Materials, methods, and systems for enhanced protease targeting

Abstract

Materials, methods, and systems for enhanced protease targeting. For example, methods for the targeting of kallikrein related peptidase 2 (KLK2) are provided.

Description

ATTORNEY DOCKET NO. JBI6949WOPCT1MATERIALS, METHODS, AND SYSTEMS FOR ENHANCED PROTEASE TARGETINGCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority under 35 U.S.C. § 119(e) of prior U.S. Provisional Patent Application Serial Nos. 63 / 702,498 and 63 / 702,504, both filed October 2, 2024, the entire contents of which are incorporated herein.FIELD

[0002] Materials, methods, and systems for enhanced protease targeting, and more particularly methods for targeting kallikrein related peptidase 2 (KLK2).BACKGROUND

[0003] Kallikrein-related peptidase 2 (KLK2) is a serine protease predominantly expressed in prostatic tissue and secreted into prostatic fluid, wherein it cleaves semenogelin and other seminal plasma proteins. KLK2 is a member of the human tissue kallikrein family, which consists of 15 closely related serine proteases located in a cluster on chromosome 19. KLK2 shares significant sequence homology with prostate-specific antigen (PSA, KLK3), and like PSA, KLK2 is primarily expressed in the prostate under androgen regulation.

[0004] Biologically, KLK2 has also been found to activate pro-forms of other kallikreins, contributing to a proteolytic cascade within the extracellular environment. In cancer biology, KLK2 expression has been most closely associated with prostate cancer. KLK2 may be found associated with the membrane of prostate cancer tumor cells, and elevated KLK2 levels have been detected in prostate cancer tissue and in patient serum. Studies indicate that KLK2 may participate in tumor progression. However, targeting, detailed pathophysiological roles of KLK2 in prostate cancer, etc. remain unclear.SUMMARY

[0005] The inventors appreciated and addressed the need for better understanding of proteases, including KLK2, inter alia, and mechanisms of KLK2 including its proximity, forms, associations, complexity, anchoring, and the like to more effectively target KLK2, reveal more about its biology and activity, and to create more effective KLK2 interacting molecules, such as binders, such as antibodies, or other types of molecules that specifically detect, identify, target such proteases, etc.

[0006] Accordingly, against this backdrop, the present disclosure provides molecular complexes, multimers, multimeric complexes, fusion proteins, mixtures and compositions thereof, and the like.-1-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1

[0007] The fusion proteins may include a molecule comprising: (i) KLK peptide, such as a KLK2 peptide, and (ii) a SPINT peptide, such as a SPINT1 peptide and / or or a SPINT2 peptide. As used herein, the term “peptide” may encompass full length proteins or fragments thereof, e.g., peptides.

[0008] The KLK2 peptide may not comprise a pro-peptide sequence.

[0009] The SPINT 1 peptide or the SPINT2 peptide may comprise a MANEC (N) domain, a PKD-like (I) domain, a Kunitz 1 (KI) domain, a LDLRA (L) domain, a Kunitz 2 (K2) domain, or any combination thereof.

[0010] The fusion protein may comprise a tag, such as a His tag.

[0011] The KLK peptide may comprise an amino acid sequence as set forth in SEQ ID NO: 2 or 19, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0012] The SPINT peptide may comprise a SPINT1 peptide having an amino acid sequence as set forth in any of SEQ ID NOS: 3, 15, or 16, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0013] The KI domain of SPINT may comprise an amino acid sequence as set forth in SEQ ID NO: 4, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0014] The K2 domain of SPINT may comprise an amino acid sequence as set forth in SEQ ID NO: 5, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0015] The SPINT peptide may comprise a SPINT2 peptide having an amino acid sequence as set forth in any of SEQ ID NOS: 6, 17, or 18, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0016] The KI domain of SPINT2 may comprise an amino acid sequence as set forth in SEQ ID NO: 7, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0017] The K2 domain of SPINT2 may comprise an amino acid sequence as set forth in SEQ ID NO: 8, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0018] The KLK peptide and the SPINT peptide may be connected directly. The KLK2 peptide and the SPINT1 peptide and / or the SPINT2 peptide may be connected directly. The-2-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1KLK2 peptide and the SPINT1 peptide and / or the SPINT2 peptide may be connected via a linker. The linker may be a peptide linker. The peptide linker may comprise an amino acid sequence GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS as set forth in SEQ ID NO: 13, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.

[0019] The fusion protein may comprise an amino acid sequence as set forth in any of SEQ ID NOS: 9, 10, 11, or 12, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.

[0020] The fusion protein may be immunogenic such as, for example, strongly immunogenic.

[0021] The molecular complex may include complexes of a KLK2 peptide with SPINT1 or SPINT2 peptide, or complexes of KLK2 peptide with a peptide fragment of SPINT1 or SPINT2. Exemplary peptide fragments of SPINT1 and SPINT2 include peptides comprising a MANEC (N) domain, a PKD-like (I) domain, a Kunitz 1 (KI) domain, a LDLRA (L) domain, a Kunitz 2 (K2) domain, or any combination thereof. As such, the molecular complex may also include KLK2 complexed with a peptide comprising any one or more of the KI domain of SPINT1, the K2 domain of SPINT1, the KI domain of SPINT2, the K2 domain of SPINT2, the MANEC (N) domain of SPINT1, the PKD-like (I) domain of SPINT1, and the LDLRA (L) domain of SPINT1.

[0022] The KLK peptide may comprise an amino acid sequence as set forth in SEQ ID NO: 2 or 19, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0023] The SPINT peptide may comprise a SPINT1 peptide having an amino acid sequence as set forth in any of SEQ ID NOS: 3, 15, or 16, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0024] The KI domain of SPINT1 may comprise an amino acid sequence as set forth in SEQ ID NO: 4, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0025] The K2 domain of SPINT 1 may comprise an amino acid sequence as set forth in SEQ ID NO: 5, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0026] The SPINT peptide may comprise a SPINT2 peptide having an amino acid sequence as set forth in any of SEQ ID NOS: 6, 17, or 18, or a sequence having 60%, 65%,-3-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT170%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0027] The KI domain of SPINT2 may comprise an amino acid sequence as set forth in SEQ ID NO: 7, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0028] The K2 domain of SPINT2 may comprise an amino acid sequence as set forth in SEQ ID NO: 8, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0029] The molecular complex may be immunogenic such as, for example, strongly immunogenic.

[0030] The molecular complex may comprise a tag, such as a His tag.

[0031] Multimeric complexes may include complexes of KLK peptide, such as KLK2 peptide, with a multimerized, e.g., dimerized or concatemerized (multimers of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptide segments), peptide fragment of SPINT1 or SPINT2. Multimeric complexes may include complexes of KLK2 protein or KLK2 peptide with two or more of the peptide fragments of SPINT1 and / or SPINT2 described herein, e.g., dimerized or concatemerized. Multimeric complexes may include complexes of two or more KLK2 peptides, e.g., dimerized or concatemerized (multimers of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptide segments), with one or more of the peptide fragments of SPINT1 or SPINT2 described herein. Multimeric complexes may include complexes of two or more KLK2 peptides, e.g., dimerized or concatemerized (multimers of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptide segments), with two or more of the peptide fragments of SPINT1 and / or SPINT2 described herein.

[0032] KLK peptides may comprise the amino acid sequence as set forth in SEQ ID NO: 2 or 19, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6). Peptides or fragment thereof of SPINT1 or SPINT2 in the multimeric complexes may comprise a MANEC (N) domain, a PKD-like (I) domain, a Kunitz 1 (KI) domain, a LDLRA (L) domain, a Kunitz 2 (K2) domain, or any combination thereof. Peptides or fragment thereof of SPINT1 or SPINT2 in the multimeric complexes may comprise the amino acid sequence as set forth in any of SEQ ID NOS: 3, 4, 5, 6, 7, 8, 15, 16, 17, or 18or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0033] The various multimerized peptides of the multimeric complexes may be connected directly, i.e., concatemerized, or via a linker. The linker may be a peptide linker.-4-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1

[0034] The multimeric complex may be immunogenic such as, for example, strongly immunogenic.

[0035] The multimeric complex may comprise a tag, such as a His tag.

[0036] Also provided are compositions comprising (i) a KLK peptide, and (ii) a SPINT peptide. Such compositions may comprise KLK2 peptide and SPINT1 peptide and / or a SPINT2 peptide. The KLK2 peptide may be absent a pro-peptide sequence and may be non-naturally occurring. The SPINT 1 peptide and / or the SPINT2 peptide may be non-naturally occurring. Non-naturally occurring may be understood to mean an isolated, purified, synthetic, or recombinant peptide. Any of the KLK2 peptide, the SPINT1 peptide, and / or the SPINT2 peptide may comprise a tag, such as a His tag.

[0037] The compositions may include a KLK2 peptide comprising an amino acid sequence as set forth in SEQ ID NO: 2 or 19, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and either or both of a SPINT 1 peptide comprising an amino acid sequence as set forth in any of SEQ ID NOS: 3, 4, 5, 15, or 16, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto and a SPINT2 peptide comprising an amino acid sequence as set forth in any of SEQ ID NOS: 6, 17, or 18, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0038] The composition may comprise complexes formed between the KLK2 peptide and the SPINT 1 and / or SPINT2 peptide.

[0039] Also provided are compositions comprising any of the molecular complexes, multimeric complexes, and / or fusion proteins disclosed herein. The compositions may comprise single species of the molecular complex, multimeric complex, or fusion protein, or may comprise mixtures of any of the molecular complexes, multimeric complexes, or fusion proteins disclosed herein.

[0040] Also provided is an antibody or antigen-binding fragment that specifically recognizes any of the molecular complexes, multimeric complexes, or fusion proteins disclosed herein.

[0041] The antibody or antigen-binding fragment may have a high affinity for the molecular complexes, multimeric complexes, or fusion proteins disclosed herein and a low affinity for free KLK2.-5-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1

[0042] The antibody or antigen-binding fragment may have a high affinity for KLK2 protein when directly or indirectly (e.g., via a linker as disclosed) bound to SPINT1 or SPINT2, or to any of the SPINT1 or SPINT2 peptides disclosed herein, and a low affinity for free KLK2.

[0043] The antibody or antigen-binding fragment may have a high affinity for the KLK2 peptide when bound directly or indirectly (e.g., via a linker as disclosed) to SPINT1 or SPINT2, or to any of the SPINT1 or SPINT2 peptides disclosed herein, and a low affinity for free KLK2.

[0044] Provided is an antibody or antigen-binding fragment that binds KLK2.

[0045] Provided is an antibody or antigen-binding fragment that has a high affinity for any of the molecular complexes, multimeric complexes, or fusions proteins disclosed herein (e.g., KLK2 when bound to SPINT1 or SPINT2), and a low affinity for free KLK2.

[0046] Any of the antibody or antigen-binding fragments disclosed herein may be generated by phage-display.

[0047] Any of the antibody or antigen-binding fragments disclosed herein may be generated by in silico methods.

[0048] Any of the antibody or antigen-binding fragments disclosed herein may be used with any modality. Any of the antibody or antigen-binding fragments disclosed herein may be linked, directly or indirectly, to a therapeutic moiety. The therapeutic moiety may comprise a cytotoxic drug or a radioisotope. See, for example, Lu, Ruei-Min et al. Journal of Biomedical Science vol. 27(1): 1 (2020), for some of the many ways that the antibodies or antigen-binding fragments may be used.

[0049] Provided is a nucleic acid encoding any one of the molecular complexes, multimeric complexes, or fusions proteins disclosed herein, optionally operably linked to a heterologous promoter.

[0050] Provided is a nucleic acid encoding any of the antibodies or antigen-binding fragments disclosed herein, or a fragment thereof, optionally linked to a heterologous promoter.

[0051] Provided is a recombinant host cell comprising any of the fusion proteins, the antibodies or antigen-binding fragments, or the nucleic acids disclosed herein.

[0052] Provided is, inter alia, a cell redirector, such as a T cell, NK cell, or other immune cell, etc. redirector and / or chimeric antigen receptor (CAR), and the like, that specifically recognizes any of the molecular complexes, multimeric complexes, or fusion proteins disclosed herein. The T-cell redirector, NK cell redirector, or CAR may have a high affinity for any of the molecular complexes, multimeric complexes, or fusion proteins disclosed herein and a low affinity for free KLK2. The T-cell redirector, NK cell redirector, or CAR may-6-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 have a high affinity for KLK2 when bound to SPINT1 or SPINT2 or when bound to any of the SPINT1 / 2 peptides disclosed herein, and a low affinity for free KLK2.

[0053] The cell redirector may comprise a soluble bispecific antibody (bsAb) or a membrane-anchored CAR, or a combination thereof. The cell redirector may be a T-cell redirector, wherein an exemplary bispecific antibody binds any of the molecular complexes, multimeric complexes, or fusion proteins disclosed herein and CD3. An exemplary bispecific antibody binds KLK2 when bound to or complexed with SPINT1, SPINT2, or any of the SPINT1 / 2 peptides disclosed herein, and CD3, i.e., a KLK2xCD3 T-cell redirector.

[0054] Non-limiting examples of CARs include a T cell expressing CAR (CAR-T cell), a natural killer cell expressing CAR (CAR-NK cell), and a macrophage expressing CAR (CAR- M cell). The CAR-T cell, CAR-NK cell, or CAR-M cell may be allogeneic. The CAR may comprise an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The extracellular antigen-binding domain binds any of the molecular complexes, multimeric complexes, or fusion proteins disclosed herein, such as KLK2 when bound to or complexed with SPINT1, SPINT2, or any of the SPINT1 / 2 peptides disclosed herein. The intracellular signaling domain may comprise a T-cell surface glycoprotein CD3 zeta chain component.

[0055] Provided is a nucleic acid encoding any of the T-cell redirectors or CARs disclosed herein, or a fragment thereof, optionally operably linked to a heterologous promoter.

[0056] Provided is a recombinant host cell comprising any of the nucleic acids disclosed herein. The cell may be a CAR-T cell.

[0057] Provided is a composition comprising any of the fusion proteins, the antibodies or antigen-binding fragments, or the T-cell redirectors or CAR-T cells disclosed herein.

[0058] Provided is a method of inducing an immune response in a non-human subject, the method comprising administering to the subject any of the complexes disclosed herein or the compositions disclosed herein. The subject may be a mammal, for example a simian, a mouse, a rat, a cow, or a llama.

[0059] Provided is a system for inducing an immune response in a non-human subject, comprising any of the complexes disclosed herein, or the compositions disclosed herein, and a delivery system. The delivery system may comprise a syringe. The compositions may be administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration. The subject may be a mammal, for example a non-human, a simian, a mouse, a rat, a cow, or a llama.-7-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1

[0060] Provided is a system for active immunization to prevent a disease in a subject, the system comprising any of the complexes disclosed herein, or the compositions disclosed herein. The subject may be a mammal, for example a non-human, a simian, a mouse, a rat, a cow, or a llama. The system may comprise a delivery system. The delivery system may be a syringe. The fusion protein may be administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration. The subject may be a mammal, for example a non-human, human, a simian, a mouse, a rat, a cow, or a llama.BRIEF DESCRIPTION OF THE FIGURES

[0061] FIG. 1A shows volcano plots depicting the global dose responsive association analysis of isotype control (left panel), KLK2-AB2 (middle panel), and KLK2-AB1 (right panel) on the VcaP cell surface.

[0062] FIG. IB shows dose responsive association curves of putative targets SPINT1 (top left panel), SPINT2 (top right panel), KLK2 (bottom left panel), and PTPRF (bottom right panel) with different antibodies of interest (• Isotype Control; o KLK2-AB2; ® KLK2-AB1).

[0063] FIG. 2 illustrates an exemplary modifier screen used to identify SPINT1 and SPINT2 as resistance hits to a KLK2xCD3 T-cell redirector.

[0064] FIG. 3A shows co-immunoprecipitation with KLK2-AB3 anti-KLK2 antibody and detection by KLK2, SPINT1, or SPINT2 via western blot.

[0065] FIG. 3B shows KLK2 surface expression determined by flow cytometry on wild type cells (top left panel), a KLK2 knock-out (top right panel), a SPINT1 knock-out (bottom left panel), and a SPINT2 knock-out (bottom right panel).

[0066] FIG. 3C shows KLK2, SPINT1, and SPINT2 knockouts characterization by Western Blot.

[0067] FIG. 4A shows a sequence alignment of the N-terminal region between KLK2 and KLK3 noting the pro-peptide in the box and nearby Trypsin and LysC cleavage sites.

[0068] FIG. 4B lists peptides monitored in the proteomics analysis of biotinylated enriched cell-surface samples from VcaP cells.

[0069] FIG. 5 A shows the domain organization of SPINT1.

[0070] FIG. 5B shows a manual docking model of KLK2 / SPINT1 complex based on serine protease and Kunitz domain complex from PDB: 1YCO2; KLK2 (PDF: 4NFE3) and KI from SPINT1 (PDB: 5H7V4) aligned to 1YCO.-8-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1

[0071] FIG. 5C shows that a multimer folding model of KLK2 with SPINT1 KI or K2 using AlphaFold2 matches well with manual docking models of fragments from complex crystal structures.

[0072] FIG. 5D shows a schematic of an antibody that binds to a KLK2 / K1 complex but does not bind to soluble (free) KLK2.

[0073] FIG. 6 shows a series of chromatograms in a study of complexing between mature KLK2 and SPINT1.

[0074] FIG. 7A shows single point binding data obtained with single B cell supernatants from mice immunized with KLK2 / SPINT1 protein complexes (top panel) and KLK2 / SPINT2 protein complexes (bottom panel) of the present disclosure.

[0075] FIG. 7B shows KLK2 monomer protein binding vs cell binding from 536 mAbs identified for mice immunized with KLK2 / SPINT complexes. Antibodies binding to protein and VCaP cells endogenously expressing KLK2 are in the blue circle (*Cluster < 6 residues differ across ALL CDR, Family < 22 residues differ across all CDRs).

[0076] FIG. 8 shows flow cytometry cell binding data for an antibody panel isolated from mice immunized with the KLK2 monomer (top panel) or a KLK2 / SPINT complex (bottom panel).

[0077] FIG. 9 illustrates structures of KLK2 in complex with SPINT1 (at left) and SPINT2 (at right), respectively. Tool Fabs are omitted from experimental structures in the figures for clarity of the KLK2 interfaces with SPINT1 and SPINT2.DETAILED DESCRIPTION

[0078] Definitions and Abbreviations

[0079] While the general inventive concepts are susceptible of embodiment in many forms, there are shown in the drawings, and will be described herein in detail, specific embodiments thereof with the understanding that the present disclosure is to be considered an exemplification of the principles of the general inventive concepts. Accordingly, the general inventive concepts are not intended to be limited to the specific embodiments illustrated herein.

[0080] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0081] In the present disclosure the singular forms “a,” “an,” and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “a material” is a reference to at least one of such materials and equivalents thereof known to those skilled in the art, and so forth.-9-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1

[0082] When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list and every combination of that list is to be interpreted as a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C.”

[0083] It is to be appreciated that certain features of the disclosure that are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. That is, unless obviously incompatible or excluded, each individual embodiment is deemed to be combinable with any other embodiments, and such a combination is considered to be another embodiment. Conversely, various features of the disclosure that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation. Finally, while an embodiment may be described as part of a series of steps or part of a more general structure, each said step may also be considered an independent embodiment in itself.

[0084] “About” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the Examples or elsewhere in the Specification in the context of a particular assay, result, or embodiment, “about” means within one standard deviation per the practice in the art, or a range of up to 5%, whichever is larger.

[0085] The conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or.”

[0086] The terms “antibody” and "antibodies" as used herein are meant in a broad sense and include immunoglobulin molecules including polyclonal antibodies, monoclonal antibodies including murine, human, human- adapted, humanized and chimeric monoclonal-10-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 antibodies, antibody fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, and single chain antibodies.

[0087] Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (K) and lambda (X), based on the amino acid sequences of their constant domains.

[0088] The term "antibody fragment" or "antigen-binding fragment" refers to a portion of an immunoglobulin molecule that retains the heavy chain and / or the light chain antigen binding site, such as heavy chain complementarity determining regions (HCDR) 1, 2 and 3, light chain complementarity determining regions (LCDR) 1, 2 and 3, a heavy chain variable region (VH), or a light chain variable region (VL). Antibody fragments include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a domain antibody (dAb) fragment, which consists of a VH domain. VH and VL domains can be engineered and linked together via a synthetic linker to form various types of single chain antibody designs where the VH / VL domains pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody; described for example in PCT Inti. Publ. Nos. WO1998 / 44Q01, WO1988 / 01649, WO1994 / 13804, and W01992 / 01047. These antibody fragments are obtained using well known techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are full length antibodies.

[0089] The phrase "isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody specifically binding KLK2 or a SPINTLKLK2 or a SPINT2:KLK2 complex is substantially free of antibodies that specifically bind antigens other than human KLK2 or a SPINTLKLK2 or a SPINT2:KLK2 complex respectively). An isolated antibody that specifically binds KLK2 or a SPINTLKLK2 or a SPINT2:KLK2 complex, however, can have cross-reactivity to other antigens, such as orthologs of human KLK2, such as Macaca-11-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 fascicularis (cynomolgus monkey) KLK2. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0090] " Humanized antibody" refers to an antibody in which the antigen binding sites are derived from non-human species and the variable region frameworks are derived from human immunoglobulin sequences. Humanized antibodies may include substitutions in the framework regions so that the framework may not be an exact copy of expressed human immunoglobulin or germline gene sequences.

[0091] " Human antibody" refers to an antibody having heavy and light chain variable regions in which both the framework and the antigen binding sites are derived from sequences of human origin. If the antibody contains a constant region, the constant region also is derived from sequences of human origin. A human antibody comprises heavy or light chain variable regions that are "derived from" sequences of human origin wherein the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such systems include human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals such as mice carrying human immunoglobulin loci as described herein. A human antibody may also contain amino acid differences when compared to the human germline or rearranged immunoglobulin sequences due to for example naturally occurring somatic mutations or intentional introduction of substitutions in the framework or antigen binding sites. Typically, a human antibody is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical in amino acid sequence to an amino acid sequence encoded by a human germline or rearranged immunoglobulin gene.

[0092] Isolated humanized antibodies may be synthetic. Human antibodies, while derived from human immunoglobulin sequences, may be generated using systems such as phage display incorporating synthetic CDRs and / or synthetic frameworks, or can be subjected to in vitro mutagenesis to improve antibody properties, resulting in antibodies that do not naturally exist within the human antibody germline repertoire in vivo.

[0093] The term "recombinant antibody" as used herein, includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the antibody, antibodies isolated from a recombinant, combinatorial antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences,-12-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 sequences, or antibodies that are generated in vitro using Fab arm exchange such as bispecific antibodies.

[0094] The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope, or in a case of a bispecific monoclonal antibody, a dual binding specificity to two distinct epitopes.

[0095] The term "epitope" as used herein means a portion of an antigen to which an antibody specifically binds. Epitopes usually consist of chemically active (such as polar, nonpolar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope can be composed of contiguous and / or discontiguous amino acids that form a conformational spatial unit. For a discontiguous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3-dimensional space through the folding of the protein molecule.

[0096] The term “chimeric antigen receptor” or “CAR” as used herein means a synthetic or recombinant receptor comprising an antigen specific domain, a costimulatory domain and an intracellular signaling domain. The CAR further comprises an extracellular hinge or spacer region, a transmembrane domain, or combinations thereof. The antigen specific domain may be an scFv.

[0097] The term “chimeric antigen receptor T cell” or “CAR-T” as used herein means a T cell expressing a CAR. The term “chimeric antigen receptor NK cell” or “CAR-NK” as used herein means an NK cell expressing a CAR.

[0098] Unless specifically indicated otherwise, the term “complex” may be understood to include molecular complexes, such as complexes between a KEK2 peptide and a SPINT1 peptide and / or a SPINT2 peptide, and fusion proteins or peptides, such as any of the fusions proteins disclosed herein (i.e., fusions between a KEK2 peptide and a SPINT1 peptide and / or a SPINT2 peptide, or fragment thereof).

[0099] Other than in the examples, or where otherwise indicated, all numbers expressing, for example, quantities of ingredients used in the specification are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification are approximations that may vary depending upon the desired properties to be obtained by the methods of the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be-13-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0100] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosed methods are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard variation found in their respective testing measurements. When ranges are given, any endpoints of those ranges and / or numbers within those ranges can be combined within the scope of the present disclosure. That is, a range is intended to comprise every integer or fraction or value within the range and endpoints of the range.

[0101] “Comprising,” “consisting essentially of,” and “consisting of’ are intended to connote their generally accepted meanings in the patent vernacular; that is, (i) “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; (ii) “consisting of’ excludes any element, step, or ingredient not specified in the claim; and (iii) “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristics” of the claimed invention. Embodiments described in terms of the phrase “comprising” (or its equivalents) also provide as embodiments those independently described in terms of “consisting of’ and “consisting essentially of.” Prostate cancer is the most common form of cancer among men. There remains a need for effective tools for studying and treating prostate cancer.

[0102] Aspects of the Disclosure

[0103] Kallikrein-related peptidase 2 (KLK2) is complex. KLK2 has been reported to exist in both membrane-bound and free (soluble) forms that exhibit distinct biological and clinical characteristics. The soluble KLK2 represents a secreted form that is released into extracellular fluids, including seminal plasma and blood. This soluble form is the type most commonly detected in diagnostic assays, such as those employed for prostate cancer biomarker testing. Membrane-bound KLK2 is thought to exert localized activity, including modulation of pericellular proteolysis, mediation of cell-cell signaling, and potential involvement in processes such as cancer cell invasion and adhesion.

[0104] To date, and prior to the invention disclosed herein, the manner by which KLK2 is anchored to the surface of prostate cells was an enigma. The ability to detect and differentiate between membrane-bound and free KLK2 may provide insight into tumor biology, such as aggressiveness or invasiveness, while also supporting systemic biomarker applications. This-14-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 unknown aspect of KLK2 biology had challenged efforts to develop improved molecules which recognized KLKs, including diagnostic, therapeutic, etc. antibodies targeting KLK2. Accordingly, the present invention addresses a long-felt and unmet need in the field.FUSION PROTEINS

[0105] Provided is a fusion protein comprising: (i) a KLK2 peptide, and (ii) a SPINT1 peptide, a SPINT2 peptide, or a combination thereof, wherein the KLK2 peptide does not comprise a pro-peptide sequence.

[0106] The SPINT1 peptide and / or the SPINT2 peptide may comprise a MANEC (N) domain, a PKD-like (I) domain, a Kunitz 1 (KI) domain, a LDLRA (L) domain, a Kunitz 2 (K2) domain, or any combination thereof. The fusion protein may comprise a tag, such as a His tag.

[0107] The KLK2 peptide may comprise the amino acid sequence as set forth in SEQ ID NO: 2, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto. See Table 6.

[0108] The SPINT1 peptide may comprise the amino acid sequence as set forth in SEQ ID NO: 3, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto. The KI domain of SPINT1 comprises the amino acid sequence as set forth in SEQ ID NO: 4, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto. The K2 domain of SPINT1 comprises the amino acid sequence as set forth in SEQ ID NO: 5, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.

[0109] The SPINT2 peptide comprises the amino acid sequence as set forth in SEQ ID NO: 6, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto. The KI domain of SPINT2 comprises the amino acid sequence as set forth in SEQ ID NO: 7, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto. The K2 domain of SPINT2 comprises the amino acid sequence as set forth in SEQ ID NO: 8, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.

[0110] The KLK2 peptide and the SPINT1 peptide or the SPINT2 peptide may be connected directly.-15-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1

[0111] The KLK2 peptide and the SPINT1 peptide or the SPINT2 peptide may be connected via a linker. The linker may be a peptide linker. The peptide linker may comprise from about 2 to about 100 amino acids, such as from about 2, 5, 7, 10, 15 amino acids to about 50, 60, 70, 75, 80, 85, 90, 85, or 100 amino acids. The peptide linker may comprise predominantly G and S amino acids.

[0112] The peptide linker may comprise the amino acid sequence GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 13), or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.

[0113] The fusion protein may comprise the amino acid sequence as set forth in SEQ ID NO: 9, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; SEQ ID NO: 10, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto, SEQ ID NO: 11; or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; or SEQ ID NO: 12, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.

[0114] The fusion protein may be immunogenic, for example strongly immunogenic.

[0115] Provided is a nucleic acid encoding any of the molecular complexes, multimer complexes, or fusion proteins disclosed herein, optionally operably linked to a heterologous promoter.

[0116] Provided is a recombinant host cell comprising any of the molecular complexes, multimer complexes, or fusion proteins disclosed herein; the antibody or antigen-binding fragments that bind to any of the molecular complexes, multimer complexes, or fusion proteins disclosed herein; or the nucleic acid of any of the molecular complexes, multimer complexes, or fusion proteins disclosed herein; or the antibody or antigen-binding fragments that bind thereto.MOLECULAR AND MULTIMERIC COMPLEXES

[0117] Provided are complexes of the KLK2 peptide and any of the SPINT1 or SPINT2 peptides, or fragments thereof. For example, provided is a molecular complex that generally includes complexes of a KLK2 peptide with SPINT1 or SPINT2 protein, or complexes of KLK2 peptide with a peptide fragment of SPINT1 or SPINT2. Exemplary peptide fragments of SPINT1 and SPINT2 include peptides comprising a MANEC (N) domain, a PKD-like (I)-16-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 domain, a Kunitz 1 (KI) domain, a LDLRA (L) domain, a Kunitz 2 (K2) domain, or any combination thereof. As such, the molecular complex may also include KLK2 complexed with a peptide comprising any one or more of the KI domain of SPINT1, the K2 domain of SPINT1, the KI domain of SPINT2, the K2 domain of SPINT2, the MANEC (N) domain of SPINT1, the MANEC (N) domain of SPINT2, the PKD-like (I) domain of SPINT1, the PKD-like (I) domain of SPINT2, the LDLRA (L) domain of SPINT1, and the LDLRA (L) domain of SPINT2.

[0118] The KLK2 peptide may comprise an amino acid sequence as set forth in SEQ ID NO: 2, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0119] The SPINT1 protein may comprise an amino acid sequence as set forth in SEQ ID NO: 3, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0120] The KI domain of SPINT1 may comprise an amino acid sequence as set forth in SEQ ID NO: 4, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0121] The K2 domain of SPINT1 may comprise an amino acid sequence as set forth in SEQ ID NO: 5, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0122] The SPINT2 protein may comprise an amino acid sequence as set forth in SEQ ID NO: 6, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0123] The KI domain of SPINT2 may comprise an amino acid sequence as set forth in SEQ ID NO: 7, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0124] The K2 domain of SPINT2 may comprise an amino acid sequence as set forth in SEQ ID NO: 8, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0125] Multimeric complexes may include complexes of KLK2 protein or KLK2 peptide with a multimerized, e.g., dimerized or concatemerized (multimers of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptide segments), peptide fragment of SPINT1 or SPINT2. Multimeric complexes may include complexes of KLK2 protein or KLK2 peptide with two or more of the peptide fragments of SPINT1 and / or SPINT2 described herein, e.g., dimerized or concatemerized. Multimeric complexes may include complexes of two or more KLK2-17-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 peptides, e.g., dimerized or concatemerized (multimers of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptide segments), with one or more of the peptide fragments of SPINT1 or SPINT2 described herein. Multimeric complexes may include complexes of two or more KLK2 peptides, e.g., dimerized or concatemerized (multimers of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more peptide segments), with two or more of the peptide fragments of SPINT1 and / or SPINT2 described herein.

[0126] KLK2 peptides may comprise the amino acid sequence as set forth in SEQ ID NO: 2, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6). Peptides or fragment thereof of SPINT1 or SPINT2 in the multimeric complexes may comprise a MANEC (N) domain, a PKD-like (I) domain, a Kunitz 1 (KI) domain, a LDLRA (L) domain, a Kunitz 2 (K2) domain, or any combination thereof. Peptides or fragment thereof of SPINT1 or SPINT2 in the multimeric complexes may comprise the amino acid sequence as set forth in any of SEQ ID NO: 3, 4, 5, 6, 7, or 8, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto (see, e.g., Table 6).

[0127] The various multimerized peptides of the multimeric complexes may be connected directly, i.e., concatemerized, or via a linker. The linker may be a peptide linker.

[0128] The molecular and multimeric complexes may be immunogenic such as, for example, strongly immunogenic.

[0129] The molecular and multimeric complex may comprise a tag, such as a His tag.ANTIBODIES AND ANTIGEN-BINDING FRAGMENTS

[0130] Also provided is an antibody or antigen-binding fragment that specifically recognizes any of the molecular complexes, multimer complexes, or fusion proteins disclosed herein. The antibody or antigen-binding fragment has high affinity for the molecular complex, multimer complex, or fusion protein and has low affinity for free KLK2.

[0131] The antibody or antigen-binding fragment has an affinity for the molecular complex, multimer complex, or fusion protein from 1 pM to 100 nM kD. The antibody or antigen-binding fragment has >5 -fold lower affinity for free KLK2 versus the molecular complex, multimer complex, or fusion protein. The antibody or antigen-binding fragment does not bind free KLK2.

[0132] The antibody or antigen-binding fragment has high affinity for KLK2 when bound to SPINT1, SPINT2, or any of the fragments thereof disclosed herein, and has low affinity for free KLK2.-18-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1

[0133] The antibody or antigen-binding fragment has an affinity for KLK2 when bound to SPINT1, SPINT2, or any of the fragments thereof disclosed herein of from 1 pM to 100 nM kD. The antibody or antigen-binding fragment has >5 -fold lower affinity for free KLK2 versus KLK2 bound to SPINT1, SPINT2, or any of the fragments thereof disclosed herein.

[0134] Provided is an antibody or antigen-binding fragment that binds KLK2.

[0135] Provided is an antibody or antigen-binding fragment that has high affinity for KLK2 when bound to SPINT1, SPINT2, or any of the fragments thereof disclosed herein, and has low affinity for free KLK2.

[0136] The antibody or antigen-binding fragments disclosed herein may be generated by phage-display. The antibody or antigen-binding fragments disclosed herein may be generated by in silico methods.

[0137] The antibodies or antigen-binding fragments disclosed herein may be used with any modality. The antibodies or antigen-binding fragments disclosed herein may be linked, directly or indirectly, to a therapeutic moiety. The therapeutic moiety may comprise a cytotoxic drug or a radioisotope. See, for example, Lu, Ruei-Min et al. Journal of biomedical science vol. 27(1): 1 (2020) for some of the many ways that the antibodies or antigen-binding fragments may be used.

[0138] The antibodies or antigen-binding fragments described herein may be radiolabeled, for example with90Y,32P,186Re / 188Re,76As / 77As,89Sr,153Sm,131I,67Cu,161Tb,105Rh,45Ca,35S,51Cr,67Ga, "mTc,U1ln,141mIn,123I,125I,2O1T1,212Bi,213Bi,223Ac,227Th,212Pb,255Fm,223Ra,149Tb,211At,177Lu,225Ac, orU1ln.

[0139] The antibodies or antigen-binding fragments described herein may be conjugated to a drug or cytotoxic agent.

[0140] The antibodies or antigen-binding fragments may be chimeric. As used herein, the term “chimeric” refers to an antibody, or antigen-binding fragment thereof, having at least some portion of at least one variable domain derived from the antibody amino acid sequence of a non-human mammal, a rodent, or a reptile, while the remaining portions of the antibody, or antigen-binding fragment thereof, are derived from a human.

[0141] The antibodies may include monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies, polyclonal, antigen-binding fragments, bispecific or multispecific antibodies, monomeric, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity. The antibody can be a naturally occurring antibody, e.g., an antibody isolated and / or purified-19-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 from a mammal, e.g., a murine, primate, mouse, rabbit, goat, horse, chicken, hamster, human, etc. Alternatively, the antibody can be an engineered (e.g., genetically engineered) antibody.

[0142] The antibodies may be humanized antibodies. Humanized antibodies may be chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2, scFv, spFv, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. The humanized antibody may include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0143] Humanized antibodies have antigen binding sites derived from non-human species and the variable region frameworks are derived from human immunoglobulin sequences. Human antibodies have heavy and light chain variable regions in which both the framework and the antigen binding site are derived from sequences of human origin.

[0144] Provided is a nucleic acid encoding any of the antibodies or antigen-binding fragments disclosed herein, of a fragment of the nucleic acid, optionally linked to a heterologous promoter.

[0145] Provided is a recombinant host cell comprising any of the antibodies or antigenbinding fragments disclosed herein, or any of the nucleic acids disclosed herein.

[0146] The antibodies or antigen-binding fragments described herein may be used as a PET tracer. The antibodies or antigen-binding fragments described herein may be used as a diagnostic tool. The antibodies or antigen-binding fragments described herein may be used to select patient populations for treatment.T-CELL REDIRECTORS AND CARS

[0147] Provided is a cell redirector or CAR that specifically recognizes any of the molecular complexes, multimeric complexes, or fusion proteins disclosed herein. The cell redirector or CAR has high affinity for the molecular complexes, multimeric complexes, or fusion proteins and has low affinity for free KLK2. The cell redirector or CAR has high affinity-20-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 for KLK2 when bound to SPINT1 or SPINT2, or when bound to any of the SPINT1 or SPINT2 peptides disclosed herein, and has low affinity for KLK2.

[0148] The cell redirector may comprise a soluble bispecific antibody (bsAb) or a membrane-anchored CAR, or a combination thereof. The cell redirector may be a T-cell redirector, wherein an exemplary bispecific antibody binds any of the molecular complexes, multimeric complexes, or fusion proteins disclosed herein and CD3. An exemplary bispecific antibody binds KLK2 when bound to or complexed with SPINT1, SPINT2, or any of the SPINT1 or SPINT2 peptides disclosed herein, and CD3, i.e., a KLK2xCD3 T-cell redirector.

[0149] Non-limiting examples of CARs include a T-cell expressing CAR (CAR-T cell), a natural killer cell expressing CAR (CAR-NK cell), and a macrophage expressing CAR (CAR-M cell). The CAR-T cell, CAR-NK cell, or CAR-M cell may be allogeneic. The CAR may comprise an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The extracellular antigen-binding domain binds any of the molecular complexes, multimeric complexes, or fusion proteins disclosed herein, such as KLK2 when bound to or complexed with SPINT1, SPINT2, or any of the SPINT1 or SPINT2 peptides disclosed herein. The intracellular signaling domain may comprise a T-cell surface glycoprotein CD3 zeta chain component.

[0150] Provided is a nucleic acid encoding any of the cell redirectors or CARs disclosed herein, or a fragment thereof, optionally operably linked to a heterologous promoter.

[0151] Provided is a recombinant host cell comprising any of the cell redirectors or CARs disclosed herein, or the nucleic acid encoding any of the cell redirectors or CARs disclosed herein.

[0152] Provided is a method of making a CAR-T or CAR-NK cell by transducing a T cell or NK cell, respectively, with a vector comprising the isolated nucleic acids encoding the CARs as disclosed herein. Also provided is a method of producing the CAR-T or CAR-NK cell as disclosed herein. The method comprising culturing T-cells or NK-cells comprising a nucleic acid encoding a chimeric antigen receptor (CAR) as disclosed herein under conditions to produce the CAR-T cell or CAR-NK cell, respectively, and recovering the CAR cell.COMPOSITIONS

[0153] Provided is a composition comprising (i) a KLK peptide, and (ii) a SPINT peptide. The KLK peptide may be absent a pro-peptide sequence. The KLK peptide and / or SPINT peptide may be non-naturally occurring, such as purified, isolated, recombinant, or synthetic.-21-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1

[0154] Provided is a composition comprising (i) a KLK2 peptide, and (ii) a SPINT1 peptide and / or a SPINT2 peptide, wherein the KLK2 peptide does not comprise a pro-peptide sequence and is non-naturally occurring and the SPINT1 peptide and / or the SPINT2 peptide are non-naturally occurring. Non-naturally occurring may be understood to mean an isolated, purified, synthetic, or recombinant peptide. Any of the KLK2 peptide, the SPINT1 peptide, and / or the SPINT2 peptide may comprise a tag, such as a His tag.

[0155] The composition may comprise complexes formed between the KLK2 peptide and the SPINT1 and / or SPINT2 peptide. The KLK2 peptide, and the SPINT1 and / or a SPINT2 peptide may be included in the composition in equimolar amounts, i.e., 1:1 KLK2:SPINT1 or KLK2:SPINT2 or even l:0.5:0.5 KLK2:SPINT1:SPINT2, etc. The KLK2 peptide, and the SPINT1 and / or a SPINT2 peptide may be included in the composition in non-equimolar amounts, i.e., an excess of the SPINT1 or SPINT2 peptide may be included in the composition to ensure complex formation or to reduce free KLK2.

[0156] Also provided is a composition comprising the molecular complexes, multimeric complexes, or fusion proteins disclosed herein, and optionally a pharmaceutically acceptable diluent, carrier, or excipient.

[0157] Provided is a method of producing a pharmaceutical composition comprising any of the molecular complexes, multimeric complexes, or fusion proteins disclosed herein, the method comprising combining the molecular complex(es), multimeric complex(es), or fusion protein(s) with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0158] Provided is a composition comprising any of the antibody or antigen-binding fragments disclosed herein, i.e., antibodies or fragments thereof that bind to any of the molecular complexes, multimeric complexes, or fusion proteins disclosed herein, and a pharmaceutically acceptable diluent, carrier, or excipient.

[0159] Provided is a method of producing a pharmaceutical composition comprising any of the antibody or antigen-binding fragments disclosed herein, comprising combining the antibody or antigen-binding fragments with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0160] Provided is a composition comprising any of the cell redirectors or CARs of any disclosed herein, and optionally a pharmaceutically acceptable diluent, carrier, or excipient.

[0161] Provided is a method of producing a pharmaceutical composition comprising any of the cell redirectors or CARs disclosed herein, comprising combining the cell redirector-22-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 or CAR cell with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0162] Additional compounds may also be included in the pharmaceutical compositions, including chelating agents such as EDTA, citrate, EGTA or glutathione.

[0163] The pharmaceutical compositions may be prepared in a manner known in the art that is sufficiently storage stable and suitable for administration to humans and animals. For example, the pharmaceutical compositions may be lyophilized, e.g., through freeze drying, spray drying, spray cooling, or through use of particle formation from super critical particle formation.

[0164] By “pharmaceutically acceptable” is meant a non-toxic material that does not decrease the effectiveness of the material of the present disclosure. Such pharmaceutically acceptable buffers, carriers or excipients are well-known in the art (see Remington’s Pharmaceutical Sciences, 18thedition, A.R. Gennaro, Ed., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rdedition, A. Kibbe, Ed. Pharmaceutical Press (2000), the disclosures of which are incorporated herein by reference in their entirety).

[0165] The term “buffer” is intended to mean an aqueous solution containing an acidbase mixture with the purpose of stabilizing pH. Examples of buffers include Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazolelactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES.

[0166] The term “diluent” is intended to mean an aqueous or non-aqueous solution with the purpose of diluting the agent in the pharmaceutical preparation. The diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol, or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).

[0167] The term “adjuvant” is intended to mean any compound added to the formulation to increase the biological effect of the fusion protein, antibody or antigen-binding fragment, or T-cell (each independently referred to as the “agent”) of the present disclosure. The adjuvant may be one or more of zinc, copper or silver salts with different anions, for example, but not limited to fluoride, chloride, bromide, iodide, thiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate, and acetates of different acyl composition. The adjuvant may also be cationic polymers such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic-23-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 synthetic polymers such as poly (vinyl imidazole), and cationic polypeptides such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.

[0168] The excipient may be one or more of carbohydrates, polymers, lipids, and minerals. Examples of carbohydrates include lactose, glucose, sucrose, mannitol, and cyclodextrines, which may be added to the composition, e.g., for facilitating lyophilization. Examples of polymers include starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polysulphonate, polyethylenglycol / polyethylene oxide, polyethyleneoxide / polypropylene oxide copolymers, poly vinylalcohol / poly vinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone, all of different molecular weight, which are added to the composition, e.g., for viscosity control, for achieving bioadhesion, or for protecting the lipid from chemical and proteolytic degradation. Examples of lipids include fatty acids, phospholipids, mono-, di-, and triglycerides, ceramides, sphingolipids and glycolipids, all of different acyl chain length and saturation, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, which are added to the composition for reasons similar to those for polymers. Examples of minerals include talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduction of liquid accumulation or advantageous pigment properties.

[0169] The agents of the present disclosure may be formulated into any type of pharmaceutical composition known in the art to be suitable for the delivery thereof.

[0170] The pharmaceutical compositions of the present disclosure may be in the form of a liposome, in which the agent is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids, which exist in aggregated forms as micelles, insoluble monolayers and liquid crystals. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Suitable lipids also include the lipids above modified by poly(ethylene glycol) in the polar headgroup for prolonging bloodstream circulation time. Preparation of such liposomal formulations can be found in for example U.S. Pat. No. 4,235,871, the disclosures of which are incorporated herein by reference.

[0171] The pharmaceutical compositions of the present disclosure may also be in the form of biodegradable microspheres. Aliphatic polyesters, such as poly (lactic acid) (PL A), poly(gly colic acid) (PGA), copolymers of PL A and PGA (PLGA) or poly(carprolactone) (PCL), and poly anhydrides have been widely used as biodegradable polymers in the production-24-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 of microspheres. Preparations of such microspheres can be found in U.S. Pat. No. 5,851,451 and in EP 0 213 303, the disclosures of which are incorporated herein by reference.

[0172] The pharmaceutical compositions disclosed herein may be provided in the form of polymer gels, where polymers such as starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polyvinyl imidazole, polysulphonate, polyethylenglycol / polyethylene oxide, polyethyleneoxide / polypropylene oxide copolymers, polyvinylalcohol / polyvinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone are used for thickening of the solution containing the agent. The polymers may also comprise gelatin or collagen.

[0173] Alternatively, the agents may simply be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol, or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth gum, and / or various buffers.

[0174] It will be appreciated that the pharmaceutical compositions disclosed herein may include ions and a defined pH for potentiation of action of the active agent. Additionally, the compositions may be subjected to conventional pharmaceutical operations such as sterilization and / or may contain conventional adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, fillers, etc.

[0175] The pharmaceutical compositions according to the present disclosure may be administered via any suitable route known to those skilled in the art. Thus, possible routes of administration include parenteral (intravenous, subcutaneous, and intramuscular), topical, ocular, nasal, pulmonar, buccal, oral, parenteral, and rectal. Also administration from implants is possible. Infusion may be a desired route because of the potentially high cytotoxicity of the administered agent.

[0176] The pharmaceutical compositions may be administered parenterally, for example, intravenously, intracerebroventricularly, intraarticularly, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly, or subcutaneously, or they may be administered by infusion techniques. They may be conveniently used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (for example, to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.-25-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1

[0177] Formulations suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

[0178] Thus, the pharmaceutical compositions of the present disclosure are particularly suitable for parenteral, e.g., intravenous, administration.

[0179] Alternatively, the pharmaceutical compositions may be administered intranasally or by inhalation (for example, in the form of an aerosol spray presentation from a pressurized container, pump, spray or nebulizer with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoro-methane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A3 or 1, 1,1, 2, 3,3,3- heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas). In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurized container, pump, spray, or nebulizer may contain a solution or suspension of the active polypeptide, e.g., using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g., sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the present disclosure and a suitable powder base such as lactose or starch.

[0180] The pharmaceutical compositions will be administered to a patient in a pharmaceutically effective dose. A ‘therapeutically effective amount,’ or ‘effective amount,’ or ‘therapeutically effective,’ as used herein, refers to that amount which provides a therapeutic effect for a given condition and administration regimen. This is generally a predetermined quantity of active material calculated to produce a desired therapeutic effect in association with the required additive and diluent, i.e., a carrier or administration vehicle. Further, it is generally intended to mean an amount sufficient to reduce and / or prevent, a clinically significant deficit in the activity, function, and response of the host. Alternatively, a therapeutically effective amount may be sufficient to cause an improvement in a clinically significant condition in a host. As is appreciated by those skilled in the art, the amount of a compound may vary-26-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 depending on its specific activity. Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent. In the methods and use for manufacture of compositions of the present disclosure, a therapeutically effective amount of the active component may be provided. A therapeutically effective amount can be determined by the ordinary skilled medical worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art. The administration of the pharmaceutically effective dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administrations of subdivided doses at specific intervals. Alternatively, the dose may be provided as a continuous infusion over a prolonged period.

[0181] It will be appreciated by persons skilled in the art that the pharmaceutical compositions of the present disclosure may be administered alone or in combination with other therapeutic agents used in the treatment of a cancer. The cancer may be liver cancer, kidney cancer, breast cancer, stomach cancer, spleen cancer, pancreatic cancer, lymphatic cancer, or prostate cancer. The cancer may be prostate cancer.

[0182] The present disclosure also provides a kit comprising an agent as defined above in respect of the first aspect of the present disclosure or a pharmaceutical composition as defined above.

[0183] The present disclosure also provides an agent for use in medicine substantially as described herein. The present disclosure also provides a pharmaceutical composition substantially as described herein. The present disclosure also provides for the use of an agent substantially as described herein. The present disclosure also provides a method of treatment substantially as described herein. The present disclosure also provides a kit substantially as defined herein.METHODS OF INDUCING AN IMMUNE RESPONSE

[0184] Provided is a method of inducing an immune response in a subject, the method comprising administering to the subject any of the molecular complexes, multimer complexes, or fusion proteins disclosed herein, or a composition thereof. The molecular complex, multimer complex, fusion protein, or composition thereof may be administered in an immunogenically effective amount. The subject may be a mammal, for example a human, or a non-human, such as a simian, a mouse, a rat, a cow, or a llama.-27-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1

[0185] The phrase “inducing an immune response” when used with reference to the methods described herein encompasses any of (i) causing a desired immune response or effect in a subject in need thereof against an antigen, such as against any of the molecular complexes, multimer complexes, or fusion proteins disclosed herein, (ii) providing a therapeutic immunity for treating against the antigen, such as producing an immunity in a subject in need thereof, e.g., to provide a therapeutic effect against a disease, such as prostate cancer, (iii) causing or improving cellular immunity, e.g., T cell response, against the antigen, (iv) causing or improving a humoral immune response against the antigen, or (v) causing or improving a cellular and a humoral immune response against the antigen.

[0186] As used herein, the term “therapeutic immunity” or “therapeutic immune response” means that the vaccinated subject is able to control the production of the noted antigens within a cell. As used herein, an “immunogenically effective amount” means an amount of an antigen, composition, polynucleotide, vector, vaccine, etc. sufficient to induce a desired immune effect or immune response in a subject in need thereof. Thus, an immunogenically effective amount can be an amount sufficient to induce an immune response in a subject in need thereof, or produce immunity in a subject in need thereof, e.g., provide a therapeutic effect against a disease. An immunogenically effective amount can vary depending upon a variety of factors, such as, the physical condition of the subject, age, weight, health, etc., and the particular application, e.g., providing protective immunity or therapeutic immunity. An immunogenically effective amount can readily be determined by one of skill in the art in view of the present disclosure.SYSTEMS

[0187] Provided is a system for inducing an immune response in a subject, comprising any of the molecular complexes, multimer complexes, or fusion proteins disclosed herein, or compositions comprising any of those molecular complexes, multimer complexes, or fusion proteins, and a delivery system. The delivery system may comprise a syringe. The molecular complex(es), multimer complex(es), fusion protein(s) or compositions thereof may be administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.

[0188] The subject is a mammal, for example a human, or a non-human, such as a simian, a mouse, a rat, a cow, or a llama.

[0189] Provided is a system for active immunization to prevent a disease in a subject, the system comprising any of the molecular complexes, multimer complexes, or fusion proteins-28-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 disclosed herein, or compositions comprising any of those molecular complexes, multimer complexes, or fusion proteins.

[0190] The system may further comprise a delivery system. The delivery system may comprise a syringe. The fusion protein may be administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.EXEMLEPARY EMBODIMENTS

[0191] Provided below are enumerated embodiments. These embodiments are illustrative only and do not limit the scope of the present disclosure or of the claims attached hereto.

[0192] Embodiment Section 1 :1. A composition comprising:(i) a KLK peptide; and(ii) a SPINT peptide.2. The composition of embodiment 1, wherein the KLK peptide does not comprise a propeptide sequence.3. The composition of embodiments 1 or 2, wherein the KLK peptide, and the SPINT peptide are isolated, purified, synthetic, non-naturally occurring, or recombinant peptides.4. The composition of embodiments 1 or 2, wherein the KLK peptide forms a complex with the SPINT peptide.5. The composition of embodiments 1 or 2, wherein the KLK peptide comprises a KLK2 peptide, and the SPINT peptide comprises a SPINT 1 peptide, a SPINT2 peptide, or a combination thereof.6. The composition of embodiment 5, wherein the KLK2 peptide, and the SPINT1 peptide and / or SPINT2 peptide, are isolated, purified, synthetic, non-naturally occurring, or recombinant peptides.7. The composition of embodiment 5, wherein the KLK2 peptide forms a complex with the SPINT 1 and / or SPINT2 peptide.8. The composition of embodiment 1, wherein the SPINT peptide comprises a MANEC (N) domain, a PKD-like (I) domain, a Kunitz 1 (KI) domain, a LDLRA (L) domain, a Kunitz 2 (K2) domain, or a combination thereof.9. The composition of embodiment 1, wherein (ii) comprises a SPINT 1 peptide or a fragment thereof.-29-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT110. The composition of embodiment 1, wherein (ii) comprises a SPINT2 peptide or a fragment thereof.11. The composition of embodiment 9, wherein the SPINT1 peptide comprises the amino acid sequence as set forth in SEQ ID NO: 3, 15, or 16, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.12. The composition of embodiment 9, wherein the fragment of the SPINT1 peptide comprises the amino acid sequence as set forth in SEQ ID NO: 4 or 5, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.13. The composition of embodiment 10, wherein the SPINT2 peptide comprises the amino acid sequence as set forth in SEQ ID NO: 6, 17, or 18, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.14. The composition of embodiment 10, wherein the fragment of the SPINT2 peptide comprises the amino acid sequence as set forth in SEQ ID NO: 7 or 8, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.15. The composition of embodiment 1, wherein (ii) comprises a multimer of the SPINT peptide or a fragment thereof.16. The composition of embodiment 15, wherein the multimer is concatenated.17. The composition of embodiment 5, wherein (ii) comprises a multimer of any combination of the SPINT 1 peptide, a fragment of the SPINT 1 peptide, the SPINT2 peptide, or a fragment of the SPINT2 peptide.18. The composition of embodiment 17, wherein the multimer is concatenated.19. The composition of embodiment 1 , wherein the KLK peptide comprises the amino acid sequence as set forth in SEQ ID NO: 2, 14, 19, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.20. The composition of any one of embodiments 1 to 19, wherein (i) and (ii) are connected directly to form a fusion protein.21. The composition of any one of embodiments 1 to 19, wherein (i) and (ii) are connected via a linker to form a fusion protein.22. The composition of embodiment 21, wherein the linker is a peptide linker.-30-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT123. The composition of embodiment 21, wherein the linker comprises the amino acid sequence as set forth in SEQ ID NO: 13, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.24. The composition of embodiment 21, wherein the fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 9, 10, 11, or 12, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.25. The complex of any one of embodiments 1 to 24, wherein the complex comprises a tag.26. The complex of embodiment 25, wherein the tag is a His tag.27. The composition of embodiment 1, wherein (i) the KLK peptide comprises the amino acid sequence as set forth in SEQ ID NO: 19, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto, and (ii) the SPINT peptide comprises the amino acid sequence as set forth in SEQ ID NO: 15, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.28. The composition of embodiment 1, wherein (i) the KLK peptide comprises the amino acid sequence as set forth in SEQ ID NO: 19, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto, and (ii) the SPINT peptide comprises the amino acid sequence as set forth in SEQ ID NO: 17, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.29. The composition of embodiments 27 or 28, wherein (i) and (ii) are connected via a linker to form a fusion protein.30. The composition of embodiment 29, wherein the linker is a peptide linker.31. The composition of embodiment 30, wherein the linker comprises the amino acid sequence as set forth in SEQ ID NO: 13, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.32. The composition of any one of embodiments 1 to 31, wherein the complex is immunogenic.33. The composition of any one of embodiments 1 to 32 used to generate molecules that differentially bind to membrane associated KLK.-31-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT134. The composition of any one of embodiments 5 to 32 used to generate antibodies that differentially bind to membrane associated KLK2 over free KLK2.35. A nucleic acid encoding (i) or (ii) according to any of embodiments 1 to 31, operably linked to a heterologous promoter.36. A recombinant host cell comprising (i) and / or (ii) of the composition according to any one of embodiments 1 to 32, or the nucleic acid embodiment 35.37. A method of inducing an immune response in a subject, the method comprising administering to the subject the composition according to any one of embodiments 1- 32, wherein the subject is a human or a non-human mammal.38. A system for inducing an immune response in a subject, comprising the composition according to any one of embodiments 1 to 32, and a delivery system.39. The system of embodiment 38, wherein the delivery system comprises a syringe.40. The system of embodiments 38 or 39, wherein the composition is administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.41. A means for inducing an immune response in a subject, the means comprising the composition according to any one of embodiments 1 to 32, and a delivery system, wherein the subject is a human or a non-human mammal42. The means of embodiment 41, wherein the delivery system comprises a syringe.43. The means of embodiment 41, wherein composition is administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.44. A system for active immunization to prevent a disease in a subject, the system comprising the composition according to any one of embodiments 1 to 32, wherein the subject is a human or a non-human mammal, for example a non-human, a simian, a mouse, a rat, a cow, or a llama.45. The system of embodiment 44, further comprising a delivery system.46. The system of embodiment 45, wherein the delivery system is a syringe.47. The system of embodiment 44, wherein the composition is administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.48. A means for active immunization to prevent a disease in a subject, the means configured to specifically target membrane associated KLK2 peptide.-32-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT149. The means of embodiment 48, wherein the means has high affinity for the membrane associated KLK2 peptide and low affinity for free KLK2 peptide.50. The means of embodiment 48, wherein the means has high affinity for KLK2 when bound to a SPINT1 peptide or a SPINT2 peptide, and has low affinity for free KLK2.51. The means of embodiment 48, wherein the means comprises the composition according to any one of embodiments 1 to 32.52. The means of embodiment 51, further comprising a delivery system.53. The means of embodiment 52, wherein the delivery system is a syringe.54. The means of embodiment 51, wherein the means is administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.EXAMPLESExample 1: Proximity labeling using anti-KLK2 antibodies

[0001] Surface proteome mapping of monoclonal antibodies was performed following manufacturer’s protocols with slight modifications. In summary, a goat anti-human IgG secondary antibody was conjugated to an Ir-catalyst following the manufacturer’s protocols. Live VCaP cells (Vertebral-Cancer of the Prostate (VCaP) cell line established from prostate cancer tissue harvested from a metastatic lesion) were plated into a 96-deep well plate, pelleted by centrifugation, and supernatant was removed by vacuum aspiration. Wells were treated with monoclonal primary antibodies in a dose response format (0 to 420 ng / pL) and incubated at 4 °C for 30 minutes with gentle shaking. After 30 minutes, cells were washed twice through cycles of centrifugation, supernatant aspiration, and addition of Dulbecco's phosphate-buffered saline (DBPS) to each well.

[0002] The Ir-catalyst conjugated secondary antibody was then applied to each well at 25 ng / pL and incubated at 4 °C for 30 minutes with gentle shaking. After 30 minutes, cells were again washed twice through cycles of centrifugation, supernatant aspiration, and addition of DBPS to each well. Each well was then treated with a solution of 250 pM diazirine biotin probe in DPBS and immediately irradiated with 450 nm light for 10 minutes.

[0003] After irradiation, cells were then washed twice through cycles of centrifugation, supernatant aspiration, and addition DBPS to each well. Cells were then lysed with the addition of Membrane Permeabilization buffer containing protease inhibitors. Plates were then incubated for 20 minutes at 4 °C with gentle shaking. The membrane fraction was pelleted by-33-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 centrifugation and washed three times with cycles of supernatant aspiration, addition of DPBS, and centrifugation.

[0004] Precipitated membrane proteins were resolubilized with the addition of 1% sodium dodecyl sulfate (SDS) in DPBS and sonicated at 50% amplitude for 5 minutes at 4 °C with a cuphorn sonicator. Magnetic streptavidin beads were added to each well and the plate was incubated at RT for 1 hour with gentle shaking. After 1 hour, the supernatant was removed and the magnetic beads were washed twice with 1% SDS, 4 M urea in DPBS and twice with water using the assistance of a magnetic rack. The beads were then resuspended in LYSE-BCT and heated to 95 °C for 10 minutes. Trypsin / LysC was then added to each well and incubated overnight at 37 °C. The supernatant was removed and further processed using the manufacturer’s protocol for iST cleanup.

[0005] Peptide content quantification was measured by assessing sample fluorescence on a microplate reader with a 295 / 340 nm em / ex filter set. Samples were dried using a vacuum concentrator and then resuspended in 20 pL of LC-LOAD (PreOmics®) and 1 pg was injected onto the Cis analytical column (75 pm x 150 mm). Peptides were separated by applying 4- 4.5% solvent B for 0.7 minutes at 1.3 pL / min, 4.5-5% solvent B for 0.3 minutes at 1.3 pL / min, 5-20% for 17 minutes at 0.6 pL / min, 20-35% solvent B for 8.9 minutes at 0.6 pL / min, 35-55% solvent B for 0.5 minutes at 1.3 pL / min, 55-99% solvent B for 0.2 minutes at 1.3 pL / min, 99% solvent B for 1.7 minutes at 1.3 pL / min, 99-4% solvent B for 0.1 minutes at 1.3 pL / min, and 4% solvent B for 1 minutes at 1.3 pL / min (Solvent A = 0.1% formic acid, Solvent B = 80% MeCN / 0.1% formic acid).

[0006] Eluted peptides were analyzed by a mass spectrometer using the following MS OT settings: [resolution: 120000, scan range: 350-1400 m / z, normalized AGC target: 300%, maximum injection time: 50 milliseconds], and DIA settings: [precursor mass range: 500-900 m / z, isolation window: 13.7 m / z, window overlap: 1 m / z, number of scan events: 30, HCD collision energy: 30%, orbitrap resolution: 30000, scan range: 145-1450, normalized AGC target: 1000%, maximum injection time: 54 milliseconds, scan loop time: 3 seconds].

[0007] Proteomic data were analyzed using DIA-NN v 1.8.1 using default settings except protein inference was turned off, neural network classifier was set to double-pass mode, match-between-runs (MBR) was enabled, and mass accuracies were set to 15 ppm. Raw data were searched against internal, data-dependent acquisition (DDA) generated libraries. For dose response curve (DRC) analysis, overall data analysis for protein group-level abundances was performed by R through Mass Dynamics. DRCs were fitted by a four-parameter log-logistic model in the “drc” package.-34-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1

[0008] FIGS. 1A-1B show that proximity labeling using anti-KLK2 antibodies identified SPINT1, SPINT2 and PTPRF as potential surface interaction partners of KLK2. FIG. 1A shows volcano plots depicting the global dose responsive association analysis of isotype control, KLK2-AB2, and KLK2-AB1 on the VcaP cell surface. FIG. IB shows dose responsive association curves of putative targets: SPINT1 (top left panel), SPINT2 (top right panel), KLK2 (bottom left panel), and PTPRF (bottom right panel) with different antibodies of interest (• Isotype Control; ® KLK2-AB1; o KLK2-AB2). Quantified parameters of the dose response curves are reported in Table 1.Table 1

[0009] The results described herein were surprising and unexpected. Prior to the present disclosure, the interaction between KLK2 and SPINT1 or SPINT2 was not known. SPINT1 and SPINT2 primarily inhibit other serine proteases, specifically matriptase and prostasin. As such, it was surprising and unexpected that KLK2 was found to bind to the partners identified in the experiments described herein, such as SPINT1 and SPINT2, for example.Example 2: Modifier Screen

[0010] CRISPR Library

[0011] A genome-wide modifier screen was performed with the Brunello lentiviral guide Library (CP0041, Broad Institute, Sanson et al. Nat Commun 9: 5416, 2018). This library contains 77,441 total sgRNA with approximately 4 guides targeting each gene in the human genome, and 1000 non-targeting sgRNA as controls.

[0012] CRISPR Screen Design

[0013] A genome-wide CRISPR screen was performed at lOOOx representation with three replicates per each treatment arm. VCaP cells expressing Cas9 protein were transduced with viral library at 0.66 multiplicity of infection (MOI) to limit the chance of cells receiving more than one sgRNA. Virus was added directly to freshly detached cells in suspension and let rest in growth vessels for two days prior to adding puromycin selection. After five days of selection (seven days post-transduction) cells were split into replicates and initial pool sample collected. Two days after seeding, cells were treated with 1:1 effector-to-target ratio (E:T) of pan T-cells, and either l.lnM of KLK2xCD3 or l.lnM of Isotype control. After four days of-35-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 treatment, cells were washed twice with phosphate buffered saline (PBS) to remove dead VCaP cells, T-cells, and antibodies. Fresh growth media was placed back on cells for two days to allow cells to recover prior to sample collection.

[0014] Sample collection, gDNA preparation, and Next Generation Sequencing (NGS)

[0015] Cells were collected after the rest period by trypsinization. An average of 94.3% cell killing was achieved in KLK2xCD3 treated samples compared to Isotype treated samples. Genomic DNA (gDNA) was prepared from cell pellets for isotype and KLK2xCD3 samples. Samples prepared for KLK2xCD3 were also run through a PCR inhibitor clean-up kit. All kits were performed using manufacturer’s instructions. Purified gDNA was sent to the Broad Institute (Cambridge, MA) for Next Generation Sequencing and deconvolution (Doench et al., Nat Biotechnol 2016, 34: 184-191). Read-counts were normalized to reads per million and log2 transformed, then a log2 fold change from the plasmid DNA (pDNA) was calculated for each sgRNA.

[0016] Data Anal sis / Hit-Calling

[0017] The log2 fold changes between KLK2xCD3 treated and isotype control cells were calculated per sgRNA. Statistical analysis of the log2 fold changes (LFC) were conducted using the STARS algorithm from the Broad Institute (Doench et al., Nat Biotechnol 2016, 34: 184-191). The STARS algorithm calculates a score using the probability mass function of a binomial distribution. The calculation was performed for all sgRNAs that ranked in the top 5% of the log2 fold changes. Then, for genes with at least two sgRNAs ranked in the top 5%, a score was assigned to the gene based on the lowest ranking guide above the 5% cutoff. P-values and false discovery rates (FDR) were calculated for each gene based on a null distribution generated by permutation of the original library. To call hits, genes with a STARS FDR <0.15 were identified as genes in which knockout mutations or functional inhibition would lead to resistance (positive LFC) or sensitization (negative LFC) to KLK2xCD3 treatment.Table 2-36-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1LFC: log2 fold changes; FDR: False Discovery Rate (statistical confidence).

[0018] FIG. 2 illustrates that a CRISPR modifier screen identified SPINT1 and SPINT2 as resistance hits to a KLK2xCD3 T-cell redirector. Specifically, FIG. 2 shows an overview of a CRISPR modifier screen to determine mechanisms of sensitization and resistance to a KLK2xCD3 T-cell redirector. Results reported in Table 2 show that resistance hits reveal SPINT1 and SPINT2 along with the positive control, KLK2, LFC: Log Fold Change (magnitude of effect). PTPRF was not a hit in this screen.Example 3: CoImmunoprecipitation and Targeted CRISPR Knockout of Putative KLK2 Anchors

[0019] Coimmunoprecipitation

[0020] Coimmunoprecipitation of KLK2 and putative KLK2 interacting partners was performed using a co-Immunoprecipitation kit. In brief, a proprietary anti-KLK2 IgGl antibody, or an anti-actin IgGl, were coupled to magnetic, M-270 epoxy beads following the manufacturer’s instructions. Whole cell lysates of VCaP cells were prepared using a detergent lysis method according to the manufacturer’s protocol. For co-immunoprecipitation, 20 mg of cell lysate per 1 mg of antibody-coupled beads was incubated, washed, and eluted according to the manufacturer’s instructions. Low volume elution was required for SPINT2.

[0021] Western blotting for SPINT1 or SPINT2 was performed to assess coimmunoprecipitation of each putative interacting partner with KLK2. Acrylamide gel electrophoresis was performed under reduced, non-denaturing conditions, and proteins were transferred to nitrocellulose using traditional wet transfer methods. Proteins were detected by immunoblotting using commercially available primary antibodies for KLK2, SPINT1, and SPINT2, followed by detection with secondary antibodies, and visualization via an imaging system. The results shown in FIG. 3A demonstrate that SPINT1 or SPINT2 coimmunoprecipitate with KLK2 but not the anti-actin IgGl control.

[0022] Generation of targeted CRISPR genetic knockout cells and characterization by flow cytometry and western blotting

[0023] Prior to knockout, 100 mg Poly-L-Lysine vial is dissolved in 20 mL of sterile dH 0. 500x solution is saved in 100 pL aliquots at -20 °C. 500X vials are reconned in 50 mL of dFLO to make IX solution. A 2 mL aliquot of IX Poly-L-Lysine solution is added to 6 well plates to begin Poly-L-Lysine coating procedure and given 30 minutes at room temperature for-37-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 coating to complete. Solution is removed via aspiration and once knockout is complete cells can be plated within Poly-L-Lysine coated wells to improve cell adherence and growth.

[0024] To generate the CRISPR knockouts, media is aspirated from flask and a gentle rinse of PBS is performed to remove remaining media. Trypsin was added to flasks and cells were placed back in incubator for 5 minutes. After 5 minutes, cells are dislodged from plate and corresponding media (DMEM+15% FBS) was added to stop the trypsin reaction. Cells were counted and 200,000 cells per reaction were aliquoted.

[0025] An electroporation system 10-pL kit, which contains the necessary buffers, cuvette, and pipette tips was utilized for the remaining steps. Each electroporation reaction contains the following, 5 pL buffer, 0.5 pL spCas9 (1000 pmol), 1 pL sgRNA for the target gene of interest (3 nmol), and 200,000 tumor cells in 5 pL of buffer. The electroporation system was updated with the corresponding settings; VCaP: 1500 V, 20 ms, 1 pulse.

[0026] Prior to electroporation, a 3 mL aliquot of E buffer is inserted into cuvette which is placed into slot on the electroporation system to produce the necessary conductivity for electroporation. 10 pL of sample (lx reaction) is drawn into the pipette with the corresponding pipette tip. The pipette is placed into the corresponding slot within electroporation system and electroporation protocol is run. After successful electroporation, cells are added to a 6 well plate coated in Poly-L-Lysine and containing DMEM+15% FBS and allowed 10 days to grow before confirming knockout via Flow Cytometry analysis. For VCaP cells, 6X reactions were conducted per 1 well within 6 well plate bringing total number of cells electroporated to 1.2xl06allowing for improved cell growth and kinetics.

[0027] For flow cytometry analysis, media is aspirated from wells and a gentle rinse of PBS is performed to remove remaining media. Trypsin is added to wells and cells are placed back in incubator for 5 minutes. After 5 minutes, cells are dislodged from plate and corresponding media (DMEM+15% FBS) is added to stop the trypsin reaction. Cell counts are performed using a Vi-cell cell counting instrument and 150,000 cells per well are aliquoted. All flow cytometry analysis is conducted within a 96 well u-bottom plates.

[0028] The cells are stained with fixable LIVE / DEAD Violet by adding 100 pL of the working solution (prepared by adding 50 pL of dimethyl sulfoxide (DMSO) to the lyophilized vial and then diluting the stock solution 1:1,000 in DPBS) and incubated for 15 minutes at RT in the dark. Plates are centrifuged at 1 ,300 rpm for 3 minutes at RT, and the supernatants were discarded. Cell pellets are washed twice with 100 pL stain buffer, resuspended with 100 pL / well of the staining antibodies (isotype or either of 2 proprietary KLK2 antibodies recognizing different KLK2 epitopes) at a fixed 300 nM concentration and incubated for-38-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT145 minutes at 4°C in the dark. Plates are centrifuged at 1 ,300 rpm for 3 minutes at RT, and the supernatants are discarded. Cell pellets are washed twice with 100 pL stain buffer, and resuspended with 100 pL / well of the secondary staining antibodies at a fixed 3 pg / mL concentration and incubated for 30 minutes at 4°C in the dark. Finally, the cells are washed twice and resuspended in 100 pL of stain buffer prior to analysis on a flow cytometer.

[0029] Results for the test antibodies are analyzed in FlowJo and the following gating strategy is applied: cell population (FSC-area x SSC-area), single cells (FSC-area x FSC-height), live cells (histogram with live events), KLK2 (histogram of fluorescence intensity). Histograms of KLK2 expression are overlayed within Flowjo assessing fluorescence intensity of Isotype Control vs. the 2 proprietary KLK2 antibodies. FIG. 3B shows KLK2 surface expression determined by flow cytometry on wild type cells (top left panel), a KLK2 knock-out (top right panel), a SPINT1 knock-out (bottom left panel), and a SPINT2 knock-out (bottom right panel). Western blotting for KLK2, SPINT1 , SPINT2, and GAPDH is performed using standard procedures and techniques according to instrument instructions.

[0030] Results are illustrated in FIG. 3C, which shows the CRISPR knockouts of KLK2, SPINT1, and SPINT2 were successful. That is, SPINT1 and SPINT2 in WT VCaP, VCaP-SPINT2 KO, or VCaP SPINT2 overexpression cells, demonstrate the expected SPINT1 or SPINT2 banding pattern in relation to the bands observed in Co-IP. Overall, the coimmunoprecipitation experiments demonstrated a potential interaction between KLK2 and SPINT1 or SPINT2.Example 4: Targeted Proteomics Analysis

[0031] Sample Preparation

[0032] VCaP cells were either biotinylated with sulfo-NHS-SS-biotin or incubated with buffer containing no NHS-biotin reagent. Each sample of each condition contained 2xl07cells. The samples were lysed with 600 |1L IxRIPA buffer (Sigma p / n 20-188) supplemented with nuclease, lx protease inhibitor cocktail and 4 mM phenylmethyl sulfonyl fluoride (PMSF). The cells were mixed with repeated aspiration and dispense steps and sonicated using a bath ultrasonicator at room temperature (RT) for 5 minutes. The cellular debris was then cleared from the solution by centrifugation at 18,000 ref at RT for 10 minutes.

[0033] Aliquots (2x) of 240 |1L for each sample, were then placed in separate wells of a 96-well microtiter plate and loaded onto the deck of a sample prep system. Two-hundred microliter of each sample (the equivalent of 6.7xl06cells) were then applied to streptavidin (SA) cartridges at a flow rate of 5 |lL / min. The captured material was then washed sequentially-39-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 with (1) RIPA buffer supplemented with lx protease inhibitor cocktail and 4 mM PMSF, (2) 0.25 % SDS in RIPA buffer, and (3) 0.25% SDS in 50 mM triethylammonium bicarbonate (TEAB) buffer, with a total volume of 250 pL / wash at a flow rate of 20 pL / min.

[0034] The material still bound was then eluded from the SA cartridges using 50 pL of the elution buffer containing 50 mM TEAB, 25 mM TCEP, and 0.25% SDS. The eluted proteins were then alkylated with iodoacetamide at a concentration of 25mM on a mixer for 60 minutes at RT while mixing at 850 rpm and protected from light. During the alkylation reaction, 60 pL of SP3 magnetic beads were washed 3x with 200 pL of HPLC-grade water and resolubilized in 30 pL of HPLC-grade water. An SP3 method was then used to isolate the proteins and remove detergent according to a modified literature method (Mol Syst Biol (2020)16: e9111).

[0035] Briefly, the samples were combined with 6 pL of SP3 beads and 80 pL acetonitrile. The beads were then incubated for 20 minutes on a mixer at 1200 rpm at RT. The beads were then isolated on magnetic separator and the supernatant discarded. The SP3 beads were washed with 2x 200 pL 80% ethanol and lx 180 pL acetonitrile, and suspended in 20 pL 50 mM TEAB. Proteins remaining on the SP3 beads were deglycosylated by the addition of 1 pL PNGaseF and incubation for 60 minutes on a mixer at 37 °C with mixing at 1200 rpm. One sample per condition was then digested with either trypsin or LysC, with the addition of 1 pL of enzyme at an enzyme concentration of 1 mg / mL in water. The samples were incubated overnight on the mixer at 37 °C and 1200 rpm.

[0036] Addition of Internal Standard Peptides and Sample Desalting

[0037] In order to determine the pro- and active-forms of KLK2 at the cell surface, a targeted mass spectrometric method was developed to look at specific peptides that are produced with LysC or Trypsin digestion of KLK2 and the potential interfering protein, KLK3. For each of the measured peptides, a stable-isotope labeled (SIL) peptide of the same sequence were custom synthesized and purchase from Biosynth (Garner, MA). The specific peptide sequences, the original proteins, protein forms, and enzymes used to produce the peptides, are shown in Table 3 below, where KArepresents 13C6, 15N2-Lysine, RArepresents 13C6, 15N4- Arginine, and (CAM) represents carbamidomethyl-cysteine.

[0038] The SIL peptides were weighed individually and diluted in DMF to a concentration of 1.0 mM. The SIL peptides were then combined and diluted serially with 0.05 mg / mL BSA trypsin digest until all SIL peptides had a final concentration of 5 nM (i.e., 5 fmole / pL).-40-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1

[0039] The digested samples were then spiked with 2 pL formic acid and applied to a magnetic separator. The supernatant was transferred to a 96-well PCR plate and combined with 20 pL (i.e., 100 fmole / SIL Peptide) of the 5 nM SIL peptide mixture. The samples were then applied to a Cl 8 solid-phase extraction cartridge on the sample prep system at a flow rate of 2 pL / min. The cartridge was then washed with 50 pL of HPLC-grade water and eluted from the cartridge with 50 pL of 75% acetonitrile 0.1 % formic acid buffer. The samples were dried down in a vacuum centrifuge and reconstituted in 10 pL 2% acetonitrile 0.25% formic acid.Table 3. SIL Peptidesrepresents carbamidomethyl-cysteine.

[0040] Liquid Chromatography / Mass Spectrometry Analysis

[0041] Two-microliter of each sample was then injected onto the LC / MS system for targeted MS analysis. The chromatographic separation was performed using direct flow injection onto a 0.075 mm x 150 mm C18 Column at a flow rate of 0.3 pL / min. The initial mobile-phase composition was 2 % mobile phase B (mobile-phase B = 0.1 % formic acid in acetonitrile; mobile phase A = 0.1 % formic acid in water) with the %B gradient progressing from 2% to 28% over 90 minutes with no initial hold. The column was then washed for 10 minutes at 85% B and returned to the initial mobile-phase conditions for 20 minutes to equilibrate the column in preparation for the next sample injection.

[0042] The mass spectrometer was operated in the targeted-MS2 (tMS2) acquisition mode using the following specific parameters; MS2 Activation Mode = HCD, MS2 Analyzer = Orbitrap, Isolation mode = Quadrupole, Isolation Window = 1.6 m / z, Collision Energy Mode-41-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1= Fixed (for each precursor), Orbitrap Resolution = 15,000, Maximum Injection Time = 22 ms, AGC Target = 5e4, Normalized AGC Target = 100 %, Microscans = 1, Maximum Injection Time Type = Dynamic, Polarity = Positive, Time Mode = Retention Time Window. The precursor ion, precursor charge, collision energy, retention time window, and MS2 ion used for analyte quantification for each peptide, is given in Table 4.

[0043] Fragment ion intensities for each peptide, and their corresponding SIL internal standards, were integrated using Skyline Software (Mass Spectrom. Rev. 2020 May, 39(3), 229-244. skyline.ms / project / home / begin.view). Light-to-heavy (i.e., endogenous-to-SIL peptide) area ratios were used to quantify the amount of each protein form. This was deemed acceptable because the response for each peptide was experimentally determined to be linear over the concentration range encompassing both the Light and Heavy peptide area responses.Table 4-42-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1represents carbamidomethyl-cysteine.

[0044] The results of the biotinylated / surfacome samples showed that there was very little KLK3 on the surface of the VCaP cells so any contribution of active-form KLK3 to activeform KLK2, because of the shared active-form specific peptide, IVGGWECEK, was negligible. Additionally, because very little pro-form KLK2 was detected in the surfaceome fraction, and the molar amount of active-KLK2 was nearly equivalent to the total molar KLK2 measured (active-form was measured to be roughly 83% of the total KLK2 measured in the surfaceome fraction), it can be concluded that KLK2 exists predominantly as active-form on the surface of the VCaP cells.

[0045] FIGS. 4A-4B illustrate that targeted proteomics analysis detected the only active form of KLK2 to be present on the cell surface. Specifically, FIG. 4A shows a sequence alignment of the N-terminal region between KLK2 and KLK3 showing the pro-peptide in the box and nearby Trypsin and LysC cleavage sites. FIG. 4B shows peptides monitored in the proteomics analysis of biotinylated enriched cell-surface samples from VCaP. The signal for KEK3 was very low compared to KLK2, therefore the measured KEK2 / 3 active form is predominantly due to active KEK2.Example 5: In Silico Modeling

[0046] Manual docking model of KEK2 / SPINT1 complex based on serine protease and Kunitz domain complex from PDB: 1YCO. KLK2 (PDB: 4NFE3) and KI from SPINT1 (PDB: 5H7V4) were aligned to 1YCO. N, I, E, and K2 domains from 5H7V were manually adjusted to the extended form similar to SAXS structure determined by Eiu et al., 2017 (7. Biol. Chem. (2017) 292(20) 8412-8423).

[0047] Multimer folding models of SPINT1 K1 / KEK2 and K2 / KEK2 complexes were generated by providing sequences of KI, K2, and KEK2 to AlphaFold2 (AF2). A relaxed model from the output was analyzed without further manipulation.

[0048] Steered molecular dynamics (SMD) simulations were performed using AF2 models of SPINT1 K1 / KEK2, SPINT1 K1-R225A / KEK2, and SPINT1 K2 / KEK2 complexes in Desmond (Schrodinger). Complexed models were prepared to 1.2 nanosecond MD equilibration and minimization with Desmond with a standard protocol. Separated KI and K2 models were translated 15 A away from KLK2 and used as tethers during SMD simulation. The pre-equilibrated complex models were then subjected to SMD with KEK2 and K1 / K2-43-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 domains tethered to the separated complex models at 25, 10, and 5 kcal / mol for 5, 25, and 50 ns, respectively. Table 5 notes time of separation determined by SMD simulation on AlphaFold2 complex models of SPINT1 K1 / KLK2, K1-R225A / KLK2, and K2 / KLK2. Equilibrated models were tethered to the separated models with a 25 kcal / mol force and a simulation time of 5 ns.Table 5

[0049] FIGS. 5A-5D show that in silico modeling of KLK2 / SPINT1 interactions predicted KLK2 to likely bind the Kunitz domain 1 of SPINT1.

[0050] FIG. 5A shows the domain organization of SPINT1. FIG. 5B shows a manual docking model of KEK2 / SPINT1 complex based on serine protease and Kunitz domain complex from PDB: 1YCO2. KEK2 (PDF: 4NFE3) and KI from SPINT1 (PDB: 5H7V4) were aligned to 1YCO. The N, I, E, and K2 domains from 5H7V were manually adjusted to the extended form similar to SAXS structure. The expanded box highlights a putative salt bridge between R225 of SPINT1 KI and D174 of KEK2. K2 has a Eys instead of Arg at this position.

[0051] FIG. 5C shows that a multimer folding model of KLK2 with SPINT1 KI or K2 using AlphaFold2 matches well with manual docking models of fragments from complex crystal structures. FIG. 5D shows a schematic of an antibody that binds to a KLK2 / K1 complex but does not bind to soluble (free) KLK2. Binding of a KLK2 / K1 complex to the antibody is illustrated at left arm of the antibody while the lack of recognition of soluble KLK2 (sKLK2), i.e., no binding, is illustrated at the right arm of the antibody.Example 6: Study of Complexing of Mature KLK2 with SPINT1

[0052] Human KLK2 binding to human SPINT1 was assessed by observing the formation of a new larger eluting complex via HPLC. An equimolar ratio (20 pM) of full- length KLK2 with pro-peptide or mature KLK2, was mixed with 20 pM of SPINT1 ECD, extracellular domain (Uniprot 043278, A28-K197, Sino Biological) and incubated for 5 minutes at room temperature. The sample mixtures were then queued up in an autosampler along with KLK2 and SPINT1 protein sample controls separately and gel filtration molecular-44-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 weight protein standards. A 10 pl aliquot of each sample was injected onto a pre-equilibrated increase 5 / 150 size exclusion column running an isocratic gradient of Dubelcco’s Phosphate- Buffered saline pH 7.4 at a flow rate of 0.35ml / min for 2 column volumes. Peak elution was monitored using A280nm absorbance.

[0053] FIG. 6 shows a series of chromatograms in a study of complexing between mature KLK2 and SPINT1. The KLK2 / SPINT1 complex is formed only when mature KLK2 is used, as shown in the first two chromatograms. It appears that pro-peptide KLK2 does not complex with SPINT1, as seen in the third chromatogram.Example 7: Generation of KLK2, SPINT1, SPINT2 proteins and complexes

[0054] Generation of KLK2 protein

[0055] Two mutations were made in the pro-peptide of KLK2 to introduce a Factor Xa site and a 6xHis tag was added to the C-terminus of mature KLK2. The KLK2 construct was subcloned into a mammalian expression vector under a CMV promoter using its native KLK2 signal sequence and transiently expressed in Expi293 cells. The expressed protein was purified from the media using Ni-NTA affinity followed by gel filtration on a preparative column. Benzamidine was included throughout the purification process to prevent auto-proteolytic damage. Final storage buffer was IxDPBS pH 7.2 containing ImM benzamidine.

[0056] Mature KLK2 was obtained either by auto-processing during purification or by buffer exchanging out the benzamidine and incubating the pro-peptide form at 4 °C for 3-5 days. In the case of the latter scenario, conversion to mature KLK2 can be followed by sampling daily and performing mass spectrometry analysis to look for removal of the pro-peptide. When conversion was complete, benzamidine was added back to the buffer to stop further degradation. The sample was then aliquoted and frozen at -80 °C for later use.

[0057] Generation ofSPINTl and SPINT2 proteins

[0058] The full-length extracellular domains (ECDs) or truncated versions containing the first Kunitz 1 domain of SPINT1 or SPINT2 were subcloned into a mammalian expression vector under a CMV promoter with a custom signal sequence and a C-terminal lOxHis tag. The constructs were transiently expressed in HEK293 cells, and the expressed protein was purified from the media by Ni-NTA affinity followed by preparative SEC.

[0059] Generation of KLK2 / SPINT1 and KLK2 / SPINT2 complexes

[0060] Excess mature KLK2 was mixed with SPINT1 or SPINT2 proteins in a 1.3 to 1 molar ratio in IxDPBS, lOmM Benzamidine to generate complexes. Extra benzamidine was then added to reduce proteolysis of the SPINT proteins. The protein mixture was incubated on-45-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 ice for 30 min to allow complex formation. To remove any unbound KLK2 or SPINT protein, the complex mixture was concentrated and loaded onto a preparative Superdex 200 Increase 16 / 40 GL column equilibrated in IxDPBS, ImM Benzamidine. Elution fractions were analyzed by SDS-PAGE to locate the complex fractions. Isolated complexes were further checked by analytical SEC to determine purity. Purified complexes were frozen and stored at -80 °C for later use.Example 8: Antibody Generation

[0061] Immunization

[0062] Ablexis Kappa mice were immunized with recombinant human KLK2 / SPINT immunogens. Specifically, Group 1 (Mice 41-50) received KLK2 / SPINT1 complex, while Group 2 (Mice 51-55) received KLK2 / SPINT2 complex. Each mouse was immunized six times (bi-weekly) with 10 pg of recombinant KLK2 / SPINT proteins loaded onto proprietary in-house nanoparticles via RIMMS-IP, using a combination of subcutaneous (S.C.), intradermal (I.D.), and intraperitoneal (I.P.) injections. All animals received a final boost consisting of 10 pg of KLK2 / SPINT complexes plus 50 pg of an anti-CD40 monoclonal antibody.

[0063] Immune sera samples were collected at Days 28 and 56 during immunization and analyzed for binding activity via ELISA against KLK2 / SPINT1 and KLK2 / SPINT2 complexes. All immunized animals demonstrated a specific response to either KLK2 / SPINT1 complexes or KLK2 / SPINT2 complexes. These animals were subsequently euthanized, and tissues — including lymph nodes, spleens, and bone marrow — were harvested and processed for single-cell analysis.

[0064] B cell sorting

[0065] On Day 7 of the immunization schedule, bone marrow, lymph nodes and spleens were harvested from each mouse in Group 2 (#51-55). On Days 75 and 85 of the immunization schedule, bone marrow, lymph nodes and spleens were harvested from each mouse in Group 1 (#41-50). All lymph nodes, bone marrow, and spleens were pooled three days post-final boost, homogenized into a single-cell suspension, and sorted into individual wells of 384-well destination plates via a FACS instrument. The cells were sorted to identify IgG-i- and antigen specific B cell populations. The cells from group 1 were sorted using biotinylated recombinant human KLK2 / SPINT1 and AF647 labeled recombinant human SPINT1 as sorting baits. The cells from group 2 were sorted using biotinylated recombinant human KLK2 / SPINT2 and AF647 labeled recombinant human SPINT2 as sorting baits. This strategy used the-46-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1KLK2 / SPINT complexes to select B cell binding to the complexes, while the SPINT protein monomers were used to identify the SPINT only binders.

[0066] A total of 36, 384-well SBC plates were generated for group 1, and 18, 384- well plates for group 2. The single B cell populations were cultured for six days before supernatants were harvested for screening.

[0067] B cell screening

[0068] Single B cell supernatants were screened for KLK2 / SPINTs binding by protein Meso Scale Discovery (MSD) assay. Plates from Group 1 were tested for binding to biotinylated recombinant KLK2 / SPINT1 complex for on target binding; and biotinylated recombinant SPINT1 monomer to identify irrelevant SPINT1 binders. Plates from Group 2 were tested for binding to biotinylated recombinant KLK2 / SPINT2 complex for on target binding; biotinylated recombinant SPINT2 monomer to identify irrelevant SPINT2 binders (FIG. 7A). From this initial screening, 10,779 hits binding to the KLK2 / SPINT complexes were identified.

[0069] The V-gene regions of these hits were recovered, sequenced and analyzed. 731 clusters from 220 families were identified (clusters < 6 residues differ across all CDRs, family < 22 residues differ across all CDRs). 536 unique antibodies were selected for expression in human IgGl format. After expression, the monoclonal antibody panel was tested again to confirm KLK2 binding. This panel of 536 antibodies was screened against soluble antigens by MSD and cell lines by flow cytometry.

[0070] A summary of the cell and binding data is presented in FIGS. 7A and 7B. Specifically, FIG. 7A illustrates MSD single point binding data obtained with single B cell supernatants from the immunized mice. Results for mice immunized with SPINT 1 protein (o) or KLK2 / SPINT1 protein complex (•) are shown in the top panel of FIG. 7A, and results for mice immunized with SPINT2 protein (o) or KLK2 / SPINT2 protein complex (•) are shown in the bottom panel of FIG. 7A. Binding is represented as a function of the IgG concentration. FIG. 7B illustrates KLK2 monomer protein binding vs cell binding from the 536 mAbs identified from mice immunized with the KLK2 / SPINT complexes. Antibodies binding to protein and VCaP cells endogenously expressing KLK2 are in the circled region.

[0071] Using the methods of the present disclosure, 193 binders were identified (63 KLK2 binders and 130 KLK2 / SPINT binders).

[0072] Flow Cytometry Cell Binding

[0073] Flow cytometry cell binding data was found to depend on the immunization strategy used to vaccinate mice against KLK2. The antibody panel isolated from mice-47-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 immunized with the KLK2 monomer demonstrated binding to DU145 cells not expressing KLK2 (FIG. 8, top panel). Few antibodies from this panel preferentially bind to VCaP cells. Upon further characterization, none of these antibodies were specifically binding to KLK2 at the surface of VCaP cells. The antibody panel isolated from mice immunized with the KLK2 / SPINT complexes was found to only bind to VcaP cells endogenously expressing KLK2. No binding was detected on DU145 cells (FIG. 8, bottom panel). Upon further characterization, this panel of antibodies was confirmed to specifically bind the KLK2 / SPINT complexes.Example 9: Structure Determination

[0074] Cryo-EM grid preparation and data acquisition ofKLK2 / SPINTl complex

[0075] KLK2 and SPINT1 proteins were incubated at 4 °C overnight at 1.5:1 ratio (KLK2:SPINT1). Two tool fabs were added to the mixture at equal molar of SPINT1 and incubated at 4 °C for another 24 hours. The final mixture was concentrated to 0.5 mg / ml. Ultrastable gold support mesh grid was freshly glow-discharged at 15 mA for 60 seconds using a glow discharge cleaning system. Cryo-EM grid preparation was performed using a cryofixation system operated at 4 °C with 100% humidity in the inner chamber. A volume of 2.5 pl of protein complex was applied to the grid. The grid was blotted with the following settings: blot time 10 seconds, blot force 2 and wait time 0 seconds. Subsequently, the grids were plunged into liquid ethane cooled by liquid nitrogen.

[0076] Datasets were acquired using EPU software (Thermo Fisher Scientific) on a 200 kV microscope equipped with a direct electron detector. The micrographs were taken at xl30,000 magnification with binning 2 in counting mode (0.91 A pixel size). The defocus range was set from -1.4 to -2.5 pm. A total of 12,917 micrographs were collected with the total exposure of 40 e A .

[0077] Cryo-EM data processing of KLK2 / SPINT1 complex

[0078] 12,917 micrographs were imported into cryoSPARC live (Punjani et al., Nat Methods 2017, 14(3): 290-296) for preprocessing. After patch motion correction and patch contrast function (CTF) estimation, particles were picked using blob picker tool with a size range of 100 - 160 A, followed by template picking. A total of 6,976,666 particles were picked and extracted from dose-weighted micrographs using a box size of 384 px, which was downsampled by a factor of 4 to 96 px for initial processing. Multiple rounds of 2D classification were performed to discard poor quality particles. The selected particles were reextracted at full box size and used for ab-initio 3D model reconstruction generating two classes.-48-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1The reconstructed models were refined using heterogenous refinement, resulting in one major class comprising -60% of the particles. These particles belonging to the major class were subjected to two additional rounds of 2D classification and re-extracted with a box size of 392 px. The finally selected 1,112,040 particles underwent global and local CTF refinement to enhance particle quality. The refined particles were processed using non-uniform refinement, resulting in a final map with a resolution of 3.11 A.

[0079] The crystal structure of KLK2 complexed with tool Fabs was determined internally previously and a predicted model of full-length SPINT1 from AlphaFold2 (Jumper et al., Nature 2021, 596(7873): 583-589) were used as initial models for rigid-body docking into the refined cryo-EM density map using UCSF ChimeraX (Meng et al., Pro Sci 2023, 32(11): e4792). Density corresponding to the SPINT1 kunitz domain 1, KLK2 serine protease domain and both Fab fragments were observed and modeled. The structure was manually adjusted and iteratively built using COOT (Emsley et al., Acta Crystallogr D Biol Crystallogr 2010, 66(Pt 4): 486-501), followed by real-space refinement in Phenix (Liebschner et al., Acta Crystallogr D Struct Biol 2019, 75(Pt 10): 861-877).

[0080] Crystallization of KLK2 / SPINT2 complex

[0081] KLK2 and SPINT2 were incubated at 4 °C overnight at 1.5:1 ratio (KLK2:SPINT2). Two tool fabs were added to the mixture at equal molar of SPINT2 and incubated at 4 °C degree for another 24 hours. The final mixture was purified by size exclusion chromatography with column buffer (20mM Tris and 150mM NaCl, 5mM Benzamidine, pH 7.2) and the purified complex was concentrated to 14.4mg / ml for crystallization. Crystals were grown by the vapor diffusion method in sitting drops in crystallization 96-well low-profile plates. The volume of reservoir for the 96-well plate was 60 (11. The drop volume was 0.2 pl. Crystallization was screened at three proteimprecipitant ratios by volume (2:1, 1:1, 1:2). Harvestable crystals formed under well solution 2 M (NEL^hSC 4% PEG400, 0.1 M Na acetate, pH 5. Crystals were cryo-protected in LVCO oil and flash frozen in liquid nitrogen for data collection.

[0082] X-ray data collection and structure determination of KLK2 / SPINT2 complex

[0083] Diffraction dataset was collected at the Brookhaven National Laboratory National Synchrotron Light Source II AMX 17-ID-l using 0.920187 A X-rays and a DECTRIS Eiger2 X 9M detector. Dataset was processed using autoPROC (Vonrhein et al., Acta Crystallogr D Biol Crystallogr 2011, 67(Pt 4): 293-302). Anisotropic data was truncated-49-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1 elliptically using local I / sigI >1.2 as criteria to determine diffraction limits (2.14, 2.14 and 2.86 A on a, b and c axes, respectively).

[0084] Diffracted crystal belonged to space group P41212 with unit cell dimensions a=97.7 A, b=97.7 A, c=291 .9 A, a=90". P=90". Y=90" with 1 protein complex in the asymmetric unit. The orientations and positions of individual protein subunits within the asymmetric unit was solved by molecular replacement using PHASER (McCoy et al., J Appl Crystallogr 2007, 40(Pt 4): 658-674). Experimental models of KLK2 complexed with tool Fabs (internal data) and SPINT2 kunitz domain 1 (PDB ID: 4U32) were used as search templates. Subsequent iterative model modification was carried out in Coot (Emsley et al., Acta Crystallogr D Biol Crystallogr 2010, 66(Pt 4): 486-501) and refined in PHENIX (Afonine et al., Acta Crystallogr D Biol Crystallogr 2012, 68(Pt 4): 352-367). The structures shown in FIG. 9 were generated using PyMOL (Schrodinger, LLC), where the KLK2 / SPINT1 complex is depicted on the left and the KLK2 / SPINT2 complex is depicted on the right.

[0085] All publications and patents referred to herein are incorporated by reference. Various modifications and variations of the described subject matter will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to these embodiments. Indeed, various modifications for carrying out the invention are obvious to those skilled in the art and are intended to be within the scope of the following claims.-50-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1Table 6. Sequences-51-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1Table 6. Sequences (cont.)-52-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT1Table 6. Sequences (cont.)-53-#124029155vl

Claims

1. ATTORNEY DOCKET NO. JBI6949WOPCT1CLAIMSWhat is claimed is:

1. A composition comprising:(i) a KLK peptide; and(ii) a SPINT peptide.

2. The composition of claim 1, wherein the KLK peptide does not comprise a pro-peptide sequence.

3. The composition of claim 1 or 2, wherein the KLK peptide, and the SPINT peptide are isolated, purified, synthetic, non-naturally occurring, or recombinant peptides.

4. The composition of claim 1 or 2, wherein the KLK peptide forms a complex with the SPINT peptide.

5. The composition of claim 1 or 2, wherein the KLK peptide comprises a KLK2 peptide, and the SPINT peptide comprises a SPINT 1 peptide, a SPINT2 peptide, or a combination thereof.

6. The composition of claim 5, wherein the KLK2 peptide, and the SPINT 1 peptide and / or SPINT2 peptide, are isolated, purified, synthetic, non-naturally occurring, or recombinant peptides.

7. The composition of claim 5, wherein the KLK2 peptide forms a complex with the SPINT 1 and / or SPINT2 peptide.

8. The composition of claim 1, wherein the SPINT peptide comprises a MANEC (N) domain, a PKD-like (I) domain, a Kunitz 1 (KI) domain, a LDLRA (L) domain, a Kunitz 2 (K2) domain, or a combination thereof.

9. The composition of claim 1, wherein (ii) comprises a SPINT1 peptide or a fragment thereof.

10. The composition of claim 1, wherein (ii) comprises a SPINT2 peptide or a fragment thereof.

11. The composition of claim 9, wherein the SPINT 1 peptide comprises the amino acid sequence as set forth in SEQ ID NO: 3, 15, or 16, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.

12. The composition of claim 9, wherein the fragment of the SPINT1 peptide comprises the amino acid sequence as set forth in SEQ ID NO: 4 or 5, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.-54-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT113. The composition of claim 10, wherein the SPINT2 peptide comprises the amino acid sequence as set forth in SEQ ID NO: 6, 17, or 18, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.

14. The composition of claim 10, wherein the fragment of the SPINT2 peptide comprises the amino acid sequence as set forth in SEQ ID NO: 7 or 8, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.

15. The composition of claim 1, wherein (ii) comprises a multimer of the SPINT peptide or a fragment thereof.

16. The composition of claim 15, wherein the multimer is concatenated.

17. The composition of claim 5, wherein (ii) comprises a multimer of any combination of the SPINT1 peptide, a fragment of the SPINT1 peptide, the SPINT2 peptide, or a fragment of the SPINT2 peptide.

18. The composition of claim 17, wherein the multimer is concatenated.

19. The composition of claim 1, wherein the KLK peptide comprises the amino acid sequence as set forth in SEQ ID NO: 2, 14, 19, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.

20. The composition of any one of claims 1 to 19, wherein (i) and (ii) are connected directly to form a fusion protein.

21. The composition of any one of claims 1 to 19, wherein (i) and (ii) are connected via a linker to form a fusion protein.

22. The composition of claim 21, wherein the linker is a peptide linker.

23. The composition of claim 21, wherein the linker comprises the amino acid sequence as set forth in SEQ ID NO: 13, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.

24. The composition of claim 21, wherein the fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 9, 10, 11, or 12, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.

25. The complex of any one of claims 1 to 24, wherein the complex comprises a tag.

26. The complex of claim 25, wherein the tag is a His tag.-55-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT127. The composition of claim 1, wherein (i) the KLK peptide comprises the amino acid sequence as set forth in SEQ ID NO: 19, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto, and (ii) the SPINT peptide comprises the amino acid sequence as set forth in SEQ ID NO: 15, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.

28. The composition of claim 1, wherein (i) the KLK peptide comprises the amino acid sequence as set forth in SEQ ID NO: 19, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto, and (ii) the SPINT peptide comprises the amino acid sequence as set forth in SEQ ID NO: 17, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.

29. The composition of claim 27 or 28, wherein (i) and (ii) are connected via a linker to form a fusion protein.

30. The composition of claim 29, wherein the linker is a peptide linker.

31. The composition of claim 30, wherein the linker comprises the amino acid sequence as set forth in SEQ ID NO: 13, or a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.

32. The composition of any one of claims 1 to 31, wherein the complex is immunogenic.

33. The composition of any one of claims 1 to 32 used to generate molecules that differentially bind to membrane associated KLK.

34. The composition of any one of claims 5 to 32 used to generate antibodies that differentially bind to membrane associated KLK2 over free KLK2.

35. A nucleic acid encoding (i) or (ii) according to any of claims 1 to 31, operably linked to a heterologous promoter.

36. A recombinant host cell comprising (i) and / or (ii) of the composition according to any one of claims 1 to 32, or the nucleic acid claim 35.

37. A method of inducing an immune response in a subject, the method comprising administering to the subject the composition according to any one of claims 1-32, wherein the subject is a human or a non-human mammal.

38. A system for inducing an immune response in a subject, comprising the composition according to any one of claims 1 to 32, and a delivery system.

39. The system of claim 38, wherein the delivery system comprises a syringe.-56-#124029155vlATTORNEY DOCKET NO. JBI6949WOPCT140. The system of claims 38 or 39, wherein the composition is administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.

41. A means for inducing an immune response in a subject, the means comprising the composition according to any one of claims 1 to 32, and a delivery system, wherein the subject is a human or a non-human mammal42. The means of claim 41, wherein the delivery system comprises a syringe.

43. The means of claim 41, wherein composition is administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.

44. A system for active immunization to prevent a disease in a subject, the system comprising the composition according to any one of claims 1 to 32, wherein the subject is a human or a non-human mammal, for example a non-human, a simian, a mouse, a rat, a cow, or a llama.

45. The system of claim 44, further comprising a delivery system.

46. The system of claim 45, wherein the delivery system is a syringe.

47. The system of claim 44, wherein the composition is administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.

48. A means for active immunization to prevent a disease in a subject, the means configured to specifically target membrane associated KLK2 peptide.

49. The means of claim 48, wherein the means has high affinity for the membrane associated KLK2 peptide and low affinity for free KLK2 peptide.

50. The means of claim 48, wherein the means has high affinity for KLK2 when bound to a SPINT1 peptide or a SPINT2 peptide, and has low affinity for free KLK2.

51. The means of claim 48, wherein the means comprises the composition according to any one of claims 1 to 32.

52. The means of claim 51, further comprising a delivery system.

53. The means of claim 52, wherein the delivery system is a syringe.

54. The means of claim 51, wherein the means is administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, or transdermal route of administration.-57-#124029155vl

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