WO2025040567A1

WO2025040567A1 - Protease activatable fc domain binding molecules

Info

Publication number: WO2025040567A1
Application number: PCT/EP2024/073041
Authority: WO
Inventors: Diana Binia DAROWSKI; Christian Klein; Marlena SURÓWKA
Original assignee: F Hoffmann La Roche AG; Hoffmann La Roche Inc
Current assignee: F Hoffmann La Roche AG; Hoffmann La Roche Inc
Priority date: 2023-08-18
Filing date: 2024-08-16
Publication date: 2025-02-27
Anticipated expiration: 2026-02-18

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

PROTEASE ACTIVATABLE FC DOMAIN BINDING MOLECULES

BACKGROUND

The selective destruction of an individual cell or a specific cell type is often desirable in a variety of clinical settings. For example, it is a primary goal of cancer therapy to specifically destroy tumor cells, while leaving healthy cells and tissues intact and undamaged, or to destroy certain cell subsets identified by a specific surface antigen.

An attractive way of achieving this is by inducing an immune response against the cells of interest, by recruiting immune effector cells such as cytotoxic T lymphocytes (CTLs) to attack and destroy tumor cells. T cells can be recruited for the killing of target cells via T cell bispecific antibodies designed to bind with a first binding moiety to CD3 , an activating, invariant component of the T cell receptor (TCR) complex, and with the second binding moiety to a surface antigen on target cells (R. A. Clynes at al. Annu. Rev. Med. (2019), doi: 10.1146/annurev-med-062617-035821). Several bispecific formats, including BiTEs (bispecific T cell engagers) (D. Nagorsen, et al. Exp. Cell Res. (2011), doi: 10.1016/j.yexcr.2011.03.010), diabodies (K. Fitzgerald et al. Protein Eng. (1997), doi: 10.1093/protein/10.10.1221), DART (dual Affinity retargeting) (C. Rader. Blood (2011), doi: 10.1182/blood-2011-02-337691) or so-called 2+1 T cell bispecific antibodies (TCB) (M. Bacac et al. Clin. Cancer Res. (2016), doi: 10.1158/1078-0432. CCR-15-1696) have been developed and their suitability for T cell mediated immunotherapy is being investigated.

Not only can T cells be redirected and activated against various tumor types, but also against different antigens on the same tumor (H. Einsele et al. Cancer (2020), doi: 10.1002/cncr.32909). This is of therapeutic interest due to tumor cells’ ability to downregulate certain antigens, also known as antigen escape or antigen loss, upon therapeutic pressure (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).

Targeting two or more antigens might therefore circumvent this resistance mechanism, ensuring the cell killing that is not dependent on presence of only one antigen (M. Ruella et al. J. Clin. Invest. (2016), doi: 10.1172/JCI87366; E. Zah, et al. Cancer Immunol. Res. (2016), doi: 10.1158/2326-6066. CIR-15-0231; Y. Zhao et al. Blood. (2021), doi: 10.1182/blood.2020006287; J. A. Park et al. J. Immunother. Cancer (2022), doi: 10.1136/JITC-2021-003771). Additionally, within the same tumor type, different patients might show different antigen expression profiles on their tumor cells (A. Jimenez-Sanchez .et al. Nat. Genet. (2020), doi: 10.1038/s41588-020-0630-5; N. G. Alkema et al. Drug Resist. Updat. (2016), doi: 10.1016/j.drup.2015.11.005; A. T. Byrne et al. Nat. Rev. Cancer (2017), doi: 10.1038/nrc.2016.140; O. Kopper c/ . Nat. Med. (2019), doi: 10.1038/s41591-019-0422- 6; J. A. Park et al. J. Immunother. Cancer (2022), doi: 10.1136/JITC-2021-003771). In this scenario, targeting two antigens increases the chances of the therapy matching the patient’s tumor profile. Alternatively, a full personalization of the therapeutic antibodies to the antigens present on the patient’s tumor aims to both circumvent the antigen escape resistance mechanism, and increase the chances of the tumor being targeted.

So far, developed bispecific antibodies directly engage with the desired antigen of interest, thereby linking target cell and CTL resulting in target cell lysis (J. Ma et al. Front. Immunol. (2021), doi: 10.3389/fimmu.2021.626616). Those bispecific antibody formats face challenges, such as the necessity to design, develop, characterize and produce a separate molecule for every target antigen, despite the T cell binding and activating moiety remaining the same in T cell bispecific molecules targeting various antigens. The ability of T cells to be redirected against different antigens on various tumors is therefore exploited. However, what remains necessary is the substantial investment of time and resources for each target, limiting the opportunity to personalize the therapy and combine antibodies against various targets.

Therefore, a universal T cell bispecific antibody platform could offer solutions to these issues, where an antigen targeting moiety is separated from the T cell targeting moiety in production and development. These two parts are then able to form a functional molecule, in the body or before injection into the patient, via Affinity to each other. Such a system would offer a mix-and-match platform. An adaptor, the antigen targeting moiety, consisting of a robust and easily produced antibody or several antibodies, can be adapted to the patient’s tumor profile. Whereas the T cell targeting moiety, an antibody binding both the CD3 on the T cell and the adaptor antibody, is the same regardless of the targeted antigen - therefore allowing for the concentration of resources and expertise on creating a safer, better characterized T cell bispecific antibody therapeutic across targets.

One of the safety issues for T cell engaging bispecific antibodies is so called on-target off-tumor effect. Up to date, the majority of the known tumor targets are not fully specific to the tumor tissue, but are rather overexpressed in the tumor while being present in smaller amount on healthy tissue (M. A. Cheever et al. Clin. Cancer Res. (2009), doi: 10.1158/1078- 0432.CCR-09-0737). This phenomenon means that antibodies targeting tumor associated antigens (TAA) are bound to also target healthy cells expressing the same antigen, rendering the therapy potentially toxic to the healthy tissue and therefore to the patient (N. Parker et al. Anal. Biochem. (2005), doi: 10.1016/j.ab.2004. 12.026; Y. Kinoshita et al. World J. Surg. (2006), doi: 10.1007/s00268-005-0544-5; D. C. Palmer et al. Proc. Natl. Acad. Sci. U. S. A. (2008), doi: 10.1073/pnas.0710929105; M. A. Cheever et al. Clin. Cancer Res. (2009), doi: 10.1158/1078-0432. CCR-09-0737; M. R. Parkhurst et al. Mol. Ther. (2011), doi: 10.1038/mt.2010.272). Therefore, to avoid the on-target off-tumor binding and toxicity, additional tumor-specific activating factors are needed. One of the characteristics of solid tumors is the increased presence of certain proteases within the tumor tissue (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). Hence, creating T cell bispecific antibodies that are only activatable by the tumor proteases, and inactive without them, could allow for additional safety feature of the therapy, by rendering the therapeutic antibody inactive in the healthy tissue without proteases despite the presence of the antigen. Such an approach could potentially prevent the safety liability of on-target off-tumor cytotoxicity, and create a therapy that is safer for the patients.

BRIEF SUMMARY

Provided herein is an improved protease-activatable T cell engaging antibody platform. In a first aspect, the present disclosure provides a bispecific protease -activatable Fc domain binding molecule capable of binding to CD3 and 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 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 CD3 (see Figure 1) and activate T cells. 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 (see Figure 1, Molecule 2). 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.

In a first aspect, the present disclosure provides a protease -activatable Fc domain binding molecule comprising a. a first antigen binding moiety capable of binding to CD3; b. a second antigen binding moiety capable of binding to a variant CH2 domain comprising G329 according to EU numbering, wherein the second antigen binding moiety is not capable of binding to a reference CH2 domain comprising P329 according to EU numbering, wherein the second antigen binding moiety is an antibody or fragment thereof; and c. a masking moiety covalently attached to the protease-activatable Fc domain binding molecule through a protease-cleavable linker, wherein the masking moiety comprises the variant CH2 domain comprising G329 according to EU numbering, wherein the second antigen binding moiety binds to the variant CH2 domain, wherein the variant CH2 domain reversibly conceals the second antigen binding moiety.

In some embodiments, the first antigen binding moiety and/or the second antigen binding moiety is an antibody or antigen-binding fragment thereof.

In some embodiments, the first antigen-binding moiety comprises:

(i) a VH region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO: 24;

HC-CDR2 having the amino acid sequence of SEQ ID NO: 25; and

HC-CDR3 having the amino acid sequence of SEQ ID NO: 26; and

(ii) a VL region incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO: 5;

LC-CDR2 having the amino acid sequence of SEQ ID NO: 6; and

LC-CDR3 having the amino acid sequence of SEQ ID NO: 7; or

(i) a VH region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO: 24;

HC-CDR2 having the amino acid sequence of SEQ ID NO: 25; and

HC-CDR3 having the amino acid sequence of SEQ ID NO: 28; and

(ii) a VL region incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO: 5;

LC-CDR2 having the amino acid sequence of SEQ ID NO: 6; and

LC-CDR3 having the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the first antigen-binding moiety comprises:

(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 27; and

(ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 8; or

(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 29; and

(ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 8.

In some embodiments, 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: 17;

HC-CDR2 having the amino acid sequence of SEQ ID NO: 18; and HC-CDR3 having the amino acid sequence of SEQ ID NO: 19; and

(ii) a VL region incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO: 21;

LC-CDR2 having the amino acid sequence of SEQ ID NO: 6; and

LC-CDR3 having the amino acid sequence of SEQ ID NO: 22.

In some embodiments, the second antigen-binding moiety comprises:

(i) a VH having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 20; and

(ii) a VL having an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 23.

In some embodiments, the masking moiety is covalently attached to the heavy chain variable region of the second antigen binding moiety.

In some embodiments, the first antigen binding moiety is a crossover Fab molecule wherein either the variable or the constant regions of the Fab light chain and the Fab heavy chain are exchanged.

In some embodiments, the second antigen binding moiety is a Fab molecule.

In some embodiments, the protease-activatable Fc domain binding molecule comprises a third antigen binding moiety which is a Fab molecule capable of binding to a variant CH2 domain comprising G329 according to EU numbering, wherein the third antigen binding moiety is not capable of binding to a reference CH2 domain comprising P329 according to EU numbering.

In some embodiments, the third antigen binding moiety is identical to the second antigen binding moiety.

In some embodiments, the first antigen binding moiety and the second antigen binding moiety, and where present the third antigen binding moiety are fused to each other, optionally via a peptide linker. In some embodiments, the variant CH2 domain comprises or consists of the amino acid sequence of SEQ ID NO: 77.

In some embodiments, the masking moiety comprises or consists of the amino acid sequence of SEQ ID NO: 77.

In some embodiments, the reference CH2 domain comprises or consists of the amino acid sequence of SEQ ID NO: 78.

In some embodiments, the protease cleavable linker comprises at least one protease recognition sequence.

In some embodiments, the protease cleavable linker comprises the protease recognition sequence PQARK (SEQ ID NO: 72) or PMAKK (SEQ ID NO: 73)

In some embodiments, the protease-activatable Fc domain binding molecule additionally comprises (d) an Fc domain composed of a first and a second subunit capable of stable association.

In some embodiments, the Fc domain is an IgG, specifically an IgGi, Fc domain.

In some embodiments, the Fc domain comprises a variant CH2 domain comprising R329 according to EU numbering.

In some embodiments, the Fc domain comprises one or two amino acid sequences selected from the group consisting of SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO:85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO:89

In some embodiments, the Fc domain comprises an amino acid sequence of SEQ ID NO: 89 and an amino acid sequence of SEQ ID NO: 90.

In a further aspect, the present disclosure provides nucleic acid, or a plurality of nucleic acids, encoding the protease-activatable Fc domain binding molecule as described hereinabove.

In a further aspect, the present disclosure provides an expression vector, or a plurality of expression vectors, comprising a nucleic acid or a plurality of nucleic acids as described hereinabove. In a further aspect, the present disclosure provides a host cell comprising the nucleic acid or the plurality of nucleic acids as described hereinabove, or the expression vector or plurality of expression vectors as described hereinabove.

In a further aspect, the present disclosure provides a method of producing a protease - activatable Fc domain binding molecule, comprising the steps of a) culturing the host cell as described herein above under conditions suitable for the expression of the protease- activatable Fc domain binding molecule and b) recovering the protease -activatable Fc domain binding molecule.

In a further aspect, the present disclosure provides a protease -activatable Fc domain binding molecule produced by the method as described hereinabove.

In a further aspect, the present disclosure provides a pharmaceutical composition comprising the protease-activatable Fc domain binding molecule as described hereinabove and a pharmaceutically acceptable carrier

In a further aspect, the present disclosure provides the protease-activatable Fc domain binding molecule as described hereinabove, or the pharmaceutical composition as described hereinabove for use in a method of medical treatment or prophylaxis.

In a further aspect, the present disclosure provides the protease-activatable Fc domain binding molecule as described hereinabove or the pharmaceutical composition as described hereinabove, 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 G329 according to EU numbering; and wherein the second antigen-binding moiety of the protease-activatable Fc domain binding molecule, binds to the variant Fc domain.

In a further aspect, the present disclosure provides a kit, comprising:

(i) a protease-activatable Fc domain binding molecule as described hereinabove or the pharmaceutical composition as described hereinabove; and (ii) 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 G329 according to EU numbering; and wherein the second antigen-binding moiety of the protease-activatable Fc domain binding molecule, binds to the variant Fc domain.

In a further aspect, the present disclosure provides the use as described hereinabove or the kit as described hereinabove, wherein the variant Fc domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77.

In a further aspect, the present disclosure provides the use as described herein above, or the kit as described hereinabove, wherein the target antigen is FolRl or CEACAM5.

DETAILED DESCRIPTION

Definitions

Terms are used herein as generally used in the art, unless otherwise defined in the following.

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.

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. 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.

An “activating T cell antigen” as used herein refers to an antigenic determinant expressed on the surface of a T lymphocyte, particularly a cytotoxic T lymphocyte, which is capable of inducing T cell activation upon interaction with an antigen binding molecule. Specifically, interaction of an antigen binding molecule with an activating T cell antigen may induce T cell activation by triggering the signaling cascade of the T cell receptor complex. In a particular embodiment the activating T cell antigen is CD3, particularly the epsilon subunit of CD3 (see UniProt no. P07766 (version 130), NCBI RefSeq no. NP_000724.1; or UniProt no. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1).

“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.

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.

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.

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.

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).

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).

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. 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.

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.

“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).

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.

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.

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.

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.

“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.

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.

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.

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.

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.

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.

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).

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.

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.

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: 74. 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.

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

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.

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).

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.

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.

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.

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”).

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:

(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));

(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)).

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. An “immune activating moiety” as used herein refers to one or more polypeptide(s) inducing activation of an immune cell (e.g. a T cell) upon interaction with an antigen, receptor or ligand (or other elements of the cells inducing activation) on the immune cell. An example of an immune activating moiety is antigen binding molecule capable of binding to an activating T cell antigen triggering the signaling cascade of the T cell receptor complex. In a particular embodiment the immune activating moiety is an antigen binding moiety capable of binding to CD3, particularly the epsilon subunit of CD3 (see UniProt no. P07766 (version 130), NCBI RefSeq no. NP 000724.1; or UniProt no. Q95LI5 (version 49), NCBI GenBank no. BAB71849.1).

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

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.

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).

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.

“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.

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.

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.

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.

“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, 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.

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).

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).

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.

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.

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.

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.

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.

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.

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 activate T cells due to a masking moiety that reduces or abrogates the protease -activatable Fc domain binding molecule's ability to bind to and activate a T cell via a target antigen binding molecule 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 antigen binding molecule and ultimately T cell activation is restored.

“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.

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 or molecule such as to prevent the antigen binding moiety or molecule from 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 or molecule, e.g., by protease cleavage, and thereby freeing the antigen-binding moiety or molecule to bind to its antigen.

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 is less than about 10% of the binding of the antigen binding moiety to the antigen 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. 10'⁸ M or less, e.g. from 10'⁸ M to 10'¹³ M, e.g., from 10'⁹ M to IO’¹³ M).

“T cell activation” as used herein refers to one or more cellular response of a T lymphocyte, particularly a cytotoxic T lymphocyte, selected from: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. The immune activating Fc domain binding molecules of the invention are capable of inducing T cell activation. Suitable assays to measure T cell activation are known in the art described herein.

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 a particular embodiment, the target cell antigen is CEACAM5, particularly human CEACAM5.

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.

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.

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.

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, 6^th ed., 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).

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”.

Protease-activatable Fc domain binding molecule

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. 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) an immune activating (Fc domain binding) molecule that specifically recognizes the Fc-part of the target antigen binding molecule, thereby recruiting immune effector cells and activating them e.g. via establishing a immunological synapse to redirect CTLs and subsequent lysis of the target cell. In a particular aspect, the present disclosure provides protease-activatable Fc domain binding molecule comprising a CD3 binding moiety, an Fc domain binding moiety, a masking moiety and a protease-cleavable linker wherein the masking moiety reversibly conceals the Fc domain binding moiety. Upon cleavage of the 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 CD3 on T cell, particularly cytotoxic T cells. 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.

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

Variant CH2 domain

In one aspect, provided are protease-activatable Fc domain binding molecules which are masked by an variant CH2 domain or a fragment thereof. The protease -activatable Fc domain binding molecules of the present disclosure comprise an antigen binding moiety and a masking 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 second antigen binding moiety is not capable of binding to a reference CH2 domain comprising P329 according to EU numbering. In one aspect, 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.

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. Moreover, the co-activation of Fc receptor signaling pathways may lead to cytokine release which can result in excessive activation of cytokine receptors and severe side effects upon systemic administration. Activation of (Fc receptor-bearing) immune cells other than T cells may even reduce efficacy of antibodies (e.g. T cell activating antibodies) due to the potential destruction of T cells e.g., by NK cells.

Accordingly, preferably, antibodies 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 antibodies.

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 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.

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.

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.

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.

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: 77, or a fragment thereof that the second 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: 77.

In one aspect, the masking moiety comprises or consists of the amino acid sequence of SEQ ID NO: 77 or a fragment thereof that the second antigen binding moiety as described herein below can bind.

Suitable methods to measure the binding of the masking moiety and the second 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. In one embodiment, binding of the second 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 MNaCl, 0.005% Surfactant P20 (BR-1006-69, GE Healthcare)). The second 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).

In one particular aspect, provides is a protease -activatable Fc domain binding molecule comprising a. a first antigen binding moiety capable of binding to CD3; b. a second antigen binding moiety capable of binding to a variant CH2 domain comprising G329 according to EU numbering, wherein the second antigen binding moiety is not capable of binding to a reference CH2 domain comprising P329 according to EU numbering, wherein the second antigen binding moiety is an antibody or fragment thereof; and c. 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 the variant CH2 domain comprising G329 according to EU numbering, wherein the second antigen binding moiety binds to the variant CH2 domain, wherein the variant CH2 domain reversibly conceals the second antigen binding moiety.

In one preferred aspect, provides is a a protease -activatable Fc domain binding molecule comprising a. a first antigen binding moiety capable of binding to CD3; b. 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: 77, 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: 78, wherein the second antigen binding moiety is an antibody or fragment thereof; and c. 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: 77. In some aspect, the protease-activatable Fc domain binding molecule comprises a third antigen binding moiety. In one such aspect, the protease -activatable Fc domain binding molecule comprises a third antigen binding moiety which is a Fab molecule capable of binding to a variant CH2 domain comprising G329 according to EU numbering, wherein the third antigen binding moiety is not capable of binding to a reference CH2 domain comprising P329 according to EU numbering. In aspects where the protease -activatable Fc domain binding molecule comprises the third antigen binding moiety, the protease -activatable Fc domain binding molecules further comprises a second masking moiety as hereinbefore described. In a preferred aspect, the third antigen binding moiety is identical to the second antigen binding moiety.

In one particular aspect, provides is a a protease -activatable Fc domain binding molecule comprising a. a first antigen binding moiety capable of binding to CD3; b. a second antigen binding moiety capable of binding to a variant CH2 domain comprising G329 according to EU numbering, wherein the second antigen binding moiety is not capable of binding to a reference CH2 domain comprising P329 according to EU numbering, wherein the second antigen binding moiety is an antibody or fragment thereof; and c. 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 the variant CH2 domain comprising G329 according to EU numbering, wherein the second antigen binding moiety binds to the variant CH2 domain, wherein the variant CH2 domain reversibly conceals the second antigen binding moiety; d. a third antigen binding moiety capable of binding to the variant CH2 domain comprising G329 according to EU numbering, wherein the third antigen binding moiety is not capable of binding to the reference CH2 domain comprising P329 according to EU numbering, wherein the third antigen binding moiety is an antibody or fragment thereof; and e. 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 the variant CH2 domain comprising G329 according to EU numbering, wherein the third antigen binding moiety binds to the variant CH2 domain, wherein the variant CH2 domain reversibly conceals the third antigen binding moiety. In one preferred aspect, provides is a a protease-activatable Fc domain binding molecule comprising a. a first antigen binding moiety capable of binding to CD3; b. 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: 77, 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: 78, wherein the second antigen binding moiety is an antibody or fragment thereof; and c. 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: 77; d. a third antigen binding moiety capable of binding to a variant CH2 domain comprising or consisting of the amino acid sequence of SEQ ID NO: 77, wherein the third 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: 78, wherein the third antigen binding moiety is an antibody or fragment thereof; and e. 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: 77.

In one aspect, the second masking moiety is covalently attached to the heavy chain variable region of the third antigen binding moiety.

In one preferred aspect, the first and second masking moiety are identical and the first and second protease-cleavable linker are identical.

Protease-cleavable linker

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 second antigen binding moiety is capable of binding to the target antigen binding molecule comprising the relevant variant CH2 domain.

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.

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 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 72 or SEQ ID NO: 73. In one aspect, the protease recognition site comprises the polypeptide sequence of SEQ ID NO: 72 or SEQ ID NO: 73. In a preferred aspect, the protease recognition site comprises the polypeptide sequence of SEQ ID NO: 72.

In some aspects, the protease-cleavable linker comprises a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 80 or SEQ ID NO: 81. In one aspect, the protease-cleavable linker comprises the polypeptide sequence of SEQ ID NO: 80 or SEQ ID NO: 81. In a preferred aspect, the protease -cleavable linker comprises the polypeptide sequence of SEQ ID NO: 80.

In one embodiment the relevant protease is matriptase.

Antigen binding moiety capable of binding to variant CH2 domain

The antigen binding moiety capable of binding to a variant CH2 domain of the present disclosure comprise an antigen binding moiety, or a component thereof. The essential function of the antigen binding moiety is to provide for binding to a variant CH2 domain, as described herein below.

Antigen binding moieties include antibodies (i.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. 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 etal., 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).

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.

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.

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.

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 10'⁶ M. 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 10'⁶ M. 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 10'⁹ M. 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 10'⁹ M. 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 10'¹² M. 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’¹² M.

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.

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.

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.

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).

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.

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.

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.

There are several different conventions for defining antibody CDRs and FRs, such as (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);

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

(iii) the international IMGT (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.

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.

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.

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

HC-CDR1 having the amino acid sequence of SEQ ID NO: 17

HC-CDR2 having the amino acid sequence of SEQ ID NO: 18

HC-CDR3 having the amino acid sequence of SEQ ID NO: 19, 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.

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: 20.

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

LC-CDR1 having the amino acid sequence of SEQ ID NO: 21

LC-CDR2 having the amino acid sequence of SEQ ID NO: 6

LC-CDR3 having the amino acid sequence of SEQ ID NO: 22, 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: 23.

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

Row Shared property Amino acids

1 Hydrophobic Met, Ala, Vai, Leu, He, Trp, Tyr, Phe,

Norleucine

2 Neutral hydrophilic Cys, Ser, Thr, Asn, Gin

3 Acidic or negatively-charged Asp, Glu

4 Basic or positively-charged His, Lys, Arg

Orientation influencing Gly, Pro

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.

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:

Amino Acid Side-chain polarity Side-chain charge (pH

7.4)

Alanine nonpolar neutral Amino Acid Side-chain polarity Side-chain charge (pH

7.4)

Arginine basic polar positive

Asparagine polar neutral

Aspartic acid acidic polar negative

Cysteine nonpolar neutral

Glutamic acid acidic polar negative

Glutamine polar neutral

Glycine nonpolar neutral

Histidine basic polar positive (10%) neutral (90%)

Isoleucine nonpolar neutral

Leucine nonpolar neutral

Lysine basic polar positive

Methionine nonpolar neutral

Phenylalanine nonpolar neutral

Proline nonpolar neutral

Serine polar neutral

Threonine polar neutral Amino Acid Side-chain polarity Side-chain charge (pH

7.4)

Tryptophan nonpolar neutral

Tyrosine polar neutral

Valine nonpolar neutral

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.

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.

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. 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: 17, HC-CDR2 according to SEQ ID NO: 18, and HC-CDR3 according to SEQ ID NO: 19, and (ii) a VL region comprising LC-CDR1 according to SEQ ID NO: 21, LC-CDR2 according to SEQ ID NO: 6, and LC-CDR3 according to SEQ ID NO: 22.

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: 20. 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: 23.

In a preferred embodiment, the antigen binding moiety comprises or consists of a VH region having the amino acid sequence of SEQ ID NO: 20 and the VL region having the amino acid sequence of SEQ ID NO: 23.

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.

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).

Antigen binding moiety capable of binding to CD3

The protease-activatable Fc domain binding molecule of the present disclosure comprises and antigen binding moiety capable of binding to CD3, or a component thereof capable of binding to CD3. The essential function of the antigen binding moiety is to provide for binding to CD3, as described herein below.

Antigen binding moieties capable of binding to CD3 include antibodies (i.e. immunoglobulins (Igs)), and antigen-binding fragments and derivatives thereof. In some embodiments, an antigen binding moiety 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.

Antigen binding moieties capable of binding to CD3 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).

The antigen binding moieties described herein preferably display specific binding to CD3, in particular to CD3e. Specific binding refers to binding which is selective for the target antigen, and which can be discriminated from non-specific 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.

The ability of a given moiety to bind specifically to CD3 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 CD3 can be measured and quantified. In some embodiments, the level of binding may be the response detected in a given assay.

In some embodiments, the antigen binding moiety described herein binds to CD3 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 10'⁶ M. In some embodiments, the antigen binding moiety described herein binds to CD3 with sub-micromolar affinity, i.e. KD < 1 X 10'⁶ M. In some embodiments, the antigen binding moiety described herein binds to CD3 with an affinity in the nanomolar range, i.e. KD = 9.9 x IO'⁷"' to 1 x 10'⁹ M. In some embodiments, the antigen binding moiety described herein binds to CD3 with sub -nanomolar affinity, i.e. KD < 1 x 10" ⁹ M. In some embodiments, the antigen binding moiety described herein binds to CD3 with an affinity in the picomolar range, i.e. KD = 9.9 x IO'¹⁰'" to 1 x 10'¹² M. In some embodiments, the antigen binding moiety described herein binds to CD3 with sub -picomolar affinity, i.e. KD < 1 X 10'¹² M.

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 CD3. 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 CD3. 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 CD3.

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

(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);

(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.

In some embodiments, the antigen binding moiety comprises the CDRs of an antigen binding moiety that binds to CD3. In some embodiments, the antigen binding moiety comprises the FRs of an antigen binding moiety that binds to CD3. In some embodiments, the antigen binding moiety comprises the CDRs and the FRs of an antigen binding moiety that binds to CD3. 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 CD3 according to the present disclosure. 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 CD3, 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 CD3. In some embodiments, an antigen binding molecule that binds to CD3 is referred to as anti-huCD3E P035.093 or anti-huCD3E clone 22.

In some embodiments, the antigen-binding moiety comprises a VH region according to (1) or (2) below:

(1) a VH region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO: 24

HC-CDR2 having the amino acid sequence of SEQ ID NO: 25

HC-CDR3 having the amino acid sequence of SEQ ID NO: 26, 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.

(2) a VH region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO: 24

HC-CDR2 having the amino acid sequence of SEQ ID NO: 25

HC-CDR3 having the amino acid sequence of SEQ ID NO: 28, 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.

In some embodiments, the antigen binding moiety comprises a VH region according to one of (3) or (4) below:

(3) 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: 27.

(4) 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: 29.

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

LC-CDR1 having the amino acid sequence of SEQ ID NO: 5

LC-CDR2 having the amino acid sequence of SEQ ID NO: 6

LC-CDR3 having the amino acid sequence of SEQ ID NO: 7, 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.

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

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:

Row Shared property Amino acids 1 Hydrophobic Met, Ala, Vai, Leu, He, Trp, Tyr, Phe,

Norleucine

2 Neutral hydrophilic Cys, Ser, Thr, Asn, Gin

3 Acidic or negatively-charged Asp, Glu

4 Basic or positively-charged His, Lys, Arg

Orientation influencing Gly, Pro

Amino Acid Side-chain polarity Side-chain charge (pH

7.4)

Alanine nonpolar neutral

Arginine basic polar positive

Asparagine polar neutral

Aspartic acid acidic polar negative

Cysteine nonpolar neutral Amino Acid Side-chain polarity Side-chain charge (pH

7.4)

Glutamic acid acidic polar negative

Glutamine polar neutral

Glycine nonpolar neutral

Histidine basic polar positive (10%) neutral (90%)

Isoleucine nonpolar neutral

Leucine nonpolar neutral

Lysine basic polar positive

Methionine nonpolar neutral

Phenylalanine nonpolar neutral

Proline nonpolar neutral

Serine polar neutral

Threonine polar neutral

Tryptophan nonpolar neutral

Tyrosine polar neutral

Valine nonpolar neutral 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.

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.

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: 24, HC-CDR2 according to SEQ ID NO: 25, and HC-CDR3 according to SEQ ID NO: 26, and (ii) a VL region comprising LC-CDR1 according to SEQ ID NO: 5, LC-CDR2 according to SEQ ID NO: 6, and LC-CDR3 according 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: 27. 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.

In some embodiments, an antigen binding moiety of the present disclosure comprises a VH region 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: 27. In some embodiments, an antigen binding moiety of the present disclosure comprises a VL region 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.

In a preferred embodiment, the antigen binding moiety comprises or consists of a VH region having the amino acid sequence of SEQ ID NO: 27 and the VL region having the amino acid sequence of SEQ ID NO: 8.

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: 24, HC-CDR2 according to SEQ ID NO: 25, and HC-CDR3 according to SEQ ID NO: 28, and (ii) a VL region comprising LC-CDR1 according to SEQ ID NO: 5, LC-CDR2 according to SEQ ID NO: 6, and LC-CDR3 according 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: 29. 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.

In some embodiments, an antigen binding moiety of the present disclosure comprises a VH region 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: 29. In some embodiments, an antigen binding moiety of the present disclosure comprises a VL region 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.

In a preferred embodiment, the antigen binding moiety comprises or consists of a VH region having the amino acid sequence of SEQ ID NO: 29 and the VL region having the amino acid sequence of SEQ ID NO: 8.

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 CD3 (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 CD3 (e.g. as described herein). In preferred embodiments, the VH region and VL region may be from the same antigen binding moiety.

Variant Fc domain of the protease-activatable Fc domain binding molecule

In some aspect, the protease-activatable Fc domain binding molecule does not comprise an Fc domain for example if a short half-life of the protease-activatable Fc domain binding molecule is preferred. Accordingly, in some aspects, provided are protease - activatable Fc domain binding molecules devoid of an Fc domain . However, in many instances it will be preferred to include an Fc domain in the protease-activatable Fc domain binding molecule. The Fc domain confers 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.

Accordingly, in some aspects, provided is a protease-activatable Fc domain binding molecule comprising (d) an Fc domain composed of a first and a second subunit capable of stable association. Notably, it may be desirable to ensure that the Fc domain binding moiety is not capable of binding to the Fc domain of (d). Binding of the Fc domain binding moiety to the Fc domain of (d) can lead to self-binding of the protease-activatable Fc domain binding molecules, i.e. one protease-activatable Fc domain binding molecule binds to another (identical) protease-activatable Fc domain binding molecule via the Fc domain of (d). Selfbinding can lead to cross-linking of multiple protease-activatable Fc domain binding molecules, which can be undesirable.

Accordingly, in a preferred aspect, provided a protease -activatable Fc domain binding molecule comprising

(a) a first antigen binding moiety capable of binding to CD3;

(b) a second antigen binding moiety capable of binding to a variant CH2 domain comprising G329 according to EU numbering, wherein the second antigen binding moiety is not capable of binding to a reference CH2 domain comprising P329 according to EU numbering, wherein the second antigen binding moiety is an antibody or fragment thereof;

(c) a masking moiety covalently attached to the protease-activatable Fc domain binding molecule through a protease-cleavable linker, wherein the masking moiety comprises the variant CH2 domain comprising G329 according to EU numbering, wherein the second antigen binding moiety binds to the variant CH2 domain, wherein the variant CH2 domain reversibly conceals the second antigen binding moiety; and

(d) Fc domain composed of a first and a second subunit capable of stable association, wherein the second antigen binding moiety does not specifically bind to the Fc domain of (d).

An Fc domain as herein described in (d) 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. In one aspect, the protease-activatable Fc domain binding molecule comprises not more than one Fc domain.

As herein before described, the Fc domain confers to an antibody favorable pharmacokinetic properties, including a long serum half-life. At the same time it may, however, lead to undesirable targeting to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. Moreover, the co-activation of Fc receptor signaling pathways may lead to cytokine release which, in combination with the T cell activating properties and the long half-life of the protease-activatable Fc domain binding molecule, results in excessive activation of cytokine receptors and severe side effects upon systemic administration. Activation of (Fc receptor-bearing) immune cells other than T cells may even reduce efficacy of the protease-activatable Fc domain binding molecule due to the potential destruction of T cells e.g. by NK cells.

In some aspects, the Fc domain of (d) comprise at least one amino acid substitution that reduce binding to an Fc receptor and/or reduce effector function. Fc mutations (e.g. amino acid substitutions) conferring such reduced binding to Fc receptors and/or effector function are known in the art and herein below described. In one embodiment, the Fc domain an IgG Fc domain, specifically an IgGi or IgG4 Fc domain. In one embodiment, the Fc domain exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgGi Fc domain. In one such embodiment the Fc domain individually exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the binding affinity to an Fc receptor, as compared to a native IgGi Fc domain (or a molecule comprising a native IgGi Fc domain), and/or less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the effector function, as compared to a native IgGi Fc domain (or a molecule comprising a native IgGi Fc domain). In one embodiment, the Fc domain does not substantially bind to an Fc receptor and/or induce effector function. In a particular embodiment the Fc receptor is an Fey receptor. In one embodiment the Fc receptor is a human Fc receptor. In one embodiment the Fc receptor is an activating Fc receptor. In a specific embodiment the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa. In one embodiment the effector function is one or more selected from the group of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment the effector function is ADCC. In one embodiment the Fc domain exhibits substantially similar binding affinity to neonatal Fc receptor (FcRn), as compared to a native IgGi Fc domain. Substantially similar binding to FcRn is achieved when Fc domain exhibits greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of the binding affinity of a native IgGi Fc domain (or molecule comprising a native IgGi Fc domain) to FcRn.

In certain embodiments the Fc domain of (d) is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a non-engineered Fc domain. In particular embodiments, the Fc domain individually comprises one or more amino acid substitution that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function. Typically, the same one or more amino acid substitution is present in each of the two subunits of the Fc domain. In one embodiment the amino acid substitution reduces the binding affinity of the Fc domain to an Fc receptor. In one embodiment the amino acid substitution reduces the binding affinity of the Fc domain to an Fc receptor by at least 2- fold, at least 5 -fold, or at least 10-fold. In embodiments where there is more than one amino acid substitution that reduces the binding affinity of the Fc domain to the Fc receptor, the combination of these amino acid substitutions may reduce the binding affinity of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment the protease-activatable Fc domain binding molecule comprises an engineered Fc domain that exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor as compared to molecule comprising a nonengineered Fc domain. In a particular embodiment the Fc receptor is an Fey receptor. In some embodiments the Fc receptor is a human Fc receptor. In some embodiments the Fc receptor is an activating Fc receptor. In a specific embodiment the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments binding affinity to a complement component, specifically binding affinity to Clq, is also reduced. In one embodiment binding affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e. preservation of the binding affinity of the Fc domain to said receptor, is achieved when the Fc domain (or a molecule comprising said Fc domain) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc domain (or a molecule comprising said non-engineered form of the Fc domain) to FcRn. The Fc domain, or molecules of the invention comprising said Fc domain, may individually exhibit greater than about 80% and even greater than about 90% of such affinity. In certain embodiments the Fc domain is engineered to have reduced effector function, as compared to a non-engineered Fc domain. The reduced effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced Antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibodydependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced crosslinking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment the reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a particular embodiment the reduced effector function is reduced ADCC. In one embodiment the reduced ADCC is less than 20% of the ADCC induced by a non-engineered Fc domain (or a molecule comprising a non-engineered Fc domain).

In one embodiment the Fc domain of (d) 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 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.

As described above, it might be important to prevent self-binding of the protease- activatable Fc domain binding molecules. Hence, it is preferred that the Fc domain of (d) comprises an amino acid substitution at position P329 by an amino acid other than glycine (G) (numbering according to Kabat EU index). In one aspect, the Fc domain of (d) comprises at least one amino acid substitution at position P329 by an amino acid other than glycine (G) (numbering according to Kabat EU index). In one embodiment, the Fc domain of (d) comprises a substitution at position P329 (numbering according to Kabat EU index) by an amino acid other than glycine (G) wherein such amino acid is not able to form a proline sandwich between two conserved tryptophan sidechains within a Fc gamma receptor, in particular within FcgRIIIa. In one embodiment, Fc domain of (d) comprises a substitution at position P329 (numbering according to Kabat EU index) by an amino acid selected from the list consisting of arginine (R), leucine (L), isoleucine (I), and alanine (A). In particular such embodiments, the Fc domain of (d) comprises a substitution at position P329 (numbering according to Kabat EU index) by arginine (R). The “P329R”, the “P329L”, the “P329I” and the “P329A” amino acid substitutions each individually combined with the “LALA” amino acid substitutions almost completely abolishes Fey receptor (as well as complement) binding. Such Fc domain mutants and their Fey receptor binding properties are described in PCT publication no. WO 2021/255138, incorporated herein by reference in its entirety. In one embodiment, the protease-activatable Fc domain binding molecule comprises an Fc domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90.

In a specific embodiment, the Fc domain of (d) is an IgGl comprising the P329L substitution (numbering according to Kabat EU index). In one particular such embodiment, the Fc domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 83.

In a specific embodiment, the Fc domain of (d) is an IgGl comprising the P329I substitution (numbering according to Kabat EU index). In one particular such embodiment, the Fc domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 84.

In a preferred embodiment, the Fc domain of (d) is an IgGl comprising the P329R substitution (numbering according to Kabat EU index). In one particular such embodiment, the Fc domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 85.

In a specific embodiment, the Fc domain of (d) is an IgGl comprising the P329A substitution (numbering according to Kabat EU index). In one particular such embodiment, the Fc domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 86. On another preferred embodiment, the Fc domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 88.

In certain embodiments N-glycosylation of the Fc domain of (d) has been eliminated. In one such embodiment the Fc domain of (d) 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).

Variant (substituted) 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.

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 some embodiments, binding of the Fc domain of (d) to a complement component, specifically to Clq, is reduced. Accordingly, in some embodiments wherein 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 protease-activatable Fc domain 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)).

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.

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).

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. 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.

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).

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)).

In a particular embodiment the antigen binding moiety capable of binding to CD3 is fused (optionally via the antigen binding moiety capable of binding to a target cell antigen) to the first subunit of the Fc domain (comprising the “knob” modification). Without wishing to be bound by theory, fusion of the antigen binding moiety capable of binding to CD3 to the knob-containing subunit of the Fc domain will (further) minimize the generation of antigen binding molecules comprising two antigen binding moieties capable of binding to CD3 (steric clash of two knob-containing polypeptides).

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:88 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: 89

In an alternative 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. Exemplary protease-activatable Fc domain binding molecules

The variant CH2 domain, the protease-cleavable linker, the antigen binding moiety capable of binding to the variant CH2 domain, the antigen binding moiety capable of binding to CD3, and where present, the variant Fc domain of the protease -activatable Fc domain binding molecule as hereinbefore described can be fused to each other in a variety of configurations.

In particular embodiments, the protease-activatable Fc domain binding molecule comprises an Fc domain composed of a first and a second subunit capable of stable association. 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 one such embodiment, the second antigen binding moiety is fused at the C- terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety. In a specific such embodiment, the protease-activatable Fc domain binding molecule essentially consists of a first and a second antigen binding moiety, a masking moiety covalently attached to the protease-activatable Fc domain binding molecule through a protease-cleavable linker, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, and 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. Optionally, the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety may additionally be fused to each other.

In another such embodiment, the second antigen binding moiety is fused at the C- terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In a specific such embodiment, the protease-activatable Fc domain binding molecule essentially consists of a first and a second antigen binding moiety, a masking moiety covalently attached to the protease-activatable Fc domain binding molecule through a protease-cleavable linker, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the first and the second antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. In other embodiments, the second antigen binding moiety is fused at the C -terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.

In a particular such embodiment, the first antigen binding moiety is fused at the C- terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety. In a specific such embodiment, the protease-activatable Fc domain binding molecule essentially consists of a first and a second antigen binding moiety, a masking moiety covalently attached to the protease-activatable Fc domain binding molecule through a protease-cleavable linker, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the first antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding moiety, and the second 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. Optionally, the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety may additionally be fused to each other

The antigen binding moieties may be fused to the Fc domain or to each other directly or through a peptide linker, comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and are described herein. Suitable, non- immunogenic peptide linkers include, for example, (G4S)_n, (SG4)n, (G4S)_n or

G4(SG4)_n peptide linkers, “n” is generally a number between 1 and 10, typically between 2 and 4. A particularly suitable peptide linker for fusing the Fab light chains of the first and the second antigen binding moiety to each other is (G4S)2. Additionally, linkers may comprise (a portion of) an immunoglobulin hinge region. Particularly where an antigen binding moiety is fused to the N-terminus of an Fc domain subunit, it may be fused via an immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.

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.

In many other cases, however, it will be advantageous to have a protease-activatable Fc domain binding molecule comprising two or more antigen binding moieties specific for a a variant CH2 domain comprising G329 according to EU numbering, for example to optimize crosslinking of target antigen binding molecules. Accordingly, in certain embodiments, the protease-activatable Fc domain binding molecule of the invention further comprises a third antigen binding moiety which is a Fab molecule capable of binding to a variant CH2 domain comprising G329 according to EU numbering, wherein the third antigen binding moiety is not capable of binding to a reference CH2 domain comprising P329 according to EU numbering. In one embodiment, the third antigen binding moiety is a conventional Fab molecule. In one embodiment, the third antigen binding moiety is capable of binding to the same variant CH2 domain as the second antigen binding moiety. In a particular embodiment, the first antigen binding moiety is capable of binding to CD3, and the second and third antigen binding moieties are capable of binding to a variant CH2 domain comprising G329 according to EU numbering, wherein the second and third antigen binding moieties are not capable of binding to a reference CH2 domain comprising P329 according to EU numbering. In a particular embodiment, the second and the third antigen binding moiety are identical (i.e. they comprise the same amino acid sequences).

In a particular embodiment, the first and the third antigen binding moiety are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits 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 Fab heavy chain of the first antigen binding moiety. In a specific such embodiment, the protease-activatable Fc domain binding molecule essentially consists of a first, a second and a third antigen binding moiety, a masking moiety covalently attached to the protease-activatable Fc domain binding molecule through a protease-cleavable linker, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, wherein the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding moiety, and 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 wherein the third 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. Optionally, the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety may additionally be fused to each other.

In some of the protease-activatable Fc domain binding molecules of the invention, the Fab light chain of the first antigen binding moiety and the Fab light chain of the second antigen binding moiety are fused to each other, optionally via a linker peptide. Depending on the configuration of the first and the second antigen binding moiety, the Fab light chain of the first antigen binding moiety may be fused at its C -terminus to the N-terminus of the Fab light chain of the second antigen binding moiety, or the Fab light chain of the second antigen binding moiety may be fused at its C-terminus to the N-terminus of the Fab light chain of the first antigen binding moiety. Fusion of the Fab light chains of the first and the second antigen binding moiety further reduces mispairing of unmatched Fab heavy and light chains, and also reduces the number of plasmids needed for expression of some of the protease-activatable Fc domain binding molecule of the invention.

In certain embodiments the protease-activatable Fc domain binding molecule comprises a polypeptide wherein the Fab light chain variable region of the first antigen binding moiety shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first antigen binding moiety (i.e. a the first antigen binding moiety comprises a crossover Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VL(i)-CHl(i)-CH2-CH3(-CH4)), and a polypeptide wherein a the Fab heavy chain of the second antigen binding moiety shares a carboxy-terminal peptide bond with an Fc domain subunit (VH(2)-CH1(2)-CH2-CH3(-CH4)). In some embodiments the protease- activatable Fc domain binding molecule further comprises a polypeptide wherein the Fab heavy chain variable region of the first antigen binding moiety shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first antigen binding moiety (VH(i)-CL(i)) and the Fab light chain polypeptide of the second antigen binding moiety (VL(2)-CL(2)). In certain embodiments the polypeptides are covalently linked, e.g., by a disulfide bond.

In alternative embodiments the protease-activatable Fc domain binding molecule comprises a polypeptide wherein the Fab heavy chain variable region of the first antigen binding moiety shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first antigen binding moiety (i.e. the first antigen binding moiety comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH(i)-CL(i)-CH2-CH3(-CH4)), and a polypeptide wherein the Fab heavy chain of the second antigen binding moiety shares a carboxy-terminal peptide bond with an Fc domain subunit (VH(2)-CH1(2)-CH2-CH3(-CH4)). In some embodiments the protease- activatable Fc domain binding molecule further comprises a polypeptide wherein the Fab light chain variable region of the first antigen binding moiety shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first antigen binding moiety (VL(i)-CHl(i)) and the Fab light chain polypeptide of the second antigen binding moiety (VL(2)-CL(2)). In certain embodiments the polypeptides are covalently linked, e.g., by a disulfide bond.

In some embodiments, the protease-activatable Fc domain binding molecule comprises a polypeptide wherein the Fab light chain variable region of the first antigen binding moiety shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first antigen binding moiety (i.e. the first antigen binding moiety comprises a crossover Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the second antigen binding moiety, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VL(i)-CHl(i)-VH(2)-CHl(2)-CH2-CH3(-CH4)). In other embodiments, the protease-activatable Fc domain binding molecule comprises a polypeptide wherein the Fab heavy chain variable region of the first antigen binding moiety shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first antigen binding moiety (i.e. the first antigen binding moiety comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the second antigen binding moiety, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH(i)-CL(i)-VH(2)-CHl(2)-CH2-CH3(-CH4)). In still other embodiments, the protease-activatable Fc domain binding molecule comprises a polypeptide wherein the Fab heavy chain of the second antigen binding moiety shares a carboxy- terminal peptide bond with the Fab light chain variable region of the first antigen binding moiety which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first antigen binding moiety (i.e. the first antigen binding moiety comprises a crossover Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH(2)-CHl(2)-VL(i)-CHl(i)-CH2-CH3(-CH4)). In other embodiments, the protease-activatable Fc domain binding molecule comprises a polypeptide wherein the Fab heavy chain of the second antigen binding moiety shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first antigen binding moiety which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first antigen binding moiety (i.e. the first antigen binding moiety comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH(2)-CHl(2)-VH(i)-CL(i)-CH2-CH3(-CH4)).

In some of these embodiments the protease-activatable Fc domain binding molecule further comprises a crossover Fab light chain polypeptide of the first antigen binding moiety, wherein the Fab heavy chain variable region of the first antigen binding moiety shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first antigen binding moiety (VH(i)-CL(i)), and the Fab light chain polypeptide of the second antigen binding moiety (VL(2)-CL(2)). In others of these embodiments the protease- activatable Fc domain binding molecule further comprises a crossover Fab light chain polypeptide, wherein the Fab light chain variable region of the first antigen binding moiety shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first antigen binding moiety (VL(i)-CHl(i)), and the Fab light chain polypeptide of the second antigen binding moiety (VL(2)-CL(2)). In still others of these embodiments the protease- activatable Fc domain binding molecule further comprises a polypeptide wherein the Fab light chain variable region of the first antigen binding moiety shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first antigen binding moiety which in turn shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of the second antigen binding moiety (VL(i)-CHl(i)-VL(2)-CL(2)}, a polypeptide wherein the Fab heavy chain variable region of the first antigen binding moiety shares a carboxy- terminal peptide bond with the Fab light chain constant region of the first antigen binding moiety which in turn shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of the second antigen binding moiety (VH(i)-CL(i)-VL(2)-CL(2)}, a polypeptide wherein the Fab light chain polypeptide of the second antigen binding moiety shares a carboxy-terminal peptide bond with the Fab light chain variable region of the first antigen binding moiety which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first antigen binding moiety (VL(2)-CL(2)-VL(i)-CHl(i)), or a polypeptide wherein the Fab light chain polypeptide of the second antigen binding moiety shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first antigen binding moiety which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first antigen binding moiety (VL(2)-CL(2)-VH(i)-CL(i)}.

The protease-activatable Fc domain binding molecule according to these embodiments may further comprise (i) an Fc domain subunit polypeptide (CH2-CH3(- CH4)), or (ii) a polypeptide wherein the Fab heavy chain of a third antigen binding moiety shares a carboxy-terminal peptide bond with an Fc domain subunit (VH(3)-CH1(3)-CH2- CH3(-CH4)) and the Fab light chain polypeptide of a third antigen binding moiety (VL(3)- CL(3)). In certain embodiments the polypeptides are covalently linked, e.g., by a disulfide bond.

According to any of the above embodiments, components of the protease -activatable Fc domain binding molecule (e.g., antigen binding moiety, Fc domain) may be fused directly or through various linkers, particularly peptide linkers comprising one or more amino acids, typically about 2-20 amino acids, that are described herein or are known in the art. Suitable, non-immunogenic peptide linkers include, for example, (G4S)_n, (SG4)n, (G4S)_n or G4(SG4)_n peptide linkers, wherein n is generally a number between 1 and 10, typically between 2 and 4.

Protease-activatable Fc domain binding molecules provided herein are multispecific antibodies, e.g., bispecific antibodies. “Multispecific antibodies” are monoclonal antibodies that have binding specificities for at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain aspects, the multispecific antibody has three or more binding specificities. In certain aspects, one of the binding specificities is for a variant CH2 domain comprising G329 according to EU numbering and the other specificity is for CD3. Multispecific antibodies may be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and “knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731,168, and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (see, e.g., WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992) and WO 2011/034605); using the common light chain technology for circumventing the light chain mis-pairing problem (see, e.g., WO 98/50431); using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991) Engineered antibodies with three or more antigen binding sites, including for example, “Octopus antibodies”, or DVD-Ig are also included herein (see, e.g., WO 2001/77342 and WO 2008/024715). Other examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO 2010/145792, and WO 2013/026831. The bispecific antibody or antigen binding fragment thereof also includes a “Dual Acting FAb” or “DAF” comprising an antigen binding site that binds to [[PRO]] as well as another different antigen, or two different epitopes of [[PRO]] (see, e.g., US 2008/0069820 and WO 2015/095539)

Multi-specific antibodies may also be provided in an asymmetric form with a domain crossover in one or more binding arms of the same antigen specificity, i.e. by exchanging the VH/VL domains (see e.g., WO 2009/080252 and WO 2015/150447), the CH1/CL domains (see e.g., WO 2009/080253) or the complete Fab arms (see e.g., WO 2009/080251, WO 2016/016299, also see Schaefer et al, PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20). In one aspect, the multispecific antibody comprises a cross-Fab fragment. The term “cross-Fab fragment” or “xFab fragment” or “crossover Fab fragment” refers to a Fab fragment, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. A cross-Fab fragment comprises a polypeptide chain composed of the light chain variable region (VL) and the heavy chain constant region 1 (CHI), and a polypeptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL). Asymmetrical Fab arms can also be engineered by introducing charged or non-charged amino acid mutations into domain interfaces to direct correct Fab pairing. See e.g., WO 2016/172485

Various further molecular formats for multispecific antibodies are known in the art and are included herein (see e.g., Spiess et al., Mol Immunol 67 (2015) 95-106).

Exemplary configuration are shown in figures 3 and 4. 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: 56, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 67, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 68, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 63. In one embodiment the protease-activatable Fc domain binding molecule comprises the polypeptide sequence of SEQ ID NO: 56, the polypeptide sequence of SEQ ID NO: 67, the polypeptide sequence of SEQ ID NO: 68, and the polypeptide sequence of SEQ ID NO: 63. This molecule is referred to as P1AJ1126 herein.

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: 56, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 64, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 67, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 63. In one embodiment the protease-activatable Fc domain binding molecule comprises the polypeptide sequence of SEQ ID NO: 56, the polypeptide sequence of SEQ ID NO: 64, the polypeptide sequence of SEQ ID NO: 67, and the polypeptide sequence of SEQ ID NO: 63. This molecule is referred to as P1AI5354 herein.

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: 56, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 68, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 63. In one embodiment the protease-activatable Fc domain binding molecule comprises the polypeptide sequence of SEQ ID NO: 56, the polypeptide sequence of SEQ ID NO: 68, and the polypeptide sequence of SEQ ID NO: 63. This molecule is referred to as Pl AI5356 herein.

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: 56, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 59, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 60, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 63. In one embodiment the protease-activatable Fc domain binding molecule comprises the polypeptide sequence of SEQ ID NO: 56, the polypeptide sequence of SEQ ID NO: 59, the polypeptide sequence of SEQ ID NO: 60, and the polypeptide sequence of SEQ ID NO: 63. This molecule is referred to as P1AJ2527 herein.

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: 56, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 59, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 64, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 63. In one embodiment the protease-activatable Fc domain binding molecule comprises the polypeptide sequence of SEQ ID NO: 56, the polypeptide sequence of SEQ ID NO: 59, the polypeptide sequence of SEQ ID NO: 63, and the polypeptide sequence of SEQ ID NO: 63. This molecule is referred to as P1AJ2528 herein.

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: 56, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 69, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 63. In one embodiment the protease-activatable Fc domain binding molecule comprises the polypeptide sequence of SEQ ID NO: 56, the polypeptide sequence of SEQ ID NO: 69, and the polypeptide sequence of SEQ ID NO: 63. This molecule is referred to as Pl AK0742 herein.

In one preferred 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: 48, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 56, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 70, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 71. In one preferred embodiment the protease-activatable Fc domain binding molecule comprises the polypeptide sequence of SEQ ID NO: 48, the polypeptide sequence of SEQ ID NO: 56, the polypeptide sequence of SEQ ID NO: 70, and the polypeptide sequence of SEQ ID NO: 71. This molecule is referred to as P1AK0750 herein.

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: 48, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 56, a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 59, and a polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 60. In one embodiment the protease-activatable Fc domain binding molecule comprises the polypeptide sequence of SEQ ID NO: 48, the polypeptide sequence of SEQ ID NO: 56, the polypeptide sequence of SEQ ID NO: 59, and the polypeptide sequence of SEQ ID NO: 60. This molecule is referred to as P1AF7732 herein.

Compositions

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.

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).

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.

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.

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.

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. 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.

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.

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.

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).

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. By way of illustration, the intervention may comprise administering a protease -activatable Fc domain binding molecule comprising the polypeptides according to SEQ ID NO: 56, SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 63 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. By way of illustration, the intervention may comprise administering a protease -activatable Fc domain binding molecule comprising the polypeptides according to SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 67, and SEQ ID NO: 63 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. By way of illustration, the intervention may comprise administering a protease-activatable Fc domain binding molecule comprising the polypeptides according to SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 68, and SEQ ID NO: 63 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. By way of illustration, the intervention may comprise administering a protease- activatable Fc domain binding molecule comprising the polypeptides according to SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 63 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. By way of illustration, the intervention may comprise administering a protease-activatable Fc domain binding molecule comprising the polypeptides according to SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 64, and SEQ ID NO: 63 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. By way of illustration, the intervention may comprise administering a protease-activatable Fc domain binding molecule comprising the polypeptides according to SEQ ID NO: 56, SEQ ID NO: 69, and SEQ ID NO: 63 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. By way of illustration, the intervention may comprise administering a protease-activatable Fc domain binding molecule comprising the polypeptides according to SEQ ID NO: 48, SEQ ID NO: 56, SEQ ID NO: 70, and SEQ ID NO: 71 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. By way of illustration, the intervention may comprise administering a protease-activatable Fc domain binding molecule comprising the polypeptides according to SEQ ID NO: 48, SEQ ID NO: 56, SEQ ID NO: 59, and SEQ ID NO: 60 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.

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).

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: 48, SEQ ID NO: 56, SEQ ID NO: 59, and SEQ ID NO: 60 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 10A). 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: 56, SEQ ID NO: 64, SEQ ID NO: 67, and SEQ ID NO: 63 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 10B). 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: 55, SEQ ID NO: 56, SEQ ID NO: 68, and SEQ ID NO: 63 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 10C). 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: 56, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 63 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 19B).

By way of further illustration, in the Examples of the present disclosure, a protease- activatable Fc domain binding molecule comprising the polypeptides of SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 63 is employed with an anti-CEACAM5 antibody comprising an Fc domain comprising P329G, such that the T cells are directed against CEACAM5 -expressing cells (see Figure 15 A). 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: 56, SEQ ID NO: 59, SEQ ID NO: 64, and SEQ ID NO: 63 is employed with an anti-CEACAM5 antibody comprising an Fc domain comprising P329G, such that the T cells are directed against CEACAM5 -expressing cells (see Figure 15B). 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: 55, SEQ ID NO: 56, SEQ ID NO: 69, and SEQ ID NO: 63 is employed with an anti- CEACAM5 antibody comprising an Fc domain comprising P329G, such that the T cells are directed against CEACAM5 -expressing cells (see Figure 15C). 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: 56, SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 63 is employed with an anti-CEACAM5 antibody comprising an Fc domain comprising P329G, such that the T cells are directed against CEACAM5 -expressing cells (see Figure 16A). 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: 56, SEQ ID NO: 64, SEQ ID NO: 67, and SEQ ID NO: 63 is employed with an anti- CEACAM5 antibody comprising an Fc domain comprising P329G, such that the T cells are directed against CEACAM5 -expressing cells (see Figure 16B). 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: 55, SEQ ID NO: 56, SEQ ID NO: 68, SEQ ID NO: 63 is employed with an anti-CEACAM5 antibody comprising an Fc domain comprising P329G, such that the T cells are directed against CECAM5 -expressing cells (see Figure 16C). 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: 48, SEQ ID NO: 56, SEQ ID NO: 70, and SEQ ID NO: 71 is employed with an anti-CEACAM5 antibody comprising an Fc domain comprising P329G, such that the T cells are directed against CEACAM5 -expressing cells (see Figure 17). 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: 48, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 60 is employed with an anti-CEACAM5 antibody comprising an Fc domain comprising P329G, such that the T cells are directed against CEACAM5 -expressing cells (see Figure 17).

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.

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 (e.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.

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.

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.

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.

IgG₄ antibodies 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 IgG₄ Fc domain, particularly a human IgG₄ Fc domain. In one embodiment the IgG₄ Fc 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 IgG4 Fc 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 IgG4 Fc 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.

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).

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).

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.

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)).

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.

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.

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.

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

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

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.

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. 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.

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.

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.

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.

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.

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:

(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

(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)-

Subjects

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).

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. 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.

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 (z.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.

Kits

The present disclosure also provides kits of parts.

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).

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).

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.

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.

EXEMPLARY SEQUENCES

BRIEF DESCRIPTION OF THE FIGURES

Embodiments and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying figures. FIGURE 1. Illustration of the mode of action of the standard, control anti-P329G x anti-CD3 TCB. 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 target antigen binding molecule is combined with an anti-P329G x anti-CD3 T cell bispecific antibody. This system of two molecules creates a functional anti -target T cell engager, and enables universal off-the-shelf platform for cancer therapy.

FIGURE 2. Illustration of the concept of the present invention - a protease- activatableanti-P329G x anti-CD3 TCB (pro-TCB). A target antigen binding molecule 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 target antigen binding molecule is combined with a protease-activatable anti-P329G x anti-CD3 T cell bispecific antibody, whereas the anti-P329G binder is masked by a P329G-containing CH2, attached by a linker that can be cleaved by a protease. In the environment with both target and T cells present, the pro-TCB remains masked, and does not form a functional T cell engager with the target antigen binding molecule. In the tumor microenvironment with abundance of proteases, the linker is cleaved, enabling the mask to dissociate from the anti-P329G binder, thus also enabling binding of the pro-TCB to the targeting adaptor antibody, in turn forming a functional T cell engager, which triggers antitumor T cell cytotoxicity.

FIGURE 3. Structure of an anti-P329G x anti-CD3 pro-TCB 2+1 with described antibody parts.

FIGURE 4. Structure, description and IDs of the antibodies described in this invention. The figures detail different formats of the anti-P329G (VH3xVLl) x anti-CD3 pro-TCBs: clone 22 CD3 binders and PQARK cleavable linkers (Figure 4A), clone 22 CD3 binders and PMAKK cleavable linkers (Figure 4B), clone 22 CD3 binders and non-cleavable linkers as controls (Figure 4C), P035.093 CD3 binders and PQARK, PMAKK, non-cleavable linkers (Figure 4D).

FIGURE 5. Structure, description and IDs of the antibodies used as non-protease activatable control anti-P329G (VH3xVLl) x anti-CD3 TCBs. The figures detail different formats of the TCBs: clone 22 CD3 binders (Figure 5A), and P035.093 CD3 binders (Figure 5B). FIGURE 6. SDS-PAGE analysis of the matriptase- or buffer-pretreated (pro-) TCBs showing matriptase-dependent removal of the masks from the masked pro-TCBs. The tested TCBs had either masks with cleavable linkers or masks with non-cleavable linkers or were unmasked controls, in TCB format 1 + 1 and 1 + 1 OA with clone 22 CD3 binder(Figure 6 A) or in TCB format 2+1 with P035.093 CD3 binder (Figure 6B). Depicted are results of an SDS- PAGE gel ran on reduced antibodies. Each band represents a chain from a tested antibody that can be matched to its molecular weight based on the placement relative to the molecular weight ladder. In the right text box, expected molecular weight value of each chain is written, calculated based on its amino acid sequence.

FIGURE 7. Principle of the Jurkat NF AT Luc2P (CD3) assay used for testing the anti- P329G x anti-CD3 pro-TCBs. Depicted are tumor cells and Jurkat NF AT Luc2P (CD3) reporter cells, treated with masked anti-P329G x anti-CD3 pro-TCBs with a tumor-targeted adaptor P329G LALA IgG. On the left, shown are pro-TCBs without protease cleavage. With masks present due to intact linkers, the pro-TCBs cannot bind the P329G epitope on the targetbound adaptor antibody, so CD3 crosslinking on the reporter cell does not take place, CD3 signaling does not take place and luminescence is not produced. On the right, shown are pro- TCBs after protease cleavage. With masks dissociated due to cleaved linkers, the pro-TCBs can bind the P329G epitope on the target-bound adaptor antibody. This leads to crosslinking of CD3 on the reporter cell and CD3 signaling, which in turn leads to luminescent signal.

FIGURE 8. Activation of Jurkat NF AT Luc2P (CD3) reporter cells by anti-FOLRl adaptor P329G IgGs combined with anti-P329G x anti-CD3 (pro-) TCBs on HeLa (FOLR1+) as target cells. The tested (pro-) TCBs had either masks with cleavable linkers or masks with non-cleavable linkers or were unmasked controls, and were tested with and without matriptase (protease) pre-treatment. Adaptor P329G IgGs and TCBs were used in a molar ratio adaptorTCB 2: 1. The antibody formats tested were 2+1 TCBs with P035.093 CD3 binders (Figure 8A), 1+1 TCBs with clone 22 CD3 binders (Figure 8B) and 1 + 1 OA TCBs with clone 22 CD3 binders (Figure 8C). Assessed by quantification of the intensity of CD3 downstream signaling using Jurkat-NFAT Luc2P (CD3) reporter assay. Depicted are technical average values from triplicates; error bars indicate SD.

FIGURE 9. Principle of the T cell activation and killing assay used for testing the anti- P329G x anti-CD3 pro-TCBs. Depicted are tumor cells and primary human T cells, treated with masked anti-P329G x anti-CD3 pro-TCBs with a tumor-targeted adaptor P329G LALA IgG. On the left, shown are pro-TCBs without protease cleavage. With masks present due to intact linkers, the pro-TCBs cannot bind the P329G epitope on the target -bound adaptor antibody, so CD3 crosslinking on the primary human T cell does not take place. T cells are not activated, they do not upregulate CD69 and CD25 proteins, do not produce cytokines, and do not lyse the tumor cells. On the right, shown are pro-TCBs after protease cleavage. With masks dissociated due to cleaved linkers, the pro-TCBs can bind the P329G epitope on the target-bound adaptor antibody. This leads to crosslinking of CD3 on the primary human T cell. T cells are then activated, upregulate CD69 and CD25 proteins, produce cytokines, and lyse the tumor cells, The dying tumor cells have their membrane disrupted, and release dead cell proteases into the supernatant of the assay.

FIGURE 10. CD4+ T cell activation assessed by CD25 expression induced by anti- FOLR1 adaptor P329G IgGs combined with anti-P329G x anti-CD3 (pro-) TCBs with HeLa (FOLR1+) as target cells, and human primary PBMCs from a healthy donor as effector cells. The tested (pro-) TCBs had either masks with cleavable linkers or masks with non-cleavable linkers or were unmasked controls, and were tested with and without matriptase (protease) pre-treatment. Adaptor P329G IgGs and TCBs were used in a molar ratio adaptorTCB 2: 1. Depicted are: 2+1 TCBs with P035.093 CD3 binders (Figure 10A), 1 + 1 TCBs with clone 22 CD3 binders (Figure 10B) and 1 + 1 OA TCBs with clone 22 CD3 binders (Figure 10C). Assessed after 48h by flow cytometry. Depicted are technical average values from triplicates; error bars indicate SD.

FIGURE 11. CD4+ T cell activation assessed by CD69 expression induced by anti- FOLR1 adaptor P329G IgGs combined with anti-P329G x anti-CD3 (pro-) TCBs with HeLa (FOLR1+) as target cells, and human primary PBMCs from a healthy donor as effector cells. The tested (pro-) TCBs had either masks with cleavable linkers or masks with non-cleavable linkers or were unmasked controls, and were tested with and without matriptase (protease) pre-treatment. Adaptor P329G IgGs and TCBs were used in a molar ratio adaptorTCB 2: 1. Depicted are: 2+1 TCBs with P035.093 CD3 binders (Figure 11 A), 1 + 1 TCBs with clone 22 CD3 binders (Figure 11B) and 1 + 1 OA TCBs with clone 22 CD3 binders (Figure 11C). Assessed after 48h by flow cytometry. Depicted are technical average values from triplicates; error bars indicate SD.

FIGURE 12. CD8+ T cell activation assessed by CD25 expression induced by anti- FOLR1 adaptor P329G IgGs combined with anti-P329G x anti-CD3 (pro-) TCBs with HeLa (FOLR1+) as target cells, and human primary PBMCs from a healthy donor as effector cells. The tested (pro-) TCBs had either masks with cleavable linkers or masks with non-cleavable linkers or were unmasked controls, and were tested with and without matriptase (protease) pre-treatment. Adaptor P329G IgGs and TCBs were used in a molar ratio adaptorTCB 2: 1. Depicted are: 2+1 TCBs with P035.093 CD3 binders (Figure 12A), 1 + 1 TCBs with clone 22 CD3 binders (Figure 12B) and 1 + 1 OA TCBs with clone 22 CD3 binders (Figure 12C). Assessed after 48h by flow cytometry. Depicted are technical average values from triplicates; error bars indicate SD.

FIGURE 13. CD8+ T cell activation assessed by CD69 expression induced by anti- FOLR1 adaptor P329G IgGs combined with anti-P329G x anti-CD3 (pro-) TCBs with HeLa (FOLR1+) as target cells, and human primary PBMCs from a healthy donor as effector cells. The tested (pro-) TCBs had either masks with cleavable linkers or masks with non-cleavable linkers or were unmasked controls, and were tested with and without matriptase (protease) pre-treatment. Adaptor P329G IgGs and TCBs were used in a molar ratio adaptorTCB 2: 1. Depicted are: 2+1 TCBs with P035.093 CD3 binders (Figure 13A), 1 + 1 TCBs with clone 22 CD3 binders (Figure 13B) and 1 + 1 OA TCBs with clone 22 CD3 binders (Figure 13C). Assessed after 48h by flow cytometry. Depicted are technical average values from triplicates; error bars indicate SD.

FIGURE 14. Tumor cell lysis induced by anti-FOLRl adaptor P329G IgGs combined with anti-P329G x anti-CD3 (pro-) TCBs with HeLa (FOLR1+) as target cells, and human primary PBMCs from a healthy donor as effector cells. The tested (pro-) TCBs had either masks with cleavable linkers or masks with non-cleavable linkers or were unmasked controls, and were tested with and without matriptase (protease) pre-treatment. Adaptor P329G IgGs and TCBs were used in a molar ratio adaptorTCB 2: 1. Depicted are: 2+1 TCBs with P035.093 CD3 binders (Figure 14A), 1 + 1 TCBs with clone 22 CD3 binders (Figure 14B) and 1+1 OA TCBs with clone 22 CD3 binders (Figure 14C). Assessed after 48h, by quantification of dead cell proteases released into the assay supernatants. Depicted are technical average values from triplicates; error bars indicate SD.

FIGURE 15. Activation of Jurkat NFAT Luc2P (CD3) reporter cells by anti- CEACAM5 adaptor P329G IgGs combined with anti-P329G x anti-CD3 (pro-) TCBs on MKN-45 (CEACAM5+) as target cells. The tested (pro-) TCBs had either masks with PMAKK cleavable linkers or masks with non-cleavable linkers or were unmasked controls, and were tested with and without matriptase (protease) pre-treatment. Adaptor P329G IgGs and TCBs were used in a molar ratio adaptorTCB 2: 1. The antibody formats tested were 2+1 TCBs with clone 22 CD3 binders (Figure 15 A), 1+1 TCBs with clone 22 CD3 binders (Figure 15B) and 1 + 1 OA TCBs with clone 22 CD3 binders (Figure 15C). Assessed by quantification of the intensity of CD3 downstream signaling using Jurkat-NFAT Luc2P (CD3) reporter assay. Depicted are technical average values from triplicates; error bars indicate SD.

FIGURE 16. Activation of Jurkat NFAT Luc2P (CD3) reporter cells by anti- CEACAM5 adaptor P329G IgGs combined with anti-P329G x anti-CD3 (pro-) TCBs on MKN-45 (CEACAM5+) as target cells. The tested (pro-) TCBs had either masks with PQARK cleavable linkers or masks with non-cleavable linkers or were unmasked controls, and were tested with and without matriptase (protease) pre -treatment. Adaptor P329G IgGs and TCBs were used in a molar ratio adaptorTCB 2: 1. The antibody formats tested were 2+1 TCBs with clone 22 CD3 binders (Figure 16A), 1+1 TCBs with clone 22 CD3 binders (Figure 16B) and 1 + 1 OA TCBs with clone 22 CD3 binders (Figure 16C). Assessed by quantification of the intensity of CD3 downstream signaling using Jurkat-NFAT Luc2P (CD3) reporter assay. Depicted are technical average values from triplicates; error bars indicate SD.

FIGURE 17. Activation of Jurkat NFAT Luc2P (CD3) reporter cells by anti- CEACAM5 adaptor P329G IgGs combined with anti-P329G x anti-CD3 (pro-) TCBs on MKN-45 (CEACAM5+) as target cells. The tested (pro-) TCBs had either masks with PMAKK cleavable linkers or masks with PQARK cleavable linkers or masks with non- cleavable linkers or were unmasked controls, and were tested with and without matriptase (protease) pre-treatment. Adaptor P329G IgGs and TCBs were used in a molar ratio adaptorTCB 2: 1. The antibody formats tested were 2+1 TCBs with P035.093 CD3 binders. Assessed by quantification of the intensity of CD3 downstream signaling using Jurkat-NFAT Luc2P (CD3) reporter assay. Depicted are technical average values from triplicates; error bars indicate SD.

FIGURE 18. Activation of Jurkat NFAT Luc2P (CD3) reporter cells by anti- CEACAM5 adaptor P329G IgGs combined with anti-P329G x anti-CD3 (pro-) TCBs, and compared to direct anti-CEACAM5 x anti-CD3 (pro-) TCBs, on MKN-45 (CEACAM5+) as target cells. The tested (pro-) TCBs had masks with PMAKK cleavable linkers or masks with non-cleavable linkers or were unmasked controls, and were tested with and without matriptase (protease) pre-treatment. Adaptor P329G IgGs and TCBs were used in a molar ratio adaptorTCB 2: 1. The anti-P329G x anti-CD3 (pro-) TCB antibody formats tested were 2+1 TCBs with P035.093 CD3 binders (Figure 18A) or with clone 22 CD3 binders (Figure 18B). The anti-CEACAM5 x anti-CD3 (pro-) TCB antibody formats tested were 2+1 TCBs with P035.093 CD3 binders (Figure 18A, 18B). Assessed by quantification of the intensity of CD3 downstream signaling using Jurkat-NFAT Luc2P (CD3) reporter assay. Depicted are technical average values from triplicates; error bars indicate SD.

FIGURE 19. Activation of Jurkat NF AT Luc2P (CD3) reporter cells by anti-FOLRl adaptor P329G IgGs combined with anti-P329G x anti-CD3 (pro-) TCBs, and compared to direct anti-FOLRl x anti-CD3 (pro-) TCBs, on HeLa (FOLR1+) as target cells. The tested (pro-) TCBs had masks with PMAKK cleavable linkers or masks with non-cleavable linkers or were unmasked controls, and were tested with and without matriptase (protease) pre- treatment. Adaptor P329G IgGs and TCBs were used in a molar ratio adaptorTCB 2: 1. The anti-P329G x anti-CD3 (pro-) TCB antibody formats tested were 2+1 TCBs with P035.093 CD3 binders (Figure 19A) or with clone 22 CD3 binders (Figure 19B). The anti-FOLRl x anti-CD3 (pro-) TCB antibody formats tested were 2+1 TCBs with P035.093 CD3 binders (Figure 18A, 18B). Assessed by quantification of the intensity of CD3 downstream signaling using Jurkat-NFAT Luc2P (CD3) reporter assay. Depicted are technical average values from triplicates; error bars indicate SD.

EXAMPLES

In the following Examples, the inventors describe the production and characterization of universal adaptor-based T cell bispecific antibodies. In particular, masked protease-activatable anti-P329G x anti-CD3 T cell bispecific (TCB) antibodies ("pro-TCBs") are evaluated, and are unexpectedly found to be provide an improved protease-dependent activation. For example, the pro-TCBs 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.

Example 1

Description o f the mode o f action and structure o f the molecules in the current invention

Principle of the non-masked anti-P329G x anti-CD3 TCB (universal adaptor-based T cell bispecific antibody) (Figure 1) and the current invention, the protease-activatable anti- P329G x anti-CD3 TCB (universal protease-activatable adaptor-based T cell bispecific antibody) (Figure 2) are shown. Detailed description of antibody structure of the protease - activatableanti-P329G x anti-CD3 TCB is shown in Figure 3.

Figure 4 depicts antibody pictograms, IDs and descriptions of example molecules of the current invention. Figure 4A shows anti-P329G (VH3xVLl) x anti-CD3 (clone 22) pro- TCBs in the formats 2+1, 1 + 1 and 1 + 1 OA, with PQARK cleavable linkers. Figure 4B shows anti-P329G (VH3xVLl) x anti-CD3 (clone 22) pro-TCBs in the formats 2+1, 1+1 and 1 + 1 OA, with PMAKK cleavable linkers. Figure 4C shows anti-P329G (VH3xVLl) x anti-CD3 (clone 22) pro-TCBs in the formats 2+1, 1 + 1 and 1 + 1 OA, with non-cleavable linkers. Figure 4D shows anti-P329G (VH3xVLl) x anti-CD3 (P035.093) pro-TCBs in the formats 2+1, 1 + 1 and 1+1 OA, with PQARK cleavable linkers.

Figure 5 depicts antibody pictograms, IDs and descriptions of unmasked molecules, which serve as control antibodies. Figure 5A shows anti-P329G (VH3xVLl) x anti-CD3 (clone 22) TCBs in the formats 2+1, 1+1 and 1 + 1 OA, without masks. Figure 5B shows anti- P329G (VH3xVLl) x anti-CD3 (P035.093) TCB in the format 2+1, without a mask.

Example 2

SDS-PAGE analysis of matriptase -pretreated anti-P329G x anti-CD 3 pro-TCBs - assessment o f PMAKK and PQARK linker cleavage dependent on matriptase pre -treatment

The capacity of matriptase to cleave linkers between the CH2 (P329G) masks and the P329G binders on the anti-P329G x anti-CD3 pro-TCBs was assessed with matriptase- pretreatment of antibodies and subsequent SDS-PAGE analysis of resulting separate antibody chains.

The tested antibodies (anti-P329G x anti-CD3 pro-TCBs with cleavable linker, non- cleavable linker and non-masked) were prepared in eppendorf tubes (Eppendorf, #030.121.589) in protein buffer (Roche, internal) at the concentration of 0.2 mg/ml, and then diluted 4x with matriptase buffer (Roche, internal) to reach the concentration of 0.05 mg/ml. Then, the antibodies were divided into matriptase pre-treatment and no pretreatment groups. Freshly thawed recombinant matriptase (Enzo Life Sciences, #ALX-201-246-U250) was added to the antibodies from the matriptase pre-treatment group for the final concentration of 5 nM. All tubes were closed and incubated at room temperature (22 °C) overnight. On the next day, antibodies were reduced via mixing 10 pl of the sample, 5 pl of the 4x NuPAGE 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. As the next step, 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). Subsequently, 10 pl of the protein ladder (Bio-Rad Laboratories, #1610375) or 15 pl of the reduced antibody samples were pipetted to their respective wells in the gel. Then, the electrophoresis was performed at a constant voltage of 200 V and for 40 to 50 minutes. After the finished run, the gel was gently removed from its cassette and stained by submersing it into a Coomassie blue-based staining solution (Expedeon, #ISB1L) in a plastic box, and placing the box on a plate rocker (IKA, #0002980203) switched to 50 rounds per minute and incubating for Ih. After Ih, the gel was rinsed 3x with tap water, and then destained via submersing it into tap water-filled plastic box, and placing the box on a plate rocker switched to 50 rounds per minute and incubating for Ih. After destaining, the gel was placed between two transparent plastic sheets and imaged with a scanner (HP, #G2710). The molecular weight of the bands was determined via comparison to the protein ladder bands, as well as predictions of molecular weight based on the amino acid structure of antibody chains.

Figure 6A depicts the results of the experiment performed with matriptase or buffer pre-treated anti-P329G x anti-CD3 pro-TCBs in the 1 + 1 and 1 + 1OA formats, in the masked PQARK cleavable versions, masked non-cleavable versions and unmasked versions, and with clone 22 CD3 binders. The table to the right of the gel shows predicted molecular weight of antibody chains based on their amino acid sequence. For the cleavable 1+1 pro-TCB and 1+1 OA pro-TCB, matriptase pre-treatment resulted in a full cleavage of the linker, and in turn separation of the CH2 mask from the antibody. This is evident by absent bands representing the masked chains (64 kDa for the 1 + 1 pro-TCB and 87 kDa for the 1 + 1 OA pro-TCB), which are present in the non-treated samples. For the non-cleavable 1+1 pro-TCB and 1+1 OA pro- TCB, matriptase did not induce the cleavage, as evident by the same bands being seen in matriptase-pretreated and non-treated samples. Additionally, matriptase also did not impact the unmasked TCBs, as evident by the same bands being seen in matriptase-pretreated and non-treated samples. Figure 6B depicts the results of the experiment performed with matriptase or buffer pre-treated anti-P329G x anti-CD3 pro-TCBs in the 2+1 format, in the masked PMAKK cleavable versions, masked non-cleavable versions and unmasked versions, and with P035.093 CD3 binders. The table to the right of the gel shows predicted molecular weight of antibody chains based on their amino acid sequence For the cleavable 2+1 pro- TCB, matriptase pre-treatment resulted in a full cleavage of the linker, and in turn separation of the CH2 mask from the antibody. This is evident by absent bands representing the masked chains (87 kDa for the long arm, and 64 kDa for the short arm), which are present in the non- treated samples. For the non-cleavable 2+1 pro-TCB, matriptase did not induce the cleavage, as evident by the same bands being seen in matriptase -pretreated and non-treated samples. Additionally, matriptase also did not impact the unmasked TCBs, as evident by the same bands being seen in matriptase-pretreated and non-treated samples.

This experiment provided evidence that matriptase was able to cleave both the PQARK and PMAKK linkers in the tested anti-P329G x anti-CD3 pro-TCBs, allowing the CH2 mask to dissociate from the rest of the antibody in each cleavable masked pro-TCB.

Example 3

Jurkat NF AT Luc2P reporter assay) induced by adaptor P329G IgGs with protease - activatable anti-P329G x anti-CD3 pro-TCBs on HeLa tumor cell line expressing a tumor- associated antigen FOLR1 - assessment of T cell activation capacity, matriptase dependency and masking efficiency

T cell activation capacity of the adaptor P329G IgGs with protease -activatable anti- P329G x anti-CD3 pro-TCBs was assessed with Jurkat NF AT Luc2P assay (GloResponse Jurkat NFAT-RE-luc2P, Promega, #CS176501) on FOLR1 -expressing tumor cell line HeLa. The principle of the assay is depicted on Figure 7.

As a tumor-targeting molecule (adaptor P329G IgG), anti-FOLRl P329G IgG was used and mixed with anti-P329G x anti-CD3 pro-TCB (cleavable, non-cleavable or unmasked, and in format 2+1, 1+1 or 1+1 OA) in the ratio of adaptorTCB 2: 1. The molecules were pretreated with matriptase or left untreated and titrated together. As negative control, unmasked anti-P329G x anti-CD3 TCB without an adaptor was used. The tested tumor cell line was HeLa (FOLR1+).

As a preparation for the assay, HeLa human tumor cells were harvested. Growth medium was removed from the cell culture flask and cells were washed once with phosphate - buffered saline (PBS, Gibco #10010023). After removing PBS, cells were trypsinised with TrypLE Express Enzyme (Gibco, #12605010). Cell count and viability was determined using a Countess Automated Cell Counter (Invitrogen, #C10227). 0.002 x 10⁶ cells/well (20 pl/well) were plated in a white flat bottom 384-well-plate (Corning, #353988) in assay medium (Advanced RPMI 1640, 2% 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 TCBs) 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 PBS) was added to the respective wells to reach the final concentration of 5.3 nM. The solutions were mixed, and the plate was centrifuged for 5 s at 200 g. The plate was sealed and incubated at room temperature (22 °C) overnight. On the next day, the solutions were diluted 5x with assay media (Advanced RPMI 1640, 2% FBS, 1% GlutaMAX), achieving the concentrations of 400 nM (for TCBs) and 800 nM (for adaptors). Subsequently, 10 pl of antibody dilutions were added to the assay plate. The plate was centrifuged for 5 s at 200 g. Additionally, Jurkat NF AT Luc2P reporter cells were harvested. The cells were counted and assessed for viability using the Countess device. The necessary amount was harvested by centrifugation for 5 min at 350 g. Next, 0.01 x 10⁶ cells/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 TCBs and 200 nM for adaptors). The plate was centrifuged for 5 s at 200 g. The assay components were incubated for 6h in a humidified atmosphere at 37°C and 5% CO2. After the incubation time, a luciferase substrate, ONE-Glo™ Luciferase Assay reagent (Promega, #E6120) was used according to the manufacturer’s protocol, allowing for a measurement of relative luminescence units (RLU). Readout was performed using a Tecan Spark 10M reader. The luminescent signal was acquired for 300 ms/well, and calculated to reflect RLU/s per well.

Figure 8 depicts matriptase-dependent T cell activation capacity of different anti- P329G x anti-CD3 pro-TCB formats when combined with the anti-FOLRl P329G IgG adaptor. Figure 8A shows matriptase-dependent and dose-dependent T cell activation induced by anti-FOLRl P329G IgGs combined with anti-P329G x anti-CD3 pro-TCBs in 2+1 format, with PMAKK linker and P035.093 CD3 binders. The 2+1 pro-TCB with a cleavable (PMAKK) linker is active when pre-treated with matriptase, but remains inactive without matriptase. Additionally, the matching masked non-cleavable 2+1 pro-TCB remains inactive in conditions both with and without matriptase pre -treatment. The unmasked control, anti- P329G x anti-CD3 TCB 2+1 is active when combined with the adaptor anti-FOLRl P329G IgG, but inactive when used without the adaptor, and this activity is matriptase independent in both cases. Figure 8B shows matriptase-dependent and dose-dependent T cell activation induced by anti-FOLRl P329G IgGs combined with anti-P329G x anti-CD3 pro-TCBs in 1+1 format, with PQARK linker and clone 22 CD3 binders. The 1 + 1 pro-TCB with a cleavable (PQARK) linker is active when pre-treated with matriptase, but remains inactive without matriptase. Additionally, the matching masked non-cleavable 1+1 pro-TCB remains inactive in conditions both with and without matriptase pre-treatment. The unmasked control, anti- P329G x anti-CD3 TCB 1 + 1 is active when combined with the adaptor anti-FOLRl P329G IgG, and this activity is matriptase independent. The unmasked control, anti-P329G x anti- CD3 TCB 1+1 is inactive without the adaptor in the matriptase pre-treated conditions; however, it shows non-specific activity in the untreated condition at the high concentration. This could be due to batch impurities, however still leaves the therapeutic window to the unmasked control, anti-P329G x anti-CD3 TCB 1 + 1 combined with the adaptor. Figure 8C shows matriptase-dependent and dose-dependent T cell activation induced by anti-FOLRl P329G IgGs combined with anti-P329G x anti-CD3 pro-TCBs in 1 + 1 one-armed (OA) format, with PQARK linker and clone 22 CD3 binders. The 1 + 1 OA pro-TCB with a cleavable (PQARK) linker is active when pre-treated with matriptase, but remains inactive without matriptase. Additionally, the matching masked non-cleavable 1+1 OA pro-TCB remains inactive in conditions both with and without matriptase pre-treatment. The unmasked control, anti-P329G x anti-CD3 TCB 1 + 1 OA is active when combined with the adaptor anti-FOLRl P329G IgG, and this activity is matriptase independent. The unmasked control, anti-P329G x anti-CD3 TCB 1+1 OA is inactive without the adaptor in the matriptase pre-treated conditions; however, it shows non-specific activity in the untreated condition at the high concentration. This could be due to batch impurities, however still leaves the therapeutic window to the unmasked control, anti-P329G x anti-CD3 TCB 1 + 1 combined with the adaptor.

In this experiment, the matriptase-dependency of TCB-induced T cell activation was assessed. The matriptase cutting the cleavable linkers allowed all cleavable masked pro-TCBs to gain T cell activating activity, while remaining inactive in the environment without matriptase. Additionally, the masking capacity of the CH2 (P329G) mask was shown, as evident by no T cell activation induced by adaptors combined with non-cleavable masked pro- TCBs with or without matriptase. Thus, both masking capacity and matriptase dependency was shown for the tested anti-P329G x anti-CD3 pro-TCBs. Example 4

Primary human T cell activation induced by adaptor P329G IgGs with protease - activatable anti-P329G x anti-CD 3 pro-TCBs on HeLa tumor cell line expressing a tumor - associated antigen F0LR1 - assessment of T cell activation capacity, matriptase dependency and masking efficiency

Primary human T cell activation capacity of the adaptor P329G IgGs with protease - activatable anti-P329G x anti-CD3 pro-TCBs was assessed with pan T cells from a heathy donor on FOLR1 -expressing tumor cell line HeLa. The principle of the assay is depicted on Figure 9.

As a preparation for the assay, HeLa human tumor cells were harvested. Growth medium was removed from the cell culture flask and cells were washed once with phosphate - buffered saline (PBS, Gibco #10010023). After removing PBS, cells were trypsinised with TrypLE Express Enzyme (Gibco, #12605010). Cell count and viability was determined using a Countess Automated Cell Counter (Invitrogen, #C10227). 0.015 x 10⁶ cells/well (30 pl/well) were plated in a white flat bottom 384-well-plate (Corning, #353988) in assay medium (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 TCBs) 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 PBS) was added to the respective wells to reach the final concentration of 5.3 nM. The solutions were mixed, and the plate was centrifuged for 5 s at 200 g. The plate was sealed and incubated at room temperature (22 °C) overnight. On the next day, the solutions were diluted 5x with assay media (RPMI 1640, 10% FBS, 1% GlutaMAX), achieving the concentrations of 400 nM (for TCBs) and 800 nM (for adaptors). Subsequently, 25 pl of antibody dilutions were added to the assay. The plate was centrifuged for 5 s at 200 g. Additionally, frozen human PBMCs obtained from a healthy donor were thawed and pan T cells were isolated using MACS technology (Miltenyi, #130-096-535). The cells were counted and assessed for viability using the Countess device. The necessary amount was harvested by centrifugation for 5 min at 350 g. 0.15 x 10⁶ cells/well (45 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 100 pl per well (achieving the concentrations of 100 nM for TCBs and 200 nM for adaptors). The plate was centrifuged for 5 s at 200 g. The assay components were incubated for 48h in a humidified atmosphere at 37°C and 5% CO2. After the incubation time, T cells were harvested and analyzed for CD25 and CD69 ad markers of T cell activation. In detail, the supernatant was removed from the plates, 60 pl of PBS was added to each well and cells were transferred to a 384-well V bottom plate (Eppendorf, #951040421) for FACS staining. The plates were centrifuged for 3 min at 600 g, supernatant was removed and cells were washed with 80 pl of PBS per well. The plate was again centrifuged for 3 min at 600 g and supernatant was removed. Subsequently, 25 pl of the PBS-based antibody mix containing Zombie Aqua™ Fixable Viability Kit (Biolegend, #423102), anti-huCD4 PerCP/Cy5.5 (Biolegend, #344608), anti-huCD8a BV711 (Biolegend, #301044), anti-huCD25 PE (Biolegend, #302606) and anti-huCD69 FITC (Biolegend, #310904) was added to each well. The plates were incubated for 30 min at 4°C. Afterwards, the cells were washed twice with FACS buffer (Roche, internal) and re-suspended in 65 pl of FACS buffer per well. The cells were acquired using a BD FACSymphony A3 flow cytometer. Raw data was analyzed using Flowjo vlO.8.1 software.

Figure 10 depicts matriptase dependent expression of CD25 T cell activation markers on CD4+ T cells, reflecting the human CD4+ T cell activation capacity of different anti- P329G x anti-CD3 pro-TCB formats. Figure 10A shows matriptase-dependent and dosedependent percentage of CD25+ out of CD4+ T cells, reflective of the T cell activation induced by anti-FOLRl P329G IgGs combined with anti-P329G x anti-CD3 pro-TCBs in 2+1 format, with PMAKK linker and P035.093 CD3 binders. Figure 10B shows matriptase- dependent and dose-dependent percentage of CD25+ out of CD4+ T cells, reflective of the T cell activation induced by anti-FOLRl P329G IgGs combined with anti-P329G x anti-CD3 pro-TCBs in 1 + 1 format, with PQARK linker and clone 22 CD3 binders. Figure 10C shows matriptase-dependent and dose-dependent percentage of CD25+ out of CD4+ T cells, reflective of the T cell activation induced by anti-FOLRl P329G IgGs combined with antiPS 29G x anti-CD3 pro-TCBs in 1 + 1 OA format, with PQARK linker and clone 22 CD3 binders.

The 2+1, 1 + 1 and 1 + 1 OA pro-TCBs with a cleavable (PMAKK or PQARK) linkers are active when pre-treated with matriptase, but remain mostly inactive without matriptase, showing small induction of CD25 expression at the highest concentrations and in the 2+1 with the PMAKK linker only. Additionally, the matching masked non-cleavable pro-TCBs remain inactive in conditions both with and without matriptase pre -treatment. The unmasked control, anti-P329G x anti-CD3 TCBs are active when combined with the adaptor anti-FOLRl P329G IgG and are matriptase independent. The unmasked controls, anti-P329G x anti-CD3 TCB in the 2+1 and 1+1 OA formats without the adaptor show residual non-specific activity, which, however, allow for a therapeutic window when compared with unmasked TCB used with the adaptor. The unmasked TCB in the 1+1 format without the adaptor does not have a significant therapeutic window.

Figure 11 depicts matriptase dependent expression of CD69 T cell activation markers on CD4+ T cells, reflecting the human CD4+ T cell activation capacity of different anti- P329G x anti-CD3 pro-TCB formats. Figure 11A shows matriptase-dependent and dosedependent percentage of CD69+ out of CD4+ T cells, reflective of the T cell activation induced by anti-FOLRl P329G IgGs combined with anti-P329G x anti-CD3 pro-TCBs in 2+1 format, with PMAKK linker and P035.093 CD3 binders. Figure 11B shows matriptase- dependent and dose-dependent percentage of CD69+ out of CD4+ T cells, reflective of the T cell activation induced by anti-FOLRl P329G IgGs combined with anti-P329G x anti-CD3 pro-TCBs in 1 + 1 format, with PQARK linker and clone 22 CD3 binders. Figure 11C shows matriptase-dependent and dose-dependent percentage of CD69+ out of CD4+ T cells, reflective of the T cell activation induced by anti-FOLRl P329G IgGs combined with antiPS 29G x anti-CD3 pro-TCBs in 1 + 1 OA format, with PQARK linker and clone 22 CD3 binders.

The 2+1 and 1+1 OA pro-TCBs with a cleavable (PMAKK or PQARK) linkers are active when pre-treated with matriptase, but remain mostly inactive without matriptase. The 1 + 1 OA pro-TCB is not inducing CD69 expression at this time point, which could be due to the transient nature of the induction of this marker. Additionally, the matching masked non- cleavable pro-TCBs remain inactive in conditions both with and without matriptase pre- treatment. The unmasked control, anti-P329G x anti-CD3 TCBs are active when combined with the adaptor anti-FOLRl P329G IgG and are matriptase independent. The unmasked controls, anti-P329G x anti-CD3 TCB in the 2+1 and 1 + 1 OA formats without the adaptor show residual non-specific activity, which, however, allow for a therapeutic window when compared with unmasked TCB used with the adaptor. The unmasked TCB in the 1 + 1 format without the adaptor does not have a significant therapeutic window. Figure 12 depicts matriptase dependent expression of CD25 T cell activation markers on CD8+ T cells, reflecting the human CD8+ T cell activation capacity of different anti- P329G x anti-CD3 pro-TCB formats. Figure 12A shows matriptase-dependent and dosedependent percentage of CD25+ out of CD8+ T cells, reflective of the T cell activation induced by anti-FOLRl P329G IgGs combined with anti-P329G x anti-CD3 pro-TCBs in 2+1 format, with PMAKK linker and P035.093 CD3 binders. Figure 12B shows matriptase- dependent and dose-dependent percentage of CD25+ out of CD8+ T cells, reflective of the T cell activation induced by anti-FOLRl P329G IgGs combined with anti-P329G x anti-CD3 pro-TCBs in 1 + 1 format, with PQARK linker and clone 22 CD3 binders. Figure 12C shows matriptase-dependent and dose-dependent percentage of CD25+ out of CD8+ T cells, reflective of the T cell activation induced by anti-FOLRl P329G IgGs combined with antiPS 29G x anti-CD3 pro-TCBs in 1 + 1 OA format, with PQARK linker and clone 22 CD3 binders.

The 2+1, 1 + 1 and 1 + 1 OA pro-TCBs with a cleavable (PMAKK or PQARK) linkers are active when pre-treated with matriptase, but remain mostly inactive without matriptase, showing small induction of CD25 expression at the highest concentrations and in the 2+1 with the PMAKK linker only. Additionally, the matching masked non-cleavable pro-TCBs remain inactive in conditions both with and without matriptase pre-treatment. The unmasked control, anti-P329G x anti-CD3 TCBs are active when combined with the adaptor anti-FOLRl P329G IgG and are matriptase independent. The unmasked controls, anti-P329G x anti-CD3 TCB in the 2+1 and 1+1 OA formats without the adaptor show residual non-specific activity, which, however, allow for a therapeutic window when compared with unmasked TCB used with the adaptor. The unmasked TCB in the 1+1 format without the adaptor does not have a significant therapeutic window.

Figure 13 depicts matriptase dependent expression of CD69 T cell activation markers on CD8+ T cells, reflecting the human CD8+ T cell activation capacity of different anti- P329G x anti-CD3 pro-TCB formats. Figure 13A shows matriptase-dependent and dosedependent percentage of CD69+ out of CD8+ T cells, reflective of the T cell activation induced by anti-FOLRl P329G IgGs combined with anti-P329G x anti-CD3 pro-TCBs in 2+1 format, with PMAKK linker and P035.093 CD3 binders. Figure 13B shows matriptase- dependent and dose-dependent percentage of CD69+ out of CD8+ T cells, reflective of the T cell activation induced by anti-FOLRl P329G IgGs combined with anti-P329G x anti-CD3 pro-TCBs in 1 + 1 format, with PQARK linker and clone 22 CD3 binders. Figure 13C shows matriptase-dependent and dose-dependent percentage of CD69+ out of CD8+ T cells, reflective of the T cell activation induced by anti-FOLRl P329G IgGs combined with antiPS 29G x anti-CD3 pro-TCBs in 1 + 1 OA format, with PQARK linker and clone 22 CD 3 binders.

The 2+1 and 1+1 OA pro-TCBs with a cleavable (PMAKK or PQARK) linkers are active when pre-treated with matriptase, but remain mostly inactive without matriptase. The 1 + 1 OA pro-TCB is not inducing CD69 expression at this time point, which could be due to the transient nature of the induction of this marker. Additionally, the matching masked non- cleavable pro-TCBs remain inactive in conditions both with and without matriptase pre- treatment. The unmasked control, anti-P329G x anti-CD3 TCBs are active when combined with the adaptor anti-FOLRl P329G IgG and are matriptase independent. The unmasked controls, anti-P329G x anti-CD3 TCB in the 2+1 and 1 + 1 OA formats without the adaptor show residual non-specific activity, which, however, allow for a therapeutic window when compared with unmasked TCB used with the adaptor. The unmasked TCB in the 1 + 1 format without the adaptor does not have a significant therapeutic window.

In this experiment, the matriptase-dependency of TCB-induced T cell activation of primary human T cells was assessed. The matriptase cutting the cleavable linkers allowed all cleavable masked pro-TCBs to gain T cell activating activity, while remaining inactive in the environment without matriptase. Additionally, the masking capacity of the CH2 (P329G) mask was shown, as evident by no T cell activation induced by adaptors combined with non- cleavable masked pro-TCBs with or without matriptase. Thus, both masking capacity and matriptase dependency was shown for the tested anti-P329G x anti-CD3 pro-TCBs. The pro- TCB in 2+1 format bearing the PMAKK linker showed residual activity without matriptase, which could be due to HeLa cells expressing proteases or a general non-specificity. Additionally, the pro-TCB format 1+1 was shown to be insufficiently active as compared to other formats in this readout, and the pro-TCB 2+1 format was shown to have the best activity, and was chosen as the format form the lead molecules.

Example 5

Primary T cell-mediated tumor cell lysis induced by adaptor P329G IgGs with protease-activatable anti-P329G x anti-CD3 pro-TCBs on HeLa tumor cell line expressing a tumor-associated antigen F0LR1 - assessment o f T cell activation capacity, matriptase dependency and masking efficiency Capacity of inducing of the tumor cell lysis by the adaptor P329G IgGs with protease- activatable anti-P329G x anti-CD3 pro-TCBs was assessed with pan T cells from a heathy donor on FOLR1 -expressing tumor cell line HeLa. The principle of the assay is depicted on Figure 9.

The assay was performed as described above (Example 4, Figures 10-13). After the 48h incubation, the plate was centrifuged for 3 min at 600 g, and 20 pl/well of the supernatant was transferred to a white flat bottom 384 -well-plate (Corning, #353988). Then, 7 pl/well of the Cytotox-Glo reagent (Promega, #G9291) was added to the supernatants and mixed. 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 300 ms/well, and calculated to reflect RLU/s per well.

Figure 14 depicts matriptase dependent T cell-mediated tumor cell lysis, induced different anti-P329G x anti-CD3 pro-TCB formats. Figure 14A shows matriptase-dependent and dose-dependent tumor cell lysis, induced by anti-FOLRl P329G IgGs combined with anti-P329G x anti-CD3 pro-TCBs in 2+1 format, with PMAKK linker and P035.093 CD3 binders. Figure 12B shows matriptase-dependent and dose-dependent tumor cell lysis induced by anti-FOLRl P329G IgGs combined with anti-P329G x anti-CD3 pro-TCBs in 1+1 format, with PQARK linker and clone 22 CD3 binders. Figure 12C shows matriptase- dependent and dose-dependent tumor cell lysis induced by anti-FOLRl P329G IgGs combined with anti-P329G x anti-CD3 pro-TCBs in 1 + 1 OA format, with PQARK linker and clone 22 CD3 binders.

The 2+1, 1 + 1 and 1 + 1 OA pro-TCBs with a cleavable (PMAKK or PQARK) linkers are active when pre-treated with matriptase, but remain mostly inactive without matriptase, showing small induction of tumor cell lysis at the highest concentrations only. Additionally, the matching masked non-cleavable pro-TCBs remain inactive in conditions both with and without matriptase pre-treatment. The unmasked control, anti-P329G x anti-CD3 TCBs are active when combined with the adaptor anti-FOLRl P329G IgG and are matriptase independent. The unmasked controls, anti-P329G x anti-CD3 TCB in the 2+1 and 1 + 1 OA formats without the adaptor show residual non-specific activity, which, however, allow for a therapeutic window when compared with unmasked TCB used with the adaptor. The unmasked TCB in the 1+1 format without the adaptor does not have a significant therapeutic window.

In this experiment, the matriptase-dependency of TCB-induced T cell mediated tumor cell lysis was assessed. The matriptase cutting the cleavable linkers allowed all cleavable masked pro-TCBs to gain T cell activating activity, while remaining inactive in the environment without matriptase. Additionally, the masking capacity of the CH2 (P329G) mask was shown, as evident by no T cell activation induced by adaptors combined with non- cleavable masked pro-TCBs with or without matriptase. Thus, both masking capacity and matriptase dependency was shown for the tested anti-P329G x anti-CD3 pro-TCBs. The cleavable pro-TCBs showed residual activity without matriptase, which could be due to HeLa cells expressing proteases or a general non-specificity. The pro-TCB format 1 + 1 was shown to be insufficiently active as compared to other formats in this readout, and the pro-TCB 2+1 format was shown to have the best activity, and was confirmed it as the format form the lead molecules. The results from the tumor cell lysis experiments match the T cell activation experiments.

Example 6

Jurkat NF A T Luc2P reporter assay (T cell activation assay) induced by adaptor P329G IgGs with protease-activatable anti-P329G x anti-CD3 pro-TCBs (PMAKK cleavable linkers, clone 22 CD 3 binders) on MKN-45 tumor cell line expressing a tumor- associated antigen CEACAM5 - assessment of activity on another target

T cell activation capacity of the adaptor P329G IgGs with protease -activatable anti- P329G x anti-CD3 pro-TCBs on a second target and with PMAKK linker was assessed with Jurkat NF AT Luc2P assay (GloResponse Jurkat NFAT-RE-luc2P, Promega, #CS176501) on CEACAM5-expressing tumor cell line MKN-45. The principle of the assay is depicted on Figure 7.

As a tumor-targeting molecule (adaptor P329G IgG), anti-CEACAM5 P329G IgG was used and mixed with anti-P329G x anti-CD3 pro-TCB (cleavable - PMAKK, non-cleavable or unmasked, and in format 2+1, 1 + 1 or 1 + 1 OA) in the ratio of adaptorTCB 2: 1. The molecules were pre-treated with matriptase or left untreated and titrated together. As negative control, unmasked anti-P329G x anti-CD3 TCB without an adaptor was used, as well as anti- CEACAM5 P329G IgG without a TCB. The tested tumor cell line was MKN-45 (CEACAM5+).

The assay was performed as described above (Example 3, Figure 8).

Figure 15 depicts matriptase-dependent T cell activation capacity of different anti- P329G x anti-CD3 pro-TCB formats (with PMAKK cleavable linkers and clone 22 CD3 binders) when combined with anti-CEACAM5 P329G IgG adaptor. Figure 15A shows matriptase-dependent and dose-dependent T cell activation induced by anti-CEACAM5 P329G IgGs combined with anti-P329G x anti-CD3 pro-TCBs in 2+1 format, with PMAKK linker and clone 22 CD3 binders. The 2+1 pro-TCB with a cleavable (PMAKK) linker is active when pre-treated with matriptase, but remains inactive without matriptase. Additionally, the matching masked non-cleavable 2+1 pro-TCB remains inactive in conditions both with and without matriptase pre -treatment. The unmasked control, anti- P329G x anti-CD3 TCB 2+1 is active when combined with the adaptor anti-FOLRl P329G IgG, but both are inactive when used separately, and this activity is matriptase independent in both cases. Figure 15B shows matriptase-dependent and dose-dependent T cell activation induced by anti-CEACAM5 P329G IgGs combined with anti-P329G x anti-CD3 pro-TCBs in 1 + 1 format, with PMAKK linker and clone 22 CD3 binders. The 1 + 1 pro-TCB with a cleavable (PMAKK) linker is active when pre-treated with matriptase, but remains inactive without matriptase. Additionally, the matching masked non-cleavable 1+1 pro-TCB remains inactive in conditions both with and without matriptase pre -treatment. The unmasked control, anti-P329G x anti-CD3 TCB 1 + 1 is active when combined with the adaptor anti-FOLRl P329G IgG, but both are inactive when used separately, and this activity is matriptase independent in both cases. Figure 15C shows matriptase-dependent and dose-dependent T cell activation induced by anti-CEACAM5 P329G IgGs combined with anti-P329G x anti- CD3 pro-TCBs in 1 + 1 OA format, with PMAKK linker and clone 22 CD3 binders. The 1 + 1 OA pro-TCB with a cleavable (PMAKK) linker is active when pre -treated with matriptase, but remains inactive without matriptase. Additionally, the matching masked non-cleavable 1 + 1 OA pro-TCB remains inactive in conditions both with and without matriptase pre- treatment. The unmasked control, anti-P329G x anti-CD3 TCB 1 + 1 OA is active when combined with the adaptor anti-FOLRl P329G IgG, but both are inactive when used separately, and this activity is matriptase independent in both cases. In this experiment, the matriptase-dependency of TCB-induced T cell activation on another target than FOLR1 - CEACAM5 - was assessed, using the PMAKK linker. The matriptase cutting the cleavable linkers allowed all cleavable masked pro-TCBs to gain T cell activating activity, while remaining inactive in the environment without matriptase. Additionally, the masking capacity of the CH2 (P329G) mask was shown, as evident by no T cell activation induced by adaptors combined with non-cleavable masked pro-TCBs with or without matriptase. Thus, both masking capacity and matriptase dependency was shown for the tested anti-P329G x anti-CD3 pro-TCBs. This activity shown on an additional target shows the universality of the pro-TCBs, in terms of targeting and antigen of choice by changing the adaptor P329G IgG but using the same pro-TCB, and retaining the matriptase- dependent T cell engaging activity.

Example 7

Jurkat NF AT Luc2P reporter assay (T cell activation assay) induced by adaptor P329G IgGs with protease-activatable anti-P329G x anti-CD 3 pro-TCBs (PQARK cleavable linkers, clone 22 CD 3 binders) on MKN-45 tumor cell line expressing a tumor - associated antigen CEACAM5.

T cell activation capacity of the adaptor P329G IgGs with protease-activatable anti- P329G x anti-CD3 pro-TCBs on a second target and with PQARK linker was assessed with Jurkat NF AT Luc2P assay (GloResponse Jurkat NFAT-RE-luc2P, Promega, #CS176501) on CEACAM5-expressing tumor cell line MKN-45. The principle of the assay is depicted on Figure 7.

As a tumor-targeting molecule (adaptor P329G IgG), anti-CEACAM5 P329G IgG was used and mixed with anti-P329G x anti-CD3 pro-TCB (cleavable - PQARK, non-cleavable or unmasked, and in format 2+1, 1+1 or 1+1 OA) in the ratio of adaptorTCB 2: 1. The molecules were pre-treated with matriptase or left untreated and titrated together. As negative control, unmasked anti-P329G x anti-CD3 TCB without an adaptor was used, as well as anti- CEACAM5 P329G IgG without a TCB. The tested tumor cell line was MKN-45 (CEACAM5+).

The assay was performed as described above (Example 3, Figure 8). Figure 16 depicts matriptase-dependent T cell activation capacity of different anti- P329G x anti-CD3 pro-TCB formats (with PQARK cleavable linkers and clone 22 CD3 binders) when combined with anti-CEACAM5 P329G IgG adaptor. Figure 16A shows matriptase-dependent and dose-dependent T cell activation induced by anti-CEACAM5 P329G IgGs combined with anti-P329G x anti-CD3 pro-TCBs in 2+1 format, with PQARK linker and clone 22 CD3 binders. The 2+1 pro-TCB with a cleavable (PQARK) linker is active when pre-treated with matriptase, but remains inactive without matriptase. Additionally, the matching masked non-cleavable 2+1 pro-TCB remains inactive in conditions both with and without matriptase pre-treatment. The unmasked control, anti-P329G x anti-CD3 TCB 2+1 is active when combined with the adaptor anti-FOLRl P329G IgG, but both are inactive when used separately, and this activity is matriptase independent in both cases. Figure 16B shows matriptase-dependent and dose-dependent T cell activation induced by anti-CEACAM5 P329G IgGs combined with anti-P329G x anti-CD3 pro-TCBs in 1 + 1 format, with PQARK linker and clone 22 CD3 binders. The 1 + 1 pro-TCB with a cleavable (PQARK) linker is active when pre-treated with matriptase, but remains inactive without matriptase. Additionally, the matching masked non-cleavable 1 + 1 pro-TCB remains inactive in conditions both with and without matriptase pre-treatment. The unmasked control, anti-P329G x anti-CD3 TCB 1 + 1 is active when combined with the adaptor anti-FOLRl P329G IgG, but both are inactive when used separately, and this activity is matriptase independent in both cases. Figure 16C shows matriptase-dependent and dose-dependent T cell activation induced by anti-CEACAM5 P329G IgGs combined with anti-P329G x anti-CD3 pro-TCBs in 1 + 1 OA format, with PQARK linker and clone 22 CD3 binders. The 1+1 OA pro-TCB with a cleavable (PQARK) linker is active when pre-treated with matriptase, but remains inactive without matriptase. Additionally, the matching masked non-cleavable 1 + 1 OA pro-TCB remains inactive in conditions both with and without matriptase pre-treatment. The unmasked control, anti- P329G x anti-CD3 TCB 1 + 1 OA is active when combined with the adaptor anti-FOLRl P329G IgG, but both are inactive when used separately, and this activity is matriptase independent in both cases.

In this experiment, the matriptase-dependency of TCB-induced T cell activation on another target than FOLR1 - CEACAM5 - was assessed, using the PQARK linker. The matriptase cutting the cleavable linkers allowed all cleavable masked pro-TCBs to gain T cell activating activity, while remaining inactive in the environment without matriptase. Additionally, the masking capacity of the CH2 (P329G) mask was shown, as evident by no T cell activation induced by adaptors combined with non-cleavable masked pro-TCBs with or without matriptase. Thus, both masking capacity and matriptase dependency was shown for the tested anti-P329G x anti-CD3 pro-TCBs. This activity shown on an additional target further confirm the universality of the pro-TCBs, in terms of targeting and antigen of choice by changing the adaptor P329GIgG but using the same pro-TCB, and retaining the matriptase- dependent T cell engaging activity. Additionally, when compared to the matching experiments with PMAKK linkers (Example 6, Figure 15), these results show universality of the linker on the cleavable masked pro-TCB, in terms of adjusting the linker sequence based on the desired biological activity, and retaining the masking capacity, and in the case of PMAKK and PQARK - retaining matriptase dependency.

Example 8

Jurkat NF A T Luc2P reporter assay (T cell activation assay) induced by adaptor P329G IgGs with protease-activatableanti-P329G x anti-CD3 pro-TCBs (PMAKK and PQARK cleavable linker, P035.093 CD 3 binder) on MKN-45 tumor cell line expressing a tumor-associated antigen CEACAM5 - comparison of 2 linkers in one experiment

T cell activation capacity of the adaptor P329G IgGs with protease -activatable anti- P329G x anti-CD3 pro-TCBs with either PMAKK or PQARK linker was assessed with Jurkat NFAT Luc2P assay (GloResponse Jurkat NFAT-RE-luc2P, Promega, #CS176501) on CEACAM5-expressing tumor cell line MKN-45. The principle of the assay is depicted on Figure 7.

As a tumor-targeting molecule (adaptor P329G IgG), anti-CEACAM5 P329G IgG was used and mixed with anti-P329G x anti-CD3 pro-TCB (cleavable - PQARK or PMAKK, non- cleavable or unmasked, and in format 2+1, with P035.093 CD3 binder) in the ratio of adaptorTCB 2: 1. The molecules were pre-treated with matriptase or left untreated and titrated together. As negative control, unmasked anti-P329G x anti-CD3 TCB without an adaptor was used, as well as anti-CEACAM5 P329G IgG without a TCB. The tested tumor cell line was MKN-45 (CEACAM5+).

The assay was performed as described above (Example 3, Figure 8).

Figure 17 depicts matriptase-dependent T cell activation capacity of different anti- P329G x anti-CD3 pro-TCB formats (with PQARK or PMAKK cleavable linkers and P035.093 CD3 binders) when combined with anti-CEACAM5 P329G IgG adaptor. Both 2+1 pro-TCBs with a cleavable linker: PMAKK and PQARK are active when pre-treated with matriptase, but remain inactive without matriptase. Both show comparable activity. Additionally, the matching masked non-cleavable 2+1 pro-TCB remains inactive in conditions both with and without matriptase pre -treatment with only residual activity at the highest concentrations. The unmasked control, anti-P329G x anti-CD3 TCB 2+1 is active when combined with the adaptor anti-FOLRl P329G IgG, but both are inactive when used separately, and this activity is matriptase independent in both cases.

In this experiment, the matriptase-dependency of TCB-induced T cell activation was assessed for 2 cleavable linkers - PQARK and PMAKK. The matriptase cutting the cleavable linkers allowed both cleavable masked pro-TCBs to gain T cell activating activity to a comparable extent, while remaining inactive in the environment without matriptase. Additionally, the masking capacity of the CH2 (P329G) mask was shown, as evident by no T cell activation induced by adaptors combined with non-cleavable masked pro-TCBs with or without matriptase. Thus, both masking capacity and matriptase dependency was shown for the tested anti-P329G x anti-CD3 pro-TCBs. These results confirm universality of the linker choice on the cleavable masked pro-TCB that retains masking capacity, which was discussed in Example 7, Figure 16. As an additional conclusion, when compared with results from Example 7, Figure 16, the anti-P329G x anti-CD3 non-cleavable masked pro-TCB 2+1, the P035.093 CD3 binder shows more non-specific induction of T cell activation than the clone 22 CD3 binder. Due to these results, clone 22 was deemed less non-specific, and was chosen as the binder for the lead molecules.

Example 9

Jurkat NF AT Luc2P reporter assay (T cell activation assay) induced by adaptor anti- CEACAM5 or anti-FOLRl P329G IgGs with protease-activatable anti-P329G x anti-CD3 pro-TCBs, assessed in contrast to direct anti-CEACAM5 x anti-CD3 pro-TCBs or anti- FOLRl x anti-CD3 pro-TCBs - comparison of protease -dependency and masking capacity.

T cell activation capacity of the antigen-targeting adaptor P329G IgGs with protease- activatable anti-P329G x anti-CD3 pro-TCBs was compared with direct antigen-targeting protease-activatable pro-TCBs, via assessment with Jurkat NF AT Luc2P assay (GloResponse Jurkat NFAT-RE-luc2P, Promega, #CS176501) on CE AC AM5 -expressing tumor cell line MKN-45 or FOLR1 -expressing cell line HeLa. The principle of the assay is depicted on Figure 7.

In the first part, as a tumor-targeting molecule (adaptor P329G IgG), anti-CEACAM5 P329G IgG was used and mixed with anti-P329G x anti-CD3 pro-TCB (cleavable - PMAKK, non-cleavable or unmasked, and in format 2+1, with P035.093 or clone 22 CD3 binder) in the ratio of adaptorTCB 2: 1. As a control, a direct anti-CEACAM5 x anti-CD3 pro-TCB was used (cleavable - PMAKK, non-cleavable or unmasked, with anti-idiotypic CD3 binder mask 4.24.72, in format 2+1, with P035.093 CD3 binder). The molecules were pre-treated with matriptase or left untreated and titrated. As negative control, anti-P329G x anti-CD3 (pro-) TCBs without an adaptor were used, as well as anti-CEACAM5 P329G IgG without any TCB. The tested tumor cell line was MKN-45 (CEACAM5+).

In the second part, as a tumor-targeting molecule (adaptor P329G IgG), anti-FOLRl P329G IgG was used and mixed with anti-P329G x anti-CD3 pro-TCB (cleavable - PMAKK, non-cleavable or unmasked, and in format 2+1, with P035.093 or clone 22 CD3 binder) in the ratio of adaptorTCB 2: 1. As a control, a direct anti-FOLRl x anti-CD3 pro-TCB was used (cleavable - PMAKK, non-cleavable or unmasked, with anti-idiotypic CD3 binder mask 4.24.72, in format 2+1, with P035.093 CD3 binder). The molecules were pre-treated with matriptase or left untreated and titrated. As negative control, anti-P329G x anti-CD3 (pro-) TCBs without an adaptor were used, as well as anti-FOLRl P329G IgG without any TCB. The tested tumor cell line was HeLa (FOLR1+).

The assay was performed as described above (Example 3, Figure 8).

Figure 18 depicts matriptase-dependent T cell activation capacity of control direct anti-CEACAM5 x anti-CD3 pro-TCBs and compared to capacity of anti-CEACAM5 P329G IgG adaptor mixed with different anti-P329G x anti-CD3 pro-TCB formats, with PMAKK cleavable linkers and P035.093 CD3 binders (Figure 18A) or clone 22 CD3 binders (Figure 18B)

Figure 19 depicts matriptase-dependent T cell activation capacity of control direct anti-FOLRl x anti-CD3 pro-TCBs and compared to capacity of anti-FOLRl P329G IgG adaptor mixed with different anti-P329G x anti-CD3 pro-TCB formats, with PMAKK cleavable linkers and P035.093 CD3 binders (Figure 19A) or clone 22 CD3 binders (Figure 19B) For both CD3 versions of the anti-P329G x anti-CD3 pro-TCBs, and for both targets, the cleavable molecules are active when pretreated with matriptase, and display low activity without matriptase pretreatment, providing a therapeutic window of matriptase -dependency. Moreover, the anti-P329G x anti-CD3 pro-TCBs in the non-cleavable format show low to no activity, indicating proper masking of the binders. In contrast, both control molecules, cleavable direct anti-CEACAM5 x anti-CD3 pro-TCBs and cleavable direct anti-FOLRl x anti-CD3 pro-TCBs, display high dose-dependent activity without matriptase, providing evidence for lower matriptase-dependency than the ant-P329G x anti-CD3 pro-TCBs. Additionally, the direct molecules in the non-cleavable format show high dose-dependent activity both with and without matriptase pretreatment, indicating insufficient masking. This insufficient masking may provide an explanation of the activity of the molecule in the cleavable format without matriptase.

In this experiment, the masking capacity of anti-P329G x anti-CD3 pro-TCBs via the CH2 (P329G) mask was superior to the masking of direct controls, the anti-CEACAM5 x anti-CD3 pro-TCBs and anti-FOLRl x anti-CD3 pro-TCBs via the anti-idiotypic CD3 binder mask 4.24.72. Additionally, the cleavable formats of the direct pro-TCBs without matriptase had higher dose-dependent activity than anti-P329G x anti-CD3 pro-TCBs without matriptase, indicating lower matriptase-dependency. Altogether, these results provide evidence of an improved masking capacity and a wider therapeutic window of the anti-P329G x anti-CD3 pro-TCBs in comparison to the controls.

Claims

CLAIMS What is claimed is:

1. A protease-activatable Fc domain binding molecule comprising

(a) a first antigen binding moiety capable of binding to CD3;

(b) a second antigen binding moiety capable of binding to a variant CH2 domain comprising G329 according to EU numbering, wherein the second antigen binding moiety is not capable of binding to a reference CH2 domain comprising P329 according to EU numbering, wherein the second antigen binding moiety is an antibody or fragment thereof; and

(c) a masking moiety covalently attached to the protease-activatable Fc domain binding molecule through a protease-cleavable linker, wherein the masking moiety comprises the variant CH2 domain comprising G329 according to EU numbering, wherein the second antigen binding moiety binds to the variant CH2 domain, wherein the variant CH2 domain reversibly conceals the second antigen binding moiety.

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

3. The protease-activatable Fc domain binding molecule of claim 1 or 2, wherein the first antigen-binding moiety comprises:

(i) a VH region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO: 24;

HC-CDR2 having the amino acid sequence of SEQ ID NO: 25; and

HC-CDR3 having the amino acid sequence of SEQ ID NO: 26; and

(ii) a VL region incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO: 5;

LC-CDR2 having the amino acid sequence of SEQ ID NO: 6; and

LC-CDR3 having the amino acid sequence of SEQ ID NO: 7; or

(i) a VH region incorporating the following CDRs:

HC-CDR1 having the amino acid sequence of SEQ ID NO: 24;

HC-CDR2 having the amino acid sequence of SEQ ID NO: 25; and

HC-CDR3 having the amino acid sequence of SEQ ID NO: 28; and

(ii) a VL region incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO: 5;

LC-CDR2 having the amino acid sequence of SEQ ID NO: 6; and

LC-CDR3 having the amino acid sequence of SEQ ID NO: 7.

4. The protease-activatable Fc domain binding molecule of any one of claims 1 to 3, wherein the first antigen-binding moiety comprises:

5. The protease-activatable Fc domain binding molecule of any one of claims 1 to 4, wherein 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: 17;

HC-CDR2 having the amino acid sequence of SEQ ID NO: 18; and

HC-CDR3 having the amino acid sequence of SEQ ID NO: 19; and

(ii) a VL region incorporating the following CDRs:

LC-CDR1 having the amino acid sequence of SEQ ID NO: 21;

LC-CDR2 having the amino acid sequence of SEQ ID NO: 6; and

LC-CDR3 having the amino acid sequence of SEQ ID NO: 22.

6. The protease-activatable Fc domain binding molecule of any one of claims 1 to 5, wherein the second antigen-binding moiety comprises:

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

8. The protease-activatable Fc domain binding molecule of any one of claims 1 to 7, wherein the first antigen binding moiety is a crossover Fab molecule wherein either the variable or the constant regions of the Fab light chain and the Fab heavy chain are exchanged.

9. The protease-activatable Fc domain binding molecule of any one of claims 1 to 8, wherein the second antigen binding moiety is a Fab molecule.

10. The protease-activatable Fc domain binding molecule of any one of claims 1 to 9, comprising a third antigen binding moiety which is a Fab molecule capable of binding to a variant CH2 domain comprising G329 according to EU numbering, wherein the third antigen binding moiety is not capable of binding to a reference CH2 domain comprising P329 according to EU numbering.

11. The protease-activatable Fc domain binding molecule of any one of claims 1 to 10, wherein the third antigen binding moiety is identical to the second antigen binding moiety.

12. The protease-activatable Fc domain binding molecule of any one of claims 1 to 11, wherein the first antigen binding moiety and the second antigen binding moiety, and where present the third antigen binding moiety are fused to each other, optionally via a peptide linker.

13. The protease-activatable Fc domain binding molecule of any one of claims 1 to 12, wherein the variant CH2 domain comprises or consists of the amino acid sequence of SEQ ID NO: 77.

14. The protease-activatable Fc domain binding molecule of any one of claims 1 to 13, wherein the masking moiety comprises or consists of the amino acid sequence of SEQ ID NO: 77.

15. The protease-activatable Fc domain binding molecule of any one of claims 1 to 14, wherein the reference CH2 domain comprises or consists of the amino acid sequence of SEQ ID NO: 78.

16. The protease-activatable Fc domain binding molecule of any one of claims 1 to 15, wherein the protease cleavable linker comprises at least one protease recognition sequence.

17. The protease-activatable Fc domain binding molecule of any one of claims 1 to 16, wherein the protease cleavable linker comprises the protease recognition sequence PQARK (SEQ ID NO: 72) or PMAKK (SEQ ID NO: 73)

18. The protease-activatable Fc domain binding molecule of any one of claims 1 to 17, additionally comprising (d) an Fc domain composed of a first and a second subunit capable of stable association.

19. The protease-activatable Fc domain binding molecule of claim 18, wherein the Fc domain is an IgG, specifically an IgGi, Fc domain.

20. The protease-activatable Fc domain binding molecule of claim 18 or 19, wherein the Fc domain comprises a variant CH2 domain comprising R329 according to EU numbering.

21. The protease-activatable Fc domain binding molecule of any one of claims 18 to 20, wherein the Fc domain comprises one or two amino acid sequences selected from the group consisting of SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO:85, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO:89, in particular wherein the Fc domain comprises an amino acid sequence of SEQ ID NO: 89 and an amino acid sequence of SEQ ID NO: 90.

22. A nucleic acid, or a plurality of nucleic acids, encoding the protease -activatable Fc domain binding molecule of any one of claims 1 to 21.

23. An expression vector, or a plurality of expression vectors, comprising a nucleic acid or a plurality of nucleic acids according to claim 22.

24. A host cell comprising the nucleic acid or the plurality of nucleic acids of claim 22, or the expression vector or plurality of expression vectors according to claim 23.

25. A method of producing a protease-activatable Fc domain binding molecule, comprising the steps of a) culturing the host cell of claim 24 under conditions suitable for the expression of the protease-activatable Fc domain binding molecule and b) recovering the protease-activatable Fc domain binding molecule.

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

25.

27. A pharmaceutical composition comprising the protease-activatable Fc domain binding molecule of any one of claims 1-21 or 26 and a pharmaceutically acceptable carrier

28. The protease-activatable Fc domain binding molecule of any one of claims 1 -21 or 26, or the pharmaceutical composition of claim 27 for use in a method of medical treatment or prophylaxis.

29. The protease-activatable Fc domain binding molecule of any one of claims 1 -21 or 26 or the pharmaceutical composition of claim 27, 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 antigen-binding domain that binds to the target antigen, and (b) a variant Fc domain comprising a variant CH2 domain comprising G329 according to EU numbering; and wherein the second antigen-binding moiety of the protease-activatable Fc domain binding molecule, binds to the variant Fc domain.

30. A kit, comprising:

(i) a protease-activatable Fc domain binding molecule of any one of claims 1 -21 or 26 or the pharmaceutical composition of claim 27; and

(ii) 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 G329 according to EU numbering; and wherein the second antigen-binding moiety of the protease-activatable Fc domain binding molecule, binds to the variant Fc domain.

31. The use of claim 29 or the kit of claim 30, wherein the variant Fc domain comprises an amino acid sequence of SEQ ID NO: 75 or SEQ ID NO: 76.

32. The use of claim 29 or claim 31, or the kit of claim 30 or claim 31, wherein the target antigen is FolRl or CEACAM5.