HK40119423A - Improved folr1 protease-activatable t cell bispecific antibodies - Google Patents

Description

Improved FOLR1 protease can activate T cell bispecific antibodies

Technical Field

This invention generally relates to improved protease-activated antigen-binding molecules comprising an anti-idiotype binding portion that reversibly masks the CD3 antigen-binding portion of the molecule. Furthermore, this invention relates to novel protease-cleavable peptide linkers and their use in such protease-activated antigen-binding molecules. Additionally, this invention relates to polynucleotides encoding such protease-activated T-cell binding molecules, and to vectors and host cells comprising such polynucleotides. The invention further relates to methods for generating the protease-activated T-cell binding molecules of the invention, and to methods for using these molecules, for example, in the treatment of diseases.

Background Technology

In various clinical settings, it is often necessary to selectively destroy individual target cells or specific target cell types. For example, a primary goal of cancer therapy is to specifically destroy tumor cells while leaving healthy cells and tissues intact.

One attractive approach to achieving this goal is to induce an immune response against tumors, causing immune effector cells (such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs)) to attack and destroy tumor cells. In this regard, bispecific antibodies designed with one "arm" binding to a surface antigen on the target cell and a second "arm" binding to the activated, non-variant component of the T cell receptor (TCR) complex have attracted attention in recent years. The simultaneous binding of such an antibody to both of its targets forces a transient interaction between the target cell and T cells, thereby activating any cytotoxic T cells and subsequently lysing the target cell. Thus, the immune response is redirected to the target cell, independent of peptide antigen presentation on the target cell or T cell specificity, and is associated with the activation of normally MHC-restricted CTLs.

In this context, it is crucial that CTLs are activated only upon proximity to target cells, i.e., mimicking immune synapses. Specifically, the desired T-cell activation of bispecific molecules does not require lymphocyte pretreatment or co-stimulation to induce effective lysis of target cells. Several bispecific antibody formulations have been developed, and their applicability to T-cell-mediated immunotherapy has been investigated. These bispecific antibody types include BiTE (bispecific T-cell conjugate) molecules (Nagorsen and Exp Cell Res 317, 1255-1260 (2011)), bispecific antibodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives such as tandem biantibodies (Kipriyanov et al., J Mol Biol 293, 41-66 (1999)), DART (biaffinity retargeting) molecules (Moore et al., Blood 117, 4542-51 (2011)) and triomab (Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)).

The task of generating therapeutically suitable bispecific molecules presents several technical challenges related to efficacy, toxicity, applicability, and manufacturability that must be met. When bispecific molecules target antigens expressed in tumor cells but also in normal tissues, on-target/off-target toxicity may occur. Therefore, there is a need for effective T-cell activating bispecific molecules that can initiate complete T-cell activation in the presence of target cells but in the absence of normal cells or tissues.

Summary of the Invention

This invention provides an improved T-cell activation bispecific molecule. Specifically, this invention provides a protease-activated T-cell activation bispecific molecule that has reduced or absent activity before reaching its site of action, such as, for example, the tumor microenvironment. This results in improved safety, such as lower toxicity and more efficient activation of the molecule at the site of action.

In one embodiment, a protease-activated T-cell activation bispecific molecule is provided, comprising:

(a) The first antigen-binding region capable of binding to CD3;

(b) a second antigen-binding moiety capable of binding to target cell antigens; and

(c) Covalently linked to the masking portion of a T-cell activation bispecific molecule via a peptide linker, wherein the masking portion can bind idiotype to either the first antigen-binding portion or the second antigen-binding portion, thereby reversibly masking either the first antigen-binding portion or the second antigen-binding portion.

The linker contains a protease recognition sequence XQARK (SEQ ID NO:39), where X is histidine (H) or proline (P).

In one embodiment, the masking portion is covalently connected to the first antigen-binding portion and reversibly conceals the first antigen-binding portion.

In one embodiment, the masking portion is covalently linked to the heavy chain variable region of the first antigen-binding portion.

In one embodiment, the masking portion is scFv.

In one embodiment, (i) the second antigen-binding portion is a conventional Fab, or (ii) the second antigen-binding portion is a cross-Fab molecule, wherein the Fab light chain exchanges with the variable or constant regions of the Fab heavy chain.

In one embodiment, the first antigen-binding portion is a conventional Fab molecule.

In one embodiment, the protease-activated T-cell bispecific molecule includes a third antigen-binding portion, which is a Fab molecule capable of binding to target cell antigens.

In one embodiment, the third antigen-binding portion is the same as the second antigen-binding portion.

In one embodiment, the target cell antigen is FolR1.

In one embodiment, the first antigen-binding portion and the second antigen-binding portion are fused together, optionally via peptide linkers.

In one embodiment, the second antigen-binding portion 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 portion.

In one embodiment, the protease-activated T-cell activation bispecific molecule additionally includes an Fc domain consisting of a first subunit and a second subunit capable of stable association.

In one embodiment, the Fc domain is IgG, specifically the IgG1 or IgG4 Fc domain.

In one embodiment, the Fc domain exhibits reduced binding affinity to the Fc receptor and/or reduced effector function compared to the native IgG1 Fc domain.

In one embodiment, the antigen-binding moiety capable of binding to CD3 includes a heavy chain variable (VH) region.

(a) The amino acid sequence of the heavy chain complementarity-determining region (HCDR) 1 of SYAMN (SEQ ID NO:1);

(b) The HCDR2 amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO:2);

(c) The HCDR3 amino acid sequence of ASNFPASYVSYFAY (SEQ ID NO:3);

And the light chain variable (VL) region, which includes:

(d) The amino acid sequence of the light chain complementarity-determining region (LCDR) 1 of GSSTGAVTTSNYAN (SEQ ID NO:7);

(e) The LCDR2 amino acid sequence of GTNKRAP (SEQ ID NO:8); and

(f) The LCDR3 amino acid sequence selected from ALWYSNLWV (SEQ ID NO:9).

In one embodiment, the antigen-binding portion capable of binding to CD3 comprises: a VH region containing 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:5; and/or a VL region containing 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:10.

In one embodiment, the antigen-binding portion capable of binding to CD3 includes: a heavy chain variable (VH) region, which contains...

(a) The amino acid sequence of the heavy chain complementarity-determining region (HCDR) 1 of SYAMN (SEQ ID NO:1);

(b) The HCDR2 amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO:2);

(c) The HCDR3 amino acid sequence of HTTFPSSYVSYYGY (SEQ ID NO:4);

And the light chain variable (VL) region, which includes:

(d) The amino acid sequence of the light chain complementarity-determining region (LCDR) 1 of GSSTGAVTTSNYAN (SEQ ID NO:7);

(e) The LCDR2 amino acid sequence of GTNKRAP (SEQ ID NO:8); and

(f) The LCDR3 amino acid sequence selected from ALWYSNLWV (SEQ ID NO:9).

In one embodiment, the antigen-binding portion capable of binding to CD3 comprises: a VH region containing at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to that of SEQ ID NO:6; and/or a VL region containing at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to that of SEQ ID NO:10.

In one embodiment, the masking portion includes: a VH region, which includes:

(a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15),

(b) The HCDR2 amino acid sequence selected from the group consisting of WINTETGEPRYTDDFKG (SEQ ID NO:16), WINTETGEPRYTDDFTG (SEQ ID NO:17) and WINTETGEPRYTQGFKG (SEQ ID NO:18);

(c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19);

And the VL zone, which includes:

(d) The LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO:25) or KSSKSVSTSSYSYMH (SEQ ID NO:26);

(e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and

(f) The LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28) or QQSREFPYT (SEQ ID NO:29).

In one embodiment, the masking portion includes: a VH region, which includes:

(a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15);

(b) The HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO:16);

(c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19);

And the VL zone, which includes:

(d) The LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO:25);

(e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and

(f) The LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28).

In one embodiment, the masking portion includes: a VH region, which includes:

(a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15);

(b) The HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO:16);

(c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19);

And the VL zone, which includes:

(d) The LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO:26);

(e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and

(f) The LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28).

In one embodiment, the masking portion includes: a VH region, which includes:

(a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15);

(b) The HCDR2 amino acid sequence of WINTETGEPRYTDDFTG (SEQ ID NO:17);

(c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19);

And the VL zone, which includes:

(d) The LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO:26);

(e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and

(f) The LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28).

In one embodiment, the masking portion includes: a VH region, which includes:

(a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15);

(b) The HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO:18);

(c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19);

And the VL zone, which includes:

(d) The LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO:26);

(e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and

(f) The LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28).

In one embodiment, the masking portion includes: a VH region, which includes:

(a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15);

(b) The HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO:18);

(c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19);

And the VL zone, which includes:

(d) The LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO:25);

(e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and

(f) The LCDR3 amino acid sequence of QQSREFPYT (SEQ ID NO:29).

In one embodiment, the second antigen-binding portion is capable of binding to FolR1 and includes: a VH region comprising:

a) The HCDR1 amino acid sequence of NAWMS (SEQ ID NO: 11);

b) The HCDR2 amino acid sequence of RIKSKTDGGTTDYAAPVKG (SEQ ID NO:12); and

c) The HCDR3 amino acid sequence of PWEWSWYDY (SEQ ID NO:13);

And the VL zone, which includes:

d) LCDR1 of GSSTGAVTTSNYAN (SEQ ID NO:7);

e) The LCDR2 amino acid sequence of GTNKRAP (SEQ ID NO: 8); and

f) The LCDR3 amino acid sequence of ALWYSNLWV (SEQ ID NO:9).

In one embodiment, the antigen-binding portion capable of binding to FolR1 comprises: a VH region containing 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: 14; and/or a VL region containing 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: 10.

In one embodiment, the protease-cleavable linker comprises a protease recognition sequence PQARK (SEQ ID NO:41).

In one embodiment, a unique type-specific polypeptide is provided for reversibly concealing the anti-CD3 antigen binding site of a molecule, wherein the unique type-specific polypeptide is covalently linked to the molecule via a peptide linker, wherein the linker comprises a protease recognition sequence (XQARK SEQ ID NO:39), wherein X is histidine (H) or proline (P).

In one embodiment, the idiotype-specific polypeptide is an anti-idiotype scFv.

In one embodiment, the molecule is a bispecific molecule for T cell activation.

In one embodiment, the adapter comprises a protease recognition sequence PQARK (SEQ ID NO:41).

In one embodiment, a pharmaceutical composition is provided comprising a protease-activated T-cell activation bispecific molecule as described herein and a pharmaceutical carrier.

In one embodiment, a pharmaceutical composition is provided comprising a unique type-specific polypeptide and a pharmaceutical carrier as described herein.

In one embodiment, a separated polynucleotide is provided that encodes a protease-activated T-cell-activated bispecific antigen-binding molecule as described herein.

In one embodiment, a separate polynucleotide is provided that encodes a unique type-specific polypeptide as described herein.

In one embodiment, a vector, particularly an expression vector, is provided that comprises polynucleotides as described herein.

In one embodiment, a host cell is provided that comprises a vector as described herein.

In one embodiment, a method for producing a protease-activated T-cell activation bispecific molecule is provided, comprising the steps of: a) culturing host cells as described herein under conditions suitable for expressing the protease-activated T-cell activation bispecific molecule, and b) recovering the protease-activated T-cell activation bispecific molecule.

In one embodiment, a protease-activated T-cell activation bispecific molecule as described herein is provided for use as a drug.

In one embodiment, the drug is used to treat cancer or delay its progression, treat an immune-related disease or delay its progression, or enhance or stimulate an individual's immune response or function.

In one embodiment, the use of a protease-activated T-cell-activating bispecific molecule as described herein for the manufacture of a medicament for the treatment of a disease is provided.

In one embodiment, the use of a protease-activated T-cell activation bispecific molecule as described herein is provided, wherein the disease is cancer.

In one embodiment, a method of treating a disease in an individual is provided, comprising administering to the individual a therapeutically effective amount of a composition comprising a protease-activated T-cell activating bispecific molecule as described herein.

In one embodiment, the method is used to treat cancer or delay its progression.

Other technical features will be apparent to those skilled in the art from the following figures, description and claims.

Attached Figure Description

Figure 1. A schematic diagram depicting exemplary protease-activated FolR1 TCB molecules (SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:53).

Figure 2. Study design for single-dose PK and stability studies. Female NSG mice were intravenously injected with protease-activated FolR1 TCB molecules containing HQARK or PQARK linkers (groups A and B), and compared with typical FolR1 TCB molecules (group C). Blood samples were collected from mice at 24 hours, 7 days, and 10 days post-injection. Serum was prepared and the total amount and active form of FolR1 TCB molecules were analyzed by ELISA.

Figure 3. Depicts the quantification of active pro-TCB in the serum of tumor-free mice. Activity and total TCB concentrations in serum over time were measured by ELISA following a single intravenous injection of protease-activated FolR1 TCB or typical FolR1 TCB. Activity and total TCB were quantified by ELISA using anti-PG antibody (protease-activated FolR1 TCB) and anti-idiotypic anti-CD3 antibody (active FolR1 TCB). The percentage of active TCB to total TCB is shown. Dose correction was not required because equimolar doses of protease-activated FolR1 TCB and typical FolR1 TCB were used in the individual studies.

Figure 4. Study design for in vivo efficacy studies. Female NSG mice were subcutaneously injected with human breast cancer PDX (BC004) and received their first treatment when the tumor reached approximately 200 mm³ (day 28). Mice were treated weekly intravenously with protease-activated FolR1 TCBs containing cleavage sites of PMAKK or PQARK molecules (groups D and E), or typical FolR1 TCB molecules (group B), and masked FolR1 TCBs containing an uncleavable linker (group C). One group received only histidine buffer as a control (group A; solvent). Tumor growth was measured using a caliper, and the study was terminated on day 58, with tumors harvested and weighed.

Figure 5. Depicts tumor growth inhibition and tumor weight at study termination. (A) Depicts mean +/- SEM of tumor volume over time across all treatment groups. FolR1 TCB molecules containing a PQARK cleavage site and capable of protease activation resulted in tumor growth inhibition comparable to that seen with typical FolR1 TCB. Masked FolR1 TCBs containing an uncleavable linker and molecules containing a PMAKK cleavage site did not result in tumor growth inhibition. (B to F) Show individual tumor growth kinetics in a single mouse in the solvent (B), typical FolR1 TCB (C), FolR1 TCB containing a PMAKK site and capable of protease activation (D), masked FolR1 TCB containing an uncleavable linker (E), and FolR1 TCB containing a PQARK cleavage site and capable of protease activation (F). (G) Tumor weight of all treatment groups at study termination.

Figure 6. Depicts Jurka NFAT activation induced by the FolR1 TCB molecule, which is activated by the protease containing CD3-binding clone 22.

Detailed Implementation

definition

Unless otherwise defined below, the terminology used herein is as it is used in the art.

For the purposes of this document, a “recipient human frame” is a frame that comprises an amino acid sequence of a light chain variable domain (VL) frame or a heavy chain variable domain (VH) frame derived from the human immunoglobulin frame or the human common frame as defined below. A recipient human frame “derived from” the human immunoglobulin frame or the human common frame may contain the same amino acid sequence as said human immunoglobulin frame or human common frame, or it may contain amino acid sequence variations. In some aspects, the number of amino acid variations is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some aspects, the VL recipient human frame is sequenceally identical to the VL human immunoglobulin frame sequence or the human common frame sequence.

“Affinity” refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, 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 molecule X for its partner Y can generally be represented by the dissociation constant (K_{_D} ). Affinity can be measured by commonly used methods known in the art, including those described herein. Specific illustrative and exemplary methods for measuring binding affinity are described below.

"Affinity-mature" antibodies are those that have one or more alterations in one or more complementarity-determining regions (CDRs), which result in improved affinity of the antibody for the antigen compared to parental antibodies without such alterations.

The term “antibody” is used in the broadest sense and includes a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

"Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody and binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; bisomatic antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAb); and multispecific antibodies formed from antibody fragments. For a review of some antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

Screening for antibody binding can be performed using methods conventional in the art, such as, but not limited to, alanine scanning, Western blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496) and cross-blocking (see “Antibodies”, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY).

Antigen structure-based antibody profiling (ASAP), also known as modification-assisted profiling (MAP), allows for the binning of numerous monoclonal antibodies that specifically bind to antigens based on the individual antibody bindings from numerous chemically or enzymatically modified antigen surfaces (see, for example, US 2004/0101920). Antibodies in each bin bind to the same epitope, which can be a unique epitope that is distinctly different from or partially overlaps with the epitope represented by another bin.

In some respects, such as as measured in competitive binding assays, if one antibody inhibits the binding of another antibody by at least 50%, at least 75%, at least 90%, or even 99% or higher by 1, 5, 10, 20, or 100-fold excess, the two antibodies are considered to bind to the same or overlapping epitopes (see, for example, Junghans et al., Cancer Res. 50 (1990) 1495-1502).

In some respects, if virtually all amino acid mutations in an antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of another antibody, then the two antibodies are considered to bind to the same epitope. If only a subset of amino acid mutations that reduce or eliminate the binding of one antibody reduces or eliminates the binding of another antibody, then the two antibodies are considered to have "overlapping epitopes".

Terminal epitope portion]]

The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and/or light chain originates from a specific source or species, while the remainder of the heavy chain and/or light chain originates from a different source or species.

An antibody's "class" 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 some of these can be further subdivided into subclasses (isotypes), such as _IgG1 , _IgG2 , _IgG3 , _IgG4 , _IgA1 , and _IgA2 . In some respects, an antibody is an _IgG1 isotype. In some respects, an antibody is an _IgG1 isotype with P329G, L234A, and L235A mutations to reduce the function of the Fc region effector. In other respects, an antibody is an _IgG2 isotype. In some respects, an antibody is an _IgG4 isotype with an S228P mutation in the hinge region to improve the stability of _IgG4 antibodies. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are designated a, d, e, g, and m, respectively. The light chain of an antibody, based on the amino acid sequence of its constant structural domain, can be classified into one of two types, known as Kappa (κ) and Lambda (λ).

As used herein, the term “human-derived constant region” or “human constant region” refers to the constant heavy chain region and/or constant light chain kappa or lambda region of human antibodies against subclasses IgG1, IgG2, IgG3, or IgG4. Such constant regions are well known in the art and are described, for example, by the following: Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also, for example, 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, the amino acid residues in the constant region are numbered according to the EU numbering system (also known as Kabat's EU index), as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

"Effective functions" refer to those biological activities attributable to the Fc region of an antibody that vary with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

The “effective amount” of a pharmaceutical agent (such as a pharmaceutical composition) refers to the amount that is sufficient to effectively achieve the desired therapeutic or preventative outcome at the required dose for the required period of time.

The term "Fc region" used herein is used to define the C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of a constant region. This term includes both native sequence Fc regions and variant Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, antibodies produced by host cells can undergo post-translational cleavage of one or more (particularly one or two) amino acids from the C-terminus of the heavy chain. Therefore, antibodies produced by host cells by expressing a specific nucleic acid molecule encoding the full-length heavy chain can comprise the full-length heavy chain, or the antibody can comprise a cleaved variant of the full-length heavy chain. This could be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering system). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. Unless otherwise stated, the amino acid sequence of the heavy chain including the Fc region herein indicates the absence of a C-terminal glycine-lysine dipeptide. In one aspect, a heavy chain including the Fc region as specified herein is included in an antibody according to the invention, said heavy chain comprising an additional C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system). In another aspect, a heavy chain including the Fc region as specified herein is included in an antibody according to the invention, said heavy chain comprising an additional C-terminal glycine residue (G446, according to EU index number). Unless otherwise stated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also known as the EU index), as described by Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991) (see also above).

"Frame" or "FR" refers to the variable domain residues other than the complementarity-determining region (CDR). A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences usually appear in the VH (or VL) as follows: FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.

The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably herein to refer to antibodies having a structure substantially similar to that of natural antibodies or having a heavy chain containing an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including progeny cells. Host cells include “transformations” and “transformed cells,” which include primary transformed cells and progeny derived from those primary transformed cells, regardless of passage number. Progeny cells may not have completely identical nucleic acid contents to the parent cells and may contain mutations. This article includes mutant progeny with the same function or biological activity as those screened or selected from the original transformed cells.

A "human antibody" is an antibody whose amino acid sequence corresponds to that of an antibody produced by a human or human cell, or to a non-human antibody derived from a complete library of human antibodies or other antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

The “human common framework” is a framework that represents the amino acid residues most frequently present in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is derived from a subgroup of variable domain sequences. Generally, the subgroup of sequences is as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one aspect, for VL, this subgroup is as described in Kabat et al., ibid., subgroup κI. In another aspect, for VH, this subgroup is as described in Kabat et al., ibid., subgroup III. [[Adjusted as needed to refer to the actual subgroup of VH/VL in this invention]]

"Humanized" antibodies refer to chimeric antibodies that contain amino acid residues from non-human CDRs and amino acid residues from human FRs. In some respects, humanized antibodies will substantially contain at least one, typically two, variable domains, wherein all or substantially all CDRs correspond to the CDRs of the non-human antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Humanized antibodies may optionally contain at least a portion of the antibody constant region derived from a human antibody. Antibodies in a "humanized form," such as non-human antibodies, refer to antibodies that have undergone humanization.

As used herein, the term "hypervariant region" or "HVR" refers to the regions within the variable domain of an antibody that are hypervariable in sequence and determine antigen-binding specificity, such as the "complementarity-determining region" ("CDR").

Typically, an antibody contains six CDRs; three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). In this document, exemplary CDRs include:

(a) Highly variable rings appearing at the following amino acid residues: 26 to 32 (L1), 50 to 52 (L2), 91 to 96 (L3), 26 to 32 (H1), 53 to 55 (H2) and 96 to 101 (H3) (Chothia and Lesk, J.Mol.Biol.196:901-917(1987));

(b) CDRs appearing at the following amino acid residues: 24–34 (L1), 50–56 (L2), 89–97 (L3), 31–35b (H1), 50–65 (H2), and 95–102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and

(c) Antigen contact sites appearing at the following amino acid residues: 27c to 36 (L1), 46 to 55 (L2), 89 to 96 (L3), 30 to 35b (H1), 47 to 58 (H2) and 93 to 101 (H3) (MacCallum et al. J.Mol.Biol.262:732-745 (1996)).

Unless otherwise specified, the CDR is determined according to the method described by Kabat et al., citing above. Those skilled in the art will understand that the CDR name may also be determined according to the method described by Chothia, citing above, the method described by McCallum, citing above, or any other scientifically accepted naming system.

"Immune conjugates" are antibodies that are conjugated to one or more heterologous molecules (including but not limited to cytotoxic agents).

The “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., human and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In some respects, the individual or subject is a human.

"Isolated" antibodies are antibodies that have been separated from components of their natural environment. In some respects, antibodies are purified to a purity greater than 95% or 99% by methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

The terms "nucleic acid molecule" or "polynucleotide" include any compound and/or substance comprising a polymer of nucleotides. Each nucleotide consists of bases, particularly purine or pyrimidine bases (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate ester group. Typically, nucleic acid molecules are described by a base sequence, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. Base sequences are typically represented from 5' to 3'. In this document, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA) (particularly messenger RNA (mRNA)), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes sense and antisense strands, as well as single-stranded and double-stranded forms. Additionally, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derived sugars, phosphate backbones, or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct in vitro and/or in vivo (e.g., in a host or patient) expression of antibodies used in this invention. 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 the expression of the encoding molecule, enabling the mRNA to be injected into a subject to generate in vivo antibodies (see, for example, Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038/nm.4356 or EP 2 101 823 B1).

"Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are contained in cells that normally contain nucleic acid molecules, but which are located outside the chromosome or at a chromosomal location different from their natural chromosomal location.

"Isolated nucleic acid encoding polypeptide" refers to one or more nucleic acid molecules that encode, for example, antibody heavy and light chains (or fragments thereof) or idiotype-specific polypeptides, including such nucleic acid molecules in a single vector or a separate vector, and such nucleic acid molecules are present at one or more locations in a host cell.

As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homologous population of antibodies, meaning that the individual antibodies comprising the population are identical and/or bind to the same epitopes, except for possible variant antibodies, such as those containing naturally occurring mutations or those generated during the production of the monoclonal antibody formulation. Such variants are typically present in small quantities. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized by being derived from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies according to the invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, 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 preparing monoclonal antibodies are described herein.

"Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabeled part. Naked antibodies may be present in pharmaceutical compositions.

"Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains bonded by disulfides. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy chain domain or heavy chain variable region, followed by three constant heavy chain domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called a variable light chain domain or light chain variable region, followed by a constant light chain (CL) domain.

The term "packaging insert" is used to refer to the instruction leaflet typically included in the commercial packaging of a therapeutic product, which contains information concerning the indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings related to the use of such therapeutic products.

The "percentage of amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after aligning the candidate sequence with the reference polypeptide sequence and introducing vacancies (if necessary) to achieve the maximum percentage of sequence identity, and for alignment purposes without considering any conserved substitutions as part of sequence identity. Alignment used to determine the percentage of amino acid sequence identity can be performed in various ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. Alternatively, the sequence comparison computer program ALIGN-2 can be used to generate the percentage of identity values. The ALIGN-2 sequence comparison computer program was written by Genentech, and the source code has been submitted with the user documentation to the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registry No. TXU510087 and described in WO 2001/007611.

Unless otherwise specified, for the purposes of this article, the ggsearch program of FASTA package version 36.3.8c or later was used to generate the amino acid sequence identity percentage values using the BLOSUM50 comparison matrix. The FASTA package was created 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 at www.fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml or www.ebi.ac.uk/Tools/sss/fasta. Alternatively, a public server accessible via fasta.bioch.virginia.edu/fasta_www2/index.cgi can be used, employing the ggsearch(global protein:protein) procedure with default options (BLOSUM50; open:-10; ext:-2; Ktup=2) to ensure a global rather than local alignment is performed. The percentage of amino acid identity is given in the output alignment header.

The terms “pharmaceutical composition” or “pharmaceutical formulation” refer to a formulation in which the active ingredient contained therein is in a biologically effective form and does not contain any additional components that would have unacceptable toxicity to a subject to whom the pharmaceutical composition will be administered.

"Pharmaceutical carrier" refers to a component in a pharmaceutical composition or formulation other than the active ingredient, which is non-toxic to the subject. Pharmaceutical carriers include, but are not limited to, buffer solutions, excipients, stabilizers, or preservatives.

Unless otherwise stated, as used herein, the term "FoR1" or "folate receptor 1" refers to any naturally occurring FolR1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term includes "full-length" unprocessed FolR1, as well as any form of FolR1 produced through cellular processing. The term also covers naturally occurring variants of FolR1, such as splice variants or allele variants.

As used herein, “treatment” (and its grammatical variations such as treat or treating) refers to an attempt to alter the natural course of a disease in the treated individual and is a clinical intervention that can be performed for prevention or may be performed during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis. In some aspects, the antibodies of the present invention are used to delay the development of disease or slow its progression.

The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007).) A single VH or VL domain is sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to a specific antigen can be isolated using either the VH or VL domain from the antibody binding that antigen to screen libraries for complementary VL or VH domains. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of the nucleic acid to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

As used herein, the term "antigen-binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, the antigen-binding moiety is capable of directing its attached entity (e.g., a second antigen-binding moiety) to a target site, such as to a specific type of tumor cell or tumor stroma bearing an antigenic determinant. In another embodiment, the antigen-binding moiety is capable of activating signal transduction via its target antigen (e.g., a T-cell receptor complex antigen). The antigen-binding moiety includes antibodies and fragments thereof as further defined herein. A particular antigen-binding moiety includes an antigen-binding domain of an antibody comprising a variable region of the antibody heavy chain and a variable region of the antibody light chain. In some embodiments, the antigen-binding moiety may include an antibody constant region as further defined herein and known in the art. Available heavy chain constant regions include any of five isoforms: α, δ, ε, γ, or μ. Available light chain constant regions include any of two isoforms: κ and λ.

As used herein, "T cell activation" refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. The protease-activated "T cell activation bispecific molecule" of the present invention can induce T cell activation. Suitable assays for measuring T cell activation are known in the art as described herein.

As used herein, “target cell antigen” refers to antigenic determinants present on the surface of target cells, such as cells in a tumor (e.g., cancer cells or cells in the tumor stroma).

As used herein, the terms “first” and “second” regarding antigen-binding moieties, etc., are used for convenience when there is more than one of each type of moieties. Unless explicitly stated otherwise, the use of these terms is not intended to assign a specific sequence or direction to the activation of bispecific molecules by proteases that can be activated by T cells.

"Fab molecule" refers to a protein composed of the VH and CH1 domains of the heavy chain ("Fab heavy chain") of an immunoglobulin and the VL and CL domains of the light chain ("Fab light chain").

"Fusion" means that components (e.g., Fab molecules and Fc domain subunits) 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 some embodiments, one of the antigen-binding portions is a single-chain Fab molecule, i.e., a Fab molecule in which the Fab light chain and Fab heavy chain are linked by peptide linkers to form a single peptide chain. In one particular such embodiment, the C-terminus of the Fab light chain in the single-chain Fab molecule is attached to the N-terminus of the Fab heavy chain.

A “crossfab” molecule (also known as a “cross-fab”) refers to a Fab molecule in which the variable or constant regions of the Fab heavy and light chains are exchanged. Specifically, a crossfab molecule comprises a peptide chain consisting of a variable region of the light chain and a constant region of the heavy chain, and a peptide chain consisting of a variable region of the heavy chain and a constant region of the light chain. For clarity, in a crossfab molecule in which the variable regions of the Fab light and Fab heavy chains are exchanged, the peptide chain containing the constant region of the heavy chain is referred to herein as the “heavy chain” of the crossfab molecule. Conversely, in a crossfab molecule in which the constant regions of the Fab light and Fab heavy chains are exchanged, the peptide chain containing the variable region of the heavy chain is referred to herein as the “heavy chain” of the crossfab molecule.

In contrast, “conventional” Fab molecules refer to Fab molecules in their natural form, namely, heavy chains consisting of variable and constant regions of heavy chains (VH-CH1) and light chains consisting of variable and constant regions of light chains (VL-CL).

As used herein, a “idiotype-specific polypeptide” refers to a polypeptide that recognizes a idiotype of an antigen-binding moiety (e.g., an antigen-binding moiety specific to CD3). “Idiotype” can be defined as a specific combination of idiotopes present within the complementarity-determining region (CDR) of an antigen-binding moiety. Idiotype-specific polypeptides can specifically bind to the variable region of an antigen-binding moiety, thereby reducing or preventing specific binding of the antigen-binding moiety to its homologous antigen. When bound to a molecule containing an antigen-binding moiety, idiotype-specific polypeptides can act as a masking portion of the molecule. Specifically, this document discloses anti-idiotype antibodies or anti-idiotype-binding antibody fragments specific to idiotypes of anti-CD3 binding molecules.

As used herein, "protease" or "proteolytic enzyme" refers to any proteolytic enzyme that cleaves a linker at a recognition site and is expressed by a target cell. Such proteases may be secreted by the target cell or maintained associated with the target cell, for example, on the surface of the target cell. Examples of proteases include, but are not limited to, metalloproteinases (e.g., matrix metalloproteinases 1 to 28, detegrin metalloproteinases (ADAM) 2, 7 to 12, 15, 17 to 23, 28 to 30, and 33), serine proteases (e.g., urokinase-type plasminogen activator and matriptase), cysteine proteases, aspartic proteases, and members of the cathepsin family.

As used herein, regarding T-cell activation bispecific molecules, "protease-activated" refers to a T-cell activation bispecific molecule that possesses the ability to reduce or eliminate T-cell activation due to a masking portion (which reduces or eliminates the ability of the T-cell activation bispecific molecule to bind to CD3). Upon dissociation of the masking portion via proteolytic cleavage (e.g., by proteolytic cleavage of the connector to the T-cell activation bispecific molecule), binding to CD3 is restored, thereby activating the T-cell activation bispecific molecule.

As used herein, “reversible masking” refers to the masking of the binding of a portion or idiotype-specific polypeptide to an antigen-binding portion or molecule, such as preventing the antigen-binding portion or molecule from binding to its antigen (e.g., CD3). This masking is reversible because the idiotype-specific polypeptide can be released from the antigen-binding portion or molecule, for example, by protease cleavage, thereby releasing the antigen-binding portion or molecule to bind to its antigen.

Protease-activated T cell activation dual-specificity molecule

This invention provides an improved T-cell activation bispecific molecule. Specifically, this invention provides a protease-activated T-cell activation bispecific molecule that has reduced or absent activity before reaching its site of action, such as, for example, the tumor microenvironment. This results in improved safety, such as lower toxicity and more efficient activation of the molecule at the site of action.

In one aspect, the present invention relates to a protease-activated T-cell activation bispecific molecule comprising...

1. The first antigen-binding portion, which can bind to CD3;

2. A second antigen-binding portion, which can bind to target cell antigens; and

3. A masking portion, which is covalently linked to a T-cell bispecific binding molecule via a protease-cleavable linker, wherein the masking portion is capable of binding idiotype to either the first antigen-binding portion or the second antigen-binding portion, thereby reversibly masking the first antigen-binding portion or the second antigen-binding portion.

The first antigen-binding moiety capable of binding to CD3 contains an idiotype. In one embodiment, a masking portion of a protease-activated T-cell activation bispecific molecule is covalently linked to the first antigen-binding moiety. In one embodiment, the masking portion is covalently linked to a heavy chain variable region of the first antigen-binding moiety. In one embodiment, the masking portion is covalently linked to a light chain variable region of the first antigen-binding moiety. This covalent bond and the masking portion are separate from the specific binding (preferably non-covalent) of the idiotype first antigen-binding site. The idiotype of the first antigen-binding moiety contains its variable region. In one embodiment, when the first antigen-binding moiety binds to CD3, the masking portion binds to an amino acid residue that contacts CD3. In a preferred embodiment, the masking portion is not a homologous antigen of the first antigen-binding moiety or a fragment thereof, i.e., the masking portion is not CD3 or a fragment thereof. In one embodiment, the masking portion is an anti-idiotype antibody or a fragment thereof. In one embodiment, the masking portion is an anti-idiotype scFv. Exemplary embodiments of the masking portion of the anti-idiotype scFv and protease-activated T-cell activation molecules containing such masking portions are described in detail below and in examples.

Exemplary antigen-binding portion

The antigen-binding molecule of the present invention is bispecific, that is, it comprises at least two antigen-binding moieties capable of specifically binding to two different antigenic determinants. According to the present invention, the antigen-binding moieties are Fab molecules (i.e., antigen-binding domains composed of heavy and light chains, wherein each chain contains a variable region and a constant region). In one embodiment, the Fab molecule is a human Fab molecule. In another embodiment, the Fab molecule is a humanized Fab molecule. In yet another embodiment, these Fab molecules comprise human heavy and light chain constant regions.

At least one of the antigen-binding portions is a cross-Fab molecule. This modification prevents mismatch between heavy and light chains from different Fab molecules, thereby improving the yield and purity of the recombinant production of the protease-activated T-cell-activating bispecific molecule of the present invention. In a specific cross-Fab molecule usable in the protease-activated T-cell-activating bispecific molecule of the present invention, the constant regions of the Fab light chain and the Fab heavy chain are exchanged. In another cross-Fab molecule usable in the protease-activated T-cell-activating bispecific molecule of the present invention, the variable regions of the Fab light chain and the Fab heavy chain are exchanged.

In a specific embodiment of the invention, the protease-activated T-cell activation bispecific molecule is capable of simultaneously binding to target cell antigens (particularly tumor cell antigens) and CD3. In one embodiment, the protease-activated T-cell activation bispecific molecule can crosslink T cells with target cells by simultaneously binding to target cell antigens and CD3. In even more specific embodiments, such simultaneous binding leads to the lysis of target cells (particularly tumor cells). In one embodiment, such simultaneous binding leads to T-cell activation. In other embodiments, such simultaneous binding leads to a cellular response of T lymphocytes, particularly cytotoxic T lymphocytes, selected from: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. In one embodiment, the protease-activated T-cell activation bispecific molecule binds to CD3 without simultaneously binding to target cell antigens, and does not lead to T-cell activation.

In one embodiment, a protease-activated T cell-activating bispecific molecule can redirect the cytotoxic activity of T cells to target cells. In a particular embodiment, this redirection is independent of target cell-mediated MHC-mediated peptide antigen presentation and/or T cell specificity.

In particular, the T cells according to any embodiment of the invention are cytotoxic T cells. In some embodiments, the T cells are CD4 ⁺ or CD8 ⁺ T cells, especially CD8 ⁺ T cells.

CD3 Combination Part

The protease-activated T-cell activation bispecific molecule of the present invention comprises at least one antigen-binding moiety capable of binding to CD3 (also referred to herein as a "CD3 antigen-binding moiety" or "first antigen-binding moiety"). In one particular embodiment, the protease-activated T-cell activation bispecific molecule comprises no more than one antigen-binding moiety capable of binding to CD3. In one embodiment, the protease-activated T-cell activation bispecific molecule provides monovalent binding to CD3. The CD3 antigen-binding moiety is a cross-Fab molecule, i.e., a Fab molecule in which the variable or constant regions of the Fab heavy and light chains are exchanged. In embodiments in which more than one antigen-binding moiety capable of binding to a target cell antigen contained in the protease-activated T-cell activation bispecific molecule is present, the antigen-binding moiety capable of binding to CD3 is preferably a cross-Fab molecule, and the antigen-binding moiety capable of binding to the target cell antigen is a conventional Fab molecule.

In one particular embodiment, CD3 is human CD3 or cynomolgus monkey CD3, most particularly human CD3. In one particular embodiment, the CD3 antigen-binding moiety cross-reacts (i.e., specifically binds) with human and cynomolgus monkey CD3. In some embodiments, the first antigen-binding moiety is capable of binding to the ε subunit of CD3.

The CD3 antigen-binding region includes at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, and at least one light chain CDR selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.

In a preferred embodiment, the CD3 antigen binding portion comprises the heavy chain CDR1 of SEQ ID NO:1, the heavy chain CDR2 of SEQ ID NO:2, the heavy chain CDR3 of SEQ ID NO:3, the light chain CDR1 of SEQ ID NO:7, the light chain CDR2 of SEQ ID NO:8, and the light chain CDR3 of SEQ ID NO:9.

In one embodiment, the CD3 antigen-binding portion comprises: a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:5; and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:10.

In a preferred embodiment, the CD3 antigen-binding portion comprises the heavy chain variable region sequence of SEQ ID NO:5 and the light chain variable region sequence of SEQ ID NO:10.

In one embodiment, the CD3 antigen binding portion comprises the heavy chain CDR1 of SEQ ID NO:1, the heavy chain CDR2 of SEQ ID NO:2, the heavy chain CDR3 of SEQ ID NO:4, the light chain CDR1 of SEQ ID NO:7, the light chain CDR2 of SEQ ID NO:8, and the light chain CDR3 of SEQ ID NO:9.

In one embodiment, the CD3 antigen-binding portion comprises: a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 6; and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10.

In one embodiment, the CD3 antigen-binding portion comprises the heavy chain variable region sequence of SEQ ID NO:6 and the light chain variable region sequence of SEQ ID NO:10.

Target cell antigen binding portion

The protease-activated T-cell activation bispecific molecule of the present invention comprises at least one antigen-binding moiety capable of binding to a target cell antigen (also referred to herein as a "target cell antigen-binding moiety" or a "second" or "third" antigen-binding moiety). In some embodiments, the protease-activated T-cell activation bispecific molecule comprises two antigen-binding moieties capable of binding to a target cell antigen. In one particular such embodiment, each of these antigen-binding moieties specifically binds to the same antigenic determinant. In one or even more particular embodiments, all of these antigen-binding moieties are identical. In one embodiment, the protease-activated T-cell activation bispecific molecule comprises an immunoglobulin molecule capable of binding to a target cell antigen. In one embodiment, the protease-activated T-cell activation bispecific molecule comprises no more than two antigen-binding moieties capable of binding to a target cell antigen.

In a preferred embodiment, the target cell antigen-binding portion is a Fab molecule, specifically a conventional Fab molecule that binds to a specific antigenic determinant and is capable of directing a protease-activated T cell-activating bispecific molecule to a target site (e.g., to a specific type of tumor cell with an antigenic determinant).

In some embodiments, the target cell antigen-binding portion specifically binds to cell surface antigens. In a particular embodiment, the target cell antigen-binding portion specifically binds to folic acid receptor 1 (FolR1) on the surface of target cells.

In some embodiments, the target cell antigen binding portion targets antigens associated with a pathological condition, such as antigens presented on tumor cells or virus-infected cells. Suitable antigens are cell surface antigens, such as, but not limited to, cell surface receptors. In a particular embodiment, the antigen is a human antigen. In one specific embodiment, the target cell antigen is folate receptor 1 (FolR1).

In a particular embodiment, the protease-activated T-cell activation bispecific molecule comprises at least one antigen-binding moiety specific to FolR1. In one embodiment, FolR1 is human FolR1. In one embodiment, the protease-activated T-cell activation bispecific molecule comprises at least one antigen-binding moiety specific to human FolR1 and not binding to human FolR2 or human FolR3. In one embodiment, the FolR1-specific antigen-binding moiety comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, and at least one light chain CDR selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

In one embodiment, the antigen-binding portion specific to FolR1 comprises the heavy chain CDR1 of SEQ ID NO:11, the heavy chain CDR2 of SEQ ID NO:12, the heavy chain CDR3 of SEQ ID NO:13, the light chain CDR1 of SEQ ID NO:7, the light chain CDR2 of SEQ ID NO:8, and the light chain CDR3 of SEQ ID NO:9.

In another embodiment, the antigen-binding portion specific to FolR1 comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% of the same heavy chain variable region sequence as SEQ ID NO:14 and at least about 95%, 96%, 97%, 98%, 99%, or 100% of the same light chain variable region sequence as SEQ ID NO:10, or retains a functional variant thereof.

In one embodiment, the antigen-binding portion specific to FolR1 comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:14; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:10.

Concealed parts

The protease-activated T-cell activation bispecific molecule of the present invention comprises at least one masking moiety. Others have attempted to mask antibody binding by capping the binding moiety with an antigen fragment recognized by the binding moiety (e.g., WO2013128194). This approach has several limitations. For example, the use of antigens offers less flexibility in reducing the affinity of the binding moiety. This is because the affinity must be high enough to be reliably masked by the antigen masker. Furthermore, the dissociated antigen may bind to and interact with its homologous receptor in vivo, generating undesirable signals to cells expressing such receptors. In contrast, the method described herein uses anti-idiotype antibodies or fragments thereof as a mask. Two conflicting considerations in designing an effective masking moiety are 1. the effectiveness of the masking and 2. the reversibility of the masking. If the affinity is too low, the masking will be inefficient. However, if the affinity is too high, the masking process may not be easily reversed. It is impossible to predict whether a high-affinity anti-idiotype mask or a low-affinity anti-idiotype mask will work better. As described herein, higher affinity masking portions generally perform better in masking the antigen-binding side while being effectively removed to activate the molecule. In one embodiment, the anti-idiotype mask has a KD of 1 to 8 nM. In one embodiment, the anti-idiotype mask has a KD of 2 nM at 37°C. In one specific embodiment, the masking portion recognizes the idiotype of a first antigen-binding portion capable of binding to CD3 (e.g., human CD3). In one specific embodiment, the masking portion recognizes the idiotype of a second antigen-binding portion capable of binding to a target cell antigen.

In one embodiment, the masking portion masks the CD3 bonding portion and includes at least one of the following: the heavy chain complementarity determination region (HCDR) 1 of SEQ ID NO:15, the HCDR 2 of SEQ ID NO:16, the HCDR 2 of SEQ ID NO:17, the HCDR 2 of SEQ ID NO:18, the HCDR 3 of SEQ ID NO:19, the light chain complementarity determination region (LCDR) 1 of SEQ ID NO:23, the LCDR 1 of SEQ ID NO:26, the LCDR 2 of SEQ ID NO:27, the LCDR 3 of SEQ ID NO:28, and the LCDR 3 of SEQ ID NO:29.

In one embodiment, the masking portion comprises: a VH region containing the HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15), the HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO:16), and the HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19); and a VL region containing the LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO:25), the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27), and the LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28).

In one embodiment, the masking portion comprises: a VH region containing the HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15), the HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO:16), and the HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19); and a VL region containing the LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO:26), the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27), and the LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28).

In a preferred embodiment, the masking portion comprises: a VH region containing the HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15), the HCDR2 amino acid sequence of WINTETGEPRYTDDFTG (SEQ ID NO:17), and the HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19); and a VL region containing the LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO:26), the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27), and the LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28).

In one embodiment, the masking portion comprises: a VH region containing the HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15), the HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO:18), and the HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19); and a VL region containing the LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO:26), the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27), and the LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28).

In one embodiment, the masking portion comprises: a VH region containing the HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15), the HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO:18), and the HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19); and a VL region containing the LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO:25), the LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27), and the LCDR3 amino acid sequence of QQSREFPYT (SEQ ID NO:29).

In one embodiment, the masking portion masks the CD3 binding portion and comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% the same polypeptide sequence as SEQ ID NO:20. In one embodiment, the masking portion masks the CD3 binding portion and comprises the polypeptide sequence of SEQ ID NO:30.

In a preferred embodiment, the masking portion is humanized. Methods for humanizing immunoglobulins are well known in the art and are described herein. Humanized masking portions H1L1, H1L2, H2L2, H3L2, H3L3, and H7L5 are provided herein. Corresponding sequences are provided below.

In one embodiment, the masking portion comprises: a heavy chain variable (VH) region 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:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24; and a light chain variable (VL) region 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:31, SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34.

In one embodiment, the masking portion comprises a VH region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:21 and a VL region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:31.

In a preferred embodiment, the masking portion comprises a VH region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:21 and a VL region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:32.

In one embodiment, the masking portion comprises a VH region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:22 and a VL region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:32.

In one embodiment, the masking portion comprises a VH region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:23 and a VL region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:32.

In one embodiment, the masking portion comprises a VH region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:23 and a VL region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:33.

In one embodiment, the masking portion comprises a VH region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:24 and a VL region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:34.

In one embodiment, the masking portion or idiotype-specific polypeptide used to reversibly conceal the antigen-binding molecule is scFc. Such idiotype-specific polypeptides for reversibly concealing the anti-CD3 antigen binding site must be able to bind idiotype-specifically to the anti-CD3 antigen binding site, thereby reducing or eliminating the binding of the anti-CD3 antigen binding site to CD3. In one embodiment, the idiotype scFc is used.

In one embodiment, the masking portion comprises a unique type-specific polypeptide for reversibly concealing antigen binding of the molecule. In one embodiment, the masking portion comprises a unique type-specific polypeptide. In a preferred embodiment, the unique type-specific polypeptide is scFv. In a preferred embodiment, the masking portion is scFv.

In a preferred embodiment, the scFv comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% the same polypeptide sequence as SEQ ID NO:35. In one embodiment, the anti-idiotype scFv comprises the polypeptide sequence of SEQ ID NO:35.

In one embodiment, the scFv comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequence to SEQ ID NO:36. In a preferred embodiment, the anti-idiotype scFv comprises the polypeptide sequence of SEQ ID NO:36.

In one embodiment, the scFv comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% the same polypeptide sequence as SEQ ID NO:37. In one embodiment, the anti-idiotype scFv comprises the polypeptide sequence of SEQ ID NO:37.

In one embodiment, the scFv comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% the same polypeptide sequence as SEQ ID NO:38. In one embodiment, the anti-idiotype scFv comprises the polypeptide sequence of SEQ ID NO:38.

Protease-cleavable adapters

The protease-activated T-cell activation bispecific molecule of the present invention comprises at least one protease-activated linker. Preferably, the protease-activated T-cell activation bispecific molecule of the present invention is inactive prior to cleavage by the protease-activated linker, for example, in the tumor microenvironment. In one embodiment, a masking portion (e.g., a idiotype-specific polypeptide) is covalently linked to the molecule via a linker. In one embodiment, the idiotype-specific polypeptide is covalently linked to the molecule via more than one linker. In one embodiment, the idiotype-specific polypeptide is covalently linked to the molecule via two linkers. In one embodiment, the linker is a peptide linker. In one embodiment, the linker is a protease-cleavable linker.

In one embodiment, the protease-cleavable adapter includes a protease recognition site. In one embodiment, the protease is a protein lyase. In a preferred embodiment, the protease-cleavable adapter includes a protein lyase recognition site.

In one embodiment, the protease-activated T-cell activation bispecific molecule comprises a linker having a protease recognition site containing the polypeptide sequence XQARK (SEQ ID NO: 39), where X is histidine (H) or proline (P). In one embodiment, the protease recognition site contains the polypeptide sequence HQARK (SEQ ID NO: 43). In a preferred embodiment, the protease recognition site contains the polypeptide sequence PQARK (SEQ ID NO: 41).

PQARK (SEQ ID NO:41) and HQARK (SEQ ID NO:43) are protein lyase recognition sites with advantageous and surprising properties. Ideally, protease-activated (therapeutic) molecules should be inactive before reaching their site of action (e.g., tumor). An advantageous property of the protein lyase recognition sites of the present invention (e.g., PQARK and HQARK) is that they are stable in vivo before reaching their site of action (see, for example, Figure 3). Furthermore, such activatable molecules should be effectively activated at the site of action (e.g., tumor). It is well known that the tumor microenvironment can exhibit a pH as low as 5.6 compared to physiological pH (approximately pH 7.4) (see, for example, Boedtkjer et al., 2020, Annual Review of Physiology, Vol. 82, 2020, pp. 103-126).

Importantly, compared to the published protein lyase recognition sites PMAKK, the protein lyase recognition sites of the present invention (e.g., PQARK and HQARK) are more strongly activated at physiological pH (see, for example, Table 4). Surprisingly, the protein lyase recognition sites of the present invention (e.g., PQARK and FOLR1 proTCB P035.093 HQARK 4.24.72 heavy chain 2P1AF5419) are strongly activated at pH as low as 5.6.

In one embodiment, the protein lyase recognition site is embedded in a linker, such as an (unstructured) peptide. In one embodiment, the peptide comprises one or more unstructured peptide linkers. In one embodiment, the isolated peptide comprises at least one peptide linker, particularly wherein the at least one peptide linker does not exhibit secondary structure. In one embodiment, the peptide comprises an amino acid sequence of at least 5 amino acids, preferably 5 to 100 amino acids, more preferably 10 to 50 amino acids, and most preferably 20 to 40 amino acids.

In one embodiment, the protease-cleavable linker is a polypeptide of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids in length. In a preferred embodiment, the protease-cleavable linker is a peptide of 33 amino acids in length. In one embodiment, the polypeptide contains a protease recognition site. In one embodiment, the protease recognition sequence is a substrate of a protease. In one embodiment, the protease recognition site contains or consists of the sequence PQARK (SEQ ID NO: 41) or HQARK (SEQ ID NO: 43).

In one embodiment, the protease-cleavable linker is an unstructured polypeptide. In one embodiment, the protease-cleavable linker does not exhibit secondary structure. In one embodiment, the protease-cleavable linker comprises at least one linker that facilitates the identification of unstructured polypeptides. In one embodiment, the linker comprises serine (S) and/or glycine (G). In one embodiment, at least one linker of the protease-cleavable linker comprises the amino acid sequence (GxS)n or (GxS)nGm, where G = glycine, S = serine, and (x = 3, n = 3, 4, 5, or 6, and m = 0, 1, 2, or 3) or (x = 4, n = 2, 3, 4, or 5, and m = 0, 1, 2, or 3), preferably x = 4 and n = 2 or 3, more preferably x = 4 and n = 2. In one embodiment, the protease-cleavable linker comprises (G ₄ S) _2. In one embodiment, the protease-cleavable linker comprises (G ₄ S) _3. In one embodiment, the protease-cleavable linker comprises G ₂ S. Protease-cleavable adapters contain protease recognition sites at any location (e.g., at the beginning, within, or end of the adapter).

In one embodiment, the isolated polypeptide contains a sequence

SGGGSGGGGSPQARKGGGGSGGGGSGGGGSGGS (SEQ ID NO:42) or composed thereof. In one embodiment, the isolated polypeptide comprises the sequence

SGGGSGGGGSHQARKGGGGSGGGGSGGGGSGGS (SEQ ID NO:44) or composed of it.

Bispecific molecular form of protease-activated T cell activation

Components of a protease-activated T-cell activation dual-specific molecule can be fused together in multiple configurations. An exemplary configuration is depicted in Figure 1.

In certain embodiments, the protease-activated T-cell activation bispecific molecule comprises an Fc domain consisting of a first subunit and a second subunit capable of stable association. In some embodiments, the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of either the first or second subunit of the Fc domain.

In one 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-activated T-cell activation bispecific molecule essentially consists of a first antigen-binding moiety and a second antigen-binding moiety, an Fc domain composed of a first subunit 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 either the first or second subunit of the Fc domain. Additionally, optionally, the Fab light chains of the first antigen-binding moiety and the Fab light chains of the second antigen-binding moiety may be fused to each other.

In another such embodiment, the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of either the first or second subunit of the Fc domain. In a specific such embodiment, the protease-activated T-cell activation bispecific molecule substantially comprises a first antigen-binding portion and a second antigen-binding portion, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, wherein each of the first and second antigen-binding portions is 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 first antigen-binding portion 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 one particular 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 embodiment of this kind, the protease-activated T-cell activation bispecific molecule essentially consists of a first antigen-binding moiety and a second antigen-binding moiety, an Fc domain composed of a first subunit 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 either the first or second subunit of the Fc domain. Additionally, optionally, the Fab light chains of the first antigen-binding moiety and the Fab light chains of the second antigen-binding moiety may be fused to each other.

The antigen-binding moieties may fuse directly to or to each other via a peptide linker comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art and described herein. Suitable non-immunogenic peptide linkers include _, for example, ( _G4S ) _n , (SG4) _n , ( _G4S ) _n , or _G4 ( _SG4 ) _n peptide linkers. “n” is typically a number between 1 and 10, and usually between 2 and 4. A particularly suitable peptide linker for fusing the Fab light chains of the first and second antigen-binding moieties to each other is ( _G4S ) ₂ . Additionally, the linker may include a portion of an immunoglobulin hinge region. Specifically, at the N-terminus where the antigen-binding moieties fuse to the Fc domain subunit, fusion may be made via the immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.

T-cell activation bispecific molecules with a single antigen-binding moiety capable of binding to target cell antigens are useful, particularly in cases where the internalization of the target cell antigen is expected to occur after the binding of a high-affinity antigen-binding moiety. In such cases, the presence of more than one antigen-binding moiety specific to the target cell antigen may enhance the internalization of the target cell antigen, thereby reducing its availability.

However, in many other cases, T-cell activation bispecific molecules containing two or more antigen-binding moieties that are specific to target cell antigens are advantageous, for example, for optimizing targeting of target sites or for cross-linking target cell antigens.

Therefore, in some embodiments, the protease-activated T-cell activation bispecific molecule of the present invention further comprises a third antigen-binding moiety, which is a Fab molecule capable of binding to a target cell antigen. 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 the same target cell antigen 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 the target cell antigen. In a particular embodiment, the second and third antigen-binding moieties are identical (i.e., they contain the same amino acid sequence).

In a particular embodiment, the first antigen-binding portion is capable of binding to CD3, and the second and third antigen-binding portions are capable of binding to FolR1, wherein the second and third antigen-binding portions include at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13 and at least one light chain CDR selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.

In one embodiment, the protease-activated T-cell activation bispecific molecule comprises

(i) A first antigen-binding portion, which is a Fab molecule capable of binding to CD3, and comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 and at least one light chain CDR selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9;

(ii) The second antigen-binding part is a Fab molecule that can bind to the target cell antigen.

In one embodiment, the first antigen-binding portion comprises: a heavy chain variable region containing at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to that of SEQ ID NO:5; and a light chain variable region containing at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to that of SEQ ID NO:10.

In one embodiment, the first antigen-binding portion comprises: a heavy chain variable region containing the amino acid sequence of SEQ ID NO:5; and a light chain variable region containing the amino acid sequence of SEQ ID NO:10.

In one specific embodiment, the second antigen-binding portion is capable of binding to FolR1 and includes at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13 and at least one light chain CDR selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.

In another specific embodiment, the second antigen-binding portion is capable of binding to FolR1 and comprises: a heavy chain variable region containing at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to that of SEQ ID NO:14; and a light chain variable region containing at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to that of SEQ ID NO:10.

In one embodiment, the present invention provides a protease-activated T-cell activation bispecific molecule comprising...

(i) A first antigen-binding portion, which is a Fab molecule capable of binding to CD3, comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:30 and at least one light chain CDR selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9;

(ii) A second antigen-binding portion, which is a Fab molecule capable of specifically binding to FolR1, and comprising at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13 and at least one light chain CDR selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.

In one embodiment, the present invention provides a protease-activated T-cell activation bispecific molecule comprising...

(i) A first antigen-binding region, which is a Fab molecule capable of binding to CD3, comprising: a heavy chain variable region containing 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:5; and a light chain variable region containing 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:10.

(ii) A second antigen-binding region, which is a Fab molecule capable of binding to FolR1, comprising: a heavy chain variable region comprising 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:14; and a light chain variable region comprising 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:10.

In one embodiment, the second antigen-binding portion is a conventional Fab molecule.

In one specific embodiment, the first antigen-binding portion is a cross-Fab molecule, wherein constant regions of the Fab light chain and the Fab heavy chain are exchanged, and the second antigen-binding portion is a conventional Fab molecule. In another specific embodiment, the first and second antigen-binding portions are fused together, optionally via peptide linkers.

In a particular embodiment, the protease-activated T-cell activation bispecific molecule further comprises an Fc domain consisting of a first subunit and a second subunit capable of stable association.

In another specific embodiment, the protease-activated T-cell activation bispecific molecule contains no more than one antigen-binding moiety capable of binding to CD3 (i.e., the protease-activated T-cell activation bispecific molecule provides monovalent binding to CD3).

In one specific embodiment, the first antigen-binding portion is capable of binding to CD3 and includes at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 and at least one light chain CDR selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9; and the second and third antigen-binding portions are capable of binding to FolR1, wherein the second and third antigen-binding portions include at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13 and at least one light chain CDR selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.

In one specific embodiment, the first antigen-binding portion is capable of binding to CD3 and includes at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 and at least one light chain CDR selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9; and the second and third antigen-binding portions are capable of binding to FolR1, wherein the second and third antigen-binding portions include at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13 and at least one light chain CDR selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.

In one specific embodiment, the first antigen-binding portion is capable of binding to CD3 and comprises: a heavy chain variable region containing at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO:5, and a light chain variable region containing at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO:10; and the second and third antigen-binding portions are capable of binding to FolR1, wherein the second and third antigen-binding portions comprise: a heavy chain variable region containing at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO:14, and a light chain variable region containing at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO:10.

The second and third antigen-binding portions may be fused to the Fc domain directly or via peptide linkers. In one specific embodiment, the second and third antigen-binding portions are each fused to the Fc domain via an immunoglobulin hinge region. In one specific embodiment, the immunoglobulin hinge region is a human _IgG1 hinge region. In one embodiment, the second and third antigen-binding portions and the Fc domain are portions of an immunoglobulin molecule. In one specific embodiment, the immunoglobulin molecule is an IgG class immunoglobulin. In one or more specific embodiments, the immunoglobulin is an _IgG1 subclass immunoglobulin. In another embodiment, the immunoglobulin is an _IgG4 subclass immunoglobulin. In another specific embodiment, the immunoglobulin is a human immunoglobulin. In other embodiments, the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin. In one embodiment, the protease-activated T-cell bispecific molecule is essentially composed of an immunoglobulin molecule capable of binding to a target cell antigen and an antigen-binding moiety capable of binding to CD3, wherein the antigen-binding moiety is a Fab molecule, particularly a cross-Fab molecule, which is fused to the N-terminus of one of the immunoglobulin heavy chains, optionally via a peptide linker.

In one particular embodiment, the first antigen-binding portion and the third antigen-binding portion 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 portion 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 portion. In a specific embodiment of this kind, the protease-activated T-cell activation bispecific molecule substantially comprises a first antigen-binding portion, a second antigen-binding portion, a third antigen-binding portion, an Fc domain composed of a first subunit and a second subunit, and optionally one or more peptide linkers, wherein the second antigen-binding portion 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 portion, and the first antigen-binding portion 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 portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Additionally, optionally, the Fab light chain of the first antigen-binding portion and the Fab light chain of the second antigen-binding portion may be fused to each other.

In one embodiment, the present invention provides a protease-activated T-cell activation bispecific molecule comprising...

(i) The first antigen-binding portion is a Fab molecule capable of binding to CD3, comprising the heavy chain complementarity-determining region (CDR) 1 of SEQ ID NO:1, the heavy chain CDR 2 of SEQ ID NO:2, the heavy chain CDR 3 of SEQ ID NO:3, the light chain CDR 1 of SEQ ID NO:7, and the light chain CDR 3 of SEQ ID NO:8 and SEQ ID NO:9, wherein the first antigen-binding portion is a cross-Fab molecule in which the variable or constant regions, particularly the constant regions, of the Fab light chain and the Fab heavy chain are exchanged;

(ii) The second antigen-binding portion and the third antigen-binding portion are each Fab molecules capable of binding to FolR1, comprising the heavy chain CDR 1 of SEQ ID NO:11, the heavy chain CDR 2 of SEQ ID NO:12, the heavy chain CDR 3 of SEQ ID NO:13, the light chain CDR 1 of SEQ ID NO:7, the light chain CDR 2 of SEQ ID NO:8, and the light chain CDR 3 of SEQ ID NO:9.

In one embodiment, the present invention provides a protease-activated T-cell activation bispecific molecule comprising...

(i) A first antigen-binding portion, which is a Fab molecule capable of binding to CD3, comprising: a heavy chain variable region comprising 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:5; and a light chain variable region comprising 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:10, wherein the first antigen-binding portion is a cross-Fab molecule, wherein the variable or constant regions, particularly the constant regions, of the Fab light chain and the Fab heavy chain are exchanged;

(ii) The second antigen-binding portion and the third antigen-binding portion, each being a Fab molecule capable of binding to FolR1, comprising: a heavy chain variable region comprising 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:14; and a light chain variable region comprising 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:10.

The protease-activated T-cell activation bispecific molecule according to any of the ten embodiments above may further include (iii) an Fc domain consisting of a first subunit and a second subunit capable of stable association, wherein the second antigen-binding portion 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 portion, and the first antigen-binding portion 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 portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

In some of the protease-activated T-cell activation bispecific molecules of the present invention, the Fab light chain of the first antigen-binding moiety and the Fab light chain of the second antigen-binding moiety are fused together, optionally via a linker peptide. Depending on the configuration of the first and second antigen-binding moieties, 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. The fusion of the Fab light chains of the first and second antigen-binding moieties further reduces the mismatch between the Fab heavy chain and the light chain, and also reduces the number of plasmids required to express some of the protease-activated T-cell activation bispecific molecules of the present invention.

In some embodiments, the protease-activated T-cell activation bispecific molecule comprises: a polypeptide wherein the Fab light chain variable region of the first antigen-binding moiety shares a carboxyl-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 cross-linked Fab heavy chain, wherein the heavy chain variable region is replaced by the light chain variable region), and the Fab heavy chain constant region of the first antigen-binding moiety subsequently shares a carboxyl-terminal peptide bond with the Fc domain subunit (VL ₍₁₎ -CH1 ₍₁₎ -CH2-CH3(-CH4)); and a polypeptide wherein the Fab heavy chain of the second antigen-binding moiety shares a carboxyl-terminal peptide bond with the Fc subunit (VH ₍₂₎ -CH1 ₍₂₎ -CH2-CH3(-CH4)). In some embodiments, the protease-activated T-cell activation bispecific molecule further comprises: a polypeptide wherein the variable region of the Fab heavy chain of the first antigen-binding moiety shares a carboxyl-terminal peptide bond (VH ₍₁₎ -CL ₍₁₎ ) with the constant region of the Fab light chain of the first antigen-binding moiety, and shares a carboxyl-terminal peptide bond (VL ₍₂₎ -CL ₍₂₎ ) with the Fab light chain polypeptide of the second antigen-binding moiety. In some embodiments, the polypeptide is covalently linked, for example, by a disulfide bond.

In an alternative embodiment, the protease-activated T-cell activation bispecific molecule comprises: a polypeptide wherein the variable region of the Fab heavy chain of the first antigen-binding moiety shares a carboxyl-terminal peptide bond with the constant region of the Fab light chain of the first antigen-binding moiety (i.e., the first antigen-binding moiety comprises a cross-Fab heavy chain, wherein the heavy chain constant region is replaced by the light chain constant region), and the constant region of the Fab light chain of the first antigen-binding moiety subsequently shares a carboxyl-terminal peptide bond with an Fc domain subunit (VH ₍₁₎ -CL ₍₁₎ -CH2-CH3(-CH4)); and a polypeptide wherein the Fab heavy chain of the second antigen-binding moiety shares a carboxyl-terminal peptide bond with an Fc domain subunit (VH ₍₂₎ -CH1 ₍₂₎ -CH2-CH3(-CH4)). In some embodiments, the protease-activated T-cell activation bispecific molecule further comprises: a polypeptide wherein the variable region of the Fab light chain of the first antigen-binding moiety shares a carboxyl-terminal peptide bond (VL ₍₁₎ -CH1 ₍₁₎ ) with the constant region of the Fab heavy chain of the first antigen-binding moiety, and shares a carboxyl-terminal peptide bond (VL ₍₂₎ -CL ₍₂₎ ) with the Fab light chain polypeptide of the second antigen-binding moiety. In some embodiments, the polypeptide is covalently linked, for example, by a disulfide bond.

In some embodiments, the protease-activated T-cell activation bispecific molecule comprises: a polypeptide wherein the Fab light chain variable region of the first antigen-binding portion shares a carboxyl-terminal peptide bond with the Fab heavy chain constant region of the first antigen-binding portion (i.e., the first antigen-binding portion comprises a cross-linked Fab heavy chain, wherein the heavy chain variable region is replaced by the light chain variable region), the Fab heavy chain constant region of the first antigen-binding portion then shares a carboxyl-terminal peptide bond with the Fab heavy chain of the second antigen-binding portion, and the Fab heavy chain of the second antigen-binding portion then shares a carboxyl-terminal peptide bond with the Fc domain subunit (VL ₍₁₎ -CH1 ₍₁₎ -VH ₍₂₎ -CH1 ₍₂₎ -CH2-CH3(-CH4)). In other embodiments, the protease-activated T-cell activation bispecific molecule comprises: a polypeptide wherein the variable region of the Fab heavy chain of the first antigen-binding moiety shares a carboxyl-terminal peptide bond with the constant region of the Fab light chain of the first antigen-binding moiety (i.e., the first antigen-binding moiety comprises a cross-linked Fab heavy chain, wherein the heavy chain constant region is replaced by the light chain constant region), the constant region of the Fab light chain of the first antigen-binding moiety then shares a carboxyl-terminal peptide bond with the Fab heavy chain of the second antigen-binding moiety, and the Fab heavy chain of the second antigen-binding moiety then shares a carboxyl-terminal peptide bond with the Fc domain subunit (VH ₍₁₎ -CL ₍₁₎ -VH ₍₂₎ -CH1 ₍₂₎ -CH2-CH3(-CH4)). In yet another embodiment, the protease-activated T-cell activation bispecific molecule comprises: a polypeptide wherein the Fab heavy chain of the second antigen-binding portion shares a C-terminal peptide bond with the variable region of the Fab light chain of the first antigen-binding portion, the variable region of the Fab light chain of the first antigen-binding portion subsequently shares a C-terminal peptide bond with the constant region of the Fab heavy chain of the first antigen-binding portion (i.e., the first antigen-binding portion comprises a cross-Fab heavy chain, wherein the heavy chain variable region is replaced by the light chain variable region), and the constant region of the Fab heavy chain of the first antigen-binding portion subsequently shares a C-terminal peptide bond with an Fc domain subunit (VH ₍₂₎ -CH1 ₍₂₎ -VL ₍₁₎ -CH1 ₍₁₎ -CH2-CH3(-CH4)). In other embodiments, the protease-activated T-cell activation bispecific molecule comprises: a polypeptide wherein the Fab heavy chain of the second antigen-binding moiety shares a C-terminal peptide bond with the variable region of the Fab heavy chain of the first antigen-binding moiety, the variable region of the Fab heavy chain of the first antigen-binding moiety subsequently shares a C-terminal peptide bond with the constant region of the Fab light chain of the first antigen-binding moiety (i.e., the first antigen-binding moiety comprises a cross-Fab heavy chain, wherein the heavy chain constant region is replaced by the light chain constant region), and the constant region of the Fab light chain of the first antigen-binding moiety subsequently shares a C-terminal peptide bond with an Fc domain subunit (VH ₍₂₎ -CH1 ₍₂₎ -VH ₍₁₎ -CL ₍₁₎ -CH2-CH3(-CH4)).

In some of these embodiments, the protease-activated T-cell activation bispecific molecule further comprises: a cross-Fab light chain polypeptide of the first antigen-binding moiety, wherein the variable region of the Fab heavy chain of the first antigen-binding moiety shares a C-terminal peptide bond (VH ₍₁₎ -CL ₍₁₎ ) with the constant region of the Fab light chain of the first antigen-binding moiety, and shares a C-terminal peptide bond (VL ₍₂₎ -CL ₍₂₎ ) with the Fab light chain polypeptide of the second antigen-binding moiety. In other such embodiments, the protease-activated T-cell activation bispecific molecule further comprises: a cross-Fab light chain polypeptide, wherein the variable region of the Fab light chain of the first antigen-binding moiety shares a C-terminal peptide bond (VL ₍₁₎ -CH1 ₍₁₎ ) with the constant region of the Fab heavy chain of the first antigen-binding moiety, and shares a C-terminal peptide bond (VL ₍₂₎ -CL ₍₂₎ ) with the Fab light chain polypeptide of the second antigen-binding moiety. In other such embodiments, the protease-activated T-cell activation bispecific molecule further comprises: a polypeptide wherein the variable region of the Fab light chain of the first antigen-binding moiety shares a C-terminal peptide bond with the constant region of the Fab heavy chain of the first antigen-binding moiety, and the constant region of the Fab heavy chain of the first antigen-binding moiety subsequently shares a C-terminal peptide bond with the Fab light chain polypeptide of the second antigen-binding moiety (VL ₍₁₎ -CH1 ₍₁₎ -VL ₍₂₎ -CL ₍₂₎ ); and a polypeptide wherein the variable region of the Fab heavy chain of the first antigen-binding moiety shares a C-terminal peptide bond with the constant region of the Fab light chain of the first antigen-binding moiety, and the constant region of the Fab light chain of the first antigen-binding moiety subsequently shares a peptide bond with the Fab light chain polypeptide of the second antigen-binding moiety (VH ₍₁₎ -CL ₍₁₎ -VL ₍₂₎ -CL _(2)). ); polypeptide, wherein the Fab light chain polypeptide of the second antigen-binding part shares a carboxyl-terminal peptide bond with the variable region of the Fab light chain of the first antigen-binding part, and the variable region of the Fab light chain of the first antigen-binding part then shares a carboxyl-terminal peptide bond with the constant region of the Fab heavy chain of the first antigen-binding part (VL ₍₂₎ -CL ₍₂₎ -VL ₍₁₎ -CH1 ₍₁₎ ); or polypeptide, wherein the Fab light chain polypeptide of the second antigen-binding part shares a carboxyl-terminal peptide bond with the variable region of the Fab heavy chain of the first antigen-binding part, and the variable region of the Fab heavy chain of the first antigen-binding part then shares a carboxyl-terminal peptide bond with the constant region of the Fab light chain of the first antigen-binding part (VL ₍₂₎ -CL ₍₂₎ -VH ₍₁₎ -CL ₍₁₎ ).

The protease-activated T-cell activation bispecific 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 the third antigen-binding moiety shares a carboxyl-terminal peptide bond with the Fc domain subunit (VH ₍₃₎ -CH1 ₍₃₎ -CH2-CH3(-CH4)) and shares a carboxyl-terminal peptide bond with the Fab light chain polypeptide of the third antigen-binding moiety (VL ₍₃₎ -CL ₍₃₎ ). In some embodiments, the polypeptide is covalently linked, for example, by a disulfide bond.

According to any of the above embodiments, components of the protease-activated T-cell activation bispecific molecule (e.g., antigen-binding moiety, Fc domain) can be directly fused or fused via various linkers, particularly via peptide linkers containing one or more amino acids (typically about 2 to 20 amino acids) as described herein or known in the domain. Suitable non-immunogenic peptide linkers include, for example, ( _G4S ) _n , ( _SG4 ) _n , ( _G4S ) _n , or _G4 ( _SG4 ) _n peptide linkers, where "n" is typically a number between 1 and 10, and typically between 2 and 4.

Fc structural domain

The Fc domain of the protease-activated T-cell activation bispecific molecule consists of a pair of polypeptide chains containing the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which contains CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain can stably associate with each other. In one embodiment, the protease-activated T-cell activation bispecific molecule of the present invention contains no more than one Fc domain.

In one embodiment of the invention, the Fc domain of the protease-activated T-cell activation bispecific molecule is an IgG Fc domain. In a particular embodiment, the Fc domain is an IgG ₁ Fc domain. In another embodiment, the Fc domain is an IgG ₄ Fc domain. In a more specific embodiment, the Fc domain is an IgG ₄ Fc domain containing an amino acid substitution (Kabat number) at position S228, specifically amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of the IgG ₄ antibody (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In another particular embodiment, the Fc domain is a human Fc domain.

Fc domain modification that promotes heterodimerization

The protease-activated T-cell activation bispecific molecule according to the invention comprises different antigen-binding moieties fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically contained in two different polypeptide chains. Recombinant co-expression and subsequent dimerization of these polypeptides result in several possible combinations of the two polypeptides. To improve the yield and purity of the protease-activated T-cell activation bispecific molecule in recombinant production, it would be advantageous to introduce modifications into the Fc domain of the protease-activated T-cell activation bispecific molecule that promote the association of the desired polypeptide.

Therefore, in a particular embodiment, the Fc domain of the protease-activated T-cell activation bispecific molecule according to the invention includes modifications that promote the association of the first and second subunits of the Fc domain. The most extensive protein-protein interaction site between the two subunits of the human IgG Fc domain is located in the CH3 domain of the Fc domain. Therefore, in one embodiment, the modification is located in the CH3 domain of the Fc domain.

In one specific embodiment, the modification is a so-called "knob-into-hole" modification, which includes a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain.

The pestle-and-mortar structure technique is described, for example, 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). Typically, this method involves introducing a protrusion (“pestle”) at the interface of a first polypeptide and a corresponding cavity (“mortar”) at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote the formation of a heterodimer and inhibit the formation of a homodimer. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensating cavity having the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

Therefore, in a specific embodiment, in the CH3 domain of the first subunit of the Fc domain of the protease-activated T cell activation dual-specificity molecule, amino acid residues are replaced by amino acid residues with larger side chain volumes, thereby generating a protrusion within the CH3 domain of the first subunit. This protrusion can be positioned in a cavity within the CH3 domain of the second subunit. Furthermore, in the CH3 domain of the second subunit of the Fc domain, amino acid residues are replaced by amino acid residues with smaller side chain volumes, thereby generating a cavity within the CH3 domain of the second subunit. The protrusion within the CH3 domain of the first subunit can be positioned within this cavity.

Protrusions and cavities can be prepared by altering the nucleic acid encoding the polypeptide (e.g., through site-specific mutagenesis or through 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 by 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 by a valine residue (Y407V). In one embodiment, additionally, in the second subunit of the Fc domain, the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A).

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

In one particular embodiment, the CD3-binding antigen-binding moiety (optionally via an antigen-binding moiety capable of binding to target cell antigens) is fused to a first subunit of the Fc domain (containing a "clublet" modification). Not wishing to be bound by theory, the fusion of the CD3-binding antigen-binding moiety with the clublet-containing subunit of the Fc domain will (further) minimize the generation of antigen-binding molecules comprising two CD3-binding antigen-binding moieties (spatial collision of two clublet-containing polypeptides).

In an alternative embodiment, the modifications that promote association between the first and second subunits of the Fc domain include modifications that mediate electrostatic reorientation effects, such as those described in PCT Publication WO 2009/089004. Typically, this method involves replacing one or more amino acid residues at the interface between the two Fc domain subunits with charged amino acid residues, making homodimer formation electrostatically unfavorable but heterodimerization electrostatically favorable.

Fc domain modifications that reduce Fc receptor binding and/or effector function

The Fc domain endows the protease-activated T-cell activation bispecific molecule with favorable pharmacokinetic properties, including a long serum half-life, which contributes to a good accumulation ratio and favorable tissue-blood partition ratio in the target tissue. However, this may simultaneously lead to undesirable targeting of the protease-activated T-cell activation bispecific molecule to cells expressing the Fc receptor, rather than to preferred antigen-bearing cells. Furthermore, co-activation of the Fc receptor signaling pathway may result in cytokine release, which, combined with the T-cell activation properties of the antigen-binding molecule and its long half-life, leads to overactivation of cytokine receptors and severe side effects after systemic administration. Activation of immune cells other than T cells (carrying the Fc receptor) may even reduce the efficacy of the protease-activated T-cell activation bispecific molecule due to its potential damage to T cells (e.g., by NK cells).

Therefore, in certain embodiments, the Fc domain of the protease-activated T-cell activation bispecific molecule according to the invention exhibits reduced binding affinity to the Fc receptor and/or reduced effector function compared to the native _IgG1 Fc domain. In one such embodiment, the Fc domain (or the protease-activated T-cell activation bispecific molecule containing said Fc domain) exhibits a binding affinity to the Fc receptor of less than ₅₀ %, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% compared to the native _{IgG1 Fc} domain (or the protease-activated T-cell activation bispecific molecule containing the native IgG1 Fc domain), and/or an effector function of less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% compared to the native IgG1 Fc domain (or the protease-activated T-cell activation bispecific molecule containing the native _IgG1 Fc domain). In one embodiment, the Fc domain (or a protease-activated T-cell activation bispecific molecule containing said Fc domain) substantially does not bind to the Fc receptor and/or induce effector function. In a particular embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In a particular embodiment, the Fc receptor is an activated human Fcγ receptor, more particularly human FcγRIIIa, FcγRI, or FcγRIIa, and most particularly human FcγRIIIa. In one embodiment, the effector function is one or more effector functions selected from the group consisting 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 the nascent Fc receptor (FcRn) compared to the native _IgG1 Fc domain. When the Fc domain (or a protease-activated T-cell activation bispecific molecule containing the Fc domain) exhibits a binding affinity greater than about 70%, particularly greater than about 80%, and more particularly greater than about 90% of the binding affinity of the native IgG ₁ Fc domain (or a protease-activated T-cell activation bispecific molecule containing the native IgG ₁ Fc domain) for FcRn, substantially similar binding to FcRn is achieved.

In some embodiments, the Fc domain is engineered to have reduced binding affinity for the Fc receptor and/or reduced effector function compared to an unengineered Fc domain. In a particular embodiment, the Fc domain of a protease-activated T-cell activation bispecific molecule contains one or more amino acid mutations that reduce the binding affinity of the Fc domain to the Fc receptor and/or effector function. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where more than one amino acid mutation reducing the binding affinity of the Fc domain to the Fc receptor is present, the combination of these amino acid mutations can reduce the binding affinity of the Fc domain to the Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment, a protease-activated T-cell activation bispecific molecule containing an engineered Fc domain exhibits a binding affinity of less than 20%, particularly less than 10%, and more particularly less than 5%, for the Fc receptor compared to a protease-activated T-cell activation bispecific molecule containing a non-engineered Fc domain. In a particular embodiment, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activated Fc receptor. In a particular embodiment, the Fc receptor is an activated human Fcγ receptor, more particularly human FcγRIIIa, FcγRI, or FcγRIIa, and most particularly human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, binding affinity to complement components, particularly to C1q, is also reduced. In one embodiment, binding affinity to the nascent Fc receptor (FcRn) is not reduced. When the Fc domain (or a protease-activated T-cell activation bispecific molecule containing said Fc domain) exhibits a binding affinity to FcRn greater than about 70% for the unengineered form of the Fc domain (or a protease-activated T-cell activation bispecific molecule containing said unengineered Fc domain), substantially similar binding to FcRn is achieved, i.e., the binding affinity of the Fc domain to the receptor is maintained. The Fc domain or the protease-activated T-cell activation bispecific molecule of the present invention containing said Fc domain can exhibit such affinity greater than about 80% and even greater than about 90%. In some embodiments, the Fc domain of the protease-activated T-cell activation bispecific molecule is engineered to have reduced effector function compared to the unengineered Fc domain. The reduced effector function may 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 antibody-dependent 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 signal transduction-induced apoptosis, reduced cross-linking of target-binding antibodies, reduced dendritic cell maturation, or reduced T cell sensitization. In one embodiment, the reduced effector function is one or more of the reduced effector functions selected from the group consisting 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 ADCC induced by a non-engineered Fc domain (or a protease-activated T cell activation bispecific molecule containing a non-engineered Fc domain).

In one embodiment, the amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor and/or the effector function is an amino acid substitution. In one embodiment, the Fc domain contains an amino acid substitution at a position selected from the group consisting of E233, L234, L235, N297, P331, and P329. In a more specific embodiment, the Fc domain contains an amino acid substitution at a position selected from the group consisting of L234, L235, and P329. In some embodiments, the Fc domain contains amino acid substitutions for L234A and L235A. In one such embodiment, the Fc domain is an IgG ₁ Fc domain, particularly the human IgG ₁ Fc domain. In one embodiment, the Fc domain contains an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment, the Fc domain contains an amino acid substitution at position P329, and further amino acid substitutions at positions selected from E233, L234, L235, N297, and P331. In a more specific embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a particular embodiment, the Fc domain includes amino acid substitutions at positions P329, L234, and L235. In a more specific embodiment, the Fc domain includes amino acid mutations L234A, L235A, and P329G (“P329GLALA”). In one such embodiment, the Fc domain is the IgG ₁ Fc domain, particularly the human IgG ₁ Fc domain. The “P329G LALA” combination of amino acid substitutions almost completely eliminates Fcγ receptor (and complement) binding of the human IgG ₁ Fc domain, as described in PCT Publication WO 2012/130831, which is incorporated herein by reference in its entirety. WO 2012/130831 also describes methods for preparing such mutant Fc domains and methods for determining their properties (such as Fc receptor binding or effector function).

Compared to _IgG1 antibodies, _IgG4 antibodies exhibit reduced binding affinity to the Fc receptor and reduced effector function. Therefore, in some embodiments, the Fc domain of the protease-activated T-cell activation bispecific molecule of the present invention is the _IgG4 Fc domain, particularly the human _IgG4 Fc domain. In one embodiment, the _IgG4 Fc domain contains an amino acid substitution at position S228, particularly the S228P amino acid substitution. To further reduce its binding affinity to the Fc receptor and/or its effector function, in one embodiment, the _IgG4 Fc domain contains an amino acid substitution at position L235, particularly the L235E amino acid substitution. In another embodiment, the _IgG4 Fc domain contains an amino acid substitution at position P329, particularly the P329G amino acid substitution. In a specific embodiment, the _IgG4 Fc domain contains amino acid substitutions at positions S228, L235, and P329, particularly the S228P, L235E, and P329G amino acid substitutions. These IgG ₄ Fc domain mutants and their Fcγ receptor binding properties are described in PCT Publication No. WO 2012/130831, the entire contents of which are incorporated herein by reference.

In one particular embodiment, the Fc domain exhibiting reduced binding affinity to the Fc receptor and/or reduced effector function compared to the native _IgG1 Fc domain is a human _IgG1 Fc domain containing amino acid substitutions of L234A, L235A, and optionally P329G, or a human _IgG4 Fc domain containing amino acid substitutions of S228P, L235E, and optionally P329G.

In some embodiments, N-glycosylation of the Fc domain has been eliminated. In one such embodiment, the Fc domain contains an amino acid mutation at position N297, specifically replacing the amino acid substitution of asparagine with alanine (N297A) or aspartic acid (N297D).

In addition to the Fc domains described above and in PCT Publication WO 2012/130831, Fc domains with reduced Fc receptor binding and/or reduced effector function also include those Fc domains with mutations in one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant, in which residues 265 and 297 are substituted with alanine (US Patent No. 7,332,581).

Mutant Fc domains can be prepared using genetic or chemical methods well-known in the art, through amino acid deletion, substitution, insertion, or modification. Genetic methods may include site-specific mutagenesis of coding DNA sequences, PCR, gene synthesis, etc. Correct nucleotide changes can be verified, for example, by sequencing.

Binding to Fc receptors can be readily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard instruments such as BIAcore instruments (GE Healthcare) and Fc receptors can be obtained, for example, through recombinant expression. Suitable binding assays of this kind are described in this article. Alternatively, the binding affinity of Fc domains or cell-activating bispecific antigen-binding molecules containing Fc domains to Fc receptors can be evaluated using cell lines known to express specific Fc receptors, such as human NK cells expressing the FcγIIIa receptor.

The effector function of Fc domain- or protease-containing proteases that can activate T cell activation bispecific molecules can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays for assessing the ADCC activity of molecules of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al., ProcNatl Acad Sci USA 83,7059-7063 (1986) and Hellstrom et al., ProcNatl 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 can be used (see, for example, the ACTI ^™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc., Mountain View, CA); and the CytoTox non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the target molecule can be assessed in vivo, for example, in animal models such as those disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

In some embodiments, the binding of the Fc domain to complement components, particularly C1q, is reduced. Therefore, in some embodiments, the Fc domain is engineered to have reduced effector functions, including reduced CDC. C1q binding assays can be performed to determine whether a protease-activated T-cell activation bispecific molecule is able to bind C1q and thus have CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006/029879 and WO 2005/100402. To assess complement activation, CDC assays can 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)).

Exemplary proteases capable of binding to CD3 and FolR1 can activate T cell activation bispecific molecules.

The first antigen-binding portion capable of binding to CD3, the second antigen-binding portion capable of binding to FolR1, the Fc domain, the masking portion, and the protease-cleavable linker of the present invention, as described above, can be fused together in various configurations.

An exemplary configuration is shown in Figure 1. An exemplary sequence is shown below.

In one embodiment, the protease-activated T-cell activation bispecific molecule comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:45, at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:46, and at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:48. In one embodiment, the protease-activated T-cell activation bispecific molecule comprises the polypeptide sequences of SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:48.

In one embodiment, the protease-activated T-cell activation bispecific molecule comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:45, at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:46, and at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:49. In one embodiment, the protease-activated T-cell activation bispecific molecule comprises the polypeptide sequences of SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:49.

In one embodiment, the protease-activated T-cell activation bispecific molecule comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:45, at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:46, and at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:50. In one embodiment, the protease-activated T-cell activation bispecific molecule comprises the polypeptide sequences of SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:50.

In one embodiment, the protease-activated T-cell activation bispecific molecule comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:45, at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:46, and at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:51. In one embodiment, the protease-activated T-cell activation bispecific molecule comprises the polypeptide sequences of SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:51.

In one embodiment, the protease-activated T-cell activation bispecific molecule comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:45, at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:46, and at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:52. In one embodiment, the protease-activated T-cell activation bispecific molecule comprises the polypeptide sequences of SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:52.

In one embodiment, the protease-activated T-cell activation bispecific molecule comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:45, at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:46, and at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:53. In one embodiment, the protease-activated T-cell activation bispecific molecule comprises the polypeptide sequences of SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:53.

In a preferred embodiment, the protease-activated T-cell activation bispecific molecule comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:45, at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:46, and at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:54. In a more preferred embodiment, the protease-activated T-cell activation bispecific molecule comprises the polypeptide sequences of SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:54.

In another preferred embodiment, the protease-activated T-cell activation bispecific molecule comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:45, at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:46, and at least about 95%, 96%, 97%, 98%, 99%, or 100% identical polypeptide sequences to SEQ ID NO:55. In a more preferred embodiment, the protease-activated T-cell activation bispecific molecule comprises the polypeptide sequences of SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:55.

Polynucleotides

The present invention further provides isolated polynucleotides encoding a protease-activated T-cell activation bispecific molecule or a fragment thereof as described herein. In some embodiments, the fragment is an antigen-binding fragment.

The polynucleotide encoding the protease-activated T-cell-activated bispecific molecule of the present invention can be expressed as a single polynucleotide encoding the entire protease-activated T-cell-activated bispecific molecule or as co-expressed multiple (e.g., two or more) polynucleotides. The polypeptides encoded by the co-expressed polynucleotides can associate, for example, via disulfide bonds or other means, to form the protease-activated T-cell-activated bispecific molecule. For example, the light chain portion of the antigen-binding moiety can be encoded by a polynucleotide separate from the portion of the protease-activated T-cell-activated bispecific molecule, which comprises the heavy chain portion of the antigen-binding moiety, an Fc domain subunit, and optionally a portion of another Fab fragment. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form the antigen-binding moiety. In another example, a portion of the protease-activated T-cell-activated bispecific molecule comprising one of the two Fc domain subunits and optionally a portion of one or more Fab molecules can be encoded by a polynucleotide separate from the portion of the protease-activated T-cell-activated bispecific molecule comprising the other of the two Fc domain subunits and optionally a portion of the antigen-binding moiety. When co-expressed, the Fc domain subunits will associate to form the Fc domain.

In some embodiments, the isolated polynucleotide encodes a T-cell activation bispecific molecule that is activatable by the entire protease according to the invention as described herein. In other embodiments, the isolated polynucleotide encodes a polypeptide contained in a T-cell activation bispecific molecule that is activatable by the protease according to the invention as described herein.

In another embodiment, the present invention relates to an isolated polynucleotide encoding a protease-activated T-cell activation bispecific molecule or a fragment thereof, wherein the polynucleotide comprises a sequence encoding a variable region sequence. In another embodiment, the present invention relates to an isolated polynucleotide encoding a protease-activated T-cell activation bispecific molecule or a fragment thereof, wherein the polynucleotide comprises a sequence encoding a polypeptide sequence or a fragment thereof as shown in SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55.

The polynucleotide encoding the idiotype-specific polypeptide of the present invention can be expressed as a single polynucleotide encoding the entire idiotype-specific polypeptide, or as multiple (e.g., two or more) polynucleotides co-expressed. The polypeptides encoded by the co-expressed polynucleotides can associate, for example, via disulfide bonds or other means, to form a functional idiotype-specific polypeptide, such as a masking portion. For example, in one embodiment, the idiotype-specific polypeptide is an anti-idiotype scFv (single-chain variable fragment), wherein the light chain variable portion of the anti-idiotype scFv can be encoded by a polynucleotide separate from a portion of the anti-idiotype scFv containing the heavy chain variable portion of the anti-idiotype scFv. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form the anti-idiotype scFv. In some embodiments, the separate polynucleotides encode the idiotype-specific polypeptide according to the present invention as described herein.

In some embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). The RNA of the present invention can be single-stranded or double-stranded.

Recombination method

The protease-activated T-cell activation bispecific molecule of the present invention can be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding a protease-activated T-cell activation bispecific molecule (fragment), such as those described above, are isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such polynucleotides can be readily isolated and sequenced using conventional methods. In one embodiment, a vector, preferably an expression vector, is provided containing one or more polynucleotides of the present invention. Expression vectors containing the coding sequence of the protease-activated T-cell activation bispecific molecule (fragment) and appropriate transcription/translation control signals can be constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination/genetic recombination. See, for example, the techniques described below: Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y. (1989). Expression vectors can be plasmids, part of a virus, or a fragment of nucleic acid. An expression vector comprises an expression cassette into which a multinucleotide (i.e., coding region) encoding a protease-activated T-cell activation bispecific molecule (fragment) operatively associated with a promoter and/or other transcriptional or translational control element is cloned. As used herein, a “coding region” is a portion of a nucleic acid consisting of codons that translate into amino acids. Although the "stop codon" (TAG, TGA, or TAA) is not translated into amino acids, it (if present) can be considered part of the coding region, while any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5' and 3' untranslated regions, are not part of the coding region. Two or more coding regions may be present in a single polynucleotide construct (e.g., on a single vector) or in separate polynucleotide constructs (e.g., on separate (different) vectors). Furthermore, any vector may contain a single coding region or may contain two or more coding regions; for example, the vectors of the present invention may encode one or more polypeptides that are cleaved into the final protein post-translation or during translation by proteolytic cleavage. Additionally, the vectors, polynucleotides, or nucleic acids of the present invention may encode heterologous coding regions that are fused or not fused with a polynucleotide or a variant or derivative thereof encoding a protease-activated T-cell activation bispecific molecule (fragment) of the present invention. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretory signal peptides or heterologous functional domains. Operable association occurs when the coding region of a gene product (e.g., a polypeptide) is associated in a manner that brings the expression of the gene product under the influence or control of the regulatory sequence. Two DNA segments (such as a polypeptide coding region and its associated promoter) are “operably associated” if the induction of promoter function leads to transcription of the mRNA encoding the desired gene product, and if the binding nature between the two DNA segments does not interfere with the ability of the expression regulatory sequence to direct the expression of the gene product or the ability to interfere with the DNA template to be transcribed. Therefore, if a promoter can influence the transcription of the nucleic acid, the promoter region will operably associate with the nucleic acid encoding the polypeptide. The promoter can be a cell-specific promoter that directs the substantial transcription of DNA only in a predetermined cell. In addition to promoters, other transcriptional control elements, such as enhancers, operons, repressors, and transcription termination signals, can be operably associated with polynucleotides to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein. Various transcriptional control regions are known to those skilled in the art. These regions include (but are not limited to) transcriptional control regions that function in vertebrate cells, such as (but not limited to) promoter and enhancer segments from cytomegaloviruses (e.g., the immediate early promoter, along with intron A), simian virus 40 (e.g., the early promoter), and retroviruses (e.g., Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes (e.g., actin, heat shock protein, bovine growth hormone, and rabbit globin), as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers, and inducible promoters (e.g., tetracycline-inducible promoters). Similarly, various translational control elements are known to those skilled in the art. These translational control elements include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly internal ribosome entry sites, or IRES, also known as CITE sequences). Expression cassettes may also include other features such as origin of replication and/or chromosomal integration elements, such as retroviral long terminal repeats (LTRs) or adeno-associated virus (AAV) inverted terminal repeats (ITRs).

The polynucleotide and nucleic acid coding regions of the present invention can associate with additional coding regions encoding secretory peptides or signal peptides, which in turn direct the secretion of polypeptides encoded by the polynucleotides of the present invention. For example, if it is desired to secrete a protease-activated T-cell activation bispecific molecule, DNA encoding a signal sequence can be placed upstream of a nucleic acid encoding a protease-activated T-cell activation bispecific molecule of the present invention or a fragment thereof. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once the protein chain has begun to export across the rough endoplasmic reticulum. Those skilled in the art know that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce the secretory or “mature” form of the polypeptide. In some embodiments, a natural signal peptide (e.g., an immunoglobulin heavy or light chain signal peptide) or a functional derivative of the sequence that retains the ability to direct the secretion of a polypeptide with which it operably associates is used. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof may be used. For example, the wild-type leader sequence can be replaced by the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

DNA encoding short protein sequences that can be used to facilitate later purification (e.g., histidine tags) or to help label protease-activated T-cell activation bispecific molecules may be included inside or at the end of a protease-activated T-cell activation bispecific molecule (fragment) encoding a polynucleotide.

In another embodiment, a host cell comprising one or more polynucleotides of the present invention is provided. In some embodiments, a host cell comprising one or more vectors of the present invention is provided. The polynucleotides and vectors may be incorporated individually or in combination with any features described herein with respect to the polynucleotides and vectors, respectively. In one such embodiment, the host cell comprises a vector (e.g., transformed or transfected using a vector) containing a polynucleotide encoding (a portion of) the protease-activated T-cell-activated bispecific molecule of the present invention. As used herein, the term “host cell” refers to any type of cellular system that can be engineered to produce the protease-activated T-cell-activated bispecific molecule of the present invention or fragments thereof. Host cells suitable for replicating and supporting the expression of the protease-activated T-cell-activated bispecific molecule are well known in the art. Such cells can be transfected or transduced with specific expression vectors and can be grown in large quantities containing vectors for inoculation of large fermenters to obtain sufficient quantities of the protease-activated T-cell-activated bispecific molecule for clinical applications. Suitable host cells include prokaryotic microorganisms, such as *Escherichia coli*, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, etc. For example, peptides can be produced in bacteria, especially when glycosylation is not required. After expression, peptides can be separated from bacterial cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for vectors encoding peptides, including fungal and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of peptides with partially or fully human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-1414 (2004) and Li et al., Nat Biotech 24, 210-215 (2006). Suitable host cells for expressing (glycosylated) peptides also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used with insect cells, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLATNIBODIES ^™ technology for generating antibodies in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspensions may be useful. Other examples of useful mammalian host cell lines include the monkey kidney CV1 line (COS-7) transformed from SV40; human embryonic kidney lines (293 or 293T cells, as described, for example, in Graham et al., J Gen Virol 36, 59 (1977)); young hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather, Biol Reprod 23, 243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells (as described, for example, in Mather et al., Annals NYAcad Sci 383, 44-68 (1982)); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr ^- CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NSO, P3X63, and Sp2/O. For a review of certain mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by BKCLo, Humana Press, Totowa, NJ), pp. 255–268 (2003). Host cells include cultured cells, such as mammalian cultured cells, yeast cells, insect cells, bacterial cells, and plant cells, as well as cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or lymphocytes (e.g., Y0, NSO, Sp20 cells).

Standard techniques for expressing exogenous genes in these systems are known in the art. Cells expressing polypeptides containing heavy or light chains of antibodies, such as antigen-binding domains, can be engineered to also express another antibody chain, such that the expressed product is an antibody having both heavy and light chains.

In one embodiment, a method for producing a protease-activated T-cell activation bispecific molecule according to the invention is provided, wherein the method comprises culturing a host cell containing a polynucleotide encoding a protease-activated T-cell activation bispecific molecule as provided herein, under conditions suitable for expressing the protease-activated T-cell activation bispecific molecule, and recovering the protease-activated T-cell activation bispecific molecule from the host cell (or host cell culture medium).

The components of a protease-activated T-cell activation bispecific molecule are genetically fused together. Protease-activated T-cell activation bispecific molecules can be designed so that their components fuse directly with each other or indirectly via adapter sequences. The composition and length of the adapter can be determined using methods well known in the art, and the efficacy of the adapter can be tested. Examples of adapter sequences between different components of a protease-activated T-cell activation bispecific molecule can be found in the sequences provided herein. Additional sequences (e.g., endopeptidase recognition sequences) may be included, if desired, to incorporate cleavage sites to separate the fused components.

In some embodiments, one or more antigen-binding portions of the protease-activated T-cell bispecific molecule of the present invention comprise at least an antibody variable region capable of binding antigenic determinants. The variable region can form and be derived from a portion of naturally occurring or non-naturally occurring antibodies and fragments thereof. Methods for generating polyclonal and monoclonal antibodies are well known in the art (see, for example, Harlow and Lane, "Antibodies, a laboratory manual", Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid-phase peptide synthesis, can be recombinantly generated (e.g., as described in U.S. Patent No. 4,186,567), or can be obtained, for example, by screening a combined library containing variable heavy and variable light chains (see, for example, U.S. Patent No. 5,969,108 to McCafferty).

Antibodies, antibody fragments, antigen-binding domains, or variable regions from any animal species can be used in the protease-activated T-cell-activating bispecific molecule of the present invention. Non-limiting antibodies, antibody fragments, antigen-binding domains, or variable regions that can be used in the present invention can be of mouse, primate, or human origin. If the protease-activated T-cell-activating bispecific molecule is intended for human use, a chimeric form of antibody can be used, wherein the constant region of the antibody is derived from a human. Humanized or fully human antibodies can also be prepared according to methods well known in the art (see, for example, U.S. Patent No. 5,565,332 to Winter). Humanization can be achieved through a variety of methods, including but not limited to (a) transplanting a non-human (e.g., donor antibody) CDR onto a human (e.g., receptor antibody) architecture and constant region, which may or may not retain key architectural residues (e.g., key architectural residues important for maintaining good antigen-binding affinity or antibody function), (b) transplanting only the non-human specificity determining region (SDR or a-CDR; residues essential for antibody-antigen interaction) onto the human architecture and constant region, or (c) transplanting the entire non-human variable domain, but “hiding” them with human-like segments by replacing surface residues. Humanized antibodies and their preparation methods are reviewed, for example, in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332, 323-329 (1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (19 89); US Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Jones et al., Nature 321,522-525 (1986); Morrison et al., Proc Natl Acad Sci 81,6851-6855 (1984); Morrison and Oi, Adv Immunol 44,65-92 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, Molec Immun 31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005) (describes SDR(a-CDR) transplantation); Padlan, Mol Immunol 28, 489-498 (1991) (describes “surface reshaping”); Dall’Acqua et al., Methods 36, 43-60 (2005) (describes “FR shuffling”); and Osbourn et al., Methods 36, 61-68 (2005) and Klimka et al., Br J Cancer 83, 252-260 (2000) (describes a “guided selection” approach for FR shuffling). Human antibodies and human variable regions can be generated using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg, Curr Opin Immunol 20, 450-459 (2008). The human variable region can form part of and be derived from a human monoclonal antibody prepared by a hybridoma method (see, for example, Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Human antibodies and human variable regions can also be prepared by administering an immunogen to a transgenic animal that has been modified to produce a complete human antibody or a complete antibody with a human variable region that responds to antigen stimulation (see, for example, Lonberg, Nat Biotech 23, 1117-1125 (2005)). Human antibodies and human variable regions can also be generated by isolating the variable region sequence of an Fv clone selected from a human-derived phage display library (see, for example, Hoogenboom et al., Methods in Molecular Biology 178, 1-37 (O’Brien et al., editor, Human Press, Totowa, NJ, 2001); and McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments.

In some embodiments, the antigen-binding portion of the present invention may be engineered to have enhanced binding affinity according to, for example, the method disclosed in U.S. Patent Application Publication No. 2004/0132066, the entire contents of which are incorporated herein by reference. The ability of the protease-activated T-cell-activating bispecific molecule of the present invention to bind to specific antigenic determinants can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art (e.g., surface plasmon resonance assay (analyzed on a BIACORE T100 system) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). Competitive analysis This can be used to identify antibodies, antibody fragments, antigen-binding domains, or variable domains that compete with a reference antibody for binding to a specific antigen, such as an antibody competing with a V9 antibody for binding to CD3. In some embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) that the reference antibody binds to. Detailed exemplary methods for mapping the epitopes bound by antibodies are provided in Morris (1996) "Epi" in Methods in Molecular Biology, Volume 66 (Humana Press, Totowa, NJ). In “Tope Mapping Protocols”, an exemplary competitive assay is performed. An immobilized antigen (e.g., CD3) is incubated in a solution containing a first labeled antibody (e.g., the V9 antibody described in US 6,054,297) that binds to the antigen and a second unlabeled antibody whose ability to compete with the first antibody for binding to the antigen is being tested. This second antibody may be present in the hybridoma supernatant. As a control, the immobilized antigen is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to the antigen, excess unbound antibody is removed, and the amount of label associated with the immobilized antigen is measured. If the amount of label associated with the immobilized antigen in the test sample is significantly reduced relative to the control sample, it indicates that the second antibody is competing with the first antibody for binding to the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

The protease-activated T-cell activation bispecific molecule prepared as described herein can be purified using techniques known in the art, such as high-performance liquid chromatography (HPLC), ion-exchange chromatography (IEC), gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The specific conditions used for purifying a particular protein will depend in part on factors such as net charge, hydrophobicity, and hydrophilicity, and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors, or antigens that bind to the protease-activated T-cell activation bispecific molecule can be used. For example, for affinity chromatography purification of the protease-activated T-cell activation bispecific molecule of the present invention, a matrix containing protein A or protein G can be used. Essentially as described in the examples, the protease-activated T-cell activation bispecific molecule can be separated using sequence protein A or G affinity chromatography and size exclusion chromatography. The purity of the protease-activated T-cell activation bispecific molecule can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high-performance liquid chromatography, etc. For example, the heavy chain fusion protein expressed as described in the examples showed to be fully and correctly assembled, as demonstrated by reductive SDS-PAGE (see, for example, Figures 8 through 12). The three bands resolved at approximately Mr 25,000, Mr 50,000, and Mr 75,000, corresponding to the predicted molecular weights of the protease-activated T cell activation bispecific molecules: the light chain, the heavy chain, and the heavy chain/light chain fusion protein.

Measurement

The protease-activated T-cell activation bispecific molecules provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activities using various assays known in the art.

Affinity Measurement

The affinity of a protease-activated T-cell-activated bispecific molecule for an Fc receptor or target antigen can be determined using standard instruments such as BIAcore instruments (GE Healthcare) and receptors or target proteins that can be obtained through recombinant expression, via surface plasmon resonance (SPR) according to the methods illustrated in the examples. Alternatively, the binding of a protease-activated T-cell-activated bispecific molecule to different receptors or target antigens can be assessed using cell lines expressing specific receptors or target antigens, for example, by flow cytometry (FACS). Specific illustrative and exemplary embodiments for measuring binding affinity are described below and in the examples below.

According to one embodiment, _KD was measured at 25°C using a T100 instrument (GE Healthcare) via surface plasmon resonance.

To analyze the interaction between the Fc moiety and the Fc receptor, a His-labeled recombinant Fc receptor was captured by an anti-pentahistidine antibody (Qiagen) immobilized on a CM5 chip, with a bispecific construct used as the analyte. Briefly, the carboxymethylated dextran biosensor chip (CM5, GE Healthcare) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The anti-pentahistidine antibody was diluted to 40 μg/ml with 10 mM sodium acetate at pH 5.0 and then injected at a flow rate of 5 μl/min to obtain approximately 6500 response units (RU) of the conjugate protein. Following ligand injection, 1 M ethanolamine was injected to block unreacted groups. The Fc receptor was subsequently captured for 60 s at 4 or 10 nM. For kinetic measurements, a four-fold serial dilution of the bispecific construct (ranging from 500 nM to 4000 nM) was injected into HBS-EP (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4) at 25 °C for 120 seconds at a flow rate of 30 μl/min.

To determine affinity for the target antigen, the bispecific construct was captured by an anti-human Fab-specific antibody (GE Healthcare) immobilized on the surface of an activated CM5 sensor chip, as described for the anti-pentahistidine antibody. The final amount of the conjugate was approximately 12,000 RU. The bispecific construct was captured at 300 nM for 90 s. The target antigen was passed through the flow cell at a concentration ranging from 250 to 1000 nM at a flow rate of 30 μl/min for 180 s. Dissociation was monitored for 180 s.

The bulk refractive index difference was corrected by subtracting the response obtained in the reference flow cell. The steady-state response was used to derive the dissociation constant K_{_D} by fitting a nonlinear curve of the Langmuir bound isotherm. The association rate (k_{_on} ) and dissociation rate (k_{_off} ) were calculated by simultaneously fitting the association and dissociation sensor maps using a simple one-to-one Langmuir bound model (T100 Evaluation Software version 1.1.1). The equilibrium dissociation constant (K_{_D}) was calculated as the ratio k _{_off} /k _{_on} . See, for example, Chen et al., J Mol Biol 293, 865-881 (1999).

Activity assay

The bioactivity of the protease-activated T cell activation bispecific molecule of the present invention can be measured by various assays as described in the examples. Bioactivity may include, for example, induction of T cell proliferation, induction of T cell signaling, induction of expression of activation markers in T cells, induction of T cell cytokine secretion, induction of target cell (e.g., tumor cell) lysis, and induction of tumor regression and/or improved survival.

Composition, Formulation and Administration

In a further aspect, the present invention provides pharmaceutical compositions comprising any of the protease-activated T-cell activation bispecific molecules provided herein, for example, for any of the following therapeutic methods. In one embodiment, the pharmaceutical composition comprises any of the protease-activated T-cell activation bispecific molecules provided herein, and a pharmaceutical carrier. In another embodiment, the pharmaceutical composition comprises any of the protease-activated T-cell activation bispecific molecules provided herein, and at least one additional therapeutic agent, such as described below.

A method is further provided for producing the protease-activated T-cell activation bispecific molecule of the present invention in a form suitable for in vivo administration, the method comprising (a) obtaining the protease-activated T-cell activation bispecific molecule according to the present invention, and (b) formulating the protease-activated T-cell activation bispecific molecule with at least one pharmaceutical carrier, wherein the formulation of the protease-activated T-cell activation bispecific molecule is prepared for in vivo administration.

The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of one or more protease-activated T-cell activating bispecific molecules dissolved or dispersed in a pharmaceutical carrier. The phrase “pharmaceutically or pharmacologically acceptable” means that the molecular entity and composition are generally non-toxic to the recipient at the doses and concentrations employed, i.e., that they do not produce adverse, allergic, or other adverse reactions when administered to animals (such as, for example, humans), as appropriate. Preparation of pharmaceutical compositions containing at least one protease-activated T-cell activating bispecific molecule and optionally additional active ingredients will be known to those skilled in the art in light of this disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, which is incorporated herein by reference. Furthermore, for animal (e.g., human) administration, it should be understood that the preparation should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biostandards or other relevant national authorities. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, “pharmaceutical carrier” includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delay agents, salts, preservatives, antioxidants, proteins, pharmaceuticals, pharmaceutical stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and similar substances and combinations thereof, as should be known to a person skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, pp. 1289-1329, which is incorporated herein by reference). Except in cases where any conventional carrier is incompatible with the active ingredient, the use of this carrier in therapeutic or pharmaceutical compositions is contemplated.

The composition may contain different types of carriers, depending on whether it is applied in solid, liquid or aerosol form, and whether sterility is required for routes of application such as injection. The protease-activated T-cell activating bispecific molecule of the present invention (and any additional therapeutic agent) can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraprostatically, intraspleurally, intrarenally, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intracysticly, intramucosally, intraperitoneally, intraumbilically, intraocularly, orally, topically, locally, by inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, direct bathing of target cells by local perfusion, via catheter, via irrigation, in the form of cream, in the form of lipid composition (e.g., liposome), or by other methods known to those skilled in the art or any combination thereof (see, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, which is incorporated herein by reference). Parenteral administration, especially intravenous injection, is most commonly used for administering polypeptide molecules, such as the protease-activated T-cell bispecific molecule of the present invention.

Parenteral compositions include those designed for administration by injection (e.g., subcutaneous, intradermal, intralesional, intravenous, intra-arterial, intramuscular, intrathecal, or intraperitoneal injection). For injection, the protease-activated T-cell activation bispecific molecule of the present invention can be formulated in aqueous solution, preferably in a physiologically compatible buffer (such as Hanks' solution, Ringer's solution, or physiological saline buffer). The solution may contain a formulation agent, such as a suspending agent, stabilizer, and/or dispersant. Alternatively, the protease-activated T-cell activation bispecific molecule can be in powder form for use prior to preparation with a suitable solvent (e.g., sterile pyrogen-free water). A sterile injectable solution is prepared by incorporating the protease-activated T-cell activation bispecific molecule of the present invention in the desired amount with various other ingredients listed below into a suitable solvent as needed. For example, sterility can be readily achieved by filtration through a sterile filter membrane. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile solvent containing a base dispersion medium and/or other components. In the case of sterile powders used to prepare sterile injectable solutions, suspensions, or emulsions, a preferred preparation method is vacuum drying or lyophilization, which produces a powder of the active ingredient from a previously sterile filtered liquid medium, plus any additional desired components. If necessary, the liquid medium should be appropriately buffered, and the liquid diluent should be isotonic with sufficient saline or glucose before injection. The composition must be stable under the conditions of manufacture and storage and preserved against contamination by microorganisms such as bacteria and fungi. It should be understood that endotoxin contamination should be kept to a safe level, for example, below 0.5 ng/mg protein. Suitable pharmaceutical carriers include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight (less than about 10 residues) peptides; proteins, such as... Serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc protein complexes); and/or nonionic surfactants, such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, etc. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to allow for the preparation of high-concentration solutions. Additionally, suspensions of the active compound can be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or media include fatty oils, such as sesame oil; or synthetic fatty acid esters, such as ethyl oleate or triglycerides; or liposomes.

The active ingredient can be encapsulated in microcapsules (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared by, for example, cohesive techniques or interfacial polymerization; encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules); or encapsulated in crude emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th edition, Mack Printing Company, 1990). Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include a semi-permeable matrix containing a solid hydrophobic polymer of a polypeptide, in the form of a molded article, such as a membrane or microcapsule. In certain embodiments, prolonged absorption of the injectable composition can be achieved by using a delayed-absorption agent in the composition (such as, for example, aluminum monostearate, gelatin, or combinations thereof).

In addition to the compositions previously described, protease-activated T-cell activation bispecific molecules can also be formulated into long-acting formulations. Such long-acting formulations can be administered via implantation (e.g., subcutaneous or intramuscular implantation) or intramuscular injection. Thus, for example, protease-activated T-cell activation bispecific molecules can be formulated with suitable polymeric or hydrophobic materials (e.g., as emulsions in acceptable oils) or with ion exchange resins, or formulated as slightly soluble derivatives, such as slightly soluble salts.

Pharmaceutical compositions comprising the protease-activated T-cell activation bispecific molecule of the present invention can be manufactured using conventional mixing, dissolving, emulsifying, encapsulating, embedding, or lyophilizing processes. The pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants that facilitate the processing of the protein into a pharmaceutically usable formulation. Appropriate formulation depends on the chosen route of administration.

Protease-activated T-cell activation bispecific molecules can be formulated into compositions in free acid or base, neutral or salt form. Pharmaceutical salts are salts that essentially retain the biological activity of the free acid or free base. These pharmaceutical salts include acid addition salts, such as those formed with the free amino group of a protein composition, or those formed with inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, or mandelic acid). Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents compared to their corresponding free base forms.

Treatment methods and compositions

Any protease-activated T-cell activating bispecific molecule provided herein can be used in therapeutic applications. The protease-activated T-cell activating bispecific molecule of the present invention can be used as an immunotherapeutic agent, for example, for the treatment of cancer.

For use in therapeutic applications, the protease-activated T-cell activating bispecific molecule of the present invention will be formulated, dispensed, and administered in accordance with good medical practice. Factors to be considered in this context include the specific disease being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disease, the site of drug delivery, the method of administration, the timing of administration, and other factors known to the practicing physician.

In one aspect, the present invention provides a protease-activated T-cell activation bispecific molecule for use as a medicament. In a further aspect, the present invention provides a protease-activated T-cell activation bispecific molecule for treating a disease. In some embodiments, the present invention provides a protease-activated T-cell activation bispecific molecule for treatment of a disease. In one embodiment, the present invention provides a protease-activated T-cell activation bispecific molecule as described herein for treating a disease in an individual with this need. In some embodiments, the present invention provides a protease-activated T-cell activation bispecific molecule for treating an individual with a disease, the method comprising administering to the individual a therapeutically effective amount of the protease-activated T-cell activation bispecific molecule. In some embodiments, the disease to be treated is a proliferative disorder. In a particular embodiment, the disease is cancer. In some embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, such as an anticancer agent if the disease to be treated is cancer. In a further embodiment, the present invention provides a protease-activated T-cell activation bispecific molecule as described herein for inducing the lysis of target cells, particularly tumor cells. In some embodiments, the present invention provides a protease-activated T-cell activation bispecific molecule for use in a method of inducing lysis of target cells, particularly tumor cells, in an individual, the method comprising administering an effective amount of the protease-activated T-cell activation bispecific molecule to the individual to induce lysis of the target cells. "Individual" according to any of the above embodiments is a mammal, preferably a human.

In a further aspect, the present invention provides the use of the protease-activated T-cell activation bispecific molecule of the present invention in the manufacture or preparation of a medicament. In one embodiment, the medicament is used to treat a disease in an individual with this need. In another embodiment, the method of using the medicament to treat a disease includes administering a therapeutically effective amount of the medicament to an individual having the disease. In some embodiments, the disease to be treated is a proliferative disorder. In a particular embodiment, the disease is cancer. In one embodiment, the method further includes administering a therapeutically effective amount of at least one additional therapeutic agent to the individual, such as an anticancer agent if the disease to be treated is cancer. In a further embodiment, the medicament is used to induce the lysis of target cells, particularly tumor cells. In yet another embodiment, the method of using the medicament to induce the lysis of target cells, particularly tumor cells, in an individual includes administering an effective amount of the medicament to the individual to induce the lysis of target cells. The “individual” according to any of the above embodiments may be a mammal, preferably a human.

In a further aspect, the present invention provides a method for treating a disease. In one embodiment, the method includes administering to an individual suffering from such a disease a therapeutically effective amount of the protease-activated T-cell-activating bispecific molecule of the present invention. In one embodiment, the individual is given a composition comprising the protease-activated T-cell-activating bispecific molecule of the present invention in a pharmaceutical form. In some embodiments, the disease to be treated is a proliferative disorder. In a particular embodiment, the disease is cancer. In some embodiments, the method further includes administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, such as an anticancer agent if the disease to be treated is cancer. The “individual” according to any of the above embodiments may be a mammal, preferably a human.

In another aspect, the present invention provides a method for inducing the lysis of target cells, particularly tumor cells. In one embodiment, the method includes, in the presence of T cells, particularly cytotoxic T cells, contacting the target cells with a protease-activated T cell-activating bispecific molecule of the present invention. In another aspect, a method for inducing the lysis of target cells, particularly tumor cells, in an individual is provided. In one such embodiment, the method includes administering an effective amount of the protease-activated T cell-activating bispecific molecule to the individual to induce the lysis of the target cells. In one embodiment, the individual is a human.

In some embodiments, the disease to be treated is a proliferative disorder, particularly cancer. Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, leukemia, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other proliferative disorders that can be treated with the protease-activated T-cell activation bispecific molecule of the present invention include, but are not limited to, tumors located in the following sites: abdomen, bones, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid gland), eyes, head and neck, nervous system (central and peripheral nervous systems), lymphatic system, pelvis, skin, soft tissue, spleen, chest, and genitourinary system. Precancerous conditions or lesions and metastases are also included. In some embodiments, the cancer is selected from the group consisting of: renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, and head and neck cancer. Those skilled in the art will readily recognize that, in many cases, the protease-activated T-cell-activating bispecific molecule may not provide a cure, but may only provide partial benefit. In some embodiments, physiological changes that have some benefit are also considered therapeutically beneficial. Therefore, in some embodiments, the amount of the protease-activated T-cell-activating bispecific molecule that provides the physiological change is considered an "effective amount" or a "therapeuticly effective amount." The subject, patient, or individual requiring treatment is typically a mammal, and more particularly a human.

In some embodiments, an effective amount of the protease-activated T-cell-activating bispecific molecule of the present invention is applied to cells. In other embodiments, a therapeutically effective amount of the protease-activated T-cell-activating bispecific molecule of the present invention is applied to an individual to treat a disease.

For the prevention or treatment of disease, the appropriate dose of the protease-activated T-cell activating bispecific molecule of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the patient's weight, the type of T-cell activating bispecific antigen-binding molecule, the severity and progression of the disease, whether the T-cell activating bispecific antigen-binding molecule is administered for prophylactic or therapeutic purposes, prior or concurrent therapeutic interventions, the patient's clinical history and response to the protease-activated T-cell activating bispecific molecule, and the attending physician's discretion. In any case, the practitioner responsible for administration will determine the concentration and appropriate dose of the active ingredient in the composition for the individual subject. Various dosing schedules are considered herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulse infusion.

The protease-activated T-cell activating bispecific molecule is appropriately administered to the patient either once or in a series of treatments. Depending on the type and severity of the disease, an initial candidate dose of about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg to 10 mg/kg) of the protease-activated T-cell activating bispecific molecule may be administered to the patient, for example, by single or multiple administrations alone or by continuous infusion. Depending on the above factors, a typical daily dose range may be about 1 μg/kg to 100 mg/kg or more. For repeated administration over several days or longer, depending on the condition, treatment usually continues until the desired suppression of disease symptoms occurs. An exemplary dose range for the T-cell activating bispecific antigen-binding molecule is about 0.005 mg/kg to about 10 mg/kg. In other non-limiting examples, the dosage may also include administration of about 1 μg/kg body weight, about 5 μg/kg body weight, about 10 μg/kg body weight, about 50 μg/kg body weight, about 100 μg/kg body weight, about 200 μg/kg body weight, about 350 μg/kg body weight, about 500 μg/kg body weight, about 1 mg/kg body weight, about 5 mg/kg body weight, about 10 mg/kg body weight, about 50 mg/kg body weight, about 100 mg/kg body weight, about 200 mg/kg body weight, about 350 mg/kg body weight, about 500 mg/kg body weight, up to about 1000 mg/kg body weight or more, and any range derived therefrom. In non-limiting examples of ranges that can be derived from the figures listed herein, ranges such as about 5 mg/kg body weight to about 100 mg/kg body weight, about 5 μg/kg body weight to about 500 mg/kg body weight, etc., may be administered based on the above values. Therefore, patients can be administered one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg, or 10 mg/kg (or any combination thereof). Such doses can be administered intermittently, for example weekly or every three weeks (e.g., so that the patient receives about two to about twenty, or for example about six, doses of a protease-activated T-cell activation bispecific molecule). An initial higher loading dose can be administered, followed by one or more lower doses. However, other dosing regimens may be useful. Progression of the therapy can be easily monitored using routine techniques and assays.

The protease-activated T-cell activating bispecific molecule of the present invention will generally be used in an amount that effectively achieves the intended purpose. For the treatment or prevention of a condition, the protease-activated T-cell activating bispecific molecule of the present invention, or a pharmaceutical composition thereof, will be administered or applied in a therapeutically effective amount. The determination of a therapeutically effective amount is entirely within the competence of those skilled in the art, particularly based on the detailed disclosure provided herein.

For systemic administration, the effective therapeutic dose can initially be estimated based on in vitro assays (such as cell culture assays). The dose can then be formulated in animal models to achieve a range of circulating concentrations including the _IC50 , as determined in cell cultures. This information can be used to more accurately determine the useful dose for humans.

The initial dose can also be estimated using techniques known in the art, based on in vivo data (e.g., animal models). Those skilled in the art can easily optimize human administration based on animal data.

Dosage and intervals can be individually adjusted to provide sufficient plasma levels of the protease-activated T-cell-activating bispecific molecule to maintain therapeutic efficacy. The usual patient dose range by injection is approximately 0.1 to 50 mg/kg/day, typically approximately 0.5 to 1 mg/kg/day. Therapeuticly effective plasma levels can be achieved by administering multiple doses daily. Plasma levels can be measured, for example, by HPLC.

In cases of topical application or selective uptake, the effective local concentration of the protease-activated T-cell activation bispecific molecule may be independent of plasma concentration. Those skilled in the art will be able to optimize the therapeutically effective local dose without requiring extensive experimentation.

The therapeutically effective doses of the protease-activated T-cell activation bispecific molecule described herein will generally provide therapeutic benefit without causing substantial toxicity. The toxicity and therapeutic efficacy of the protease-activated T-cell activation bispecific molecule can be determined using standard pharmaceutical methods in cell culture or laboratory animals. Cell culture assays and animal studies can be used to determine the _LD50 (the dose at which 50% of the population is lethal) and _ED50 (the dose at which 50% of the population is therapeutically effective). The dose ratio between toxicity and efficacy is the therapeutic index, which can be expressed as the ratio _LD50 / _ED50 . Protease-activated T-cell activation bispecific molecules exhibiting a large therapeutic index are preferred. In one embodiment, the protease-activated T-cell activation bispecific molecule according to the invention exhibits a high therapeutic index. Data obtained from cell culture assays and animal studies can be used to formulate a range of doses suitable for humans. The dose is preferably within a range including circulating concentrations with little or no toxicity at the _ED50 . The dose can vary within this range depending on various factors, such as the dosage form used, the route of administration employed, the patient's condition, etc. The exact formulation, route of administration, and dosage can be chosen by individual physicians based on the patient's condition (see, for example, Fingl et al., 1975, in The Pharmacological Basis of Therapeutics, Chapter 1, page 1, the entire contents of which are incorporated herein by reference).

The attending physician of a patient treated with the protease-activated T-cell bispecific molecular therapy of the present invention should know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, etc. Conversely, if the clinical response is inadequate (excluding toxicity), the attending physician will also know to adjust the treatment to a higher level. The dosage administered in the management of the target disease will vary depending on the severity of the disease being treated, the route of administration, etc. For example, the severity of the disease can be assessed in part by standard prognostic assessment methods. Furthermore, the dosage and possible dosing frequency will also vary according to the individual patient's age, weight, and response.

Other medications and treatments

The protease-activated T-cell activation bispecific molecule of the present invention can be administered in combination with one or more other agents in a therapy. For example, the protease-activated T-cell activation bispecific molecule of the present invention can be administered with at least one additional therapeutic agent. The term "therapeutic agent" includes any agent administered to treat symptoms or diseases of an individual in need of such treatment. Such additional therapeutic agents may contain any active ingredient suitable for the specific indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other. In some embodiments, the additional therapeutic agent is an immunomodulator, a cell growth inhibitor, a cell adhesion inhibitor, a cytotoxic agent, an apoptosis activator, or an agent that increases the sensitivity of cells to apoptosis inducers. In a particular embodiment, the additional therapeutic agent is an anticancer agent, such as a microtubule disruptor, antimetabolite, topoisomerase inhibitor, DNA intercalating agent, alkylating agent, hormone therapy, kinase inhibitor, receptor antagonist, tumor cell apoptosis activator, or antiangiogenic agent.

Other such agents are appropriately combined in amounts effective for the intended purpose. The effective amount of such other agents depends on the amount of the protease-activated T-cell activating bispecific molecule used, the type of disease or treatment, and other factors discussed above. The protease-activated T-cell activating bispecific molecule is generally used at the same dosage and route of administration as described herein, or at about 1% to 99% of the dosage described herein, or at any dosage and route of administration empirically/clinically determined to be appropriate.

The combination therapies described above encompass both combined administration (where two or more therapeutic agents are included in the same or different compositions) and single administration. In the case of single administration, the administration of the protease-activated T-cell activating bispecific molecule of the present invention can be performed before, simultaneously with, and/or after the administration of additional therapeutic agents and/or adjuvants. The protease-activated T-cell activating bispecific molecule of the present invention can also be used in combination with radiotherapy.

Products

In another aspect of the invention, an article is provided containing a substance that can be used to treat, prevent, and/or diagnose the aforementioned conditions. The article includes a container and a label or packaging insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous (IV) solution bags, etc. The container can be formed from a variety of materials such as glass or plastic. The container contains a composition that, on its own or in combination with another composition, is effective for treating, preventing, and/or diagnosing the condition, and the container may have a sterile inlet (e.g., the container may be an IV solution bag or vial with a stopper capable of being punctured by a hypodermic needle). At least one active agent in the composition is a protease-activated T-cell activating bispecific molecule of the present invention. The label or packaging insert indicates that the composition is intended to treat the selected condition. Furthermore, the article may include (a) a first container containing the composition, wherein the composition comprises the protease-activated T-cell activating bispecific molecule of the present invention; and (b) a second container containing the composition, wherein the composition comprises additional cytotoxic agents or other therapeutic agents. The article of manufacture in this embodiment of the invention may further include a packaging insert indicating that the compositions can be used to treat a specific condition. Alternatively or additionally, the article of manufacture may further include a second (or third) container containing pharmaceutical buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. The article of manufacture may further include other substances required from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

Exemplary embodiments

1. A protease-activated T-cell activation bispecific molecule, comprising:

(a) The first antigen-binding region capable of binding to CD3;

(b) a second antigen-binding moiety capable of binding to target cell antigens; and

(c) A masking portion of the T-cell bispecific binding molecule is covalently linked to a peptide linker, wherein the masking portion is capable of binding idiotype to either the first antigen-binding portion or the second antigen-binding portion, thereby reversibly masking either the first antigen-binding portion or the second antigen-binding portion.

The linker contains a protease recognition sequence XQARK (SEQ ID NO:39), where X is histidine (H) or proline (P).

2. The protease-activated T-cell activation bispecific molecule according to Example 1, wherein the masking portion is covalently linked to the first antigen-binding portion and reversibly masks the first antigen-binding portion.

3. A protease-activated T-cell activation bispecific molecule according to Example 1 or 2, wherein the masking portion is covalently linked to the heavy chain variable region of the first antigen-binding portion.

4. A protease-activated T-cell activation bispecific molecule according to Example 1 or 2, wherein the masking portion is covalently linked to the light chain variable region of the first antigen-binding portion.

5. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 4, wherein the masking portion is scFv.

6. The protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 5, wherein the protease-activated T-cell activation bispecific molecule comprises a second masking portion that reversibly conceals the second antigen-binding portion.

7. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 6, wherein the protease is expressed by a target cell.

8. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 7, wherein (i) the second antigen-binding portion is a conventional Fab, or (ii) the second antigen-binding portion is a cross-Fab molecule, wherein the variable or constant regions of the Fab light chain and the Fab heavy chain are exchanged.

9. A protease-activated T-cell bispecific molecule according to any one of Examples 1 to 8, wherein the second antigen-binding portion is a cross-Fab molecule, wherein the Fab light chain and the Fab heavy chain constant region are exchanged.

10. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 9, wherein the first antigen-binding portion is a conventional Fab molecule.

11. The protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 10, comprising no more than one antigen-binding moiety capable of binding to CD3.

12. The protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 11, comprising a third antigen-binding portion, wherein the third antigen-binding portion is a Fab molecule capable of binding to a target cell antigen.

13. The protease-activated T-cell activation bispecific molecule according to Example 12, wherein the third antigen-binding portion is the same as the second antigen-binding portion.

14. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 13, wherein the second antigen-binding portion is capable of binding to FolR1.

15. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 14, wherein the first antigen-binding portion and the second antigen-binding portion are fused to each other, optionally via a peptide linker.

16. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 15, wherein the second antigen-binding portion 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 portion.

17. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 15, wherein the first antigen-binding portion 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 portion.

18. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 17, wherein the Fab light chain of the first antigen-binding portion and the Fab light chain of the second antigen-binding portion are fused to each other, optionally fused to each other via a peptide linker.

19. The protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 18, further comprising an Fc domain consisting of a first subunit and a second subunit capable of stable association.

20. The protease-activated T-cell activation bispecific molecule according to Example 19, wherein the Fc domain is an IgG Fc domain, specifically an IgG1 Fc domain or an IgG4 Fc domain.

21. A protease-activated T-cell activation bispecific molecule according to Example 19 or 20, wherein the Fc domain is a human Fc domain.

22. A protease-activated T-cell activation bispecific molecule according to any one of Examples 19 to 21, wherein the Fc domain exhibits reduced binding affinity to the Fc receptor and/or reduced effector function compared to the native IgG1 Fc domain.

23. The protease-activated T-cell activation bispecific molecule according to Example 22, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to the Fc receptor and/or effector function.

24. The protease-activated T-cell activation bispecific molecule according to Example 23, wherein one or more amino acids are substituted at one or more positions selected from the group consisting of L234, L235 and P329 (Kabat number).

25. The protease-activated T-cell activation bispecific molecule according to Example 24, wherein each subunit of the Fc domain comprises three amino acid substitutions that reduce binding to and/or effector function of the activated Fc receptor, wherein the amino acid substitutions are L234A, L235A, and P329G.

26. A protease-activated T-cell activation bispecific molecule according to any one of Examples 22 to 25, wherein the Fc receptor is an Fcγ receptor.

27. A protease-activated T-cell activation bispecific molecule according to any one of Examples 22 to 26, wherein the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).

28. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 27, wherein the portion capable of binding to CD3 comprises

(i) a heavy chain variable (VH) region comprising the heavy chain complementarity determination region (HCDR) 1 of SEQ ID NO:1, HCDR 2 of SEQ ID NO:2, and HCDR 3 of SEQ ID NO:3, and

(ii) Light chain variable (VL) regions, which include light chain complementarity determination region (LCDR) 1 of SEQ ID NO:7, LCDR 2 of SEQ ID NO:8 and LCDR 3 of SEQ ID NO:9.

29. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 28, wherein the portion capable of binding to CD3 comprises: a VH region containing 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: 5; and/or the VL region containing 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: 10.

30. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 27, wherein the portion capable of binding to CD3 comprises

(i) a heavy chain variable (VH) region comprising the heavy chain complementarity determination region (HCDR) 1 of SEQ ID NO:1, HCDR 2 of SEQ ID NO:2, and HCDR 3 of SEQ ID NO:4, and

(ii) Light chain variable (VL) regions, which include light chain complementarity determination region (LCDR) 1 of SEQ ID NO:7, LCDR 2 of SEQ ID NO:8 and LCDR 3 of SEQ ID NO:9.

31. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 27 or 30, wherein the portion capable of binding to CD3 comprises: a VH region containing 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: 6; and/or the VL region containing 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: 10.

32. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 31, wherein the masking portion comprises a heavy chain variable region comprising at least one of the following:

(a) The amino acid sequence of the heavy chain complementarity-determining region (HCDR) 1 of DYSMN (SEQ ID NO:15);

(b) The HCDR2 amino acid sequence selected from the group consisting of WINTETGEPRYTDDFKG (SEQ ID NO:16), WINTETGEPRYTDDFTG (SEQ ID NO:17) and WINTETGEPRYTQGFKG (SEQ ID NO:18);

(c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19).

33. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 32, wherein the masking portion comprises a light chain variable region comprising at least one of the following:

(d) The amino acid sequence of the light chain complementarity-determining region (LCDR) of RASKSVSTSSYSYMH (SEQ ID NO:25) or RASKSVSTSSYSYMH (SEQ ID NO:26);

(e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and

(f) The amino acid sequence of LCDR3 is selected from the group consisting of QHSREFPYT (SEQ ID NO:28) or QQSREFPYT (SEQ ID NO:29).

34. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 33, wherein the masking portion comprises a heavy chain variable region, the heavy chain variable region comprising:

(a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15);

(b) The HCDR2 amino acid sequence selected from the group consisting of WINTETGEPRYTDDFKG (SEQ ID NO:16), WINTETGEPRYTDDFTG (SEQ ID NO:17) and WINTETGEPRYTQGFKG (SEQ ID NO:18);

(c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO: 60); and a light chain variable region comprising:

(d) The amino acid sequence of LCDR1 is selected from the group consisting of RASKSVSTSSYSYMH (SEQ ID NO:25) and KSSKSVSTSSYSYMH (SEQ ID NO:26);

(e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and

(f) The amino acid sequence of LCDR3 is selected from the group consisting of QHSREFPYTSEQ ID NO:28 and QQSREFPYT (SEQ ID NO:29).

35. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 33, wherein the masking portion comprises: a VH region comprising:

(a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15);

(b) The HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO:16);

(c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19);

And the VL zone, which includes:

(d) The LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO:25);

(e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and

(f) The LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28).

36. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 33, wherein the masking portion comprises: a VH region comprising:

(a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15);

(b) The HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO:16);

(c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19);

And the VL zone, which includes:

(d) The LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO:26);

(e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and

(f) The LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28).

37. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 33, wherein the masking portion comprises: a VH region comprising:

(a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15);

(b) The HCDR2 amino acid sequence of WINTETGEPRYTDDFTG (SEQ ID NO:17);

(c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19);

And the VL zone, which includes:

(d) The LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO:26);

(e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and

(f) The LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28).

38. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 33, wherein the masking portion comprises: a VH region comprising:

(a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15);

(b) The HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO:18);

(c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19);

And the VL zone, which includes:

(d) The LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO:26);

(e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and

(f) The LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28).

39. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 33, wherein the masking portion comprises: a VH region comprising:

(a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15);

(b) The HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO:18);

(c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19);

And the VL zone, which includes:

(d) The LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO:25);

(e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and

(f) The LCDR3 amino acid sequence of QQSREFPYT (SEQ ID NO:29).

40. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 39, wherein the masking portion is humanized.

41. The protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 40, wherein the masking portion comprises: a VH region containing at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 21; and a VL region containing at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 32.

42. The protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 33, wherein the masking portion comprises: a VH region containing the amino acid sequence of SEQ ID NO: 21; and a VL region containing the amino acid sequence of SEQ ID NO: 32.

43. The protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 40, wherein the second antigen-binding portion is capable of binding to FolR1 and comprises at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13 and/or at least one light chain CDR selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.

44. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 43, wherein the second antigen-binding portion is capable of binding to FolR1 and includes a heavy chain variable region comprising:

a) The HCDR1 amino acid sequence of NAWMS (SEQ ID NO: 11);

b) The HCDR2 amino acid sequence of RIKSKTDGGTTDYAAPVKG (SEQ ID NO:12); and

c) The HCDR3 amino acid sequence of PWEWSWYDY (SEQ ID NO:13);

And the VL zone, which includes:

d) LCDR1 of GSSTGAVTTSNYAN (SEQ ID NO:7);

e) The LCDR2 amino acid sequence of GTNKRAP (SEQ ID NO: 8); and

f) The LCDR3 amino acid sequence of ALWYSNLWV (SEQ ID NO:9).

45. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 44, wherein the second antigen-binding portion comprises: a heavy chain variable region comprising 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: 14, and a light chain variable region comprising 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: 10.

46. The protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 44, wherein the second antigen-binding portion is capable of binding to FolR1 and comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10.

47. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 46, wherein the protease-cleavable linker comprises a protease recognition sequence PQARK (SEQ ID NO: 41).

48. A protease-activated T cell activation bispecific molecule, comprising:

(a) The first heavy chain, which contains the amino acid sequence of SEQ ID NO:46;

(b) A second chain comprising the amino acid sequence of SEQ ID NO:48; and

(c) A light chain containing the amino acid sequence of SEQ ID NO:45.

49. A protease-activated T cell activation bispecific molecule, comprising:

(a) The first heavy chain, which contains the amino acid sequence of SEQ ID NO:46;

(b) A second chain comprising the amino acid sequence of SEQ ID NO:49; and

(c) A light chain containing the amino acid sequence of SEQ ID NO:45.

50. A protease-activated T cell activation bispecific molecule, comprising:

(a) The first heavy chain, which contains the amino acid sequence of SEQ ID NO:46;

(b) A second chain comprising the amino acid sequence of SEQ ID NO:53; and

(c) A light chain containing the amino acid sequence of SEQ ID NO:45.

51. A protease-activated T cell activation bispecific molecule, comprising:

(a) The first heavy chain, which contains the amino acid sequence of SEQ ID NO:46;

(b) A second chain comprising the amino acid sequence of SEQ ID NO:54; and

(c) A light chain containing the amino acid sequence of SEQ ID NO:45.

52. A protease-activated T cell activation bispecific molecule, comprising:

(a) The first heavy chain, which contains the amino acid sequence of SEQ ID NO:46;

(b) A second chain comprising the amino acid sequence of SEQ ID NO:55; and

(c) A light chain containing the amino acid sequence of SEQ ID NO:45.

53. A unique type-specific polypeptide for reversibly concealing the anti-CD3 antigen binding site of a molecule, wherein the unique type-specific polypeptide is covalently linked to the molecule via a peptide linker, wherein the linker comprises a protease recognition sequence XQARK (SEQ ID NO: 39), wherein X is histidine (H) or proline (P).

54. The idiotype-specific polypeptide according to Example 52, wherein the idiotype-specific polypeptide is an anti-idiotype scFv, an anti-idiotype Fab, or an anti-idiotype scFab.

55. The idiotype-specific polypeptide according to Example 53 or 54, wherein the idiotype-specific polypeptide is scFv.

56. The unique type-specific polypeptide according to any one of Examples 53 to 55, wherein the molecule is a T-cell activation bispecific molecule.

57. The idiotype-specific polypeptide according to any one of Examples 53 to 56, wherein the idiotype-specific polypeptide comprises: a heavy chain variable (VH) region, which includes:

(a) The amino acid sequence of the heavy chain complementarity-determining region (HCDR) 1 of DYSMN (SEQ ID NO: 15),

(b) The HCDR2 amino acid sequence selected from the group consisting of WINTETGEPRYTDDFKG (SEQ ID NO:16), WINTETGEPRYTDDFTG (SEQ ID NO:17) and WINTETGEPRYTQGFKG (SEQ ID NO:18);

(c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19);

And the light chain variable (VL) region, which includes:

(d) The amino acid sequence of the light chain complementarity-determining region (LCDR) 1 of RASKSVSTSSYSYMH (SEQ ID NO:25) or KSSKSVSTSSYSYMH (SEQ ID NO:26);

(e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and

(f) The LCDR3 amino acid sequences of QHSREFPYT (SEQ ID NO:28) and QQSREFPYT (SEQ ID NO:29).

58. The idiotype-specific polypeptide according to any one of Examples 53 to 57, wherein the idiotype-specific polypeptide comprises: a VH region comprising:

(a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15);

(b) The HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO:16);

(c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19);

And the VL zone, which includes:

(d) The LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO:25);

(e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and

(f) The LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28).

59. The idiotype-specific polypeptide according to any one of Examples 53 to 57, wherein the idiotype-specific polypeptide comprises: a VH region comprising:

(a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15);

(b) The HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO:16);

(c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19);

And the VL zone, which includes:

(d) The LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO:26);

(e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and

(f) The LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28).

60. The idiotype-specific polypeptide according to any one of Examples 53 to 57, wherein the idiotype-specific polypeptide comprises: a VH region comprising:

(a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15);

(b) The HCDR2 amino acid sequence of WINTETGEPRYTDDFTG (SEQ ID NO:17);

(c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19);

And the VL zone, which includes:

(d) The LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO:26);

(e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and

(f) The LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28).

61. The idiotype-specific polypeptide according to any one of Examples 53 to 57, wherein the idiotype-specific polypeptide comprises: a VH region comprising:

(a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15);

(b) The HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO:18);

(c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19);

And the VL zone, which includes:

(d) The LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO:26);

(e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and

(f) The LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28).

62. The idiotype-specific polypeptide according to any one of Examples 53 to 57, wherein the idiotype-specific polypeptide comprises: a VH region comprising:

(a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15);

(b) The HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO:18);

(c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19);

And the VL zone, which includes:

(d) The LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO:25);

(e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and

(f) The LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28).

63. The unique type-specific polypeptide according to any one of Examples 53 to 62, wherein the protease-cleavable linker comprises a protease recognition sequence PQARK (SEQ ID NO: 41).

64. The idiotype-specific polypeptide according to any one of Examples 53 to 63, wherein the idiotype-specific polypeptide comprises: a heavy chain variable region 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:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24; and a light chain variable region 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:31, SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34.

65. The idiotype-specific polypeptide according to any one of Examples 53 to 64, wherein the idiotype-specific polypeptide comprises at least about 95%, 96%, 97%, 98%, 99% or 100% of the same heavy chain variable region sequence as SEQ ID NO:21 and at least about 95%, 96%, 97%, 98%, 99% or 100% of the same light chain variable region sequence as SEQ ID NO:31.

66. The idiotype-specific polypeptide according to any one of Examples 53 to 64, wherein the idiotype-specific polypeptide comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% of the same heavy chain variable region sequence as SEQ ID NO:21 and at least about 95%, 96%, 97%, 98%, 99%, or 100% of the same light chain variable region sequence as SEQ ID NO:32.

67. The idiotype-specific polypeptide according to any one of Examples 53 to 64, wherein the idiotype-specific polypeptide comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% of the same heavy chain variable region sequence as SEQ ID NO:22 and at least about 95%, 96%, 97%, 98%, 99%, or 100% of the same light chain variable region sequence as SEQ ID NO:32.

68. The idiotype-specific polypeptide according to any one of Examples 53 to 64, wherein the idiotype-specific polypeptide comprises at least about 95%, 96%, 97%, 98%, 99% or 100% of the same heavy chain variable region sequence as SEQ ID NO:23 and at least about 95%, 96%, 97%, 98%, 99% or 100% of the same light chain variable region sequence as SEQ ID NO:32.

69. The idiotype-specific polypeptide according to any one of Examples 53 to 64, wherein the idiotype-specific polypeptide comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% of the same heavy chain variable region sequence as SEQ ID NO:23 and at least about 95%, 96%, 97%, 98%, 99%, or 100% of the same light chain variable region sequence as SEQ ID NO:29.

70. The idiotype-specific polypeptide according to any one of Examples 53 to 64, wherein the idiotype-specific polypeptide comprises at least about 95%, 96%, 97%, 98%, 99%, or 100% of the same heavy chain variable region sequence as SEQ ID NO:24 and at least about 95%, 96%, 97%, 98%, 99%, or 100% of the same light chain variable region sequence as SEQ ID NO:34.

71. The idiotype-specific polypeptide according to any one of Examples 53 to 70, wherein the idiotype-specific polypeptide is a portion of a T-cell activation bispecific molecule.

72. The idiotype-specific polypeptide according to Examples 53 to 71, wherein the idiotype-specific polypeptide is humanized.

73. An isolated polynucleotide encoding a protease-activated T-cell-activating bispecific antigen-binding molecule according to any one of Examples 1 to 51 or a unique type-specific polypeptide according to any one of Examples 52 to 72.

74. A polypeptide encoded by a polynucleotide isolated according to Example 73.

75 A vector, particularly an expression vector, comprising the polynucleotide as described in Example 73.

76. A host cell comprising the polynucleotide described in Example 73 or the vector described in Example 75.

77. A method for producing a protease-activated T-cell activation bispecific molecule, the method comprising the steps of: a) culturing a host cell according to Example 76 under conditions suitable for expressing the protease-activated T-cell activation bispecific molecule, and b) recovering the protease-activated T-cell activation bispecific molecule.

78. A protease-activated T-cell activation bispecific molecule, which is produced by the method described in Example 77.

79. A method for producing a unique type-specific polypeptide, comprising the steps of: a) culturing a host cell according to Example 76 under conditions suitable for expressing the unique type-specific polypeptide, and b) recovering the unique type-specific polypeptide.

80. A unique type-specific polypeptide produced by the method described in Example 79.

81. A pharmaceutical composition comprising a protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 51 and a pharmaceutical carrier.

82. A pharmaceutical composition comprising a unique type-specific polypeptide according to any one of Examples 52 to 72 and a pharmaceutical carrier.

83. The protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 51 or the composition described in Example 81 or 82, for use as a medicine.

84. A protease-activated T-cell activation bispecific molecule used according to Example 83, wherein the drug is used in an individual to treat cancer or delay its progression, treat immune-related diseases or delay their progression, or enhance or stimulate immune responses or functions.

85. A protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 51 or a unique type-specific polypeptide according to any one of Examples 52 to 72, for the treatment of a disease in an individual with this need.

86. The protease-activated T-cell activating bispecific molecule or idiotype-specific polypeptide according to Example 85, for the treatment of a disease in an individual with this need, wherein the disease is cancer.

87. Use of the protease-activated T-cell activation bispecific molecule according to any one of Examples 1 to 51 or the unique type-specific polypeptide according to any one of Examples 52 to 72 for the manufacture of a medicament for the treatment of a disease.

88. The use according to Example 87, wherein the disease is cancer.

89. A method of treating a disease in an individual, the method comprising administering to the individual a therapeutically effective amount of a composition comprising a protease-activated T-cell activating bispecific molecule according to any one of Examples 1 to 51 or the composition according to Example 81.

90. A method for inducing target cell lysis, comprising contacting the target cell with a protease-activated T cell-activating bispecific molecule according to any one of Examples 1 to 51 or a composition according to Example 81 in the presence of T cells.

91. The method according to Example 90, wherein the target cell is a cancer cell.

92. The method according to Example 90 or 91, wherein the target cell expresses a protease capable of activating the protease-activated T-cell dual-specific molecule.

93. A method for reducing the in vivo toxicity of a T-cell activation bispecific molecule, comprising linking a unique type-specific polypeptide according to any one of Examples 52 to 72 to a T-cell activation bispecific molecule using a protease-cleavable linker to form a protease-activated T-cell activation bispecific molecule, wherein the in vivo toxicity of the protease-activated T-cell activation bispecific molecule is reduced compared to the toxicity of the T-cell activation bispecific molecule.

116. The present invention as described above.

Exemplary sequence

Table 2: CDR definition according to Kabat

Example

The following are examples of the methods and compositions of the present invention. It should be understood that various other embodiments may be practiced given the general description provided above.

Example 1

Expression builder:

All T-cell bispecific molecules are generated in the form of a proprietary 2+1 heterodimer based on a mortise and tenon technique (two binding moieties for the target antigen and one for CD3). The anti-CD3 conjugate blocks scFv (stabilized via an H44/L100 disulfide bridge) from fusing with the N-terminus of the VH of the CD3-binding Fab in the VHVL sequence (Figure 1). The linker between scFv and Fab is 33 amino acids long and consists of a proteolytic enzyme site embedded in the GS linker sequence.

The genes of each strand of proTCB are inserted separately into a mammalian expression vector. The expression of all genes is controlled by the human CMV promoter-intron A-5'UTR box. The BGH polyadenylation signal is located downstream of the gene.

Production of FolR1 proTCB with a protein-cleavable adaptor by protein lysin.

Bispecific proTCB molecules with different protein lysin linkers were generated by transient transfection of Expi293F ^™ cells. Cells were seeded at a density of 2.5 x ^10⁶ /ml in Expi293 ^™ medium (Gibco, catalog 1435101). The expression vector cassette ExpiFectamine (Gibco, ExpiFectamine ^™ Transfection Kit, catalog 13385544) was separately mixed in OptiMEM ^™ serum-depleted medium (Gibco, catalog 11520386). After 5 minutes, the two solutions were combined, mixed by pipetting, and incubated at room temperature for 25 minutes. Cells were added to the expression vector/ExpiFectamine solution and incubated at 37°C for 24 hours in a shaking incubator with 5% _CO₂ . One day after transfection, supplements (transfection enhancers 1 and 2, ExpiFectamine ^™ Transfection Kit) were added. Four to five days later, the cell supernatant was harvested by centrifugation and subsequent filtration (0.2 μm filter), and the protein was purified from the harvested supernatant using the standard method shown below.

Purification of IgG-like proteins

Proteins were purified from filtered cell culture supernatant according to a standard protocol. In short, Fc-containing proteins were purified from the filtered cell culture supernatant using protein A affinity chromatography (equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5; elution buffer: 20 mM sodium citrate, pH 3.0). Elution was performed at pH 3.0, followed by immediate pH neutralization of the sample. Proteins were concentrated by centrifugation (Millipore ULTRA-15 (Art. Nr.: UFC903096), and then size exclusion chromatography was used to separate aggregated proteins from monomeric proteins in 20 mM histidine, 140 mM sodium chloride (pH 6.0) (or as otherwise specified).

Table 3: Production and Purification

Example 2

Use SPR to determine the mastriptase cleavage rate

The cleavage rate of recombinant matriptase was investigated using surface plasmon resonance (SPR) on a Biacore T200 instrument (Cytiva). Biotinylated CD3ε was immobilized on an S-series Sensorchip SA (Cytiva, 29104992) to a final surface density of 2000–4000 resonance units (RU). 10 nM FOLR1 proTCB and 50 pM recombinant matriptase (R&D Systems, 3946-SE) were incubated together at 37 °C in PBS-T pH 7.4 and PBS-T pH 6.5. The CD3ε binding response and proTCB activation rate were monitored by continuously injecting the proTCB/matriptase mixture onto the surface at a flow rate of 5 μl/min for 30 s for up to 10 h. The CD3ε surface was regenerated by continuously injecting 10 mM glycine (pH 1.5) at a flow rate of 5 μl/min for 60 s. In the same experiment, concentration series of 0.16, 0.31, 0.63, 1.25, and 2.5 nM FOLR1 TCB were injected to generate calibration lines, and the obtained proTCB binding responses were converted from resonance units (RU) to molar concentrations (nM). The molar concentrations of activated proTCB were plotted against incubation time, and the cleavage rate (pM/min) was calculated by determining the slope of each derived line.

Table 4: Initial cleavage rate at different pH values:

Example 3

Developability of FOLR1proTCB including different humanized cover variants

The selected FORL1proTCB molecules with different humanization masks were produced as described in Example 1, and their stability and exploitability were analyzed.

Table 5: Production and Purification

thermal stability

Thermal stability was investigated using static light scattering (SLS) on the Uncle platform (Unchained Labs). Simply put, 9 μl of a 1 mg/mL proTCB solution was transferred to the instrument's sample holder. A temperature gradient from 30 °C to 90 °C was applied at a rate of 0.1 °C/min. Static light scattering was monitored at a wavelength of 266 nm to determine the settling temperature (Tagg).

Apparent hydrophobicity

Apparent hydrophobicity was investigated using high-performance liquid chromatography (HPLC) via hydrophobic interaction chromatography (HIC). In short, 20 μl of proTCB at a concentration of 1 mg/mL was injected onto a TSKgel ether-5PW column (Tosoh Bioscience 0008641). A linear gradient of 0 to 1.5 _{M NH₄SO₄} in 25 mM sodium phosphate was applied at a flow rate of 0.8 mL/min for 20 minutes at a column temperature of 40 °C. The detection wavelength was set to 214 nm. Relative retention time was calculated using an appropriate reference antibody.

FcRn chromatography

Relative FcRn binding affinity was determined by high-performance liquid chromatography (HPLC). Simply put, 30 μl of proTCB at a concentration of 1 mg/mL was injected onto an FcRn streptavidin agarose gel column (Roche Diagnostics 08128057001). Following the manufacturer's recommendations, a stepwise gradient of 20 mM sodium MES, 140 mM NaCl, pH 5.5, and pH 8.8 was applied at a column temperature of 25 °C. The detection wavelength was set to 280 nm. The relative retention time was calculated using an appropriate reference antibody.

Heparin chromatography

Relative heparin binding affinity was determined by high-performance liquid chromatography (HPLC). In short, 100 μl of proTCB at a concentration of 0.35 mg/mL was injected onto a TSK-Gel heparin-5PW column (Tosoh Bioscience 13064). Stepwise gradients were applied using 50 mM Tris at pH 7.4 and 50 mM Tris, 1 M NaCl, pH 7.4, respectively. The flow rate was set to 0.8 mL/min, and the column temperature to 25 °C. The detection wavelength was set to 280 nm. The relative retention time was calculated using an appropriate reference antibody.

Table 6: Results

parameter P1AF5794 P1AF65795 P1AF5796 <![CDATA[Thermal stability(SLS Tagg)]]> 63.5 62.4 62.5 Apparent hydrophobicity (HIC) 0.22 0.21 0.21 FcRn chromatography 0.25 0.25 0.29 Heparin chromatography 0.64 0.64 0.69

All tested molecules exhibited good thermal stability (>60℃) and showed completely acceptable values regarding hydrophobicity and in FcRn and heparin chromatography.

Example 4

SPR was used to determine the cleavage rate of different proteases.

The cleavage rates of recombinant protein lyase (R&D Systems 3946-SEB), protein lyase-2 (Enzo ALX-201-752), hepsin (R&D Systems 4776-SE), uPA (Sigma-Aldrich 6273), Legumin (R&D Systems 2199-CY), and furin protease (R&D Systems 1503-SE) were investigated using surface plasmon resonance (SPR) on a Biacore T200 instrument (Cytiva). Biotinylated CD3ε was immobilized on S-series Sensorchip SA (Cytiva, 29104992) to a final surface density of 2000–4000 resonance units (RU). FOLR1proTCB was incubated in PBS-TpH 7.4 at 37°C with the following concentrations of the different proteases listed above:

i. 10 nM proTCB + 50 pM protein lyase

ii. 10 nM proTCB + 1 U protein lyase-2

iii. 10 nM proTCB + 300 pM activated Hepsin

IV. 10 nM proTCB + 5.5 nM uPA

v. 10nM proTCB + 5nM activated Legumin

vi. 10 nM proTCB + 3 nM furin protease (with 1 mM CaCl2 added)

The CD3ε binding response and proTCB activation rate were monitored for up to 10 hours by continuously injecting a proTCB/protease mixture onto the surface at a flow rate of 5 μl/min for 30 seconds. CD3ε surface regeneration was achieved by injecting 10 mM glycine (pH 1.5) at a flow rate of 5 μl/min for 60 seconds. In the same experiment, a series of concentrations of 0.16, 0.31, 0.63, 1.25, and 2.5 nM FOLR1TCB were injected to generate calibration lines, and the obtained proTCB binding responses were converted from resonance units (RU) to molar concentrations (nM). The molar concentration of activated proTCB was plotted against incubation time, and the cleavage rate (pM/min) was calculated by determining the slope of each derivation line.

Table 7: Protein lysin cleavage rate

Table 8: Cutting rate of protein lyase-2

Table 9: Hepsin Cutting Rate

Table 10: uPA Cutting Rate

Table 11: Legumin Cutting Rate

Table 12: Frin protease cleavage rate

Example 5

Developability of FOLR1proTCB (different protein cleavage sites; humanized masking)

The stability and exploitability of the selected FOLR1proTCB molecule were analyzed. The same methods as in Example 3 were used.

Table 13: Results

parameter P1AG0882 P1AG0884 <![CDATA[Thermal stability(SLS Tagg)]]> 63.0 62.2 Apparent hydrophobicity (HIC) 0.22 0.22 FcRn chromatography 0.27 0.22 Heparin chromatography 0.65 0.63

All tested molecules exhibited good thermal stability (>60℃) and showed completely acceptable values regarding hydrophobicity and in FcRn and heparin chromatography.

Example 6

Developability of humanized anti-idiotype IgG

The binding of anti-idiotype antibodies to 20 mM Hmis/HCl, 140 mM NaCl at pH 6.0 at 40 °C, or 1 x PBS at pH 7.4 at 37 °C for 14 days was investigated using a Biacore T200 instrument (Cytiva). In short, following the manufacturer's instructions, biotinylated anti-human CD3ε antibody and biotinylated anti-human IgG (Capture Select, Thermoscientific, 7103302500) were immobilized on the s-series sensor chip CAP (Biotin Capture Kit, Cytiva 28920234) after injection of the capture reagent. The obtained surface densities were approximately 1000 RU and 1500 RU, respectively. Anti-idiotype antibodies were injected onto the chip surface at a concentration of 1 μg/ml for 30 seconds at a flow rate of 5 μl/min. Dissociation was monitored for 30 seconds. After each injection, the chip surface was regenerated by injecting 2M guanidine hydrochloride and 0.5M NaOH for 120 seconds. The difference in bulk refractive index was corrected by subtracting the response obtained from the simulated surface.

To standardize the binding signal of anti-idiotype antibodies, the binding response on the surface of anti-human CD3ε antibody was divided by the binding response on the surface of anti-human IgG. The relative activity concentration was obtained by dividing the normalized response of the stressed sample by the normalized response of each molecule in a non-stressed reference sample.

Table 14: Results

Example 7

proTCB's developability

Binding of proTCB was investigated using a Biacore T200 instrument (Cytiva) via surface plasmon resonance after incubation at 40°C in 20 mM Hmis/HCl, 140 mM NaCl, pH 6.0, or at 37°C in 1xPBS, for 14 days. In short, mouse anti-huIgG CH2 PG-LALA antibody (P1AE2335) and human CD3ε (P1AA6119) were immobilized on the CM5 (Cytiva) s-series sensor chip using standard amine coupling chemistry. The resulting ligand densities were approximately 8500 RU and 7000 RU, respectively. For FolR1 binding assessment, proTCB was captured onto the anti-human IgG PG-LALA surface at a concentration of 2 μg/ml and a flow rate of 10 μl/min for 75 seconds. Subsequently, human FolR1 (P1AD6798) was injected at a concentration of 900 nM at a flow rate of 10 μl/min for 120 seconds. Dissociation was monitored for 120 seconds. After each injection of human FolR1, the surface was regenerated by injecting 20 mM NaOH for 35 seconds. For CD3ε binding assessment, proTCB was injected into the CD3ε surface at a concentration of 10 μg/ml at a flow rate of 10 μg/ml for 90 seconds. Dissociation was monitored for 90 seconds. After each injection, the surface was regenerated by injecting 10 mM glycine at pH 2.1 for 70 seconds. The bulk refractive index difference was corrected for each interaction by subtracting the response obtained from the simulated surface.

To normalize the proTCB binding signal, the binding responses of FolR1 and CD3ε were divided by the binding response on the surface of anti-human IgG PG-LALA. The relative activity concentration was obtained by dividing the normalized response of the stressed sample by the normalized response of each molecule in the unstressed reference sample (FolR1) or the unmasked control molecule (CD3ε).

Table 15: Relative FolR1 binding

Table 16: Relative CD3 binding

Example 8

In vivo stability and pharmacokinetic characteristics of different linkers after a single injection in NSG mice.

A single dose of 5 mg/kg pro-FolR1-TCB molecules containing selective cleavage sites of different protein lysins was injected into NSG mice. As shown in Figure 2, 200 μl of the appropriate solution was administered intravenously to all mice. To obtain an appropriate amount of compound per 200 μl, the stock solution was diluted with histidine buffer (Table 17). Blood samples were collected from two mice in each group at each time point: 24 hours, day 7, and day 10. Serum samples were analyzed for the injected compound using ELISA.

Molecular assays were performed using LBA (ligand binding assay) as follows. Serum samples from mice treated with P1AF5419 (cleavage site: HQARK), P1AF5420 (cleavage site: PQARK), or P1AE6554 (typical FolR1 2+1TCB) were analyzed using an ECLIA method (“total assay”) with specificity for the human CH1/PGLALA domain and an ECLIA method (“activity assay”) using CD3 anti-ID antibody capture and detection with anti-PGLALA specific antibody on a cobas e411 instrument.

For the “Total Assay”, test samples of P1AF5419 (cleavage site: HQARK) (004-09), P1AF5420 (cleavage site: PQARK) (004-06), or P1AE6554 (typical FolR1 2+1TCB), the first detection antibody mAb<H-IgG>11-1.19.31-IgG-Bi, the second detection antibody mAb<H-Fc(PGLALA)>M-1.7.24-IgG-Ru, and SA beads are added stepwise to the test container and incubated for 9 minutes in each step.

For the "activity assay," the test sample of FolR1 TCB (007-19), the first detection antibody mAb<CH2527>rH-4.24.72-IgG()-Bi, the second detection antibody mAb<H-Fc(PGLALA)>M-1.7.24-IgG-Ru, and SA beads were progressively added to the test container, and incubated for 9 minutes in each step. Finally, the SA bead-bound complex was detected by a measurement unit that repeatedly counted the SA beads. The count was proportional to the analyte concentration in the test sample.

Table 17: Preparation of Stock Solutions

Serum analyses of mice administered 5 mg/kg of FOLR1-TCB or FOLR1 pro-TCB showed low levels of active TCB, less than 5% of total pro-TCB, across all time points measured against both FOLR1 pro-TCB molecules containing HQARK or PQARK cleavage sites. For typical (unmasked) FOLR1-TCB, 100% active TCB was detected (Figure 3).

Example 9

Efficacy study of the pro-FOLR1-TCB construct containing selective cleavage sites of different protein lysins in the humanized mouse BC004 PDX.

The HER2+ER- xenograft model BC004, derived from human breast cancer patients, was purchased from OncoTest (Freiburg, Germany). Tumor fragments were digested with collagenase D and DNase I (Roche), counted, and 1× ^10⁶ BC004 cells were injected into a 100 μl mixture of RPMI and matrix gel. The mixture was then subcutaneously injected into the flank of anesthetized mice using a 22G to 30G needle.

According to the guidelines (GV-Solas; Felasa; Tiersch G), female NSG mice (Jackson laboratory) aged 4-5 weeks at the start of the experiment were kept in a pathogen-free environment with a 12-hour light/12-hour dark cycle. The experimental protocol was reviewed and approved by the local government (P 2011/128). Upon arrival, the animals were kept in the new environment for one week for acclimatization and observation. Regular and continuous health monitoring was conducted.

Female NSG mice were intraperitoneally injected with 15 mg/kg busulfan, followed one day later by intravenous injection of ¹ x 10⁵ human hematopoietic stem cells isolated from umbilical cord blood. At weeks 14–16 post-stem cell injection, mice were exsanguinated sublingually, and blood was analyzed by flow cytometry for successful humanization. Effectively transplanted mice were randomized to different treatment groups based on their human T cell frequency. At this time, mice were injected with tumor PDX cells, and when the tumor size reached approximately 200 mm³ (day 28), they were treated weekly with either the compound or histidine buffer (solvent). All mice were intravenously injected with 200 μl of the appropriate solution. To obtain an appropriate amount of compound per 200 μl, the stock solution was diluted with histidine buffer as needed (Table 18).

Measure tumor growth twice a week using calipers (Figure 2), and calculate tumor volume using the following formula:

T _v ：(W ² /2)x L (W: width, L: length)

At termination (day 58), the mice were euthanized, and the tumors and spleens were removed and weighed.

Figure 5A shows tumor growth kinetics (mean, +/- SEM) and individual tumor growth per mouse in the most effective treatment group. As described here, the FOLR1 pro-TCB containing the PQARK cleavage site was identified as the optimal pro-TCB tested in this study. Furthermore, no efficacy was observed in the groups treated with the FOLR1 pro-TCB containing an uncuttable connector. In Figures 5A through 5G, tumor weight at termination is plotted for all treatment groups. This reading clearly supports the findings in tumor growth kinetics and shows that the pro-TCB containing the PQARK cleavage site results in a comparable tumor weight at termination compared to the typical FolR1 TCB.

Table 18: Preparation of Stock Solutions

Example 10

Production and purification of FOLR1proTCB using clone 22 as a CD3 binding agent

P1AI3541 (FOLR1proTCB with a PQARK cleavable adapter, SEQ ID No: 45, 46, 56), P1AI3542 (FOLR1proTCB with an uncleavable adapter, SEQ ID No: 45, 46, 59), and P1AI3543 (unmasked TCB, SEQ ID No: 45, 57, 58) were transiently produced in HEK293 cells. Purification was performed according to standard procedures using protein A affinity chromatography followed by size exclusion chromatography.

Table 19: Production Quantity and Quality

Example 11

Jurka NFAT activation induced by FOLR1 TCB, a protease that can be activated by the CD3-binding clone 22.

After incubation with TCB and huFOLR1-coated beads used for crosslinking, Jurkat NFAT activation mediated by FORL1TCB (SEQ ID NO: 45, 57, 58) or FORL1 pro-TCB (SEQ ID No: 45, 46, 56) was measured (Figure 6). FOLR1 TCB was used as a positive control because it did not contain a mask that blocked CD3 binding. FORL1 pro-TCB containing a CD3 mask with an uncleavable G4S linker was used as a negative control. Cleavable FORL1 pro-TCB containing a PQARK cleavage site was incubated overnight at room temperature with human rec protein lyase to cleave the CD3 mask. Jurkat NFAT activation was measured by luminescent readings using a Tecan Spark Reader with a sampling time of 0.5 seconds after incubation at 37°C in a humidified incubator for 24 hours. Each point represents the mean of three replicates. Standard deviation is indicated by error bars (n = 1).

Claims (44)

1. A protease-activated T-cell activation bispecific molecule, comprising: (a) The first antigen-binding region capable of binding to CD3; (b) a second antigen-binding moiety capable of binding to target cell antigens; and (c) A masking portion of the T cell activation bispecific molecule is covalently linked via a peptide linker, wherein the masking portion is capable of binding idiotype to either the first antigen-binding portion or the second antigen-binding portion, thereby reversibly masking either the first antigen-binding portion or the second antigen-binding portion. The adapter contains a protease recognition sequence XQARK (SEQ ID NO:39), where X is histidine (H) or proline (P). 2. The protease-activated T-cell activation bispecific molecule according to claim 1, wherein the masking portion is covalently linked to the first antigen-binding portion and reversibly masks the first antigen-binding portion. 3. The protease-activated T-cell activation bispecific molecule according to claim 1 or 2, wherein the masking portion is covalently linked to the heavy chain variable region of the first antigen-binding portion. 4. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 3, wherein the masking portion is scFv. 5. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 4, wherein (i) the second antigen-binding portion is a conventional Fab, or (ii) the second antigen-binding portion is a cross-Fab molecule, wherein the variable or constant regions of the Fab light chain and the Fab heavy chain are exchanged. 6. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 5, wherein the first antigen-binding portion is a conventional Fab molecule. 7. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 6, comprising a third antigen-binding portion, said third antigen-binding portion being a Fab molecule capable of binding to target cell antigens. 8. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 7, wherein the third antigen-binding portion is the same as the second antigen-binding portion. 9. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 8, wherein the target cell antigen is FolR1. 10. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 9, wherein the first antigen-binding portion and the second antigen-binding portion are fused to each other, optionally via a peptide linker. 11. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 10, wherein the second antigen-binding portion 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 portion. 12. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 11, further comprising an Fc domain consisting of a first subunit and a second subunit capable of stable association. 13. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 12, wherein the Fc domain is an IgG Fc domain, specifically an IgG1 Fc domain or an IgG4 Fc domain. 14. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 13, wherein the Fc domain exhibits reduced binding affinity to the Fc receptor and/or reduced effector function compared to the native IgG1 Fc domain. 15. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 14, wherein the antigen-binding portion capable of binding to CD3 comprises: a heavy chain variable (VH) region. (a) The amino acid sequence of the heavy chain complementarity-determining region (HCDR) 1 of SYAMN (SEQ ID NO:1); (b) The HCDR2 amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO:2); (c) The HCDR3 amino acid sequence of ASNFPASYVSYFAY (SEQ ID NO:3); And the light chain variable (VL) region, which includes: (d) The amino acid sequence of the light chain complementarity-determining region (LCDR) 1 of GSSTGAVTTSNYAN (SEQ ID NO:7); (e) The LCDR2 amino acid sequence of GTNKRAP (SEQ ID NO:8); and (f) The LCDR3 amino acid sequence selected from ALWYSNLWV (SEQ ID NO:9). 16. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 15, wherein the antigen-binding portion capable of binding to CD3 comprises: a VH region containing 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: 5; and/or a VL region containing 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: 10. 17. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 14, wherein the antigen-binding portion capable of binding to CD3 comprises: a heavy chain variable (VH) region, which includes (a) The amino acid sequence of the heavy chain complementarity-determining region (HCDR) 1 of SYAMN (SEQ ID NO:1); (b) The HCDR2 amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ ID NO:2); (c) The HCDR3 amino acid sequence of HTTFPSSYVSYYGY (SEQ ID NO:4); And the light chain variable (VL) region, which includes: (d) The amino acid sequence of the light chain complementarity-determining region (LCDR) 1 of GSSTGAVTTSNYAN (SEQ ID NO:7); (e) The LCDR2 amino acid sequence of GTNKRAP (SEQ ID NO:8); and (f) The LCDR3 amino acid sequence selected from ALWYSNLWV (SEQ ID NO:9). 18. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 15, wherein the antigen-binding portion capable of binding to CD3 comprises: a VH region containing 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: 6; and/or a VL region containing 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: 10. 19. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 18, wherein the masking portion comprises: a VH region comprising: (a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15), (b) The HCDR2 amino acid sequence selected from the group consisting of WINTETGEPRYTDDFKG (SEQ ID NO:16), WINTETGEPRYTDDFTG (SEQ ID NO:17) and WINTETGEPRYTQGFKG (SEQ ID NO:18); (c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19); And the VL zone, which includes: (d) The LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO:25) or KSSKSVSTSSYSYMH (SEQ ID NO:26); (e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and (f) The LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28) or QQSREFPYT (SEQ ID NO:29). 20. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 19, wherein the masking portion comprises: a VH region comprising: (a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15); (b) The HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO:16); (c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19); And the VL zone, which includes: (d) The LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO:25); (e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and (f) The LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28). 21. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 19, wherein the masking portion comprises: a VH region comprising: (a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15); (b) The HCDR2 amino acid sequence of WINTETGEPRYTDDFKG (SEQ ID NO:16); (c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19); And the VL zone, which includes: (d) The LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO:26); (e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and (f) The LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28). 22. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 19, wherein the masking portion comprises: a VH region comprising: (a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15); (b) The HCDR2 amino acid sequence of WINTETGEPRYTDDFTG (SEQ ID NO:17); (c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19); And the VL zone, which includes: (d) The LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO:26); (e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and (f) The LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28). 23. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 19, wherein the masking portion comprises: a VH region comprising: (a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15); (b) The HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO:18); (c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19); And the VL zone, which includes: (d) The LCDR1 amino acid sequence of KSSKSVSTSSYSYMH (SEQ ID NO:26); (e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and (f) The LCDR3 amino acid sequence of QHSREFPYT (SEQ ID NO:28). 24. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 19, wherein the masking portion comprises: a VH region comprising: (a) The HCDR1 amino acid sequence of DYSMN (SEQ ID NO:15); (b) The HCDR2 amino acid sequence of WINTETGEPRYTQGFKG (SEQ ID NO:18); (c) The HCDR3 amino acid sequence of EGDYDVFDY (SEQ ID NO:19); And the VL zone, which includes: (d) The LCDR1 amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO:25); (e) The LCDR2 amino acid sequence of YVSYLES (SEQ ID NO:27); and (f) The LCDR3 amino acid sequence of QQSREFPYT (SEQ ID NO:29). 25. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 24, wherein the second antigen-binding portion is capable of binding to FolR1 and comprises: a VH region comprising: a) The HCDR1 amino acid sequence of NAWMS (SEQ ID NO: 11); b) The HCDR2 amino acid sequence of RIKSKTDGGTTDYAAPVKG (SEQ ID NO:12); and c) The HCDR3 amino acid sequence of PWEWSWYDY (SEQ ID NO:13); And the VL zone, which includes: d) LCDR1 of GSSTGAVTTSNYAN (SEQ ID NO:7); e) The LCDR2 amino acid sequence of GTNKRAP (SEQ ID NO: 8); and f) The LCDR3 amino acid sequence of ALWYSNLWV (SEQ ID NO:9). 26. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 25, wherein the antigen-binding portion capable of binding to FolR1 comprises: a VH region containing at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 14; and/or a VL region containing at least about 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 10. 27. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 26, wherein the protease-cleavable linker comprises the protease recognition sequence PQARK (SEQ ID NO: 41). 28. A unique type-specific polypeptide for reversibly concealing the anti-CD3 antigen binding site of a molecule, wherein the unique type-specific polypeptide is covalently linked to the molecule via a peptide linker, wherein the linker comprises a protease recognition sequence XQARK (SEQ ID NO: 39), wherein X is histidine (H) or proline (P). 29. The idiotype-specific polypeptide of claim 28, wherein the idiotype-specific polypeptide is an anti-idiotype scFv. 30. The unique type-specific polypeptide according to claim 29, wherein the molecule is a T cell activation bispecific molecule. 31. The unique type-specific polypeptide of claim 30, wherein the linker comprises a protease recognition sequence PQARK (SEQ ID NO: 41). 32. A pharmaceutical composition comprising a protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 27 and a pharmaceutical carrier. 33. A pharmaceutical composition comprising a unique type-specific polypeptide according to any one of claims 28 to 31 and a pharmaceutical carrier. 34. An isolated polynucleotide encoding a protease-activated T-cell-activating bispecific antigen-binding molecule according to any one of claims 1 to 27. 35. An isolated polynucleotide encoding a unique type-specific polypeptide according to any one of claims 28 to 31. 36. A vector, particularly an expression vector, comprising the polynucleotide according to claim 34 or 35. 37. A host cell comprising the vector according to claim 36. 38. A method for producing a protease-activated T-cell activation bispecific molecule, the method comprising the steps of: a) culturing a host cell according to claim 37 under conditions suitable for expressing the protease-activated T-cell activation bispecific molecule, and b) recovering the protease-activated T-cell activation bispecific molecule. 39. The protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 27, for use as a drug. 40. The protease-activated T-cell activation bispecific molecule used according to claim 39, wherein the drug is used in an individual to treat cancer or delay its progression, treat an immune-related disease or delay its progression, or enhance or stimulate an immune response or function. 41. Use of the protease-activated T-cell-activating bispecific molecule according to any one of claims 1 to 27 for the manufacture of a medicament for the treatment of a disease. 42. Use of the protease-activated T-cell activation bispecific molecule according to claim 41, wherein the disease is cancer. 43. A method of treating a disease in an individual, the method comprising administering to the individual a therapeutically effective amount of a composition comprising a protease-activated T-cell activation bispecific molecule according to any one of claims 1 to 27. 44. The method of claim 43, used to treat cancer or delay its progression.