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

Abstract

Described herein are compositions and formulations that include the recombinant polypeptides. Also described herein are methods and uses relating to these compositions and formulations.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 327,322, filed April 4, 2022, and U.S. Provisional Patent Application No. 63 / 338,220, filed May 4, 2022, each of which is incorporated by reference in its entirety.

[0002] Sequence Listing This application has been submitted electronically in XML file format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. A copy of said XML, created on Mar. 24, 2023, is named 52426-743_601 SL.xml and is 24,891 bytes in size. Summary of the Invention

[0003] In one aspect, disclosed herein is an isolated recombinant polypeptide complex comprising a first chain having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:1, and a second chain having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:2, the isolated recombinant polypeptide complex having the following characteristics: (a) at least one disulfide bond formed by a pair of cysteine ​​residues in the first chain or the second chain, or a pair of cysteine ​​residues in the first chain and the second chain, (b) a secondary structure composition comprising a β-sheet or a random coil, (c) at least one pyroglutamine in the second chain, (d) a melting temperature (T) of about 65° C. to about 85° C. 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. 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 at 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 at 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 comprises at least two of the foregoing characteristics. In some embodiments, the polypeptide comprises at least three of the aforementioned features. In some embodiments, the polypeptide comprises at least four of the aforementioned features. In some embodiments, the polypeptide comprises at least five of the aforementioned features. In some embodiments, the polypeptide comprises at least six of the aforementioned features. In some embodiments, the polypeptide comprises at least seven of the aforementioned features. In some embodiments, the polypeptide comprises at least eight of the aforementioned features. In some embodiments, the first strand comprises at least 85% sequence identity to SEQ ID NO:1. In some embodiments, the first strand comprises at least 90% sequence identity to SEQ ID NO:1. In some embodiments, the first strand comprises at least 95% sequence identity to SEQ ID NO:1. In some embodiments, the first strand comprises at least 99% sequence identity to SEQ ID NO:1. In some embodiments, the first strand comprises the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the second strand comprises at least 85% sequence identity to SEQ ID NO:2. In some embodiments, the second strand comprises at least 90% sequence identity to SEQ ID NO:2. In some embodiments, the second strand 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 chain comprises the amino acid sequence set forth in SEQ ID NO:2.

[0004] In some embodiments, at least one disulfide bond is an intrachain disulfide bond formed between cysteine ​​22 and cysteine ​​96 of the first chain, 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 pair of cysteine ​​residues between cysteine ​​22 and cysteine ​​96 of the second chain, or between cysteine ​​150 and cysteine ​​206 of the second chain. In some embodiments, the at least one disulfide bond is an interchain disulfide bond formed between the first and second chains 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 pairs of cysteine ​​residues. In some embodiments, the polypeptide comprises at least three disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the polypeptide comprises at least four disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the polypeptide comprises at least five disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the polypeptide comprises at least six disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the polypeptide comprises at least seven disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the polypeptide comprises at least eight disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the pair of cysteine ​​residues comprises cysteine ​​22 and cysteine ​​96 of SEQ ID NO: 1. In some embodiments, the pair of cysteine ​​residues comprises cysteine ​​138 and cysteine ​​148 of SEQ ID NO: 1. In some embodiments, the pair of cysteine ​​residues comprises cysteine ​​199 and cysteine ​​275 of SEQ ID NO: 1.In some embodiments, the pair of cysteine ​​residues comprises cysteine ​​339 and cysteine ​​407 of SEQ ID NO:1. In some embodiments, the pair of cysteine ​​residues comprises cysteine ​​454 and cysteine ​​519 of SEQ ID NO:1. In some embodiments, the pair of cysteine ​​residues comprises cysteine ​​565 and cysteine ​​625 of SEQ ID NO:1. In some embodiments, the pair of cysteine ​​residues comprises cysteine ​​645 and cysteine ​​226 of SEQ ID NO:2. In some embodiments, the pair of cysteine ​​residues comprises cysteine ​​150 and cysteine ​​206 of SEQ ID NO:2. In some embodiments, the pair of cysteine ​​residues comprises cysteine ​​22 and cysteine ​​96 of SEQ ID NO:2. In some embodiments, at least one cysteine ​​residue is a free sulfhydryl. In some embodiments, at least one cysteine ​​residue is a free sulfhydryl, 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. In some embodiments, at least one cysteine ​​residue is a free sulfhydryl, 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. In some embodiments, at least one pair of cysteine ​​residues includes cysteine ​​22 and cysteine ​​96 of SEQ ID NO:1, cysteine ​​138 and cysteine ​​148 of SEQ ID NO:1, cysteines 199 and 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.

[0005] In some embodiments, the secondary structural composition comprises β-sheet and random coil. In some embodiments, the isolated recombinant polypeptide complex has a melting temperature (T) of about 71° C. to about 81° C. m In some embodiments, the isolated recombinant polypeptide complex has a melting temperature (T) of about 71.4° C. to about 79.5° C. m In some embodiments, the isolated recombinant polypeptide complex has a melting temperature (T) of about 65° C. to about 85° C. m In some embodiments, the isolated recombinant polypeptide complex has a far-UV circular dichroism peak at a wavelength of 195 nm or less. In some embodiments, the isolated recombinant polypeptide complex has a far-UV circular dichroism peak at a wavelength of 205 nm or less. In some embodiments, the isolated recombinant polypeptide complex has a far-UV circular dichroism dip at a wavelength of 220 nm or less. In some embodiments, the isolated recombinant polypeptide complex has a near-UV circular dichroism peak at a wavelength of 273 nm or less. In some embodiments, the isolated recombinant polypeptide complex has a near-UV circular dichroism peak at a wavelength of 279 nm or less. In some embodiments, the isolated recombinant polypeptide complex has a near-UV circular dichroism peak at a wavelength of 290 nm or less. In some embodiments, the isolated recombinant polypeptide complex has a near-UV circular dichroism peak at a wavelength of 295 nm or less.

[0006] In another aspect, disclosed herein is a method of treating cancer comprising administering to a subject in need of treatment an isolated recombinant polypeptide complex disclosed herein.

[0007] In another aspect, disclosed herein are a plurality of isolated recombinant polypeptide complexes comprising a first chain having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:1 and a second chain having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:2, said plurality comprising greater than 90% monomers of the isolated recombinant polypeptide complexes. In some embodiments, the plurality of isolated recombinant polypeptide complexes comprises greater than 95% monomers. In some embodiments, the plurality of isolated recombinant polypeptide complexes comprises greater than 99% monomers. In some embodiments, the plurality of isolated recombinant polypeptide complexes comprises a concentration of 1.0 mg / mL or greater. In some embodiments, the plurality of isolated recombinant polypeptide complexes comprises a concentration of at least 2.0 mg / mL. In some embodiments, the plurality of isolated recombinant polypeptide complexes comprises a pH of 5.0 mg / mL or greater. In some embodiments, the plurality of isolated recombinant polypeptide complexes comprises greater than 90% monomers at a concentration of 2.0 mg / mL or greater and a pH of 6.3.

[0008] In another aspect, disclosed herein is a method of treating prostate cancer, comprising administering to a subject in need of treatment 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 at a dose of at least 0.1 μg / kg once a week. 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: administering to the subject a dose of at least 0.1 μg / kg of the antibody or antigen-binding fragment in week 1, administering to the subject a dose of at least 0.3 mg / kg of the antibody or antigen-binding fragment in week 2, administering to the subject a dose of at least 1.5 mg / kg of the antibody or antigen-binding fragment in week 3, followed by a 4 week antibody or antigen-binding fragment non-treatment interval.

[0009] In some embodiments, the isolated recombinant polypeptide complex provides a maximum plasma concentration (Cmax) in a subject within about 0.1 hours after intravenous administration. In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 2500-3500 ng / ml in a subject within about 0.1 hours 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 7500-10500 ng / ml in a subject within about 0.1 hours 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 37500-52500 ng / ml in a subject within about 0.1 hours after intravenous administration of a dose of about 1.5 mg / kg. In some embodiments, the isolated recombinant polypeptide complex provides a maximum plasma concentration half-life (T1 / 2) of about 60-120 hours after intravenous administration in a subject. In some embodiments, the value of Cmax correlates with the dose of isolated recombinant polypeptide complex administered. In some embodiments, the value of Cmax is proportional to the dose of 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 daily. 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).

[0010] In another aspect, disclosed herein is a method for treating cancer, comprising administering to a subject in need of treatment an isolated recombinant polypeptide complex as disclosed herein in a therapeutically effective amount. In some embodiments, the method comprises administering to the subject an isolated recombinant polypeptide complex in a first dose and a second dose, the second dose being equal to or greater than the first dose, and the first or second dose being at least 100 μg. 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, the second dose being equal to or greater than the first dose, the third dose being equal to or greater than the second dose, and the first, second, or third dose being at least 100 μg. In some embodiments, the method comprises a first treatment course and a second treatment course, the first dose being administered to the subject during the first treatment course, and the second dose being administered to the subject during the second treatment course. In some embodiments, the method comprises a 21 day treatment course, wherein a first dose is administered to the subject in week 1 of the treatment course, a second dose is administered to the subject in week 2 of the treatment course, and a third dose is administered to the subject in week 3 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 greater than the first dose of the first 21 day treatment course, the second dose of the second 21 day treatment course is equal to or greater than the second dose of the first 21 day treatment course, and the third dose of the second 21 day treatment course is equal to or greater than the third dose of the second 21 day treatment course. In some embodiments, the method comprises a 21 day treatment cycle, the treatment cycle comprising: (a) administering to the subject a first dose of an isolated recombinant polypeptide complex during week 1 of the treatment cycle; (b) administering to the subject a second dose of an isolated recombinant polypeptide complex during week 2 of the treatment cycle; and (c) administering to the subject a third dose of an isolated recombinant polypeptide complex during week 3 of the treatment cycle.In some embodiments, the method comprises: (a) administering to the subject a first dose of an isolated recombinant polypeptide complex on day 1 of a 21-day treatment cycle; (b) administering to the subject a second dose of an isolated recombinant polypeptide complex on day 8 of a 21-day treatment cycle; and (c) administering to the subject a third dose of an isolated recombinant polypeptide complex on day 15 of a 21-day treatment cycle. In some embodiments, the treatment cycle is repeated 6 times over 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 greater than the first dose. In some embodiments, the third dose is equal to or greater than the second dose. In some embodiments, the third dose is equal to or greater 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 greater than the first dose of the first 21-day treatment cycle, the second dose of the second 21-day treatment cycle is equal to or greater than the second dose of the first 21-day treatment cycle, and the third dose of the second 21-day treatment cycle is equal to or greater than the third dose of the first 21-day treatment cycle. In some embodiments, the administering step includes administering by intravenous infusion. In some embodiments, the cancer includes prostate cancer. In some embodiments, the cancer includes mCRPC. In some embodiments, the method further includes treating the subject with an infusion-associated reaction therapy prior to administering. In some embodiments, the infusion-associated reaction therapy includes an antipyretic, an antihistamine, an antiemetic, or a corticosteroid. In some embodiments, the infusion-associated reaction therapy includes acetaminophen, paracetamol, or diphenhydramine. In some embodiments, the method further comprises treating the subject with a corticosteroid prior to step (a). In some embodiments, the method further comprises treating the subject with a corticosteroid prior to step (a). In some embodiments, the method further comprises treating the subject with a therapy for cytokine release syndrome (CRS) prior to or after administration.In some embodiments, the treatment for CRS includes intravenous hydration procedures, oxygen therapy, corticosteroids, immunosuppressants, vasopressors, or antiepileptic drugs. In some embodiments, the oxygen therapy includes mechanical ventilation. In some embodiments, the immunosuppressant includes an IL-6 receptor inhibitor. In some embodiments, the IL-6 receptor inhibitor includes tocilizumab. In some embodiments, the treatment for CRS includes high doses of corticosteroids. In some embodiments, the isolated recombinant polypeptide complex is cleaved by a tumor-specific protease to generate an enzymatic product of the isolated recombinant polypeptide complex after administration. In some embodiments, the tumor-specific protease includes two or more proteases, the isolated recombinant polypeptide complex is cleaved by a first protease of the two or more proteases to generate a first metabolic product of the isolated recombinant polypeptide complex, and the isolated recombinant polypeptide complex is cleaved by a second protease of the two or more proteases to generate a second metabolic product of the isolated recombinant polypeptide complex. In some embodiments, the first protease comprises a serine protease and the second protease comprises a matrix metalloprotease. In some embodiments, the serine protease comprises a recombinant human matriptase (MTSP1) and the matrix metalloprotease comprises a recombinant human matrix metalloprotease 9 (MMP9). In some embodiments, the first metabolite comprises an amino acid sequence having at least 90% sequence identity to 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 to the amino acid sequence of SEQ ID NO:6. In some embodiments, the subject is a non-human primate. In some embodiments, the non-human primate is a cynomolgus monkey.

[0011] In another aspect, disclosed herein is a pharmaceutical composition comprising (a) an isolated recombinant polypeptide complex disclosed herein, and (b) a pharma- ceutically acceptable excipient. In some embodiments, the pharma- ceutically 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 both the L-histidine and L-histidine monohydrochloride monohydrate forms. 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 of about 5 to about 7. In some embodiments, the pharmaceutical composition comprises a pH of about 6.3.

[0012] 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 which is 484 amino acids in length.

[0013] 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 which is 476 amino acids in length.

[0014] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0015] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0016] In another aspect, disclosed herein is a method for treating cancer, comprising administering to a subject in need of treatment a therapeutically effective amount of the pharmaceutical composition disclosed herein.In some embodiments, the cancer comprises mCRPC.In some embodiments, the subject is a human.

[0017] Incorporation by Reference All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with disclosures contained herein, the present specification is intended to supersede and / or take precedence over such conflicting material. [Brief description of the drawings]

[0018] The novel features of the present disclosure are set forth with particularity in the appended claims. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the disclosure are utilized, and the accompanying drawings (also referred to herein as "Figures" and "FIG.").

[0019] [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 that possesses PSMA- and CD3-binding domains, an albumin-binding domain to extend 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 (TME) separates the tandem mask and albumin-binding domain from PC-1. This allows TME-restricted CD3 binding and subsequent T-cell activation to PSMA expressing prostate cancer cells. Loss of the albumin-binding domain likely ensures that any activated PC-1 that migrates from the tumor is rapidly cleared, reducing its potential accumulation in healthy tissues that may contribute to safety risks. [Figure 2A] We describe the discovery of masks by peptide phage display. Phages displaying peptide libraries were screened for binding to surface-immobilized anti-CD3 scFv. After multiple cycles of binding, elution, and amplification, clonal phages were screened for CD3 competitive binding by ELISA. Selected clonal phage sequences were synthesized as peptides and screened for binding and inhibitory properties against anti-CD3 scFv. Peptide inhibitors were then incorporated into the polypeptide complex design. [Figure 2B]We describe the discovery of masks by peptide phage display. Phages displaying peptide libraries were screened for binding to surface-immobilized anti-CD3 scFv. After multiple cycles of binding, elution, and amplification, clonal phages were screened for CD3 competitive binding by ELISA. Selected clonal phage sequences were synthesized as peptides and screened for binding and inhibitory properties against anti-CD3 scFv. Peptide inhibitors were then incorporated into the polypeptide complex design. [Figure 2C] We describe the discovery of masks by peptide phage display. Phages displaying peptide libraries were screened for binding to surface-immobilized anti-CD3 scFv. After multiple cycles of binding, elution, and amplification, clonal phages were screened for CD3 competitive binding by ELISA. Selected clonal phage sequences were synthesized as peptides and screened for binding and inhibitory properties against anti-CD3 scFv. Peptide inhibitors were then incorporated into the polypeptide complex design. [Figure 2D] We describe the discovery of masks by peptide phage display. Phages displaying peptide libraries were screened for binding to surface-immobilized anti-CD3 scFv. After multiple cycles of binding, elution, and amplification, clonal phages were screened for CD3 competitive binding by ELISA. Selected clonal phage sequences were synthesized as peptides and screened for binding and inhibitory properties against anti-CD3 scFv. Peptide inhibitors were then incorporated into the polypeptide complex design. [Figure 3A] We demonstrate that PC-1 binding to CD3 is cleavage- and dose-dependent. PC-1 CD3 target engagement is cleavage-dependent, with masking reducing CD3 binding by >600-fold. Treatment of PC-1 with protease enzymes allows potent CD3 binding equivalent to unmasked TCE-1. PC-1 shows potent binding to human and monkey PSMA and albumin. [Figure 3B] We demonstrate that PC-1 binding to CD3 is cleavage- and dose-dependent. PC-1 CD3 target engagement is cleavage-dependent, with masking reducing CD3 binding by >600-fold. Treatment of PC-1 with protease enzymes allows potent CD3 binding equivalent to unmasked TCE-1. PC-1 shows potent binding to human and monkey PSMA and albumin. [Figure 3C] We demonstrate that PC-1 binding to CD3 is cleavage- and dose-dependent. PC-1 CD3 target engagement is cleavage-dependent, with masking reducing CD3 binding by >600-fold. Treatment of PC-1 with protease enzymes allows potent CD3 binding equivalent to unmasked TCE-1. PC-1 shows potent binding to human and monkey PSMA and albumin. [Figure 3D] We demonstrate that PC-1 binding to CD3 is cleavage- and dose-dependent. PC-1 CD3 target engagement is cleavage-dependent, with masking reducing CD3 binding by >600-fold. Treatment of PC-1 with protease enzymes allows potent CD3 binding equivalent to unmasked TCE-1. PC-1 shows potent binding to human and monkey PSMA and albumin. [Figure 3E] We demonstrate that PC-1 binding to CD3 is cleavage- and dose-dependent. PC-1 CD3 target engagement is cleavage-dependent, with masking reducing CD3 binding by >600-fold. Treatment of PC-1 with protease enzymes allows potent CD3 binding equivalent to unmasked TCE-1. PC-1 shows potent binding to human and monkey PSMA and albumin. [Figure 3F] We demonstrate that PC-1 binding to CD3 is cleavage- and dose-dependent. PC-1 CD3 target engagement is cleavage-dependent, with masking reducing CD3 binding by >600-fold. Treatment of PC-1 with protease enzymes allows potent CD3 binding equivalent to unmasked TCE-1. PC-1 shows potent binding to human and monkey PSMA and albumin. [Figure 4] We explain that TCE-1 potency is structure and orientation dependent. TCE-1 activity depends on the combined geometry of the PSMA and CD3 binding domains. [Figure 5A] We demonstrate that 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 demonstrates robust T cell functional conversion demonstrating the potential for an enhanced safety profile. [Figure 5B] We demonstrate that 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 demonstrates robust T cell functional conversion demonstrating the potential for an enhanced safety profile. [Figure 5C] We demonstrate that 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 demonstrates robust T cell functional conversion demonstrating the potential for an enhanced safety profile. [Figure 5D] We demonstrate that 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 demonstrates robust T cell functional conversion demonstrating the potential for an enhanced safety profile. [Figure 5E]We demonstrate that 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 demonstrates robust T cell functional conversion demonstrating the potential for an enhanced safety profile. [Figure 5F] We demonstrate that 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 demonstrates robust T cell functional conversion demonstrating the potential for an enhanced safety profile. [Figure 6A] We describe that PC-1 has a prolonged half-life and enhanced safety profile in NHPs. [Figure 6B] We describe that PC-1 has a prolonged half-life and enhanced safety profile in NHPs. [Figure 6C] We describe that PC-1 has a prolonged half-life and enhanced safety profile in NHPs. [Figure 7] FIG. 1 illustrates a flow diagram of the upstream cell culture process involved in the production of PC-1. [Figure 8] FIG. 1 illustrates a flow diagram of the downstream purification process associated with the production of PC-1. [Figure 9] The circular dichroism (CD) spectrum of PC-1 in the far-ultraviolet region is described. [Figure 10] The CD spectrum of PC-1 in the near-ultraviolet region is described. [Figure 11] 1 illustrates differential scanning calorimetry data for PC-1. [Figure 12A] SEC-MALS chromatogram of PC-1 is illustrated. [Figure 12B] SEC-MALS chromatogram of PC-1 is illustrated. [Figure 13]The arrangement of light and heavy chains in PC-1 is illustrated. [Figure 14A] FIG. 14 describes ELISA binding of test articles to PSMA. [Figure 14B] FIG. 14 describes ELISA binding of test articles to PSMA. [Figure 14C] FIG. 14 describes ELISA binding of test articles to PSMA. [Figure 14D] FIG. 14 describes ELISA binding of test articles to PSMA. [Figure 14E] FIG. 14 describes ELISA binding of test articles to PSMA. [Figure 14F] FIG. 14 describes ELISA binding of test articles to PSMA. [Figure 15A] FIG. 1 illustrates ELISA binding of PC-1 to albumin. [Figure 15B] FIG. 1 illustrates ELISA binding of PC-1 to albumin. [Figure 15C] FIG. 1 illustrates ELISA binding of PC-1 to albumin. [Figure 15D] FIG. 1 illustrates ELISA binding of PC-1 to albumin. [Figure 16A] FIG. 1 illustrates ELISA binding of test substances to CD3. [Figure 16B] FIG. 1 illustrates ELISA binding of test substances to CD3. [Figure 16C] FIG. 1 illustrates ELISA binding of test substances to CD3. [Figure 16D] FIG. 1 illustrates ELISA binding of test substances to CD3. [Figure 16E] FIG. 1 illustrates ELISA binding of test substances to CD3. [Figure 16F] FIG. 1 illustrates ELISA binding of test substances to CD3. [Figure 17A] 1 illustrates an example of CD3 octet binding data for PC-1 over time after incubation in serum. [Figure 17B] Illustrates example CD3 octet binding data for PC-1 over time after incubation in serum [Figure 18A] Describe the PC-1 cleavage rate in serum of cynomolgus monkeys, healthy humans, or human mCRPC donors. [Figure 18B] Describe the PC-1 cleavage rate in serum of cynomolgus monkeys, healthy humans, or human mCRPC donors. [Figure 18C] Describe the PC-1 cleavage rate in serum of cynomolgus monkeys, healthy humans, or human mCRPC donors. [Figure 18D] Describe the PC-1 cleavage rate in serum of cynomolgus monkeys, healthy humans, or human mCRPC donors. [Figure 18E] Describe the PC-1 cleavage rate in serum of cynomolgus monkeys, healthy humans, or human mCRPC donors. [Figure 18F] Describe the PC-1 cleavage rate in serum of cynomolgus monkeys, healthy humans, or human mCRPC donors. [Figure 18G] Describe the PC-1 cleavage rate in serum of cynomolgus monkeys, healthy humans, or human mCRPC donors. [Figure 18H] Describe the PC-1 cleavage rate in serum of cynomolgus monkeys, healthy humans, or human mCRPC donors. [Figure 19A] FIG. 1 illustrates dose-dependent test substance-induced production of IFNγ, TNF, and IL-6 by PBMCs co-cultured with LNCaP tumor cells. [Figure 19B] FIG. 1 illustrates dose-dependent test substance-induced production of IFNγ, TNF, and IL-6 by PBMCs co-cultured with LNCaP tumor cells. [Figure 19C] FIG. 1 illustrates dose-dependent test substance-induced production of IFNγ, TNF, and IL-6 by PBMCs co-cultured with LNCaP tumor cells. [Figure 19D] FIG. 1 illustrates dose-dependent test substance-induced production of IFNγ, TNF, and IL-6 by PBMCs co-cultured with LNCaP tumor cells. [Figure 19E]FIG. 1 illustrates dose-dependent test substance-induced production of IFNγ, TNF, and IL-6 by PBMCs co-cultured with LNCaP tumor cells. [Figure 19F] FIG. 1 illustrates dose-dependent test substance-induced production of IFNγ, TNF, and IL-6 by PBMCs co-cultured with LNCaP tumor cells. [Figure 19G] FIG. 1 illustrates dose-dependent test substance-induced production of IFNγ, TNF, and IL-6 by PBMCs co-cultured with LNCaP tumor cells. [Figure 19H] FIG. 1 illustrates dose-dependent test substance-induced production of IFNγ, TNF, and IL-6 by PBMCs co-cultured with LNCaP tumor cells. [Figure 19I] FIG. 1 illustrates dose-dependent test substance-induced production of IFNγ, TNF, and IL-6 by PBMCs co-cultured with LNCaP tumor cells. [Figure 20A] FIG. 1 illustrates dose-dependent test substance-induced production of IFNγ, TNF, and IL-6 by PBMCs co-cultured with 22Rv1 tumor cells. [Figure 20B] FIG. 1 illustrates dose-dependent test substance-induced production of IFNγ, TNF, and IL-6 by PBMCs co-cultured with 22Rv1 tumor cells. [Figure 20C] FIG. 1 illustrates dose-dependent test substance-induced production of IFNγ, TNF, and IL-6 by PBMCs co-cultured with 22Rv1 tumor cells. [Figure 20D] FIG. 1 illustrates dose-dependent test substance-induced production of IFNγ, TNF, and IL-6 by PBMCs co-cultured with 22Rv1 tumor cells. [Figure 20E] FIG. 1 illustrates dose-dependent test substance-induced production of IFNγ, TNF, and IL-6 by PBMCs co-cultured with 22Rv1 tumor cells. [Figure 20F]FIG. 1 illustrates dose-dependent test substance-induced production of IFNγ, TNF, and IL-6 by PBMCs co-cultured with 22Rv1 tumor cells. [Figure 20G] FIG. 1 illustrates dose-dependent test substance-induced production of IFNγ, TNF, and IL-6 by PBMCs co-cultured with 22Rv1 tumor cells. [Figure 20H] FIG. 1 illustrates dose-dependent test substance-induced production of IFNγ, TNF, and IL-6 by PBMCs co-cultured with 22Rv1 tumor cells. [Figure 20I] FIG. 1 illustrates dose-dependent test substance-induced production of IFNγ, TNF, and IL-6 by PBMCs co-cultured with 22Rv1 tumor cells. [Figure 21A] Test substance-induced cytokine production by PBMC in the absence of tumor cells is described. [Figure 21B] Test substance-induced cytokine production by PBMC in the absence of tumor cells is described. [Figure 21C] Test substance-induced cytokine production by PBMC in the absence of tumor cells is described. [Figure 21D] Test substance-induced cytokine production by PBMC in the absence of tumor cells is described. [Figure 21E] Test substance-induced cytokine production by PBMC in the absence of tumor cells is described. [Figure 21F] Test substance-induced cytokine production by PBMC in the absence of tumor cells is described. [Figure 22A] FIG. 1 illustrates dose-dependent LNCaP tumor cell killing by PBMC stimulated with test substances. [Figure 22B] FIG. 1 illustrates dose-dependent LNCaP tumor cell killing by PBMC stimulated with test substances. [Figure 22C] FIG. 1 illustrates dose-dependent LNCaP tumor cell killing by PBMC stimulated with test substances. [Figure 22D]FIG. 1 illustrates dose-dependent LNCaP tumor cell killing by PBMC stimulated with test substances. [Figure 22E] FIG. 1 illustrates dose-dependent LNCaP tumor cell killing by PBMC stimulated with test substances. [Figure 22F] FIG. 1 illustrates dose-dependent LNCaP tumor cell killing by PBMC stimulated with test substances. [Figure 22G] FIG. 1 illustrates dose-dependent LNCaP tumor cell killing by PBMC stimulated with test substances. [Fig. 22H] FIG. 1 illustrates dose-dependent LNCaP tumor cell killing by PBMC stimulated with test substances. [Figure 23A] FIG. 1 illustrates dose-dependent 22Rv1 tumor cell killing by PBMC stimulated with test substances. [Figure 23B] FIG. 1 illustrates dose-dependent 22Rv1 tumor cell killing by PBMC stimulated with test substances. [Figure 23C] FIG. 1 illustrates dose-dependent 22Rv1 tumor cell killing by PBMC stimulated with test substances. [Figure 23D] FIG. 1 illustrates dose-dependent 22Rv1 tumor cell killing by PBMC stimulated with test substances. [Figure 23E] FIG. 1 illustrates dose-dependent 22Rv1 tumor cell killing by PBMC stimulated with test substances. [Figure 23F] FIG. 1 illustrates dose-dependent 22Rv1 tumor cell killing by PBMC stimulated with test substances. [Figure 23G] FIG. 1 illustrates dose-dependent 22Rv1 tumor cell killing by PBMC stimulated with test substances. [Figure 23H] FIG. 1 illustrates dose-dependent 22Rv1 tumor cell killing by PBMC stimulated with test substances. [Figure 24A] FIG. 1 illustrates the absence of PC3 tumor cell killing by PBMC stimulated with test substances. [Figure 24B] FIG. 1 illustrates the absence of PC3 tumor cell killing by PBMC stimulated with test substances. [Figure 24C] FIG. 1 illustrates the absence of PC3 tumor cell killing by PBMC stimulated with test substances. [Figure 24D] FIG. 1 illustrates the absence of PC3 tumor cell killing by PBMC stimulated with test substances. [Diagram 25] A sample preparation workflow using immunocapture purification is described. [Figure 26] FIG. 1 illustrates the TK profile of PC-1 in monkey plasma samples collected from Group 2 on day 22 of the TK study. [Figure 27] The correlation between intact PC-1 and anti-drug antibody (ADA) concentrations in monkeys in the high dose group is described. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] In the context of this application, the following terms have the meanings ascribed to them unless specifically stated otherwise.

[0021] As used throughout this specification and claims, the terms "a", "an" and "the" are generally used in the sense of meaning "at least one", "at least a first", "one or more", or "multiple" of the components or steps to which they refer, except in instances where an upper limit is subsequently specified. For example, "cleavage sequence" as used herein means "at least a first cleavage sequence", but includes multiple cleavage sequences. Operable limits and parameters of combinations, as well as amounts of any single agent, will be known to those of skill in the art in light of this application.

[0022] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to generally refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term further encompasses 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 labeling component.

[0023] As used herein in the context of a polypeptide structure, the "N-terminus" (or "amino terminus") and the "C-terminus" (or "carboxyl terminus") generally refer to the extreme amino and carboxyl termini of a polypeptide, respectively.

[0024] As used herein, the term "therapeutically effective amount" generally refers to an amount of a polypeptide variant or polypeptide composition that, when administered to a patient to treat a disease or other undesirable medical condition, is sufficient to have a beneficial effect with respect to the disease or condition. Therapeutically effective amounts vary depending on the polypeptide variant or polypeptide composition, the disease or condition and its severity, and the age, weight, etc., of the patient being 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 does not require routine experimentation.

[0025] As used herein, the terms "about" and "approximately" are used interchangeably. Any numbers used herein with or without about / approximately are intended to cover any normal variation understood by those of ordinary skill in the art. For example, the term "about" can refer to a range of values ​​of ±10%, ±5%, ±2%, or ±1% of a particular value. By way of example, the phrase "about 50%" can include 45%-55%, 48%-52%, or 49%-51%.

[0026] The above definitions supersede any conflicting definitions in any references incorporated herein by reference. However, the fact that a particular term is defined should not be taken to indicate that any term not defined is indefinite. Rather, all terms used are believed to describe the present disclosure in terms that allow one skilled in the art to understand and implement the scope of the present application.

[0027] Recombinant Polypeptide Compositions Metastatic castration-resistant prostate cancer (mCRPC) remains an intractable 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 the treatment of mCRPC. These TCEs suffer from cytokine release syndrome (CRS) and poor pharmacokinetic (PK) profiles. There remains a need for new immunotherapies. Disclosed herein is a tumor-activating T cell engager (TRACTr) targeting PSMA that features enhanced safety and pharmacokinetic profiles.

[0028] FIG. 1 illustrates the design, structure, and mechanism of action of Polypeptide Complex 1 (PC-1). PC-1 is a tumor-activated T-cell engager with PSMA- and CD3-binding domains, an albumin-binding domain to extend circulating half-life, a peptide mask to inhibit CD3 engagement on 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 domain from PC-1. This allows TME-restricted CD3 binding and subsequent T-cell activation to PSMA expressing prostate cancer cells. Loss of the albumin-binding domain likely ensures that any activated PC-1 that migrates out of the tumor is rapidly cleared, reducing its potential accumulation in healthy tissues that may contribute to safety risks.

[0029] Recombinant polypeptides are disclosed herein. In some embodiments, the recombinant polypeptide comprises a tumor-activating T cell engager having a PSMA-binding domain and a CD3-binding domain, an albumin-binding domain for extending 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. As described herein, the recombinant polypeptide can comprise chain 1 and chain 2. In some embodiments, chain 1 comprises an amino acid sequence with 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:1. In some embodiments, chain 2 comprises an amino acid sequence with 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.

[0030] 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.

[0031] In some embodiments, the recombinant polypeptide comprises the amino acid sequence set forth in SEQ ID NO:1.

[0032] In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:2. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:2. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO:2. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO:2. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO:2. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO:2. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:2. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 97% sequence identity to 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.

[0033] In some embodiments, the recombinant polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0034] [Table 1-1]

[0035] [Table 1-2]

[0036] Disulfide bonds In some embodiments, the recombinant polypeptide comprises at least one disulfide bond formed by a pair of cysteine ​​residues. In some embodiments, the recombinant polypeptide comprises at least one disulfide bond formed by a pair of cysteine ​​residues in the first chain. In some embodiments, the recombinant polypeptide comprises at least one disulfide bond formed by a pair of cysteine ​​residues in the second chain. In some embodiments, the recombinant polypeptide comprises at least one disulfide bond formed by a pair of cysteine ​​residues 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 a pair of cysteine ​​residues comprising the pair of cysteine ​​residues. In some embodiments, the recombinant polypeptide comprises at least two disulfide bonds formed by a pair of cysteine ​​residues comprising the pair of cysteine ​​residues. In some embodiments, the recombinant polypeptide comprises at least three disulfide bonds formed by a pair of cysteine ​​residues comprising the pair of cysteine ​​residues. In some embodiments, the recombinant polypeptide comprises at least four disulfide bonds formed by pairs of cysteine ​​residues comprising the pair of cysteine ​​residues. In some embodiments, the recombinant polypeptide comprises at least five disulfide bonds formed by pairs of cysteine ​​residues comprising the pair of cysteine ​​residues. In some embodiments, the recombinant polypeptide comprises at least six disulfide bonds formed by pairs of cysteine ​​residues comprising the pair of cysteine ​​residues. In some embodiments, the recombinant polypeptide comprises at least seven disulfide bonds formed by pairs of cysteine ​​residues comprising the pair of cysteine ​​residues. In some embodiments, the recombinant polypeptide comprises at least eight disulfide bonds formed by pairs of cysteine ​​residues comprising the pair of cysteine ​​residues.

[0037] In some embodiments, the pair of cysteine ​​residues that form a disulfide bond corresponds to 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.

[0038] In some embodiments of the recombinant peptide, at least one disulfide bond formed by a pair of cysteine ​​residues is formed by and corresponds to a cysteine ​​residue selected from positions 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 a pair of cysteine ​​residues is formed by and corresponds to a cysteine ​​residue selected from positions cysteine ​​645 of SEQ ID NO: 1 and cysteine ​​226 of SEQ ID NO:2. In some embodiments of the recombinant peptide, at least one disulfide bond formed by a pair of cysteine ​​residues is formed by and corresponds to a cysteine ​​residue selected from positions cysteine ​​22, cysteine ​​96, cysteine ​​150, and cysteine ​​206 of SEQ ID NO: 2. The presence of a disulfide bond may be determined by mass spectrometry (MS).

[0039] In some embodiments, the recombinant polypeptide comprises at least one cysteine ​​residue that is a free sulfhydryl. The presence of a free sulfhydryl may be determined by mass spectrometry (MS).

[0040] In some embodiments, the isolated recombinant polypeptide complex is administered to a subject at about 0.001 to about 1 hour, e.g., 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, 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 .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, 0.540, 0.545 ,0.550,0.555,0.560,0.565,0.570,0.575,0.580,0.585,0.590,0.595,0.600,0.605,0.610,0.615,0.620,0.625,0.630,0.635,0.640,0.645,0.650,0.655,0.660,0.665,0.670,0.675,0.680,0.685,0.690,0.695,0.700,0.705,0.7 10, 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, Provides a maximum plasma concentration (Cmax) within about 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 hours, or any time therebetween. In some embodiments, the isolated recombinant polypeptide complex provides a Cmax in a subject within about 0.1 hours after intravenous administration.

[0041] In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 2000-4000 ng / ml, e.g., about 2000-4000 ng / ml, 2000-3500 ng / ml, 2000-3000 ng / ml, 2000-2500 ng / ml, 2500-4000 ng / ml, 2500-3500 ng / ml, 2500-3000 ng / ml, 3000-4000 ng / ml, 3000-3500 ng / ml, or 3500-4000 ng / ml, or any concentration therebetween, in a subject within about 0.1 hours 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 2500-3500 ng / ml in a subject within about 0.1 hours after intravenous administration of a dose of about 0.1 mg / kg.

[0042] In some embodiments, the isolated recombinant polypeptide complex is administered to a subject within about 0.1 hours after intravenous administration of a dose of about 0.3 mg / kg, at a concentration of about 7000-12000 ng / ml, e.g., about 7000-12000 ng / ml, 7000-11500 ng / ml, 7000-11000 ng / ml, 7000-7000 ng / ml, 7000-10000 ng / ml, 10500-9500 ng / ml, 7000-9000 ng / ml, 7000-8500 ng / ml, 7000-8000 ng / ml, 7000-7500 ng / ml, 7500- 12000ng / ml, 7500~11500ng / ml, 7500~11000ng / ml, 7500~10500ng / ml, 7500~10000ng / ml, 7500~9500ng / ml, 7500~9000ng / ml, 7500~8500ng / ml, 7500 ~8000ng / ml, 8000~12000ng / ml, 8000~11500ng / ml, 8000~11000ng / ml, 8000~10500ng / ml, 8000~10000ng / ml, 8000~9500ng / ml, 8000~9000ng / ml, 800 0~8500ng / ml, 8500~12000ng / ml, 8500~11500ng / ml, 8500~11000ng / ml, 8500~10500ng / ml, 8500~10000ng / ml, 8500~9500ng / ml, 8500~9000ng / ml, 90 00~12000ng / ml, 9000~11500ng / ml, 9000~11000ng / ml, 9000~10500ng / ml, 9000~10000ng / ml, 9000~9500ng / ml, 9500~12000ng / ml, 9500~11500ng / ml , 9500-11000ng / ml, 9500-10500ng / ml, 9500-10000ng / ml, 10000-12000ng / ml, 10000-11500ng / ml, 10000-11000ng / ml, 10000-10500ng / ml, 10500-12000ng / ml, 10500-11500ng / ml, 10500-11000ng / ml, 11000-12000ng / ml, 11000-11500ng / ml, or about 11500-12000ng / ml, or any range therebetween.In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 7500-10500 ng / ml in a subject within about 0.1 hours after intravenous administration of a dose of about 0.3 mg / kg.

[0043] In some embodiments, the isolated recombinant polypeptide complex is administered to a subject within about 0.1 hours after intravenous administration of a dose of about 1.5 mg / kg, at a concentration of about 32500-57500 ng / ml, e.g., about 32500-57500 ng / ml, 32500-55000 ng / ml, 32500-52500 ng / ml, 32500-32500 ng / ml, 32500-47500 ng / ml, 50000-45000 ng / ml, 32500-42500 ng / ml, 32500-40000 ng / ml, 32500-37500 ng / ml, 32500 ~35000ng / ml, 35000~57500ng / ml, 35000~55000ng / ml, 35000~52500ng / ml, 35000~50000ng / ml, 35000~47500ng / ml, 35000~45000ng / ml, 35000~4250 0ng / ml, 35000~40000ng / ml, 35000~37500ng / ml, 37500~75000ng / ml, 37500~72500ng / ml, 37500~70000ng / ml, 37500~67500ng / ml, 37500~65000ng / m l, 37500~62500ng / ml, 37500~60000ng / ml, 37500~57500ng / ml, 37500~55000ng / ml, 37500~52500ng / ml, 37500~50000ng / ml, 37500~47500ng / ml, 37 500~45000ng / ml, 37500~42500ng / ml, 37500~40000ng / ml, 40000~57500ng / ml, 40000~55000ng / ml, 40000~52500ng / ml, 40000~50000ng / ml, 40000~4 7500ng / ml, 40000~45000ng / ml, 40000~42500ng / ml, 42500~57500ng / ml, 42500~55000ng / ml, 42500~52500ng / ml, 42500~50000ng / ml, 42500~47500n g / ml, 42500~45000ng / ml, 45000~57500ng / ml, 45000~55000ng / ml, 45000~52500ng / ml, 45000~50000ng / ml, 45000~47500ng / ml, 47500~57500ng / ml,Provides a Cmax of 47500-55000ng / ml, 47500-52500ng / ml, 47500-50000ng / ml, 50000-57500ng / ml, 50000-55000ng / ml, 50000-52500ng / ml, 52500-57500ng / ml, 52500-55000ng / ml, or 55000-57500ng / ml, or a concentration range therebetween. In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 37500-52500ng / ml in a subject within about 0.1 hours after intravenous administration of a dose of about 1.5mg / kg.

[0044] In some embodiments, the isolated recombinant polypeptide complex is administered to a subject within about 50 to about 130 hours (T1 / 2) after intravenous administration, 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, 70 to about 130 hours, about 70 to about 120 hours, about 70 to about 120 hours, about 70 to about 140 hours, about 70 to about 160 hours, about 70 to about 180 hours, about 70 to about 190 hours, about 80 to about 190 hours, about 80 to about 190 hours, about 90 to about 190 hours, about 190 to about 20 ... The compound provides a maximum plasma concentration half-life of 0 to about 110 hours, about 70 to about 100 hours, about 70 to about 90 hours, about 70 to about 80 hours, about 80 to about 130 hours, about 80 to about 120 hours, about 80 to about 110 hours, about 80 to about 100 hours, about 80 to about 90 hours, about 90 to about 130 hours, about 90 to about 120 hours, about 90 to about 110 hours, about 90 to about 100 hours, about 100 to about 130 hours, about 100 to about 120 hours, about 100 to about 110 hours, about 110 to about 130 hours, about 110 to about 120 hours, or about 120 to about 130 hours, or any duration therebetween.

[0045] In some embodiments, the value of C is correlated with the dose of the isolated recombinant polypeptide complex administered. In some embodiments, the value of C is proportional to the dose of the isolated recombinant polypeptide complex administered.

[0046] In some embodiments, the isolated recombinant polypeptide complex is less than about 10%, e.g., 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%,3.6%,3.5%,3.4%,3.3%,3.2%,3.1%,3%,2.9%,2.8%,2.7%,2.6%,2.5%,2.4%,2.3%,2.2%, Less than 2.1%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or less than about 0.1%, or any percentage therebetween, is degraded in the serum of the subject on a daily basis. In some embodiments, less than 1% of the isolated recombinant polypeptide complex is degraded in the serum of the subject on a daily basis.

[0047] 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).

[0048] In some embodiments, the isolated recombinant polypeptide complex is cleaved by a tumor-specific protease to generate an enzymatic product of the isolated recombinant polypeptide complex after administration. In some embodiments, the tumor-specific protease comprises two or more proteases. In some embodiments, the isolated recombinant polypeptide complex is cleaved by a first protease of the two or more proteases to generate a first metabolic product of the isolated recombinant polypeptide complex. In some embodiments, the isolated recombinant polypeptide complex is cleaved by a second protease of the two or more proteases to generate a second metabolic product of the isolated recombinant polypeptide complex. In some embodiments, the first protease comprises a serine protease. In some embodiments, the second protease comprises a matrix metalloprotease. In some embodiments, the serine protease comprises recombinant human matriptase (MTSP1). In some embodiments, the matrix metalloprotease comprises recombinant human matrix metalloprotease 9 (MMP9).

[0049] 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 first metabolite of the isolated 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 the amino acid sequence of SEQ ID NO:5.

[0050] In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 80% 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 81% 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 82% 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 83% 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 84% 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 85% 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 86% 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 87% 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 88% 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 89% 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 90% 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 91% 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 92% 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 93% 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 94% 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 95% 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 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 the amino acid sequence of SEQ ID NO:6.

[0051] In some embodiments, the subject is a non-human primate, hi some embodiments, the non-human primate is a cynomolgus monkey.

[0052] 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 which is 484 amino acids in length.

[0053] 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 which is 484 amino acids in length.

[0054] 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 which is 484 amino acids in length.

[0055] 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 which is 484 amino acids in length.

[0056] 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 which is 484 amino acids in length.

[0057] 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 which is 484 amino acids in length.

[0058] 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 which is 484 amino acids in length.

[0059] 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 which is 484 amino acids in length.

[0060] 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 which is 484 amino acids in length.

[0061] 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 which is 484 amino acids in length.

[0062] 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 which is 484 amino acids in length.

[0063] 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 which is 484 amino acids in length.

[0064] 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 which is 484 amino acids in length.

[0065] 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 which is 484 amino acids in length.

[0066] 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 which is 484 amino acids in length.

[0067] 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 which is 484 amino acids in length.

[0068] 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 which is 484 amino acids in length.

[0069] 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 which is 484 amino acids in length.

[0070] 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 which is 484 amino acids in length.

[0071] 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 which is 484 amino acids in length.

[0072] In another aspect, disclosed herein is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:5, and which is 484 amino acids in length.

[0073] 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 which is 476 amino acids in length.

[0074] 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 which is 476 amino acids in length.

[0075] 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 which is 476 amino acids in length.

[0076] 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 which is 476 amino acids in length.

[0077] 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 which is 476 amino acids in length.

[0078] 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 which is 476 amino acids in length.

[0079] 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 which is 476 amino acids in length.

[0080] 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 which is 476 amino acids in length.

[0081] 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 which is 476 amino acids in length.

[0082] 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 which is 476 amino acids in length.

[0083] 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 which is 476 amino acids in length.

[0084] 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 which is 476 amino acids in length.

[0085] 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 which is 476 amino acids in length.

[0086] 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 which is 476 amino acids in length.

[0087] 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 which is 476 amino acids in length.

[0088] 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 which is 476 amino acids in length.

[0089] 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 which is 476 amino acids in length.

[0090] 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 which is 476 amino acids in length.

[0091] 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 which is 476 amino acids in length.

[0092] 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 which is 476 amino acids in length.

[0093] In another aspect, disclosed herein is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:6, and which is 476 amino acids in length.

[0094] Amino acid modifications In some embodiments, at least one glutamine of SEQ ID NO:1 or SEQ ID NO:2 is cyclized to form pyroglutamine. In some embodiments, the pyroglutamine is on 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 may be determined by mass spectrometry (MS).

[0095] thermal stability In some embodiments, the recombinant polypeptide has a melting temperature (T m In some embodiments, the recombinant polypeptide complex is characterized by a melting temperature (T) of about 70° C. to about 81° C. m In some embodiments, the recombinant polypeptide complex is characterized by a melting temperature (T) of about 71° C. to about 72° C. m In some embodiments, the recombinant polypeptide complex is characterized by a melting temperature (T) of about 78° C. to about 80° C. m In some embodiments, the recombinant polypeptide complex is characterized by a melting temperature (T m In some embodiments, the recombinant polypeptide complex is characterized by a melting temperature (T m In some embodiments, the recombinant polypeptide complex is characterized by an onset melting temperature (T) of about 55° C. to about 60° C. onset In some embodiments, the recombinant polypeptide complex is characterized by an onset melting temperature (T) of about 58° C. to about 60° C. onset In some embodiments, the recombinant polypeptide complex is characterized by an onset melting temperature (T) of about 59° C. onset ) is characterized by its melting temperature (T m ) may be determined by differential scanning calorimetry (DSC). onset ) may be determined by differential scanning calorimetry (DSC).

[0096] structural composition In some embodiments, the recombinant polypeptide comprises a secondary structural composition comprising a β-sheet and a random coil. The secondary structural composition may comprise a β-sheet. The secondary structural composition may comprise a random coil.

[0097] In some embodiments, the recombinant polypeptide is characterized by a near-UV circular dichroism peak at a wavelength of 300 nm, 295 nm, 290 nm, 285 nm, 280 nm, or 275 nm or less. In some embodiments, the recombinant polypeptide is characterized by a near-UV circular dichroism peak at a wavelength of 275 nm, 280 nm, 285 nm, 290 nm, or 300 nm or more. 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.

[0098] In some embodiments, the recombinant polypeptide is characterized by a near-UV circular dichroism peak at a wavelength of 220 nm, 210 nm, or 205 nm or less. In some embodiments, the recombinant polypeptide is characterized by a near-UV circular dichroism peak at a wavelength of 205 nm, 210 nm, or 220 nm or more. In some embodiments, the recombinant polypeptide is characterized by a near-UV circular dichroism peak at a wavelength of 200 nm to 210 nm, or 205 nm to 220 nm.

[0099] One or more characteristics A polypeptide or recombinant polypeptide described herein may have one or more of the characteristics as described previously in this section.

[0100] In some embodiments, the recombinant polypeptide has characteristics (a)-(g): (a) at least one disulfide bond formed by a pair of cysteine ​​residues in the first chain or the second chain, or a pair of cysteine ​​residues in the first chain and the second chain, or (b) a secondary structure composition comprising a β-sheet or a random coil, (c) at least one pyroglutamine in the second chain, (d) a melting temperature (T) of about 65° C. to about 85° C. 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. 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 at 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 at 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 10 mM histidine, 8% (w / v) sucrose, 0.01% (w / v) polysorbate 20 at pH 6.3.

[0101] In some embodiments, the recombinant polypeptide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, or 7) of features (a)-(g). In some embodiments, the recombinant polypeptide comprises at least two (e.g., 2, 3, 4, 5, 6, or 7) of features (a)-(g). In some embodiments, the recombinant polypeptide comprises at least three (e.g., 3, 4, 5, 6, or 7) of features (a)-(g). In some embodiments, the recombinant polypeptide comprises at least four (e.g., 4, 5, 6, or 7) of features (a)-(g). In some embodiments, the recombinant polypeptide comprises at least five (e.g., 5, 6, or 7) of features (a)-(g). In some embodiments, the recombinant polypeptide comprises at least six (e.g., 6 or 7) of features (a)-(g). In some embodiments, the recombinant polypeptide comprises all seven of features (a)-(g).

[0102] formulation The disclosure herein includes formulations that include populations of polypeptides or recombinant polypeptides, such as those that include any one or combination of the polypeptides or recombinant polypeptides as described herein.

[0103] In some embodiments of the above formulations, at least about 80%, 85%, 90%, or 95% (e.g., mol % or mass %) of the polypeptides of the population comprise at least one pyroglutamine residue (such as any of those described above). In some embodiments, at least about 80% (e.g., mol % or mass %) of the polypeptides of the population comprise at least one pyroglutamine residue, e.g., at an amino acid position corresponding to glutamine 1 of SEQ ID NO:2. In some embodiments, at least about 85% (e.g., mol % or mass %) of the polypeptides of the population comprise at least one pyroglutamine residue, e.g., at an amino acid position corresponding to glutamine 1 of SEQ ID NO:2. In some embodiments, at least about 90% (e.g., mol % or mass %) of the polypeptides of the population comprise at least one pyroglutamine residue, e.g., at an amino acid position corresponding to glutamine 1 of SEQ ID NO:2. In some embodiments, at least about 95% (e.g., mol % or mass %) of the polypeptides of the population comprise at least one pyroglutamine residue, e.g., at an amino acid position corresponding to glutamine 1 of SEQ ID NO:2.

[0104] In some embodiments of the above formulations, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (e.g., mol % or mass %) of the polypeptides of the population are monomeric. In some embodiments of the above formulations, no more than 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% (e.g., mol % or mass %) of the polypeptides of the population are aggregated.

[0105] In some embodiments of the above 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 the amino acid sequence set forth in SEQ ID NO:1.

[0106] In some embodiments of the above 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 the amino acid sequence set forth in SEQ ID NO:2.

[0107] Disclosed herein is a pharmaceutical composition comprising one or more pharma- ceutically acceptable excipients, the one or more pharma-ceutically acceptable excipients comprising histidine, sucrose, polysorbate 20, sodium phosphate, citrate, glutamate, glycine, arginine, sorbitol, arginine hydrochloride, acetate, sodium chloride, potassium chloride, magnesium chloride, and calcium chloride. Disclosed herein is a pharmaceutical composition comprising a recombinant polypeptide (such as any of those described herein), sodium phosphate monobasic monohydrate, sodium phosphate dibasic, heptahydrate, sodium chloride, potassium chloride, histidine, citrate, acetate, sucrose, polysorbate 20, polysorbate 80, magnesium chloride hexahydrate, and calcium chloride dihydrate. The pharmaceutical composition may have a pH of 7.0, 6.5, 6.0, or 5.5 or less. The pharmaceutical composition may have a pH of 5.5, 6.0, 6.5, or 7.0 or more. The pharmaceutical composition may comprise 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM or more of histidine. The pharmaceutical composition may comprise 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, or 5 mM or less of histidine. The pharmaceutical composition may comprise 5% (w / v), 6% (w / v), 7% (w / v), or 8% (w / v) or more of sucrose. The pharmaceutical composition may comprise 9% (w / v), 8% (w / v), 7% (w / v), 6% (w / v), or 5% (w / v) or less of sucrose. The pharmaceutical composition may comprise 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 less than 0.01% (w / v) polysorbate 20. The pharmaceutical composition may comprise 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 more than 0.05% (w / v) polysorbate 20.Disclosed herein is a pharmaceutical composition, the pharmaceutical composition comprising a recombinant polypeptide (such as any of those described herein), 10 mM histidine, 8% (w / v) sucrose, and 0.01% (w / v) polysorbate 20.

[0108] In another aspect, a pharmaceutical composition is disclosed herein. In some embodiments, the pharmaceutical composition comprises (a) an isolated recombinant polypeptide complex disclosed herein, and (b) a pharma- ceutical acceptable excipient.

[0109] In some embodiments, the pharma- ceutically acceptable excipient comprises a buffer, a stabilizer, an isotonicity 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 isotonicity agent comprises a sugar. In some embodiments, the sugar comprises sucrose. In some embodiments, the surfactant comprises a polysorbate. In some embodiments, the surfactant comprises polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. In some embodiments, the surfactant comprises 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. 1mM, 3.2mM, 3.3mM, 3.4mM, 3.5mM, 3.6mM, 3.7mM, 3.8mM, 3.9mM, 4mM, 4.1mM, 4.2mM, 4.3mM, 4.4 mM, 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, 8.4 mM, 8.5mM, 8.6mM, 8.7mM, 8.8mM, 8.9mM, 9mM, 9.1mM, 9.2mM, 9.3mM, 9.4mM, 9.5mM, 9.6mM, 9.7m M, 9.8mM, 9.9mM, 10mM, 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、13.6mM、13.7mM、13.8mM、13.9mM、14mM、14.1mM、14.2mM、14.3mM、14.4mM、14.5mM、14.6mM、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、17.4mM、17.5mM、17.6mM、17.7mM、17.8mM、17.9mM、18mM、18.1mM、18.2mM、18.3mM、18.4mM、18.5mM、18.6mM、18.7mM、18.8mM、18.9mM、19mM、19.1mM、19.2mM、19.3mM、19.4mM、19.5mM、19.6mM、19.7mM、19.8mM、19.9mM, or 20mM, or any concentration therebetween. 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 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 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).

[0110] In some embodiments, the pharmaceutical composition is 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), 0.04 4%(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), 0.0475%(w / v), 0.048% (w / v), 0.0485% (w / v), 0.049% (w / v), 0.0495% (w / v), 0.05% (w / v), 0.055% (w / v), 0.06% (w / v), 0.065% (w / v), 0.07% (w / v), 0.075% (w / v), 0.08% (w / v), 0.085% (w / v), 0.09% (w / v), 0.095% (w / v), or about 0.1% (w / v), or any concentration therebetween. 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.

[0111] In some embodiments, 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, 12.1 mg / 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, 13.2mg / 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, 1 6.9mg / 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. The pharmaceutical composition comprises an amount of the isolated recombinant polypeptide complex of 1 mg / ml, 18.2 mg / ml, 18.3 mg / ml, 18.4 mg / ml, 18.5 mg / ml, 18.6 mg / ml, 18.7 mg / ml, 18.8 mg / ml, 18.9 mg / ml, 19 mg / ml, 19.1 mg / ml, 19.2 mg / ml, 19.3 mg / ml, 19.4 mg / ml, 19.5 mg / ml, 19.6 mg / ml, 19.7 mg / ml, 19.8 mg / ml, 19.9 mg / ml, or 20 mg / ml, or any concentration therebetween. In some embodiments, the pharmaceutical composition comprises about 2 mg / ml of the isolated recombinant polypeptide complex.

[0112] 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 of about 5 to about 7. In some embodiments, the pharmaceutical composition comprises a pH of about 6.3.

[0113] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0114] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0115] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0116] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0117] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0118] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0119] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0120] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0121] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0122] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0123] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0124] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0125] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0126] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0127] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0128] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0129] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0130] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0131] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0132] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0133] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:5 and which is 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0134] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0135] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0136] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0137] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0138] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0139] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0140] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0141] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0142] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0143] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0144] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0145] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0146] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0147] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0148] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0149] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0150] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0151] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0152] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0153] In another aspect, disclosed herein is a pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0154] In another aspect, disclosed herein is a pharmaceutical composition comprising an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:6 and being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0155] In another aspect, disclosed herein is a method for treating cancer, comprising administering to a subject in need of treatment an effective amount of a pharmaceutical composition disclosed herein.

[0156] In some embodiments, the cancer comprises mCRPC. In some embodiments, the subject is a human.

[0157] kit In some embodiments, provided herein is a kit comprising a recombinant polypeptide (such as any of those described herein) or composition (such as any of those described herein), a container, and a label or package insert on or associated with the container.

[0158] Treatment In some embodiments, the method of treating cancer in a subject in need of treatment comprises administering to the subject an isolated recombinant polypeptide complex as described herein. In some embodiments, the cancer has cells that express PSMA. In some examples, 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, colon cancer, pancreatic cancer, or gastric cancer.

[0159] In some embodiments, a method of treating prostate cancer in a subject in need of treatment, comprising administering to the subject an isolated recombinant polypeptide complex as described herein. In some embodiments, a method of treating metastatic castration-resistant prostate cancer (mCRPC) in a subject in need of treatment, comprising administering to the subject an isolated recombinant polypeptide complex as described herein.

[0160] In some embodiments, the method includes administering a recombinant polypeptide (such as any of those described herein) or formulation (such as any of those described herein) to the subject in need of treatment for metastatic castration-resistant prostate cancer (mCRPC). In some embodiments, the subject has at least one symptom of mCRPC. In some embodiments, the subject has been diagnosed with mCRPC.

[0161] For administration to a subject, the recombinant polypeptides disclosed herein may be provided in a pharmaceutical composition together with one or more pharma- ceutically 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 not toxic to the patient to which it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile liquids, and the like. Such carriers can be formulated by conventional methods and administered to a subject in appropriate dosages. Preferably, the compositions are sterile. These compositions may further include adjuvants such as preservatives, emulsifiers, and dispersants. Prevention of microbial action can be ensured by the inclusion of various antibacterial and antifungal agents.

[0162] The pharmaceutical composition may be in any suitable form (depending on the desired method of administration). The pharmaceutical composition may be provided in a unit dosage form, in a sealed container, or as part of a kit. Such a kit may include instructions for use. The kit may include a plurality of the unit dosage forms described above.

[0163] The pharmaceutical composition can be adapted for administration by any suitable route, including parenteral (e.g., subcutaneous, intramuscular, or intravenous) route. Such compositions can be prepared by any method known in the art of pharmacy, for example, by mixing the active ingredient with a carrier or excipient under sterile conditions. In some embodiments, the pharmaceutical composition disclosed herein is administered intravenously to a subject in need of administration.

[0164] The dosage of the substances of the present disclosure may vary between 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.

[0165] In another aspect, disclosed herein is a method for treating cancer, comprising administering to a subject in need of treatment 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 in a first dose and a second dose. In some embodiments, the second dose is equal to or greater 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.

[0166] In some embodiments, the method comprises administering the isolated recombinant polypeptide complex to a subject in a first dose, a second dose, and a third dose. In some embodiments, the second dose is equal to or greater than the first dose. In some embodiments, the third dose is equal to or greater 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.

[0167] In some embodiments, the method includes 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 includes a 21 day treatment course. In some embodiments, the first dose is administered to the subject during the first week of the treatment course. In some embodiments, the second dose is administered to the subject during the second week of the treatment course. In some embodiments, the third dose is administered to the subject during the third week of the treatment course. In some embodiments, the method includes 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 greater 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 greater than the second dose of the first 21 day treatment course. In some embodiments, the second dose of the third 21-day treatment course is equal to or greater 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 to the subject a first dose of an isolated recombinant polypeptide complex in week 1 of the treatment cycle; (b) administering to the subject a second dose of an isolated recombinant polypeptide complex in week 2 of the treatment cycle; and (c) administering to the subject a third dose of an isolated recombinant polypeptide complex in week 3 of the treatment cycle. In some embodiments, the method comprises: (a) administering to the subject a first dose of an isolated recombinant polypeptide complex in day 1 of the 21-day treatment cycle; (b) administering to the subject a second dose of an isolated recombinant polypeptide complex in day 8 of the 21-day treatment cycle; and (c) administering to the subject a third dose of an isolated recombinant polypeptide complex in day 15 of the 21-day treatment cycle. In some embodiments, the treatment cycle is repeated six times over an 18 week period.

[0168] 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 greater than the first dose. In some embodiments, the third dose is equal to or greater than the second dose. In some embodiments, the third dose is equal to or greater than the first dose.

[0169] 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 greater 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 greater 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 greater than the third dose of the first 21-day treatment cycle. In some embodiments, the administering step includes administering by intravenous infusion. In some embodiments, the cancer includes prostate cancer. In some embodiments, the cancer includes mCRPC. In some embodiments, the method further includes treating the subject with an infusion-associated reaction therapy prior to administering. In some embodiments, the infusion-associated reaction therapy includes an antipyretic, an antihistamine, an antiemetic, or a corticosteroid. In some embodiments, the infusion-associated reaction therapy includes acetaminophen, paracetamol, or diphenhydramine. In some embodiments, the method further comprises (a) treating the subject with a corticosteroid prior to administering to the subject a first dose of the isolated recombinant polypeptide complex on week 1 of a 21-day treatment cycle. In some embodiments, the method further comprises (a) treating the subject with a corticosteroid prior to administering to the subject a first dose of the isolated recombinant polypeptide complex on day 1 of a 21-day treatment cycle. In some embodiments, the method further comprises treating the subject with a treatment for cytokine release syndrome (CRS) prior to or after administration. In some embodiments, the treatment for CRS comprises intravenous hydration procedures, oxygen therapy, corticosteroids, immunosuppressants, vasopressors, or antiepileptic drugs. In some embodiments, the oxygen therapy comprises mechanical ventilation. In some embodiments, the immunosuppressant comprises an IL-6 receptor inhibitor. In some embodiments, the IL-6 receptor inhibitor comprises tocilizumab. In some embodiments, the treatment for CRS comprises a high dose of a corticosteroid.

[0170] In some embodiments, the isolated recombinant polypeptide complex is cleaved by a tumor-specific protease to generate an enzymatic product of the isolated recombinant polypeptide complex after administration. In some embodiments, the tumor-specific protease comprises two or more proteases. In some embodiments, the isolated recombinant polypeptide complex is cleaved by a first protease of the two or more proteases to generate a first metabolic product of the isolated recombinant polypeptide complex. In some embodiments, the isolated recombinant polypeptide complex is cleaved by a second protease of the two or more proteases to generate a second metabolic product of the isolated recombinant polypeptide complex. In some embodiments, the first protease comprises a serine protease. In some embodiments, the second protease comprises a matrix metalloprotease. In some embodiments, the serine protease comprises recombinant human matriptase (MTSP1). In some embodiments, the matrix metalloprotease comprises recombinant human matrix metalloprotease 9 (MMP9).

[0171] In some embodiments, the first metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 80% 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 81% 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 82% 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 83% 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 84% 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 85% 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 86% 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 87% 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 88% 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 89% 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 90% 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 91% 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 92% 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 93% 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 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 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 96% 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 97% 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 98% sequence identity to the amino acid sequence of SEQ ID NO:5. In some embodiments, the first metabolic product of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the first metabolic product of the isolated recombinant polypeptide complex comprises the amino acid sequence of SEQ ID NO: 5.

[0172] In some embodiments, the second metabolite of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 80% 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 81% 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 82% 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 83% 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 84% 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 85% 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 86% 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 87% 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 88% 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 89% 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 90% 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 91% 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 92% 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 93% 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 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 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 96% 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 97% 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 98% sequence identity to the amino acid sequence of SEQ ID NO:6. In some embodiments, the second metabolic product of the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the second metabolic product of the isolated recombinant polypeptide complex comprises the amino acid sequence of SEQ ID NO: 6.

[0173] In some embodiments, the subject is a non-human primate, hi some embodiments, the non-human primate is a cynomolgus monkey.

[0174] 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 which is 484 amino acids in length.

[0175] 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 which is 484 amino acids in length.

[0176] 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 which is 484 amino acids in length.

[0177] 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 which is 484 amino acids in length.

[0178] 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 which is 484 amino acids in length.

[0179] 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 which is 484 amino acids in length.

[0180] 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 which is 484 amino acids in length.

[0181] 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 which is 484 amino acids in length.

[0182] 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 which is 484 amino acids in length.

[0183] 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 which is 484 amino acids in length.

[0184] 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 which is 484 amino acids in length.

[0185] 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 which is 484 amino acids in length.

[0186] 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 which is 484 amino acids in length.

[0187] 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 which is 484 amino acids in length.

[0188] 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 which is 484 amino acids in length.

[0189] 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 which is 484 amino acids in length.

[0190] 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 which is 484 amino acids in length.

[0191] 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 which is 484 amino acids in length.

[0192] 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 which is 484 amino acids in length.

[0193] 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 which is 484 amino acids in length.

[0194] In another aspect, disclosed herein is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:5, and which is 484 amino acids in length.

[0195] 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 which is 476 amino acids in length.

[0196] 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 which is 476 amino acids in length.

[0197] 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 which is 476 amino acids in length.

[0198] 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 which is 476 amino acids in length.

[0199] 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 which is 476 amino acids in length.

[0200] 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 which is 476 amino acids in length.

[0201] 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 which is 476 amino acids in length.

[0202] 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 which is 476 amino acids in length.

[0203] 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 which is 476 amino acids in length.

[0204] 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 which is 476 amino acids in length.

[0205] 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 which is 476 amino acids in length.

[0206] 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 which is 476 amino acids in length.

[0207] 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 which is 476 amino acids in length.

[0208] 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 which is 476 amino acids in length.

[0209] 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 which is 476 amino acids in length.

[0210] 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 which is 476 amino acids in length.

[0211] 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 which is 476 amino acids in length.

[0212] 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 which is 476 amino acids in length.

[0213] 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 which is 476 amino acids in length.

[0214] 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 which is 476 amino acids in length.

[0215] In another aspect, disclosed herein is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:6, and which is 476 amino acids in length.

[0216] Antibody production In some embodiments, the polypeptides (e.g., antibodies and binding fragments thereof) described herein are produced using any method known in the art to aid in the synthesis of polypeptides (e.g., antibodies), inter alia, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques.

[0217] In some examples, antibodies or binding fragments thereof are recombinantly expressed and nucleic acids encoding the antibodies or binding fragments thereof are assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves synthesis of overlapping oligonucleotides containing portions of the antibody encoding sequence, annealing and ligation of the oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

[0218] Alternatively, nucleic acid molecules encoding antibodies are optionally produced 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 capable of hybridizing to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for the particular gene sequence.

[0219] In some examples, the antibody or binding thereof is optionally produced by immunizing an animal such as a mouse to produce polyclonal antibodies, or more preferably, by producing monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497), or 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 the antibody are optionally obtained by screening Fab expression libraries for clones of FAb fragments that bind to a specific antigen (e.g., as described in Huse et al., 1989, Science 246:1275-1281) or by screening antibody libraries (see Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

[0220] In some embodiments, techniques developed for the production of "chimeric antibodies" (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) are used by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as an animal species having a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region.

[0221] In some embodiments, techniques described for the production of single chain antibodies (US Pat. 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) are suitable for producing single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region by an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli are also optionally used (Skerra et al., 1988, Science 242:1038-1041).

[0222] In some embodiments, an expression vector containing the nucleotide sequence of an antibody or fragment thereof, or the nucleotide sequence of an antibody or fragment thereof, is introduced into a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In certain embodiments, expression of the antibody is regulated by a constitutive, inducible, or tissue-specific promoter.

[0223] In some embodiments, a variety of host-expression vector systems are utilized to express the antibodies or binding fragments thereof described herein. Such host-expression systems represent not only the vehicles in which the antibody coding sequences are produced and subsequently purified, but also cells which, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibodies or binding fragments thereof in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody or binding fragment thereof coding sequence, yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing the antibody or binding fragment thereof coding sequence, insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody or binding fragment thereof coding sequence, plant cell systems infected with recombinant viral expression vectors (e.g., Cauliflower Mosaic Virus (CaMV) and Tobacco Mosaic Virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody or binding fragment thereof coding sequence, or mammalian cell systems (e.g., COS, CHO, BH, HEK293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0224] For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some instances, cell lines that stably express the antibody are optionally engineered. Rather than using expression vectors containing viral origins of replication, host cells are transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA, engineered cells are grown in enriched medium for 1-2 days and then switched to selective medium. The selectable marker on the recombinant plasmid confers resistance to selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci that are then cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express antibodies or binding fragments thereof.

[0225] In some examples, a number of selection systems are used, including, but not limited to, blasticidin, zeocin, herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes utilized in tk-, hgprt-, or aprt-cells, respectively. Similarly, antimetabolite resistance has been used as a selection criterion 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); and neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy for Cancer Research 12:131-135). 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 30:14 7).Methods generally known in the art 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 Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1).

[0226] In some instances, expression levels of a recombinant polypeptide are increased by vector amplification (for review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). If the marker in the vector system expressing the recombinant polypeptide is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Because the amplified region is associated with the nucleotide sequence of the recombinant polypeptide, production of the recombinant polypeptide will also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0227] In some examples, any method for purification of recombinant polypeptides known in the art is used, for example, by chromatography (e.g., ion exchange, affinity, especially affinity to a specific antigen followed by Protein A, and sizing column chromatography), centrifugation, differential solubility, or other standard techniques for purification of proteins.

[0228] Expression vector In some embodiments, the vector comprises any suitable vector derived from a eukaryotic or prokaryotic source. In some cases, the vector is derived from a bacterial (e.g., E. coli), insect, yeast (e.g., Pichia pastoris), algae, or mammalian source. Exemplary bacterial vectors include pACYC177, pASK75, pBAD vector system, pBADM vector system, pET vector system, pETM vector system, pGEX vector system, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector system, pRSET A, pRSET B, pRSET C, pTrcHis2 system, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

[0229] 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, pVL1393 M11, pVL1393 M12, a FLAG vector, e.g., pPolh-FLAG1 or pPolh-MAT 2, or a MAT vector, e.g., pPolh-MAT1 or pPolh-MAT2.

[0230] In some cases, the yeast vector includes a Gateway® pDEST™ 14 vector, a Gateway® pDEST™ 15 vector, a Gateway® pDEST™ 17 vector, a Gateway® pDEST™ 24 vector, a Gateway® pYES-DEST52 vector, a pBAD-DEST49 Gateway® destination vector, a pAO815 Pichia vector, a pFLD1 Pichi pastoris vector, a pGAPZA, B, and C Pichia pastoris vector, a pPIC3.5K Pichia vector, a pPIC6 A, B, and C Pichia vector, a pPIC9K Pichia vector, a pTEF1 / Zeo, a pYES2 yeast vector, a pYES2 / CT yeast vector, a pYES2 / NT A, B, and C yeast vector, or a pYES3 / CT yeast vector.

[0231] Exemplary algal vectors include the pChlamy-4 vector or the MCS vector.

[0232] Examples of mammalian vectors include transient expression vectors or stable expression vectors.Mammalian transient expression vectors can include pRK5, p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a,b,c, pFLAG-CMV 5.1, pFLAG-CMV 5a,b,c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3 or pBICEP-CMV 4. Mammalian stable expression vectors 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-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2.

[0233] In some instances, 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 either prokaryotic or eukaryotic components. Often, nucleic acid synthesis is obtained in a cell-free system based on, for example, Drosophila cells, Xenopus 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 PURExpress®.

[0234] host cell

[0235] In some embodiments, the host cell includes any suitable cell, for example, a naturally occurring cell or a genetically engineered cell. In some examples, the host cell is a production host cell. In some examples, the host cell is a eukaryotic cell. In other examples, the host cell is a prokaryotic cell. In some cases, the eukaryotic cell includes a fungus (e.g., a yeast cell), an animal cell, or a plant cell. In some cases, the prokaryotic cell is a bacterial cell. Examples of bacterial cells include gram-positive or gram-negative bacteria. Gram-negative bacteria are often anaerobic, rod-shaped, or both.

[0236] In some examples, the Gram-positive bacteria include Actinobacteria, Firmicutes, or Tenericutes. 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. The other bacteria may be from the phylum Acidobacteria, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Dictyoglomi, Thermodesulfobacteria, or Thermotogae. The bacterial cell may be Escherichia coli, Clostridium botulinum, or E. coli.

[0237] Exemplary prokaryotic host cells include, but are not limited to, BL21, Mach1™, DH10B™, TOP10, DH5α, DH10Bac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F', INVαF, TOP10 / P3, ccdB Survival, PIR1, PIR2, Stbl2™, Stbl3™, or Stbl4™.

[0238] In some instances, the animal cells include cells derived from vertebrates or invertebrates. In some instances, the animal cells include cells derived from marine invertebrates, fish, insects, amphibians, reptiles, or mammals. In some instances, the fungal cells include yeasts, such as brewer's yeast, baker's yeast, or wine yeast.

[0239] Fungi include ascomycetes, such as yeasts, molds, filamentous fungi, basidiomycetes, or zygomycetes. In some instances, yeasts include ascomycetes or basidiomycetes. In some instances, ascomycetes include Saccharomycotina (true yeasts, e.g., Saccharomyces cerevisiae (baker's yeast)) or Taphrinicotina (e.g., Schizosaccharomycetes (fission yeast)). In some instances, basidiomycetes include Agaricomycotina (e.g., Tremellomycetes) or Pucciniomycotina (e.g., Microbotryomycetes).

[0240] Exemplary yeasts or filamentous fungi include, for example, the following genera: Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula, Kluyveromyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidium, Aspergillus, Fusarium, or Trichoderma. Exemplary yeast or filamentous fungi include, for example, 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 ... utilis, Candida boidini, Candida albicans, Candida utilis, Candida boidini, Candida albicans, Candida utilis, Candida boidini, Candida albicans, Candida utilis, Candida boidini, Candida albicans, Candida utilis, Candida boidini, Candida albicans, Candida utilis, Candida boidini, Candida albicans, Candida utilis, Candida boidini, Candida albicans, Can mucilaginosa, Pichia methanolica, Pichia angusta, Pichia pastoris, Pichia anomala, Hansenula polymorpha, Kluyveromyces lactis, Zygosaccharomyces rouxii, Yarrowia lipolytica, Trichosporon pullulans, Rhodosporidium toru-Aspergillus niger, Aspergillus nidulans nidulans, Aspergillus awamoriExamples of suitable microbial strains include Aspergillus oryzae, Aspergillus oryzae, Trichoderma reesei, Yarrowia lipolytica, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii, Zygosaccharomyces bailii, Cryptococcus neoformans, Cryptococcus gattii, and Saccharomyces boulardii.

[0241] Exemplary yeast host cells include, but are not limited to, Pichia pastoris yeast strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33, and Saccharomyces cerevisiae yeast strains such as INVSc1.

[0242] In some embodiments, the additional animal cell comprises a cell obtained from a mollusc, an arthropod, an annelid, or a sponge. In some embodiments, the additional animal cell is a mammalian cell derived from, for example, a primate, an ape, an equine, a bovine, a porcine, a canine, a feline, or a rodent. In some cases, the rodent comprises a mouse, a rat, a hamster, a gerbil, a hamster, a chinchilla, a fancy rat, or a guinea pig.

[0243] Exemplary mammalian host cells include 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(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, F lp-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, GripTite(TM) 293 These include, but are not limited to, MSR cell line, GS-CHO cell line, HepaRG™ cells, T-REx™ Jurkat cell line, Per.C6 cells, T-REx™-293 cell line, T-REx™-CHO cell line, and T-REx™-HeLa cell line.

[0244] In some instances, 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 product of the genetic material after many generations of cell division. In some instances, 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 product of the genetic material after many generations of cell division.

[0245] Exemplary insect host cells include, but are not limited to, Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five™ cells, and expresSF+® cells.

[0246] In some examples, the plant cell comprises a cell from algae. Exemplary insect cell lines include, but are not limited to, strains from Chlamydomonas reinhardtii 137c or Synechococcus elongatus PPC 7942.

[0247] product

[0248] In another aspect of the present disclosure, a product is provided that contains materials useful for treating, preventing, and / or diagnosing the above disorders.The product comprises a container and a label or package insert on or attached to the container.Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.The container may be formed from a variety of materials, such as glass or plastic.

[0249] The label or package insert indicates that the composition is used to treat the condition of choice. The article of manufacture in this embodiment of the present invention may further include a package insert indicating that the composition can be used to treat a particular condition.

[0250] Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution, and may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. EXAMPLES

[0251] Example 1: PC-1 characterization and toxicity testing Figures 2A-2D illustrate the discovery of masks by peptide phage display. Phages displaying peptide libraries were screened for binding to surface-immobilized anti-CD3 scFv. After multiple cycles of binding, elution, and amplification, clonal phages were screened for CD3 competitive binding by ELISA. Selected clonal phage sequences were synthesized as peptides and screened for binding and inhibitory properties against anti-CD3 scFv. Peptide inhibitors were then incorporated into the polypeptide complex design.

[0252] Figures 3A-3F illustrate that PC-1 binding to CD3 is cleavage- and dose-dependent. PC-1 CD3 target engagement is cleavage-dependent, with masking reducing CD3 binding by over 600-fold. Treatment of PC-1 with protease enzymes allows for potent CD3 binding comparable to unmasked TCE-1. PC-1 shows potent binding to human and monkey PSMA and albumin.

[0253] 4A-4B illustrate that TCE-1 potency is conformation and orientation dependent. TCE-1 activity depends on the combined geometry of the PSMA and CD3 binding domains.

[0254] Figures 5A-5F illustrate that 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 demonstrates robust T cell functional conversion, demonstrating potential for an enhanced safety profile.

[0255] Table 2 illustrates that the cleavable linker features rapid proteolysis and high serum stability. The linker is cleaved faster than that possessed by pacmilimab (CL-1) and maintains stability in human serum. The proteolytic activity of serum is greater than blood, representing a conservative assessment of in vivo stability. The cleavable linker is rapidly cleaved by a panel of recombinant tumor proteases, which enhances unmasking and antitumor activity in the TME. PC-1 shows high stability with 1% or less cleavage per day in healthy human donor serum and in serum of mCRPC human donors. While proteolytic cleavage of PC-1 in the TME is expected to drive antitumor activity, a critical safety attribute 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.

[0256] [Table 2]

[0257] 6A-6C and Table 3 illustrate that PC-1 has an extended half-life and enhanced safety profile in NHPs.

[0258] [Table 3]

[0259] PC-1 toxicity studies support PK, safety, and enhanced drug design. PC-1 administered at 1.5 mg / kg IV bolus, QW×5 in cynomolgus monkeys achieved high exposure and long half-life without significant changes in clinical signs or clinical pathology measures. PC-1 was highly stable in vivo with minimal detectable cleavage. Cleaved PC-1 reached the LLoQ, while intact (masked) PC-1 achieved 1,500-fold exposure over its minimally detectable metabolites. Total (masked+unmasked) and intact PC-1 showed overlapping PK profiles further supporting the in vivo stability of PC-1. Rapid clearance of TCE minimizes cleaved PC-1 accumulation and, in turn, the risk of on-target healthy tissue toxicity and cytokine release.

[0260] PC-1 PSMA GLP Toxicity Study Summary in Non-Human Primates describes weekly dosing of 0.1, 0.3, and 1.5 mg / kg with 4 weeks recovery. No microscopic histopathological findings were observed. Lack of TCE accumulation in vivo resulted in mitigated on-target healthy tissue toxicity and minimal cytokine release. Clinical chemistry, hematology, and pathology data packages support a no observed adverse effect level (NOAEL) of ≥1.5 mg / kg / dose.

[0261] PC-1 exhibits enhanced safety and PK properties relative to TCE-1. The defining safety profile of PC-1 is a tumor protease-cleavable inhibitory peptide mask that reduces PC-1 binding to human CD3 by >600-fold while limiting T cell activation to the TME. In vitro, PC-1 exhibits up to a 500-fold reduction in potency to activate T cells and induce T cell-mediated tumor cell killing relative to unmasked TCE-1. PC-1 exhibits an enhanced safety profile in NHPs, characterized by a reduction in cytokine CRS-associated proinflammatory cytokines at a NOAEL of ≥1.5 mg / kg / dose, IV bolus QW × 5. The albumin binding domain extends the circulating half-life of PC-1 to approximately 120 hours in NHPs versus the half-life of unmasked TCE of <2 hours, supporting the planned weekly clinical dosing of PC-1.

[0262] PC-1's cleavage-dependent activity, extended half-life PK, excellent safety potential, and manufacturability characteristics support its further development as an attractive mCRPC treatment.

[0263] Example 2: Production and purification process The manufacturing process is divided into upstream (cell culture), harvest, and downstream (purification) processes. The upstream process consists of upstream fed-batch expression in Chinese Hamster Ovary (CHO)-K1 cell culture at a 200 L cell culture scale, followed by a harvest step. The downstream process uses multiple purification steps to produce bulk drug substance. The drug substance is then subjected to bulk filtration after final formulation and addition of excipients and filled into polycarbonate bottles.

[0264] summary PC-1 was produced and purified according to the steps shown in Figures 7-8. The upstream process starts with the thawing of 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 with glutamine, hypoxanthine, and thymidine, was inoculated with one vial of the master cell bank to produce approximately (1.30-4.00) × 10 6 The cultures are grown in shake flasks to a defined viable cell density of cells / milliliter (mL). The cultures are then transferred to a 20 liter (L) single-use bioreactor supplemented with basal medium 2 consisting of ActiPro medium with glutamine, hypoxanthine, and thymidine, followed by cultivation in a 50 L single-use bioreactor filled with the same medium. The viable cell density was determined to be (3.60-6.00) × 10 6 When the approximate viable cell density (VCD) of cells / mL is reached and viability is greater than 90%, the cell culture is transferred to a 250 L single-use bioreactor for fed-batch production at a cell culture process scale of 200 L. The cell culture proceeds for approximately 10 days, at which point harvest is performed by depth filtration.

[0265] The downstream process begins with capture of PC-1 from harvested cell culture fluid (HCCF) by protein A affinity chromatography, followed by steps of viral inactivation at low pH, neutralization, and depth filtration. Host cell impurities are removed by an intermediate polishing step using an anion exchange chromatography resin. PC-1 is collected in the flow-through and further polished by binding to a multimodal chromatography resin using an elution step. A viral nanofiltration step is performed to remove any potential adventitious agents. The product is then subjected to ultrafiltration and diafiltration using a histidine buffer at pH 6.3, with the addition of sucrose and polysorbate 20 to improve bulk stability. The formulated product is filtered and aseptically dispensed into single-use sterilized polycarbonate bottles, and the bulk drug substance is stored at -70°C ± 10°C.

[0266] Upstream Cell Culture Process With reference to Figure 7, the upstream manufacturing process begins with the thawing of one vial from a master cell bank. The cells are gradually grown using a chemically defined growth medium to achieve an appropriate cell density before inoculating a 20 L disposable bioreactor unit, followed by a 50 L and final 250 L production bioreactor. During cell growth and culture, cell density, dissolved oxygen, pH, and environmental conditions are controlled. Upon completion of cell culture (approximately 10 days), the cells are harvested. Further details of each of the steps in Figure 7 are provided below.

[0267] Vial thawing and cell culture in shake flasks The manufacturing process began when one vial from the master cell bank was thawed in a 37.0°C ± 1°C water bath and the cells were aseptically transferred to a shake flask and resuspended in fresh basal medium 1 consisting of CD CHO medium with glutamine, hypoxanthine, and thymidine. The cell suspension was diluted to 100 mM NaCl, with a VCD of approximately (1.10-3.00) × 10 6 When the VCD and viability were greater than 85%, the cells were incubated at 36.5° C. with 6.0% carbon dioxide (CO2) and a shaking speed of 120 revolutions per minute (rpm) for approximately 2 days. The cell cultures are further subcultured into shaking flasks of increasing size under controlled environmental conditions with the addition of fresh basal medium 1 at the defined VCD and viability. Backup inoculum growth is generated at the N-3 stage and maintained for up to 3 passages.

[0268] Cell growth in 20L bioreactor After two rounds of growth in shake flasks, the VCD of the cell culture was approximately (1.30–4.00) × 10 6 When the viability reached 90.0% and the cell culture was diluted to approximately 0.35 × 10 6 Transfer the culture to a 20 L disposable bioreactor with basal medium 2 consisting of ActiPro medium with glutamine, hypoxanthine, and thymidine at a cell density of 1000 cells / mL. The culture should grow to approximately (3.50-5.50) x 10 6Proceed at 36.5 °C with 6.0% CO2 and a rocker speed of approximately 20 rpm until a VCD of cells / mL and a viability of >90%.

[0269] Cell Growth in 50L Bioreactor Cells from a 20 L bioreactor were cultured at approximately 0.40 x 10 6 Inoculate a 50 L single-use bioreactor filled with basal medium 2 with a target VCD of cells / mL and a viability higher than 90%. Set the temperature and agitation speed at 36.5 °C and 140 rpm, respectively. Maintain pH at 6.95 with CO2 sparging and control dissolved oxygen (DO) at 40% with air / oxygen sparging. The cell culture will grow to approximately (3.60-6.00) x 10 6 Continue growing to a VCD of cells / mL and viability of >90%. Once this criterion is achieved, transfer the contents to the final 250 L production bioreactor filled with Basal Medium 2.

[0270] Cell Culture in 250L Bioreactor The cell culture is carried out in a 250 L single-use bioreactor with a target production volume of 200 L. Approximately 1.00 x 10 6 After inoculation at the target VCD in cells / mL, transfer approximately 1.5 kilograms (kg) of culture from the 250 L bioreactor to a 3 L bioreactor satellite run. Set the temperature at 36.5 °C at the time of inoculation and allow the VCD to reach approximately (10.00 - 14.00) x 10 6 Shift to 33.0 °C when cell / mL is reached or on day 5. pH and dissolved oxygen are monitored and controlled at approximately 6.90 and 40.0%, respectively, and the 250 L bioreactor is operated with 100 rpm agitation and air / oxygen and CO2 sparging. Cell culture proceeds with periodic addition of nutrient feed. Glucose concentration is tested daily and maintained at approximately 6 g / L by addition of 400 grams (g) / kg glucose stock solution. Cultures are harvested on day 10 or when viability falls below 85%.

[0271] Cell harvesting and clarification Once harvest criteria are met, the cell culture is harvested and clarified by depth filtration using a combination of filters to remove cells and cell debris. The depth filtration system consists of two-stage, two-layered regenerated cellulose filters with nominal retentions of 6-30 micrometers (μm) and 0.10-0.85 μm, respectively. The cell culture is pumped through the filters at approximately 50 liters per square meter per hour (LMH), with the initial effluent discarded. Filtration proceeds until all of the cell culture has been filtered, and the filters are chased with a wash buffer of 50 millimolar (mM) Tris-Acetate (Tris-HAc), 150 mM sodium chloride (NaCl), pH 7.4. The filtrate is collected in a disposable storage bag, followed by 0.5 / 0.2 μm filtration into a mixing bag. The final filtrate is mixed before proceeding to the next step.

[0272] Downstream Refining Process Referring to FIG. 8, the downstream process begins with capture of PC-1 from the clarified cell culture fluid by affinity chromatography and elution at approximately pH 4.2. A step of viral inactivation is then performed using a low pH treatment step, followed by neutralization and removal of any precipitates by intermediate depth filtration. Additional impurities are further removed by an intermediate polishing step using an anion exchange chromatography resin. PC-1 is collected in the chromatography flow-through and further polished by a bind-elute multimodal chromatography resin using the steps of the elution scheme. The viral nanofiltration step is followed by an ultrafiltration and diafiltration (UF / DF) step into a histidine buffer at pH 6.3. Sucrose and polysorbate 20 are then added at final target concentrations of 8% weight / volume (w / v) and 0.01% (w / v), respectively, to improve bulk stability. The formulated bulk is filtered and aseptically dispensed into sterilized polycarbonate bottles. The drug substance is stored at -70°C ± 10°C. Further details of the steps shown in FIG. 8 are provided below.

[0273] Affinity chromatography The principle of this chromatography step is affinity binding, using MabSelect PrismA Protein A resin to capture the target protein PC-1 while allowing impurities to be removed by flowing through a packed column. PC-1 is then eluted from the resin using a low pH buffer. Protein A chromatography resin is dedicated to the production of PC-1 drug substance.

[0274] The column is rinsed and sanitized with sodium hydroxide (NaOH) with a normality (N) of 0.5 and equilibrated with a buffer of 50 mM Tris-HAc, 150 mM NaCl, pH 7.4. The clarified cell culture fluid is 0.5 / 0.2 μm filtered during column loading at approximately 20-50 g protein per liter of resin with a maximum flow rate of 300 centimeters per hour (cm / h) and a minimum residence time of 5 minutes. The column is then washed with high / low salt and pH buffers to remove impurities. The bound PC-1 is eluted with 30 mM sodium acetate acetate (NaAc-HAc) at pH 4.2 at the same flow rate. Eluate peak collection begins at approximately 50 mAU / mm and ends 50 milliabsorbance units per millimeter (mAU / mm) after the peak. If multiple cycles are performed, the eluates are combined and mixed before proceeding to the virus inactivation step.

[0275] Low pH viral inactivation and neutralization The pH of the Protein A eluate is adjusted to pH 3.6 ± 0.1 using 1 molar (M) acetic acid (HAc) and maintained at 18-26 °C with stirring to achieve robust viral inactivation. After 1-2 h under these conditions, the solution is neutralized to pH 5.5 ± 0.2 using 1 M Tris base and held at ambient temperature for 30-240 min before proceeding to the intermediate depth filtration step.

[0276] Intermediate Depth Filtration The intermediate depth filtration step removes any precipitate that may have formed during the low pH viral inactivation and neutralization process steps. Regenerated cellulose depth filters with a nominal retention of 0.10-0.85 μm are equilibrated with 30 mM NaAc-HAc buffer and loaded with approximately 100 LMH of neutralization product pool while maintaining a filter pressure drop (dP) of less than 2 bar. After loading, the filters are stripped with equilibration buffer and the combined filtrate is further 0.5 / 0.2 μm polyethersulfone (PES) filtered into a sterile mixing and storage bag prior to the next step.

[0277] Anion exchange chromatography The anion exchange (Capto Q) chromatography step is carried out in flow-through mode. The filtrate from the intermediate depth filtration step is adjusted to pH 6.8-7.2 and conductivity 3.0-8.0 millisiemens per centimeter (mS / cm), followed by 0.5 / 0.2 μm PES filtration into a storage bag. The anion exchange column is sanitized with 1 M NaOH and equilibrated with 50 mM Tris-HAc at pH 7.0, 1 M NaCl, followed by 50 mM Tris-HAc at pH 7.0. Loading proceeds with a maximum linear flow rate of 300 cm / h and a minimum residence time of 5 minutes, maintaining a loading capacity of 100-200 g / L CaptoQ resin. After loading is complete, the column is washed with 50 mM Tris-HAc at pH 7.0. The eluate is 0.5 / 0.2 μm PES filtered during collection starting at approximately 50 mAU / mm during loading and ending at 50 mAU / mm after the peak.

[0278] Multimodal cation exchange chromatography Multimodal cation exchange (Capto MMC ImpRes) chromatography is used in bind-elute mode as a polishing step to further remove impurities. Prior to loading, the column is rinsed and sanitized with 1 M NaOH and equilibrated with 50 mM Tris-HAc at pH 7.0. The anion exchange eluate is loaded onto the column at a maximum linear flow rate of 300 cm / h and a minimum residence time of 5 min. UV 280The column is washed with equilibration / wash buffer until the absorbance is less than 25 mAU / mm. Following the wash, an additional wash of 50 mM Tris-HAc, pH 7.0, 60 mM NaCl is performed and PC-1 is eluted from the column at the same flow rate with 50 mM Tris-HAc, pH 7.0, 205 mM NaCl elution buffer. Eluate collection begins at approximately 500 mAU / mm or higher and ends at 125 mAU / mm or higher after the peak. The eluate is 0.5 / 0.2 μm PES filtered during collection and if more than one cycle is performed, the eluates are combined and mixed before proceeding to the virus filtration step.

[0279] Viral Filtration The viral filtration step removes potential viral particles and consists of a 0.5 / 0.2 μm prefilter, a 20 nanometer (nm) viral-retentive filter, and a 0.5 / 0.2 μm filter in series. The prefilter and viral filter are equilibrated with 50 mM Tris-HAc, 205 mM NaCl buffer at pH 7.0 before loading with the multimodal cation exchange eluate. The maximum loading capacity of the filters is 254 liters per square meter (L / m2). During loading, the differential pressure is maintained below 2 bar for the prefilter, 0.7-1.0 for the viral-retentive nanofilter, and below 2 bar for the final 0.5 / 0.2 μm filter. After loading is complete, the filters are stripped using a wash buffer. The final combined filtrate is mixed before proceeding to the next step.

[0280] Ultrafiltration and Diafiltration Ultrafiltration and diafiltration serve to adjust in-process drug substance protein concentration and exchange buffers prior to final bulk formulation.

[0281] An ultrafiltration / diafiltration unit with a PES 30 kilodalton (kDa) molecular weight cutoff filter cassette is equilibrated with 50 mM Tris-HAc, 205 mM NaCl buffer at pH 7.0 until the determined pH and conductivity are met. The filtrate from the previous step is pumped along the surface membrane at less than 500 grams per square meter (g / m2) and concentrated to approximately 15 grams per liter (g / L). The concentrated solution is then diafiltered with 10 mM histidine hydrochloride (His-HCl) at pH 6.3 for a minimum of 6 diavolumes until the pH and conductivity criteria are met. The flux and transmembrane pressure are maintained below approximately 300 LMH and 1.25 bar, respectively, throughout the ultrafiltration and diafiltration processes. After circulation at low flow rates, the DF pool is collected and stripped with 10 mM His-HCl at pH 6.3. The product is 0.5 / 0.2 μm filtered and the filtrate is kept at ambient conditions for no more than 3 days before further processing.

[0282] Excipient addition, bulk formulation and filling During bulk formulation, sucrose and polysorbate 20 are added from stock solutions to the intermediate material at final concentrations of 8% (w / v) and 0.01% (w / v), respectively, to improve product stability. Subsequent dilutions to the target concentration range are performed with 10 mM His-HCl, pH 6.3. The formulated product is filtered through a 0.22 μm polyvinylidene fluoride (PVDF) membrane filter and approximately 4 kg of the filtered product is aseptically dispensed into individual single-use 5 L sterile polycarbonate bottles. The bulk is stored at -70°C ± 10°C. All operations, including excipient addition, bulk filtration, and filling, are performed in a laminar flow hood.

[0283] Example 3: Disulfide bond linkage confirmation by LC-MS / MS Disulfide bond linkages are important in protein folding and play a crucial role in both protein structure and function. The number of disulfide bonds and their location are important attributes for ensuring the safety and efficacy of biopharmaceuticals.

[0284] In PC-1, there are 18 cysteine ​​residues, cross-linked by one interchain disulfide bond and eight intrachain disulfide bonds. Eight disulfide bond-associated peptides (DS1–DS3 and DS5–DS9) were predicted by non-reducing Lys-C / trypsin sequential digestion. One disulfide bond-associated peptide (DS4) was predicted by non-reducing Lys-C / trypsin / thermolysin sequential digestion. The liquid chromatography with tandem mass spectrometry (LC-MS / MS) results are provided in Table 4.

[0285] [Table 4-1]

[0286] [Table 4-2]

[0287] Example 4: Secondary structure determination by far-UV CD Protein far-UV circular dichroism (far-UV CD) spectra can reveal characteristic secondary structures, i.e., α-helix, β-sheet, random coil, etc. Prior to measurements, protein samples were 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-815 CD spectrometer and Spectra Manager software.

[0288] The CD spectrum of PC-1 in the far-UV region (190-260 nm) is shown in Figure 9. β-sheet and random coil were the main secondary structure compositions.

[0289] Example 5: Tertiary structure determination by near-UV CD Protein near-UV CD spectra provide information about protein tertiary structure. The CD spectral pattern in the 250-350 nm region is determined by the absorption, dipole orientation, and nature of the surrounding environment of phenylalanine (250-270 nm), tyrosine (270-290 nm), and tryptophan (290-305 nm), respectively. For measurements, protein samples were diluted to 1.0 mg / mL with 10 mM His, 8 (w / v)% sucrose, 0.01 (w / v)% polysorbate 20 at pH 6.3. Data collection and analysis were performed by a JASCO / J-815 CD spectrometer and Spectra Manager software, respectively.

[0290] The CD spectrum of PC-1 in the near-UV region (250–350 nm) is shown in Figure 10. Spectral similarity / structural consistency analysis was achieved by calculation of the correlation coefficient of the two spectra using the “Quick Compare” tool of the OPUS spectroscopy software.

[0291] Example 6: Thermal stability analysis by DSC Differential scanning calorimetry (DSC) is an experimental technique for obtaining thermal transition profiles of materials. It is widely used in protein characterization in the investigation of protein folding / unfolding transitions under programmed temperature ramps. The onset of the thermal transition (T onset ) and the midpoint (T m ) temperature (T m ) is commonly used as an indicator of thermal stability, and DSC thermograms reveal the thermal transition profile.

[0292] PC-1 was analyzed using a MicroCal DSC from Malvern. Protein samples were diluted to 1 mg / mL with 10 mM His, 8% (w / v) sucrose, 0.01% (w / v) polysorbate 20 at pH 6.3 before analysis. 400 microliters (μL) of the corresponding formulation buffer was added to a 96-well plate as a reference, followed by 400 μL of protein sample. Samples were heated from 10° C. to 95° C. at a heating rate of 90° C. / h in a capillary DSC system. DSC data were analyzed and fitted with MicroCal PEAQ-DSC software 1.51. Results are shown in FIG. 11. For PC-1, T onset was determined to be 59.0°C, and the midpoint temperature (T m1 and T m2 ) were determined to be 71.4°C and 79.5°C.

[0293] Example 7: Molar mass and size analysis by SEC-MALS Size exclusion chromatography coupled with a multi-angle light scattering (SEC-MALS) detector separates proteins based on their size, and then the molecular weight of the separated components is measured via the MALS detector. Larger proteins elute at earlier retention times, while smaller proteins elute later from the SEC column, resulting in separation between 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 the solution are calculated using the intensity and angle 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, which is represented by the Zimm equation. The intensity of the laser scattering is directly proportional to the molecular weight and protein concentration of proteins larger than 10 nm. Therefore, their protein molecular weights are calculable according to the relationship between the scattered light intensity and the angle P(θ), the protein size Rg, and the protein concentration c.

[0294] SEC-MALS chromatograms of PC-1 are shown in Figures 12A and 12B. The molecular weight of the main peak (peak 1) is 90.2 kDa (RT = 3.111 min), which corresponds to the monomer.

[0295] Example 8: Clinical Efficacy of Polypeptide Complex 1 (PC-1) This example illustrates the clinical efficacy of Polypeptide Complex 1 (PC-1) disclosed herein.

[0296] PC-1 is a tumor-activating 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).

[0297] Pharmacology In vitro nonclinical pharmacology studies with PC-1 investigated: 1. PC-1 binds to PSMA, cluster of differentiation 3 (CD3), and albumin antigens from mouse, rat, cynomolgus monkey, and human. 2. PC-1 stability in serum of healthy human donors, mCRPC patients, and cynomolgus monkeys 3. Ability of PC-1 to induce cytokine production 4. Ability of PC-1 to induce T cell-mediated antitumor cytotoxic activity Results from binding studies showed that PC-1 bound to PSMA, CD3, and albumin in humans and cynomolgus monkeys with low nanomolar affinity, while showing minimal binding to mouse or rat antigens, concluding that CD3 binding affinity was cleavage-dependent. Stability studies showed that PC-1 showed a similar cleavage rate in healthy human serum (1%) compared to serum from mCRPC patients (2%), and that cleavage of PC-1 in cynomolgus monkey serum was 6% / day. Cytokine induction studies showed that cleaved and unmasked forms of PC-1 showed a dose-dependent ability to induce release of interferon gamma (IFNγ), tumor necrosis factor (TNF), and interleukin (IL)-6 by immune cells, whereas masking of the PC-1 CD3-binding domain reduced its ability to induce cytokine production, suggesting that cytokine release required proteolytic PC-1 unmasking. Cytokine production was not detected in the absence of target cells, suggesting that the activity of PC-1 was dependent on the presence of PSMA-expressing target cells. The presence of the mask also reduced the ability of PC-1 to induce T cell-mediated tumor cell killing. Furthermore, the ability of PC-1 to induce T cell-mediated tumor cell killing was dose- and PSMA-dependent. A non-clinical pharmacological study investigated the ability of cleaved PC-1 and PC-1-serine protease (SP) to induce cytokine release in human whole blood samples. The study examined cytokine release under two conditions, using soluble and wet-coated (plate-bound) test substances. No IL-2, IL-6, IL-10, TNF, and IFNγ release above the levels of untreated controls (very low to no signal) was observed following stimulation with either soluble or wet-coated (plate-bound) cleaved PC-1-SP in any of the samples. No IL-2 or IFNγ cytokine release was observed in any of the samples following PC-1 stimulation. 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 small number of samples after treatment with soluble PC-1 at the highest concentrations tested (10 nM [917.4 ng / mL] and 100 nM [9174 ng / mL]). Nonclinical safety pharmacological parameters included cardiovascular, CNS, and respiratory safety endpoints incorporated in a 4-week repeated-dose intravenous (IV) toxicity study in cynomolgus monkeys. There were no functional effects on the CNS or respiratory systems based on clinical observations of the animals and detailed weekly examinations at doses of PC-1 ≤ 1.5 mg / kg / week. In addition, there were no clearly defined effects from PC-1 administration to the cardiovascular system of the animals on qualitative or quantitative electrocardiogram (ECG) parameters. All ECGs were within normal limits, except for a few episodes of non-test substance-related arrhythmias.

[0298] Pharmacokinetics The pharmacokinetic (PK) properties of PC-1 were evaluated after single and multiple dose studies in cynomolgus monkeys. The PK of PC-1 was evaluated at 0.1 mg / kg, 0.3 mg / kg, and 1.5 mg / kg after a single IV dose to male cynomolgus monkeys. Four analytes were measured (intact PC-1 [uncleaved PC-1], Met1 [also called cleaved PC-1-SP], Met2 [also called cleaved PC-1-MMP], and total PC-1 [intact (uncleaved) PC-1 + Met1 + Met2]). However, PK parameters were derived from three analytes (intact PC-1, total PC-1, and Met1) because Met2 was below the level of quantification (BLQ). Additionally, the clearance of the analytes is likely affected by the presence of anti-drug antibodies (ADA), as will be further investigated in the following toxicity studies. With regard to PK parameters, the clearance of intact and total PC-1 remained relatively constant among the three dose groups. The faster clearance observed at the highest dose (1.5 mg / kg) was likely caused by the presence of ADA. The intact PC-1 half-life (T1 / 2) was 111, 78.5, and 70.4 hours at the three doses, respectively, whereas the T1 / 2 of total PC-1 was 110, 105, and 62.1 hours, respectively. Systemic exposure was measured by area under the curve (AUC0-inf), and the maximum concentrations (Cmax) of intact PC-1 and total PC-1 increased dose-proportionally across the entire dose range. The AUC0-last of Met1 decreased dose-proportionally as the dose level increased from 0.1 mg / kg to 1.5 mg / kg, whereas Cmax increased dose-proportionally across the entire dose range. Overall systemic exposure to Met1 was minimal and appeared to be affected by cleavage of intact PC-1 during sample processing in the PK assay. Using a validated PK assay, sample processing induced conversion of an average of 1.87% of intact PC-1 to Met1. However, native cleavage in vivo is expected to be much lower. In a multiple-dose toxicity study, PC-1 was administered IV at 0.1, 0.3, or 1.5 mg / kg / week to male and female monkeys for a total of five doses per animal.Minimal or no systemic exposure differences were observed between intact and total PC-1.

[0299] In general, accumulation was not observed after multiple doses, but could not be assessed in animals with confirmed ADA. In ADA-negative animals, median T1 / 2 for intact PC-1 and total PC-1 on days 1 and 22 ranged from 67.7 to 109.1 hours and 60.3 to 156.4 hours, respectively. No significant sex differences in systemic exposure as measured by Cmax and AUC0-168h were observed on days 1 and 22 in ADA-negative animals at either dose. Overall exposure of Met1 was minimal, which was affected by an average of 1.87% cleavage of intact PC-1 during the sample preparation process, as described above. Natural in vivo cleavage is expected to be much lower. No tissue distribution studies were performed for PC-1. No specific studies were performed on PC-1 metabolites, as classical drug metabolic elimination does not represent a significant clearance mechanism for monoclonal antibodies. Therapeutic antibodies such as PC-1 are normally catabolized to small peptides, carbohydrates, and amino acids, which are either returned to the nutrient pool without any biological effect or excreted via the kidney.

[0300] toxicology The nonclinical toxicity profile of PC-1 was evaluated in a 4-week Good Laboratory Practice (GLP) repeat-dose IV toxicity study in cynomolgus monkeys, in vitro and in vivo human and / or monkey cytokine release assays, and in serum stability assays using serum from cynomolgus monkeys, healthy human donors, and patients diagnosed with mCRPC. In addition, the immunogenicity of PC-1 was evaluated in all animals during the repeat-dose toxicity study in cynomolgus monkeys. In the repeat-dose toxicity study, PC-1 was administered IV slow bolus to female and male monkeys at doses of 0.1, 0.3, and 1.5 mg / kg / week (the control group received vehicle control) for a total of five doses per animal. No non-adverse changes to hematology, clinical chemistry, and microscopic findings were observed at 0.1 mg / kg / week or higher. PC-1 induced a transient, dose-dependent increase in cytokines within 24 hours after the first dose at 0.1 mg / kg / week or higher. Most, if not all, of the changes could be attributed to the mechanism of action of PC-1 (i.e., activation of immune cell populations) and / or a proinflammatory response consistent with cytokine release observed following administration of the first dose of PC-1. Based on the lack of adverse effects noted during the dosing and recovery phase of the study, the highest non-severe toxic dose (HNSTD) / no observed adverse effect level (NOAEL) was 1.5 mg / kg / week. Immunogenicity assessment revealed that 1 of 6 animals receiving PC-1 at 0.1 mg / kg / week, 5 of 6 animals receiving PC-1 at 0.3 mg / kg / week, and all 10 animals receiving PC-1 at 1.5 mg / kg / week were ADA positive by the last dose. No ADA positive animals were found in Group 1 (vehicle control). Toxicokinetic (TK) profiles were affected by ADA in all animals with confirmed antibodies, although in some animals, ADA was less affected following multiple dose exposure. Furthermore, PC-1 induced a dose- or concentration-dependent release of cytokines (ie, IL-6, TNF, IFNγ) in the presence of tumor cells in vitro and in healthy monkeys in vivo.In vivo, cytokine release was observed primarily after the first dose and correlated with clinical signs and clinical chemistry changes indicative of cytokine release syndrome (CRS) and potential vascular leakage. PC-1 was shown to be stable in vitro in human serum with minimal cleavage. An increased daily in vitro cleavage rate was observed in serum from cynomolgus monkeys, but the cleavage rate was still considered to be overall low in this species.

[0301] Phase 1 Clinical Trial Design This study is a first-in-human (FIH), Phase 1, open-label, multicenter study 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 three parts: dose escalation with approximately 20-30 subjects (Part 1), cohort backfill expansion with approximately 30 subjects enrolled across three dose levels (Part 2), and dose expansion with approximately 30 subjects enrolled at the recommended phase 2 dose (Part 3). 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 safety and dose-level activity. Dose expansion (part 3) will determine additional safety, tolerability, PK, PD, and preliminary clinical activity data for PC-1 at a dose and schedule to be determined by the Safety Review Committee after reviewing all available safety, PK, PD, and preliminary efficacy data.

[0302] Rationale for Evaluation of JANX007 in Patients with Metastatic Castration-Resistant Prostate Cancer While TCEs are a potentially powerful class of PSMA-targeted therapy, attempts to use TCEs to recruit the immune system to recognize PC cells by priming them against PSMA have so far been unsuccessful. Systemic activation and redirection of T cells can result in on-target off-tumor cytotoxicity and cytokine release, leading to severe CRS and severely limiting dosing and ultimately efficacy (Tran et al., 2020; Bono et al., 2021). In addition, first-generation TCEs suffer from poor PK, resulting in short half-lives and cumbersome dosing regimens. Thus, there is a critical unmet need to optimally leverage T cell-mediated cytotoxicity in targeting tumor cells. Products that can selectively accumulate and activate within the tumor microenvironment (TME) would have significant advantages in developing a favorable risk-to-benefit profile. PC-1-based approaches are designed to provide a more focused method to activate T cells in the tumor, minimize systemic activation, and allow for higher dosing, thereby increasing antitumor efficacy.

[0303] Physical, Chemical, and Pharmaceutical Properties and Formulations Physical and biochemical properties: PC-1 is a 92 kDa humanized trispecific protein composed of an anti-PSMA antigen-binding fragment (Fab), an anti-CD3 single-chain variable fragment (scFv), and an 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 heavy chain (HC) of TRACTr, and eight intramolecular disulfide bonds. The peptide mask contains a single internal disulfide bond. The arrangement of the LC and HC of PC-1 TRACTr is provided in Figure 13. The LC of the CD3 scFv is fused to the N-terminal LC of the PSMA Fab via a short flexible amino acid linker. The tandem albumin-binding sdAb, peptide mask, and tumor protease-cleavable amino acid linker are fused to the amino terminus of the HC of the CD3 scFv. The albumin-binding sdAb is attached to the mask via a short flexible linker (Figure 1).

[0304] Pharmaceuticals PC-1 solution for infusion, specifically IV infusion (referred to as PC-1 DP), is supplied 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). PC-1 drug product is contained in a 2R, Type 1 borosilicate glass vial with a 13 mm nested cap consisting of a polypropylene snap-on cap and a recessed Flurotec® laminated elastomeric stopper closure. The formulation of PC-1 drug product is outlined in Table 5. The physical and chemical properties of PC-1 are summarized in Table 6.

[0305] [Table 5]

[0306] [Table 6]

[0307] Storage and handling PC-1 DP vials are stored and shipped frozen (-20°C).

[0308] Non-clinical trials

[0309] Introduction: Preclinical studies for PC-1 are designed to support a Phase 1 clinical program in subjects diagnosed with mCRPC. Preclinical pharmacology studies were performed in in vitro and ex vivo model systems. In vitro studies investigated: PC-1 binding to PSMA, CD3, and albumin antigens from mouse, rat, cynomolgus monkey, and human; PC-1 stability in serum of healthy human donors, mCRPC patients, and cynomolgus monkeys; the ability of PC-1 to induce cytokine production; and the ability of PC-1 to induce T cell-mediated antitumor cytotoxicity activity.

[0310] Ex vivo studies investigated the ability of PC-1 to induce cytokine release in human whole blood samples from human donors. Further details of the studies are provided in the following sections. In vivo studies included a nonclinical single-dose PK study in cynomolgus monkeys. The nonclinical PK data provided the rationale for the proposed weekly dosing schedule in Phase 1 studies. A GLP repeat-dose study evaluated the safety, tolerability, and associated systemic exposure of PC-1 over 4 weeks of weekly dosing in cynomolgus monkeys (a total of 5 doses per animal) and assessed the reversibility of any effects after a 4-week recovery period.

[0311] Nonclinical Pharmacology Tests were performed using not only PC-1 (uncleaved or intact PC-1), but also the cleaved metabolites, cleaved PC-1-SP (referred to as Met1 in PK studies) and cleaved PC-1-MMP (referred to as Met2 in PK studies), as well as the active unmasked molecule PC-1-TCE (Table 7). Total PC-1 (uncleaved PC-1+Met1+Met2) was also measured in the PK studies. The cleaved PC-1-SP and cleaved PC-1-MMP used in the in vitro pharmacological tests were obtained from PC-1 by enzymatic treatment with recombinant human matriptase (MTSP1) and recombinant human matrix metalloproteinase 9 (MMP9), respectively.

[0312] [Table 7]

[0313] In vitro studies to support efficacy PC-1 binding affinity for prostate-specific membrane antigen, CD3, and albumin (PC-1-001-TD): The affinity of PC-1 for 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 cleaved PC-1-SP, cleaved PC-1-MMP, and PC-1-TCE. Briefly, serial dilutions of PC-1, cleaved PC-1-SP, cleaved PC-1-MMP, or PC-1-TCE were added to plates containing immobilized mouse, rat, cynomolgus monkey, or human antigen. 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 fitted to a four-parameter variable slope regression from which half-maximal effective concentrations (EC50) were determined and reported as affinities (Tables 8-11).

[0314] All test agents (PC-1, truncated PC-1-SP, truncated PC-1-MMP, and PC-1-TCE) bound to human and cynomolgus PSMA (Figures 14A-14F) and albumin (Figures 15A-15D) with low nanomolar affinity. Truncated PC-1-SP, truncated PC-1-MMP, and PC-1-TCE showed low nanomolar binding to CD3. In contrast, PC-1 bound to CD3 several orders of magnitude weaker due to inhibition by the peptide mask (Figures 16A-16F). All test agents showed minimal binding to mouse and rat antigens, respectively (Figures 14A-16F). The observed differences in binding of test agents to mouse and rat antigens versus human and cynomolgus antigens are consistent with the low percentage of sequence homology of rodent antigens versus human and cynomolgus antigens.

[0315] [Table 8]

[0316] [Table 9]

[0317] [Table 10]

[0318] [Table 11]

[0319] In summary, PC-1 binds with low nanomolar affinity to human and cynomolgus PSMA and albumin. PC-1 CD3 binding affinity is cleavage-dependent. Treatment of PC-1 with MTSP1 or MMP9 to form cleaved PC-1-SP or cleaved PC-1-MMP, respectively, results in strong CD3 binding similar to that observed for PC-1-TCE. PC-1 shows minimal binding to mouse or rat PSMA, CD3, and albumin. The observed differences in binding of test substances to mouse and rat antigens versus human and cynomolgus PSMA, CD3, and albumin are consistent with the low percentage of sequence homology of rodent antigens to human and cynomolgus antigens.

[0320] Stability of PC-1 in serum of healthy cynomolgus monkeys PC-1 stability in serum from human donors, and metastatic castration-resistant prostate cancer patients (PC-1-002-TD): cynomolgus monkeys, healthy human donors, and humans diagnosed with mCRPC was assessed using a kinetic binding assay with an 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 was calculated at various time points based on the initial slope of the binding curve to the CD3-coated sensor. The relative concentration of cleaved PC-1 over time was used to calculate the cleavage rate in serum as an index of PC-1 stability compared to the unmasked PC-1-TCE reference sample. The resulting data is reported as percent cleavage per day (Table 12). An example of a CD3 kinetic binding curve is shown in Figures 17A-17B. A linear regression of percent cleavage versus time is shown in Figures 18A-18H. PC-1 was measured in pooled healthy human serum and mCRPC human serum and was stable for 168 hours. Less than 1% cleavage per day was observed in pooled healthy human serum, and 2% or less cleavage per day was observed in mCRPC serum. A somewhat higher cleavage rate of 6% per day was observed in serum from healthy cynomolgus monkeys.

[0321] [Table 12]

[0322] In summary, PC-1 showed similar cleavage rates in healthy human serum compared to sera from mCRPC patients, less than 1% and 2% cleavage per day, respectively. PC-1 cleavage in cynomolgus monkey serum was 6% per day.

[0323] PC-1-induced cytokine production by peripheral blood mononuclear cells cocultured with target tumor cells expressing prostate-specific membrane antigen 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 blood from eight consented healthy human donors were cultured for 72 h with or without human PC cell lines expressing high (LNCaP), intermediate (22Rv1), and minimally detectable (PC3) levels of PSMA in the presence of increasing concentrations of test substances (Dang et al., 2021; Gorges et al., 2016). While truncated (cleaved PC-1-SP and cleaved PC-1-MMP) and unmasked (PC-1-TCE) test substances induced strong dose-dependent production of IFNγ, TNF, and IL-6, masked PC-1 was shown to have a lower ability to induce cytokine production as illustrated by the high EC50 for the three evaluated cytokines (Figures 19A-19I: each data point represents the average of two replicates. EC50 values ​​(concentration of test substance required to induce cytokine levels corresponding to 50% of the maximal level) were extrapolated using the generated nonlinear regression response curves. Abbreviations: IFN = interferon, IL = interferon, Figures 20A-20I: Each data point represents the average of two replicates. EC50 values ​​(concentration of test substance required to induce cytokine levels equivalent to 50% of the maximal level) were extrapolated using the generated nonlinear regression response curves. Abbreviations: IFN = interferon, IL = interleukin, MMP = matrix metalloprotease, PBMC = peripheral blood mononuclear cells, SP = serine protease, TCE = T cell engager, TNF = tumor necrosis factor). Cytokine induction was not detected in the absence of PSMA-expressing target cells. (Figures 21A-21F: Abbreviations: IFN = interferon, IL = interleukin, MMP = matrix metalloprotease, PBMC = peripheral blood mononuclear cells, SP = serine protease, TCE = T cell engager, TNF = tumor necrosis factor).

[0324] In summary, masking of the CD3-binding domain of PC-1 reduced its ability to induce cytokine production, suggesting that cytokine release required proteolytic PC-1 unmasking. The truncated (cleaved PC-1-SP, cleaved PC-1-MMP) and unmasked (PC-1-TCE) versions of PC-1 showed a dose-dependent ability to induce cytokine production, although PC-1 had a higher EC50 for all cytokines evaluated. Cytokine production was not detected in the absence of target cells, suggesting that the activity of PC-1 is dependent on the presence of PSMA.

[0325] In vitro T cell-mediated killing of PC-1-induced prostate-specific membrane antigen-expressing target tumor cells The ability of PC-1 to induce T cell-mediated target cell killing was evaluated using PBMCs derived from the blood of eight consenting healthy human donors co-cultured with prostate tumor cell lines expressing high (LNCaP), moderate (22Rv1), or minimally detectable (PC3) levels of PSMA in the presence of increasing concentrations of test substances. Results showed that PC-1-induced T cell-mediated tumor cell killing was dose-dependent and reduced in the presence of mask (Figures 22A-22H: each symbol represents the average of the appropriate treatment replicates. Abbreviations: MMP = matrix metalloproteinase, PBMC = peripheral blood mononuclear cells, SP = serine protease, TCE = T cell engager, and Figures 23A-23H: each symbol represents the average of the appropriate treatment replicates. Abbreviations: MMP = matrix metalloproteinase, PBMC = peripheral blood mononuclear cells, SP = serine protease, TCE = T cell engager). Compared to cleaved PC-1-SP, cleaved PC-1-MMP, and PC-1-TCE, PC-1 reduced the ability of T cells to induce cytotoxic activity in the presence of LNCaP and 22Rv1 cells by up to 30-fold and 160-fold, respectively. Furthermore, the magnitude of induced tumor cell killing was dependent on the level of PSMA expression, as none of the test agents induced PBMC-mediated killing of PC3 cells (representative plots shown in Figures 24A-24D: each symbol represents the average of the appropriate treatment replicates. Abbreviations: MMP = matrix metalloprotease, PBMC = peripheral blood mononuclear cell, SP = serine protease, TCE = T cell engager).

[0326] In summary, masking of the CD3 binding domain reduces the ability of PC-1 to induce T cell-mediated killing of target tumor cells, with up to 160-fold lower cytotoxic activity compared to cleaved (cleaved PC-1-SP and cleaved PC-1-MMP) and unmasked (PC-1-TCE) test substances. The ability of PC-1 to induce T cell-mediated tumor cell killing is dose- and cleavage-dependent and requires PSMA expression.

[0327] Ex vivo drug efficacy testing Cytometric bead array analysis of human cytokines using whole blood from healthy human donors

[0328] The ability of PC-1 and cleaved PC-1-SP to induce cytokine release was evaluated in whole blood samples from 10 healthy human donors using a cytometric bead array approach. Stimulation was performed for 24 hours in soluble and wet-coated (plate-bound) formats. Stimulation with Staphylococcal Enterotoxin B (SEB), a positive control, in soluble format, resulted in cytokine production in samples from all donors, demonstrating that these cells have the ability to release cytokines in response to stimulation. Cytokine release was not observed for any sample in the absence of treatment. No cytokine release was observed when stimulation was performed in wet-coated (plate-bound) format. No release of IL-2, IL-6, IL-10, TNF, and IFNγ above the levels of untreated controls (low to undetectable) was detected in any of the samples following stimulation with cleaved PC-1-SP in either format. No release of IL-2 or IFNγ was observed following PC-1 stimulation for all donors in either format. No IL-6, IL-10, or TNF cytokine release was observed following PC-1 stimulation in the plate-bound format. After treatment with the highest concentrations of soluble PC-1 tested (10 nM [917.4 ng / mL] and 100 nM [9174 ng / mL]), IL-6, IL-10, and TNF release was observed in some samples.

[0329] Safety Pharmacology No stand-alone safety pharmacology studies were conducted in PC-1 according to ICH S6(R1) and ICH S9 guidelines. A 4-week repeat-dose IV toxicity study in cynomolgus monkeys incorporated cardiovascular, CNS, and respiratory safety pharmacology endpoints.

[0330] No clearly defined effects of PC-1 administration were observed on qualitative or quantitative ECG parameters. Sinus tachycardia, defined as a mean heart rate above 270 beats / min, occurred in four instances in three animals receiving 1.5 mg / kg / week post-dose on day 1, and one instance was observed in one animal post-dose in the control group. Sinus tachycardia is often a variant of normal in cynomolgus monkeys. Although the majority of post-dose cases occurred after the high dose, there was no significant effect of PC-1 administration on group mean heart rate. Thus, the tachycardia was probably not PC-1 related. One animal receiving 1.5 mg / kg / week had three atrial premature complexes (APCs) on the ECG on day 1. Low frequency APCs have been reported to occur sporadically in non-experimental cynomolgus monkeys, and because APCs were observed in a single animal, the arrhythmia was not considered test article related. No PC-1-related respiratory rate or temperature changes were observed during the 4-week repeated dose toxicity study. There were no PC-1-related CNS changes based on daily clinical signs and weekly detailed clinical observations during the study.

[0331] Pharmacokinetics and metabolism in animals Pharmacokinetics in Cynomolgus Monkeys: The PK properties of PC-1 were evaluated following a single-dose (non-GLP) PK study and a repeat-dose GLP toxicity study in Cynomolgus monkeys. Determination of the analytes intact PC-1 (also called uncleaved PC-1), total PC-1 (PC-1+Met1+Met2), and metabolites Met1 (cleaved PC-1-SP) and Met2 (cleaved PC-1-MMP) in non-human primate K2EDTA plasma was performed.

[0332] Single-dose PK study In a single-dose PK study, the PK of PC-1 was evaluated at dose levels of 0.1 mg / kg, 0.3 mg / kg, and 1.5 mg / kg administered to male cynomolgus monkeys. Four analytes (intact PC-1, total PC-1, Met1, and Met2) were measured, but PK parameters were derived from three analytes (intact PC-1, total PC-1, and Met1). Met2 was excluded from the TK parameter calculations because its concentration was at the BLQ for most samples. Additionally, analyte clearance was likely affected by the presence of ADA. Intact and total PC-1 clearance remained relatively constant among the three dose groups. The faster clearance observed at the highest dose (1.5 mg / kg) was likely caused by the presence of ADA. The mean half-life (T1 / 2) of intact PC-1 was 111, 78.5, and 70.4 hours, respectively, for the three dose groups, whereas total PC-1 T1 / 2 was 110, 105, and 62.1 hours, respectively. Systemic exposure of intact PC-1 and total PC-1, as measured by AUC0-inf and Cmax, increased with increasing dose across the entire dose range. Met1 AUC0-last increased less proportionally with increasing dose levels from 0.1 mg / kg to 0.3 mg / kg and from 0.1 mg / kg to 1.5 mg / kg, whereas Cmax increased proportionally with dose. Systemic exposure, as measured by AUC0-last and Cmax of Met1, increased proportionally with increasing dose levels from 0.3 mg / kg to 1.5 mg / kg. The overall exposure of Met1 was minimal, and cleavage of intact PC-1 during sample processing for the PK assay likely affected Met1 generation. Using a validated PK assay, sample processing was demonstrated to induce, on average, 1.87% conversion of intact PC-1 to Met1. Thus, native cleavage in vivo is expected to be much lower. This study also analyzed cytokine production in cynomolgus serum by flow cytometry. In general, the elevation of cytokines was transient and dose-dependent.In animals receiving 0.1 mg / kg or more of PC-1, levels of IL-6 were elevated 8 hours after dosing and declined to pre-study levels by 48 hours after dosing. In animals receiving 0.3 mg / kg or more, levels of IL-2 were variably elevated 2 hours after dosing and declined to baseline by 24 hours after dosing. Increases in IL-5, IL-10, TNF, and IFNγ were only observed in animals receiving 1.5 mg / kg (the highest dose) of PC-1, and these values ​​returned to baseline 24 to 48 hours after dosing.

[0333] Repeated dose GLP toxicity study In this GLP toxicity study, male and female cynomolgus monkeys were administered weekly IV injections of vehicle control (Group 1), or 0.1 mg / kg / dose (Group 2), 0.3 mg / kg / dose (Group 3), or 1.5 mg / kg / dose (Group 4) of PC-1 for a total of five doses per animal. In the immunogenicity study, ADAs were detected in a number of animals in each dose group (summarized in section 4.4.1). ADAs influenced the TK profile of the test article as discussed below. Overall, minimal to no differences in systemic exposure were observed between intact and total PC-1. Accumulation was not observed after multiple doses, but could not be fully assessed in either male Group 3 or Group 4 animals due to the presence of ADA. No significant accumulation was observed in both sexes at 0.1 mg / kg / week (Group 2) and in females at 0.3 mg / kg / week (Group 3), who were ADA negative. In ADA-negative animals, the median intact PC-1 T1 / 2 on days 1 and 22 ranged from 67.7 to 109.1 hours across all doses tested. In ADA-negative animals, the median total PC-1 T1 / 2 on days 1 and 22 ranged from 60.3 to 156.4 hours across all doses tested. At 0.1 mg / kg / week on days 1 and 22, Cmax and AUC 0-168hNo significant differences in systemic exposure were observed between the sexes as measured by . At 0.3 and 1.5 mg / kg / week, no significant differences were observed between the sexes on day 1, but exposure on day 22 could not be compared between the sexes due to the presence of ADA and its effect on the total PC-1 TK profile. Overall exposure of Met1 was minimal, which was affected 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 of the group 1 animals. Immunogenicity was assessed in all animals. No animals from group 1 (vehicle control) were found to be ADA positive, while one of six animals from group 2 (0.1 mg / kg / week), five of six animals from group 3 (0.3 mg / kg / week), and all ten animals from group 4 (1.5 mg / kg / week) were confirmed to be ADA positive by the last dose. The TK profile was affected in all animals that were ADA positive, although ADA had a lesser effect on exposure after multiple doses in some animals. Two of the ADA positive female animals in group 3 still achieved substantial exposure after the day 22 dose, with an approximate doubling of AUC0-168h (2.0-fold increase) and Cmax (1.9-fold increase) compared to the day 1 dose. In addition, two ADA positive female animals in group 4 also had substantial exposure as measured by AUC0-168h and Cmax after the day 22 dose, with AUC0-168h values ​​being approximately 54% and Cmax values ​​being approximately 96% of the respective values ​​observed after the day 1 dose.

[0334] metabolism No specific studies were performed on PC-1 metabolism because classical drug metabolic elimination does not represent a significant clearance mechanism for monoclonal antibodies. Antibodies such as PC-1 are generally catabolized to small peptides, carbohydrates, and amino acids, which are either returned to the nutrient pool or excreted via the kidney without any biological effect (Wang et al., 2008). Therefore, in accordance with the ICH S6(R1) guidance, no metabolism studies were performed on PC-1.

[0335] excretion Renal excretion is relatively unimportant for monoclonal antibodies because their large size limits the extent of their glomerular filtration, therefore, in accordance with ICH S6(R1) guidance, excretion studies were not performed for PC-1.

[0336] Pharmacokinetic Drug Interactions 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 or transported by P-glycoprotein (Pgp) or related adenosine triphosphate-binding cassette membrane transporters. Cytokines produced by activated lymphocytes can affect the levels of Pgp and the activity of CYP enzymes (Harvey and Morgan, 2014). The clinical relevance of PC-1 immunomodulation and potential cytokine production that may affect Pgp and CYP is unclear, but clinically relevant drug-drug interaction effects are considered highly unlikely (Seitz and Zhou, 2007; Huang et al., 2010).

[0337] toxicology Repeated-dose toxicity study: PC-1 was evaluated in a GLP, 4-week, weekly, repeated-dose toxicity study in cynomolgus monkeys (Table 13). Consistent with the intended clinical route of administration, toxicity studies were performed using the IV route of administration. Cynomolgus monkeys were selected as a pharmacologically relevant species due to the high PSMA, CD3, and albumin protein sequence homology with human antigens, and the nearly equivalent binding of PC-1 to its target antigens (PSMA, CD3, and albumin) in monkeys and humans. Additionally, PC-1 was evaluated for cytokine release and serum stability in vitro and in vivo.

[0338] [Table 13]

[0339] PC-1 was administered by IV bolus to male and female monkeys (3 / sex / group) at doses of 0 (vehicle control), 0.1, 0.3, or 1.5 mg / kg / week (total of 5 doses). Additional animals (2 / sex / group) at 0 and 1.5 mg / kg / week were evaluated after a 4-week recovery period for reversibility of any PC-1-related effects. Dosing regimens were selected based on the half-life of PC-1 in cynomolgus monkeys and were designed to have similar or more intensive dosing regimens as clinical dosing regimens. No PC-1-related mortality, food consumption or weight changes, body temperature, respiration rate, coagulation, urinalysis, or gross findings were observed during the dosing or recovery phase. No PC-1-related ophthalmological and electrocardiographic findings were observed.

[0340] Clinical Signs Chilliness and reduced activity were observed in one male after the first dose of PC-1 at 1.5 mg / kg / week, with clinical signs gradually resolving before the second dose. The clinical signs appeared to be related to the cytokine induction observed after the first dose. No other clinical signs were noted in other animals during the dosing or recovery phase.

[0341] hematology PC-1-associated non-adverse hematological changes included erythroid changes (reduction in red blood cell [RBC] mass, regenerative erythroid response, mean corpuscular hemoglobin [MCH], and mean corpuscular hemoglobin concentration [MCHC]) consistent with decreased erythropoiesis and hemolysis, and increases in leukocyte and platelet counts consistent with a proinflammatory response at 0.1 mg / kg / week and above. The decrease in RBC mass was partially reversed, the changes in reticulocyte count were fully reversed, and the changes in MCH and MCHC showed limited or no reversibility at the end of the 4-week recovery period in animals dosed with 1.5 mg / kg / week. The non-detrimental, minimally to significantly decreased lymphocyte counts in animals dosed at 0.1 mg / kg / week or more correlated with immunophenotyping findings of decreased mean absolute numbers of total T cells, cytotoxic T cells, helper T cells, and natural killer (NK) cells 24 hours after dosing in animals dosed at 0.1 mg / kg / week or more, consistent with the expected pharmacology of the test article (i.e., transient sequestration of activated lymphocytes). A mild decrease in monocyte counts was also noted on day 2 in animals dosed at 0.1 mg / kg / week or more, although the cause was unclear. The minimally decreased platelet counts on day 2 were consistent with a transient nonspecific effect of the large molecule test article on platelets in monkeys or may be due to a PC-1-associated increase in proinflammatory cytokines within 24 hours after the first dose. On or after day 15, PC-1-associated white blood cell changes included minimal to mild increases in neutrophil counts in females receiving 0.1 mg / kg / week or more, minimal or mild increases in lymphocyte counts in males and females receiving 1.5 mg / kg / week, and moderate increases in eosinophil counts in males receiving 1.5 mg / kg / week and females receiving 0.3 mg / kg / week or more, consistent with a proinflammatory response. PC-1-associated mild to moderately increased platelet counts on or after day 8 in animals receiving 0.1 mg / kg / week or more were 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.

[0342] clinical chemistry PC-1-related non-adverse clinical chemistry findings noted on day 2 (16–24 h post-dose) were consistent with a transient, more pronounced proinflammatory response after the first dose, likely resulting in vascular leakage and / or a drop in blood pressure (not measured), which correlated with increased cytokine release within 24 h after the first dose. Mild to markedly increased urea and creatinine on day 2 in animals dosed with 0.3 mg / kg / week or higher was consistent with renal dysfunction potentially due to reduced renal tissue perfusion; however, a direct effect on the kidney could not be excluded, and correlative microscopic renal findings on day 31 were limited to minimal to moderate mononuclear cell infiltration. Increased urea and creatinine had evidence of reversibility by day 8. More marked decreases in serum albumin in animals dosed with 0.1 mg / kg / week or higher and minimal decreases in sodium and chloride on day 2 in animals dosed with 0.3 mg / kg / week or higher were also consistent with potential vascular leakage or potential renal dysfunction. Less pronounced decreases in serum albumin were noted by day 31, which was mostly reversed by the end of the 4-week recovery period. The decreased albumin at these later time points, along with minimally or mildly increased globulins, were consistent with a proinflammatory response. Further PC-1-associated serum chemistry changes included reversible minimally or mildly decreased total cholesterol.

[0343] Organ weight PC-1-related organ weight differences at day 31 consisted of increased spleen weights in animals dosed at 0.1 mg / kg / week or higher and increased liver weights in animals dosed at 1.5 mg / kg / week. The increased spleen weights did not appear to correlate with dose. However, the magnitude of the differences was consistent with test substance-related findings and correlated with the microscopic findings of minimal or mild increases in lymphocytic cellularity in the splenic white pulp. The increased liver weights correlated with minimal perivascular mononuclear cell infiltration. PC-1-related organ weight differences at the end of the recovery period consisted of increased spleen weights in males dosed at 1.5 mg / kg / week, consistent with partial reversibility and lacking microscopic correlation.

[0344] Microscopic findings PC-1-associated non-adverse microscopic findings on day 31 consisted of minimal to prominent mononuclear cell infiltrates, often with a perivascular pattern, in the liver, gallbladder, adrenal medulla, cervix, vagina, epididymis, seminal vesicles, and urinary bladder, as well as increased lymphocytic cellularity in the white pulp of the spleen in animals administered 0.1 mg / kg / week or higher. However, these microscopic findings were often poorly dose-related. The microscopic findings were consistent with a proinflammatory response but were not associated with evidence of tissue damage and were therefore not considered adverse. The microscopic findings had largely reversed by the end of the recovery period, and the remaining changes at the end of the recovery period were of uncertain relationship to PC-1 treatment.

[0345] Immunophenotyping of T / B / natural killer cells In monkeys receiving PC-1 at 0.1 mg / kg / week or higher, the mean absolute numbers of total T cells, cytotoxic T cells, helper T cells, and NK cells were decreased 24 hours after dosing compared to pre-dose. A transient increase in B cell numbers was noted at 1.5 mg / kg / week after the first dose. A transient and minimal increase in the percentage of CD69 helper T cells, CD69 cytotoxic T cells, and Ki67 helper T cells after the first dose induced by PC-1 treatment. All changes observed in peripheral white blood cell (WBC) cell populations were transient, returning to levels similar to pre-dose values ​​before the third or fifth dose.

[0346] Cytokine levels PC-1 induced a transient, dose-dependent increase 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 or higher. Cytokine levels returned to pre-study values ​​by 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 some animals.

[0347] Effect of anti-drug antibodies Anti-PC-1 antibodies were detected in all groups, and the incidence and titers 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 by 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 until the end of the dosing phase, and most animals showed a substantial reduction in exposure as assessed by the third or fourth dose. Two ADA-positive female animals dosed with 0.3 mg / kg / week still achieved substantial exposure after the 22nd dose, with AUC 0-168h (2.0-fold increase) and Cmax (1.9-fold increase). In addition, two ADA-positive female animals dosed at 1.5 mg / kg / week also showed a significant increase in AUC 0-168h and maintained substantial exposure as measured by Cmax and AUC 0-168h There was approximately 54% exposure as measured by Cmax and approximately 96% exposure as measured by Cmax versus day 1. Exposures at 0.1 mg / kg / week were similar or higher in males and females on day 22 compared to day 1. There were no differences in toxicity findings between females maintained at 0.3 mg / kg / week or higher and animals not maintained at exposure. Therefore, evaluation of potential toxicity in this study at all dose levels was considered valid.

[0348] conclusion In conclusion, 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. No adverse hematology (decreased RBC mass and regenerative erythroid response, transiently decreased then increased WBC population, changes in platelet count), clinical chemistry (transiently increased urea, creatinine, globulin, and bilirubin, transiently decreased sodium and chloride, decreased albumin and cholesterol), organ weight (increased spleen and liver weight), and microscopic findings (minimal to marked mononuclear cell infiltration in some tissues) were observed at 0.1 mg / kg / week and above. PC-1 induced a transient, dose-dependent increase in cytokines within 24 hours after the first dose at 0.1 mg / kg / week and above. Most, if not all, changes can be attributed to a proinflammatory response consistent with the mechanism of action of PC-1 and / or cytokine release observed after administration of the first dose of PC-1. Based on the lack of adverse effects observed during the dosing and recovery phases of the study, the HNSTD / NOAEL was 1.5 mg / kg / week. Systemic exposure (Cmax and AUC 0-168h ) were 43,500 ng / mL and 2,340,000 h·ng / mL, respectively, after the first dose at the NOAEL in both sexes combined.

[0349] Genotoxicity, Carcinogenicity, and Reproductive Toxicity Studies According to the ICH S6(R1) guidelines, the scope and type of genotoxicity studies routinely performed on small molecule drug products are generally not applicable to biotechnology-derived products. Humanized trispecific proteins such as PC-1 are not expected to directly interact with DNA or other chromosomal material. Thus, mutagenicity studies are not considered relevant and are not planned. No carcinogenicity studies have been performed with PC-1. No carcinogenicity studies are planned (per ICH S9 guidelines) if the intended patient population is subjects with mCRPC. No nonclinical reproductive and developmental toxicity studies have been performed with PC-1. As its effects on pregnancy are unknown at this time, all subjects and their female partners are required to practice at least two highly effective protocol-specified birth control methods while participating in clinical trials of PC-1.

[0350] Effects in humans Introduction: There is no clinical experience with PC-1. The current study is an FIH Phase 1 clinical trial of PC-1. Please see the clinical trial protocol for complete patient eligibility criteria. An overview of the planned clinical trial is provided below.

[0351] Phase 1 Clinical Trial Design Key Points Study Design: This is an FIH, Phase 1, blinded, multicenter study 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 three parts: dose escalation with approximately 20-30 subjects (Part 1), cohort backfill expansion with approximately 30 subjects enrolled across three dose levels (Part 2), and dose expansion with approximately 30 subjects enrolled at the RP2D (Part 3). Dose escalation (Part 1) will evaluate the safety, tolerability, PK, PD, and preliminary efficacy of PC-1 administered by IV infusion. Dose escalation decisions will be made upon recommendation of the Safety Review Committee based on review of safety data and available PK, PD, and preliminary efficacy data. Dose escalation may continue to a dose where at least two dose-limiting toxicities are observed (above the MTD). Cohort backfill expansion (Part 2) will allow further characterization of safety and dose-level activity. Dose expansion (Part 3) will enroll up to approximately 30 subjects to generate additional safety, tolerability, PK, PD, and preliminary clinical activity data for PC-1 at a dose and schedule to be determined by the Safety Review Committee after review of all available safety, PK, PD, and preliminary efficacy data.

[0352] Data Summary and Guidance Indications and Use: PC-1 is in development for the treatment of mCRPC and is not currently approved for any indication.

[0353] Dosage and Administration: In the FIH study, the starting dose is 100 μg with subsequent dose escalation. PC-1 is administered IV on days 1, 8, and 15 of a 21-day cycle. Subjects are treated for 6 cycles (3 weeks each) for a total of 18 scheduled infusions over 18 weeks.

[0354] Preparation Instructions: Parenteral drug products should be visually inspected for particulate matter and discoloration prior to administration whenever the solution and container permit. Refer to the drug manual (or test protocol) for further details.

[0355] Dosage Form and Strength: PC-1 DP is provided as a solution for injection for IV administration at a single concentration presentation of 2 mg / mL.

[0356] Contraindications: The use of PC-1 is contraindicated in the following conditions: known hypersensitivity to any of the drug components.

[0357] WARNINGS AND PRECAUTIONS FOR USE: There has been no prior clinical experience with PC-1. PC-1 is an antibody protein construct. Like other molecules of this class, it is highly specific for its target. Antibody therapeutics are well tolerated, but are "foreign" proteins and some patients may experience infusion-related reactions (IRR) or develop an immune response against them. However, PC-1 was designed to carry less downstream risk of CD3 activation compared to other T-cell binding therapies. PC-1 is an experimental drug that should only be administered to patients in the context of a clinical trial.

[0358] Potential Adverse Reactions Because PC-1 is a T-cell redirecting antibody, CRS, neurotoxicity, and / or tumor lysis syndrome may occur, but likely with a lower frequency / severity than other T-cell activating therapies based on the PC-1 design. Published literature on experience with other monoclonal antibody therapies indicates that fever, immunogenic reactions (i.e., formation of ADA), and / or hypersensitivity reactions may be observed. These reactions may be both severe and systemic (e.g., anaphylaxis) and may occur acutely or may be delayed. For regulatory reporting purposes, all adverse events (AEs) are assessed as unexpected at this stage of the development program. Thus, AEs that are severe and possibly related to PC-1 will be reported to health authorities in accordance with applicable regulations.

[0359] Infusion-related reactions PC-1 is a recombinant protein-based therapeutic, and administration of the therapeutic protein has been associated with infusion reactions with symptoms and signs including fever, chills, rash, urticaria, dyspnea, hypotension, and / or nausea. To minimize the risk of infusion reactions, premedicate all subjects with acetaminophen / paracetamol and diphenhydramine, as well as corticosteroids for the first infusion. Mild infusion reactions should be managed according to institutional standards with antipyretics, antihistamines, and antiemetics, and corticosteroids for more severe reactions.

[0360] Management of infusion-related reactions IRR is defined by the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) v5.0 (under the category "Injury, Poisoning, and Procedural Complications") as "a disorder characterized by an adverse reaction to an infusion of a pharmacological or biological agent." For the purposes of this study, the time frame for infusion reaction assessment is a 24-hour period beginning with the start of the infusion. Recommendations for management of IRR are provided below. For Grade 1 IRR: described as mild and reaction, infusion interruption not indicated, no intervention indicated: · Remain at bedside and monitor subject until symptomatic recovery. For Grade 2 IRR: described as infusion interruption indicated, but responds promptly to treatment for symptoms (e.g., antihistamines, nonsteroidal anti-inflammatory drugs, anesthetics, IV fluids), and prophylactic medications for 4 hours or less: · Stop PC-1 infusion, begin IV infusion of saline, and treat subject with diphenhydramine 50 mg IV (or equivalent) and / or 500-750 mg oral acetaminophen. ·At the investigator's discretion, corticosteroid therapy may be given. Remain at the bedside and monitor the subject until resolution of symptoms. ·If the infusion is interrupted, wait until symptoms resolve, then resume 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 device. ·No further PC-1 will be administered at that visit. ·The amount of infusion of PC-1 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 resolve during that period must be observed until the AE has resolved or stabilized, with vital signs measurement every 4 hours and further evaluation as medically indicated for management of the AE.

[0361] For Grade 3 or Grade 4 IRR: Grade 3 is described as a prolonged (e.g., not rapidly responsive to symptomatic medication and / or brief interruption of infusion) recurrence of symptoms after initial improvement, and hospitalization is indicated for other clinical sequelae (e.g., renal impairment, pulmonary infiltrates). Grade 4 is described as a life-threatening outcome, and urgent intervention is indicated. ·Immediately discontinue infusion of PC-1. ·Investigators should follow institutional guidelines for the treatment of anaphylaxis or high-grade infusion reactions. ·Treat the subject with an IV infusion of normal saline and consider administering corticosteroids, H1 and / or H2 blockers, bronchodilators, oxygen, or vasopressors as needed. ·Subjects should be monitored until the investigator agrees that symptoms will not recur. ·Observe all subjects with a Grade 3 or higher IRR until the AE has resolved or stabilized, with vital signs measurements and further evaluation as medically indicated for management of the AE. Subjects with a Grade 3 IRR during Week 1 of Cycle 1 may also be considered for dose reduction and may be hospitalized during or after administration of the subsequent dose.

[0362] Cytokine release syndrome The identified risks of treatment with T cell engagers (TCEs) are primarily related to cytokine release and CRS. PC-1 requires protease cleavage for activation and is designed to reduce the risk of CRS by focusing molecular activity in 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 (of unmasking TCEs) have shown that extensive T cell activation can result in the release of cytokines into the blood, which can cause acute effects of fever, hypotension, and hypoxia, requiring care in hospitals. These side effects are short-lived and can be treated with IV fluids, tocilizumab, and supportive care, but sometimes have the potential to be severe. These vital signs and adverse effects will be closely monitored for their appearance and will be closely followed in this clinical trial. In the nonclinical toxicology studies described in section 4, PC-1 can be dosed at 50-150 times higher doses than the unmasked active form of TCE with comparable safety and cytokine induction activity. This safety margin observed in animal studies offers the promise that higher active doses can be achieved in patients and provide increased levels of antitumor activity. Guidelines for the treatment of CRS have been driven primarily by the CAR-T field, where CRS seen with anti-CD19 CAR-T cells can be severe and more prolonged (Neelapu, 2018). With bispecific antibodies, CRS is of shorter duration and generally less severe, but can still be dose limiting and is usually mitigated by a priming / staged dosing approach. Cytokine release is primarily a first dose or first cycle phenomenon, and resistance to cytokine induction occurs with subsequent infusions of TCE. Prophylactic measures for cytokine release and CRS include glucocorticoid premedication, IV prehydration, and retention of antihypertensive medications on the day of infusion. If CRS is observed during dose escalation, priming and step dosing will be initiated.

[0363] CRS Treatment: Cytokine release may result in fever and tachycardia, and may become more severe, resulting in hypotension and hypoxia, and must be followed closely. CRS may also be associated with fatigue, nausea, headache, dyspnea, chills, myalgia / arthralgia, and anorexia. CRS is graded using the American Society for Transplantation and Cellular Therapy (ASTCT) consensus grading scale (Table 14), adapted from (Lee et al., 2019). CRS is managed with increased IV hydration and other supportive care, as well as monitoring, as needed. 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, anticonvulsants, high-dose corticosteroids, and / or mechanical ventilation. Recommended guidelines for the treatment of CRS are primarily driven by the CAR-T field, and CRS seen with anti-CD19 CAR-T cells can be severe and more prolonged (Lee et al., 2019). With immunoglobulin G format bispecific antibodies, CRS can occur and be dose-limiting.

[0364] [Table 14]

[0365] For any grade of CRS: ·Monitor clinical laboratory evaluations. ·Monitor C-reactive protein levels and serum PD cytokine laboratory assessments. Extract additional safety laboratory samples and plasma for study safety and PD biomarkers. For grade 1 CRS: Many subjects may present with only fever and tachycardia. Careful monitoring is required to detect and respond to early signs of progression. Frequent monitoring of vital signs For grade 2 CRS: Hypotension and / or hypoxia are detected Treatment with aggressive IV hydration with normal saline Supplemental oxygen as indicated Consider ECG telemetry and pulse oximetry Consider corticosteroids (e.g., dexamethasone 10 mg every 12 hours) Consider tocilizumab or a comparable anti-IL-6 antibody (e.g., siltuximab), or an anti-IL-6 receptor antibody (e.g., salilimab)

[0366] Grade 3 CRS: Hypotension unresponsive to IV fluids Management of monitored beds or intensive care units Corticosteroids as indicated, dexamethasone 10 mg every 6 hours, or methylprednisolone 1 mg / kg IV twice daily Tocilizumab (4–8 mg / kg IV over 1 hour, not to exceed 800 mg), or an equivalent anti-IL-6 antibody (e.g., siltuximab), or an anti-IL-6 receptor antibody (e.g., salilimab) Support by boosting voltage Oxygen therapy using a mask if necessary

[0367] Other potential toxicities Tumor lysis syndrome; subjects with high disease burden may be at risk for developing tumor lysis syndrome (TLS) with PC-1 treatment. Prophylactic treatment / measurements are strongly recommended for subjects considered at risk for TLS according to institutional or clinical criteria. Subjects should be closely monitored for clinical evidence of TLS (hyperuricemia, hyperkalemia, hyperphosphatemia, and hypocalcemia). In case of evidence of TLS associated with PC-1, subjects will be admitted to the hospital as clinically indicated. Standard management includes aggressive IV hydration, hypouricemic agents, and correction of acidosis, if present. Renal function, serum uric acid, calcium, phosphorus, and electrolytes should be closely monitored. Subjects with grade 3-4 TLS during week 1 or cycle 1 may also be hospitalized for more than 24 hours after the end of administration of the subsequent dose, allowing for dose reduction as described in the study protocol.

[0368] Neurological events: Administration of solid tumor-targeting bispecifics has shown a low likelihood of severe neurotoxicity (compared to CAR-T therapy or anti-CD19 BiTE format antibodies, e.g., blinatumomab) in early clinical trials. PC-1 at active doses has not shown a trend for induction of cytokine levels that may be associated with neurotoxicity in in vitro human cell cultures or in vivo cynomolgus monkey studies, but the risk of neurotoxicity is unknown and caution is warranted. Neurological events associated with cytokine release may include tremors, mental status changes, confusion, difficulty speaking, and potentially 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 (strong word finding) may be the earliest and most specific sign. Provide supportive care as needed for any neurological events. Work-up may include head magnetic resonance imaging and EEG, and may require corticosteroids or anti-seizure medications if severe. In the event of potentially treatment-related neurotoxicity, medical monitoring should be contacted.

[0369] infection As seen with other TCE-based immunotherapies, severe infections, including lethal bacterial, fungal, and new or reactivated viral infections, may 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). PC-1 should be discontinued if a severe infection develops and appropriate anti-infective therapy implemented. PC-1 is not recommended for use in subjects with severe active infections.

[0370] Hepatitis B reactivation: HBV reactivation can occur in patients treated with drugs classified as TCE antibodies. Cases have been reported in patients who are hepatitis B surface antigen (HBsAg) negative but hepatitis B core antibody (anti-HBc) positive. HBV reactivation is defined as a sudden increase in HBV replication manifested as 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 often followed by hepatitis (i.e., increased transaminase levels). In severe cases, increased bilirubin levels, liver failure, and death can occur. It is recommended that subjects with evidence of previous HBV infection (anti-HBc positive) be monitored with monthly HBV DNA testing and for clinical and laboratory signs of hepatitis during and for several months after PC-1 therapy.

[0371] Immunization The safety of immunization with live virus vaccines during or after PC-1 therapy has not been studied. Vaccination with live virus vaccines is not recommended prior to the start of PC-1 treatment, during treatment, and for more than 2 weeks until immune recovery after the last cycle of PC-1. Subjects are encouraged to receive COVID-19 vaccination / boost ≥2 weeks prior to study initiation. Vaccination efficacy in the study is unknown, however, COVID-19 vaccination is permitted in the study after clearance of the first cycle and with 2 weeks of dose retention.

[0372] Other potential healthy tissue toxicities PSMA is most highly expressed in PC cells and is highly prostate specific, but may be expressed at lower levels in some healthy tissues. By requiring tumor proteases for activation of PC-1, there is another layer of protection against normal tissue T cell activation. Tissues must not only express PSMA, but also express specific proteases for local activation of PC-1 at non-tumor sites.

[0373] Renal, gastrointestinal, and hepatic Immunohistochemistry studies have shown that some non-prostatic tissues may express low levels of PSMA. These include renal proximal tubule cells, and dural brush border cells, and rare colonic crypt cells (Silver et al., 1997; The Protein Atlas, 2022). PSMA-PET scanning with small molecule PSMA active site binders confirmed tissue accumulation in the small intestine and liver. (Kidney accumulation cannot be resolved as the reagent is excreted through the kidney and bladder). Nephrotoxicity or gastrointestinal toxicity has not been described as an issue with small molecule PSMA binders such as 177-Lu-PSMA-617 (PLUVICTO) (Sartor et al., 2021), or with PSMA-CD3 bispecific antibodies such as AMG160. Transperitoneal inflammation has been seen with AMG160, however, mostly attributable to CRS (Tran et al., 2020).

[0374] Salivary and lacrimal glands: PSMA-PET scans using small molecule PSMA active site binders showed clear salivary and lacrimal gland uptake. It is unclear whether there are other non-specific or less understood mechanisms for this uptake (Morris et al., 2021; Sheehan et al., 2021). Small molecule PSMA binders such as 177-Lu-PSMA617 (PLUVICTO) have shown potential for dry mouth and dry eye side effects (Sartor et al., 2021), and these side effects are seen much less frequently with PSMA-CD3 bispecifics, but can occur (Tran et al., 2020).

[0375] Drug Interactions: The drug interaction profile of PC-1 is unknown, however no clinically relevant drug-drug interactions are expected in PC-1.

[0376] Use in Specific Populations

[0377] pregnancy No nonclinical reproductive and developmental toxicity studies have been conducted with PC-1. PC-1 is being developed for the treatment of advanced mCRPC, which occurs exclusively in men. Because its effects on partner fertility are unknown at this time, all sexually active subjects in this study and their female partners must practice at least two highly effective protocol-specified methods of birth control while participating in clinical trials of PC-1.

[0378] Teratogenic effects It is unknown whether PC-1 has teratogenic effects. If the subject or the subject's female partner experiences a pregnancy during the study, further treatment with PC-1 should be discontinued immediately.

[0379] Non-teratogenic effects It is unknown whether PC-1 has non-teratogenic effects. If the subject or the subject's female partner experiences a pregnancy during the study, further treatment with PC-1 should be discontinued immediately.

[0380] Use in the elderly There have been no trials in elderly patients. Because the average age at diagnosis of PC is 66 years (Cancer.org), elderly patients will be included in the initial clinical trials of PC-1. However, elderly subjects should be closely monitored.

[0381] Kidney problems The effect of renal impairment on PC-1 PK and / or function is not known. However, because PC-1 is a biological compound, PK / functional changes due to renal impairment are not expected (FDA 2020).

[0382] Liver problems The effect of hepatic impairment on the PK and / or function of PC-1 is unknown.

[0383] Overdosage The effects of overdosing on PC-1 are unknown. In case of overdosage (i.e., any dose above that defined in the protocol), supportive care will be provided.

[0384] PC-1 is a trispecific TRACTr that incorporates PSMA and CD3 binding domains, a tandem albumin binding domain with a peptide (mask) that inhibits PC-1 binding to CD3 on T cells, and a tumor protease cleavage sequence (Figure 1). Intact PC-1 has reduced binding to CD3 on T cells (compared to the unmasked version) and is designed to minimize T cell activation and pharmacology in healthy tissues.

[0385] Clinical Pharmacology Mechanism of action Target binding by PC-1 requires proteolysis of its cleavable amino acid linker by proteases present in the TME. When the amino acid cleavage sequence undergoes proteolysis by tumor proteases, the tandem mask and albumin binding domains are released to allow CD3 binding. Simultaneous binding of the bispecific components of cleaved PC-1 to CD3 and PSMA results in T cell activation in the tumor, leading to T cell-mediated killing of prostate tumor cells.

[0386] Pharmacodynamics Pharmacodynamics in humans have not been evaluated and will be evaluated in clinical trials of PC-1.

[0387] Pharmacokinetics Pharmacokinetics in humans have not been previously evaluated and will be evaluated in clinical trials of PC-1.

[0388] Non-clinical Toxicity Studies Carcinogenicity, Mutagenesis, and Impairment of Fertility: Genotoxicity studies of biotechnology-derived pharmaceuticals are not applicable according to the ICH S6(R1) guideline. No studies have been performed for carcinogenicity, reproductive and developmental toxicity, or fertility.

[0389] Animal Toxicology and / or Pharmacology: Nonclinical safety pharmacology parameters included cardiovascular, CNS, and respiratory safety endpoints incorporated in a 4-week repeated dose IV toxicity study in cynomolgus monkeys (B49-0027-TX). There were no functional effects on the CNS or respiratory systems based on clinical observations of the animals and detailed weekly examinations at doses of PC-1 ≤ 1.5 mg / kg / week. Additionally, there were no clearly defined effects of PC-1 administration on the animals' cardiovascular systems based on qualitative and quantitative ECG parameters. All ECGs were within normal limits, except for a few episodes of non-test article-related arrhythmias.

[0390] Clinical Trials: No previous clinical studies of PC-1 have been conducted.

[0391] How to Supply / Storage and Handling: PC-1 DP should be shipped frozen and stored at -20°C.

[0392] result Quantification of PC-1 and its metabolites in monkey plasma by IC-LC-MS / MS in a preclinical toxicokinetic study

[0393] PC-1 is designed to mask the scFv epitope via a peptide linker with two protease cleavage sites. Enzyme-linked immunosorbent (ELISA) assays are usually the standard method for bioarresis of biological therapeutics. However, in the case of BsAb PC-1s, its highly complex structure and the multiple components that need to be quantified in the matrix make it less capable. Instead, liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS) becomes a better choice due to its characteristics of being highly specific, multiplatform, and less dependent on specific reagents. The results demonstrated a bioanalytical method using immunocapture (IC) followed by enzymatic digestion and then LC-MS / MS analysis for this PC-1. The targets in the matrix quantification included intact PC-1, its two metabolites, and the total antibody. The lower limit of quantification (LLOQ) could be achieved at 5 ng / ml for the metabolites. This method was successfully used to facilitate the bioanalysis of monkey plasma in preclinical TK studies.

[0394] method Immunocapture purification: Immunocapture was used to extract intact PC-1 and its metabolites from monkey plasma. Briefly, 500 μL of high-capacity magnetic TM streptavidin beads were incubated with 500 μL of biotinylated human PSMA (335 μg / mL) in PBST for 1 h at room temperature with shaking (1500 rpm). 100 μL of plasma sample was incubated with 10.0 μL of the above prepared PSMA-coated beads for 1 h at room temperature with shaking (1500 rpm). After washing twice with 400 μL of PBST buffer, the analytes concentrated on the beads were eluted with 50.0 μL of 100 mM glycine-HCl (pH=2.7).

[0395] Enzymatic digestion: All eluates are transferred to a new 96-well plate and stable isotope-labeled internal standard (SIL-IS) signature peptide solution is added. Analytes are denatured with 20 μL of 1.00 mg / mL RapiGest solution, reduced with 10 μL of 100 mM dithiothreitol in 200 mM NH4HCO3, alkylated with 10 μL of 600 mM iodoacetamide in 200 mM NH4HCO3, and finally digested with 10.0 μL of 200 μg / mL Glu-C for 2 hours at 37° C. with shaking (1200 rpm). Digestion is terminated with 10.0 μL of 10% trifluoroacetic acid (TFA) (FIG. 25).

[0396] LC-MS / MS analysis: A Shimadzu-30 UPLC system coupled with an AB Sciex QQQ 6500+ was used for LC-MS / MS analysis. A Waters ACQUITY UPLC HSS T3 Column (100 Å, 2.1 * A 50 mm, 1.8 μm particle size column was used for the separation. The mobile phase was 0.1% formic acid in water (MPA) and 0.1% formic acid in acetonitrile (MPB).

[0397] result Method Validation: The LC-MS / MS method for quantification of BsAb PC-1 and its two metabolites, as well as total antibody, with lower limits of quantification reaching 100, 5.00, 5.0, 110 ng / ml, respectively, in monkey K2EDTA plasma, has been fully validated. TK Results: The concentrations of intact PC-1, its two metabolites (i.e., Met1 and Met2) and total antibody (total concentration of intact PC-1, Met1 and Met2) in the test samples collected from the TK study were measured. The metabolic curves of intact PC-1, Met1, Met2, and total PC-1 are shown in Figure 26. The median Tmax values ​​for intact PC-1 were observed at 0.1 hours for both the first dose (day 1) and the fourth dose (day 22) in both male and female animals at all three dose levels. In anti-drug antibody (ADA) negative animals, the median intact PC-1 T1 / 2 on days 1 and 22 ranged from 67.7 to 109.1 hours across all doses tested. Met1 and Met2 were either below the limit of quantification (BLQ) or were generated during sample preparation and not 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. Figure 27 shows the correlation between intact PC-1 and anti-drug antibody (ADA) concentrations in the high-dose group of monkeys. Metabolites of PC-1 were found to be very low in the plasma of healthy monkeys, indicating low off-site toxicity and a great advantage of the on-site activation mechanism of PC-1. ADA was detected in most of the mid- and high-dose animals towards the end of the dosing phase when the exposure of the prodrug was significantly reduced. The validation method is shown in Table 15.

[0398] Immunogenicity results: Intact PC-1 was detected as BLQ in many samples from the high-dose group, likely due to the large number of ADAs generated in the high-dose group (Figure 28). The presence of ADAs may cause a decrease in drug efficacy and is therefore worth extra attention during preclinical trials.

[0399] [Table 15-1]

[0400] [Table 15-2]

[0401] [Table 15-3]

[0402] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It is understood that various alternatives to the embodiments of the disclosure described herein may be utilized in implementing the present disclosure. The following claims define the scope of the disclosure, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0403] Embodiment Embodiment 1. An isolated recombinant polypeptide complex comprising a first chain having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:1, and a second chain having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:2, the complex having the following characteristics: (a) at least one disulfide bond formed by a pair of cysteine ​​residues in the first chain or the second chain, or a pair of cysteine ​​residues in the first chain and the second chain; or (b) secondary structure composition including β-sheet or random coil; (c) at least one pyroglutamine in the second chain; (d) the isolated recombinant polypeptide complex has a melting temperature (T) of about 65° C. to about 85° C. when formulated at a concentration of 1.0 mg / mL in a buffer solution containing 10 mM histidine buffer, 8% (w / v) sucrose, and 0.01% (w / v) polysorbate 20 at a pH of 6.3. m ), (e) the far-UV circular dichroism peak at wavelengths 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) far-UV circular dichroism dip 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 (g) Near-ultraviolet circular dichroism peak 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, and 0.01% (w / v) polysorbate 20 at a pH of 6.3. 2. An isolated recombinant polypeptide complex comprising at least one of:

[0404] Embodiment 2. The isolated recombinant polypeptide complex of embodiment 1, wherein the polypeptide comprises at least two of the above characteristics.

[0405] Embodiment 3. The isolated recombinant polypeptide complex of embodiment 1, wherein the polypeptide comprises at least three of the above characteristics.

[0406] Embodiment 4. The isolated recombinant polypeptide complex of embodiment 1, wherein the polypeptide comprises at least four of the above characteristics.

[0407] Embodiment 5. The isolated recombinant polypeptide complex of embodiment 1, wherein the polypeptide comprises at least five of the above characteristics.

[0408] Embodiment 6. The isolated recombinant polypeptide complex of embodiment 1, wherein the polypeptide comprises at least six of the above characteristics.

[0409] Embodiment 7. The isolated recombinant polypeptide complex of embodiment 1, wherein the polypeptide comprises at least seven of the above characteristics.

[0410] Embodiment 8. The isolated recombinant polypeptide complex of embodiment 1, wherein the polypeptide comprises at least eight of the above characteristics.

[0411] Embodiment 9. The isolated recombinant polypeptide complex of any one of embodiments 1 to 7, wherein the first chain comprises at least 85% sequence identity to SEQ ID NO:1.

[0412] Embodiment 10. The isolated recombinant polypeptide complex of any one of embodiments 1 to 7, wherein the first chain comprises at least 90% sequence identity to SEQ ID NO:1.

[0413] Embodiment 11. The isolated recombinant polypeptide complex of any one of embodiments 1 to 7, wherein the first chain comprises at least 95% sequence identity to SEQ ID NO:1.

[0414] Embodiment 12. The isolated recombinant polypeptide complex of any one of embodiments 1 to 7, wherein the first chain comprises at least 99% sequence identity to SEQ ID NO:1.

[0415] Embodiment 13. An isolated recombinant polypeptide complex according to any one of embodiments 1 to 11, wherein the first chain comprises the amino acid sequence set forth in SEQ ID NO:1.

[0416] Embodiment 14. The isolated recombinant polypeptide complex of any one of embodiments 1 to 13, wherein the second chain comprises at least 85% sequence identity to SEQ ID NO:2.

[0417] Embodiment 15. The isolated recombinant polypeptide complex of any one of embodiments 1 to 13, wherein the second chain comprises at least 90% sequence identity to SEQ ID NO:2.

[0418] Embodiment 16. The isolated recombinant polypeptide complex of any one of embodiments 1 to 13, wherein the second chain comprises at least 95% sequence identity to SEQ ID NO:2.

[0419] Embodiment 17. The isolated recombinant polypeptide complex of any one of embodiments 1 to 13, wherein the second chain comprises at least 99% sequence identity to SEQ ID NO:2.

[0420] Embodiment 18. An isolated recombinant polypeptide complex according to any one of embodiments 1 to 17, wherein the second chain comprises the amino acid sequence set forth in SEQ ID NO:2.

[0421] Embodiment 19. The isolated recombinant polypeptide complex according to embodiment 1, wherein 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.

[0422] Embodiment 20. The isolated recombinant polypeptide complex of embodiment 1, wherein at least one disulfide bond is an intrachain disulfide bond formed by a pair of cysteine ​​residues between cysteine ​​22 and cysteine ​​96, or between cysteine ​​150 and cysteine ​​206 of the second chain.

[0423] Embodiment 21. The isolated recombinant polypeptide complex of embodiment 1, wherein 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.

[0424] Embodiment 22. An 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.

[0425] Embodiment 23. An isolated recombinant polypeptide complex according to any one of the above embodiments, wherein the polypeptide comprises at least three disulfide bonds formed by pairs of cysteine ​​residues.

[0426] Embodiment 24. An isolated recombinant polypeptide complex according to any one of the previous embodiments, wherein the polypeptide comprises at least four disulfide bonds formed by pairs of cysteine ​​residues.

[0427] Embodiment 25. An isolated recombinant polypeptide complex according to any one of the above embodiments, wherein the polypeptide comprises at least five disulfide bonds formed by pairs of cysteine ​​residues.

[0428] Embodiment 26. An isolated recombinant polypeptide complex according to any one of the above embodiments, wherein the polypeptide comprises at least six disulfide bonds formed by pairs of cysteine ​​residues.

[0429] Embodiment 27. An isolated recombinant polypeptide complex according to any one of the above embodiments, wherein the polypeptide comprises at least seven disulfide bonds formed by pairs of cysteine ​​residues.

[0430] Embodiment 28. An isolated recombinant polypeptide complex according to any one of the above embodiments, wherein the polypeptide comprises at least eight disulfide bonds formed by pairs of cysteine ​​residues.

[0431] Embodiment 29. An isolated recombinant polypeptide complex according to any one of the preceding embodiments, wherein the pair of cysteine ​​residues comprises cysteine ​​22 and cysteine ​​96 of SEQ ID NO:1.

[0432] Embodiment 30. An isolated recombinant polypeptide complex according to any one of the previous embodiments, wherein the pair of cysteine ​​residues comprises cysteine ​​138 and cysteine ​​148 of SEQ ID NO:1.

[0433] Embodiment 31. An isolated recombinant polypeptide complex according to any one of the previous embodiments, wherein the pair of cysteine ​​residues comprises cysteine ​​199 and cysteine ​​275 of SEQ ID NO:1.

[0434] Embodiment 32. An isolated recombinant polypeptide complex according to any one of the preceding embodiments, wherein the pair of cysteine ​​residues comprises cysteine ​​339 and cysteine ​​407 of SEQ ID NO:1.

[0435] Embodiment 33. An isolated recombinant polypeptide complex according to any one of the previous embodiments, wherein the pair of cysteine ​​residues comprises cysteine ​​454 and cysteine ​​519 of SEQ ID NO:1.

[0436] Embodiment 34. An isolated recombinant polypeptide complex according to any one of the previous embodiments, wherein the pair of cysteine ​​residues comprises cysteine ​​565 and cysteine ​​625 of SEQ ID NO:1.

[0437] Embodiment 35. An isolated recombinant polypeptide complex according to any one of the previous embodiments, wherein the pair of cysteine ​​residues comprises cysteine ​​645 of SEQ ID NO:1 and cysteine ​​226 of SEQ ID NO:2.

[0438] Embodiment 36. An isolated recombinant polypeptide complex according to any one of the preceding embodiments, wherein the pair of cysteine ​​residues comprises cysteine ​​150 and cysteine ​​206 of SEQ ID NO:2.

[0439] Embodiment 37. An isolated recombinant polypeptide complex according to any one of the previous embodiments, wherein the pair of cysteine ​​residues comprises cysteine ​​22 and cysteine ​​96 of SEQ ID NO:2.

[0440] Embodiment 38. An isolated recombinant polypeptide complex according to any one of the above embodiments, wherein at least one cysteine ​​residue is a free sulfhydryl.

[0441] Embodiment 39. The isolated recombinant polypeptide complex according to any one of the previous embodiments, wherein at least one cysteine ​​residue is a free sulfhydryl, and wherein 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.

[0442] Embodiment 40. An isolated recombinant polypeptide complex according to any one of the previous embodiments, wherein at least one cysteine ​​residue is a free sulfhydryl, and wherein 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.

[0443] Embodiment 41. The isolated recombinant polypeptide complex according to any one of the above embodiments, wherein at least one pair of cysteine ​​residues 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.

[0444] Embodiment 42. An isolated recombinant polypeptide complex according to any one of the above embodiments, wherein the secondary structure composition comprises beta-sheet and random coil.

[0445] Embodiment 43. A melting temperature (T m 5. The isolated recombinant polypeptide complex of any one of the preceding embodiments, comprising:

[0446] Embodiment 44. A melting temperature (T m 5. The isolated recombinant polypeptide complex of any one of the preceding embodiments, comprising:

[0447] Embodiment 45. A melting temperature (T m 5. The isolated recombinant polypeptide complex of any one of the preceding embodiments, comprising:

[0448] Embodiment 46. The isolated recombinant polypeptide complex of any one of the preceding embodiments, having a far-UV circular dichroism peak at a wavelength of 195 nm or less.

[0449] Embodiment 47. The isolated recombinant polypeptide complex of any one of the previous embodiments, having a far-UV circular dichroism peak at a wavelength of 205 nm or less.

[0450] Embodiment 48. An isolated recombinant polypeptide complex according to any one of the previous embodiments, having a far-UV circular dichroism dip at a wavelength of 220 nm or less.

[0451] Embodiment 49. An isolated recombinant polypeptide complex according to any one of the preceding embodiments, having a near-UV circular dichroism peak at a wavelength of 273 nm or less.

[0452] Embodiment 5. An isolated recombinant polypeptide complex according to any one of the preceding embodiments, having a near-UV circular dichroism peak at a wavelength of 0.279 nm or less.

[0453] Embodiment 51. The isolated recombinant polypeptide complex of any one of the previous embodiments, having a near-UV circular dichroism peak at a wavelength of 290 nm or less.

[0454] Embodiment 52. An isolated recombinant polypeptide complex according to any one of the preceding embodiments, having a near-UV circular dichroism peak at a wavelength of 295 nm or less.

[0455] Embodiment 53. A method of treating cancer comprising administering to a subject in need thereof an isolated recombinant polypeptide complex according to any one of the preceding embodiments.

[0456] Embodiment 54. A plurality of isolated recombinant polypeptide complexes comprising a first chain having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:1, and a second chain having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:2, wherein the plurality of isolated recombinant polypeptide complexes comprises more than 90% monomers of the isolated recombinant polypeptide complex.

[0457] Embodiment 55. The plurality of isolated recombinant polypeptide complexes of embodiment 54, comprising greater than 95% monomer.

[0458] Embodiment 56. The plurality of isolated recombinant polypeptide complexes of embodiment 55, comprising greater than 99% monomer.

[0459] Embodiment 57. A plurality of isolated recombinant polypeptide complexes according to any one of embodiments 54 to 56, having a concentration of 1.0 mg / mL or greater.

[0460] Embodiment 58. A plurality of recombinant polypeptides according to any one of embodiments 54 to 57, having a concentration of at least 2.0 mg / mL.

[0461] Embodiment 59. A plurality of isolated recombinant polypeptide complexes according to any one of embodiments 54 to 57, having a pH of 5.0 or greater.

[0462] Embodiment 60. The plurality of isolated recombinant polypeptide complexes of embodiment 54, comprising greater than 90% monomer at a concentration of 2.0 mg / mL or greater and a pH of 6.3.

[0463] Embodiment 61. A method of 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.

[0464] 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.

[0465] 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.

[0466] Embodiment 64. The method of any one of embodiments 61 to 63, wherein the antibody or antigen-binding fragment is administered to the subject at a dose of at least 1.0 mg / kg.

[0467] Embodiment 65. The method of any one of embodiments 61 to 64, wherein the antibody or antigen-binding fragment is administered to the subject at a dose of at least 1.5 mg / kg.

[0468] Embodiment 66. The method of any one of embodiments 61-65, wherein the antibody or antigen-binding fragment is administered to the subject according to the following treatment regimen: administering to the subject a dose of at least 0.1 μg / kg of the antibody or antigen-binding fragment in week 1, administering to the subject a dose of at least 0.3 mg / kg of the antibody or antigen-binding fragment in week 2, administering to the subject a dose of at least 1.5 mg / kg of the antibody or antigen-binding fragment in week 3, followed by a 4 week antibody or antigen-binding fragment non-treatment interval.

[0469] Embodiment 67. An isolated recombinant polypeptide complex according to any one of embodiments 1 to 52, which provides a maximum plasma concentration (Cmax) in a subject within about 0.1 hours after intravenous administration.

[0470] Embodiment 68. An isolated recombinant polypeptide complex according to any one of embodiments 1 to 52, which provides a Cmax of about 2500 to 3500 ng / ml in a subject within about 0.1 hours after intravenous administration of a dose of about 0.1 mg / kg.

[0471] Embodiment 69. An isolated recombinant polypeptide complex according to any one of embodiments 1 to 52, which provides a Cmax of about 7500 to 10500 ng / ml in a subject within about 0.1 hours after intravenous administration of a dose of about 0.3 mg / kg.

[0472] Embodiment 70. An isolated recombinant polypeptide complex according to any one of embodiments 1 to 52, which provides a Cmax of about 37500 to 52500 ng / ml in a subject within about 0.1 hours after intravenous administration of a dose of about 1.5 mg / kg.

[0473] Embodiment 71. An isolated recombinant polypeptide complex according to any one of embodiments 1 to 52, which provides a maximum plasma half-life (T1 / 2) of about 60 to 120 hours after intravenous administration in a subject.

[0474] Embodiment 72. The isolated recombinant polypeptide complex of embodiment 67, wherein the value of Cmax correlates with the dose of the isolated recombinant polypeptide complex administered.

[0475] Embodiment 73. An isolated recombinant polypeptide complex according to embodiment 72, wherein the value of Cmax is proportional to the dose of the isolated recombinant polypeptide complex administered.

[0476] Embodiment 74. The isolated recombinant polypeptide complex according to any one of embodiments 1 to 52, wherein less than 1% of the isolated recombinant polypeptide complex is degraded in the serum of the subject daily.

[0477] Embodiment 75. The isolated recombinant polypeptide complex of embodiment 74, wherein the subject has been diagnosed with cancer.

[0478] Embodiment 76. The isolated recombinant polypeptide complex of embodiment 75, wherein the cancer comprises prostate cancer.

[0479] Embodiment 77. The isolated recombinant polypeptide complex of embodiment 75, wherein the cancer comprises metastatic castration-resistant prostate cancer (mCRPC).

[0480] Embodiment 78. A method for treating cancer, comprising administering to a subject in need of such treatment a therapeutically effective amount of an isolated recombinant polypeptide complex described in any one of embodiments 1-52 and 67-77.

[0481] Embodiment 79. The method of embodiment 78, comprising administering to a subject an 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, and wherein the first dose or the second dose is at least 100 μg.

[0482] 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, the third dose is equal to or greater than the second dose, and wherein the first dose, the second dose, or the third dose is at least 100 μg.

[0483] Embodiment 81. The method of embodiment 79, wherein the method includes a first treatment course and a second treatment course, and wherein the first dose is administered to the subject during the first treatment course and the second dose is administered to the subject during the second treatment course.

[0484] Embodiment 82. The method of embodiment 80, wherein the method comprises a 21-day treatment course, wherein a first dose is administered to the subject during week 1 of the treatment course, a second dose is administered to the subject during week 2 of the treatment course, and a third dose is administered to the subject during week 3 of the treatment course.

[0485] 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 greater than the first dose of the first 21 day treatment course, the second dose of the second 21 day treatment course is equal to or greater than the second dose of the first 21 day treatment course, and the third dose of the second 21 day treatment course is equal to or greater than the third dose of the second 21 day treatment course.

[0486] Embodiment 84. The method comprises a 21 day treatment cycle, the treatment cycle comprising: (a) administering to a subject a first dose of an isolated recombinant polypeptide complex during week 1 of a treatment cycle; (b) administering to the subject a second dose of the isolated recombinant polypeptide complex during the second week of the treatment cycle; and (c) administering to the subject a third dose of the isolated recombinant polypeptide complex during the third week of the treatment cycle. 79. The method of embodiment 78, comprising:

[0487] 85. (a) administering to a subject a first dose of an isolated recombinant polypeptide complex on day 1 of a 21 day treatment cycle; (b) on day 8 of the 21 day treatment cycle, administering to the subject a second dose of the isolated recombinant polypeptide complex; and (c) on day 15 of the 21-day treatment cycle, administering to the subject a third dose of the isolated recombinant polypeptide complex. 85. The method of embodiment 84, comprising:

[0488] Embodiment 86. The method of embodiment 84 or 85, wherein the treatment cycle is repeated 6 times over 18 weeks.

[0489] Embodiment 87. The method of any one of embodiments 84-86, wherein the first dose, the second dose, or the third dose is at least 100 μg.

[0490] Embodiment 88. The method of any one of embodiments 84-87, wherein the second dose is equal to or greater than the first dose.

[0491] Embodiment 89. The method of any one of embodiments 84-88, wherein the third dose is equal to or greater than the second dose.

[0492] Embodiment 90. The method of any one of embodiments 84-89, wherein the third dose is equal to or greater than the first dose.

[0493] Embodiment 91. The method of any one of embodiments 84-90, 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 greater than the first dose of the first 21 day treatment cycle, the second dose of the second 21 day treatment cycle is equal to or greater than the second dose of the first 21 day treatment cycle, and the third dose of the second 21 day treatment cycle is equal to or greater than the third dose of the first 21 day treatment cycle.

[0494] Embodiment 92. The method of any one of embodiments 78-91, wherein the administering step comprises administering by intravenous infusion.

[0495] Embodiment 93. The method of any one of embodiments 78 to 92, wherein the cancer comprises prostate cancer.

[0496] Embodiment 94. The method of any one of embodiments 78 to 92, wherein the cancer comprises mCRPC.

[0497] Embodiment 95. The method of embodiment 92, further comprising treating the subject with a therapy for infusion-associated reactions prior to the administering step.

[0498] Embodiment 96. The method of embodiment 95, wherein the treatment for an infusion-related reaction comprises an antipyretic, an antihistamine, an antiemetic, or a corticosteroid.

[0499] Embodiment 97. The method of embodiment 95 or 96, wherein the treatment for an infusion-related reaction comprises acetaminophen, paracetamol, or diphenhydramine.

[0500] Embodiment 98. The method of embodiment 84, further comprising treating the subject with a corticosteroid prior to step (a).

[0501] Embodiment 99. The method of embodiment 85, further comprising treating the subject with a corticosteroid prior to step (a).

[0502] Embodiment 100. The method of embodiment 84, further comprising treating the subject with a therapy for cytokine release syndrome (CRS) before or after the administering step.

[0503] Embodiment 101. The method of embodiment 100, wherein the treatment for CRS comprises intravenous hydration procedures, oxygen therapy, corticosteroids, immunosuppressants, vasopressors, or antiepileptic drugs.

[0504] Embodiment 102. The method of embodiment 101, wherein the oxygen therapy includes mechanical ventilation.

[0505] Embodiment 103. The method of embodiment 101, wherein the immunosuppressant comprises an IL-6 receptor inhibitor.

[0506] Embodiment 104. The method of embodiment 103, wherein the IL-6 receptor inhibitor comprises tocilizumab.

[0507] Embodiment 105. The method of embodiment 100, wherein the treatment for CRS comprises high doses of corticosteroids.

[0508] Embodiment 106. The method of embodiment 78, wherein the isolated recombinant polypeptide complex is cleaved by a tumor-specific protease to generate an enzyme product of the isolated recombinant polypeptide complex after the administering step.

[0509] Embodiment 107. The method of embodiment 106, wherein the tumor-specific protease comprises two or more proteases, and the isolated recombinant polypeptide complex is cleaved by a first protease of the two or more proteases to generate a first metabolic product of the isolated recombinant polypeptide complex, and the isolated recombinant polypeptide complex is cleaved by a second protease of the two or more proteases to generate a second metabolic product of the isolated recombinant polypeptide complex.

[0510] Embodiment 108. The method of embodiment 106 or 107, wherein the first protease comprises a serine protease and the second protease comprises a matrix metalloprotease.

[0511] Embodiment 109. The method of embodiment 108, wherein the serine protease comprises recombinant human matriptase (MTSP1) and the matrix metalloprotease comprises recombinant human matrix metalloprotease 9 (MMP9).

[0512] Embodiment 120. The method of any one of embodiments 107 to 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.

[0513] Embodiment 111. The method of any one of embodiments 107 to 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.

[0514] Embodiment 112. The isolated recombinant polypeptide complex according to any one of embodiments 67 to 77 or the method according to any one of embodiments 78 to 111, wherein the subject is a non-human primate or a human.

[0515] Embodiment 113. The method of embodiment 112, wherein the non-human primate is a cynomolgus monkey.

[0516] Embodiment 114. A pharmaceutical composition comprising (a) an isolated recombinant polypeptide complex according to any one of embodiments 1 to 52 and 67 to 77, and (b) a pharma- ceutically acceptable excipient.

[0517] Embodiment 115. The pharmaceutical composition of embodiment 114, wherein the pharma- ceutically acceptable excipient comprises a buffer, a stabilizer, an isotonicity agent, a surfactant, or a combination thereof.

[0518] Embodiment 116. The pharmaceutical composition of embodiment 115, wherein the buffer comprises an amino acid or a derivative thereof.

[0519] Embodiment 117. The pharmaceutical composition of embodiment 116, wherein the amino acid or derivative thereof comprises L-histidine, L-histidine monohydrochloride monohydrate, or a combination thereof.

[0520] Embodiment 118. The pharmaceutical composition of embodiment 115, wherein the stabilizer comprises a sugar.

[0521] Embodiment 119. The pharmaceutical composition of embodiment 118, wherein the sugar comprises sucrose.

[0522] Embodiment 120. The pharmaceutical composition of embodiment 115, wherein the tonicity agent comprises a sugar.

[0523] Embodiment 121. The pharmaceutical composition of embodiment 120, wherein the sugar comprises sucrose.

[0524] Embodiment 122. The pharmaceutical composition of embodiment 115, wherein the surfactant comprises polysorbate 20.

[0525] 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 both L-histidine and L-histidine monohydrochloride monohydrate.

[0526] Embodiment 124. The pharmaceutical composition of embodiment 123, wherein the molar ratio of L-histidine to L-histidine monohydrochloride monohydrate is about 3:2.

[0527] Embodiment 125. The pharmaceutical composition of embodiment 119 or 121, comprising about 8% (w / v) sucrose.

[0528] Embodiment 126. The pharmaceutical composition of embodiment 122, comprising at least 0.01% (w / v) polysorbate 20.

[0529] Embodiment 127. A pharmaceutical composition according to any one of embodiments 114 to 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.

[0530] Embodiment 128. A pharmaceutical composition according to any one of embodiments 114 to 127, comprising about 2 mg / ml of the isolated recombinant polypeptide complex.

[0531] Embodiment 129. The pharmaceutical composition according to any one of embodiments 114 to 128, comprising a pH of about 5 to about 7.

[0532] Embodiment 130. The pharmaceutical composition of any one of embodiments 114 to 129, comprising a pH of about 6.3.

[0533] 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 which is 484 amino acids in length.

[0534] 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 which is 476 amino acids in length.

[0535] Embodiment 133. A pharmaceutical composition comprising 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 being 484 amino acids in length, and a pharma- ceutically acceptable excipient.

[0536] Embodiment 134. A pharmaceutical composition comprising 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 being 476 amino acids in length, and a pharma- ceutically acceptable excipient.

[0537] Embodiment 135. A method for treating cancer, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition according to any one of embodiments 114 to 130.

[0538] Embodiment 136. The method of embodiment 135, wherein the cancer comprises mCRPC.

[0539] Embodiment 137. The method of embodiment 135, wherein the subject is a human.

[0540] [Table 16-1]

[0541] [Table 16-2]

[0542] [Table 16-3]

[0543] [Table 16-4]

[0544]

Table 16-5

[0545]

Table 16-6

[0546]

Table 16-7

[0547]

Table 16-8

Claims

1. An isolated recombinant polypeptide complex comprising a first chain having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 1, and a second chain having an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 2, characterized by the following: (a) a pair of cysteine ​​residues in the first chain or the second chain, or at least one disulfide bond formed by the pair of cysteine ​​residues in the first chain and the second chain, (b) Secondary structural composition including β-sheets or random coils, (c) at least one pyroglutamine in the second chain, (d) When the isolated recombinant polypeptide complex is formulated at a concentration of 1.0 mg / mL in a pH 6.3 buffer containing 10 mM histidine buffer, 8% (w / v) sucrose, and 0.01% (w / v) polysorbate 20, the melting temperature (T) is approximately 65°C to approximately 85°C. ｍ ), (e) When the isolated recombinant polypeptide complex is formulated at a concentration of 0.1 mg / mL in 10 mM potassium phosphate buffer at pH 7.0, the far-ultraviolet circular dichroism peak at wavelengths between 190 nm and 205 nm, (f) When the isolated recombinant polypeptide complex is formulated at a concentration of 0.1 mg / mL in 10 mM potassium phosphate buffer at pH 7.0, a far-ultraviolet circular dichroism dip at wavelengths between 210 nm and 220 nm is observed, or (g) When the isolated recombinant polypeptide complex is formulated at a concentration of 1.0 mg / mL in a pH 6.3 buffer containing 10 mM histidine buffer, 8% (w / v) sucrose, and 0.01% (w / v) polysorbate 20, the near-ultraviolet circular dichroism peak at wavelengths between 250 nm and 300 nm is observed. An isolated recombinant polypeptide complex containing at least one of the following.

2. The isolated recombinant polypeptide complex according to claim 1, wherein the isolated recombinant polypeptide complex comprises at least four of the features described above.

3. The isolated recombinant polypeptide complex according to claim 1, wherein the isolated recombinant polypeptide complex comprises at least eight of the features described above.

4. The isolated recombinant polypeptide complex according to claim 1, wherein the first chain contains at least 90% sequence identity with respect to SEQ ID NO:

1.

5. The isolated recombinant polypeptide complex according to claim 1, wherein the second chain contains at least 90% sequence identity with respect to SEQ ID NO:

2.

6. The isolated recombinant polypeptide complex according to 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.

7. The isolated recombinant polypeptide complex of claim 1, wherein the at least one disulfide bond is an intrachain disulfide bond formed between 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.

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

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

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

11. The isolated recombinant polypeptide complex according to claim 1, wherein at least one cysteine ​​residue is a free sulfhydryl.

12. The isolated recombinant polypeptide complex according to claim 1, wherein at least one cysteine ​​residue is a free sulfhydryl, and at least one cysteine ​​is 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, cysteine ​​625, and cysteine ​​645 of SEQ ID NO:

1.

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

2.

14. The isolated recombinant polypeptide complex according to claim 1, wherein at least one pair of cysteine ​​residues 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.

15. The isolated recombinant polypeptide complex according to claim 1, wherein the secondary structural composition comprises a β-sheet and a random coil.

16. The isolated recombinant polypeptide complex according to claim 1, having a melting temperature (T m) of approximately 71.4°C to approximately 79.5°C.

17. The isolated recombinant polypeptide complex according to claim 1, having a far-ultraviolet circular dichroism peak with a wavelength of 195 nm or less.

18. The isolated recombinant polypeptide complex according to claim 1, having a near-ultraviolet circular dichroism peak with a wavelength of 279 nm or less.

19. The isolated recombinant polypeptide complex according to claim 1, having a near-ultraviolet circular dichroism peak with a wavelength of 290 nm or less.

20. Use of an isolated recombinant polypeptide complex according to any one of claims 1 to 19 in the manufacture of a drug for the treatment of cancer.

Images