Anti-KLK2 antibodies, bispecific binding proteins and uses thereof

Abstract

Provided are antibodies capable of binding KLK2, and bispecific binding proteins in FIT-Ig format that binds KLK2 and CD3. The antibodies and bispecific KLK2×CD3 binding proteins are useful for treating or preventing a KLK2-associated disorder, particularly a prostate cancer.

Description

WSGR Docket No. 65457-712.602ANTI-KLK2 ANTIBODIES, BISPECIFIC BINDING PROTEINS AND USES THEREOFCROSS-REFERENCE

[0001] This application claims the benefit of PCT / CN2025 / 085783 filed March 28, 2025, which is incorporated herein by reference in its entirety and for all purposes.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on March 25, 2026, is named 65457-712_602_SL.xml and is 92,272 bytes in size.TECHNICAL FIELD

[0003] The present disclosure relates to anti-KLK2 antibodies specifically recognizing the kallikrein-related peptidase 2 (KLK2), and bispecific binding proteins comprising said anti-KLK2 antibodies, such as those capable of binding KLK2 and CD3. The present disclosure further relates to use of said antibodies and bispecific binding proteins disclosed herein for treating diseases or disorders such as hematopoietic cancers and solid tumors.BACKGROUND

[0004] Human Kallikrein-2 (KLK2, also named hK2) is a trypsin-like serine protease and highly expressed in prostate tissue. It belongs to the glandular kallikrein protein family (KLK family) proteins and shares 78% homology with KLK3 (also named PSA) which is a chymotrypsin-like serine protease. Both KLK2 and KLK3 are highly expressed in prostate tissue produced by prostate gland and KLK2 is about 1% of the total KLK3 concentration in seminal plasma. As a trypsin-like serine protease with high catalytic activity, KLK2 is secreted and activated into the seminal fluid to liquefy the seminal gel by degrading the gel-forming proteins (SEMG1 and SEMG2). Moreover, KLK2 is also involved in degrade cellular matrix proteins including activation of urokinase-type plasminogen activator (uPA), activation of matrix metalloproteases (MMPs) and proteolytic degradation of extracellular matrix proteins (Khosroabadi, G., & Yousefnia, S. (2023). Personalized Medicine Journal, 5(28), 5-12). The KLK2 expression in patients with prostate cancer is higher than the healthy people and the expression in prostate cancer is 106times higher compared to concentrations in blood. TheWSGR Docket No. 65457-712.602expression levels of KLK2 correlate with tumor stage, degree of differentiation and total tumor volume (Kohli, Manish, et al. Cancer Biomarkers 7.2 (2010): 101-108).

[0005] KLK2 has been proposed as a target for cancer treatment. US patent publication 2021 / 0040210 has disclosed an anti-KLK2*CD3 bispecific antibody generated by engineering of a humanized parental anti-KLK2 clonal hul 1B6, and a parental anti-CD3 antibody. This KLK2*CD3 bispecific antibody shows high affinity to human KLK2 or CD3 and has good thermal stability. It exhibits in vitro cytotoxicity to VCaP, a KLK2 positive human prostate cancer cell line, as well as T cell activation and proliferation with functional IFN-y and TNF-a release. In vivo model demonstrates robust tumor growth inhibition of this I<LI<2 CD3 bispecific antibody at the dose of 5mg / kg. The anti-KLK2 parental antibody hul 1B6 has also been used for the development of chimeric antigen receptor T-cells (CAR-T) therapy and antibody radio-conjugate therapy for the treatment of prostate cancer according to public information.

[0006] Given that KLK2 is a promising target in prostate cancer treatment, there remains a need in the art to develop diversified anti-KLK2 antibody-based therapies with improved therapeutic potency, safety or drug-like developability for large scale manufacturing.SUMMARY OF THE INVENTION

[0007] The present disclosure provides anti-KLK2 antibodies, and binding proteins that specifically bind to KLK2 and CD3. The disclosure also provides KLK2*CD3 bispecific Fabs-in-Tandem immunoglobulins (FIT-Igs) that are reactive with both CD3 and KLK2. Bispecific binding proteins of the present disclosure are capable of activating the TCR-CD3 complex. The bispecific, multivalent binding proteins described herein will be useful for the treatment of a KLK2 -positive solid tumor, such as a prostate cancer, especially a castration-resistant prostate cancer (CRPC), through redirected T cell cytotoxicity.

[0008] In particular, in some embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to KLK2, e.g., with high binding potency to KLK2.

[0009] In some embodiments, the present disclosure provides a bispecific binding protein that specifically binds KLK2 and CD3, comprising a first antigen-binding site that specifically binds KLK2, and a second antigen-binding site that specifically binds CD3. In an embodiment, theWSGR Docket No. 65457-712.602bispecific binding protein of the present disclosure is a bispecific Fabs-in-Tandem immunoglobulin (FIT-Ig) binding protein comprising first, second, and third polypeptide chains, wherein the first polypeptide chain comprises, from amino terminus to carboxyl terminus, 1) VLA-CL-VHB-CHl-hinge-CH2-CH3, or 2) VHB-CHl-VLA-CL-hinge-CH2-CH3; the second polypeptide chain comprises, from amino to carboxyl terminus, VHA-CH1; the third polypeptide chain comprises, from amino to carboxyl terminus, VLB-CL;

[0010] wherein the VLA-CL pairs with VHA-CH1 to form a first Fab that specifically binds antigen A, and VLB-CL pairs with VHB-CH1 to form a second Fab that specifically binds antigen B, and

[0011] wherein antigen A is KLK2, and antigen B is CD3, or antigen A is CD3, and antigen B is KLK2;

[0012] wherein said three polypeptide chains as a half molecule associates with another said three polypeptide chains as the other half molecule to form a FIT-Ig protein.

[0013] In some embodiments, such FIT-Ig binding proteins have the structure as shown in FIG. 1

[0014] In some embodiments, the bispecifc binding proteins described herein are useful to activate T cells to kill tumor cells expressing KLK2 protein, while induce lower cytokine (such as human IL-2, IL-6, IL-10, TNFa and IFNy) release by T cells in vivo and / or in vitro. In some embodiments, the bispecifc binding proteins described herein are useful to reduce tumor burden / growth / cell expansion.

[0015] In some embodiments, the present disclosure also provides methods of making and using the anti-KLK2 antibodies and KLK2*CD3 bispecific binding proteins described herein. Various compositions, e.g., those that may be used in methods of treating or preventing a KLK2-associated disorder in an individual are also provided herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 is a schematic view of FIT-Ig constructions.

[0017] FIG. 2 shows in a KLK2 protein-bound reporter gene assay (RGA), FIT2023-8ac (red triangle), FIT2023-10ac (green inverted triangle), FIT2023-20ac (purple diamond), FIT2023-22ac (orange circle), and FIT2023-23ac (dark green square), concentration dependently activate CD3 on T cells in the presence of KLK2 protein, while the parental anti-CD3 antibodyWSGR Docket No. 65457-712.602huEM0006-01-24 (circle with a cross) exhibits minimum to no activation in the assay.

[0018] FIG. 3 shows KLK2*CD3 chimeric bispecific antibodies, i.e. FIT2023-8ac (red triangle), FIT2023-10ac (green inverted triangle), FIT2023-20ac (purple diamond), FIT2023-22ac (orange circle), and FIT2023-23ac (dark green square), concentration dependently activate T cells to kill VCaP cell line in-vitro (upper panel); FIG. 3 further shows KLK2*CD3 chimeric bispecific antibodies, i.e. FIT2023-8ac (red triangle), FIT2023-10ac (green inverted triangle), FIT2023-20ac (purple diamond), FIT2023-22ac (orange circle), and FIT2023-23ac (dark green square), concentration dependently activate T cells to kill LNCaP-KLK2 cell line in-vitro (lower panel).

[0019] FIG. 4 shows FIT2023-10ah-4(h) (blue circle), FIT2023-20ah-l(h) (red triangle), and FIT2023-22ah- 10(h) (green square), concentration dependently activate T cells in-vitro to kill both VCaP cell line (upper panel) and LNCaP-KLK2 cell line (lower panel). The reference molecule Bi2023-4a (purple triangle) is used as positive control, and the parental CD3 monoclonal antibody huEM0006-01-24 (black diamond) is used as control.

[0020] FIGS. 5A-5J show compared to the reference molecule Bi2023-4a (green), FIT2023-10ah-4(h) (blue), FIT2023-20ah-l(h) (red), and FIT2023-22ah- 10(h) (orange) in-vitro induces lower cytokine (human IL-2 (FIG. 5A and FIG. 5F), IL-6 (FIG. 5B and FIG. 5G), IL- 10 (FIG.5C and FIG. 5H), TNFa (FIG. 5D and FIG. 51) and IFNy (FIG. 5E and FIG. 5J)) release by T cells after coculturing with target cells expressing KLK2 protein for 48 hours. The parental CD3 monoclonal antibody huEM0006-01-24 (purple) is used as control.

[0021] FIGS. 6A-6E show FIT2023-10ah-4(h) (blue), FIT2023-20ah-l(h) (red), and FIT2023-22ah- 10(h) (brown), in-vitro induces lower cytokine (human IL-6 (FIG. 6A), IL-2 (FIG. 6B), IL- 10 (FIG. 6C), IFNy (FIG- 6D) and TNFa (FIG. 6E)) release by PBMCs in the absence of target cells expressing KLK2 protein. The parental CD3 monoclonal antibody huEM0006-01-24 (black) and OKT-3 (purple) are used as controls, and hlgG (orange) as isotype control.

[0022] FIG. 7 shows the in vivo effect of FIT2023-10ah-4(h) (green inverted triangle), FIT2023-20ah-l(h) (purple square), and FIT202322ah- 10(h) (orange diamond), at a higher dose of lOmg / kg on VCaP tumor volume in mice. The reference molecule Bi2023-4a (red triangle) and Vehicle (blue circle) are used as controls.

[0023] FIG. 8 shows the in vivo effect of FIT2023-10ah-4(h) (green inverted triangle) and FIT2023-20ah-l(h) (purple square), at a lower dose of 2mg / kg on VCaP tumor volume in mice.WSGR Docket No. 65457-712.602The reference molecule Bi2023-4a (red triangle) and Vehicle (blue circle) are used as controls.

[0024] FIG. 9 shows the in vivo effect of FIT2023-10ah-4(h) (green inverted triangle) and FIT2023-20ah-l(h) (purple square), at a higher dose of lOmg / kg on LNCaP-KLK2 tumor volume in mice. The reference molecule Bi2023-4a (red triangle) , and Vehicle (blue circle) are used as controls.

[0025] FIG. 10 shows the results from the stability assay. Upper panel shows the accelerated stabilities of FIT2023-10ah-4(h) (blue circle) and FIT2023-20ah-l(h) (red square) after incubating at 40°C for up to 14 days. Lower panel shows compared with the stabilities (blue) at 4°C starting point, the freeze-thaw stabilities (red) of FIT2023-10ah-4(h) and FIT2023-20ah-1(h) after 5 freeze-thaw cycles.DETAILED DESCRIPTION

[0026] Bispecific T cell engagers (TCE) targeting tumor-associated antigens have demonstrated therapeutic potential, especially in cancer immunotherapy. They function by redirecting T cell cytotoxicity toward malignant cells through engaging both a T-cell constant component (like CD3) and a tumor-associated antigen (TAA). This technology offers various benefits, including redirecting cytotoxic T-cells in a non-MHC restricted manner, and has made significant strides in clinical advancements. Illustrative examples include ubamatamab (REGN4018) for ovarian cancer (Crawford et al., AMucin 16 bispecific T cell-engaging antibody for the treatment of ovarian cancer, Sci. Transl. Med. 11, eaau7534 (2019) 19 June 2019), tarlatamab for small cell lung cancer (Apaydin et al., Taking it up a notch: a promising immunotherapy against small cell lung cancer, Transl Lung Cancer Res 2023, https: / / dx.doi.org / 10.21037 / tlcr-23-230), and teclistamab for multiple myeloma (Pillarisetti et al., Teclistamab is an active T cell-redirecting bispecific antibody against B-cell maturation antigen for multiple myeloma, Blood Adv. 2020 Sep 22; 4(18): 4538-4549).

[0027] While bispecific T cell engagers offer significant promise for cancer treatment, their clinical development is challenged by issues related to efficacy, toxicity profiles, and manufacturability. Tumors often demonstrate considerable heterogeneity in tumor-associated antigen (TAA) expression, both within a single tumor and across different patients with the same type of cancer. This variability can result in varying sensitivities to T cell engager therapies targeting different TAAs. Moreover, in contrast to TCEs used for treating hematologicalWSGR Docket No. 65457-712.602malignancies, those targeting solid tumors typically require higher peripheral exposure, which increases the risk of off-target toxicities, commonly manifesting as cytokine release syndrome (CRS). Addressing these challenges presented by such as tumor heterogeneity and solid tumors requires the exploration of new targets, novel formats, and strategies for T cell engagers to enhance specificity, efficacy, and safety and improve manufacturability.

[0028] To meet such need, in an aspect, the present disclosure provides an anti-CD3 antibody, an anti-KLK2 antibody, and a T cell engager comprising at least one KLK2 -binding site and at least one CD3-binding site. In another aspect, the present disclosure also provides an anti-CD3 antibody, an anti-KLK2 antibody, and fragments thereof, which are useful to create the T cell engager of the present disclosure. In a preferred embodiment, the T cell engager of the present disclosure is a bispecific binding protein that specifically binds KLK2 and CD3. In another preferred embodiment, the T cell engager of the present disclosure is a FIT-Ig binding protein.

[0029] In other aspects, the present disclosure also provides pharmaceutical compositions, as well as nucleic acids, recombinant expression vectors and host cells for making such antibodies, functional antibody fragments, and binding proteins. Methods of using the antibodies, functional antibody fragments, and bispecific binding proteins of the disclosure to detect human CD3, human KLK2, or both; to inhibit human KLK2 activity, either in vitro or in vivo, and to treat diseases, especially cancer, that are associated with expression of KLK2, or medicated by KLK2 binding to its ligand are also encompassed by the disclosure.Definitions

[0030] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[0031] Unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.

[0032] The term “about” used in conjunction with a numerical value is intended to encompass the numerical values in a range from a lower limit less than the specified numerical value by 5% to an upper limit greater than the specified numerical value by 5%.WSGR Docket No. 65457-712.602

[0033] The use of “or” means “and / or”, unless stated otherwise. As used herein, the term “and / or”, when used in conjunction with more than one options, refers to any one of the options, or any two or more or all the options.

[0034] As used herein, the term “comprise”, “include” or the variants thereof is intended to mean that the described elements, integers or steps are included, but not to the exclusion of any other elements, integers or steps. The term “comprise” or “include” used herein, unless otherwise specified, also encompasses the situation where the entirety consists of the described elements, integers or steps. For example, when referring to an antibody variable region “comprising” a particular sequence, it is also intended to encompass an antibody variable region consisting of the particular sequence.

[0035] The term “isolated protein” or “isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation is at least partially separated from naturally associated components that accompany it in its native state, is substantially free of other proteins from the same species, is expressed by a cell from a different species, or does not occur in nature. A polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates may be “isolated” from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. As measured by Size Exclusion Chromatography SEC-HPLC, an isolated protein may be of more than 80%, 85%, 90%, 95% or 99% purity.

[0036] The term “CD3”, as used herein, refers to an antigen which is expressed on T cells as part of the multimolecular T cell receptor (TCR) and which consists of a homodimer or heterodimer formed from the association of two of four receptor chains: CD3 -epsilon, CD3-delta, CD3-zeta, and CD3-gamma. An amino acid sequence of a human CD3-epsilon is set forth under UniProtKB accession number P07766; an amino acid sequence of a human CD3-delta under UniProtKB accession number P04234; an amino acid sequence of a human CD3-zeta under UniProtKB accession number P20963; and an amino acid sequence of a CD3 -gamma under UniProtKB accession number P09693. All references to CD3 proteins, polypeptides and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide or protein fragment unless explicitly specified as being from a non-human species.

[0037] The term “KLK2” or “hK2”, as used herein, refers to kallikrein-related peptidase 2.WSGR Docket No. 65457-712.602KLK2 also known as kallikrein-2, grandular kallikrein 2, or HK2. KLK2 is produced as a preproprotein and cleaved during proteolysis to generate active protease. All KLK2 isoforms and variants are encompassed in “KLK2”. The amino acid sequences of the various isoforms are retrievable from GenBank accession numbers NP_005542.1, NP_001002231.1 andNP 001243009. The amino acid sequence of a full length KLK2 is shown in SEQ ID NO: 13. The sequence includes the signal peptide (residues 1-18) and the pro-peptide region (residues 19-24).MWDLVLS IALSVGCTGAVPLIQSRIVGGWECEKHSQPWQVAVYSHGWAHCGGVLVHPQWVLTAA HCLKKNSQVWLGRHNLFEPEDTGQRVPVSHSFPHPLYNMSLLKHQSLRPDEDSSHDLMLLRLSE PAKITDWKVLGLPTQEPALGTTCYASGWGSIEPEEFLRPRSLQCVSLHLLSNDMCARAYSEKV TEFMLCAGLWTGGKDTCGGDSGGPLVCNGVLQGITSWGPEPCALPEKPAVYTKWHYRKWIKDT IAANP (SEQ ID NO: 13)

[0038] The term “specific binding” or “specifically bind” in reference to the interaction of an antibody, a binding protein, or a peptide with a second chemical species, means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the second chemical species. For example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. In general, if an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0039] As used herein, the term “antibody” refers to a binding protein comprising at least a light chain or heavy chain immunoglobulin variable region that specifically recognizes and binds to a particular antigen (e.g., CD3 or KLK2). The term encompasses various antibody structures, including, but not limited to, monoclonal antibodies, single-chain antibodies or multichain antibodies, single valent or multivalent antibodies, monospecific or multispecific antibodies, chimeric or humanized antibodies, complete antibodies, and antibody fragments, as long as they exhibit the desired antigen-binding activity. Therefore, the term “antibody” includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). The term “antibody” also includes any functional fragment, mutant, variant, or derivation of such immunoglobulin molecules, which retains the essential epitope binding features. SuchWSGR Docket No. 65457-712.602functional fragment, mutant, variant, or derivative antibody formats are known in the art, and non-limiting embodiments (e.g., FIT-Ig binding proteins) are discussed below.

[0040] As used herein, the term “immunoglobulin (Ig)” refers to heterodimeric proteins composed of two heavy (H) and two light (L) chains. The heavy chains can be classified as p, 8, y, a or a, and the isotypes of an immunoglobulin are defined as IgM, IgD, IgG (e.g., IgGl, IgG2, IgG3 or IgG4 subtype), IgA and IgE, respectively. The light chains can be classified as K and 1 light chains. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three or four domains: CHI, CH2, CH3, and optionally CH4. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. VK is the variable region part of the K-type light chain, and VI is the variable region part of the 1-type light chain. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is comprised of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. First, second and third CDRs of a VH domain are commonly enumerated as CDR-H1, CDR-H2, and CDR-H3; likewise, first, second and third CDRs of a VL domain are commonly enumerated as CDR-L1, CDR-L2, and CDR-L3. Depending on the sequences of heavy chain constant regions, immunoglobulins can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), subtype (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass. The term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, which may be generated by papain digestion of an intact antibody. The Fc region may be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally comprises two constant domains, i.e., a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain, for example, as in the case of the Fc regions of IgM and IgE antibodies. The Fc region of IgG, IgA, and IgD antibodies comprises a hinge region, a CH2 domain, and a CH3 domain. In contrast, the Fc region of IgM and IgE antibodies lacks a hinge region but comprises a CH2 domain, a CH3 domain and a CH4 domain. In one embodiment, a human IgG immunoglobulin Fc region is from Asp221 or from Cys226 or from Asp231 to the C-terminus of heavy chain. However, the lysine (Lys447) or glycine-lysine (Gly446Lys447) at the C-terminal end of the FcWSGR Docket No. 65457-712.602region may be present or absent. Variant Fc regions having substitutions of amino acid residues in the Fc portion to alter antibody effector function are known in the art (see, e.g., Winter et al., US Patent Nos. 5,648,260 and 5,624,821). The Fc portion of an antibody may mediate one or more effector functions, for example, cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC), and / or half-life / clearance rate of antibody and antigen-antibody complexes. In some cases, these effector functions are desirable for therapeutic antibody but in other cases might be unnecessary or even deleterious, depending on the therapeutic objectives. Certain human IgG isotypes, particularly IgGl and IgG3, mediate ADCC and CDC via binding to FcyRs and complement Clq, respectively. In still another embodiment at least one amino acid residue is replaced in the constant region of the antibody, for example the Fc region of the antibody, such that effector functions of the antibody are altered. The dimerization of two immunoglobulin Fc regions or two antibody chains may be mediated by the dimerization of CH3 domains, and further stabilized by the disulfide bonds within the hinge region that connects CHI constant domains to the Fc constant domains (e.g., CH2 and CH3), and / or artificially introduced into the Fc domains. The anti-inflammatory activity of IgG is dependent on sialylation of the N-linked glycan of the IgG Fc fragment. The precise glycan requirements for anti-inflammatory activity have been determined, such that an appropriate IgGl Fc fragment can be created, thereby generating a fully recombinant, sialylated IgGl Fc with greatly enhanced potency (see, Anthony et al., Science, 320:373-376 (2008)).

[0041] The terms “antigen-binding portion (ABP)”, “antigen-binding domain (ABD)” and “antigen-binding fragment” or “functional fragment” of an antibody are used interchangeably and refer to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen, i.e., the same antigen as the full-length antibody from which the portion or fragment is derived. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also be bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens. Examples of binding fragments encompassed within the term “antigenbinding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a singleWSGR Docket No. 65457-712.602arm of an antibody, (v) a dAb fragment (Ward et al., Nature, 341 : 544-546 (1989); PCT Publication No. WO 90 / 05144), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al., Science, 242: 423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988)). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody and equivalent terms given above. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993). Such antibody binding portions are known in the art (Kontermann and Diibel eds., Antibody Engineering (Springer- Verlag, New York, 2001), p.790 (ISBN 3-540-41354-5)). In addition, single chain antibodies also include “linear antibodies” comprising a pair of tandem Fv segments (VH-CHl-VH-CHl) which, together with complementary light chain polypeptides, form a pair of antigen binding portions (Zapata et al., Protein Eng., 8(10): 1057-1062 (1995); and US Patent No. 5,641,870)).

[0042] An immunoglobulin constant domain refers to a heavy (CH) or light (CL) chain constant domain. Murine and human IgG heavy chain and light chain constant domain amino acid sequences are known in the art.

[0043] The term “human sequence” or “from human”, in relation to the light chain constant domain CL, heavy chain constant domain CH, and Fc region of the antibody or the binding protein according to the present disclosure, means the sequence is of, or from, human immunoglobulin sequence. The human sequence of the present disclosure may be native human sequence, or a variant thereof including one or more (for example, up to 20, 15, 10) amino acid residue changes.

[0044] The term “linker” as used herein refers to an artificial molecule, including a peptide linker (e.g., having less than 50 amino acid residues) and a chemical non-peptide connector.WSGR Docket No. 65457-712.602Typically, linkers are used to conjugate or fuse one moiety to another, enabling each moiety to execute its individual function. In some embodiments of a FIT-Ig binding protein of the present disclosure, typically, there is no linker inserted between the immunological domains of their tandem Fab elements. However, as appreciated by persons skilled in the art, an antibody or a binding protein of the present disclosure, including a FIT-Ig binding protein, may be conjugated or fused via a linker to an additional proteinaceous and non-proteinaceous moiety.

[0045] The term “monoclonal antibody” or “mAb” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method.

[0046] An “antibody that competes with a reference antibody for binding to its antigen” refers to an antibody that blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the reference antibody to its antigen in a competition assay. Conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the antibody to its antigen in a competition assay. Numerous types of competitive binding assays can be used to determine whether an antibody competes with another antibody, such as direct or indirect solid-phase radioimmunoassay (RIA), direct or indirect solid-phase enzyme immunoassay (EIA) and sandwich competition assay.

[0047] An antibody that shows the same or similar binding affinity and / or specificity as a reference antibody refers to an antibody that is capable of having at least 50%, 60%, 70%, 80%, 90% or 95% or more of the binding affinity and / or specificity of the reference antibody. This can be determined by any method known in the art for determining binding affinity and / or specificity.

[0048] The term “KD”, as used herein, is intended to refer to the “equilibrium dissociation constant”, and refers to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (koff) by the association rate constant (kon). The association rate constant (kon), the dissociation rate constant (koff), and the equilibrium dissociation constant (KD) are used to represent the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art. Using fluorescence-based techniques offers high sensitivity and the ability to examine samplesWSGR Docket No. 65457-712.602in physiological buffers at equilibrium. Other experimental approaches and instruments such as a BIAcore® (biomolecular interaction analysis) assay can be used (e.g., instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). Biolayer interferometry (BLI) using, e.g., the Octet® RED96 system (Pall ForteBio LLC), is another affinity assay technique. Additionally, a KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Idaho) can also be used. In some embodiments, KD values of antibodies or binding proteins of the present disclosure are measured by a grating-coupled interferometry (GCI) based assay.

[0049] As used herein, the term “variable region” or “variable domain” refers to the domain of the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. Typically, a heavy chain variable domain (VH) is paired with a light chain variable domain (VL) to confer antigen binding specificity; however, in some cases, a single VH domain (e.g., a single VHH domain from a heavy chain antibody) is also sufficient to confer antigen binding specificity. A heavy chain variable region and a light chain variable region both comprise four conserved framework regions (FRs) and three complementary determining regions (CDRs), which are arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CD3-FR4. In some aspects, the variable domain(s) of an antibody may be modified. For example, substitution, deletion and / or addition of one or more amino acid residues, especially conservative residue substitution, may be introduced into one or more CDR regions and / or one or more framework regions to obtain an antibody variant. Alternatively, the variable domain(s) of an antibody may be modified by CDR grafting. As known in the art, CDRs are primarily responsible for antibody-antigen interactions. Antibody variants can be constructed by grafting CDR sequences from a known antibody onto the framework regions of a different antibody having a desired property. In addition, one to several mutations can be further performed, such as back mutations, to refine the desired properties of the antibody variant. The properties of antibody variants can be assessed in vitro or in vivo assays. Preferably, antibody variants substantially retain at least one desired biological property (e.g., antigen binding ability) of their parent antibodies.

[0050] The term “CDR” refers to the complementarity determining regions within antibody variable domain sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3.WSGR Docket No. 65457-712.602

[0051] The growth and analysis of extensive public databases of amino acid sequences of variable heavy and light regions over the past twenty years have led to the understanding of the typical boundaries between framework regions (FRs) and CDR sequences within variable region sequences and have enabled persons skilled in the art to accurately determine the CDRs. The CDR definition schemes available in the art include e.g., Chothia scheme based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature, 342:877-883; Al-Lazikani et al., Standard conformations for the canonical structures of immunoglobulins, Journal of Molecular Biology, 273:927-948 (1997)), Kabat scheme based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4thEd., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM scheme (University of Bath), Contact scheme (University College London), International ImMunoGeneTics database (IMGT) scheme (imgt.cines.fr / on the World Wide Web), and North scheme based on the affinity propagation clustering using a large number of crystal structures. The boundaries of each CDRs according to several CDR definition schemes are presented below:Kabat AbM Chothia Contact IMGT CDRscheme scheme scheme scheme scheme CDR-L1 (Kabat andL24-L34 L24-L34 L26-L32 L30-L36 L27-L32 Chothia numbering system)CDR-L2 (Kabat andL50-L56 L50-L56 L50-L52 L46-L55 L50-L52 Chothia numbering system)CDR-L3 (Kabat andL89-L97 L89-L97 L91-L96 L89-L96 L89-L96 Chothia numbering system)CDR-H1 (Kabat numberingH31-H35B H26-H35B H26-H32 H30-H35B H26-H35B system)CDR-H1 (ChothiaH31-H35 H26-H35 H26-H32 H30-H35 H26-H35 numbering system)CDR-H2 (Kabat andH50-H65 H50-H58 H53-H55 H47-H58 H51-H57 Chothia numbering system)CDR-H3 (Kabat andH95-H102 H95-H102 H96-H101 H93-H101 H93-H102 Chothia numbering system)

[0052] Unless otherwise indicated, when referring to CDRs contained in a variable domain, the CDRs can be determined using any one or a combination of the antibody CDR definition schemes mentioned above.

[0053] It should be noted that boundaries of CDRs in variable domains of an antibodyWSGR Docket No. 65457-712.602determined by different definition schemes may differ. Therefore, the CDR sequences of variable regions of the same antibody defined by different schemes may differ. Accordingly, when it comes to defining an antibody with specific CDR sequences according to a specific definition scheme (e.g., Kabat Scheme) in the present disclosure, the scope of antibody encompasses any antibodies whose variable region sequences comprise the specific CDR sequences, but may have different CDR boundaries assigned by application of a different scheme.

[0054] As known in the art, CDRs vary from antibody to antibody. However, a limited number of the amino acid positions in a set of CDRs are directly involved in antigen binding. Using at least two of Kabat, Chothia, AbM and IMGT schemes, the minimum overlapping regions for each CDRs can be determined, thereby providing a "minimum binding unit" for antigen binding. Such a minimum binding unit can be a subset of CDR residues of an antibody, while the remaining CDR residues, as those skilled in the art will appreciate, can be determined by analyzing the structure and folding of the antibody. Therefore, in some instances, CDR amino acid sequences as described herein may comprise a modification(s). The modification may be amino acid substitution, deletion, and / or addition. Antibodies and antigen-binding fragments obtained in such manner can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced biological properties, etc.

[0055] As used herein, unless otherwise indicated, the amino acid positions of CDRs and variable domains of an antibody are numbered according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

[0056] As used herein, the amino acid positions of all constant regions and domains of the heavy and light chain of an antibody are numbered according to the Kabat numbering system described in Kabat, etal., Sequences of Proteins of Immunological Interest, 5thed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) and is referred to as “numbering according to Kabat” or “Kabat numbering” herein. Specifically, the Kabat numbering system (see pages 647-660) of Kabat, et al., Sequences of Proteins of Immunological Interest, 5thed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) is used for the light chain constant domain CL of kappa and lambda isotype, and the Kabat EU index numbering system (see pages 661-723) is used for the constant heavy chain domainsWSGR Docket No. 65457-712.602(CHI, Hinge, CH2 and CH3, which is herein further clarified by referring to “numbering according to Kabat EU index” or “EU numbering” in this case).

[0057] General information regarding the sequences of human immunoglobulins light and heavy chains is given in: Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5thed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0058] The term "multivalent binding protein" denotes a binding protein comprising two or more antigen binding domains. A multivalent binding protein is, in certain cases, engineered to have three or more antigen binding domains, and is generally not a naturally occurring antibody. The term "multispecific (e.g., bispecific) binding protein" (which can be used interchangeably with the term “multispecific (e.g., bispecific) antibody”, unless stated otherwise) refers to a binding protein capable of binding at least two different target antigens or different epitopes. Hence, a multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope. FIT-Ig binding proteins of the present disclosure comprise four antigen binding sites and are typically tetravalent binding proteins. A FIT-Ig according to this disclosure binds both KLK2 and CD3 and is bispecific.

[0059] As used herein, the term “FIT-Ig binding protein” is used interchangeably with “FIT-Ig antibody”, “FIT-Ig protein” and “FIT-Ig”, and refers to a multivalent antibody comprising two long (heavy) V-C-V-C-Fc chain polypeptides and four short (light) V-C chain polypeptides, which form a hexamer exhibiting four Fab antigen binding sites (VH-CH1 paired with VL-CL, sometimes notated VH-CH1 : : VL-CL). Each half of a FIT-Ig comprises a long chain polypeptide and two short chain polypeptides, and complementary immunoglobulin pairing of the VH-CH1 and VL-CL elements of the three chains results in two Fab -structured antigen binding sites, arranged in tandem. In the present disclosure, it is preferred that the immunoglobulin domains comprising the Fab elements are directly fused in the long chain polypeptide, without the use of interdomain linkers. That is, the N-terminal V-C element of the long (heavy) polypeptide chains is directly fused at its C-terminus to the N-terminus of another V-C element, which in turn is linked to a C-terminal Fc region. In bispecific FIT-Ig binding proteins, the tandem Fab elements may be reactive with different antigens. Each Fab antigen binding site comprises a heavy chain variable domain and a light chain variable domain with a total of six CDRs per antigen binding site.WSGR Docket No. 65457-712.602

[0060] A description of the design, expression, and characterization of FIT-Ig molecules is provided in PCT Publication WO 2015 / 103072. An example of such FIT-Ig molecules comprises a heavy (long) chain and two different light (short) chains. The long chain comprises the structural formula VLA-CL-VHB-CH1-FC where CL is directly fused to VHB (namely “Format LH”) or VHB-CH1-VLA-CL-FC where CHI is fused directly to VLA (namely “Format HL”), and the two short polypeptide chains of the FIT-Ig correspondingly have the formulas VHA-CHI and VLB-CL , respectively; wherein VLA is a variable light domain from a parental antibody that binds antigen A, VLB is a variable light domain from a parental antibody that binds antigen B, VHA is a variable heavy domain from a parental antibody that binds antigen A, VHB is a variable heavy domain from a parental antibody that binds antigen B, CL is a light chain constant domain, CHI is a heavy chain constant domain 1, and Fc is an immunoglobulin Fc region (e.g., the C-terminal hinge-CH2-CH3 portion of a heavy chain of an IgGl antibody). In bispecific FIT-Ig embodiments, antigen A and antigen B are different antigens, or different epitopes of the same antigen. In the present disclosure, one of antigen A and antigen B is KLK2 and the other is CD3, for example, antigen A is KLK2, and antigen B is CD3. FIG. 1 provides schematic illustration of the domain structure of a FIT-Ig bispecific antibody.

[0061] “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm needed to achieve maximal alignment over the full length of the sequences being compared.

[0062] When percentages of sequence identity are referred to in this application, these percentages are calculated relative to the full length of the longer sequence, unless otherwise specifically indicated. The calculation relative to the full length of the longer sequence applies to both the nucleic acid sequence and the polypeptide sequence.

[0063] The term “pharmaceutical composition” refers to such a composition that exists in aWSGR Docket No. 65457-712.602form allowing effective biological activity of the active ingredient contained therein, and does not contain additional ingredients having unacceptable toxicity to an individual to which the composition is administered.

[0064] The term “a pharmaceutically acceptable carrier” refers to diluents, adjuvants (e.g., Freund’s adjuvants (complete and incomplete)), excipients, vehicles, stabilizers, or the like, which are administered with the active substance.

[0065] The term “cancer” refers to or describes a physiological disease in mammals that is typically characterized by unregulated cell growth. In certain embodiments, cancers suitable for treatment with the antibody or the binding protein of the present disclosure include metastatic forms of such cancers. The cancer can be solid cancer or hematopoietic cancer.

[0066] The term “tumor” refers to all neoplastic cell growth and proliferation, whether being malignant or benign, and all pre-cancerous and cancerous cells and tissues. Tumor includes both solid and hematopoietic tumors.

[0067] The terms “cancer” and “tumor” are not mutually exclusive when referred to herein.

[0068] The term “individual” or “subject” as used herein includes mammals. The mammals include, but are not limited to, domestic animals (e.g., cattle, goats, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0069] As used herein, the term “therapeutically effective amount” refers to the amount of a therapy that is sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof; prevent the advancement of a disorder; cause regression of a disorder; prevent the recurrence, development, or progression of one or more symptoms associated with a disorder; or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., prophylactic or therapeutic agent).

[0070] As used herein, “treatment” (or “treat” or “treating”) refers to slowing, interrupting, arresting, alleviating, stopping, lowering, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

[0071] As used herein, “prevention” (or “prevent” or “preventing”) includes the inhibition of the development or progression of symptoms of a disease or disorder, or a specific disease or disorder. In some embodiments, individuals with family history of cancer are candidates for preventive regimens. Generally, in the context of cancer, the term “prevention” refers to theWSGR Docket No. 65457-712.602administration of a drug prior to the onset of signs or symptoms of a cancer, particularly in individuals at risk of cancer.

[0072] As used herein, the term “administer”, “administering”, “administration” or “administered” refers to the physical introduction of a composition comprising a therapeutic agent to an individual, using any of the various methods and delivery systems known to those skilled in the art. Routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.Anti-KLK2 and Anti-CD3 monoclonal antibodies

[0073] Anti-KLK2 and anti-CD3 antibodies of the present disclosure may be produced by any of a number of techniques known in the art, for example, expression from host cells, wherein expression vector(s) encoding the heavy and light chains is (are) transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection, and the like. Although it is possible to express the antibodies of the present disclosure in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, for instance, in mammalian host cells, is particularly contemplated, as widely used for assembly and secretion of properly folded and immunologically active antibodies.

[0074] In some embodiments, mammalian host cells for expressing the recombinant antibodies of the present disclosure is Chinese Hamster Ovary (CHO cells) (including dhfr CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Set. USA, 77: 4216-4220 (1980), used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp, J. Mol. BioL, 159: 601-621 (1982)), NS0 myeloma cells, COS cells, and SP2 cells. When recombinant expression vectorsWSGR Docket No. 65457-712.602encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells, or further secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0075] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedure are within the scope of the present disclosure. For example, it may be desirable to transfect a host cell with DNA encoding functional fragments of either the light chain and / or the heavy chain of an antibody of this disclosure. Recombinant DNA technology may also be used to remove some, or all, of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigens of interest. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the present disclosure.

[0076] In an exemplary system for recombinant expression of an antibody, or antigen-binding portion thereof, of the present disclosure, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to CMV enhancer / AdMLP promoter regulatory elements to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection / amplification. The selected transfected host cells are cultured to allow expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transfectants, culture the host cells and recover the antibody from the culture medium. The present disclosure further provides a method of making a recombinant anti-KLK2 or anti-CD3 antibody by culturing a transfected host cell of the present disclosure in a suitable culture medium until a recombinant antibody of the present disclosure is produced. The method can further comprise isolating the recombinant antibody from the culture medium.Anti-KLK2 monoclonal antibodies

[0077] In some embodiments, the present disclosure provides proteins that bind to humanWSGR Docket No. 65457-712.602KLK2. In some embodiments, the present disclosure discloses an isolated anti-KLK2 antibody or antigen-binding fragment thereof that specifically binds to human KLK2. In a further embodiment, the anti-KLK2 antibody or antigen-binding fragment thereof comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, wherein:

[0078] CDR-H1 comprises the sequence of DTYMH (SEQ ID NO: 62);

[0079] CDR-H2 comprises the sequence of RIDPANGNTKYDPKFQG (SEQ ID NO: 63);

[0080] CDR-H3 comprises the sequence of GGYRYDDYFDY (SEQ ID NO: 65);

[0081] CDR-L1 comprises the sequence of RASETVDSYGSSFMH (SEQ ID NO: 66);

[0082] CDR-L2 comprises the sequence of RASTLES (SEQ ID NO: 68); and

[0083] CDR-L3 comprises the sequence of QQNNEDPFT (SEQ ID NO: 69),

[0084] CDR-H1 comprises the sequence of DYYMH (SEQ ID NO: 70);

[0085] CDR-H2 comprises the sequence of WIDPENGNTIYDPRFQG (SEQ ID NO: 71);

[0086] CDR-H3 comprises the sequence of SFYYGYLRFDY (SEQ ID NO: 73);

[0087] CDR-L1 comprises the sequence of SASSSVSYMH (SEQ ID NO: 74);

[0088] CDR-L2 comprises the sequence of EISKLAS (SEQ ID NO: 75); and

[0089] CDR-L3 comprises the sequence of QQWNYPLFT (SEQ ID NO: 76), or

[0090] CDR-H1 comprises the sequence of SYVMH (SEQ ID NO: 77);

[0091] CDR-H2 comprises the sequence of YISPYNDGTKYNEKFKG (SEQ ID NO: 78);

[0092] CDR-H3 comprises the sequence of GGGLEYSFDY (SEQ ID NO: 80);

[0093] CDR-L1 comprises the sequence of RASQSIGTSIH (SEQ ID NO: 81);

[0094] CDR-L2 comprises the sequence of YASESIS (SEQ ID NO: 82); and

[0095] CDR-L3 comprises the sequence of QQTNSWPWT (SEQ ID NO: 83),

[0096] wherein the CDRs are defined according to Kabat numbering.

[0097] In some embodiments, the anti-KLK2 antibody or antigen-binding fragment thereof comprises at least one, two, three, four, but not more than five residue modifications in 6 CDR sequences of above i), ii), or iii). The amino acid modifications may be amino acid substitutions, deletions, and / or additions, for instance, conservative substitution.

[0098] In one embodiment, an anti-KLK2 antibody or antigen-binding fragment thereof according to the present disclosure comprises CDR-H1, CDR-H2, and CDR-H3, of a heavy chain variable domain VH of SEQ ID NO: 21, as well as CDR-L1, CDR-L2, and CDR-L3 of a light chain variable domain VL of SEQ ID NO: 22. The CDRs can be determined by a personWSGR Docket No. 65457-712.602skilled in the art using the most widely CDR definition schemes, for example, Kabat, Chothia or IMGT definitions.

[0099] In one embodiment, an anti-KLK2 antibody or antigen-binding fragment thereof according to the present disclosure comprises CDR-H1, CDR-H2, and CDR-H3, of a heavy chain variable domain VH of SEQ ID NO: 23, as well as CDR-L1, CDR-L2, and CDR-L3 of a light chain variable domain VL of SEQ ID NO: 24. The CDRs can be determined by a person skilled in the art using the most widely CDR definition schemes, for example, Kabat, Chothia or IMGT definitions.

[0100] In one embodiment, an anti-KLK2 antibody or antigen-binding fragment thereof according to the present disclosure comprises CDR-H1, CDR-H2, and CDR-H3, of a heavy chain variable domain VH of SEQ ID NO: 25, as well as CDR-L1, CDR-L2, and CDR-L3 of a light chain variable domain VL of SEQ ID NO: 26.

[0101] In one embodiment, an anti-KLK2 antibody or antigen-binding fragment thereof according to the present disclosure comprises a CDR-H1 comprising the sequence of DYYMH (SEQ ID NO: 70); a CDR-H2 comprising the sequence of WIDPENANTIYDPRFQG (SEQ ID NO: 72); a CDR-H3 comprising the sequence of SFYYGYLRFDY (SEQ ID NO: 73); a CDR-L1 comprising the sequence of SASSSVSYMH (SEQ ID NO: 74); a CDR-L2 comprising the sequence of EISKLAS (SEQ ID NO: 75); and a CDR-L3 comprising the sequence of QQWNYPLFT (SEQ ID NO: 76).

[0102] In one embodiment, an anti-KLK2 antibody or antigen-binding fragment thereof according to the present disclosure comprises a CDR-H1 comprising the sequence of DTYMH (SEQ ID NO: 62); a CDR-H2 comprising the sequence of RIDPANANTKYDPKFQG (SEQ ID NO: 64); a CDR-H3 comprising the sequence of GGYRYDDYFD Y (SEQ ID NO: 65); a CDR-L1 comprising the sequence of RASETVDAYGSSFMH (SEQ ID NO: 67); a CDR-L2 comprising the sequence of RASTLES (SEQ ID NO: 68); and a CDR-L3 comprising the sequence of QQNNEDPFT (SEQ ID NO: 69).

[0103] In one embodiment, an anti-KLK2 antibody or antigen-binding fragment thereof according to the present disclosure comprises a CDR-H1 comprising the sequence of SYVMH (SEQ ID NO: 77); a CDR-H2 comprising the sequence of YISPYNDATKYNEKFKG (SEQ ID NO: 79); a CDR-H3 comprising the sequence of GGGLEYSFDY (SEQ ID NO: 80); a CDR-L1 comprising the sequence of RASQSIGTSIH (SEQ ID NO: 81); a CDR-L2 comprising theWSGR Docket No. 65457-712.602sequence of YASESIS (SEQ ID NO: 82); and a CDR-L3 comprising the sequence of QQTNAWPWT (SEQ ID NO: 84). The CDRs can be determined by a person skilled in the art using the most widely CDR definition schemes, for example, Kabat, Chothia or IMGT definitions.

[0104] In one embodiment, an anti-KLK2 antibody or antigen-binding fragment thereof according to the present disclosure comprises a heavy chain variable domain VH and a light chain variable domain VL, wherein:

[0105] the VH domain comprises the sequence of SEQ ID NO: 21, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and / or

[0106] the VL domain comprises the sequence of SEQ ID NO: 22, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith.

[0107] In one embodiment, an anti-KLK2 antibody or antigen-binding fragment thereof according to the present disclosure comprises a heavy chain variable domain VH and a light chain variable domain VL, wherein:

[0108] the VH domain comprises the sequence of SEQ ID NO: 23, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and / or

[0109] the VL domain comprises the sequence of SEQ ID NO: 24, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith.

[0110] In one embodiment, an anti-KLK2 antibody or antigen-binding fragment thereof according to the present disclosure comprises a heavy chain variable domain VH and a light chain variable domain VL, wherein:[OHl] the VH domain comprises the sequence of SEQ ID NO: 25, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and / or

[0112] the VL domain comprises the sequence of SEQ ID NO: 26, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith.WSGR Docket No. 65457-712.602

[0113] In some embodiments, an anti-KLK2 antibody comprises a VH sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, while retains the ability to bind to the KLK2 with the same or improved binding properties, such as Kd. In a certain embodiment, substitutions, insertions, or deletions occur in CDRs; in another embodiment, substitutions, insertions, or deletions occur in regions outside the CDRs (e.g, in the FRs). In some embodiments, an anti-KLK2 antibody comprises amino acid modifications of DG(Asp-Gly) / DS(Asp-Ser) or NG(Asn-Gly) / NS(Asn-Ser) substituted by DA(Asp-Ala) or NA(Asn-Ala) in CDRs, such as “DA” in CDR-L1 of SEQ ID NO: 67 and in CDR-H2 of SEQ ID NO: 79, and “NA” in CDR-H2 of SEQ ID NOs: 64 and 72 and in CDR-L3 of SEQ ID NO: 84; to mitigate post-translational modification (PTM) that may result in heterogeneity during recombinant antibody manufacturing.

[0114] In a further embodiment, the anti-KLK2 antibody or antigen-binding fragment thereof according to the present disclosure comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, wherein:

[0115] CDR-H1 comprises the sequence of DTYMH (SEQ ID NO: 62);

[0116] CDR-H2 comprises the sequence of RIDPANGNTKYDPKFQG (SEQ ID NO: 63) or RIDPANANTKYDPKFQG (SEQ ID NO: 64);

[0117] CDR-H3 comprises the sequence of GGYRYDDYFDY (SEQ ID NO: 65);

[0118] CDR-L1 comprises the sequence of RASETVDSYGSSFMH (SEQ ID NO: 66) or RASETVDAYGSSFMH (SEQ ID NO: 67);

[0119] CDR-L2 comprises the sequence of RASTLES (SEQ ID NO: 68);

[0120] CDR-L3 comprises the sequence of QQNNEDPFT (SEQ ID NO: 69);

[0121] CDR-H1 comprises the sequence of DYYMH (SEQ ID NO: 70);

[0122] CDR-H2 comprises the sequence of WIDPENGNTIYDPRFQG (SEQ ID NO: 71) or WIDPENANTIYDPRFQG (SEQ ID NO: 72);

[0123] CDR-H3 comprises the sequence of SFYYGYLRFDY (SEQ ID NO: 73);

[0124] CDR-L1 comprises the sequence of SASSSVSYMH (SEQ ID NO: 74);

[0125] CDR-L2 comprises the sequence of EISKLAS (SEQ ID NO: 75); and

[0126] CDR-L3 comprises the sequence of QQWNYPLFT (SEQ ID NO: 76);

[0127] or

[0128] CDR-H1 comprises the sequence of SYVMH (SEQ ID NO: 77);WSGR Docket No. 65457-712.602

[0129] CDR-H2 comprises the sequence of YISPYNDGTKYNEKFKG (SEQ ID NO: 78) or YISPYNDATKYNEKFKG (SEQ ID NO: 79);

[0130] CDR-H3 comprises the sequence of GGGLEYSFDY (SEQ ID NO: 80);

[0131] CDR-L1 comprises the sequence of RASQSIGTSIH (SEQ ID NO: 81);

[0132] CDR-L2 comprises the sequence of YASESIS (SEQ ID NO: 82); and

[0133] CDR-L3 comprises the sequence of QQTNSWPWT (SEQ ID NO: 83) or QQTNAWPWT (SEQ ID NO: 84),

[0134] optionally wherein the CDRs are defined according to Kabat numbering.

[0135] In some embodiments, an anti-KLK2 antibody or antigen-binding fragment thereof according to the present disclosure comprises a variable heavy chain domain VH and a variable light chain domain VL, wherein:i) the VH domain comprises the sequence of SEQ ID NO: 21, 27, 28, 29, 30, 31, 32, or 33, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and / or the VL domain comprises the sequence of SEQ ID NO: 22, 34, 35, 36, 37, 38, or 39, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith;ii) the VH domain comprises the sequence of SEQ ID NO: 23, 40, 41, 42, 43, 44, 45, or 46, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and / or the VL domain comprises the sequence of SEQ ID NO: 24, 47, or 48, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith; oriii) the VH domain comprises the sequence of SEQ ID NO: 25, 49, 50, 51, 52, 53, 54, or 55, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and / or the VL domain comprises the sequence of SEQ ID NO: 26, 56, 57, 58, 59, 60, or 61, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith.

[0136] In some embodiments, an anti-KLK2 antibody or antigen-binding fragment thereof according to the present disclosure comprises a variable heavy chain domain VH and a variable light chain domain VL, wherein:

[0137] the VH domain comprises the sequence of SEQ ID NO: 21, 27, 28, 29, 30, 31, 32, or

[0138] 33 and / or the VL domain comprises the sequence of SEQ ID NO: 22, 34, 35, 36, 37, 38,WSGR Docket No. 65457-712.602or 39;

[0139] the VH domain comprises the sequence of SEQ ID NO: 23, 40, 41, 42, 43, 44, 45, or 46 and / or the VL domain comprises the sequence of SEQ ID NO: 24, 47, or 48; or

[0140] the VH domain comprises the sequence of SEQ ID NO: 25, 49, 50, 51, 52, 53, 54, or 55 and / or the VL domain comprises the sequence of SEQ ID NO: 26, 56, 57, 58, 59, 60, or 61.

[0141] In one embodiment, the isolated anti-KLK2 antibody or antigen-binding fragment according to the present disclosure is a chimeric antibody or a humanized antibody. In some embodiments, the anti-KLK2 antibody or antigen-binding fragment is a humanized antibody, and optionally comprises one or more back mutations at positions in framework regions to improve the binding property, such as selected from the group consisting ofiv) the VH domain of the antibody comprises amino acid residues IE, 72A, and 0 to 10 residues selected from 27F, 28N, 291, 30D, 44G, 481, 67K, 68 A, 77N, 79D, according to Kabat numbering; and the VL domain comprises 0 to 3 amino acid residues selected from 4L, 621, and 72R, according to Kabat numbering;v) the VH domain of the antibody comprises amino acid residue 98R, and 0 to 10 residues selected from 24 A, 27F, 28N, 291, 3 OK, 481, 67K, 68 A, 69N, 77N, according to Kabat numbering; and the VL domain comprises 0 to 3 amino acid residues selected from 42S, 45P, and 46W, according to Kabat numbering; orvi) the VH domain of the antibody comprises amino acid residues IE, 72S, and 0 to 7 residues selected from 38K, 44G, 481, 67K, 68 A, 70L,74K, according to Kabat numbering; and the VL domain comprises 0 to 3 amino acid residues selected from ID, 22S, and 581, according to Kabat numbering.

[0142] In some embodiments, the isolated anti-KLK2 antibody or antigen-binding fragment according to the present disclosure comprises a combination of VH and VL sequences selected from the group consisting of:Combination VH sequence VL sequence1 SEQ ID NO 27 SEQ ID NO 34 2 SEQ ID NO 28 SEQ ID NO 34 3 SEQ ID NO 29 SEQ ID NO 34 4 SEQ ID NO 30 SEQ ID NO 34 i) 5 SEQ ID NO 31 SEQ ID NO 346 SEQ ID NO 27 SEQ ID NO 35 7 SEQ ID NO 28 SEQ ID NO 358 SEQ ID NO 29 SEQ ID NO 35WSGR Docket No. 65457-712.6029 SEQ ID NO: 30 SEQ ID NO: 35 10 SEQ ID NO: 31 SEQ ID NO: 35 11 SEQ ID NO: 27 SEQ ID NO: 36 12 SEQ ID NO: 28 SEQ ID NO: 36 13 SEQ ID NO: 29 SEQ ID NO: 36 14 SEQ ID NO: 30 SEQ ID NO: 36 15 SEQ ID NO: 31 SEQ ID NO: 36 16 SEQ ID NO: 27 SEQ ID NO: 37 17 SEQ ID NO: 28 SEQ ID NO: 37 18 SEQ ID NO: 29 SEQ ID NO: 37 19 SEQ ID NO: 30 SEQ ID NO: 37 20 SEQ ID NO: 31 SEQ ID NO: 37 21 SEQ ID NO: 31 SEQ ID NO: 22 22 SEQ ID NO: 21 SEQ ID NO: 37 23 SEQ ID NO: 32 SEQ ID NO: 22 24 SEQ ID NO: 21 SEQ ID NO: 38 25 SEQ ID NO: 21 SEQ ID NO: 22 26 SEQ ID NO: 33 SEQ ID NO: 39 1 SEQ ID NO 40 SEQ ID NO 47 2 SEQ ID NO 41 SEQ ID NO 47 3 SEQ ID NO 42 SEQ ID NO 47 4 SEQ ID NO 43 SEQ ID NO 47 5 SEQ ID NO 44 SEQ ID NO 47 6 SEQ ID NO 40 SEQ ID NO 48 7 SEQ ID NO 41 SEQ ID NO 48 ii) 8 SEQ ID NO 42 SEQ ID NO 489 SEQ ID NO 43 SEQ ID NO 48 10 SEQ ID NO 44 SEQ ID NO 48 11 SEQ ID NO 44 SEQ ID NO 24 12 SEQ ID NO 23 SEQ ID NO 48 13 SEQ ID NO 45 SEQ ID NO 24 14 SEQ ID NO 23 SEQ ID NO 24 15 SEQ ID NO 46 SEQ ID NO 47 1 SEQ ID NO 49 SEQ ID NO 56 2 SEQ ID NO 50 SEQ ID NO 56 3 SEQ ID NO 51 SEQ ID NO 56 4 SEQ ID NO 52 SEQ ID NO 56 5 SEQ ID NO 53 SEQ ID NO 56 6 SEQ ID NO 49 SEQ ID NO 57 7 SEQ ID NO 50 SEQ ID NO 57 8 SEQ ID NO 51 SEQ ID NO 57 iii) 9 SEQ ID NO 52 SEQ ID NO 5710 SEQ ID NO 53 SEQ ID NO 57 11 SEQ ID NO 49 SEQ ID NO 58 12 SEQ ID NO 50 SEQ ID NO 58 13 SEQ ID NO 51 SEQ ID NO 58 14 SEQ ID NO 52 SEQ ID NO 58 15 SEQ ID NO 53 SEQ ID NO 58 16 SEQ ID NO 49 SEQ ID NO 5917 SEQ ID NO 50 SEQ ID NO 59WSGR Docket No. 65457-712.60218 SEQ ID NO 51 SEQ ID NO 59 19 SEQ ID NO 52 SEQ ID NO 59 20 SEQ ID NO 53 SEQ ID NO 59 21 SEQ ID NO 53 SEQ ID NO 26 22 SEQ ID NO 25 SEQ ID NO 59 23 SEQ ID NO 54 SEQ ID NO 26 24 SEQ ID NO 25 SEQ ID NO 60 25 SEQ ID NO 25 SEQ ID NO 2626 SEQ ID NO 55 SEQ ID NO 61

[0143] In some embodiments, the antibody comprises a VH domain comprising the sequence of SEQ ID NO: 33 and a VL domain comprising the sequence of SEQ ID NO: 39, or wherein the antibody comprises a VH domain comprising the sequence of SEQ ID NO: 46 and a VL domain comprising the sequence of SEQ ID NO: 47; or wherein the antibody comprises a VH domain comprising the sequence of SEQ ID NO: 55 and a VL domain comprising the sequence of SEQ ID NO: 61.

[0144] In some embodiments of an anti-KLK2 antibody or antigen-binding fragment according to the present disclosure, the antibody comprises a VH domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 46 and a VL domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 47. In some embodiments of an anti-KLK2 antibody or antigenbinding fragment according to the present disclosure, the antibody comprises a VH domain comprising the sequence of SEQ ID NO: 46 and a VL domain comprising the sequence of SEQ ID NO: 47.

[0145] In some embodiments of an anti-KLK2 antibody or antigen-binding fragment according to the present disclosure, the antibody comprises a VH domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 33 and a VL domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 39. In some embodiments of an anti-KLK2 antibody or antigenbinding fragment according to the present disclosure, the antibody comprises a VH domain comprising the sequence of SEQ ID NO: 33 and a VL domain comprising the sequence of SEQ ID NO: 39.

[0146] In some embodiments of an anti-KLK2 antibody or antigen-binding fragment according to the present disclosure, the antibody comprises a VH domain having at least 80%, 85%, 90%,WSGR Docket No. 65457-712.60291%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 55 and a VL domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 61. In some embodiments of an anti-KLK2 antibody or antigenbinding fragment according to the present disclosure, the antibody comprises a VH domain comprising the sequence of SEQ ID NO: 55 and a VL domain comprising the sequence of SEQ ID NO: 61.

[0147] In some embodiments of an anti-KLK2 antibody or antigen-binding fragment according to the present disclosure, the antibody or antigen-binding fragment comprises an Fc region, which may be a native or a variant Fc region. In particular embodiments, the Fc region is a human Fc region from IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. Depending on the utility of the antibody, it may be desirable to use a variant Fc region to change (for example, reduce or eliminate) at least one effector function, for example, ADCC and / or CDC. In some embodiments, the present disclosure provides an anti-KLK2 antibody or antigen-binding fragment comprising an Fc region with one or more mutation to change at least one effector function, for example, L234A and L235A, and / or P329A.

[0148] In some embodiments, antigen-binding fragments of an anti-KLK2 antibody according to the present disclosure may be for example, Fv, Fab, Fab', Fab’-SH, F(ab')2; diabodies; linear antibodies; or single-chain antibody molecules (e.g. scFv).

[0149] In some embodiments, anti-KLK2 antibodies described herein or antigen-binding fragments thereof bind to same or different epitopes on KLK2 antigen.

[0150] In one embodiment, an anti-KLK2 antibody described herein or an antigen-binding fragment thereof has a dissociation constant (KD) to KLK2 in the nanomolar (10’8to IO'10) range, for example, less than about lOnM, less than about 5nM, or less than about InM, preferably 0. InM to 0.95nM, and is not detected to bind to human KLK3, as measured by a grating-coupled interferometry (GCI) based assay.

[0151] Some cancers show elevated levels of KLK2 relative to noncancerous tissue of the same type, preferably from the same patient. Therefore, the anti-KLK2 antibody described herein or an antigen-binding fragment thereof can be applied to measure KLK2 at the protein level, (e.g., by immunoassay).Anti-CD3 monoclonal antibodies

[0152] In some embodiments, the present disclosure provides proteins that bind to CD3. InWSGR Docket No. 65457-712.602some embodiments, the present disclosure discloses an isolated anti-CD3 antibody or antigenbinding fragment thereof that specifically binds to CD3. In a further embodiment, the anti-CD3 antibody or antigen-binding fragment thereof comprises a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, wherein:

[0153] CDR-H1 comprises the sequence of NYYVH(SEQ ID NO: 3);

[0154] CDR-H2 comprises the sequence of WISPGSDNTKYNEKFKG (SEQ ID NO: 4);

[0155] CDR-H3 comprises the sequence of DDYGNYYFDY (SEQ ID NO: 5);

[0156] CDR-L1 comprises the sequence of KSSQSLLNARTRKNYLA (SEQ ID NO: 7);

[0157] CDR-L2 comprises the sequence of WASTRES (SEQ ID NO: 8); and

[0158] CDR-L3 comprises the sequence of KQSYILRT (SEQ ID NO: 9),

[0159] wherein the CDRs are defined according to Kabat numbering.

[0160] In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof comprises at least one, two, three, four, but not more than five residue modifications in the CDR sequences of SEQ ID NOs: 3-5 and 7-9. The amino acid modifications may be amino acid substitutions, deletions, and / or additions, for instance, conservative substitution.

[0161] In one embodiment, an anti-CD3 antibody or antigen-binding fragment thereof according to the present disclosure comprises CDR-H1, CDR-H2, and CDR-H3 of a heavy chain variable domain VH of SEQ ID NO: 2, as well as CDR-L1, CDR-L2, and CDR-L3 of a light chain variable domain VL of SEQ ID NO: 6. The CDRs can be determined by a person skilled in the art using the most widely CDR definition schemes, for example, Kabat, Chothia or IMGT definitions.

[0162] In one embodiment, an anti-CD3 antibody or antigen-binding fragment thereof according to the present disclosure comprises a heavy chain variable domain VH and a light chain variable domain VL, wherein:

[0163] the VH domain comprises the sequence of SEQ ID NO: 2, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and / or

[0164] the VL domain comprises the sequence of SEQ ID NO: 6, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith.

[0165] In some embodiments, an anti-CD3 antibody comprises a VH sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containsWSGR Docket No. 65457-712.602substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, while retains the ability to bind to the CD3 with the same or improved binding properties, such as Kd. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).

[0166] In one embodiment, the isolated anti-CD3 antibody or antigen-binding fragment according to the present disclosure is a chimeric antibody or a humanized antibody. In some embodiments, the anti-CD3 antibody or antigen-binding fragment is a humanized antibody.

[0167] In one embodiment, the isolated anti-CD3 antibody or antigen-binding fragment according to the present disclosure is a humanized antibody, comprising a set of six CDRs of above SEQ ID NOs: 3-5 and 7-9.

[0168] In some embodiments, the antibody comprises a VH domain comprising or consisting of the sequence of SEQ ID NO: 2, and a VL domain comprising or consisting of the sequence of SEQ ID NO: 6.

[0169] In some embodiments of an anti-CD3 antibody or antigen-binding fragment according to the present disclosure, the antibody or antigen-binding fragment comprises an Fc region, which may be a native or a variant Fc region. In particular embodiments, the Fc region is a human Fc region from IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. Depending on the utility of the antibody, it may be desirable to use a variant Fc region to change (for example, reduce or eliminate) at least one effector function, for example, ADCC and / or CDC. In some embodiments, the present disclosure provides an anti-CD3 antibody or antigen-binding fragment comprising an Fc region with one or more mutation to change at least one effector function, for example, L234A and L235A and / or P329A.

[0170] In some embodiments, antigen-binding fragments of an anti-CD3 antibody according to the present disclosure may be for example, Fv, Fab, Fab', Fab’-SH, F(ab')2; diabodies; linear antibodies; or single-chain antibody molecules (e.g. scFv).KLK2xCD3 bispecific binding proteins

[0171] In another aspect, the present disclosure provides KLK2*CD3 bispecific binding proteins, for example, Fabs-in-Tandem immunoglobulins (FIT-Ig), that are capable of binding to both KLK2 and CD3. Each variable domain (VH or VL) in a FIT-Ig may be obtained from one or more "parental" monoclonal antibodies that bind one of the target antigens, i.e., KLK2 or CD3. FIT-Ig binding proteins may be produced using variable domain sequences of anti-KLK2WSGR Docket No. 65457-712.602and anti-CD3 monoclonal antibodies as disclosed herein. For instance, the parental antibodies are humanized antibodies.

[0172] An aspect of the present disclosure pertains to selecting parental antibodies with at least one or more properties desired in the FIT-Ig molecule. In one embodiment, the antibody properties are selected from the group consisting of antigen specificity, affinity to antigen, cell binding potency, biological function, epitope recognition, stability, solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, tissue cross reactivity, and orthologous antigen binding.

[0173] In some embodiments, bispecific FIT-Ig proteins according to the present disclosure are configured without any interdomain peptide linker. Whereas in multivalent engineered immunoglobulin formats having tandem binding sites, it was commonly understood in the field that the adjacent binding sites would interfere with each other unless a flexible linker was used to separate the binding sites spatially. It has been discovered for the KLK2*CD3 FIT-Ig of the present disclosure, however, that the arrangement of the immunoglobulin domains according to the chain formulas disclosed herein results in polypeptide chains that are well-expressed in transfected mammalian cells, assemble appropriately, and are secreted as bispecific, multivalent immunoglobulin-like binding proteins that bind the target antigens KLK2 and CD3. See, Examples, infra. Moreover, omission of synthetic linker sequences from the binding proteins can avoid the creation of antigenic sites recognizable by mammalian immune systems, and in this way the elimination of linkers decreases possible immunogenicity of the FIT-Igs and leads to a half-life in circulation that is like a natural antibody, that is, the FIT-Igs are not rapidly cleared through immune opsonization and capture in the liver.

[0174] In some embodiments, an KLK2*CD3 bispecific binding protein according to the present application comprises:a) a first antigen-binding site that specifically binds KLK2; andb) a second antigen-binding site that specifically binds CD3.

[0175] In one embodiment, the bispecific binding proteins as described herein comprise a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 derived from any anti-KLK2 antibody or antigen-binding fragment thereof according to the present disclosureand described herein in the previous section to form the KLK2 binding site of the bispecific binding protein. In some further embodiments, the bispecific binding proteins as described hereinWSGR Docket No. 65457-712.602comprise a VH / VL pair derived from any anti-KLK2 antibody or antigen-binding fragment thereof according to the present disclosureand described herein in the previous section to form the KLK2 binding site of the bispecific binding protein.

[0176] In one embodiment, the bispecific binding proteins as described herein further comprise a set of six CDRs, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 derived from any anti-CD3 antibody or antigen-binding fragment thereof according to the present disclosureand described herein in the previous section to form the CD3 binding site of the bispecific binding protein. In some further embodiments, the bispecific binding proteins as described herein comprise a VH / VL pair derived from any anti-CD3 antibody or antigen-binding fragment thereof according to the present disclosureand described in the previous section herein to form the CD3 binding site of the bispecific binding protein.

[0177] In one embodiment, the KLK2 binding site and the CD3 binding site in a bispecific KLK2*CD3 binding protein according to the present disclosureare humanized, comprising humanized VH / VL sequences, respectively.

[0178] In one embodiment, an KLK2*CD3 bispecific binding protein according to the present application is a bispecific FIT-Ig binding protein capable of binding KLK2 and CD3. AFabs-in-Tandem immunoglobulin (FIT-Ig) binding protein is a monomeric, dual-specific, tetravalent binding protein comprising six polypeptide chains, and having four functional Fab binding regions with two outer Fab binding regions and two inner Fab binding regions. As shown in FIG. 1, the binding protein adopts the format (outer Fab-inner Fab-Fc)*2, and binds both antigen A and antigen B. In one aspect, the KLK2*CD3 bispecific binding protein according to the present application is a bispecific FIT-Ig binding protein, wherein two Fab domains of the FIT-Ig protein form the first antigen-binding site that specifically binds KLK2; and the other two Fab domains of the FIT-Ig protein form the second antigen-binding site that specifically binds CD3. In some embodiments, a FIT-Ig binding protein according to the present disclosure employs no linker between immunoglobulin domains.

[0179] In a further embodiment, the present disclosure provides a bispecific Fabs-in-Tandem immunoglobulin (FIT-Ig) binding protein comprises a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, wherein(i) in Format LH, the first polypeptide chain comprises, from amino terminus to carboxyl terminus, VLA-CL-VHB-CH1-FC wherein CL is fused directly to VHB; the second polypeptideWSGR Docket No. 65457-712.602chain comprises, from amino to carboxyl terminus, VHA-CHI; the third polypeptide chain comprises, from amino to carboxyl terminus, VLB-CL; or(ii) in Format HL, the first polypeptide chain comprises, from amino terminus to carboxyl terminus, VHB-CH1-V A-CL-FC wherein CHI is fused directly to VLA; the second polypeptide chain comprises, from amino to carboxyl terminus, VHA-CL; the third polypeptide chain comprises, from amino to carboxyl terminus, VLB-CHI;

[0180] wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CHI is a heavy chain constant domain, Fc is an immunoglobulin Fc region, for example, the Fc of IgGl (for instance, the Fc comprising, from amino terminus to carboxyl terminus, hinge-CH2-CH3),

[0181] wherein VLA-CL pairs with VHA-CHI to form a first Fab that specifically binds a first antigen A, and VLB-CL pairs with VHB-CHI to form a second Fab that specifically binds a second antigen B, and

[0182] wherein the antigen A is KLK2, and the antigen B is CD3, or wherein the antigen A is CD3, and the antigen B is KLK2,

[0183] wherein two of the first polypeptide chains, two of the second polypeptide chains, and two of the third polypeptide chains are associated to form a FIT-Ig binding protein.

[0184] In some embodiments of the bispecific FIT-Ig binding protein according to the present application, the first polypeptide chain comprises, from amino terminus to carboxyl terminus, VLA-CL-VHB-CH1-FC, wherein antigen A is KLK2 and antigen B is CD3, or antigen A is CD3 and antigen B is KLK2.

[0185] In some embodiments of the FIT-Ig binding protein, the KLK2 binding site is a Fab formed by VLKLK2-CL pairing with VHKLK2-CH1 (for example, when A is KLK2, formed by VLA-CL and VHA-CHI; or when B is KLK2, formed by VLB-CL and VHB-CHI), and comprises a set of six CDRs, namely CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 derived from any anti-KLK2 antibody or antigen-binding fragment thereof according to the present disclosure.

[0186] In some embodiments, the Fab binding to KLK2 in the FIT-Ig binding protein comprises a VH / VL pair derived from any anti-KLK2 antibody or antigen-binding fragment thereof according to the present application and described herein. In some embodiments, the Fab binding to KLK2 in the FIT-Ig binding protein comprises a VH sequence of SEQ ID NO: 33 andWSGR Docket No. 65457-712.602a VL sequence of SEQ ID NO: 39. In some embodiments, the Fab binding to KLK2 in the FIT-Ig binding protein comprises a VH sequence of SEQ ID NO: 46 and a VL sequence of SEQ ID NO: 47. In some embodiments, the Fab binding to KLK2 in the FIT-Ig binding protein comprises a VH sequence of SEQ ID NO: 55 and a VL sequence of SEQ ID NO: 61.

[0187] In some embodiments of the FIT-Ig binding protein, the CD3 binding site is a Fab formed by VLCD3-CL pairing with VHCD3-CH1 (for example, when A is CD3, formed by VLA-CL and VHA-CHI; or when B is CD3, formed by VLB-CL and VHB-CHI), and comprises a set of six CDRs, namely CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, derived from any anti-CD3 antibody or antigen-binding fragment thereof according to the present disclosure.

[0188] In some embodiments, the Fab fragments of such FIT-Ig binding proteins incorporate VLA-CL and VHA-CHI domains from a parental antibody binding to one of the antigens KLK2 and CD3, and incorporate VLB-CL and VHB-CHI domains from a different parental antibody binding to the other of the antigens KLK2 and CD3. In some embodiments, Fab moieties in tandem recognizing KLK2 and CD3 are respectively formed by VH-CH1::VL-CL pairing.

[0189] In accordance with the present disclosure, an KLK2*CD3 FIT-Ig binding protein comprises first, second, and third polypeptide chains; wherein the first polypeptide chain comprises, from amino to carboxyl terminus, VLKLK2-CL-VHcD3-CHl-hinge-CH2-CH3 wherein CL is directly fused to VHCD3; wherein the second polypeptide chain comprises, from amino to carboxyl terminus, VHKLK2-CH1; and wherein the third polypeptide chain comprises, from amino to carboxyl terminus, VLCD3-CL. In alternative embodiments, an KLK2*CD3 FIT-Ig binding protein comprises first, second, and third polypeptide chains, wherein the first polypeptide chain comprises, from amino to carboxyl terminus, VHKLK2-CHl-VLcD3-CL-hinge-CH2-CH3 wherein CHI is directly fused to VLCD3, wherein the second polypeptide chain comprises, from amino to carboxyl terminus, VHCD3-CH1; and wherein the third polypeptide chain comprises, from amino to carboxyl terminus, VLKLK2-CL. In some embodiments, VLKLK2 is a light chain variable domain of an anti-KLK2 antibody, CL is a light chain constant domain, VHKLK2 is a heavy chain variable domain of an anti-KLK2 antibody, CHI is a heavy chain constant domain, VLCD3 is a light chain variable domain of an anti-CD3 antibody, VHCD3 is a heavy chain variable domain of an anti-CD3 antibody; and optionally, the domains VLCD3-CL are the same as the light chain of an anti-CD3 parental antibody, the domains VHCD3-CH1 areWSGR Docket No. 65457-712.602the same as the heavy chain variable and heavy chain constant domains of an anti-CD3 parental antibody, the domains VLKLK2-CL are the same as the light chain of an anti-KLK2 parental antibody, and the domains VHKLK2-CH1 are the same as the heavy chain variable and heavy chain constant domains of an anti-KLK2 parental antibody.

[0190] In the foregoing formulas for a FIT-Ig binding protein, an Fc region may be a native or a variant Fc region. In particular embodiments, the Fc region is a human Fc region from IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. In particular embodiments, the Fc is a human Fc from IgGl, or a modified human Fc comprising one or more mutations to reduce or eliminate at least one Fc effector function, for example the binding of the Fc to FcyR, ADCC and / or CDC; in a certain embodiment, the mutations may be for example, L234A / L235A (numbering according to Kabat EU index). In another embodiment, the mutations may be for example, L234A / L235A and P329A mutation (numbering according to Kabat EU index). In one embodiment, the Fc region corresponding to CHl-hinge-CH2-CH3 derived from human constant IgGl with L234A / L235 A mutations comprises an amino acid sequence of residues 104 to 330 of the following sequence:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 14).

[0191] In another embodiment, the Fc region corresponding to CHl-hinge-CH2-CH3 derived from human constant IgGl with L234A / L235A and P329A mutations comprises an amino acid sequence of SEQ ID NO: 20.

[0192] In some embodiments of a FIT-Ig binding protein according to the present disclosure, CHI, CL and Fc domains are of or from human sequences. In some embodiments of a FIT-Ig binding protein according to the present disclosure, CHI is a human IgGl constant CHI domain, or a sequence having at least 90%, 95%, 97%, 98%, 99% or more identity herewith. In the foregoing formulas for a FIT-Ig binding protein, CL is a human constant kappa CL domain, or a sequence having at least 90%, 95%, 97%, 98%, 99% or more identity herewith.

[0193] In one embodiment, FIT-Ig binding proteins of the present disclosure retain one orWSGR Docket No. 65457-712.602more properties of the parental antibodies. In some embodiments, the FIT-Ig retains binding affinity for the target antigens (i.e., CD3 and KLK2) comparable to that of the parental antibodies, meaning that the binding affinity of the FIT-Ig binding protein for the KLK2 and CD3 antigen targets does not vary by greater than 10-fold in comparison to the binding affinity of the parental antibodies for their respective target antigens, as measured by surface plasmon resonance or biolayer interferometry.

[0194] In one embodiment, a FIT-Ig binding protein of the present disclosure binds KLK2 and CD3, and is comprised of a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein:

[0195] the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 85, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith,

[0196] the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 86, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and

[0197] the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 87, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith; or

[0198] the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 88, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith,

[0199] the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 89, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and

[0200] the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 90, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith; or

[0201] the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 91, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith,

[0202] the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 92, orWSGR Docket No. 65457-712.602a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and

[0203] the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 93, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith; or

[0204] In one embodiment, a FIT-Ig binding protein of the present disclosure binds KLK2 and CD3, and is comprised of a first polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 85; a second polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 86; and a third polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 87.

[0205] In one embodiment, a FIT-Ig binding protein of the present disclosure binds KLK2 and CD3, and is comprised of a first polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 88; a second polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 89; and a third polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 90.

[0206] In one embodiment, a FIT-Ig binding protein of the present disclosure binds KLK2 and CD3, and is comprised of a first polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 91; a second polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 92; and a third polypeptide chain comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO: 93.

[0207] In one embodiment, a bispecific binding protein comprises a first polypeptide chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 88 and a second polypeptide chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 89. In one embodiment, a bispecific binding protein comprises a first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 88 and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 89.WSGR Docket No. 65457-712.602

[0208] In one embodiment, a bispecific binding protein comprises a first polypeptide chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 85 and a second polypeptide chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 86. In one embodiment, a bispecific binding protein comprises a first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 85 and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 86.

[0209] In one embodiment, a bispecific binding protein comprises a first polypeptide chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 91 and a second polypeptide chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 92. In one embodiment, a bispecific binding protein comprises a first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 91 and a second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 92.Properties of bispecific binding proteins

[0210] In one embodiment, a bispecific KLK2*CD3 FIT-Ig binding protein capable of binding both CD3 and KLK2 as described herein comprises a humanized KLK2 binding domain. In one embodiment, a FIT-Ig binding protein antibody described herein or antigen-binding fragment thereof has a dissociation constant (KD) to KLK2 in the 10'8to IO'10range, for example, less than about lOnM, less than about 5nM, or less than about InM, preferably O.lnM to 0.95nM, and is not detected to bind to human KLK3, as measured by a grating-coupled interferometry (GCI) based assay.

[0211] In one embodiment, a bispecific KLK2*CD3 FIT-Ig binding protein capable of binding both CD3 and KLK2 as described herein comprises a humanized CD3 binding domain. In one embodiment, a FIT-Ig binding protein antibody described herein or antigen-binding fragment thereof has a dissociation constant (KD) to CD3 in the 10'8to IO'10range, for example, less than 8 x 10'8M, less than 5 x 10'8M, less than 3 x 10’8M, less than 2 x 10’8M, less than 1 x 10'8M, less than 8 x 10'9M, less than 6 x 10'9M, less than 4 x 10'9M, less than 2 x 1 O’9M, or less than 1 x 10'9M, less than 8 x 10'10M, less than 6 x 10'10M, less than 4 x 10'10M, less than 2 x 10’10WSGR Docket No. 65457-712.602M, or less than 1 x 1 O’10M.

[0212] In some embodiments, the KD values are measured by a GCI based assay. Grating-coupled interferometry (GCI) is a surface-based, label-free optical sensing technique used for real-time binding kinetic measurements. Typically, GCI is used to detect binding events between a target molecule and an analyte. When a protein immobilised on the sensor surface binds to an analyte, there is a light phase shift, which is read out interferometrically. The binding kinetics are analyzed by measuring these phase-shift signals in real time. See, e.g., Patko, D., et al., (2012). Optics Express , 20(21), p. 23162. https: / / doi.org / 10.1364 / oe.20.023162. In some further embodiments, the KD values are measured by a GCI based assay as described in Example 4.

[0213] In one embodiment, a bispecific KLK2 CD3 FIT-Ig binding protein capable of binding CD3 and KLK2 as described herein can be expressed in cultures of transfected mammalian host cells such as CHO cells or HEK293 cells at levels greater than 10 mg of KLK2xCD3 binding protein per liter of cell culture (>10 mg / L). In one embodiment, the expression level of the FIT-Ig binding protein is greater than 15 mg / L, for example, 15 mg / L to 100 mg / L, or more. In another embodiment, the expression level of FIT-Ig binding protein is greater than 60 mg / L.

[0214] In one embodiment, a bispecific KLK2 CD3 FIT-Ig binding protein capable of binding CD3 and KLK2 as described herein, after a one-step purification from cell culture media using a Protein A affinity chromatography, have a purity of no less than 90% as detected by SEC-HPLC. In one embodiment, the one-step purified binding proteins have a purity of no less than 91%, 92%, 93%, 95%, 97%, 99% as detected by SEC-HPLC.

[0215] In one embodiment, a bispecific KLK2 CD3 FIT-Ig binding protein as described herein is capable of binding both CD3 -expressing cells and KLK2-expressing cells. In one embodiment, the CD3 -expressing cells are human TCR / CD3 complex transfected CHO cell lines, or human T cells. In one embodiment, the KLK2-expressing cells are KLK2-expressing tumor cells, for example, human prostate cancer cells.

[0216] In one embodiment, as measured by flow cytometry in a cell-based assay, the binding potency of the bispecific FIT-Ig binding protein to the KLK2-expressing cells are equivalent to or comparable to the corresponding parental anti-KLK2 monoclonal IgG antibody comprising the same VH / VL sequence pairs for KLK2 binding as the bispecific FIT-Ig protein. In one embodiment, the binding potency of the bispecific FIT-Igbinding protein to the CD3 -expressing cells are equivalent to, or relatively lower than (but no more than a 10-fold difference, forWSGR Docket No. 65457-712.602instance, no more than 2-fold, 1-fold, or 50% decrease) the corresponding parental anti-CD3 monoclonal IgG antibody comprising the same VH / VL sequence pairs for CD3 binding as the bispecific binding protein, as measured by flow cytometry.

[0217] In one embodiment, a bispecific binding protein described herein is capable of modulating a biological function of KLK2, CD3, or both. In one embodiment, the bispecific KLK2*CD3 FIT-Ig binding protein as described herein is capable of activating CD3 signaling in terms of KLK2 dependence. In one embodiment, the bispecific binding proteins of the present disclosure exhibit KLK2-dependent activation of T cells. In one embodiment, a bispecific KLK2*CD3 FIT-Ig binding protein as described herein exhibits KLK2 -redirected T cell cytotoxicity. In one embodiment, the bispecific binding proteins of the present disclosure is used for redirecting the cytotoxic activity of T-cells towards KLK2 expressing cells in a non-MHC restricted fashion.

[0218] In one embodiment, a bispecific KLK2*CD3 FIT-Ig binding protein as described herein exhibits KLK2-dependent CD3 activation. In one embodiment, upon binding to KLK2-expressing cells, the bispecific KLK2*CD3 antibodies induce the crosslink of CD3 / TCR complex on T cells and activation of CD3 signaling. In a further embodiment, the bispecific KLK2*CD3 binding proteins exhibit increased T cell activation in the presence of KLK2-expressing target cells, and much less non-target redirected CD3 activation in the absence of KLK2-expressing target cells, in comparison to corresponding parental anti-CD3 monoclonal IgG antibodies comprising the same VH / VL sequence pairs for CD3 binding as the bispecific FIT-Ig proteins.

[0219] In one embodiment, a bispecific KLK2*CD3 FIT-Ig binding protein as described herein redirect T cell cytotoxicity to KLK2-expressing tumor cells. In another embodiment, a bispecific KLK2*CD3 FIT-Ig binding protein as described herein exhibits anti-tumor activities, such as reducing tumor burden, inhibiting tumor growth, or suppressing neoplastic cell expansion.

[0220] In one embodiment, a bispecific KLK2*CD3 FIT-Ig binding protein as described herein has one or more of the following properties:(a) the first antigen-binding domain binds to a human KLK2, with a KD value of less than about lOnM, less than about 5nM, or less than about InM, preferably O.lnM to 0.95nM, and is not detected to bind to human KLK3, as measured by a grating-coupled interferometry (GCI)WSGR Docket No. 65457-712.602based assay;(b) the first antigen-binding domain binds to the same or overlapping epitope, and / or competes with a reference antigen-binding domain comprising a VH sequence of SEQ ID NO: 11 and a VL sequence of SEQ ID NO: 12 for binding to a human KLK2; or the first antigenbinding domain binds to the different epitope, and / or does not compete with a reference antigenbinding domain comprising a VH sequence of SEQ ID NO: 11 and a VL sequence of SEQ ID NO: 12 for binding to a human KLK2;(c) the second antigen-binding domain binds to a human CD3 (especially, CD3s / 6 antigen), with a KD value of less than about 50nM, less than about 30nM, or less than about 20nM, preferably 5nM to 20nM, as measured by a grating-coupled interferometry (GCI) based assay;(d) the bispecific binding protein activates CD3 on T cells in vitro in a concentrationdependent manner at a concentration of 0.01 nM to 100 nM in the presence of KLK2 protein, as measured by a KLK2 protein-bound reporter gene assay (RGA);(e) the bispecific binding protein activates T cells in vitro in a concentration-dependent manner at a concentration of 0.01 nM to lOOnM to kill tumor cells expressing KLK2 protein, inducing lower cytokine (such as human IL-2, IL-6, IL-10, TNFa and IFNy) release by T cells;(f) the bispecific binding protein has in vivo anti-tumor effect on KLK2-expressing tumor; (g) the bispecific binding protein remains stable during storage.Nucleic acid, vector, and host cells

[0221] The present disclosure provides isolated nucleic acids, recombinant expression vectors and host cells, which is useful for making the antibodies, functional antibody fragments, and binding proteins described herein.

[0222] The term "isolated nucleic acid", as used herein, means a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or some combination thereof) that, by human intervention, is not associated with all or a portion of the polynucleotides with which it is found in nature; is operably linked to a polynucleotide that it is not linked to in nature; or does not occur in nature as part of a larger sequence.

[0223] The term "vector", as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNAWSGR Docket No. 65457-712.602segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0224] The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence. "Operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term "expression control sequence" as used herein refers to polynucleotide sequences that are necessary to affect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term "control sequences" is intended to include components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader or signal sequences and fusion partnerWSGR Docket No. 65457-712.602sequences.

[0225] The vector(s) comprising nucleic acid(s) encoding the antibodies, functional antibody fragments, and binding proteins described herein can be introduced into a host cell(s) by transformation. The term "transformation", as defined herein, refers to any process by which exogenous DNA enters a host cell. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, transfection, viral infection, electroporation, lipofection, and particle bombardment. Such "transformed" cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. They also include cells which transiently express the inserted DNA or RNA for limited periods of time.

[0226] The term "recombinant host cell" (or simply "host cell"), is intended to refer to a cell into which exogenous DNA has been introduced. In an embodiment, the host cell comprises two or more (e.g., multiple) nucleic acids encoding antibodies, such as the host cells described in US Patent No. 7,262,028, for example. Such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. In an embodiment, host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life. In another embodiment, eukaryotic cells include protist, fungal, plant and animal cells. In another embodiment, host cells include but are not limited to the prokaryotic cell line Escherichia coli mammalian cell lines CHO, HEK 293, COS, NSO, SP2 and PER.C6; the insect cell line Sf9; and the fungal cell Saccharomyces cerevisiae.

[0227] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in theWSGR Docket No. 65457-712.602art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y, 1989).

[0228] Antibodies, functional fragments thereof, and binding proteins according to the present disclosure may be purified (for an intended use) by using one or more of a variety of methods and materials available in the art for purifying antibodies and binding proteins. Such methods and materials include, but are not limited to, affinity chromatography (e.g., using resins, particles, or membranes conjugated to Protein A, Protein G, Protein L, or a specific ligand of the antibody, functional fragment thereof, or binding protein), ion exchange chromatography (for example, using ion exchange particles or membranes), hydrophobic interaction chromatography ("HIC"; for example, using hydrophobic particles or membranes), ultrafiltration, nanofiltration, diafiltration, size exclusion chromatography ("SEC"), low pH treatment (to inactivate contaminating viruses), and combinations thereof, to obtain an acceptable purity for an intended use. A non-limiting example of a low pH treatment to inactivate contaminating viruses comprises reducing the pH of a solution or suspension comprising an antibody, functional fragment thereof, or binding protein of the present disclosure to pH 3.5 with 0.5 M phosphoric acid, at 18°C - 25°C, for 60 to 70 minutes.Method of production

[0229] In another aspect, the present disclosure provides a method of producing a bispecific, multivalent binding protein capable of binding KLK2 and CD3, specifically a FIT-Ig binding protein binding KLK2 and CD3, comprising culturing a host cell comprising an expression vector encoding the FIT-Ig binding protein in culture medium under conditions sufficient to cause the host cell to express the binding protein capable of binding KLK2 and CD3. The proteins produced by the methods disclosed herein can be isolated and used in various compositions and methods described herein.Pharmaceutical compositions

[0230] The present disclosure provides pharmaceutical compositions comprising an antibody, or antigen-binding portion thereof, or a bispecific multivalent binding protein of the present disclosure (i.e., the primary active ingredient) and a pharmaceutically acceptable carrier. In a specific embodiment, a composition comprises one or more antibodies or binding proteins of theWSGR Docket No. 65457-712.602present disclosure. The present disclosure also provides pharmaceutical compositions comprising a combination of anti-KLK2 and anti-CD3 antibodies as described herein, or antigen-binding fragment(s) thereof, and a pharmaceutically acceptable carrier. In particular, the present disclosure provides pharmaceutical compositions comprising at least one FIT-Ig binding protein capable of binding KLK2 and CD3 and a pharmaceutically acceptable carrier.Pharmaceutical compositions of the present disclosure may further comprise at least one additional active ingredient. In some embodiments, such an additional ingredient includes, but is not limited to, a prophylactic and / or therapeutic agent, a detection agent, such as an anti-tumor drug, a cytotoxic agent, an antibody of different specificity or functional fragment thereof, a detectable label or reporter. In an embodiment, the pharmaceutical composition comprises one or more additional prophylactic or therapeutic agents, i.e., agents other than the antibodies or binding proteins of the present disclosure, for treating a disorder in which KLK2 activity is detrimental. In an embodiment, the additional prophylactic or therapeutic agents are known to be useful for, have been used, or are currently being used in the prevention, treatment, management, or amelioration of, a disorder or one or more symptoms thereof.

[0231] The pharmaceutical compositions comprising proteins of the present disclosure are for use in, but not limited to, diagnosing, detecting, or monitoring a disorder; treating, managing, or ameliorating a disorder or one or more symptoms thereof; and / or research. In some embodiments, the composition may further comprise a carrier, diluent, or excipient. An excipient is generally any compound or combination of compounds that provides a desired feature to a composition other than that of the primary active ingredient (i.e., other than an antibody, functional portion thereof, or binding protein of the present disclosure).Conjugate and fusion

[0232] The present disclosure provides conjugates and fusions comprising antibodies, functional antibody fragments, and binding proteins described herein.

[0233] The terms “conjugate”, “immunoconjugate”, “fusion” and “immunofusion” can be used interchangeably herein, and refer to an antibody or a binding protein or functional fragments (e.g., Fab fragments of FIT-Ig) of the present disclosure conjugated to one or more other substances, including but not limited to a therapeutic agent, a diagnostic agent, a detectable agent, a cytotoxic agent, and small molecule drug.

[0234] In an embodiment, the present disclosure provides a conjugate or a fusion comprisingWSGR Docket No. 65457-712.602an anti-KLK2 antibody or a binding protein or functional fragments (e.g., Fab fragments of FIT-Ig) of the present disclosure conjugated to a therapeutic agent. The term “therapeutic agent” used herein encompasses any substance that is effective in preventing or treating a tumor, e.g., cancer, including a chemotherapeutic agent, a cytokine, a cytotoxic agent, an antibody of different specificity or functional fragment thereof, a small molecule drug or an immunosuppressant e.g., an immunosuppressive agent). In some embodiments, the therapeutic agent to be conjugated is a cytotoxic agent that inhibits or prevents cell functions and / or causes cell death or cell destruction. In some embodiments, the therapeutic agent to be conjugated is a chemotherapeutic agent useful in the treatment of immune system diseases. In some embodiments, the therapeutic agent to be conjugated is a small molecule drug having a molecular weight of less than 10 Kd, usually less than 2 Kd and preferably less than 1 Kd. As therapeutic agents, small molecules penetrate cells better, are less susceptible to degradation and are less likely to induce an immune response compared with large molecules.

[0235] In an embodiment, the present disclosure provides a conjugate or a fusion comprising an anti-KLK2 antibody or a binding protein or functional fragments (e.g., Fab fragments of FIT-Ig) of the present disclosure conjugated to a diagnostic agent or a detectable agent.Medical uses

[0236] In a further aspect, the present disclosure provides the anti-KLK2 antibody, the KLK2*CD3 binding protein or the fragment thereof described herein for use as medicament, or for use in manufacture of a medicament, preferably the medicament is for treating or preventing a KLK2-associated disease or disorder. In another further aspect, the present disclosure provides methods for treating a KLK2 -associated disease or disorder in an individual in need thereof, the method comprising administering to the individual an antibody, a binding protein or a functional fragment thereof as described herein. In one embodiment, the disease or disorder is a condition where KLK2-mediated signaling activity is detrimental (e.g., a KLK2+solid tumor) in an individual in need thereof.

[0237] In another embodiment, the present disclosure provides use of an effective amount of an anti-KLK2 antibody, the KLK2*CD3 binding protein or antigen-binding fragment thereof described herein in the treatment of a disease or disorder. In another embodiment, the present disclosure provides use of an anti-KLK2 antibody, the KLK2 / CD3 antibody or antigen-binding fragment thereof described herein in the manufacture of a composition for the treatment of suchWSGR Docket No. 65457-712.602a disorder. In another embodiment, the present disclosure provides an anti-KLK2 antibody, the KLK2*CD3 binding protein or antigen-binding fragment thereof described herein for use in the treatment of a disease or disorder.

[0238] In a further embodiment of the method or use described herein, the KLK2*CD3 binding protein or antigen binding fragment of the present disclosure is a FIT-Ig binding protein comprising a first, second, and third polypeptide chains comprising or consisting of the sequences as shown in SEQ ID NOs: 85, 86 and 87 respectively.

[0239] In a further embodiment of the method or use described herein, an anti-KLK2 x anti-CD3 antibody or antigen binding fragment of the present disclosure is a FIT-Ig binding protein comprising a first, second, and third polypeptide chains comprising or consisting of the sequences as shown in SEQ ID NOs: 88, 89 and 90 respectively.

[0240] In a further embodiment of the method or use described herein, an anti-KLK2 x anti-CD3 antibody or antigen binding fragment of the present disclosure is a FIT-Ig binding protein comprising a first, second, and third polypeptide chains comprising or consisting of the sequences as shown in SEQ ID NOs: 91, 92 and 93 respectively.

[0241] In some further embodiments, said disease or disorder include various hematopoietic and solid malignancies expressing KLK2 on the cell surface of the malignant cells. In another embodiment, the antibody or the binding protein described herein inhibits the growth or survival of malignant cells. In another embodiment, the antibody or the binding protein described herein reduces the tumor burden.

[0242] In some embodiments, said disease or disorder is cancer. In one embodiment, said cancer is a cancer typically responsive to immunotherapy. In another embodiment, said cancer is a cancer that has not been associated with immunotherapy. In another embodiment, said cancer is a cancer that is a refractory or a recurring malignancy. In another embodiment, the cancer is a KLK2-positive cancer. In another embodiment, the cancer is selected from the group consisting of breast cancer, and prostate cancer, and other neoplastic malignancies. In another embodiment, said KLK2-positive cancer is an androgen receptor (AR) expressing breast cancer. In another embodiment, said KLK2-positive cancer is a relapsed, refractory, malignant or castration-resistant prostate cancer (CRPC), or any combination thereof. Methods of treatment described herein may further comprise administering to an individual in need thereof, of additional active ingredient, which is suitably present in combination with the present antibodyWSGR Docket No. 65457-712.602or binding protein described herein for the treatment purpose intended, for example, another drug having antitumor activity. In a method of treatment of the present disclosure, the additional active ingredient may be incorporated into a composition comprising an antibody or binding protein of the present disclosure, and the composition administered to an individual in need of treatment. In another embodiment, a method of treatment of the present disclosure may comprise a step of administering to an individual in need of treatment an antibody of binding protein described herein and a separate step of administering the additional active ingredient to the individual before, concurrently, or after the step of administering to the individual an antibody of binding protein of the present disclosure.

[0243] In a further aspect, the present disclosure provides the anti-KLK2 antibody, the KLK2*CD3 binding protein or the fragment thereof, or the pharmaceutical composition described herein for use as a method of targeting a T-cell to a tumor cell, the method comprising administering to an individual in need thereof a therapeutically effective amount of the antibody, thereby targeting the T-cell to the tumor cell.

[0244] In other aspects, the present disclosure provides the anti-KLK2 antibody, the KLK2*CD3 binding protein or the fragment thereof, or the pharmaceutical composition described herein for use as a method of killing or inhibiting growth of a tumor cell, the method comprising contacting the tumor cell with an effective amount of the bispecific binding protein or the pharmaceutical composition, thereby killing or inhabiting growth of the tumor cell.EXAMPLES

[0245] The following examples further illustrate the present disclosure, however, it should be understood that Examples are described in an illustrative rather than a limiting manner, and that various modifications may be made by those skilled in the art.

[0246] Unless expressly stated to the contrary, conventional methods from chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology will be used. Experimental methods, unless otherwise specified, are conventional methods in art using default parameters, steps, etc.; the experimental materials used, unless otherwise specified, are all commercially available products. If no specific techniques or conditions are noted, they shall be executed according to the techniques or conditions described in the literature in the art, or according to corresponding product instructions. The reagents or instruments used without indicating the manufacturer areWSGR Docket No. 65457-712.602all standard products available through the usual channels.

[0247] Generally, in the present disclosure, conventional hybridoma technology was used to generate de-novo anti-KLK2 monoclonal antibodies, which were later humanized using CDR-grafting method. Novel KLK2*CD3 bispecific antibodies were constructed in FIT-Ig format (as published in W02015103072, incorporated herein in entirety by reference).Example 1. Generation of anti-KLK2 antibodies, construction and characterization of KLK2xCD3 FIT-Ig chimeric bispecific antibodies1.1: Generation of anti-KLK2 antibodies

[0248] Anti-KLK2 antibodies were obtained by immunizing Balb / c mice with a human KLK2 (NP_005542.1) encoded mRNA-LNP complex. A sequence of HUMAN KLK2 CDS is shown as follows:>HUMAN_KLK2_CDS atgtgggacctggttctctccatcgccttgtctgtggggtgcactggtgccgtgcccctcatccagtctcggattgtgggaggctgggagtgtgagaagc attcccaaccctggcaggtggctgtgtacagtcatggatgggcacactgtgggggtgtcctggtgcacccccagtgggtgctcacagctgcccattgcc taaagaagaatagccaggtctggctgggtcggcacaacctgtttgagcctgaagacacaggccagagggtccctgtcagccacagcttcccacaccc gctctacaatatgagccttctgaagcatcaaagccttagaccagatgaagactccagccatgacctcatgctgctccgcctgtcagagcctgccaagatc acagatgttgtgaaggtcctgggcctgcccacccaggagccagcactggggaccacctgctacgcctcaggctggggcagcatcgaaccagaggag ttcttgcgccccaggagtcttcagtgtgtgagcctccatctcctgtccaatgacatgtgtgctagagcttactctgagaaggtgacagagttcatgttgtgtg ctgggctctggacaggtggtaaagacacttgtgggggtgattctgggggtccacttgtctgtaatggtgtgcttcaaggtatcacatcatggggccctgag ccatgtgccctgcctgaaaagcctgctgtgtacaccaaggtggtgcattaccggaagtggatcaaggacaccatcgcagccaacccctga (SEQ ID NO: 1)

[0249] Mice were immunized at 2-week intervals and monitored for serum titer once a week after the second immunization injection. After 2 immunizations, splenocytes derived from the immunized mice were harvested and fused with mouse myeloma cells to form hybridoma cell lines by conventional electrofusion technology. Hybridoma cells were plated in 384-well plates with selection media comprising hypoxanthine-aminopterin-thymidine (HAT) at a density of 2.5* 104cells per well. Seven to ten days later, supernatants of each well were screened against a recombinant human KLK2 protein by enzyme-linked immunosorbent assay (ELISA) or human KLK2-expressing cell lines by flowcytometry. Binding positive hybridoma clones that produced specific anti-KLK2 antibodies were then cherry picked and expanded for following immunoglobulin variable gene (IgV) sequencing.WSGR Docket No. 65457-712.6021.2: Selection of anti-KLK2 antibodies

[0250] To amplify heavy and light chain variable regions, total RNA was extracted from the hybridoma cells obtained in Example 1.1 with HiPure RNA Mini Columns (Magen) according to the manufacturer’s instructions. NanoDrop and gel electrophoresis were employed to measure the RNA concentration and integrity. cDNA was synthesized from RNA with SMART Scribe Reverse Transcriptase (Takara) with oligo-dT and template switch oligo (TSO) according to the manufacturer’s instructions. The double-stranded cDNA was then diluted for amplification. The forward primer was anchored to the TSO and the reverse primers were binding to the constant regions of heavy chain or light chain. The 5’ ends of the forward primer and the reverse primer were tagged with partial P5 and P7 adaptors, respectively. Heavy-chain and light-chain fragments were amplified in separate reactions in the first stage PCR. PCR products were purified with Magnetic beads. During the second stage PCR, index primers were attached to both ends of the first stage PCR products to form TruSeq dual index library. The libraries were purified with Magnetic beads and quantified by Qubit and finally sequenced on Illumina MiSeq PE300 following the manufacturer’s instructions (Illumina). Raw fastq files were first individualed to quality assessment. Adapters and bases with poor quality scores (Q value lower than 20) were removed using cutadapt (vl.9.1) to generate clean data (trimmed data). Pandaseq (v2.10) was used to merge pair-end reads. Merged sequences were processed by NCBI-IgBLAST (vl.17.0) software to identify the best matched germline V(D)J sequences and sequences in CDR1, CDR2, CDR3 region. The reference sequences were obtained in IMGT database (IMGT, https: / / www.imgt.org / ).

[0251] Five of the colonies (designated as EM1031-K126, EM1031-K31, EM1031-K97, EM1031-K112, and EM1031-K117, respectively) from each transformation were selected and used to construct bi specific binding proteins for further screening.1.3 Generation of KLK2*CD3 FIT-Ig chimeric bispecific antibodies

[0252] A group of chimeric FIT-Ig proteins recognizing both human KLK2 and human CD3 were constructed utilizing variable domains of five clones obtained in Example 1.2 as anti-KLK2 moiety, and VH / VL sequences published in WO2022 / 042488 as anti-CD3 moiety (Table 1), as well as human constant region sequences, as shown in Table 2. The obtained antibodies are named with prefix “FIT”.WSGR Docket No. 65457-712.602Table 1: Amino acid sequences of anti-CD3 variable domainsAnti-CD3 Amino acid sequenceDomain SEQIDclone NO: 1234567890123456789012345678901234567890VHCD3 E VQ L VQ S GAE VKK P GAS VKVS C KAS G F S FTNYYVHWMRQAP G (EM0006- 2 Q GL EWMGWISPGSDNTKYNEKFKGRVTMT RDT S I S T AYME L S OlVH.lh) RLRS DDTAVYYCARDDYGNYYFDYWGQGTTVTVS S CDR-H1 3 NYYVHCDR-H2 4 WISPGSDNTKYNEKFKGhuEM0006 CDR-H3 5 DDYGNYYFDY-01-24 VLCD3 D I VMT Q S P D S LAVS L GE RAT I N CKSSQSLLNARTRKNYLAWY (EM0006- 6 QQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSL 01VK.l(S31aA)) QAEDVAVYYCKQSYILRTFGGGTKVEIKCDR-L1 7 KSSQSLLNARTRKNYLACDR-L2 8 WAS T RESCDR-L3 9 KQSYILRTCDRs defined according to Kabat scheme are underlined.Table 2: Amino acid sequences of human IgG constant regionsSEQ ID Amino acid sequenceRegionNO: 1234567890123456789012345678901234567890 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKHuman IgGl heavy PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT chain constant domain 15 KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI (wild type) EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN CHl-hinge-CH2-CH3 HKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPK (human IgGl heavy PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT chain constant, with 16 KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPI LALA + P329A EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS mutations) DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK CL RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV(human kappa constant, 17 DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CK) CEVTHQGLSSPVTKSFNRGECCHI ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS(human IgGl constant 18 GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN region 1) HKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLHuman IgGl Fc TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY (hinge-CH2-CH3), 19 TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK wild-type TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKWSGR Docket No. 65457-712.602SEQ ID Amino acid sequenceRegionNO: 1234567890123456789012345678901234567890 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLHuman IgGl Fc, withTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVY LALA + P329A 20TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKmutations TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKFc mutations are shown in bold italic.

[0253] FIT-Ig molecules were constructed following the general procedures described in PCT Publication No. W02015 / 103072. Each FIT-Ig consists of two long chains (long chain), two first short chains (short chain 1) and two second short chains (short chain 2). These polypeptide chains have the following structures:Chain #1 (long chain): VLA-CL-VHB-CHl-hinge-CH2-CH3;Chain #2 (short chain 1): VHA-CHI;Chain #3 (short chain 2): VLB-CL;wherein A stands for antigen KLK2 and B stands for antigen CD3, therefore, VLAand VLK K2 are used interchangeably and means the light chain variable domain of a mouse derived antibody recognizing KLK2, VHB and VHCDS are used interchangeably and means the heavy chain variable domain of a humanized monoclonal antibody recognizing CD3 (for example, those recorded in Table 1 shown above), VHAand VHKLK2 are used interchangeably and means the heavy chain variable domain of a mouse derived antibody recognizing KLK2, VLB and VLCDS are used interchangeably and means the light chain variable domain of a humanized monoclonal antibody recognizing CD3 (for example, those recorded in Table 1 shown above); each CL is a light chain constant domain (for example, as shown in SEQ ID NO: 17), each CHI is a first heavy chain constant domain (for example, as shown in SEQ ID NO: 18), and CH1-hinge-CH2-CH3 is the C-terminal heavy chain constant region from CHI through the terminus of the Fc region (for example, as shown in SEQ ID NO: 16).

[0254] To construct the long chain, cDNA encoding the VLKLK2-CL-VHCD3 segment was synthesized de novo and inserted into the multiple cloning site (MCS) of a vector comprising coding sequences for human CHl-hinge-CH2-CH3. In the resulting vector, the MCS sequence was eliminated during homologous recombination to ensure that all the domain fragments were in the correct reading frame. Similarly, to construct the first and second short chains, cDNA encoding the VHKLK2 or the VLCD3 segment was de novo synthesized and inserted into the MCSWSGR Docket No. 65457-712.602of the appropriate vectors including coding segments for human CHI and CL domains, respectively.

[0255] The pairing of the VH and the VL creating chimeric KLK2*CD3 FIT-Ig binding proteins were listed in Table 3.Table 3: FIT-Ig proteins with chimeric anti-KLK2 mabsFIT-Ig Identifier Anti-KLK2 Anti-CD3FIT2023-8ac EM1031-K126 HuEM0006-01-24FIT2023-10ac EM1031-K31 HuEM0006-01-24FIT2023-20ac EM1031-K97 HuEM0006-01-24FIT2023-22ac EM1031-K112 HuEM0006-01-24FIT2023-23ac EM1031-K117 HuEM0006-01-241.4: Binding kinetics of KLK2xCD3 chimeric bispecific antibodies

[0256] Binding affinities and kinetics constants of KLK2*CD3 FIT-Igs in Table 3 were determined using a grating-coupled interferometry (GCI) (WAVEsystem, Creoptix AG). Briefly, a goat anti-human IgG Fc antibody was immobilized onto a PCP WAVEchip (Creoptix AG) at a density of 4059.7 pg / mm2on Channel 1, at a density of 3923.6 pg / mm2on Channel 2, at a density of 4720.3pg / mm2on Channel 3 and at a density of 4625.4 pg / mm2on Channel 4 by amine coupling. FIT-Igs were then captured onto the PCP WAVEchip at a density of 3091.8-8763.2 pg / mm2. Recombinant human KLK2 or recombinant human KLK3 were injected at 3-fold serial diluted concentrations (1.23 nM, 3.7 nM, 11.11 nM, 33.33 nM, 100 nM) in kinetics buffer (BRI 00669, Cat. #22064428-AD). The series diluted recombinant proteins were then injected for 250s at a flow rate of 60 pL / min for the association period. Immediately after the association step, running buffer was injected for 1000s at 60 pL / min for the dissociation period. After each round of association and dissociation, WAVEchip surface was regenerated by injection of the Glycine-HCl buffer at pH 1.5 for 120s at a flow rate of 80 pL / min. All sensorgrams were recorded at 25°C and data were analyzed with the WAVEcontrol software (Creoptix AG). Data was double referenced by subtracting the signals from blank injections and from the reference channel. A 1 : 1 binding kinetics model was used for KLK2 affinity data fitting. All the KLK2xCD3 chimeric bispecific antibodies showed high affinity to human KLK2 and no detectable binding to human KLK3. Kinetics constants were shown in Table 4.WSGR Docket No. 65457-712.602Table 4: Binding kinetics of KLK2xCD3 chimeric bispecific antibodiesKLK2 KLK3Sample IDKa (1 / Ms) Kd (1 / s) KD (M) Ka (1 / Ms) Kd (1 / s) KD (M) FIT2023-8ac 7.64E+06 9.43E-04 1.23E-10 ND ND ND FIT2023-10ac 2.45E+06 2.32E-03 9.48E-10 ND ND ND FIT2023-20ac 7.70E+06 1.81E-03 2.35E-10 ND ND ND FIT2023-22ac 5.50E+06 1.40E-03 2.54E-10 ND ND NDFIT2023-23ac 3.44E+06 4.45E-04 1.29E-10 ND ND ND ND: No detectable binding1.5: Epitope binning of KLK2*CD3 chimeric bispecific antibodies

[0257] The binding epitope of KLK2*CD3 antibodies to KLK2 were identified with a competition ELISA. Briefly, 96 well plates were coated with 1 pg / mL purified KLK2*CD3 antibodies in Table 3 and incubated overnight at 4°C. After washing with PBS containing 0.05% Tween 20, plates were blocked with blocking buffer (PBS containing 0.05% Tween 20 and 2% BSA) at 37°C for 1 hour. Biotinylated human KLK2 protein pre-mixed with KLK2*CD3 antibodies in Table 3 (as sample) or irrelevant human IgG (as baseline) was added into plate wells and incubated at 37°C for 1 hour, then wells were washed 3 times. Streptavidin-HRP (1:5000 dilution) was then added into each well and incubated at 37°C for 1 hour, then wells were washed another 3 times. Tetramethylbenzidine (TMB) chromogenic solution was added for color development, 5 minutes later the reaction was stopped with IM HC1. Absorbance at 450 nm (OD450) was measured on a microplate reader. The OD450 baseline represents the level of human KLK2 binding to KLK2 antibodies in the absence of competition, while the difference between OD450baseiine and OD450sampie reflects the competition between the KLK2*CD3 antibodies in Table 3 coated on plate and the KLK2*CD3 antibodies in Table 3 in solution. The inhibition percentage was calculated by the following equation:Inhibition=(1 - OD450sample / OD450basellne) x 100%

[0258] The results of the competition ELISA were shown in Table 5. According to the competition analysis, we can group the related antibodies into 4 different epitope bins, wherein the ref molecule hullB6 binds to epitope bin #4.VH amino acid sequence of the ref molecule hullB6 (SEQ ID NO: 11) QVQLQESGPGLVKPSDTLSLTCAVSGNSITSDYAWNWIRQPPGKGLEWIGYISYSGSTTYNPSL KSRVTMSRDTSKNQFSLKLSSVTAVDTAVYYCATGYYYGSGFWGQGTLVTVSSWSGR Docket No. 65457-712.602VL amino acid sequence of the ref molecule hullB6 (SEQ ID NO: 12) DIVLTQSPDSLAVSLGERATINCKASESVEYFGTSLMHWYQQKPGQPPKLLIYAASNRESGVPD RFSGSGSGTDFTLTISSLQAEDVAVYYCQQTRKVPYTFGQGTKLEIKTable 5: Competition ELISA result of anti-KLK2*CD3 antibodiesCoatingFIT2023 FIT2023 FIT2023 FIT2023 FIT2023 Epitope hlgG-8ac -lOac -20ac -22ac -23ac Bin Competition""-^FIT2023-8ac 95.00% 7.91% 25.16% 91.89% 30.71% 2.75% 1 FIT2023-10ac 22.25% 92.03% 18.70% 27.53% 9.01% 0.16% 4 FIT2023-20ac 6.63% -5.55% 63.93% 0.30% 22.62% 0.94% 3 FIT2023-22ac 67.16% 8.96% 25.11% 95.49% 20.76% 2.96% 1 FIT2023-23ac 71.07% 44.65% 47.59% 74.40% 94.32% 6.62% 2 hullB6 3.20% 73.34% 19.13% 5.20% -24.74% -7.82% 4hlgG 5.69% 8.15% 7.40% 7.30% 7.08% 5.47% NA NA: Not applicable1.6: Redirected CD3 activation of chimeric FIT-Ig bispecific antibodies

[0259] To measure redirected CD3 activation by KLK2*CD3 FIT-Igs, a NFAT-luciferase reporter gene assay (RGA) was used. In this assay, Jurkat-NFAT-luc cells trigger downstream luciferase signal when cell surface CD3 is activated. Plate coated KLK2 protein can crosslink CD3 / TCR complex on T cells via bispecific KLK2*CD3 antibodies upon KLK2 binding.

[0260] The 96-well plates (Costar #3903) were coated with 1 pg / mL KLK2 protein and incubated overnight at 4°C. After washing with PBS containing 0.05% Tween 20, plates were blocked with blocking buffer (PBS containing 0.05% Tween 20 and 2% BSA) at 37°C for 1 hour. Jurkat-NFAT-luc cells (ChemPartner) were washed and resuspended in assay medium (RPMI1640 with 2% FBS) and seeded at 4* 104cells per well. FIT-Ig antibodies in Table 3 were added and mixed with the cells and incubated for 4 hours at 37°C. At the end of incubation, ONE-Glo™ luminescence assay kit (Promega, Cat. #E6130) reagents were prepared and added into wells according to the manufacturer's instructions. Plates were read for luminescence signals with Varioskan™ LUX microplate reader (ThermoFisher Scientific). The results were shown in FIG. 2.

[0261] As shown in FIG.2, bispecific KLK2*CD3 antibodies showed concentration dependent activation of CD3 in the presence of KLK2 protein (FIG.2), while the parental anti-CD3 antibody (huEM0006-01-24) showed minimum to no activation of CD3 in this assay.WSGR Docket No. 65457-712.6021.7: In vitro killing of chimeric FIT-Ig bispecific antibodies

[0262] An assay using cocultured tumor cells and human primary CD3+ T-cells was utilized to evaluate in-vitro redirected T-cell cytotoxicity of KLK2*CD3 chimeric bispecific antibodies in Table 3. A prostate cancer cell line VCaP (ATCC, CRL-2876) and a human KLK2-overexpressing prostate cancer cell line LNCaP (ATCC, CRL-1740), namely LNCaP-KLK2 were used in this assay.

[0263] Briefly, 2* 104tumor cells / well and 1 * 105human primary PBMC cells / well were seeded into 96-well plates (Costar #3903) at 5:1 effector-to-target [E / T] ratio. Then serial dilutions of test articles (i.e. KLK2*CD3 FIT-Igs in Table 3), negative control (i.e. anti-CD3 antibody huEM0006-01-24) were added to cells, respectively. Following incubation for 48h or 72h at 37°C, 5% CO2, the supernatant and cells in the supernatant were discarded. Wells were gently washed twice, then the CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega) was used to quantitate the number of viable cells present in wells according to the manufacturer's instructions. The results were shown in FIG.3.

[0264] As shown in FIG.3, KLK2*CD3 chimeric bispecific antibodies showed concentrationdependent killing activity to both VCaP and LNCaP -KLK2 cell lines.Example 2: Humanization of anti-KLK2 antibodies

[0265] In view of evaluations in Example 1, three of the five clones obtained in Example 1.2 were selected for further humanization. The variable domain sequences for the selected three anti-KLK2 antibody were set out in Table 6. Complementarity determining regions (CDRs) were underlined according to Kabat numbering.Table 6: Amino acid sequences of variable regions of anti-KLK2 antibodiesAntibody SEQ ID Amino acid sequenceDomainIdentifier NO: 1234567890123456789012345678901234567890 EVQLQQSGAEFVKPGASVKLSCTASGFNIDDTYMHWVKQR VH 21 PEQGLY WI GRIDPANGNTKYDPKFQGKAT I TADT S SNT DY EM1031- L Q L S S L T S E D T A V Y Y C AR GGYRYDDYFDY W G Q G T T L T V S S K31 DIVLTQSPASLAVSLGQRATISCRASETVDSYGSSFMHWY VL 22 QQKSGQPPKLLIYRASTLESGIPARFSGSGSRTDFTLTIN AVEADDVATYYCQQNNEDPFTFGGGTKLEIK EVQLQQSGAELVRPGALLKLSCKASGFNIKDYYMHWVKQR VH 23 PEQGLEWI GWIDPENGNTIYDPRFQGKAN ITADTSSNTAY EM1031- LQLSSLTS E DT AVY Y C AR SFYYGYLRFDY WGQ GT L VAV SA K97 EIVLTQSPAITAASLGQKVTITCSASSSVSYMHWYQQKSG VL 24 T S P KP W I Y EISKLAS G VP AR F S G S G S GT S F S L T I S SME AE D AA I Y Y C QQWNYPLFTFG AG T KL E L KWSGR Docket No. 65457-712.602Antibody SEQ ID Amino acid sequenceDomainIdentifier NO: 1234567890123456789012345678901234567890 EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQK VH 25 PGQGLE WI GYISPYNDGTKYNEKFKGKATLT S DKS S STAY EM1031- MYLSSLTSEDSAVYYCARGGGLEYSFDYWGQGTTLTVSS K112 DILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRT VL 26 NGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVES E D I ADY YCQQTNSWPWTFGGGT KL E I K* CDRs defined according to Kabat Numbering underlined.

[0266] The anti-KLK2 variable region-encoding genes of the anti-KLK2 antibodies were employed for humanization design. In the first step of this process, the amino acid sequences of the VH and VL domains of the anti-KLK2 antibodies were compared against the available database of human Ig V-gene sequences to find the overall best-matching human germline Ig V-gene sequences. Additionally, J-region sequences of the VH or VL were compared against the J-region database to find the human framework having the highest homology to the murine VH and VL regions, respectively. Afterward, the CDRs (Kabat numbering) of anti-KLK2 VH / VL were grafted to selected human acceptors (human V, J regions) to obtain primary humanized VH / VLs. Three-dimensional Fv models of parental murine anti-KLK2 antibodies were then generated and analyzed to determine if there were any framework positions involved in supporting loop structures or the VH / VL interface. The primary humanized sequences could be back mutated to mouse residues at the same positions to retain affinity / activity. The obtained humanized antibodies, humanized VH or VL were all named based on corresponding parent antibodies with prefix “hu”.

[0267] Several desirable back-mutations were introduced to EM1031-K31-VH / VK, EM1031-K97-VH and EM1031-K112-VH / VK, thereby several modified VH or VK with mutations were constructed, as shown in Table 7. As the “NG” (Asn-Gly) pattern in CDR-H2 or “NS” (Asn-Ser) in CDR-L3 is prone to deamination, “DG” (Asp-Gly) in CDR-H2, or “DS” (Asp-Ser) in CDR-L1 is prone to isomerization, which may result in heterogeneity during manufacturing, VH and VL domains containing amino acid modifications of NG (Asn-Gly) to NA (Asn-Ala), NS (Asn-Ser) to NA (Asn-Ala), DG (Asp-Gly) to DA (Asp-Ala), and / or DS (Asp-Ser) to DA (Asp-Ala) (highlighted in bold italic in Table 7) were also designed and evaluated. The obtained corresponding VH / VL was named by specific mutations in parenthesis, for example, “EMI 031-K31-VH(G55A)” means EM1031-K31-VH with an extra mutation G55A, and “EM1031-K112-VH(G56A)” represents EM1031-K112-VH with an extra mutation G56A.WSGR Docket No. 65457-712.602Table 7: Humanized VH / VL sequences of anti-KLK2 antibodiesSEQ ID Amino acid sequenceVariable DomainNO: 1234567890123456789012345678901234567890EVQLQQSGAEFVKPGASVKLSCTASGFNIDDTYMHWVKQR EM1031-K31-VH 21 P EQGL Y W I GRIDPANGNTKYDPKFQGKAT I T ADT S SNT D Y LQLSSLTSEDTAVYYCARGGYRYDDYFDYWGQGTTLTVSS E VQL VQ S GAE VKKPGAS VKVS CKAS GY T FTDTYMH WVRQ AHuEM1031-K31-VH.la 27 PGQ RLEWMGRIDPANGNTKYDPKFQGRVT I T ADT SAS T AY MELSSLRSEDTAVYYCARGGYRYDDYFDYWGQGTLVTVSS E VQL VQ S GAE VKKPGAS VKVS CKAS GY T FTDTYMH WVRQ AHuEM1031-K31-VH.lb 28 PGQ RLEWMGRIDPANGNTKYDPKFQGRVT I T ADT SANT DY MELSSLRSEDTAVYYCARGGYRYDDYFDYWGQGTLVTVSS E VQL VQ S GAE VKKPGAS VKVS CKAS GY T FTDTYMHWVRQAHuEM1031-K31-VH.lc 29 PGQ RL E WZGRIDPANGNTKYDPKFQGKAT I T ADT S ANT D Y MELSSLRSEDTAVYYCARGGYRYDDYFDYWGQGTLVTVSS EVOLVOSGAEVKKPGASVKVSCKASGFNIDDTYMHWVRQAHuEM1031-K31-VH.ld 30 PGQ RL E WZGRIDPANGNTKYDPKFQGKAT I T ADT S ANT D Y MELSSLRSEDTAVYYCARGGYRYDDYFDYWGQGTLVTVSS EVOLVOSGAEVKKPGASVKVSCKASGFNIDDTYMHWVRQAHuEM1031-K31-VH.le 31 PGQGL E WZGRIDPANGNTKYDPKFQGKAT I T ADT S ANT D Y MELSSLRSEDTAVYYCARGGYRYDDYFDYWGQGTLVTVSS EVQLQQSGAEFVKPGASVKLSCTASGFNIDDTYMHWVKQR EM1031-K31-VH (G55A) 32 P EQGL Y W I GRIDPAN ANTKYDPKFQGKAT I T ADT S SNT D Y LQLSSLTSEDTAVYYCARGGYRYDDYFDYWGQGTTLTVSS EVOLVOSGAEVKKPGASVKVSCKASGFNIDDTYMHWVRQAHuEM1031-K31-VH.ld33 PGQ RL E WZGRIDPAN ANTKYDPKFQGKAT I T ADT S ANT D Y (G55A) MELSSLRSEDTAVYYCARGGYRYDDYFDYWGQGTLVTVSS DI VLTQS PASLAVSLGQRAT I SCRASETVDSYGSSFMHWY EM1031-K31-VK 22 QQKSGQPPKLLIYRASTLESGIPARFSGSGSRTDFTLTIN AVEADDVATYYCQQNNEDPFTFGGGTKLEIK DIVMTQSPDSLAVSLGERATI NCRASETVDSYGSSFMHW Y HUEM1031-K31-VK.1 34 QQKPGQPPKLLIYRASTLESGVPDRFSGSGSGTDFTLTIS SLQAEDVAVYYCQQNNEDPFTFGQGTKLEIK D I VLT QSPDSLAVSLGE RAT I NCRASETVDSYGSSFMHWYHuEM1031-K31-VK.la 35 QQKPGQPPKLLIYRASTLESGVPDRFSGSGSGTDFTLTIS SLQAEDVAVYYCQQNNEDPFTFGQGTKLEIK D I VLT QSPDSLAVSLGE RAT I NCRASETVDSYGSSFMHWYHuEM1031-K31-VK.lb 36 QQKPGQPPKLLIYRASTLESGZPDRFSGSGSGTDFTLTIS SLQAEDVAVYYCQQNNEDPFTFGQGTKLEIK D I VLT QSPDSLAVSLGE RAT I NCRASETVDSYGSSFMHWY HUEM1031-K31-VK.1C 37 QQKPGQPPKLLIYRASTLESGZPDRFSGSGSRTDFTLTIS SLQAEDVAVYYCQQNNEDPFTFGQGTKLEIK DI VLTQS PASLAVSLGQRAT I SCRASETVDAYGSSFMHWY EM1031-K31-VK (S31A) 38 QQKSGQPPKLLIYRASTLESGIPARFSGSGSRTDFTLTIN AVEADDVATYYCQQNNEDPFTFGGGTKLEIK DIVMTQSPDSLAVSLGERATI NCRASETVDAYGSSFMHW Y HUEM1031-K31-VK.139 QQKPGQPPKLLIYRASTLESGVPDRFSGSGSGTDFTLTIS (S31A) SLQAEDVAVYYCQQNNEDPFTFGQGTKLEIK EVQLQQSGAELVRPGALLKLSCKASGFNIKDYYMHWVKQR EM1031-K97-VH 23 P EQGL E W I GWIDPENGNTIYDPRFQGKAN I T ADT S SNT AYLQLSSLTSEDTAVYYCARSFYYGYLRFDYWGQGTLVAVSAWSGR Docket No. 65457-712.602SEQ ID Amino acid sequenceVariable DomainNO: 1234567890123456789012345678901234567890EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAHuEM1031-K97-VH.la 40 PGKGL E WMGWIDPENGNTIYDPRFQGRVT I T ADT S T DT AY MELSSLRSEDTAVYYCARSFYYGYLRFDYWGQGTLVTVSS EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAHuEM1031-K97-VH.lb 41 PGKGL EWMGWIDPENGNTIYDPRFQGRVT I T ADT S TNT AY MELSSLRSEDTAVYYCARSFYYGYLRFDYWGQGTLVTVSS EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAHuEM1031-K97-VH.lc 42 PGKGL E WPGWIDPENGNTIYDPRFQGKANI T ADT S TNT AY MELSSLRSEDTAVYYCARSFYYGYLRFDYWGQGTLVTVSS EVOLVQSGAEVKKPGATVKISCKVSGFNIKDYYMHWVOOAHuEM1031-K97-VH.ld 43 PGKGL E WPGWIDPENGNTIYDPRFQGKANI T ADT S TNT AY MELSSLRSEDTAVYYCARSFYYGYLRFDYWGQGTLVTVSS EVOLVQSGAEVKKPGATVKISCKASGFNIKDYYMHWVOOAHuEM1031-K97-VH.le 44 PGKGL E WPGWIDPENGNTIYDPRFQGKANI T ADT S TNT AY MELSSLRSEDTAVYYCARSFYYGYLRFDYWGQGTLVTVSS EVQLQQSGAELVRPGALLKLSCKASGFNIKDYYMHWVKQR EM1031-K97-VH (G55A) 45 P EQGL E W I GWIDPENANTIYDPRFQGKAN I T ADT S SNT AY LQL S S LT S E DT AVY Y CARSFYYGYLRFDY WGQGTL VAVS A EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAHuEM1031-K97-VH.la46 PGKGL EWMGWIDPENGNTIYDPRFQGRVT I T ADT S T DT AY (G55A) MELSSLRSEDTAVYYCARSFYYGYLRFDYWGQGTLVTVSS E I VLT Q S P A I T AAS L GQ KVT I T C SASSSVSYMH W Y QQ KS G EM1031-K97-VK 24 TSPKPWIYEISKLASGVPARFSGSGSGTSFSLTISSMEAE DAAIYYCQQWNYPLFTFGAGTKLELK D I QLT Q S P S FL SAS VGDRVT I TC SASSSVSYMH WY QQ KPG HUEM1031-K97-VK.1 47 KAPKLLIYEISKLASGVPSRFSGSGSGTEFTLTISSLQPE DFATYYCQQWNYPLFTFGQGTKLEIK D I QLT Q S P S FL SAS VGDRVT I TC SASSSVSYMH WY QQ KPGHuEM1031-K97-VK.la 48 KSPKPWIYEISKLASGVPSRFSGSGSGTEFTLTISSLQPE DFATYYCQQWNYPLFTFGQGTKLEIK EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQK EM1031-K112-VH 25 PGQGLEWI GYISPYNDGTKYNEKFKGKATLT SDKSSSTAY MYLSSLTSEDSAVYYCARGGGLEYCFDYWGQGTTLTVSS E VQL VQ S GAE VKKPGAS VKVS CKAS GY T FTSYVMHWVRQAHuEM1031-K112-VH.la 49 PGQ RLEWMGYISPYNDGTKYNEKFKGRVT I T S DT SAS T AY MELSSLRSEDTAVYY CARGGGLEYCFDYWGQ GTLVTVSS E VQL VQ S GAE VKKPGAS VKVS CKAS GY T FT SYVMHWVRQAHuEM1031-K112-VH.lb 50 PGQ RLEWMGYISPYNDGTKYNEKFKGRVTLTSDT SAS T AY MELSSLRSEDTAVYYCARGGGLEYCFDYWGQGTLVTVSS E VQL VQ S GAE VKKPGAS VKVS CKAS GY T FT SYVMHWVRQAHuEM1031-K112-VH.lc 51 PGQ RLEWMGYISPYNDGTKYNEKFKGRVTLTSDKSAS T AY MELSSLRSEDTAVYY CARGGGLEYCFDYWGQ GTLVTVSS E VQL VQ S GAE VKKPGAS VKVS CKAS GY T FT SYVMHWVRQAHuEM1031-K112-VH.ld 52 PGQ RL E W PG YISPYNDGTKYNEKFKGKAT LT S DKS AS T AY MELSSLRSEDTAVYY CARGGGLEYCFDYWGQ GTLVTVSS E VQL VQ S GAE VKKPGAS VKVS CKAS GY T FT SYVMHWVRQAHuEM1031-K112-VH.le 53 PGQGL E W PG YISPYNDGTKYNEKFKGKAT LT S DKS AS T AY MELSSLRSEDTAVYY CARGGGLEYCFDYWGQ GTLVTVSS EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQK EM1031-K112-VH PGQGLEWI G YISPYNDATKYNEKFKGKAT LT SDKSSSTAY (G56A) 54MYLSSLTSEDSAVYYCARGGGLEYCFDYWGQGTTLTVSSWSGR Docket No. 65457-712.602SEQ ID Amino acid sequenceVariable DomainNO: 1234567890123456789012345678901234567890E VQL VQ S GAE VRRPGAS VRVS CRAS GY T FT SYVMH WVRQ AHuEM1031-K112- 55 PGQGL E WIG YISPYNDATRYNERFRGRAT LT S DRS AS T AY VH.le (G56A) MELSSLRSEDTAVYY C ARGGGLEYSFDY WGQ GTLVTVSSD I L L T Q S P A I L S V S P G E R V S F S C RASQSIGTSIH W Y Q Q R T EM1031-K112-VK 26 NGSPRLLIRYASESISGIPSRFSGSGSGTDFTLSINSVES EDIADYYCQQTNSWPWTFGGGTRLEIR E I VLTQS PD FQ S VT PRE KVT I TCRASQSIGTSIHWYQQRP HUEM1031-K112-VK.1 56 DQSPRLLIRYASESISGVPSRFSGSGSGTDFTLTINSLEA EDAATYYCQQTNSWPWT FGQGTRLE IR E I VLTQS PD FQ S VT PRE RVT I SCRASQSIGTSIHWYQQRPHuEM1031-K112-VK.la 57 DQSPRLLIRYASESISGVPSRFSGSGSGTDFTLTINSLEA EDAATYYCQQTNSWPWT FGQGTRLE IR E I VLTQS PD FQ S VT PRE RVT I SCRASQSIGTSIHWYQQRP HUEM1031-K112-VK. lb 58 DQSPRLLIRYASESISGZPSRFSGSGSGTDFTLTINSLEA EDAATYYCQQTNSWPWT FGQGTRLE IR PIVLTQSPDFQSVTPRERVTI SCRASQSIGTSIHWYQQRP HUEM1031-K112-VK.1C 59 DQSPRLLIRYASESISGZPSRFSGSGSGTDFTLTINSLEA EDAATYYCQQTNSWPWT FGQGTRLE IR DILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRT EM1031-K112-VK (S93A) 60 NGSPRLLIRYASESISGIPSRFSGSGSGTDFTLSINSVES EDIADYYCQQTNAWPWTFGGGTRLEIR EIVLTQSPDFQSVTPRERVTISCRASQSIGTSIHWYQQRHuEM1031-K112- 61 PDQSPRLLIRYASESISGVPSRFSGSGSGTDFTLTINSL VK.la (S93A) EAEDAATYYCQQTNAWPWT FGQGTRLE I RCDR sequences according to the Kabat Numbering are single underlined; framework back-mutations are double underlined; NG (Asn-Gly) to NA (Asn-Ala), NS (Asn-Ser) to NA (Asn-Ala), DG (Asp-Gly) to DA (Asp-Ala) and DS (Asp-Ser) to DA (Asp-Ala) mutations are in bold italic.Example 3: Generation and characterization of humanized KLK2*CD3 FIT-Ig bispecific antibodies

[0268] A group of FIT-Ig proteins recognizing both human KLK2 and human CD3 were constructed utilizing VH / VL sequences in Table 7 as anti-KLK2 moiety, VH / VL sequences in Table 1 as anti-CD3 moiety, and human constant region sequences in Table 2.

[0269] The specific methods referred to those recorded in Example 1.2 above, except that anti- KLK2 VH and VL used are humanized sequences as shown in Table 7.

[0270] Information of constructed humanized KLK2*CD3 FIT-Ig proteins were listed in Table 8.Table 8: Production of FIT-Ig proteins with humanized anti-KLK2 VH / VLFIT-Ig Identifier VHKLKZ VLKLKZ VHCDS VLCDS HuEM1031-K31- HuEM1031-K31- EM0006- EM0006- FIT2023-10ah-lVH.la VK.1 OlVH.lh 01VK.l(s31aA) HuEM1031-K31- HuEM1031-K31- EM0006- EM0006- FIT2023-10ah-2VH.lb VK.1 OlVH.lh 01VK.l(s31aA)WSGR Docket No. 65457-712.602HuEM1031-K31- HuEM1031-K31- EM0006- EM0006- FIT2023-10ah-3VH.lc VK.1 OlVH.lh 01VK.l(s31aA) HuEM1031-K31- HuEM1031-K31- EM0006- EM0006- FIT2023-10ah-4VH.ld VK.1 OlVH.lh 01VK.l(s31aA) HuEM1031-K31- HuEM1031-K31- EM0006- EM0006- FIT2023-10ah-5VH.le VK.1 OlVH.lh 01VK.l(s31aA) HuEM1031-K31- HuEM1031-K31- EM0006- EM0006- FIT2023-10ah-6VH.la VK.la OlVH.lh 01VK.l(s31aA) HuEM1031-K31- HuEM1031-K31- EM0006- EM0006- FIT2023-10ah-7VH.lb VK.la OlVH.lh 01VK.l(s31aA) HuEM1031-K31- HuEM1031-K31- EM0006- EM0006- FIT2023-10ah-8VH.lc VK.la OlVH.lh 01VK.l(s31aA) HuEM1031-K31- HuEM1031-K31- EM0006- EM0006- FIT2023-10ah-9VH.ld VK.la OlVH.lh 01VK.l(s31aA) HuEM1031-K31- HuEM1031-K31- EM0006- EM0006- FIT2023-10ah-10VH.le VK.la OlVH.lh 01VK.l(s31aA) HuEM1031-K31- HuEM1031-K31- EM0006- EM0006- FIT2023-10ah-llVH.la VK.lb OlVH.lh 01VK.l(s31aA) HuEM1031-K31- HuEM1031-K31- EM0006- EM0006- FIT2023-10ah-12VH.lb VK.lb OlVH.lh 01VK.l(s31aA) HuEM1031-K31- HuEM1031-K31- EM0006- EM0006- FIT2023-10ah-13VH.lc VK.lb OlVH.lh 01VK.l(s31aA) HuEM1031-K31- HuEM1031-K31- EM0006- EM0006- FIT2023-10ah-14VH.ld VK.lb OlVH.lh 01VK.l(s31aA) HuEM1031-K31- HuEM1031-K31- EM0006- EM0006- FIT2023-10ah-15VH.le VK.lb OlVH.lh 01VK.l(s31aA) HuEM1031-K31- HuEM1031-K31- EM0006- EM0006- FIT2023-10ah-16VH.la VK.1C OlVH.lh 01VK.l(s31aA) HuEM1031-K31- HuEM1031-K31- EM0006- EM0006- FIT2023-10ah-1701VK.l(s31aA) VH.lb VK.1C OlVH.lhHuEM1031-K31- HuEM1031-K31- EM0006- EM0006- FIT2023-10ah-18VH.lc VK.1C OlVH.lh 01VK.l(s31aA) HuEM1031-K31- HuEM1031-K31- EM0006- EM0006- FIT2023-10ah-19VH.ld VK.1C OlVH.lh 01VK.l(s31aA) HuEM1031-K31- HuEM1031-K31- EM0006- EM0006- FIT2023-10ah-20VH.le VK.1C OlVH.lh 01VK.l(s31aA) FIT2023-10ac HuEM1031-K31- EM0006- EM0006- EM1031-K31-VK(VH.le) VH.le OlVH.lh 01VK.l(s31aA)HuEM1031-K31- EM0006- EM0006- FIT2023-10acEM1031-K31-VH(VK.IC) VK.1C OlVH.lh 01VK.l(s31aA) FIT2023-10ac EM1031-K31-VH EM0006- EM0006- EM1031-K31-VK(G55A) (G55A) OlVH.lh 01VK.l(s31aA) FIT2023-10ac EM0006- EM0006- EM1031-K31-VH EM1031-K31-VK(S31A) (S31A) OlVH.lh 01VK.l(s31aA)EM0006- EM0006- FIT2023-10ac EM1031-K31-VH EM1031-K31-VKOlVH.lh 01VK.l(s31aA) FIT2023-10ah- HuEM1031-K31- HuEM1031-K31- EM0006- EM0006- VH.ld (G55A) VK.1 (S31A) 01VK.l(s31aA) OlVH.lh 4(h)HuEM1031-K97- HuEM1031-K97- EM0006- EM0006- FIT2023-20ah-lVK.1 01VK.l(s31aA) VH.la OlVH.lhHuEM1031-K97- HuEM1031-K97- EM0006- EM0006- FIT2023-20ah-2VK.1 01VK.l(s31aA) VH.lb OlVH.lhFIT2023-20ah-3 HuEM1031-K97- HuEM1031-K97- EM0006- EM0006-WSGR Docket No. 65457-712.602VH.le VK.1 OlVH.lh 01VK.l(s31aA) HuEM1031-K97- HuEM1031-K97- EM0006- EM0006- FIT2023-20ah-4VH.ld VK.1 OlVH.lh 01VK.l(s31aA) HuEM1031-K97- HuEM1031-K97- EM0006- EM0006- FIT2023-20ah-5VH.le VK.1 OlVH.lh 01VK.l(s31aA) HuEM1031-K97- HuEM1031-K97- EM0006- EM0006- FIT2023-20ah-6VH.la VK.la OlVH.lh 01VK.l(s31aA) HuEM1031-K97- HuEM1031-K97- EM0006- EM0006- FIT2023-20ah-7VH.lb VK.la OlVH.lh 01VK.l(s31aA) HuEM1031-K97- HuEM1031-K97- EM0006- EM0006- FIT2023-20ah-8VH.lc VK.la OlVH.lh 01VK.l(s31aA) HuEM1031-K97- HuEM1031-K97- EM0006- EM0006- FIT2023-20ah-9VH.ld VK.la OlVH.lh 01VK.l(s31aA) HuEM1031-K97- HuEM1031-K97- EM0006- EM0006- FIT2023-20ah-10VH.le VK.la OlVH.lh 01VK.l(s31aA) FIT2023-20ac HuEM1031-K97- EM0006- EM0006- EM1031-K97-VK(VH.le) VH.le OlVH.lh 01VK.l(s31aA) FIT2023-20ac HuEM1031-K97- EM0006- EM0006- EM1031-K97-VH(VK.la) VK.la OlVH.lh 01VK.l(s31aA) FIT2023-20ac EM1031-K97-VH EM0006- EM0006- EM1031-K97-VK(G55A) (G55A) OlVH.lh 01VK.l(s31aA)EM0006- EM0006- FIT2023-20ac EM1031-K97-VH EM1031-K97-VKOlVH.lh 01VK.l(s31aA) FIT2023-20ah- HuEM1031-K97- HuEM1031-K97- EM0006- EM0006- 1(h) VH.la (G55A) VK.1 OlVH.lh 01VK.l(s31aA)HuEM1031-K112- HuEM1031-K112- EM0006- EM0006- FIT2023-22ah-lVH.la VK.1 OlVH.lh 01VK.l(s31aA) HuEM1031-K112- HuEM1031-K112- EM0006- EM0006- FIT2023-22ah-2VH.lb VK.1 OlVH.lh 01VK.l(s31aA) HuEM1031-K112- HuEM1031-K112- EM0006- EM0006- FIT2023-22ah-3VH.lc VK.1 OlVH.lh 01VK.l(s31aA) HuEM1031-K112- HuEM1031-K112- EM0006- EM0006- FIT2023-22ah-4VH.ld VK.1 OlVH.lh 01VK.l(s31aA) HuEM1031-K112- HuEM1031-K112- EM0006- EM0006- FIT2023-22ah-5VH.le VK.1 OlVH.lh 01VK.l(s31aA) HuEM1031-K112- EM0006- EM0006- FIT2023-22ah-6 HuEM1031-K112- VH.la VK.la OlVH.lh 01VK.l(s31aA) HuEM1031-K112- HuEM1031-K112- EM0006- EM0006- FIT2023-22ah-7VH.lb VK.la OlVH.lh 01VK.l(s31aA) HuEM1031-K112- HuEM1031-K112- EM0006- EM0006- FIT2023-22ah-8VH.lc VK.la OlVH.lh 01VK.l(s31aA) HuEM1031-K112- HuEM1031-K112- EM0006- EM0006- FIT2023-22ah-9VH.ld VK.la OlVH.lh 01VK.l(s31aA) HuEM1031-K112- HuEM1031-K112- EM0006- EM0006- FIT2023-22ah-10VH.le VK.la OlVH.lh 01VK.l(s31aA) HuEM1031-K112- EM0006- EM0006- FIT2023-22ah-ll HuEM1031-K112- VH.la VK.lb OlVH.lh 01VK.l(s31aA) HuEM1031-K112- HuEM1031-K112- EM0006- EM0006- FIT2023-22ah-12VH.lb VK.lb OlVH.lh 01VK.l(s31aA) HuEM1031-K112- HuEM1031-K112- EM0006- EM0006- FIT2023-22ah-13VH.lc VK.lb OlVH.lh 01VK.l(s31aA) HuEM1031-K112- HuEM1031-K112- EM0006- EM0006- FIT2023-22ah-14VH.ld VK.lb OlVH.lh 01VK.l(s31aA)WSGR Docket No. 65457-712.602HuEM1031-K112- HuEM1031-K112- EM0006- EM0006- FIT2023-22ah-15VH.le VK.lb OlVH.lh 01VK.l(s31aA) HuEM1031-K112- EM0006- EM0006- FIT2023-22ah-16 HuEM1031-K112- VH.la VK.1C OlVH.lh 01VK.l(s31aA) HuEM1031-K112- HuEM1031-K112- EM0006- EM0006- FIT2023-22ah-17VH.lb VK.1C OlVH.lh 01VK.l(s31aA) HuEM1031-K112- HuEM1031-K112- EM0006- EM0006- FIT2023-22ah-18VH.lc VK.1C OlVH.lh 01VK.l(s31aA) HuEM1031-K112- HuEM1031-K112- EM0006- EM0006- FIT2023-22ah-19VH.ld VK.1C OlVH.lh 01VK.l(s31aA) HuEM1031-K112- HuEM1031-K112- EM0006- EM0006- FIT2023-22ah-20VH.le VK.1C OlVH.lh 01VK.l(s31aA) FIT2023-22ac HuEM1031-K112- HuEM1031-K112- EM0006- EM0006- (VH.le) VH.le VK OlVH.lh 01VK.l(s31aA) FIT2023-22ac HuEM1031-K112- HuEM1031-K112- EM0006- EM0006- (VK.IC) VH VK.1C OlVH.lh 01VK.l(s31aA) FIT2023-22ac EM1031-K112-VH HuEM1031-K112- EM0006- EM0006- (G56A) (G56A) VK OlVH.lh 01VK.l(s31aA) FIT2023-22ac HuEM1031-K112- EM1031-K112-VK EM0006- EM0006- (S93A) VH (S93A) OlVH.lh 01VK.l(s31aA) HuEM1031-K112- HuEM1031-K112- EM0006- EM0006- FIT2023-22acVH VK OlVH.lh 01VK.l(s31aA) FIT2023-22ah- HuEM1031-K112- HuEM1031-K112- EM0006- EM0006-10(h) VH.le (G56A) VK.la (S93A) OlVH.lh 01VK.l(s31aA)

[0271] Table 9 exemplified the sequences of several humanized KLK2*CD3 FIT-Ig proteins.Table 9: Sequences of humanized KLK2xCD3 FIT-IgsSEQID Amino Acid SequenceDescriptionNO: 12345678901234567890123456789012345678901234567890D I VMTQS PD S LAVS LGERATINCRASETVDAYGS S FMHWYQQKPGQPPKL LIYRASTLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNEDPF TFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLS S PVTKS FNRGECEVQLVQSGAEVKKPGASVKVSCKASGFSFTNY FIT2023-10ah-4(h) YVHWMRQAPGQGLEWMGWISPGSDNTKYNEKFKGRVTMTRDTSISTAYME LSRLRSDDTAVYYCARDDYGNYYFDYWGQGTTVTVS SAS T KGP S VFP LAP85 Chain #1 SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY (VLKLK2-CL- SLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP VHCD3S-CH1-FC) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK EVQLVQSGAEVKKPGASVKVSCKASGFNIDDTYMHWVRQAPGQRLEWIGR FIT2023-10ah-4(h) IDPANANTKYDPKFQGKATITADTSANTDYMELSSLRSEDTAVYYCARGG 86 YRYDDYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK Chain #2 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQT (VHKLK2-CH1) YICNVNHKPSNTKVDKKVEPKSC DIVMTQSPDSLAVSLGERATINCKSSQSLLNARTRKNYLAWYQQKPGQPP FIT2023-10ah-4(h) KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYIL 87 RTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAK Chain #3 VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE (VLCD3e-CL) VTHQGLSS PVTKS FNRGECFIT2023-20ah-l(h) DIQLTQSPSFLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLLIYEI 88SKLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQWNYPLFTFGQGWSGR Docket No. 65457-712.602SEQID Amino Acid SequenceDescriptionNO: 12345678901234567890123456789012345678901234567890Chain #1 TKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVD (VLKLK2-CL- NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL VHCD3E-CH1-FC) S S PVTKS FNRGECEVQLVQSGAEVKKPGASVKVSCKASGFSFTNYYVHWM RQAPGQGLEWMGWISPGSDNTKYNEKFKGRVTMTRDTSISTAYMELSRLR SDDTAVYYCARDDYGNYYFDYWGQGTTVTVS SAS T KGP S VFP LAP S S KS T SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAA GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK EVQLVQS GAEVKKPGATVKI S CKVS GYTFTD Y YMHWVQQAPGKGLEWMGW FIT2023-20ah-l(h) IDPENANTIYDPRFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCARSF 89 YYGYLRFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK Chain #2 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQT (VHKLK2-CH1) YICNVNHKPSNTKVDKKVEPKSC DIVMTQSPDSLAVSLGERATINCKSSQSLLNARTRKNYLAWYQQKPGQPP FIT2023-20ah-l(h) KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYIL 90 RTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAK Chain #3 VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE (VLCD3e-CL) VTHQGLSS PVTKS FNRGEC EIVLTQSPDFQSVTPKEKVTISCRASQSIGTSIHWYQQKPDQSPKLLIKY ASESISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQTNAWPWTFGQ GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LS S PVTKS FNRGECEVQLVQSGAEVKKPGASVKVSCKASGFSFTNYYVHW FIT2023-22ah-10(h) MRQAPGQGLEWMGWISPGSDNTKYNEKFKGRVTMTRDTSISTAYMELSRL RSDDTAVYYCARDDYGNYYFDYWGQGTTVTVS SAS T KGP S VFP LAP S S KS91 Chain #1 TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS (VLKLK2-CL- WTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEA VHCD3E-CH1-FC) AGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALAAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVKQAPGQGLEWIGY FIT2023-22ah-10(h) ISPYNDATKYNEKFKGKATLTSDKSASTAYMELSSLRSEDTAVYYCARGG 92 GLEYSFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD Chain #2 YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTY (VHKLK2-CH1) ICNVNHKPSNTKVDKKVEPKSC DIVMTQSPDSLAVSLGERATINCKSSQSLLNARTRKNYLAWYQQKPGQ FIT2023-22ah-10(h) PPKLLI YWAS TRE S GVPDRFS GSGS GTDFTLT I S SLQAEDVAVYYCKQ 93 SYILRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNF Chain #3YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE(VLCD3E-CL)KHKVYACEVT HQGL S S PVTKS FNRGECVariable domains in bold.

[0272] Recombinant FIT-Igs listed in Table 8 were transiently expressed and purified.Specifically, for each FIT-Ig construct, 3 plasmids encoding each of the 3 polypeptide chains were co-transfected into Expi293 cells and cell culture was performed. After approximately six days of post-transfection, supernatants of each of cell cultures were separately harvested and individualed to Protein A affinity chromatography, followed by size exclusion chromatography (SEC) using a TSKgel SuperSW3000, 300 x 4.6 mm, SEC column (TOSOH). A DIONEX™WSGR Docket No. 65457-712.602UltiMate 3000 HPLC instrument (Thermo Scientific) was used for SEC using UV detection at 280 nm and 214 nm. The expression and SEC-HPLC results were shown in Table 10.

[0273] The FIT-Ig proteins were assayed for and ranked by dissociation rate constant (koff, "off-rate") using an Octet®RED96 biolayer interferometry (Pall ForteBio LLC). Specifically, anti-hlgG Fc Capture (AHC) Biosensors (Pall) were first exposed to KLK2*CD3 FIT-Ig bispecific antibodies in Table 8 at a concentration of 100 nM for 120 seconds to capture the antibodies, then dipped into running buffer (IX PBS pH 7.2, 0.05% Tween 20, 0.1% BSA) for 30 seconds to check baseline. Sensors with captured antibodies were dipped into 3.097 ug / ml recombinant human KLK2 protein solutions for 200 seconds to measure association, followed by dipping into running buffer for 500 seconds to measure dissociation. The association and dissociation curves were fitted to a 1:1 Langmuir binding model using ForteBio Data Analysis software (Pall). Results were shown in Table 10. The off-rate ratios were calculated by the off-rate of the antibody to that of the chimeric bispecific FIT-Ig in Example 1, respectively. Lower ratio indicated slower dissociation of the antibody in comparison with the parental chimeric antibodies in Example 1.Table 10: Expression and off-rate ranking of humanized and chimeric FIT-Ig proteins Purity% (SEC- FIT-Ig Identifier Expression Titer (mg / L) Off-rate Ratio HPLC)FIT2023-10ah-l 61.67 84.67 NAFIT2023-10ah-2 37.67 79.07 NAFIT2023-10ah-3 7.67 77.71 NAFIT2023-10ah-4 15.00 90.77 31%FIT2023-10ah-5 30.33 85.71 63%FIT2023-10ah-6 54.00 66.17 NAFIT2023-10ah-7 67.67 73.34 NAFIT2023-10ah-8 17.67 91.39 NAFIT2023-10ah-9 50.67 94.78 106%FIT2023-10ah-10 98.00 88.56 204%FIT2023-10ah-ll 60.33 92.53 NAFIT2023-10ah-12 72.00 91.35 NAFIT2023-10ah-13 45.67 85.35 NAFIT2023-10ah-14 96.33 86.68 201%FIT2023-10ah-15 53.33 78.31 111%FIT2023-10ah-16 45.67 75.57 NAFIT2023-10ah-17 27.33 74.01 NAFIT2023-10ah-18 15.33 71.08 NAFIT2023-10ah-19 36.00 77.05 75%FIT2023-10ah-20 44.33 77.11 92%FIT2023-10ac(VH.le) 70.67 84.65 147%FIT2023-10ac(VK.lc) 36.67 85.3 76%FIT2023-10ac(G55A) 47.33 81.64 99%FIT2023-10ac(S31A) 40.67 67.29 85%WSGR Docket No. 65457-712.602Purity% (SEC- FIT-Ig Identifier Expression Titer (mg / L) Off-rate Ratio HPLC)FIT2023-10ac 48.00 69.38 100%FIT2023-10ah-4(h) 8.55 88.65 100%FIT2023-20ah-l 103.73 86.03 85%FIT2023-20ah-2 81.33 87.31 67%FIT2023-20ah-3 113.60 82.04 93%FIT2023-20ah-4 100.00 86.75 82%FIT2023-20ah-5 181.33 86.48 149%FIT2023-20ah-6 90.13 83.22 74%FIT2023-20ah-7 26.67 64.81 22%FIT2023-20ah-8 123.20 86.18 101%FIT2023-20ah-9 69.60 86.48 57%FIT2023-20ah-10 175.73 83.65 145%FIT2023-20ac(VH.le) 135.20 90.43 111%FIT2023-20ac(VK.la) 216.00 84.49 178%FIT2023-20ac(G55A) 140.53 91.72 116%FIT2023-20ac 121.60 71.32 100%FIT2023-20ah-l(h) 116.4 82.74 100%FIT2023-22ah-l 136.00 94.4 120%FIT2023-22ah-2 157.33 93.4 138%FIT2023-22ah-3 27.33 96.48 24%FIT2023-22ah-4 15.00 96.39 13%FIT2023-22ah-5 95.33 95.68 84%FIT2023-22ah-6 37.33 94.25 33%FIT2023-22ah-7 65.67 94.84 58%FIT2023-22ah-8 98.00 91.28 86%FIT2023-22ah-9 69.67 96.04 61%FIT2023-22ah-10 63.00 94.54 55%FIT2023-22ah-ll 14.67 96.55 13%FIT2023-22ah-12 62.67 95.16 55%FIT2023-22ah-13 36.00 95.09 32%FIT2023-22ah-14 61.33 94.63 54%FIT2023-22ah-15 60.67 93.52 53%FIT2023-22ah-16 73.00 92.04 64%FIT2023-22ah-17 57.00 91.88 50%FIT2023-22ah-18 62.67 92.73 55%FIT2023-22ah-19 70.67 92.28 62%FIT2023-22ah-20 38.00 92.57 33%FIT2023-22ac(VH.le) 58.00 84.1 51%FIT2023-22ac(VK.lc) 49.33 94.24 43%FIT2023-22ac(G56A) 77.33 94.78 68%FIT2023-22ac(S93A) 20.67 96.01 18%FIT2023-22ac 113.67 92.65 100%FIT2023-22ah- 10(h) 105.96 92.56 100%NA: Not Applicable; ND: No Data.Example 4: Binding kinetics characterization of humanized KLK2*CD3 FIT-Ig antibodies

[0274] Binding affinities and kinetics constants of humanized KLK2 FIT-Igs were determined using a grating-coupled interferometry (GCI) based WAVEsystem (Creoptix AG, Switzerland), a label-free biosensor. Briefly, a goat anti-human IgGFc antibody was immobilized onto a PCPWSGR Docket No. 65457-712.602WAVEchip (Creoptix AG) at a density of 4059.7 pg / mm2on Channel 1, 3923.6 pg / mm2on Channel 2, 4720.3pg / mm2on Channel 3 and 4625.4 pg / mm2on Channel 4 by amine coupling. KLK2*CD3 FIT-Ig bispecific antibodies in Table 8 were then captured onto the PCP WAVEchip at a density of 3091.8-8763.2 pg / mm2. Recombinant human KLK2, KLK3, or CD3a & CD35 proteins were injected at 3-fold serial diluted concentrations (1.23 nM, 3.7 nM, 11.11 nM, 33.33 nM, 100 nM for KLK2 and KLK3; 0.25 nM, 0.74 nM, 2.22 nM, 6.67 nM, 20 nM for CD3s&CD36) in kinetics buffer (BR100669Cat No 22064428-AD). The recombinant proteins were injected for 250s at a flow rate of 60 pL / min and dissociation was set to 1000s. After each round of association and dissociation, WAVEchip surface was regenerated by injection of Glycine-HCl buffer at pH 1.5 for 120s at a flow rate of 80 pL / min. All sensorgrams were recorded at 25°C and the data analyzed on the WAVEcontrol (Creoptix AG). Data was double referenced by subtracting the signals from blank injections and from the reference channel. A 1:1 Kinetic model was used for KLK2 affinity data fitting, and conformational change model was used for CD3s & CD36 affinity data fitting. All the FIT-Igs showed no detectable binding activity to human KLK3. Kinetics constants were shown in Table 11.Table 11: Binding kinetics of humanized FIT-Ig bispecific antibodiesKLK2 CD3s & CD36Sample IDka (M-1s-1) kd (s'1) Kd(M) ka (M-1s-1) kd (s'1) Kd(M) FIT2023-10ah-4(h) 7.78E+05 3.80E-03 4.89E-09 1.12E+06 1.84E-02 1.65E-08 FIT2023-20ah-l(h) 2.50E+06 1.61E-03 6.41E-10 2.73E+06 1.48E-02 5.41E-09FIT2023-22ah- 10(h) 1.23E+06 1.83E-03 1.49E-09 ND ND NDND: No DataExample 5: Redirected T cell cytotoxicity of humanized FIT-Ig

[0275] A cocultured killing assay as described in Example 1.7 was used for the comparison of humanized anti-KLK><CD3 FIT-Igs with the reference molecule Bi2023-4a (reference BsAb (KL2B30) targeting human KLK2 and CD3, as disclosed in US20210040210A1). The parental CD3 monoclonal antibody huEM0006-01-24 was used as negative control. The results were showed in FIG. 4.

[0276] The ECso of FIT2023-10ah-4(h), FIT2023-20ah-l(h), FIT2023-22ah- 10(h), and Bi2023-4a were 0.0007078 nM, 0.06165 nM, 0.1356 nM and 0.4206 nM, respectively. As shown in FIG. 4, FIT2023-10ah-4(h) and FIT2023-20ah-l(h) showed concentration-dependent killing activity to both VCaP and LNCaP-KLK2 cell lines.WSGR Docket No. 65457-712.602Example 6: Cytokine release of T cells cocultured with target cells and induced by humanized KLK2xCD3 bispecific antibodies

[0277] To measure the cytokine release in the redirected T cells cytotoxicity assay as described in Example 5, the supernatants were harvested and measured for human IL-2 (FIG. 5A and FIG. 5F), IL-6 (FIG. 5B and FIG. 5G), IL- 10 (FIG. 5C and FIG. 5H), TNFa (FIG. 5D and FIG. 51) and IFNy (FIG. 5E and FIG. 5J) using an antibody captured bead-based kit (LEGENDplex™ HU Thl Panel (5-plex) w / VbP V02, Biolegend) with flowcytometry for readout. The results were showed in FIG. 5A-5J.

[0278] As shown in FIG. 5A-5J, FIT2023-10ah-4(h) showed similar cytokine levels as reference molecule Bi2023-4a; meanwhile, FIT2023-20ah-l(h) and FIT2023-22ah- 10(h) induced much lower cytokine release compared to Bi2023-4a.Example 7: Cytokine release from PBMCs induced by humanized KLK2xCD3 bispecific antibodies

[0279] The ability of test articles to induce in vitro cytokine release from PBMCs was assessed. Cryopreserved PBMCs from normal healthy human volunteers were thawed and cultured in RPMI1640 medium (Gibco RPML1640 Medium supplemented with 10% FCS, 100 lU / mL penicillin, 100 pg / mL streptomycin) with viability >85%, as determined using an automated cell counter prior to experiments. PBMCs were seeded at a density of 1 x 107cells per well into 12-well-plate, followed by incubation for 48 hours at 37°C, 5% CO2. After 48h incubation, PBMCs were transferred into a 96 well plate at a density of 2 105cells per well, and then the cells were stimulated by the test articles, positive controls (i.e. anti-CD3 monoclonal antibodies OKT-3 and HuEM0006-01-24), or isotype control (hlgG) for 24 hours at 37°C, 5% CO2. Then 96-well plates were centrifuged, supernatants were harvested, respectively. Detection of the cytokines in the supernatants was performed using an antibody capture bead-based kit (LEGENDplex™ HU Thl Panel (5-plex) w / VbP V02, Biolegend) and flow cytometric analysis. The results were showed in FIGS. 6A-6E.

[0280] FIGS. 6A-6E showed that FIT2023-10ah-4(h) and FIT2023-20ah-l(h) induced much lower cytokines release in the absence of target cells.WSGR Docket No. 65457-712.602Example 8: In vivo anti-tumor activity of KLK2xCD3 bispecific antibodies8.1: VCaP tumor volume in human PBMC engrafted NCG mice treated with KLK2*CD3bispecific antibodies

[0281] The antitumor efficacy was evaluated in VCaP human prostate cancer xenograft model in PBMC engraft NCG mice, which is an immunodeficient strain lacking T cells, B cells and natural killer cell. 8-10 weeks male NCG mice (GemPharmatech) were used for human PBMC reconstitution and VCaP tumor model establishment. Briefly, the mice received a single intraperitoneal injection of 8* 106human PBMCs and on the same day 10x 106VCaP cells suspended in 0.2ml DPBS (Dulbecco's Phosphate-Buffered Saline) containing 50% ABW Matrigel per animal were subcutaneously injected into the right flank of male NCG mice. 15 days post tumor cell inoculation, huCD45 cells in mouse peripheral blood were detected using FACS, and tumor bearing mice with tumor size at an average of around 100 mm3were selected. Based on the tumor size and huCD45 cell counts, the selected mice were randomized (Stratified blocked randomization) divided into 5 groups, with 8 mice in each group, and treatment was initiated on the same day (Day 0). Mice were treated with 10 mg / kg of FIT2023-10ah-4(h), FIT2023-20ah-l(h), FIT2023-22ah- 10(h), and 5 mg / kg Bi2023-4a or DPBS (as vehicle) respectively by single intraperitoneal (i.p.) injection.

[0282] Tumor volumes were measured 2-3 times per week, and determined by using the following formula:Tumor volume (mm3) = length x width2x 0.5

[0283] For all groups, the percentage of tumor growth inhibition (TGI) compared to vehicle control was defined as follows: / I mean net increased volume of treated tumors •.TGI(%) = ( I - ; - - — - - - - - - ) X 100%mean net increased volume of control tumors

[0284] All the treatment groups were well tolerated (data not shown). FIG. 7 showed the average tumor volumes of the mice on different time points, indicating the inhibition of tumor growth by various treatments. TGI% values based on tumor volume were 127.98%, 120.18%, 93.56% and 114.11% for groups of FIT2023-10ah-4(h), FIT2023-20ah-l(h), FIT2023-22ah-10(h), Bi2023-4a on Day 17, respectively.WSGR Docket No. 65457-712.6028.2: VCaP tumor volume in human PBMC engrafted NCG mice treated with low dose KLK2xCD3 bispecific antibodies

[0285] The antitumor efficacy was evaluated in VCaP human prostate cancer xenograft model in PBMC engraft NCG mice, which is an immunodeficient strain lacking T cells, B cells and natural killer cell. 8-10 weeks male NCG mice (GemPharmatech) were used for human PBMC reconstitution and VCaP tumor model establishment. Briefly, the mice received a single intraperitoneal injection of 8* 106human PBMCs and on the same day 10x 106VCaP cells suspended in 0.2ml DPBS (Dulbecco's Phosphate-Buffered Saline) containing 50% ABW Matrigel per animal were subcutaneously injected into the right flank of male NCG mice. 15 days post tumor cell inoculation, huCD45 cells in mouse peripheral blood were detected using FACS, and tumor bearing mice with tumor size at an average of around 110 mm3were selected. Based on the tumor size and huCD45 cell counts, the selected mice were randomized (Stratified blocked randomization) divided into 4 groups, with 8 mice in each group, and treatment was initiated on the same day (Day 0). Mice were treated with 2 mg / kg of FIT2023-10ah-4(h), 2 mg / kg FIT2023-20ah-l(h), and 1 mg / kg Bi2023-4a or DPBS (as vehicle) respectively by intraperitoneal (i.p.) injection once a week (QW injection).

[0286] All the treatment groups were well tolerated (data not shown). FIG. 8 showed the average tumor volumes of the mice on different time points, indicating the inhibition of tumor growth by various treatments. TGI% values based on tumor volume were 43.7%, 82.27% and 66.23% for groups of FIT2023-10ah-4(h), FIT2023-20ah-l(h), and Bi2023-4a on Day 22, respectively.8.3: LNCaP-KLK2 tumor volume in human PBMC engrafted NCG mice treated with KLK2xCD3 bispecific antibodies

[0287] The antitumor efficacy was also evaluated in LNCaP-KLK2 human prostate cancer xenograft model in PBMC engraft NCG mice, which is an immunodeficient strain lacking T cells, B cells and natural killer cell. 6-8 weeks male NCG mice (Gempharmatech Co., Ltd) were used for human PBMC reconstitution and LNCAP-KLK2 tumor model establishment. Briefly, the mice received a single intraperitoneal injection of 8 106human PBMCs and on the same day, 5 106LNCaP-KLK2 cells suspended in 0.2ml DPBS containing 50% ABW Matrigel per animal were subcutaneously injected into the right flank of male NCG mice. 13 days post tumor cell inoculation, huCD45 cells in mouse peripheral blood were detected using FACS, and tumorsWSGR Docket No. 65457-712.602bearing mice with tumor size at an average of around 120 mm3were selected. Based on the tumor size and huCD45 cell counts, the selected mice were randomized (blocked randomization) divided into 4 groups, with 8 mice in each group, and treatment was initiated on the same day (Day 0). Mice were treated with 10 mg / kg of FIT2023-10ah-4(h), 10 mg / kg of FIT2023-20ah-1(h), 5 mg / kg of Bi2023-4a, or DPBS (as vehicle) respectively by single intraperitoneal (i.p.) injection.

[0288] Tumor volumes and the percentages of tumor growth inhibition (TGI) compared to vehicle control were measured and defined following the method mentioned in Example 8.1.

[0289] All the treatment groups were well tolerated (data not shown). FIG. 9 showed the average tumor volumes of the mice on different time points, indicating the inhibition of tumor growth by various treatments. TGI% values based on tumor volume were 69.41%, 137.52%, and 144.21% for groups of FIT2023-10ah-4(h), FIT2023-20ah-l(h), and Bi2023-4a on Day 16 respectively.Example 9: Stability of humanized KLK2*CD3 FIT-Igs

[0290] To measure the stability of the humanized FIT-Igs, an accelerated stability assay and a freezing and thawing assay were performed.

[0291] In the accelerated stability assay, FIT2023-10ah-4(h) and FIT2023-20ah-l(h) were reconstituted to 1 mg / ml, 50pl of the samples were incubated at 40°C for 1, 3, 7, 10, and 14 days, respectively. Samples were detected by SEC-HPLC and BioDrop for purity and content identification.

[0292] In the freeze-thaw assay, 50pl of the samples were freeze-thawed for 1 to 5 cycles and their purities and contents were detected by SEC-HPLC and BioDrop, respectively.

[0293] Results were shown in FIG. 10 and indicated that all tested antibodies were stable in high temperature and freezing-thawing stress.

[0294] All patents, applications, and publications cited in the above text are incorporated herein by reference.

[0295] Other variations and embodiments of the disclosure described herein will now be apparent to those of skill in the art without departing from the present disclosure or the claims below.WSGR Docket No. 65457-712.602List of SequenceSequenceSEQID Description 1234567890123456789012345678901234567890123456789 NO: 0atgtgggacctggttctctccatcgccttgtctgtggggtgcactggtg ccgtgcccctcatccagtctcggattgtgggaggctgggagtgtgagaa gcattcccaaccctggcaggtggctgtgtacagtcatggatgggcacac tgtgggggtgtcctggtgcacccccagtgggtgctcacagctgcccatt gcctaaagaagaatagccaggtctggctgggtcggcacaacctgtttga gcctgaagacacaggccagagggtccctgtcagccacagcttcccacac ccgctctacaatatgagccttctgaagcatcaaagccttagaccagatg aagactccagccatgacctcatgctgctccgcctgtcagagcctgccaa 1 HUMAN KLK2 CDS gatcacagatgttgtgaaggtcctgggcctgcccacccaggagccagca ctggggaccacctgctacgcctcaggctggggcagcatcgaaccagagg agttcttgcgccccaggagtcttcagtgtgtgagcctccatctcctgtc caatgacatgtgtgctagagcttactctgagaaggtgacagagttcatg ttgtgtgctgggctctggacaggtggtaaagacacttgtgggggtgatt ctgggggtccacttgtctgtaatggtgtgcttcaaggtatcacatcatg gggccctgagccatgtgccctgcctgaaaagcctgctgtgtacaccaag gtggtgcattaccggaagtggatcaaggacaccatcgcagccaacccct ga EVQLVQSGAEVKKPGASVKVSCKASGFSFTNYYVHWMRQAPGQGLEWMG2 Anti-CD3 VHCD3 WISPGSDNTKYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR (EM0006-01VH.lh) DDYGNYYFDYWGQGTTVTVS SAnti-CD3 HCDR13 NYYVH(EM0006-01VH.lh)Anti-CD3 HCDR24 WISPGSDNTKYNEKFKG(EM0006-01VH.lh)Anti-CD3 HCDR35 DDYGNYYFDY(EM0006-01VH.lh)DIVMTQSPDSLAVSLGERATINCKSSQSLLNARTRKNYLAWYQQKPGQP6 Anti-CD3 VLCDS PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSY (EM0006-01VK.l(S31aA)) ILRTFGGGTKVEIKAnti-CD3 LCDR17 KSSQSLLNARTRKNYLA(EM0006-01VK.l(S31aA))Anti-CD3 LCDR28 WAS T RES(EM0006-01VK.l(S31aA))Anti-CD3 LCDR39 KQSYILRT(EM0006-01VK.l(S31aA))EVQLVQSGAEVKKPGASVKVSCKASGFSFTNYYVHWMRQAPGQGLEWMG10 Anti-CD3 VHCD3 WISPGSDNTKYNEKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR (EM0006-01VH.lh) DDYGNYYFDYWGQGTTVTVS S QVQLQESGPGLVKPSDTLSLTCAVSGNSITSDYAWNWIRQPPGKGLEWI11 VH amino acid sequence GYISYSGSTTYNPSLKSRVTMSRDTSKNQFSLKLSSVTAVDTAVYYCAT of the ref molecule hullB6 GYYYGSGFWGQGTLVTVSS DIVLTQSPDSLAVSLGERATINCKASESVEYFGTSLMHWYQQKPGQPPK12 VL amino acid sequence of LLIYAASNRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQTRKV the ref molecule hullB6 PYTFGQGTKLEIK MWDLVLSIALSVGCTGAVPLIQSRIVGGWECEKHSQPWQVAVYSHGWAH CGGVLVHPQWVLTAAHCLKKNSQVWLGRHNLFEPEDTGQRVPVSHSFPH PLYNMSLLKHQSLRPDEDSSHDLMLLRLSEPAKITDWKVLGLPTQEPA13 Amino acid sequence of afull length KLK2 LGTTCYASGWGSIEPEEFLRPRSLQCVSLHLLSNDMCARAYSEKVTEFM LCAGLWTGGKDTCGGDSGGPLVCNGVLQGITSWGPEPCALPEKPAVYTK WHYRKWIKDTIAANP ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVHuman constant IgGl Fc EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVW 14 region with L234A / L235A DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDW mutations LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKWSGR Docket No. 65457-712.602SequenceSEQIDDescription 1234567890123456789012345678901234567890123456789 NO: 0ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVHuman IgGl heavy chain EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVW 15 constant domain (wild DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDW type) LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVHuman IgGl heavy chain EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVW 16 constant, with LALA + DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDW P329A mutations LNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQS17 CL (human kappa constant, GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV CK) TKSFNRGEC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG18 CHI (human IgGl VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKV constant region 1) EPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE19 Human IgGl Fc (hinge- YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC CH2-CH3), wild-type LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEHuman IgGl Fc, with20 YKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LALA + P329A mutations LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK EVQLQQSGAEFVKPGASVKLSCTASGFNIDDTYMHWVKQRPEQGLYWIG EM1031-K3121 RIDPANGNTKYDPKFQGKATITADTSSNTDYLQLSSLTSEDTAVYYCAR VH GGYRYDDYFDYWGQGTTLTVSS DIVLTQSPASLAVSLGQRATISCRASETVDSYGSSFMHWYQQKSGQPPK EM1031-K3122 LLIYRASTLESGIPARFSGSGSRTDFTLTINAVEADDVATYYCQQNNED VL PFTFGGGTKLEIK EVQLQQSGAELVRPGALLKLSCKASGFNIKDYYMHWVKQRPEQGLEWIG EM1031-K9723 WIDPENGNTIYDPRFQGKANITADTSSNTAYLQLSSLTSEDTAVYYCAR VH SFYYGYLRFDYWGQGTLVAVSA EIVLTQSPAITAASLGQKVTITCSASSSVSYMHWYQQKSGTSPKPWIYE EM1031-K9724 ISKLASGVPARFSGSGSGTSFSLTISSMEAEDAAIYYCQQWNYPLFTFG VL AGTKLELK EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIG EM1031-K11225 YISPYNDGTKYNEKFKGKATLTSDKSSSTAYMYLSSLTSEDSAVYYCAR VH GGGLEYS FDYWGQGTTLTVS S DILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNGSPRLLIK EM1031-K11226 YASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQTNSWPWTF VL GGGTKLEIK E VQ L VQ S GAE VKK P GAS VKVS C KAS G YT FT DT YMHWVRQAP GQ RL EWMG27 HuEM1031-K31-VH.la RIDPANGNTKYDPKFQGRVTITADTSASTAYMELSSLRSEDTAVYYCAR GGYRYDDYFDYWGQGTLVTVSS E VQ L VQ S GAE VKK P GAS VKVS C KAS G YT FT DT YMHWVRQAP GQ RL EWMG28 HuEM1031-K31-VH.lb RIDPANGNTKYDPKFQGRVTITADTSANTDYMELSSLRSEDTAVYYCAR GGYRYDDYFDYWGQGTLVTVSS E VQ L VQ S GAE VKK P GAS VKVS C KAS G YT FT DT YMHWVRQAP GQ RL EW I G29 HuEM1031-K31-VH.lc RIDPANGNTKYDPKFQGKATITADTSANTDYMELSSLRSEDTAVYYCAR GGYRYDDYFDYWGQGTLVTVSS E VQ L VQ S GAE VKK P GAS VKVS C KAS G FN I D DT YMHWVRQAP GQ RL EW I G30 HuEM1031-K31-VH.ld RIDPANGNTKYDPKFQGKATITADTSANTDYMELSSLRSEDTAVYYCARGGYRYDDYFDYWGQGTLVTVSSWSGR Docket No. 65457-712.602SequenceSEQIDDescription 1234567890123456789012345678901234567890123456789 NO: 0E VQ L VQ S GAE VKK P GAS VKVS C KAS G FN I D DT YMHWVRQAP GQ GL EW I G31 HuEM1031-K31-VH.le RIDPANGNTKYDPKFQGKATITADTSANTDYMELSSLRSEDTAVYYCAR GGYRYDDYFDYWGQGTLVTVSS EVQLQQSGAEFVKPGASVKLSCTASGFNIDDTYMHWVKQRPEQGLYWIG32 EM1031-K31-VH (G55A) RIDPANANTKYDPKFQGKATITADTSSNTDYLQLSSLTSEDTAVYYCAR GGYRYDDYFDYWGQGTTLTVSS E VQ L VQ S GAE VKK P GAS VKVS C KAS G FN I D DT YMHWVRQAP GQ RL EW I G33 HuEM1031-K31-VH.ld RIDPANANTKYDPKFQGKATITADTSANTDYMELSSLRSEDTAVYYCAR (G55A) GGYRYDDYFDYWGQGTLVTVSS DIVMTQSPDSLAVSLGERATINCRASETVDSYGSSFMHWYQQKPGQPPK34 HUEM1031-K31-VK.1 LLIYRASTLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNED PFTFGQGTKLEIK DIVLTQSPDSLAVSLGERATINCRASETVDSYGSSFMHWYQQKPGQPPK35 HuEM1031-K31-VK.la LLIYRASTLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNED PFTFGQGTKLEIK DIVLTQSPDSLAVSLGERATINCRASETVDSYGSSFMHWYQQKPGQPPK36 HuEM1031-K31-VK.lb LLIYRASTLESGIPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNED PFTFGQGTKLEIK DIVLTQSPDSLAVSLGERATINCRASETVDSYGSSFMHWYQQKPGQPPK37 HUEM1031-K31-VK.1C LLIYRASTLESGIPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQNNED PFTFGQGTKLEIK DIVLTQSPASLAVSLGQRATISCRASETVDAYGSSFMHWYQQKSGQPPK38 EM1031-K31-VK (S31A) LLIYRASTLESGIPARFSGSGSRTDFTLTINAVEADDVATYYCQQNNED PFTFGGGTKLEIK DIVMTQSPDSLAVSLGERATINCRASETVDAYGSSFMHWYQQKPGQPPK HUEM1031-K31-VK.139 LLIYRASTLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNED (S31A) PFTFGQGTKLEIK EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMG40 HuEM1031-K97-VH.la WIDPENGNTIYDPRFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCAR S FYYGYLRFDYWGQGTLVTVS S EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMG41 HuEM1031-K97-VH.lb WIDPENGNTIYDPRFQGRVTITADTSTNTAYMELSSLRSEDTAVYYCAR S FYYGYLRFDYWGQGTLVTVS S EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWIG42 HuEM1031-K97-VH.lc WIDPENGNTIYDPRFQGKANITADTSTNTAYMELSSLRSEDTAVYYCAR S FYYGYLRFDYWGQGTLVTVS S EVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYMHWVQQAPGKGLEWIG43 HuEM1031-K97-VH.ld WIDPENGNTIYDPRFQGKANITADTSTNTAYMELSSLRSEDTAVYYCAR S FYYGYLRFDYWGQGTLVTVS S EVQLVQSGAEVKKPGATVKISCKASGFNIKDYYMHWVQQAPGKGLEWIG44 HuEM1031-K97-VH.le WIDPENGNTIYDPRFQGKANITADTSTNTAYMELSSLRSEDTAVYYCAR S FYYGYLRFDYWGQGTLVTVS S EVQLQQSGAELVRPGALLKLSCKASGFNIKDYYMHWVKQRPEQGLEWIG45 EM1031-K97-VH (G55A) WIDPENANTIYDPRFQGKANITADTSSNTAYLQLSSLTSEDTAVYYCAR SFYYGYLRFDYWGQGTLVAVSA EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYMHWVQQAPGKGLEWMG46 HuEM1031-K97-VH.la WIDPENANTIYDPRFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCAR (G55A) S FYYGYLRFDYWGQGTLVTVS S DIQLTQSPSFLSASVGDRVTITCSASSSVSYMHWYQQKPGKAPKLLIYE47 HUEM1031-K97-VK.1 ISKLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQWNYPLFTFG QGTKLEIK DIQLTQSPSFLSASVGDRVTITCSASSSVSYMHWYQQKPGKSPKPWIYE48 HuEM1031-K97-VK.la ISKLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQWNYPLFTFG QGTKLEIK EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIG25 EM1031-K112-VH YISPYNDGTKYNEKFKGKATLTSDKSSSTAYMYLSSLTSEDSAVYYCAR GGGLEYCFDYWGQGTTLTVSS E VQ L VQ S GAE VKK P GAS VKVS C KAS G YT FT S YVMHWVRQAP GQ RL EWMG49 HuEM1031-K112-VH.la YISPYNDGTKYNEKFKGRVTITSDTSASTAYMELSSLRSEDTAVYYCARGGGLEYCFDYWGQGTLVTVSSWSGR Docket No. 65457-712.602SequenceSEQID Description 1234567890123456789012345678901234567890123456789 NO: 0E VQ L VQ S GAE VKK P GAS VKVS CKASGYTFTS YVMHWVRQAP GQ RL EWMG50 HuEM1031-K112-VH.lb YISPYNDGTKYNEKFKGRVTLTSDTSASTAYMELSSLRSEDTAVYYCAR GGGLEYCFDYWGQGTLVTVSS E VQ L VQ S GAE VKK P GAS VKVS CKASGYTFTS YVMHWVRQAP GQ RL EWMG51 HuEM1031-K112-VH.lc YISPYNDGTKYNEKFKGRVTLTSDKSASTAYMELSSLRSEDTAVYYCAR GGGLEYCFDYWGQGTLVTVSS E VQ L VQ S GAE VKK P GAS VKVS CKASGYTFTS YVMHWVRQAP GQ RL EW I G52 HuEM1031-K112-VH.ld YISPYNDGTKYNEKFKGKATLTSDKSASTAYMELSSLRSEDTAVYYCAR GGGLEYCFDYWGQGTLVTVSS E VQ L VQ S GAE VKK P GAS VKVS CKASGYTFTS YVMHWVKQAP GQ GL EW I G53 HuEM1031-K112-VH.le YISPYNDGTKYNEKFKGKATLTSDKSASTAYMELSSLRSEDTAVYYCAR GGGLEYCFDYWGQGTLVTVSS EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIG54 EM1031-K112-VH YISPYNDATKYNEKFKGKATLTSDKSSSTAYMYLSSLTSEDSAVYYCAR (G56A) GGGLEYCFDYWGQGTTLTVSSE VQ L VQ S GAE VKK P GAS VKVS CKASGYTFTS YVMHWVKQAP GQ GL EW I G55 HuEM1031-K112-VH.le YISPYNDATKYNEKFKGKATLTSDKSASTAYMELSSLRSEDTAVYYCAR (G56A) GGGLEYS FDYWGQGTLVTVS S EIVLTQSPDFQSVTPKEKVTITCRASQSIGTSIHWYQQKPDQSPKLLIK56 HUEM1031-K112-VK.1 YASESISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQTNSWPWTF GQGTKLEIK EIVLTQSPDFQSVTPKEKVTISCRASQSIGTSIHWYQQKPDQSPKLLIK57 HuEM1031-K112-VK.la YASESISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQTNSWPWTF GQGTKLEIK EIVLTQSPDFQSVTPKEKVTISCRASQSIGTSIHWYQQKPDQSPKLLIK58 HUEM1031-K112-VK. lb YASESISGIPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQTNSWPWTF GQGTKLEIK DIVLTQSPDFQSVTPKEKVTISCRASQSIGTSIHWYQQKPDQSPKLLIK59 HUEM1031-K112-VK.1C YASESISGIPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQTNSWPWTF GQGTKLEIK DILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNGSPRLLIK60 EM1031-K112-VK (S93A) YASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQTNAWPWTF GGGTKLEIK EIVLTQSPDFQSVTPKEKVTISCRASQSIGTSIHWYQQKPDQSPKLLIK61 HuEM1031-K112-VK.la YASESISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQTNAWPWTF (S93A) GQGTKLEIKAnti-KLK2 CDR-H1(EM1031-K31-VH,HuEM1031-K31-VH.la,HuEM1031-K31-VH.lb,62 HuEM1031-K31-VH.lc, DTYMHHuEM1031-K31-VH.ld,HuEM1031-K31-VH.le,EM1031-K31-VH (G55A),HuEM1031-K31-VH.ld(G55A))Anti-KLK2 CDR-H2(EM1031-K31-VH,HuEM1031-K31-VH.la,63 HuEM1031-K31-VH.lb, RIDPANGNTKYDPKFQGHuEM1031-K31-VH.lc,HuEM1031-K31-VH.ld,HuEM1031-K31-VH.le)Anti-KLK2 CDR-H2(EM1031-K31-VH64 (G55A), RIDPANANTKYDPKFQGHuEM1031-K31-VH.ld(G55A))65 Anti-KLK2 CDR-H3 GGYRYDDYFDYWSGR Docket No. 65457-712.602SequenceSEQID Description 1234567890123456789012345678901234567890123456789 NO: 0(EM1031-K31-VH,HuEM1031-K31-VH.la,HuEM1031-K31-VH.lb,HuEM1031-K31-VH.lc,HuEM1031-K31-VH.ld,HuEM1031-K31-VH.le,EM1031-K31-VH (G55A),HuEM1031-K31-VH.ld(G55A))Anti-KLK2 CDR-L1(EM1031-K31-VK,66 HUEM1031-K31-VK.1, RASETVDSYGSSFMHHuEM1031-K31-VK.la,HuEM1031-K31-VK.lb,HUEM1031-K31-VK.1C)Anti-KLK2 CDR-L167 (EM1031-K31-VK (S31A), RASETVDAYGSSFMHHUEM1031-K31-VK.1(S31A))Anti-KLK2 CDR-L2(EM1031-K31-VK,HUEM1031-K31-VK.1,HuEM1031-K31-VK.la,68 HuEM1031-K31-VK.lb, RASTLESHUEM1031-K31-VK.1C,EM1031-K31-VK (S31A),HUEM1031-K31-VK.1(S31A))Anti-KLK2 CDR-L3(EM1031-K31-VK,HUEM1031-K31-VK.1,HuEM1031-K31-VK.la,69 HuEM1031-K31-VK.lb, QQNNEDPFTHUEM1031-K31-VK.1C,EM1031-K31-VK (S31A),HUEM1031-K31-VK.1(S31A))Anti-KLK2 CDR-H1(EM1031-K97-VH,HuEM1031-K97-VH.la,HuEM1031-K97-VH.lb,70 HuEM1031-K97-VH.lc, DYYMHHuEM1031-K97-VH.ld,HuEM1031-K97-VH.le,EM1031-K97-VH (G55A),HuEM1031-K97-VH.la(G55A))Anti-KLK2 CDR-H2(EM1031-K97-VH,HuEM1031-K97-VH.la,71 HuEM1031-K97-VH.lb, WIDPENGNTIYDPRFQGHuEM1031-K97-VH.lc,HuEM1031-K97-VH.ld,HuEM1031-K97-VH.le)Anti-KLK2 CDR-H272 (EM1031-K97-VH WIDPENANTIYDPRFQG(G55A), HuEM1031-K97- VH.la(G55A))73 Anti-KLK2 CDR-H3 SFYYGYLRFDYWSGR Docket No. 65457-712.602SequenceSEQID Description 1234567890123456789012345678901234567890123456789 NO: 0(EM1031-K97-VH,HuEM1031-K97-VH.la,HuEM1031-K97-VH.lb,HuEM1031-K97-VH.lc,HuEM1031-K97-VH.ld,HuEM1031-K97-VH.le,EM1031-K97-VH (G55A),HuEM1031-K97-VH.la(G55A))Anti-KLK2 CDR-L174 (EM1031-K97-VK, SASSSVSYMHHUEM1031-K97-VK.1,HuEM1031-K97-VK.la)Anti-KLK2 CDR-L275 (EM1031-K97-VK, EISKLASHUEM1031-K97-VK.1,HuEM1031-K97-VK.la)Anti-KLK2 CDR-L376 (EM1031-K97-VK, QQWNYPLFTHUEM1031-K97-VK.1,HuEM1031-K97-VK.la)Anti-KLK2 CDR-H1(EM1031-K112-VH,HuEM1031-K112-VH.la,HuEM1031-K112-VH.lb,77 HuEM1031-K112-VH.lc, SYVMHHuEM1031-K112-VH.ld,HuEM1031-K112-VH.le,EM1031-K112-VH(G56A), HuEM1031- K112-VH.le (G56A))Anti-KLK2 CDR-H2(EM1031-K112-VH,HuEM1031-K112-VH.la,78 HuEM1031-K112-VH.lb, YISPYNDGTKYNEKFKGHuEM1031-K112-VH.lc,HuEM1031-K112-VH.ld,HuEM1031-K112-VH. le)Anti-KLK2 CDR-H279 (EM1031-K112-VH YISPYNDATKYNEKFKG(G56A), HuEM1031- K112-VH.le (G56A))Anti-KLK2 CDR-H3(EM1031-K112-VH,HuEM1031-K112-VH.la,HuEM1031-K112-VH.lb,80 HuEM1031-K112-VH.lc, GGGLEYSFDYHuEM1031-K112-VH.ld,HuEM1031-K112-VH.le,EM1031-K112-VH(G56A), HuEM1031- K112-VH.le (G56A))Anti-KLK2 CDR-L1(EM1031-K112-VK,HUEM1031-K112-VK.1,81 HuEM1031-K112-VK.la, RASQSIGTSIHHUEM1031-K112-VK. lb,HUEM1031-K112-VK.1C,EM1031-K112-VK (S93A),WSGR Docket No. 65457-712.602SequenceSEQID Description 1234567890123456789012345678901234567890123456789 NO: 0HuEM1031-K112-VK.la(S93A))Anti-KLK2 CDR-L2(EM1031-K112-VK,HUEM1031-K112-VK.1,HuEM1031-K112-VK.la,82 HUEM1031-K112-VK. lb, YASESISHUEM1031-K112-VK.1C,EM1031-K112-VK (S93A),HuEM1031-K112-VK.la(S93A))Anti-KLK2 CDR-L3(EM1031-K112-VK,HUEM1031-K112-VK.1,83 QQTNSWPWTHuEM1031-K112-VK.la,HUEM1031-K112-VK. lb,HUEM1031-K112-VK.1C)Anti-KLK2 CDR-L3(EM1031-K112-VK84 QQTNAWPWT(S93A), HuEM1031-K112- VK.la (S93A))FIT2023-10ah-4(h)85 Chain #1 (VLKLK2-CL- see Table 9VHCD3S-CH1-FC)FIT2023-10ah-4(h)86 see Table 9Chain #2 (VHKLK2-CH1)FIT2023-10ah-4(h)87 see Table 9Chain #3 (VLCD3e-CL)FIT2023-20ah-l(h)88 Chain #1 (VLKLK2-CL- see Table 9VHCD3S-CH1-FC)FIT2023-20ah-l(h)89 see Table 9Chain #2 (VHKLK2-CH1)FIT2023-20ah-l(h)90 see Table 9Chain #3 (VLCD3e-CL)FIT2023-22ah-10(h)91 Chain #1 (VLKLK2-CL- see Table 9VHCD3S-CH1-FC)FIT2023-22ah-10(h)92 see Table 9Chain #2 (VHKLK2-CH1)FIT2023-22ah-10(h)93 see Table 9Chain #3 (VLCD3e-CL)

Claims

WSGR Docket No. 65457-712.602CLAIMS1. An isolated antibody or antigen-binding fragment thereof that specifically binds to KLK2, comprising a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, wherein:i) CDR-H1 comprises the sequence of DYYMH (SEQ ID NO: 70);CDR-H2 comprises the sequence of WIDPENGNTIYDPRFQG (SEQ ID NO: 71) or WIDPENANTIYDPRFQG (SEQ ID NO: 72);CDR-H3 comprises the sequence of SFYYGYLRFDY (SEQ ID NO: 73);CDR-L1 comprises the sequence of SASSSVSYMH (SEQ ID NO: 74);CDR-L2 comprises the sequence of EISKLAS (SEQ ID NO: 75); andCDR-L3 comprises the sequence of QQWNYPLFT (SEQ ID NO: 76), or ii) CDR-H1 comprises the sequence of DTYMH (SEQ ID NO: 62);CDR-H2 comprises the sequence of RIDPANGNTKYDPKFQG (SEQ ID NO: 63) or RIDPANANTKYDPKFQG (SEQ ID NO: 64);CDR-H3 comprises the sequence of GGYRYDDYFD Y (SEQ ID NO: 65); CDR-L1 comprises the sequence of RASETVDSYGSSFMH (SEQ ID NO: 66) or RASETVDAYGSSFMH (SEQ ID NO: 67);CDR-L2 comprises the sequence of RASTLES (SEQ ID NO: 68); and CDR-L3 comprises the sequence of QQNNEDPFT (SEQ ID NO: 69), or iii) CDR-H1 comprises the sequence of SYVMH (SEQ ID NO: 77);CDR-H2 comprises the sequence of YISPYNDGTKYNEKFKG (SEQ ID NO: 78) or YISPYNDATKYNEKFKG (SEQ ID NO: 79);CDR-H3 comprises the sequence of GGGLEYSFDY (SEQ ID NO: 80);CDR-L1 comprises the sequence of RASQSIGTSIH (SEQ ID NO: 81);CDR-L2 comprises the sequence of YASESIS (SEQ ID NO: 82); andCDR-L3 comprises the sequence of QQTNSWPWT (SEQ ID NO: 83) or QQTNAWPWT (SEQ ID NO: 84),optionally wherein the CDRs are defined according to Kabat numbering.

2. The antibody or antigen-binding fragment of claim 1, wherein CDR-H1 comprises the sequence of DYYMH (SEQ ID NO: 70); CDR-H2 comprises the sequence of WIDPENANTIYDPRFQG (SEQ ID NO: 72); CDR-H3 comprises the sequence of SFYYGYLRFDY (SEQ ID NO: 73); CDR-L1 comprises the sequence of SASSSVSYMH (SEQWSGR Docket No. 65457-712.602ID NO: 74); CDR-L2 comprises the sequence of EISKLAS (SEQ ID NO: 75); and CDR-L3 comprises the sequence of QQWNYPLFT (SEQ ID NO: 76).

3. The antibody or antigen-binding fragment of claim 1, wherein CDR-H1 comprises the sequence of DTYMH (SEQ ID NO: 62); CDR-H2 comprises the sequence of RIDPANANTKYDPKFQG (SEQ ID NO: 64); CDR-H3 comprises the sequence of GGYRYDDYFDY (SEQ ID NO: 65); CDR-L1 comprises the sequence of RASETVDAYGSSFMH (SEQ ID NO: 67); CDR-L2 comprises the sequence of RASTLES (SEQ ID NO: 68); and CDR-L3 comprises the sequence of QQNNEDPFT (SEQ ID NO: 69).

4. The antibody or antigen-binding fragment of claim 1, wherein CDR-H1 comprises the sequence of SYVMH (SEQ ID NO: 77); CDR-H2 comprises the sequence of YISPYNDATKYNEKFKG (SEQ ID NO: 79); CDR-H3 comprises the sequence of GGGLEYSFDY (SEQ ID NO: 80); CDR-L1 comprises the sequence of RASQSIGTSIH (SEQ ID NO: 81); CDR-L2 comprises the sequence of YASESIS (SEQ ID NO: 82); and CDR-L3 comprises the sequence of QQTNAWPWT (SEQ ID NO: 84).

5. The isolated antibody or antigen -binding fragment of claim 1, wherein the antibody comprises a variable heavy chain domain VH and a variable light chain domain VL, wherein:i) the VH domain comprises the sequence of SEQ ID NO: 21, 27, 28, 29, 30, 31, 32, or 33, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and / or the VL domain comprises the sequence of SEQ ID NO: 22, 34, 35, 36, 37, 38, or 39, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith;ii) the VH domain comprises the sequence of SEQ ID NO: 23, 40, 41, 42, 43, 44, 45, or 46, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and / or the VL domain comprises the sequence of SEQ ID NO: 24, 47, or 48, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith; oriii) the VH domain comprises the sequence of SEQ ID NO: 25, 49, 50, 51, 52, 53, 54, or 55, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and / or the VL domainWSGR Docket No. 65457-712.602comprises the sequence of SEQ ID NO: 26, 56, 57, 58, 59, 60, or 61, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith.

6. The isolated antibody or antigen-binding fragment of claim 1, wherein the antibody comprises a variable heavy chain domain VH and a variable light chain domain VL, wherein:i) the VH domain comprises the sequence of SEQ ID NO: 21, 27, 28, 29, 30, 31, 32, or 33 and / or the VL domain comprises the sequence of SEQ ID NO: 22, 34, 35, 36, 37, 38, or 39;ii) the VH domain comprises the sequence of SEQ ID NO: 23, 40, 41, 42, 43, 44, 45, or 46 and / or the VL domain comprises the sequence of SEQ ID NO: 24, 47, or 48; oriii) the VH domain comprises the sequence of SEQ ID NO: 25, 49, 50, 51, 52, 53, 54, or 55 and / or the VL domain comprises the sequence of SEQ ID NO: 26, 56, 57, 58, 59, 60, or 61.

7. The isolated antibody or antigen-binding fragment of claim 1, wherein the antibody is a chimeric or humanized antibody, optionally the antibody is a humanized antibody, and further optionally,i) the VH domain of the antibody comprises amino acid residues IE, 72A, and 0 to 10 residues selected from 27F, 28N, 291, 30D, 44G, 481, 67K, 68 A, 77N, 79D, according to Kabat numbering; and the VL domain comprises 0 to 3 amino acid residues selected from 4L, 621, and 72R, according to Kabat numbering;ii) the VH domain of the antibody comprises amino acid residue 98R, and 0 to 10 residues selected from 24 A, 27F, 28N, 291, 3 OK, 481, 67K, 68 A, 69N, 77N, according to Kabat numbering; and the VL domain comprises 0 to 3 amino acid residues selected from 42S, 45P, and 46W, according to Kabat numbering; or iii) the VH domain of the antibody comprises amino acid residues IE, 72S, and 0 to 7 residues selected from 38K, 44G, 481, 67K, 68 A, 70L,74K, according to Kabat numbering; and the VL domain comprises 0 to 3 amino acid residues selected from ID, 22S, and 581, according to Kabat numbering.

8. The isolated antibody or antigen -binding fragment of claim 1, wherein the antibody comprises a combination of VH and VL sequences selected from the group consisting of:WSGR Docket No. 65457-712.602i) SEQ ID NO: 27 and SEQ ID NO: 34, SEQ ID NO: 28 and SEQ ID NO: 34, SEQ ID NO: 29 and SEQ ID NO: 34, SEQ ID NO: 30 and SEQ ID NO: 34, SEQ ID NO: 31 and SEQ ID NO: 34, SEQ ID NO: 27 and SEQ ID NO: 35, SEQ ID NO: 28 and SEQ ID NO: 35, SEQ ID NO: 29 and SEQ ID NO: 35, SEQ ID NO: 30 and SEQ ID NO: 35, SEQ ID NO: 31 and SEQ ID NO: 35, SEQ ID NO: 27 and SEQ ID NO: 36, SEQ ID NO: 28 and SEQ ID NO: 36, SEQ ID NO: 29 and SEQ ID NO: 36, SEQ ID NO: 30 and SEQ ID NO: 36, SEQ ID NO: 31 and SEQ ID NO: 36, SEQ ID NO: 27 and SEQ ID NO: 37, SEQ ID NO: 28 and SEQ ID NO: 37, SEQ ID NO: 29 and SEQ ID NO: 37, SEQ ID NO: 30 and SEQ ID NO: 37, SEQ ID NO: 31 and SEQ ID NO: 37, SEQ ID NO: 31, SEQ ID NO: 22, SEQ ID NO: 21 and SEQ ID NO: 37, SEQ ID NO: 32 and SEQ ID NO: 22, SEQ ID NO: 21 and SEQ ID NO: 38, SEQ ID NO: 21 and SEQ ID NO: 22, and SEQ ID NO: 33 and SEQ ID NO: 39;ii) SEQ ID NO: 40 and SEQ ID NO: 47, SEQ ID NO: 41 and SEQ ID NO: 47, SEQ ID NO: 42 and SEQ ID NO: 47, SEQ ID NO: 43 and SEQ ID NO: 47, SEQ ID NO: 44 and SEQ ID NO: 47, SEQ ID NO: 40 and SEQ ID NO: 48, SEQ ID NO: 41 and SEQ ID NO: 48, SEQ ID NO: 42 and SEQ ID NO: 48, SEQ ID NO: 43 and SEQ ID NO: 48, SEQ ID NO: 43 and SEQ ID NO: 48, SEQ ID NO: 44 and SEQ ID NO: 48, SEQ ID NO: 44 and SEQ ID NO: 24, SEQ ID NO: 23 and SEQ ID NO: 48, SEQ ID NO: 45 and SEQ ID NO: 24, SEQ ID NO: 23 and SEQ ID NO: 24, and SEQ ID NO: 46 and SEQ ID NO: 47;iii) SEQ ID NO: 49 and SEQ ID NO: 56, SEQ ID NO: 50 and SEQ ID NO: 56, SEQ ID NO: 51 and SEQ ID NO: 56, SEQ ID NO: 52 and SEQ ID NO: 56, SEQ ID NO: 53 and SEQ ID NO: 56, SEQ ID NO: 49 and SEQ ID NO: 57, SEQ ID NO: 50 and SEQ ID NO: 57, SEQ ID NO: 51 and SEQ ID NO: 57, SEQ ID NO: 52 and SEQ ID NO: 57, SEQ ID NO: 53 and SEQ ID NO: 57, SEQ ID NO: 49 and SEQ ID NO: 58, SEQ ID NO: 50 and SEQ ID NO: 58, SEQ ID NO: 51 and SEQ ID NO: 58, SEQ ID NO: 52 and SEQ ID NO: 58, SEQ ID NO: 53 and SEQ ID NO: 58, SEQ ID NO: 49 and SEQ ID NO: 59, SEQ ID NO: 50 and SEQ ID NO: 59, SEQ ID NO: 51 and SEQ ID NO: 59, SEQ ID NO: 52 and SEQ ID NO: 59, SEQ ID NO: 53 and SEQ ID NO: 59, SEQ ID NO: 53 and SEQ ID NO: 26, SEQWSGR Docket No. 65457-712.602ID NO: 25 and SEQ ID NO: 59, SEQ ID NO: 54 and SEQ ID NO: 26, SEQ ID NO: 25 and SEQ ID NO: 60, SEQ ID NO: 25 and SEQ ID NO: 26, and SEQ ID NO: 55 and SEQ ID NO: 61.

9. The isolated antibody or antigen-binding fragment of claim 1, wherein the antibody comprises a VH domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 46 and a VL domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:

47.

10. The isolated antibody or antigen-binding fragment of claim 1, wherein the antibody comprises a VH domain comprising the sequence of SEQ ID NO: 46 and a VL domain comprising the sequence of SEQ ID NO: 47.

11. The isolated antibody or antigen -binding fragment of claim 1, wherein the antibody comprises a VH domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 33 and a VL domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:

39.

12. The isolated antibody or antigen-binding fragment of claim 1, wherein the antibody comprises a VH domain comprising the sequence of SEQ ID NO: 33 and a VL domain comprising the sequence of SEQ ID NO: 39.

13. The isolated antibody or antigen -binding fragment of claim 1, wherein the antibody comprises a VH domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 55 and a VL domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:

61.

14. The isolated antibody or antigen-binding fragment of claim 1, wherein the antibody comprises a VH domain comprising the sequence of SEQ ID NO: 55 and a VL domain comprising the sequence of SEQ ID NO: 61.

15. The antibody or antigen biding fragment of any one of claims 1 to 14, wherein the antibody or antigen binding fragment comprises an Fv, Fab, Fab', Fab’-SH, F(ab')2.

16. The antibody or antigen biding fragment of claim 15, wherein the antibody or antigen binding fragment comprises an IgG.

17. The isolated antibody or antigen-binding fragment of any one of claims 1-14, wherein the antibody comprises an Fc region, for example, the Fc region is of IgG type, or of IgGl, IgG2, IgG3, or IgG4 subtype, optionally, the Fc region is of IgGl subtype, for example, the Fc regionWSGR Docket No. 65457-712.602has an amino acid sequence of SEQ ID NO: 19 or 20.

18. A fusion or a conjugate comprising the isolated antibody or antigen-binding fragment of any one of claims 1-17.

19. A method of detecting KLK2 in a biological sample, comprising contacting the biological sample with the isolated antibody or antigen-binding fragment of any one of claims 1-17 or the fusion or conjugate of claim 18.

20. A nucleic acid molecule encoding the isolated antibody or antigen-binding fragment of any one of claims 1-17.

21. A vector comprising the nucleic acid molecule of claim 20.

22. A host cell expressing the nucleic acid of claims 20 or the vector of claim 21.

23. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment of any one of claims 1-17, the fusion or conjugate of claim 18, the nucleic acid molecule of claim 20, the vector of claim 21, or the host cell of claim 22.

24. Abispecific binding protein that specifically binds KLK2 and CD3, comprising a first antigen-binding site that specifically binds KLK2, and a second antigen-binding site that specifically binds CD3, wherein the first antigen-binding site comprises a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as defined in any one of claims 1-4, optionally, the first antigen-binding site comprises a VH domain and a VL domain as defined in any one of claims 5-17; wherein the CDRs are defined according to Kabat numbering.

25. The bispecific binding protein of claim 24, wherein the second antigen -binding site comprises a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, wherein: CDR-H1 comprises the sequence of NYYVH (SEQ ID NO: 3); CDR-H2 comprises the sequence of WISPGSDNTKYNEKFKG (SEQ ID NO: 4); CDR-H3 comprises the sequence of DDYGNYYFDY (SEQ ID NO: 5); CDR-L1 comprises the sequence of KSSQSLLNARTRKNYLA (SEQ ID NO: 7); CDR-L2 comprises the sequence of WASTRES (SEQ ID NO: 8); and CDR-L3 comprises the sequence of KQSYILRT (SEQ ID NO: 9).

26. The bispecific binding protein of claim 24, wherein the second antigen-binding site comprises a VH domain and a VL domain, wherein the VH domain comprises the sequence of SEQ ID NO: 2, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and / or the VL domain comprises the sequence of SEQ ID NO: 6, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,WSGR Docket No. 65457-712.60296%, 97%, 98%, 99% or more identity therewith.

27. The bispecific binding protein of claim 24, wherein the second antigen-binding site comprises a VH domain comprising the sequence of SEQ ID NO: 2, and a VL domain comprising the sequence of SEQ ID NO: 6; wherein the CDRs are defined according to Kabat numbering.

28. The bispecific binding protein of any one of claims 24-27, comprising a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, wherein the first polypeptide chain comprises, from amino terminus to carboxyl terminus, 1) VLA-CL-VHB-CHl-hinge-CH2-CH3 or 2) VHB-CHl-VLA-CL-hinge-CH2-CH3; the second polypeptide chain comprises, from amino to carboxyl terminus, VHA-CHI; the third polypeptide chain comprises, from amino to carboxyl terminus, VLB-CL; wherein the VLA-CL pairs with VHA-CHI to form a first Fab that specifically binds a first antigen A, and VLB-CL pairs with VHB-CHI to form a second Fab that specifically binds a second antigen B, and wherein the antigen A is KLK2, and the antigen B is CD3, or the antigen A is CD3, and the antigen B is KLK2; wherein said three polypeptide chains as a half molecule associates with another said three polypeptide chains as the other half molecule to form a FIT-Ig protein.

29. The bispecific binding protein of any one of claims 24-27, wherein the protein comprises a Fc comprising, from amino terminus to carboxyl terminus, hinge-CH2-CH3, and optionally: the Fc is of IgG type, or of IgGl, IgG2, IgG3, or IgG4 subtype, optionally, said Fc is of IgGl subtype, the Fc is from human, the Fc comprises a mutation that alters, increases or reduces an effector function thereof, or comprises L234A and L235A substitutions and / or P329A substitution (EU numbering); and / or the Fc comprises a sequence of SEQ ID NO: 19 or a sequence having at least 95%, 96%, 97%, 98%, 99%, or more identity therewith, for example, SEQ ID NO: 20.

30. The bispecific binding protein of claim 26 or 27, wherein:i) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 85, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 86, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and the third polypeptide chain comprises an amino acidWSGR Docket No. 65457-712.602sequence of SEQ ID NO: 87, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith; or ii) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 88, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 89, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 90, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith; or iii) the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 91, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 92, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 93, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity therewith.

31. The bispecific binding protein of claim 24, wherein the first polypeptide chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 88 and the second polypeptide chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 89.

32. The bispecific binding protein of claim 24, wherein the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 88 and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 89.

33. The bispecific binding protein of claim 24, wherein the first polypeptide chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 85 and the second polypeptide chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 86.WSGR Docket No. 65457-712.60234. The bispecific binding protein of claim 24, wherein the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 85 and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 86.

35. The bispecific binding protein of claim 24, wherein the first polypeptide chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 91 and the second polypeptide chain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 92.

36. The bispecific binding protein of claim 24, wherein the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 91 and the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 92.

37. The bispecific binding protein of any one of claims 24-36, wherein the first antigen-binding domain binds to a human KLK2, with a KD value of less than about lOnM, less than about 5nM, or less than about InM, preferably O.lnM to 0.95nM, and is not detected to bind to human KLK3, as measured by a grating-coupled interferometry (GCI) based assay.

38. The bispecific binding protein of any one of claims 24-37, wherein the first antigen-binding domain binds to the same or overlapping epitope, and / or competes with a reference antigenbinding domain comprising a VH sequence of SEQ ID NO: 11 and a VL sequence of SEQ ID NO: 12 for binding to a human KLK2; or the first antigen-binding domain binds to the different epitope, and / or does not compete with a reference antigen -binding domain comprising a VH sequence of SEQ ID NO: 11 and a VL sequence of SEQ ID NO: 12 for binding to a human KLK2.

39. The bispecific binding protein of any one of claims 24-38, wherein the second antigenbinding domain binds to a human CD3 (especially, CD3s / 6 antigen), with a KD value of less than about 50nM, less than about 30nM, or less than about 20nM, preferably 5nM to 20nM, as measured by a grating-coupled interferometry (GCI) based assay.

40. The bispecific binding protein of any one of claims 24-39, wherein the bispecific binding protein activates CD3 on T cells in vitro in a concentration-dependent manner at a concentration of 0.01 nM to 100 nM in the presence of KLK2 protein, as measured by a KLK2 protein-bound reporter gene assay.

41. The bispecific binding protein of any one of claims 24-40, wherein the bispecific bindingWSGR Docket No. 65457-712.602protein activates T cells in vitro in a concentration-dependent manner at a concentration of 0.01 nM to 100 nM to kill tumor cells expressing KLK2 protein, inducing lower cytokine (such as human IL-2, IL-6, IL-10, TNFa and IFNy) release by T cells.

42. The bispecific binding protein of any one of claims 24-41, wherein the bispecific binding protein has in vivo anti-tumor effect on KLK2-expressing tumor.

43. The bispecific binding protein of any one of claims 24-42, wherein the bispecific binding protein remains stable during storage.

44. The bispecific binding protein of any one of claims 24-43, wherein the first and / or the second antigen binding site comprises an Fv, Fab, Fab', Fab’-SH, F(ab')2.

45. A nucleic acid molecule encoding the bispecific binding protein of any one of claims 21-44.

46. A vector comprising the nucleic acid molecule of claim 45.

47. A host cell comprising the nucleic acid molecule of claim 45, or the vector of claim 46.

48. A method of preparing the isolated antibody or antigen -binding fragment of any one of claims 1-17, or the bispecific binding protein of any one of claims 24-44, comprising: culturing the host cell of claim 22 or claim 47 under conditions that allow the production of the antibody, antigen-binding fragment, or bispecific binding protein; and recovering the antibody, antigenbinding fragment, or bispecific binding protein from the culture.

49. A pharmaceutical composition comprising the bispecific binding protein of any one of claims 24-44, the nucleic acid of claim 45, the vector of claim 46, or the host cell of claim 47.

50. A method of treating or preventing a KLK2-associated disorder, comprising administering to an individual in need thereof a therapeutically effective amount of the antibody of any one of claims 1-17, the bispecific binding protein of any one of claims 24-44, or the pharmaceutical composition of claim 49.

51. The method of claim 50, wherein the individual is a human.

52. The method of claim 50, wherein the disorder is a KLK2 -positive cancer, optionally wherein: the cancer is a prostate cancer or a breast cancer, e.g., the KLK2 -positive cancer is an androgen receptor (AR) expressing breast cancer, or is a relapsed, refractory, malignant or castration-resistant prostate cancer (CRPC), or any combination thereof.

53. A method of targeting a T-cell to a tumor cell, comprising administering to an individual in need thereof a therapeutically effective amount of the isolated antibody or antigen-binding fragment of any one of claims 1-17, the bispecific binding protein of any one of claims 24-44, orWSGR Docket No. 65457-712.602the pharmaceutical composition of claim 49, thereby targeting the T-cell to the tumor cell.

54. A method of killing or inhibiting growth of a tumor cell, comprising contacting the tumor cell with an effective amount of the isolated antibody or antigen-binding fragment protein of any one of claims 1-17, the bispecific binding protein of any one of claims 24-44 or the pharmaceutical composition of claim 49, thereby killing or inhabiting growth of the tumor cell.

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