Protease activatable FC domain binding molecules

Abstract

The present invention generally relates to novel protease activatable Fc domain binding molecules, polynucleotides encoding such molecules, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the molecules of the invention, and to methods of using these molecules in the treatment of disease.

Description

[0001] PROTEASE ACTIVATABLE FC DOMAIN BINDING MOLECULES

[0002] BACKGROUND

[0003] Novel antibody-engineering approaches have enabled the selective depletion of a specific cell type in human body. For instance, in cancer immunotherapy field, these engineered antibodies are used to redirect immune cells to eliminate cancer or suppressive immune cells specifically, while leaving healthy tissues untouched.

[0004] One of the first methods used in cancer immunotherapy involved engaging innate immune cells such as natural killer (NK) cells or macrophages against the cells of interest, activating antibody-dependent cellular cytotoxicity (ADCC) or phagocytosis (ADCP) mechanisms (S. Varchetta et al. Cancers (2001), doi:10.3390 / cancersl3081988; Y. Li et al. Oncology Letters (2018), doi: 10.3892 / ol.2018.9630). To eliminate cells of interests, several antibodies or antibody-derivatives are engineered to engage activating receptors on innate immune cells such as Fc receptors, while simultaneously binding to surface antigens on target cells (S. Pinto et al. Trends in Immunology (2022), doi: 10.1016 / j.it.2022.09.007).

[0005] Over the course of cancer immunotherapy history, several innate immune cell engagers including monoclonal antibodies (mAbs) with or without Fc -binding improvements, bispecific single-chain variable fragments (BiKEs), tri specific single-chain variable fragments (TriKEs), innate cell engagers (ICEs), as well as multispecific antibody-based NK cell engager technologies (ANKETs) have been designed and being investigated for their capacity of activating innate immune cells and mediating anti-cancer therapy (S. Pinto et al. Trends in Immunology (2022), doi: 10.1016 / j.it.2022.09.007).

[0006] However, currently existing innate immune cell engagers suffers from two major drawbacks, which is first, antigen downregulation by tumor cells, and second, presence of antigen of interest in the healthy tissue or in other words, on-target off-tumor effect.

[0007] New antibody-engineering approaches are needed to overcome these challenges.

[0008] BRIEF SUMMARY

[0009] Provided herein is an improved protease-activatable innate cell engaging (ICE) antibody platform. In a first aspect, the present disclosure provides a bispecific protease - activatable Fc domain binding molecule capable of binding to a variant CH2 domain. In a particular aspect, the variant CH2 domain according to the present disclosure comprises G329 according to EU numbering. In a one preferred aspect, the bispecific protease- activatable Fc domain binding molecule is glycoengineered. In another preferred aspect, the bispecific protease-activatable Fc domain binding molecule is bivalent for the variant CH2 domain. In a further aspect, the present disclosure provides a (tumor) target antigen binding molecule comprising a variant CH2 domain comprising G329 according to EU numbering. In a further aspect, the protease-activatable Fc domain binding molecule is masked by the variant CH2 domain which is attached to the molecule through a protease -cleavable linker (see Figure 2, Molecule 2). After cleavage of the protease -cleavable linker in the proximity to a target cell, such as in a tumor, the antigen binding moiety capable of binding to the variant CH2 domain becomes accessible and the protease -activatable Fc domain binding molecule can bind to the target antigen binding molecule. Taken together, the present disclosure provides a versatile platform wherein a target cell (e.g. a tumor cell) is recognized by a target antigen binding molecule (see Figure 1, Molecule 1) and wherein the target antigen binding molecule is specifically recognized by the protease-activatable Fc domain binding molecule after release of the masking moiety. The herein disclosed therapeutic platform improves efficacy of treatment, for example based on personalization and multiple antigen targeting, and improved safety due to increased tumor specificity.

[0010] In one aspect, provided is a protease-activatable Fc domain binding molecule comprising a first antigen binding moiety capable of binding to a variant CH2 domain comprising or consisting of the amino acid sequence of SEQ ID NO: 76, wherein the first antigen binding moiety is not capable of binding to a reference CH2 domain comprising or consisting of the amino acid sequence of SEQ ID NO: 75; a first masking moiety covalently attached to the protease-activatable Fc domain binding molecule through a first protease- cleavable linker, wherein the first masking moiety comprises or consists of the amino acid sequence of SEQ ID NO: 76, wherein the first antigen binding moiety binds to the first masking moiety, wherein the first masking moiety reversibly conceals the first antigen binding moiety; and an Fc domain composed of a first and a second subunit capable of stable association, wherein the Fc domain comprises non-fucosylated oligosaccharides and / or bisected oligosacharides.

[0011] In some aspects, the protease-activatable Fc domain binding molecule comprises a second antigen binding moiety capable of binding to a variant CH2 domain comprising or consisting of the amino acid sequence of SEQ ID NO: 76, wherein the second antigen binding moiety is not capable of binding to a reference CH2 domain comprising or consisting of the amino acid sequence of SEQ ID NO: 75; and a second masking moiety covalently attached to the protease-activatable Fc domain binding molecule through a second protease-cleavable linker, wherein the second masking moiety comprises or consists of the amino acid sequence of SEQ ID NO: 76, wherein the second antigen binding moiety binds to the second masking moiety, wherein the second masking moiety reversibly conceals the second antigen binding moiety.

[0012] In some aspects, the first antigen binding moiety and, where present, the second antigen binding moiety is an antibody or antigen-binding fragment thereof.

[0013] In some aspects, the first antigen binding moiety and, where present, the second antigen binding moiety comprises (i) a VH region incorporating the following CDRs HC- CDR1 having the amino acid sequence of SEQ ID NO: 1, HC-CDR2 having the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 having the amino acid sequence of SEQ ID NO: 3. The domain binding molecule may also comprise (ii) a VL region incorporating the following CDRs LC-CDR1 having the amino acid sequence of SEQ ID NO: 4, LC-CDR2 having the amino acid sequence of SEQ ID NO: 5, and LC-CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0014] In some aspects, the first antigen binding moiety and, where present, the second antigen binding moiety comprises (i) a VH having an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO: 7, and (ii) a VL having an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO: 8.

[0015] In some aspects, the first masking moiety is covalently attached to the heavy chain variable region of the first antigen binding moiety and, where present, the second masking moiety is covalently attached to the heavy chain variable region of the second antigen binding moiety.

[0016] In some aspects, the first antigen binding moiety and, where present, the second antigen binding moiety is a Fab molecule.

[0017] In some aspects, the second antigen binding moiety is identical to the first antigen binding moiety.

[0018] In some aspects, the second masking moiety is identical to the first masking moiety. In some aspects, the first antigen binding moiety is fused to the first subunit of the Fc domain, optionally via a peptide linker.

[0019] In some aspects, the second antigen binding moiety is fused to the second subunit of the Fc domain, optionally via a peptide linker.

[0020] In some aspects, the first protease cleavable linker comprises at least one protease recognition sequence.

[0021] In some aspects, the second protease cleavable linker comprises at least one protease recognition sequence.

[0022] In some aspects, the first protease cleavable linker and, where present, the second protease cleavable linker comprises the protease recognition sequence PQARK (SEQ ID NO: 64) or PMAKK (SEQ ID NO: 66)

[0023] In some aspects, the Fc domain is an IgG, specifically an IgGl, Fc domain.

[0024] Further provided is nucleic acid, or a plurality of nucleic acids, encoding the protease-activatable Fc domain binding molecule as hereinabove described.

[0025] Further provided is an expression vector, or a plurality of expression vectors, comprising a nucleic acid or a plurality of nucleic acids as hereinabove described.

[0026] Further provided is a host cell comprising the nucleic acid or the plurality of nucleic acids as hereinabove described or the expression vector or plurality of expression vectors as hereinabove described.

[0027] Further provided is a method of producing a protease -activatable Fc domain binding molecule, comprising the steps of a) culturing a host cell as hereinabove described under conditions suitable for the expression of the protease -activatable Fc domain binding molecule and b) recovering the protease-activatable Fc domain binding molecule.

[0028] Further provided is a protease-activatable Fc domain binding molecule produced by a method as hereinabove described.

[0029] Further provided is a pharmaceutical composition comprising the protease - activatable Fc domain binding molecule as hereinabove described and a pharmaceutically acceptable carrier Further provided is a kit, comprising (i) a protease -activatable Fc domain binding molecule as hereinabove described or a pharmaceutical composition as hereinabove described, and (ii) an antigen-binding molecule comprising: (a) an antigen-binding domain that binds to a target antigen, and (b) a variant Fc domain comprising a variant CH2 domain comprising or consisting of the amino acid sequence of SEQ ID NO: 76.

[0030] Further provided is a protease-activatable Fc domain binding molecule as hereinabove described, a pharmaceutical composition as hereinabove described, or a kit as hereinabove described for use in a method of medical treatment or prophylaxis.

[0031] Further provided is a protease-activatable Fc domain binding molecule as hereinabove described, a pharmaceutical composition as hereinabove described, or a kit as hereinabove described, for use in a method of treating or preventing a disease in which cells comprising or expressing a target antigen are pathologically-implicated, wherein the method comprises administering the protease-activatable Fc domain binding molecule or pharmaceutical composition to a subject to which an antigen-binding molecule has been or is to be administered; wherein the antigen-binding molecule comprises: (a) an antigenbinding domain that binds to the target antigen, and (b) a variant Fc domain comprising a variant CH2 domain comprising or consisting of the amino acid sequence of SEQ ID NO: 76.

[0032] Further provided is use of a protease-activatable Fc domain binding molecule as hereinabove described, a pharmaceutical composition as hereinabove described, or a kit as hereinabove described, in the manufacture of a medicament for the treatment of a disease in which cells comprising or expressing a target antigen are pathologically-implicated.

[0033] Further provided is a method of treating an individual having a disease in which cells comprising or expressing a target antigen are pathologically-implicated, comprising administering to the individual an effective amount of a protease-activatable Fc domain binding molecule as hereinabove described or a pharmaceutical composition as hereinabove described, wherein the individual has been or is to be administered an antigen-binding molecule comprising: (a) an antigen-binding domain that binds to the target antigen, and (b) a variant Fc domain comprising a variant CH2 domain comprising or consisting of the amino acid sequence of SEQ ID NO: 76.

[0034] In some aspects, the target antigen is selected from the group consisting of FolRl, EPCAM, HER2, and CD25. Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.

[0035] DETAILED DESCRIPTION

[0036] Currently existing innate immune cell engagers suffers from several major drawbacks, for example antigen downregulation by tumor cells and the presence of antigen of interest in the healthy tissue which often leads to on-target off-tumor effect. To overcome these challenges, the present inventors herein disclose new protease activatable Fc domain binding immune cell engager (pro-ICE) molecules.

[0037] The new protease activatable Fc domain binding molecules disclosed herein are based on a two-molecule approach combining an antigen targeting adaptor molecule and a protease activatable Fc domain binding innate immune cell engager (ICE) molecule (see for example Figure 2). In one aspect, an Fc-silenced adaptor moiety comprising P329G LALA mutations is used to target the desired antigen in the tumor microenvironment. In another aspect, a protease activatable Fc domain binding immune cell engager (pro-ICE) targeting the P329G mutation on the adaptor moiety is used as an effector molecule. Upon activation of the pro- ICE molecule in the tumor microenvironment, unmasked universal innate cell engager molecules bind to the adaptor molecule and recruits innate cells through the Fc domain, leading to ADCC and / or ADCP directed against the target cells. In preferred embodiments, the Fc portion of the universal innate cell engager are glycoengineered for enhanced FcyRIII affinity. In a most preferred embodiment, the protease -activatable Fc domain binding molecule is glycoengineered and bivalent for the variant CH2 domain.

[0038] One of the major advantages of the new protease activatable Fc domain binding molecules disclosed herein compared to conventional innate cell engagers is their ability to mitigate antigen downregulation by cancer cells. Frequently, cancer cells accumulate random mutations in their DNA due to their abnormal DNA-repair mechanisms (P. G. Pilie et al. Nature Review Clinical Oncology (2018), doi: 10.1038 / s41571 -018-0114-z). As a result, they can upregulate certain proteins which can be beneficial for their survival (R. K. Bright et al. Human Vac. & Immunotherapeutics (2014), doi: 10.4161 / hv.29475; C. L. Arteaga et al. Cancer Cell (2014), doi: 10.1016 / j.ccr.2014.02.025; S. E. Moddy et al. Cancer Cell (2002), doi : 10.1016 / sl535-6108(02)00212-x). Such upregulated proteins, also known as tumor antigens, are one the major targets of cancer immunotherapy, as they provide a way to identify and target cancer cells (L. M. Weiner et al. Clin. Cancer Res. (2009), doi: 10.1158 / 1078- 0432.CCR-09-0737). However, upon therapeutic pressure, cancer cells can also downregulate their antigens, a mechanism called antigen escape or antigen loss (E. Zah, et al. Cancer Immunol. Res. (2016), doi: 10.1158 / 2326-6066. CIR-15-0231; F. Braig et al. Blood. (2017), doi: 10.1182 / blood-2016-05-718395; V. Prima et al. Proc. Natl. Acad. Sci. U. S. A. (2017), doi: 10.1073 / pnas.1612920114; Y. Zhao et al. Blood. (2021), doi: 10.1182 / blood.2020006287).

[0039] To circumvent this resistance mechanism, the new protease-activatable Fc domain binding molecule disclosed herein enable targeting two or more tumor antigens, by combining different adaptor moieties simultaneously. As a result, antigen escape is reduced.

[0040] A further major advantage of the new protease-activatable Fc domain binding molecule disclosed herein is their improved safety profile. In cancer, the majority of the tumor antigens are not fully specific to the malignant tissue, in other words, tumor antigens are mostly overexpressed in the tumor while being still present in lesser extend on healthy tissues (R. K. Bright et al. Human Vac. & Immunotherapeutics (2014), doi: 10.4161 / hv.29475). As a results, drugs targeting tumor antigens can also bind to some extend to healthy cells expressing the same antigen, which leads to toxicity against healthy tissue. This phenomenon is called on-target off-tumor effect and is one of the major toxicity concern of the cancer immunotherapy field. (L. M. Weiner et al. Clin. Cancer Res. (2009), doi: 10.1158 / 1078-0432. CCR-09-0737).

[0041] The new protease-activatable Fc domain binding molecules disclosed herein are capable of reducing or omitting the on-target off-tumor binding. This prevents systemic toxicity and is ensured by masking the molecules with a protease-cleavable protein masks. In healthy tissues, the masks will stay bound to molecules of the present invention, preventing their binding to the adaptor moieties. However, as solid tumors show increased accumulation of certain proteases (H. Tanimoto et al. Tumor Biol. (2001), doi: 10.1159 / 000050604; K. A. Autio et al. Clin. Cancer Res. (2020), doi: 10.1158 / 1078-0432. CCR-19-1457), the masks will be cleaved off, specifically only in the tumor tissue followed by cross -linking of innate immune cells with target cells triggering ADCC and / or ADCP.

[0042] Definitions Terms are used herein as generally used in the art, unless otherwise defined in the following.

[0043] As used herein, the term “antigen binding molecule” refers in its broadest sense to a molecule that specifically binds an antigenic determinant. Examples of antigen binding molecules are immunoglobulins and derivatives, e.g. fragments, thereof.

[0044] An “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some aspects, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some aspects, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0045] The term “bispecific” means that the antigen binding molecule is able to specifically bind to at least two distinct antigenic determinants. Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of which is specific for a different antigenic determinant. In certain embodiments the bispecific antigen binding molecule is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells.

[0046] “Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary methods for measuring binding affinity are described in the following.

[0047] An “affinity matured” antibody refers to an antibody with one or more alterations in one or more complementary determining regions (CDRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.

[0048] The term “amino acid mutation” as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., reduced binding to an Fc receptor, or increased association with another peptide. Amino acid sequence deletions and insertions include amino- and / or carboxy-terminal deletions and insertions of amino acids. Particular amino acid mutations are amino acid substitutions. For the purpose of altering e.g. the binding characteristics of an Fc region, non-conservative amino acid substitutions, i.e. replacing one amino acid with another amino acid having different structural and / or chemical properties, are particularly preferred. Amino acid substitutions include replacement by non- naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard amino acids (e.g. 4-hydroxyproline, 3 -methylhistidine, ornithine, homoserine, 5- hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful. Various designations may be used herein to indicate the same amino acid mutation.

[0049] The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0050] An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab’ -SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23: 1126-1136 (2005).

[0051] The term “antigen binding domain” refers to the part of an antibody that comprises the area which specifically binds to and is complementary to part or all of an antigen. An antigen binding domain may be provided by, for example, one or more antibody variable domains (also called antibody variable regions). Particularly, an antigen binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0052] An “antigen binding site” refers to the site, i.e. one or more amino acid residues, of an antigen binding molecule which provides interaction with the antigen. For example, the antigen binding site of an antibody comprises amino acid residues from the complementarity determining regions (CDRs). A native immunoglobulin molecule typically has two antigen binding sites, a Fab molecule typically has a single antigen binding site.

[0053] As used herein, the term “antigen binding moiety” refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen binding moiety is able to direct the entity to which it is attached (e.g. a second antigen binding moiety) to a target site, for example to a specific type of tumor cell or tumor stroma bearing the antigenic determinant. In another embodiment an antigen binding moiety is able to activate signaling through its target antigen, for example a T cell receptor complex antigen. Antigen binding moieties include antibodies and fragments thereof as further defined herein. Particular antigen binding moieties include an antigen binding domain of an antibody, comprising an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen binding moieties may comprise antibody constant regions as further defined herein and known in the art. Useful heavy chain constant regions include any of the five isotypes: a, 5, a, y, or p. Useful light chain constant regions include any of the two isotypes: K and X.

[0054] As used herein, the term “antigenic determinant” is synonymous with “antigen” and “epitope” and refers to a site (e.g. a contiguous stretch of amino acids or a conformational configuration made up of different regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen binding moiety binds, forming an antigen binding moiety - antigen complex. Useful antigenic determinants can be found, for example, on the surfaces of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in blood serum, and / or in the extracellular matrix (ECM). The proteins referred to as antigens herein can be any native form the proteins from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g. mice and rats), unless otherwise indicated. In a particular embodiment the antigen is a human protein. Where reference is made to a specific protein herein, the term encompasses the “full- length”, unprocessed protein as well as any form of the protein that results from processing in the cell. The term also encompasses naturally occurring variants of the protein, e.g. splice variants or allelic variants.

[0055] “Antibody-dependent cell-mediated cytotoxicity” (“ADCC”) is an immune mechanism leading to the lysis of antibody-coated target cells by immune effector cells. The target cells are cells to which antibodies or derivatives thereof comprising an Fc region specifically bind, generally via the protein part that is N-terminal to the Fc region. As used herein, the term “reduced ADCC” is defined as either a reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody in the medium surrounding the target cells, by the mechanism of ADCC defined above, and / or an increase in the concentration of antibody in the medium surrounding the target cells, required to achieve the lysis of a given number of target cells in a given time, by the mechanism of ADCC. The reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cells, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art), but that has not been engineered. For example the reduction in ADCC mediated by an antibody comprising in its Fc domain an amino acid substitution that reduces ADCC, is relative to the ADCC mediated by the same antibody without this amino acid substitution in the Fc domain. Suitable assays to measure ADCC are well known in the art (see e.g. PCT publication no. WO 2006 / 082515 or PCT publication no. WO 2012 / 130831).

[0056] The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG2, IgGs, IgG4, IgAi, and IgA2. In certain aspects, the antibody is of the IgGi isotype. In certain aspects, the antibody is of the IgGi isotype with the P329G, L234A and L235A mutation to reduce Fc-region effector function. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, a, y, and p, respectively. The light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain.

[0057] The terms “constant region derived from human origin” or “human constant region” as used in the current application denotes a constant heavy chain region of a human antibody of the subclass IgGi, IgG2, IgG3, or IgG4 and / or a constant light chain kappa or lambda region. Such constant regions can be used in human or humanized antibodies and are well known in the state of the art and e.g. described by Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also e.g. Johnson, G., and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218; Kabat, E.A., et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). Unless otherwise specified herein, numbering of amino acid residues in the constant region is according to the EU numbering system, also called the EU index of Kabat, as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91 -3242.

[0058] By a “crossover” Fab molecule (also termed “Crossfab”) is meant a Fab molecule wherein the variable domains of the Fab heavy and light chain are exchanged (i.e. replaced by each other), i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable domain VL and the heavy chain constant domain 1 CHI (VL-CH1, in N- to C-terminal direction), and a peptide chain composed of the heavy chain variable domain VH and the light chain constant domain CL (VH-CL, in N- to C-terminal direction). For clarity, in a crossover Fab molecule wherein the variable domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant domain 1 CHI is referred to herein as the “heavy chain” of the crossover Fab molecule.

[0059] An “effective amount” of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0060] “Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0061] As used herein, the terms “engineer, engineered, engineering”, are considered to include any manipulation of the peptide backbone or the post -translational modifications of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications of the amino acid sequence, of the glycosylation pattern, or of the side chain group of individual amino acids, as well as combinations of these approaches.

[0062] As used herein, the terms “first”, “second” or “third” with respect to Fab molecules etc., are used for convenience of distinguishing when there is more than one of each type of moiety. Use of these terms is not intended to confer a specific order or orientation of the immune activating Fc domain binding molecule unless explicitly so stated.

[0063] A “Fab molecule” refers to a protein consisting of the VH and CHI domain of the heavy chain (the “Fab heavy chain”) and the VL and CL domain of the light chain (the “Fab light chain”) of an immunoglobulin.

[0064] By “fused” is meant that the components (e.g. a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0065] As used herein, the term “single-chain” refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain embodiments, one of the antigen binding moieties is a single-chain Fab molecule, i.e. a Fab molecule wherein the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain. In a particular such embodiment, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule.

[0066] In contrast thereto, by a “conventional Fab molecule" is meant a Fab molecule in its natural format, i.e. comprising a heavy chain composed of the heavy chain variable and constant domains (VH-CH1, in N- to C-terminal direction), and a light chain composed of the light chain variable and constant domains (VL-CL, in N- to C-terminal direction).

[0067] The terms “full length antibody”, “intact antibody”, and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0068] The term “Fc domain” or “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the carboxyl -terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain (also referred to herein as a “cleaved variant heavy chain”). This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to Kabat EU index). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447), of the Fc region may or may not be present. Amino acid sequences of heavy chains including Fc domains (or a subunit of an Fc domain as defined herein) are denoted herein without C-terminal glycine-lysine dipeptide if not indicated otherwise. In one embodiment of the invention, a heavy chain including a subunit of an Fc domain as specified herein, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). In one embodiment of the invention, a heavy chain including a subunit of an Fc domain as specified herein, comprises an additional C- terminal glycine residue (G446, numbering according to EU index of Kabat). Compositions of the invention, such as the pharmaceutical compositions described herein, comprise a population of antigen binding molecules of the invention. The population of antigen binding molecule may comprise molecules having a full-length heavy chain and molecules having a cleaved variant heavy chain. The population of antigen binding molecules may consist of a mixture of molecules having a full-length heavy chain and molecules having a cleaved variant heavy chain, wherein at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the antigen binding molecules have a cleaved variant heavy chain. In one embodiment of the invention a composition comprising a population of antigen binding molecules of the invention comprises an antigen binding molecule comprising a heavy chain including a subunit of an Fc domain as specified herein with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). In one embodiment of the invention a composition comprising a population of antigen binding molecules of the invention comprises an immune activating Fc domain binding molecule comprising a heavy chain including a subunit of an Fc domain as specified herein with an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat). In one embodiment of the invention such a composition comprises a population of antigen binding molecules comprised of molecules comprising a heavy chain including a subunit of an Fc domain as specified herein; molecules comprising a heavy chain including a subunit of a Fc domain as specified herein with an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat); and molecules comprising a heavy chain including a subunit of an Fc domain as specified herein with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above). A “subunit” of an Fc domain as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association. For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.

[0069] The term “CH2 domain” or "CH2 constant domain", as used herein refers to the second constant domain (or region) of the heavy chain of an antibody. The constant region of the antibody heavy chain has three or four domains, depending on the class of the antibody, which are named CHI, CH2, CH3 and CH4 domain. For a human IgG antibody, the CH2 domain usually begins around amino acid residue 231 and ends around residue 340 of the human IgG. However, these values can differ slightly among various antibodies and isotypes, and in certain engineered antibodies. The CH2 domain is part of the Fc domain, which for IgGl antibodies consists of the CH2 domain and the CH3 domain. An exemplary sequence for human IgGl Fc domains is provided in SEQ ID NO: 75. The skilled person can readily determine where the CH2 domain starts and ends in a heavy chain sequence on interest, for example based on the EU index.

[0070] An “Fc domain binding moiety” as herein used is an antigen binding moiety capable of binding to an Fc domain.

[0071] The terms “host cell”, “host cell line”, and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells”, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0072] An “activating Fc receptor” is an Fc receptor that following engagement by an Fc domain of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions. Human activating Fc receptors include FcyRIIIa (CD 16a), FcyRI (CD64), FcyRIIa (CD32), and FcaRI (CD89).

[0073] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0074] A “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one aspect, for the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one aspect, for the VH, the subgroup is subgroup III as in Kabat et al., supra.

[0075] A “humanized antibody" refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0076] The term “ hyp ervari able region” or “HVR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence and which determine antigen binding specificity, for example “complementarity determining regions” (“CDRs”).

[0077] Generally, antibodies comprise six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3), and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include:

[0078] (a) hypervariable loops occurring at amino acid residues 26-32 (LI), 50-52 (L2), 91- 96 (L3), 26-32 (Hl), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901- 917 (1987));

[0079] (b) CDRs occurring at amino acid residues 24-34 (LI), 50-56 (L2), 89-97 (L3), 31- 35b (Hl), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) antigen contacts occurring at amino acid residues 27c-36 (LI), 46-55 (L2), 89-96 (L3), 30-35b (Hl), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732- 745 (1996)).

[0080] Unless otherwise indicated, the CDRs are determined according to Kabat et al., supra. One of skill in the art will understand that the CDR designations can also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

[0081] An “immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.

[0082] An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain aspects, the individual or subject is a human.

[0083] An “isolated antibody” is one which has been separated from a component of its natural environment. In some aspects, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0084] The term “immunoglobulin molecule” refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant domains (CHI, CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain, also called a light chain constant region. The heavy chain of an immunoglobulin may be assigned to one of five types, called a (IgA), 5 (IgD), a (IgE), y (IgG), or p (IgM), some of which may be further divided into subtypes, e.g. yi (IgGi), 72 (IgG?), 73 (IgGs), 74 (IgG4), ai (IgAi) and a? (IgA?). The light chain of an immunoglobulin may be assigned to one of two types, called kappa (K) and lambda (X), based on the amino acid sequence of its constant domain. An immunoglobulin essentially consists of two Fab molecules and an Fc domain, linked via the immunoglobulin hinge region.

[0085] “Framework” or “FR” refers to variable domain residues other than complementary determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1-CDR-H1(CDR-L1)-FR2- CDR-H2(CDR-L2)- FR3- CDR-H3(CDR-L3)-FR4.

[0086] A “modification promoting the association of the first and the second subunit of the Fc domain” is a manipulation of the peptide backbone or the post -translational modifications of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer. A modification promoting association as used herein particularly includes separate modifications made to each of the two Fc domain subunits desired to associate (i.e. the first and the second subunit of the Fc domain), wherein the modifications are complementary to each other so as to promote association of the two Fc domain subunits. For example, a modification promoting association may alter the structure or charge of one or both of the Fc domain subunits so as to make their association sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which might be non-identical in the sense that further components fused to each of the subunits (e.g. antigen binding moieties) are not the same. In some embodiments the modification promoting association comprises an amino acid mutation in the Fc domain, specifically an amino acid substitution. In a particular embodiment, the modification promoting association comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two subunits of the Fc domain.

[0087] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

[0088] A “naked antibody” refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical composition.

[0089] “Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant heavy domains (CHI, CH2, and CH3). Similarly, firom N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain.

[0090] The term “nucleic acid molecule” or “polynucleotide” includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine- or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group. Often, the nucleic acid molecule is described by the sequence of bases, whereby said bases represent the primary structure (linear structure) of a nucleic acid molecule. The sequence of bases is typically represented from 5’ to 3’. Herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. In addition, the term nucleic acid molecule includes both, sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the herein described nucleic acid molecule can contain naturally occurring or non- naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules which are suitable as a vector for direct expression of an antibody of the invention in vitro and / or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors, can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or expression of the encoded molecule so that mRNA can be injected into a subject to generate the antibody in vivo (see e.g., Stadler ert al, Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356 or EP 2 101 823 Bl).

[0091] By a nucleic acid or polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the 5’ or 3’ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs, such as the ones discussed above for polypeptides (e.g. ALIGN-2).

[0092] The term “expression cassette” refers to a polynucleotide generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette of the invention comprises polynucleotide sequences that encode bispecific antigen binding molecules of the invention or fragments thereof. “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity for the purposes of the alignment. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST -2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Alternatively, the percent identity values can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087 and is described in WO 2001 / 007611.

[0093] Unless otherwise indicated, for purposes herein, percent amino acid sequence identity values are generated using the ggsearch program of the FASTA package version 36.3.8c or later with a BLOSUM50 comparison matrix. The FASTA program package was authored by W. R. Pearson and D. J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448; W. R. Pearson (1996) “Effective protein sequence comparison” Meth. Enzymol. 266:227- 258; and Pearson et. al. (1997) Genomics 46:24-36 and is publicly available from www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www. ebi.ac.uk / Tools / sss / fastaAlternatively, a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi can be used to compare the sequences, using the ggsearch (global proteimprotein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure a global, rather than local, alignment is performed. Percent amino acid identity is given in the output alignment header. As used herein, term “polypeptide” refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein”, “amino acid chain”, or any other term used to refer to a chain of two or more amino acids, are included within the definition of polypeptide, and the term polypeptide may be used instead of, or interchangeably with any of these terms. The term polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis. A polypeptide of the invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded.

[0094] The term “pharmaceutical composition” or “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

[0095] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0096] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0097] The term “protease” or “proteolytic enzyme” as used herein refers to any proteolytic enzyme that cleaves the linker at a recognition site and that is expressed by a target cell or by a cell in the vicinity of the target cell (e.g. in a tumor microenvironment). Such proteases might be secreted by the target cell or remain associated with the target cell, e.g., on the target cell surface. Examples of proteases include but are not limited to metalloproteinases, e.g., matrix metalloproteinase 1-28 and A Disintegrin And Metalloproteinase (ADAM) 2, 7-12, 15, 17-23, 28-30 and 33, serine proteases, e.g., urokinase-type plasminogen activator and Matriptase, cysteine protease, aspartic proteases, and members of the cathepsin family.

[0098] The term “protease-activatable” as used herein, with respect to the protease-activatable Fc domain binding molecule, refers to a molecule having reduced or abrogated ability to bind it's target Fc domain as herein described. Upon dissociation of the masking moiety by proteolytic cleavage, e.g., by proteolytic cleavage of a linker connecting the masking moiety to the protease-activatable Fc domain binding molecule, binding to the target Fc domain and ultimately immune cell activation is restored.

[0099] “Reduced binding”, for example reduced binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, as measured for example by SPR. For clarity, the term includes also reduction of the affinity to zero (or below the detection limit of the analytic method), i.e. complete abolishment of the interaction. Conversely, “increased binding” refers to an increase in binding affinity for the respective interaction.

[0100] The term “reversibly conceals” or “reversibly concealing” as used herein refers to the binding of a masking moiety to an antigen binding moiety (such as an Fc domain binding moiety) to prevent the antigen binding moiety from binding its antigen (such as a variant CH2 domain according to the disclosure). This concealing is reversible in that the masking moiety can be released from the antigen binding moiety, e.g., by protease cleavage, and thereby freeing the antigen binding moiety to bind to its antigen.

[0101] By “specific binding” is meant that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antigen binding moiety to bind to a specific antigenic determinant can be measured either through an enzyme - linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. surface plasmon resonance (SPR) technique (analyzed on a Biacore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of an antigen binding moiety to an unrelated protein (such as e.g. a reference CH2 domain) is less than about 10% of the binding of the antigen binding moiety to the antigen (such as a variant CH2 domain) as measured, e.g., by SPR. In certain embodiments, an antigen binding moiety that binds to the antigen, or an antigen binding molecule comprising that antigen binding moiety, has a dissociation constant (KD) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 10'8M or less, e.g. from 10'8M to 10'13M, e.g., from 10'9M to 10'13M). A “target cell antigen” or “target antigen” as used herein refers to an antigenic determinant presented on the surface of a target cell, for example a cell in a tumor such as a cancer cell or a cell of the tumor stroma. In a particular embodiment, the target cell antigen is FolRl, particularly human FolRl . In another particular embodiment, the target cell antigen is EPCAM, particularly human EPCAM. In another particular embodiment, the target cell antigen is CD25, particularly human CD25. In another particular embodiment, the target cell antigen is HER2, particularly human HER2.

[0102] A “therapeutically effective amount” of an agent, e.g. a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent for example eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.

[0103] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some aspects, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.

[0104] The term “valent” as used herein denotes the presence of a specified number of antigen binding sites in an antigen binding molecule. As such, the term “monovalent binding to an antigen” denotes the presence of one (and not more than one) antigen binding site specific for the antigen in the antigen binding molecule.

[0105] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementary determining regions (CDRs). (See, e.g., Kindt et al. Kuby Immunology, 6thed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0106] The term “vector”, as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.

[0107] Protease-activatable Fc domain binding molecule

[0108] The present invention provides a modular antibody based platform for flexible antigen targeting and individual immune cell stimulation that can be adapted to desired indications. Compared to the conventional bispecific formats that directly engage with their target of interest, the present invention consists of two components that can be individually adapted and used in a plug and play manner (see Figure 2). This modular platform mainly focuses on two parts: (i) an easy to produce target antigen binding molecule for precise and selective antigen targeting, and (ii) a masked immune activating (Fc domain binding) molecule that specifically recognizes the Fc-part of the target antigen binding molecule specifically in the tumor microenvironment, thereby recruiting immune effector cells and activating them leading to lysis of the target cell.

[0109] Upon cleavage of a protease-cleavable linker in the vicinity of a target cell (such as in a tumor where the relevant protease activity is higher compared to healthy tissue), the Fc domain binding molecule becomes activated and can bind to a target antigen binding molecule (see Figure 1 and 2) and to immune effector cells through it's Fc domain. Through the combination of the target antigen binding molecule and the protease -activatable Fc domain binding molecule an individualized, customizable off-the-shelf approach to stimulate individual immune cells is possible without the need to generate different effector molecules for each and every target cell antigen.

[0110] In one embodiment, the immune effector cells are natural killer (NK) cells and / or macrophages. NK cell and macrophages are involved in innate immunity and activation of these cells leads to ADCC and / or ADCP. In a preferred embodiment, the protease-activatable Fc domain binding molecule is glycoengineered to maximize ADCC and / or ADCP as will be described further below..

[0111] In a particular aspect, the present disclosure provides a protease-activatable Fc domain binding molecule comprising a) a first antigen binding moiety capable of binding to a variant CH2 domain, wherein the first antigen binding moiety is not capable of binding to a reference CH2 domain; b) first masking moiety covalently attached to the protease-activatable Fc domain binding molecule through a first protease -cleavable linker, wherein the first antigen binding moiety binds to the first masking moiety, wherein the first masking moiety reversibly conceals the first antigen binding moiety; and c) an Fc domain composed of a first and a second subunit capable of stable association, wherein the Fc domain comprises non-fucosylated oligosaccharides and / or bisected oligosacharides.

[0112] Individual components of the protease-activatable Fc domain binding molecule are now further described.

[0113] Variant CH2 domain

[0114] In one aspect, provided are protease-activatable Fc domain binding molecules which are masked by a variant CH2 domain or a fragment thereof. The protease-activatable Fc domain binding molecules of the present disclosure comprise at least one antigen binding moiety capable of binding to a variant CH2 domain and at least one masking moiety that reversibly conceals the antigen binding moiety. In one aspect, the masking moiety is a variant CH2 domain or fragment thereof. In one aspect, the variant CH2 domain comprises G329 according to EU numbering. In one aspect, the antigen binding moiety is capable of binding to a variant CH2 domain comprising G329 according to EU numbering, wherein the antigen binding moiety is not capable of binding to a reference CH2 domain comprising P329 according to EU numbering. In one aspect, the variant CH2 domain masks the antigen binding moiety capable of binding to a variant CH2 domain comprising G329 according to EU numbering. Suitable variant CH2 domains are known in the art and also further described herein below. The CH2 domain is part of the fragment crystallizable (Fc) domain binding molecule which is well known in the art. The Fc domain consists of a pair of polypeptide chains comprising heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other.

[0115] The Fc domain confers to antibodies favorable pharmacokinetic properties, including a long serum half-life which contributes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio. At the same time it may, however, lead to undesirable targeting to cells expressing Fc receptors rather than to the preferred antigenbearing cells.

[0116] Accordingly, preferably, target antigen binding molecules (Molecule 1 in Figure 1 and 2) used according to the present invention, exhibit reduced binding affinity to an Fc receptor and / or reduced effector function, as compared to a native IgGi Fc domain. Reduced binding affinity to an Fc receptor and / or reduced effector function is achieved by modification of the Fc of the target antigen binding molecules.

[0117] According to one aspect of the present invention, the variant CH2 domain that masks the protease-activatable Fc domain binding molecule of the present disclosure is modified / engineered to match the modification of the Fc domain of the target antigen binding molecule with which the protease-activatable Fc domain binding molecule may be used in combination. However, the modification in the masking moiety are not necessary identical to the modifications in the Fc domain of the target antigen binding molecule as long as the masking moiety is capable of binding to both the masking moiety and the Fc domain of the target antigen binding molecule. In the appended examples which are included as proof of concept, the antigen binding moiety (in Molecule 2 of Figure 1 and 2) binds to the CH2 masking domain comprising the P329G mutation (according to EU numbering). A matching target antigen binding molecule comprises the P329G mutation in the Fc domain, however, the therapeutic antibody and / or the masking moiety may comprise additional mutations.

[0118] According to this concept, a modified / engineered CH2 domain or fragments thereof is used as a masking moiety to mask an antigen binding moiety of the protease -activatable Fc domain binding molecule of the present disclosure. Once the masking moiety is released from the protease-activatable Fc domain binding molecule (e.g., by protease cleavage in the vicinity of the target cell, for example in a tumor microenvironment), the antigen binding moiety can bind to the target antigen binding molecule comprising the variant CH2 domain comprising G329 according to EU numbering (see Figure 1).

[0119] In a further aspect, the masking moiety may comprise further substitutions. In some aspects, the masking moiety comprises the amino acid substitutions L234A and L235A according to EU numbering. In one such embodiment, the masking moiety is an IgGi CH2 domain, particularly a human IgGi CH2 domain. In one aspect, the masking moiety comprises the amino acid mutations L234A, L235A and P329G (“P329G LAL A”) according to EU numbering. The “P329G LALA” combination of amino acid substitutions almost completely abolishes Fey receptor (as well as complement) binding of a human IgGi Fc domain, as described in PCT publication no. WO 2012 / 130831, incorporated herein by reference in its entirety. WO 2012 / 130831 also describes methods of preparing such mutant Fc domains or fragments thereof and methods for determining its properties such as Fc receptor binding or effector functions.

[0120] Binding to Fc receptors can be measured by methods known in the art for example in WO2021 / 255138 (e.g. Example 2) which is hereby incorporated by reference in its entirety. For example, binding to Fc receptors can be easily determined e.g., by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a Biacore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression or using cell lines known to express particular Fc receptors, such as human NK cells expressing Fcyllla receptor.

[0121] Effector function of an Fc domain or fragments thereof can be measured by methods known in the art. For example a suitable assay for measuring ADCC is described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351 -1361 (1987). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® nonradioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998). In a particular aspect, the variant CH2 domain comprises the amino acid sequence of SEQ ID NO: 76, or a fragment thereof that the at least one antigen binding moiety as described herein below can bind. In one aspect, the variant CH2 domain consists of the amino acid sequence of SEQ ID NO: 76.

[0122] Suitable methods to measure the binding of the masking moiety and the at least one antigen binding moiety are known in the art for example in W02022 / 029051 (e.g. Example 1) which is incorporated herein by reference it its entirety.

[0123] In one embodiment, binding of the at least one antigen binding moiety to the masking moiety is measured by SPR at 25°C on a Biacore T200 with HBS-EP+ as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% Surfactant P20 (BR-1006-69, GE Healthcare)). The antigen binding moiety in Fab format is directly immobilized by amine coupling on a CM5 chip (GE Healthcare). A two-fold dilution series of the masking moiety is passed over the ligand at 30 pl / min for 240 sec to record the association phase. The dissociation phase is monitored for 800 s and triggered by switching from the sample solution to HBS-EP+. Bulk refractive index differences are corrected for by subtracting the response obtained on a reference flow cell. The affinity constants are derived from the kinetic rate constants by fitting to a 1 : 1 Langmuir binding using the Biaeval software (GE Healthcare).

[0124] Binding of the at least one antigen binding moiety to a variant CH2 domain can be compared to binding of the at least one antigen binding moiety to a reference CH2 domain to assess specificity of binding of the at least one antigen binding moiety to the variant CH2 domain. Preferably, the reference CH2 domain is identical to the variant CH2 domain with the exception of the specific mutations incorporated in the variant CH2 domain.

[0125] In a preferred embodiment, the reference CH2 domain comprises or consists of the amino acid sequence of SEQ ID NO: 75.

[0126] Protease-cleavable linker

[0127] The protease-activatable Fc domain binding molecule of the present disclosure comprises at least one protease cleavable linker. In the absence of the relevant protease, the masking moiety (i.e. the variant CH2 domain or fragment thereof) masks the second antigen binding moiety, i.e., the antigen binding moiety binds to the masking moiety and can therefore not bind to the target antigen binding molecule. In the presence of the relevant protease, the protease cleavable linker connecting the variant CH2 domain or fragment thereof and the second antigen binding moiety is cleaved and the masking moiety is released / detached from the protease-activatable Fc domain binding molecule. After cleavage, the at least one antigen binding moiety is capable of binding to the target antigen binding molecule comprising the relevant variant CH2 domain.

[0128] Accordingly, in some aspects the masking moiety is covalently attached to the protease-activatable Fc domain binding molecule through a linker. In some aspects the linker is a peptide linker. In some aspects the linker is a protease-cleavable (peptide) linker.

[0129] In some aspects, the protease-activatable Fc domain binding molecule comprises a linker (having a protease recognition site) comprising a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 64 or SEQ ID NO: 66. In one aspect, the protease recognition site comprises the polypeptide sequence of SEQ ID NO: 63 or SEQ ID NO: 65. In a preferred aspect, the protease recognition site comprises the polypeptide sequence of SEQ ID NO: 63.

[0130] In one embodiment the relevant protease is matriptase.

[0131] Antigen binding moiety capable of binding to variant CH2 domain

[0132] The protease-activatable Fc domain binding molecules according to the present invention comprise at least one antigen binding moiety capable of binding to a variant CH2 domain. In some aspects, the protease-activatable Fc domain binding molecules comprise a first antigen binding moiety capable of binding to a variant CH2 domain. In preferred aspects, the protease-activatable Fc domain binding molecules comprise a first and a second antigen binding moiety capable of binding to a variant CH2 domain. The latter molecules are bivalent regarding their capability to bind to a variant CH2 domain. In a preferred embodiment, the first and second antigen binding moiety capable of binding to a variant CH2 domain are identical.

[0133] The essential function of the antigen binding moiety is to provide for binding to a variant CH2 domain, as described herein below.

[0134] Antigen binding moieties include antibodies (z.e. immunoglobulins (Igs)), and antigen-binding fragments and derivatives thereof. In some embodiments, an antigen binding moiety capable of binding to variant CH2 domain according to the present disclosure comprises, or consists of, a monoclonal antibody, a monospecific antibody, a multispecific (e.g., bispecific, trispecific, etc.) antibody, a variable fragment (Fv) moiety, a single-chain Fv (scFv) moiety, a fragment antigen-binding (Fab) moiety, a single-chain Fab moiety (scFab), a CrossFab moiety, a Fab’ moiety, a Fab’-SH moiety, a F(ab’)2 moiety, a diabody moiety, a triabody moiety, an scFv-Fc moiety, a minibody moiety, a heavy chain only antibody (HCAb) moiety, or a single domain antibody (dAb, VHH) moiety.

[0135] Antigen binding moieties capable of binding to a variant CH2 domain according to the present disclosure also include further target antigen-binding peptides / polypeptides such as peptide aptamers, thioredoxins, anticalins, Kunitz domains, avimers, knottins, fynomers, atrimers, DARPins, affibodys, affilins, armadillo repeat proteins (ArmRPs), OBodys and adnectins (reviewed e.g. in Reverdatto et aL, Curr Top Med Chem. 2015; 15(12): 1082- 1101, which is hereby incorporated by reference in its entirety (see also e.g. Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48)). Antigen binding moieties according to the present disclosure also include target antigen-binding nucleic acids, e.g. nucleic acid aptamers (reviewed, for example, in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3): 181-202). Antigen-binding moieties according to the present disclosure also include target antigen-binding small molecules (e.g. low molecular weight (< 1000 daltons, typically between -300-700 daltons) organic compounds).

[0136] The antigen binding moiety capable of binding to a variant CH2 domain of the present disclosure are capable of binding to a variant CH2 domain according to the present disclosure. Antigen binding moieties that are capable of binding to a variant CH2 domain according to the present disclosure may also be described as antigen binding moieties that bind to a variant CH2 domain according to the present disclosure.

[0137] The antigen binding moieties described herein preferably display specific binding to a variant CH2 domain according to the present disclosure. Specific binding refers to binding which is selective for the target antigen, and which can be discriminated from nonspecific binding to non-target antigen. An antigen-binding moiety that specifically binds to a given target antigen preferably binds the target antigen with greater affinity, and / or with greater duration than it binds to other, non-target antigens.

[0138] The ability of a given moiety to bind specifically to a variant CH2 domain can be determined by analysis according to methods known in the art, such as by ELISA, Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol (2012) 907:411-442), Bio-Layer Interferometry (BLI; see e.g. Lad et al., (2015) J Biomol Screen 20(4): 498-507), flow cytometry, or by a radiolabeled antigen-binding assay (RIA) enzyme-linked immunosorbent assay. Through such analysis binding to a given variant CH2 domain can be measured and quantified. In some embodiments, the level of binding may be the response detected in a given assay.

[0139] In some embodiments, the antigen binding moiety described herein binds to a variant CH2 domain according to the present disclosure with an affinity (e.g. determined by SPR or BLI) in the micromolar range, i.e. KD = 9.9 x IO'4"' to 1 x 10'6M. In some embodiments, the antigen binding moiety described herein binds to a variant CH2 domain according to the present disclosure with sub-micromolar affinity, i.e. KD < 1 x 10'6M. In some embodiments, the antigen binding moiety described herein binds to a variant CH2 domain according to the present disclosure with an affinity in the nanomolar range, i.e. KD = 9.9 x IO'7"' to 1 x 10'9M. In some embodiments, the antigen binding moiety described herein binds to a variant CH2 domain according to the present disclosure with sub -nanomolar affinity, i.e. KD < 1 x 10'9M. In some embodiments, the antigen binding moiety described herein binds to a variant CH2 domain according to the present disclosure with an affinity in the picomolar range, i.e. KD = 9.9 x IO'10'" to 1 x 10'12M. In some embodiments, the antigen binding moiety described herein binds to a variant CH2 domain according to the present disclosure with sub-picomolar affinity, i.e. KD < 1 x IO’12M.

[0140] The antigen binding moieties according to the present disclosure preferably do not display specific binding to a reference CH2 domain according to the present disclosure. In some embodiments, the antigen binding moiety does not bind, or displays substantially no binding, to a reference CH2 domain according to the present disclosure.

[0141] An antigen binding moiety that does not bind or that displays substantially no binding to a given CH2 domain displays a level of binding to the given CH2 domain which is similar to the level of binding to an antigen that the antigen binding moiety is known not to bind, or known to not to bind specifically, e.g. a non-target antigen. In some embodiments, the level of binding of an antigen binding moiety that does not bind, or that displays substantially no binding, to a given CH2 domain is > 0.5 times and < 2 times, e.g. one of > 0.75 times and < 1.5 times, > 0.8 times and < 1.4 times, > 0.85 times and < 1.3 times, > 0.9 times and < 1.2 times, > 0.95 times and < 1.1 times the level of binding displayed by the antigen binding moiety to an antigen that the antigen binding immune activating moiety is known not to bind, or known to not to bind specifically, e.g. a nontarget antigen.

[0142] In some embodiments, the level of binding of the antigen binding moiety to a reference CH2 domain according to the present disclosure is <10% of the binding of the antigen binding moiety to a variant CH2 domain according to the present disclosure as determined e.g. by ELISA, SPR, BLI or RIA. In some embodiments, the antigen binding moiety binds to a reference CH2 domain according to the present disclosure with an equilibrium dissociation constant (KD; e.g. determined by SPR or BLI) that is at least 0.1 order of magnitude greater than the KD of the antigen binding moiety for a variant CH2 domain according to the present disclosure. Preferably, the reference CH2 domain is identical to the variant CH2 domain with the exception of the specific mutations incorporated in the variant CH2 domain. In a preferred embodiment, the reference CH2 domain comprises or consists of the amino acid sequence of SEQ ID NO: 75.

[0143] An antigen binding moiety according to the present disclosure may be, or may comprise, an antigen binding peptide / polypeptide, or an antigen binding peptide / polypeptide complex. An antigen binding moiety may comprise more than one peptide / polypeptide that together form an antigen binding domain. The peptides / polypeptides may associate covalently or non-covalently. In some embodiments, the peptides / polypeptides form part of a larger polypeptide comprising the peptides / polypeptides e.g. in the case of an scFv moiety comprising a VH region and a VL region, or in the case of a scFab moiety comprising VH-CH1 and VL-CL).

[0144] In some embodiments, the antigen binding moiety of the present disclosure comprises an antibody heavy chain variable (VH) region and an antibody light chain variable (VL) region of an antibody capable of binding to a given variant CH2 domain. In some embodiments, the antigen binding moiety comprises, or consists of, an Fv moiety formed by the VH region and a VL region of an antibody capable of binding to a given variant CH2 domain. In some embodiments, the VH region and a VL region may be provided in the same polypeptide, and joined by a linker sequence. In some embodiments, the antigen binding moiety comprises, or consists of, an scFv moiety that binds to a given variant CH2 domain.

[0145] Antigen binding moieties of the present disclosure generally comprise six complementarity-determining regions CDRs; three in the heavy chain variable (VH) region: HC-CDR1, HC-CDR2 and HC-CDR3, and three in the light chain variable (VL) region: LC- CDR1, LC-CDR2, and LC-CDR3. The six CDRs together define the paratope of the antigen binding moiety, which is the part of the moiety that binds to the target antigen.

[0146] The VH region and VL region comprise framework regions (FRs) either side of each CDR, which provide a scaffold for the CDRs. From N-terminus to C-terminus, VH regions comprise the following structure: N term-[HC-FRl]-[HC-CDRl]-[HC-FR2]-[HC-CDR2]- [HC-FR3]-[HC-CDR3]-[HC-FR4]-C term; and VL regions comprise the following structure: N term-[LC-FRl]-[LC-CDRl]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C term.

[0147] There are several different conventions for defining antibody CDRs and FRs, such as

[0148] (i) the Kabat system, described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991);

[0149] (ii) the Chothia system, described in Chothia et al., J. Mol. Biol. 196:901-917 (1987); and

[0150] (iii) the international IM GT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue):D413-22), which uses the IMGT V-DOMAIN numbering rules as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77.

[0151] The CDRs and FRs of the VH regions and VL regions of the antigen binding moieties described herein are defined according to the Kabat system.

[0152] In some embodiments, the antigen binding moiety comprises the CDRs of an antigen binding moiety that binds to a variant CH2 domain according to the present disclosure. In some embodiments, the antigen binding moiety comprises the FRs of an antigen binding moiety that binds to a variant CH2 domain according to the present disclosure. In some embodiments, the antigen binding moiety comprises the CDRs and the FRs of an antigen binding moiety that binds to a variant CH2 domain according to the present disclosure. That is, in some embodiments, the antigen binding moiety comprises the VH region and the VL region of an antigen binding moiety that binds to a variant CH2 domain according to the present disclosure.

[0153] Wessels et al. Bioanal. (2017) 9(l l):849-59 describes the identification of an antibody that binds to antibodies comprising a CH2 domain derived from human IgGl comprising P329G, but that does not bind to antibodies comprising the equivalent CH2 domain lacking the P329G substitution. The antibody also binds to antibodies having a hlgGl -derived Fc region comprising P329G and further comprising L234A and L235A. Darowski et al., Protein Eng. Des. Sei. (2019) 32(5):207-218 and Stock et al., Journal for ImmunoTherapy of Cancer (2022) 10:e005054 provide the structure of the anti-P329G Fab with Fc comprising P329G, L234A and L235A. The anti-P329G Fab interacts with Fc comprising P329G, L234A and L235A with 1: 1 stoichiometry. The epitope is disclosed to include positions N325 to P331 (including G329), and also S267 to E272. In some embodiments, the antigen binding moiety comprises the CDRs, FRs and / or the VH and / or VL regions of an antigen binding molecule described herein that binds to a variant CH2 domain according to the present disclosure, or comprises CDRs, FRs and / or VH and / or VL regions which are derived from those of an antigen binding molecule described herein that binds to a variant CH2 domain according to the present disclosure. In some embodiments, an antigen binding molecule that binds to a variant CH2 domain according to the present disclosure is referred to as anti-P329G or anti-CH2 PG VH3xVLl.

[0154] In some embodiments, the antigen binding moiety comprises a VH region incorporating the following CDRs:

[0155] HC-CDR1 having the amino acid sequence of SEQ ID NO: 1

[0156] HC-CDR2 having the amino acid sequence of SEQ ID NO: 2

[0157] HC-CDR3 having the amino acid sequence of SEQ ID NO: 3, or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1, and / or in which 1 or 2 or 3 amino acids in HC-CDR2, and / or in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid.

[0158] In some embodiments, the antigen binding moiety comprises a VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%, sequence identity, to the amino acid sequence of SEQ ID NO: 7.

[0159] In some embodiments, the antigen-binding moiety comprises a VL region incorporating the following CDRs:

[0160] LC-CDR1 having the amino acid sequence of SEQ ID NO: 4

[0161] LC-CDR2 having the amino acid sequence of SEQ ID NO: 5

[0162] LC-CDR3 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1, and / or in which 1 or 2 or 3 amino acids in LC-CDR2, and / or in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid. In some embodiments, the antigen-binding moiety comprises a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%, sequence identity, to the amino acid sequence of SEQ ID NO: 8.

[0163] In some embodiments, the antigen-binding moiety comprises a VH region as described above, and a VL region as described above.

[0164] Substitutions of amino acids in accordance with the present disclosure may be biochemically conservative. In some embodiments, where an amino acid to be substituted is provided in one of rows 1 to 5 of the table below, the replacement amino acid of the substitution is another, non-identical amino acid provided in the same row:

[0165] By way of illustration, in some embodiments wherein substitution is of a Met residue, the replacement amino acid may be selected from Ala, Vai, Leu, He, Trp, Tyr, Phe and Norleucine.

[0166] In some embodiments, a replacement amino acid in a substitution may have the same side chain polarity as the amino acid residue it replaces. In some embodiments, a replacement amino acid in a substitution may have the same side chain charge (at pH 7.4) as the amino acid residue it replaces:

[0167]

[0168] That is, in some embodiments, a nonpolar amino acid is substituted with another, non-identical nonpolar amino acid. In some embodiments, a polar amino acid is substituted with another, non-identical polar amino acid. In some embodiments, an acidic polar amino acid is substituted with another, non-identical acidic polar amino acid. In some embodiments, a basic polar amino acid is substituted with another, non-identical basic polar amino acid. In some embodiments, a neutral amino acid is substituted with another, non- identical neutral amino acid. In some embodiments, a positive amino acid is substituted with another, non-identical positive amino acid. In some embodiments, a negative amino acid is substituted with another, non-identical negative amino acid.

[0169] In some embodiments, substitution(s) may be functionally conservative. That is, in some embodiments, the substitution may not affect (or may not substantially affect) one or more functional properties (e.g. target antigen binding) of the antigen-binding moiety comprising the substitution, as compared to the equivalent unsubstituted molecule.

[0170] In some embodiments, an antigen binding moiety of the present disclosure comprises a VH as described herein. In some embodiments, an antigen binding moiety comprises a VL as described herein. In some embodiments, an antigen binding moiety comprises one or more antibody heavy chain constant regions (CH). In some embodiments, an antigen binding moiety comprises one or more antibody light chain constant regions (CL). In some embodiments, an antigen binding moiety comprises a CHI, CH2 region and / or a CH3 region of an immunoglobulin (Ig). In some embodiments, an antigen binding moiety comprises a linker sequence as described herein.

[0171] In some embodiments, the antigen binding moiety of the present disclosure comprises a polypeptide or polypeptides comprising: (i) a VH region comprising HC-CDR1 according to SEQ ID NO: 1, HC-CDR2 according to SEQ ID NO: 2, and HC-CDR3 according to SEQ ID NO: 3, and (ii) a VL region comprising LC-CDR1 according to SEQ ID NO: 4, LC-CDR2 according to SEQ ID NO: 5, and LC-CDR3 according to SEQ ID NO: 6.

[0172] In some embodiments, an antigen binding moiety of the present disclosure comprises an amino acid having at least 70%, preferably one of >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to SEQ ID NO: 7. In some embodiments, an antigen binding moiety of the present disclosure comprises an amino acid having at least 70%, preferably one of >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to SEQ ID NO: 8.

[0173] In a preferred embodiment, the antigen binding moiety comprises or consists of a VH region having the amino acid sequence of SEQ ID NO: 7 and the VL region having the amino acid sequence of SEQ ID NO: 8.

[0174] In some aspects and embodiments of the present disclosure, first and second components of an antigen binding moiety are provided. In accordance with such aspects and embodiments, it will be appreciated that the first and second components of an antigen binding moiety are complementary, and capable of associating to form the (complete, functional) antigen binding moiety.

[0175] In some embodiments according to the present disclosure, a component of an antigen binding moiety may be or comprise the VH region of an antigen binding moiety specific for a variant CH2 domain (e.g. as described herein). In some embodiments, a component of an antigen binding moiety may be or comprise the VL region of an antigen binding moiety specific for a variant CH2 domain (e.g. as described herein). In preferred embodiments, the VH region and VL region may be from the same antigen binding moiety. In some embodiments, a component of an antigen binding moiety comprises, or consists of, a VH as described herein. In some embodiments, a component of an antigen binding moiety comprises, or consists of, a VL as described herein. In some embodiments, a component of an antigen binding moiety comprises one or more antibody heavy chain constant regions (CH). In some embodiments, a component of an antigen binding moiety comprises one or more antibody light chain constant regions (CL). In some embodiments, a component of an antigen binding moiety comprises a CHI, CH2 region and / or a CH3 region of an immunoglobulin (Ig).

[0176] Glycosylation variants

[0177] In certain aspects, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

[0178] For example, in a preferred embodiment, the protease -activatable Fc domain binding molecule according to the present disclosure is altered to increase the extent to which the molecule is glycosylated.

[0179] Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some aspects, modifications of the oligosaccharide in a protease-activatable Fc domain binding molecule of the invention may be made in order to create antibody variants with certain improved properties.

[0180] In one aspect, protease-activatable Fc domain binding molecules are provided having a non-fucosylated oligosaccharide, i.e. an oligosaccharide structure that lacks fucose attached (directly or indirectly) to an Fc region. Such non-fucosylated oligosaccharide (also referred to as “afucosylated” oligosaccharide) particularly is an N-linked oligosaccharide which lacks a fucose residue attached to the first GlcNAc in the stem of the biantennary oligosaccharide structure. In one aspect, protease -activatable Fc domain binding molecules are provided having an increased proportion of non-fucosylated oligosaccharides in the Fc region as compared to a native or parent antibody. For example, the proportion of non- fucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e. no fucosylated oligosaccharides are present). The percentage of non-fucosylated oligosaccharides is the (average) amount of oligosaccharides lacking fucose residues, relative to the sum of all oligosaccharides attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI- TOF mass spectrometry, as described in WO 2006 / 082515, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Antibodies having an increased proportion of non-fucosylated oligosaccharides in the Fc region may have improved FcyRIIIa receptor binding and / or improved effector function, in particular improved ADCC function. See, e.g., US 2003 / 0157108; US 2004 / 0093621.

[0181] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lee 13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003 / 0157108; and WO 2004 / 056312, especially at Example 11), and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614- 622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107), or cells with reduced or abolished activity of a GDP -fucose synthesis or transporter protein (see, e.g., US2004259150, US2005031613, US2004132140, US2004110282).

[0182] In a further aspect, protease-activatable Fc domain binding molecules are provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the protease-activatable Fc domain binding molecule is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function as described above. Examples of such antibody variants are described, e.g., in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99 / 54342; WO 2004 / 065540, WO 2003 / 011878.

[0183] Protease-activatable Fc domain binding molecules with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764. Fc domain modifications promoting heterodimerization

[0184] The protease-activatable Fc domain binding molecule may further comprise additional Fc domain modifications promoting heterodimerization. In a specific such aspect said modification is a so-called “knob-into-hole” modification, comprising a “knob” modification in one of the two subunits of the Fc domain and a “hole” modification in the other one of the two subunits of the Fc domain.

[0185] The knob-into-hole technology is described e.g., in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine).

[0186] Accordingly, in a particular embodiment, in the CH3 domain of the first subunit of the Fc domain of the protease-activatable Fc domain binding molecule an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.

[0187] The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g., by site-specific mutagenesis, or by peptide synthesis.

[0188] In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain additionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A).

[0189] In yet a further embodiment, in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C). Introduction of these two cysteine residues results in formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0190] In one preferred embodiment, the first subunit of the Fc domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 73 and the second subunit of the Fc domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 74.

[0191] In the alternative preferred embodiment, the first subunit of the Fc domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 74 and the second subunit of the Fc domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 73.

[0192] In an further embodiment a modification promoting association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g., as described in PCT publication WO 2009 / 089004. Generally, this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable.

[0193] Exemplary protease-activatable Fc domain binding molecules

[0194] The variant CH2 domain, the protease-cleavable linker, the antigen binding moiety capable of binding to the variant CH2 domain, and the Fc domain of the protease -activatable Fc domain binding molecule as hereinbefore described can be fused to each other in a variety of configurations.

[0195] In some embodiments, the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or the second subunit of the Fc domain. In some embodiments, the protease-activatable Fc domain binding molecules comprise a second antigen binding moiety. In one embodiment, the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain and the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. In one embodiment, the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain and the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain.

[0196] In some embodiments, the first masking moiety is fused at the C-terminus to the N- terminus of the first antigen binding moiety. In some embodiments, the first masking moiety is fused at the C-terminus to the N-terminus of the Fab heavy chain of the first antigen binding moiety. In some embodiments, the first masking moiety is fused at the C-terminus to the N-terminus of the Fab heavy chain of the first antigen binding moiety through the first protease-cleavable linker.

[0197] In some embodiments, the second masking moiety is fused at the C-terminus to the N-terminus of the second antigen binding moiety. In some embodiments, the second masking moiety is fused at the C-terminus to the N-terminus of the Fab heavy chain of the second antigen binding moiety. In some embodiments, the first masking moiety is fused at the C-terminus to the N-terminus of the Fab heavy chain of the first antigen binding moiety through the second protease-cleavable linker.

[0198] A protease-activatable Fc domain binding molecule with a single antigen binding moiety capable of binding to a variant CH2 domain comprising G329 according to EU numbering is useful, particularly in cases where excessive crosslinking of molecules is to be avoided.

[0199] In many other cases, however, it will be advantageous to have a protease-activatable Fc domain binding molecule comprising two antigen binding moieties specific for a variant CH2 domain comprising G329 according to EU numbering, for example to optimize crosslinking of target antigen binding molecules.

[0200] Exemplary configuration are shown in Figures 3. Exemplary sequences are shown herein below. In one embodiment the protease-activatable Fc domain binding molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 49, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 55. In one embodiment the protease -activatable Fc domain binding molecule comprises the polypeptide sequence of SEQ ID NO: 49, and the polypeptide sequence of SEQ ID NO: 55. This molecule is referred to as P1AJ4817 herein.

[0201] In one embodiment the protease-activatable Fc domain binding molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 49, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 56. In one embodiment the protease -activatable Fc domain binding molecule comprises the polypeptide sequence of SEQ ID NO: 49, and the polypeptide sequence of SEQ ID NO: 56. This molecule is referred to as Pl AJ4819 herein.

[0202] In one embodiment the protease-activatable Fc domain binding molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 49, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 53, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 58. In one embodiment the protease-activatable Fc domain binding molecule comprises the polypeptide sequence of SEQ ID NO: 49, the polypeptide sequence of SEQ ID NO: 53, and the polypeptide sequence of SEQ ID NO: 58. This molecule is referred to as Pl AJ4833 herein.

[0203] In one embodiment the protease-activatable Fc domain binding molecule comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 49, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 53, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 59. In one embodiment the protease-activatable Fc domain binding molecule comprises the polypeptide sequence of SEQ ID NO: 49, the polypeptide sequence of SEQ ID NO: 53, and the polypeptide sequence of SEQ ID NO: 59. This molecule is referred to as Pl AJ4835 herein.

[0204] Compositions

[0205] The present disclosure also provides compositions comprising the protease - activatable Fc domain binding molecule described herein. The protease-activatable Fc domain binding molecules described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may comprise a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant. In preferred aspects and embodiments, the present disclosure provides a pharmaceutical composition or medicament comprising protease-activatable Fc domain binding molecule according to the present disclosure. Thus, the present disclosure also provides a pharmaceutical composition / medicament comprising a protease-activatable Fc domain binding molecule described herein.

[0206] The pharmaceutical compositions / medicaments of the present disclosure may comprise one or more pharmaceutically-acceptable carriers (e.g. liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g. starch, cellulose, a cellulose derivative, a polyol, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methyl paraben, propyl paraben), anti-oxidants (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (e.g. magnesium stearate, talc, silica, stearic acid, vegetable stearin), binders (e.g. sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents or colouring agents (e.g. titanium oxide).

[0207] The term “pharmaceutically-acceptable” as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g. a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent of a composition according to the present disclosure must also be acceptable in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilisers, solubilisers, surfactants, masking agents, colouring agents, flavouring agents or sweetening agents can be found in standard pharmaceutical texts, for example, Remington’s ‘The Science and Practice of Pharmacy’ (Ed. A. Adejare), 23rd Edition (2020), Academic Press. Pharmaceutical compositions and medicaments of the present disclosure may be formulated for topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal routes of administration. In some embodiments, a pharmaceutical composition / medicament may be formulated for administration by injection or infusion, or administration by ingestion.

[0208] Suitable formulations may comprise the protease -activatable Fc domain binding molecule provided in a sterile or isotonic medium. Medicaments and pharmaceutical compositions may be formulated in fluid, including gel, form. Fluid formulations may be formulated for administration by injection or infusion (e.g. via catheter) to a selected region of the human or animal body.

[0209] In some embodiments, the pharmaceutical compositions / medicament is formulated for injection or infusion, e.g. into a blood vessel, tissue / organ of interest, or a tumor.

[0210] The present disclosure also provides methods for the production of pharmaceutically useful compositions, such methods of production may comprise one or more steps selected from: producing a protease-activatable Fc domain binding molecule described herein; isolating / purifying a protease-activatable Fc domain binding molecule described herein; and / or mixing a protease-activatable Fc domain binding molecule described herein with a pharmaceutically-acceptable carrier, adjuvant, excipient or diluent.

[0211] For example, a further aspect the present disclosure relates to a method of formulating or producing a medicament or pharmaceutical composition for use in the treatment of a disease / condition (e.g. a disease / condition described herein), the method comprising formulating a pharmaceutical composition or medicament by mixing a protease- activatable Fc domain binding molecule described herein with a pharmaceutically- acceptable carrier, adjuvant, excipient or diluent.

[0212] Therapeutic and prophylactic application The articles of the present disclosure find use in therapeutic and prophylactic methods. In particular, a protease-activatable Fc domain binding molecule according to the present disclosure, finds use in therapeutic and prophylactic methods. Similarly, a composition according to the present disclosure, e.g. a pharmaceutical composition comprising a protease-activatable Fc domain binding molecule according to the present disclosure finds use in such methods.

[0213] Accordingly, the present disclosure provides a protease-activatable Fc domain binding molecule or composition described herein for use in a method of medical treatment or prophylaxis. Also provided is a protease-activatable Fc domain binding molecule or composition described herein for use in a method of treating or preventing a disease or condition described herein. Also provided is the use of a protease-activatable Fc domain binding molecule or composition described herein in the manufacture of a medicament for treating or preventing a disease or condition described herein. Also provided is a method of treating or preventing a disease or condition described herein, comprising administering to a subject a therapeutically- or prophylactically- effective amount of a protease-activatable Fc domain binding molecule or composition described herein.

[0214] The intervention described in the preceding paragraph may be effective to reduce the development or progression of a disease / condition, alleviate the symptoms of a disease / condition or reduce the pathology of a disease / condition. The intervention may be effective to prevent progression of the disease / condition, e.g. to prevent worsening of, or to slow the rate of development of, the disease / condition. In some embodiments, the intervention may lead to an improvement in the disease / condition, e.g. a reduction in the symptoms of the disease / condition or reduction in some other correlate of the severity / activity of the disease / condition. In some embodiments, the intervention may prevent progression / development of the disease / condition a later stage e.g. a chronic stage or metastasis).

[0215] Therapeutic or prophylactic intervention in accordance with the present disclosure generally comprises administering a protease-activatable Fc domain binding molecule or pharmaceutical composition according to the present disclosure to a subject to which a target antigen binding molecule comprising: (a) an antigen binding domain that binds to the target antigen, and (b) a variant Fc domain comprising a variant CH2 domain according to the present disclosure, has been or is to be administered. It will be appreciated that in accordance with such intervention, the protease - activatable Fc domain binding molecule comprises an antigen binding moiety that binds to the variant Fc domain of the target antigen binding molecule.

[0216] By way of illustration, the intervention may comprise administering a protease- activatable Fc domain binding molecule comprising the polypeptides according to SEQ ID NO: 49, and SEQ ID NO: 55 to a subject that has been, or is to be, administered a target antigen binding molecule comprising an Fc domain according to SEQ ID NO: 82.

[0217] By way of illustration, the intervention may comprise administering a protease- activatable Fc domain binding molecule comprising the polypeptides according to SEQ ID NO: 49, and SEQ ID NO: 56 to a subject that has been, or is to be, administered a target antigen binding molecule comprising an Fc domain according to SEQ ID NO: 82.

[0218] By way of illustration, the intervention may comprise administering a protease- activatable Fc domain binding molecule comprising the polypeptides according to SEQ ID NO: 49, SEQ ID NO: 53, and SEQ ID NO: 58 to a subject that has been, or is to be, administered a target antigen binding molecule comprising an Fc domain according to SEQ ID NO: 82.

[0219] By way of illustration, the intervention may comprise administering a protease- activatable Fc domain binding molecule comprising the polypeptides according to SEQ ID NO: 49x, SEQ ID NO: 53, and SEQ ID NO: 59 to a subject that has been, or is to be, administered a target antigen binding molecule comprising an Fc domain according to SEQ ID NO: 82.

[0220] In the therapeutic / prophylactic intervention of the present disclosure, the target antigen binding molecule comprising the variant Fc domain comprising the variant CH2 domain serves as an adaptor molecule, and directs the activity of a cell according to the present disclosure against the antigen to which the target antigen binding molecule binds. That is, in embodiments wherein the cell is an immune cell (e.g. a T cell), the variant Fc domain-bearing target antigen binding molecule directs a cell-mediated immune response (e.g. a T cell-mediated immune response) against cells expressing the antigen to which the target antigen binding molecule binds (see for example Figure 1 and 2).

[0221] By way of illustration, in the Examples of the present disclosure, a protease- activatable Fc domain binding molecule comprising the polypeptides of SEQ ID NO: 49, and SEQ ID NO: 55 (P1AJ4817) is employed with an anti-FolRl antibody comprising an Fc domain comprising P329G, such that the T cells are directed against FolRl -expressing cells (see e.g. Figure 8, 13, 14). By way of illustration, in the Examples of the present disclosure, a protease-activatable Fc domain binding molecule comprising the polypeptides of SEQ ID NO: 49, and SEQ ID NO: 56 (P1AJ4819) is employed with an anti-FolRl antibody comprising an Fc domain comprising P329G, such that the T cells are directed against FolRl -expressing cells (see Figure 8, 13, 14).

[0222] By way of illustration, in the Examples of the present disclosure, a protease- activatable Fc domain binding molecule comprising the polypeptides of SEQ ID NO: 49, and SEQ ID NO: 55 (P1AJ4817) is employed with an anti-EpCAM antibody comprising an Fc domain comprising P329G, such that the T cells are directed against EpCAM-expressing cells (see e.g. Figure 8). By way of illustration, in the Examples of the present disclosure, a protease-activatable Fc domain binding molecule comprising the polypeptides of SEQ ID NO: 49, and SEQ ID NO: 56 (P1AJ4819) is employed with an anti-EpCAM antibody comprising an Fc domain comprising P329G, such that the T cells are directed against EpCAM-expressing cells (see Figure 8).

[0223] By way of illustration, in the Examples of the present disclosure, a protease- activatable Fc domain binding molecule comprising the polypeptides of SEQ ID NO: 49, and SEQ ID NO: 55 (P1AJ4817) is employed with an anti-CD25 antibody comprising an Fc domain comprising P329G, such that the T cells are directed against CD25 -expressing cells (see e.g. Figure 9). By way of illustration, in the Examples of the present disclosure, a protease-activatable Fc domain binding molecule comprising the polypeptides of SEQ ID NO: 49, and SEQ ID NO: 56 (P1AJ4819) is employed with an anti-CD25 antibody comprising an Fc domain comprising P329G, such that the T cells are directed against CD25-expressing cells (see Figure 9).

[0224] By way of illustration, in the Examples of the present disclosure, a protease- activatable Fc domain binding molecule comprising the polypeptides of SEQ ID NO: 49, SEQ ID NO: 53, and SEQ ID NO: 58 (Pl AJ4833) is employed with an anti-FolRl antibody comprising an Fc domain comprising P329G, such that the T cells are directed against FolRl -expressing cells (see Figure 11). By way of illustration, in the Examples of the present disclosure, a protease-activatable Fc domain binding molecule comprising the polypeptides of SEQ ID NO: 49, SEQ ID NO: 53, and SEQ ID NO: 59 (P1AJ4835) is employed with an anti-FolRl antibody comprising an Fc domain comprising P329G, such that the T cells are directed against FolRl -expressing cells (see Figure 11). The variant Fc domain-bearing antigen-binding molecule employed with a protease- activatable Fc domain binding molecule or composition according to the present disclosure may bind to any given target antigen.

[0225] The target antigen may be any target antigen expressed by a cell that is desired to be killed / depleted in order to attain a therapeutic / prophylactic effect. In some embodiments, the target antigen is an antigen whose expression / activity, or whose upregulated expression / activity, is positively associated with a disease / condition ( .g. a cancer, an infectious disease or an autoimmune disease). The target antigen is preferably expressed at the cell surface of a cell expressing the target antigen.

[0226] In some embodiments, the target antigen may be a cancer cell antigen. A cancer cell antigen is an antigen which is expressed or over-expressed by a cancer cell. A cancer cell antigen may be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. A cancer cell antigen’s expression may be associated with a cancer. A cancer cell antigen may be abnormally expressed by a cancer cell (e.g. the cancer cell antigen may be expressed with abnormal localization), or may be expressed with an abnormal structure by a cancer cell. A cancer cell antigen may be capable of eliciting an immune response. In some embodiments, the antigen is expressed at the cell surface of the cancer cell (i.e. the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the part of the antigen which is bound by the antigen-binding molecule described herein is displayed on the external surface of the cancer cell (z.e. is extracellular). The cancer cell antigen may be a cancer-associated antigen. In some embodiments the cancer cell antigen is an antigen whose expression is associated with the development, progression or severity of symptoms of a cancer. The cancer-associated antigen may be associated with the cause or pathology of the cancer, or may be expressed abnormally as a consequence of the cancer. In some embodiments, the cancer cell antigen is an antigen whose expression is upregulated (e.g. at the RNA and / or protein level) by cells of a cancer, e.g. as compared to the level of expression of by comparable non-cancerous cells (e.g. non- cancerous cells derived from the same tissue / cell type). In some embodiments, the cancer- associated antigen may be preferentially expressed by cancerous cells, and not expressed by comparable non-cancerous cells (e.g. non-cancerous cells derived from the same tissue / cell type). In some embodiments, the cancer-associated antigen may be the product of a mutated oncogene or mutated tumor suppressor gene. In some embodiments, the cancer-associated antigen may be the product of an overexpressed cellular protein, a cancer antigen produced by an oncogenic virus, an oncofetal antigen, or a cell surface glycolipid or glycoprotein. Cancer cell antigens are reviewed by Zarour HM, DeLeo A, Finn OJ, et al. Categories of Tumor Antigens. In: Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton (ON): BC Decker; 2003. Cancer cell antigens include oncofetal antigens: CEA, Immature laminin receptor, TAG-72; oncoviral antigens such as HPV E6 and E7; overexpressed proteins: BING-4, calcium-activated chloride channel 2, cyclin-Bl, 9D7, Ep-CAM, EphA3, HER2 / neu, telomerase, mesothelin, SAP-1, survivin; cancer-testis antigens: BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1, PRAME, SSX-2; lineage restricted antigens: MARTI, GplOO, tyrosinase, TRP-1 / 2, MC1R, prostate specific antigen; mutated antigens: P-catenin, BRCA1 / 2, CDK4, CML66, Fibronectin, MART -2, p53, Ras, TGF-PRII; post-translationally altered antigens: MUC1, idiotypic antigens: Ig, TCR. Other cancer cell antigens include heat-shock protein 70 (HSP70), heat-shock protein 90 (HSP90), glucose-regulated protein 78 (GRP78), vimentin, nucleolin, feto-acinar pancreatic protein (FAPP), alkaline phosphatase placental-like 2 (ALPPL-2), siglec-5, stress-induced phosphoprotein 1 (STIP1), protein tyrosine kinase 7 (PTK7), and cyclophilin B. In some embodiments the cancer cell antigen is a cancer cell antigen described in Zhao and Cao, Front Immunol. (2019) 10: 2250, which is hereby incorporated by reference in its entirety.

[0227] In some embodiments, the target antigen is selected from: FAP (fibroblast activation protein), CEA (carcinoembryonic antigen), p95 (p95HER2), BCMA (B-cell maturation antigen), EpCAM (epithelial cell adhesion molecule), MSLN (mesothelin), MCSP (melanoma chondroitin sulfate proteoglycan), HER-1 (human epidermal growth factor 1), HER-2 (human epidermal growth factor 2), HER-3 (human epidermal growth factor 3), CD 19, CD20, CD22, CD33, CD38, CD52Flt3, folate receptor 1 (FOLR1), human trophoblast cell-surface antigen 2 (Trop-2) cancer antigen 12-5 (CA-12-5), human leukocyte antigen - antigen D related (HLA-DR), MUC-1 (Mucin-1), A33-antigen, PSMA (prostatespecific membrane antigen), FMS-like tyrosine kinase 3 (FLT-3), PSCA (prostate stem cell antigen), transferrin-receptor, TNC (tenascin), carbon anhydrase IX (CA-IX), and / or a peptide bound to a molecule of the human major histocompatibility complex (MHC). In some embodiments, the target antigen is FolRl. In some embodiments, the target antigen is CEACAM5. In some embodiments, the target antigen is CD25.

[0228] The variant Fc domain-bearing antigen-binding molecule employed with a protease- activatable Fc domain binding molecule or composition according to the present disclosure may comprise additional amino acid substitutions in the Fc domain, as long as the antigen binding moiety capable of binding to the variant CH2 domain retains the ability to bind. In one embodiment the variant Fc domain-bearing antigen-binding molecule comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numberings according to Kabat EU index). In a more specific embodiment the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numberings according to Kabat EU index). In some embodiments the Fc domain comprises the amino acid substitutions L234A and L235A (numberings according to Kabat EU index). In one such embodiment, the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain. In one embodiment the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment the amino acid substitution is P329A or P329G, particularly P329G (numberings according to Kabat EU index). In one embodiment the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numberings according to Kabat EU index). In a more specific embodiment the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In particular embodiments the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numberings according to Kabat EU index). In more particular embodiments the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (“P329G LALA”). In one such embodiment, the Fc domain is an IgGi Fc domain, particularly a human IgGi Fc domain. The “P329G LALA” combination of amino acid substitutions almost completely abolishes Fey receptor (as well as complement) binding of a human IgGi Fc domain, as described in PCT publication no. WO 2012 / 130831, incorporated herein by reference in its entirety. WO 2012 / 130831 also describes methods of preparing such mutant Fc domains and methods for determining its properties such as Fc receptor binding or effector functions.

[0229] IgG4antibodies exhibit reduced binding affinity to Fc receptors and reduced effector functions as compared to IgGi antibodies. Hence, in some embodiments the Fc domain of the variant CH2 domain-bearing antigen-binding molecule is an IgG4Fc domain, particularly a human IgG4Fc domain. In one embodiment the IgG4Fc domain comprises amino acid substitutions at position S228, specifically the amino acid substitution S228P (numberings according to Kabat EU index). To further reduce its binding affinity to an Fc receptor and / or its effector function, in one embodiment the IgG4Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E (numberings according to Kabat EU index). In another embodiment, the IgG4Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (numberings according to Kabat EU index). In a particular embodiment, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically amino acid substitutions S228P, L235E and P329G (numberings according to Kabat EU index). Such IgG4 Fc domain mutants and their Fey receptor binding properties are described in PCT publication no. WO 2012 / 130831, incorporated herein by reference in its entirety.

[0230] In a particular embodiment the Fc domain of the variant Fc domain-bearing antigenbinding molecule exhibiting reduced binding affinity to an Fc receptor and / or reduced effector function, as compared to a native IgGi Fc domain, is a human IgGi Fc domain comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG4 Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G (numberings according to Kabat EU index).

[0231] In certain embodiments N-glycosylation of the Fc domain of the variant Fc domainbearing antigen-binding molecule has been eliminated. In one such embodiment the target Fc domain comprises an amino acid substitution at position N297, particularly an amino acid substitution replacing asparagine by alanine (N297A) or aspartic acid (N297D) (numberings according to Kabat EU index).

[0232] Variant (mutant) Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing.

[0233] Binding to Fc receptors can be measured by methods known in the art for example in WO2021 / 255138 (e.g. Example 2) which is hereby incorporated by reference in its entirety. For example, binding to Fc receptors can be easily determined e.g., by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a Biacore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression or using cell lines known to express particular Fc receptors, such as human NK cells expressing Fcyllla receptor.

[0234] Effector function of an Fc domain or fragments thereof can be measured by methods known in the art. For example a suitable assay for measuring ADCC is described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351 -1361 (1987). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® nonradioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0235] In some embodiments, binding of the Fc domain of the variant Fc domain-bearing antigen-binding molecule to a complement component, specifically to Clq, is reduced. Accordingly, in some embodiments the Fc domain is engineered to have reduced effector function, said reduced effector function includes reduced CDC. Clq binding assays may be carried out to determine whether the variant Fc domain-bearing antigen-binding molecule is able to bind Clq and hence has CDC activity. See e.g., Clq and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano- Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

[0236] It will be appreciated that the protease-activatable Fc domain binding molecule and composition of the present disclosure may be used for the treatment / prevention of any disease / condition that would derive therapeutic or prophylactic benefit from a reduction in the level / activity of a given target antigen, or a reduction in the number / proportion / activity of cells comprising / expressing a given target antigen.

[0237] For example, the disease / condition may be a disease / condition in which the target antigen, or cells comprising / expressing target antigen are pathologically -implicated, e.g. a disease / condition in which an increased level / activity of the target antigen, or an increase in the number / proportion / activity of cells comprising / expressing target antigen is positively associated with the onset, development or progression of the disease / condition, and / or severity of one or more symptoms of the disease / condition. In some embodiments, an increased level / activity of the target antigen, or an increase in the number / proportion / activity of cells comprising / expressing target antigen may be a risk factor for the onset, development or progression of the disease / condition. In some embodiments, the disease / condition to be treated / prevented in accordance with the present disclosure is a disease / condition characterised by an increase in the level of expression or activity of the target antigen, e.g. as compared to the level of expression / activity in the absence of the disease / condition. In some embodiments, the disease / condition to be treated / prevented is a disease / condition characterised by an increase in the number / proportion / activity of cells expressing target antigen, e.g. as compared to the level / number / proportion / activity in the absence of the disease / condition (e.g. in a healthy subject, or in equivalent non-diseased tissue). Where the disease / condition is a cancer, the level of expression or activity of the target antigen may be greater than the level of expression or activity of the target antigen in equivalent non-cancerous cells / non-tumor tissue. A cancer / cell thereof may comprise one or more mutations (e.g. relative to equivalent non-cancerous cells / non-tumor tissue) causing upregulation of expression or activity of the target antigen.

[0238] Therapeutic / prophylactic intervention in accordance with the present disclosure may achieve one or more of the following in a subject (compared to an equivalent untreated subject, or subject treated with an appropriate control): a reduction in the level of the target antigen; a reduction in the activity of the target antigen; and / or a reduction in the number / proportion / activity of cells comprising / expressing the target antigen.

[0239] The present disclosure provides methods comprising administering protease - activatable Fc domain binding molecules or compositions according to the present disclosure to a subject.

[0240] In some embodiments, the methods further comprise: administering an antigen-binding molecule comprising a variant Fc domain according to the present disclosure to the subject, wherein the protease -activatable Fc domain binding molecule comprises an antigen-binding moiety that binds to the variant Fc domain of the antigen-binding molecule.

[0241] It will be appreciated that the method steps recited in the preceding two paragraphs may be performed in any suitable order.

[0242] Administration of the articles of the present disclosure is preferably in a therapeutically-effective or prophylactically-effective amount, this being sufficient to show therapeutic or prophylactic benefit to the subject. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease / condition and the particular article administered. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disease / disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s ‘The Science and Practice of Pharmacy’ (ed. A. Adejare), 23rd Edition (2020), Academic Press.

[0243] Administration of the articles of the present disclosure may be parenteral, systemic, intravenous, intra-arterial, intramuscular, intracavitary, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal, topical or transdermal. Administration may be by injection or infusion.

[0244] Administration of the articles of the present disclosure may be intratumoral. In some cases, the articles of the present disclosure may be formulated for targeted delivery to specific cells, a tissue, an organ and / or a tumor.

[0245] Multiple doses of an article of the present disclosure may be provided. Multiple doses may be separated by a predetermined time interval, which may be selected to be one of 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, or 31 days, or 1, 2, 3, 4, 5, or 6 months.

[0246] Administration of a protease-activatable Fc domain binding molecule or composition according to the present disclosure with an antigen-binding molecule described herein to a subject in accordance with the therapeutic and prophylactic intervention described herein may be simultaneous or sequential.

[0247] Simultaneous administration refers to administration of (i) a protease -activatable Fc domain binding molecule or composition according to the present disclosure, and (ii) an antigen-binding molecule described herein together, for example as a pharmaceutical composition containing both agents (i.e. a combined preparation), or immediately after one another, and optionally via the same route of administration, e.g. to the same artery, vein or other blood vessel.

[0248] Sequential administration refers to administration of one of (i) a protease-activatable Fc domain binding molecule or composition according to the present disclosure, and (ii) an antigen-binding molecule described herein, followed after a given time interval by separate administration of the other agent. It is not required that the two agents are administered by the same route, although this is the case in some embodiments. The time interval may be any time interval.

[0249] The present disclosure also provides methods for depleting or killing cells comprising or expressing a target antigen, comprising contacting cells comprising / expressing a target antigen with:

[0250] (i) an antigen-binding molecule comprising:(a) an antigen-binding domain that binds to the target antigen, and (b) a variant Fc domain according to the present disclosure; and

[0251] (ii) a protease-activatable Fc domain binding molecule according to the present disclosure; wherein the protease-activatable Fc domain binding molecule of (ii) comprises an antigen-binding moiety that binds to the variant Fc domain of the antigen-binding molecule of (i).

[0252] Subjects

[0253] A subject in accordance with the various aspects of the present disclosure may be any animal or human. Therapeutic and prophylactic applications may be in human or animals (veterinary use).

[0254] The subject to be administered with an article of the present disclosure (e.g. in accordance with therapeutic or prophylactic intervention) may be a subject in need of such intervention. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal, but is more preferably human. The subject may be male or female. The subject may be a patient.

[0255] A subject may have (e.g. may have been diagnosed with) a disease or condition described herein, may be suspected of having such a disease / condition, or may be at risk of developing / contracting such a disease / condition. In embodiments according to the present disclosure, a subject may be selected for treatment according to the methods based on characterisation for one or more markers of such a disease / condition.

[0256] In some embodiments, a subject may be selected for therapeutic or prophylactic intervention as described herein based on the detection of cells / tissue expressing a target antigen (i.e. the target antigen of an antigen-binding molecule to be employed in conjunction with a cell or composition according to the present disclosure), or of cells / tissue overexpressing the target antigen, e.g. in a sample obtained from the subject.

[0257] Kits

[0258] The present disclosure also provides kits of parts.

[0259] In some aspects and embodiments, a kit of parts according to the present disclosure comprises (i) a protease-activatable Fc domain binding molecule according to the present disclosure, and (ii) an antigen-binding molecule comprising: (a) an antigen-binding domain that binds to the target antigen, and (b) a variant Fc domain according to the present disclosure. It will be appreciated that in accordance with such aspects and embodiments, the protease-activatable Fc domain binding molecule of (i) comprises an antigen-binding moiety that binds to the variant Fc domain of the antigen-binding molecule of (ii).

[0260] In some aspects and embodiments, a kit of parts according to the present disclosure comprises (i) a composition according to the present disclosure, and (ii) an antigen-binding molecule comprising:(a) an antigen-binding domain that binds to the target antigen, and (b) a variant Fc domain according to the present disclosure. It will be appreciated that in accordance with such aspects and embodiments, the composition of (i) comprises a protease-activatable Fc domain binding molecule comprising an antigen-binding moiety that binds to the variant Fc domain of the antigen-binding molecule of (ii).

[0261] Kits of parts according to the present disclosure may comprise a predetermined quantity of articles according to (i) and / or (ii), as described in the preceding paragraphs. In some embodiments, articles according to (i) and / or (ii) are provided in containers (e.g. in vials or bottles). The kit may provide articles according to (i) and / or (ii) together with instructions (e.g. a protocol) as to how to employ them in accordance with a therapeutic or prophylactic intervention as described herein.

[0262] The manufacture of kits of parts according to the present disclosure preferably follows standard procedures which are known to the person skilled in the art.

[0263] EXEMPLARY SEQUENCES

[0264]

[0265]

[0266]

[0267] BRIEF DESCRIPTION OF THE FIGURES

[0268] Embodiments and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying figures.

[0269] FIGURE 1. Illustration of the mode of action of the standard, control anti-P329G immune cell engager (ICE) molecules. A targeting adaptor antibody in a human IgGl format comprises at least one binding moiety and P329G Fc-silencing mutations in the CH2 part of the Fc. The binding moiety can be designed for any antigen of choice. The P329G-containing targeting antibody is combined with an anti-P329G GE or WT innate cell engager antibody comprising at least one binding moiety to P329G mutation on adaptor antibody. This system of two molecules creates a functional anti-target innate cell engager, and enables universal off-the-shelf platform for cancer therapy.

[0270] FIGURE 2. Illustration of the concept of the present invention - a protease-activated anti-P329G immune cell engager (pro-ICE) molecule. A targeting antibody in a human IgGl format comprises at least one binding moiety and P329G Fc-silencing mutations in the CH2 part of the Fc. The binding moiety can be designed for any antigen of choice. The P329G- containing targeting antibody is combined with a protease-activated anti-P329G GE or WT innate cell engager antibody comprising at least one binding moiety to P329G mutation, while the anti-P329G binders are masked by P329G-containing CH2, attached by linkers that can be cleaved by a protease. In the periphery with both target and ADCC mediating cells presence, the pro-ICE remains masked, and does not form a functional innate cell engager with the targeting antibody. In the tumor microenvironment with abundance of proteases, linker is cleaved, enabling the mask to dissociate from the anti-P329G binder, thus also enabling binding of the ICE to the targeting adaptor antibody, in turn forming a functional innate engager, which triggers antibody-dependent cellular cytotoxicity (ADCC) against target cells.

[0271] FIGURE 3. Structures of selected anti-P329G pro-ICE variants with annotated antibody parts.

[0272] FIGURE 4. Structure, description and IDs of the antibodies described in this invention. The figures detail different formats of the (pro-) ICEs: comprising anti-P329G (VH3xVLl) bivalent binding moieties, GE Fc with enhanced FcyRIII binding capacity and P329G- containing CH2 masks linked either with PQARK or PMAKK cleavable linkers or with non- cleavable linkers as control (Figure 4A); comprising bivalent anti-P329G (VH3xVLl) binding moieties, WT Fc and P329G-containing CH2 masks linked either with PQARK or PMAKK cleavable linkers or with non-cleavable linkers as control (Figure 4B); comprising anti-P329G (VH3xVLl) monovalent binding moiety, GE Fc with enhanced FcyRIII binding capacity and P329G-containing CH2 masks linked either with PQARK or PMAKK cleavable linkers or with a non-cleavable linker as control (Figure 4C); comprising anti-P329G (VH3xVLl) monovalent binding moiety, WT Fc with and P329G-containing CH2 masks linked either with PQARK or PMAKK cleavable linkers or with a non-cleavable linker as control (Figure 4D)

[0273] FIGURE 5. Structure, description and IDs of the antibodies used in this invention as unmasked controls. The figures detail different formats of the unmasked P329G ICEs: comprising unmasked bivalent, GE or WT Fc control ICEs (Figure 5 A); comprising unmasked monovalent, GE or WT Fc control ICEs (Figure 5B).

[0274] FIGURE 6. SDS-PAGE analysis of the protease (i.e matriptase)- or buffer- pretreated (pro-) ICEs showing protease-dependent removal of the masks from the pro-ICEs. The tested (pro-) ICEs had either masks with cleavable linkers or masks with non-cleavable linkers or were unmasked controls, in bivalent (Figure 6A) or monovalent (Figure 6B) format. Depicted are results of SDS-PAGE gels loaded with pre-reduced (pro-) ICEs. As positive and negative controls, anti-P329G (VH3xVLl) x anti-CD3 pro-TCBs in 2+1 TCB format with P035.093 CD3 binder carrying either PMAKK cleavable linker or non-cleavable linker were used. Each band represents a chain from a tested (pro-) ICE or pro-TCBs which can be matched to its molecular weight based on the placement relative to the ladder containing molecules with known molecular weight. In the right text box, expected molecular weights of different chains calculated based on their amino acid sequences can be found.

[0275] FIGURE 7. Principle of the Jurkat NF AT Luc (ADCC) reporter assay used for proof of concept studies of anti-P329G pro-ICEs. Depicted are target cells and Jurkat NF AT Luc (ADCC) reporter cells, treated with masked anti-P329G pro-ICEs and an Fc-silenced adaptor IgG comprising P329G LALA mutation. On the left, pro-ICEs peripheral crosslinking capacity are depicted. As periphery lacks abundant protease presence, masks stay intact and pro-ICEs cannot bind the P329G epitope on the target -bound adaptor antibody, preventing crosslinking on the Jurkat NFAT Luc (ADCC) reporter cells and luminescence is not produced. On the right, pro-ICEs crosslinking capacity in the tumor microenvironment are depicted. As solid tumors have abundant protease presence, masks are cleaved and disassociated, allowing binding of pro-ICEs to the P329G epitope in adaptor antibody. This consequently leads to crosslinking of FcyRIII on the Jurkat NF AT Luc (ADCC) reporter cells and downstream signaling, which in turn results to luminescent signal.

[0276] FIGURE 8. Activation of Jurkat NF AT Luc (ADCC) reporter cells by different tumortargeting bivalent adaptor P329G IgG combined with anti-P329G (pro-) GE ICEs. Figure 8A shows activation Jurkat NF AT Luc (ADCC) reporter cells by anti-EPCAM P329G adaptor IgG combined with anti-P329G (pro-) ICEs on MKN45 NLR (EPCAM+) tumor cells. Figure 8B shows activation of Jurkat NF AT Luc (ADCC) reporter cells by anti-FOLRl bivalent adaptor P329G IgG combined with anti-P329G (pro-) GE bivalent ICEs on OVMANA (F0LR1+) cells. The tested (pro-) GE ICE molecules had either masks with cleavable linkers or masks with non-cleavable linkers or were unmasked controls. All tested molecules were pre-incubated with or without matriptase (protease) and used in molar ratio adaptor IgG: pro- ICE 2: 1. Assessed by quantification of the intensity of FcyRIII downstream signaling using Jurkat NF AT Luc (ADCC) reporter assay. Depicted are representative of values of triplicates, error bars indicate SD.

[0277] FIGURE 9. Activation of Jurkat NF AT Luc (ADCC) reporter cells by anti-CD25 bivalent adaptor P329G IgG combined with anti-P329G (pro-) GE ICEs on immune- suppressive iTreg (CD25+) cells. The tested (pro-) GE bivalent ICE molecules had either masks with cleavable linkers or masks with non-cleavable linkers or were unmasked controls. All tested molecules were pre-incubated with or without matriptase (protease) and used in molar ratio adaptor IgG: pro-ICE 2: 1. Assessed by quantification of the intensity of FcyRIII downstream signaling using Jurkat NF AT Luc (ADCC) reporter assay. Depicted are representative of values of triplicates, error bars indicate SD.

[0278] FIGURE 10. Activation of Jurkat NF AT Luc (ADCC) reporter cells by anti-HER2 bivalent adaptor P329G IgG combined with anti-P329G (pro-) WT or GE ICEs on SK-Br3 (HER2+) cells. The tested (pro-) WT or GE bivalent ICE molecules had either masks with cleavable linkers or masks with non-cleavable linkers or were unmasked controls. All tested molecules were pre-incubated with or without matriptase (protease) and used in molar ratio adaptor IgG: pro-ICE 2: 1. Assessed by quantification of the intensity of FcyRIII downstream signaling using Jurkat NF AT Luc (ADCC) reporter assay. Depicted are representative of values of triplicates, error bars indicate SD. FIGURE 11. Activation of Jurkat NF AT Luc (ADCC) reporter cells by anti-FOLRl bivalent adaptor P329G IgG combined with anti-P329G monovalent pro-ICEs on OVMANA (FOLR1+) cells. The tested GE monovalent pro-ICE molecules had masks with cleavable linkers. All tested molecules were pre-incubated with or without matriptase (protease) and used in molar ratio adaptor IgG: pro-ICE 2: 1. Assessed by quantification of the intensity of FcyRIII downstream signaling using Jurkat NF AT Luc (ADCC) reporter assay. Depicted are representative of values of triplicates, error bars indicate SD.

[0279] FIGURE 12. Principle of the NK cell activation and killing assay used for testing P329G pro-ICEs. Depicted are target cells and primary human NK cells, treated with anti P329G Pro-ICEs with an Fc-silenced adaptor IgG comprising P329G LALA mutation. On the left, pro-ICEs peripheral NK cell activation capacity are depicted. As periphery lacks abundant protease presence, masks stay intact and pro-ICEs cannot bind the P329G epitope on the target-bound adaptor antibody, crosslinking of FcyRIII on the primary NK cells does not take place. Preventing NK cell activation and target cell lysis. On the right, pro-ICEs’ crosslinking capacity in the tumor microenvironment are depicted. As solid tumors have abundant protease presence, masks are cleaved and disassociated, allowing binding of pro- ICEs to the P329G epitope in adaptor antibody. This leads to crosslinking of FcyRIII on the primary NK cells and consequent NK cell activation. Upon crosslinking, NK cells release cytotoxic molecules into the synapse resulting into target cell lysis in the tumor microenvironment. The dying target cells have their membrane disrupted and release dead cell proteases into the supernatant of the assay. NK cells activation also results in upregulation of CD107a and CD25 but downregulation of FcyRIII on their cell surface.

[0280] FIGURE 13. NK cell-mediated tumor cell lysis induced by anti-FOLRl bivalent adaptor P329G IgGs combined with anti-P329G (pro-) GE ICEs. Primary human NK cells isolated from two healthy donors (Figure 13A and Figure 13B, respectively) used as effector cells and OVMANA (FOLR1+) cells used as target cells. All tested molecules were preincubated with or without matriptase (protease) and used in molar ratio adaptor IgG: pro-ICE 2: 1. Depicted are tumor cell lysis by NK cells from healthy Donor 1 (FIGURE 13 A) and from healthy Donor 2 (FIGURE 13B). Tumor cell lysis assessed after 22h co -culturing, by quantification of dead cell protease released into the assay supernatants Depicted are average values from technical triplicates; error bars indicate SD.

[0281] FIGURE 14. NK cell-activation assessed by the regulation of several activation markers induced by anti-FOLRl bivalent adaptor P329G IgGs combined with anti-P329G (pro-) GE ICEs. Primary human NK cells isolated from healthy Donor 1 used as effector cells and OVMANA (FOLR1+) cells used as target cells. Figure 14A depicts CD107a granulation marker upregulation by anti-P329G (pro-) GE ICEs, whereas Figure 14B focusses on CD25 activation marker upregulation by the same anti-P329G (pro-) GE ICEs, on primary NK cells. Figure 14C shows FcyRIII receptor downregulation upon engagement with anti-P329G (pro- ) GE ICEs on primary NK cells. Tested molecules were pre-incubated with or without matriptase (protease) and used in molar ratio adaptor IgG: pro-ICE 2:1. Assessed after 22h co-culturing, using flow cytometry. Depicted are average values from technical triplicates; error bars indicate SD.

[0282] EXAMPLES

[0283] In the following Examples, the inventors describe the production and characterization of universal adaptor-based innate cell engaging antibodies. In particular, masked protease-activatable anti-P329G innate cell engager ("pro-ICE") antibodies are evaluated, and are unexpectedly found to provide improved properties. For example, the pro-ICE antibodies of the present invention display no activation in absence of the relevant protease and robust activation in the presence of the relevant protease. This leads to a more specific activation and a potentially improved therapeutic window due to the absence of unspecific activation.

[0284] Example 1

[0285] Description of the mode of action and structure of the molecules in the current invention

[0286] Principle of the non-masked anti-P329G ICE (universal adaptor-based innate cell engager antibody) depicted in Figure 1. As an advancement, the current invention, the protease-activatable anti-P329G pro-ICE (universal protease-activatable adaptor-based innate cell engager antibody) is shown in Figure!. Detailed description of antibody structures of the protease-activatable anti-P329G pro-ICE formats are shown in Figure 3.

[0287] Figure 4 depicts antibody pictograms, IDs and descriptions of example molecules of the current invention. Figure 4A shows anti-P329G (VH3xVLl) bivalent pro-ICEs with glycoengineered (GE) Fc domains masked either with PQARK or PMAKK cleavable linkers or with non-cleavable linker as control. Figure 4B shows anti-P329G (VH3xVLl) bivalent pro-ICEs with wildtype (WT)-Fc domains masked either with PQARK or PMAKK cleavable linkers or with non-cleavable linker as control. Figure 4C shows anti-P329G (VH3xVLl) monovalent pro-ICEs with glycoengineered (GE) Fc domains masked either with PQARK or PMAKK cleavable linkers or with non-cleavable linker as control. Figure 4D shows anti- P329G (VH3xVLl) monovalent pro-ICEs with WT-Fc domains masked either with PQARK or PMAKK cleavable linkers or with non-cleavable linker as control.

[0288] Figure 5 depicts antibody pictograms, IDs and descriptions of unmasked ICEs which served as control antibodies. Figure 5A shows anti-P329G (VH3xVLl) bivalent non-masked ICEs with glycoengineered (GE) or WT-Fc domains used as control. Figure 5B shows anti- P329G (VH3xVLl) monovalent non-masked ICEs with glycoengineered (GE) or WT-Fc domains used again as control.

[0289] Example 2

[0290] SDS-PAGE analysis of matriptase (protease) -pretreated anti-P329G pro-ICEs - assessment of PMAKK and PQARK linker cleavage and mask release dependent on matriptase (protease) pretreatment

[0291] To prove the cleavage of linkers between the CH2 (P329G) masks and the P329G binders on the anti-P329G pro-ICEs, SDS-PAGE analysis was performed after overnight matriptase (protease) pre-treatment of the antibodies.

[0292] Prior the SDS-PAGE analysis, antibodies described in the current invention (anti- P329G pro-ICEs with cleavable linkers or non-cleavable linker) and from previous inventions (unmasked anti-P329G ICE and anti-P329G x anti-CD3 pro-TCB with cleavable linker) were diluted with matriptase buffer (Roche, internal) and brought to the concentration of 0.05 mg / ml in different wells of an U-bottom 96 well plate (TPP, #TPP92097). Then, antibodies were divided into two groups comprising matriptase pre-treatment and buffer pre-treatment groups in the same U-bottom 96 well plate (TPP, #TPP92097). Recombinant matriptase aliquots were freshly thawed on ice (Enzo Life Sciences, #ALX-201-246-U250) and added onto antibodies in the matriptase pre-treatment group at final matriptase concentration of 5 nM. For the buffer pre-treatment group, only matriptase buffer was added onto the antibodies without any protease addition. After that, U-bottom 96 well plate (TPP, #TPP92097) was sealed and incubated overnight at room temperature. Next day, antibodies from both groups were reduced their chains via mixing 10 pl of the sample, 5 pl of the 4xNuPAGE LDS Sample Buffer (Thermo Fisher Scientific, #NP007), 2 pl of the lOx NuPAGE Sample Reducing Agent (Thermo Fisher Scientific, #NP0004) and 3 pl of deionized water (Roche, internal), and subsequently incubating the mixes for 10 min at 70 °C in a heating block. After reduction of antibodies, NuPAGE 4 to 12% Bis-Tris Gel (Thermo Fisher Scientific, # NP0323BOX) was mounted into the vertical electrophoresis cell (Bio-Rad Laboratories, #1658003FC). The cell was filled with lx NuPAGE™ MOPS SDS Running Buffer (Thermo Fisher Scientific, #NP0001). From each reduced antibody sample, 15 pl was pipetted to the corresponding wells of the gel. To be able determine, antibody chains’ molecular weights, 10 pl of kaleidoscope protein (Bio-Rad Laboratories, #1610375) ladder was also pipetted into a separate well in the gel. After loading of samples, the electrophoresis was performed at a constant voltage of 200 V for 30 to 40 minutes. When the run was finished, the gel was removed from its cassette and stained Ih by submerging using Coomassie blue-based staining solution (Expedeon, #ISB1L) inside a plastic box, placed on a plate rocker at 50 round per minute (IKA, #0002980203). After 1 h of staining, to remove excess dye, gel was washed with fresh tap water 3 times and destained Ih by submerging into the tap water inside a plastic box, placed on a plate rocker at 50 round per minute (IKA, #0002980203). After destaining, gel was gently placed inside a transparent plastic folder and imaged a scanner (HP, #G2710). The molecular weight of anti- P329G pro-ICEs’ chains were compared with the protein ladder and also with anti-P329G x anti-CD3 pro-TCBs’ chains which was used as positive control for mask cleavage.

[0293] Figure 6A depicts the results of the experiment performed with anti-P329G (VH3xVLl) bivalent GE pro-ICEs either with PQARK / PMAKK cleavable linkers or with non-cleavable linkers. As controls, unmasked anti-P329G ICE and anti-P329G x anti-CD3 pro-TCBs either with PMAKK cleavable linkers or with non-cleavable linkers were used. The table to the right of the gel shows predicted molecular weight of antibody chains based on their amino acid sequence. As it can be concluded from the picture, matriptase resulted in a full mask cleavage in anti-P329G (VH3xVLl) bivalent GE pro-ICEs with PQARK and PMAKK cleavable linkers. This is evident by the absence of bands representing the masked chains (64 kDa) but the presence of the cleaved masks (15 kDa) in matriptase treated samples only. For the non-cleavable anti-P329G pro-ICE, as expected, matriptase did not induce any cleavage of the mask, as evident by the same bands being seen in matriptase pre -treated and buffer pre-treated samples. Also, matriptase pre-treatment did not have any impact on the unmasked anti-P329G ICE. Figure 6B depicts the results of the experiment performed with anti-P329G (VH3xVLl) monovalent GE pro-ICEs either with PQARK and PMAKK cleavable linkers. As controls, unmasked anti-P329G ICE and anti-P329G x anti-CD3 pro-TCBs either with PMAKK cleavable linkers or with non-cleavable linkers were used. The table to the right of the gel shows predicted molecular weight of antibody chains based on their amino acid sequence. As it can be concluded from the picture, matriptase resulted in a full mask cleavage in anti-P329G (VH3xVLl) monovalent GE pro-ICEs with PQARK and PMAKK cleavable linkers. This is evident by the absence of bands representing the masked chains (64 kDa) but the presence of the cleaved masks (15 kDa) in matriptase treated samples only. For the non- cleavable anti-P329G pro-ICE, as expected, matriptase did not induce any cleavage of the mask, as evident by the same bands being seen in matriptase pre-treated and buffer pre-treated samples. Also, matriptase pre-treatment did not have any impact on the unmasked anti-P329G ICE, as it can be seen that matriptase pre-treatment resulted in the same band as non-treated samples.

[0294] This method provided evidence that anti-P329G pro-ICEs with PQARK and PMAKK cleavable linkers were indeed cleaved by matriptase, a known tumor abundant protease, disassociating the CH2 mask from the rest of anti-P329G pro-ICEs.

[0295] Example 3

[0296] Jurkat NF A T Luc (ADCC) reporter assay principle

[0297] The Jurkat NF AT Luc (ADCC) reporter assay (ADCC Reporter Bioassay F variant, #G9302) is a bioluminescent reporter assay for quantifying biological activity of therapeutic antibody drugs dependent on antibody-dependent cellular cytotoxicity (ADCC) pathway. For this, assay uses acute lymphatic leukemia reporter cell line stably expressing FcyRIIIa Fl 58 (low affinity) genetically engineered Jurkat NFAT Luc (ADCC) reporter cells (ADCC Reporter Bioassay F variant, #G9302) and an NFAT -response element driving the expression of firefly luciferase. Upon crosslinking of the Fc part of target -bound antibodies with FcyRIIIa F158 receptors on Jurkat NFAT Luc (ADCC) reporter cells (ADCC Reporter Bioassay F variant, #G9302), the NFAT promoter is activated leading to a dose-dependent expression of firefly luciferase. Adding a luciferase substrate results in a luminescent signal, reflecting the strength of the Jurkat NFAT Luc (ADCC) reporter cells (ADCC Reporter Bioassay F variant, #G9302) activation or in other words, ADCC capacity of the tested therapeutic antibody candidate.

[0298] Figure 7 represents the principle of Jurkat NF AT Luc (ADCC) reporter assay (ADCC Reporter Bioassay F variant, #G9302), used to assess the capacity of protease -cleaved anti- P329G pro-ICEs to activate ADCC pathway upon cross-linking when binding to adaptor IgG and target cell. The pictograms show cross -linking events between adaptor P329G IgGs and protease-cleaved anti-P329G pro-ICEs leading to the signal production by the ADCC reporter cells, the strength of which corresponds to the strength of NF AT transcription, which in turn reflects the strength of NK cell activation.

[0299] Example 4

[0300] Jurkat NFAT Luc (ADCC) reporter cell activation induced by adaptor P329G IgGs with protease activatable anti-P329G bivalent glycoengineered pro-ICEs on tumor cells lines expressing different tumor -associated antigens - assessment of ADCC activation capacity against tumor cells, matriptase dependency and masking efficiency

[0301] In Figure 8, ADCC activation capacity of the adaptor P329G IgGs combined with anti-P329G bivalent GE (pro-ICEs) assessed by Jurkat NFAT Luc (ADCC) reporter assay (ADCC Reporter Bioassay F variant, #G9302) on two different tumor-associated antigen expressing cell lines. The principle of the assay is depicted on Figure 7.

[0302] For preparation of the assays, both tumor lines were harvested by removing the growth media from the cell culture flask, washing cells with phosphate-buffered saline (Gibco #10010023), removing the remaining cell culture media and PBS (Gibco #10010023) traces and incubating cell with TrypLE Express Enzyme (Gibco, #12605010) for 5 min at 37 , 5% CO2. Cell count and viability of the tumor lines were determined using Cedex HiRes (Roche, #05650216001) cell counter and 0.002 x 106cells / well (20 pl / well) were plated in a white flat bottom 384-well-plate (Corning, #353988) in the assay medium (Advanced RPMI 1640, 10% FBS, 1% GlutaMAX), one day before the assay and incubated overnight in a humidified atmosphere at 37°C and 5% CO2. The molecules were prepared at the concentration of 2000 nM (for (pro-) ICEs) or 4000 nM (for adaptors) in protein buffer (Roche, internal) in a U- bottom 96 well plate (TPP, #TPP92097). Freshly thawed matriptase (Enzo Life Sciences, #ALX-201-246-U250) or an equivalent volume of protein buffer was added to the respective molecules on wells to reach the final matriptase concentration of 5.3 nM. The molecules were mixed and the plate was centrifuged for 5 s at 200 g. The plate was sealed and incubated overnight at RT. On the next day, overnight incubated molecules were diluted 5x with assay media (Advanced RPMI 1640, 10% FBS, 1% GlutaMAX) to achieve the concentration of 400 nM for (pro-) ICEs and 800 nM for adaptors. Then, 10 pl of antibody dilutions were added to the assay plate to the respective wells. The assay plate was centrifuged for 5 min at 200g. Meanwhile, Jurkat NF AT Luc (ADCC) reporter cells (ADCC Reporter Bioassay F variant, #G9302) were harvested. The cells were counted using Cedex HiRes (Roche, #05650216001) cell counter and 0.01 x 106cells / well (10 pl / well) were plated in assay medium to obtain a final effector-to-target cell ratio (E:T) of 5: 1, and a final assay volume of 40 pl per well - achieving the concentrations of 100 nM for (pro-) ICEs and 200 nM for adaptors. The assay plate was incubated in 37°C and 5% CCL for 6h. After the incubation time, a luciferase substrate, ONE-Glo™ Luciferase Assay reagent (Promega, #E6120) was added onto the assay plate (40 pl / well) and incubated for 5 min in dark at RT, before luminescence signal was measured using Tecan Spark 10M. The luminescent signal was acquired for 500 ms / well, and calculated to reflect RLU / s per well.

[0303] In Figure 8A, EPC AM tumor antigen expressing human cell line MKN-45 NLR was used as target cells. As a tumor targeting adaptor molecule, anti-EPCAM adaptor P329G IgG was mixed with anti-P329G (VH3 VL1) bivalent GE pro -ICEs either with PQARK or PMAKK cleavable linkers in a molar ratio adaptor: pro-ICE 2: 1. As a negative control, adaptor P329G IgGs were mixed with anti-P329G (VH3VL1) bivalent GE pro-ICE with non-cleavable linkers. As positive controls, anti-P329G (VH3VL1) bivalent GE unmasked ICE was combined with anti-EPCAM adaptor P329G IgG. Direct anti-EPCAM bivalent GE ICE was also added as an additional positive control. As expected, anti-P329G (VH3VL1) bivalent GE pro-ICEs either with PQARK or PMAKK cleavable linkers led to Jurkat NF AT Luc (ADCC) reporter cell activation in a dose response dependent manner, when they were pre-treated with matriptase, but they remained inactive without matriptase pre -treatment. Additionally, anti- P329G bivalent GE pro-ICE with non-cleavable linkers remained fully inactive in both with and without matriptase pre-treatment conditions. The unmasked positive control anti-P329G bivalent GE ICE combined with anti-EPCAM P329G adaptor IgG remained active regardless of pretreatment conditions.

[0304] In Figure 8B, FOLR1 tumor-antigen expressing human cell line OVMANA was used as target cells. As a tumor targeting adaptor molecule, anti-FOLRl adaptor P329G IgG was mixed with anti-P329G (VH3VL1) bivalent GE pro-ICEs either with PQARK or PMAKK cleavable linkers in a molar ratio adaptor: pro-ICE 2: 1. As a negative control, bivalent adaptor P329G IgGs were mixed with anti-P329G (VH3 VL1) bivalent GE pro-ICE with non-cleavable linkers. As positive controls, unmasked anti-P329G (VH3VL1) bivalent GE ICE was combined with anti-FOLRl adaptor P329G IgG. Direct anti-FOLRl bivalent GE ICE was also added as an additional positive control. As expected, anti-P329G (VH3VL1) bivalent GE pro-ICEs either with PQARK or PMAKK cleavable linkers led to Jurkat NF AT Luc (ADCC) reporter cell activation in a dose response dependent manner, when they were pre-treated with matriptase, but they remained inactive without matriptase pre-treatment. Additionally, anti- P329G bivalent GE pro-ICE with non-cleavable linkers remained fully inactive in both with and without matriptase pre-treatment conditions. The unmasked positive control anti-P329G bivalent GE ICE combined with anti-FOLRl P329G adaptor IgG remained active regardless of pretreatment conditions.

[0305] This experiment provided a proof-of-concept that matriptase was able to cleave both the PQARK and PMAKK linkers in the tested anti-P329G bivalent GE pro-ICEs, allowing the CH2 mask to dissociate from the rest of the antibody in each cleavable masked pro-ICEs, creating functional universal ICEs which can be used against different tumor -associated antigens. It also showed that P329G pro-ICE can be more potent than the previous art, unmasked P329G ICE, depending on the tumor-associated antigen of choice (Figure 8B).

[0306] Example 5

[0307] Jurkat NF AT Luc (ADCC) reporter cell activation induced by anti-CD25 adaptor P329G IgG with protease activated anti-P329G GE bivalent pro-ICE on immunosuppressive iTreg cells expressing high levels of CD25 - assessment of ADCC activation capacity against immunosuppressive cells , matriptase dependency and masking efficiency

[0308] ADCC activation capacity of the anti-CD25 adaptor P329G IgGs combined with anti- P329G bivalent GE (pro-) ICEs assessed by Jurkat NF AT Luc (ADCC) reporter assay (ADCC Reporter Bioassay F variant, #G9302) on immunosuppressive iTreg cells (Figure 9). The principle of the assay is depicted on Figure 7.

[0309] Couple weeks prior to the assay day, induced regulatory T cells (iTregs) were generated from healthy donors’ isolated CD4 T cells. For this, first, the buffy coat donated by a healthy donor was pre-diluted 1 : 1 ratio with PBS (Gibco, #10010023) and then layered on 15 mL Lymphoprep density gradient (StemCell Technologies, #07581) in SepMate-50 tubes (StemCell Technologies, #15460). Prepared tubes were centrifuged at 1200g for 20 min. PBMC layers were transferred to a fresh Falcon™ 50 mL High Clarity Conical Centrifuge Tube (Corning, #14-432-22) and washed with PBS (Gibco #10010023) two times, via centrifuging at 300g for 6 min. Then, isolated PBMCs were counted using Beckman Coulter DxH 500 (Beckman Coulter) and applied to CD4 T cell isolation using Naive CD4 T cell Isolation kit (Miltenyi Biotec, #130-094-131), by following manufacturer’s recommendations. After naive CD4 T cells were isolated from the healthy donor’s buffy coat, iTreg differentiation protocol was applied. Isolated CD4 T cells were brought to 1.0 x 106cells / mL in iTreg media (X-Vivo 15, 10% FBS, 1% GlutaMAX, l%Na-Py, 1% Hepes, 1% NEAA, 1% Pen / Strep, 2 mg / mL N-acetylcysteine, 50 pM final B-mercaptoenhanol, 300 u / mL Preleukin (IL2) (Novartis), 109 nM Rapamycin, 10 ng / mL Final rhTGFbl, Dynabeads Human T-Activator CD3 / CD28 (1.0 x 106beads / mL)) and cultured for 14 days, by bringing the cells back to 1.0 x 106cells / mL in every 24-48 hours. Generated iTregs were first disassociated from Dynabeads Human T-Activator CD3 / CD28 by vigorous pipetting, washing with PBS (Gibco #10010023) and placing the iTreg suspension on DynaMag-2 magnet (Invitrogen, #12321D) and then, they were frozen in FBS+10%DMSO and stored in LN.

[0310] The day before assay was performed, molecules were prepared at the concentration of 2000 nM (for (pro-) ICEs) or 4000 nM (for adaptors) in protein buffer (Roche, internal) in a U-bottom 96 well plate (TPP, #TPP92097). Freshly thawed matriptase (Enzo Life Sciences, #ALX-201-246-U250) or an equivalent volume of protein buffer was added to the respective molecules on wells to reach the final matriptase concentration of 5.3 nM. The molecules were mixed and the plate was centrifuged for 5 s at 200 g. The plate was sealed and incubated overnight at RT. In addition, two vials of iTreg stocks were thawed and iTregs were cultured in Advanced RPMI 1640, 10% FBS, 1% GlutaMAX with 400U / mL addition of Proleukin (IL2) (Novartis) overnight, in a cell culture incubator with humidified atmosphere at 37°C and 5% CO2.

[0311] For preparation of the assay, both target cells (iTregs) and Jurkat NF AT Luc (ADCC) cells were collected into Falcon™ 50 mL High Clarity Conical Centrifuge Tubes (Corning, #14-432-22) and washed once with the assay media (Advanced RPMI 1640, 10% FBS, 1% GlutaMAX), removing the remaining cell culture media traces. Cell count and viability of the cells were determined using Cedex HiRes (Roche, #05650216001) cell counter. As target cells, 0.002 x 106iTreg cells / well (20 pl / well) were plated in a white flat bottom 384-well- plate (Corning, #353988) in assay medium (Advanced RPMI 1640, 10% FBS, 1% GlutaMAX), and plate was shortly centrifuged at 200g to ensure settlement of the cells to the bottom of the wells. To the same plate, Jurkat NF AT Luc (ADCC) reporter cells (ADCC Reporter Bioassay F variant, #G9302) were plated in assay medium as 0.01 x 106cells / well (10 jj.l / well) to achieve a final effector-to-target cell ratio (E:T) of 5: 1. Overnight incubated molecules were diluted 5x with assay media (Advanced RPMI 1640, 10% FBS, 1% GlutaMAX) to achieve the concentration of 400 nM for (pro-) ICEs and 800 nM.for adaptors. Then, 10 p.1 of antibody dilutions were added to the assay plate to the respective wells. The assay plate was centrifuged for 5 min at 200g. Meanwhile, Jurkat NF AT Luc (ADCC) reporter cells (ADCC Reporter Bioassay F variant, #G9302) were harvested. The cells were counted using Cedex HiRes (Roche, #05650216001) cell counter and 0.01 x 106cells / well (10 pl / well) were plated in assay medium to obtain a final effector-to-target cell ratio (E:T) of 5: 1, and a final assay volume of 40 pl per well - achieving the concentrations of 100 nM for (pro-) ICEs and 200 nM for adaptors. The assay plate was incubated in 37°C and 5% CCL for 6h. After the incubation time, a luciferase substrate, ONE-Glo™ Luciferase Assay reagent (Promega, #E6120) was added onto the assay plate (40 pl / well) and incubated for 5 min in dark at RT, before luminescence signal was measured using Tecan Spark 10M. The luminescent signal was acquired for 500 ms / well, and calculated to reflect RLU / s per well.

[0312] In Figure 9, immunosuppressive iTreg cells expressing high levels of CD25 antigen was used as target cells. As a tumor targeting adaptor molecule, anti-CD25 adaptor P329G IgG was mixed with anti-P329G (VH3VL1) bivalent GE pro-ICEs either with PQARK or PMAKK cleavable linkers in a molar ratio adaptor: pro-ICE 2: 1. As a negative control, bivalent adaptor P329G IgGs were mixed with anti-P329G (VH3VL1) bivalent GE pro-ICE with non-cleavable linkers. As a positive control, anti-P329G (VH3VL1) bivalent GE unmasked ICE was combined with anti-EPCAM bivalent adaptor P329G IgG. Direct anti- EPCAM bivalent GE ICE was also added as an additional positive control. As expected, anti- P329G (VH3VL1) bivalent GE pro-ICEs either with PQARK or PMAKK cleavable linkers led to Jurkat NF AT Luc (ADCC) reporter cell activation in a dose response dependent manner, when they were pre-treated with matriptase, but they remained inactive without matriptase pre-treatment. Additionally, anti-P329G bivalent GE pro-ICE with non-cleavable linkers remained fully inactive in both with and without matriptase pre-treatment conditions. The unmasked positive control anti-P329G bivalent GE ICE combined with anti-CD25 P329G adaptor IgG remained active regardless of pretreatment conditions.

[0313] This experiment provided a proof-of-concept that protease activatable P329G pro- ICEs can be used to eliminate not only cancer cells but also immune suppressive cells in the tumor microenvironment. Example 6

[0314] Jurkat NFAT Luc (ADCC) reporter cell activation induced by anti-HER2 adaptor P329G IgG with protease activated anti-P329G GE or WT bivalent pro-ICEs on SK-Br-3 cell line expressing a tumor -associated antigen HER2- assessment of ADCC activation capacity of WT anti-P 329G pro-ICEs, matriptase dependency and masking efficiency

[0315] In Figure 10, ADCC activation capacity of the anti-P329G bivalent WT (pro-ICEs) combined with the adaptor P329G IgGs assessed by Jurkat NFAT Luc (ADCC) reporter assay (ADCC Reporter Bioassay F variant, #G9302) on HER2 tumor-associated antigen expressing cell line, SK-Br-3. The principle of the assay is depicted on Figure 7.

[0316] For preparation of the assays, HER2+ tumor line SK-Br-3 was harvested by removing the growth media from the cell culture flask, washing cells with phosphate -buffered saline (Gibco #10010023), removing the remaining cell culture media and PBS (Gibco #10010023) traces and incubating cell with TrypLE Express Enzyme (Gibco, #12605010) for 5 min at 37 , 5% CO2. Cell count and viability of the tumor line were determined using Cedex HiRes (Roche, #05650216001) cell counter and 0.002 x 106cells / well (20 pl / well) were plated in a white flat bottom 384-well-plate (Corning, #353988) in the assay medium (Advanced RPMI 1640, 10% FBS, 1% GlutaMAX), one day before the assay and incubated in a humidified atmosphere at 37°C and 5% CO2. The molecules were prepared at the concentration of 2000 nM (for (pro-) ICEs) or 4000 nM (for adaptors) in protein buffer (Roche, internal) in a U- bottom 96 well plate (TPP, #TPP92097). Freshly thawed matriptase (Enzo Life Sciences, #ALX-201-246-U250) or an equivalent volume of protein buffer was added to the respective molecules on wells to reach the final matriptase concentration of 5.3 nM. The molecules were mixed and the plate was centrifuged for 5 s at 200 g. The plate was sealed and incubated overnight at RT. On the next day, overnight incubated molecules were diluted 5x with assay media (Advanced RPMI 1640, 10% FBS, 1% GlutaMAX) to achieve the concentration of 400 nM for (pro-) ICEs and 800 nM.for adaptors. Then, 10 pl of antibody dilutions were added to the assay plate to the respective wells. The assay plate was centrifuged for 5 min at 200g. Meanwhile, Jurkat NFAT Luc (ADCC) reporter cells (ADCC Reporter Bioassay F variant, #G9302) were harvested. The cells were counted using Cedex HiRes (Roche, #05650216001) cell counter and 0.01 x 106cells / well (10 pl / well) were plated in assay medium to obtain a final effector-to-target cell ratio (E:T) of 5: 1, and a final assay volume of 40 pl per well - achieving the concentrations of 100 nM for (pro-) ICEs and 200 nM for adaptors. The assay plate was incubated in 37°C and 5% CCL for 6h. After the incubation time, a luciferase substrate, ONE-Glo™ Luciferase Assay reagent (Promega, #E6120) was added onto the assay plate (40 pl / well) and incubated for 5 min in dark at RT, before luminescence signal was measured using Tecan Spark 10M. The luminescent signal was acquired for 500 ms / well, and calculated to reflect RLU / s per well.

[0317] In Figure 10, HER2 tumor antigen expressing human cell line SK-Br-3 was used as target cells. As a tumor targeting adaptor molecule, anti-HER2 adaptor P329G IgG was mixed either with GE or WT anti-P329G (VH3VL1) bivalent pro-ICEs carrying PQARK cleavable linker, in a molar ratio of adaptor: pro-ICE 2: 1. As a negative control, adaptor P329G IgGs were mixed with anti-P329G (VH3VL1) bivalent GE pro-ICE with non-cleavable linker. As positive controls, anti-P329G (VH3VL1) bivalent unmasked GE or WT ICEs were combined with anti-HER2 adaptor P329G IgG. As expected, anti-P329G (VH3VL1) bivalent GE pro- ICEs with PQARK cleavable linker led to Jurkat NF AT Luc (ADCC) reporter cell activation in a dose response dependent manner, when they were pre-treated with matriptase. However, anti-P329G (VH3VL1) bivalent WT pro-ICE with PQARK cleavable linker failed in Jurkat NF AT Luc (ADCC) reporter cell activation regardless of matriptase pre -treatment. On the other hand, unmasked anti-P329G bivalent GE or WT ICEs, used as positive controls, combined with anti-HER2 P329G adaptor IgG led to in Jurkat NF AT Luc (ADCC) reporter cell activation regardless of pretreatment conditions. Additionally, anti-P329G bivalent GE pro-ICE with non-cleavable linkers remained fully inactive in both with and without matriptase pre-treatment conditions.

[0318] This experiment provided a proof-of-concept that protease activatable P329G pro- ICEs require to have GE Fc portion to be able to engage innate immune cells and eliminate target cells in the tumor microenvironment.

[0319] Example 7

[0320] Jurkat NF AT Luc (ADCC) reporter cell activation induced by anti-FOLRl P329G IgGs with protease activatable anti-P329G monovalent glycoengineered pro-ICEs on OVMANA cell line expressing FOLR1 as tumor -associated antigen- assessment of ADCC activation capacity of monovalent P329G pro-ICEs, matriptase dependency and masking efficiency

[0321] In Figure 11, ADCC activation capacity of the adaptor P329G IgGs combined with anti-P329G monovalent GE (pro-ICEs) assessed by Jurkat NF AT Luc (ADCC) reporter assay (ADCC Reporter Bioassay F variant, #G9302) on OVMANA cell line expressing FOLRlas tumor-associated antigen. The principle of the assay is depicted on Figure 7.

[0322] For preparation of the assays, FOLR1+ tumor line OVMANA was harvested by removing the growth media from the cell culture flask, washing cells with phosphate-buffered saline (Gibco #10010023), removing the remaining cell culture media and PBS (Gibco #10010023) traces and incubating cell with TrypLE Express Enzyme (Gibco, #12605010) for 5 min at 37 , 5% CO2. Cell count and viability of the tumor line were determined using Cedex HiRes (Roche, #05650216001) cell counter and 0.002 x 106cells / well (20 pl / well) were plated in a white flat bottom 384-well-plate (Corning, #353988) in the assay medium (Advanced RPMI 1640, 10% FBS, 1% GlutaMAX), one day before the assay and incubated in a humidified atmosphere at 37°C and 5% CO2. The molecules were prepared at the concentration of 2000 nM (for (pro-) ICEs) or 4000 nM (for adaptors) in protein buffer (Roche, internal) in a U-bottom 96 well plate (TPP, #TPP92097). Freshly thawed matriptase (Enzo Life Sciences, #ALX-201-246-U250) or an equivalent volume of protein buffer was added to the respective molecules on wells to reach the final matriptase concentration of 5.3 nM. The molecules were mixed and the plate was centrifuged for 5 s at 200 g. The plate was sealed and incubated overnight at RT. On the next day, overnight incubated molecules were diluted 5x with assay media (Advanced RPMI 1640, 10% FBS, 1% GlutaMAX) to achieve the concentration of 400 nM for (pro-) ICEs and 800 nM.for adaptors. Then, 10 pl of antibody dilutions were added to the assay plate to the respective wells. The assay plate was centrifuged for 5 min at 200g. Meanwhile, Jurkat NF AT Luc (ADCC) reporter cells (ADCC Reporter Bioassay F variant, #G9302) were harvested. The cells were counted using Cedex HiRes (Roche, #05650216001) cell counter and 0.01 x 106cells / well (10 pl / well) were plated in assay medium to obtain a final effector-to-target cell ratio (E:T) of 5: 1, and a final assay volume of 40 pl per well - achieving the concentrations of 100 nM for (pro-) ICEs and 200 nM for adaptors. The assay plate was incubated in 37°C and 5% CCL for 6h. After the incubation time, a luciferase substrate, ONE-Glo™ Luciferase Assay reagent (Promega, #E6120) was added onto the assay plate (40 pl / well) and incubated for 5 min in dark at RT, before luminescence signal was measured using Tecan Spark 10M. The luminescent signal was acquired for 500 ms / well, and calculated to reflect RLU / s per well.

[0323] In Figure 11, FOLR1 tumor antigen expressing human cell line OVMANA was used as target cells. As a tumor targeting adaptor molecule, anti-FOLRl adaptor P329G IgG was mixed with GE anti-P329G (VH3VL1) monovalent pro-ICEs carrying PQARK or PMAKK cleavable linkers, in a molar ratio of adaptor: pro-ICE 2: 1. As expected, anti-P329G (VH3VL1) monovalent GE pro-ICEs with PQARK and PMAKK cleavable linkers led to Jurkat NF AT Luc (ADCC) reporter cell activation in a dose response dependent manner, when they were pre-treated with matriptase. Whereas in the absence of matriptase pre-treatment anti-P329G (VH3VL1) monovalent GE pro-ICEs did not lead to any unspecific activation of Jurkat NF AT Luc (ADCC) reporter cells.

[0324] This experiment provided a proof-of-concept that protease activatable P329G pro- ICEs can be also designed and used in monovalent formats to eliminate target cells in the tumor microenvironment.

[0325] Example 8

[0326] Primary NK cell-mediated killins assay principle - Cytotoxicity measurement via Cytotox-Glo kit and NK-cell activation measurement via Flow -cytometry

[0327] Figure 12 shows the principle of primary NK cell -mediated killing assay used to assess the capacity of the therapeutic antibodies to activate NK cell-mediated ADCC upon crosslinking, when binding to the target simultaneously. The pictogram depicts antibodydependent immune synapse formation and subsequent cytokine and cytotoxic protein release by NK cells which leads to membrane disruption and death of the target cell. The pictogram also depicts activation of primary human NK cells upon cross-linking via upregulation and downregulation of specific markers on the cell surface.

[0328] In order to measure the target cell killing by primary NK cell engagement, Cytotox- glo luminescent assay was used. In principle, the assay measures extracellular activity of a distinct intracellular protease activity (dead-cell protease) when the protease is released from membrane-compromised cells. Addition of a luminogenic cell-impermeant peptide substrate (AAF-aminoluciferin) onto cell populations results in liberation of luciferin from AAF- aminoluciferin by dead-cell protease. Subsequently, liberated luciferin gets catalyzed by Ultra-Gio Recombinant Luciferase provided in the assay reagent, resulting in a luminescent signal which in turn reflects the relative number of dead cells in the population. Dead-cell protein released from membrane-disrupted target cells liberates luciferin from AAF-luciferin. Luciferin gets catalyzed by Ultra-Gio Recombinant Luciferase resulting in a luminescent signal, of which strength correlates the NK cell -mediated ADCC capacity of the tested antibody drugs. To measure activation status of primary NK cells upon cross-linking, upregulation of CD25, CD69 and downregulation of CD 16 surface markers were measured via flowcytometry. The level of surface markers’ regulations are expected to correlate with the NK cell activation capacity of the tested therapeutic antibodies in a dose -response manner.

[0329] Example 9

[0330] Primary NK cell-mediated tumor cell lysis induced by F0LR1 adaptor P329G IgGs with protease-activatable anti-P329G pro-ICEs on OVMANA tumor cell line expressing a tumor-associated antigen F0LR1- assessment of tumor cell lysis capacity, matriptase dependency and masking efficiency

[0331] In Figure 13, capacity of inducing the tumor cell lysis by the adaptor P329G IgGs with protease-activatable anti-P329G pro-ICEs was assessed with primary human NK cells isolated from two healthy donors on F0LR1 -expressing cancer cell line OVMANA. The principle of the assay was depicted on Figure 12.

[0332] As a tumor targeting molecule (adaptor P329G adaptor IgG), anti-FOLRl P329G IgGs was used and mixed with protease-activatable anti-P329G pro-ICEs (cleavable, non-cleavable and unmasked formats) in ratio of adaptorTCEs 2: 1. The molecules were pre-treated with matriptase or left untreated and titrated together.

[0333] For preparation of the assays, F0LR1 -expressing OVMANA cell lines was harvested by removing the growth media from the cell culture flask, washing cells with phosphate - buffered saline (Gibco #10010023), removing the remaining cell culture media and PBS (Gibco #10010023) traces and incubating cell with TrypLE Express Enzyme (Gibco, #12605010) for 5 min at 37 , 5% CO2. Cell count and viability of the tumor lines were determined using Cedex HiRes (Roche, #05650216001) cell counter and 0.015 x 106cells / well (50 pl / well) were plated in a white flat bottom 384 -well-plate (Corning, #353988) in the assay medium (Advanced RPMI 1640, 10% FBS, 1% GlutaMAX), one day before the assay and incubated in a humidified atmosphere at 37°C and 5% CO2. The molecules were prepared at the concentration of 2000 nM (for (pro-) ICEs) or 4000 nM (for adaptors) in protein buffer (Roche, internal) in a U-bottom 96 well plate (TPP, #TPP92097). Freshly thawed matriptase (Enzo Life Sciences, #ALX-201-246-U250) or an equivalent volume of protein buffer was added to the respective molecules on wells to reach the final matriptase concentration of 5.3 nM. The molecules were mixed and the plate was centrifuged for 5 s at 200 g. The plate was sealed and incubated overnight at RT. Additionally, primary human NK cells were isolated from healthy donors' huffy coats. For NK cell isolation, huffy coats were pre-diluted 1 : 1 ratio with PBS (Gibco #10010023) and then layered on 15 mL Lymphoprep density gradient (StemCell Technologies, #07581) in SepMate-50 tubes (StemCell Technologies, #15460). Prepared tubes were centrifuged at 1200g for 20 min w / o break. PBMC layers were transferred to fresh Falcon™ 50 mL High Clarity Conical Centrifuge Tube (Corning, #14-432-22) and washed with PBS (Gibco #10010023) two times, via centrifuging at 300g for 6 min. Then, isolated PBMCs were counted using Beckman Coulter DxH 500 (Beckman Coulter) and applied to NK cell isolation using NK cell Isolation kit (Miltenyi Biotec, #130-092-657), by following manufacturer’s recommendations. Isolated NK cells were brought to 1.0 x 106cells / mL in (RPMI 1640, 10%FBS, 1% GlutaMAX) media and cultured overnight in the presence of 100 U / mL Proleukin (IL2) (Novartis).

[0334] On the assay day, overnight incubated molecules were diluted 5x with assay media (Advanced RPMI 1640, 10% FBS, 1% GlutaMAX) to achieve the concentration of 400 nM for (pro-) ICEs and 800 nM.for adaptors. Then, 25 pl of antibody dilutions were added to the assay plate to the respective wells. The assay plate was centrifuged for 5 min at 200g. Meanwhile, primary NK cells were harvested. The cells were counted using Cedex HiRes (Roche, #05650216001) cell counter and 0.09 x 106cells / well (25 pl / well) were plated in assay medium to obtain a final effector-to-target cell ratio (E:T) of 6: 1, and a final assay volume of 100 pl per well - achieving the concentrations of 100 nM for (pro-) ICEs and 200 nM for adaptors. The assay plate was incubated in 37°C and 5% CCL for 20h. After the incubation time, the plate was centrifuged at 400g for 3 min to ensure cell settlement to the bottom of the wells. Then, 20 pl of the assay supernatant was removed from each well and transferred to a new white flat bottom 384-well-plate (Corning, #353988). Then, 10 pl / well of the Cytotox-Glo reagent (Promega, #G9291) was added to supernatants from each well and centrifuged at 200g for 1 min. After 15 min of incubation at room temperature (22 °C), produced luminescence (reflective of the amount of dead cell proteases) was measured. Readout was performed using a Tecan Spark 10M reader. The luminescent signal was acquired for 500 ms / well, and calculated to reflect RLU / s per well.

[0335] In this experiment, primary human NK cells from two donors (Figure 13A and Figure 13B, respectively) were used as effector cells, while FOLR1 tumor antigen expressing human cell line OVMANA was used as target cells. As a tumor targeting adaptor molecule, anti- FOLR1 adaptor P329G IgG was mixed with anti-P329G (VH3VL1) bivalent GE pro-ICEs either with PQARK or PMAKK cleavable linkers in a molar ratio adaptor: pro-ICE 2:1. As a negative control, adaptor P329G IgGs were mixed with anti-P329G (VH3VL1) bivalent GE pro-ICE with non-cleavable linkers. As positive controls, anti-P329G (VH3VL1) bivalent GE unmasked ICE was combined with anti-FOLRl adaptor P329G IgG. Direct anti-FOLRl bivalent GE ICE was also added as an additional positive control. As expected, anti-P329G (VH3VL1) bivalent GE pro-ICEs either with PQARK or PMAKK cleavable linkers led to tumor cell lysis response dependent manner, when they were pre -treated with matriptase, but they remained silent without matriptase pre -treatment. Additionally, anti-P329G bivalent GE pro-ICE with non-cleavable linkers remained fully inactive in both with and without matriptase pre-treatment conditions. The unmasked positive control anti-P329G bivalent GE ICE combined with anti-FOLRl P329G adaptor IgG remained active regardless of pretreatment conditions.

[0336] By performing primary NK-cell mediated killing assays, protease-activatable anti- P329G pro-ICEs capacity to induce NK-cell mediated tumor cell lysis was confirmed ex vivo. In addition, pro-ICEs’ protease-dependency were also assessed. These results from NK cell- mediated tumor cell lysis experiments correlated nicely with the results of previously shown reporter cell assays and confirmed that anti-P329G pro-ICEs can indeed engage primary human innate cells and lead to target cell killing in a protease-dependent manner.

[0337] Example 10

[0338] Primary NK cell activation induced by FOLR1 adaptor P329G IgGs with protease - activatable anti-P329G pro-ICEs on OVMANA tumor cell line expressing a tumor -associated antigen FOLR1- assessment of NK cell activation capacity, matriptase dependency and masking efficiency

[0339] Primary human NK cell activation capacity of protease -activatable P329G pro-ICEs combined with tumor-antigen targeting FOLR1 adaptor IgG was assessed on FOLR1 expressing tumor cell line OVMANA in Figure 14. The principle of the assay was depicted on Figure 12.

[0340] As a tumor targeting molecule (adaptor P329G adaptor IgG), anti-FOLRl P329G IgGs was used and mixed with protease-activatable anti-P329G pro-ICEs (cleavable, non-cleavable and unmasked formats) in ratio of adaptorTCEs 2: 1. The molecules were pre-treated with matriptase or left untreated and titrated together. As effector cells, primary human NK cells were isolated from a healthy donors’ huffy coat.

[0341] For preparation of the assays, F0LR1 -expressing OVMANA cell lines was harvested by removing the growth media from the cell culture flask, washing cells with phosphate- buffered saline (Gibco #10010023), removing the remaining cell culture media and PBS (Gibco #10010023) traces and incubating cell with TrypLE Express Enzyme (Gibco, #12605010) for 5 min at 37 , 5% CO2. Cell count and viability of the tumor lines were determined using Cedex HiRes (Roche, #05650216001) cell counter and 0.015 x 106cells / well (50 pl / well) were plated in a white flat bottom 384 -well-plate (Corning, #353988) in the assay medium (Advanced RPMI 1640, 10% FBS, 1% GlutaMAX), one day before the assay and incubated in a humidified atmosphere at 37°C and 5% CO2. The molecules were prepared at the concentration of 2000 nM (for (pro-) ICEs) or 4000 nM (for adaptors) in protein buffer (Roche, internal) in a U-bottom 96 well plate (TPP, #TPP92097). Freshly thawed matriptase (Enzo Life Sciences, #ALX-201-246-U250) or an equivalent volume of protein buffer was added to the respective molecules on wells to reach the final matriptase concentration of 5.3 nM. The molecules were mixed and the plate was centrifuged for 5 s at 200 g. The plate was sealed and incubated overnight at RT. Additionally, primary human NK cells were isolated from healthy donors' buffy coats. For NK cell isolation, buffy coat from an healthy donor was pre-diluted 1 : 1 ratio with PBS (Gibco #10010023) and then layered on 15 mL Lymphoprep density gradient (StemCell Technologies, #07581) in SepMate-50 tubes (StemCell Technologies, #15460). Prepared tubes were centrifuged at 1200g for 20 min w / o break. PBMC layers were transferred to a fresh Falcon™ 50 mL High Clarity Conical Centrifuge Tube (Corning, #14-432-22) and washed with PBS (Gibco #10010023) two times, via centrifuging at 300g for 6 min. Then, isolated PBMCs were counted using Beckman Coulter DxH 500 (Beckman Coulter) and applied to NK cell isolation using NK cell Isolation kit (Miltenyi Biotec, #130-092-657), by following manufacturer’s recommendations. Isolated NK cells were brought to 1.0 x 106cells / mL in (RPMI 1640, 10%FBS, 1% GlutaMAX) media and cultured overnight in the presence of 100 U / mL Proleukin (IL2) (Novartis).

[0342] On the assay day, overnight incubated molecules were diluted 5x with assay media (Advanced RPMI 1640, 10% FBS, 1% GlutaMAX) to achieve the concentration of 400 nM for (pro-) ICEs and 800 nM.for adaptors. Then, 25 pl of antibody dilutions were added to the assay plate to the respective wells. The assay plate was centrifuged for 5 min at 200g. Meanwhile, primary NK cells were harvested. The cells were counted using Cedex HiRes (Roche, #05650216001) cell counter and 0.09 x 106cells / well (25 pl / well) were plated in assay medium to obtain a final effector-to-target cell ratio (E:T) of 6: 1, and a final assay volume of 100 pl per well - achieving the concentrations of 100 nM for (pro-) ICEs and 200 nM for adaptors. The assay plate was incubated in 37°C and 5% CCh for 20h. After the incubation time, the plate was centrifuged at 400g for 3 min to ensure cell settlement to the bottom of the wells. Then, supernatant was removed from the plate and 60 pl of PBS (Gibco, #10010023) was added to the assay plate (Corning, #353988) before cells in PBS (Gibco, #10010023) were transferred to a 384-well U-bottom plate (Corning, #CLS3656) for flow cytometry staining. The plates were centrifuged for 3 min at 420 g, supernatant was removed and cells were washed with 80 pl of PBS per well. The plate was again centrifuged for 3 min at 420 g and supernatant was removed. Subsequently, 20 pl of the PBS -based staining mix containing LIVE / DEAD™ Fixable Blue Dead Cell Stain Kit (Invitrogen, #L23105) and Human TruStain FcX™ (Biolegend, #422302) was added onto the cells and incubated for 15 min at RT. Then, the plate was washed with 60 pl of PBS per well and centrifuged for 3 min at 420 g. After the removal of first staining mix, 20 pl of PBS-based second staining mix comprising anti-huCD45 PerCP / Cy5.5 (Biolegend, #304028), anti-huNK AF488 (Biolegend, #318312), anti-huCD25 PE (Biotechne, #FAB1020P), anti-huCD69 BV711 (Biolegend, #300944), anti-huCD107a BV605 (Biolegend, #328634) and anti-huCD16 APC (Biolegend, #302012) were added to each well and incubated at 4°C for 20 min. Afterwards, the cells were washed twice with PBS (Gibco, #10010023) and re-suspended in 80 pl of PBS (Gibco, #10010023) per well. The cells were acquired using a BD FACSymphony A3 flow cytometer. Raw data was analyzed using Flowjo vl0.8.1 software.

[0343] Figure 14 depicts regulation ofNK cell activation markers on primary human NK cells engaged with protease-activatable P329G pro-ICEs. Figure 14A shows protease-dependent and dose-dependent percentage of CD107a+ out of CD45+ CD56+ NK cells, reflecting the NK cell granulation induced by anti-FOLRl P329G IgG combined with protease-activatable P329G bivalent GE pro-ICEs with PQARK and PMAKK cleavable linkers. Figure 14B proves protease-dependent and dose-dependent percentage of CD25a+ out of CD45+ CD56+ NK cells, reflecting the NK cell activation induced by anti-FOLRl P329G IgG combined with protease-activatable P329G bivalent GE pro-ICEs with PQARK and PMAKK cleavable linkers. Figure 14C protease-dependent and dose-dependent percentage of CD16+ out of CD45+ CD56+ NK cells, reflecting the NK cell engagement induced by anti-FOLRl P329G IgG combined with protease-activatable P329G bivalent GE pro-ICEs with PQARK and PMAKK cleavable linkers. In this experiment, primary human NK cells from a healthy donor (Figurel4) were used as effector cells, while FOLR1 tumor antigen expressing human cell line OVMANA was used as target cells. As a tumor targeting adaptor molecule, anti-FOLRl adaptor P329G IgG was mixed with anti-P329G (VH3 VL1) bivalent GE pro-ICEs either with PQARK or PMAKK cleavable linkers in a molar ratio adaptor: pro-ICE 2: 1. As a negative control, adaptor P329G IgGs were mixed with anti-P329G (VH3VL1) bivalent GE pro-ICE with non-cleavable linkers. As positive controls, anti-P329G (VH3VL1) bivalent GE unmasked ICE was combined with anti-FOLRl adaptor P329G IgG. Direct anti-FOLRl bivalent GE ICE was also added as an additional positive control. As expected, anti-P329G (VH3VL1) bivalent GE pro-ICEs either with PQARK or PMAKK cleavable linkers led to NK cell activation in a dose-dependent manner, when they were pre-treated with matriptase. But they remained mainly inactive without matriptase pre -treatment, only in the highest two concentrations a negligible amount of activation was observed. Additionally, anti-P329G bivalent GE pro-ICE with non-cleavable linkers remained inactive in both with and without matriptase pre- treatment conditions. The unmasked positive control anti-P329G bivalent GE ICE combined with anti-FOLRl P329G adaptor IgG led to activation regardless of pretreatment conditions.

[0344] By performing primary NK-cell mediated killing assays, protease-activatable anti- P329G pro-ICEs capacity to induce NK-cell activation was also confirmed ex vivo. In addition, pro-ICEs’ protease-dependency were also assessed. These results from NK cell activation experiments correlated nicely with the results of NK-cell mediated killing assays as well as previously shown reporter cell assays, confirming that anti-P329G pro-ICEs can indeed engage and activate primary human innate cells in a protease -dependent manner.

Claims

CLAIMSWhat is claimed is:

1. A protease-activatable Fc domain binding molecule comprising(a) a first antigen binding moiety capable of binding to a variant CH2 domain comprising or consisting of the amino acid sequence of SEQ ID NO: 76 wherein the first antigen binding moiety is not capable of binding to a reference CH2 domain comprising or consisting of the amino acid sequence of SEQ ID NO: 75;(b) a first masking moiety covalently attached to the protease-activatable Fc domain binding molecule through a first protease -cleavable linker, wherein the first masking moiety comprises or consists of the amino acid sequence of SEQ ID NO: 76, wherein the first antigen binding moiety binds to the first masking moiety, wherein the first masking moiety reversibly conceals the first antigen binding moiety; and(c) an Fc domain composed of a first and a second subunit capable of stable association, wherein the Fc domain comprises non-fucosylated oligosaccharides and / or bisected oligosacharides.

2. The protease-activatable Fc domain binding molecule of claim 1, further comprising a second antigen binding moiety capable of binding to a variant CH2 domain comprising or consisting of the amino acid sequence of SEQ ID NO: 76, wherein the second antigen binding moiety is not capable of binding to a reference CH2 domain comprising or consisting of the amino acid sequence of SEQ ID NO: 75; and a second masking moiety covalently attached to the protease-activatable Fc domain binding molecule through a second protease-cleavable linker, wherein the second masking moiety comprises or consists of the amino acid sequence of SEQ ID NO: 76, wherein the second antigen binding moiety binds to the second masking moiety, wherein the second masking moiety reversibly conceals the second antigen binding moiety.

3. The protease-activatable Fc domain binding molecule of claim 1 or 2, wherein the first antigen binding moiety and, where present, the second antigen binding moiety is an antibody or antigen-binding fragment thereof.

4. The protease-activatable Fc domain binding molecule of any one of claims 1 to 3, wherein the first antigen binding moiety and, where present, the second antigen binding moiety comprises:(i) a VH region incorporating the following CDRs:HC-CDR1 having the amino acid sequence of SEQ ID NO: 1;HC-CDR2 having the amino acid sequence of SEQ ID NO: 2; andHC-CDR3 having the amino acid sequence of SEQ ID NO: 3; and(ii) a VL region incorporating the following CDRs:LC-CDR1 having the amino acid sequence of SEQ ID NO: 4;LC-CDR2 having the amino acid sequence of SEQ ID NO: 5; andLC-CDR3 having the amino acid sequence of SEQ ID NO: 6.

5. The protease-activatable Fc domain binding molecule of any one of claims 1 to 4, wherein the first antigen binding moiety and, where present, the second antigen binding moiety comprises:(i) a VH having an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO: 7; and(ii) a VL having an amino acid sequence having at least 80% amino acid sequence identity to SEQ ID NO: 8.

6. The protease-activatable Fc domain binding molecule of any one of claims 1 to 5, wherein the first masking moiety is covalently attached to the heavy chain variable region of the first antigen binding moiety and, where present, the second masking moiety is covalently attached to the heavy chain variable region of the second antigen binding moiety.

7. The protease-activatable Fc domain binding molecule of any one of claims 1 to 6, wherein the first antigen binding moiety and, where present, the second antigen binding moiety is a Fab molecule.

8. The protease-activatable Fc domain binding molecule of any one of claims 1 to 7, wherein the second antigen binding moiety is identical to the first antigen binding moiety.

9. The protease-activatable Fc domain binding molecule of any one of claims 1 to 8, wherein the second masking moiety is identical to the first masking moiety.

10. The protease-activatable Fc domain binding molecule of any one of claims 1 to 9, wherein the first antigen binding moiety is fused to the first subunit of the Fc domain, optionally via a peptide linker.

11. The protease-activatable Fc domain binding molecule of any one of claims 2 to 10, wherein the second antigen binding moiety is fused to the second subunit of the Fc domain, optionally via a peptide linker.

12. The protease-activatable Fc domain binding molecule of any one of claims 1 to 11, wherein the first protease cleavable linker comprises at least one protease recognition sequence.

13. The protease-activatable Fc domain binding molecule of any one of claims 2 to 12, wherein the second protease cleavable linker comprises at least one protease recognition sequence.

14. The protease-activatable Fc domain binding molecule of any one of claims 1 to 13, wherein the first protease cleavable linker and, where present, the second protease cleavable linker comprises the protease recognition sequence PQARK (SEQ ID NO: 64) or PMAKK (SEQ ID NO: 66)15. The protease-activatable Fc domain binding molecule of any one of claims 1 to 14, wherein the Fc domain is an IgG, specifically an IgGi, Fc domain.

16. A nucleic acid, or a plurality of nucleic acids, encoding the protease-activatable Fc domain binding molecule of any one of claims 1 to 15.

17. An expression vector, or a plurality of expression vectors, comprising a nucleic acid or a plurality of nucleic acids according to claim 16.

18. A host cell comprising the nucleic acid or the plurality of nucleic acids of claim 16, or the expression vector or plurality of expression vectors according to claim 17.

19. A method of producing a protease-activatable Fc domain binding molecule, comprising the steps of a) culturing the host cell of claim 18 under conditions suitable for the expression of the protease-activatable Fc domain binding molecule and b) recovering the protease-activatable Fc domain binding molecule.

20. A protease-activatable Fc domain binding molecule produced by the method of claim 19.

21. A pharmaceutical composition comprising the protease -activatable Fc domain binding molecule of any one of claims 1-15 or 20 and a pharmaceutically acceptable carrier22. A kit, comprising:(i) a protease-activatable Fc domain binding molecule of any one of claims 1 -15 or 20 or the pharmaceutical composition of claim 21 ; and(ii) an antigen-binding molecule comprising: (a) an antigen-binding domain that binds to a target antigen, and (b) a variant Fc domain comprising a variant CH2 domain comprising or consisting of the amino acid sequence of SEQ ID NO: 76.

23. The protease-activatable Fc domain binding molecule of any one of claims 1 -15 or 20, the pharmaceutical composition of claim 21, or the kit of claim 24 for use in a method of medical treatment or prophylaxis.

24. The protease-activatable Fc domain binding molecule of any one of claims 1 -15 or 20, the pharmaceutical composition of claim 21 or the kit of claim 24, for use in a method of treating or preventing a disease in which cells comprising or expressing a target antigen are pathologically-implicated, wherein the method comprises administering the protease- activatable Fc domain binding molecule or pharmaceutical composition to a subject to which an antigen-binding molecule has been or is to be administered; wherein the antigenbinding molecule comprises: (a) an antigen-binding domain that binds to the target antigen, and (b) a variant Fc domain comprising a variant CH2 domain comprising or consisting of the amino acid sequence of SEQ ID NO: 76.

25. Use of the protease-activatable Fc domain binding molecule of any one of claims 1 -15 or 20, the pharmaceutical composition of claim 21, or the kit of claim 22, in the manufacture of a medicament for the treatment of a disease in which cells comprising or expressing a target antigen are pathologically-implicated.

26. A method of treating an individual having a disease in which cells comprising or expressing a target antigen are pathologically-implicated, comprising administering to the individual an effective amount of the protease -activatable Fc domain binding molecule of any one of claims 1-15 or 20 or the pharmaceutical composition of claim 21, wherein the individual has been or is to be administered an antigen-binding molecule comprising: (a) an antigen-binding domain that binds to the target antigen, and (b) a variant Fc domain comprising a variant CH2 domain comprising or consisting of the amino acid sequence of SEQ ID NO: 76.

27. The kit of claim 22, the use of any one of claims 23-25, or the method of claim 26, wherein the target antigen is selected from the group consisting of FolRl, EPCAM, HER2, and CD25.

Images