Compositions and methods related to tumor activating antibodies targeting PSMA and effector cell antigens

Abstract

Described herein are compositions and formulations comprising recombinant polypeptides. Related methods and uses of these compositions and formulations are also described herein. The isolated recombinant polypeptide complexes include features comprising disulfide bonds, pyroglutamine, UV circular dichroism depression.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 327,322, filed on April 4, 2022, and U.S. Provisional Application No. 63 / 338,220, filed on May 4, 2022, each of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in XML file format and is incorporated herein by reference in its entirety. The XML copy was created on March 24, 2023, is named 52426-743_601sl.xml, and is 24,891 bytes in size. Summary of the invention

[0005] In one aspect, disclosed herein is an isolated recombinant polypeptide complex comprising a first chain having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1 and a second chain having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2, wherein the isolated recombinant polypeptide complex comprises at least one of the following features: (a) at least one disulfide bond formed by a pair of cysteine ​​residues in the first chain or the second chain or by a pair of cysteine ​​residues in the first chain and the second chain; (b) a secondary structure comprising a β-sheet or a random coil; (c) at least one pyroglutamine in the second chain; (d) a melting temperature (T) between about 65° C. and about 85° C. when the isolated recombinant polypeptide complex is formulated at a concentration of 1.0 mg / mL in a buffer comprising 10 mM histidine buffer, 8% (w / v) sucrose, 0.01% (w / v) polysorbate 20, pH 6.3. m); (e) a far-UV circular dichroism peak at a wavelength between 190 nm and 205 nm when the isolated recombinant polypeptide complex is formulated at a concentration of 0.1 mg / mL in 10 mM potassium phosphate buffer, pH 7.0; (f) a far-UV circular dichroism dip at a wavelength between 210 nm and 220 nm when the isolated recombinant polypeptide complex is formulated at a concentration of 0.1 mg / mL in 10 mM potassium phosphate buffer, pH 7.0; or (g) a near-UV circular dichroism peak at a wavelength between 250 nm and 300 nm when the isolated recombinant polypeptide complex is formulated at a concentration of 1.0 mg / mL in a buffer containing 10 mM histidine buffer, 8% (w / v) sucrose, 0.01% (w / v) polysorbate 20, pH 6.3. In some embodiments, the polypeptide includes at least two of the features. In some embodiments, the polypeptide includes at least three of the features. In some embodiments, the polypeptide includes at least four of the features. In some embodiments, the polypeptide includes at least five of the features. In some embodiments, the polypeptide includes at least six of the features. In some embodiments, the polypeptide includes at least seven of the features. In some embodiments, the polypeptide includes at least eight of the features. In some embodiments, the first chain comprises at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the first chain comprises at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the first chain comprises at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the first chain comprises at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the first chain comprises an amino acid sequence according to SEQ ID NO: 1. In some embodiments, the second chain comprises at least 85% sequence identity to SEQ ID NO: 2. In some embodiments, the second chain comprises at least 90% sequence identity to SEQ ID NO: 2. In some embodiments. The second chain comprises at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, the second strand comprises at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the second strand comprises the amino acid sequence according to SEQ ID NO:2.

[0006] In some embodiments, at least one disulfide bond is an intrachain disulfide bond formed between cysteine ​​22 and cysteine ​​96, between cysteine ​​138 and cysteine ​​148, between cysteine ​​199 and cysteine ​​275, between cysteine ​​339 and cysteine ​​407, between cysteine ​​454 and cysteine ​​519, or between cysteine ​​565 and cysteine ​​625 of the first chain. In some embodiments, at least one disulfide bond is an intrachain disulfide bond formed by a cysteine ​​residue pair of the second chain between cysteine ​​22 and cysteine ​​96 of the second chain, or between cysteine ​​150 and cysteine ​​206. In some embodiments, at least one disulfide bond is an interchain disulfide bond formed between the first chain and the second chain between cysteine ​​645 of the first chain and cysteine ​​226 of the second chain. In some embodiments, the polypeptide comprises at least two disulfide bonds formed by cysteine ​​residue pairs. In some embodiments, the polypeptide comprises at least three disulfide bonds formed by cysteine ​​residue pairs. In some embodiments, the polypeptide comprises at least four disulfide bonds formed by cysteine ​​residue pairs. In some embodiments, the polypeptide comprises at least five disulfide bonds formed by cysteine ​​residue pairs. In some embodiments, the polypeptide comprises at least six disulfide bonds formed by cysteine ​​residue pairs. In some embodiments, the polypeptide comprises at least seven disulfide bonds formed by cysteine ​​residue pairs. In some embodiments, the polypeptide comprises at least eight disulfide bonds formed by cysteine ​​residue pairs. In some embodiments, the cysteine ​​residue pairs comprise cysteine ​​22 and cysteine ​​96 of SEQ ID NO: 1. In some embodiments, the cysteine ​​residue pairs comprise cysteine ​​138 and cysteine ​​148 of SEQ ID NO: 1. In some embodiments, the cysteine ​​residue pairs comprise cysteine ​​199 and cysteine ​​275 of SEQ ID NO: 1. In some embodiments, the cysteine ​​residue pairs comprise cysteine ​​339 and cysteine ​​407 of SEQ ID NO: 1. In some embodiments, the cysteine ​​residue pairs comprise cysteine ​​454 and cysteine ​​519 of SEQ ID NO: 1. In some embodiments, the cysteine ​​residue pair comprises cysteine ​​565 and cysteine ​​625 of SEQ ID NO: 1. In some embodiments, the cysteine ​​residue pair comprises cysteine ​​645 of SEQ ID NO: 1 and cysteine ​​226 of SEQ ID NO: 2. In some embodiments, the cysteine ​​residue pair comprises cysteine ​​150 and cysteine ​​206 of SEQ ID NO: 2. In some embodiments, the cysteine ​​residue pair comprises cysteine ​​22 and cysteine ​​96 of SEQ ID NO: 2. In some embodiments, at least one cysteine ​​residue is a free sulfhydryl group.In some embodiments, at least one cysteine ​​residue is a free sulfhydryl group, and at least one cysteine ​​is selected from the group consisting of and corresponds to cysteine ​​22, cysteine ​​96, cysteine ​​138, cysteine ​​148, cysteine ​​199, cysteine ​​275, cysteine ​​339, cysteine ​​407, cysteine ​​454, cysteine ​​519, cysteine ​​565, cysteine ​​625, and cysteine ​​645 of SEQ ID NO: 1. In some embodiments, at least one free cysteine ​​residue is a free sulfhydryl group, and at least one cysteine ​​is selected from the group consisting of and corresponds to cysteine ​​22, cysteine ​​96, cysteine ​​150, cysteine ​​226, and cysteine ​​206 of SEQ ID NO:2. In some embodiments, at least one cysteine ​​residue pair includes cysteine ​​22 and cysteine ​​96 of SEQ ID NO:1, cysteine ​​138 and cysteine ​​148 of SEQ ID NO:1, cysteine ​​199 and cysteine ​​275 of SEQ ID NO:1, cysteine ​​454 and cysteine ​​519 of SEQ ID NO:1, cysteine ​​565 and cysteine ​​625 of SEQ ID NO:1, cysteine ​​22 and cysteine ​​96 of SEQ ID NO:2, cysteine ​​150 and cysteine ​​206 of SEQ ID NO:2, and cysteine ​​645 of SEQ ID NO:1 and cysteine ​​226 of SEQ ID NO:2.

[0007] In some embodiments, the secondary structure composition includes β-sheets and random coils. In some embodiments, the isolated recombinant polypeptide complex has a melting temperature (T) between about 71°C and about 81°C. m In some embodiments, the isolated recombinant polypeptide complex has a melting temperature (T) between about 71.4°C and about 79.5°C. m In some embodiments, the isolated recombinant polypeptide complex has a melting temperature (T) between about 65°C and about 85°C. m). In some embodiments, the isolated recombinant polypeptide complex has a far-UV circular dichroism peak at a wavelength less than or equal to 195 nm. In some embodiments, the isolated recombinant polypeptide complex has a far-UV circular dichroism peak at a wavelength less than or equal to 205 nm. In some embodiments, the isolated recombinant polypeptide complex has a far-UV circular dichroism dip at a wavelength less than or equal to 220 nm. In some embodiments, the isolated recombinant polypeptide complex has a near-UV circular dichroism peak at a wavelength less than or equal to 273 nm. In some embodiments, the isolated recombinant polypeptide complex has a near-UV circular dichroism peak at a wavelength less than or equal to 279 nm. In some embodiments, the isolated recombinant polypeptide complex has a near-UV circular dichroism peak at a wavelength less than or equal to 290 nm. In some embodiments, the isolated recombinant polypeptide complex has a near-UV circular dichroism peak at a wavelength less than or equal to 295 nm.

[0008] In another aspect, disclosed herein is a method of treating cancer comprising administering to a subject in need thereof an isolated recombinant polypeptide complex disclosed herein.

[0009] In another aspect, disclosed herein are multiple isolated recombinant polypeptide complexes comprising a first chain having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1 and a second chain having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2, wherein the multiple comprises greater than 90% of the monomers of the isolated recombinant polypeptide complexes. In some embodiments, the multiple isolated recombinant polypeptide complexes comprise greater than 95% of the monomers. In some embodiments, the multiple isolated recombinant polypeptide complexes comprise greater than 99% of the monomers. In some embodiments, the multiple isolated recombinant polypeptide complexes comprise a concentration greater than or equal to 1.0 mg / mL. In some embodiments, the multiple isolated recombinant polypeptide complexes comprise a concentration of at least 2.0 mg / mL. In some embodiments, the multiple isolated recombinant polypeptide complexes comprise a pH greater than or equal to 5.0. In some embodiments, the multiple isolated recombinant polypeptide complexes comprise greater than 90% of the monomers at a concentration greater than or equal to 2.0 mg / mL and a pH of 6.3.

[0010] In another aspect, disclosed herein is a method for treating prostate cancer, comprising administering to a subject in need thereof an antibody or antigen-binding fragment comprising a CD3 binding domain and a PSMA binding domain, wherein the antibody or antibody-binding fragment is administered to the subject once a week at a dose of at least 0.1 μg / kg. In some embodiments, the antibody or antigen-binding fragment is administered to the subject at a dose of at least 0.1 mg / kg. In some embodiments, the antibody or antigen-binding fragment is administered to the subject at a dose of at least 0.3 mg / kg. In some embodiments, the antibody or antigen-binding fragment is administered to the subject at a dose of at least 1.0 mg / kg. In some embodiments, the antibody or antigen-binding fragment is administered to the subject at a dose of at least 1.5 mg / kg. In some embodiments, the antibody or antigen-binding fragment is administered to the subject according to the following treatment regimen: the antibody or antigen-binding fragment is administered to the subject at a dose of at least 0.1 μg / kg in the first week, the antibody or antigen-binding fragment is administered to the subject at a dose of at least 0.3 mg / kg in the second week, and the antibody or antigen-binding fragment is administered to the subject at a dose of at least 1.5 mg / kg in the third week, followed by a 4-week non-treatment interval of the antibody or antigen-binding fragment.

[0011] In some embodiments, the isolated recombinant polypeptide complex provides a maximum plasma concentration (Cmax) in a subject within about 0.1 hour after intravenous administration. In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 2500 to 3500 ng / ml in a subject within about 0.1 hour after intravenous administration of a dose of about 0.1 mg / kg. In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 7500 to 10500 ng / ml in a subject within about 0.1 hour after intravenous administration of a dose of about 0.3 mg / kg. In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 37500 to 52500 ng / ml in a subject within about 0.1 hour after intravenous administration of a dose of about 1.5 mg / kg. In some embodiments, the isolated recombinant polypeptide complex provides a half-maximal plasma concentration in a subject within about 60 to about 120 hours (T1 / 2) after intravenous administration. In some embodiments, the value of Cmax is related to the dose of the isolated recombinant polypeptide complex administered. In some embodiments, the value of Cmax is proportional to the dose of the isolated recombinant polypeptide complex administered. In some embodiments, less than 1% of the isolated recombinant polypeptide complex is degraded in the serum of the subject every day. In some embodiments, the subject is diagnosed with cancer. In some embodiments, the cancer includes prostate cancer. In some embodiments, the cancer includes metastatic castration-resistant prostate cancer (mCRPC).

[0012] On the other hand, disclosed herein is a method for treating cancer, comprising administering an effective amount of an isolated recombinant polypeptide complex disclosed herein to a subject in need thereof. In some embodiments, the method comprises administering the isolated recombinant polypeptide complex to the subject at a first dose and a second dose, wherein the second dose is equal to or higher than the first dose, wherein the first dose or the second dose is at least 100 μg. In some embodiments, the method comprises administering the isolated recombinant polypeptide complex to the subject at a first dose, a second dose, and a third dose, wherein the second dose is equal to or higher than the first dose, and the third dose is equal to or higher than the second dose, wherein the first dose, the second dose, or the third dose is at least 100 μg. In some embodiments, the method comprises a first treatment course and a second treatment course, wherein the first dose is administered to the subject during the first treatment course, wherein the second dose is administered to the subject during the second treatment course. In some embodiments, the method comprises a 21-day treatment course, wherein the first dose is administered to the subject within the first week of the treatment course, wherein the second dose is administered to the subject within the second week of the treatment course, and wherein the third dose is administered to the subject within the third week of the treatment course. In some embodiments, the method comprises a first 21-day treatment course and a second 21-day treatment course, wherein the first dose of the second 21-day treatment course is equal to or higher than the first dose of the first 21-day treatment course, wherein the second dose of the second 21-day treatment course is equal to or higher than the second dose of the first 21-day treatment course, wherein the third dose of the second 21-day treatment course is equal to or higher than the third dose of the first 21-day treatment course. In some embodiments, the method comprises a 21-day treatment cycle, the treatment cycle comprising: (a) administering the isolated recombinant polypeptide complex to the subject at a first dose in the first week of the treatment cycle; (b) administering the isolated recombinant polypeptide complex to the subject at a second dose in the second week of the treatment cycle; (c) administering the isolated recombinant polypeptide complex to the subject at a third dose in the third week of the treatment cycle. In some embodiments, the method comprises: (a) administering to the subject an isolated recombinant polypeptide complex at a first dose on the first day of a 21-day treatment cycle; (b) administering to the subject an isolated recombinant polypeptide complex at a second dose on the eighth day of the 21-day treatment cycle; and (c) administering to the subject an isolated recombinant polypeptide complex at a third dose on the fifteenth day of the 21-day treatment cycle. In some embodiments, the treatment cycle is repeated 6 times within 18 weeks. In some embodiments, the first dose, the second dose, or the third dose is at least 100 μg. In some embodiments, the second dose is equal to or higher than the first dose. In some embodiments, the third dose is equal to or higher than the second dose. In some embodiments, the third dose is equal to or higher than the first dose.In some embodiments, the method includes a first 21-day treatment cycle and a second 21-day treatment cycle, wherein the first dose of the second 21-day treatment cycle is equal to or higher than the first dose of the first 21-day treatment cycle, wherein the second dose of the second 21-day treatment cycle is equal to or higher than the second dose of the first 21-day treatment cycle, wherein the third dose of the second 21-day treatment cycle is equal to or higher than the third dose of the first 21-day treatment cycle. In some embodiments, administration includes administration by intravenous infusion. In some embodiments, cancer includes prostate cancer. In some embodiments, cancer includes mCRPC. In some embodiments, the method also includes treating the subject with a therapy for infusion-related reactions before administration. In some embodiments, the therapy for infusion-related reactions includes antipyretics, antihistamines, antiemetics, or corticosteroids. In some embodiments, the therapy for infusion-related reactions includes acetaminophen, paracetamol, or diphenhydramine. In some embodiments, the method also includes treating the subject with a corticosteroid before step (a). In some embodiments, the method also includes treating the subject with a corticosteroid before step (a). In some embodiments, the method further includes treating the subject with a therapy for cytokine release syndrome (CRS) before or after administration. In some embodiments, the therapy for CRS includes an intravenous rehydration program, oxygen therapy, corticosteroids, immunosuppressants, vasopressors, or anti-epileptic drugs. In some embodiments, oxygen therapy includes mechanical ventilation. In some embodiments, immunosuppressants include IL-6 receptor inhibitors. In some embodiments, IL-6 receptor inhibitors include tocilizumab. In some embodiments, the therapy for CRS includes high doses of corticosteroids. In some embodiments, the recombinant polypeptide complex separated after administration is cleaved by a tumor-specific protease to generate an enzyme product of the separated recombinant polypeptide complex. In some embodiments, the tumor-specific protease includes two or more proteases, wherein the separated recombinant polypeptide complex is cleaved by a first protease of two or more proteases to generate a first metabolite of the separated recombinant polypeptide complex, wherein the separated recombinant polypeptide complex is cleaved by a second protease of two or more proteases to generate a second metabolite of the separated recombinant polypeptide complex. In some embodiments, the first protease includes a serine protease, wherein the second protease includes a matrix metalloproteinase. In some embodiments, the serine protease comprises recombinant human membrane serine protease (MTSP1), wherein the matrix metalloprotease comprises recombinant human matrix metalloprotease 9 (MMP9). In some embodiments, the first metabolite comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the second metabolite comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 6.In some embodiments, the subject is a non-human primate or a human. In some embodiments, the non-human primate is a cynomolgus monkey.

[0013] In another aspect, disclosed herein is a pharmaceutical composition comprising: (a) an isolated recombinant polypeptide complex disclosed herein; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises a buffer, a stabilizer, a tonicity agent, a surfactant, or a combination thereof. In some embodiments, the buffer comprises an amino acid or a derivative thereof. In some embodiments, the amino acid or a derivative thereof comprises L-histidine, L-histidine monohydrochloride monohydrate, or a combination thereof. In some embodiments, the stabilizer comprises a sugar. In some embodiments, the sugar comprises sucrose. In some embodiments, the tonicity agent comprises a sugar. In some embodiments, the sugar comprises sucrose. In some embodiments, the surfactant comprises polysorbate 20. In some embodiments, the total amount of L-histidine in the pharmaceutical composition is about 10 mM in the form of both L-histidine and L-histidine monohydrochloride monohydrate. In some embodiments, the molar ratio of L-histidine to L-histidine monohydrochloride monohydrate is about 3:2. In some embodiments, the pharmaceutical composition comprises about 8% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises at least 0.01% (w / v) polysorbate 20. In some embodiments, the pharmaceutical composition comprises about 6 mM L-histidine, about 4 mM L-histidine monohydrochloride monohydrate, about 8% (w / v) sucrose, and about 0.01% (w / v) polysorbate 20. In some embodiments, the pharmaceutical composition comprises about 2 mg / ml of the isolated recombinant polypeptide complex. In some embodiments, the pharmaceutical composition comprises a pH between about 5 and about 7. In some embodiments, the pharmaceutical composition comprises a pH of about 6.3.

[0014] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 5, and wherein the isolated polypeptide is 484 amino acids in length.

[0015] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6, and wherein the isolated polypeptide is 476 amino acids in length.

[0016] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 5, and wherein the isolated polypeptide is 484 amino acids in length.

[0017] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 6, and wherein the isolated polypeptide is 476 amino acids in length.

[0018] In another aspect, disclosed herein is a method for treating cancer, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition disclosed herein. In some embodiments, the cancer comprises mCRPC. In some embodiments, the subject is a human.

[0019] Incorporation by reference

[0020] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. If a publication, patent, or patent application incorporated by reference contradicts the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such contradictory material. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The novel features of the present disclosure are set forth in detail in the appended claims. The patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawings will be provided by the Patent Office upon request and payment of the necessary fee. The features and advantages of the present invention will be better understood by reference to the following detailed description and accompanying drawings (also referred to herein as "Figures" and "FIG.") which illustrate illustrative embodiments utilizing the principles of the present disclosure, in which:

[0022] Figure 1 The design, structure, and mechanism of action of polypeptide complex 1 (PC-1) are described. PC-1 is a tumor-activated T-cell engager with PSMA-binding domains and CD3-binding domains, an albumin-binding domain that prolongs the circulating half-life, a peptide mask that inhibits CD3 engagement with T cells, and a tumor protease-cleavable linker. Tumor-specific proteolysis of the cleavable linker in the tumor microenvironment (TME) separates the tandem mask and albumin-binding domains from PC-1. It enables TME-restricted CD3 binding and subsequent T-cell activation against PSMA-expressing prostate cancer cells. The loss of the albumin-binding domain ensures that any activated PC-1 that migrates out of the tumor will be rapidly cleared and reduces its potential accumulation in healthy tissues that could contribute to safety risks.

[0023] Figures 2A-2DDescription Mask discovery by peptide phage display. Phage displayed peptide libraries are screened against binding surface-immobilized anti-CD3 scFv. After several cycles of binding, elution and amplification, clonal phage are screened for competitive binding to CD3 by ELISA. Selected clonal phage sequences are synthesized as peptides and screened for binding and inhibitory properties against anti-CD3 scFv. Peptide inhibitors are then incorporated into peptide complex designs.

[0024] Figures 3A-3F The binding of PC-1 to CD3 was shown to be cleavage- and dose-dependent. PC-1 CD3 target engagement was cleavage-dependent, with masking reducing CD3 binding by >600-fold. Treatment of PC-1 with proteases achieved potent CD3 binding comparable to unmasked TCE-1. PC-1 exhibited potent binding to human and monkey PSMA and albumin.

[0025] Figure 4A-4B This indicates that TCE-1 potency depends on structure and orientation. TCE-1 activity depends on the connection geometry of the PSMA and CD3 binding domains.

[0026] Figures 5A-5F The activity of PC-1 is cleavage-, dose-, and PSMA expression-dependent. Masking of the PC-1 CD3 binding domain reduces its ability to induce cytokine release. Functional activity in prostate cancer and T cell co-culture assays is dependent on masking and PSMA expression. PC-1 exhibits a robust shift in T cell function, which shows the potential for an enhanced safety profile.

[0027] Figures 6A-6C This indicates that PC-1 has a prolonged half-life and enhanced safety profile in NHPs.

[0028] Figure 7 Flowchart illustrating the upstream cell culture processes involved in PC-1 production.

[0029] Figure 8 Flowchart illustrating the downstream purification processes associated with PC-1 production.

[0030] Fig. 9 The circular dichroism (CD) spectrum of PC-1 in the far-UV region is illustrated.

[0031] Fig.10 The CD spectrum of PC-1 in the near-UV region is illustrated.

[0032] Fig.11 Differential Scanning Calorimetry data for PC-1 are illustrated.

[0033] Figures 12A-12B Illustrate the SEC-MALS chromatogram of PC-1.

[0034] Fig.13 Illustrate the light and heavy chain arrangement in PC-1.

[0035] Figures 14A-14F ELISA binding of test article to PSMA is shown.

[0036] Figures 15A-15D This shows the ELISA binding of PC-1 to albumin.

[0037] Figures 16A-16F ELISA binding of test article to CD3.

[0038] Figures 17A-17B Example illustrating CD3 Octet binding data for PC-1 over time following incubation in serum.

[0039] Figures 18A-18H PC-1 cleavage rates in cynomolgus monkey, healthy human or human mCRPC donor sera are shown.

[0040] Figures 19A-19I Dose-dependent test article-induced production of IFNγ, TNF, and IL-6 by PBMCs co-cultured with LNCaP tumor cells is demonstrated.

[0041] Figures 20A-20I Dose-dependent test article-induced production of IFNγ, TNF, and IL-6 by PBMCs co-cultured with 22Rv1 tumor cells is demonstrated.

[0042] Figures 21A-21F This indicates that in the absence of tumor cells, there is no test article-induced cytokine production in PBMCs.

[0043] Figures 22A-22H This demonstrates the dose-dependent killing of LNCaP tumor cells by PBMCs stimulated by the test articles.

[0044] Figures 23A-23H This demonstrates the dose-dependent killing of 22Rv1 tumor cells by PBMCs stimulated by the test articles.

[0045] Figures 24A-24D This indicates the lack of PC3 tumor cell killing by PBMCs stimulated by the test articles.

[0046] Fig.25 Describes the sample preparation workflow using immunocapture purification.

[0047] Fig.26 Illustration of the TK signature of PC-1 in monkey plasma samples collected from Group 2 on Day 22 of the TK study.

[0048] Fig. 27The results showed that the correlation between intact PC-1 concentration and anti-drug antibody (ADA) concentration in the high-dose group of monkeys was significant. DETAILED DESCRIPTION

[0049] In the context of this application, unless otherwise stated, the following terms have the meanings ascribed to them:

[0050] As used in the specification and claims, the terms "a", "an", and "the" generally mean "at least one", "at least a first", "one or more", or "a plurality" of the referenced components or steps, unless an upper limit is explicitly stated later. For example, "a cleavage sequence" as used herein means "at least a first cleavage sequence", but includes multiple cleavage sequences. One of ordinary skill in the art will be aware of the operable limits and parameters of the combination, as well as the amount of any single agent, based on this application.

[0051] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein and generally refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, or it may be interrupted by non-amino acids. These terms also include amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a tagging component.

[0052] As used herein in the context of polypeptide structures, the "N-terminus" (or "amino terminus") and the "C-terminus" (or "carboxyl terminus") generally refer to the extreme amino and carboxyl termini, respectively, of a polypeptide.

[0053] The term "therapeutically effective amount" as used herein generally refers to an amount of a polypeptide variant or polypeptide composition that, when administered to a patient to treat a disease or other adverse medical condition, is sufficient to produce a beneficial effect on the disease or condition. The therapeutically effective amount will vary depending on the polypeptide variant or polypeptide composition, the disease or condition and its severity, and the age, weight, etc. of the patient to be treated. Determining a therapeutically effective amount of a given polypeptide variant or a given polypeptide composition is generally within the ordinary skill in the art and requires only routine experimentation.

[0054] As used herein, the terms "about" and "approximately" are used interchangeably. Any number used herein, whether or not with about / approximately, is intended to encompass any normal fluctuations understood by a person of ordinary skill in the relevant art. For example, the term "about" may refer to a range of ±10%, ±5%, ±2%, or ±1% of a particular value. For example, the phrase "about 50%" may include 45% to 55%, 48% to 52%, or 49% to 51%.

[0055] The above definitions supersede any conflicting definitions in any references incorporated herein by reference. However, the fact that certain terms are defined should not be taken to indicate that any undefined terms are undefined. Instead, all terms used are considered to describe the present disclosure in such a manner that a person of ordinary skill can understand the scope of the application and practice the application.

[0056] Recombinant polypeptide composition

[0057] Metastatic castration-resistant prostate cancer (mCRPC) remains an incurable disease. Bispecific T cell engagers (TCEs) targeting prostate-specific membrane antigen (PSMA) on prostate tumor cells and cluster of differentiation 3 (CD3) on T cells have clinical efficacy for mCRPC treatment. These TCEs have problems with cytokine release syndrome (CRS) and poor pharmacokinetic (PK) profiles. New immunotherapies are still needed. Disclosed herein is a PSMA-targeted tumor-activated T cell engager (TRACTr) with enhanced safety and pharmacokinetic profiles.

[0058] Figure 1 The design, structure, and mechanism of action of polypeptide complex 1 (PC-1) are described. PC-1 is a tumor-activated T-cell engager with PSMA-binding domains and CD3-binding domains, an albumin-binding domain that prolongs the circulating half-life, a peptide mask that inhibits CD3 engagement with T cells, and a tumor protease-cleavable linker. Tumor-specific proteolysis of the cleavable linker in the tumor microenvironment (TME) separates the tandem mask and albumin-binding domains from PC-1. It enables TME-restricted CD3 binding and subsequent T-cell activation against PSMA-expressing prostate cancer cells. The loss of the albumin-binding domain may ensure that any activated PC-1 that migrates out of the tumor will be rapidly cleared and reduce its accumulation in healthy tissues that could lead to safety risks.

[0059] A recombinant polypeptide is disclosed herein. In some embodiments, the recombinant polypeptide includes a tumor-activated T cell engager having a PSMA binding domain and a CD3 binding domain, an albumin binding domain that extends the circulating half-life, a peptide mask that inhibits CD3 engagement on T cells, and a tumor protease cleavable linker. Tumor-specific proteolysis of the cleavable linker in the tumor microenvironment can separate the tandem mask and the albumin binding domain from the recombinant polypeptide. The recombinant polypeptide may include chain 1 and chain 2 as described herein. In some embodiments, chain 1 comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1. In some embodiments, chain 2 comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:2.

[0060] In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 1. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 1. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 1. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 1. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 1. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 1. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 1. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:1.

[0061] In some embodiments, the recombinant polypeptide comprises the amino acid sequence according to SEQ ID NO:1.

[0062] In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:2. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO:2. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:2. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 91% sequence identity with SEQ ID NO:2. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 92% sequence identity with SEQ ID NO:2. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 93% sequence identity with SEQ ID NO:2. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 94% sequence identity with SEQ ID NO:2. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:2. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 96% sequence identity with SEQ ID NO:2. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 97% sequence identity with SEQ ID NO:2. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 2. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:2.

[0063] In some embodiments, the recombinant polypeptide comprises the amino acid sequence according to SEQ ID NO:2.

[0064] Table 1: Amino acid sequence of recombinant polypeptide

[0065]

[0066]

[0067] Disulfide bond(s)

[0068] In some embodiments, the recombinant polypeptide comprises at least one disulfide bond formed by one cysteine ​​residue pair. In some embodiments, the recombinant polypeptide comprises at least one disulfide bond formed by one cysteine ​​residue pair in the first chain. In some embodiments, the recombinant polypeptide comprises at least one disulfide bond formed by one cysteine ​​residue pair in the second chain. In some embodiments, the recombinant polypeptide comprises at least one disulfide bond formed by one cysteine ​​residue pair in the first chain and the second chain. In some embodiments, the recombinant polypeptide comprises at least two, at least three, or at least four disulfide bonds formed by the cysteine ​​residue pair comprising the cysteine ​​residue pair. In some embodiments, the recombinant polypeptide comprises at least two disulfide bonds formed by the cysteine ​​residue pair comprising the cysteine ​​residue pair. In some embodiments, the recombinant polypeptide comprises at least three disulfide bonds formed by the cysteine ​​residue pair comprising the cysteine ​​residue pair. In some embodiments, the recombinant polypeptide comprises at least four disulfide bonds formed by the cysteine ​​residue pair comprising the cysteine ​​residue pair. In some embodiments, the recombinant polypeptide comprises at least five disulfide bonds formed by the cysteine ​​residue pair comprising the cysteine ​​residue pair. In some embodiments, the recombinant polypeptide comprises at least six disulfide bonds formed by the cysteine ​​residue pairs comprising the cysteine ​​residue pair. In some embodiments, the recombinant polypeptide comprises at least seven disulfide bonds formed by the cysteine ​​residue pairs comprising the cysteine ​​residue pair. In some embodiments, the recombinant polypeptide comprises at least eight disulfide bonds formed by the cysteine ​​residue pairs comprising the cysteine ​​residue pair.

[0069] In some embodiments, one pair of cysteine ​​residues that form a disulfide bond corresponds to the following amino acid positions: (i) cysteine ​​22 and cysteine ​​96 of SEQ ID NO: 1, or (ii) cysteine ​​138 and cysteine ​​148 of SEQ ID NO: 1, or (iii) cysteine ​​199 and cysteine ​​275 of SEQ ID NO: 1, or (iv) cysteine ​​339 and cysteine ​​407 of SEQ ID NO: 1, or (v) cysteine ​​454 and cysteine ​​519 of SEQ ID NO: 1, or (vi) cysteine ​​565 and cysteine ​​625 of SEQ ID NO: 1, or (vii) cysteine ​​645 of SEQ ID NO: 1 and cysteine ​​226 of SEQ ID NO: 2, or (viii) cysteine ​​22 and cysteine ​​96 of SEQ ID NO: 2, or (ix) cysteine ​​150 and cysteine ​​206 of SEQ ID NO: 2.

[0070] In some embodiments of the recombinant peptide, at least one disulfide bond formed by the pair of cysteine ​​residues is formed by a cysteine ​​residue selected from and corresponding to cysteine ​​22, cysteine ​​96, cysteine ​​138, cysteine ​​148, cysteine ​​199, cysteine ​​275, cysteine ​​339, cysteine ​​407, cysteine ​​454, cysteine ​​519, cysteine ​​565, or cysteine ​​625 of SEQ ID NO: 1. In some embodiments of the recombinant polypeptide, at least one disulfide bond formed by the pair of cysteine ​​residues is formed by a cysteine ​​residue selected from and corresponding to cysteine ​​645 of SEQ ID NO: 1 and cysteine ​​226 of SEQ ID NO: 2. In some embodiments of the recombinant polypeptide, at least one disulfide bond formed by the pair of cysteine ​​residues is formed by a cysteine ​​residue selected from and corresponding to cysteine ​​22, cysteine ​​96, cysteine ​​150, cysteine ​​206 of SEQ ID NO: 2. The presence of disulfide bond(s) can be determined by mass spectrometry (MS).

[0071] In some embodiments, the recombinant polypeptide comprises at least one cysteine ​​residue that is a free thiol.The presence of free thiol(s) can be determined by mass spectrometry (MS).

[0072] In some embodiments, the isolated recombinant polypeptide complex provides a maximum plasma concentration (Cmax) in a subject within about 0.001 to about 1 hour after intravenous administration, for example, about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.010, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.020, 0.021, 0.022, 0.023, 0.024, 0.025, 0.026, 0.027, 0.028, 0.029, 0.030, 0.031, 0.032, 32, 0.033, 0.034, 0.035, 0.036, 0.037, 0.038, 0.039, 0.040, 0.041, 0.042, 0.043, 0.044, 0.045, 0.046, 0.047, 0.048, 0.049, 0.050, 0.051, 0.052, 0. 0.053、0.054、0.055、0.056、0.057、0.058、0.059、0.060、0.061、0.062、0.063、0.064、0.065、0.066、0.067、0.068、0.069、0.070、0.071、0.072、0.073、0 .074, 0.075, 0.076, 0.077, 0.078, 0.079, 0.080, 0.081, 0.082, 0.083, 0.084, 0.085, 0.086, 0.087, 0.088, 0.089, 0.090, 0.091, 0.092, 0.093, 0.094 , 0.095, 0.096, 0.097, 0.098, 0.099, 0.100, 0.105, 0.110, 0.115, 0.120, 0.125, 0.130, 0.135, 0.140, 0.145, 0.150, 0.155, 0.160, 0.165, 0.170, 0.17 5. 0.180, 0.185, 0.190, 0.195, 0.200, 0.205, 0.210, 0.215, 0.220, 0.225, 0.230, 0.235, 0.240, 0.245, 0.250, 0.255, 0.260, 0.265, 0.270, 0.275, 0.2 80, 0.285, 0.290, 0.295, 0.300, 0.305, 0.310, 0.315, 0.320, 0.325, 0.330, 0.335, 0.340, 0.345, 0.350, 0.355, 0.360, 0.365, 0.370, 0.375, 0.380, 0.385, 0.390, 0.395, 0.400, 0.405, 0.410, 0.415, 0.420, 0.425, 0.430, 0.435, 0.440, 0.445, 0.450, 0.455, 0.460, 0.465, 0.470, 0.475, 0.480, 0.485, 0.490, 0.495, 0.500, 0.505, 0.510, 0.515, 0.520, 0.525, 0.530, 0.535, 0.54 0, 0.545, 0.550, 0.555, 0.560, 0.565, 0.570, 0.575, 0.580, 0.585, 0.590, 0.595, 0.600, 0.605, 0.610, 0.615, 0.620, 0.625, 0.630, 0.635, 0.640, 0.645, 0.650, 0.655, 0.660, 0.665, 0.670, 0.675, 0.680, 0.685, 0.690, 0.695, 0.700, 0.705, 0.710, 0.715, 0.720, 0.725, 0.730, 0.735, 0.740, 0.745, 0.750, 0.755, 0.760, 0.765, 0.770, 0.775, 0.780, 0.785, 0.790, 0.795, 0.800, 0.805, 0.810, 0.815, 0.820, 0.825, 0.830, 0.835, 0.840, 0.845, 0.850, 0. 855, 0.860, 0.865, 0.870, 0.875, 0.880, 0.885, 0.890, 0.895, 0.900, 0.905, 0.910, 0.915, 0.920, 0.925, 0.930, 0.935, 0.940, 0.945, 0.950, 0.955, 0.960, 0.965, 0.970, 0.975, 0.980, 0.985, 0.990, 0.995, or about 1.0 hour, or any time therebetween. In some embodiments, the isolated recombinant polypeptide complex provides a Cmax in a subject within about 0.1 hour after intravenous administration.

[0073] In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 2000 to 4000 ng / ml in a subject within about 0.1 hour after intravenous administration of a dose of about 0.1 mg / kg, such as about 2000 to 4000 ng / ml, 2000 to 3500 ng / ml, 2000 to 3000 ng / ml, 2000 to 2500 ng / ml, 2500 to 4000 ng / ml, 2500 to 3500 ng / ml, 2500 to 3000 ng / ml, 3000 to 4000 ng / ml, 3000 to 3500 ng / ml, or about 3500 to 4000 ng / ml, or any concentration therebetween. In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 2500 to 3500 ng / ml in a subject within about 0.1 hour after intravenous administration of a dose of about 0.1 mg / kg.

[0074] In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 7000 to 12000 ng / ml in a subject within about 0.1 hour after intravenous administration of a dose of about 0.3 mg / kg, e.g., about 7000 to 12000 ng / ml, 7000 to 11500 ng / ml, 7000 to 11000 ng / ml, 7000 to 10500 ng / ml, 7000 to 10000 ng / ml, 7000 to 9500 ng / ml, 7000 to 9000 ng / ml, 7000 to 8500 ng / ml, 7000 to 8000 ng / ml, 7000 to 7500 ng / ml, 7500 to 12000 ng / ml. 0ng / ml, 7500 to 11500ng / ml, 7500 to 11000ng / ml, 7500 to 10500ng / ml, 7500 to 10000ng / ml, 7500 to 9500ng / ml, 7500 to 9000ng / ml, 7500 to 8500ng / ml, 7500 to 8000ng / ml, 8000 to 12000ng / ml, 8000 to 11500ng / ml, 8000 to 11000ng / ml, 8000 to 10500ng / ml, 8000 to 10000ng / ml, 8000 to 9500ng / ml, 8000 to 9000ng / ml, 8000 to 8500 ng / ml, 8500 to 12000 ng / ml, 8500 to 11500 ng / ml, 8500 to 11000 ng / ml, 8500 to 10500 ng / ml, 8500 to 10000 ng / ml, 8500 to 9500 ng / ml, 8500 to 9000 ng / ml, 9000 to 12000 ng / ml, 9000 to 11500 ng / ml, 9000 to 11000 ng / ml, 9000 to 10500 ng / ml, 9000 to 10000 ng / ml, 9000 to 9500 ng / ml, 9500 to 12000 ng / ml, 9500 To 11500ng / ml, 9500 to 11000ng / ml, 9500 to 10500ng / ml, 9500 to 10000ng / ml, 10000 to 12000ng / ml, 10000 to 11500ng / ml, 10000 to 11000ng / ml, 10000 to 11000ng / ml, 10500 to 12000ng / ml, 10500 to 11500ng / ml, 10500 to 11000ng / ml, 11000 to 12000ng / ml, 11000 to 11500ng / ml, or about 11500 to 12000ng / ml, or any range therebetween.In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 7500 to 10500 ng / ml in a subject within about 0.1 hour following intravenous administration of a dose of about 0.3 mg / kg.

[0075] In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 32,500 to 57,500 ng / ml in a subject within about 0.1 hour after intravenous administration of a dose of about 1.5 mg / kg, e.g., about 32,500 to 57,500 ng / ml, 32,500 to 55,000 ng / ml, 32,500 to 52,500 ng / ml, 32,500 to 50,000 ng / ml, 32,500 to 47,500 ng / ml, 32,500 to 42,500 ng / ml, 32,500 to 40,000 ng / ml, 32,500 to 5 ... 00 to 37500ng / ml, 32500 to 35000ng / ml, 35000 to 57500ng / ml, 35000 to 55000ng / ml, 35000 to 52500ng / ml, 35000 to 50000ng / ml, 35000 to 47500ng / ml, 35000 to 45000ng / ml, 35000 to 42500ng / ml, 35000 to 40000ng / ml, 35000 to 37500ng / ml, 37500 to 75000ng / ml, 37500 to 70000ng / ml, 37500 to 675 00ng / ml, 37500 to 65000ng / ml, 7500 to 62500ng / ml, 37500 to 60000ng / ml, 37500 to 57500ng / ml, 37500 to 55000ng / ml, 37500 to 52500ng / ml, 37500 to 50000ng / ml, 37500 to 47500ng / ml, 37500 to 45000ng / ml, 37500 to 42500ng / ml, 37500 to 40000ng / ml l, 40000 to 57500 ng / ml, 40000 to 55000 ng / ml, 40000 to 52500 ng / ml, 40000 to 50000 ng / ml, 40000 to 47500 ng / ml, 40000 to 45000 ng / ml, 40000 to 42500 ng / ml, 42500 to 57500 ng / ml, 42500 to 55000 ng / ml, 42500 to 52500 ng / ml, 425In some embodiments, the present invention relates to a concentration range of about 5000 ng / ml to about 47,500 ng / ml, about 42,500 ng / ml to about 45,000 ng / ml, about 42,500 ng / ml to about 45,000 ng / ml, about 45,000 ng / ml to about 55,000 ng / ml, about 45,000 ng / ml to about 52,500 ng / ml, about 45,000 ng / ml to about 50,000 ng / ml, about 45,000 ng / ml to about 47,500 ng / ml, about 47,500 ng / ml to about 57,500 ng / ml, about 47,500 ng / ml to about 55,000 ng / ml In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 37,500 to 52,500 ng / ml in a subject within about 0.1 hour following intravenous administration of a dose of about 1.5 mg / kg.

[0076] In some embodiments, the isolated recombinant polypeptide complex is about 50 to about 130 hours (T 1 / 2 ) to provide a half-maximal plasma concentration in a subject, e.g., about 50 to about 130 hours, about 50 to about 120 hours, about 50 to about 110 hours, about 50 to about 100 hours, about 50 to about 90 hours, about 50 to about 80 hours, about 50 to about 70 hours, about 50 to about 60 hours, about 60 to about 130 hours, about 60 to about 120 hours, about 60 to about 110 hours, about 60 to about 100 hours, about 60 to about 90 hours, about 60 to about 80 hours, about 60 to about 70 hours, about 50 to about 60 hours, about 60 to about 130 hours, about 60 to about 120 hours, about 60 to about 110 hours, about 60 to about 100 hours, about 60 to about 90 hours, about 60 to about 80 hours, about 60 to about 70 hours, about 70 to about 130 hours, about 70 to about 120 hours, about 70 to about 110 hours, about 70 to about 100 hours. In some embodiments, the present invention relates to a method for producing a therapeutically effective amount of ...

[0077] In some embodiments, the value of Cmax is related to the dose of the isolated recombinant polypeptide complex administered. In some embodiments, the value of Cmax is proportional to the dose of the isolated recombinant polypeptide complex administered.

[0078] In some embodiments, less than about 10% of the isolated recombinant polypeptide complex is degraded in the serum of a subject per day, for example, less than about 10%, 9.9%, 9.8%, 9.7%, 9.6%, 9.5%, 9.4%, 9.3%, 9.2%, 9.1%, 9%, 8.9%, 8.8%, 8.7%, 8.6%, 8.5%, 8.4%, 8.3%, 8.2%, %, 8.1%, 8%, 7.9%, 7.8%, 7.7%, 7.6%, 7.5%, 7.4%, 7.3%, 7.2%, 7.1%, 7%, 6.9%, 6.8%, 6.7%, 6.6%, 6.5%, 6.4%, 6.3%, 6.2%, 6.1%, 6%, 5.9%, 5.8%, 5.7%, 5.6%, 5.5%, 5.4% , 5.3%, 5.2%, 5.1%, 5%, 4.9%, 4.8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3%, 4.2%, 4.1%, 4%, 3.9%, 3.8%, 3.7%, 3.6%, 3.5%, 3.4%, 3.3%, 3.2%, 3.1%, 3%, 2.9%, 2.8%, 2.7%, 2.6%, In some embodiments, the isolated recombinant polypeptide complex is degraded in the serum of the subject per day.

[0079] In some embodiments, the subject is diagnosed with cancer. In some embodiments, the cancer comprises prostate cancer. In some embodiments, the cancer comprises metastatic castration-resistant prostate cancer (mCRPC).

[0080] In some embodiments, after administration, the separated recombinant polypeptide complex is cleaved by a tumor-specific protease to generate an enzyme product of the separated recombinant polypeptide complex. In some embodiments, the tumor-specific protease includes two or more proteases. In some embodiments, the separated recombinant polypeptide complex is cleaved by a first protease of two or more proteases to generate a first metabolite of the separated recombinant polypeptide complex. In some embodiments, the separated recombinant polypeptide complex is cleaved by a second protease of two or more proteases to generate a second metabolite of the separated recombinant polypeptide complex. In some embodiments, the first protease includes a serine protease. In some embodiments, the second protease includes a matrix metalloprotease. In some embodiments, the serine protease includes a recombinant human membrane serine protease (MTSP1). In some embodiments, the matrix metalloprotease includes a recombinant human matrix metalloprotease 9 (MMP9).

[0081] In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 81% sequence identity to SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 82% sequence identity to SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 83% sequence identity to SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 84% sequence identity to SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 86% sequence identity to SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 87% sequence identity to SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 88% sequence identity to SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 89% sequence identity to SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5. In some embodiments, the isolated first metabolite of the recombinant polypeptide complex comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:5.In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence of SEQ ID NO: 5.

[0082] In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 81% sequence identity with SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 82% sequence identity with SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 83% sequence identity with SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 84% sequence identity with SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 86% sequence identity with SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 87% sequence identity with SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 88% sequence identity with SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 89% sequence identity with SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 91% sequence identity with SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 92% sequence identity with SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 93% sequence identity with SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 94% sequence identity with SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 6. In some embodiments, the isolated second metabolite of the recombinant polypeptide complex comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:6.In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence of SEQ ID NO: 6.

[0083] In some embodiments, the subject is a non-human primate or a human. In some embodiments, the non-human primate is a cynomolgus monkey.

[0084] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0085] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 81% sequence identity to SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0086] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 82% sequence identity to SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0087] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 83% sequence identity to SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0088] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 84% sequence identity to SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0089] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0090] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 86% sequence identity to SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0091] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 87% sequence identity to SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0092] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 88% sequence identity to SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0093] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 89% sequence identity to SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0094] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0095] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0096] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0097] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0098] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0099] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0100] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0101] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0102] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0103] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0104] In another aspect, disclosed herein is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0105] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0106] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 81% sequence identity to SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0107] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 82% sequence identity to SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0108] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 83% sequence identity to SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0109] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 84% sequence identity to SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0110] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0111] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 86% sequence identity to SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0112] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 87% sequence identity to SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0113] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 88% sequence identity to SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0114] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 89% sequence identity to SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0115] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0116] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0117] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0118] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0119] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0120] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0121] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0122] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0123] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0124] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0125] In another aspect, disclosed herein is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0126] Amino acid modification

[0127] In some embodiments, at least one glutamine in SEQ ID NO: 1 or SEQ ID NO: 2 is cyclized to form pyroglutamine. In some embodiments, the pyroglutamine is located at the N-terminus of SEQ ID NO: 2. In some embodiments, the recombinant polypeptide comprises a pyroglutamine residue at the amino acid position corresponding to glutamine 1 of SEQ ID NO: 2. The presence and amount of the pyroglutamine residue(s) can be determined by mass spectrometry (MS).

[0128] Thermal stability

[0129] In some embodiments, the recombinant polypeptide is characterized by a melting temperature (T m ) is about 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C or 80°C, or a range between any two of the foregoing. In some embodiments, the recombinant polypeptide is characterized by a melting temperature (T m ) is about 70° C. to about 81° C. In some embodiments, the recombinant polypeptide is characterized by a melting temperature (T m ) is about 71° C. to about 72° C. In some embodiments, the recombinant polypeptide is characterized by a melting temperature (T m) is about 78° C. to about 80° C. In some embodiments, the recombinant polypeptide is characterized by a melting temperature (T m ) is about 71.4° C. In some embodiments, the recombinant polypeptide is characterized by a melting temperature (T m ) is about 79.5° C. In some embodiments, the recombinant polypeptide is characterized by a melting onset temperature (T 起始 ) is about 55° C. to about 60° C. In some embodiments, the recombinant polypeptide is characterized by a melting onset temperature (T 起始 ) is about 58°C to about 60°C. In some embodiments, the recombinant polypeptide is characterized by a melting onset temperature (T 起始 ) is about 59°C. The melting temperature (T m ) can be measured by differential scanning calorimetry (DSC). 起始 ) can be determined by differential scanning calorimetry (DSC).

[0130] Structural composition

[0131] In some embodiments, the recombinant polypeptide has a secondary structural composition comprising a beta-sheet or a random coil. The secondary structural composition may include a beta-sheet. The secondary structural composition may include a random coil.

[0132] In some embodiments, the recombinant polypeptide is characterized by a near UV circular dichroism peak at a wavelength less than or equal to 300 nm, 295 nm, 290 nm, 285 nm, 280 nm, or 275 nm. In some embodiments, the recombinant polypeptide is characterized by a near UV circular dichroism peak at a wavelength greater than or equal to 275 nm, 280 nm, 285 nm, 290 nm, or 300 nm. In some embodiments, the recombinant polypeptide is characterized by a near UV circular dichroism peak at a wavelength between 275 nm and 285 nm, between 280 nm and 290 nm, between 285 nm and 295 nm, or between 290 nm and 300 nm.

[0133] In some embodiments, the recombinant polypeptide is characterized by a far-UV circular dichroism peak at a wavelength less than or equal to 220 nm, 210 nm, or 205 nm. In some embodiments, the recombinant polypeptide is characterized by a far-UV circular dichroism peak at a wavelength greater than or equal to 205 nm, 210 nm, or 220 nm. In some embodiments, the recombinant polypeptide is characterized by a far-UV circular dichroism peak at a wavelength between 200 nm and 210 nm or between 205 nm and 220 nm.

[0134] One or more features

[0135] The polypeptides or recombinant polypeptides described herein may have one or more of the characteristics described above in this section.

[0136] In some embodiments, the recombinant polypeptide has at least one, at least two, at least three, at least four, at least five, at least six, or all seven of features (a)-(g): (a) at least one disulfide bond formed by a pair of cysteine ​​residues in the first chain or the second chain or by a pair of cysteine ​​residues in the first chain and the second chain; or

[0137] (b) secondary structure consisting of β-sheets or random coils;

[0138] (c) at least one pyroglutamine in the second chain;

[0139] (d) a melting temperature (Tm) between about 65°C and about 85°C when the isolated recombinant polypeptide complex is formulated at a concentration of 1.0 mg / mL in 10 mM His, 8 (w / v)% sucrose, 0.01 (w / v)% polysorbate-20, pH 6.3;

[0140] (e) a far-UV circular dichroism peak at a wavelength between 190 nm and 205 nm when the isolated recombinant polypeptide complex is formulated at a concentration of 0.1 mg / mL in 10 mM potassium phosphate buffer, pH 7.0;

[0141] (f) a deep UV circular dichroism depression at wavelengths between 210 nm and 220 nm when the isolated recombinant polypeptide complex is formulated at a concentration of 0.1 mg / mL in 10 mM potassium phosphate buffer, pH 7.0; or

[0142] (g) Near-UV circular dichroism peaks at wavelengths between 250 nm and 300 nm when the isolated recombinant polypeptide complex is formulated at a concentration of 1.0 mg / mL in 10 mM His, 8% (w / v) sucrose, 0.01% (w / v) polysorbate-20, pH 6.3.

[0143] In some embodiments, the recombinant polypeptide includes at least one (e.g., one, two, three, four, five, six, or seven) of features (a)-(g). In some embodiments, the recombinant polypeptide includes at least two (e.g., two, three, four, five, six, or seven) of features (a)-(g). In some embodiments, the recombinant polypeptide includes at least three (e.g., three, four, five, six, or seven) of features (a)-(g). In some embodiments, the recombinant polypeptide includes at least four (e.g., four, five, six, or seven) of features (a)-(g). In some embodiments, the recombinant polypeptide includes at least five (e.g., five, six, or seven) of features (a)-(g). In some embodiments, the recombinant polypeptide includes at least six (e.g., six or seven) of features (a)-(g). In some embodiments, the recombinant polypeptide includes all seven of features (a)-(g).

[0144] preparation

[0145] Disclosed herein are formulations (one or more) comprising a population of polypeptides or recombinant polypeptides, such as any one or a combination of the polypeptides (one or more) or recombinant polypeptides (one or more) described herein.

[0146] In some embodiments of the preparations, at least about 80%, 85%, 90%, or 95% (e.g., by mole or by mass) of the polypeptides of the population comprise at least one pyroglutamine residue (such as any of the residues described above). In some embodiments, at least about 80% (e.g., by mole or by mass) of the polypeptides of the population comprise at least one pyroglutamine residue, for example, at an amino acid position corresponding to glutamine 1 of SEQ ID NO: 2. In some embodiments, at least about 85% (e.g., by mole or by mass) of the polypeptides of the population comprise at least one pyroglutamine residue, for example, at an amino acid position corresponding to glutamine 1 of SEQ ID NO: 2. In some embodiments, at least about 90% (e.g., by mole or by mass) of the polypeptides of the population comprise at least one pyroglutamine residue, for example, at an amino acid position corresponding to glutamine 1 of SEQ ID NO: 2. In some embodiments, at least about 95% (e.g., by mole or by mass) of the polypeptides of the population comprise at least one pyroglutamine residue, for example, at an amino acid position corresponding to glutamine 1 of SEQ ID NO: 2.

[0147] In some embodiments of the preparation, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% (e.g., by mole or by mass) of the polypeptides in the population are monomers. In some embodiments of the preparation, less than or equal to 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% (e.g., by mole or by mass) of the polypeptides in the population are aggregated.

[0148] In some embodiments of the formulations, the polypeptides of the population comprise at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the polypeptides of the population comprise at least about 85% sequence identity to SEQ ID NO: 1. In some embodiments, the polypeptides of the population comprise at least about 90% sequence identity to SEQ ID NO: 1. In some embodiments, the polypeptides of the population comprise at least about 95% sequence identity to SEQ ID NO: 1. In some embodiments, the polypeptides of the population comprise at least about 99% sequence identity to SEQ ID NO: 1. In some embodiments, the polypeptides of the population comprise an amino acid sequence according to SEQ ID NO: 1.

[0149] In some embodiments of the formulations, the polypeptides of the population comprise at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2. In some embodiments, the polypeptides of the population comprise at least about 85% sequence identity to SEQ ID NO: 2. In some embodiments, the polypeptides of the population comprise at least about 90% sequence identity to SEQ ID NO: 2. In some embodiments, the polypeptides of the population comprise at least about 95% sequence identity to SEQ ID NO: 2. In some embodiments, the polypeptides of the population comprise at least about 99% sequence identity to SEQ ID NO: 2. In some embodiments, the polypeptides of the population comprise an amino acid sequence according to SEQ ID NO: 2.

[0150] A pharmaceutical composition is disclosed herein, comprising one or more pharmaceutically acceptable excipients, wherein the one or more pharmaceutically acceptable excipients include histidine, sucrose, polysorbate-20, sodium phosphate, citrate, glutamic acid, glycine, arginine, sorbitol, arginine hydrochloride, acetate, sodium chloride, potassium chloride, magnesium chloride and calcium chloride. A pharmaceutical composition is disclosed herein, wherein the pharmaceutical composition comprises a recombinant polypeptide (such as any recombinant polypeptide described herein), sodium dihydrogen phosphate monohydrate, sodium hydrogen phosphate, heptahydrate, sodium chloride, potassium chloride, histidine, citrate, acetate, sucrose, polysorbate-20, polysorbate-80, magnesium chloride hexahydrate and calcium chloride dihydrate. The pH of the pharmaceutical composition may be less than or equal to 7.0, 6.5, 6.0 or 5.5. The pH of the pharmaceutical composition may be greater than or equal to 5.5, 6.0, 6.5 or 7.0. The pharmaceutical composition may comprise greater than or equal to 5mM, 6mM, 7mM, 8mM, 9mM or 10mM histidine. The pharmaceutical composition may comprise less than or equal to 10mM, 9mM, 8mM, 7mM, 6mM or 5mM histidine. The pharmaceutical composition may comprise greater than or equal to 5% (w / v), 6% (w / v), 7% (w / v) or 8% (w / v) sucrose. The pharmaceutical composition may comprise less than or equal to 9% (w / v), 8% (w / v), 7% (w / v), 6% (w / v) or 5% (w / v) sucrose. The pharmaceutical composition may comprise less than or equal to 0.05% (w / v) polysorbate 20, 0.04% (w / v) polysorbate 20, 0.03% (w / v) polysorbate 20, 0.02% (w / v) polysorbate 20, or 0.01% polysorbate 20. The pharmaceutical composition may comprise greater than or equal to 0.01% (w / v) polysorbate 20, 0.02% (w / v) polysorbate 20, 0.03% (w / v) polysorbate 20, 0.04% (w / v) polysorbate 20, or 0.05% polysorbate 20. Disclosed herein is a pharmaceutical composition, wherein the pharmaceutical composition comprises a recombinant polypeptide (such as any recombinant polypeptide described herein), 10 mM histidine, 8% (w / v) sucrose, and 0.01% (w / v) polysorbate-20.

[0151] In another aspect, the present invention discloses a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises: (a) the isolated recombinant polypeptide complex disclosed in the present invention; and (b) a pharmaceutically acceptable excipient.

[0152] In some embodiments, pharmaceutically acceptable excipients include buffers, stabilizers, tonicity agents, surfactants, or combinations thereof. In some embodiments, buffers include amino acids or derivatives thereof. In some embodiments, amino acids or derivatives thereof include L-histidine, L-histidine monohydrochloride monohydrate, or combinations thereof. In some embodiments, stabilizers include sugars. In some embodiments, sugars include sucrose. In some embodiments, tonicity agents include sugars. In some embodiments, sugars include sucrose. In some embodiments, surfactants include polysorbates. In some embodiments, surfactants include polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. In some embodiments, surfactants include polysorbate-20. In some embodiments, the total amount of L-histidine in the pharmaceutical composition is about 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, 2.5 mM, 2.6 mM, 2.7 mM, 2.8 mM, 2.9 mM, 3 mM, 3.1 mM, 3.2 mM, 3.3 mM, 3.4 mM, 3.5 mM, 3.6 mM, 3.7 mM, 3.8 mM, 3.9 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM 7mM, 3.8mM, 3.9mM, 4mM, 4.1mM, 4.2mM, 4.3mM, 4.4mM, 4.5mM, 4.6mM, 4.7mM, 4.8mM, 4.9mM, 5mM, 5.1mM, 5.2mM, 5 .3mM, 5.4mM, 5.5mM, 5.6mM, 5.7mM, 5.8mM, 5.9mM, 6mM, 6.1mM, 6.2mM, 6.3mM, 6.4mM, 6.5mM, 6.6mM, 6.7mM, 6.8mM , 6.9mM, 7mM, 7.1mM, 7.2mM, 7.3mM, 7.4mM, 7.5mM, 7.6mM, 7.7mM, 7.8mM, 7.9mM, 8mM, 8.1mM, 8.2mM, 8.3mM, 8.4m M, 8.5mM, 8.6mM, 8.7mM, 8.8mM, 8.9mM, 9mM, 9.1mM, 9.2mM, 9.3mM, 9.4mM, 9.5mM, 9.6mM, 9.7mM, 9.8mM, 9.9mM, 10 mM, 10.1mM, 10.2mM, 10.3mM, 10.4mM, 10.5mM, 10.6mM, 10.7mM, 10.8mM, 10.9mM, 11mM, 11.1mM, 11.2mM, 11.3mM, 11.4mM, 11.5mM, 11.6mM, 11.7mM, 11.8mM, 11.9mM, 12mM, 12.1mM, 12.2mM, 12.3mM, 12.4mM, 12.5mM, 12.6mM, 12.7mM, 12.8mM, 12.9mM, 13mM, 13.1mM, 13.2mM, 13.3mM, 13.4mM, 13.5mM, 13.6m M, 13.7mM, 13.8mM, 13.9mM, 14mM, 14.1mM, 14.2mM, 14.3mM, 14.4mM, 14.5mM, 1 4.6mM, 14.7mM, 14.8mM, 14.9mM, 15mM, 15.1mM, 15.2mM, 15.3mM, 15.4mM, 15. 5mM, 15.6mM, 15.7mM, 15.8mM, 15.9mM, 16mM, 16.1mM, 16.2mM, 16.3mM, 16.4mM , 16.5mM, 16.6mM, 16.7mM, 16.8mM, 16.9mM, 17mM, 17.1mM, 17.2mM, 17.3mM, 1 7.4mM, 17.5mM, 17.6mM, 17.7mM, 17.8mM, 17.9mM, 18mM, 18.1mM, 18.2mM, 18.3 In some embodiments, the total amount of L-histidine in the pharmaceutical composition is about 10mM. In some embodiments, the total amount of L-histidine in the pharmaceutical composition is in the form of L-histidine. In some embodiments, the total amount of L-histidine in the pharmaceutical composition is in the form of L-histidine monohydrochloride monohydrate. In some embodiments, the total amount of L-histidine in the pharmaceutical composition is in the form of L-histidine monohydrochloride monohydrate. In some embodiments, the total amount of L-histidine in the pharmaceutical composition is in the form of both L-histidine and L-histidine monohydrochloride monohydrate. In some embodiments, the molar ratio of L-histidine to L-histidine monohydrochloride monohydrate is about 1: 1, 1:2, 1:3, 1:4, 1:5, 1:6, 2:1, 2:3, 2:5, 3:1, 3:2, 3:4, 3:5, 4:1, 4:3, 4:5, 5:1, 5:2, 5:3, 5:4, 5:6, 6:1, or 6:5. In some embodiments, the molar ratio of L-histidine to L-histidine monohydrochloride monohydrate is about 3:2. In some embodiments, the pharmaceutical composition comprises sucrose in an amount of about 1% (w / v), 1.5% (w / v), 2% (w / v), 2.5% (w / v), 3% (w / v), 3.5% (w / v), 4% (w / v), 4.5% (w / v), 5% (w / v), 5.5% (w / v), 6% (w / v), 6.5% (w / v), 7% (w / v), 7.5% (w / v), 8% (w / v), 8.5% (w / v), 9% (w / v), 9.5% (w / v), 10% (w / v), 10.5% (w / v), 11% (w / v), 11.5% (w / v), 12% (w / v), 12.5% ​​(w / v), 13% (w / v), 13.5% (w / v), 14% (w / v), 14.5% (w / v), 15% (w / v), 15.5% (w / v), 16% (w / v), 16.5% (w / v), 17% (w / v), 17.5% (w / v), 18% (w / v), 18.5% (w / v), 19% (w / v), 19.5% (w / v), or 20% (w / v), or any concentration therebetween. In some embodiments, the pharmaceutical composition comprises sucrose in an amount of about 8% (w / v). .

[0153] In some embodiments, the pharmaceutical composition comprises polysorbate 20 in an amount of at least 0.001% (w / v), 0.0015% (w / v), 0.002% (w / v), 0.0025% (w / v), 0.003% (w / v), 0.0035% (w / v), 0.004% (w / v), 0.0045% (w / v), 0.005% (w / v), 0.0055% (w / v), 0.006% (w / v), 0.0065% (w / v), 0.007% (w / v), 0.0075% (w / v), 0.008% (w / v), 0.0085% (w / v), 0.009% (w / v), 0.0095% (w / v), 0.01% (w / v), 0.0105% (w / v), 0.011% (w / v), 0.0115% (w / v), 0.012% (w / v), 0.0125% (w / v), 0.013% (w / v), 0.0135% (w / v), 0.014% (w / v), 0.0145% (w / v), 0.015% (w / v), 0.0155% (w / v), 0.016% (w / v), 0.0165% (w / v), 0.017% (w / v), 0.0175% (w / v), 0.018% (w / v), 0.0185% (w / v), 0.019% (w / v), 0.0195% (w / v), 0.02% (w / v), 0.0205% (w / v), 0.021% (w / v), 0.0215% (w / v), 0.022% (w / v), 0.0225% (w / v), 0.023% (w / v), 0.0235% (w / v), 0.024% (w / v), 0.0245% (w / v), 0.025% (w / v), 0.0255% (w / v), 0.026% (w / v), 0.0265% (w / v), 0.027% (w / v), 0.0275% (w / v), 0.028% (w / v), 0.0285% (w / v), 0.029% (w / v), 0.0295% (w / v), 0.03% (w / v), 0.0305% (w / v), 0.031% (w / v), 0.0315% (w / v), 0.032% (w / v), 0.0325% (w / v), 0.033% (w / v), 0.0335% (w / v), 0.034% (w / v), 0.0345% (w / v), 0.035% (w / v), 0.0355% (w / v), 0.036% (w / v), 0.0365% (w / v), 0.037% (w / v), 0.0375% (w / v), 0.038% (w / v), 0.0385% (w / v), 0.039% (w / v), 0.0395% (w / v), 0.04% (w / v), 0.0405% (w / v), 0.041% (w / v), 0.0415% (w / v), 0.042% (w / v), 0.0425% (w / v), 0.043% (w / v), 0 .0435% (w / v), 0.044% (w / v), 0.0445% (w / v), 0.045% (w / v), 0.0455% (w / v), 0.046% (w / v), 0.0465% (w / v), 0.047% (w / v), In some embodiments, the pharmaceutical composition comprises polysorbate 20 in an amount of at least 0.01% (w / v). In some embodiments, the pharmaceutical composition comprises polysorbate 20 in an amount of about 0.01% (w / v). In some embodiments, the pharmaceutical composition comprises about 6 mM L-histidine. In some embodiments, the pharmaceutical composition comprises about 4 mM L-histidine monohydrochloride monohydrate. In some embodiments, the pharmaceutical composition comprises about 8% (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 0.01% (w / v) polysorbate 20. In some embodiments, the pharmaceutical composition comprises about 6 mM L-histidine, about 4 mM L-histidine monohydrochloride monohydrate, about 8% (w / v) sucrose, and about 0.01% (w / v) polysorbate 20. .

[0154] In some embodiments, the amount of the isolated recombinant polypeptide complex comprised in the pharmaceutical composition is about 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1 mg / ml, 1.1 mg / ml, 1.2 mg / ml, 1.3 mg / ml, 1.4 mg / ml, 1.5 mg / ml, 1.6 mg / ml, 1.7 mg / ml, 1.8 mg / ml, 1.9 mg / ml, 2 mg / ml, 2.1 mg / ml, 2.2 mg / ml, 2.3 mg / ml, 2.4 mg / ml, 2.5 mg / ml, 2.6 mg / ml, 2.7 mg / ml, 2.8 mg / ml, 2.9 mg / ml, 3 mg / ml, 3.1 mg / ml, 3.2 mg / ml, 3.3 mg / ml, 3.4 mg / ml, 3.5 mg / ml, 3.6 mg / ml, 3.7 mg / ml, 3.8 mg / ml, 3.9 mg / ml, 4 mg / ml, 4.1 mg / ml, 4.2 mg / ml, 4.3 mg / ml, 4.4 mg / ml, 4.5 mg / ml, 4.6 mg / ml, 4.7 mg / ml, 4.8 mg / ml, 4.9 mg / ml, 5 mg / ml, 5.1 mg / ml, 5.2 mg / ml, 5.3 mg / ml, 5.4 mg / ml, 5.5 mg / ml, 5.6 mg / ml, 5.7 mg / ml, 5.8 mg / ml, 5.9 mg / ml, 6 mg / ml, 6.1 mg / ml, 6.2 mg / ml, 6.3 mg / ml, 6.4 mg / ml, 6.5 mg / ml, 6.6 mg / ml, 6.7 mg / ml, 6.8 mg / ml, 6.9 mg / ml, 7 mg / ml, 7.1 mg / ml, 7.2 mg / ml, 7.3 mg / ml, 7.4 mg / ml, 7.5 mg / ml, 7.6 mg / ml, 7.7 mg / ml, 7.8 mg / ml, 7.9 mg / ml, 8 mg / ml, 8.1 mg / ml, 8.2 mg / ml, 8.3 mg / ml, 8.4 mg / ml, 8.5 mg / ml, 8.6 mg / ml, 8.7 mg / ml, 8.8 mg / ml, 8.9 mg / ml, 9 mg / ml, 9.1 mg / ml, 9.2 mg / ml, 9.3 mg / ml, 9.4 mg / ml, 9.5 mg / ml, 9.6 mg / ml, 9.7 mg / ml, 9.8 mg / ml, 9.9 mg / ml, 10 mg / ml, 10.1 mg / ml, 10.2 mg / ml, 10.3 mg / ml, 10.4 mg / ml, 10.5 mg / ml, 10.6 mg / ml, 10.7 mg / ml, 10.8 mg / ml, 10.9mg / ml, 11mg / ml, 11.1mg / ml, 11.2mg / ml, 11.3mg / ml, 11.4mg / ml, 11.5mg / ml, 11.6mg / ml, 11.7mg / ml, 11.8mg / ml, 11.9mg / ml, 12mg / ml, 12.1mg / ml, 12.2mg / ml, 12.3mg / ml, 12.4mg / ml, 12.5mg / ml, 12.6mg / ml, 12.7mg / ml, 12.8mg / ml, 12.9mg / ml, 13mg / ml, 13.1mg / ml, 13.2m g / ml, 13.3mg / ml, 13.4mg / ml, 13.5mg / ml, 13.6mg / ml, 13.7mg / ml, 13.8mg / ml, 13.9mg / ml, 14mg / ml, 14.1mg / ml, 14.2mg / ml, 14.3mg / ml, 14.4mg / ml, 14.5mg / ml, 14.6mg / ml, 14.7mg / ml, 14.8mg / ml, 14.9mg / ml, 15mg / ml, 15.1mg / ml, 15.2mg / ml, 15.3mg / ml, 15.4mg / ml, 15.5mg / ml, 15.6mg / ml, 15.7mg / ml, 15.8mg / ml, 15.9mg / ml, 16mg / ml, 16.1mg / ml, 16.2mg / ml, 16.3mg / ml, 16.4mg / ml, 16.5mg / ml, 16.6mg / ml, 16.7mg / ml, 16.8mg / ml, 16.9mg / ml, 17mg / ml, 17.1mg / ml, 17.2mg / ml, 17.3mg / ml, 17.4mg / ml, 17.5mg / ml, 17.6mg / ml, 17.7mg / ml, 17.8mg / ml, 17.9mg / ml, 18mg / ml, 18.1mg / ml, 18.2mg / ml, 18.3mg / ml, 18.4mg / ml, 18.5mg / ml, 18.6mg / ml, 18.7mg / ml, 18.8mg / ml, 18.9mg / ml, 19mg / ml, 19.1mg / ml, 19.2mg / ml, 19.3mg / ml, 19.4mg / ml, 19.5mg / ml, 19.6mg / ml, 19.7mg / ml, 19.8mg / ml, 19.9mg / ml or 20mg / ml, or any concentration therebetween. In some embodiments, the pharmaceutical composition comprises about 2mg / ml of the isolated recombinant polypeptide complex. .

[0155] In some embodiments, the pharmaceutical composition comprises a pH of about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or about 9.0 or any value therebetween. In some embodiments, the pharmaceutical composition comprises a pH between about 5 and about 7. In some embodiments, the pharmaceutical composition comprises a pH of about 6.3.

[0156] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0157] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 81% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0158] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 82% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0159] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 83% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0160] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 84% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0161] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0162] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 86% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0163] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 87% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0164] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 88% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0165] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 89% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0166] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0167] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 91% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0168] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 92% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0169] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 93% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0170] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 94% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0171] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0172] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0173] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0174] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0175] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0176] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0177] On the other hand, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0178] On the other hand, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 81% sequence identity with the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0179] On the other hand, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 82% sequence identity with the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0180] On the other hand, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 83% sequence identity with the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0181] On the other hand, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 84% sequence identity with the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0182] On the other hand, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0183] On the other hand, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 86% sequence identity with the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0184] On the other hand, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 87% sequence identity with the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0185] On the other hand, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 88% sequence identity with the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0186] On the other hand, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 89% sequence identity with the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0187] On the other hand, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0188] On the other hand, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 91% sequence identity with the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0189] On the other hand, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 92% sequence identity with the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0190] On the other hand, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 93% sequence identity with the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0191] On the other hand, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 94% sequence identity with the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0192] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0193] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0194] On the other hand, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0195] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0196] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0197] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence of the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0198] In another aspect, disclosed herein is a method for treating cancer, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition disclosed herein.

[0199] In some embodiments, the cancer comprises mCRPC.In some embodiments, the subject is a human.

[0200] Reagent test kit

[0201] In some embodiments, provided herein is a kit comprising a recombinant polypeptide (e.g., any recombinant polypeptide described herein) or a composition (e.g., any composition described herein), a container, and a label or package insert on or associated with the container.

[0202] Treatment

[0203] In some embodiments, there is a method of treating cancer in a subject in need thereof, comprising administering to the subject an isolated recombinant polypeptide complex as described herein. In some embodiments, the cancer has cells that express PSMA. In some cases, the cancer is a solid tumor cancer. In some embodiments, the cancer is lung cancer, breast cancer (e.g., HER2+; ER / PR+; TNBC), cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, or gastric cancer.

[0204] In some embodiments, there is a method of treating prostate cancer in a subject in need thereof, comprising administering to the subject an isolated recombinant polypeptide complex as described herein. In some embodiments, there is a method of treating metastatic castration-resistant prostate cancer (mCRPC) in a subject in need thereof, comprising administering to the subject an isolated recombinant polypeptide complex as described herein.

[0205] In some embodiments, provided herein is a method for treating metastatic castration-resistant prostate cancer (mCRPC) in a subject in need thereof, comprising administering a recombinant polypeptide (e.g., any recombinant polypeptide described herein) or a formulation (e.g., any formulation described herein) to the subject. In some embodiments, the subject has at least one mCRPC symptom. In some embodiments, the subject has received a diagnosis of mCRPC.

[0206] For administration to a subject, the recombinant polypeptide disclosed herein can be provided in a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the component and is nontoxic to the patient to which it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions (e.g., oil / water emulsions), various types of wetting agents, sterile solutions, etc. Such carriers can be prepared by conventional methods and can be administered to a subject in a suitable dose. Preferably, the composition is sterile. These compositions can also contain adjuvants, such as preservatives, emulsifiers, and dispersants. By including various antibacterial and antifungal agents, it can be ensured that the action of microorganisms is prevented.

[0207] The pharmaceutical composition can be in any suitable form (depending on the desired method of administration). It can be provided in unit dosage form, can be contained in a sealed container, and can also be provided as part of a kit. Such a kit can include instructions for use. It can include a plurality of said unit dosage forms.

[0208] The pharmaceutical composition can be adjusted for administration by any appropriate route, including parenteral (e.g., subcutaneous, intramuscular or intravenous) routes. Such compositions can be prepared by any method known in the pharmaceutical field, such as mixing the active ingredient with a carrier (one or more) or excipient (one or more) under aseptic conditions. In some embodiments, the pharmaceutical compositions disclosed herein are administered intravenously to a subject in need.

[0209] The dosage of the substances of the present disclosure may vary within wide limits depending on the disease or disorder being treated, the age and condition of the individual being treated, etc., and the physician will ultimately determine the appropriate dosage to be used.

[0210] In another aspect, disclosed herein is a method for treating cancer, comprising administering to a subject in need thereof an effective amount of an isolated recombinant polypeptide complex disclosed herein. In some embodiments, the method comprises administering to the subject an isolated recombinant polypeptide complex at a first dose and a second dose. In some embodiments, the second dose is equal to or higher than the first dose. In some embodiments, the first dose or the second dose is at least 100 μg. In some embodiments, the first dose or the second dose is about 100 μg.

[0211] In some embodiments, the method comprises administering to the subject an isolated recombinant polypeptide complex in a first dose, a second dose, and a third dose. In some embodiments, the second dose is equal to or higher than the first dose. In some embodiments, the third dose is equal to or higher than the second dose. In some embodiments, the first dose, the second dose, or the third dose is at least 100 μg. In some embodiments, the first dose, the second dose, or the third dose is about 100 μg.

[0212] In some embodiments, the method comprises a first treatment course and a second treatment course. In some embodiments, the first dose is administered to the subject during the first treatment course. In some embodiments, the second dose is administered to the subject during the second treatment course. In some embodiments, the method comprises a 21-day treatment course. In some embodiments, the first dose is administered to the subject within the first week of the treatment course. In some embodiments, the second dose is administered to the subject within the second week of the treatment course. In some embodiments, the third dose is administered to the subject within the third week of the treatment course. In some embodiments, the method comprises a first 21-day treatment course and a second 21-day treatment course. In some embodiments, the first dose of the second 21-day treatment course is equal to or higher than the first dose of the first 21-day treatment course. In some embodiments, the second dose of the second 21-day treatment course is equal to or higher than the second dose of the first 21-day treatment course. In some embodiments, the third dose of the second 21-day treatment course is equal to or higher than the third dose of the first 21-day treatment course. In some embodiments, the method comprises a 21-day treatment cycle, the treatment cycle comprising: (a) administering the isolated recombinant polypeptide complex to the subject at a first dose in the first week of the treatment cycle; (b) administering the isolated recombinant polypeptide complex to the subject at a second dose in the second week of the treatment cycle; and (c) administering the isolated recombinant polypeptide complex to the subject at a third dose in the third week of the treatment cycle. In some embodiments, the method comprises: (a) administering the isolated recombinant polypeptide complex to the subject at a first dose on the first day of the 21-day treatment cycle; (b) administering the isolated recombinant polypeptide complex to the subject at a second dose on the eighth day of the 21-day treatment cycle; and (c) administering the isolated recombinant polypeptide complex to the subject at a third dose on the fifteenth day of the 21-day treatment cycle. In some embodiments, the treatment cycle is repeated 6 times within 18 weeks.

[0213] In some embodiments, the first dose, the second dose, or the third dose is at least 100 μg. In some embodiments, the first dose, the second dose, or the third dose is about 100 μg. In some embodiments, the second dose is equal to or higher than the first dose. In some embodiments, the third dose is equal to or higher than the second dose. In some embodiments, the third dose is equal to or higher than the first dose.

[0214] In some embodiments, the method includes a first 21-day treatment cycle and a second 21-day treatment cycle. In some embodiments, the first dose of the second 21-day treatment cycle is equal to or higher than the first dose of the first 21-day treatment cycle. In some embodiments, the second dose of the second 21-day treatment cycle is equal to or higher than the second dose of the first 21-day treatment cycle. In some embodiments, the third dose of the second 21-day treatment cycle is equal to or higher than the third dose of the first 21-day treatment cycle. In some embodiments, administration includes administration by intravenous infusion. In some embodiments, cancer includes prostate cancer. In some embodiments, cancer includes mCRPC. In some embodiments, the method also includes treating the subject with a therapy for infusion-related reactions before administration. In some embodiments, the therapy for infusion-related reactions includes antipyretics, antihistamines, antiemetics, or corticosteroids. In some embodiments, the therapy for infusion-related reactions includes acetaminophen, paracetamol, or diphenhydramine. In some embodiments, the method also includes treating the subject with a corticosteroid before administering the separated recombinant polypeptide complex to the subject at a first dose in the first week of the 21-day treatment cycle in step (a). In some embodiments, the method further comprises treating the subject with a corticosteroid before administering the separated recombinant polypeptide complex to the subject in a first dose in step (a) on the first day of a 21-day treatment cycle. In some embodiments, the method further comprises treating the subject with a therapy for cytokine release syndrome (CRS) before or after administration. In some embodiments, the therapy for CRS includes an intravenous rehydration program, oxygen therapy, corticosteroids, immunosuppressants, vasopressors, or anti-epileptic drugs. In some embodiments, oxygen therapy includes mechanical ventilation. In some embodiments, immunosuppressants include IL-6 receptor inhibitors. In some embodiments, IL-6 receptor inhibitors include tocilizumab. In some embodiments, the therapy for CRS includes high doses of corticosteroids.

[0215] In some embodiments, after administration, the separated recombinant polypeptide complex is cleaved by a tumor-specific protease to generate an enzyme product of the separated recombinant polypeptide complex. In some embodiments, the tumor-specific protease includes two or more proteases. In some embodiments, the separated recombinant polypeptide complex is cleaved by a first protease of two or more proteases to generate a first metabolite of the separated recombinant polypeptide complex. In some embodiments, the separated recombinant polypeptide complex is cleaved by a second protease of two or more proteases to generate a second metabolite of the separated recombinant polypeptide complex. In some embodiments, the first protease includes a serine protease. In some embodiments, the second protease includes a matrix metalloprotease. In some embodiments, the serine protease includes a recombinant human membrane serine protease (MTSP1). In some embodiments, the matrix metalloprotease includes a recombinant human matrix metalloprotease 9 (MMP9).

[0216] In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 81% sequence identity with the amino acid sequence of SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 82% sequence identity with the amino acid sequence of SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 83% sequence identity with the amino acid sequence of SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 84% sequence identity with the amino acid sequence of SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 86% sequence identity with the amino acid sequence of SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 87% sequence identity with the amino acid sequence of SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 88% sequence identity with the amino acid sequence of SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 89% sequence identity with the amino acid sequence of SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 91% sequence identity with the amino acid sequence of SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 92% sequence identity with the amino acid sequence of SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 93% sequence identity with the amino acid sequence of SEQ ID NO:5. In some embodiments, the isolated first metabolite of the recombinant polypeptide complex comprises an amino acid sequence having at least 94% sequence identity to the amino acid sequence of SEQ ID NO:5.In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO:5. In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence of SEQ ID NO:5.

[0217] In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 81% sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 82% sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 83% sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 84% sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 86% sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 87% sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 88% sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 89% sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 91% sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 92% sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 93% sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 94% sequence identity to the amino acid sequence of SEQ ID NO:6.In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence of SEQ ID NO: 6.

[0218] In some embodiments, the subject is a non-human primate or a human. In some embodiments, the non-human primate is a cynomolgus monkey.

[0219] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0220] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 81% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0221] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 82% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0222] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 83% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0223] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 84% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0224] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0225] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 86% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0226] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 87% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0227] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 88% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0228] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 89% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0229] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0230] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 91% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0231] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 92% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0232] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 93% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0233] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 94% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0234] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0235] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0236] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0237] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0238] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0239] In another aspect, disclosed herein is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 5, and the isolated polypeptide is 484 amino acids in length.

[0240] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0241] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 81% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0242] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 82% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0243] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 83% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0244] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 84% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0245] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0246] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 86% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0247] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 87% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0248] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 88% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0249] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 89% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0250] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0251] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 91% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0252] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 92% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0253] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 93% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0254] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 94% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0255] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0256] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0257] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0258] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0259] In another aspect, disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0260] In another aspect, disclosed herein is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 6, and the isolated polypeptide is 476 amino acids in length.

[0261] Antibody production

[0262] In some embodiments, the polypeptides described herein (e.g., antibodies and binding fragments thereof) are produced using any method known in the art for synthesizing polypeptides (e.g., antibodies), in particular by chemical synthesis or recombinant expression, and preferably by recombinant expression technology.

[0263] In some cases, the antibody or its binding fragment is expressed recombinantly and the nucleic acid encoding the antibody or its binding fragment is assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves synthesizing overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating those oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0264] Alternatively, nucleic acid molecules encoding antibodies are optionally generated from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell that expresses immunoglobulins) by PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of the sequence or by cloning using oligonucleotide probes specific for a particular gene sequence.

[0265] In some cases, the antibody or its binding partner is optionally generated by immunizing an animal (e.g., a mouse) to generate a polyclonal antibody, or more preferably, by generating a monoclonal antibody, for example, as described by Kohler and Milstein (1975, Nature 256:495-497), or as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, clones encoding at least the Fab portion of an antibody are optionally obtained by screening a Fab expression library for Fab fragment clones that bind to a specific antigen (e.g., as described in Huse et al., 1989, Science 246:1275-1281), or by screening an antibody library (see, e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

[0266] In some embodiments, a technique developed for producing "chimeric antibodies" is used by splicing together the genes of mouse antibody molecules of appropriate antigen specificity with the genes of human antibody molecules of appropriate biological activity (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81: 851-855; Neuberger et al., 1984, Nature 312: 604-608; Takeda et al., 1985, Nature 314: 452-454). Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having variable regions derived from mouse monoclonal antibodies and human immunoglobulin constant regions.

[0267] In some embodiments, the technology described for producing single-chain antibodies (U.S. Patent No. 4,694,778; Bird, 1988, Science 242: 423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; and Ward et al., 1989, Nature 334: 544-54) is adjusted to produce single-chain antibodies. Single-chain antibodies are formed by connecting the heavy chain and light chain fragments of the Fv region by amino acid bridges, thereby producing a single-chain polypeptide. The technology for assembling functional Fv fragments in Escherichia coli (E. coli) (Skerra et al., 1988, Science 242: 1038-1041) is also optionally used.

[0268] In some embodiments, an expression vector comprising the nucleotide sequence of an antibody or its fragment or the nucleotide sequence of an antibody or its fragment is transported into a host cell by conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In a specific embodiment, the expression of the antibody is regulated by a constitutive, inducible, or tissue-specific promoter.

[0269] In some embodiments, a variety of host expression vector systems are used to express the antibodies or binding fragments thereof described herein. Such host expression systems represent vehicles for producing and subsequently purifying antibody coding sequences, but also represent cells that express antibodies or binding fragments thereof in situ when transformed or transfected with appropriate nucleotide coding sequences. These include, but are not limited to, microorganisms, such as bacteria (e.g., E. coli and Bacillus subtilis (B. subtilis)) transformed with recombinant bacterial phage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody or binding fragment coding sequences; yeast (e.g., Pichia) transformed with recombinant yeast expression vectors containing antibody or binding fragment coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing antibody or binding fragment coding sequences; infected heavy A plant cell system transformed with a recombinant plasmid expression vector (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or a recombinant plasmid expression vector (e.g., Ti plasmid) containing an antibody or binding fragment encoding sequence; or a mammalian cell system (e.g., COS, CHO, BH, HEK293, 293T, 3T3 cells) containing a recombinant expression construct, the recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0270] In order to produce recombinant protein for a long time and in high yield, stable expression is preferred. In some cases, the cell line of the optionally engineered stably expressed antibody. Host cell is not to use the expression vector containing the viral replication origin, but to transform with the DNA and the selectable marker controlled by the appropriate expression control element (for example, promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.). After introducing foreign DNA, the cell of the engineered approach is allowed to grow 1-2 days in the enrichment medium, and then switch to the selective medium. The selectable marker in the recombinant plasmid gives the resistance to selection, and allows the cell to stably integrate the plasmid into its chromosome and grow to form colonies, and then clone and expand into cell line. This method can be advantageously used for the cell line of the engineered expression antibody or its binding fragment.

[0271] In some cases, a number of selection systems are used, including, but not limited to, blasticidin, bleomycin, herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes in tk cells, hgprt cells, or aprt cells, respectively. In addition, antimetabolite resistance has been used as the basis for selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 12:577-614). 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May 1993, TIB TECH 11(5):155-215) and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).Methods well known in the field of recombinant DNA technology that can be used are described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13 of Dracopoli et al. (eds.), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1).

[0272] In some cases, the expression level of the recombinant polypeptide is increased by vector amplification (for review, see Bebbington and Hentschel, the use of vectors based on gene augmentation for the expression of cloned gene in maternal cells in DNA cloning, Volume 3 (Academic Press, New York, 1987)). When the marker in the vector system expressing the recombinant polypeptide is amplifiable, an increase in the inhibitor level in the host cell culture will increase the copy number of the marker gene. Since the amplified region is related to the nucleotide sequence of the recombinant polypeptide, the production of the recombinant polypeptide will also increase (Crouse et al., 1983, Mol. Cell Biol. 3: 257).

[0273] In some cases, the recombinant polypeptide is purified using any method known in the art, for example, by chromatography (e.g., ion exchange, affinity, particularly for a specific antigen following protein A, and fractionated column chromatography), centrifugation, differential solubility, or by any other standard technique to purify the protein.

[0274] Expression vector

[0275] In some embodiments, the vector includes any suitable vector derived from a eukaryotic or prokaryotic source. In some cases, the vector is obtained from bacteria (e.g., Escherichia coli), insects, yeast (e.g., Pichia pastoris), algae, or mammalian sources. Exemplary bacterial vectors include pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

[0276] Exemplary insect vectors include pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393M11, pVL1393 M12, a FLAG vector (e.g., pPolh-FLAG1 or pPolh-MAT 2), or a MAT vector (e.g., pPolh-MAT1 or pPolh-MAT2).

[0277] In some cases, the yeast vector includes pDEST TM 14 carriers, pDEST TM 15 carriers, pDEST TM 17 carriers, pDEST TM 24 carriers, pYES-DEST52 vector, pBAD-DEST49 Destination vector, pAO815 Pichia pastoris vector, pFLD1 Pichia pastoris vector, pGAPZA, pGAPZB & pGAPZC Pichia pastoris vector, pPIC3.5K Pichia pastoris vector, pPIC6 A, pPIC6B & pPIC6 C Pichia pastoris vector, pPIC9K Pichia pastoris vector, pTEF1 / Zeo, pYES2 yeast vector, pYES2 / CT yeast vector, pYES2 / NT A, pYES2 / NT B & pYES2 / NT C yeast vector or pYES3 / CT yeast vector.

[0278] Exemplary algal vectors include pChlamy-4 vectors or MCS vectors.

[0279] Examples of mammalian vectors include transient expression vectors or stable expression vectors. Mammalian transient expression vectors may include pRK5, p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a, pFLAG-CMV 6b, pFLAG-CMV 6c, pFLAG-CMV 5.1, pFLAG-CMV 5a, pFLAG-CMV5b, pFLAG-CMV 5c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4. The mammalian stable expression vector may include pFLAG-CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV26, pBICEP-CMV 1, or pBICEP-CMV 2.

[0280] In some cases, the cell-free system is a mixture of cytoplasmic and / or nuclear components from cells and is used for in vitro nucleic acid synthesis. In some cases, the cell-free system utilizes prokaryotic cell components or eukaryotic cell components. Sometimes, nucleic acid synthesis is obtained in a cell-free system based on, for example, Drosophila cells, African clawed frog eggs, or HeLa cells. Exemplary cell-free systems include, but are not limited to, E. coli S30 extract system, E. coli T7 S30 system, or

[0281] Host cells

[0282] In some embodiments, host cells include any suitable cells, such as cells of natural origin or genetically modified cells. In some cases, host cells are production host cells. In some cases, host cells are eukaryotic cells. In other cases, host cells are prokaryotic cells. In some cases, eukaryotic cells include fungi (e.g., yeast cells), animal cells or plant cells. In some cases, prokaryotic cells are bacterial cells. Examples of bacterial cells include gram-positive bacteria or gram-negative bacteria. Sometimes gram-negative bacteria are anaerobic, rod-shaped or both.

[0283] In some cases, the gram-positive bacteria include actinomycetes, firmicutes or soft wall bacteria. In some cases, the gram-negative bacteria include Aquificae, Deinococcus-Thermus, Fibrobacteres-Chlorobi / Bacteroidetes (FCB group), Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes-Verrucomicrobia / Chlamydiae (PVC group), Proteobacteria, Spirochaetes or Synergistetes. Other bacteria can be Acidobacteria, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Dictyoglomi, Thermodesulfobacteria or Thermotogae. The bacterial cell can be Escherichia coli, Clostridium botulinum or Escherichia coli.

[0284] Exemplary prokaryotic host cells include, but are not limited to, BL21, Mach1 TM 、DH10B TM 、TOP10、DH5α、DH10Bac TM 、OmniMax TM 、MegaX TM DH12S TM , INV120, TOP10F', INVαF, TOP10 / P3, ccdB Survival, PIR1, PIR2, Stbl2TM 、Stbl3 TM or Stbl4 TM .

[0285] In some cases, animal cells include cells from vertebrates or from invertebrates. In some cases, animal cells include cells from marine invertebrates, fish, insects, amphibians, reptiles, or mammals. In some cases, fungal cells include yeast cells, such as brewer's yeast, baker's yeast, or wine yeast.

[0286] Fungi include ascomycetes, such as yeast, mold, filamentous fungi, basidiomycetes or zygomycetes. In some cases, yeast includes Ascomycota or Basidiomycota. In some cases, Ascomycota includes Saccharomycotina (true yeast, such as Saccharomyces cerevisiae (baker's yeast)) or Taphrinomycotina (such as Schizosaccharomycetes (fission yeast)). In some cases, Basidiomycetes include Agaricomycota (such as Tremellomycetes) or Pucciniomycotina (such as Microbotryomycetes).

[0287] Exemplary yeasts or filamentous fungi include, for example, the genera Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula, Kluyveromyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidi, Aspergillus, Fusarium, or Trichoderma. Exemplary yeasts or filamentous fungi include, e.g., the following species: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, Candida boidini, Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida viswanathii, Candida lusitaniae, Rhodotorula mucilaginosa, Pichia metanolica, Pichia angusta, Pichia pastoris, Pichia anomala, and Candida spp. anomala), Hansenula polymorpha, Kluyveromyces lactis, Zygosaccharomyces rouxii, Yarrowia lipolytica, Trichosporon pullulans, Rhodosporidium toru-Aspergillus niger, Aspergillus nidulans, Aspergillus awamoriawamori, Aspergillus oryzae, Trichoderma reesei, Yarrowialipolytica, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii, Zygosaccharomyces bailii, Cryptococcus neoformans, Cryptococcus gattii, or Saccharomyces boulardii.

[0288] Exemplary yeast host cells include, but are not limited to, Pichia pastoris strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33; and Saccharomyces cerevisiae strains such as INVSc1.

[0289] In some cases, the additional animal cells include cells obtained from molluscs, arthropods, annelids, or sponges. In some cases, the additional animal cells are mammalian cells, such as from primates, apes, horses, cattle, pigs, dogs, cats, or rodents. In some cases, rodents include mice, rats, hamsters, gerbils, hamsters, chinchillas, spotted rats, or guinea pigs.

[0290] Exemplary mammalian host cells include, but are not limited to, 293A cell line, 293FT cell line, 293F cells, 293H cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, FUT8 KO CHOK1, ExpiCHO-S cells, Expi293F cells, TM Cells, Flp-In TM T-REx TM 293 cell line, Flp-In TM -293 cell line, Flp-In TM -3T3 cell line, Flp-In TM -BHK cell line, Flp-In TM -CHO cell line, Flp-In TM -CV-1 cell line, Flp-In TM -Jurkat cell line, FreeStyle TM 293-F cells, FreeStyle TM CHO-S cells, GripTiteTM 293MSR cell line, GS-CHO cell line, HepaRG TM Cells, T-REx TM Jurkat cell line, Per.C6 cell, T-REx TM -293 cell line, T-REx TM -CHO cell lines and T-REx TM -HeLa cell line.

[0291] In some cases, the mammalian host cell is a stable cell line, or a cell line that has integrated the genetic material of interest into its own genome and has the ability to express the genetic material product after multiple generations of cell division. In some cases, the mammalian host cell is a transient cell line, or a cell line that has not integrated the genetic material of interest into its own genome and does not have the ability to express the genetic material product after multiple generations of cell division.

[0292] Exemplary insect host cells include, but are not limited to, Drosophila S2 cells, Sf9 cells, Sf21 cells, HighFive TM Cells and cell.

[0293] In some cases, the plant cell comprises a cell from algae.Exemplary insect cell lines include, but are not limited to, strains from Chlamydomonas reinhardtii 137c or Synechococcuse longatus PPC 7942.

[0294] Products

[0295] In another aspect of the present disclosure, an article of manufacture is provided that contains materials useful for treating, preventing and / or diagnosing the above-mentioned conditions. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be made of a variety of materials (e.g., glass or plastic).

[0296] The label or package insert indicates that the composition is used to treat the selected condition.The article of manufacture in this embodiment of the disclosure may also include a package insert indicating that the composition can be used to treat a particular condition.

[0297] Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may also include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes.

[0298] Example 1: PC-1 Characterization and Toxicity Studies

[0299] Figures 2A-2D Description Mask discovery by peptide phage display. Phage displayed peptide libraries are screened against binding surface-immobilized anti-CD3 scFv. After several cycles of binding, elution and amplification, clonal phage are screened for competitive binding to CD3 by ELISA. Selected clonal phage sequences are synthesized as peptides and screened for binding and inhibitory properties against anti-CD3 scFv. Peptide inhibitors are then incorporated into peptide complex designs.

[0300] Figures 3A-3F The binding of PC-1 to CD3 was shown to be cleavage- and dose-dependent. PC-1 CD3 target engagement was cleavage-dependent, with masking reducing CD3 binding by >600-fold. Treatment of PC-1 with proteases achieved potent CD3 binding comparable to unmasked TCE-1. PC-1 exhibited potent binding to human and monkey PSMA and albumin.

[0301] Figure 4A-4B This indicates that TCE-1 potency depends on structure and orientation. TCE-1 activity depends on the connection geometry of the PSMA and CD3 binding domains.

[0302] Figures 5A-5F The activity of PC-1 is cleavage-, dose-, and PSMA expression-dependent. Masking of the PC-1 CD3 binding domain reduces its ability to induce cytokine release. Functional activity in prostate cancer and T cell co-culture assays is dependent on masking and PSMA expression. PC-1 exhibits a robust shift in T cell function, which shows the potential for an enhanced safety profile.

[0303] Table 2 illustrates that the cleavable linker is characterized by rapid proteolysis and high serum stability. The linker is cleaved faster than the linker contained in pamilizumab (CL-1) and maintains stability in human serum. Serum proteolytic activity is greater than blood-represents a conservative assessment of in vivo stability. The cleavable linker is rapidly cleaved by a panel of recombinant tumor proteases, which results in enhanced unmasking and antitumor activity in the TME. PC-1 exhibits high stability in healthy and mCRPC human donor serum, with cleavage of ≤1% per day. While proteolytic cleavage of PC-1 in the TME is expected to drive antitumor activity, a key safety feature of PC-1 is its stability in the blood compartment, where maintenance of masking is expected to mitigate safety risks associated with potential healthy tissue toxicity and cytokine release syndrome.

[0304] Table 2: Apparent cleavage rates of PC-1 containing different cleavable linkers

[0305]

[0306] Rate: R greater than 10 5 M -1 S -1 ; Y between 10 4 M -1 S -1 and 10 5 M -1 S -1 Between; G between 2.5x 10 3 M -1 S -1 and 10 4 M -1 S -1 B is equal to or less than 2.5x 10 3 M -1 S -1

[0307] Figures 6A-6C and Table 3 illustrate that PC-1 has a prolonged half-life and enhanced safety profile in NHPs.

[0308] Table 3: Pharmacokinetics of PC-1

[0309]

[0310] PC-1 toxicity studies support enhanced PK, safety, and drug design. PC-1 administered as 1.5 mg / kg IV bolus QW x5 in cynomolgus monkeys achieved high exposure and long half-life with no significant changes in clinical signs or clinical pathology measurements. PC-1 is highly stable in vivo with minimal detectable cleavage. Cleaved PC-1 is close to the LLoQ, while intact (masked) PC-1 achieves 1,500x exposure multiples above its minimal detectable metabolite. Total (masked + unmasked) and intact PC-1 exhibit overlapping PK profiles, further supporting the stability of PC-1 in vivo. Rapid clearance of TCE minimizes accumulation of cleaved PC-1, thereby minimizing the risk of toxicity and cytokine release in target healthy tissues.

[0311] Summary of PC-1PSMA-GLP Toxicity Study in Non-Human PrimatesDescription Weekly dosing at 0.1, 0.3, 1.5 mg / kg with 4-week recovery.No microscopic histopathological findings were observed.Lack of TCE accumulation in vivo mitigated toxicity in target healthy tissues and minimized cytokine release.The clinical chemistry, hematology, and pathology data packages support a no observed adverse effect level (NOAEL) ≥ 1.5 mg / kg / dose.

[0312] PC-1 exhibits enhanced safety and PK properties compared to TCE-1. The key safety feature of PC-1 is a tumor protease-cleavable inhibitory peptide mask that reduces PC-1 binding to human CD3 by >600x, limiting T cell activation to the TME. In vitro, PC-1 exhibited up to 500x reduction in potency to activate T cells and induce T cell-mediated tumor cell killing compared to unmasked TCE-1. PC-1 demonstrated an enhanced safety profile in NHPs, characterized by reductions in cytokine CRS-associated proinflammatory cytokines and a NOAEL ≥ 1.5 mg / kg / dose IV bolus QW x5. The albumin binding domain extends the circulating half-life of PC-1 in NHPs to ~120h, compared to <2h for unmasked TCE, supporting the projected once-weekly clinical dosing of PC-1.

[0313] The cleavage-dependent activity, half-life-extended PK, superior safety potential, and manufacturability properties of PC-1 support its further development as an attractive mCRPC therapeutic.

[0314] Example 2: Production and purification process

[0315] The manufacturing process is divided into upstream (cell culture) process, harvesting and downstream (purification) process. The upstream process consists of upstream fed-batch expression in Chinese hamster ovary (CHO)-K1 cell culture at 200L cell culture scale until the harvesting step. The downstream process uses multiple purification steps to produce batch drug substance. The drug substance is then final formulated and excipients are added, followed by batch filtration and filling into polycarbonate bottles.

[0316] summary

[0317] The production and purification of PC-1 followed Figure 7-8 The steps are shown. The upstream process starts with thawing a single vial of the master cell bank, followed by cell culture expansion and fed-batch production of PC-1 in harvested cell culture fluid (HCCF). Basal medium 1 consisting of CD CHO medium containing glutamine, hypoxanthine, and thymidine was inoculated with one vial of the master cell bank and expanded in shake flasks to approximately (1.30-4.00)×10 6 The culture was then transferred to a 20-liter (L) disposable bioreactor filled with basal medium 2 consisting of ActiPro medium containing glutamine, hypoxanthine, and thymidine and then cultured in a 50-L disposable bioreactor filled with the same medium. When the viable cell density reached approximately (3.60-6.00)×10 6When the viable cell density (VCD) reached 10 cells / mL and the viability was above 90%, the cell culture was transferred to a 250 L single-use bioreactor for fed-batch production at a 200 L cell culture process scale. The cell culture was carried out for about 10 days, at which time it was harvested by depth filtration.

[0318] The downstream process begins with the capture of PC-1 from harvested cell culture fluid (HCCF) by protein A affinity chromatography, followed by viral inactivation, neutralization, and depth filtration steps at low pH. Host cell impurities are removed by a moderate polishing step using an anion exchange chromatography resin. PC-1 is recovered in the flow-through and further refined by step elution in conjunction with a multimodal chromatography resin. A viral nanofiltration step is performed to remove any potential adventitious agents. The product is then ultrafiltered and diafiltered using histidine buffer pH 6.3, followed by the addition of sucrose and polysorbate 20 to improve batch stability. The formulated product is filtered and aseptically dispensed into single-use sterile polycarbonate bottles, and the bulk drug substance is stored at -70°C ± 10°C.

[0319] Upstream cell culture process

[0320] See also Figure 7 , the upstream manufacturing process begins with thawing a vial from the master cell bank. Using chemically defined growth medium, the cells are gradually expanded to reach the appropriate cell density and then inoculated into 20L disposable bioreactor units, and subsequently inoculated into 50L and finally 250L production bioreactors. During cell expansion and culture, cell density, dissolved oxygen, pH and environmental conditions are controlled. When the cell culture (about 10 days) is completed, the cells are harvested. The following is provided Figure 7 More details on each step in .

[0321] Thawing of vials and cell culture in shake flasks

[0322] The manufacturing process begins with thawing a vial from the master cell bank in a 37.0°C ± 1°C water bath and aseptically transferring the cells to a shake flask followed by resuspension in fresh basal medium 1 consisting of CD CHO medium containing glutamine, hypoxanthine, and thymidine. The cell suspension is maintained at 36.5°C with 6.0% carbon dioxide (CO 2 ) and incubate at a shaking speed of 120 revolutions per minute (rpm) for about 2 days, at which time the VCD is approximately (1.10 -3.00)×10 6 cells / mL and viability above 85%. Under controlled environmental conditions, the cell culture was further subcultured into shake flasks of increasing size by adding fresh basal medium 1 at a defined VCD and viability. A backup inoculum expansion was generated at the N-3 stage and maintained for up to three passages.

[0323] Cell expansion in a 20L bioreactor

[0324] After two rounds of expansion in shake flasks, and when the VCD of the cell culture reached approximately (1.30–4.00)×10 6 When the viability was higher than 90.0%, basal medium 2 consisting of ActiPro medium containing glutamine, hypoxanthine and thymidine was used at a rate of approximately 0.35 × 10 6 The cell culture was transferred to a 20 L disposable bioreactor at a cell density of 10 cells / mL. The culture was maintained at 36.5 °C with 6.0% CO. 2 and a shaking speed of about 20 rpm until the VCD is approximately (3.50-5.50)×10 6 cells / mL and the viability was higher than 90%. Cell Expansion in a 50L Bioreactor

[0325] Cells from the 20 L bioreactor were cultured at approximately 0.40 × 10 6 The target VCD of 10 cells / mL and viability above 90% were inoculated into a 50 L single-use bioreactor filled with basal medium 2. The temperature and stirring speed were set at 36.5 °C and 140 rpm, respectively. 2 The pH was maintained at 6.95 by sparging, and the dissolved oxygen (DO) was controlled at 40% by air / oxygen sparging. The cell culture was expanded to a VCD of approximately (3.60-6.00)×10 6 Once this criterion is reached, the contents will be transferred to a final 250 L production bioreactor filled with basal medium 2.

[0326] Cell culture in a 250L bioreactor

[0327] Cell culture was performed in a 250 L single-use bioreactor with a target production volume of 200 L. 6 After inoculation with a target VCD of 10 cells / mL, approximately 1.5 kg of culture was transferred from the 250L bioreactor to the 3L bioreactor satellite. The temperature was set at 36.5°C during inoculation and the VCD reached approximately (10.00-14.00)×10 6 At 10 cells / mL or day 5, the temperature was shifted to 33.0°C. pH and dissolved oxygen were monitored and controlled at approximately 6.90% and 40.0%, respectively, with stirring at 100 rpm and air / oxygen and CO 2The 250L bioreactor was operated by bubbling. Cell culture was carried out by regular addition of feed. Glucose concentration was checked every day and maintained at about 6 g / L by adding 400 g / kg glucose stock solution. The culture was harvested on the 10th day or when the viability dropped below 85%, whichever came first.

[0328] Cell harvesting and clarification

[0329] When meeting the harvest criteria, harvest cell culture and clarify by depth filtration, use filter combination to remove cells and cell debris. Depth filtration system is composed of 2 levels of double-layer regenerated cellulose filters, and nominal retention levels are respectively 6 to 30 microns (μm) and 0.10 to 0.85 μm. Cell culture is pumped through the filter with about 50 liters / square meter hour (LMH), and the initial effluent is discarded. Filtration continues until all cell cultures are filtered, and with 50 millimoles (mM) Tris-acetic acid (Tris-HAc), 150mM sodium chloride (NaCl), pH 7.4 washing buffer rinse filter. Filtrate is collected in a single use storage bag, filtered into a disposable mixing bag by 0.5 / 0.2 μm subsequently. Final filtrate is mixed, and then next step is carried out.

[0330] Downstream purification process

[0331] See also Figure 8 , the downstream process begins with capturing PC-1 from clarified cell culture fluid by affinity chromatography and elution at approximately pH 4.2. A viral inactivation step is then performed using a low pH process step, followed by a neutralization step and removal of any precipitate by medium depth filtration. Additional impurities are further removed by a medium polishing step using an anion exchange chromatography resin. PC-1 is recovered in the chromatography flowthrough and further refined by a bind-elute multimodal chromatography resin using a step elution scheme. A viral nanofiltration step is performed prior to the ultrafiltration and diafiltration (UF / DF) steps into histidine buffer, pH 6.3. Next, sucrose and polysorbate 20 are added at a final target concentration of 8% weight / volume (w / v) and 0.01% (w / v), respectively, to improve batch stability. The formulated batch is filtered and dispensed into sterile polycarbonate bottles under aseptic conditions. The drug substance is stored at -70°C ± 10°C. Figure 8 More details of the steps shown in are provided below.

[0332] Affinity chromatography

[0333] The principle of this chromatography step is to use MabSelect PrismA Protein A resin for affinity binding to capture the target protein PC-1 while allowing impurities to be removed by flow through the packed column. PC-1 is then eluted from the resin using a low pH buffer. Protein A chromatography resins are specifically designed for the manufacture of PC-1 drug substances.

[0334] The column is rinsed, sterilized with 0.5 equivalent (N) sodium hydroxide (NaOH), and balanced with 50mM Tris-HAc, 150mM NaCl, pH7.4 buffer. During the loading of the column with a maximum flow rate of about 20 to 50g protein per liter of resin and 300 cm / h and a minimum residence time of 5 minutes, the clarified cell culture fluid is subjected to 0.5 / 0.2 μm filtration. Then, the column is cleaned with high / low salt and pH buffer to remove impurities. The bound PC-1 is eluted with 30mM sodium acetate-acetic acid (NaAc-HAc), pH 4.2 at the same flow rate. The eluent peak collection begins at about 50mAU / mm and ends at 50 milli-absorbance units per millimeter (mAU / mm) after the peak. If multiple cycles are performed, the eluent is merged and mixed before the virus inactivation step is performed.

[0335] Low pH Virus Inactivation and Neutralization

[0336] The pH of the protein A eluate was adjusted to pH 3.6 ± 0.1 with 1 molar (M) acetic acid (HAc) and maintained at 18-26°C while stirring to achieve robust viral inactivation. After 1-2 hours under these conditions, the solution was neutralized to pH 5.5 ± 0.2 with 1 M Tris-base and maintained at ambient temperature for 30-240 minutes before a medium depth filtration step.

[0337] Medium depth filtration

[0338] The mid-depth filtration step removes precipitates that can form during the low pH virus inactivation and neutralization process steps. Before loading the neutralized product pool at approximately 100 LMH, a regenerated cellulose depth filter with a nominal 0.10 to 0.85 μm retention rating is equilibrated with 30 mM NaAc-HAc buffer while maintaining the filter pressure drop (dP) below 2 bar. After loading, the filter is rinsed with equilibration buffer, and the combined filtrate is further filtered through 0.5 / 0.2 μm polyethersulfone (PES) into a sterile mixing and storage bag before proceeding to the next step.

[0339] Anion exchange chromatography

[0340] Anion exchange (Capto Q) chromatography step is carried out in flow-through mode. The filtrate from the mid-depth filtration step is adjusted to pH 6.8-7.2 and conductivity is 3.0-8.0 millisiemens per centimeter (mS / cm), then filtered into a storage bag by 0.5 / 0.2 μm PES. Anion exchange column is sterilized with 1M NaOH, and balanced with 50mM Tris-HAc, 1M NaCl, pH 7.0, then balanced with 50mM Tris-HAc, pH 7.0. Loading is carried out with the maximum linear flow rate of 300cm / h and the minimum residence time of 5 minutes, and the loading capacity remains between 100 and 200g / L CaptoQ resin. After loading is completed, use 50mMTris-Hac, pH7.0 washing column. The eluate was subjected to 0.5 / 0.2 μm PES filtration during collection, with filtration starting at approximately 50 mAU / mm during loading and ending at 50 mAU / mm post-peak.

[0341] Multimodal Cation Exchange Chromatography

[0342] Multimodal cation exchange (Capto MMC ImpRes) chromatography in bind-elute mode was used as a polishing step to further remove impurities. Prior to loading, the column was rinsed, sanitized with 1 M NaOH and equilibrated with 50 mM Tris-HAc, pH 7.0. The anion exchange eluent was loaded onto the column at a maximum linear flow rate of 300 cm / h and a minimum residence time of 5 minutes. The column was washed with equilibration / wash buffer until UV 280 The absorbance was less than 25 mAU / mm. Washing was followed by an additional wash with 50 mM Tris-HAc, 60 mM NaCl, pH 7.0, and PC-1 was eluted from the column with 50 mM Tris-HAc, 205 mM NaCl, pH 7.0 elution buffer at the same flow rate. Eluate collection began at approximately ≥500 mAU / mm and ended at ≥125 mAU / mm after the peak. Eluates were filtered through 0.5 / 0.2 μm PES during collection and, if more than one cycle was performed, the eluates were combined and mixed before the virus filtration step.

[0343] Virus Filtration

[0344] The virus filtration step removes potential viral particles and consists of a 0.5 / 0.2μm pre-filter, a 20 nanometer (nm) virus retention filter, and a 0.5 / 0.2μm filter in series. Before loading the multimodal cation exchange eluent, the pre-filter and virus filter are balanced with 50mM Tris-HAc, 205mM NaCl pH 7.0 buffer. The maximum loading capacity of the filter is 254 liters per square meter (L / m2). During the loading process, the pressure difference of the pre-filter is maintained at ≤2 bar, the pressure difference of the virus retention nanofilter is maintained between 0.7-1.0 bar, and the pressure difference of the final 0.5 / 0.2μm filter is maintained at ≤2 bar. After loading is completed, the filter is rinsed with wash buffer. The final combined filtrate is mixed and then proceed to the next step.

[0345] Ultrafiltration and diafiltration

[0346] Ultrafiltration and diafiltration are used to adjust in-process drug substance protein concentration and exchange buffers prior to final batch formulation.

[0347] The ultrafiltration / diafiltration unit with PES 30 kilodalton (kDa) molecular weight cut-off filter box is balanced with 50mM Tris-HAc, 205mM NaCl, pH 7.0 buffer, until reaching definite pH value and conductivity. The filtrate of the previous step is pumped along the membrane surface with ≤500 grams / square meter (g / m2), and concentrated to about 15 grams / liter (g / L). Then with 10mM histidine hydrochloride (His-HCl), pH 6.3, the concentrated solution is filtered by diafiltration of at least 6 times of volume, until pH and conductivity criteria are met. In the whole ultrafiltration and diafiltration process, flux and transmembrane pressure remain at about 300LMH and below 1.25 bar respectively. Reclaim the DF pool after circulation at low flow rate, then rinse with 10mM His-HCl, pH 6.3. The product is filtered with 0.5 / 0.2 μm, and the filtrate remains less than 3 days under ambient conditions, until the next step.

[0348] Excipient addition, bulk formulation and filling

[0349] During batch formulation, sucrose and polysorbate 20 were added to the in-process material from the stock solution to a final concentration of 8% (w / v) and 0.01% (w / v), respectively, to improve product stability. Then diluted to the target concentration range with 10mM His-HCl, pH 6.3. The formulated product was filtered through a 0.22μm polyvinylidene fluoride (PVDF) membrane filter, and approximately 4kg of the filtered product was aseptically dispensed into a single, single-use 5L sterile polycarbonate bottle. Batch storage was at -70°C ± 10°C. All operations (including excipient addition, batch filtration, and filling) were performed in a laminar flow hood.

[0350] Example 3: Confirmation of disulfide bonds by LC-MS / MS

[0351] Disulfide bonds are important in protein folding, and they play a major role in both protein structure and function. The number of disulfide bonds and their location are important properties for ensuring the safety and efficacy of biopharmaceuticals.

[0352] There are 18 cysteine ​​residues in PC-1, which are cross-linked by one interchain disulfide bond and eight intrachain disulfide bonds. Non-reducing Lys-C / trypsin sequential digestion is expected to produce eight disulfide-related peptides (DS1 to DS3 and DS5 to DS9). Non-reducing Lys-C / trypsin / thermolysin sequential digestion is expected to produce one disulfide-related peptide (DS4). Liquid chromatography and tandem mass spectrometry (LC-MS / MS) results are listed in Table 4.

[0353] Table 4: LC-MS / MS results of DS1-DS9

[0354]

[0355]

[0356] PTM = post-translational modification

[0357] Example 4: Secondary structure determination by far-UV CD

[0358] Protein far-UV circular dichroism (far-UV CD) spectra can reveal characteristic secondary structures, i.e., α-helix, β-sheet, random coil, etc. Before measurement, the protein sample was buffer exchanged with 10 mM potassium phosphate buffer (pH 7.0) and diluted to 0.1 mg / mL. Data collection and analysis were performed using a JASCO / J-815CD spectrometer and Spectra Manager software.

[0359] Fig. 9 The CD spectrum of PC-1 in the far UV region (190-260 nm) is shown. β-sheet and random coil are the main secondary structure components.

[0360] Example 5: Tertiary structure determination by near UV-CD

[0361] Protein near-UV CD spectroscopy provides information about the tertiary structure of proteins. The CD spectral patterns in the 250-350nm region are determined by the absorption, dipole orientation and properties of the surrounding environment of phenylalanine (250-270nm), tyrosine (270-290nm) and tryptophan (290-305nm), respectively. For measurement, protein samples were diluted to 1.0mg / mL with 10mM His, 8(w / v)% sucrose, 0.01(w / v)% polysorbate-20, pH 6.3. Data collection and analysis were performed by JASCO / J-815CD spectrometer and Spectra Manager software, respectively.

[0362] Fig.10 The CD spectrum of PC-1 in the near UV region (250-350 nm) is shown. Spectral similarity / structural identity analysis was performed by calculating the correlation coefficient of two spectra using the "Quick Compare" tool of OPUS spectroscopy software.

[0363] Example 6: Thermal stability analysis by DSC

[0364] Differential Scanning Calorimetry (DSC) is an experimental technique to characterize the thermal transitions of materials. It is widely used in protein characterization to study the folding / unfolding transitions of proteins under programmed temperature. m ) starts (T 起始 ) and midpoint (T m ) temperature is often used as an indicator of thermal stability, and the DSC thermogram reveals the thermal transition characteristics.

[0365] PC-1 was analyzed using Malvern's MicroCal DSC. Prior to analysis, the protein sample was diluted to 1 mg / mL with 10 mM His, 8 (w / v)% sucrose, 0.01 (w / v)% polysorbate-20, pH 6.3. 400 microliters (μL) of the corresponding formulation buffer was added to a 96-well plate as a reference, and 400 μL of the protein sample was added. The sample was heated from 10°C to 95°C at a heating rate of 90°C / h in a capillary DSC system. The DSC data was analyzed and fitted using MicroCal PEAQ-DSC software 1.51. The results are shown in Figure 2. Fig.11 As shown. For PC-1, T 起始 The midpoint temperatures (T m1 and T m2 ) were determined to be 71.4°C and 79.5°C.

[0366] Example 7: Molar mass and size analysis by SEC-MALS

[0367] Size exclusion chromatography coupled with a multi-angle light scattering (SEC-MALS) detector separates proteins based on their size, and the molecular weight of the separated components is then measured by the MALS detector. Smaller proteins elute later from the SEC column, while larger proteins elute at earlier retention times and result in the separation of proteins based on their size differences. The separated components, including monomers, high molecular weight species (HMWS), and low molecular weight species (LMWS), are quantified by a UV detector. The absolute molar mass and size of the molecules in solution are calculated using the intensity and angular dependence of the scattered light signal from the MALS detector. The static multi-angle light scattering method characterizes the absolute molecular weight of proteins based on the principle of static light scattering, expressed in terms of Zimm's equation. For proteins larger than 10 nm, the laser scattering intensity is directly proportional to the molecular weight and protein concentration. Therefore, the intensity of the scattered light can be determined based on the relationship between the angle P (θ), the protein size R g The molecular weights of those proteins were calculated from the relationship between the protein concentration c.

[0368] The SEC-MALS chromatogram of PC-1 is shown in Figures 12A-12B The molecular weight of the main peak (peak 1) was 90.2 kDa (RT=3.111 min), which corresponded to the monomer.

[0369] Example 8: Clinical effects of polypeptide complex 1 (PC-1)

[0370] This example illustrates the clinical effects of the polypeptide complex 1 (PC-1) disclosed herein.

[0371] PC-1 is a tumor-activated T cell engager (TRACTr) molecule designed to improve the therapeutic profile of prostate-specific membrane antigen (PSMA)-targeted T cell engagers (TCEs) in patients with metastatic castration-resistant prostate cancer (mCRPC).

[0372] Pharmacology

[0373] Nonclinical in vitro pharmacology studies of PC-1 examined the following:

[0374] 1. Binding of PC-1 to PSMA, cluster of differentiation 3 (CD3), and albumin antigens in mouse, rat, cynomolgus monkey, and human

[0375] 2. Stability of PC-1 in sera of healthy human donors, mCRPC patients, and cynomolgus monkeys

[0376] 3. PC-1 ability to induce cytokine production

[0377] 4. The ability of PC-1 to induce T cell-mediated anti-tumor cytotoxic activity was concluded from the results of binding studies that PC-1 binds to human and cynomolgus PSMA, CD3, and albumin with low nanomolar affinity, while exhibiting minimal binding to mouse or rat antigens, and CD3 binding affinity is cleavage-dependent. Stability studies showed that PC-1 exhibited similar cleavage rates in healthy human serum (1%) relative to serum of mCRPC patients (2%), and PC-1 cleavage in cynomolgus serum was 6% per day. Cytokine induction studies showed that while cleaved and unmasked versions of PC-1 exhibited a dose-dependent ability to induce immune cells to release interferon gamma (IFNγ), tumor necrosis factor (TNF), and interleukin (IL)-6, masking of the PC-1 CD3 binding domain reduced its ability to induce cytokine production. This suggests that cytokine release requires proteolytic PC-1 unmasking. Cytokine production was not detected in the absence of target cells, indicating that the activity of PC-1 depends on the presence of target cells expressing PSMA. The presence of masking material also reduces the ability of PC-1 to induce T cell-mediated tumor cell killing. In addition, the ability of PC-1 to induce T cell-mediated tumor cell killing is dose- and PSMA-dependent. Non-clinical pharmacology studies have examined the ability of PC-1 and PC-1-serine protease (SP) cleavage products to induce cytokine release in human whole blood samples. The study examined the cytokine release of soluble and wet-coated (plate-bound) test articles under 2 conditions. After stimulation with soluble or wet-coated (plate-bound) PC-1-SP cleavage products in any sample, IL-2, IL-6, IL-10, TNF and IFNγ release above untreated control levels (very low to no signal) were not observed. After PC-1 stimulation, IL-2 or IFNγ cytokine release was not observed in any sample. No IL-6, IL-10, or TNF cytokine release was observed following PC-1 stimulation in the plate-bound format; however, IL-6, IL-10, and TNF cytokine release was observed in a few samples following treatment with the highest tested concentrations of soluble PC-1 (10 nM [917.4 ng / mL] and 100 nM [9174 ng / mL]). Nonclinical safety pharmacology parameters included cardiovascular, CNS, and respiratory safety endpoints that were included in a 4-week repeated dose intravenous (IV) toxicity study in cynomolgus monkeys. At doses of PC-1 ≤ 1.5 mg / kg / week, no functional effects on the CNS or respiratory systems were found based on clinical observations and detailed weekly examinations of the animals. In addition, PC-1 administration had no clear effects on the cardiovascular system of the animals on qualitative or quantitative electrocardiographic (ECG) parameters. All ECGs were within normal limits, except for a few arrhythmic episodes that were not related to the test article.

[0378] Pharmacokinetics

[0379] The pharmacokinetic (PK) properties of PC-1 were evaluated after single-dose and multiple-dose studies in cynomolgus monkeys. The PK of PC-1 at 0.1 mg / kg, 0.3 mg / kg and 1.5 mg / kg was evaluated after a single IV administration to male cynomolgus monkeys. Four analytes (intact PC-1 [uncleaved PC-1], Met 1 [also known as PC-1-SP cleavage], Met 2 [also known as PC-1-MMP cleavage] and total PC-1 [intact (uncleaved) PC-1 + Met 1 + Met 2]) were measured; however, since Met 2 was below the quantitative level (BLQ), the PK parameters were derived from three analytes (intact PC-1, total PC-1 and Met 1). In addition, the clearance of the analytes may be affected by anti-drug antibodies (ADA), as further discussed in the toxicity studies below. In terms of PK parameters, the clearance of intact and total PC-1 remained relatively stable in the 3 dose groups. The faster clearance observed at the highest dose (1.5 mg / kg) may be caused by the presence of ADA. The half-life (T1 / 2) of intact PC-1 was 111, 78.5, and 70.4 hours at the three doses, while the T1 / 2 of total PC-1 was 110, 105, and 62.1 hours, respectively. Systemic exposure measured by the area under the curve (AUC0-inf) and maximum concentration (Cmax) of intact PC-1 and total PC-1 increased in a dose-proportional manner across all dose ranges. The AUC0-last of Met 1 increased less than dose-proportional with increasing dose levels from 0.1 mg / kg to 1.5 mg / kg, while Cmax increased in a dose-proportional manner across all dose ranges. The overall systemic exposure of Met 1 was minimal and may be affected by the cleavage of intact PC-1 during sample processing in the PK assay. Using a validated PK assay, the conversion of intact PC-1 to Met 1 induced by sample processing averaged 1.87%. However, native cleavage in vivo is expected to be much lower. In a multiple-dose toxicity study, PC-1 was administered IV to male and female monkeys at 0.1, 0.3, or 1.5 mg / kg / week for a total of 5 administrations per animal. Little to no difference in systemic exposure was observed between intact PC-1 and total PC-1.

[0380] In general, accumulation was not observed after multiple doses, although this could not be assessed in animals with confirmed ADA. In ADA-negative animals, median intact PC-1 and total PC-1 T1 / 2 ranged from 67.7 to 109.1 hours and 60.3 to 156.4 hours on days 1 and 22, respectively. No significant sex differences were observed in systemic exposure measured by Cmax and AUC0-168h at any dose in ADA-negative animals on days 1 and 22. Total exposure to Met 1 was minimal and was affected by an average of 1.87% cleavage of intact PC-1 during the sample preparation steps described above. Native cleavage in vivo is expected to be much lower. Tissue distribution studies were not performed on PC-1. PC-1 metabolism was not specifically studied because classical drug metabolic elimination does not represent an important clearance mechanism for monoclonal antibodies. Therapeutic antibodies, such as PC-1, are typically broken down into small peptides, carbohydrates, and amino acids that return to nutrient pools or are excreted via the kidneys without any biological effect.

[0381] toxicology

[0382] The nonclinical toxicity profile of PC-1 was evaluated in the following studies: in a Good Laboratory Practice (GLP) 4-week repeated dose IV toxicity study in cynomolgus monkeys, in in vitro and in vivo human and / or monkey cytokine release assays, and in serum stability assays using sera from cynomolgus monkeys, healthy human donors, and patients diagnosed with mCRPC. In addition, PC-1 immunogenicity was evaluated in all animals during the repeated dose toxicity study in cynomolgus monkeys. In the repeated dose toxicity study, PC-1 was administered to male and female monkeys by IV slow push at doses of 0.1, 0.3, and 1.5 mg / kg / week (control group received vehicle control), for a total of 5 administrations per animal. No non-adverse changes in hematology, clinical chemistry, and microscopic findings were observed at ≥0.1 mg / kg / week. After the first dose of ≥0.1 mg / kg / week, PC-1 induced a transient, dose-dependent cytokine increase within 24 hours. Most, if not all, of the changes can be attributed to a pro-inflammatory response consistent with the mechanism of action of PC-1 (i.e., activation of immune cell populations) and / or cytokine release observed following administration of the first dose of PC-1. No adverse effects were observed based on the dosing and recovery period of the study, with the highest no serious toxicity dose (HNSTD) / no observed adverse effect level (NOAEL) being 1.5 mg / kg / week. Immunogenicity assessments showed that 1 of 6 animals receiving PC-1 0.1 mg / kg / week, 5 of 6 animals receiving 0.3 mg / kg / week, and all 10 animals receiving 1.5 mg / kg / week were ADA positive at the last dose. No ADA-positive animals were found in Group 1 (vehicle control). The toxicokinetic (TK) profiles of all animals with confirmed antibodies were affected by ADA; however, in some animals, ADA had less effect on exposure after multiple doses. Furthermore, PC-1 induced a dose- or concentration-dependent release of cytokines (i.e., IL-6, TNF, IFNγ) in vitro in the presence of tumor cells and in vivo in healthy monkeys. In vivo, cytokine release was observed primarily after the first dose and correlated with clinical signs and clinical chemistry changes that were suggestive of cytokine release syndrome (CRS) and potential vascular leakage. PC-1 was shown to be stable in human serum with minimal in vitro cleavage. An increase in the in vitro cleavage rate was observed daily in cynomolgus monkey serum, but the cleavage rate remained low overall in this species.

[0383] Phase 1 Clinical Study Design

[0384] This study is a first-in-human (FIH), Phase 1, open-label, multicenter study designed to evaluate the safety, tolerability, PK, pharmacodynamics (PD), and preliminary efficacy of PC-1 administered as a single agent in adult subjects with mCRPC. The study will be conducted in 3 parts: dose escalation (Part 1), approximately 20 to 30 subjects; cohort backfill expansion (Part 2), approximately 30 subjects enrolled at three dose levels; and dose expansion (Part 3), approximately 30 subjects enrolled at the recommended Phase 2 dose. Dose escalation (Part 1) will evaluate the safety, tolerability, PK, PD, and preliminary efficacy of PC-1 administered by IV infusion. Cohort backfill expansion (Part 2) will allow further characterization of the safety and activity of dose levels. Dose expansion (Part 3) will determine additional safety, tolerability, PK, PD, and preliminary clinical activity data for PC-1 at doses and schedules determined by the Safety Review Committee after reviewing all available safety, PK, PD, and preliminary efficacy data. JANX007 in transfer Rationale for evaluation in men with castration-resistant prostate cancer

[0385] While TCEs can be a powerful class of PSMA-targeted therapeutics, attempts to recruit the immune system to recognize PC cells by using TCEs to prime them against PSMA have so far been unsuccessful. Systemic activation and redirection of T cells can lead to cytotoxicity and cytokine release outside the target tumor, resulting in severe CRS, significantly limiting dosing and ultimately limiting efficacy (Tran et al., 2020; Bono et al., 2021). In addition, first-generation TCEs have poor PK, resulting in short half-lives and cumbersome dosing regimens. Therefore, there is a significant unmet need to fully exploit T cell-mediated cytotoxicity in targeting tumor cells. Products that can selectively accumulate and activate within the tumor microenvironment (TME) appear to have significant advantages in developing a favorable risk-benefit profile. Designed based on PC-1 to provide a more targeted approach to activate T cells in tumors, minimize systemic activation, and achieve higher dosing, thereby improving anti-tumor efficacy.

[0386] Physical, chemical and pharmaceutical properties and preparations

[0387] Physical and biochemical properties: PC-1 is a 92 kDa humanized trispecific protein composed of: anti-PSMA antigen binding fragment (Fab), anti-CD3 single chain variable fragment (scFv) and anti-albumin single domain antibody (sdAb). The molecular formula of PC-1 is C 4050 H 6203 N 1101 O 1285 S 27The calculated average molecular weight of PC-1 is 91,742 Da. PC-1 TRACTr consists of two protein chains connected by a single intermolecular disulfide bond between the light chain (LC) and the heavy chain (HC) of TRACTr and eight intramolecular disulfide bonds. The peptide mask contains a single internal disulfide bond. Fig.13 The LC and HC arrangement of PC-1TRACTr is provided. The LC of CD3 scFv is fused to the N-terminal LC of PSMA Fab via a short flexible amino acid linker. The albumin-bound sdAb, peptide mask, and tumor protease cleavable amino acid linker are fused to the amino terminus of CD3 scFv HC in tandem. The albumin-bound sdAb is attached to the mask via a short flexible linker ( Figure 1 ).

[0388] Drug products

[0389] PC-1 solution for injection (particularly IV infusion) (referred to as PC-1DP) is provided as a sterile aqueous solution formulated in 10 mM histidine, 8% (w / v) sucrose, 0.01% (w / v) polysorbate 20 at a nominal concentration of 2 mg / mL (Table 5). The PC-1 drug product is packaged in 2R, Type 1 borosilicate glass vials with 13 mm nesting caps consisting of a polypropylene snap cap and an insert. Laminated elastomeric plug composition. The formulation of PC-1 drug product is listed in Table 5. The physical and chemical properties of PC-1 are summarized in Table 6.

[0390] Table 5: Formulation composition of PC-1 drug product

[0391]

[0392] Abbreviations: cGMP = Current Good Manufacturing Practice; BP = British Pharmacopoeia; Ch.P = Chinese Pharmacopoeia; JP = Japanese Pharmacopoeia; Ph Eur = European Pharmacopoeia; QS = Quantitative Sufficient; USP / NF = US Pharmacopoeia / National Formulary

[0393] Table 6: Physical and Chemical Properties of JANX007 Drug Product

[0394]

[0395] Abbreviations: CD3 = cluster of differentiation 3; DSC = differential scanning calorimetry; ELISA = enzyme-linked immunosorbent assay; NTU = adrenal turbidity unit; TM = melting temperature.

[0396] Storage and handling

[0397] PC-1DP vials are stored and transported frozen (-20°C).

[0398] Nonclinical studies

[0399] Introduction: Nonclinical studies of PC-1 were designed to support a Phase 1 clinical program in subjects diagnosed with mCRPC. Nonclinical pharmacology studies were conducted in in vitro and ex vivo model systems. In vitro studies examined the following: binding of PC-1 to PSMA, CD3, and albumin antigens in mice, rats, cynomolgus monkeys, and humans; PC-1 stability in sera from healthy human donors, mCRPC patients, and cynomolgus monkeys; the ability of PC-1 to induce cytokine production; the ability of PC-1 to induce T cell-mediated anti-tumor cytotoxic activity.

[0400] An in vitro study examined the ability of PC-1 to induce cytokine release in human whole blood samples from human donors. These studies are described in further detail in the following sections. In vivo studies included a nonclinical single-dose PK study in cynomolgus monkeys. The nonclinical PK data provided the basis for the once-weekly dosing schedule proposed in the Phase 1 study. A GLP repeated-dose study evaluated the safety, tolerability, and relevant systemic exposure of PC-1 in cynomolgus monkeys over 4 weeks of once-weekly dosing (5 administrations per animal total) and evaluated the reversibility of any effects after a 4-week recovery period.

[0401] Nonclinical Pharmacology

[0402] Studies were conducted using PC-1 (uncleaved or intact PC-1) as well as cleaved metabolites, PC-1-SP cleavage (referred to as Met 1 in PK studies) and PC-1-MMP cleavage (referred to as Met 2 in PK studies) as well as the active, unmasked molecule PC-1-TCE (Table 7). Total PC-1 (uncleaved PC-1 + Met 1 + Met 2) was also measured in the PK studies. The PC-1-SP cleavage and PC-1-MMP cleavage used in the in vitro pharmacology studies were obtained from PC-1 by enzymatic treatment with recombinant human membrane serine protease (MTSP1) and recombinant human matrix metalloproteinase 9 (MMP9), respectively.

[0403] Table 7: Brief description of PC-1 and related molecules

[0404]

[0405] Abbreviations: BD = binding domain; CD3 = cluster of differentiation 3; MMP9 = matrix metalloproteinase 9; MTSP1 = membrane-type serine protease; PSMA = prostate-specific membrane antigen; SP = serine protease; TCE = T cell engager; TRACTr = tumor-activated T cell engager.

[0406] Preliminary in vitro pharmacodynamics

[0407] Binding affinity of PC-1 to prostate-specific membrane antigen, CD3, and albumin (PC-1–001–TD): The affinity of PC-1 to mouse, rat, cynomolgus monkey, and human PSMA, CD3, or albumin proteins was determined by standard enzyme-linked immunosorbent assay (ELISA) and compared to the corresponding binding affinities of PC-1-SP cleavage, PC-1-MMP cleavage, and PC-1-TCE. Briefly, serial dilutions of PC-1, PC-1-SP cleavage, PC-1-MMP cleavage, or PC-1-TCE were added to plates containing immobilized mouse, rat, cynomolgus monkey, or human antigens. Bound molecules were then detected using an anti-human horseradish peroxidase-conjugated secondary antibody that recognizes the PSMA-binding fragment. The resulting dose-response curves were fit to a 4-parameter variable slope regression, from which the half-maximal effective concentration (EC50) was determined and reported as the affinity (Tables 8 to 11).

[0408] All tested agents (PC-1, PC-1-SP cleavage, PC-1-MMP cleavage, and PC-1-TCE) bound to human and cynomolgus PSMA with low nanomolar affinity. Figures 14A-14F ) and albumin ( Figures 15A-15D ) binding. PC-1-SP cleavage, PC-1-MMP cleavage, and PC-1-TCE exhibited low nanomolar binding to CD3. In contrast, PC-1 exhibited several orders of magnitude weaker binding to CD3 due to inhibition by the peptide mask ( Figures 16A-16F All tested agents showed minimal binding to the corresponding mouse and rat antigens ( FIG. 14A to FIG. 16F ). The observed differences in binding of the test articles to mouse and rat antigens relative to human and cynomolgus monkey antigens are consistent with the lower percentage of sequence identity of the rodent antigens relative to human and cynomolgus monkey antigens.

[0409] Table 8: Binding affinity of PC-1 to PSMA, CD3 and albumin

[0410]

[0411] Abbreviations: CD3 = cluster of differentiation 3; EC50 = half maximal effective concentration; PSMA = prostate-specific membrane antigen.

[0412] Table 9: Binding affinity of PC-1-SP cleavage products to PSMA, CD3 and albumin

[0413]

[0414] Abbreviations: CD3 = cluster of differentiation 3; EC50 = half maximal effective concentration; MTSP1 = membrane serine protease; NA = not available; PSMA = prostate-specific membrane antigen

[0415] Table 10: Binding affinity of PC-1-MMP cleavage products to PSMA, CD3 and albumin

[0416]

[0417] Abbreviations: CD3 = cluster of differentiation 3; EC50 = half maximal effective concentration; MMP9 = matrix metalloproteinase 9; NA = not available; PSMA = prostate-specific membrane antigen.

[0418] Table 11: Binding affinity of PC-1-TCE to PSMA, CD3 and albumin

[0419]

[0420] Abbreviations: CD3 = cluster of differentiation 3; EC50 = half maximal effective concentration; NA = not available; PSMA = prostate specific membrane antigen TCE = T cell engager.

[0421] In summary, PC-1 binds human and cynomolgus PSMA and albumin with low nanomolar affinity. PC-1 CD3 binding affinity is cleavage dependent. Treatment of PC-1 with MTSP1 or MMP9 to form PC-1-SP cleavage or PC-1-MMP cleavage, respectively, resulted in potent CD3 binding similar to that observed with PC-1-TCE. PC-1 exhibited minimal binding to mouse or rat PSMA, CD3, and albumin. The observed differences in binding of the test articles to mouse and rat relative to human and cynomolgus PSMA, CD3, and albumin are consistent with the lower percentage of sequence identity of the rodent antigens relative to the human and cynomolgus antigens.

[0422] Stability of PC-1 in serum of healthy cynomolgus monkeys

[0423] Human Donors and Patients with Metastatic Castration-Resistant Prostate Cancer (PC-1–002–TD): The stability of PC-1 in sera from cynomolgus monkeys, healthy human donors, and humans diagnosed with mCRPC was evaluated using a kinetic binding assay using the Octet RED instrument (Octet). The Octet is an instrument used to measure the concentration of proteins and other biomolecules, measure kinetics and affinity, and screen protein-protein and protein-small molecule interactions. The relative concentration of cleaved PC-1 at different time points was calculated based on the initial slope of the binding curve against the CD3-coated sensor. The relative concentration of cleaved PC-1 over time was used to calculate the cleavage rate in serum as an indicator of PC-1 stability compared to an unmasked PC-1-TCE reference sample. The resulting data are reported as percentage cleavage per day (Table 12). Figures 17A-17B Examples of CD3 kinetic binding curves are shown. Figures 18A-18H Linear regression of percent cleavage versus time is shown. PC-1 was stable in pooled healthy human serum and mCRPC human serum measured over 168 hours. Cleavage was observed to be less than 1% per day in pooled healthy human serum and 2% or less per day in mCRPC serum. A slightly higher cleavage rate of 6% per day was observed in healthy cynomolgus monkey serum.

[0424] Table 12: Serum stability of PC-1

[0425]

[0426] Abbreviations: BD = binding domain; CD3 = cluster of differentiation 3; mCRPC = metastatic castration-resistant prostate cancer.

[0427] In summary, PC-1 exhibited similar cleavage rates in healthy human sera relative to mCRPC patient sera, <1% and ≤2% cleavage per day, respectively. PC-1 cleavage in cynomolgus monkey sera was 6% per day.

[0428] PC-1 induction in peripheral blood mononuclear cells co-cultured with target tumor cells expressing prostate-specific membrane antigen Cytokine production

[0429] The ability of PC-1 to induce cytokine production was evaluated in co-cultures of peripheral blood mononuclear cells (PBMCs) and tumor cells treated with PC-1. PBMCs isolated from the blood of eight consenting healthy human donors were cultured in the presence or absence of human PC cell lines expressing high (LNCaP), moderate (22Rv1), and minimally detectable (PC3) levels of PSMA for 72 h in the presence of increasing concentrations of the test article (Dang et al., 2021; Gorges et al., 2016). The cleaved (PC-1-SP cleavage and PC-1-MMP cleavage) and unmasked (PC-1-TCE) test articles induced potent, dose-dependent production of IFNγ, TNF, and IL-6, whereas masked PC-1 showed a lower ability to induce cytokine production, as indicated by higher EC50s for all three cytokines evaluated ( Figures 19A-19I : Each data point represents the mean of two replicates. The generated nonlinear regression response curves were used to extrapolate EC50 values ​​(the concentration of the test article required to induce cytokine levels equivalent to 50% of the maximal level). Abbreviations: IFN = interferon; IL = interleukin; MMP = matrix metalloproteinase; PBMC = peripheral blood mononuclear cells; SP = serine protease; TCE = T cell engager; TNF = tumor necrosis factor; Figures 20A-20I : Each data point represents the mean of two replicates. The generated nonlinear regression response curves were used to extrapolate EC50 values ​​(the concentration of the test article required to induce cytokine levels equivalent to 50% of the maximal level). Abbreviations: IFN = interferon; IL = interleukin; MMP = matrix metalloproteinase; PBMC = peripheral blood mononuclear cells; SP = serine protease; TCE = T cell engager; TNF = tumor necrosis factor. ). No cytokine induction was detected in the absence of PSMA-expressing target cells ( Figures 21A-21F Abbreviations: IFN = interferon; IL = interleukin; MMP = matrix metalloproteinase; PBMC = peripheral blood mononuclear cell; SP = serine protease; TCE = T cell engager; TNF = tumor necrosis factor. ).

[0430] In summary, masking of the CD3 binding domain of PC-1 reduced its ability to induce cytokine production, indicating that proteolytic PC-1 unmasking is required for cytokine release. Cleaved (PC-1-SP cleavage, PC-1-MMP cleavage) and unmasked (PC-1-TCE) versions of PC-1 exhibited a dose-dependent ability to induce cytokine production, with PC-1 having a higher EC50 for all cytokines evaluated. Cytokine production was not detected in the absence of target cells, indicating that the activity of PC-1 depends on the presence of PSMA.

[0431] PC-1-induced T cell-mediated killing of target tumor cells expressing prostate-specific membrane antigen in vitro

[0432] The ability of PC-1 to induce T cell-mediated killing of target cells was evaluated using PBMCs from eight consenting healthy human donors co-cultured with prostate tumor cell lines expressing high (LNCaP), moderate (22Rv1), or minimally detectable (PC3) PSMA levels in the presence of increasing concentrations of the test article. Results showed that PC-1-induced T cell-mediated killing of tumor cells was dose-dependent and decreased in the presence of the masking agent ( Figures 22A-22H : Each symbol represents the mean of the appropriate treatment replicates. Abbreviations: MMP = matrix metalloproteinase; PBMC = peripheral blood mononuclear cell SP = serine protease; TCE = T cell engager; and Figures 23A-23H : Each symbol represents the mean of the appropriate treatment replicates. Abbreviations: MMP = matrix metalloproteinase; PBMC = peripheral blood mononuclear cell; SP = serine protease; TCE = T cell engager). PC-1 exhibited up to 30-fold and 160-fold reductions in the ability to induce T cell cytotoxic activity in the presence of LNCaP and 22Rv1 cells relative to PC-1-SP cleavage, PC-1-MMP cleavage, and PC-1-TCE, respectively. Furthermore, the extent of induced tumor cell killing depended on the level of PSMA expression, as none of the tested articles induced PBMC-mediated killing of PC3 cells ( Figures 24A-24D Representative graphs shown in : Each symbol represents the mean of the appropriate treatment replicates. Abbreviations: MMP = matrix metalloproteinase; PBMC = peripheral blood mononuclear cells; SP = serine protease; TCE = T cell engager. )

[0433] In summary, masking of the CD3 binding domain reduced the ability of PC-1 to induce T cell-mediated killing of target tumor cells, with up to 160-fold reduction in cytotoxic activity compared to cleaved (PC-1-SP cleavage and PC-1-MMP cleavage) and unmasked (PC-1-TCE) test articles. The ability of PC-1 to induce T cell-mediated killing of tumor cells was dose- and cleavage-dependent and required PSMA expression.

[0434] Preliminary in vitro pharmacodynamics

[0435] Flow Cytometric Bead Array Analysis of Human Cytokines Using Whole Blood from Healthy Human Donors

[0436] The ability of PC-1 and PC-1-SP cleavage products to induce cytokine release in whole blood samples from 10 healthy human donors was assessed using a flow cytometry bead array method. Stimulation was performed for 24 hours in soluble and wet coating (plate binding) forms. Stimulation with soluble forms of Staphylococcus enterotoxin B (SEB) (positive control) resulted in cytokine production in samples from all donors, indicating that these cells have the ability to release cytokines in response to stimulation. In the absence of treatment, cytokine release was not observed in any sample. When stimulating in a wet coating (plate binding) form, cytokine release was not observed. In any form, after stimulation with PC-1-SP cleavage products, IL-2, IL-6, IL-10, TNF and IFNγ release higher than untreated control levels (low to undetected) were not detected in any sample. In any form, all donors did not observe IL-2 or IFNγ release after PC-1 stimulation. In the plate binding form, IL-6, IL-10 or TNF cytokine release was not observed after PC-1 stimulation. After treatment with the highest tested concentrations of soluble PC-1 (10 nM [917.4 ng / mL] and 100 nM [9174 ng / mL]), IL-6, IL-10, and TNF release was observed in a minority of samples.

[0437] Safety Pharmacology

[0438] In accordance with ICH S6(R1) and ICH S9 guidelines, no independent safety pharmacology studies were conducted for PC-1. Cardiovascular, CNS, and respiratory safety pharmacology endpoints were included in a 4-week repeat-dose IV toxicity study in cynomolgus monkeys.

[0439] No clear effects of PC-1 administration on qualitative or quantitative ECG parameters were observed. Sinus tachycardia (defined as mean heart rate greater than 270 beats per minute) occurred 4 times in 3 animals after dosing at 1.5 mg / kg / week on day 1, while one time was observed in 1 animal after dosing in the control group. Sinus tachycardia is usually a normal variant in cynomolgus monkeys. Although most post-dose events occurred after high doses, PC-1 administration had no significant effect on group mean heart rate. Therefore, tachycardia may not be related to PC-1. One animal receiving 1.5 mg / kg / week had 3 atrial premature beat syndromes (APCs) on the ECG on day 1. Rare APCs have been reported to occur occasionally in young cynomolgus monkeys, and since APCs were only observed in one animal, the arrhythmias are not considered to be related to the test article. No changes in respiratory rate or body temperature related to PC-1 were observed during the 4-week repeat dose toxicity study. Based on daily clinical signs and detailed weekly clinical observations during the study, there were no CNS changes related to PC-1.

[0440] Pharmacokinetics and product metabolism in animals

[0441] Pharmacokinetics in Cynomolgus Monkeys: The PK properties of PC-1 were evaluated following a single-dose (non-GLP) PK study and a repeated-dose GLP toxicity study in cynomolgus monkeys. The analytes, intact PC-1 (also known as uncleaved PC-1), total PC-1 (PC-1+Met 1+Met 2), and the metabolites Met 1 (PC-1-SP cleavage) and Met2 (PC-1-MMP cleavage) were determined in nonhuman primate K2EDTA plasma.

[0442] Single-dose PK study

[0443] In a single-dose PK study, the PK of PC-1 administered to male cynomolgus monkeys at 0.1 mg / kg, 0.3 mg / kg and 1.5 mg / kg dose levels was evaluated. Four analytes (intact PC-1, total PC-1, Met 1 and Met 2) were measured; however, PK parameters were derived from three analytes (intact PC-1, total PC-1 and Met 1). Met 2 was excluded from the TK parameter calculation because the concentration of most samples was BLQ. In addition, the clearance of analytes may be affected by the presence of ADA. The clearance of intact and total PC-1 remained relatively constant in the 3 dose groups. The faster clearance observed at the highest dose (1.5 mg / kg) may be caused by the presence of ADA. The mean half-life (T1 / 2) of intact PC-1 in the 3 dose groups was 111, 78.5 and 70.4 hours, respectively, while the total PC-1 T1 / 2 was 110, 105 and 62.1 hours, respectively. The systemic exposure of intact and total PC-1 increased with dose over all dose ranges, as measured by AUC0-inf and Cmax. The increase in Met 1 AUC0-last was less than dose-proportional, while the increase in Cmax was dose-proportional when the dose level increased from 0.1 mg / kg to 0.3 mg / kg and from 0.1 mg / kg to 1.5 mg / kg. The increase in systemic exposure of Met 1 AUC0-last and Cmax was dose-proportional when the dose level increased from 0.3 mg / kg to 1.5 mg / kg. The overall exposure of Met 1 was minimal, and cleavage of intact PC-1 during sample processing for the PK assay may affect Met 1 generation. A validated PK assay was used, which showed that the conversion of intact PC-1 to Met 1 induced by sample processing averaged 1.87%. Therefore, native cleavage in vivo is expected to be much lower. Cytokine production in cynomolgus monkey serum was also analyzed by flow cytometry in this study. In general, cytokine elevation was transient and dose-dependent. In animals administered ≥0.1 mg / kg of PC-1, IL-6 levels increased 8 hours after dosing and decreased to pre-test levels 48 hours after dosing. In animals administered ≥0.3 mg / kg, IL-2 levels increased to varying degrees 2 hours after dosing and decreased to baseline 24 hours after dosing. Increases in IL-5, IL-10, TNF, and IFNγ were observed only in animals administered 1.5 mg / kg (the highest dose) of PC-1, and these values ​​returned to baseline 24 to 48 hours after dosing.

[0444] Repeated-dose GLP toxicity studies

[0445] In this GLP toxicity study, male and female cynomolgus monkeys received vehicle control (Group 1) or PC-1 injected IV once a week at 0.1 mg / kg / dose (Group 2), 0.3 mg / kg / dose (Group 3) or 1.5 mg / kg / dose (Group 4), for a total of 5 injections per animal. In the immunogenicity study, ADA was detected in many animals in each dose group (summarized in Section 4.4.1). ADA has an effect on the TK characteristics of the test article, as discussed below. Overall, there was little or no difference in systemic exposure observed between intact PC-1 and total PC-1. No accumulation was observed after multiple doses, but due to the presence of ADA, it could not be fully evaluated in males in Group 3 and any animals in Group 4. No significant accumulation was observed in both sexes at 0.1 mg / kg / week (Group 2) and females at 0.3 mg / kg / week (Group 3) that were ADA negative. In ADA-negative animals, the median intact PC-1 T1 / 2 ranged from 67.7 to 109.1 hours on days 1 and 22 at all doses tested. In ADA-negative animals, the median total PC-1 T1 / 2 ranged from 60.3 to 156.4 hours on days 1 and 22 at all doses tested. The median total PC-1 T1 / 2 ranged from 60.3 to 156.4 hours on days 1 and 22 at 0.1 mg / kg / week. 0-168hSystemic exposure was not significantly different between the sexes when measured. No significant differences were observed between the sexes on Day 1 at the 0.3 and 1.5 mg / kg / week doses; however, exposure on Day 22 could not be compared between the sexes due to the presence of ADA and its effect on the overall PC-1 TK profile. Overall exposure to Met 1 was minimal and this was influenced by an average of 1.87% cleavage of intact PC-1 during sample preparation for the TK assay. Native cleavage is expected to be much lower. No exposure to any analyte was observed in any Group 1 animal. Immunogenicity was assessed in all animals. No animals were found to be ADA positive in Group 1 (vehicle control), while 1 of 6 animals in Group 2 (0.1 mg / kg / week), 5 of 6 animals in Group 3 (0.3 mg / kg / week), and all 10 animals in Group 4 (1.5 mg / kg / week) were confirmed to be ADA positive at the last dose. The TK profile of all ADA positive animals was affected; however, in some animals, ADA had a minor effect on exposure after multiple doses. The two ADA-positive female animals in Group 3 still achieved significant exposure after the Day 22 dose, with AUC0-168h (increased to 2.0-fold) and Cmax (increased to 1.9-fold) approximately doubling compared to the Day 1 dose. In addition, the two ADA-positive female animals in Group 4 also achieved significant exposure after the Day 22 dose, measured by AUC0-168h and Cmax, with AUC0-168h values ​​approximately 54% of the corresponding values ​​observed after the Day 1 dose and Cmax values ​​approximately 96% of the corresponding values ​​observed after the Day 1 dose.

[0446] metabolism

[0447] PC-1 metabolism has not been specifically studied, as classical drug elimination by metabolism does not represent an important clearance mechanism for monoclonal antibodies. Antibodies, such as PC-1, are typically broken down into small peptides, carbohydrates, and amino acids that return to nutrient pools or are excreted via the kidneys without any biological effects (Wang et al., 2008). Therefore, in accordance with the ICH S6(R1) guideline, metabolism studies have not been performed on PC-1.

[0448] excretion

[0449] Renal elimination is relatively unimportant for monoclonal antibodies because their large size limits the extent of their glomerular filtration. Therefore, in accordance with the ICH S6(R1) guideline, excretion studies have not been performed for PC-1.

[0450] Pharmacokinetic drug interactions

[0451] Pharmacokinetic drug interaction studies have not been performed for PC-1. In general, antibodies such as PC-1 are not metabolized by cytochrome P450 (CYP) enzymes and are not transported by P-glycoprotein (Pgp) or related ATP-binding cassette membrane transporters. Cytokines produced by activated lymphocytes can affect Pgp levels and CYP enzyme activity (Harvey and Morgan, 2014). The clinical relevance of PC-1 immunomodulation and potential cytokine production that can affect Pgp and CYP is unclear, but clinically relevant drug-drug interaction effects are considered extremely unlikely (Seitz and Zhou, 2007; Huang et al., 2010).

[0452] toxicology

[0453] Repeated Dose Toxicity Studies: PC-1 was evaluated in a GLP, 4-week, weekly, repeat-dose toxicity study in cynomolgus monkeys (Table 13). Consistent with the intended clinical route of administration, the toxicity studies were performed using an IV route of administration. The cynomolgus monkey was selected as a pharmacologically relevant species because the PSMA, CD3, and albumin protein sequences have high identity to human antigens and PC-1 binds to target antigens (PSMA, CD3, and albumin) in monkeys and humans almost equally. In addition, PC-1 was evaluated for in vitro and in vivo cytokine release and serum stability.

[0454] Table 13: Overview of PC-1 Toxicology Program

[0455]

[0456] Abbreviation: GLP = Good Laboratory Practice.

[0457] PC-1 was administered to male and female monkeys (3 / sex / group) by IV bolus at doses of 0 (vehicle control), 0.1, 0.3, or 1.5 mg / kg / week (5 doses total). Additional animals (2 / sex / group) were evaluated at 0 and 1.5 mg / kg / week for the reversibility of any PC-1-related effects after a 4-week recovery period. The dosing regimen was selected based on the half-life of PC-1 in cynomolgus monkeys and was designed to have a dosing regimen similar to or more intensive than the clinical dosing regimen. No mortality, food consumption or weight changes, body temperature, respiratory rate, coagulation, urinalysis, or macroscopic findings associated with PC-1 were observed during dosing or the recovery period. No ophthalmic and electrocardiographic findings associated with PC-1 were observed.

[0458] Clinical signs

[0459] After the first dose of PC-1, one male animal at 1.5 mg / kg / week developed coldness to the touch and decreased activity; clinical signs gradually resolved before the second dose. The clinical signs may be related to the cytokine induction observed after the first dose. No other clinical signs were observed in any other animals during dosing or the recovery period.

[0460] hematology

[0461] PC-1-related non-adverse hematologic changes included erythrocyte changes consistent with decreased erythropoiesis and hemolysis at ≥0.1 mg / kg / week (decreased red blood cell [RBC] mass, regenerative erythroid response, mean corpuscular hemoglobin [MCH], and mean corpuscular hemoglobin concentration [MCHC]) and increases in leukocyte and platelet counts consistent with a proinflammatory response. In animals administered 1.5 mg / kg / week, the reduction in RBC mass was partially reversed, the changes in reticulocyte counts were completely reversed, and the changes in MCH and MCHC showed limited or irreversible reversibility at the end of the 4-week recovery period. The non-adverse, minimally reduced to significantly reduced lymphocyte counts in animals administered ≥0.1 mg / kg / week were associated with immunophenotypic findings of reduced mean absolute numbers of total T cells, cytotoxic T cells, helper T cells, and natural killer (NK) cells 24 hours after dosing in animals administered ≥0.1 mg / kg / week, and were consistent with the expected pharmacology of the test article (i.e., transient sequestration of activated lymphocytes). A slight decrease in monocyte counts was also observed in animals administered ≥0.1 mg / kg / week on Day 2; however, the reason is unknown. The minimally decreased platelet counts on Day 2 are consistent with transient nonspecific effects of the macromolecular test article on monkey platelets, or potentially due to an increase in PC-1-related proinflammatory cytokines within 24 hours after the first dose. On or after Day 15, PC-1-related leukocyte changes included minimal to slight increases in neutrophil counts in females administered ≥0.1 mg / kg / week, minimal or slight increases in lymphocyte counts in males and females administered 1.5 mg / kg / week, and moderate increases in eosinophil counts in males administered 1.5 mg / kg / week and females administered ≥0.3 mg / kg / week, which are consistent with a proinflammatory response. PC-1-related mild or moderate increases in platelet counts in animals administered ≥0.1 mg / kg / week on or after Day 8 are also consistent with a proinflammatory response. Changes in lymphocyte, monocyte, and platelet counts were largely reversible by the end of the 4-week recovery period.

[0462] Clinical Chemistry

[0463] The PC-1-related non-adverse clinical chemistry findings observed on day 2 (16-24 hours post-dose) are consistent with a transient, more pronounced pro-inflammatory response following the first dose that may result in vascular leak and / or a decrease in blood pressure (not measured), and are associated with increased cytokine release within 24 hours after the first dose. On day 2, urea and creatinine were mildly to markedly increased in animals dosed ≥0.3 mg / kg / week, consistent with renal dysfunction potentially due to decreased renal tissue perfusion; however, a direct effect on the kidney cannot be excluded, and correlative microscopic renal findings on day 31 were limited to minimal to moderate mononuclear cell infiltration. By day 8, there was evidence of reversibility of the increased urea and creatinine. The decrease in serum albumin was more pronounced in animals dosed ≥0.1 mg / kg / week, and there was minimal decrease in sodium and chloride on day 2 in animals dosed ≥0.3 mg / kg / week, also consistent with potential vascular leak or potential renal dysfunction. The decrease in serum albumin was less pronounced by day 31 and was largely reversed by the end of the 4-week recovery period. The decrease in albumin at these later time points, coupled with minimal or mild increases in globulins, is consistent with a proinflammatory response. Other PC-1-associated serum chemistry changes included a reversible minimal or mild decrease in total cholesterol.

[0464] Organ weight

[0465] PC-1-related organ weight differences on Day 31 included increased spleen weights in animals dosed ≥ 0.1 mg / kg / week, and increased liver weights in animals dosed at 1.5 mg / kg / week. The spleen weight increases appeared to be unrelated to dose. However, the magnitude of the differences was consistent with test article-related findings and correlated with microscopic findings of minimal or slightly increased lymphocyte cell density in the white pulp of the spleen. The increased liver weights were associated with minimal perivascular mononuclear infiltration. PC-1-related organ weight differences at the end of the recovery period included increased spleen weights in males dosed at 1.5 mg / kg / week, consistent with partial reversibility and lack of microscopic correlation.

[0466] Microscope Discovery

[0467] Nonadverse microscopic findings associated with PC-1 on day 31 included minimal to marked mononuclear infiltrates in the liver, gallbladder, adrenal medulla, cervix, vagina, epididymis, seminal vesicles, and bladder that generally had a perivascular pattern, and increased lymphocytic cell density in the white pulp of the spleen in animals administered ≥ 0.1 mg / kg / week. However, these microscopic findings were generally poorly dose-related. Microscopic findings were consistent with a proinflammatory response but were not associated with evidence of tissue damage and were therefore considered nonadverse. The microscopic findings were largely reversed by the end of the recovery period, and the relationship of the remaining changes at the end of the recovery period to PC-1 treatment is uncertain.

[0468] T / B / NK cell immunophenotyping

[0469] In monkeys administered PC-1 ≥ 0.1 mg / kg / week, mean absolute counts of total T cells, cytotoxic T cells, helper T cells, and NK cells decreased 24 hours after dosing compared to pretest values. After the first dose, transient increases in B cell counts were observed at 1.5 mg / kg / week. After the first dose, PC-1 treatment induced transient minimal increases in the percentages of CD69+ helper T cells, CD69+ cytotoxic T cells, and Ki67+ helper T cells. All changes observed in peripheral white blood cell (WBC) cell populations were transient and returned to levels similar to pretest values ​​by the third or fifth dose.

[0470] Cytokine levels

[0471] PC-1 induced transient, dose-dependent increases in IL-2, IL-5, IL-6, IL-10, TNF, and IFNγ within 24 hours after the first dose at ≥0.1 mg / kg / week. Cytokine levels returned to pre-test values ​​within 24 hours after the first dose or before the second dose. At 1.5 mg / kg / week, the fourth dose induced a transient, minimal increase in IL-6 levels in a few animals.

[0472] Impact of antidrug antibodies

[0473] Anti-PC-1 antibodies were detected in all groups, and the incidence and titer of ADA were dose-dependent (1 / 6 animals at 0.1 mg / kg / week, 5 / 6 animals at 0.3 mg / kg / week, and 10 / 10 animals at 1.5 mg / kg / week). All animals in the high-dose group (1.5 mg / kg / week) were ADA positive at the end of the study, and these animals also had the highest ADA titers. Due to the presence of ADA, most animals at 1.5 mg / kg / week did not maintain exposure at the end of the dosing period, and most animals showed a significant decrease in exposure when evaluated at the third or fourth dose. The two ADA-positive female animals administered 0.3 mg / kg / week still achieved significant exposure after the 22nd day dose, with AUC 0-168h In addition, the AUC of 2.5 mg / kg / week was approximately doubled in two ADA-positive female animals after administration on day 22. 0-168h and Cmax measurements also achieved significant exposure, with AUC 0-168hExposures were measured to be approximately 54% compared to Day 1 and approximately 96% compared to Day 1 as measured by Cmax. Exposures at 0.1 mg / kg / week were similar or higher in males and females on Day 22 compared to Day 1. No differences in toxicity were found in females maintained at ≥0.3 mg / kg / week compared to animals not maintained at exposure. Therefore, all dose levels assessed for potential toxicity in this study were considered to be valid.

[0474] in conclusion

[0475] In summary, male and female cynomolgus monkeys were administered vehicle control or 0.1, 0.3 or 1.5 mg / kg / week PC-1 by IV injection for 4 weeks, followed by a 4-week recovery period. Non-adverse hematology (reduced RBC mass and regenerative erythroid response; transient reduction followed by increased WBC population; platelet count changes), clinical chemistry (transiently reduced urea, creatinine, globulin and bilirubin; transiently reduced sodium and chloride, and reduced albumin and cholesterol), organ weights (increased spleen and liver weights) and microscopic findings (minimal to significant mononuclear cell infiltration in several tissues) were observed at ≥0.1 mg / kg / week. PC-1 induced a dose-dependent increase in cytokine transients within 24 hours after the first dose at ≥0.1 mg / kg / week. Most, if not all, of the changes can be attributed to proinflammatory responses consistent with the PC-1 mechanism of action and / or cytokine release observed after administration of the first dose of PC-1. No adverse effects were observed based on the dosing and recovery period studied, and the HNSTD / NOAEL was 1.5 mg / kg / week. At the NOAEL, systemic exposure (Cmax and AUC 0-168h ) were 43,500 ng / mL and 2,340,000 h·ng / mL, respectively, for both sexes combined.

[0476] Genotoxicity, carcinogenicity and reproductive toxicity studies

[0477] According to the ICH S6(R1) guideline, the scope and type of genotoxicity studies routinely conducted on small molecule drug products are generally not applicable to biotechnology-derived products. Humanized trispecific proteins such as PC-1 are not expected to interact directly with DNA or other chromosomal material. Therefore, mutagenicity studies are not considered relevant and are not planned. Carcinogenicity studies have not been conducted on PC-1. Given that the intended patient population is patients with mCRPC, carcinogenicity studies are not planned (according to the ICH S9 guideline). Non-clinical reproductive and developmental toxicity studies have not been conducted on PC-1. Because its effects on pregnancy are currently unknown, all subjects and their female partners must use at least 2 highly effective, protocol-specified methods of contraception while participating in PC-1 clinical trials.

[0478] Effect on people

[0479] Introduction: There is no clinical experience with PC-1. The current study will be a FIH Phase 1 clinical trial of PC-1. Please refer to the clinical study protocol for complete patient eligibility guidelines. A brief summary of the planned clinical study is provided below.

[0480] Summary of Phase 1 Trial Design

[0481] Study Design: This study is a FIH, Phase 1, open-label, multicenter study designed to evaluate the safety, tolerability, PK, PD, and preliminary efficacy of PC-1 administered as a single agent in adult subjects with mCRPC. The study will be conducted in 3 parts: dose escalation (Part 1), approximately 20 to 30 subjects; cohort backfill expansion (Part 2), approximately 30 subjects enrolled at three dose levels; and dose expansion (Part 3), approximately 30 subjects enrolled at RP2D. Dose escalation (Part 1) will evaluate the safety, tolerability, PK, PD, and preliminary efficacy of PC-1 administered by IV infusion. The dose escalation decision will be based on the recommendation of the Safety Review Committee, based on a review of safety data and available PK, PD, and preliminary efficacy data. Dose escalation can continue to the dose at which at least 2 dose-limiting toxicities are observed (exceeding the MTD). The cohort backfill expansion (Part 2) will allow further characterization of the safety and activity of the dose level. In dose expansion (Part 3), up to approximately 30 subjects will be enrolled to obtain additional safety, tolerability, PK, PD, and preliminary clinical activity data for PC-1, with the dose and schedule to be determined by the Safety Review Committee after review of all available safety, PK, PD, and preliminary efficacy data.

[0482] Summary of Data and Guidelines

[0483] Indications and Usage: PC-1 is being developed for the treatment of mCRPC and is not currently approved for any indication.

[0484] Dosage and Administration: In the FIH study, the starting dose will be 100 μg, followed by dose escalation. PC-1 will be administered IV on days 1, 8, and 15 of a 21-day cycle. Subjects will receive treatment for 6 cycles (3 weeks each), with a total of 18 infusions planned over 18 weeks.

[0485] Preparation Instructions: Parenteral drug products should be visually inspected for particulate matter and discoloration prior to administration, where solution and container permit. Refer to the Dosage Manual (or study protocol) for further details.

[0486] Dosage Form and Strength: PC-1DP will be provided as an injection solution presented as a single strength of 2 mg / mL for IV administration.

[0487] Contraindications: PC-1 is contraindicated in the following cases: Known hypersensitivity to any of the components of the drug.

[0488] WARNINGS AND PRECAUTIONS: There is no prior clinical experience with PC-1. PC-1 is an antibody protein construct. Like other molecules in this class, it is highly specific for its target. Although antibody therapeutics are well tolerated, they are "foreign" proteins, and some patients can experience infusion-related reactions (IRRs) or develop immune responses against them. However, PC-1 is designed to have less downstream risk of CD3 activation than other T cell engaging therapies. PC-1 is an experimental drug and should be administered to patients only in the context of clinical studies.

[0489] Potential adverse reactions

[0490] Because PC-1 is a T cell redirecting antibody, CRS, neurotoxicity, and / or tumor lysis syndrome may occur, although based on the PC-1 design, their frequency / severity may be lower than other T cell activating therapies. Published experience with other monoclonal antibody therapies indicates that fever, immunogenic reactions (i.e., formation of ADAs), and / or hypersensitivity reactions may be observed. These reactions can be both severe and systemic (e.g., anaphylaxis) and can occur acutely or delayed. For regulatory reporting purposes, all adverse events (AEs) will be assessed as unexpected at this stage of the development program. Therefore, AEs that are serious and potentially related to PC-1 will be reported to health authorities in accordance with applicable regulations.

[0491] Infusion-related reactions

[0492] PC-1 is a recombinant protein-based therapeutic, and administration of therapeutic proteins has been associated with infusion reactions, signs and symptoms of which include fever, chills, rash, urticaria, dyspnea, hypotension, and / or nausea. To minimize the risk of infusion reactions, all subjects will be premedicated with acetaminophen / paracetamol and diphenhydramine, and corticosteroids will be administered with the first infusion. Mild infusion reactions should be managed with antipyretics, antihistamines, and antiemetics per institutional standards, and for more severe reactions, corticosteroids.

[0493] Management of infusion-related reactions

[0494] The National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) v5.0 (under the category “Injury, Poisoning, and Procedural Complications”) defines an IRR as “a condition characterized by an adverse reaction to an infusion of a pharmacological or biologic substance.” For the purposes of this study, the time frame for evaluation of infusion reactions was the 24-hour period from the start of the infusion. Recommendations for the management of IRRs are provided below. For Grade 1 IRRs: described as mild, transient reactions; no interruption of the infusion is required; no intervention is required: · Remain at the bedside and monitor the subject until recovery from symptoms. For Grade 2 IRRs: described as requiring interruption of the infusion but responding promptly to symptomatic treatment (e.g., antihistamines, nonsteroidal anti-inflammatory drugs, narcotics, IV fluids); and prophylactic medications for ≤4 hours: · Stop the PC-1 infusion, start an IV infusion of normal saline, and treat the subject with diphenhydramine 50 mg IV (or equivalent) and / or 500 to 750 mg oral acetaminophen. · Corticosteroid therapy may also be administered at the investigator’s discretion. Remain at the bedside and monitor the subject until symptoms resolve. · If the infusion is interrupted, wait until symptoms subside and then restart the infusion at 50% of the original infusion rate. · If no further complications occur after 60 minutes, the rate may be increased to 100% of the original infusion rate. Monitor the subject closely. · If symptoms recur, stop the infusion and disconnect the subject from the infusion set. · No further PC-1 will be administered at this visit. · The amount of PC-1 infused must be recorded on the case report form. · Subjects who experience a Grade 2 infusion reaction during the post-infusion observation period that does not subside within that time should be observed until the AE subsides or stabilizes, with vital signs measured every 4 hours and other assessments performed as medically indicated for the management of the AE.

[0495] For Grade 3 or 4 IRRs: Grade 3 is described as prolonged recurrence of symptoms after initial improvement (e.g., not responsive to symptomatic medications and / or transient interruption of infusion); hospitalization for other clinical sequelae (e.g., renal impairment, pulmonary infiltrates). Grade 4 is described as life-threatening consequences; urgent intervention required. · Immediately stop PC-1 infusion. · Investigators should follow their institution's guidelines for the treatment of anaphylaxis or high-grade infusion reactions. · Consider treating subjects with normal saline IV infusion and administer corticosteroids, H1 and / or H2 blockers, bronchodilators, oxygen, or vasopressors as needed. · Subjects should be monitored until the investigator is confident that symptoms will not recur. · All subjects with an IRR ≥ Grade 3 should be observed until the AE resolves or stabilizes, with vital sign measurements and additional assessments for the management of the AE as medically indicated. · Subjects experiencing a Grade 3 IRR during Week 1 of Cycle 1 may also be hospitalized during or after administration of subsequent doses, and dose reductions should be considered.

[0496] Cytokine Release Syndrome

[0497] The known risks of treatment with T cell engagers (TCEs) are primarily related to cytokine release and CRS. PC-1 is designed to reduce the risk of CRS by requiring protease cleavage for activation, focusing the activity of the molecule to the TME where proteases are overexpressed, dysregulated, and activated. This approach has been shown to significantly reduce systemic cytokine exposure in preclinical models. Data from other clinical development programs (unmasked TCEs) show that widespread T cell activation can lead to cytokine release into the blood, which can cause acute effects of fever, hypotension, and hypoxia and may require hospitalization. These side effects are transient and can be treated with IV infusions, tocilizumab, and supportive care, but occasionally have the potential to become severe. In this clinical study, we will carefully monitor the emergence of these vital signs and adverse effects and follow closely. In the nonclinical toxicology studies described in Section 4, the dose of PC-1 can be 50 to 150 times higher than the unmasked TCE active form with equivalent safety and cytokine induction activity. This safety window observed in animal studies is expected to enable higher active doses in patients and provide higher levels of anti-tumor activity. Guidelines for the treatment of CRS are primarily driven by the CAR-T field, where CRS seen with anti-CD19 CAR-T cells can be severe and last longer (Neelapu, 2018). For bispecific antibodies, CRS is shorter in duration and generally less severe, but can still be dose-limiting and is often mitigated by a priming / step-dosing approach. Cytokine release is primarily a first-dose or first-cycle phenomenon, and tolerance to cytokine induction occurs with subsequent TCE infusions. Measures to prevent cytokine release and CRS include premedication with glucocorticoids, IV prehydration, and discontinuation of antihypertensive medications on the day of infusion. If CRS occurs during dose escalation, priming and step-dosing will be initiated.

[0498] CRS Management: Cytokine release can cause fever and tachycardia, and can also become more severe and cause hypotension and hypoxia, and require close attention. CRS can also be associated with fatigue, nausea, headache, dyspnea, chills, myalgia / arthralgia, and anorexia. CRS will be graded using the American Society for Transplantation and Cellular Therapy (ASTCT) consensus grading scale (Table 14) (adapted from (Lee et al., 2019)). CRS will be managed with IV fluids and other supportive care measures (if necessary) and increased monitoring. More severe CRS may require oxygen, corticosteroids, and / or tocilizumab (anti-IL-6 receptor). Severe CRS may also require intensive care unit monitoring and management with vasopressors, anti-seizure medications, high-dose corticosteroids, and / or mechanical ventilation. The recommended guidelines for the treatment of CRS are primarily driven by the CAR-T field, and CRS seen in anti-CD19 CAR-T cells can be severe and last longer (Lee et al., 2019). When using bispecific antibodies in immunoglobulin G format, CRS can occur and is dose-limiting.

[0499] Table 14: Grading of cytokine release syndrome (ASTCT consensus scale)

[0500]

[0501] Abbreviations: ASTCT = American Society for Transplantation and Cellular Therapy; BiPAP = bilevel positive airway pressure; CPAP = continuous positive airway pressure; IV = intravenous.

[0502] For any level of CRS:

[0503] Monitor clinical laboratory evaluations.

[0504] Monitor C-reactive protein levels and serum PD cytokine laboratory assessments.

[0505] Additional safety laboratory samples and plasma will be drawn for exploration of safety and PD biomarkers.

[0506] For Grade 1 CRS: Many subjects may present with only fever and tachycardia. Close monitoring is required to detect and respond to early signs of progression.

[0507] Frequent monitoring of vital signs

[0508] For grade 2 CRS: Hypotension and / or hypoxia detected

[0509] Aggressive IV fluid therapy with saline

[0510] Supplemental oxygen as needed

[0511] Consider cardiac telemetry and pulse oximetry

[0512] Consider corticosteroids (eg, dexamethasone 10 mg every 12 hours)

[0513] Consider tocilizumab or an equivalent anti-IL-6 antibody (eg, siltuximab) or an anti-IL-6 receptor antibody (eg, sarilimab)

[0514] For grade 3 CRS: Hypotension unresponsive to IV fluids

[0515] Managed in a monitored bed or intensive care unit

[0516] Corticosteroids: dexamethasone 10 mg every 6 hours or methylprednisolone 1 mg / kg IV twice a day as needed

[0517] Tocilizumab (4 to 8 mg / kg IV over 1 hour, not to exceed 800 mg), or an equivalent anti-IL-6 antibody (eg, sildenafil) or anti-IL-6 receptor antibody (eg, salimumab)

[0518] Vasopressor support

[0519] Oxygen therapy with a mask if necessary

[0520] Other potential toxicities

[0521] Tumor Lysis Syndrome; Subjects with a higher disease burden may be at risk for developing tumor lysis syndrome (TLS) while receiving PC-1. Prophylactic treatment / measures are strongly recommended for subjects considered at risk for TLS based on institutional or clinical criteria. Subjects should be closely monitored for laboratory evidence of TLS (hyperuricemia, hyperkalemia, hyperphosphatemia, and hypocalcemia). If there is evidence of TLS related to PC-1, the subject should be admitted to the hospital as clinically indicated. Standard treatment includes vigorous IV hydration, uric acid-lowering agents, and correction of acidosis if present. Renal function, serum uric acid, calcium, phosphorus, and electrolytes should be closely monitored. Subjects who experience Grade 3 to 4 TLS during Week 1 or Cycle 1 may also be hospitalized for ≥24 hours after the end of subsequent dose administration and considered for dose reduction as described in the study protocol.

[0522] Neurological Events: In early clinical studies, administration of bispecific antibodies against solid tumors showed a lower potential for severe neurological toxicity (compared to CAR-T therapy or anti-CD19 BiTE formats such as blinatumomab). Although active doses of PC-1 did not show a tendency to induce cytokine levels that could be associated with neurological toxicity in in vitro human cell culture or in vivo cynomolgus monkey studies, the risk of neurotoxicity is unknown and caution is required. Neurological events associated with cytokine release may include tremor, mental status changes, confusion, speech difficulties, and potential seizures. Monitor subjects for neurological events and rule out other causes of neurological symptoms. Neurological events should be graded according to NCI CTCAE v5.0. Mild disorientation or expressive aphasia (difficulty finding words) may be the earliest and most specific symptoms. Provide supportive care for any neurological events as needed. Examination may include head magnetic resonance imaging and electroencephalogram, and corticosteroids or anti-epileptic drugs may be required if severe. The medical monitor should be contacted if there is any potential for treatment-related neurotoxicity.

[0523] Infect

[0524] As with other TCE-based immunotherapies, serious infections, including fatal bacterial, fungal, and new or reactivated viral infections, can occur during and / or after completion of PC-1-based therapy. New or reactivated viral infections may include cytomegalovirus, herpes simplex virus, parvovirus B19, varicella-zoster virus, West Nile virus, hepatitis B virus (HBV), and hepatitis C virus (HCV). If a serious infection develops, PC-1 should be discontinued and appropriate anti-infective therapy instituted. PC-1 is not recommended for use in patients with serious active infections.

[0525] Hepatitis B Reactivation: HBV reactivation can occur in patients receiving drugs classified as TCE antibodies. This condition has also been reported in patients who are negative for hepatitis B surface antigen (HBsAg) but positive for hepatitis B core antibody (anti-HBc). HBV reactivation refers to an abrupt increase in HBV replication, manifested by a rapid increase in serum HBV DNA levels or detection of HBsAg in a person who was previously HBsAg negative and anti-HBc positive. Reactivation of HBV replication is typically followed by hepatitis (i.e., elevated transaminase levels). In severe cases, elevated bilirubin levels, liver failure, and death may occur. Monthly HBV DNA testing is recommended for subjects with evidence of prior HBV infection (anti-HBc positive) and monitoring for clinical and laboratory signs of hepatitis during PC-1 therapy and for several months thereafter.

[0526] Immunization

[0527] The safety of immunization with live virus vaccines during or following treatment with PC-1 has not been studied. Live virus vaccines are not recommended ≥2 weeks prior to starting PC-1 treatment, during treatment, and until immune recovery after the last cycle of PC-1. Subjects are encouraged to receive COVID-19 vaccine / booster >2 weeks prior to starting the study. The efficacy of vaccination in the study is unknown; however, COVID-19 vaccination is permitted in the study after washout of the first cycle and a 2-week dosing pause.

[0528] Other potential healthy tissue toxicity

[0529] Although PSMA is most highly expressed in PC cells and is highly prostate restricted, it can be expressed at lower levels in some healthy tissues. By requiring tumor proteases to activate PC-1, normal tissue T cell activation has another layer of protection. Tissues need to express PSMA, as well as express specific proteases to activate PC-1 locally at non-tumor sites.

[0530] Kidneys, stomach, and liver

[0531] Immunohistochemical studies have shown that several non-prostate tissues can express low levels of PSMA. These tissues include renal proximal tubule cells, duodenal brush border cells, and colonic crypt rare cells (Silver et al., 1997; The Protein Atlas, 2022). PSMA-PET scans using small molecule PSMA active site conjugates have confirmed tissue accumulation in the small intestine and liver. (Kidney accumulation cannot be resolved because the agent is excreted through the kidneys and bladder). Renal or gastrointestinal toxicity has not been described as a problem for small molecule PSMA conjugates (such as 177-Lu-PSMA-617 (PLUVICTO) (Sartor et al., 2021)) or PSMA-CD3 bispecific antibodies (such as AMG160). However, transaminase elevations have occurred with AMG160, but were primarily attributed to CRS (Tran et al., 2020).

[0532] Salivary and lacrimal glands: PSMA-PET scans using small molecule PSMA active site conjugates have shown clear salivary and lacrimal gland uptake. It is not clear whether there are other nonspecific or less understood mechanisms for this uptake (Morris et al., 2021; Sheehan et al., 2021). Small molecule PSMA conjugates such as 177-Lu-PSMA617 (PLUVICTO) have shown potential for dry mouth and dry eyes as side effects (Sartor et al., 2021); these side effects are rare with PSMA-CD3 bispecifics, but they can occur. (Tran et al., 2020).

[0533] Drug Interactions: The drug interaction profile of PC-1 is unknown, but PC-1 is not expected to cause clinically relevant drug-drug interactions.

[0534] Use by specific groups

[0535] pregnancy

[0536] Nonclinical reproductive and developmental toxicity studies have not been conducted on PC-1. PC-1 is being developed for the treatment of progressive mCRPC that occurs only in males. Because its effect on partner pregnancy is currently unknown, all sexually active subjects in this study and their female partners must use at least two highly effective, protocol-specified contraceptive methods while participating in the PC-1 clinical trial.

[0537] Teratogenic effect

[0538] It is unknown whether PC-1 is teratogenic. If a subject or the subject's female partner becomes pregnant during the study, further treatment with PC-1 should be discontinued immediately.

[0539] Non-teratogenic effect

[0540] It is unknown whether PC-1 is non-teratogenic. If a subject or the subject's female partner becomes pregnant during the study, further treatment with PC-1 should be discontinued immediately.

[0541] Use by the elderly

[0542] Elderly patients have not been studied. Because the average age at diagnosis of PC is 66 years (Cancer.org), elderly patients will be included in the initial clinical trial of PC-1. However, elderly subjects should be carefully monitored.

[0543] Renal insufficiency

[0544] The effect of renal impairment on PC-1 PK and / or function is unknown. However, since PC-1 is a biological compound, alterations in PK / function due to renal impairment are not expected (FDA 2020).

[0545] Hepatic insufficiency

[0546] The effects of hepatic impairment on PC-1 PK and / or function are unclear.

[0547] Overdose

[0548] The role of PC-1 overdose is unknown. If an overdose occurs (i.e., any dose greater than that specified in the protocol), supportive care will be provided.

[0549] describe

[0550] PC-1 is a trispecific TRACTr that incorporates PSMA and CD3 binding domains, tandem albumin binding domains and a peptide that inhibits PC-1 binding to CD3 on T cells (mask), and a tumor protease cleavage sequence ( Figure 1 Intact PC-1 is engineered to reduce binding to CD3 on T cells (relative to the unmasked version) and minimize T cell activation and pharmacology in healthy tissues.

[0551] Clinical Pharmacology

[0552] Mechanism of action

[0553] Target engagement of PC-1 requires proteolysis of its cleavable amino acid linker by proteases present in the TME. Once the amino acid cleavage sequence is proteolytically cleaved by tumor proteases, the tandem mask and albumin binding domains are released to enable CD3 binding. The bispecific component of the cleaved PC-1 binds to both CD3 and PSMA simultaneously, resulting in T cell activation in the tumor, leading to T cell-mediated killing of prostate tumor cells.

[0554] Pharmacodynamics

[0555] The pharmacodynamics in humans has not been evaluated yet and will be evaluated in the PC-1 clinical study.

[0556] Pharmacokinetics

[0557] The pharmacokinetics in humans have not been evaluated previously and will be evaluated in the clinical study of PC-1.

[0558] Nonclinical toxicology

[0559] Carcinogenesis, Mutagenesis and Impairment of Fertility: According to the ICH S6(R1) guideline, genotoxicity studies for biotechnology-derived pharmaceuticals are not applicable. Carcinogenicity, reproductive and developmental toxicity, and fertility studies have not been conducted.

[0560] Animal Toxicology and / or Pharmacology: Parameters of nonclinical safety pharmacology included cardiovascular, CNS, and respiratory safety endpoints, which were included in a 4-week, repeat-dose, IV toxicity study in cynomolgus monkeys (B49-0027-TX). Based on clinical observations and weekly detailed examinations of the animals, PC-1 doses ≤ 1.5 mg / kg / week had no functional effects on the CNS or respiratory systems. In addition, based on qualitative and quantitative ECG parameters, PC-1 administration had no clear effects on the cardiovascular system of the animals. With the exception of a few arrhythmic episodes unrelated to the test article, all ECGs were within normal limits.

[0561] Clinical Studies: No previous clinical studies of PC-1 have been conducted.

[0562] Supplied / Storage and Handling: PC-1DP will be shipped frozen and should be stored at -20°C.

[0563] result

[0564] Quantification of PC-1 and its metabolites in monkey plasma by IC-LC-MS / MS in preclinical toxicokinetic studies

[0565] PC-1 was designed to mask the scFv epitope through a peptide linker with two protease cleavage sites. Enzyme-linked immunosorbent (ELISA) assays are typically the standard method for bioanalysis of biotherapeutics. However, it is less capable in the case of BsAb PC-1 due to its highly complex structure and the need to quantify multiple components in the matrix. In contrast, liquid chromatography combined with tandem mass spectrometry (LC-MS / MS) is a better choice due to its high specificity, multiplexed platform, and low dependence on specific reagents. The results showed that the bioanalysis method for this PC-1 used immunocapture (IC) followed by enzymatic digestion and then LC-MS / MS analysis. The targets in the matrix quantified included intact PC-1, its two metabolites, and total antibody. The lower limit of quantification (LLOQ) of the metabolites was able to reach 5 ng / ml. This method has been successfully used to facilitate the bioanalysis of monkey plasma in preclinical TK studies.

[0566] method

[0567] Immunocapture purification: Intact PC-1 and its metabolites were extracted from monkey plasma using immunocapture. Briefly, 500 μL of high capacity magnetic TM streptavidin microbeads were incubated with 500 μL of biotinylated human PSMA (335 ug / mL) in PBST for one hour at room temperature with shaking (1500 rpm). 100 μL of plasma sample was incubated with 10.0 μL of PSMA-coated microbeads prepared above for one hour at room temperature with shaking (1500 rpm). After two cycles of washing with 400 μL PBST buffer, the enriched analytes on the microbeads were eluted with 50.0 μL of 100 mM glycine-HCl (pH = 2.7).

[0568] Enzymatic digestion: All eluates were transferred to a new 96-well plate and the stable isotope labeled internal standard (SIL-IS) signature peptide solution was added. The analytes were denatured with 20 μL of 1.00 mg / mL rapigest solution and 10 μL of 200 mM NH 4 HCO 3 Reduce the 100 mM dithiothreitol with 10 μL of 200 mM NH 4 HCO 3 The cells were alkylated with 600 mM iodoacetamide in 4% iodine and finally digested with 10.0 μL of 200 μg / mL Glu-C at 37°C with shaking (1200 rpm) for 2 h. The digestion was terminated with 10.0 μL of 10% trifluoroacetic acid (TFA). Fig.25 ).

[0569] LC-MS / MS analysis: Shimadzu-30UPLC system was used in conjunction with AB Sciex QQQ6500+ for LC-MS / MS analysis. Waters ACQUITY UPLC HSS T3 column ( The mobile phases were 0.1% formic acid in water (MPA) and 0.1% formic acid in acetonitrile (MPB).

[0570] result

[0571] Method Validation: The LC-MS / MS method was fully validated for the quantitative determination of BsAb PC-1, its two metabolites, and total antibody in monkey K2 EDTA plasma with lower limits of quantification of 100, 5.00, 5.0, and 110 ng / ml, respectively. TK Results: The concentrations of intact PC-1, its two metabolites (called Met 1 and Met 2), and total antibody (total concentration of intact PC-1, Met 1, and Met 2) were measured in study samples collected from the TK study. Fig.26Metabolic profiles of intact PC-1, Met 1, Met 2, and total PC-1 are shown. At all three dose levels, in both male and female animals, the median Tmax value of intact PC-1 at the first dose (day 1) and the fourth dose (day 22) was observed at 0.1 hours. In anti-drug antibody (ADA) negative animals, the median intact PC-1 T1 / 2 at days 1 and 22 ranged from 67.7 to 109.1 hours at all doses tested. Met1 and Met 2 were either below the limit of quantification (BLQ) or were produced during sample pretreatment rather than in vivo. LLOQ in plasma: 5 ng / ml for metabolites 1 and 2, 100 ng / ml for intact PC-1, and 110 ng / ml for total antibodies. Fig. 27 The correlation between intact PC-1 concentration and anti-drug antibody (ADA) concentration in high-dose monkeys was shown. PC-1 metabolites were found to be very low in the plasma of healthy monkeys, indicating that PC-1 has low ectopic toxicity and that the activation mechanism at the site of PC-1 is significantly advantageous. At the end of the dosing period, ADA was detected in most of the medium-dose and high-dose animals, at which time the exposure of the prodrug was significantly reduced. The identification method is shown in Table 15.

[0572] Immunogenicity results: Intact PC-1 was detected as BLQ in a large number of samples from the high-dose group, which may be due to the production of a large amount of ADA in the high-dose group (Figure 28). The presence of ADA can lead to decreased drug efficacy, which should be paid special attention to in preclinical studies.

[0573] Table 15: Verification methods

[0574]

[0575]

[0576]

[0577] Although preferred embodiments of the present disclosure have been shown and described herein, it will be appreciated by those skilled in the art that these embodiments are provided by way of example only. Those skilled in the art will now appreciate that many variations, changes, and substitutions will not depart from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing the present disclosure. It is intended that the scope of the present disclosure be limited by the appended claims, and that methods and structures within the scope of these claims and their equivalents are thereby covered.

[0578] Implementation

[0579] Embodiment 1. An isolated recombinant polypeptide complex comprising a first chain having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1 and a second chain having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2, wherein the isolated recombinant polypeptide complex comprises at least one of the following features:

[0580] (a) at least one disulfide bond formed by a pair of cysteine ​​residues in the first chain or the second chain or by a pair of cysteine ​​residues in the first chain and the second chain; or

[0581] (b) secondary structure consisting of β-sheets or random coils;

[0582] (c) at least one pyroglutamine in the second chain;

[0583] (d) when the isolated recombinant polypeptide complex is formulated at a concentration of 1.0 mg / mL in a buffer comprising 10 mM histidine buffer, 8% (w / v) sucrose, 0.01% (w / v) polysorbate 20, pH 6.3, the melting temperature (Tm) is between about 65°C and about 85°C.

[0584] (e) a far-UV circular dichroism peak at a wavelength between 190 nm and 205 nm when the isolated recombinant polypeptide complex is formulated at a concentration of 0.1 mg / mL in 10 mM potassium phosphate buffer, pH 7.0;

[0585] (f) a deep UV circular dichroism depression at wavelengths between 210 nm and 220 nm when the isolated recombinant polypeptide complex is formulated at a concentration of 0.1 mg / mL in 10 mM potassium phosphate buffer, pH 7.0; or

[0586] (g) Near-UV circular dichroism peaks at wavelengths between 250 nm and 300 nm when the isolated recombinant polypeptide complex is formulated at a concentration of 1.0 mg / mL in a buffer containing 10 mM histidine buffer, 8% (w / v) sucrose, 0.01% (w / v) polysorbate 20, pH 6.3.

[0587] Embodiment 2. The isolated recombinant polypeptide complex of embodiment 1, wherein the polypeptide comprises at least two of the characteristics.

[0588] Embodiment 3. The isolated recombinant polypeptide complex of embodiment 1, wherein the polypeptide comprises at least three of the characteristics.

[0589] Embodiment 4. The isolated recombinant polypeptide complex of embodiment 1, wherein the polypeptide comprises at least four of the characteristics.

[0590] Embodiment 5. The isolated recombinant polypeptide complex of embodiment 1, wherein the polypeptide comprises at least five of the characteristics.

[0591] Embodiment 6. The isolated recombinant polypeptide complex of embodiment 1, wherein the polypeptide comprises at least six of the characteristics.

[0592] Embodiment 7. The isolated recombinant polypeptide complex of embodiment 1, wherein the polypeptide comprises at least seven of the characteristics.

[0593] Embodiment 8. The isolated recombinant polypeptide complex of embodiment 1, wherein the polypeptide comprises at least eight of said features.

[0594] Embodiment 9. The isolated recombinant polypeptide complex according to any one of embodiments 1-7, wherein the first chain comprises at least 85% sequence identity to SEQ ID NO:1.

[0595] Embodiment 10. The isolated recombinant polypeptide complex according to any one of embodiments 1-7, wherein the first chain comprises at least 90% sequence identity to SEQ ID NO:1.

[0596] Embodiment 11. The isolated recombinant polypeptide complex according to any one of embodiments 1-7, wherein the first chain comprises at least 95% sequence identity to SEQ ID NO:1.

[0597] Embodiment 12. The isolated recombinant polypeptide complex according to any one of embodiments 1-7, wherein the first chain comprises at least 99% sequence identity to SEQ ID NO:1.

[0598] Embodiment 13. The isolated recombinant polypeptide complex according to any one of embodiments 1-11, wherein the first chain comprises the amino acid sequence according to SEQ ID NO:1.

[0599] Embodiment 14. The isolated recombinant polypeptide complex according to any one of embodiments 1-13, wherein the second chain comprises at least 85% sequence identity to SEQ ID NO:2.

[0600] Embodiment 15. The isolated recombinant polypeptide complex according to any one of embodiments 1-13, wherein the second chain comprises at least 90% sequence identity to SEQ ID NO:2.

[0601] Embodiment 16. The isolated recombinant polypeptide complex according to any one of embodiments 1-13, wherein the second chain comprises at least 95% sequence identity to SEQ ID NO:2.

[0602] Embodiment 17. The isolated recombinant polypeptide complex according to any one of embodiments 1-13, wherein the second chain comprises at least 99% sequence identity to SEQ ID NO:2.

[0603] Embodiment 18. The isolated recombinant polypeptide complex according to any one of embodiments 1-17, wherein the second chain comprises the amino acid sequence according to SEQ ID NO:2.

[0604] Embodiment 19. The isolated recombinant polypeptide complex of embodiment 1, wherein the at least one disulfide bond is an intrachain disulfide bond formed between cysteine ​​22 and cysteine ​​96, between cysteine ​​138 and cysteine ​​148, between cysteine ​​199 and cysteine ​​275, between cysteine ​​339 and cysteine ​​407, between cysteine ​​454 and cysteine ​​519, or between cysteine ​​565 and cysteine ​​625 of the first chain.

[0605] Embodiment 20. The isolated recombinant polypeptide complex of embodiment 1, wherein the at least one disulfide bond is an intrachain disulfide bond formed by a pair of cysteine ​​residues of the second chain between cysteine ​​22 and cysteine ​​96, or between cysteine ​​150 and cysteine ​​206 of the second chain.

[0606] Embodiment 21. The isolated recombinant polypeptide complex of embodiment 1, wherein the at least one disulfide bond is an interchain disulfide bond between the first chain and the second chain formed between cysteine ​​645 of the first chain and cysteine ​​226 of the second chain.

[0607] Embodiment 22. The isolated recombinant polypeptide complex according to any one of the above embodiments, wherein the polypeptide comprises at least two disulfide bonds formed by pairs of cysteine ​​residues.

[0608] Embodiment 23. The isolated recombinant polypeptide complex according to any of the above embodiments, wherein the polypeptide comprises at least three disulfide bonds formed by pairs of cysteine ​​residues

[0609] Embodiment 24. The isolated recombinant polypeptide complex according to any of the above embodiments, wherein the polypeptide comprises at least four disulfide bonds formed by pairs of cysteine ​​residues

[0610] Embodiment 25. The isolated recombinant polypeptide complex according to any of the above embodiments, wherein the polypeptide comprises at least five disulfide bonds formed by pairs of cysteine ​​residues

[0611] Embodiment 26. The isolated recombinant polypeptide complex according to any of the above embodiments, wherein the polypeptide comprises at least six disulfide bonds formed by pairs of cysteine ​​residues.

[0612] Embodiment 27. The isolated recombinant polypeptide complex according to any of the above embodiments, wherein the polypeptide comprises at least seven disulfide bonds formed by pairs of cysteine ​​residues.

[0613] Embodiment 28. The isolated recombinant polypeptide complex according to any of the above embodiments, wherein the polypeptide comprises at least eight disulfide bonds formed by pairs of cysteine ​​residues.

[0614] Embodiment 29. The isolated recombinant polypeptide complex according to any of the above embodiments, wherein the cysteine ​​residue pair comprises cysteine ​​22 and cysteine ​​96 of SEQ ID NO:1.

[0615] Embodiment 30. The isolated recombinant polypeptide complex according to any of the above embodiments, wherein the cysteine ​​residue pair comprises cysteine ​​138 and cysteine ​​148 of SEQ ID NO:1.

[0616] Embodiment 31. The isolated recombinant polypeptide complex according to any of the above embodiments, wherein the cysteine ​​residue pair comprises cysteine ​​199 and cysteine ​​275 of SEQ ID NO:1.

[0617] Embodiment 32. The isolated recombinant polypeptide complex according to any of the above embodiments, wherein the cysteine ​​residue pair comprises cysteine ​​339 and cysteine ​​407 of SEQ ID NO:1.

[0618] Embodiment 33. The isolated recombinant polypeptide complex according to any of the above embodiments, wherein the cysteine ​​residue pair comprises cysteine ​​454 and cysteine ​​519 of SEQ ID NO:1.

[0619] Embodiment 34. The isolated recombinant polypeptide complex according to any of the above embodiments, wherein the cysteine ​​residue pair comprises cysteine ​​565 and cysteine ​​625 of SEQ ID NO:1.

[0620] Embodiment 35. The isolated recombinant polypeptide complex according to any of the above embodiments, wherein the cysteine ​​residue pair comprises cysteine ​​645 of SEQ ID NO:1 and cysteine ​​226 of SEQ ID NO:2.

[0621] Embodiment 36. The isolated recombinant polypeptide complex according to any of the above embodiments, wherein the cysteine ​​residue pair comprises cysteine ​​150 and cysteine ​​206 of SEQ ID NO:2.

[0622] Embodiment 37. The isolated recombinant polypeptide complex according to any one of the above embodiments, wherein the cysteine ​​residue pair comprises cysteine ​​22 and cysteine ​​96 of SEQ ID NO:2.

[0623] Embodiment 38. The isolated recombinant polypeptide complex according to any of the above embodiments, wherein at least one cysteine ​​residue is a free thiol group.

[0624] Embodiment 39. The isolated recombinant polypeptide complex according to any of the above embodiments, wherein at least one cysteine ​​residue is a free sulfhydryl group, and at least one cysteine ​​is selected from and corresponds to cysteine ​​22, cysteine ​​96, cysteine ​​138, cysteine ​​148, cysteine ​​199, cysteine ​​275, cysteine ​​339, cysteine ​​407, cysteine ​​454, cysteine ​​519, cysteine ​​565, cysteine ​​625 and cysteine ​​645 of SEQ ID NO:1.

[0625] Embodiment 40. The isolated recombinant polypeptide complex according to any of the above embodiments, wherein at least one cysteine ​​residue is a free sulfhydryl group, and at least one cysteine ​​is selected from and corresponds to cysteine ​​22, cysteine ​​96, cysteine ​​150, cysteine ​​226 and cysteine ​​206 of SEQ ID NO:2.

[0626] Embodiment 41. The isolated recombinant polypeptide complex according to any of the above embodiments, wherein at least one cysteine ​​residue pair comprises cysteine ​​22 and cysteine ​​96 of SEQ ID NO: 1, cysteine ​​138 and cysteine ​​148 of SEQ ID NO: 1, cysteine ​​199 and cysteine ​​275 of SEQ ID NO: 1, cysteine ​​454 and cysteine ​​519 of SEQ ID NO: 1, cysteine ​​565 and cysteine ​​625 of SEQ ID NO: 1, cysteine ​​22 and cysteine ​​96 of SEQ ID NO: 2, cysteine ​​150 and cysteine ​​206 of SEQ ID NO: 2, and cysteine ​​645 of SEQ ID NO: 1 and cysteine ​​226 of SEQ ID NO: 2.

[0627] Embodiment 42. The isolated recombinant polypeptide complex according to any of the above embodiments, wherein the secondary structure composition comprises β-sheets and random coils.

[0628] Embodiment 43. The isolated recombinant polypeptide complex according to any of the above embodiments, having a melting temperature (Tm) between about 71°C and about 81°C.

[0629] Embodiment 44. The isolated recombinant polypeptide complex according to any of the above embodiments, having a melting temperature (Tm) between about 71.4°C and about 79.5°C.

[0630] Embodiment 45. The isolated recombinant polypeptide complex according to any of the above embodiments, having a melting temperature (Tm) between about 65°C and about 85°C.

[0631] Embodiment 46. The isolated recombinant polypeptide complex according to any of the above embodiments, having a far-UV circular dichroism peak at a wavelength less than or equal to 195 nm.

[0632] Embodiment 47. The isolated recombinant polypeptide complex according to any of the above embodiments, having a far-UV circular dichroism peak at a wavelength less than or equal to 205 nm.

[0633] Embodiment 48. The isolated recombinant polypeptide complex according to any of the above embodiments, having a far-UV circular dichroism dip at a wavelength less than or equal to 220 nm.

[0634] Embodiment 49. The isolated recombinant polypeptide complex according to any of the above embodiments, having a near-UV circular dichroism peak at a wavelength less than or equal to 273 nm.

[0635] Embodiment 50. The isolated recombinant polypeptide complex according to any of the above embodiments, having a near-UV circular dichroism peak at a wavelength less than or equal to 279 nm.

[0636] Embodiment 51. The isolated recombinant polypeptide complex according to any of the above embodiments, having a near-UV circular dichroism peak at a wavelength less than or equal to 290 nm.

[0637] Embodiment 52. The isolated recombinant polypeptide complex according to any of the above embodiments, having a near-UV circular dichroism peak at a wavelength less than or equal to 295 nm.

[0638] Embodiment 53. A method for treating cancer, comprising administering the isolated recombinant polypeptide complex according to any of the above embodiments to a subject in need thereof.

[0639] Embodiment 54. A plurality of isolated recombinant polypeptide complexes comprising a first chain having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1 and a second chain having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2, wherein the plurality comprises greater than 90% of the monomers of the isolated recombinant polypeptide complexes.

[0640] Embodiment 55. The plurality of isolated recombinant polypeptide complexes of embodiment 54, comprising greater than 95% monomers.

[0641] Embodiment 56. The plurality of isolated recombinant polypeptide complexes of embodiment 55, comprising greater than 99% monomers.

[0642] Embodiment 57. The plurality of isolated recombinant polypeptide complexes of any one of Embodiments 54-56, having a concentration greater than or equal to 1.0 mg / mL.

[0643] Embodiment 58. The plurality of recombinant polypeptides of any one of Embodiments 54-57, having a concentration of at least 2.0 mg / mL.

[0644] Embodiment 59. The plurality of isolated recombinant polypeptide complexes of any one of Embodiments 54-57, having a pH greater than or equal to 5.0.

[0645] Embodiment 60. The plurality of isolated recombinant polypeptide complexes of embodiment 54, comprising greater than 90% monomers at a concentration greater than or equal to 2.0 mg / mL and a pH of 6.3.

[0646] Embodiment 61. A method for treating prostate cancer, comprising administering to a subject in need thereof an antibody or antigen binding fragment comprising a CD3 binding domain and a PSMA binding domain, wherein the antibody or antibody binding fragment is administered to the subject once a week at a dose of at least 0.1 μg / kg.

[0647] Embodiment 62. The method of Embodiment 61, wherein the antibody or antigen-binding fragment is administered to the subject at a dose of at least 0.1 mg / kg.

[0648] Embodiment 63. The method of any one of Embodiments 61-62, wherein the antibody or antigen-binding fragment is administered to the subject at a dose of at least 0.3 mg / kg.

[0649] Embodiment 64. The method of any one of Embodiments 61-63, wherein the antibody or antigen-binding fragment is administered to the subject at a dose of at least 1.0 mg / kg.

[0650] Embodiment 65. The method of any one of Embodiments 61-64, wherein the antibody or antigen-binding fragment is administered to the subject at a dose of at least 1.5 mg / kg.

[0651] Embodiment 66. The method of any of Embodiments 61-65, wherein the antibody or antigen-binding fragment is administered to the subject according to the following treatment regimen: the antibody or antigen-binding fragment is administered to the subject at a dose of at least 0.1 μg / kg in the first week, the antibody or antigen-binding fragment is administered to the subject at a dose of at least 0.3 mg / kg in the second week, the antibody or antigen-binding fragment is administered to the subject at a dose of at least 1.5 mg / kg in the third week, followed by a 4-week non-treatment interval of the antibody or antigen-binding fragment.

[0652] Embodiment 67. The isolated recombinant polypeptide complex of any one of embodiments 1-52, wherein the isolated recombinant polypeptide complex provides a maximum plasma concentration (Cmax) in a subject within about 0.1 hour after intravenous administration.

[0653] Embodiment 68. The isolated recombinant polypeptide complex according to any one of embodiments 1-52, wherein the isolated recombinant polypeptide complex provides a Cmax of about 2500 to 3500 ng / ml in a subject within about 0.1 hour after intravenous administration of a dose of about 0.1 mg / kg.

[0654] Embodiment 69. The isolated recombinant polypeptide complex according to any one of embodiments 1-52, wherein the isolated recombinant polypeptide complex provides a Cmax of about 7500 to 10500 ng / ml in a subject within about 0.1 hour after intravenous administration of a dose of about 0.3 mg / kg.

[0655] Embodiment 70. The isolated recombinant polypeptide complex according to any one of embodiments 1-52, wherein the isolated recombinant polypeptide complex provides a Cmax of about 37500 to 52500 ng / ml in a subject within about 0.1 hour after intravenous administration of a dose of about 1.5 mg / kg.

[0656] Embodiment 71. The isolated recombinant polypeptide complex according to any one of embodiments 1-52, wherein the isolated recombinant polypeptide complex provides a half-maximal plasma concentration in a subject within about 60 to about 120 hours (T1 / 2) after intravenous administration.

[0657] Embodiment 72. The isolated recombinant polypeptide complex of embodiment 67, wherein the value of Cmax is related to the dose of the isolated recombinant polypeptide complex administered.

[0658] Embodiment 73. The isolated recombinant polypeptide complex of embodiment 72, wherein the value of Cmax is proportional to the dose of the isolated recombinant polypeptide complex administered.

[0659] Embodiment 74. The isolated recombinant polypeptide complex according to any one of embodiments 1-52, wherein less than 1% of the isolated recombinant polypeptide complex is degraded in the serum of the subject per day.

[0660] Embodiment 75. The isolated recombinant polypeptide complex of Embodiment 74, wherein the subject is diagnosed with cancer.

[0661] Embodiment 76. The isolated recombinant polypeptide complex of Embodiment 75, wherein the cancer comprises prostate cancer.

[0662] Embodiment 77. The isolated recombinant polypeptide complex of Embodiment 75, wherein the cancer comprises metastatic castration-resistant prostate cancer (mCRPC).

[0663] Embodiment 78. A method for treating cancer, comprising administering to a subject in need thereof an effective amount of the isolated recombinant polypeptide complex according to any one of embodiments 1-52 and 67-77.

[0664] Embodiment 79. The method of embodiment 78, comprising administering to the subject the isolated recombinant polypeptide complex in a first dose and a second dose, wherein the second dose is equal to or greater than the first dose, wherein the first dose or the second dose is at least 100 μg.

[0665] Embodiment 80. The method of embodiment 78, comprising administering to the subject the isolated recombinant polypeptide complex in a first dose, a second dose, and a third dose, wherein the second dose is equal to or greater than the first dose, and the third dose is equal to or greater than the second dose, wherein the first dose, the second dose, or the third dose is at least 100 μg.

[0666] Embodiment 81. The method of Embodiment 79, wherein the method comprises a first treatment course and a second treatment course, wherein the first dose is administered to the subject during the first treatment course, and wherein the second dose is administered to the subject during the second treatment course.

[0667] Embodiment 82. The method of Embodiment 80, wherein the method comprises a 21 day treatment course, wherein the first dose is administered to the subject during the first week of the treatment course, wherein the second dose is administered to the subject during the second week of the treatment course, wherein the third dose is administered to the subject during the third week of the treatment course.

[0668] Embodiment 83. The method of Embodiment 82, wherein the method comprises a first 21 day treatment course and a second 21 day treatment course, wherein the first dose of the second 21 day treatment course is equal to or higher than the first dose of the first 21 day treatment course, wherein the second dose of the second 21 day treatment course is equal to or higher than the second dose of the first 21 day treatment course, wherein the third dose of the second 21 day treatment course is equal to or higher than the third dose of the first 21 day treatment course.

[0669] Embodiment 84. The method of embodiment 78, wherein the method comprises a 21-day treatment cycle comprising:

[0670] (a) administering the isolated recombinant polypeptide complex to the subject at a first dose in the first week of the treatment cycle;

[0671] (b) administering the isolated recombinant polypeptide complex to the subject at a second dose in the second week of the treatment cycle; and

[0672] (c) administering the isolated recombinant polypeptide complex to the subject at a third dose in the third week of the treatment cycle.

[0673] Embodiment 85. The method of Embodiment 84, wherein the method comprises:

[0674] (a) administering to a subject an isolated recombinant polypeptide complex at a first dose on the first day of a 21-day treatment cycle;

[0675] (b) administering to the subject the isolated recombinant polypeptide complex at a second dose on day eight of the 21-day treatment cycle; and

[0676] (c) on day 15 of the 21-day treatment cycle, administering the isolated recombinant polypeptide complex to the subject at a third dose.

[0677] Embodiment 86. The method of embodiment 84 or 85, wherein the treatment cycle is repeated 6 times within 18 weeks.

[0678] Embodiment 87. The method of any one of Embodiments 84 to 86, wherein the first dose, the second dose, or the third dose is at least 100 μg.

[0679] Embodiment 88. The method of any one of Embodiments 84 to 87, wherein the second dose is equal to or higher than the first dose.

[0680] Embodiment 89. The method of any one of Embodiments 84 to 88, wherein the third dose is equal to or higher than the second dose.

[0681] Embodiment 90. The method of any one of Embodiments 84 to 89, wherein the third dose is equal to or higher than the first dose.

[0682] Embodiment 91. The method of any one of Embodiments 84 to 90, wherein the method comprises a first 21 day treatment cycle and a second 21 day treatment cycle, wherein the first dose of the second 21 day treatment cycle is equal to or higher than the first dose of the first 21 day treatment cycle, wherein the second dose of the second 21 day treatment cycle is equal to or higher than the second dose of the first 21 day treatment cycle, wherein the third dose of the second 21 day treatment cycle is equal to or higher than the third dose of the first 21 day treatment cycle.

[0683] Embodiment 92. The method of any one of Embodiments 78-91, wherein administering comprises administering by intravenous infusion.

[0684] Embodiment 93. The method of any one of Embodiments 78-92, wherein the cancer comprises prostate cancer.

[0685] Embodiment 94. The method of any one of Embodiments 78-92, wherein the cancer comprises mCRPC.

[0686] Embodiment 95. The method of Embodiment 92, further comprising treating the subject with a therapy for an infusion-related reaction prior to administration.

[0687] Embodiment 96. The method of Embodiment 95, wherein the therapy for the infusion-related reaction comprises an antipyretic, an antihistamine, an antiemetic, or a corticosteroid.

[0688] Embodiment 97. The method of Embodiment 95 or 96, wherein the therapy for the infusion-related reaction comprises acetaminophen, paracetamol, or diphenhydramine.

[0689] Embodiment 98. The method of Embodiment 84, further comprising treating the subject with a corticosteroid prior to step (a).

[0690] Embodiment 99. The method of Embodiment 85, further comprising treating the subject with a corticosteroid prior to step (a).

[0691] Embodiment 100. The method of Embodiment 84, further comprising treating the subject with a therapy for cytokine release syndrome (CRS) prior to or after the administering.

[0692] Embodiment 101. The method of embodiment 100, wherein the therapy for CRS comprises an intravenous fluid program, oxygen therapy, corticosteroids, immunosuppressants, vasopressors, or anti-epileptic drugs.

[0693] Embodiment 102. The method of Embodiment 101, wherein the oxygen therapy comprises mechanical ventilation.

[0694] Embodiment 103. The method of embodiment 101, wherein the immunosuppressant comprises an IL-6 receptor inhibitor.

[0695] Embodiment 104. The method of Embodiment 103, wherein the IL-6 receptor inhibitor comprises tocilizumab.

[0696] Embodiment 105. The method of embodiment 100, wherein the therapy for CRS comprises high-dose corticosteroids.

[0697] Embodiment 106. The method of embodiment 78, wherein after administration the separated recombinant polypeptide complex is cleaved by a tumor-specific protease to generate an enzyme product of the separated recombinant polypeptide complex.

[0698] Embodiment 107. The method of embodiment 106, wherein the tumor-specific protease comprises two or more proteases, wherein the separated recombinant polypeptide complex is cleaved by a first protease of the two or more proteases to generate a first metabolite of the separated recombinant polypeptide complex, and wherein the separated recombinant polypeptide complex is cleaved by a second protease of the two or more proteases to generate a second metabolite of the separated recombinant polypeptide complex.

[0699] Embodiment 108. The method of Embodiment 106 or 107, wherein the first protease comprises a serine protease and wherein the second protease comprises a matrix metalloprotease.

[0700] Embodiment 109. The method of Embodiment 108, wherein the serine protease comprises recombinant human membrane serine protease (MTSP1), and wherein the matrix metalloprotease comprises recombinant human matrix metalloprotease 9 (MMP9).

[0701] Embodiment 120. The method of any one of Embodiments 107-109, wherein the first metabolite comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:5.

[0702] Embodiment 111. The method of any one of Embodiments 107-120, wherein the second metabolite comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:6.

[0703] Embodiment 112. The isolated recombinant polypeptide complex according to any one of embodiments 67-77 or the method according to any one of embodiments 78-111, wherein the subject is a non-human primate or a human.

[0704] Embodiment 113. The method of Embodiment 112, wherein the non-human primate is a cynomolgus monkey.

[0705] Embodiment 114. A pharmaceutical composition comprising: (a) the isolated recombinant polypeptide complex according to any one of embodiments 1-52 and 67-77; and (b) a pharmaceutically acceptable excipient.

[0706] Embodiment 115. The pharmaceutical composition of embodiment 114, wherein the pharmaceutically acceptable excipient comprises a buffer, a stabilizer, a tonicity agent, a surfactant, or a combination thereof.

[0707] Embodiment 116. The pharmaceutical composition of Embodiment 115, wherein the buffer comprises an amino acid or a derivative thereof.

[0708] Embodiment 117. The pharmaceutical composition of Embodiment 116, wherein the amino acid or its derivative comprises L-histidine, L-histidine monohydrochloride monohydrate, or a combination thereof.

[0709] Embodiment 118. The pharmaceutical composition of Embodiment 115, wherein the stabilizer comprises a sugar.

[0710] Embodiment 119. The pharmaceutical composition of Embodiment 118, wherein the sugar comprises sucrose.

[0711] Embodiment 120. The pharmaceutical composition of Embodiment 115, wherein the tonicity agent comprises a sugar.

[0712] Embodiment 121. The pharmaceutical composition of Embodiment 120, wherein the sugar comprises sucrose.

[0713] Embodiment 122. The pharmaceutical composition of Embodiment 115, wherein the surfactant comprises polysorbate 20.

[0714] Embodiment 123. The pharmaceutical composition of embodiment 117, wherein the total amount of L-histidine in the pharmaceutical composition is about 10 mM, in the form of L-histidine and L-histidine monohydrochloride monohydrate.

[0715] Embodiment 124. The pharmaceutical composition of Embodiment 123, wherein the molar ratio of L-histidine to L-histidine monohydrochloride monohydrate is about 3:2.

[0716] Embodiment 125. The pharmaceutical composition of Embodiment 119 or 121, comprising about 8% (w / v) sucrose.

[0717] Embodiment 126. The pharmaceutical composition of Embodiment 122, comprising at least 0.01% (w / v) polysorbate 20.

[0718] Embodiment 127. The pharmaceutical composition of any one of Embodiments 114-126, comprising about 6 mM L-histidine, about 4 mM L-histidine monohydrochloride monohydrate, about 8% (w / v) sucrose, and about 0.01% (w / v) polysorbate 20.

[0719] Embodiment 128. The pharmaceutical composition of any one of Embodiments 114-127, comprising about 2 mg / ml of the isolated recombinant polypeptide complex.

[0720] Embodiment 129. The pharmaceutical composition of any one of Embodiments 114-128, wherein the pharmaceutical composition comprises a pH between about 5 and about 7.

[0721] Embodiment 130. The pharmaceutical composition of any one of Embodiments 114-129, wherein the pharmaceutical composition comprises a pH of about 6.3.

[0722] Embodiment 131. An isolated polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 5, and wherein the isolated polypeptide is 484 amino acids in length.

[0723] Embodiment 132. An isolated polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6, and wherein the isolated polypeptide is 476 amino acids in length.

[0724] Embodiment 133. A pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, the isolated polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 5, and wherein the isolated polypeptide is 484 amino acids in length.

[0725] Embodiment 134. A pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, the isolated polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 6, and wherein the isolated polypeptide is 476 amino acids in length.

[0726] Embodiment 135. A method for treating cancer, comprising administering an effective amount of the pharmaceutical composition according to any one of Embodiments 114-130 to a subject in need thereof.

[0727] Embodiment 136. The method of Embodiment 135, wherein the cancer comprises mCRPC.

[0728] Embodiment 137. The method of Embodiment 135, wherein the subject is a human.

[0729]

[0730]

[0731]

[0732]

[0733]

[0734]

[0735]

[0736]

Claims

1. An isolated recombinant polypeptide complex comprising a first chain having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1 and a second chain having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2, wherein the isolated recombinant polypeptide complex comprises at least one of the following features: (a) at least one disulfide bond formed by a pair of cysteine ​​residues in the first chain or the second chain or by a pair of cysteine ​​residues in the first chain and the second chain; (b) secondary structure consisting of β-sheets or random coils; (c) at least one pyroglutamine in said second chain; (d) when the isolated recombinant polypeptide complex is formulated at a concentration of 1.0 mg / mL in a buffer comprising 10 mM histidine buffer, 8% (w / v) sucrose, 0.01% (w / v) polysorbate 20, pH 6.3, the melting temperature (T) between about 65°C and about 85°C m ) (e) a far-UV circular dichroism peak at a wavelength between 190 nm and 205 nm when the isolated recombinant polypeptide complex is formulated at a concentration of 0.1 mg / mL in 10 mM potassium phosphate buffer, pH 7.0; (f) a deep UV circular dichroism depression at a wavelength between 210 nm and 220 nm when the isolated recombinant polypeptide complex is formulated at a concentration of 0.1 mg / mL in 10 mM potassium phosphate buffer, pH 7.0; or (g) A near-UV circular dichroism peak at a wavelength between 250 nm and 300 nm when the isolated recombinant polypeptide complex is formulated at a concentration of 1.0 mg / mL in a buffer containing 10 mM histidine buffer, 8% (w / v) sucrose, 0.01% (w / v) polysorbate 20, pH 6.

3.

2. The isolated recombinant polypeptide complex according to claim 1, wherein the polypeptide comprises at least two of the characteristics.

3. The isolated recombinant polypeptide complex of claim 1, wherein the polypeptide comprises at least three of the characteristics.

4. The isolated recombinant polypeptide complex of claim 1, wherein the polypeptide comprises at least four of the characteristics.

5. The isolated recombinant polypeptide complex of claim 1, wherein the polypeptide comprises at least five of the characteristics.

6. The isolated recombinant polypeptide complex of claim 1, wherein the polypeptide comprises at least six of the characteristics.

7. The isolated recombinant polypeptide complex of claim 1, wherein the polypeptide comprises at least seven of the characteristics.

8. The isolated recombinant polypeptide complex of claim 1, wherein the polypeptide comprises at least eight of the features.

9. The isolated recombinant polypeptide complex of claim 1, wherein the first chain comprises at least 85% sequence identity to SEQ ID NO:

1.

10. The isolated recombinant polypeptide complex of claim 1, wherein the first chain comprises at least 90% sequence identity to SEQ ID NO:

1.

11. The isolated recombinant polypeptide complex of claim 1, wherein the first chain comprises at least 95% sequence identity to SEQ ID NO:

1.

12. The isolated recombinant polypeptide complex of claim 1, wherein the first chain comprises at least 99% sequence identity to SEQ ID NO:

1.

13. The isolated recombinant polypeptide complex according to claim 1, wherein the first chain comprises the amino acid sequence according to SEQ ID NO:

1.

14. The isolated recombinant polypeptide complex of claim 1, wherein the second chain comprises at least 85% sequence identity to SEQ ID NO:

2.

15. The isolated recombinant polypeptide complex of claim 1, wherein the second chain comprises at least 90% sequence identity to SEQ ID NO:

2.

16. The isolated recombinant polypeptide complex of claim 1, wherein the second chain comprises at least 95% sequence identity to SEQ ID NO:

2.

17. The isolated recombinant polypeptide complex of claim 1, wherein the second chain comprises at least 99% sequence identity to SEQ ID NO:

2.

18. The isolated recombinant polypeptide complex according to claim 1, wherein the second chain comprises the amino acid sequence according to SEQ ID NO:

2.

19. The isolated recombinant polypeptide complex of claim 1, wherein the at least one disulfide bond is an intrachain disulfide bond formed between cysteine ​​22 and cysteine ​​96, between cysteine ​​138 and cysteine ​​148, between cysteine ​​199 and cysteine ​​275, between cysteine ​​339 and cysteine ​​407, between cysteine ​​454 and cysteine ​​519, or between cysteine ​​565 and cysteine ​​625 of the first chain.

20. The isolated recombinant polypeptide complex according to claim 1, wherein the at least one disulfide bond is an intrachain disulfide bond formed by a pair of cysteine ​​residues of the second chain between cysteine ​​22 and cysteine ​​96, or between cysteine ​​150 and cysteine ​​206 of the second chain.

21. The isolated recombinant polypeptide complex of claim 1, wherein the at least one disulfide bond is an interchain disulfide bond between the first chain and the second chain formed between cysteine ​​645 of the first chain and cysteine ​​226 of the second chain.

22. The isolated recombinant polypeptide complex of claim 1, wherein the polypeptide comprises at least two disulfide bonds formed by pairs of cysteine ​​residues.

23. The isolated recombinant polypeptide complex of claim 1, wherein the polypeptide comprises at least three disulfide bonds formed by pairs of cysteine ​​residues.

24. The isolated recombinant polypeptide complex of claim 1, wherein the polypeptide comprises at least four disulfide bonds formed by pairs of cysteine ​​residues.

25. The isolated recombinant polypeptide complex of claim 1, wherein the polypeptide comprises at least five disulfide bonds formed by pairs of cysteine ​​residues.

26. The isolated recombinant polypeptide complex of claim 1, wherein the polypeptide comprises at least six disulfide bonds formed by pairs of cysteine ​​residues.

27. The isolated recombinant polypeptide complex of claim 1, wherein the polypeptide comprises at least seven disulfide bonds formed by pairs of cysteine ​​residues.

28. The isolated recombinant polypeptide complex of claim 1, wherein the polypeptide comprises at least eight disulfide bonds formed by pairs of cysteine ​​residues.

29. The isolated recombinant polypeptide complex of claim 1, wherein the pair of cysteine ​​residues comprises cysteine ​​22 and cysteine ​​96 of SEQ ID NO:

1.

30. The isolated recombinant polypeptide complex of claim 1, wherein the pair of cysteine ​​residues comprises cysteine ​​138 and cysteine ​​148 of SEQ ID NO:

1.

31. The isolated recombinant polypeptide complex of claim 1, wherein the pair of cysteine ​​residues comprises cysteine ​​199 and cysteine ​​275 of SEQ ID NO:

1.

32. The isolated recombinant polypeptide complex of claim 1, wherein the pair of cysteine ​​residues comprises cysteine ​​339 and cysteine ​​407 of SEQ ID NO:

1.

33. The isolated recombinant polypeptide complex of claim 1, wherein the cysteine ​​residue pair comprises cysteine ​​454 and cysteine ​​519 of SEQ ID NO:

1.

34. The isolated recombinant polypeptide complex of claim 1, wherein the pair of cysteine ​​residues comprises cysteine ​​565 and cysteine ​​625 of SEQ ID NO:

1.

35. The isolated recombinant polypeptide complex of claim 1, wherein the pair of cysteine ​​residues comprises cysteine ​​645 of SEQ ID NO: 1 and cysteine ​​226 of SEQ ID NO:

2.

36. The isolated recombinant polypeptide complex of claim 1, wherein the pair of cysteine ​​residues comprises cysteine ​​150 and cysteine ​​206 of SEQ ID NO:

2.

37. The isolated recombinant polypeptide complex of claim 1, wherein the pair of cysteine ​​residues comprises cysteine ​​22 and cysteine ​​96 of SEQ ID NO:

2.

38. The isolated recombinant polypeptide complex of claim 1, wherein at least one cysteine ​​residue is a free sulfhydryl group.

39. The isolated recombinant polypeptide complex of claim 1, wherein at least one cysteine ​​residue is a free sulfhydryl group and at least one cysteine ​​is selected from and corresponds to cysteine ​​22, cysteine ​​96, cysteine ​​138, cysteine ​​148, cysteine ​​199, cysteine ​​275, cysteine ​​339, cysteine ​​407, cysteine ​​454, cysteine ​​519, cysteine ​​565, cysteine ​​625 and cysteine ​​645 of SEQ ID NO:

1.

40. The isolated recombinant polypeptide complex of claim 1, wherein at least one cysteine ​​residue is a free sulfhydryl group and at least one cysteine ​​is selected from the group consisting of and corresponding to cysteine ​​22, cysteine ​​96, cysteine ​​150, cysteine ​​226, and cysteine ​​206 of SEQ ID NO:

2.

41. The isolated recombinant polypeptide complex of claim 1, wherein the at least one cysteine ​​residue pair comprises cysteine ​​22 and cysteine ​​96 of SEQ ID NO: 1, cysteine ​​138 and cysteine ​​148 of SEQ ID NO: 1, cysteine ​​199 and cysteine ​​275 of SEQ ID NO: 1, cysteine ​​454 and cysteine ​​519 of SEQ ID NO: 1, cysteine ​​565 and cysteine ​​625 of SEQ ID NO: 1, cysteine ​​22 and cysteine ​​96 of SEQ ID NO: 2, cysteine ​​150 and cysteine ​​206 of SEQ ID NO: 2, and cysteine ​​645 of SEQ ID NO: 1 and cysteine ​​226 of SEQ ID NO:

2.

42. The isolated recombinant polypeptide complex of claim 1, wherein the secondary structure composition comprises β-sheets and random coils.

43. The isolated recombinant polypeptide complex of claim 1, having a melting temperature (T m ).

44. The isolated recombinant polypeptide complex of claim 1, having a melting temperature (T) between about 71.4°C and about 79.5°C. m ).

45. The isolated recombinant polypeptide complex according to claim 1, having a melting temperature (T) between about 65°C and about 85°C. m ).

46. ​​The isolated recombinant polypeptide complex of claim 1, having a far-UV circular dichroism peak at a wavelength less than or equal to 195 nm.

47. The isolated recombinant polypeptide complex of claim 1, having a far-UV circular dichroism peak at a wavelength less than or equal to 205 nm.

48. The isolated recombinant polypeptide complex of claim 1, having a far-UV circular dichroism dip at a wavelength less than or equal to 220 nm.

49. The isolated recombinant polypeptide complex of claim 1, having a near-UV circular dichroism peak at a wavelength less than or equal to 273 nm.

50. The isolated recombinant polypeptide complex of claim 1, having a near-UV circular dichroism peak at a wavelength less than or equal to 279 nm.

51. The isolated recombinant polypeptide complex of claim 1, having a near-UV circular dichroism peak at a wavelength less than or equal to 290 nm.

52. The isolated recombinant polypeptide complex of claim 1, having a near-UV circular dichroism peak at a wavelength less than or equal to 295 nm.

53. A method for treating cancer, comprising administering the isolated recombinant polypeptide complex according to claim 1 to a subject in need thereof.

54. A plurality of isolated recombinant polypeptide complexes comprising a first chain having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1 and a second chain having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 2, wherein the plurality comprises greater than 90% of the monomers of the isolated recombinant polypeptide complexes.

55. The plurality of isolated recombinant polypeptide complexes of claim 54, comprising greater than 95% monomers.

56. The plurality of isolated recombinant polypeptide complexes of claim 55, comprising greater than 99% monomers.

57. The plurality of isolated recombinant polypeptide complexes of claim 54, having a concentration greater than or equal to 1.0 mg / mL.

58. The plurality of recombinant polypeptides of claim 54, having a concentration of at least 2.0 mg / mL.

59. The plurality of isolated recombinant polypeptide complexes of claim 54, having a pH greater than or equal to 5.

0.

60. The plurality of isolated recombinant polypeptide complexes of claim 54, comprising greater than 90% monomers at a concentration greater than or equal to 2.0 mg / mL and a pH of 6.

3.

61. A method for treating prostate cancer, comprising administering to a subject in need thereof an antibody or antigen binding fragment comprising a CD3 binding domain and a PSMA binding domain, wherein the antibody or antibody binding fragment is administered to the subject once a week at a dose of at least 0.1 μg / kg.

62. The method of claim 61, wherein the antibody or antigen-binding fragment is administered to the subject at a dose of at least 0.1 mg / kg.

63. The method of claim 61, wherein the antibody or antigen-binding fragment is administered to the subject at a dose of at least 0.3 mg / kg.

64. The method of claim 61, wherein the antibody or antigen-binding fragment is administered to the subject at a dose of at least 1.0 mg / kg.

65. The method of claim 61, wherein the antibody or antigen-binding fragment is administered to the subject at a dose of at least 1.5 mg / kg.

66. The method of claim 61, wherein the antibody or antigen-binding fragment is administered to the subject according to the following treatment regimen: the antibody or antigen-binding fragment is administered to the subject at a dose of at least 0.1 μg / kg in the first week, the antibody or antigen-binding fragment is administered to the subject at a dose of at least 0.3 mg / kg in the second week, the antibody or antigen-binding fragment is administered to the subject at a dose of at least 1.5 mg / kg in the third week, followed by a 4-week non-treatment interval of the antibody or antigen-binding fragment.

67. The isolated recombinant polypeptide complex of claim 1, wherein the isolated recombinant polypeptide complex provides a maximum plasma concentration (Cmax) in a subject within about 0.1 hour following intravenous administration.

68. The isolated recombinant polypeptide complex of claim 1, wherein the isolated recombinant polypeptide complex provides a Cmax of about 2500 to 3500 ng / ml in a subject within about 0.1 hour following intravenous administration of a dose of about 0.1 mg / kg.

69. The isolated recombinant polypeptide complex of claim 1, wherein the isolated recombinant polypeptide complex provides a Cmax of about 7500 to 10500 ng / ml in a subject within about 0.1 hour following intravenous administration of a dose of about 0.3 mg / kg.

70. The isolated recombinant polypeptide complex of claim 1, wherein the isolated recombinant polypeptide complex provides a Cmax of about 37,500 to 52,500 ng / ml in a subject within about 0.1 hour following intravenous administration of a dose of about 1.5 mg / kg.

71. The isolated recombinant polypeptide complex of claim 1, wherein the isolated recombinant polypeptide complex is about 60 to about 120 hours (T) after intravenous administration. 1 / 2 ) provides half-maximal plasma concentration in subjects.

72. The isolated recombinant polypeptide complex of claim 67, wherein the value of Cmax is related to the dose of the isolated recombinant polypeptide complex administered.

73. The isolated recombinant polypeptide complex of claim 72, wherein the value of Cmax is proportional to the dose of the isolated recombinant polypeptide complex administered.

74. The isolated recombinant polypeptide complex of claim 1, wherein less than 1% of the isolated recombinant polypeptide complex is degraded in the serum of a subject per day.

75. The isolated recombinant polypeptide complex of claim 74, wherein the subject is diagnosed with cancer.

76. The isolated recombinant polypeptide complex of claim 75, wherein the cancer comprises prostate cancer.

77. The isolated recombinant polypeptide complex of claim 75, wherein the cancer comprises metastatic castration-resistant prostate cancer (mCRPC).

78. A method for treating cancer, comprising administering an effective amount of the isolated recombinant polypeptide complex according to claim 1 to a subject in need thereof.

79. The method of claim 78, comprising administering to the subject the isolated recombinant polypeptide complex in a first dose and a second dose, wherein the second dose is equal to or greater than the first dose, wherein the first dose or the second dose is at least 100 μg.

80. The method of claim 78, comprising administering to the subject the isolated recombinant polypeptide complex in a first dose, a second dose, and a third dose, wherein the second dose is equal to or higher than the first dose, and the third dose is equal to or higher than the second dose, wherein the first dose, the second dose, or the third dose is at least 100 μg.

81. The method of claim 79, wherein the method comprises a first treatment course and a second treatment course, wherein the first dose is administered to the subject during the first treatment course, and wherein the second dose is administered to the subject during the second treatment course.

82. The method of claim 80, wherein the method comprises a 21 day treatment course, wherein the first dose is administered to the subject within a first week of the treatment course, wherein the second dose is administered to the subject within a second week of the treatment course, and wherein the third dose is administered to the subject within a third week of the treatment course.

83. The method of claim 82, wherein the method comprises a first 21 day treatment course and a second 21 day treatment course, wherein the first dose of the second 21 day treatment course is equal to or higher than the first dose of the first 21 day treatment course, wherein the second dose of the second 21 day treatment course is equal to or higher than the second dose of the first 21 day treatment course, wherein the third dose of the second 21 day treatment course is equal to or higher than the third dose of the first 21 day treatment course.

84. The method of claim 78, wherein the method comprises a 21 day treatment cycle comprising: (a) administering the isolated recombinant polypeptide complex to the subject at a first dose in the first week of the treatment cycle; (b) administering the isolated recombinant polypeptide complex to the subject at a second dose in a second week of the treatment cycle; and (c) administering the isolated recombinant polypeptide complex to the subject at a third dose in a third week of the treatment cycle.

85. The method of claim 84, wherein the method comprises: (a) administering the isolated recombinant polypeptide complex to the subject at the first dose on the first day of the 21-day treatment cycle; (b) administering the isolated recombinant polypeptide complex to the subject at the second dose on day eight of the 21-day treatment cycle; and (c) administering the isolated recombinant polypeptide complex to the subject at the third dose on day 15 of the 21-day treatment cycle.

86. The method of claim 84, wherein the treatment cycle is repeated 6 times within 18 weeks.

87. The method of claim 84, wherein the first dose, the second dose, or the third dose is at least 100 μg.

88. The method of claim 84, wherein the second dose is equal to or higher than the first dose.

89. The method of claim 84, wherein the third dose is equal to or higher than the second dose.

90. The method of claim 84, wherein the third dose is equal to or higher than the first dose.

91. The method of claim 84, wherein the method comprises a first 21 day treatment cycle and a second 21 day treatment cycle, wherein the first dose of the second 21 day treatment cycle is equal to or higher than the first dose of the first 21 day treatment cycle, wherein the second dose of the second 21 day treatment cycle is equal to or higher than the second dose of the first 21 day treatment cycle, wherein the third dose of the second 21 day treatment cycle is equal to or higher than the third dose of the first 21 day treatment cycle.

92. The method of claim 78, wherein said administering comprises administering by intravenous infusion.

93. The method of claim 78, wherein the cancer comprises prostate cancer.

94. The method of claim 78, wherein the cancer comprises mCRPC.

95. The method of claim 92, further comprising treating the subject with a therapy for an infusion-related reaction prior to administration.

96. The method of claim 95, wherein the therapy for an infusion-related reaction comprises an antipyretic, an antihistamine, an antiemetic, or a corticosteroid.

97. The method of claim 95, wherein the therapy for an infusion-related reaction comprises acetaminophen, paracetamol, or diphenhydramine.

98. The method of claim 84, further comprising treating the subject with a corticosteroid prior to step (a).

99. The method of claim 85, further comprising treating the subject with a corticosteroid prior to step (a).

100. The method of claim 84, further comprising treating the subject with a therapy for cytokine release syndrome (CRS) prior to or after the administering.

101. The method of claim 100, wherein the therapy for CRS comprises an intravenous fluid program, oxygen therapy, corticosteroids, immunosuppressants, vasopressors, or anti-epileptic drugs.

102. The method of claim 101, wherein the oxygen therapy comprises mechanical ventilation.

103. The method of claim 101, wherein the immunosuppressant comprises an IL-6 receptor inhibitor.

104. The method of claim 103, wherein the IL-6 receptor inhibitor comprises tocilizumab.

105. The method of claim 100, wherein the therapy for CRS comprises high-dose corticosteroids.

106. The method of claim 78, wherein following said administering said isolated recombinant polypeptide complex is cleaved by a tumor-specific protease to generate an enzyme product of said isolated recombinant polypeptide complex.

107. The method of claim 106, wherein the tumor-specific protease comprises two or more proteases, wherein the separated recombinant polypeptide complex is cleaved by a first protease of the two or more proteases to generate a first metabolite of the separated recombinant polypeptide complex, and wherein the separated recombinant polypeptide complex is cleaved by a second protease of the two or more proteases to generate a second metabolite of the separated recombinant polypeptide complex.

108. The method of claim 106, wherein the first protease comprises a serine protease, and wherein the second protease comprises a matrix metalloprotease.

109. The method of claim 108, wherein the serine protease comprises recombinant human membrane serine protease (MTSP1), wherein the matrix metalloproteinase comprises recombinant human matrix metalloproteinase 9 (MMP9).

110. The method of claim 107, wherein the first metabolite comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

5.

111. The method of claim 107, wherein the second metabolite comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

6.

112. The isolated recombinant polypeptide complex of claim 67, wherein the subject is a non-human primate or a human.

113. The method of claim 112, wherein the non-human primate is a cynomolgus monkey.

114. A pharmaceutical composition comprising (a) the isolated recombinant polypeptide complex according to claim 1; and (b) a pharmaceutically acceptable excipient.

115. The pharmaceutical composition of claim 114, wherein the pharmaceutically acceptable excipient comprises a buffer, a stabilizer, a tonicity agent, a surfactant, or a combination thereof.

116. The pharmaceutical composition of claim 115, wherein the buffer comprises an amino acid or a derivative thereof.

117. The pharmaceutical composition of claim 116, wherein the amino acid or derivative thereof comprises L-histidine, L-histidine monohydrochloride monohydrate, or a combination thereof.

118. The pharmaceutical composition of claim 115, wherein the stabilizer comprises a sugar.

119. The pharmaceutical composition of claim 118, wherein the sugar comprises sucrose.

120. The pharmaceutical composition of claim 115, wherein the tonicity agent comprises a sugar.

121. The pharmaceutical composition of claim 120, wherein the sugar comprises sucrose.

122. The pharmaceutical composition of claim 115, wherein the surfactant comprises polysorbate 20.

123. The pharmaceutical composition of claim 117, wherein the total amount of L-histidine in the pharmaceutical composition in the form of both L-histidine and L-histidine monohydrochloride monohydrate is about 10 mM.

124. The pharmaceutical composition of claim 123, wherein the molar ratio of L-histidine to L-histidine monohydrochloride monohydrate is about 3:

2.

125. The pharmaceutical composition of claim 119, comprising about 8% (w / v) sucrose.

126. The pharmaceutical composition of claim 122, comprising at least 0.01% (w / v) polysorbate 20.

127. The pharmaceutical composition of claim 114, comprising about 6 mM L-histidine, about 4 mM L-histidine monohydrochloride monohydrate, about 8% (w / v) sucrose, and about 0.01% (w / v) polysorbate 20.

128. The pharmaceutical composition of claim 114, comprising about 2 mg / ml of the isolated recombinant polypeptide complex.

129. The pharmaceutical composition of claim 114, wherein the pharmaceutical composition comprises a pH between about 5 and about 7.

130. The pharmaceutical composition of claim 114, wherein the pharmaceutical composition comprises a pH of about 6.

3.

131. An isolated polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:5, and wherein the isolated polypeptide is 484 amino acids in length.

132. An isolated polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6, and wherein the isolated polypeptide is 476 amino acids in length.

133. A pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:5, and wherein the isolated polypeptide is 484 amino acids in length.

134. A pharmaceutical composition comprising an isolated polypeptide and a pharmaceutically acceptable excipient, wherein the isolated polypeptide comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:6, and wherein the isolated polypeptide is 476 amino acids in length.

135. A method for treating cancer, comprising administering an effective amount of the pharmaceutical composition according to claim 114 to a subject in need thereof.

136. The method of claim 135, wherein the cancer comprises mCRPC.

137. The method of claim 135, wherein the subject is a human.

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