) one (tumor specific) antigen (e.g. specifically expressed or overexpressed by (tumor) cells) preferably chosen from the group consisting of CD30, B7H3, fibronectin, CD19, CD21 , CD22, mesothelin, CD123, PDGFRb, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PDL1, PD1, CTLA4, GD2, EpCAM, Her3, Her2, VEGFR,

[0024] CD24, CD27, CD33, CD38, CD44, CD52, CD56, CD64, CD70, CD96, CD97, CD99, CD115, CD117, Her2 / neu (CD340), SLAMF7, DR5, TF, GD3, PTHR2, CD2 and EGFR; and

[0025] - a domain that binds FcaRI (CD89), wherein the construct preferably binds at most one (tumor specific) antigen moiety.

[0026] The antigen may be expressed by a tumor cell and / or any cell that may be present in a tumor microenvironment, which the skilled person will be aware of, such as immune cells, fibroblasts, endothelial cells. In case of more than one domain that bind the (tumor specific) antigen, these domains may bind different and / or overlapping epitopes of the (tumor specific) antigen. Alternatively, the one or more domain(s) that bind(s) one (tumor specific) antigen according to the present disclosure, may also be replaced by one or more domain(s) that bind(s) one or more (tumor specific) antigen (e.g. specifically expressed or overexpressed by (tumor) cells), preferably two (tumor specific) antigens, preferably chosen from the group consisting of CD30, B7H3, fibronectin, CD19, CD21 , CD22, mesothelin, CD123, PDGFRb, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PDL1, PD1 , CTLA4, GD2, EpCAM, Her3, Her2, VEGFR, CD24, CD27, CD33, CD38, CD44, CD52, CD56, CD64, CD70, CD96, CD97, CD99, CD115, CD117, Her2 / neu (CD340), SLAMF7, DR5, TF, GD3, PTHR2, CD2 and EGFR.

[0027] The construct may relate to a (combination of) binding peptide(s), comprising the recited domains. There may be one, at most one, two, at most two, or more domain(s) that bind(s) said one (tumor specific) antigen (moiety). The one or more domain(s) that bind(s) (one or more epitope of) one (tumor specific) antigen, and the domain that binds FcaRI (CD89) may be linked through any means known in the art, for example through a linker, more preferably a linker peptide and / or an enzyme-based linker. In a preferred embodiment, both the one or more domains that bind(s) the (tumor specific) antigen and the domain that binds FcaRI are binding peptides. Preferably, the one or more domains that bind(s) the (tumor specific) antigen and / or the domain that binds FcaRI is or comprises a (functional) antibody fragment. In an embodiment, the construct of the current disclosure is an antibody, preferably a (monovalent) IgA antibody. In a particularly preferred embodiment, the construct according to the current disclosure is an antibody comprising one and at most one Fab-arm, preferably an IgA antibody comprising one and at most one Fab arm. In addition or alternatively, the construct according to the current disclosure may also be a bispecific antibody and / or a biparatopic antibody which may bind e.g. two epitopes of the same antigen target. As per the current disclosure, said bispecific and / or biparatopic antibody builds on the concept of effectively doubling of the number of Fc-domains available for leukocyte activation, as compared to double armed IgA which binds two of the same antigens and / or epitopes and contains only one Fc domain.

[0028] An antibody normally comprises two heavy (H) chains and two light (L) chains, which may be inter-connected by disulfide bonds. Each heavy chain typically comprises a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region may be comprised of three domains, namely CH1 , CH2 and CH3. Each light chain may be comprised of a light chain variable region (VL) and a light chain constant region, namely CL. In a bispecific format, one arm of the antibody has a heavy chain typically comprising of CL, CH2, and CH3, and the light chain typically comprises VL and CH1 (e.g. cross-over to ensure correct pairing, i.e. CrossMab Technology) while the other follows the typical order. As used herein, a biparatopic antibody is preferably an antibody that binds two different epitopes on the same antigen. The light chain constant region typically comprises one domain, CL. The VH and VL regions can be further subdivided into regions of hyper variability, termed complementarity determining regions (CDR). These CDRs are interspersed with regions that are more conserved, named framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. An antibody may further be subdivided by its functions, wherein each Fab (antigen binding fragment) region of an antibody, which comprises one VL, CL, VH and CH1 domain, recognizes an antigen.

[0029] Herein, the term ‘functional fragment’ refers to a portion of an antibody structure, which may be smaller in size and / or may lack certain parts of the full antibody structure, yet retains the ability to bind its target. In an embodiment, the “functional fragment” is selected from the list comprising (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546), which consists of a VH domain; and (vi) an isolated CDR. Furthermore, although the two domains of the Fv fragment, VL and VH, are normally coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) ; see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibody is also intended to be encompassed within the term "functional fragment". These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. In a preferred embodiment, the construct of the current disclosure may be or comprise a monoclonal antibody, polyclonal antibody, chimeric antibody and / or (functional) fragments thereof.

[0030] In a preferred embodiment, the one or more domain(s) that bind(s) the (tumor specific) antigen is / are or comprise(s) a Fab fragment. In addition or alternatively, the domain that binds FcaRI preferably is or comprises an Fc region of an antibody, wherein the term “Fc region of an antibody” refers to a dimer complex comprising the C-terminal polypeptide sequences of an immunoglobulin heavy chain, wherein a C-terminal polypeptide sequence is that which is obtainable by papain digestion of an intact antibody.

[0031] The (tumor specific) antigen according to the current disclosure, namely preferably CD30, B7H3, fibronectin, CD19, CD21 , CD22, mesothelin, CD123, PDGFRb, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PDL1, PD1, CTLA4, GD2, EpCAM, Her3, Her2, VEGFR, CD24, CD27, CD33, CD38, CD44, CD52, CD56, CD64, CD70, CD96, CD97, CD99, CD115, CD117, Her2 / neu (CD340), SLAMF7, DR5, TF, GD3, PTHR2, CD2 and EGFR are promising tumor targets for antibody-based immunotherapy due to their specific characteristics and involvement in cancer progression. These targets have been extensively studied, leading to the development of successful therapeutic strategies.

[0032] CD30, also known as Ki-1 or TNFRSF8, is a member of the tumor necrosis factor receptor superfamily, characteristically expressed in (certain) hematopoietic malignancies, such as anaplastic large cell lymphoma and Hodgkin lymphoma. Whereas the role of glycoprotein fibronectin in tumorigenesis and malignant progression has been controversial, cancerous fibronectin is has been linked to provoking late stages of cancer metastasis and poor prognosis. The abbreviation “CD” refers to cluster of differentiation.

[0033] CD19 is a transmembrane protein belonging to the immunoglobulin (Ig) superfamily and is expressed in most acute lymphoblastic leukemias, chronic lymphocytic leukemias (CLL) and B-cell lymphomas. CD21 and CD22 are also transmembrane proteins, typically associated with B-cell lymphomas. Mesothelin is a cell surface protein identified in multiple tumor types, including lung adenocarcinomas and ovarian carcinomas. Interleukin-3 receptor alpha chain (IL-3Ra), also referred to as CD123, is overexpressed in various hematological malignancies such as acute myeloid leukemia (AML) and B-cell acute lymphoblastic leukemia. Platelet- derived growth factor Receptor b (PDGFRb) has a critical role in activating cancer-associated fibroblasts, which in turn facilitate e.g. breast cancer progression. B7 homolog 3 protein (B7H3), also known as CD276, Programmed Cell Death Protein 1 (PD-1) and cytotoxic T- lymphocyte-associated protein 4 (CTLA-4) are immune checkpoint proteins and highly expressed in cancer cells. Programmed cell death ligand 1 (PD-L1) is a transmembrane protein, commonly upregulated on the surface of tumor cells, binding to the programmed death 1 (PD-1) expressed on tumor-infiltrating lymphocytes (which may cause T-cell tolerance). Chondroitin Sulfate Proteoglycan 4 (CSPG4) has been associated with a multiple types of cancers, several examples being melanoma, breast cancer, neuroblastoma and breast cancer. Disialoganglioside GD2 is overexpressed on several solid tumors and highly expressed by e.g. brain tumors, melanomas, lung cancer and retinoblastomas. Epithelial cell adhesion molecule (EpCAM), also known as CD326, is one of the most studied tumor-specific antigens, significantly elevated in a number of human adenocarcinomas as well as squamous cell carcinomas. CD24 is a cell surface marker involved in cell adhesion and differentiation. It has been implicated in various cancers and is associated with tumor progression. CD27 is a co-stimulatory molecule on the surface of T cells. Its activation plays a crucial role in regulating immune responses and promoting the survival and expansion of T cells. CD33 is a myeloid cell surface antigen. It is commonly used as a target for antibody-based therapies in certain types of leukemia, such as acute myeloid leukemia. CD38 is a cell surface protein involved in various cellular functions, including immune response regulation. In cancer, it is often overexpressed and is a target for therapies, particularly in multiple myeloma. CD44 is a cell surface glycoprotein involved in cell adhesion and migration. It plays a role in cancer progression, particularly in metastasis and cancer stem cell function. CD52 is a glycoprotein expressed on the surface of some immune cells. It is targeted in the treatment of certain lymphomas using the monoclonal antibody alemtuzumab. CD56 is an adhesion molecule found on the surface of natural killer (NK) cells. It is used as a marker for various malignancies, including neuroendocrine tumors. CD64 is a high-affinity receptor for the Fc portion of immunoglobulin G. It is expressed on the surface of certain immune cells and is implicated in inflammatory responses. CD70 is a co-stimulatory molecule expressed on activated immune cells. Its interaction with its receptor plays a role in immune responses and is a target for cancer immunotherapy. CD96 is an immune checkpoint receptor involved in regulating T cell function. It is implicated in immune responses against cancer and is being explored as a target for immunotherapy. CD97 is a cell surface receptor involved in cell adhesion and migration. Its overexpression has been observed in various cancers, and it is associated with tumor invasion. CD99 is a cell surface glycoprotein involved in cell adhesion. It is expressed in various cancers and is associated with metastasis and tumor cell migration. CD115, also known as CSF1 R, is a receptor for colony-stimulating factor 1 (CSF-1). It plays a role in the differentiation and survival of macrophages and is implicated in cancer progression. CD117, also known as c-KIT, is a receptor tyrosine kinase. It is often mutated in gastrointestinal stromal tumors (GISTs) and is a target for therapy using tyrosine kinase inhibitors. CD274, also known as PD-L1, is an immune checkpoint protein expressed on the surface of some cancer cells. Its interaction with PD-1 on immune cells can suppress the immune response, and targeting this interaction is a strategy in cancer immunotherapy. Her2 / neu is a receptor tyrosine kinase that is overexpressed in certain breast cancers. Targeting Her2 with specific therapies has proven effective in treating Her2-positive breast cancer. SLAMF7, also known as CD319 or CS1 , is a cell surface receptor expressed on multiple myeloma cells. Monoclonal antibodies targeting SLAMF7 are used in the treatment of multiple myeloma. Death receptor 5 (DR5) is a cell surface receptor that can induce apoptosis (programmed cell death) in cancer cells. It is a target for therapies aiming to trigger apoptosis in cancer cells. Tissue factor (TF) is a cell surface protein involved in blood coagulation. It is overexpressed in various cancers and is associated with tumor angiogenesis and metastasis. GD3 is a ganglioside, a type of glycosphingolipid. It is expressed on the surface of certain cancer cells and is a target for immunotherapy in melanoma. Parathyroid hormone receptor 2 (PTHR2) is a G protein-coupled receptor. Its role in cancer is not as well-established, and it may have context-dependent functions. CD2 is a cell adhesion molecule expressed on T cells and natural killer cells. Its role in cancer is associated with immune responses and cell-to-cell interactions.

[0034] As used herein, the term ‘(tumor specific) antigen moiety’, such as of CD30, B7H3, fibronectin, CD19, CD21 , CD22, mesothelin, CD123, PDGFRb, CSPG4, PDL1 , PD1 , CTLA4, GD2, EpCAM, Her3, Her2, VEGFR, CD24, CD27, CD33, CD38, CD44, CD52, CD56, CD64, CD70, CD96, CD97, CD99, CD115, CD117, Her2 / neu (CD340), SLAMF7, DR5, TF, GD3, PTHR2, CD2 and EGFR, peptides, haptens or epitope (thereof) herein refers to a single entity of the respective antigen, e.g. single (protein) molecule of the respective antigen. Reference to an amount or density of said moieties thus refers to an amount or density in terms of entities / protein molecules (and does not refer to a number of different types of antigens).

[0035] As used herein, the terms "CD89" and "human Fc-alfa receptor I" (FcaRI) are used interchangeably and are intended to include the FCAR gene product located on human chromosome 19 (at 19q13.4). FaRI is constitutively expressed on antigen-presenting cells, such as monocytes, macrophages, eosinophils and dendritic cells, and inducible on neutrophils.

[0036] As used herein, the term ‘bind(s)’ encompasses both partial and complete binding of the one or more domain(s) that preferably bind(s) the tumor specific antigen and / or the domain that binds FcaRI. Herein, ‘bind(s)’ is intended to include all types of binding mechanisms known in the art, including but not limited to hydrogen bonds, van der Waals forces, ionic bonds and / or disulfide bonds.

[0037] In a preferred embodiment, the one or more domain(s) that bind(s) the (tumor specific) antigen(s) and / or the domain that binds FcaRI, bind specifically and / or preferentially to their target(s) i.e. with greater affinity (and / or avidity) in comparison to their binding to epitopes on other polypeptides, such as with an affinity as determined by a dissociation constant KD lower than 5x10-9M, more preferably lower than 1x10-9M, even more preferably lower than 8x10'1° M, even more preferably lower than 6x1 O'10M, yet even more preferably lower than 4x1 O'10M, yet even more preferably lower than 2x1 O'10M, yet even more preferably lower than 1x1 O'10M, yet even more preferably lower than 5x1 O'11M or lower than 1x1 O'11M, most preferably lower than 5x10'12M or lower than 1x10'12M.

[0038] Methods to determine affinity and avidity are well-known in the art and may for example comprise: enzyme-linked immunosorbent assay (ELISA), flow cytometry, surface plasmon resonance, biacore assay, equilibrium dialysis, biolayer interferometry. The binding affinity as referred to herein can be represented by the equilibrium constant for the dissociation (KD). KD is a measure for binding strength and represents the concentration of antigen at which half of the available binding sites on a target e.g. the domain(s) of the current disclosure is / are occupied. It is calculated by dividing the rate constant for dissociation (koff) by the rate constant for association (kon), and is expressed in units of molarity (M). A lower KD indicates a higher affinity of e.g. the domains(s) for the antigen.

[0039] As will be clear to a skilled person, there are several methods to determine the KD of a construct and / or a domain, such as the construct and / or the domain(s) that bind(s) the (tumor specific) antigen and / or the domain that binds FcaRI. For example, the KD of one of these domains for a tumor specific antigen(s) and / or FcaRI may be determined experimentally using LigandTracer, e.g. by using live cells (expressing the ligand, or target) in 3 mL of (RPMI 1640) medium (obtainable from Merck), and adding (microliters of) antibody directly and performing dissociation analysis through refreshing the medium, or through a process called binding affinity analysis, wherein the general process comprises the steps of: Preparing a range of concentrations of the tumor specific antigen and / or FcaRI in a buffer solution;

[0040] Preparing a fixed concentration of the domain in the same buffer solution;

[0041] Mixing of each concentration of the tumor specific antigen and / or FcaRI with the fixed concentration of the domain;

[0042] Incubating the mixture for a specific period of time to allow binding to occur;

[0043] Separating the unbound tumor specific antigen and / or FcaRI from the bound domain using a technique such as filtration or centrifugation;

[0044] Measuring the amount of bound tumor specific antigen and / or FcaRI in each sample, typically by using a labeled ligand or by detecting the amount of unbound tumor specific antigen and / or FcaRI;

[0045] Plotting the data as the fraction of bound domain versus the concentration of the tumor specific antigen and / or FcaRI;

[0046] Using a mathematical model, such as the Langmuir isotherm or the Scatchard plot, to determine the KD value from the binding data.

[0047] In an embodiment, the construct according to the current disclosure comprises one, two or more domains that binds one (and at most one moiety) of (tumor specific) antigen preferably chosen from CD30, CD37, B7H3, fibronectin, CD19, CD21 , CD22, mesothelin, CD123, PDGFRb, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PDL1, PD1, CTLA4, Her3, Her2, VEGFR CD24, CD27, CD33, CD38, CD44, CD52, CD56, CD64, CD70, CD96, CD97, CD99, CD115, CD117, Her2 / neu (CD340), SLAMF7, DR5, TF, GD3, PTHR2, CD2 and EGFR, wherein preferably said two or more domains bind different and / or non-overlapping epitopes of said one (at most one moiety) of (tumor specific) antigen preferably CD30, B7H3, fibronectin, CD19, CD21 , CD22, mesothelin, CD123, PDGFRb, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PDL1, PD1, CTLA4, GD2, EpCAM, Her3, Her2, VEGFR, CD24, CD27, CD33, CD38, CD44, CD52, CD56, CD64, CD70, CD96, CD97, CD99, CD115, CD117, Her2 / neu (CD340), SLAMF7, DR5, TF, GD3, PTHR2, CD2 and EGFR.

[0048] In an embodiment, the (one, two or more) domain(s) that binds FcaRI (each) is or is comprised in an IgA crystallizable fragment (Fc), or an IgA crystallizable fragment (Fc) CH2 domain.

[0049] In an embodiment, the one or more domain(s) that bind(s) one (tumor specific) antigen preferably chosen from CD30, CD37, B7H3, fibronectin, CD19, CD21 , CD22, mesothelin, CD123, PDGFRb, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PDL1, PD1, CTLA4, GD2, EpCAM, Her3, Her2, VEGFR, CD24, CD27, CD33, CD38, CD44, CD52, CD56, CD64, CD70, CD96, CD97, CD99, CD115, CD117, Her2 / neu (CD340), SLAMF7, DR5, TF, GD3, PTHR2, CD2 and EGFR and the domain that that binds FcaRI are connected via a linker, wherein the linker is natural or synthetic, wherein the linker is preferably chosen from IgA antibody hinge region, linker peptide, and enzyme-based linker.

[0050] In an embodiment, the one or more domain(s) that bind(s) one (tumor specific) antigen preferably e.g. chosen from CD30, CD37, B7H3, fibronectin, CD19, CD21 , CD22, mesothelin, CD123, PDGFRb, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PDL1, PD1, CTLA4, GD2, EpCAM, Her3, Her2, VEGFR, CD24, CD27, CD33, CD38, CD44, CD52, CD56, CD64, CD70, CD96, CD97, CD99, CD115, CD117, Her2 / neu (CD340), SLAMF7, DR5, TF, GD3, PTHR2, CD2 and EGFR (each) is or comprises a single domain antibody, a ligand or a ligand-mimic. Preferably, the single domain antibody is a Variable Heavy domain of Heavy chain (VHH) antibody, also known in the art as a nanobody.

[0051] In an embodiment, the construct of the invention may be used as a medicament.

[0052] The current disclosure further relates to use in prevention or treatment of disease, preferably cancer, wherein the disease is characterized by presence of (diseased / cancer) cells expressing per cell between 50.000-200.000 of (tumor specific) antigen moieties preferably chosen from CD30, CD37, B7H3, fibronectin, CD19, CD21 , CD22, mesothelin, CD123, PDGFRb, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PDL1, PD1, CTLA4, GD2, EpCAM, Her3, Her2, VEGFR, CD24, CD27, CD33, CD38, CD44, CD52, CD56, CD64, CD70, CD96, CD97, CD99, CD115, CD117, Her2 / neu (CD340), SLAMF7, DR5, TF, GD3, PTHR2, CD2 and EGFR.

[0053] In a preferred embodiment, the construct according to the disclosure may be used in the prevention or treatment of disease, preferable cancer, wherein the disease is preferably characterized by presence of (diseased / cancer) cells expressing per cell preferably (on average) between 15.000-300.000, more preferably between 25.000-250.000, even more preferably between 30.000-200.000, yet even more preferably between 40.000-200.000, yet even more preferably between 50.000-200.000, yet even more preferably between 60.000- 200.000, yet even more preferably between 60.000-150.000, yet even more preferably between 70.000-120.000, most preferably between 75.000-100.000 (tumor specific) antigen moieties, e.g. chosen of one of CD30, B7H3, fibronectin, CD19, CD21, CD22, mesothelin, CD123, PDGFRb, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PDL1, PD1, CTLA4, Her3, Her2, VEGFR, CD24, CD27, CD33, CD38, CD44, CD52, CD56, CD64, CD70, CD96, CD97, CD99, CD115, CD117, Her2 / neu (CD340), SLAMF7, DR5, TF, GD3, PTHR2, CD2 and EGFR. In a preferred embodiment, the construct according to the disclosure may be used in the prevention or treatment of disease, preferable cancer, wherein the disease is preferably characterized by presence of (diseased / cancer) cells expressing per cell preferably (on average) at least 20.000, 30.000, 40.000, 50.000, 60.000, 70.000, 80.000, 90.000, 100.000, 120.000 (tumor specific) antigen moieties, e.g. chosen of one of CD30, B7H3, fibronectin, CD19, CD21, CD22, mesothelin, CD123, PDGFRb, CSPG4, PDL1 , PD1, CTLA4, Her3, Her2, CD24, CD27, CD33, CD38, CD44, CD52, CD56, CD64, CD70, CD96, CD97, CD99, CD115, CD117, Her2 / neu (CD340), SLAMF7, DR5, TF, GD3, PTHR2, CD2, VEGFR and EGFR. In yet another preferred embodiment, the construct according to the disclosure may be used in the prevention or treatment of disease, preferably cancer, wherein the disease is preferably characterized by presence of (diseased / cancer) cells expressing per cell preferably on average at most 500.000, 400.000, 300.000, 250.000, 100.000, 75.000, 50.000 (tumor specific) antigen moieties, e.g. chosen of one of CD30, B7H3, fibronectin, CD19, CD21, CD22, mesothelin, CD123, PDGFRb, CSPG4, PDL1 , PD1 , CTLA4, GD2, EpCAM, Her3, Her2, VEGFR, CD24, CD27, CD33, CD38, CD44, CD52, CD56, CD64, CD70, CD96, CD97, CD99, CD115, CD117, Her2 / neu (CD340), SLAMF7, DR5, TF, GD3, PTHR2, CD2 and EGFR. Also foreseen, is the construct according to the disclosure may be used in the prevention or treatment of cancer, preferably in combination with (e.g. simultaneously, sequentially, and / or after other cancer treatments known in the art e.g. chemotherapy and / or radiotherapy).

[0054] In a preferred embodiment, the construct according to the disclosure may be used in the prevention or treatment of disease, preferably cancer, wherein:

[0055] - in the use the ratio between the number of (tumor specific) antigen moieties that are bound by said construct and the number of constructs that are bound to said (tumor specific) antigen moieties is preferably substantially equimolar, preferably between 1:4 and 4:1 , more preferably between 1:3 and 3:1, even more preferably between 1:2 and 2:1, yet even more preferably between 1.5:1 and 1:1.5, most preferably (about) 1:1; and / or

[0056] - in the use, for at least 99%, 95%, 90%, 85%, 80% 75%, 70%, 65%, 60% of (tumor specific) antigen moieties that are bound, the construct binds said (tumor specific) antigen moieties via monovalent binding.

[0057] In an embodiment, the construct according to the current disclosure may be used in prevention and / or treatment of cancer, preferably leukemia, multiple myeloma, lymphoma, breast cancer, head and neck cancer, lung cancer, colorectal cancer, prostate cancer, skin cancer, bladder cancer, (non-)Hodgkin lymphoma, kidney cancer, pancreatic cancer, liver cancer, ovarian cancer, brain and central nervous system (CNS) tumor, stomach cancer, or esophageal cancer.

[0058] In a preferred embodiment, the construct according to the current disclosure may be used in prevention and / or treatment of disease, with preferably one or more of the following disease I (tumor specific) antigen combinations: In a particularly preferred embodiment, the construct according to the current disclosure may be used in prevention and / or treatment of disease, wherein:

[0059] - the disease is breast cancer, and the antigen is HER2;

[0060] - the disease is colorectal cancer, and the antigen is EGFR, ALPP and / or GLICY2C;

[0061] - the disease is pancreatic cancer, and the antigen is EGFR, and / or EpCAM;

[0062] - the disease is ovarian cancer, and the antigen is EGFR and / or FOLR1;

[0063] - the disease is GD2-positive tumor (excluding neuroblastoma), and the antigen is GD2, wherein the GD2-positive tumor is preferably chosen from bladder cancer, lung cancer, melanoma, sarcoma, osteosarcoma, rhabdomyosarcoma, and Ewing sarcoma;

[0064] - the disease is testicular, ovarian, uterine, or lung adenocarcinoma, and the antigen is

[0065] CLDN6;

[0066] - the disease is anaplastic meningioma, and the antigen is CD276;

[0067] - the disease is metastatic renal cell carcinoma, and the antigen is CA9;

[0068] - the disease is hepatocellular carcinoma, and the antigen is GPC3;

[0069] - the disease is glioblastoma, and the antigen is PDPN, HER2, and / or IL13RA2;

[0070] - the disease is pancreatic carcinoma, and the antigen is PODXL;

[0071] - the disease is triple-negative breast cancer, and the antigen is ANTXR1.

[0072] - the disease is gastric cancer, and the antigen is CLDN18;

[0073] - the disease is glioblastoma or pancreatic carcinoma, and the antigen is EGFR;

[0074] - the disease is prostate cancer, and the antigen is FOLH1 and / or PSCA,

[0075] - the disease is lung cancer, and the antigen is PTK7;

[0076] - the disease is gastric cancer or pancreatic carcinoma, and the antigen is MSLN;

[0077] - the disease is MLIC1 -positive cancer, and the antigen is MUC1.

[0078] These diseases are examples of the diseases characterized by presence of (diseased / cancer) cells expressing per cell preferably (on average) between 15.000-300.000, more preferably between 25.000-250.000, even more preferably between 30.000-200.000, yet even more preferably between 40.000-200.000, yet even more preferably between 50.000-200.000, yet even more preferably between 60.000-200.000, yet even more preferably between 60.000-150.000, yet even more preferably between 70.000-120.000, most preferably between 75.000-100.000 antigen (moieties).

[0079] HER2 (Human Epidermal Growth Factor Receptor 2): A receptor tyrosine kinase overexpressed in some breast cancers and glioblastomas, promoting tumor growth and survival. EGFR (Epidermal Growth Factor Receptor): A receptor tyrosine kinase involved in cell proliferation, commonly overexpressed in colorectal, pancreatic, ovarian, and glioblastoma cancers.

[0080] ALPP (Alkaline Phosphatase Placental): An enzyme expressed in colorectal cancer and other cancers, associated with tumor growth and metastasis.

[0081] GUCY2C (Guanylate Cyclase 2C): A receptor involved in intestinal homeostasis, often overexpressed in colorectal cancer.

[0082] EpCAM (Epithelial Cell Adhesion Molecule): A glycoprotein promoting cell adhesion and proliferation, highly expressed in pancreatic and other epithelial cancers.

[0083] FOLR1 (Folate Receptor Alpha): A receptor involved in folate transport, often overexpressed in ovarian cancer.

[0084] GD2 (Disialoganglioside): A glycolipid present on the surface of neuroectoderm-derived tumors such as melanoma, osteosarcoma, and rhabdomyosarcoma.

[0085] CLDN6 (Claudin-6): A tight junction protein associated with testicular, ovarian, uterine, and lung adenocarcinomas.

[0086] CD276 (B7-H3): An immune checkpoint protein overexpressed in anaplastic meningioma and other tumors, suppressing T-cell activation.

[0087] CA9 (Carbonic Anhydrase IX): An enzyme regulating pH, overexpressed in metastatic renal cell carcinoma and promoting tumor survival.

[0088] GPC3 (Glypican-3): A cell surface proteoglycan overexpressed in hepatocellular carcinoma, involved in tumor cell signaling and growth.

[0089] PDPN (Podoplanin): A glycoprotein associated with glioblastoma, promoting tumor cell migration and invasion.

[0090] IL13RA2 (Interleukin-13 Receptor Alpha 2): A receptor overexpressed in glioblastomas, acting as a decoy to promote tumor growth.

[0091] PODXL (Podocalyxin): A sialomucin overexpressed in pancreatic carcinoma, associated with enhanced tumor cell invasiveness.

[0092] ANTXR1 (Anthrax Toxin Receptor 1): A protein overexpressed in triple-negative breast cancer, mediating cell adhesion and proliferation.

[0093] CLDN18 (Claudin-18): A tight junction protein specific to gastric cancer, promoting tumor growth and metastasis.

[0094] PSCA (Prostate Stem Cell Antigen): A glycoprotein associated with prostate cancer, involved in cell adhesion and tumor progression.

[0095] PTK7 (Protein Tyrosine Kinase 7): A pseudokinase overexpressed in lung cancer, associated with cell migration and invasion.

[0096] MSLN (Mesothelin): A cell surface protein overexpressed in gastric and pancreatic cancers, promoting tumor invasion. MUC1 (Mucin 1): A glycoprotein overexpressed in MUCI-positive cancers, contributing to tumor growth and immune evasion.

[0097] Methods for Determining Antigen Expression

[0098] 1. Flow Cytometry (Fluorescence-Activated Cell Sorting, FACS)

[0099] Flow cytometry is a widely used method to quantify antigen expression on individual cells by labeling the antigens with fluorescently conjugated antibodies.

[0100] Example protocol:

[0101] - Prepare a single-cell suspension from the diseased tissue or cancer cell culture.

[0102] - Stain the cells with a fluorescently labeled monoclonal antibody specific to the target antigen.

[0103] - Include a known quantity of calibration beads with a defined number of fluorophores or antigen-binding sites (e.g., Quantibrite beads).

[0104] - Analyze the samples using a flow cytometer, determining the median fluorescence intensity (MFI).

[0105] - Use calibration beads to correlate MFI with the number of antigen molecules per cell.

[0106] 2. Quantitative Immunoassay, e.g. Enzyme-Linked Immunosorbent Assay (ELISA)

[0107] - Homogenize the cells or tissue sample and extract surface proteins.

[0108] - Perform an ELISA using an antibody specific to the target antigen.

[0109] - Compare the signal to a standard curve generated using known quantities of the antigen.

[0110] - Estimate the number of antigens per cell based on total protein quantity and cell count.

[0111] 3. Immunohistochemistry (IHC):

[0112] IHC can semi-quantitatively assess antigen density on cells within tissue sections.

[0113] Example protocol:

[0114] - Stain tissue sections with an antibody specific to the antigen of interest.

[0115] - Use image analysis software to quantify the staining intensity and estimate antigen expression levels.

[0116] - Calibrate the method using controls with known antigen expression.

[0117] In another particularly preferred embodiment, the construct according to the current disclosure may be used in prevention and / or treatment of disease, wherein:

[0118] - the disease is Hodgkin lymphoma, peripheral T-cell lymphoma, cutaneous T cell lymphoma, CD30-expressing B-cell lymphoma, or anaplastic large cell lymphoma, and the antigen is CD30;

[0119] - the disease is neuroblastoma, osteosarcoma, cervical cancer, or medulloblastoma, and the antigen is B7-H3; - the disease is neuroblastoma, breast cancer, teratocarcinoma, ovarian cancer, or metastasized tumor, and the antigen is fibronectin;

[0120] - the disease is B-cell lymphoma, acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL), and the antigen is CD19;

[0121] - the disease is lymphoma, diffuse large B-cell lymphoma, or non-Hodgkin’s lymphoma, and the antigen is CD21;

[0122] - the disease is B-cell acute lymphoblastic leukemia (B-ALL) or B-cell non-Hodgkin lymphoma (B-cell NHL), and the antigen is CD22;

[0123] - the disease is mesothelioma, lung adenocarcinoma, ovarian adenocarcinoma, or pancreatic adenocarcinoma, and the antigen is mesothelin;

[0124] - the disease is a hematological malignancy, including acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (B-ALL), hairy cell leukemia, Hodgkin lymphoma, or blastic plasmacytoid dendritic neoplasm (BPDCN), and the antigen is CD123;

[0125] - the disease is associated with cancer-associated fibroblasts, non-small cell lung carcinoma, prostate cancer, PDGFRB-associated chronic eosinophilic leukemia, colorectal cancer, glioma, testis cancer, breast cancer, endometrial cancer, ovarian cancer, or melanoma, and the antigen is PDGFRP;

[0126] - the disease is melanoma, lung cancer, colon cancer, head and neck cancer, liver cancer, pancreatic ductal adenocarcinoma (PDAC), renal cell carcinoma (RCC), oligodendroglioma, glioma, triple-negative breast carcinoma, or squamous cell carcinoma, and the antigen is CSPG4;

[0127] - the disease is lung cancer, breast cancer, or melanoma, and the antigen is PD-L1 ;

[0128] - the disease is breast cancer, ovarian cancer, lung cancer, colon cancer, pancreatic cancer, gastric cancer, head and neck cancer, or prostate cancer, and the antigen is HER3.

[0129] These diseases are examples of the diseases characterized by presence of (diseased / cancer) cells expressing per cell preferably (on average) between 15.000-300.000, more preferably between 25.000-250.000, even more preferably between 30.000-200.000, yet even more preferably between 40.000-200.000, yet even more preferably between 50.000-200.000, yet even more preferably between 60.000-200.000, yet even more preferably between 60.000-150.000, yet even more preferably between 70.000-120.000, most preferably between 75.000-100.000 (moieties).

[0130] PD1 and CTLA4 are also expressed on regulatory T cells, that support tumoroutgrowth. Targeting them with a construct according to the present invention could eradicate them more efficiently than with a bivalent antibody, leading to better treatment of the tumor. Accordingly, the construct according to the current disclosure may be used in prevention and / or treatment of disease, preferably cancer, wherein the antigen is PD1 and / or CTLA4, preferably PD1 and / or CTLA4 (over) expressed on regulatory T cells.

[0131] The treatment may encompass administering the construct according to the present disclosure to a patient in need thereof. The patient may be a patient who has already received immunotherapy and became or is resistant to said immunotherapy. The term “resistant” in this regard means that the treatment is not (sufficiently) effective and / or does not stabilize or reduce tumor size.

[0132] The current disclosure further relates to a pharmaceutical composition comprising the construct according to the current disclosure. Herein, a pharmaceutical composition refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgement, suitable e.g. non-toxic and / or do not lead to allergic response and / or irritation when in contact with tissues of (most) human beings and animals. The pharmaceutical composition may also comprise any other therapeutically effective agents and / or other agents, such as a pharmaceutically acceptable solvent, diluent, carrier, buffer, excipient, adjuvant, carrier medium, antiseptic, filling, stabilizing or thickening agent.

[0133] Some examples of other (pharmaceutically acceptable) agents include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15 ca) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0134] The pharmaceutical composition according to the disclosure may be in any form suitable for administration, for example a solid, semisolid or liquid form. A formulation can be selected from the list consisting of, but not limited to, powder, solutions, emulsions, suspensions, spray, tablets, pellets and capsules. The pharmaceutical compositions may be produced by any conventional processes known in the art, which the skilled person will be aware of. Preferably, the pharmaceutical composition according to the disclosure is for administration by systemic route, for example by intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous or oral route. More preferably, the composition comprising the antibody or fragment thereof of the invention can be administered in separate doses that are spaced over time. Their administration routes, dosing schedules and optimal galenic forms can be determined according to the criteria generally taken into account when establishing a treatment suited to a patient such as, for example, the patient's age or body weight, the seriousness of the patient’s general state, tolerance for the treatment and the side effects experienced.

[0135] The current disclosure further relates to a nucleic acid sequence coding for the construct according to the current disclosure, particularly comprising nucleic acid sequences encoding the one or more domain(s) that bind(s) one (tumor specific) antigen preferably chosen from CD30, CD37, B7H3, fibronectin, CD19, CD21 , CD22, mesothelin, CD123, PDGFRb, CSPG4, PDL1 , PD1 , CTLA4, GD2, EpCAM, Her3, Her2, VEGFR, CD24, CD27, CD33, CD38, CD44, CD52, CD56, CD64, CD70, CD96, CD97, CD99, CD115, CD117, Her2 / neu (CD340), SLAMF7, DR5, TF, GD3, PTHR2, CD2 and EGFR and / or the domain that binds FcaRI as disclosed herein. The term "nucleic acid sequence", as used herein, is intended to include but does not exclusively relate to DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but preferably refers to double-stranded DNA.

[0136] In the foregoing description and below examples, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided. Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0137] Clauses

[0138] 1. A construct comprising:

[0139] - one or more domain(s) that bind(s) one tumor specific antigen chosen from CD30, CD37, B7H3, fibronectin, CD19, CD21 , CD22, mesothelin, CD123, PDGFRb, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PDL1, PD1, CTLA4 and Her3; and

[0140] - a domain that binds FcaRI, wherein the construct binds at most one tumor specific antigen moiety.

[0141] 2. A construct according to clause 1 , wherein the one or more domain(s) that bind(s) one tumor specific antigen chosen from CD30, CD37, B7H3, fibronectin, CD19, CD21 , CD22, mesothelin, CD123, PDGFRb, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PDL1 , PD1, CTLA4 and Her3 is a fragment antigen-binding (Fab) domain. 3. A construct according to any of the previous clauses, comprising two or more domains that bind one of CD30, CD37, B7H3, fibronectin, CD19, CD21, CD22, mesothelin, CD123, PDGFRb, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PDL1, PD1, CTLA4, and Her3, wherein said two or more domains bind different and / or non-overlapping epitopes of said one of CD30, B7H3, fibronectin, CD19, CD21, CD22, mesothelin, CD123, PDGFRb, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PDL1, PD1, CTLA4 and Her3.

[0142] 4. Construct according to any of the previous clauses, wherein the domain that binds FcaRI is or is comprised in an IgA crystallizable fragment (Fc) CH2 domain.

[0143] 5. Construct according to any of the previous clauses, wherein the domain that binds FcaRI is or is comprised in an IgA crystallizable fragment (Fc).

[0144] 6. Construct according to any of the previous clauses, wherein the construct is an antibody comprising at most one Fab-arm, preferably an IgA antibody comprising at most one Fab arm.

[0145] 7. Construct according to any of the previous clauses, wherein the one or more domain(s) that bind(s) one tumor specific antigen chosen from CD30, CD37, B7H3, fibronectin, CD19, CD21, CD22, mesothelin, CD123, PDGFRb, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PDL1 , PD1 , CTLA4 and Her3 and the domain that that binds FcaRI are connected via a linker, wherein the linker is natural or synthetic, wherein the linker is preferably chosen from IgA antibody hinge region, linker peptide, and enzyme-based linker.

[0146] 8. Construct according to any of the previous clauses, wherein the one or more domain(s) that bind(s) one tumor specific antigen chosen from CD30, CD37, B7H3, fibronectin, CD19, CD21, CD22, mesothelin, CD123, PDGFRb, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PDL1 , PD1 , CTLA4 and Her3 is or comprises a nanobody, a ligand or a ligand-mimic.

[0147] 9. Construct according to any one of the previous clauses, for use as medicament.

[0148] 10. Construct according to any of the previous clauses, for use in prevention or treatment of disease, preferably cancer, wherein the disease is characterized by presence of diseased cells expressing per cell between 50.000-200.000 of tumor specific antigen moieties chosen from CD30, CD37, B7H3, fibronectin, CD19, CD21 , CD22, mesothelin, CD123, PDGFRb, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PDL1, PD1 , CTLA4 and Her3. 11. Construct for use according to clause 10, wherein

[0149] - in the use the ratio between the number of tumor antigen moieties that are bound by said construct and the number of constructs that are bound to said tumor antigen moieties is substantially equimolar , preferably between 1:4 and 4:1 , most preferably 1:1; and / or

[0150] - in the use, for at least 80% of tumor antigen moieties that are bound, the construct binds said tumor antigen moieties via monovalent binding.

[0151] 12. Construct according to any one of the previous clauses, for use in prevention or treatment of cancer, preferably leukemia, multiple myeloma, lymphoma, breast cancer, head and neck cancer, lung cancer, colorectal cancer, prostate cancer, skin cancer, bladder cancer, (non-)Hodgkin lymphoma, kidney cancer, pancreatic cancer, liver cancer, ovarian cancer, brain and central nervous system (CNS) tumor, stomach cancer, or esophageal cancer.

[0152] 13. Pharmaceutical composition comprising the construct according to any one of the previous clauses.

[0153] 14. Nucleic acid sequence coding for the construct according to any one of the previous clauses.

[0154] Brief description of the figures

[0155] Figure 1 : Schematic overview of IgA comprising one Fab-arm. IgA comprising one Fab-arm binds a single antigen, thereby effectively doubling the number of Fc-domains available for leukocyte activation.

[0156] Figure 2: Binding of tumor cells by IgA and IgA comprising one Fab-arm, on cells with high expression (IMR32) and cells with low-medium expression of tumor target (SKNAS and SKNFI)

[0157] Figure 3: In vitro antibody-dependent cellular cytotoxicity (ADCC) by IgA and IgA comprising one Fab-arm.

[0158] Figure 4: In vivo killing of tumor cells by IgA and IgA comprising one Fab-arm

[0159] Figure 5: Binding of tumor cells by anti-CD30 IgA and IgA comprising one Fab-arm, on cells with CD30 expression

[0160] Figure 6: In vivo killing of tumor cells by IgA and IgA comprising one Fab-arm, targeting CD30

[0161] Figure 7: In vivo killing of tumor cells by IgA and IgA comprising one Fab-arm, targeting HER2

[0162] Figure 8: In vivo killing of tumor cells by IgA and IgA comprising one Fab-arm, targeting EGFR

[0163] Figure 9: In vivo killing of tumor cells by IgA and IgA comprising one Fab-arm, targeting EpCAM

[0164] Figure 10: ADCC of A431 cells by NK cells (effector / target 5:1) and neutrophils (effector / target 50:1) in the presence of 1 pg / mL TrisomAb, IgA, and IgG a-EGFR antibodies. CellTiter-Blue Cell Viability Assay was performed after 24 hours.

[0165] Figure 11: Comparison of lgG1 / 4 bsAbs with silencing mutations and control antibodies for the induction of ADCC, CDC and C5a-generation. (A) Induction of ADCC by lgG1 / 4 bsAbs with silencing mutations, ch14.18 (K322A) and ch14.18-lgA3.0 against neuroblastoma cell line GIMEN-GD2 with isolated PMNs (E:T ratio of 40:1) as effector cells after incubation for 4 hours at 37°C.

[0166] Examples

[0167] The following Examples illustrate the different embodiments of the invention.

[0168] Example 1 : Binding of IgA to and lysis of CD30+ Hodgkin cells

[0169] Methods

[0170] (A) Anti-CD30 IgG 1 and IgA were produced in Expi-CHO cells, and purified with protein A for lgG1 (1. profile), with Kappa select (2. profile) and SEC (3. profile) for IgA (yield after purification: 2.9 mg for lgG1, and 6.4 mg for IgA).

[0171] Results

[0172] Anti-CD30 IgA and I gG 1 bound specifically to the CD30+ Hodgkin cell line L-428, but not to the CD30- cell line A431 (antibody concentration: 0.1 ug / ml) when assessed by flow Cytometry. Isotype controls did not bind to both cell lines. AntiCD30 IgA and anti-CD30 IgG- mediated lysis of L-428 cells was induced and not obtained by isotype controls (anti-HER2 antibodies) or in absence of antibodies (n=2).

[0173] In sum, the IgA antibodies can bind to CD30-expressing Hodgkin Lymphoma cells and induce neutrophil- and PBMC-dependent cellular toxicity.

[0174] Example 2: The effect of single-armed IgA on binding, in vitro antibody-dependent cellular cytotoxicity (ADCC) and in vivo killing of tumor cells

[0175] Figure 1 shows the concept according to this Example. One-armed IgA (i.e. IgA antibody comprising one Fab arm) binds a single antigen. Only in low-medium tumor target expressing cells, the binding is increased, thereby effectively doubling the number of Fc-domains available for leukocyte activation, as compared to double armed IgA.

[0176] Methods

[0177] The binding of IgA or single-armed IgA (i.e. IgA antibody comprising one Fab arm) to targetexpressing cancer cell lines was assessed using flow cytometry. To evaluate ADCC, healthy donor polymorphonuclear leukocytes were assessed using a 51-chromium release assay. In vivo killing of tumor cells was studied by injecting 5x10e6 CellTrace violet-labelled targetexpressing cells intraperitoneally in human FcaR transgenic Balb / C or C57B / L6 mice. Subsequently, the mice were injected with 0.3125 mmol of antibody. After 16 hours, peritoneal lavage was performed and the remaining tumor cells were quantified using flow cytometry using TrueCount beads.

[0178] Results

[0179] Increased binding of one-armed IgA antibodies was observed compared to unmodified IgA in low-medium tumor target expressing cells (Figure 2). In this case, the target is B7H3. Using neutrophils from healthy donors as effector cells, one-armed IgA showed increased killing against target-expressing cells (Figure 3). Finally, one-armed IgA showed increased killing in vivo in a short IP model against target-expressing cells (Figure 4).

[0180] In sum, single-armed IgA shows increased binding, in vitro ADCC and in vivo killing of targetexpressing cells.

[0181] Example 3: The effect of single-armed IgA on CD30 expressing tumor cells

[0182] Methods

[0183] The binding of IgA or single-armed IgA (i.e. IgA antibody comprising one Fab arm) to targetexpressing cancer cell lines was assessed using flow cytometry. In vivo killing of tumor cells was studied by injecting 5x10e6 CellTrace violet-labelled target-expressing cells intraperitoneally in human FcaR transgenic C57B / L6 mice. Subsequently, the mice were injected with 0.625 mmol of antibody. After 16 hours, peritoneal lavage was performed and the remaining tumor cells were quantified using flow cytometry using TrueCount beads.

[0184] Results

[0185] Anti-CD30 IgA and single-armed IgA show binding to CD30 expressing tumor cell lines (L- 428, L-540, Jurkat, TALL1) when assessed by flow cytometry. (Figure 5) The binding is increased for the single-armed IgA compared to IgA. Specific killing of the CD30 expressing tumor cells was observed in vivo for the single armed IgA anti-CD30 (Figure 6)

[0186] In summary, the data support the notion that CD30 can be used as a target for IgA. Also, the CD30 monovalent IgA is capable of killing CD30 positive target cells (preliminary data).

[0187] Example 4: In vivo killing of single-armed IgA directed against multiple targets

[0188] Results

[0189] The single-armed antibody showed in vivo killing in a short IP model against multiple tumor target-expressing cell lines. For example HER2 (Figure 7), EGFR (Figure 8) and EpCAM (Figure 9). An isotype control antibody does not affect the tumor cells. Neutrophils were attracted to the tumor location for the single-armed antibody.

[0190] In summary, optimal killing was seen of both bivalent and monovalent (single armed) IgA in in vivo models, except for pertuzumab IgA, which shows suboptimal killing as a bivalent IgA molecule, but maximal killing as a one-armed molecule.

[0191] Example 5

[0192] In the present invention, having an Fc portion of IgA gives the unique property to bind and activate CD89 on neutrophils.

[0193] In contrast, with only Fab binding to a neutrophil (as in the prior art), the activation is not as strong. This is because the Fc portion of IgA binds in a 1:2 stoichiometry to its receptor CD89, e.g. explained in Brandsma et al, Front Immunol 2019, 11:10:704. Another drawback is that the arm binding to CD89 is with high affinity. When such an antibody would be injected in patients, is will bind immediately to all neutrophils, which present 50-70 % of their circulating leucocytes, potentially leading to dangerous activation of these cels, and loss of antibody, leading to short half life. Also, in bispecific molecules with an intact IgG Fc portion, this portion can bind to other immune cells such as NK cells, with the risk that they can killed the neutrophils bound by the Fab and vice versa (fratercide).

[0194] In Heemskerk (J Clin Invest. 2021;131(6):e134680), a bispecific antibody with one arm to EGFR, one arm to CD89 (receptor for IgA) and an intact IgG Fc tail was used to kill A431 cells expressing EGFR. Neutrophils from CRC patients are used in ADCC, and in all concentrations IgA outperforms the bispcific antibody, probably because IgA can bind 2 CD89 receptors, whereas the bispcific can bind only 1, see Figure 10. In Figure 11 , IgA (far right column set) outperforms all bispecific formats targeting GD2 in an ADCC assay. The wt IgG 1 based bispecific is the best option after IgA, but all silencing mutations (needed to prevent fratercide) even further decrease the killing capacity.

Claims

CLAIMS1. A construct for use in prevention or treatment of disease, preferably cancer, wherein the construct comprises:- one or more domain(s) that bind(s) one antigen; and- a domain that binds FcaRI wherein the domain that binds FcaRI is, or is comprised in, an IgA crystallizable fragment (Fc), wherein the construct binds at most one antigen moiety, and wherein the disease is characterized by presence of diseased cells expressing per cell between 50.000-200.000 antigen moieties.

2. Construct for use according to claim 1, wherein the one antigen is chosen from CD30, CD37, B7H3, fibronectin, CD19, CD21, CD22, mesothelin, CD123, PDGFRb, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PDL1, PD1, CTLA4 and Her3 is a fragment antigen-binding (Fab) domain.

3. Construct for use according to any of the previous claims, wherein the one or more domain(s) that bind(s) one antigen is a fragment antigen-binding (Fab) domain.

4. Construct for use according to claim 2, comprising two or more domains that bind one of CD30, CD37, B7H3, fibronectin, CD19, CD21 , CD22, mesothelin, CD123, PDGFRb, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PDL1, PD1 , CTLA4, and Her3, wherein said two or more domains bind different and / or non-overlapping epitopes of said one of CD30, B7H3, fibronectin, CD19, CD21 , CD22, mesothelin, CD123, PDGFRb, Chondroitin Sulfate Proteoglycan 4 (CSPG4), PDL1, PD1, CTLA4 and Her3.

5. Construct for use according to any of the previous claims, wherein the construct is an antibody comprising at most one Fab-arm, preferably an IgA antibody comprising at most one Fab arm.

6. Construct for use according to any of the previous claims, wherein the one or more domain(s) that bind(s) one antigen and the domain that that binds FcaRI are connected via a linker, wherein the linker is natural or synthetic, wherein the linker is preferably chosen from IgA antibody hinge region, linker peptide, and enzyme-based linker.

7. Construct for use according to any of the previous claims, wherein the one or more domain(s) that bind(s) one antigen is or comprises a nanobody, or a ligand.

8. Construct for use according to any of the previous claims, wherein- in the use the ratio between the number of antigen moieties that are bound by said construct and the number of constructs that are bound to said antigen moieties is substantially equimolar, preferably between 1:4 and 4:1 , most preferably 1 :1; and / or- in the use, for at least 80% of antigen moieties that are bound, the construct binds said antigen moieties via monovalent binding.

9. Construct for use according to any one of the previous claims, for use in prevention or treatment of cancer, preferably leukemia, multiple myeloma, lymphoma, breast cancer, head and neck cancer, lung cancer, colorectal cancer, prostate cancer, skin cancer, bladder cancer, (non-)Hodgkin lymphoma, kidney cancer, pancreatic cancer, liver cancer, ovarian cancer, brain and central nervous system (CNS) tumor, stomach cancer, or esophageal cancer.

10. Construct for use according to any one of the previous claims, wherein:- the disease is Hodgkin lymphoma, peripheral T-cell lymphoma, cutaneous T cell lymphoma, CD30-expressing B-cell lymphoma, or anaplastic large cell lymphoma, and the antigen is CD30;- the disease is neuroblastoma, osteosarcoma, cervical cancer, or medulloblastoma, and the antigen is B7-H3;- the disease is neuroblastoma, breast cancer, teratocarcinoma, ovarian cancer, or metastasized tumor, and the antigen is fibronectin;- the disease is B-cell lymphoma, acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL), and the antigen is CD19;- the disease is lymphoma, diffuse large B-cell lymphoma, or non-Hodgkin’s lymphoma, and the antigen is CD21;- the disease is B-cell acute lymphoblastic leukemia (B-ALL) or B-cell non-Hodgkin lymphoma (B-cell NHL), and the antigen is CD22;- the disease is mesothelioma, lung adenocarcinoma, ovarian adenocarcinoma, or pancreatic adenocarcinoma, and the antigen is mesothelin;- the disease is a hematological malignancy, including acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (B-ALL), hairy cell leukemia, Hodgkin lymphoma, or blastic plasmacytoid dendritic neoplasm (BPDCN), and the antigen is CD123;- the disease is associated with cancer-associated fibroblasts, non-small cell lung carcinoma, prostate cancer, PDGFRB-associated chronic eosinophilic leukemia, colorectal cancer,glioma, testis cancer, breast cancer, endometrial cancer, ovarian cancer, or melanoma, and the antigen is PDGFRP;- the disease is melanoma, lung cancer, colon cancer, head and neck cancer, liver cancer, pancreatic ductal adenocarcinoma (PDAC), renal cell carcinoma (RCC), oligodendroglioma, glioma, triple-negative breast carcinoma, or squamous cell carcinoma, and the antigen is CSPG4;- the disease is lung cancer, breast cancer, or melanoma, and the antigen is PD-L1 ;- the disease is breast cancer, ovarian cancer, lung cancer, colon cancer, pancreatic cancer, gastric cancer, head and neck cancer, or prostate cancer, and the antigen is HER3.

11. Construct for use according to any one of claims 1-9, wherein:- the disease is breast cancer, and the antigen is HER2;- the disease is colorectal cancer, and the antigen is EGFR, ALPP or GLICY2C;- the disease is pancreatic cancer, and the antigen is EGFR, or EpCAM;- the disease is ovarian cancer, and the antigen is EGFR or FOLR1 ;- the disease is GD2-positive tumor (excluding neuroblastoma), and the antigen is GD2, wherein the GD2-positive tumor is preferably chosen from bladder cancer, lung cancer, melanoma, sarcoma, osteosarcoma, rhabdomyosarcoma, and Ewing sarcoma;- the disease is testicular, ovarian, uterine, or lung adenocarcinoma, and the antigen isCLDN6;- the disease is anaplastic meningioma, and the antigen is CD276;- the disease is metastatic renal cell carcinoma, and the antigen is CA9;- the disease is hepatocellular carcinoma, and the antigen is GPC3;- the disease is glioblastoma, and the antigen is PDPN, HER2, or IL13RA2;- the disease is pancreatic carcinoma, and the antigen is PODXL;- the disease is triple-negative breast cancer, and the antigen is ANTXR1.- the disease is gastric cancer, and the antigen is CLDN18;- the disease is glioblastoma or pancreatic carcinoma, and the antigen is EGFR;- the disease is prostate cancer, and the antigen is FOLH1 or PSCA,- the disease is lung cancer, and the antigen is PTK7;- the disease is gastric cancer or pancreatic carcinoma, and the antigen is MSLN;- the disease is MUC1 -positive cancer, and the antigen is MUC1.

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