TW202342521A - Conditionally bispecific binding proteins - Google Patents

Abstract

The present disclosure, in some aspects, provides conditionally activable molecules comprising one single sdABD that binds to a human target tumor antigen (TTA) and uses thereof.

Description

conditional bispecific binding protein

This application relates to conditional bispecific binding proteins.

In a variety of clinical settings, selective destruction of individual cells or specific cell types is often required. For example, the main goal of cancer therapy is to specifically destroy tumor cells while leaving healthy cells and tissues intact and undamaged as much as possible. One such approach is by inducing an immune response against the tumor, allowing immune effector cells such as natural killer (NK) cells or cytotoxic T lymphocytes (CTL) to attack and destroy tumor cells.

In some aspects, the present disclosure provides methods and compositions for reducing the toxicity and/or side effects of immune cell-engaging bispecific antibodies that bind cancer cells and immune cells to stimulate immunity. Cells kill target cancer cells. Many of the proteins provided herein are prodrugs that can be activated by proteases, such as those found in the tumor microenvironment. In some embodiments, the proteins described herein are formulated such that when they are not in the tumor microenvironment, the protein is able to bind to tumor cells but not immune cells (inactive), and such that when the protein enters the tumor microenvironment, the protein Cleavage of the cleavable linker "activates" the protein, producing two "active" bispecific molecules, each of which can bind to tumor cells and immune cells.

In some embodiments, the proteins described herein comprise a single domain that binds a human target tumor antigen (TTA). In contrast to similar constructs containing more than one (e.g., two) domains that bind human TTAs (e.g., the same or different TTAs), the proteins described herein (e.g., in prodrug and active forms) may Exhibit reduced affinity and/or cell surface residence time.

In some embodiments, reducing the affinity and/or cell surface residence time of proteins described herein does not significantly reduce the in vivo tumor suppressive efficacy of such proteins. In some embodiments, enhanced in vivo tumor suppressive efficacy is unexpectedly achieved despite reduced affinity and cell surface retention of proteins described herein. In some embodiments, proteins described herein are effective against cancers with any level of HER2 expression. In some embodiments, proteins described herein are effective against cancers with low levels of HER2 expression. In some embodiments, proteins described herein are effective against cancers with intermediate levels of HER2 expression. In some embodiments, proteins described herein are effective against cancers with high levels of HER2 expression.

In some embodiments, reducing the affinity and cell surface residence time of a protein described herein results in reduced toxicity and increased individual tolerance (eg, tolerance at higher doses). In some embodiments, proteins described herein exhibit an enhanced safety and efficacy profile compared to proteins with more than one domain that binds human TTA.

Some aspects of the present disclosure provide proteins comprising: from the N-terminus to the C-terminus: (i) a first single domain antigen binding domain (sdABD) and a first domain linker at the C-terminus of the first sdABD, wherein (i) is present or absent; (ii) a first constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region linked to a light chain variable region via a first constrained non-cleavable linker (CNCL), wherein the heavy chain The variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen, and wherein the heavy chain variable region and the light chain variable region do not associate in the first restricted scFv domain , and the first restricted scFv domain does not bind the human immune cell antigen; (iii) a second domain linker and a second single domain antigen binding domain (sdABD), wherein the second domain linker is at the N-terminus of the second sdABD, and wherein (iii) is present or absent; (iv) cleavable linkers; (v) a second constrained single chain variable fragment (scFv) domain comprising a pseudo heavy chain variable region linked to a pseudo light chain variable region via a second constrained non-cleavable linker (CNCL), wherein the The pseudo heavy chain variable region and the pseudo light chain variable region do not associate in the second restricted scFv domain, and the second restricted scFv domain does not bind the human immune cell antigen of (ii); (vi) third domain linker; and (vii) a third sdABD, which binds human serum albumin (HSA); wherein the heavy chain variable region of (ii) and the pseudo light chain variable region of (v) are intramolecularly associated to form a variable fragment (Fv) that does not bind the immune cell antigen; and wherein the pseudo heavy chain variable region of (v) and the light chain variable region of (ii) are intramolecularly associated to form a variable fragment (Fv) that does not bind the immune cell antigen; Among them, (i) and (iii) do not exist; And when (i) and (iii) both exist: (a) The first sdABD binds human target tumor antigen (TTA) and the second sdABD does not bind human TTA; or (b) The first sdABD does not bind human TTA and the second sdABD binds human TTA.

In some embodiments, (i) and (iii) are both present, and wherein the first sdABD does not bind human TTA, and the second sdABD binds human TTA. In some embodiments, the first sdABD binds hen egg lysozyme (HEL). In some embodiments, the first sdABD comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, (i) and (iii) are both present, and wherein the second sdABD does not bind human TTA, and the first sdABD binds human TTA. In some embodiments, the second sdABD binds hen egg lysozyme (HEL). In some embodiments, the second sdABD comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, (i) is absent and (iii) is present, and wherein the second sdABD binds human TTA. In some embodiments, (i) is present and (iii) is not present, and wherein the first sdABD binds human TTA.

In some embodiments, the human TTA is HER2, EGFR, FOLR1, TROP2, or EpCAM. In some embodiments, the human TTA is HER2. In some embodiments, the first sdABD or the second sdABD that binds HER2 comprises the amino acid sequence of any of the following: SEQ ID NOs: 41, 45, 48-52, 54, 58, 60, 63, 66 , 68, 72, 75-78, 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121 and 165.

In some embodiments, the cleavable linker of (iv) is 8-45 amino acids in length.

In some embodiments, the cleavable linker of (iv) comprises a cleavage site for a protease present in the tumor microenvironment. In some embodiments, the protease is MMP2, MMP9, Meprin, Cathepsin, granzyme, Matriptase, thrombin, enterokinase, KLK7-6, KLK7-13, KLK7- 11. KLK7-10 or uPA.

In some embodiments, the immune cell is a T cell, natural killer (NK) cell, natural killer T (NKT) cell, macrophage, B cell, neutrophil, dendritic cell, or monocyte. In some embodiments, the human immune cell antigen is CD3, CD28, T cell receptor, programmed cell death protein 1 (PD-1), PD-L1, cytotoxic T lymphocyte associated protein 4 (CTLA-4), T cell immunoglobulin and mucin domain 3 (TIM-3), lymphocyte activating gene 3 (LAG-3), killer cell immunoglobulin-like receptor (KIR), CD137, OX40, OX40L, CD27, GITR ( TNFRSF18), TIGIT, inducible T cell costimulator (ICOS), CD16A, CD226, CD96, CD40L, CRTAM, LFA-1, NKG2D, CSF1R, CD40, MARCO, VSIG4 or CD163. In some embodiments, the human immune cell antigen is CD3.

In some embodiments, the heavy chain variable region is linked to the N-terminus of the light chain variable region in the first restricted scFv domain of (ii). In some embodiments, the pseudo heavy chain variable region is linked to the C-terminus of the pseudo light chain variable region in the second restricted scFv domain of (v).

In some embodiments, the first restricted non-cleavable linker of (ii) and/or the second restricted non-cleavable linker of (v) is 6-10 amino acids in length. In some embodiments, the first restricted non-cleavable linker of (ii) and/or the second restricted non-cleavable linker of (v) is 8 amino acids in length.

In some embodiments, the human immune cell antigen is CD3, the heavy chain variable region of (ii) includes the amino acid sequence of SEQ ID NO: 7, and the light chain variable region of (ii) includes SEQ ID NO: 8 The amino acid sequence.

In some embodiments, the pseudo heavy chain variable region of (v) comprises the amino acid sequence of SEQ ID NO: 33, and the pseudo light chain variable region of (v) comprises the amino acid sequence of SEQ ID NO: 34 sequence.

In some embodiments, the third sdABD comprises the amino acid sequence of SEQ ID NO: 22.

In some embodiments, the protein comprises the amino acid sequence of any of SEQ ID NOs: 37-40 and 173-176.

Further provided herein are nucleic acid molecules comprising a nucleotide sequence encoding any of the proteins described herein. In some embodiments, the nucleic acid molecule is a vector. In some embodiments, the vector is a realization vector.

Also provided herein are cells comprising any of the proteins described herein or the nucleic acid molecules described herein. Other aspects of the present disclosure provide methods of producing any of the proteins described herein, including culturing such cells under conditions that allow expression of the protein. In some embodiments, the methods further comprise isolating the protein.

Compositions comprising any of the proteins described herein are provided.

Other aspects of the present disclosure provide methods of treating cancer comprising administering to a subject any of the proteins described herein or a composition comprising such a protein. In some embodiments, the cancer expresses HER2. In some embodiments, the individual is a human individual.

Further provided herein are compositions comprising: The first protein and the second protein each include from the N-terminus to the C-terminus: (i) a first single domain antigen binding domain (sdABD) and a first domain linker at the C-terminus of the first sdABD, wherein (i) is present or absent; (ii) a first constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region linked to a light chain variable region via a first constrained non-cleavable linker (CNCL), wherein the heavy chain The variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen, and wherein the heavy chain variable region and the light chain variable region do not associate in the first restricted scFv domain , and the first restricted scFv domain does not bind the human immune cell antigen; (iii) a second domain linker and a second single domain antigen binding domain (sdABD), wherein the second domain linker is at the N-terminus of the second sdABD, and wherein (iii) is present or absent; (iv) cleavable linkers; (v) a second constrained single chain variable fragment (scFv) domain comprising a pseudo heavy chain variable region linked to a pseudo light chain variable region via a second constrained non-cleavable linker (CNCL), wherein the The pseudo heavy chain variable region and the pseudo light chain variable region do not associate in the second restricted scFv domain, and the second restricted scFv domain does not bind the human immune cell antigen of (ii); (vi) third domain linker; and (vii) a third sdABD, which binds human serum albumin (HSA); wherein the heavy chain variable region of (ii) and the pseudo light chain variable region of (v) are intramolecularly associated to form a variable fragment (Fv) that does not bind the immune cell antigen; and wherein the pseudo heavy chain variable region of (v) and the light chain variable region of (ii) are intramolecularly associated to form a variable fragment (Fv) that does not bind the immune cell antigen; Among them, (i) and (iii) do not exist; And when (i) and (iii) both exist: (a) The first sdABD binds human target tumor antigen (TTA) and the second sdABD does not bind human TTA; or (b) The first sdABD does not bind human TTA and the second sdABD binds human TTA.

In some embodiments, the first protein and the second protein are the same. In some embodiments, after cleavage of the cleavable linker of (iv) in the first protein and the second protein: The heavy chain variable region of the first protein associates with the light chain variable region of the second protein to form an active Fv that binds to the human immune cell antigen; The light chain variable region of the first protein associates with the heavy chain variable region of the second protein to form an active Fv that binds the human immune cell antigen.

In some embodiments, lysis occurs in the tumor microenvironment of the individual upon administration of the composition to the individual.

Further provided herein are compositions comprising a homodimer of a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are identical, and wherein the first polypeptide and the second polypeptide Each of the peptides contains: (i) a single domain antigen binding domain (sdABD) that binds a first human target tumor antigen (TTA); (ii) domain linkers; (iii) A constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region (VH) linked to a light chain variable region (VL) via a constrained non-cleavable linker (CNCL), wherein the The heavy chain variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen, and wherein the heavy chain variable region and the light chain variable region do not associate in the restricted scFv domain , and the restricted scFv domain does not bind the human immune cell antigen; wherein the VH of the first polypeptide associates with the VL of the second polypeptide, and the VL of the first polypeptide associates with the VH of the second polypeptide, forming two active variable fragments ( Fv), each active variable fragment is capable of binding to the immune antigen.

In some embodiments, the TTA is HER2, EGFR, FOLR1, TROP2, or EpCAM. In some embodiments, the human immune cell antigen is CD3.

Related applications

This application claims priority to U.S. Provisional Application No. 63/313,068, entitled "Conditional Bispecific Binding Protein", filed on February 23, 2022, the contents of which are incorporated herein by reference in their entirety. Reference electronic sequence listing

The contents of the electronic sequence listing (T083370018WO00-SEQ-ZJG.xml; size: 183,215 bytes; creation date: February 22, 2023) are incorporated by reference in their entirety.

In some aspects, the present disclosure provides methods and compositions for reducing the toxicity and side effects of immune cell-engaged bispecific antibodies that bind cancer cells and immune cells to stimulate immune cells to kill Kill target cancer cells. Many of the proteins provided herein are prodrugs that can be activated by proteases, such as those found in the tumor microenvironment. In some embodiments, the proteins described herein are formulated such that when they are not in the tumor microenvironment, the protein is able to bind to tumor cells but not immune cells (inactive), and such that when the protein enters the tumor microenvironment, the protein Cleavage of the cleavable linker "activates" the protein, producing two "active" bispecific molecules, each of which can bind to tumor cells and immune cells.

The proteins described herein comprise a single domain that binds a human target tumor antigen (TTA). Compared to similar constructs containing more than one (e.g., two) domains that bind human TTAs (e.g., the same or different TTAs), the proteins described herein (e.g., in prodrug and active forms) may exhibit Reduced affinity and cell surface residence time.

In some embodiments, reducing the affinity and cell surface residence time of proteins described herein does not significantly reduce the in vivo tumor suppressor efficacy of such proteins. In some embodiments, enhanced in vivo tumor suppressive efficacy is unexpectedly achieved despite reduced affinity and cell surface retention of proteins described herein. In some embodiments, proteins described herein are effective against cancers with any level of HER2 expression. In some embodiments, proteins described herein are effective against cancers with low levels of HER2 expression. In some embodiments, proteins described herein are effective against cancers with intermediate levels of HER2 expression. In some embodiments, proteins described herein are effective against cancers with high levels of HER2 expression.

In some embodiments, reducing the affinity and cell surface residence time of a protein described herein results in reduced toxicity and increased individual tolerance (eg, tolerance at higher doses). In some embodiments, proteins described herein exhibit an enhanced overall safety and efficacy profile compared to proteins with more than one domain that binds human TTA. I. Definition

In order to provide a more comprehensive understanding of this application, several definitions are set forth below. Such definitions are meant to cover grammatical equivalents.

"Amino acid" and "amino acid identity" as used herein mean one of the 20 naturally occurring amino acids or any non-natural analog that may be present at a specific defined position. In many embodiments, "amino acid" means one of the 20 naturally occurring amino acids. "Protein" as used herein means at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.

"Amino acid modification" as used herein means amino acid substitutions, insertions and/or deletions in the polypeptide sequence or changes in the portions chemically linked to the protein. For example, the modification may be an altered carbohydrate or PEG structure attached to the protein. For clarity, unless otherwise stated, amino acid modifications are always directed to amino acids encoded by DNA, eg, the 20 amino acids with codons in DNA and RNA. The preferred amino acid modifications herein are substitutions.

In some embodiments, as outlined herein, the protein specifically binds an immune cell antigen and a target tumor antigen (TTA) such as a target cell receptor. "Specific binding/specifically binds to" or "specific for" a specific antigen or epitope means binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is typically a structurally similar molecule that does not have binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target.

Specific binding to a particular antigen or epitope can be achieved, for example, by an antibody having a KD for the antigen or epitope of at least about 10 ^-4 M, at least about 10 ^-5 M, at least about 10 ^-6 M, at least about 10 ^{- 7} M, at least about ^10-8 M, at least about ^10-9 M, alternatively at least about ^10-10 M, at least about ^10-11 M, at least about ^10-12 M or greater, where KD refers to a specific Dissociation rate of antibody-antigen interactions. Typically, the KD of an antibody that specifically binds an antigen will be 20, 50, 100, 500, 1000, 5,000, 10,000, or more times that of the control molecule relative to the antigen or epitope.

Likewise, specific binding to a specific antigen or epitope can be achieved, for example, by an antibody having a Ka of at least 20 times, 50 times, 100 times, 500 times, 1000 times, or 1000 times that of the antigen or epitope relative to the control. 5,000-fold, 10,000-fold or more, where Ka (or KA) refers to the association rate of a specific antibody-antigen interaction. Binding affinity is typically measured using the BIACORE assay or OCTET as known in the art.

"Position" as used herein means location within a protein sequence. Positions can be numbered sequentially or according to an established format (eg, EU index for antibody numbering).

"Target antigen" as used herein means a molecule specifically bound by the variable region of a given antibody. Target antigens can be proteins, carbohydrates, lipids, or other compounds. Described herein are a series of suitable exemplary target antigens, including target tumor antigens.

As used herein, "target cell" means a cell that expresses a target antigen. In some embodiments, the target cells are diseased cells, such as tumor cells expressing TTA. In some embodiments, the target cells are immune cells, such as T cells expressing immune cell antigens.

"Fv", or "Fv domain", or "Fv region" as used herein means a polypeptide comprising a light chain variable region (VL) and a heavy chain variable region (VH) of an antigen-binding domain, usually but Not always from antibodies. Fv domains typically form an "antigen binding region" or "antigen binding domain" or "ABD" as discussed herein if they contain VH and VL domains each containing a CDR that will bind the antigen. The VH or VL of the Fv domain can be on a single polypeptide chain ("single chain variable fragment (scFv)"), or on separate polypeptide chains. The Fv domain can be an active Fv domain, a restricted Fv domain, an inactive Fv domain or a pseudo-Fv domain.

An "active Fv" is an Fv having a variable heavy domain and a variable light domain, each having CDRs that bind the same antigen, and in which the VH and VL are associated with each other such that the Fv can Binding antigen.

In some embodiments, a "restricted Fv domain" or a "restricted scFv domain" herein refers to a VH and VL that is covalently linked to a restricted linker as described herein, such that active VH and VL Uninteractive scFv domains. In some embodiments, a restricted scFv domain contains an active VH and an active VL that contain CDRs capable of binding the same antigen, but due to the restricted linker between VH and VL, they are unable to associate to form a domain that will bind the antigen. Active Fv. In some embodiments, the restricted scFv domain contains an inactive VH and an inactive VL that lack CDRs capable of binding antigen, and the VH and VL are unable to associate with each other due to restricted linkers. Due to the presence of restricted linkers, VH and VL in restricted scFv cannot associate with each other, but they can assemble with other variable domains, including those in different restricted Fv domains (e.g., molecules intra or intermolecular).

An "inactive Fv" or "inert Fv" is an Fv that has VH and VL but neither VH nor VL contains CDRs capable of binding antigen (e.g., the CDRs have been modified such that the antigen-binding ability is lost). Inactive Fv does not bind antigen. In some embodiments, the inactive Fv can be in a restricted form, where VH and VL are connected via restricted linkers that prevent them from associating with each other. In some embodiments, the VH and VL in an inactive Fv can associate with each other, but the Fv still does not bind antigen. VH or VL lacking CDRs capable of binding antigen are also referred to herein as "inert VH" or "inert VL" respectively.

A "pseudo-Fv" as used herein refers to an Fv in which VH and VL are associated with each other and in which: (i) VH contains CDRs capable of binding antigen but VL lacks CDRs capable of binding any antigen; (ii) VL contains CDRs capable of binding any antigen; CDRs that bind the antigen but VH lacks CDRs capable of binding any antigen; or (iii) both VH and VL contain CDRs that bind the antigen but different antigens. Therefore, the pseudo-Fv cannot bind to the antigen despite having VH and VL associated with each other. In some embodiments, the VH and VL of the pseudo-Fv are on a single polypeptide chain (i.e., the association is intramolecular). In some embodiments, the VH and VL of the pseudo-Fv are on a single polypeptide chain (i.e., the association is intermolecular). The VH and VL forming false Fv are also referred to herein as "false VH" and "false VL" respectively.

"Variable domain" as used herein means an immunoglobulin comprising one or more genes substantially encoded by any of the Vκ, Vλ and/or VH genes constituting the kappa, lambda and heavy chain immunoglobulin loci, respectively. region of the Ig domain. In some cases, a single variable domain may be used, such as sdFv (also referred to herein as sdABD).

In embodiments utilizing variable heavy (VH) domains and variable light (VL) domains, each VH and VL consists of three hypervariable regions ("complementarity determining regions", "CDRs") and four "frameworks" "Region" or "FR", which is arranged in the following order from the amine terminus to the carboxyl terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Thus, the VH domain has the structure vhFR1-vhCDR1-vhFR2-vhCDR2-vhFR3-vhCDR3-vhFR4, and the VL domain has the structure vlFR1-vlCDR1-vlFR2-vlCDR2-vlFR3-vlCDR3-vlFR4. As described more fully herein, the vhFR region and vlFR region self-assemble to form the Fv domain. In some embodiments, in an inactive (e.g., prodrug) version of a conditionally activatable molecule (e.g., a fusion protein), there is a "restricted scFv domain" in which the VH and VL structures within the same Fv domain The domains cannot self-associate due to the presence of restricted linkers between the VH and VL domains and the "inert Fv" of the CDRs that do not form an antigen-binding domain when self-associated.

The hypervariable region confers antigen-binding specificity and generally encompasses approximately amino acid residues 24-34 (LCDR1; "L" for light chain), 50-56 (LCDR2), and 89-97 (LCDR2) from the light chain variable region. LCDR3) and about amino acid residues 31-35B (HCDR1; "H" indicates heavy chain), 50-65 (HCDR2) and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991); and/or those residues that form a hypervariable loop (e.g., the light chain may Residues 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) in the variable region and 26-32 (HCDR1), 53-55 (HCDR2) and 96- in the heavy chain variable region 101 (HCDR3)); Chothia and Lesk (1987) J. Mol. Biol. 196:901-917. Specific CDRs of the protein are described below.

Those skilled in the art will understand that the exact numbering and placement of CDRs can vary in different numbering systems. However, it is understood that disclosure of variable heavy and/or variable light sequences includes disclosure of associated (intrinsic) CDRs. Accordingly, the exposure of each variable heavy region is the exposure of vhCDR (eg, vhCDR1, vhCDR2, and vhCDR3), and the exposure of each variable light region is the exposure of vlCDR (eg, vlCDR1, vlCDR2, and vlCDR3).

A comparison of available CDR numbers is as follows, see Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003):

Throughout this specification, the Kabat numbering system will generally be used when referring to residues in the variable domains (roughly, residues 1-107 of the light chain variable domain and residues 1-113 of the heavy chain variable domain). used, and the EU numbering system was used for the Fc region (eg, Kabat et al., supra (1991)).

This disclosure provides a number of different CDR groups. In this case, "complete CDR set" in the context of a domain that binds an immune cell antigen (e.g., a scFv that binds CD3 or CD28) means that the component contains a heavy chain variable region that contains three CDRs (e.g., vhCDR1, vhCDR2, and vhCDR3) and light chain variable regions containing CDRs (e.g., vlCDR1, vlCDR2, vlCDR3). Those skilled in the art will appreciate that each set of CDRs, VH and VL CDRs, can bind antigen individually as well as as a set. For example, in a restricted scFv domain, the vhCDR can, for example, bind CD3, and the vlCDR can bind CD3, but in the restricted version, they are unable to bind CD3.

"Single domain Fv", "sdFv" or "sdABD" as used herein means an antigen-binding domain with only three CDRs, typically based on camel antibody technology. See: Protein Engineering 9(7):1129-35 (1994); Rev Mol Biotech 74:277-302 (2001); Ann Rev Biochem 82:775-97 (2013). The sdABD typically contains a single heavy chain variable region, which contains a set of three CDRs. sdABD is sometimes referred to as "VHH" domain or Nanobody in this technology. In some embodiments, the sdABDs described herein bind TTA, as annotated (sdABD-TTA is the general term, or e.g., sdABD-EGFR is that which binds EGFR, sdABD-FOLR1 is that which binds FOLR1, etc.). In some embodiments, the sdABD described herein binds human serum albumin (HSA) and is annotated as such (sdABD-HSA).

These CDRs may be part of a larger variable light domain or variable heavy domain. Furthermore, as more fully outlined herein, the variable heavy domain and the variable light domain may be on separate polypeptide chains or, in the case of scFv sequences, on a single polypeptide chain, depending on the form and configuration of the moieties herein. Certainly.

CDRs facilitate the formation of antigen binding, or more specifically, epitope binding sites. "Antigenic determinant" refers to a determinant that interacts with a specific antigen-binding site called a paratope in the variable region. An epitope is a classification of molecules (such as amino acids or sugar side chains) and often has specific structural characteristics as well as specific charge characteristics. A single antigen can have more than one epitope.

Antigenic regions may include amino acid residues that are directly involved in binding (also known as immunodominant components of the epitope) and other amino acid residues that are not directly involved in binding, such as those effectively blocked by specific antigen-binding peptides. An amino acid residue; in other words, an amino acid is within the footprint of a specific antigen-binding peptide.

Antigenic regions may be conformational or linear. Conformational epitopes result from the spatial juxtaposition of amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes generated by adjacent amino acid residues in a polypeptide chain. The difference between conformational and non-conformational epitopes may be that in the presence of a denaturing solvent, binding to the former is lost while binding to the latter is not lost.

Antigenic regions generally include at least 3 and more usually at least 5 or 8-10 amino acids in a unique spatial configuration. Antibodies recognizing the same epitope can be verified in a simple immunoassay, which shows the ability of one antibody to block the binding of another antibody to the target antigen, such as "binning". As summarized below, proteins include not only the antigen-binding domains and antibodies listed herein, but also those that compete for binding to the epitope to which the listed antigen-binding domain binds.

The term "antigen binding domain" (ABD) is characterized as a domain that (specifically) binds/interacts with/recognizes a given target epitope or a given target site on a target molecule (antigen). The ABD can be a single VH (e.g., an sdABD as described herein), a single VL, or a scFv containing VH and VL domains. Typically, ABDs that bind target tumor antigens (TTA) and human serum albumin (HSA) are called sdABDs (“TTA-sdABD” or “HSA-sdABD”), while ABDs that bind immune cell antigens (e.g., CD3 or CD28) is a scFv containing both VH and VL domains. The terms "antigen binding domain" (ABD) and "antigen binding region" are used interchangeably herein. As described herein, an antigen-binding domain or region includes an active Fv, wherein VH and VL can associate and bind the antigen, and includes a restricted scFv that does not bind the antigen, and wherein the VH and VL each contain an Fv that is capable of binding the same antigen. CDR, but VH and VL do not associate with each other (e.g., due to a restricted linker between VH and VL).

"Domain" as used herein means a protein sequence that has structure and/or function, as summarized herein. The domains of proteins described herein include target tumor antigen binding domain (TTA domain), immune cell binding domain, linker domain and half-life extension domain.

"Domain linker" as used herein means an amino acid sequence that joins two domains, as summarized herein. Domain linkers can be cleavable linkers, restricted cleavable linkers, non-cleavable linkers, restricted non-cleavable linkers, scFv linkers, etc.

As used herein, "cleavable linker" ("CL") means an amino acid sequence that can be cleaved by a protease, preferably a human protease in diseased tissue, as summarized herein. Cleavable linkers are typically at least 3 amino acids in length, with 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids for proteins, This depends on the flexibility required. A number of cleavable linker sequences are shown in Table 1.

"Non-cleavable linker" ("NCL") as used herein means an amino acid sequence that is not cleaved by human proteases under normal physiological conditions.

As used herein, a "constrained non-cleavable linker" ("CNCL") means a short polypeptide that joins two domains as outlined herein in a manner such that the two domains do not significantly interact with each other and that in physiological Not significantly cleaved by human proteases under conditions.

"Protease cleavage site" as used herein refers to an amino acid sequence recognized and cleaved by a protease.

As used herein, "protease cleavage domain" refers to the incorporation of a "protease cleavage site" and any linkers between individual protease cleavage sites and between protease cleavage sites, and other functionality of the construct of the protein Peptide sequences of components (eg, _VH , _VL , target antigen binding domain, half-life extending domain, etc.). As outlined herein, the protease cleavage domain may also include additional amino acids if necessary, for example to confer flexibility. II. Protein

In some aspects, the present disclosure provides proteins referred to herein as "conditional bispecific redirected activation" constructs or "COBRA". In some embodiments, proteins described herein comprise a single sdABD capable of binding TTA and one or more (eg, one, two) restricted single chain variable fragment (scFv) domains. In some embodiments, proteins described herein comprise a single restricted scFv domain and an unrestricted scFv domain. In some embodiments, proteins described herein comprise two restricted scFv domains. VH and VL within a restricted scFv domain cannot associate with each other due to the presence of a short peptide linker between VH and VL (i.e., "restricted"). In addition, two scFv domains (both restricted, or one restricted and one unrestricted) associate intramolecularly to form two pseudo-Fvs that do not bind immune cell antigens. In some embodiments, the proteins described herein further comprise a second sdABD capable of binding an antigen that is not TTA. In some embodiments, the second sdABD binds an antigen that does not confer any function to the protein (i.e., is a "non-functional" sdABD). In some embodiments, the second sdABD does not bind any human protein. In some embodiments, the second sdABD binds a non-human protein (eg, a protein of non-human origin). In some embodiments, the second sdABD binds a fluorescent protein (eg, GFP). In some embodiments, the second sdABD binds hen egg lysozyme (HEL). In some embodiments, the proteins described herein further comprise a half-life extending domain (eg, an sdABD capable of binding human serum albumin (HSA)).

It is considered herein that a protein disclosed herein is inactive when the cleavable linker in the protein is not cleaved. In some embodiments, the uncleaved and inactive proteins described herein are capable of binding TTA rather than immune cell antigens. For example, in a disease-specific microenvironment or in the blood of an individual, the protein is cleaved and activated at an internal cleavable linker containing a protease cleavage site (eg, a modified MMP9 cleavage site described herein). Once cleaved, fragments of the cleavage product form dimers (eg, homodimers), which are bispecific molecules capable of binding both TTA and immune cell antigens, thereby stimulating and/or activating one or more immune cells.

In some embodiments, two dimers (eg, homodimers) bind to a TTA for HER2, EGFR, FOLR1, TROP2, or EpCAM. In some embodiments, two dimers (eg, homodimers) bind the same or different immune cell antigens. In some embodiments, the immune cell antigen is CD3, CD28, T cell receptor, programmed cell death protein 1 (PD-1), PD-L1, cytotoxic T lymphocyte associated protein 4 (CTLA-4), T Cellular immunoglobulin and mucin domain 3 (TIM-3), lymphocyte activating gene 3 (LAG-3), killer cell immunoglobulin-like receptor (KIR), CD137, OX40, OX40L, CD27, GITR (TNFRSF18 ), TIGIT, inducible T cell costimulator (ICOS), CD16A, CD226, CD96, CD40L, CRTAM, LFA-1, NKG2D, CSF1R, CD40, MARCO, VSIG4 or CD163.

The COBRA constructs described herein are constructed in a "prodrug (inactive)" form. In some embodiments, such prodrug proteins described herein comprise from the N-terminus to the C-terminus: (i) a first single domain antigen binding domain (sdABD) and a first domain linker at the C-terminus of the first sdABD, wherein (i) is present or absent; (ii) a first constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region linked to a light chain variable region via a first constrained non-cleavable linker (CNCL), wherein the heavy chain The variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen, and wherein the heavy chain variable region and the light chain variable region do not associate in the first restricted scFv domain , and the first restricted scFv domain does not bind the human immune cell antigen; (iii) a second domain linker and a second single domain antigen binding domain (sdABD), wherein the second domain linker is at the N-terminus of the second sdABD, and wherein (iii) is present or absent; (iv) cleavable linkers; (v) a second constrained single chain variable fragment (scFv) domain comprising a pseudo heavy chain variable region linked to a pseudo light chain variable region via a second constrained non-cleavable linker (CNCL), wherein the The pseudo heavy chain variable region and the pseudo light chain variable region do not associate in the second restricted scFv domain, and the second restricted scFv domain does not bind the human immune cell antigen of (ii); (vi) third domain linker; and (vii) a third sdABD, which binds human serum albumin (HSA); wherein the heavy chain variable region of (ii) and the pseudo light chain variable region of (v) are intramolecularly associated to form a variable fragment (Fv) that does not bind the immune cell antigen; and wherein the pseudo heavy chain variable region of (v) and the light chain variable region of (ii) are intramolecularly associated to form a variable fragment (Fv) that does not bind the immune cell antigen; Among them, (i) and (iii) do not exist; And when (i) and (iii) both exist: (a) The first sdABD binds human target tumor antigen (TTA) and the second sdABD does not bind human TTA; or (b) The first sdABD does not bind human TTA and the second sdABD binds human TTA.

Without being bound by scientific theory, the intramolecular association of the VH of (ii) with the pseudo VL of (v) and/or the intramolecular association of the pseudo VH of (v) with the VL of (ii) makes the precursors described herein The drug protein is stabilized (eg, results in a stable conformation) and restricted intermolecular dimerization of scFv molecules between different prodrug proteins is prevented prior to activation of the prodrug protein.

A prodrug (inactive) form of a protein that, when administered to an individual in a composition comprising one or more such prodrug proteins, is activated once the cleavable linker of (iv) is cleaved (e.g., by the tumor microenvironment) Proteases such as MMP9) can be activated. Activation of the prodrug protein involves cleavage of at least two identical prodrug proteins (a first protein and a second protein that are identical to each other) in the cleavable linker of (iv).

Cleavage of each prodrug protein in the cleavable linker of (iv) produces a first polypeptide comprising a TTA-binding sdABD, a first domain linker, a first restricted scFv domain, and a second restricted sdFv structure. The second polypeptide of the domain. In some embodiments, the first polypeptide further comprises a second sdABD that does not bind TTA. In some embodiments, the second sdABD binds an antigen that does not confer any function to the protein (i.e., is a "non-functional" sdABD). In some embodiments, the second sdABD does not bind any human protein. In some embodiments, the second sdABD binds a non-human protein (eg, a protein of non-human origin). In some embodiments, the second sdABD binds a fluorescent protein (eg, GFP). In some embodiments, the second sdABD binds hen egg lysozyme (HEL). In some embodiments, the second polypeptide may additionally comprise a half-life extending domain (eg, a third sdABD that binds HSA).

After cleavage of the cleavable linker of (iv) in the two prodrug proteins (i.e., the first prodrug protein and the second prodrug protein, which are identical before cleavage), the first constrained scFv domain of the first protein The VH associates with the VL of the first restricted scFv domain of the second protein to form an active Fv that binds the human immune cell antigen, and the VL of the first restricted scFv domain of the first protein associates with the first VL of the second protein. VH association of restricted scFv domains results in an active Fv that binds human immune cell antigens.

Thus, in some embodiments, cleaved fragments of the prodrug (inactive) protein assemble into dimers that are bispecific molecules capable of binding TTA and immune cell antigens.

Non-limiting examples of protein components described herein are provided in prodrug or active forms.

The proteins provided herein comprise a single sdABD that binds a human target tumor antigen (TTA). The sdABD that binds human TTA may be present at the N-terminus of the protein or within the protein. The protein may contain additional sdABD that does not bind human TTA.

In some embodiments, a single sdABD that binds human TTA is present at the N-terminus of the protein along with a first domain linker at the C-terminus of the sdABD (i.e., the first sdABD of (i) is present and binds human TTA). In some embodiments, the human TTA is HER2, EGFR, FOLR1, TROP2, or EpCAM. In this case, component (iii) of the protein may or may not be present. When component (iii) is present, the second sdABD of (iii) does not bind human TTA. In some embodiments, the second sdABD of (iii) does not bind human proteins when component (iii) is present. In some embodiments, the second sdABD of (iii) is non-functional. For example, the second sdABD of (iii) may bind an antigen that does not confer any function to the protein (eg, tumor binding or immune cell binding function). In some embodiments, the sdABD of (iii) binds chicken egg lysozyme and is non-functional. When component (iii) is present, the cleavable linker of (v) is typically 8-15 amino acids in length (e.g., 8, 9, 10, 11, 12, 13, 14 or 15 amino acids ). When component (iii) is absent, the cleavable linker of (iv) can be of extended length (eg, 15-45 amino acids in length). In some embodiments, when component (iii) is absent, the cleavable linker of (iv) is 17-35 amino acids in length. In some embodiments, when component (iii) is not present, the length of the cleavable linker of (iv) is 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33, 34, 35 amino acids. In some embodiments, when component (iii) is absent, the cleavable linker of (iv) is 29 or 33 amino acids in length.

In some embodiments, a single human TTA-binding sdABD is present within the protein along with a second domain linker at the N-terminus of the sdABD connecting the first restricted scFv domain to the sdABD, e.g., in the first of (ii) The C-terminus of the restricted single chain scFv domain (ie, the second sdABD of (iii) is present and binds human TTA). In some embodiments, the human TTA is HER2, EGFR, FOLR1, TROP2, or EpCAM. In this case, component (i) of the protein may or may not be present. In some embodiments, when component (i) is absent, the first restricted scFv domain of (ii) is at the N-terminus of the protein. In some embodiments, when component (i) is absent, additional amino acid sequences are present at the N-terminus of the protein, ie, at the N-terminus of the first restricted scFv domain of (ii). When component (i) is present, the first sdABD of (i) does not bind human TTA. In some embodiments, the first sdABD of (i) does not bind human protein. In some embodiments, the sdABD of (i) is non-functional. For example, the sdABD of (i) may bind an antigen that does not confer any function to the protein (eg, tumor binding or immune cell binding function). In some embodiments, the sdABD of (i) binds chicken egg lysozyme and is non-functional.

Non-limiting examples of sdABDs that bind HER2 and can be used in the first sdABD of (i) or the second sdABD of (iii) as described herein are provided in Table 1. In some embodiments, the first sdABD of (i) or the second sdABD of (iii) includes SEQ ID NOs: 45, 48-52, 54, 58, 60, 63, 66, 68, 72, 75-78 , 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121, 299 and 300 any one of the CDR1, CDR2 and CDR3 of the anti-HER2 sdABD. In some embodiments, the first sdABD of (i) or the second sdABD of (iii) comprises SEQ ID NOs: 41, 45, 48-52, 54, 58, 60, 63, 66, 68, 72, 75 -The amino acid sequence of any one of 78, 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121 and 165 has at least 80% (e.g., at least 80% , at least 85%, at least 90%, at least 95% or at least 99%) identical amino acid sequences. In some embodiments, the first sdABD of (i) or the second sdABD of (iii) comprises the amino acid sequence of any of the following: SEQ ID NO: 41, 45, 48-52, 54, 58, 60, 63, 66, 68, 72, 75-78, 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121 and 165. In some embodiments, the first sdABD of (i) and/or the second sdABD of (iii) comprises the amino acid sequence of SEQ ID NO: 96 or SEQ ID NO: 117.

Non-limiting examples of non-functional sdABDs are provided in Table 1. In some embodiments, a non-functional sdABD useful in the first sdABD of (i) or the second sdABD of (iii) as described herein binds hen egg lysozyme (HEL). In some embodiments, the non-functional sdABD that binds HEL includes CDR1 as set forth in SEQ ID NO: 23, CDR2 as set forth in SEQ ID NO: 24, and CDR3 as set forth in SEQ ID NO: 25. In some embodiments, the non-functional sdABD that binds HEL comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 26 (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99 %) identical amino acid sequence. In some embodiments, the non-functional sdABD that binds HEL comprises the amino acid sequence of SEQ ID NO: 26.

In some embodiments, the immune cell antigen is an antigen expressed on an immune cell selected from: T cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophages, B cells, neutrophils White blood cells, dendritic cells and monocytes. In some embodiments, the immune cell antigen is CD3, CD28, T cell receptor, programmed cell death protein 1 (PD-1), PD-L1, cytotoxic T lymphocyte associated protein 4 (CTLA-4), T Cellular immunoglobulin and mucin domain 3 (TIM-3), lymphocyte activating gene 3 (LAG-3), killer cell immunoglobulin-like receptor (KIR), CD137, OX40, OX40L, CD27, GITR (TNFRSF18 ), TIGIT, inducible T cell costimulator (ICOS), CD16A, CD226, CD96, CD40L, CD226, CRTAM, LFA-1, CD27, NKG2D, CSF1R, CD40, MARCO, VSIG4 or CD163. In some embodiments, the immune cell antigen is CD3. In some embodiments, any of the immune cell antigens provided herein is a human immune cell antigen.

It will also be understood that in the proteins described herein, for example, by linking the first VH and the first VL to a first "restricted non-cleavable linker (CNCL)" such that the first restricted scFv domain does not bind the first An immune cell antigen prevents the first VH from being able to associate with the first VL within the first restricted scFv domain. Similarly, for example, by connecting the second VH and the second VL to a second "restricted non-cleavable linker", causing the second restricted scFv domain not to bind the second immune cell antigen to prevent the second VH from being able to bind to the second immune cell antigen. The two VLs associate within a second restricted scFv domain. The restricted non-cleavable linker is too short to allow association of the first VH with the first VL, or the second VH with the second VL within the restricted scFv domain. In some embodiments, the first CNCL and/or the second CNCL are 6-10 amino acids long (eg, 6, 7, 8, 9, or 10 amino acids long). Non-limiting examples of amino acid sequences of the first CNCL and/or the second CNCL are provided in Table 1. In some embodiments, the first CNCL and/or the second CNCL has the sequence GGGSGGGS (SEQ ID NO: 179).

Non-limiting examples of antibodies that bind CD3 of the VH and VL of the first restricted scFv domain derivatizable in (ii) include: moromonab-CD3 (OKT3), otelixizumab (TRX4) , teplizumab (MGA031), visilizumab (NUVION), blinatumomab, foralumab, SP34 or I2C, TR- 66 or -141, Non-limiting examples of VH and VL that bind CD3 when associated and can be used in the first restricted scFv domain of (ii) are provided in Table 1. In some embodiments, the first restricted scFv domain of (ii) comprises a VH comprising vhCDR1 as set forth in SEQ ID NO: 1, vhCDR2 as set forth in SEQ ID NO: 2, and as set forth in SEQ ID NO: vhCDR3 as set forth in SEQ ID NO: 4, and VL comprising vlCDR1 as set forth in SEQ ID NO: 4, vlCDR2 as set forth in SEQ ID NO: 5, and vlCDR3 as set forth in SEQ ID NO: 6. In some embodiments, the first restricted scFv domain of (ii) comprises a VH comprising at least 80% (e.g., at least 80%, at least 85%, at least An amino acid sequence that is 90%, at least 95%, or at least 99%) identical, and a VL that contains an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%) identical to the amino acid sequence of SEQ ID NO:8 , at least 90%, at least 95%, or at least 99%) identical amino acid sequences. In some embodiments, the first restricted scFv domain of (ii) comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, and a VL comprising the amino acid sequence of SEQ ID NO: 8 .

Non-limiting examples of antibodies that bind CD28 to VH and VL of the first restricted scFv domain derivatizable in (ii) include: theralizumab, utomilumab (PF-05082566 ), ES101 (bispecific PD-L1 stated in the number. Non-limiting examples of VH and VL that bind CD28 when associated and can be used in the first restricted scFv domain of (ii) are provided in Table 1. In some embodiments, the first restricted scFv domain of (ii) comprises a VH comprising vhCDR1 as set forth in SEQ ID NO: 9, vhCDR2 as set forth in SEQ ID NO: 10 or SEQ ID NO: 17 and vhCDR3 as set forth in SEQ ID NO: 11, and a VL comprising vlCDR1 as set forth in SEQ ID NO: 12, vlCDR2 as set forth in SEQ ID NO: 13, and vlCDR3 as set forth in SEQ ID NO: 14 . In some embodiments, the first restricted scFv domain of (ii) comprises a VH comprising at least 80% (e.g., at least 80%) an amino acid sequence identical to SEQ ID NO: 15 or SEQ ID NO: 18 , at least 85%, at least 90%, at least 95%, or at least 99%) identical amino acid sequence, and VL, the VL comprising an amino acid sequence having at least 80% identity with SEQ ID NO: SEQ ID NO: 16 (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical amino acid sequences. In some embodiments, the first restricted scFv domain of (ii) comprises a VH comprising the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 18, and a VL comprising SEQ ID NO: Amino acid sequence of SEQ ID NO: 16.

Non-limiting examples of PD-1 antibodies that bind VH and VL of the first restricted scFv domain derivatizable in (ii) include: pembrolizumab, dostarlimab, and nanoparticles. Nivolumab.

Non-limiting examples of antibodies that bind CTLA-4 that bind VH and VL of the first restricted scFv domain from which (ii) can be derived include: ipilimumab.

Non-limiting examples of antibodies that bind TIM-3 that bind VH and VL of the first restricted scFv domain from which (ii) can be derived include: TSR-022 and Sym023.

Non-limiting examples of antibodies that bind LAG-3 that bind VH and VL of the first restricted scFv domain from which (ii) can be derived include: BMS-986016.

Non-limiting examples of antibodies that bind the KIR of the VH and VL of the first restricted scFv domain from which (ii) can be derived include: lirilumab.

Non-limiting examples of antibodies that bind CD137 to the VH and VL of the first restricted scFv domain from which (ii) can be derived include: utolumab and urrelumab.

Non-limiting examples of antibodies that bind OX40 to the VH and VL of the first restricted scFv domain from which (ii) can be derived include: PF-045-18600 and BMS-986178.

Non-limiting examples of antibodies that bind CD27 to the VH and VL of the first restricted scFv domain from which (ii) can be derived include: varlilumab.

Non-limiting examples of antibodies that bind GITR to the VH and VL of the first restricted scFv domain from which (ii) can be derived include: GWN323 or BMS-986156.

Non-limiting examples of antibodies that bind TIGIT of the VH and VL of the first restricted scFv domain from which (ii) can be derived include: OMP-313M32, MTIG7192A, BMS-986207 and MK-7684.

Non-limiting examples of antibodies that bind ICOS of VH and VL of the first restricted scFv domain from which (ii) can be derived include: JTX-2011.

Non-limiting examples of antibodies that bind CSF1R to the VH and VL of the first restricted scFv domain from which (ii) can be derived include: mactuzumab/RG7155 and IMC-CS4.

Non-limiting examples of antibodies that bind CD40 to the VH and VL of the first restricted scFv domain from which (ii) can be derived include: CP-870,893.

Non-limiting examples of antibodies that bind CD16A to the VH and VL of the first restricted scFv domain from which (ii) can be derived include: NTM-1633 and AFM13.

Non-limiting examples of antibodies that bind CD96 to the VH and VL of the first restricted scFv domain from which (ii) can be derived include: GSK6097608.

Non-limiting examples of antibodies that bind CD40L that bind VH and VL of the first restricted scFv domain from which (ii) can be derived include: BG9588.

Non-limiting examples of antibodies that bind LFA-1 that bind VH and VL of the first restricted scFv domain from which (ii) can be derived include: efalizumab.

In some embodiments, in the proteins described herein, the second restricted scFv domain of (v) comprises a pseudo VH linked to a pseudo VL via a second restricted cleavable linker (CNCL), wherein the pseudo heavy chain The variable region and the pseudo-light chain variable region are not associated in the second restricted scFv domain, and the second restricted scFv domain does not bind the human immune cell antigen of (ii). In some embodiments, the pseudo VH is an inactive VH (e.g., a VH lacking CDRs capable of binding the antigen), and/or the pseudo VL is an inactive VL (e.g., a VL lacking CDRs capable of binding the antigen). In some embodiments, the inactive VH and/or the inactive VL comprise human framework regions that allow self-assembly with another variable region, regardless of which amino acids are at the CDR positions of the variable region. In some cases, inactive VH/VL is said to include CDRs, but the CDRs are unable to bind any antigen, nor do VH or VL contain such CDRs.

Inactive VH or VL can be generated using methods known in the art. For example, an inactive VH or VL can be produced by altering one or more of the CDRs of an active VH or VL (including altering one or more of the three CDRs). In some embodiments, one or more amino acid substitutions are made at functionally important residues in one or more CDRs of active VH or VL to generate inactive VH or VL, respectively. In some embodiments, some or all CDR residues in active VH or VL can be replaced with random sequences, non-functional tag sequences (eg, FLAG sequences) to generate inactive VH or VL, respectively.

In some embodiments, inactive VH and VL can be engineered to promote selective binding of the prodrug form, thereby facilitating intramolecular association (eg, via intermolecular pair formation) prior to cleavage. Such engineered VH/VL and interface residue amino acid substitutions are described, for example, in Igawa et al., Protein Eng. Des. Selection 23(8):667-677 (2010).

In some embodiments, the second restricted scFv domain of (v) comprises an inactive VH comprising vhCDR1 as set forth in SEQ ID NO: 27, vhCDR2 as set forth in SEQ ID NO: 28, and as set forth in SEQ ID NO: 28 vhCDR3 as set forth in ID NO: 29, and an inactive VL comprising vlCDR1 as set forth in SEQ ID NO: 30, vlCDR2 as set forth in SEQ ID NO: 31 and vlCDR3 as set forth in SEQ ID NO: 32 . In some embodiments, the second restricted scFv domain of (v) comprises an inactive VH comprising at least 80% (e.g., at least 80%, at least 85% of the amino acid sequence of SEQ ID NO: 33) %, at least 90%, at least 95%, or at least 99%) amino acid sequence identity, and an inactive VL comprising an amino acid sequence that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical amino acid sequences. In some embodiments, the second restricted scFv domain of (v) comprises an inactive VH comprising the amino acid sequence of SEQ ID NO: 33, and an inactive VL comprising SEQ ID NO: 33. 34 amino acid sequences.

In some embodiments, in the proteins described herein, the first restricted scFv domain of (ii) comprises VH linked to the N-terminus of VL. In some embodiments, in the proteins described herein, the first restricted scFv domain of (ii) comprises VH linked to the C-terminus of VL.

In some embodiments, in the proteins described herein, the second restricted scFv domain of (v) comprises a pseudo-VH linked to the N-terminus of the pseudo-VL. In some embodiments, in the proteins described herein, the second restricted scFv domain of (v) comprises a pseudo-VH linked to the C-terminus of a pseudo-VL.

In some embodiments, in the proteins described herein, the first restricted scFv domain of (ii) comprises from N-terminus to C-terminus: VH-first CNCL-VL, and the second restricted scFv domain of (v) The scFv domain contains from N-terminus to C-terminus: pseudo VL-second CNCL-pseudo VH.

In some embodiments, the cleavable linker of (iv) of the proteins described herein comprises at least one (e.g., 1, 2, 3 or more) protease cleavage site comprising at least one Amino acid sequence cleaved by protease. It is known that proteases are secreted by certain diseased cells and tissues (eg, tumors or cancer cells), creating a protease-rich microenvironment or a protease-enriched microenvironment. In some embodiments, in the proteins described herein, the cleavable linker of (iv) is cleavable by a protease in the blood of an individual. In some embodiments, in the proteins described herein, the cleavable linker of (iv) is cleaved by a protease secreted by the tumor into the tumor microenvironment. Proteases include, but are not limited to, serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamate proteases, metalloproteases, aspartate peptide resolvase, serum proteases, cathepsins ( For example, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin S), vasodilator, hK1, hK10, hK15, KLK7, granzyme B, cytoplasm Factor XA, collagenase, type IV collagenase, stromelysin, factor XA, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidain, Bromelain, calpain, apoptotic protease (e.g., apoptotic protease 3), Mir1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, protease, Legumain, plasmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, matrix metalloproteinase (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, hypnosis Protein, urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-1β converting enzyme, thrombin, FAP (FAP-α) , dipeptidyl peptidase and dipeptidyl peptidase IV (DPPIV/CD26). In some embodiments, in the proteins described herein, the first cleavable linker of (iv) and/or the second cleavable linker of (viii) is cleavable by MMP9. Non-limiting examples of cleavable linkers that can be used as the first cleavable linker of (iv) in the proteins described herein are provided in Table 1.

In some embodiments, in the proteins described herein, the first domain linker of (i) (if (i) is present), the second domain linker of (iv), and/or the second domain linker of (viii) The third domain linker is a non-cleavable linker. In some embodiments, in the proteins described herein, the first domain linker of (i) (if (i) is present), the second domain linker of (iv), and/or the second domain linker of (viii) The third domain linker is the same. In some embodiments, in the proteins described herein, the first domain linker of (i) (if (i) is present), the second domain linker of (iv), and/or the second domain linker of (viii) The third domain linkers differ from each other. In some embodiments, domain linkers used to join domains to maintain functionality of the domains are typically longer flexible linkers that are not cleaved (eg, by individual proteases). Examples of linkers suitable for use as domain linkers for proteins described herein include, but are not limited to, (GS)n (SEQ ID NO: 166), (GGS)n (SEQ ID NO: 167), (GGGS)n (SEQ ID NO: 168), (GGSG)n (SEQ ID NO: 169), (GGSGG)n (SEQ ID NO: 170) or (GGGGS)n (SEQ ID NO: 171), where n is 1, 2 , 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the length of the first domain linker in (i) (if (i) is present), the second domain linker in (iv), and/or the third domain linker in (viii) is about 15 amino acids.

In some embodiments, in the proteins disclosed herein, the half-life extending domain of (vii) can be any known half-life extending domain known in the art, including but not limited to HSA, HSA binding domain, Fc domains and small molecules.

Human serum albumin (HSA) (molecular mass approximately 67 kDa) is the most abundant protein in plasma, is present at approximately 50 mg/ml (600 µM) and has a half-life in humans of approximately 20 days. HSA serves to maintain plasma pH, contributes to colloidal blood pressure, serves as a carrier for many metabolites and fatty acids, and serves as the primary drug transport protein in plasma. Non-covalent association with albumin extends the elimination half-life of short-lived proteins. For example, recombinant fusion of an albumin-binding domain to a Fab fragment resulted in a 25-fold and 58-fold reduction in in vivo clearance and an increase in half-life when administered intravenously to mice and rabbits, respectively, compared to administration of the Fab fragment alone. times and 37 times. In another example, when insulin was chelated with fatty acids to promote association with albumin, a prolonged effect was observed when injected subcutaneously into rabbits or pigs. Taken together, these findings demonstrate a link between albumin binding and prolonged action.

In some embodiments, in the proteins described herein, the half-life extending domain of (vii) comprises a domain that specifically binds HSA. In some embodiments, the HSA binding domain is a peptide. In other embodiments, the HSA binding domain is a small molecule. The HSA binding domain of an antigen binding protein is contemplated to be relatively small and in some embodiments no more than 25 kD, no more than 20 kD, no more than 15 kD, or no more than 10 kD. In some cases, if the HSA binding domain is a peptide or small molecule, it is 5 kD or less.

In some embodiments, the half-life extending domain is a single domain antigen binding domain from an sdABD that binds HSA. Non-limiting examples of sdABDs that bind HSA are provided in Table 1. In some embodiments, in the proteins described herein, the third sdABD of (ix) that binds HSA comprises CDR1 as set forth in SEQ ID NO: 19, CDR2 as set forth in SEQ ID NO: 20, and as set forth in SEQ ID NO: 20. NO: CDR3 described in 21. In some embodiments, the third sdABD of (ix) comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 22 (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99 %) identical amino acid sequence. In some embodiments, the third sdABD of (ix) comprises the amino acid sequence of SEQ ID NO: 22.

The half-life extension domain of the protein provides pharmacodynamic and pharmacokinetic changes to the antigen-binding protein itself. As above, the half-life extending domain extends the elimination half-life. The half-life extending domain also changes the pharmacodynamic properties, including changing the tissue distribution, penetration and diffusion of the antigen-binding protein. In some embodiments, a half-life extending domain provides improved tissue (including tumor) targeting, tissue penetration, tissue distribution, diffusion within tissues, and enhanced efficacy compared to proteins without a half-life extending binding domain. In one embodiment, the treatment method effectively and efficiently utilizes reduced amounts of the antigen-binding protein, resulting in reduced side effects, such as a reduced probability of interleukin release syndrome or interleukin storm.

Additionally, characteristics of the half-life extending domain (eg, HSA binding domain) include the binding affinity of the HSA binding domain for HSA. The affinity of the HSA binding domain can be selected to target a specific elimination half-life in a particular polypeptide construct. Thus, in some embodiments, the HSA binding domain has high binding affinity. In other embodiments, the HSA binding domain has intermediate binding affinity. In still other embodiments, the HSA binding domain has low or minimal binding affinity. Exemplary binding affinities include KD concentrations of 10 nM or less (high), between 10 nM and 100 nM (moderate), and greater than 100 nM (low). As above, binding affinity for HSA is determined by known methods such as surface plasmon resonance (SPR).

Furthermore, it is known in the art that there may be immunogenicity in humans derived from the C-terminal sequences of certain ABDs. Therefore, often, especially when the C-terminus of the protein terminates in sdABD (e.g., the sdABD-HSA domain of many constructs), a C-terminal capping sequence is added to reduce the likelihood of clearance of the protein by the patient's innate immune system. After cleavage, the remaining linker amino acids act as blocking peptides against human serum antibodies. Non-limiting examples of C-terminal capping sequences are provided in U.S. Patent No. 1,0858,418, which is incorporated herein by reference.

In some embodiments, proteins described herein comprise at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95% or at least 99%) identical amino acid sequence. In some embodiments, the proteins described herein comprise the amino acid sequence of any of SEQ ID NOs: 37-40.

In some embodiments, proteins described herein comprise at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95% or at least 99%) identical amino acid sequence. In some embodiments, the proteins described herein comprise the amino acid sequence of any of SEQ ID NOs: 173-176.

In some embodiments, a histidine tag (His6 or His10) can be used. Any of the proteins described herein (e.g., a protein comprising the amino acid sequence of any of SEQ ID NOs: 173-176 listed in Table 1) may further comprise a His6 C-terminal tag (e.g., at the C-terminus) (for purification reasons), but these sequences can also be used to reduce immunogenicity in humans, as shown by Holland et al., DOI 10.1007/s10875-013-9915-0 and US Patent No. 10808040.

In some embodiments, any of the proteins described herein (e.g., a protein comprising the amino acid sequence of any of SEQ ID NOs: 37-40 and 173-176 listed in Table 1) A signal peptide may further be included, for example, a signal peptide including the amino acid sequence of MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 177).

Non-limiting examples of different configurations of proteins described herein are provided. A. Configuration 1 (for example, Pro1225 in Table 1)

In some embodiments, a protein described herein comprises from the N-terminus to the C-terminus: (i) a first single domain antigen binding domain (sdABD) that binds a human TTA selected from HER2, EGFR, FOLR1, TROP2, or EpCAM and a first domain linker at the C-terminus of the first sdABD; (ii) a first constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region linked to a light chain variable region via a first constrained non-cleavable linker (CNCL), wherein the heavy chain can The variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen (e.g., CD3), and wherein the heavy chain variable region and the light chain variable region are not associated in the first restricted scFv domain , and the first restricted scFv domain does not bind a human immune cell antigen (e.g., CD3); (iii) does not exist; (iv) a cleavable linker (e.g., a cleavable linker comprising a protease cleavage site provided in Table 1, such as an MMP9 cleavage site or a variant thereof), wherein the length of the cleavable linker is 17-35 (e.g., 29 or 33) amino acids; (v) a second constrained single chain variable fragment (scFv) domain comprising a dummy heavy chain variable region linked to a dummy light chain variable region via a second constrained non-cleavable linker (CNCL), wherein dummy The heavy chain variable region and the pseudo-light chain variable region are not associated in the second restricted scFv domain, and the second restricted scFv domain does not bind the human immune cell antigen (e.g., CD3) of (ii); (vi) third domain linker; and (vii) a third sdABD, which binds human serum albumin (HSA); wherein the heavy chain variable region of (ii) and the pseudo light chain variable region of (v) are intramolecularly associated to form a variable fragment (Fv) that does not bind immune cell antigens; and The pseudo heavy chain variable region of (v) is intramolecularly associated with the light chain variable region of (ii) to form a variable fragment (Fv) that does not bind immune cell antigens.

In some embodiments, the first sdABD of (i) binds HER2. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of any of the following: SEQ ID NO: 41, 45, 48-52, 54, 58, 60, 63, 66, 68, 72, 75-78, 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121 and 165.

In some embodiments, the immune cell antigen is CD3 and the first restricted scFv domain of (ii) comprises a first VH comprising the amino acid sequence of SEQ ID NO: 7, the first VH being identical to the amino acid sequence comprising SEQ ID NO: : The N-terminal fusion of the first VL of the amino acid sequence 8.

In some embodiments, the second restricted scFv domain of (v) comprises an inert VL comprising the amino acid sequence of SEQ ID NO: 33, the inert VL being identical to an inert VL comprising the amino acid sequence of SEQ ID NO: 34. N-terminal fusion of inert VH.

In some embodiments, the third sdABD of (vii) comprises the amino acid sequence of SEQ ID NO: 22.

In some embodiments, the protein comprises an amine that is at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 37 amino acid sequence. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the protein consists of the amino acid sequence of SEQ ID NO: 37.

In some embodiments, the protein comprises an amine that is at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 173 amino acid sequence. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 173. In some embodiments, the protein consists of the amino acid sequence of SEQ ID NO: 173. B. Configuration 2 (for example, Pro1148 in Table 1)

In some embodiments, a protein described herein comprises from the N-terminus to the C-terminus: (i) a first single domain antigen binding domain (sdABD) that binds a human TTA selected from HER2, EGFR, FOLR1, TROP2, or EpCAM and a first domain linker at the C-terminus of the first sdABD; (ii) a first constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region linked to a light chain variable region via a first constrained non-cleavable linker (CNCL), wherein the heavy chain can The variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen (e.g., CD3), and wherein the heavy chain variable region and the light chain variable region are not associated in the first restricted scFv domain , and the first restricted scFv domain does not bind a human immune cell antigen (e.g., CD3); (iii) a second domain linker and a second single domain antigen binding domain (sdABD), wherein the second domain linker is at the N-terminus of the second sdABD, and wherein the second sdABD does not bind human TTA; (iv) cleavable linker (e.g., a cleavable linker comprising a protease cleavage site provided in Table 1 such as an MMP9 cleavage site or a variant thereof), wherein the cleavable linker is 8-15 amine groups in length acid; (v) a second constrained single chain variable fragment (scFv) domain comprising a dummy heavy chain variable region linked to a dummy light chain variable region via a second constrained non-cleavable linker (CNCL), wherein dummy The heavy chain variable region and the pseudo-light chain variable region are not associated in the second restricted scFv domain, and the second restricted scFv domain does not bind the human immune cell antigen (e.g., CD3) of (ii); (vi) third domain linker; and (vii) a third sdABD, which binds human serum albumin (HSA); wherein the heavy chain variable region of (ii) and the pseudo light chain variable region of (v) are intramolecularly associated to form a variable fragment (Fv) that does not bind immune cell antigens; and The pseudo heavy chain variable region of (v) is intramolecularly associated with the light chain variable region of (ii) to form a variable fragment (Fv) that does not bind immune cell antigens.

In some embodiments, the first sdABD of (i) binds HER2. In some embodiments, the first sdABD of (i) comprises the amino acid sequence of any of the following: SEQ ID NO: 41, 45, 48-52, 54, 58, 60, 63, 66, 68, 72, 75-78, 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121 and 165.

In some embodiments, the second sdABD of (iii) binds the antigen and does not confer any function to the protein ("non-functional"). In some embodiments, the second sdABD of (iii) binds hen egg lysozyme (HEL). In some embodiments, the second sdABD of (iii) comprises the amino acid sequence of SEQ ID NO: 26.

In some embodiments, the immune cell antigen is CD3 and the first restricted scFv domain of (ii) comprises a first VH comprising the amino acid sequence of SEQ ID NO: 7, the first VH being identical to the amino acid sequence comprising SEQ ID NO: : The N-terminal fusion of the first VL of the amino acid sequence 8.

In some embodiments, the second restricted scFv domain of (v) comprises an inert VL comprising the amino acid sequence of SEQ ID NO: 33, the inert VL being identical to an inert VL comprising the amino acid sequence of SEQ ID NO: 34. N-terminal fusion of inert VH.

In some embodiments, the third sdABD of (vii) comprises the amino acid sequence of SEQ ID NO: 22.

In some embodiments, the protein comprises an amine that is at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 39 amino acid sequence. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the protein consists of the amino acid sequence of SEQ ID NO: 39.

In some embodiments, the protein comprises an amine that is at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 175 amino acid sequence. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 175. In some embodiments, the protein consists of the amino acid sequence of SEQ ID NO: 175. C. Configuration 3 (for example, Pro1145 and Pro1168 in Table 1)

In some embodiments, a protein described herein comprises from the N-terminus to the C-terminus: (i) a first single domain antigen binding domain (sdABD) that does not bind human TTA and a first domain linker at the C-terminus of the first sdABD; (ii) a first constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region linked to a light chain variable region via a first constrained non-cleavable linker (CNCL), wherein the heavy chain can The variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen (e.g., CD3), and wherein the heavy chain variable region and the light chain variable region are not associated in the first restricted scFv domain , and the first restricted scFv domain does not bind a human immune cell antigen (e.g., CD3); (iii) a second domain linker and a second single domain antigen-binding domain (sdABD) that binds a human target tumor antigen (TTA), wherein the second domain linker is at the N-terminus of the second sdABD, and wherein human TTA is selected from HER2, EGFR, FOLR1, TROP2 or EpCAM; (iv) cleavable linker (e.g., a cleavable linker comprising a protease cleavage site provided in Table 1 such as an MMP9 cleavage site or a variant thereof), wherein the cleavable linker is 8-15 amine groups in length acid; (v) a second constrained single chain variable fragment (scFv) domain comprising a dummy heavy chain variable region linked to a dummy light chain variable region via a second constrained non-cleavable linker (CNCL), wherein dummy The heavy chain variable region and the pseudo-light chain variable region are not associated in the second restricted scFv domain, and the second restricted scFv domain does not bind the human immune cell antigen (e.g., CD3) of (ii); (vi) third domain linker; and (vii) a third sdABD, which binds human serum albumin (HSA); wherein the heavy chain variable region of (ii) and the pseudo light chain variable region of (v) are intramolecularly associated to form a variable fragment (Fv) that does not bind immune cell antigens; and The pseudo heavy chain variable region of (v) is intramolecularly associated with the light chain variable region of (ii) to form a variable fragment (Fv) that does not bind immune cell antigens.

In some embodiments, the second sdABD of (iii) binds HER2. In some embodiments, the second sdABD of (iii) comprises the amino acid sequence of any of the following: SEQ ID NO: 41, 45, 48-52, 54, 58, 60, 63, 66, 68, 72, 75-78, 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121 and 165.

In some embodiments, the first sdABD of (i) binds the antigen and does not confer any function to the protein ("non-functional"). In some embodiments, the first sdABD of (i) binds hen egg lysozyme (HEL). In some embodiments, the first sdABD of (i) comprises the amino acid sequence of SEQ ID NO: 26.

In some embodiments, the immune cell antigen is CD3 and the first restricted scFv domain of (ii) comprises a first VH comprising the amino acid sequence of SEQ ID NO: 7, the first VH being identical to the amino acid sequence comprising SEQ ID NO: : The N-terminal fusion of the first VL of the amino acid sequence 8.

In some embodiments, the second restricted scFv domain of (v) comprises an inert VL comprising the amino acid sequence of SEQ ID NO: 33, the inert VL being identical to an inert VL comprising the amino acid sequence of SEQ ID NO: 34. N-terminal fusion of inert VH.

In some embodiments, the third sdABD of (vii) comprises the amino acid sequence of SEQ ID NO: 22.

In some embodiments, the protein comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 38 or SEQ ID NO: 40 (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99 %) identical amino acid sequence. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 40. In some embodiments, the protein consists of the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 40.

In some embodiments, the protein comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 174 or SEQ ID NO: 176 (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99 %) identical amino acid sequence. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 174 or SEQ ID NO: 176. In some embodiments, the protein consists of the amino acid sequence of SEQ ID NO: 174 or SEQ ID NO: 176. D. Configuration 4

In some embodiments, a protein described herein comprises from the N-terminus to the C-terminus: (i) does not exist; (ii) a first constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region linked to a light chain variable region via a first constrained non-cleavable linker (CNCL), wherein the heavy chain can The variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen (e.g., CD3), and wherein the heavy chain variable region and the light chain variable region are not associated in the first restricted scFv domain , and the first restricted scFv domain does not bind a human immune cell antigen (e.g., CD3); (iii) a second domain linker and a second single domain antigen-binding domain (sdABD) that binds a human target tumor antigen (TTA), wherein the second domain linker is at the N-terminus of the second sdABD, and wherein human TTA is selected from HER2, EGFR, FOLR1, TROP2 or EpCAM; (iv) cleavable linker (e.g., a cleavable linker comprising a protease cleavage site provided in Table 1 such as an MMP9 cleavage site or a variant thereof), wherein the cleavable linker is 8-15 amine groups in length acid; (v) a second constrained single chain variable fragment (scFv) domain comprising a dummy heavy chain variable region linked to a dummy light chain variable region via a second constrained non-cleavable linker (CNCL), wherein dummy The heavy chain variable region and the pseudo-light chain variable region are not associated in the second restricted scFv domain, and the second restricted scFv domain does not bind the human immune cell antigen (e.g., CD3) of (ii); (vi) third domain linker; and (vii) a third sdABD, which binds human serum albumin (HSA); wherein the heavy chain variable region of (ii) and the pseudo light chain variable region of (v) are intramolecularly associated to form a variable fragment (Fv) that does not bind immune cell antigens; and The pseudo heavy chain variable region of (v) is intramolecularly associated with the light chain variable region of (ii) to form a variable fragment (Fv) that does not bind immune cell antigens.

In some embodiments, the second sdABD of (iii) binds HER2. In some embodiments, the second sdABD of (iii) comprises the amino acid sequence of any of the following: SEQ ID NO: 41, 45, 48-52, 54, 58, 60, 63, 66, 68, 72, 75-78, 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121 and 165.

In some embodiments, the immune cell antigen is CD3 and the first restricted scFv domain of (ii) comprises a first VH comprising the amino acid sequence of SEQ ID NO: 7, the first VH being identical to the amino acid sequence comprising SEQ ID NO: : The N-terminal fusion of the first VL of the amino acid sequence 8.

In some embodiments, the second restricted scFv domain of (v) comprises an inert VL comprising the amino acid sequence of SEQ ID NO: 33, the inert VL being identical to an inert VL comprising the amino acid sequence of SEQ ID NO: 34. N-terminal fusion of inert VH.

In some embodiments, the third sdABD of (vii) comprises the amino acid sequence of SEQ ID NO: 22. III. Generation method

In some aspects, the present disclosure provides nucleic acid molecules comprising a nucleotide sequence encoding any of the proteins described herein. In some embodiments, the nucleic acid molecule is a vector. In some embodiments, the nucleic acid molecule is an expression vector (eg, an expression vector suitable for expression of a protein in mammalian cells, such as human cells).

As will be understood by those skilled in the art, the nucleic acid composition will depend on the form of the protein. Generally, the proteins described herein are encoded by a single nucleic acid molecule in a single expression vector for production.

As is known in the art, nucleic acids encoding components of the protein can be incorporated into expression vectors and depending on the host cell used to produce the prodrug compositions disclosed herein. Generally, the nucleic acid is operably linked to any number of regulatory elements (promoters, origins of replication, selectable markers, ribosome binding sites, inducers, etc.). Expression vectors can be extrachromosomal or integrative vectors.

The nucleic acids and/or expression vectors encoding the prodrugs disclosed herein are then transformed into any number of different types of host cells known in the art, including mammalian cells, bacterial cells, yeast cells, insect cells, cells and/or fungal cells, with mammalian cells (eg, CHO cells, 293 cells) being used in many embodiments.

The prodrug compositions described herein are prepared by culturing host cells containing expression vectors. Once generated, traditional antibody purification steps are completed, including protein A affinity chromatography steps and/or ion exchange chromatography steps.

In some embodiments, the activity of a protein described herein can be determined via a T cell-dependent cytotoxicity assay. For example, human T cells isolated from healthy donors can be incubated with varying concentrations of the protein along with target-bearing tumor cell lines pre-labeled with firefly luciferase. Cytotoxicity can be measured by observing changes in luciferase levels using a luminometer. IV. Compositions and Methods of Use

In some aspects, the present disclosure provides compositions comprising any one or more of the proteins described herein. In some embodiments, by combining a protein of desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (such as Remington's Pharmaceutical Sciences 16th Edition, Osol, A. Ed. [ 1980]) to prepare compositions containing proteins described herein for storage in the form of lyophilized formulations or aqueous solutions. In some embodiments, compositions comprising proteins described herein are prepared for administration, eg, to an individual, to treat a disease (eg, cancer). In some embodiments, the cancer is a HER2+ cancer. In some embodiments, the cancer has any level of HER2 expression. In some embodiments, the cancer has low HER2 expression levels. In some embodiments, the cancer has intermediate HER2 expression levels. In some embodiments, the cancer has high HER2 expression levels. In some embodiments, the cancer is breast cancer, lung cancer, stomach cancer, gastric cancer, or colorectal cancer. In some embodiments, the cancer is breast, lung, gastric, gastric, or colorectal cancer and has a low HER2 expression level. In some embodiments, the cancer is breast, lung, gastric, gastric, or colorectal cancer and has an intermediate HER2 expression level. In some embodiments, the cancer is breast, lung, gastric, gastric, or colorectal cancer and has a high HER2 expression level.

Other aspects of the present disclosure provide methods of treating cancer by administering to an individual in need thereof a composition comprising a protein described herein. In some embodiments, compositions described herein for administration to an individual comprise a first protein and a second protein, wherein the first protein and the second protein are the same (same construct), wherein the first protein and each of the second protein includes from the N-terminus to the C-terminus: (i) a first single domain antigen binding domain (sdABD) and a first domain linker at the C-terminus of the first sdABD, wherein (i) is present or absent; (ii) a first constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region linked to a light chain variable region via a first constrained non-cleavable linker (CNCL), wherein the heavy chain can The variable region and the light chain variable region, if associated, are capable of binding to a human immune cell antigen, and wherein the heavy chain variable region and the light chain variable region are not associated in the first restricted scFv domain, and the first receptor The restricted scFv domain does not bind human immune cell antigens; (iii) a second domain linker and a second single domain antigen binding domain (sdABD), wherein the second domain linker is at the N-terminus of the second sdABD, and wherein (iii) is present or absent; (iv) cleavable linkers; (v) a second constrained single chain variable fragment (scFv) domain comprising a dummy heavy chain variable region linked to a dummy light chain variable region via a second constrained non-cleavable linker (CNCL), wherein dummy The heavy chain variable region and the pseudo-light chain variable region are not associated in the second restricted scFv domain, and the second restricted scFv domain does not bind the human immune cell antigen of (ii); (vi) third domain linker; and (vii) a third sdABD, which binds human serum albumin (HSA); wherein the heavy chain variable region of (ii) and the pseudo light chain variable region of (v) are intramolecularly associated to form a variable fragment (Fv) that does not bind immune cell antigens; and wherein the pseudo heavy chain variable region of (v) and the light chain variable region of (ii) are intramolecularly associated to form a variable fragment (Fv) that does not bind the immune cell antigen; Among them, (i) and (iii) do not exist; And when (i) and (iii) both exist: (a) The first sdABD binds human target tumor antigen (TTA) and the second sdABD does not bind human TTA; or (b) The first sdABD does not bind human TTA and the second sdABD binds human TTA.

In some embodiments, (i) is present and (iii) is not present, and wherein the first sdABD binds human TTA. In some embodiments, (i) is absent and (iii) is present, and wherein the second sdABD binds human TTA. In some embodiments, (i) and (iii) are both present, wherein the second sdABD does not bind human TTA, and the first sdABD binds human TTA. In some embodiments, (i) and (iii) are both present, and wherein the first sdABD does not bind human TTA, and the second sdABD binds human TTA.

In some embodiments, after cleavage of the cleavable linker of (iv) in the first protein and the second protein: the VH of the first restricted scFv domain of the first protein and the first restricted scFv of the second protein The VL of the structural domain associates to form an active binding site for binding to the human immune cell antigen; the VL of the first restricted scFv domain of the first protein associates with the VH of the first restricted scFv domain of the second protein to form Active binding site for human immune cell antigens. Therefore, the cleaved fragments of the prodrug (inactive) protein assemble into two homodimers (the first homodimer and the second homodimer), each of which is capable of binding TTA and immune cell antigens. of bispecific molecules.

In some embodiments, cleavage of the cleavable linker of (iv) in the first protein and the second protein produces a composition comprising a homodimer of the first polypeptide and the second polypeptide, wherein the first polypeptide Is the same as the second polypeptide, and wherein each of the first polypeptide and the second polypeptide comprises: (i) a single domain antigen binding domain (sdABD) that binds a first human target tumor antigen (TTA); (ii) domain linkers; (iii) A constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region (VH) linked to a light chain variable region (VL) via a constrained non-cleavable linker (CNCL), wherein the The heavy chain variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen, and wherein the heavy chain variable region and the light chain variable region do not associate in the restricted scFv domain , and the restricted scFv domain does not bind the human immune cell antigen; wherein the VH of the first polypeptide associates with the VL of the second polypeptide, and the VL of the first polypeptide associates with the VH of the second polypeptide, forming two active variable fragments ( Fv), each active variable fragment is capable of binding to the immune antigen.

In some embodiments, a composition described herein is administered to an individual in need thereof via one or more suitable routes of administration using one or more of a variety of methods known in the art. The means and/or methods of investment will vary depending on the desired results. Acceptable routes of administration may refer to any route of administration known in the art, which the clinician may consider in conjunction with the intended therapeutic use, such as by parenteral administration, which is typically associated with injection at the intended site of action. Associated with or associated with the intended site of action (e.g., intravenous administration).

In some embodiments, the composition is administered to the same subject one or more times.

As used herein, the term "treat/treating/treatment" and its grammatical variations have the same meaning as commonly understood by those of ordinary skill in the art. In some embodiments, these terms refer to methods for obtaining beneficial or desired clinical results. These terms may refer to slowing the onset or progression of a condition, disorder or disease, alleviating or alleviating symptoms associated therewith, causing complete or partial resolution of a condition, or some combination of any of the above. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to: alleviation or alleviation of symptoms, reduction in disease severity, stabilization of disease status (e.g., no worsening), delay or slowing of disease progression, reduction in disease status Improvement or alleviation, and alleviation (partial or complete), detectable or undetectable. "Treat/treating/treatment" may also include prolonging survival relative to expected survival if no treatment was received. Thus, an individual (eg, a human) in need of treatment may be an individual who has suffered from the disease or condition in question. The term "treat/treating/treatment" includes the suppression or reduction of the increase in severity of a pathological condition or symptom relative to no treatment and is not necessarily meant to imply complete cessation of the associated disease or condition.

In some embodiments, the composition is administered to the individual in an effective amount. An "effective amount" is an amount effective in treating and/or preventing a disease, disorder or condition as disclosed herein. In some embodiments, an effective amount is a composition (e.g., a therapeutic composition, compound, or dose). This amount will vary depending on a variety of factors, including, but not limited to, the characteristics of the therapeutic composition (including activity, pharmacokinetics, pharmacodynamics and bioavailability), the physiological condition of the individual (including age, gender, type of disease, stage of disease) , general physical condition, response to a given dose and type of drug), the nature of the pharmaceutically acceptable carrier for the formulation, and the route of administration. One skilled in the art of clinical and pharmacology can assess the effective amount, for example, by monitoring the individual's response to administered compositions and adjusting the dosage accordingly (see, e.g., Remington: The Science and Practice of Pharmacy (Gennaro A, Editor, Mack Publishing Co., Easton, PA, U.S., 19th Edition, 1995)).

As used herein, the term "individual" refers to any living organism, typically a mammalian individual, such as humans and animals. The terms "individual" and "patient" are used interchangeably. In some embodiments, the individual is a mammal, such as a primate (eg, a human or non-human primate), or a livestock animal (eg, a cow, horse, pig, sheep, goat, etc.). Example

A core challenge in developing cancer therapeutics is balancing tumor-killing efficacy with toxicity to patients. One strategy to achieve this balance is by designing therapeutics with specific affinity for tumor cells. For example, conditionally activatable multispecific proteins containing a tumor-targeting antigen binding domain and an immune cell binding domain can be used to specifically recruit T cells to tumors. These multispecific proteins can be activated by proteases in the tumor microenvironment to further reduce potential toxicity in non-tumor tissues. Example 1: HER2 single VHH construct (Pro1225) demonstrates tumor killing activity in vivo

The conditionally activatable multispecific protein can contain two tumor-binding single domain antibodies (sdABD) (eg, Pro1111 and Pro1118 in Figure 1). To design new constructs with improved profiles and an improved balance between tumor killing efficacy and toxicity to patients, these proteins were modified by removing one tumor-targeting sdABD to generate a single tumor-targeting sdABD. of protein. This is accomplished by replacing the second tumor-targeting sdABD with an extension of the MMP2/9 cleavable linker of poly(GGGS) (SEQ ID NO: 178) sequence (total linker length 15-41 aa) or by removing This is accomplished by targeting the first tumor-targeting sdAB at the N-terminus (e.g., Pro1225 in Figure 1). Alternatively, the first or second tumor-targeting sdABD can be replaced with a non-functional sdABD such as one targeting chicken egg lysozyme (HEL), or by extending the MMP9 protease-cleavable linker (e.g., Pro1168 in Figure 1) . The dimerized active fragment (active form) produced by proteolysis of the prodrug has reduced affinity compared to a protein with two functional sdABDs (e.g., both (i) and (iii) present). This may be due to the reduction in the number of binding sites from 4 for proteins with two tumor-targeting sdABDs to 2 for proteins with a single tumor-targeting sdABD. Reduced affinity is hypothesized to result in reduced residence time on the cell surface, resulting in reduced protein potency while increasing the protein's tolerability and safety profile.

Three additional proteins with a single HER2-targeting sdABD were generated by modifying Pro1111 or Pro1118: Pro1148 contains a HER2-targeting sdABD at the N-terminus, and the second HER2-targeting sdABD in Pro1111 was replaced with a HEL-binding sdABD; Pro1145 contains an internal HER2-targeting sdABD, and the N-terminal HER2-targeting sdABD in Pro1111 was replaced with an sdABD that binds HEL; Pro1168 contains an internal HER2-targeting sdABD, and the N-terminal HER2-targeting sdABD in Pro1118 was replaced with an sdABD that binds HEL. The amino acid sequences of Pro1225, Pro1145, Pro1148 and Pro1168 are provided in Table 1. Proteins with only a single tumor-targeting sdABD targeting other tumor antigens (eg, EGFR, CA9, B7H3, EpCAM) have also been generated.

Proteins with a single tumor-binding sdABD were tested for binding affinity using the OCTET assay and compared with their respective counterparts containing two tumor-binding sdABDs. Human tumor antigen-Fxa-hFc protein was immobilized on an anti-human Fc capture sensor (ForteBio) and washed with 0.25% casein in PBS, pH 7.4. Each protein tested was added to individual sensors at varying concentrations (concentration range 1.56 – 100 nM), and the association of the protein with its target was recorded for 180 seconds. The sensor is then placed in buffer and the dissociation of the complex is recorded. K _d is calculated as k _off /k _on of the dissociation and association curves. Each experiment was performed in duplicate, and the average of the two measurements is reported. All proteins containing a single tumor-targeting sdABD exhibited reduced binding affinity to their respective targets compared to their counterparts with two tumor-targeting sdABDs. This was observed for proteins targeting HER2, EGFR, CA9, EpCAM and B7H3.

Next, the HER2-binding activity of proteins Pro1225 and Pro1268 containing a single HER2-targeting sdABD was compared with Pro1118, as well as in vivo and in vitro tumor killing assays. Compared with Pro1118, Pro1225 and Pro1268 have reduced binding affinity to human and cynomolgus monkey HER2 (Table 2). In in vitro tumor killing experiments, Pro1225 and Pro1268 showed reduced potency (about 2-fold lower potency) relative to Pro1118 in HT29 cells (which express low levels of HER2), but in SKLOV-3 cells (which express high levels of HER2 ) showed considerable efficacy (Table 2).

Surprisingly and unexpectedly, despite Pro1225's reduced binding affinity to its target and its reduced in vitro tumor killing potency, it demonstrated greater efficacy than Pro1118 in a mouse model of human lung cancer (HCC827 cells, which express low levels of HER2). Higher in vivo tumor killing efficacy (Figure 2A), and demonstrated comparable in vivo tumor killing efficacy at a dose 3 times lower than that of Pro1118 (Figure 2A and Figure 2B). Similarly, in a mouse model of human breast cancer (JIMT-1 cells, which express intermediate levels of HER2), Pro1225 demonstrated higher in vivo tumor killing efficacy than Pro1118 (Figure 3A) and demonstrated a 3 lower efficacy than Pro1118. It has considerable in vivo tumor killing efficacy at twice the dose (Figure 3A and Figure 3B). Pro1168, similar to Pro1225, has a single HER2-targeting sdABD and has dose-dependent in vivo tumor killing efficacy in a human gastric cancer (SNU16) mouse model (Figure 4). The results demonstrate that the binding affinity, in vitro potency, and in vivo efficacy of conditionally activatable proteins can be tuned by using individual tumor-targeting sdABDs. By using a single tumor-targeting sdABD, proteins with comparable or enhanced in vivo tumor killing efficacy and improved tolerability and reduced toxicity can be achieved. Example 2: In vitro characterization of HER2 single VHH construct (Pro1225)

Pro1225 was evaluated in a T cell-dependent cytotoxicity assay (TDCC) against a gastric cancer model (Figure 8), a breast cancer model (Figure 9), and a gastric cancer model (Figure 10). Pro1303 was used as a control, which has the same configuration as Pro1225, but without the MMP9 cleavable linker as depicted in Figure 1, it contains a non-cleavable linker (NCL) (Figure 6). Another cleaved construct, Pro1225, a cleaved version of Pro1225 (always active), was also included as a control (Figure 5). IC50 values for the three tested constructs are provided in Table 3.

First, cancer cell lines were tested for HER2 performance. Harvest all adherent cell lines using enzyme-free dissociation buffer. Dissociated cells were counted, and 1.0 × ¹⁰ cells were stained with PE-conjugated anti-human HER2 antibody at a saturating concentration of 33.3 nM. Stained samples were analyzed on a BD LSR Fortessa™ according to the manufacturer's instructions, and the mean fluorescence intensity (MFI) of the PE signal was generated for each cell line. PE-labeled BD Quantibrite™ beads were analyzed in parallel using the same instrument settings. MFI from Quantibrite™ beads was used to generate a standard curve, and the number of HER2 molecules per cell was inferred from the MFI signal of each stained cell line.

The endogenous HER2 cell surface density of a panel of nine cell lines was evaluated using the BD Quantibrite™ System. The values above each bar in Figure 7 represent the average number of HER2 antibodies bound per cell. The nine cell lines examined displayed a range of HER2 cell surface densities, ranging from relatively low (iPSC cardiomyocytes, SNU-16, HCC827), to low to moderate (HT-29, HT-55, Capan-1, JIMT-1). to relatively high (SKOV-3, NCI-N87) (Figure 7).

To determine the effect of cleaved Pro1225 on a human gastric cancer model with low expression of HER2, SNU-16 (HER2 ^low ; FLuc) cells were cultured in defined TDCC medium (AIM V [Gibco, Cat. No. 12055-091]). The cells were seeded on a 96-well white transparent bottom plate at a density of 5.0 × 10 ³ cells/well. Human PBMC effector cells were then co-cultured with SNU-16 (HER2 ^low ; FLuc) target cells at an effector:target (E:T) ratio of 5:1. Subsequently, test article Pro1225, cleaved Pro1225 and Pro1303 were added to respective wells and the treated co-cultures were allowed to incubate at 37C/5% CO _for 96 hours. The viability of SNU-16 (HER2 ^low ; FLuc) target cells was assessed using the Steady- ^Glo® Luciferase Assay System to measure the number of viable cells remaining in the wells after treatment with the test article compared to untreated controls. The luminescence of each well was measured using SpectraMax i3x. A concentration response curve was generated by calculating the relative luminescence units (RLU) of the test article treated sample normalized to the mean of the untreated control and performing nonlinear regression to generate a concentration-response curve using a 4 parameter logistic The model determines the _IC50 value.

In this study, the killing activity of Pro1225 and cleaved Pro1225 against SNU-16 (HER2 ^low ; FLuc) cells was observed at picomole concentrations (Figure 8 and Table 3). Pro1225 was 15-fold less potent in vitro (IC ₅₀ = 838.0 pM) relative to cleaved Pro1225 (IC ₅₀ = 56.04 pM).

To determine the effect of cleaved Pro1225 on a human breast cancer model with intermediate HER2 expression, JIMT-1 (HER2 ^intermediate ; FLuc) cells were cultured in defined TDCC medium (AIM V [Gibco, Cat. No. 12055-091]). The density of 5.0 × 10 ³ cells/well was plated on a 96-well white transparent bottom plate. Human PBMC effector cells were then co-cultured with JIMT-1 (HER2 ^intermediate ; FLuc) target cells at an effector:target (E:T) ratio of 5:1. Subsequently, test article Pro1225, cleaved Pro1225 and Pro1303 were added to respective wells and the treated co-cultures were allowed to incubate at 37C/5% CO _for 96 hours. The viability of JIMT-1 (HER2 ^intermediate ; FLuc) target cells was assessed using the Steady- ^Glo® Luciferase Assay System to measure the number of viable cells remaining in the wells after treatment with the test article compared to untreated controls. The luminescence of each well was measured using SpectraMax i3x. A concentration response curve was generated by calculating the relative luminescence units (RLU) of the test article treated sample normalized to the mean of the untreated control and performing nonlinear regression to generate a concentration-response curve using a 4 parameter logistic The model determines the _IC50 value.

In this study, the killing activity of Pro1225 and cleaved Pro1225 against JIMT-1 (HER2 ^medium ; FLuc) cells was observed at picomole concentrations (Figure 9 and Table 3). Pro1225 was 36-fold less potent in vitro (IC ₅₀ = 119.1 pM) relative to cleaved Pro1225 (IC ₅₀ = 3.323 pM).

To determine the effect of cleaved Pro1225 on a human gastric cancer model with high levels of HER2 expression, NCI-N87 (HER2 ^high ; FLuc ) cells were plated on a 96-well white transparent bottom plate at a density of 5.0 × 10 ³ cells/well. Human PBMC effector cells were then co-cultured with NCI-N87 (HER2 ^high ; FLuc) target cells at an effector:target (E:T) ratio of 5:1. Subsequently, test article Pro1225, cleaved Pro1225 and Pro1303 were added to respective wells and the treated co-cultures were allowed to incubate at 37C/5% CO _for 96 hours. The viability of NCI-N87 (HER2 ^high ; FLuc) target cells was assessed using the Steady- ^Glo® Luciferase Assay System to measure the number of viable cells remaining in the wells after treatment with the test article compared to untreated controls. The luminescence of each well was measured using SpectraMax i3x. A concentration response curve was generated by calculating the relative luminescence units (RLU) of the test article treated sample normalized to the mean of the untreated control and performing nonlinear regression to generate a concentration-response curve using a 4 parameter logistic The model determines the _IC50 value.

In this study, the killing activity of Pro1225 and cleaved Pro1225 against NCI-N87 (HER2 ^high ; FLuc) cells was observed at subpicomolar concentrations (Figure 10 and Table 3). Pro1225 was 3.6-fold less potent in vitro (IC ₅₀ = 0.9703 pM) relative to cleaved Pro1225 (IC ₅₀ = 0.2667 pM).

Next, the cross-reactivity of Pro1225 to cynomolgus monkey HER2-expressing cells was evaluated. Serum-supplemented TDCC medium (RPMI Medium 1640 [Gibco, Catalog No. 11875-093], 5% heat-inactivated fetal bovine serum [FBS], 1% Penicillin/Streptomycin, 1% GlutaMAX, 1% Non-Essential Amine Cynomolgus macaque HER2-expressing Raji (Fluc) cells were seeded on a 96-well white transparent bottom plate at a density of 5.0 × 10 ³ cells/well in 55 mM HEPES, 1mM sodium pyruvate, and 55mM 2-mercaptoethanol. Cynomolgus PBMC effector cells were then co-cultured with cynomolgus HER2-expressing Raji (FLuc) target cells at an effector:target (E:T) ratio of 5:1. Subsequently, test article Pro1225, cleaved Pro1225 and Pro1303 were added to respective wells and the treated co-cultures were allowed to incubate at 37C/5% CO _for 96 hours. The viability of cynomolgus monkey HER2-expressing Raji (FLuc) target cells was assessed using the Steady- ^Glo® Luciferase Assay System to measure the relative number of viable cells remaining in the wells after treatment with the test article compared to untreated controls. Use CLARIOstar to measure the luminescence of each well. A concentration response curve was generated by calculating the reduction in relative luminescence units (RLU) of the test article treated sample normalized to the mean of the untreated control and performing nonlinear regression to generate a concentration-response curve using 4 parameters Logic model determines IC ₅₀ value.

In this study, the killing activity of Pro1225 and cleaved Pro1225 against NHP HER2-engineered Raji cells in the presence of NHP PBMC was observed at concentrations as low as subpicomoles (Figure 11 and Table 3). Pro1225 was 47-fold less potent in vitro (IC ₅₀ = 2.957 pM) relative to cleaved Pro1225 (IC ₅₀ = 0.06255 pM).

Table 3 provides IC50 values for the three test constructs in different cell lines. Example 3: Efficacy and conditionality of Pro1225 and Pro1303 (EC50)

Pro1225 was evaluated in a T cell-dependent cytotoxicity assay (TDCC) against a colorectal cancer model with moderate HER2 expression (Figure 12). Pro1303 was used as a control, which has the same configuration as Pro1225, but without the MMP9 cleavable linker as depicted in Figure 1, it contains a non-cleavable linker (NCL) (Figure 6). Pro1225 was administered alone or as pre-cleaved Pro1225 after overnight treatment with the MMP9 enzyme, which cleaves the cleavable MMP9 linker (Figure 5). EC50 values for the tested constructs are provided in Table 4.

HT-29 cells in defined TDCC medium (AIM V [Gibco, Catalog No. 12055-091]) were seeded on a 96-well white transparent bottom plate at a density of 5.0 × 10 ³ cells/well. Human PBMC effector cells were then co-cultured with NCI-N87 (HER2 ^high ; FLuc) target cells at an effector:target (E:T) ratio of 5:1. Subsequently, test articles Pro1225, Pro1225 + MMP9 and Pro1303 were added to their respective wells and the treated co-cultures were allowed to incubate at 37C/5% CO _for 48 hours. The viability of HT-29 target cells was assessed using the Steady- ^Glo® Luciferase Assay System to measure the number of viable cells remaining in the wells after treatment with the test article compared to untreated controls. The luminescence of each well was measured using SpectraMax i3x. A concentration response curve was generated by calculating the relative luminescence units (RLU) of the test article-treated sample, and nonlinear regression was performed to generate a concentration-response curve and the _EC50 value was determined using a 4-parameter logistic model.

In this study, the killing activity of Pro1225 and Pro1225 + MMP9 against HT-29 cells in the presence of PBMC was observed at picomole concentrations (Figure 12 and Table 4). Pro1225 was approximately 300-fold less potent in vitro (EC ₅₀ = 1047.9 pM) relative to Pro1225 + MMP9 (IC ₅₀ = 3.2 pM). In addition, all samples were stable and showed no degradation after three freeze-thaw cycles and storage at room temperature and 4°C for 10 days. Example 4: HER2 single VHH construct (Pro1225) demonstrates tumor killing activity in vivo

Multiple doses of Pro1225 were evaluated in in vivo gastric cancer tumor models, gastric cancer tumor models, lung cancer tumor models, colorectal cancer tumor models, and breast cancer tumor models. Pro1303 was used as a control, which has the same configuration as Pro1225, but without the MMP9 cleavable linker as depicted in Figure 1, it contains a non-cleavable linker (NCL) (Figure 6).

In the SNU16 (low HER2 expression level) gastric cancer model, 2.5 x 10 ⁶ SNU16 cells were implanted subcutaneously into the right side of NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice ( The Jackson Laboratory , catalog no. 005557) into the abdomen and allow it to grow until a tumor is established. Human T cells were cultured in parallel in T Cell Medium ( X-VIVO 15 [ Lonza , Cat. No. 04-418Q], 5% human serum, 1% penicillin/streptomycin, 0.01mM 2-mercaptoethanol) for 11 days and supplemented with recombinant human IL-2 protein. Tumor growth and human T cell activation/expansion in mice were coordinated so that mice were randomized (N=6) based on tumor size (mean tumor volume approximately 150 ^mm3 ) on study day 0. Mice were then injected intravenously (IV) with ^2.5x106 cultured human T cells and administered a first dose of Pro1225 or a control molecule (Pro1303). Mice were dosed every 3 days for 7 doses (day 0, day 3, day 6, day 9, day 12, day 15, and day 18) and then followed until study day 39 terminate. Each group received 0.3, 0.1 and 0.03 mg/kg of Pro1225 containing MMP9 cleavable linker, or 0.3 mg/kg of Pro1303. Tumor volume was measured every 3 days.

Administration of Pro1225 resulted in dose-dependent inhibition of tumor growth in a SNU16 HER2-low expressing gastric cancer model. Tumor regression was observed in the groups treated with 0.3 and 0.1 mg/kg (5/6 mice in the 0.3 mg/kg group and 2/6 mice in the 0.1 mg/kg group) (Figure 13) .

In the HCC827 (low HER2 expression level) lung cancer model, 5 x 10 ⁶ HCC827 cells were implanted subcutaneously into the right flank of NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice ( The Jackson Laboratory , Catalog No. 005557) , and allowed to grow until a tumor is established. Human T cells were cultured in parallel in T Cell Medium ( X-VIVO 15 [ Lonza , Cat. No. 04-418Q], 5% human serum, 1% penicillin/streptomycin, 0.01mM 2-mercaptoethanol) for 11 days and supplemented with recombinant human IL-2 protein. Tumor growth and human T cell activation/expansion in mice were coordinated so that mice were randomized (N=6) based on tumor size (mean tumor volume approximately 150 ^mm3 ) on study day 0. Mice were then injected intravenously (IV) with ^2.5x106 cultured human T cells and administered a first dose of Pro1225 or a control molecule (Pro1303). Mice were dosed every 3 days for 7 doses (day 0, day 3, day 6, day 9, day 12, day 15, and day 18) and then followed until study day 40 terminate. Each group received 0.3, 0.1 and 0.03 mg/kg of Pro1225 containing MMP9 cleavable linker, or 0.3 mg/kg of Pro1303. Tumor volume was measured every 3 days.

Administration of Pro1225 resulted in dose-dependent inhibition of tumor growth in the HCC827 HER2 low expression lung cancer model (Figure 14).

In the HT55 (low HER2 expression level) colorectal cancer model, 2.5 x ¹⁰ HT55 cells were implanted subcutaneously into the right side of NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice ( The Jackson Laboratory , catalog no. 005557) into the abdomen and allow it to grow until a tumor is established. Human T cells were cultured in parallel in T Cell Medium ( X-VIVO 15 [ Lonza , Cat. No. 04-418Q], 5% human serum, 1% penicillin/streptomycin, 0.01mM 2-mercaptoethanol) for 12 days and supplemented with recombinant human IL-2 protein. Tumor growth and human T cell activation/expansion in mice were coordinated so that mice were randomized (N=6) based on tumor size (mean tumor volume approximately 100 ^mm3 ) on study day 0. Mice were then injected intravenously (IV) with ^2.5x106 cultured human T cells and administered a first dose of Pro1225 or a control molecule (Pro1303). Mice were dosed every 3 days for 7 doses (Q3Dx7) (Day 0, Day 3, Day 6, Day 9, Day 12, Day 15, and Day 18) and then followed until study Terminates on day 27. Each group received 1, 0.3 and 0.1 mg/kg of Pro1225, or 1 mg/kg of Pro1303. Tumor volume was measured every 3 days.

Administration of 1 and 0.3 mg/kg of Pro1225 resulted in complete tumor regression in the HT55 HER2 low expression colorectal cancer model (Figure 15).

In a JIMT-1 HER2 (intermediate HER2 expression level) breast cancer model, 5 x 10 JIMT- ¹ cells were implanted subcutaneously into NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice ( The Jackson Laboratory , catalog no. 005557 ) in the right side of the abdomen and allowed to grow until a tumor was established. Human T cells were cultured in parallel in T Cell Medium ( X-VIVO 15 [ Lonza , Cat. No. 04-418Q], 5% human serum, 1% penicillin/streptomycin, 0.01mM 2-mercaptoethanol) for 11 days and supplemented with recombinant human IL-2 protein. Tumor growth and human T cell activation/expansion in mice were coordinated so that mice were randomized (N=6) based on tumor size (mean tumor volume approximately 120 ^mm3 ) on study day 0. Mice were then injected intravenously (IV) with ^2.5x106 cultured human T cells and administered a first dose of Pro1225 or a control molecule (Pro1303). Mice were dosed every 3 days for 7 doses (day 0, day 3, day 6, day 9, day 12, day 15, and day 18) and then followed until study day 40 terminate. Each group received 1, 0.3 and 0.1 mg/kg of Pro1225, or 1 mg/kg of Pro1303. Tumor volume was measured every 3 days.

Administration of Pro1225 resulted in dose-dependent inhibition of tumor growth in a JIMT-1 HER2 intermediate breast cancer model. Tumor regression was observed in 4 out of 6 mice in the 0.3 mg/kg treatment group. Complete regression was observed in the 1 mg/kg treatment group (6/6 mice) (Figure 16).

[Fig. 1] shows a schematic diagram of control proteins (Pro1111 and Pro1118 containing two HER2-binding sdABDs). Pro1225 contains a single HER2-binding sdABD at the N-terminus of the protein and the presence of an extended cleavable linker corresponding to the position of a second HER2-binding sdABD in Pro1111 or Pro1118. Pro1168 replaces the N-terminal HER2-targeting sdABD of Pro1118 with a non-functional sdABD that binds chicken egg lysozyme (HEL). The prodrug-active form of the protein is shown. [Figure 2A]-[Figure 2B] are graphs showing the tumor killing efficacy of Pro1225 in human lung cancer (HCC827) mouse model. Pro1225 is approximately 10 times more effective than Pro1118 in reducing lung cancer tumor volume. [Figure 3A]-[Figure 3B] are graphs showing the tumor killing efficacy of Pro1225 in a mouse model of human breast cancer (JIMT-1 cells). Pro1225 is approximately 3 times more effective than Pro1118 in reducing breast cancer tumor size. [Figure 4] is a graph showing the tumor killing efficacy of Pro1168 in human gastric cancer (SNU16) mouse model. [Fig. 5] Schematic diagram showing formation of full-length Pro1225, cleaved associated full-length Pro1225, cleaved Pro1225 product, and Pro1225 active dimer. [Fig. 6] shows a schematic diagram of the control protein Pro1303. [Fig. 7] is a graph showing the cell surface density of endogenous HER2 in cancer cell lines. [Figure 8] is a graph showing the tumor killing efficacy of Pro1225 in an in vitro human gastric cancer model (SNU16). [Figure 9] is a graph showing the tumor killing efficacy of Pro1225 in an in vitro human breast cancer model (JIMT-1). [Fig. 10] A graph showing the tumor killing efficacy of Pro1225 in an in vitro human gastric cancer model (NCI-N87). [Fig. 11] A graph showing the cross-reactivity of Pro1225 in an in vitro human lymphoma model (cyHER2-Raji). [Fig. 12] A graph showing the tumor killing efficacy of Pro1225 in an in vitro human colorectal cancer model (HT-29). [Fig. 13] A graph showing the tumor killing efficacy of Pro1225 in human gastric cancer (SNU16) mouse model. Pro1225 dose-dependently reduces gastric cancer tumor volume. [Fig. 14] A graph showing the tumor killing efficacy of Pro1225 in a mouse model of human lung cancer (HCC827). Pro1225 dose-dependently reduces lung cancer tumor volume. [Fig. 15] A graph showing the tumor killing efficacy of Pro1225 in human colorectal cancer (HT55) mouse model. Pro1225 dose-dependently reduces colorectal cancer tumor volume. [Fig. 16] A graph showing the tumor killing efficacy of Pro1225 in human breast cancer (JIMT-1) mouse model. Pro1225 dose-dependently reduces breast cancer tumor volume.

TW202342521A_112106790_SEQL.xml

Claims (46)

A protein comprising: from the N-terminus to the C-terminus: (i) a first single domain antigen binding domain (sdABD) and a first domain linker at the C-terminus of the first sdABD, wherein (i) is present or absent; (ii) a first constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region linked to a light chain variable region via a first constrained non-cleavable linker (NCL), wherein the heavy chain The variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen, and wherein the heavy chain variable region and the light chain variable region do not associate in the first restricted scFv domain , and the first restricted scFv domain does not bind the human immune cell antigen; (iii) a second domain linker and a second single domain antigen binding domain (sdABD), wherein the second domain linker is at the N-terminus of the second sdABD, and wherein (iii) is present or absent; (iv) cleavable linkers; (v) a second constrained single chain variable fragment (scFv) domain comprising a pseudo heavy chain variable region linked to a pseudo light chain variable region via a second constrained non-cleavable linker (CNCL), wherein the The pseudo heavy chain variable region and the pseudo light chain variable region do not associate in the second restricted scFv domain, and the second restricted scFv domain does not bind the human immune cell antigen of (ii); (vi) third domain linker; and (vii) a third sdABD, which binds human serum albumin (HSA); wherein the heavy chain variable region of (ii) and the pseudo light chain variable region of (v) are intramolecularly associated to form a variable fragment (Fv) that does not bind the immune cell antigen; and wherein the pseudo heavy chain variable region of (v) and the light chain variable region of (ii) are intramolecularly associated to form a variable fragment (Fv) that does not bind the immune cell antigen; Among them, (i) and (iii) do not exist; And when (i) and (iii) both exist: (a) The first sdABD binds human target tumor antigen (TTA) and the second sdABD does not bind human TTA; or (b) The first sdABD does not bind human TTA and the second sdABD binds human TTA. The protein of claim 1, wherein both (i) and (iii) are present, and wherein the first sdABD does not bind human TTA, and the second sdABD binds human TTA. The protein of claim 2, wherein the first sdABD binds hen egg lysozyme (HEL). The protein of claim 3, wherein the first sdABD includes the amino acid sequence of SEQ ID NO: 26. The protein of claim 1, wherein both (i) and (iii) are present, and wherein the second sdABD does not bind human TTA, and the first sdABD binds human TTA. The protein of claim 5, wherein the second sdABD binds hen egg lysozyme (HEL). The protein of claim 6, wherein the second sdABD includes the amino acid sequence of SEQ ID NO: 26. The protein of claim 1, wherein (i) is absent and (iii) is present, and wherein the second sdABD binds human TTA. The protein of claim 1, wherein (i) is present and (iii) is not present, and wherein the first sdABD binds human TTA. The protein of any one of claims 1-9, wherein the human TTA is HER2, EGFR, FOLR1, TROP2 or EpCAM. The protein of any one of claims 1-9, wherein the human TTA is HER2. The protein of any one of claims 1-9, wherein the first sdABD or the second sdABD that binds HER2 comprises the amino acid sequence of any one of the following: SEQ ID NO: 41, 45, 48-52 , 54, 58, 60, 63, 66, 68, 72, 75-78, 82, 85, 89, 95, 96, 99, 103, 104, 108, 112, 116, 117, 121 and 165. The protein of any one of claims 1-12, wherein the cleavable linker in (iv) has a length of 8-45 amino acids. The protein of any one of claims 1-13, wherein the cleavable linker of (iv) includes a cleavage site for a protease present in the tumor microenvironment. Such as the protein of claim 14, wherein the protease is MMP2, MMP9, meprin, cathepsin, granzyme, protease (Matriptase), thrombin, enterokinase, KLK7-6, KLK7-13 , KLK7-11, KLK7-10 or uPA. The protein of any one of claims 1-15, wherein the immune cells are T cells, natural killer (NK) cells, natural killer T (NKT) cells, macrophages, B cells, neutrophils, dendrites cells or mononuclear spheres. Such as the protein of any one of claims 1-16, wherein the human immune cell antigen is CD3, CD28, T cell receptor, programmed cell death protein 1 (PD-1), PD-L1, cytotoxic T lymphocyte Related protein 4 (CTLA-4), T cell immunoglobulin and mucin domain 3 (TIM-3), lymphocyte activating gene 3 (LAG-3), killer cell immunoglobulin-like receptor (KIR), CD137 , OX40, OX40L, CD27, GITR (TNFRSF18), TIGIT, inducible T cell costimulator (ICOS), CD16A, CD226, CD96, CD40L, CRTAM, LFA-1, NKG2D, CSF1R, CD40, MARCO, VSIG4, or CD163 . The protein of any one of claims 1-17, wherein the human immune cell antigen is CD3. The protein of any one of claims 1-18, wherein the heavy chain variable region is linked to the N-terminus of the light chain variable region in the first restricted scFv domain of (ii). The protein of any one of claims 1-19, wherein the pseudo heavy chain variable region is connected to the C terminus of the pseudo light chain variable region in the second restricted scFv domain of (v). The protein of any one of claims 1-20, wherein the length of the first restricted non-cleavable linker of (ii) and/or the second restricted non-cleavable linker of (v) is 6-10 Amino acids. The protein of any one of claims 1-21, wherein the length of the first restricted non-cleavable linker of (ii) and/or the second restricted non-cleavable linker of (v) is 8 amine groups acid. The protein of any one of claims 1-22, wherein the human immune cell antigen is CD3, the heavy chain variable region of (ii) includes the amino acid sequence of SEQ ID NO: 7, and the (ii) The light chain variable region includes the amino acid sequence of SEQ ID NO: 8. The protein of any one of claims 1-23, wherein the pseudo heavy chain variable region of (v) includes the amino acid sequence of SEQ ID NO: 33, and the pseudo light chain variable region of (v) includes SEQ Amino acid sequence of ID NO: 34. The protein of any one of claims 1-24, wherein the third sdABD comprises the amino acid sequence of SEQ ID NO: 22. The protein of any one of claims 1-25, which includes the amino acid sequence of any one of SEQ ID NOs: 37-40 and 173-176. A nucleic acid molecule comprising a nucleotide sequence encoding a protein according to any one of claims 1-26. The nucleic acid molecule of claim 27, wherein the nucleic acid molecule is a vector. The nucleic acid molecule of claim 28, wherein the vector is an expression vector. A cell comprising a protein as in any one of claims 1-26 or a nucleic acid molecule as in any one of claims 27-29. A method of producing a protein comprising culturing a cell as claimed in claim 30 under conditions allowing expression of the protein. The method of claim 31, further comprising isolating the protein. A composition comprising the protein of any one of claims 1-26. A method of treating cancer, comprising administering to an individual a protein according to any one of claims 1-26 or a composition according to claim 33. The method of claim 34, wherein the cancer expresses HER2. The method of claim 35, wherein the cancer has low HER2 expression. The method of claim 35, wherein the cancer has intermediate HER2 expression. The method of claim 35, wherein the cancer has high HER2 expression. The method of any one of claims 34-38, wherein the individual is a human individual. A composition comprising: The first protein and the second protein each include from the N-terminus to the C-terminus: (i) a first single domain antigen binding domain (sdABD) and a first domain linker at the C-terminus of the first sdABD, wherein (i) is present or absent; (ii) a first constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region linked to a light chain variable region via a first constrained non-cleavable linker (CNCL), wherein the heavy chain The variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen, and wherein the heavy chain variable region and the light chain variable region do not associate in the first restricted scFv domain , and the first restricted scFv domain does not bind the human immune cell antigen; (iii) a second domain linker and a second single domain antigen binding domain (sdABD), wherein the second domain linker is at the N-terminus of the second sdABD, and wherein (iii) is present or absent; (iv) cleavable linkers; (v) a second constrained single chain variable fragment (scFv) domain comprising a pseudo heavy chain variable region linked to a pseudo light chain variable region via a second constrained non-cleavable linker (CNCL), wherein the The pseudo heavy chain variable region and the pseudo light chain variable region do not associate in the second restricted scFv domain, and the second restricted scFv domain does not bind the human immune cell antigen of (ii); (vi) third domain linker; and (vii) a third sdABD, which binds human serum albumin (HSA); wherein the heavy chain variable region of (ii) and the pseudo light chain variable region of (v) are intramolecularly associated to form a variable fragment (Fv) that does not bind the immune cell antigen; and wherein the pseudo heavy chain variable region of (v) and the light chain variable region of (ii) are intramolecularly associated to form a variable fragment (Fv) that does not bind the immune cell antigen; Among them, (i) and (iii) do not exist; And when (i) and (iii) both exist: (a) The first sdABD binds human target tumor antigen (TTA) and the second sdABD does not bind human TTA; or (b) The first sdABD does not bind human TTA and the second sdABD binds human TTA. The composition of claim 40, wherein the first protein and the second protein are the same. The composition of claim 40 or claim 41, wherein after cleavage of the cleavable linker in (iv) in the first protein and the second protein: The heavy chain variable region of the first protein associates with the light chain variable region of the second protein to form an active Fv that binds to the human immune cell antigen; The light chain variable region of the first protein associates with the heavy chain variable region of the second protein to form an active Fv that binds the human immune cell antigen. The composition of claim 42, wherein upon administration of the composition to the individual, the lysis occurs in the tumor microenvironment of the individual. A composition comprising a homodimer of a first polypeptide and a second polypeptide, wherein the first polypeptide is the same as the second polypeptide, and wherein one of the first polypeptide and the second polypeptide Each includes: (i) a single domain antigen binding domain (sdABD) that binds a first human target tumor antigen (TTA); (ii) domain linkers; (iii) A constrained single chain variable fragment (scFv) domain comprising a heavy chain variable region (VH) linked to a light chain variable region (VL) via a constrained non-cleavable linker (CNCL), wherein the The heavy chain variable region and the light chain variable region, if associated, are capable of binding a human immune cell antigen, and wherein the heavy chain variable region and the light chain variable region do not associate in the restricted scFv domain , and the restricted scFv domain does not bind the human immune cell antigen; wherein the VH of the first polypeptide associates with the VL of the second polypeptide, and the VL of the first polypeptide associates with the VH of the second polypeptide, forming two active variable fragments ( Fv), each active variable fragment is capable of binding to the immune antigen. The composition of claim 44, wherein the TAA is HER2, EGFR, FOLR1, TROP2 or EpCAM. The composition of claim 44 or claim 45, wherein the human immune cell antigen is CD3.