Composition of tumor activation antibodies targeting EGFR and effector cell antigens and use thereof

Abstract

Disclosed herein are compositions and uses of tumor activating antibodies targeting EGFR and effector cell antigens. Specifically, the present application provides isolated recombinant polypeptide complexes comprising binding domains that selectively bind to effector cell antigens and EGFR wherein one or more of the binding domains are selectively activated in the tumor microenvironment, and the isolated polypeptide or polypeptide complex comprising molecules that extend half-life.

Description

[0001] Cross-references

[0002] This application claims the benefit of PCT International Application No. PCT / IB2022 / 000650, filed on November 11, 2022, and PCT International Application No. PCT / IB2023 / 000137, filed on March 9, 2023, each of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in XML file format and is incorporated herein by reference in its entirety. The XML copy was created on October 26, 2023, is named 52426-750_603SL.xml, and is 45,501 bytes in size. Summary of the Invention

[0005] In one aspect, disclosed herein is an isolated recombinant polypeptide complex comprising a first chain having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1 and a second chain having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2, wherein the isolated recombinant polypeptide complex comprises at least one of the following features: (a) at least one N-glycan moiety; (b) at least one disulfide bond; (c) a melting onset temperature (T) between about 60° C. and about 65° C. when the isolated recombinant polypeptide complex is formulated at a concentration of about 1.0 mg / mL in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) polysorbate 20 (PS20), pH about 5.3. 起始 ) and a transition midpoint temperature (T m1 ), wherein the T 起始 and the T m1(d) a far-UV circular dichroism dip at a wavelength between 210 nm and 230 nm when the isolated recombinant polypeptide complex is formulated in water at a concentration of about 0.1 mg / mL; or (e) a near-UV circular dichroism dip at a wavelength between 275 nm and 290 nm when the isolated recombinant polypeptide complex is formulated in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) PS20, pH about 5.3, at a concentration of about 1.0 mg / mL. In some embodiments, the polypeptide complex comprises at least two of the features. In some embodiments, the polypeptide complex comprises at least three of the features. In some embodiments, the polypeptide complex comprises at least four of the features. In some embodiments, the polypeptide complex comprises at least five of the features. In some embodiments, the first chain comprises at least 75% sequence identity to SEQ ID NO: 1. In some embodiments, the first strand comprises at least 80% sequence identity to SEQ ID NO: 1. 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 according to SEQ ID NO: 1. In some embodiments, the second strand comprises at least 75% sequence identity to SEQ ID NO: 2. In some embodiments, the second strand comprises at least 80% sequence identity to SEQ ID NO: 2. 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 chain comprises at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the second chain comprises an amino acid sequence according to SEQ ID NO: 2. In some embodiments, the at least one N-glycan moiety is located on the first chain. In some embodiments, the at least one N-glycan moiety is located on the second chain. In some embodiments, the at least one N-glycan moiety comprises G2F, G2FS1, or G2FS2. In some embodiments, the at least one N-glycan moiety comprises G2F.In some embodiments, the at least one N-glycan moiety comprises G2FS1. In some embodiments, the at least one N-glycan moiety comprises G2FS2. In some embodiments, the at least one asparagine deamidation moiety is located at asparagine 83 of SEQ ID NO: 1. In some embodiments, the at least one N-glycan moiety is located at asparagine 519 of SEQ ID NO: 2. In some embodiments, the isolated recombinant polypeptide complex further comprises O-xylosylation, asparagine deamidation, or succinimide formation. In some embodiments, the succinimide formation is located at asparagine 83 of SEQ ID NO: 1. In some embodiments, the polypeptide complex comprises at least two disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the polypeptide complex comprises at least three disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the polypeptide complex comprises at least four disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the polypeptide complex comprises at least five disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the polypeptide complex comprises at least six disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the polypeptide complex comprises at least seven disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the polypeptide complex comprises at least eight disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the polypeptide complex comprises at least nine disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the polypeptide complex comprises at least ten disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the cysteine ​​residue pair comprises cysteine ​​4 and cysteine ​​15 of SEQ ID NO: 1. In some embodiments, the cysteine ​​residue pair comprises cysteine ​​65 and cysteine ​​130 of SEQ ID NO: 1. In some embodiments, the cysteine ​​residue pair comprises cysteine ​​176 and cysteine ​​236 of SEQ ID NO: 1. In some embodiments, the cysteine ​​residue pair comprises cysteine ​​256 of SEQ ID NO: 1 and cysteine ​​653 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, the pair of cysteine ​​residues comprises cysteine ​​138 and cysteine ​​148 of SEQ ID NO: 2. In some embodiments, the pair of cysteine ​​residues comprises cysteine ​​199 and cysteine ​​275 of SEQ ID NO: 2. In some embodiments, the pair of cysteine ​​residues comprises cysteine ​​339 and cysteine ​​407 of SEQ ID NO: 2.In some embodiments, the pair of cysteine ​​residues comprises cysteine ​​453 and cysteine ​​526 of SEQ ID NO: 2. In some embodiments, the pair of cysteine ​​residues comprises cysteine ​​577 and cysteine ​​633 of SEQ ID NO: 2. In some embodiments, the polypeptide complex comprises 1 interchain disulfide bond and 9 intrachain disulfide bonds between the first chain and the second chain. In some embodiments, the polypeptide complex comprises 1 interchain disulfide bond between the first chain and the second chain, and the second chain comprises 6 intrachain disulfide bonds, and the first chain comprises 3 intrachain disulfide bonds. In some embodiments, the isolated recombinant polypeptide complex has a T between about 61° C. and about 64.5° C. 起始 In some embodiments, the isolated recombinant polypeptide complex has a T between about 62°C and about 64°C. 起始 In some embodiments, the isolated recombinant polypeptide complex has a T of about 62.5°C. 起始 In some embodiments, the isolated recombinant polypeptide complex has a T of about 63.2°C. 起始 In some embodiments, the isolated recombinant polypeptide complex has a T between about 71°C and about 75°C. m1 In some embodiments, the isolated recombinant polypeptide complex has a T between about 72.5°C and about 74.5°C. m1 In some embodiments, the isolated recombinant polypeptide complex has a T of about 73.8°C. m1 In some embodiments, the isolated recombinant polypeptide complex has a T of about 74.0°C. m1. In some embodiments, the secondary structure composition includes β-sheets and random coils. In some embodiments, the isolated recombinant polypeptide complex has a far-UV circular dichroism dip at a wavelength between 215 nm and 225 nm. In some embodiments, the isolated recombinant polypeptide complex has a far-UV circular dichroism dip at a wavelength between 215 nm and 220 nm. In some embodiments, the isolated recombinant polypeptide complex has a near-UV circular dichroism dip at a wavelength between 280 nm and 290 nm. In some embodiments, the isolated recombinant polypeptide complex has a near-UV circular dichroism dip at a wavelength between 280 nm and 285 nm. In some embodiments, the isolated recombinant polypeptide complex has a near-UV circular dichroism peak at a wavelength between 270 nm and 275 nm. In some embodiments, the isolated recombinant polypeptide complex has a near-UV circular dichroism peak at a wavelength between 285 nm and 290 nm. In some embodiments, the first chain comprises the amino acid sequence according to SEQ ID NO: 1, and the second chain comprises the amino acid sequence according to SEQ ID NO: 2, and the at least one N-glycan moiety comprises G2F, G2FS1, or G2FS2, and the recombinant polypeptide complex comprises disulfide bonds formed by the following pairs of cysteine ​​residues: Cysteine ​​4 and Cysteine ​​15 of SEQ ID NO: 1, Cysteine ​​65 and Cysteine ​​130 of SEQ ID NO: 1, Cysteine ​​176 and Cysteine ​​236 of SEQ ID NO: 1, Cysteine ​​256 of SEQ ID NO: 1 and Cysteine ​​653 of SEQ ID NO: 2, Cysteine ​​138 and Cysteine ​​148 of SEQ ID NO: 2, Cysteine ​​22 and Cysteine ​​96 of SEQ ID NO: 2, Cysteine ​​199 and Cysteine ​​275 of SEQ ID NO: 2, Cysteine ​​339 and Cysteine ​​407 of SEQ ID NO: 2, Cysteine ​​338 and Cysteine ​​408 of SEQ ID NO: 2, Cysteine ​​340 and Cysteine ​​409 of SEQ ID NO: 2, Cysteine ​​341 and Cysteine ​​409 of SEQ ID NO: 2, Cysteine ​​342 and Cysteine ​​409 of SEQ ID NO: 2, Cysteine ​​343 and Cysteine ​​409 of SEQ ID NO: 2, Cysteine ​​344 and Cysteine ​​409 of SEQ ID NO: 2, Cysteine ​​345 and Cysteine ​​409 of SEQ ID NO: 2, Cysteine ​​346 and Cysteine ​​409 of SEQ ID NO: 2, Cysteine ​​347 and Cysteine ​​409 of SEQ ID NO: 2, Cysteine ​​34 NO:2 cysteine ​​453 and cysteine ​​526 and cysteine ​​577 and cysteine ​​633 of SEQ ID NO:2, and when the isolated recombinant polypeptide complex is formulated at a concentration of about 1.0 mg / mL in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) PS20 pH about 5.3, the T 起始 Between about 60°C and about 65°C and the transition midpoint temperature (T m1 ) is between about 70°C and about 75°C, wherein the T 起始 and the T m1Differential scanning calorimetry (DSC) was used to measure the far-UV circular dichroism at wavelengths between 210 nm and 230 nm when the isolated recombinant polypeptide complex was formulated at a concentration of 0.1 mg / mL in water, and the near-UV circular dichroism at wavelengths between 275 nm and 290 nm was depressed when the isolated recombinant polypeptide complex was formulated at a concentration of about 1.0 mg / mL in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) PS20, pH about 5.3.

[0006] In another aspect, disclosed herein is an isolated recombinant polypeptide complex comprising a first chain having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1 and a second chain having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2, wherein the isolated recombinant polypeptide complex comprises the following features: (a) at least one N-glycan moiety; (b) at least one disulfide bond; (c) a melting onset temperature (T) between about 60° C. and about 65° C. when the isolated recombinant polypeptide complex is formulated at a concentration of about 1.0 mg / mL in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) PS20, pH about 5.3. 起始 ) and a transition midpoint temperature (T m1 ), wherein the T starts and the T m1 (d) a far-UV circular dichroism dip at a wavelength between 210 nm and 230 nm when the isolated recombinant polypeptide complex is formulated in water at a concentration of about 0.1 mg / mL; or (e) a near-UV circular dichroism dip at a wavelength between 275 nm and 290 nm when the isolated recombinant polypeptide complex is formulated in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) PS20, pH about 5.3, at a concentration of about 1.0 mg / mL.

[0007] On the other hand, disclosed herein is a pharmaceutical composition comprising: (a) an isolated recombinant polypeptide complex disclosed herein; and (b) a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier 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 derivative thereof comprises L-histidine, L-histidine hydrochloride monohydrate, or a combination thereof. In some embodiments, the surfactant is polysorbate 20. In some embodiments, the stabilizer is sucrose. In some embodiments, the pharmaceutical composition has a pH below 6.0.

[0008] Incorporation by reference

[0009] All publications, patents, and patent applications mentioned in this specification are herein 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The features of the present disclosure are particularly set forth in the appended claims. The features and advantages of the present disclosure will be better understood by reference to the following detailed description and accompanying drawings (also referred to herein as "Figures") which illustrate illustrative embodiments utilizing the principles of the present disclosure, in which:

[0011] Figure 1 The design, structure, and mechanism of action of the polypeptide complex 1 (PC-1) disclosed herein are described. PC-1 is a tumor-activated T cell engager comprising an EGFR binding domain and a CD3 binding domain, an albumin binding domain that extends circulating half-life, a peptide mask that inhibits CD3 engagement with T cells, and a tumor protease-cleavable linker. Tumor-specific proteolysis of the cleavable linker in the tumor microenvironment (TME) separates the tandem mask and albumin binding domain from PC-1. This enables TME-restricted CD3 binding and subsequent T cell activation against EGFR-expressing cancer cells. Loss of the albumin binding domain ensures that any activated PC-1 that migrates from the tumor will be rapidly cleared and reduces its potential accumulation in healthy tissues, which could pose a safety risk.

[0012] Figure 2 The design and structure of polypeptide complex 1 (PC-1) are described. PC-1 is a tumor-activated T cell engager comprising an EGFR-engaging domain and a CD3-binding domain (VH2 and VL1), an albumin-binding domain (VH1) that extends circulating half-life, a peptide mask that inhibits CD3 engagement with T cells, a peptide mask that inhibits binding to EGFR, and a tumor protease-cleavable linker.

[0013] Figure 3 Flowchart illustrating the upstream cell culture process used in the production of PC-1.

[0014] Figure 4 Flowchart illustrating the downstream purification process used in the production of PC-1.

[0015] Figure 5 The circular dichroism (CD) spectrum of PC-1 in the far UV region is illustrated.

[0016] Figure 6 The CD spectrum of PC-1 in the near-UV region is illustrated.

[0017] Figure 7 Differential scanning calorimetry data for PC-1 are illustrated.

[0018] Figure 8A 、 8B , 8C, and 8D illustrate the SEC-MALS chromatograms of PC-1.

[0019] Figure 9A and 9B Illustrate the dynamic light scattering chromatogram of PC-1.

[0020] Figure 10 Illustrate the symbolic structures of the major N-glycans.

[0021] Figure 11 Illustration of the light chain (LC) and heavy chain (HC) arrangement in PC-1.

[0022] Figure 12A and 12B Binding of PC-1, PC-1-MMP9 cleavage product, PC-1-SP cleavage product or PC-1-TCE to human EGFR and cynomolgus monkey EGFR is demonstrated.

[0023] Figure 13A and 13B Binding of PC-1, PC-1-MMP cleavage, PC-1-SP cleavage, or PC-1-TCE to human CD3 and cynomolgus monkey CD3 is demonstrated.

[0024] Figure 14A and 14B The binding of PC-1 to human albumin and cynomolgus monkey albumin is illustrated.

[0025] Figure 15A 、 15B , 15C and 15D illustrate the killing of HCT116 tumor cells by donor PBMCs stimulated with PC-1-TCE, PC-1, PC-1-SP cleavage, or PC-1-MMP cleavage.

[0026] Figure 16A 、 16B , 16C and 16D illustrate the killing of A549 tumor cells by donor PBMCs stimulated with PC-1-TCE, PC-1, PC-1-SP cleavage, or PC-1-MMP cleavage.

[0027] Figure 17A 、 17B , 17C and 17D illustrate killing of Cal27 tumor cells by donor PBMCs stimulated with PC-1-TCE, PC-1, PC-1-SP cleavage, or PC-1-MMP cleavage.

[0028] Figure 18A and 18B This figure illustrates the killing of A549 EGFR-KO tumor cells by donor PBMCs stimulated with PC-1-TCE, PC-1, PC-1-SP cleavage, or PC-1-MMP cleavage.

[0029] Figure 19A 、 19B , 19C, 19D, 19E and 19F illustrate the release of IFNγ, TNF and IL-6 from healthy donor PBMCs induced by PC-1-TCE, PC-1, PC-1-SP cleavage or PC-1-MMP cleavage in the presence of HCT116 cells.

[0030] Figure 20A 、 20B , 20C, 20D, 20E and 20F illustrate the release of IFNγ, TNF and IL-6 from healthy donor PBMCs induced by PC-1-TCE, PC-1, PC-1-SP cleavage or PC-1-MMP cleavage in the presence of A549 cells.

[0031] Figure 21A 、 21B , 21C, 21D, 21E and 21F illustrate the release of IFNγ, TNF and IL-6 from healthy donor PBMCs induced by PC-1-TCE, PC-1, PC-1-SP cleavage or PC-1-MMP cleavage in the presence of Cal27 cells.

[0032] Figure 22A 、 22B , 22C, 22D, 22E and 22F illustrate the lack of IFNγ, TNF and IL-6 release from healthy donor PBMCs induced by PC-1-TCE, PC-1, PC-1-SP cleavage or PC-1-MMP cleavage in the presence of EGFR-KO A549 cells.

[0033] Figure 23 The cleavage-dependent PC-1-His tag activity is illustrated in HCT116 tumor-bearing mice co-implanted with human PBMCs using vehicle, PC-1-NC (0.5 mg / kg), PC-1-TCE (0.5 mg / kg), PC-1-His tag (0.15 mg / kg), PC-1-His tag (0.5 mg / kg), and PC-1-His tag (1.5 mg / kg).

[0034] Figure 24 The structure of PC-1 is described.

[0035] Figure 25The structure of PC-1-SP cut product is illustrated.

[0036] Figure 26 The structure of the PC-1-MMP cleavage product is illustrated.

[0037] Figure 27 The structure of PC-1-TCE is described.

[0038] Figure 28 The structure of the PC-1-His tag is illustrated.

[0039] Figure 29 The structure of PC-1-NC is described. DETAILED DESCRIPTION

[0040] Multispecific antibodies combine the advantages of different binding specificities derived from two or more antibodies into a single composition. In preclinical and clinical studies, multispecific antibodies for redirecting T cells to cancer have shown promise. This approach relies on one antigen-interacting portion of the antibody to bind to a tumor-associated antigen or marker, while the second antigen-interacting portion can bind to an effector cell antigen (such as CD3) on the T cell, thereby triggering cytotoxic activity. One such tumor-associated antigen is the epidermal growth factor receptor (EGFR). EGFR is a transmembrane protein that is a receptor for members of the epidermal growth factor family of extracellular protein ligands. EGFR is the most common overexpressed membrane protein in cancer. However, EGFR expression is not limited to tumors, but is widely expressed throughout the body, resulting in systemic toxicity of EGFR-directed therapy.

[0041] T cell engager (TCE) therapies have several benefits, including that they are not cell therapies and therefore, unlike chimeric antigen receptor T cell (CAR T cell) therapies, can be provided as off-the-shelf therapies. Although TCE therapeutics have shown potent anti-tumor activity in blood cancers, the development of TCEs for the treatment of solid tumors is still facing challenges due to the limitations of previous TCE technology, namely: (i) excessive activation of the immune system leading to cytokine release syndrome (CRS); (ii) toxicity to target healthy tissues; and (iii) poor pharmacokinetics (PK), resulting in a short half-life. CRS is caused by systemic activation of T cells and may lead to life-threatening increases in inflammatory cytokines such as interleukin-6 (IL-6). In poor clinical studies, severe acute CRS has been observed to lead to dose-limiting toxicity and death when T cell engagers developed using other platforms are administered to treat cancer patients. This toxicity limits the maximum blood level of T cell engagers that can be safely administered. The effectiveness of T cell engagers is also limited by toxicity to target healthy tissues. T cell engagers developed using platforms that are not designed for tumor-specific activation have resulted in clinically suspended and dose-limiting toxicities due to target expression in healthy tissues. T cell engagers have also been limited by short half-lives. T cell engagers quickly reach subtherapeutic levels as they are rapidly eliminated from the body due to their short exposure half-life after administration. Therefore, T cell engagers such as Blinatumomab are typically administered via low-dose continuous infusion pumps over several weeks to overcome the challenges of short half-life and maintain therapeutic levels of the drug in vivo. Continuous dosing regimens represent a significant burden on patients.

[0042] To overcome these challenges associated with the effectiveness of T cell engagers, recombinant polypeptide complexes are described herein that include binding domains that selectively bind to effector cell antigens and EGFR, wherein one or more of the binding domains are selectively activated in the tumor microenvironment, and the isolated polypeptide or polypeptide complex includes a molecule that extends half-life. This modification reduces the risk of CRS and on-target healthy tissue toxicity and improves stability and serum half-life in the bloodstream before activation. The recombinant polypeptide complexes described herein are active at low target expression levels and can be easily manufactured and formulated.

[0043] In some embodiments, the recombinant polypeptide complexes described herein are used in methods for treating cancer. In some embodiments, the cancer has cells that express EGFR. In some embodiments, the recombinant polypeptide complexes described herein are used in methods for treating renal cell carcinoma, colorectal cancer (CRC), squamous cell carcinoma of the head and neck (SCCHN), non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, colon / rectal cancer, head and neck cancer, esophageal and gastric cancer, liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, or pancreatic cancer. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods for treating subjects who are resistant to EGFR inhibitor therapy. In some embodiments, the recombinant polypeptide complexes described herein are used in methods for treating subjects who carry KRAS mutations. In some embodiments, the recombinant polypeptide complexes described herein are used in methods for treating subjects who are resistant to EGFR inhibitor therapy and carry KRAS mutations.

[0044] One or more features

[0045] The isolated polypeptides or recombinant polypeptide complexes described herein may have one or more of the characteristics described above in this section.

[0046] In some embodiments, the isolated recombinant polypeptide complex comprises a first chain having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1 and a second chain having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2. In some embodiments, the recombinant polypeptide complex has at least one, at least two, at least three, at least four, or all five of features (a)-(e):

[0047] (a) at least one N-glycan moiety;

[0048] (b) at least one disulfide bond;

[0049] (c) When the isolated recombinant polypeptide complex is formulated at a concentration of about 1.0 mg / mL in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) PS20 pH about 5.3, the melting onset temperature (T) is between about 60°C and about 65°C. 起始 ) and a transition midpoint temperature (T m1 );

[0050] (d) a negative far-UV circular dichroism peak at a wavelength between 210 nm and 230 nm when the isolated recombinant polypeptide complex is formulated in water at a concentration of 0.1 mg / mL; or

[0051] (e) The isolated recombinant polypeptide complex has a depressed near-UV circular dichroism at wavelengths between 275 nm and 290 nm when formulated in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) PS20, pH about 5.3, at a concentration of about 1.0 mg / mL.

[0052] In some embodiments, the isolated recombinant polypeptide complex comprises a first chain having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1 and a second chain having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2. In some embodiments, the recombinant polypeptide complex has at least one, at least two, at least three, at least four, or all five of features (a)-(e):

[0053] (a) at least one N-glycan moiety;

[0054] (b) at least one disulfide bond;

[0055] (c) When the isolated recombinant polypeptide complex is formulated at a concentration of about 1.0 mg / mL in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) PS20 pH about 5.3, the melting onset temperature (T) is between about 60°C and about 65°C. 起始 ) and a transition midpoint temperature (T m1 ); where T 起始 and T m1 Measured using differential scanning calorimetry (DSC);

[0056] (d) a deep UV circular dichroism at wavelengths between 210 nm and 230 nm when the isolated recombinant polypeptide complex is formulated in water at a concentration of about 0.1 mg / mL; or

[0057] (e) The isolated recombinant polypeptide complex has a depressed near-UV circular dichroism at wavelengths between 275 nm and 290 nm when formulated in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) PS20, pH about 5.3, at a concentration of about 1.0 mg / mL.

[0058] In some embodiments, the isolated recombinant polypeptide complex comprises a first chain having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1 and a second chain having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2, wherein the isolated recombinant polypeptide complex comprises the following features:

[0059] (a) at least one N-glycan moiety;

[0060] (b) at least one disulfide bond;

[0061] (c) When the isolated recombinant polypeptide complex is formulated at a concentration of about 1.0 mg / mL in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) PS20 pH about 5.3, the melting onset temperature (T) is between about 60°C and about 65°C. 起始 ) and a transition midpoint temperature (T m1 ), where T 起始 and T m1 Measured using differential scanning calorimetry (DSC);

[0062] (d) a deep UV circular dichroism at wavelengths between 210 nm and 230 nm when the isolated recombinant polypeptide complex is formulated in water at a concentration of about 0.1 mg / mL; or

[0063] (e) The isolated recombinant polypeptide complex has a depressed near-UV circular dichroism at wavelengths between 275 nm and 290 nm when formulated in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) PS20, pH about 5.3, at a concentration of about 1.0 mg / mL.

[0064] In some embodiments, the recombinant polypeptide complex comprises at least one (e.g., one, two, three, four, five, six, or seven) of features (a)-(e). In some embodiments, the recombinant polypeptide complex comprises at least two (e.g., two, three, four, five, six, or seven) of features (a)-(e). In some embodiments, the recombinant polypeptide complex comprises at least three (e.g., three, four, five, six, or seven) of features (a)-(e). In some embodiments, the recombinant polypeptide complex comprises at least four (e.g., four, five, six, or seven) of features (a)-(e). In some embodiments, the isolated recombinant polypeptide complex comprises at least five (e.g., five, six, or seven) of features (a)-(e).

[0065] Isolated recombinant polypeptide complex composition

[0066] In some embodiments, the isolated recombinant polypeptide complex comprises a tumor-activated T cell engager having an EGFR binding domain and a CD3 binding domain, an albumin binding domain that extends circulating half-life, a peptide mask that inhibits CD3 engagement with T cells, and a tumor protease cleavable linker. Tumor-specific proteolysis of the cleavable linker in the tumor microenvironment can separate the tandem mask and albumin binding domain from the isolated recombinant polypeptide complex. The isolated recombinant polypeptide complex can comprise chain 1 and chain 2 as described herein. In some embodiments, chain 1 comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, chain 2 comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2.

[0067] In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 1. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 1. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 1. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 1. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 1. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 1. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 1. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 1.

[0068] In some embodiments, the isolated recombinant polypeptide complex comprises the amino acid sequence according to SEQ ID NO:1.

[0069] In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 2. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 2. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 2. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 2. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 2. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 2. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 2. In some embodiments, the isolated recombinant polypeptide complex comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 2.

[0070] In some embodiments, the isolated recombinant polypeptide complex comprises the amino acid sequence according to SEQ ID NO:2.

[0071] In some embodiments, the LC comprises at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises an amino acid sequence according to SEQ ID NO: 1. In some embodiments, the HC comprises at least 85% sequence identity to SEQ ID NO: 2. In some embodiments, the HC comprises at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the HC comprises at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, the HC comprises at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the HC comprises an amino acid sequence according to SEQ ID NO: 2. In some embodiments, the LC comprises at least 99% sequence identity to SEQ ID NO: 1 and the HC comprises at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the LC comprises the amino acid sequence according to SEQ ID NO: 1 and the HC comprises the amino acid sequence according to SEQ ID NO:2.

[0072] Table 1. Exemplary amino acid sequences of isolated recombinant polypeptide complexes (PC-1) that selectively bind to EGFR and effector cell antigens such as CD3 and their metabolites and derivatives.

[0073]

[0074]

[0075] PC-1 = polypeptide complex 1, EGFR = epidermal growth factor receptor, HC = heavy chain, LC = light chain, CD3 = cluster of differentiation 3, FAB = fragment antigen binding domain, scFv = single-chain variable fragment, MMP9 = matrix metallopeptidase 9, SP = serine protease, His tag = histidine tag, NC = non-cleavable, TCE = T cell engager

[0076] disulfide bonds

[0077] In some embodiments, at least one disulfide bond is an intrachain disulfide bond. In some embodiments, at least one disulfide bond is an interchain disulfide bond. In some embodiments, the interchain disulfide bond is between the LC and the HC. In some embodiments, the isolated recombinant antibody comprises at least two disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the isolated recombinant antibody comprises at least three disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the isolated recombinant antibody comprises at least four disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the isolated recombinant antibody comprises at least five disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the isolated recombinant antibody comprises at least six disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the isolated recombinant antibody comprises at least seven disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the isolated recombinant antibody comprises at least eight disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the isolated recombinant antibody comprises at least nine disulfide bonds formed by pairs of cysteine ​​residues. In some embodiments, the isolated recombinant antibody comprises at least ten disulfide bonds formed by pairs of cysteine ​​residues.

[0078] In some embodiments, the cysteine ​​residue pair comprises cysteine ​​4 and cysteine ​​15 of SEQ ID NO: 1. In some embodiments, the cysteine ​​residue pair comprises cysteine ​​65 and cysteine ​​130 of SEQ ID NO: 1. In some embodiments, the cysteine ​​residue pair comprises cysteine ​​176 of SEQ ID NO: 1 and cysteine ​​236 of SEQ ID NO: 1. In some embodiments, the cysteine ​​residue pair comprises cysteine ​​256 of SEQ ID NO: 1 and cysteine ​​653 of SEQ ID NO: 2. In some embodiments, the cysteine ​​residue pair comprises cysteine ​​22 of SEQ ID NO: 2 and cysteine ​​96 of SEQ ID NO: 2. In some embodiments, the cysteine ​​residue pair comprises cysteine ​​138 of SEQ ID NO: 2 and cysteine ​​148 of SEQ ID NO: 2. In some embodiments, the cysteine ​​residue pair comprises cysteine ​​199 of SEQ ID NO: 2 and cysteine ​​275 of SEQ ID NO: 2. In some embodiments, the pair of cysteine ​​residues comprises cysteine ​​339 of SEQ ID NO: 2 and cysteine ​​407 of SEQ ID NO: 2. In some embodiments, the pair of cysteine ​​residues comprises cysteine ​​453 of SEQ ID NO: 2 and cysteine ​​526 of SEQ ID NO: 2. In some embodiments, the pair of cysteine ​​residues comprises cysteine ​​577 of SEQ ID NO: 2 and cysteine ​​633 of SEQ ID NO: 2.

[0079] In some embodiments, the at least one disulfide bond formed by a pair of cysteine ​​residues is selected from: cysteine ​​4 of SEQ ID NO: 1 and cysteine ​​15 of SEQ ID NO: 1; cysteine ​​130 of SEQ ID NO: 1 and cysteine ​​176 of SEQ ID NO: 1; cysteine ​​236 of SEQ ID NO: 2 and cysteine ​​256 of SEQ ID NO: 1; cysteine ​​653 of SEQ ID NO: 2 and cysteine ​​22 of SEQ ID NO: 2; cysteine ​​96 of SEQ ID NO: 2 and cysteine ​​199 of SEQ ID NO: 2; cysteine ​​275 of SEQ ID NO: 2 and cysteine ​​339 of SEQ ID NO: 2; cysteine ​​407 of SEQ ID NO: 2 and cysteine ​​453 of SEQ ID NO: 2; cysteine ​​526 of SEQ ID NO: 2 and cysteine ​​577 of SEQ ID NO: 2; cysteine ​​633 of SEQ ID NO: 2.

[0080] In some embodiments, the isolated recombinant polypeptide complex comprises at least one cysteine ​​residue that is a free thiol. The presence of free thiol groups can be determined by mass spectrometry (MS).

[0081] Amino acid modification

[0082] In some embodiments, the isolated recombinant polypeptide complex further comprises O-xylosylation, asparagine deamidation, or succinimide formation. Asparagine 83 of SEQ ID NO:1 can be deamidated. Asparagine 83 of SEQ ID NO:1 can comprise succinimide formation. Asparagine 179 of SEQ ID NO:1 can be deamidated. Asparagine 233 of SEQ ID NO:2 can be deamidated. Serine 110 of SEQ ID NO:2 can comprise O-xylosylation. Serine 123 of SEQ ID NO:2 can comprise O-xylosylation. Serine 124 of SEQ ID NO:2 can comprise O-xylosylation. Serine 129 of SEQ ID NO:2 can comprise O-xylosylation. Serine 133 of SEQ ID NO:2 can comprise O-xylosylation. Serine 154 of SEQ ID NO:2 can comprise O-xylosylation. In some embodiments, the succinimide formation is located at asparagine 83 of SEQ ID NO:1.

[0083] N-glycan moiety

[0084] In some embodiments, the isolated recombinant polypeptide complex comprises at least one N-glycan moiety. The N-glycan moiety may comprise one fucose residue, four N-acetylglucosamine (GlcNAc) residues, and five hexose residues, such as Figure 10 The N-glycan portion can contain one fucose residue, four GlcNac residues, five hexose residues and one N-acetylneuraminic acid (Neu5Ac) residue, such as Figure 10 The N-glycan moiety can contain one fucose residue, four GlcNac residues, five hexose residues, one Neu5Ac residue and one Neu5Gc residue, such as Figure 10 In some embodiments, the isolated recombinant antibody comprises at least two N-glycan moieties. In some embodiments, the heavy chain sequence comprises at least one N-glycan moiety. In some embodiments, the heavy chain sequence comprises an N-glycan moiety at asparagine 83. In some embodiments, the light chain sequence comprises an N-glycan moiety at asparagine 83. In some embodiments, the N-glycan moiety is located at asparagine 83 of SEQ ID NO: 1. In some embodiments, at least one asparagine deamidation moiety is located at asparagine 83 of SEQ ID NO: 1. In some embodiments, the heavy chain sequence comprises an N-glycan moiety at asparagine 519.

[0085] In some embodiments, at least one N-glycan moiety comprises N-acetylglucosamine (GlcNAc), a hexose, fucose, N-acetylneuraminic acid (Neu5Ac), or N-glycolylneuraminic acid (Neu5Gc). In some embodiments, at least one N-glycan moiety comprises GlcNAc, a hexose, fucose, or Neu5Ac. In some embodiments, at least one N-glycan moiety comprises GlcNAc and a hexose. In some embodiments, at least one N-glycan moiety comprises GlcNAc, a hexose, and Neu5Ac. In some embodiments, at least one N-glycan moiety comprises GlcNac, a hexose, and fucose. In some embodiments, at least one N-glycan moiety comprises at least two GlcNAc moieties and at least two hexose moieties. In some embodiments, at least one N-glycan moiety comprises at least three GlcNAc moieties and at least three hexose moieties. In some embodiments, at least one N-glycan moiety comprises three GlcNAc moieties and three hexose moieties. In some embodiments, at least one N-glycan moiety comprises four GlcNAc moieties and three hexose moieties. In some embodiments, at least one N-glycan moiety comprises three GlcNAc moieties and four hexose moieties. In some embodiments, at least one N-glycan moiety comprises two GlcNAc moieties and five hexose moieties. In some embodiments, at least one N-glycan moiety comprises five GlcNAc moieties and three hexose moieties. In some embodiments, at least one N-glycan moiety comprises four GlcNAc moieties and four hexose moieties. In some embodiments, at least one N-glycan moiety comprises three GlcNAc moieties and five hexose moieties. In some embodiments, at least one N-glycan moiety comprises five GlcNAc moieties and four hexose moieties. In some embodiments, at least one N-glycan moiety comprises four GlcNAc moieties and five hexose moieties. In some embodiments, at least one N-glycan moiety comprises four GlcNAc moieties, five hexose moieties, one Neu5Ac moiety and one fucose moiety. In some embodiments, at least one N-glycan moiety comprises four GlcNAc moieties, six hexose moieties and one fucose moiety.

[0086] Tertiary and secondary structure

[0087] In some embodiments, the isolated recombinant polypeptide complex has a secondary structural composition comprising a β-sheet or a random coil. The secondary structural composition may comprise a β-sheet. The secondary structural composition may comprise a random coil.

[0088] In some embodiments, the isolated recombinant polypeptide complex is characterized by a far-UV circular dichroism peak at a wavelength less than or equal to 220 nm, 210 nm, or 205 nm. In some embodiments, the isolated recombinant polypeptide complex is characterized by a far-UV circular dichroism peak at a wavelength greater than or equal to 205 nm, 210 nm, or 220 nm. In some embodiments, the isolated recombinant polypeptide complex is characterized by a far-UV circular dichroism peak at a wavelength between 200 nm and 210 nm or between 205 nm and 220 nm.

[0089] In some embodiments, the far UV circular dichroism peak is at a wavelength between 200 nm and 235 nm. In some embodiments, the far UV circular dichroism peak is at a wavelength between 210 nm and 230 nm.

[0090] In some embodiments, the isolated recombinant polypeptide complex has a far-UV circular dichroism dip at a wavelength between about 215 nm and about 225 nm, e.g., about 215 nm, 216 nm, 217 nm, 218 nm, 219 nm, 220 nm, 221 nm, 222 nm, 223 nm, 224 nm, or about 225 nm, or any wavelength therebetween.

[0091] In some embodiments, the isolated recombinant polypeptide complex has a far-UV circular dichroism dip at a wavelength between about 215 nm and about 220 nm, e.g., about 215 nm, 216 nm, 217 nm, 218 nm, 219 nm, or about 220 nm, or any wavelength therebetween.

[0092] In some embodiments, the isolated recombinant polypeptide complex has a near-UV circular dichroism dip at a wavelength between about 280 nm and about 290 nm, e.g., about 280 nm, 281 nm, 282 nm, 283 nm, 284 nm, 285 nm, 286 nm, 287 nm, 288 nm, 289 nm, or about 290 nm, or any wavelength therebetween.

[0093] In some embodiments, the isolated recombinant polypeptide complex has a near-UV circular dichroism dip at a wavelength between about 280 nm and about 285 nm, e.g., about 280 nm, 281 nm, 282 nm, 283 nm, 284 nm, or 285 nm, or any wavelength therebetween.

[0094] In some embodiments, the isolated recombinant polypeptide complex has a near-UV circular dichroism peak at a wavelength between about 270 nm and about 275 nm, e.g., about 270 nm, 271 nm, 272 nm, 273 nm, 274 nm, or about 275 nm, or any wavelength therebetween.

[0095] In some embodiments, the isolated recombinant polypeptide complex has a near-UV circular dichroism peak at a wavelength between about 285 nm and about 290 nm, e.g., about 285 nm, 286 nm, 284 nm, 288 nm, 289 nm, or about 290 nm, or any wavelength therebetween.

[0096] In some embodiments, the isolated recombinant polypeptide complex is characterized by a near UV circular dichroism peak at a wavelength less than or equal to 300 nm, 295 nm, 290 nm, 285 nm, 280 nm, 275 nm, or 270 nm. In some embodiments, the isolated recombinant polypeptide complex is characterized by a near UV circular dichroism peak at a wavelength greater than or equal to 270 nm, 275 nm, 280 nm, 285 nm, 290 nm, or 300 nm. In some embodiments, the isolated recombinant polypeptide complex is characterized by a near UV circular dichroism peak at a wavelength between 270 nm and 275 nm, 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.

[0097] In some embodiments, the near UV circular dichroism peak is at a wavelength between 270 nm and 300 nm. In some embodiments, the near UV circular dichroism peak is at a wavelength between 275 nm and 290 nm.

[0098] metabolites

[0099] Disclosed herein are metabolites of the isolated recombinant polypeptide complex disclosed herein. In some embodiments, after administration, the isolated recombinant polypeptide complex is cleaved by a protease to generate an enzyme product of the isolated recombinant polypeptide complex. In some embodiments, after administration, the isolated recombinant polypeptide complex is cleaved by a tumor-specific protease to generate an enzyme product of the isolated recombinant polypeptide complex. 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 metabolite 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 metabolite 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 human membrane serine protease (MTSP1). In some embodiments, the matrix metalloprotease comprises human matrix metalloprotease 9 (MMP9).

[0100] In some embodiments, the enzyme product of the separated recombinant polypeptide includes a first metabolite. In some embodiments, the enzyme product of the separated recombinant polypeptide includes a second metabolite. In some embodiments, the enzyme product of the separated recombinant polypeptide includes a first metabolite and a second metabolite.

[0101] Disclosed herein is an isolated polypeptide that is an enzyme product of an isolated recombinant polypeptide complex disclosed herein. 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: 3, and wherein the isolated polypeptide is 221 amino acids in length. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3, and wherein the isolated polypeptide is 221 amino acids in length. In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 3.

[0102] 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: 4, and wherein the isolated polypeptide is 229 amino acids in length. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4, and wherein the isolated polypeptide is 229 amino acids in length. In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 4.

[0103] Disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 5, and wherein the isolated polypeptide is 484 amino acids in length. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 5, and wherein the isolated polypeptide is 484 amino acids in length. In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 5.

[0104] Disclosed herein is an isolated polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6, and wherein the isolated polypeptide is 492 amino acids in length. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 6, and wherein the isolated polypeptide is 492 amino acids in length. In some embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 6.

[0105] Disclosed herein is an isolated polypeptide comprising a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3, and a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: The amino acid sequence of NO:2 has a second amino acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity. In some embodiments, the isolated polypeptide comprises a first amino acid sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 and is 221 amino acids in length, and a first amino acid sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: NO:2 has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity and a second amino acid sequence of 653 amino acids in length. In some embodiments, the isolated polypeptide comprises a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:3 and 221 amino acids in length, and a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2 and 653 amino acids in length. In some embodiments, the isolated polypeptide comprises a first amino acid sequence having the amino acid sequence of SEQ ID NO:3, and a second amino acid sequence having the amino acid sequence of SEQ ID NO:2.

[0106] Disclosed herein is an isolated polypeptide comprising a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3, and a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: The amino acid sequence of NO:5 has a second amino acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical. In some embodiments, the isolated polypeptide comprises a first amino acid sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 and is 221 amino acids in length, and a first amino acid sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: NO:5 amino acid sequence has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity and a second amino acid sequence of 484 amino acids in length. In some embodiments, the isolated polypeptide comprises a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:3 and 221 amino acids in length, and a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:5 and 484 amino acids in length. In some embodiments, the isolated polypeptide comprises a first amino acid sequence having the amino acid sequence of SEQ ID NO:3, and a second amino acid sequence having the amino acid sequence of SEQ ID NO:5.

[0107] Disclosed herein is an isolated polypeptide comprising a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3, and a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: The amino acid sequence of NO:6 has a second amino acid sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity. In some embodiments, the isolated polypeptide comprises a first amino acid sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 and is 221 amino acids in length, and a first amino acid sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: NO:6 amino acid sequence has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity and a second amino acid sequence of 492 amino acids in length. In some embodiments, the isolated polypeptide comprises a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:3 and 221 amino acids in length, and a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:6 and 492 amino acids in length. In some embodiments, the isolated polypeptide comprises a first amino acid sequence having the amino acid sequence of SEQ ID NO:3, and a second amino acid sequence having the amino acid sequence of SEQ ID NO:6.

[0108] Disclosed herein is an isolated polypeptide comprising a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4, and a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4. The amino acid sequence of NO:2 has a second amino acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity. In some embodiments, the isolated polypeptide comprises a first amino acid sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 and is 229 amino acids in length, and a first amino acid sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 NO:2 has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity and a second amino acid sequence of 653 amino acids in length. In some embodiments, the isolated polypeptide comprises a first amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:4 and is 229 amino acids in length, and a second amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2 and is 653 amino acids in length. In some embodiments, the isolated polypeptide comprises a first amino acid sequence having the amino acid sequence of SEQ ID NO:4, and a second amino acid sequence having the amino acid sequence of SEQ ID NO:2.

[0109] Disclosed herein is an isolated polypeptide comprising a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4, and a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4. The amino acid sequence of NO:5 has a second amino acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical. In some embodiments, the isolated polypeptide comprises a first amino acid sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 and is 229 amino acids in length, and a first amino acid sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 NO:5 amino acid sequence has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity and a second amino acid sequence of 484 amino acids in length. In some embodiments, the isolated polypeptide comprises a first amino acid sequence of at least 90% sequence identity to the amino acid sequence of SEQ ID NO:4 and 229 amino acids in length, and a second amino acid sequence of at least 90% sequence identity to the amino acid sequence of SEQ ID NO:5 and 484 amino acids in length. In some embodiments, the isolated polypeptide comprises a first amino acid sequence having the amino acid sequence of SEQ ID NO:4, and a second amino acid sequence having the amino acid sequence of SEQ ID NO:5.

[0110] Disclosed herein is an isolated polypeptide comprising a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4, and a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4. The amino acid sequence of NO:6 has a second amino acid sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity. In some embodiments, the isolated polypeptide comprises a first amino acid sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 and is 229 amino acids in length, and a first amino acid sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 NO:6 amino acid sequence has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity and a second amino acid sequence of 492 amino acids in length. In some embodiments, the isolated polypeptide comprises a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:4 and 229 amino acids in length, and a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:6 and 492 amino acids in length. In some embodiments, the isolated polypeptide comprises a first amino acid sequence having the amino acid sequence of SEQ ID NO:4, and a second amino acid sequence having the amino acid sequence of SEQ ID NO:6.

[0111] Disclosed herein is an isolated polypeptide comprising a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1, and a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of NO:5 has a second amino acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical. In some embodiments, the isolated polypeptide comprises a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 and being 256 amino acids in length, and a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 NO:5 amino acid sequence has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity and a second amino acid sequence of 484 amino acids in length. In some embodiments, the isolated polypeptide comprises a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1 and 256 amino acids in length, and a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 5 and 484 amino acids in length. In some embodiments, the isolated polypeptide comprises a first amino acid sequence having the amino acid sequence of SEQ ID NO:1, and a second amino acid sequence having the amino acid sequence of SEQ ID NO:5.

[0112] Disclosed herein is an isolated polypeptide comprising a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1, and a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of NO:6 has a second amino acid sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity. In some embodiments, the isolated polypeptide comprises a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 and being 256 amino acids in length, and a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 NO:6 amino acid sequence has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity and a second amino acid sequence of 492 amino acids in length. In some embodiments, the isolated polypeptide comprises a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1 and 256 amino acids in length, and a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:6 and 492 amino acids in length. In some embodiments, the isolated polypeptide comprises a first amino acid sequence having the amino acid sequence of SEQ ID NO:1, and a second amino acid sequence having the amino acid sequence of SEQ ID NO:6.

[0113] Disclosed herein is a pharmaceutical composition comprising an isolated polypeptide disclosed herein and a pharmaceutically acceptable excipient disclosed herein.

[0114] Thermal stability

[0115] In some embodiments, the isolated recombinant polypeptide complex is characterized by a melting temperature (T) of about 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, or 80°C, or a range between any two of the above values. m In some embodiments, the isolated recombinant polypeptide complex is characterized by a T of about 69°C to about 76°C. m In some embodiments, the isolated recombinant polypeptide complex is characterized by a T of about 73°C to about 74°C. m In some embodiments, the isolated recombinant polypeptide complex is characterized by a melting temperature (T m In some embodiments, the isolated recombinant polypeptide complex is characterized by a melting onset temperature (T 起始 In some embodiments, the isolated recombinant polypeptide complex is characterized by a melting onset temperature (T 起始 In some embodiments, the isolated recombinant polypeptide complex is characterized by a melting onset temperature (T 起始 ). Melting temperature (T m ) can be determined by differential scanning calorimetry (DSC). 起始 ) can be determined by differential scanning calorimetry (DSC).

[0116] In some embodiments, the isolated recombinant polypeptide complex is characterized by one or more of the following: a first chain comprising the amino acid sequence according to SEQ ID NO: 1; a second chain comprising the amino acid sequence according to SEQ ID NO: 2; at least one N-glycan moiety comprises G2F, G2FS1, or G2FS2; the recombinant polypeptide complex comprises disulfide bonds formed by the following pairs of cysteine ​​residues: cysteine ​​4 and cysteine ​​15 of SEQ ID NO: 1, cysteine ​​65 and cysteine ​​130 of SEQ ID NO: 1, cysteine ​​176 and cysteine ​​236 of SEQ ID NO: 1, cysteine ​​256 of SEQ ID NO: 1 and cysteine ​​653 of SEQ ID NO: 2, cysteine ​​138 and cysteine ​​148 of SEQ ID NO: 2, cysteine ​​22 and cysteine ​​96 of SEQ ID NO: 2, cysteine ​​199 and cysteine ​​275 of SEQ ID NO: 2, cysteine ​​339 and cysteine ​​407 of SEQ ID NO: 2, cysteine ​​339 and cysteine ​​408 of SEQ ID NO: 2, cysteine ​​340 and cysteine ​​347 of SEQ ID NO: 2, cysteine ​​348 and cysteine ​​349 of SEQ ID NO: 2, cysteine ​​350 and cysteine ​​351 of SEQ ID NO: 2, cysteine ​​352 and cysteine ​​353 of SEQ ID NO: 2, cysteine ​​354 and cysteine ​​356 of SEQ ID NO: 1 NO:2 cysteine ​​453 and cysteine ​​526 and cysteine ​​577 and cysteine ​​633 of SEQ ID NO:2; when the isolated recombinant polypeptide complex is formulated at a concentration of about 1.0 mg / mL in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) PS20 pH about 5.3, T 起始 Between about 60°C and about 65°C and the transition midpoint temperature (T m1 ) is between about 70°C and about 75°C, wherein T 起始 and T m1 Differential scanning calorimetry (DSC) was used to measure the far-UV circular dichroism at wavelengths between 210 nm and 230 nm when the isolated recombinant polypeptide complex was formulated at a concentration of about 0.1 mg / mL in water, and the near-UV circular dichroism at wavelengths between 275 nm and 290 nm was depressed when the isolated recombinant polypeptide complex was formulated at a concentration of about 1.0 mg / mL in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) PS20, pH about 5.3.

[0117] In some embodiments, the isolated recombinant polypeptide complex is characterized by all of the following: wherein the first chain comprises the amino acid sequence according to SEQ ID NO: 1, and the second chain comprises the amino acid sequence according to SEQ ID NO: 2, and at least one N-glycan moiety comprises G2F, G2FS1, or G2FS2, and the recombinant polypeptide complex comprises disulfide bonds formed by the following pairs of cysteine ​​residues: cysteine ​​4 and cysteine ​​15 of SEQ ID NO: 1, cysteine ​​65 and cysteine ​​130 of SEQ ID NO: 1, cysteine ​​176 and cysteine ​​236 of SEQ ID NO: 1, cysteine ​​256 of SEQ ID NO: 1 and cysteine ​​653 of SEQ ID NO: 2, cysteine ​​138 and cysteine ​​148 of SEQ ID NO: 2, cysteine ​​22 and cysteine ​​96 of SEQ ID NO: 2, cysteine ​​199 and cysteine ​​275 of SEQ ID NO: 2, cysteine ​​339 and cysteine ​​407 of SEQ ID NO: 2, NO:2 cysteine ​​453 and cysteine ​​526 and cysteine ​​577 and cysteine ​​633 of SEQ ID NO:2, and when the isolated recombinant polypeptide complex is formulated at a concentration of about 1.0 mg / mL in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) PS20 pH about 5.3, T 起始 Between about 60°C and about 65°C and the transition midpoint temperature (T m1 ) is between about 70°C and about 75°C, wherein T 起始 and T m1 Measured using differential scanning calorimetry (DSC); and a dip in far-UV circular dichroism at wavelengths between 210 nm and 230 nm when the isolated recombinant polypeptide complex is formulated in water at a concentration of about 0.1 mg / mL; and a dip in near-UV circular dichroism at wavelengths between 275 nm and 290 nm when the isolated recombinant polypeptide complex is formulated in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) PS20, pH about 5.3, at a concentration of about 1.0 mg / mL.

[0118] Preparation of isolated recombinant antibodies targeting EGFR and CD3

[0119] The present disclosure includes formulations comprising a population of antibodies or recombinant antibodies, such as comprising any one or combination of the antibodies or recombinant antibodies described herein.

[0120] Disclosed herein is a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, wherein the one or more pharmaceutically acceptable excipients include histidine, sucrose, polysorbate-20, sodium phosphate, citrate, acetate, sodium chloride, potassium chloride, magnesium chloride, and calcium chloride. Disclosed herein is a pharmaceutical composition, wherein the pharmaceutical composition comprises (such as any described herein) separated recombinant polypeptide complex, sodium dihydrogen phosphate monohydrate, sodium hydrogen phosphate, citrate, acetate, histidine, 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 less than or equal to 6.5, 6.0, 5.5, 5.0, or 4.5. The pharmaceutical composition may have a pH greater than or equal to 4.5, 5.0, 5.5, 6.0, or 6.5. The pharmaceutical composition can have a pH between 4.0 and 5.0, between 4.5 and 5.5, or between 5.0 and 6.0. The pharmaceutical composition can comprise greater than or equal to 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM histidine. The pharmaceutical composition can comprise less than or equal to 20 mM, 19 mM, 18 mM, 17 mM, 16 mM, 15 mM, 14 mM, 13 mM, 12 mM, 11 mM, 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, or 1 mM histidine. The pharmaceutical composition may comprise greater than or equal to 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v) or 10% (w / v) sucrose. The pharmaceutical composition may comprise less than or equal to 10% (w / v), 9% (w / v), 8% (w / v), 7% (w / v), 6% (w / v) or 5% (w / v) sucrose. The pharmaceutical composition may comprise less than or equal to 0.05% (w / v) polysorbate 20, 0.04% (w / v) polysorbate 20, 0.03% (w / v) polysorbate 20, 0.02% (w / v) polysorbate 20 or 0.01% polysorbate 20. The pharmaceutical composition may comprise greater than or equal to 0.01% (w / v) polysorbate 20, 0.02% (w / v) polysorbate 20, 0.03% (w / v) polysorbate 20, 0.04% (w / v) polysorbate 20, or 0.05% polysorbate 20. Disclosed herein is a pharmaceutical composition comprising an isolated recombinant polypeptide complex (such as any described herein), about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) polysorbate 20, and a pH of about 5.3.

[0121] In some embodiments of the formulation, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% (e.g., by mole or by mass) of the antibody population is monomeric. In some embodiments of the formulation, less than or equal to 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% (e.g., by mole or by mass) of the antibody population is aggregated.

[0122] Disclosed herein are multiple isolated recombinant antibodies targeting EGFR and CD3, wherein the isolated recombinant antibodies comprise a light chain (LC) amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1, a heavy chain (HC) amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2, wherein multiple comprise greater than 90% of a monomer of the isolated recombinant antibody.

[0123] In some embodiments, a plurality of isolated recombinant antibodies comprise greater than 91% monomers. In some embodiments, a plurality of isolated recombinant antibodies comprise greater than 92% monomers. In some embodiments, a plurality of isolated recombinant antibodies comprise greater than 93% monomers. In some embodiments, a plurality of isolated recombinant antibodies comprise greater than 94% monomers. In some embodiments, a plurality of isolated recombinant antibodies comprise greater than 95% monomers. In some embodiments, a plurality of isolated recombinant antibodies comprise greater than 96% monomers. In some embodiments, a plurality of isolated recombinant antibodies comprise greater than 97% monomers. In some embodiments, a plurality of isolated recombinant antibodies comprise greater than 98% monomers. In some embodiments, a plurality of isolated recombinant antibodies comprise greater than 99% monomers.

[0124] In some embodiments, the LC comprises at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises the amino acid sequence according to SEQ ID NO: 1. In some embodiments, the HC comprises at least 85% sequence identity to SEQ ID NO: 2. In some embodiments, the HC comprises at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the HC comprises at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, the HC comprises at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the HC comprises the amino acid sequence according to SEQ ID NO: 2.

[0125] In some embodiments, the LC comprises at least 99% sequence identity to SEQ ID NO: 1 and the HC comprises at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the LC comprises the amino acid sequence according to SEQ ID NO: 1 and the HC comprises the amino acid sequence according to SEQ ID NO: 2.

[0126] In some embodiments, the concentration of the isolated recombinant antibody is greater than or equal to 1.0 mg / mL. In some embodiments, the concentration of the isolated recombinant antibody is at least 2.0 mg / mL. In some embodiments, the concentration of the isolated recombinant antibody is at least 5.0 mg / mL. In some embodiments, the concentration of the isolated recombinant antibody is at least 10.0 mg / mL. In some embodiments, the concentration of the isolated recombinant antibody is at least 15.0 mg / mL. In some embodiments, the concentration of the isolated recombinant antibody is at least 20.0 mg / mL. In some embodiments, the plurality of isolated recombinant antibodies are in a buffer solution having a pH less than or equal to 5.5. In some embodiments, the buffer solution comprises one or more of acetate, phosphate, or histidine. In some embodiments, the buffer solution comprises histidine at a concentration greater than 5 mM. In some embodiments, the buffer solution comprises sucrose. In some embodiments, the concentration of sucrose is greater than or equal to 5% w / v. In some embodiments, the buffer solution comprises polysorbate 20. In some embodiments, the concentration of polysorbate 20 is greater than or equal to 0.01% w / v. In some embodiments, the plurality of isolated recombinant antibodies comprises greater than 90% monomer at a concentration greater than or equal to about 20.0 mg / mL in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) PS20 pH about 5.3.

[0127] Disclosed herein is a pharmaceutical composition comprising an isolated recombinant polypeptide complex disclosed herein and a pharmaceutically acceptable excipient. In some embodiments, the isolated recombinant polypeptide complex comprises a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the isolated recombinant polypeptide complex comprises a second amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NO:2. In some embodiments, the isolated recombinant polypeptide complex comprises a first amino acid sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NO: 1, and a second amino acid sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the isolated recombinant polypeptide complex comprises a first amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1 and a second amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2. In some embodiments, the isolated recombinant polypeptide complex comprises a first amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 and a second amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the isolated recombinant polypeptide complex comprises a first amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the isolated recombinant polypeptide complex comprises a second amino acid sequence having at least 90% sequence identity to SEQ ID NO:2.In some embodiments, the isolated recombinant polypeptide complex comprises a first amino acid sequence having the amino acid sequence of SEQ ID NO: 1, and a second amino acid sequence having the amino acid sequence of SEQ ID NO: 2. In some embodiments, the isolated recombinant polypeptide complex comprises a first amino acid sequence having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the isolated recombinant polypeptide complex comprises a second amino acid sequence having the amino acid sequence of SEQ ID NO: 2.

[0128] In some embodiments, the pharmaceutical composition comprises an isolated recombinant polypeptide complex at a concentration of about 2 mg / ml. In some embodiments, the pharmaceutically acceptable excipient comprises a buffer, a stabilizer, a tonicity agent, a surfactant, or a combination thereof. In some embodiments, the pharmaceutically acceptable excipient comprises a buffer. In some embodiments, the pharmaceutically acceptable excipient comprises a tonicity agent. In some embodiments, the pharmaceutically acceptable excipient comprises a surfactant. In some embodiments, the pharmaceutically acceptable excipient comprises a buffer, a stabilizer, a tonicity agent, and a surfactant. 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 a polysorbate. In some embodiments, the surfactant comprises polysorbate 20 (PS20).

[0129] In some embodiments, the pharmaceutical composition comprises from about 1 millimolar (mM) to about 50 mM L-histidine in the form of L-histidine and / or L-histidine monohydrochloride monohydrate, for example, about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 47mM, 48mM, 49mM or about 50mM or any concentration therebetween. In some embodiments, the pharmaceutical composition comprises about 1 to about 50 mM L-histidine in the form of L-histidine and L-histidine monohydrochloride monohydrate, for example, about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, In some embodiments, the pharmaceutical composition comprises about 10 mM of L-histidine in the form of L-histidine and / or L-histidine monohydrochloride monohydrate. In some embodiments, the pharmaceutical composition comprises about 10 mM of L-histidine in the form of L-histidine or L-histidine monohydrochloride monohydrate. In some embodiments, the pharmaceutical composition comprises about 10 mM L-histidine in the form of L-histidine and L-histidine monohydrochloride monohydrate.

[0130] In some embodiments, the pharmaceutical composition comprises about 1% weight / volume (w / v) to about 20% (w / v) sucrose, for example, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or about 20%, or any concentration therebetween. In some embodiments, the pharmaceutical composition comprises about 8% (w / v) sucrose.

[0131] In some embodiments, the pharmaceutical composition comprises about 0.001% (w / v) to about 0.1% (w / v) polysorbate 20 (PS20), for example, about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.011%, 0.012%, 0.013%, 0.014%, 0.015%, 0.016%, 0.017%, 0.018%, 0.019% , 0.02%, 0.021%, 0.022%, 0.023%, 0.024%, 0.025%, 0.026%, 0.027%, 0.028%, 0.029%, 0.03%, 0.031%, 0.032%, 0.033%, 0.034%, 0.035%, 0.036%, 0.037%, 0.038%, 0.039%, 0.04%, 0.041%, 0.042%, 0.043%, 0.044%, 0.045%, 0.046%, 0. 0.047%, 0.048%, 0.049%, 0.05%, 0.051%, 0.052%, 0.053%, 0.054%, 0.055%, 0.056%, 0.057%, 0.058%, 0.059%, 0.06%, 0.061%, 0.062%, 0.063%, 0.064%, 0.065%, 0.066%, 0.067%, 0.068%, 0.069%, 0.07%, 0.071%, 0.072%, 0.073%, 0.074 In some embodiments, the pharmaceutical composition comprises about 0.01% (w / v) polysorbate 20 (PS20).

[0132] In some embodiments, the pharmaceutical composition comprises about 10 mM L-histidine in the form of L-histidine and L-histidine monohydrochloride monohydrate, about 8% (w / v) sucrose, and about 0.01% (w / v) polysorbate 20. In some embodiments, the pharmaceutical composition comprises a pH of about 5.3. In some embodiments, the pharmaceutical composition comprises an osmotic pressure of about 276 mOsmol / kg.

[0133] Disclosed herein is a pharmaceutical composition comprising an isolated polypeptide disclosed herein and a pharmaceutically acceptable excipient disclosed herein. In some embodiments, the isolated polypeptide is an enzyme product of an isolated recombinant polypeptide complex disclosed herein.

[0134] Drug delivery and pharmacology

[0135] In some embodiments, the isolated recombinant polypeptide complex disclosed herein provides a maximum plasma concentration (Cmax) in a subject following a single intravenous bolus administration of a dose of about 0.1 milligrams per kilogram of body weight (mg / kg) to about 1 mg / kg, e.g., about 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, or about 1 mg / kg, or any dose therebetween. In some embodiments, Cmax increases as the dose increases. In some embodiments, the increase in Cmax is proportional to the increase in dose. In some embodiments, the increase in Cmax is greater than a value proportional to the increase in dose. In some embodiments, the area under the drug concentration-time curve (AUC) between 0 hours (h) and 216 hours after administration is greater than a value proportional to the increase in dose. 0-216h ) is greater than a value proportional to the increase in dose. In some embodiments, AUC 0-216h In some embodiments, the subject has a maximum Cmax within 24 hours after administration. In some embodiments, the subject has a maximum AUC at 24 hours after administration. 0-24h .

[0136] In some embodiments, the dosage is about 0.1 mg / kg. In some embodiments, the dosage is about 0.3 mg / kg. In some embodiments, the dosage is about 1 mg / kg. In some embodiments, the dosage is about 0.3 mg / kg to about 1 mg / kg. In some embodiments, the dosage is about 0.1 mg / kg to about 0.3 mg / kg. In some embodiments, the dosage is about 0.1 mg / kg to about 0.3 mg / kg. In some embodiments, the dosage is about 0.3 mg / kg to about 1 mg / kg.

[0137] In some embodiments, the isolated recombinant polypeptide complex is expressed in a subject at about 88.8 h to about 101 h, e.g., about 88.8 h, 88.9 h, 89 h, 89.1 h, 89.2 h, 89.3 h, 89.4 h, 89.5 h, 89.6 h, 89.7 h, 89.8 h, 89.9 h, 90 h, 90.1 h, 90.2 h, 90.3 h, 90.4 h, 90.5 h, 90.6 h, 90.7 h, 90.8 h, 90.9 h, 91 h, 91.1 h, 91.2 h, 91.3 h, 91.4 h , 91.5h, 91.6h, 91.7h, 91.8h, 91.9h, 92h, 92.1h, 92.2h, 92.3h, 92.4h, 92.5h, 92.6h, 92.7h, 92.8h, 92.9h, 93h, 93.1h , 93.2h, 93.3h, 93.4h, 93.5h, 93.6h, 93.7h, 93.8h, 93.9h, 94h, 94.1h, 94.2h, 94.3h, 94.4h, 94.5h, 94.6h, 94.7h, 94. 8h, 94.9h, 95h, 95.1h, 95.2h, 95.3h, 95.4h, 95.5h, 95.6h, 95.7h, 95.8h, 95.9h, 96h, 96.1h, 96.2h, 96.3h, 96.4h, 96 .5h, 96.6h, 96.7h, 96.8h, 96.9h, 97h, 97.1h, 97.2h, 97.3h, 97.4h, 97.5h, 97.6h, 97.7h, 97.8h, 97.9h, 98h, 98.1h, 98 .2h, 98.3h, 98.4h, 98.5h, 98.6h, 98.7h, 98.8h, 98.9h, 99h, 99.1h, 99.2h, 99.3h, 99.4h, 99.5h, 99.6h, 99.7h, 99.8h, 99.9h, 100h, 100.1h, 100.2h, 100.3h, 100.4h, 100.5h, 100.6h, 100.7h, 100.8h, 100.9h or about 101h or any time therebetween to provide a half-maximal plasma concentration (T 1 / 2 ).

[0138] In some embodiments, the isolated recombinant polypeptide complex is administered to a subject by a single intravenous infusion over about 30 minutes (min). In some embodiments, the increase in Cmax is proportional to the increase in dose. In some embodiments, when the isolated recombinant polypeptide complex is administered to a subject by a single intravenous infusion over about 30 min, the increase in Cmax is proportional to the increase in dose. In some embodiments, the area under the curve (AUC) between 0 h and 168 h after administration is greater than or equal to 0. 0-168h) increases less than a value proportional to the increase in dose. In some embodiments, when the isolated recombinant polypeptide complex is administered to a subject by a single intravenous infusion over about 30 minutes, the area under the curve (AUC) between 0 hours and 168 hours after administration is less than a value proportional to the increase in dose. 0-168h ) increases less than a value proportional to the increase in dose. In some embodiments, when the isolated recombinant polypeptide complex is administered to a subject by a single intravenous infusion over about 30 minutes, the AUC 0-168The increase in h is proportional to the increase in dose. In some embodiments, the dose is about 0.05 mg / kg. In some embodiments, the dose is about 0.2 mg / kg. In some embodiments, the dose is about 0.6 mg / kg. In some embodiments, the dose is about 0.05 mg / kg to about 0.2 mg / kg. In some embodiments, the dosage is from about 0.2 mg / kg to about 0.6 mg / kg, for example, about 0.21 mg / kg, 0.22 mg / kg, 0.23 mg / kg, 0.24 mg / kg, 0.25 mg / kg, 0.26 mg / kg, 0.27 mg / kg, 0.28 mg / kg, 0.29 mg / kg, 0.3 mg / kg, 0.31 mg / kg, 0.32 mg / kg, 0.33 mg / kg, 0.34 mg / kg, 0.35 mg / kg, 0.36 mg / kg, 0.37 mg / kg, 0.38 mg / kg, 0.39 mg / kg. g / kg, 0.4 mg / kg, 0.41 mg / kg, 0.42 mg / kg, 0.43 mg / kg, 0.44 mg / kg, 0.45 mg / kg, 0.46 mg / kg, 0.47 mg / kg, 0.48 mg / kg, 0.49 mg / kg, 0.5 mg / kg, 0.51 mg / kg, 0.52 mg / kg, 0.53 mg / kg, 0.54 mg / kg, 0.55 mg / kg, 0.56 mg / kg, 0.57 mg / kg, 0.58 mg / kg, 0.59 mg / kg or about 0.6 mg / kg, or any dose therebetween. In some embodiments, the isolated recombinant polypeptide complex is provided at about 68.2h to about 96.5h, e.g., about 68.2h, 68.3h, 68.4h, 68.5h, 68.6h, 68.7h, 68.8h, 68.9h, 69h, 69.1h, 69.2h, 69.3h, 69.4h, 69.5h, 69.6h, 69.7h, 69.8h, 69.9h, 70h, 70.1h, 70.2h, 70.3h, 70.4h, 70.5h, 70.6h, 70.7h, 70.8h, 70.9h, 71h, 71.1h, 71.2h, 71.3h, 71.4h, 71.5h, 71.6h, 71.7h, 71.8h, 71.9h, 71.1h, 71.2h, 71. .3h, 71.4h, 71.5h, 71.6h, 71.7h, 71.8h, 71.9h, 72h, 72.1h, 72.2h, 72.3h, 72.4h, 72.5h, 72.6h, 72.7h, 72.8h, 72.9h, 73h, 73.1h, 73 .2h, 73.3h, 73.4h, 73.5h, 73.6h, 73.7h, 73.8h, 73.9h, 74h, 74.1h, 74.2h, 74.3h, 74.4h, 74.5h, 74.6h, 74.7h, 74.8h, 74.9h, 75h, 75.<h2 style=";text-align:left;direction:ltr">1h, 75.2h, 75.3h, 75.4h, 75.5h, 75.6h, 75.7h, 75.8h, 75.9h, 76h, 76.1h, 76.2h, 76.3h, 76.4h, 76.5h, 76.6h, 76.7h, 76.8h, 76.9h, 77h, 77.1h, 77.2h 77.3h, 77.4h, 77.5h, 77.6h, 77.7h, 77.8h, 77.9h, 78h, 78.1h, 78.2h, 78.3h, 78.4h, 78.5h, 78.6h, 78.7h, 78.8h, 78.9h, 79h, 79.1h, 79.2h, 79.3h, 79 4h, 79.5h, 79.6h, 79.7h, 79.8h, 79.9h, 80h, 80.1h, 80.2h, 80.3h, 80.4h, 80.5h, 80.6h, 80.7h, 80.8h, 80.9h, 81h, 81.1h, 81.2h, 81.3h, 81.4h, 81.5h 81.6h, 81.7h, 81.8h, 81.9h, 82h, 82.1h, 82.2h, 82.3h, 82.4h, 82.5h, 82.6h, 82.7h, 82.8h, 82.9h, 83h, 83.1h, 83.2h, 83.3h, 83.4h, 83.5h, 83.6h, 83 .7h, 83.8h, 83.9h, 84h, 84.1h, 84.2h, 84.3h, 84.4h, 84.5h, 84.6h, 84.7h, 84.8h, 84.9h, 85h, 85.1h, 85.2h, 85.3h, 85.4h, 85.5h, 85.6h, 85.7h, 85.8 h, 85.9h, 86h, 86.1h, 86.2h, 86.3h, 86.4h, 86.5h, 86.6h, 86.7h, 86.8h, 86.9h, 87h, 87.1h, 87.2h, 87.3h, 87.4h, 87.5h, 87.6h, 87.7h, 87.8h, 87.9h, 8 8h, 88.1h, 88.2h, 88.3h, 88.4h, 88.5h, 88.6h, 88.7h, 88.8h, 88.9h, 89h, 89.1h, 89.2h, 89.3h, 89.4h, 89.5h, 89.6h, 89.7h, 89.8h, 89.9h, 90h, 90.1h 90.2h, 90.3h, 90.4h, 90.5h, 90.6h, 90.7h, 90.8h, 90.9h, 91h, 91.1h, 91.2h, 91.3h, 91.4h, 91.5h, 91.6h, 91.7h, 91.8h, 91.9h, 92h, 92.1h, 92.2h, 92.3h, 92.4h, 92.5h, 92.6h, 92.7h, 92.8h, 92.9h, 93h, 93.1h, 93.2h, 93.3h, 93.4 h, 93.5h, 93.6h, 93.7h, 93.8h, 93.9h, 94h, 94.1h, 94.2h, 94.3h, 94.4h, 94.5h , 94.6h, 94.7h, 94.8h, 94.9h, 95h, 95.1h, 95.2h, 95.3h, 95.4h, 95.5h, 95.6h, 95.7h, 95.8h, 95.9h, 96h, 96.1h, 96.2h, 96.3h, 96.4h or about 96.5h or any duration therebetween. 1 / 2 In some embodiments, the isolated recombinant polypeptide complex provides a T of about 81.3 h at a dose of about 0.05 mg / kg. 1 / 2 In some embodiments, the isolated recombinant polypeptide complex provides a T of about 68.2 h at a dose of about 0.2 mg / kg administered. 1 / 2 In some embodiments, the isolated recombinant polypeptide complex provides a T of about 96.5 h at a dose of about 0.6 mg / kg. 1 / 2 .

[0139] In some embodiments, the isolated recombinant polypeptide complex is administered at least once a week. In some embodiments, the isolated recombinant polypeptide complex is administered once a week. In some embodiments, the isolated recombinant polypeptide complex is administered at least twice a week. In some embodiments, the isolated recombinant polypeptide complex is administered for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks. In some embodiments, the isolated recombinant polypeptide complex is administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. In some embodiments, the isolated recombinant polypeptide complex is administered for at least 1 year, 2 years, 3 years, 4 years, or 5 years. In some embodiments, the isolated recombinant polypeptide complex is administered for at most 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks. In some embodiments, the isolated recombinant polypeptide complex is administered for up to 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. In some embodiments, the isolated recombinant polypeptide complex is administered for up to 1 year, 2 years, 3 years, 4 years, or 5 years. In some embodiments, the increase in Cmax is proportional to the increase in dose after day 1, where day 1 is the day of administration. In some embodiments, AUC 0-24 h increases proportionally with increasing dose after day 1, where day 1 is the day of administration. In some embodiments, AUC0-168h The increase is proportional to the increase in dose after day 1, where day 1 is the day of administration. In some embodiments, the dose is about 0.05 mg / kg. In some embodiments, the dose is about 0.2 mg / kg. In some embodiments, the dose is about 0.6 mg / kg. In some embodiments, the isolated recombinant polypeptide complex is not metabolized by cytochrome P450 (CYP) enzymes. In some embodiments, the isolated recombinant polypeptide complex is not transported by P-glycoprotein (Pgp) or related adenosine triphosphate binding cassette membrane transporters. In some embodiments, the isolated recombinant polypeptide complex causes the release of cytokines in the subject. In some embodiments, the cytokine is interleukin 6 (IL-6), interleukin 10 (IL-10), interferon gamma (IFNγ), tumor necrosis factor (TNF), or a combination thereof. In some embodiments, cytokine release is associated with dose. In some embodiments, IL-10 release occurs at a dose of not less than 0.2 mg / kg. In some embodiments, IL-6 release occurs at a dose of not less than 0.6 mg / kg. In some embodiments, IFNy release occurs at a dose of no less than 0.6 mg / kg.

[0140] In some embodiments, the isolated recombinant polypeptide complex is administered at a single dose of about 0.05 mg / kg to about 1 mg / kg, for example, about 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.11 mg / kg, 0.12 mg / kg, 0.13 mg / kg, 0.14 mg / kg, 0.15 mg / kg, 0.16 mg / kg, 0.17 mg / kg, 0.18 mg / kg, 0.19 mg / kg, 0.2 mg / kg, 0.21 mg / kg, 0.22 mg / kg, 0.23 mg / kg, 0.24 mg / kg. g、0.25mg / kg、0.26mg / kg、0.27mg / kg、0.28mg / kg、0.29mg / kg、0.3mg / kg、0.31mg / kg、0.32mg / kg、0.33mg / kg、0.34mg / kg、0.35mg / kg、0.36mg / kg、0. 37mg / kg, 0.38mg / kg, 0.39mg / kg, 0.4mg / kg, 0.41mg / kg, 0.42mg / kg, 0.43mg / kg, 0.44mg / kg, 0.45mg / kg, 0.46mg / kg, 0.47mg / kg, 0.48mg / kg, 0.49mg / kg, 0.5mg / kg, 0.51mg / kg, 0.52mg / kg, 0.53mg / kg, 0.54mg / kg, 0.55mg / kg, 0.56mg / kg, 0.57mg / kg, 0.58mg / kg, 0.59mg / kg, 0.6mg / kg, 0.61mg / kg, 0.62mg / kg, 0.63mg / kg, 0.64mg / kg, 0.65mg / kg, 0.66mg / kg, 0.67mg / kg, 0.68mg / kg, 0.69mg / kg, 0.7mg / kg, 0.71mg / kg, 0.72mg / kg, 0.73mg / kg, 0.74 mg / kg, 0.75mg / kg, 0.76mg / kg, 0.77mg / kg, 0.78mg / kg, 0.79mg / kg, 0.8mg / kg, 0.81mg / kg, 0.82mg / kg, 0.83mg / kg, 0.84mg / kg, 0.85mg / kg, 0.86mg / k g、0.87mg / kg、0.88mg / kg、0.89mg / kg、0.9mg / kg、0.91mg / kg、0.92mg / kg、0.93mg / kg、0.94mg / kg、0.95mg / kg、0.96mg / kg、0.97mg / kg、0.98mg / kg、0.99 mg / kg or about 1 mg / kg, or any dose therebetween, providing a T of about 79 h to about 101 h, for example, about 79 h, 80 h, 81 h, 82 h, 83 h, 84 h, 85 h, 86 h, 87 h, 88 h, 89 h, 90 h, 91 h, 92 h, 93 h, 94 h, 95 h, 96 h, 97 h, 98 h, 99 h, 100 h or about 101 h, or any time therebetween. 1 / 2 In some embodiments, the isolated recombinant polypeptide complex provides a T of about 79 h to about 101 h after administration of a single dose of about 0.05 mg / kg to about 1 mg / kg. 1 / 2 .

[0141] In some embodiments, the isolated recombinant polypeptide complex is administered to a subject by a single intravenous bolus administration. In some embodiments, the isolated recombinant polypeptide complex provides a Cmax, wherein the increase in Cmax is proportional to the increase in dosage. In some embodiments, the isolated recombinant polypeptide complex provides an AUC, wherein the increase in AUC is proportional to the increase in dosage. In some embodiments, the isolated recombinant polypeptide complex is administered for at least 4 weeks. In some embodiments, the isolated recombinant polypeptide complex provides a no observed adverse effect level (NOAEL) at about 0.6 mg / kg. In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 16100 ng / ml at a dose of about 0.6 mg / kg on day 1, wherein day 1 is the day of administration. In some embodiments, the isolated recombinant polypeptide complex provides an AUC of about 775,000 hr*ng / ml when a dose of about 0.6 mg / kg is administered on day 1. 0-168h . In some embodiments, the dose is about 0.05 mg / kg, about 0.2 mg / kg, or about 0.6 mg / kg. In some embodiments, the dose is about 0.05 mg / kg. In some embodiments, the dose is about 0.2 mg / kg. In some embodiments, the dose is about 0.6 mg / kg. In some embodiments, the dose is administered on day 1, day 8, day 15, day 22, and day 29. In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 1870 ng / ml when administered at a dose of about 0.05 mg / kg on day 1. In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 6180 ng / ml when administered at a dose of about 0.2 mg / kg on day 1. In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 16100 ng / ml when administered at a dose of about 0.6 mg / kg on day 1. In some embodiments, the isolated recombinant polypeptide complex provides an AUC of about 106,000 hr*ng / ml when administered at a dose of about 0.05 mg / kg on day 1. 0-168hIn some embodiments, the isolated recombinant polypeptide complex provides an AUC of about 325,000 hr*ng / ml when administered at a dose of about 0.2 mg / kg on day 1. 0-168h In some embodiments, the isolated recombinant polypeptide complex provides an AUC of about 775,000 hr*ng / ml when administered at a dose of about 0.6 mg / kg on day 1. 0- 168h.

[0142] In some embodiments, the isolated recombinant polypeptide complex provides an AUC at a dose administered on day 22 that is similar or identical to the AUC at the same dose administered on day 1, wherein the subject exhibits no detectable levels of anti-drug antibodies after administration on day 22. In some embodiments, the isolated recombinant polypeptide complex provides an AUC at a dose administered on day 29 that is similar or identical to the AUC at the same dose administered on day 1, wherein the subject exhibits no detectable levels of anti-drug antibodies after administration on day 29. In some embodiments, the AUC is AUC 0-24h or AUC 0-168h In some embodiments, the isolated recombinant polypeptide complex, at a dose administered on day 22, provides a Cmax that is similar or identical to the Cmax of the same dose administered on day 1, wherein the subject exhibits no detectable levels of anti-drug antibodies after administration on day 22. In some embodiments, the isolated recombinant polypeptide complex, at a dose administered on day 29, provides a Cmax that is similar or identical to the Cmax of the same dose administered on day 1, wherein the subject exhibits no detectable levels of anti-drug antibodies after administration on day 29.

[0143] In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 1620 ng / ml following a single intravenous bolus administration of about 0.1 mg / kg. In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 8150 ng / ml following a single intravenous bolus administration of about 0.3 mg / kg. In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 27200 ng / ml following a single intravenous bolus administration of about 1 mg / kg. In some embodiments, the isolated recombinant polypeptide complex provides a Tmax of about 88.8 h following a single intravenous bolus administration of about 0.1 mg / kg. 1 / 2 In some embodiments, the isolated recombinant polypeptide complex provides a T of about 101 h following a single intravenous bolus administration of a dose of about 0.3 mg / kg. 1 / 2 In some embodiments, the isolated recombinant polypeptide complex provides a T of about 79 h following a single intravenous bolus administration of a dose of about 1 mg / kg. 1 / 2 .

[0144] In some embodiments, the isolated recombinant polypeptide complex provides a clearance rate of about 2.5 ml / h to about 4.47 ml / h following a single intravenous bolus administration, e.g., about 2.5 ml / h, 2.51 ml / h, 2.52 ml / h, 2.53 ml / h, 2.54 ml / h, 2.55 ml / h, 2.56 ml / h, 2.57 ml / h, 2.58 ml / h, 2.59 ml / h, 2.6 ml / h, 2.61 ml / h, 2.62 ml / h, 2.63 ml / h, 2.64 ml / h, 2.65 ml / h, 2.66 ml / h, 2.67 ml / h, 2.68 ml / h, 2.69 ml / h, 2.70 ml / h, 2.71 ml / h, 2.72 ml / h, 2.73 ml / h, 2.74 ml / h, 2.75 ml / h, 2.76 ml / h, 2.77 ml / h, 2.78 ml / h, 2.79 ml / h, 2.80 ml / h, 2.81 ml / h, 2.82 ml / h, 2.83 ml / h, 2.84 ml / h 71ml / h, 2.72ml / h, 2.73ml / h, 2.74ml / h, 2.75ml / h, 2.76ml / h, 2.77ml / h, 2.78ml / h, 2.79ml / h, 2.8ml / h, 2.81ml / h, 2.82ml / h, 2.83ml / h, 2.84ml / h, 2 .85ml / h, 2.86ml / h, 2.87ml / h, 2.88ml / h, 2.89ml / h, 2.9ml / h, 2.91ml / h, 2.92ml / h, 2.93ml / h, 2.94ml / h, 2.95ml / h, 2.96ml / h, 2.97ml / h, 2.98ml / h, 2 .99ml / h, 3ml / h, 3.01ml / h, 3.02ml / h, 3.03ml / h, 3.04ml / h, 3.05ml / h, 3.06ml / h, 3.07ml / h, 3.08ml / h, 3.09ml / h, 3.1ml / h, 3.11ml / h, 3.12ml / h, 3.1 3ml / h, 3.14ml / h, 3.15ml / h, 3.16ml / h, 3.17ml / h, 3.18ml / h, 3.19ml / h, 3.2ml / h, 3.21ml / h, 3.22ml / h, 3.23ml / h, 3.24ml / h, 3.25ml / h, 3.26ml / h, 3. 27ml / h, 3.28ml / h, 3.29ml / h, 3.3ml / h, 3.31ml / h, 3.32ml / h, 3.33ml / h, 3.34ml / h, 3.35ml / h, 3.36ml / h, 3.37ml / h, 3.38ml / h, 3.39ml / h, 3.4ml / h, 3. 41ml / h, 3.42ml / h, 3.43ml / h, 3.44ml / h, 3.45ml / h, 3.46ml / h, 3.47ml / h, 3.48ml / h, 3.49ml / h, 3.5ml / h, 3.51ml / h, 3.52ml / h, 3.53ml / h, 3.54ml / h, 3.55 ml / h, 3.56 ml / h, 3.57 ml / h, 3.58 ml / h, 3.59 ml / h, 3.6 ml / h, 3.61 ml / h, 3.62 ml / h, 3.63 ml / h, 3.64 ml / h, 3.65 ml / h, 3.66 ml / h, 3.67 ml / h, 3.68 ml / h, 3.69 ml / h, 3.7 ml / h, 3.71 ml / h, 3.72 ml / h, 3.73 ml / h, 3.74 ml / h, 3.75 ml / h, 3.76 ml / h, 3.77 ml / h, 3.78 ml / h, 3.79 ml / h, 3.8 ml / h, 3.81 ml / h, 3.82 ml / h, 3.83 ml / h, 3.84 ml / h, 3.85 ml / h, 3.86 ml / h, 3.87 ml / h, 3.88 ml / h, 3.89 ml / h, 3.9 ml / h, 3.91 ml / h, 3.92 ml / h, 3.93 ml / h, 3.94 ml / h, 3.95 ml / h, 3.96 ml / h, 3.97 ml / h, 3.98 ml / h, 3.99 ml / h, 4 ml / h, 4.01 ml / h, 4.02 ml / h, 4.03 ml / h, 4.04 ml / h, 4.05 ml / h, 4.06 ml / h, 4.07 ml / h, 4.08 ml / h, 4.09 ml / h, 4.1 ml / h, 4.11 ml / h, 4.12 ml / h, 4.13 ml / h, 4.14 ml / h, 4.15 ml / h, 4.16 ml / h, 4.17 ml / h, 4.18 ml / h, 4.19 ml / h, 4.2 ml / h, 4.21 ml / h, 4.22 ml / h, 4.23 ml / h, 4.24 ml / h, 4.25 ml / h, 4.26 ml / h, 4.27 ml / h, 4.28 ml / h, 4.29 ml / h, 4.3 ml / h, 4.31 ml / h, 4.32 ml / h, 4.33 ml / h, 4.34 ml / h, 4.35 ml / h, 4.36 ml / h, 4.37 ml / h, 4.38 ml / h, 4.39 ml / h, 4.4 ml / h, 4.41 ml / h, 4.42 ml / h, 4.43 ml / h, 4.44 ml / h, 4.45 ml / h, 4.46 ml / h, or about 4.47 ml / h, or any rate therebetween.

[0145] In some embodiments, the isolated recombinant polypeptide complex provides a volume of distribution of about 372 ml to about 576 ml following a single intravenous bolus administration, for example, about 372 ml, 373 ml, 374 ml, 375 ml, 376 ml, 377 ml, 378 ml, 379 ml, 380 ml, 381 ml, 382 ml, 383 ml, 384 ml, 385 ml, 386 ml, 387 ml, 388 ml, 389 ml, 390 ml, 391 ml, 392 ml, 393 ml, 394 ml, 395 ml, 396 ml, 397 ml, 398 ml, 399 ml, 400 ml, 401 ml, 402 ml, 403 ml, 404 ml, 405 ml, 406 ml, 407 ml, 408 ml, 409 ml, 410 ml, 411 ml, 412 ml, 413 ml, 414 ml, 415 ml, 416 ml, 417 ml, 418 ml, 419 ml, 420 ml, 421 ml, 422 ml, 423 ml, 424 ml, 425 ml, 426 ml, 427 ml, 428 ml, 04ml, 405ml, 406ml, 407ml, 408ml, 409ml, 410ml, 411ml, 412ml, 413ml, 414ml, 415ml, 416ml, 417ml, 418ml, 419ml, 420ml, 421ml, 422ml, 423ml, 424ml, 425ml, 426ml, 427ml, 428ml, 429ml, 430ml, 431ml, 432ml, 433ml, 434ml, 435ml, 436ml, 437ml, 438ml, 439ml, 440ml, 441ml, 442ml, 443ml, 444ml, 445ml , 446ml, 447ml, 448ml, 449ml, 450ml, 451ml, 452ml, 453ml, 454ml, 455ml, 456ml, 457ml, 458ml, 459ml, 460ml, 461ml, 462ml, 463ml, 464ml, 465ml, 466 ml, 467ml, 468ml, 469ml, 470ml, 471ml, 472ml, 473ml, 474ml, 475ml, 476ml, 477ml, 478ml, 479ml, 480ml, 481ml, 482ml, 483ml, 484ml, 485ml, 486ml, 48 7ml, 488ml, 489ml, 490ml, 491ml, 492ml, 493ml, 494ml, 495ml, 496ml, 497ml, 498ml, 499ml, 500ml, 501ml, 502ml, 503ml, 504ml, 505ml, 506ml, 507ml, 5 08ml, 509ml, 510ml, 511ml, 512ml, 513ml, 514ml, 515ml, 516ml, 517ml, 518ml, 519ml, 520ml, 521ml, 522ml, 523ml, 524ml, 525ml, 526ml, 527ml, 528ml,529ml, 530ml, 531ml, 532ml, 533ml, 534ml, 535ml, 536ml, 537ml, 538ml, 539ml, 540ml, 541ml, 542ml, 543ml, 544ml, 545ml, 546ml, 547ml, 548ml, 549ml, 550ml, 551ml, 552ml, 553ml, 554ml, 555ml, 556ml, 557ml, 558ml, 559ml, 560ml, 561ml, 562ml, 563ml, 564ml, 565ml, 566ml, 567ml, 568ml, 569ml, 570ml, 571ml, 572ml, 573ml, 574ml, 575ml or about 576ml, or any volume in between. 、

[0146] In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 1170 ng / ml following a single intravenous infusion of a dose of about 0.05 mg / kg over about 30 minutes. In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 5950 ng / ml following a single intravenous infusion of a dose of about 0.2 mg / kg over about 30 minutes. In some embodiments, the isolated recombinant polypeptide complex provides a Cmax of about 17300 ng / ml following a single intravenous infusion of a dose of about 0.6 mg / kg over about 30 minutes. In some embodiments, the isolated recombinant polypeptide complex provides a clearance of about 0.504 ml / hr / kg following a single intravenous infusion of a dose of about 0.05 mg / kg over about 30 minutes. In some embodiments, the isolated recombinant polypeptide complex provides a clearance of about 0.762 ml / hr / kg following a single intravenous infusion of a dose of about 0.2 mg / kg over about 30 minutes. In some embodiments, the isolated recombinant polypeptide complex provides a clearance of about 0.650 ml / hr / kg following a single intravenous infusion of a dose of about 0.6 mg / kg over about 30 minutes. In some embodiments, the isolated recombinant polypeptide complex provides a volume of distribution of about 58.8 ml / kg following a single intravenous infusion of a dose of about 0.05 mg / kg over about 30 minutes. In some embodiments, the isolated recombinant polypeptide complex provides a volume of distribution of about 75.7 ml / kg following a single intravenous infusion of a dose of about 0.2 mg / kg over about 30 minutes. In some embodiments, the isolated recombinant polypeptide complex provides a volume of distribution of about 88.9 ml / kg following a single intravenous infusion of a dose of about 0.6 mg / kg over about 30 minutes. In some embodiments, the isolated recombinant polypeptide complex provides a T of about 81.3 h following a single intravenous infusion of a dose of about 0.05 mg / kg over about 30 minutes. 1 / 2In some embodiments, the isolated recombinant polypeptide complex provides a T of about 68.2 h following a single intravenous infusion of about 0.05 mg / kg over about 30 min. 1 / 2 In some embodiments, the isolated recombinant polypeptide complex provides a T of about 96.5 h following a single intravenous infusion of about 0.05 mg / kg over about 30 min. 1 / 2 . In some embodiments, the subject is a primate. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a monkey. In some embodiments, the subject is a cynomolgus macaque. In some embodiments, the subject is a human. In some embodiments, the isolated recombinant polypeptide complex degrades in vitro in the serum of a subject at a rate of about 1% / day. In some embodiments, the isolated recombinant polypeptide complex degrades in vitro in the serum of a subject at a rate of about 2% / day. In some embodiments, the isolated recombinant polypeptide complex degrades in vitro in the serum of a subject at a rate of about 6.6% to about 11.5% / day, for example, about 6.6%, 6.7%, 6.8%, 6.9%, 7%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 11.4%, about 11.5%, or any percentage therebetween. In some embodiments, the subject is healthy.

[0147] In some embodiments, the subject has cancer. In some embodiments, the cancer comprises colorectal cancer (CRC), squamous cell carcinoma of the head and neck (SCCHN), or non-small cell lung cancer (NSCL). In some embodiments, the cancer is CRC. In some embodiments, the cancer is SCCHN. In some embodiments, the cancer is NSCLC.

[0148] Reagent test kit

[0149] In some embodiments, provided herein is a kit comprising a recombinant antibody (such as any recombinant antibody described herein) or a composition (such as any composition described herein), a container, and a label or package insert on or associated with the container.

[0150] Treatment

[0151] Disclosed herein are methods for treating a subject suffering from cancer, comprising administering to the subject any of the antibodies disclosed herein that specifically bind to EGFR and CD3. In some embodiments, the cancer comprises cancer cells that express EGFR or CD3. In some embodiments, the cancer cells that express EGFR or CD3 are lysed. In some embodiments, the antibodies induce antibody-dependent cellular phagocytosis (ADCP) of cancer cells that express EGFR or CD3.

[0152] In some embodiments, the isolated recombinant polypeptide complex described herein is used in a method for treating cancer. In some embodiments, the isolated polypeptide disclosed herein is used in a method for treating cancer, and the isolated polypeptide is an enzyme product of the recombinant polypeptide complex separated after administration. In some embodiments, the cancer has cells expressing EGFR. In some embodiments, the polypeptide or polypeptide complex described herein is used in a method for treating renal cell carcinoma, colorectal cancer (CRC), head and neck squamous cell carcinoma (SCCHN), non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, colon / rectal cancer, head and neck cancer, esophageal and gastric cancer, liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, kidney cancer or pancreatic cancer. In some embodiments, the polypeptide or polypeptide complex described herein is used in a method for treating an object resistant to EGFR inhibitor therapy. In some embodiments, the polypeptide or polypeptide complex described herein is used in a method for treating an object carrying a KRAS mutation. In some embodiments, the polypeptide or polypeptide complex described herein is used in a method for treating an object resistant to EGFR inhibitor therapy and carrying a KRAS mutation.

[0153] In some embodiments, described herein is an isolated recombinant polypeptide complex that is administered once weekly. In some embodiments, the isolated recombinant polypeptide complex is administered once weekly by intravenous, intramuscular, intralesional, topical, subcutaneous, infusion, or oral administration. In some embodiments, the isolated recombinant polypeptide complex is administered once weekly by bolus injection. In some embodiments, the isolated recombinant polypeptide complex is administered once weekly by continuous infusion. In some embodiments, the isolated recombinant polypeptide complex is administered to a subject once weekly by continuous infusion over a period of no more than 60 minutes. In some embodiments, the isolated recombinant polypeptide complex is administered to a subject once weekly by continuous intravenous infusion over a period of no more than 30 minutes. In some embodiments, the isolated recombinant polypeptide complex is administered to a subject once weekly by continuous intravenous infusion over a period of at least 10 minutes.

[0154] In some embodiments, the method further comprises administering an anticancer agent to the subject. In some embodiments, the anticancer agent is a chemotherapeutic agent or a biological agent. In some embodiments, the administration is sufficient to reduce or eliminate the cancer compared to a similar method lacking the administration.

[0155] In some embodiments, is a method of treating cancer in a subject in need thereof, comprising administering to the subject an isolated recombinant polypeptide complex as described herein. In some embodiments, the cancer has cells that express EGFR.

[0156] For administration to a subject, the isolated recombinant polypeptide complexes as disclosed herein can be provided in a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the component and is non-toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline solutions, water, emulsions (e.g., oil / water emulsions), various types of wetting agents, sterile solutions, and the like. Such carriers can be formulated by conventional methods and can be administered to a subject at a suitable dose. Preferably, the composition is sterile. These compositions may also contain adjuvants, such as preservatives, emulsifiers, and dispersants. Protection against the action of microorganisms can be ensured by including various antibacterial and antifungal agents.

[0157] The pharmaceutical composition can be in any suitable form (depending on the desired method of administration). It can be provided in unit dose form, can be provided in a sealed container, and can also be provided as part of a kit. Such a kit can include instructions for use. It can include a plurality of said unit dose forms.

[0158] Pharmaceutical compositions can be adapted for administration by any appropriate route, including parenteral (e.g., subcutaneous, intramuscular, or intravenous) routes. Such compositions can be prepared by any method known in the pharmaceutical field, such as by mixing the active ingredient with a carrier or excipient under aseptic conditions. In some embodiments, pharmaceutical compositions disclosed herein are administered intravenously to a subject in need thereof.

[0159] The dosage of the substances of the present disclosure may vary within wide limits, depending on the disease or disorder being treated, the age and condition of the individual being treated, etc., and the physician will ultimately determine the appropriate dosage to use.

[0160] In some embodiments, disclosed herein is a method for treating cancer comprising administering an effective amount of a pharmaceutical composition to a subject in need thereof. In some embodiments, the pharmaceutical composition comprises a dose of an isolated recombinant polypeptide complex disclosed herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient. In some embodiments, the cancer comprises cancer cells that express epidermal growth factor receptor (EGFR). In some embodiments, the cancer comprises cancer cells that overexpress EGFR.

[0161] In some embodiments, the cancer comprises CRC, SCCHN, NSCLC, renal cell carcinoma (RCC), breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, brain cancer, glioblastoma multiforme, or papillary carcinoma. In some embodiments, the cancer comprises CRC. In some embodiments, the cancer comprises SCCHN. In some embodiments, the cancer comprises NSCLC. In some embodiments, the cancer comprises RCC. In some embodiments, the cancer comprises breast cancer. In some embodiments, the cancer comprises pancreatic cancer. In some embodiments, the cancer comprises ovarian cancer. In some embodiments, the cancer comprises prostate cancer. In some embodiments, the cancer comprises brain cancer. In some embodiments, the cancer comprises glioblastoma multiforme. In some embodiments, the cancer comprises papillary carcinoma. In some embodiments, the cancer is metastatic, refractory, or recurrent. In some embodiments, the cancer is metastatic. In some embodiments, the cancer is refractory. In some embodiments, the cancer is recurrent. In some embodiments, the cancer is advanced or non-advanced. In some embodiments, the cancer is advanced. In some embodiments, the cancer is non-advanced.

[0162] In some embodiments, the dosage is at least about 25μg to at least about 80mg, for example, at least about 25μg, 26μg, 27μg, 28μg, 29μg, 30μg, 31μg, 32μg, 33μg, 34μg, 35μg, 36μg, 37μg, 38μg, 39μg, 40μg, 41μg, 42μg 43μg、44μg、45μg、46μg、47μg、48μg、49μg、50μg、51μg、52μg、53μg、54μg、55 μg、56μg、57μg、58μg、59μg、60μg、61μg、62μg、63μg、64μg、65μg、66μg、67μg、 68μg, 69μg, 70μg, 71μg, 72μg, 73μg, 74μg, 75μg, 76μg, 77μg, 78μg, 79μg, 80μg, 81μg, 82μg, 83μg, 84μg, 85μg, 86μg, 87μg, 88μg, 89μg, 90μg, 91μg, 92μg ,93μg,94μg,95μg,96μg,97μg,98μg,99μg,100μg,105μg,110μg,115μg,120μg,125μg,130μg,135μg,140μg,145μg,150μg,155μg,160μg,165μg,170μg ,175μg,180μg,185μg,190μg,195μg,200μg,205μg,210μg,215μg,220μg,225μg,230μg,235μg,240μg,245μg,250μg,255μg,260μg,265μg,270μg,275 μg、280μg、285μg、290μg、295μg、300μg、305μg、310μg、315μg、320μg、325μg 、330μg、335μg、340μg、345μg、350μg、355μg、360μg、365μg、370μg、375μg、38 0μg, 385μg, 390μg, 395μg, 400μg, 405μg, 410μg, 415μg, 420μg, 425μg, 430μg, 435μg, 440μg, 445μg, 450μg, 455μg, 460μg, 465μg, 470μg, 475μg, 480μg, 4 85μg, 490μg, 495μg, 500μg, 505μg, 510μg, 515μg, 520μg, 525μg, 530μg, 535μg, 540μg, 545μg, 550μg, 555μg, 560μg, 565μg, 570μg, 575μg, 580μg, 585μg,590μg、595μg、600μg、605μg、610μg、615μg、620μg、625μg、630μg、635μg、640μg、645μg、650μg、655μg、660μg、665μg、670μg、675μg、680μg、685μg、690μg、695μg、700μg、705μg、710μg、715μg、720μg、725μg、730μg、735μg、740μg、745μg、750μg、755μg、760μg、765μg、770μg、775μg、780μg、785μg、790μg、795μg、800μg、805μg、810μg、815μg、820μg、825μg、830μg、835μg、840μg、845μg、850μg、855μg、860μg、865μg、870μg、875μg、880μg、885μg、890μg、895μg、900μg、905μg、910μg、915μg、920μg、925μg、930μg、935μg、940μg、945μg、950μg、955μg、960μg、965μg、970μg、975μg、980μg、985μg、990μg、995μg、1mg、1.1 mg、1.2 mg、1.3 mg、1.4 mg、1.5 mg、1.6 mg、1.7 mg、1.8 mg、1.9 mg、2 mg、2.1mg、2.2 mg、2.3 mg、2.4 mg、2.5 mg、2.6 mg、2.7mg、2.8 mg、2.9 mg、3 mg、3.1 mg、3.2 mg、3.3 mg、3.4 mg、3.5 mg、3.6 mg、3.7 mg、3.8 mg、3.9 mg、4 mg、4.1 mg、4.2 mg、4.3 mg、4.4mg、4.5 mg、4.6 mg、4.7 mg、4.8 mg、4.9 mg、5 mg、5.1 mg、5.2 mg、5.3 mg、5.4 mg、5.5mg、5.6 mg、5.7 mg、5.8 mg、5.9 mg、6 mg、6.1mg、6.2 mg、6.3 mg、6.4 mg、6.5 mg、6.6 mg、6.7 mg、6.8 mg、6.9mg、7 mg、7.1 mg、7.2 mg、7.3 mg、7.4 mg、7.5 mg、7.6 mg、7.7 mg、7.8mg、7.9 mg、8 mg、8.1 mg、8.2 mg、8.3 mg、8.4 mg、8.5 mg、8.6mg、8.7mg、8.8mg、8.9mg、9mg、9.1mg、9.2mg、9.3mg、9.4 mg、9.5mg、9.6mg、9.7mg、9.8mg、9.9mg、10mg、11mg、12mg、13mg、14mg、15mg、16mg、17mg、18mg、19mg、20mg、21mg、22mg、23mg、24mg、25mg、26mg 、27mg、28mg、29mg、30mg、31mg、32mg、33mg、34mg、35mg、36mg、37mg、38mg、39mg、40mg、41mg、42mg、43mg、44mg、45mg、46mg、47mg、48mg、49mg、50mg mg、51mg、52mg、53mg、54mg、55mg、56mg、57mg、58mg、59mg、60mg、61mg、62mg、63mg、64mg、65mg、66mg、67mg、68mg、69mg、70mg、71mg、72mg、73mg 、74mg、75mg、76mg、77mg、78mg、79mg or at least about 80mg or any dose". In some embodiments, the dose is 25μg to 80mg, for example, 25μg, 26μg, 27μg, 28μg, 29μg, 30μg, 31μg, 32μg μg, 33μg, 34μg, 35μg, 36μg, 37μg, 38μg, 39μg, 40μg, 41μg, 42μg, 43μg, 44μg, 45μg, 46μg, 47μg, 48μg, 49μg, 50μg, 51μg, 52μg, 53μg, 54μg, 55μg, 56μg, 57μg, 58μg, 59μg, 60μg, 61μg, 62μg, 63μg, 64μg, 65μg, 66μg, 67μg, 68μg, 69μg, 70μg, 71μg, 72μg, 73μg, 74μg, 75μg, 76μg, 77μg, 78μg, 79 μg, 80μg, 81μg, 82μg, 83μg, 84μg, 85μg, 86μg, 87μg, 88μg, 89μg, 90μg, 91μg, 92μg, 93μg, 94μg, 95μg, 96μg, 97μg, 98μg, 99μg, 100μg, 105μg, 110μg, 115μg, 120μg, 125μg, 130μg, 135μg, 140μg, 145μg, 150μg, 155μg, 160μg, 165μg, 170μg, 175μg, 180μg, 185μg, 190μg, 195μg, 200μg, 205μg,210μg, 215μg, 220μg, 225μg, 230μg, 235μg, 240μg, 245μg, 250μg, 255μg, 260μg, 265μg, 270μg, 275μg, 280μg, 285μg, 290μg, 295μg, 300μg, 305μg, 310μg, 315μg, 320μg, 325μg, 330μg, 335μg, 340μg, 345μg, 350μg, 355μg, 360μg, 365μg, 370μg, 375μg, 380μg, 385μg, 390μg, 395μg, 400μg, 405 μg, 410μg, 415μg, 420μg, 425μg, 430μg, 435μg, 440μg, 445μg, 450μg, 455μg, 460μg, 465μg, 470μg, 475μg, 480μg, 485μg, 490μg, 495μg, 500μg, 505μg, 510μg, 515μg, 520μg, 525μg, 530μg, 535μg, 540μg, 545μg, 550μg, 555μg, 560μg, 565μg, 570μg, 575μg, 580μg, 585μg, 590μg, 595μg, 600μg, 605μg, 610μg, 615μg, 620μg, 625μg, 630μg, 635μg, 640μg, 645μg, 650μg, 655μg, 660μg, 665μg, 670μg, 675μg, 680μg, 685μg, 690μg, 695μg, 700μg, 705μg, 710μg, 715μg, 720μg, 725μg, 730μg, 735μg, 740μg, 745μg, 750μg, 755μg, 760μg, 765μg, 770μg, 775μg, 780μg, 785μg, 790μg, 795μg, 800 μg, 805μg, 810μg, 815μg, 820μg, 825μg, 830μg, 835μg, 840μg, 845μg, 850μg, 855μg, 860μg, 865μg, 870μg, 875μg, 880μg, 885μg, 890μg, 895μg, 900μg, 905μg, 910μg, 915μg, 920μg, 925μg, 930μg, 935μg, 940μg, 945μg, 950μg, 955μg, 960μg, 965μg, 970μg, 975μg, 980μg, 985μg, 990μg, 995μg, 1 mg、1.1 mg、1.2 mg、1.3 mg、1.4 mg、1.5 mg、1.6 mg、1.7 mg、1.8 mg、1.9 mg、2 mg、2.1 mg、2.2 mg、2.3 mg、2.4 mg、2.5 mg、2.6 mg、2.7 mg、2.8mg、2.9 mg、3 mg、3.1 mg、3.2 mg、3.3 mg、3.4 mg、3.5mg、3.6 mg、3.7 mg、3.8 mg、3.9 mg、4 mg、4.1 mg、4.2 mg、4.3 mg、4.4 mg、4.5mg、4.6 mg、4.7 mg、4.8 mg、4.9 mg、5 mg、5.1 mg、5.2 mg、5.3 mg、5.4 mg、5.5 mg、5.6 mg、5.7 mg、5.8 mg、5.9 mg、6 mg、6.1 mg、6.2mg、6.3 mg、6.4 mg、6.5 mg、6.6 mg、6.7 mg、6.8 mg、6.9mg、7 mg、7.1 mg、7.2 mg、7.3 mg、7.4 mg、7.5 mg、7.6 mg、7.7 mg、7.8 mg、7.9 mg、8 mg、8.1 mg、8.2 mg、8.3 mg、8.4 mg、8.5 mg、8.6 mg、8.7mg、8.8 mg、8.9 mg、9 mg、9.1 mg、9.2mg、9.3 mg、9.4 mg、9.5 mg、9.6 mg、9.7 mg、9.8 mg、9.9 mg、10 mg、11 mg、12 mg、13 mg、14mg、15 mg、16 mg、17 mg、18 mg、19 mg、20 mg、21 mg、22 mg、23 mg、24 mg、25 mg、26 mg、27 mg、28 mg、29 mg、30 mg、31 mg、32 mg、33 mg、34 mg、35 mg、36 mg、37 mg、38 mg、39mg、40 mg、41 mg、42 mg、43 mg、44 mg、45 mg、46 mg、47 mg、48 mg、49 mg、50 mg、51 mg、52mg、53 mg、54 mg、55 mg、56 mg、57 mg、58 mg、59 mg、60 mg、61 mg、62 mg、63 mg、64 mg、65mg、66 mg、67 mg、68mg、69mg、70mg、71mg、72mg、73mg、74mg、75mg、76mg、77mg、78mg、79 mg or up to about 80 mg or any dose therebetween. In some embodiments, the dosage is from about 25 μg to about 80 mg, for example, about 25 μg, 26 μg, 27 μg, 28 μg, 29 μg, 30 μg, 31 μg, 32 μg, 33 μg, 34 μg, 35 μg, 36 μg, 37 μg, 38 μg, 39 μg, 40 μg, 41 μg, 42 μg, 43 μg, 44 μg, 45 μg, 46 μg, 47 μg, 48 μg, 49 μg, 50 μg, 51 μg, 52 μg, 53 μg, 54 μg, 55 μg, 56 μg, 57 μg, 58 μg, 59 μg, 60 μg, 61 μg, 62 μg, 63 μg, 64 μg, 65 μg, 66 μg, 67 μg, 68 μg, 69 μg, 70 μg, 71 μg, 72 μg, 73 μg, 74 μg, 75 μg, 76 μg, 77 μg, 78 μg, 79 μg, 80 μg, 81 μg, 82 μg, 83 μg, 84 μg, 85 μg, 86 μg, 87 μg, 88 μg, 89 μg, 90 μg 8μg, 69μg, 70μg, 71μg, 72μg, 73μg, 74μg, 75μg, 76μg, 77μg, 78μg, 79μg, 80 μg, 81μg, 82μg, 83μg, 84μg, 85μg, 86μg, 87μg, 88μg, 89μg, 90μg, 91μg, 92μ g, 93μg, 94μg, 95μg, 96μg, 97μg, 98μg, 99μg, 100μg, 105μg, 110μg, 115μg, 120μg, 125μg, 130μg, 135μg, 140μg, 145μg, 150μg, 155μg, 160μg, 165μg, 17 0μg, 175μg, 180μg, 185μg, 190μg, 195μg, 200μg, 205μg, 210μg, 215μg, 220 μg, 225μg, 230μg, 235μg, 240μg, 245μg, 250μg, 255μg, 260μg, 265μg, 270μ g, 275μg, 280μg, 285μg, 290μg, 295μg, 300μg, 305μg, 310μg, 315μg, 320μg , 325μg, 330μg, 335μg, 340μg, 345μg, 350μg, 355μg, 360μg, 365μg, 370μg, 3 75μg, 380μg, 385μg, 390μg, 395μg, 400μg, 405μg, 410μg, 415μg, 420μg, 42 5μg, 430μg, 435μg, 440μg, 445μg, 450μg, 455μg, 460μg, 465μg, 470μg, 475 μg, 480μg, 485μg, 490μg, 495μg, 500μg, 505μg, 510μg, 515μg, 520μg, 525μ g, 530μg, 535μg, 540μg, 545μg, 550μg, 555μg, 560μg, 565μg, 570μg, 575μg,580μg、585μg、590μg、595μg、600μg、605μg、610μg、615μg、620μg、625μg、630μg、635μg、640μg、645μg、650μg、655μg、660μg、665μg、670μg、675μg、680μg、685μg、690μg、695μg、700μg、705μg、710μg、715μg、720μg、725μg、730μg、735μg、740μg、745μg、750μg、755μg、760μg、765μg、770μg、775μg、780μg、785μg、790μg、795μg、800μg、805μg、810μg、815μg、820μg、825μg、830μg、835μg、840μg、845μg、850μg、855μg、860μg、865μg、870μg、875μg、880μg、885μg、890μg、895μg、900μg、905μg、910μg、915μg、920μg、925μg、930μg、935μg、940μg、945μg、950μg、955μg、960μg、965μg、970μg、975μg、980μg、985μg、990μg、995μg、1mg、1.1mg、1.2mg、1.3mg、1.4mg、1.5mg、1.6mg、1.7mg、1.8mg、1.9mg、2mg、2.1mg、2.2mg、2.3mg、2.4mg、2.5mg、2.6mg、2.7mg、2.8mg、2.9mg、3mg、3.1mg、3.2mg、3.3mg、3.4mg、3.5mg、3.6mg、3.7mg、3.8mg、3.9mg、4mg、4.1mg、4.2mg、4.3mg、4.4mg、4.5mg、4.6mg、4.7mg、4.8mg、4.9mg、5mg、5.1mg、5.2mg、5.3mg、5.4mg、5.5mg、5.6mg、5.7mg、5.8mg、5.9mg、6mg、6.1mg、6.2mg、6.3mg、6.4mg、6.5mg、6.6mg、6.7mg、6.8mg、6.9mg、7mg、7.1mg、7.2mg、7.3mg、7.4mg、7.5mg、7.6mg、7.7mg、7.8mg、7.9mg、8mg、8.1mg、8.2mg、8.3mg、8.4mg、8.5mg、8.6mg、8.7mg、8.8mg、8.9mg、9mg、9.1mg、9.2mg、9.3mg、9.4mg、9.5mg, 9.6mg, 9.7mg, 9.8mg, 9.9mg, 10mg, 11mg, 12mg, 13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 19mg, 20mg, 21mg, 22mg, 23m g, 24mg, 25mg, 26mg, 27mg, 28mg, 29mg, 30mg, 31mg, 32mg, 33mg, 34mg, 35mg, 36mg, 37mg, 38mg, 39mg, 40mg, 41mg, 42mg, 43 In some embodiments, the dosage disclosed herein is any dosage administered in the method. In some embodiments, the dosage disclosed herein is the first dosage administered to the subject in the method. In some embodiments, the dosage disclosed herein is the last dosage administered to the subject in the method. In some embodiments, the dosage disclosed herein is the dosage administered to the subject between the first and last dosages in the method. In some embodiments, two or more dosages disclosed herein are administered to the subject in a dosing regimen. In some embodiments, three or more doses disclosed herein are administered to a subject in a dosing regimen. In some embodiments, more than three doses disclosed herein are administered to a subject in a dosing regimen.

[0163] In some embodiments, the dose is at least about 25 μg, at least about 50 μg, at least about 100 μg, at least about 150 μg, or at least about 200 μg. In some embodiments, the dose is at least about 25 μg. In some embodiments, the dose is at least about 50 μg. In some embodiments, the dose is at least about 100 μg. In some embodiments, the dose is at least about 150 μg. In some embodiments, the dose is at least about 200 μg. In some embodiments, the dose is at most about 25 μg, at most about 50 μg, at most about 100 μg, at most about 150 μg, or at most about 200 μg. In some embodiments, the dose is at most about 25 μg. In some embodiments, the dose is at most about 50 μg. In some embodiments, the dose is at most about 100 μg. In some embodiments, the dose is at most about 150 μg. In some embodiments, the dose is at most about 200 μg. In some embodiments, the dose is about 25 μg, about 50 μg, about 100 μg, about 150 μg, or about 200 μg. In some embodiments, the dose is about 25 μg. In some embodiments, the dose is about 50 μg. In some embodiments, the dose is about 100 μg. In some embodiments, the dose is about 150 μg. In some embodiments, the dose is about 200 μg. In some embodiments, the dose is at least about 300 μg, at least about 400 μg, at least about 500 μg, at least about 600 μg, at least about 700 μg, at least about 800 μg, at least about 900 μg, at least about 1 mg, at least about 5 mg, at least about 10 mg, at least about 20 mg, at least about 40 mg, at least about 60 mg, or at least about 80 mg. In some embodiments, the dosage is at most about 300 μg, at most about 400 μg, at most about 500 μg, at most about 600 μg, at most about 700 μg, at most about 800 μg, at most about 900 μg, at most about 1 mg, at most about 5 mg, at most about 10 mg, at most about 20 mg, at most about 40 mg, at most about 60 mg, or at most about 80 mg. In some embodiments, the dosage is about 300 μg, about 400 μg, about 500 μg, about 600 μg, about 700 μg, about 800 μg, about 900 μg, about 1 mg, about 5 mg, about 10 mg, about 20 mg, about 40 mg, about 60 mg, or about 80 mg. In some embodiments, the dosage is at least about 300 μg. In some embodiments, the dosage is at least about 400 μg. In some embodiments, the dosage is at least about 500 μg. In some embodiments, the dose is at least about 600 μg. In some embodiments, the dose is at least about 700 μg. In some embodiments, the dose is at least about 800 μg. In some embodiments, the dose is at least about 900 μg. In some embodiments, the dose is at least about 1 mg. In some embodiments, the dose is at least about 5 mg.In some embodiments, the dose is at least about 10 mg. In some embodiments, the dose is at least about 20 mg. In some embodiments, the dose is at least about 40 mg. In some embodiments, the dose is at least about 60 mg. In some embodiments, the dose is at least about 80 mg. In some embodiments, the dose is at most about 300 μg. In some embodiments, the dose is at most about 400 μg. In some embodiments, the dose is at most about 500 μg. In some embodiments, the dose is at most about 600 μg. In some embodiments, the dose is at most about 700 μg. In some embodiments, the dose is at most about 800 μg. In some embodiments, the dose is at most about 900 μg. In some embodiments, the dose is at most about 1 mg. In some embodiments, the dose is at most about 5 mg. In some embodiments, the dose is at most about 10 mg. In some embodiments, the dose is at most about 20 mg. In some embodiments, the dose is at most about 40 mg. In some embodiments, the dose is at most about 60 mg. In some embodiments, the dose is up to about 80 mg. In some embodiments, the dose is about 300 μg. In some embodiments, the dose is about 400 μg. In some embodiments, the dose is about 500 μg. In some embodiments, the dose is about 600 μg. In some embodiments, the dose is about 700 μg. In some embodiments, the dose is about 800 μg. In some embodiments, the dose is about 900 μg. In some embodiments, the dose is about 1 mg. In some embodiments, the dose is about 5 mg. In some embodiments, the dose is about 10 mg. In some embodiments, the dose is about 20 mg. In some embodiments, the dose is about 40 mg. In some embodiments, the dose is about 60 mg. In some embodiments, the dose is about 80 mg. In some embodiments, the dose disclosed herein is any dose administered in the method. In some embodiments, the dose disclosed herein is the first dose administered to the subject in the method. In some embodiments, the dose disclosed herein is the last dose administered to the subject in the method. In some embodiments, the dose disclosed herein is the dose administered to the subject between the first dose and the last dose in the method. In some embodiments, two or more doses disclosed herein are administered to the subject in a dosing regimen. In some embodiments, three or more doses disclosed herein are administered to the subject in a dosing regimen. In some embodiments, more than three doses disclosed herein are administered to the subject in a dosing regimen.

[0164] In some embodiments, the dosage comprises 1 dose, 2 doses, 3 doses, 4 doses, 5 doses, 6 doses, 7 doses, 8 doses, 9 doses, 10 doses, 11 doses, 12 doses, 13 doses, 14 doses, 15 doses, 16 doses, 17 doses, 18 doses, 19 doses, 20 doses, 21 doses, 22 doses, 23 doses, 24 doses, 25 doses. , 26 doses, 27 doses, 28 doses, 29 doses, 30 doses, 31 doses, 32 doses, 33 doses, 34 doses, 35 doses, 36 doses, 37 doses, 38 doses, 39 doses, 40 doses, 41 doses, 42 doses, 43 doses, 44 doses, 45 doses, 46 doses, 47 doses, 48 ​​doses, 49 doses, 50 doses, 51 dose, 52 doses, 53 doses, 54 doses, 55 doses, 56 doses, 57 doses, 58 doses, 59 doses, 60 doses, 61 doses, 62 doses, 63 doses, 64 doses, 65 doses, 66 doses, 67 doses, 68 doses, 69 doses, 70 doses, 71 doses, 72 doses, 73 doses, 74 doses, 75 doses, 76 doses, 77 doses, 78 doses, 79 doses, 80 doses, 81 doses, 82 doses, 83 doses, 84 doses, 85 doses, 86 doses, 87 doses, 88 doses, 89 doses, 90 doses, 91 doses, 92 doses, 93 doses, 94 doses, 95 doses, 96 doses, 97 doses, 98 doses, 99 doses, or 100 doses or more than 100 doses. In some embodiments, the doses include at least 2 doses. In some embodiments, the doses include at least 3 doses. In some embodiments, the doses include at least 4 doses. In some embodiments, the doses include at least 5 doses. In some embodiments, the doses include at least 6 doses. In some embodiments, the doses include at least 7 doses. In some embodiments, the doses include at least 8 doses. In some embodiments, the doses include at least 9 doses. In some embodiments, the doses include at least 10 doses. In some embodiments, the doses include at least 9 doses. In some embodiments, the dosage comprises at least 20 doses. In some embodiments, the dosage comprises at least 9 doses. In some embodiments, the dosage comprises at least 30 doses. In some embodiments, the dosage comprises at least 9 doses. In some embodiments, the dosage comprises at least 40 doses. In some embodiments, the dosage comprises at least 9 doses. In some embodiments, the dosage comprises at least 50 doses. In some embodiments, the dosage comprises at least 9 doses. In some embodiments, the dosage comprises at least 100 doses.In some embodiments, the dosage comprises at least 9 doses. In some embodiments, the dosage comprises at least 200 doses. In some embodiments, the dosage comprises at least 9 doses. In some embodiments, the dosage comprises at least 300 doses. In some embodiments, the dosage comprises at least 9 doses. In some embodiments, the dosage comprises at least 400 doses. In some embodiments, the dosage comprises at least 500 doses.

[0165] In some embodiments, the dose comprises a first dose and a second dose, wherein the second dose is equal to or higher than the first dose, wherein the first dose or the second dose is a dose disclosed herein. In some embodiments, the dose comprises a first dose and a second dose, wherein the second dose is equal to or higher than the first dose, wherein the first dose is at least about 25 μg. In some embodiments, the dose comprises a first dose and a second dose, wherein the second dose is equal to or higher than the first dose, wherein the first dose is at least about 50 μg. In some embodiments, the dose comprises a first dose and a second dose, wherein the second dose is equal to or higher than the first dose, wherein the first dose is at least about 100 μg. In some embodiments, the dose comprises a first dose and a second dose, wherein the second dose is equal to or higher than the first dose, wherein the first dose is at least about 150 μg. In some embodiments, the dose comprises a first dose and a second dose, wherein the second dose is equal to or higher than the first dose, wherein the first dose is at least about 200 μg. In some embodiments, the dose comprises a first dose and a second dose, wherein the second dose is equal to or higher than the first dose, wherein the first dose is a dose disclosed herein. In some embodiments, the dose comprises a first dose and a second dose, wherein the second dose is equal to or higher than the first dose, wherein the first dose is at most about 25 μg. In some embodiments, the dose comprises a first dose and a second dose, wherein the second dose is equal to or higher than the first dose, wherein the first dose is at most about 50 μg. In some embodiments, the dose comprises a first dose and a second dose, wherein the second dose is equal to or higher than the first dose, wherein the first dose is at most about 100 μg. In some embodiments, the dose comprises a first dose and a second dose, wherein the second dose is equal to or higher than the first dose, wherein the first dose is at most about 150 μg. In some embodiments, the dose comprises a first dose and a second dose, wherein the second dose is equal to or higher than the first dose, wherein the first dose is at most about 200 μg. In some embodiments, the dose comprises a first dose and a second dose, wherein the second dose is equal to or higher than the first dose, wherein the first dose is about 25 μg. In some embodiments, the dose comprises a first dose and a second dose, wherein the second dose is equal to or higher than the first dose, wherein the first dose is about 50 μg. In some embodiments, the dose comprises a first dose and a second dose, wherein the second dose is equal to or greater than the first dose, wherein the first dose is about 100 μg. In some embodiments, the dose comprises a first dose and a second dose, wherein the second dose is equal to or greater than the first dose, wherein the first dose is about 150 μg. In some embodiments, the dose comprises a first dose and a second dose, wherein the second dose is equal to or greater than the first dose, wherein the first dose is about 200 μg.

[0166] In some embodiments, the dose includes a first dose, a second dose, and a third dose, wherein the second dose is equal to or higher than the first dose, wherein the third dose is equal to or higher than the second dose, wherein the first dose, the second dose, or the third dose is a dose disclosed herein. In some embodiments, the dose includes a first dose, a second dose, and a third dose, wherein the second dose is equal to or higher than the first dose, wherein the third dose is equal to or higher than the second dose, wherein the first dose is at least about 25 μg. In some embodiments, the dose includes a first dose, a second dose, and a third dose, wherein the second dose is equal to or higher than the first dose, wherein the third dose is equal to or higher than the second dose, wherein the first dose is at least about 50 μg. In some embodiments, the dose includes a first dose, a second dose, and a third dose, wherein the second dose is equal to or higher than the first dose, wherein the third dose is equal to or higher than the second dose, wherein the first dose is at least about 100 μg. In some embodiments, the dose includes a first dose, a second dose, and a third dose, wherein the second dose is equal to or higher than the first dose, wherein the third dose is equal to or higher than the second dose, wherein the first dose is at least about 150 μg. In some embodiments, the dose comprises a first dose, a second dose, and a third dose, wherein the second dose is equal to or higher than the first dose, wherein the third dose is equal to or higher than the second dose, wherein the first dose is at least about 200 μg. In some embodiments, the dose comprises a first dose, a second dose, and a third dose, wherein the second dose is equal to or higher than the first dose, wherein the third dose is equal to or higher than the second dose, wherein the first dose is at most about 25 μg. In some embodiments, the dose comprises a first dose, a second dose, and a third dose, wherein the second dose is equal to or higher than the first dose, wherein the third dose is equal to or higher than the second dose, wherein the first dose is at most about 50 μg. In some embodiments, the dose comprises a first dose, a second dose, and a third dose, wherein the second dose is equal to or higher than the first dose, wherein the third dose is equal to or higher than the second dose, wherein the first dose is at most about 100 μg. In some embodiments, the dose comprises a first dose, a second dose, and a third dose, wherein the second dose is equal to or higher than the first dose, wherein the third dose is equal to or higher than the second dose, wherein the first dose is at most about 150 μg. In some embodiments, the dose comprises a first dose, a second dose, and a third dose, wherein the second dose is equal to or higher than the first dose, wherein the third dose is equal to or higher than the second dose, wherein the first dose is up to about 200 μg.

[0167] In some embodiments, the dose includes a first dose, a second dose, and a third dose, wherein the second dose is equal to or higher than the first dose, wherein the third dose is equal to or higher than the second dose, and wherein the first dose is about 25 μg. In some embodiments, the dose includes a first dose, a second dose, and a third dose, wherein the second dose is equal to or higher than the first dose, wherein the third dose is equal to or higher than the second dose, and wherein the first dose is about 50 μg. In some embodiments, the dose includes a first dose, a second dose, and a third dose, wherein the second dose is equal to or higher than the first dose, wherein the third dose is equal to or higher than the second dose, and wherein the first dose is about 100 μg. In some embodiments, the dose includes a first dose, a second dose, and a third dose, wherein the second dose is equal to or higher than the first dose, wherein the third dose is equal to or higher than the second dose, and wherein the first dose is about 150 μg. In some embodiments, the dose includes a first dose, a second dose, and a third dose, wherein the second dose is equal to or higher than the first dose, wherein the third dose is equal to or higher than the second dose, and wherein the first dose is about 200 μg. In some embodiments, a dose comprises a plurality of doses disclosed herein, wherein a first dose in the plurality of doses is a dose disclosed herein and is at most equal to any other dose in the plurality of doses.

[0168] In some embodiments, the dose of the isolated recombinant polypeptide complex is administered at least once a week. In some embodiments, the dose of the isolated recombinant polypeptide complex is administered at least twice a week. In some embodiments, the dose of the isolated recombinant polypeptide complex is administered at most once a week. In some embodiments, the dose of the isolated recombinant polypeptide complex is administered at most twice a week. In some embodiments, the dose of the isolated recombinant polypeptide complex is administered once a week. In some embodiments, the dose of the isolated recombinant polypeptide complex is administered twice a week.

[0169] In some embodiments, a dose of the isolated recombinant polypeptide complex is administered at least once every two weeks. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered at least twice every two weeks. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered at least three times every two weeks. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered at most once every two weeks. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered at most twice every two weeks. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered at most three times every two weeks. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered at most once every three weeks. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered at most twice every two weeks. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered at most three times every three weeks. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered at most once every three weeks. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered at most twice every three weeks. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered at most three times every three weeks. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered once every three weeks. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered twice every three weeks. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered three times every three weeks. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered at least once every four weeks. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered at least twice every four weeks. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered at most once every four weeks. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered at most twice every four weeks. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered once every four weeks. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered twice every four weeks. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered at least once every five weeks or more. In some embodiments, a dose of the isolated recombinant polypeptide complex is administered at most once every five weeks or more.

[0170] In some embodiments, the method further comprises a course of treatment. In some embodiments, the method further comprises at least two courses of treatment. In some embodiments, the method further comprises a 21-day course of treatment. In some embodiments, the method further comprises a 28-day course of treatment. In some embodiments, the method further comprises at least one 21-day course of treatment. In some embodiments, the method further comprises at least one 28-day course of treatment. In some embodiments, the method further comprises at least one 21-day course of treatment and at least one 28-day course of treatment. In some embodiments, the method further comprises at least two 21-day courses of treatment. In some embodiments, the method further comprises at least two 28-day courses of treatment. In some embodiments, the method further comprises at least two 21-day courses of treatment and at least two 28-day courses of treatment. In some embodiments, the method further comprises one 21-day course of treatment and at least one 28-day course of treatment. In some embodiments, the method further comprises at least one 21-day course of treatment and at least two 28-day courses of treatment. In some embodiments, the method further comprises at least two 21-day courses of treatment and at least one 28-day course of treatment. In some embodiments, the method further comprises at least two 21-day courses of treatment and at least one 28-day course of treatment. In some embodiments, the method further comprises at least two 21-day courses of treatment and at least two 28-day courses of treatment. In some embodiments, the method comprises at least one 28-day treatment course and does not comprise any 21-day treatment courses. In some embodiments, the method further comprises at least two 21-day treatment courses and at least four 28-day treatment courses. In some embodiments, the method further comprises two 21-day treatment courses and at least four 28-day treatment courses. In some embodiments, the method further comprises at least six 28-day treatment courses. In some embodiments, the method further comprises one 21-day treatment course and at least five 28-day treatment courses. In some embodiments, the method further comprises at least two, three, four, five, six, seven, eight, nine, ten, or more than ten 21-day treatment courses. In some embodiments, the method further comprises at most two, three, four, five, six, seven, eight, nine, ten, or more than ten 21-day treatment courses. In some embodiments, the method further comprises at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 21-day treatment courses. In some embodiments, the method further comprises up to 20, 30, 40, 50, 60, 70, 80, 90, or 100 21-day treatment courses. In some embodiments, the method further comprises at least two, three, four, five, six, seven, eight, nine, ten, or more than ten 28-day treatment courses. In some embodiments, the method further comprises up to two, three, four, five, six, seven, eight, nine, ten, or more than ten 28-day treatment courses.In some embodiments, the method further comprises at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 28-day treatment courses. In some embodiments, the method further comprises at most 20, 30, 40, 50, 60, 70, 80, 90, or 100 28-day treatment courses.

[0171] In some embodiments, a first dose is administered to a subject within the first week of the treatment course, a second dose is administered to a subject within the second week of the treatment course, and a third dose is administered to a subject within the third week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is a dose disclosed herein. In some embodiments, a first dose is administered to a subject within the first week of the treatment course, a second dose is administered to a subject within the second week of the treatment course, and a third dose is administered to a subject within the third week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is at least about 25 μg, 50 μg, 100 μg, 150 μg, or 200 μg. In some embodiments, a first dose is administered to a subject within the first week of the treatment course, a second dose is administered to a subject within the second week of the treatment course, and a third dose is administered to a subject within the third week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is at least about 25 μg. In some embodiments, a first dose is administered to a subject within the first week of the treatment course, a second dose is administered to the subject within the second week of the treatment course, and a third dose is administered to the subject within the third week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is at least about 50 μg. In some embodiments, a first dose is administered to a subject within the first week of the treatment course, a second dose is administered to the subject within the second week of the treatment course, and a third dose is administered to the subject within the third week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is at least about 100 μg. In some embodiments, a first dose is administered to a subject within the first week of the treatment course, a second dose is administered to the subject within the second week of the treatment course, and a third dose is administered to the subject within the third week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is at least about 150 μg. In some embodiments, the subject is administered a first dose within the first week of the treatment course, the subject is administered a second dose within the second week of the treatment course, and the subject is administered a third dose within the third week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is at least about 200 μg.

[0172] In some embodiments, a first dose is administered to a subject within the first week of the treatment course, a second dose is administered to the subject within the second week of the treatment course, and a third dose is administered to the subject within the third week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is at most about 25 μg, 50 μg, 100 μg, 150 μg, or 200 μg. In some embodiments, a first dose is administered to a subject within the first week of the treatment course, a second dose is administered to the subject within the second week of the treatment course, and a third dose is administered to the subject within the third week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is at most about 25 μg. In some embodiments, a first dose is administered to a subject within the first week of the treatment course, a second dose is administered to the subject within the second week of the treatment course, and a third dose is administered to the subject within the third week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is at most about 50 μg. In some embodiments, a first dose is administered to a subject within the first week of the treatment course, a second dose is administered to the subject within the second week of the treatment course, and a third dose is administered to the subject within the third week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is at most about 100 μg. In some embodiments, a first dose is administered to a subject within the first week of the treatment course, a second dose is administered to the subject within the second week of the treatment course, and a third dose is administered to the subject within the third week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is at most about 150 μg. In some embodiments, a first dose is administered to a subject within the first week of the treatment course, a second dose is administered to the subject within the second week of the treatment course, and a third dose is administered to the subject within the third week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is at most about 200 μg.

[0173] In some embodiments, a first dose is administered to a subject within the first week of the treatment course, a second dose is administered to the subject within the second week of the treatment course, and a third dose is administered to the subject within the third week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is about 25 μg. In some embodiments, a first dose is administered to a subject within the first week of the treatment course, a second dose is administered to the subject within the second week of the treatment course, and a third dose is administered to the subject within the third week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is about 50 μg. In some embodiments, a first dose is administered to a subject within the first week of the treatment course, a second dose is administered to the subject within the second week of the treatment course, and a third dose is administered to the subject within the third week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is about 100 μg. In some embodiments, a first dose is administered to a subject within the first week of the treatment course, a second dose is administered to a subject within the second week of the treatment course, and a third dose is administered to a subject within the third week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is about 150 μg. In some embodiments, a first dose is administered to a subject within the first week of the treatment course, a second dose is administered to a subject within the second week of the treatment course, and a third dose is administered to a subject within the third week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is about 200 μg. In some embodiments, the first dose and the second dose are each administered to a subject once every two weeks during the treatment course, wherein the second dose is equal to or higher than the first dose, wherein the first dose is at least about 25 μg. In some embodiments, the first dose and the second dose are each administered to a subject once every two weeks during the treatment course, wherein the second dose is equal to or higher than the first dose, wherein the first dose is at least about 50 μg. In some embodiments, the first dose and the second dose are each administered to the subject once every two weeks during the course of treatment, wherein the second dose is equal to or higher than the first dose, wherein the first dose is at least about 100 μg. In some embodiments, the first dose and the second dose are each administered to the subject once every two weeks during the course of treatment, wherein the second dose is equal to or higher than the first dose, wherein the first dose is at least about 150 μg. In some embodiments, the first dose and the second dose are each administered to the subject once every two weeks during the course of treatment, wherein the second dose is equal to or higher than the first dose, wherein the first dose is at least about 200 μg. In some embodiments, the first dose and the second dose are each administered to the subject once every two weeks during the course of treatment, wherein the second dose is equal to or higher than the first dose, wherein the first dose is at most about 25 μg.In some embodiments, the first and second doses are each administered to the subject once every two weeks during the course of treatment, wherein the second dose is equal to or higher than the first dose, wherein the first dose is at most about 50 μg. In some embodiments, the first and second doses are each administered to the subject once every two weeks during the course of treatment, wherein the second dose is equal to or higher than the first dose, wherein the first dose is at most about 100 μg. In some embodiments, the first and second doses are each administered to the subject once every two weeks during the course of treatment, wherein the second dose is equal to or higher than the first dose, wherein the first dose is at most about 150 μg. In some embodiments, the first and second doses are each administered to the subject once every two weeks during the course of treatment, wherein the second dose is equal to or higher than the first dose, wherein the first dose is at most about 200 μg. In some embodiments, the first and second doses are each administered to the subject once every two weeks during the course of treatment, wherein the second dose is equal to or higher than the first dose, wherein the first dose is about 25 μg. In some embodiments, the first and second doses are each administered to the subject once every two weeks during the course of treatment, wherein the second dose is equal to or higher than the first dose, wherein the first dose is about 50 μg. In some embodiments, the first dose and the second dose are each administered to the subject once every two weeks during the course of treatment, wherein the second dose is equal to or greater than the first dose, wherein the first dose is about 100 μg. In some embodiments, the first dose and the second dose are each administered to the subject once every two weeks during the course of treatment, wherein the second dose is equal to or greater than the first dose, wherein the first dose is about 150 μg. In some embodiments, the first dose and the second dose are each administered to the subject once every two weeks during the course of treatment, wherein the second dose is equal to or greater than the first dose, wherein the first dose is about 200 μg.

[0174] In some embodiments, a first dose and a second dose are administered to a subject within the first week of the treatment course, and a third dose is administered to the subject within the second week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is at least about 25 μg. In some embodiments, a first dose and a second dose are administered to a subject within the first week of the treatment course, and a third dose is administered to the subject within the second week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is at least about 50 μg. In some embodiments, a first dose and a second dose are administered to a subject within the first week of the treatment course, and a third dose is administered to the subject within the second week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is at least about 100 μg. In some embodiments, a first dose and a second dose are administered to a subject within the first week of the treatment course, and a third dose is administered to the subject within the second week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is at least about 150 μg. In some embodiments, the subject is administered a first dose and a second dose within the first week of the treatment course, and the subject is administered a third dose within the second week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is at least about 200 μg.

[0175] In some embodiments, a first dose and a second dose are administered to a subject within the first week of the treatment course, and a third dose is administered to the subject within the second week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is at most about 25 μg. In some embodiments, a first dose and a second dose are administered to a subject within the first week of the treatment course, and a third dose is administered to the subject within the second week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is at most about 50 μg. In some embodiments, a first dose and a second dose are administered to a subject within the first week of the treatment course, and a third dose is administered to the subject within the second week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is at most about 100 μg. In some embodiments, a first dose and a second dose are administered to a subject within the first week of the treatment course, and a third dose is administered to a subject within the second week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is at most about 150 μg. In some embodiments, the subject is administered a first dose and a second dose within the first week of the treatment course, and the subject is administered a third dose within the second week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is up to about 200 μg.

[0176] In some embodiments, a first dose and a second dose are administered to a subject within the first week of the treatment course, and a third dose is administered to the subject within the second week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is about 25 μg. In some embodiments, a first dose and a second dose are administered to a subject within the first week of the treatment course, and a third dose is administered to the subject within the second week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is about 50 μg. In some embodiments, a first dose and a second dose are administered to a subject within the first week of the treatment course, and a third dose is administered to the subject within the second week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is about 100 μg. In some embodiments, a first dose and a second dose are administered to a subject within the first week of the treatment course, and a third dose is administered to the subject within the second week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is about 150 μg. In some embodiments, the subject is administered a first dose and a second dose within the first week of the treatment course, and the subject is administered a third dose within the second week of the treatment course, wherein the second dose is equal to or higher than the first dose and the third dose is equal to or higher than the second dose, wherein the first dose is about 200 μg.

[0177] In some embodiments, a dose is administered on day 1 of a course of treatment. In some embodiments, a dose is administered on day 4 of a course of treatment. In some embodiments, a dose is administered on day 8 of a course of treatment. In some embodiments, a dose is administered on day 15 of a course of treatment. In some embodiments, a first dose is administered on day 1 of a course of treatment, a second dose is administered on day 8 of a course of treatment, and a third dose is administered on day 15 of a course of treatment. In some embodiments, a first dose is administered on day 1 of a 21-day course of treatment, a second dose is administered on day 8 of a 21-day course of treatment, and a third dose is administered on day 15 of a 21-day course of treatment. In some embodiments, a first dose is administered on day 1 of a 28-day course of treatment, a second dose is administered on day 8 of a 28-day course of treatment, and a third dose is administered on day 15 of a 28-day course of treatment. In some embodiments, a first dose is administered on day 1 of a course of treatment, and a second dose is administered on day 15 of a 28-day course of treatment. In some embodiments, a first dose is administered on day 1 of a course of treatment, and a second dose is administered on day 15 of a 28-day course of treatment. In some embodiments, the first dose is administered on day 1 of the treatment course, the second dose is administered on day 4 of the treatment course, and the third dose is administered on day 8 of the treatment course. In some embodiments, the first dose is administered on day 1 of a 21-day treatment course, the second dose is administered on day 4 of the 21-day treatment course, and the third dose is administered on day 8 of the 21-day treatment course. In some embodiments, the method can include one or more of the dosing regimens disclosed herein. In some embodiments, the dosing regimens disclosed herein can be combined with one or more of the additional dosing regimens disclosed herein.

[0178] In some embodiments, the method comprises a first 21 day treatment course and a second 21 day treatment course, wherein the first 21 day treatment course precedes the second 21 day treatment course, wherein the first dose of the second 21 day treatment course is equal to or higher than the first dose of the first 21 day treatment course, the second dose of the second 21 day treatment course is equal to or higher than the second dose of the first 21 day treatment course, and the third dose of the second 21 day treatment course is equal to or higher than the third dose of the first 21 day treatment course, wherein the first dose of the first 21 day treatment course is a dose disclosed herein. In some embodiments, the method comprises a first 21 day treatment course and a second 21 day treatment course, wherein the first 21 day treatment course precedes the second 21 day treatment course, wherein the first dose of the second 21 day treatment course is equal to or higher than the first dose of the first 21 day treatment course, the second dose of the second 21 day treatment course is equal to or higher than the second dose of the first 21 day treatment course, and the third dose of the second 21 day treatment course is equal to or higher than the third dose of the first 21 day treatment course, wherein the first dose of the first 21 day treatment course is at least 25 μg, at least 50 μg, at least 100 μg, at least 150 μg, or at least 200 μg. In some embodiments, the method comprises a first 21 day treatment course and a second 21 day treatment course, wherein the first 21 day treatment course precedes the second 21 day treatment course, wherein the first dose of the second 21 day treatment course is equal to or higher than the first dose of the first 21 day treatment course, the second dose of the second 21 day treatment course is equal to or higher than the second dose of the first 21 day treatment course, and the third dose of the second 21 day treatment course is equal to or higher than the third dose of the first 21 day treatment course, wherein the first dose of the first 21 day treatment course is at most 25 μg, at most 50 μg, at most 100 μg, at most 150 μg, or at most 200 μg. In some embodiments, the method comprises a first 21 day treatment course and a second 21 day treatment course, wherein the first 21 day treatment course precedes the second 21 day treatment course, wherein the first dose of the second 21 day treatment course is equal to or higher than the first dose of the first 21 day treatment course, the second dose of the second 21 day treatment course is equal to or higher than the second dose of the first 21 day treatment course, and the third dose of the second 21 day treatment course is equal to or higher than the third dose of the first 21 day treatment course, wherein the first dose of the first 21 day treatment course is about 25 μg, about 50 μg, about 100 μg, about 150 μg, or about 200 μg.

[0179] In some embodiments, the method comprises a first 28-day treatment course and a second 28-day treatment course, wherein the first 28-day treatment course precedes the second 28-day treatment course, wherein the first dose of the second 28-day treatment course is equal to or higher than the first dose of the first 28-day treatment course, and the second dose of the second 28-day treatment course is equal to or higher than the second dose of the first 28-day treatment course, wherein the first dose of the first 21-day treatment course is a dose disclosed herein. In some embodiments, the method comprises a first 28-day treatment course and a second 28-day treatment course, wherein the first 28-day treatment course precedes the second 28-day treatment course, wherein the first dose of the second 28-day treatment course is equal to or higher than the first dose of the first 28-day treatment course, and the second dose of the second 28-day treatment course is equal to or higher than the second dose of the first 28-day treatment course, wherein the first dose of the first 21-day treatment course is at least 25 μg, at least 50 μg, at least 100 μg, at least 150 μg, or at least 200 μg. In some embodiments, the method comprises a first 28-day treatment course and a second 28-day treatment course, wherein the first 28-day treatment course precedes the second 28-day treatment course, wherein the first dose of the second 28-day treatment course is equal to or higher than the first dose of the first 28-day treatment course, and the second dose of the second 28-day treatment course is equal to or higher than the second dose of the first 28-day treatment course, wherein the first dose of the first 21-day treatment course is at most 25 μg, at most 50 μg, at most 100 μg, at most 150 μg, or at most 200 μg. In some embodiments, the method comprises a first 28-day treatment course and a second 28-day treatment course, wherein the first 28-day treatment course precedes the second 28-day treatment course, wherein the first dose of the second 28-day treatment course is equal to or higher than the first dose of the first 28-day treatment course, and the second dose of the second 28-day treatment course is equal to or higher than the second dose of the first 28-day treatment course, wherein the first dose of the first 21-day treatment course is about 25 μg, about 50 μg, about 100 μg, about 150 μg, or about 200 μg.

[0180] In some embodiments, the method comprises at least one 21-day treatment course and at least one 28-day treatment course, wherein the doses are administered on days 1, 8, and 15 of the 21-day treatment course and on days 1 and 15 of the 28-day treatment course. In some embodiments, the 21-day or 28-day treatment course is repeated at least once. In some embodiments, the method comprises a first 28-day treatment course and a second 28-day treatment course, wherein the doses are administered on days 1, 8, and 15 of the first 28-day treatment course and on days 1 and 15 of the second 28-day treatment course. In some embodiments, either of the 28-day treatment courses is repeated at least once. In some embodiments, the method comprises at least one 21-day treatment course and at least one 28-day treatment course, wherein the doses are administered on days 1, 4, and 8 of the 21-day treatment course and on days 1 and 15 of the 28-day treatment course. In some embodiments, the 21-day or 28-day treatment course is repeated at least once.

[0181] In some embodiments, the method comprises at least two treatment courses disclosed herein, wherein administration is suspended or paused during the time period between the two treatment courses. In some embodiments, administration is continued after the suspension or pause.

[0182] In some embodiments, administering comprises administering by intravenous infusion. In some embodiments, administering comprises administering over about 30 min to about 2 h, for example, about 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47 min, 48 min, 49 min, 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58 min, 59 min, 60 min, 61 min, 62 min, 63 min, 64 min, 65 min, 66 min, 67 min, 68 min, 69 min, 70 min, 71 min, 72 min, 73 min, 74 min, 75 min, 76 min The administration may be by intravenous infusion at 120 min, 140 min, 147 min, 148 min, 149 min, 150 min, 151 min, 152 min, 153 min, 154 min, 155 min, 156 min, 157 min, 158 min, 159 min, or about 160 min, or any time period therebetween.

[0183] In some embodiments, administering comprises administering a mixture comprising the pharmaceutical composition and dextrose, wherein the mixture comprises about 5% (w / v) dextrose. In some embodiments, administering comprises administering a mixture comprising the pharmaceutical composition and dextrose, wherein the mixture comprises about 5% (v / v) dextrose.

[0184] In some embodiments, the pharmaceutical composition comprises an isolated recombinant polypeptide complex at a concentration of about 2 mg / ml. In some embodiments, the pharmaceutically acceptable excipient comprises a buffer, a stabilizer, a tonicity agent, a surfactant, or a combination thereof. In some embodiments, the pharmaceutically acceptable excipient comprises a buffer. In some embodiments, the pharmaceutically acceptable excipient comprises a tonicity agent. In some embodiments, the pharmaceutically acceptable excipient comprises a surfactant. In some embodiments, the pharmaceutically acceptable excipient comprises a buffer, a stabilizer, a tonicity agent, and a surfactant. 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 a polysorbate. In some embodiments, the surfactant comprises polysorbate 20 (PS20).

[0185] In some embodiments, the pharmaceutical composition comprises about 1 millimolar (mM) to about 50 mM L-histidine in the form of L-histidine and / or L-histidine monohydrochloride monohydrate, for example, about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 1 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM or about 50 mM or any concentration therebetween. In some embodiments, the pharmaceutical composition comprises about 1 to about 50 mM L-histidine in the form of L-histidine and L-histidine monohydrochloride monohydrate, for example, about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, In some embodiments, the pharmaceutical composition comprises about 10 mM of L-histidine in the form of L-histidine and / or L-histidine monohydrochloride monohydrate. In some embodiments, the pharmaceutical composition comprises about 10 mM of L-histidine in the form of L-histidine or L-histidine monohydrochloride monohydrate. In some embodiments, the pharmaceutical composition comprises about 10 mM L-histidine in the form of L-histidine and L-histidine monohydrochloride monohydrate.

[0186] In some embodiments, the pharmaceutical composition comprises about 1% weight / volume (w / v) to about 20% (w / v) sucrose, for example, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or about 20%, or any concentration therebetween. In some embodiments, the pharmaceutical composition comprises about 8% (w / v) sucrose.

[0187] In some embodiments, the pharmaceutical composition comprises about 0.001% (w / v) to about 0.1% (w / v) polysorbate 20 (PS20), for example, about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.011%, 0.012%, 0.013%, 0.014%, 0.015%, 0.016%, 0.017%, 0.018%, 0.019% , 0.02%, 0.021%, 0.022%, 0.023%, 0.024%, 0.025%, 0.026%, 0.027%, 0.028%, 0.029%, 0.03%, 0.031%, 0.032%, 0.033%, 0.034%, 0.035%, 0.036%, 0.037%, 0.038%, 0.039%, 0.04%, 0.041%, 0.042%, 0.043%, 0.044%, 0.045%, 0.046%, 0. 0.047%, 0.048%, 0.049%, 0.05%, 0.051%, 0.052%, 0.053%, 0.054%, 0.055%, 0.056%, 0.057%, 0.058%, 0.059%, 0.06%, 0.061%, 0.062%, 0.063%, 0.064%, 0.065%, 0.066%, 0.067%, 0.068%, 0.069%, 0.07%, 0.071%, 0.072%, 0.073%, 0.074 In some embodiments, the pharmaceutical composition comprises about 0.01% (w / v) polysorbate 20 (PS20).

[0188] In some embodiments, the pharmaceutical composition comprises about 10mM L-histidine in the form of L-histidine and L-histidine monohydrochloride monohydrate. In some embodiments, the pharmaceutical composition comprises about 8% weight / volume (w / v) sucrose. In some embodiments, the pharmaceutical composition comprises about 0.01% (w / v) polysorbate 20 (PS20). In some embodiments, the pharmaceutical composition comprises about 10mM L-histidine in the form of L-histidine and L-histidine monohydrochloride monohydrate, about 8% (w / v) sucrose and about 0.01% (w / v) polysorbate 20. In some embodiments, the pharmaceutical composition comprises a pH of about 5.3. In some embodiments, the pharmaceutical composition comprises an osmotic pressure of about 276mOsmol / kg.

[0189] In some embodiments, the method further comprises treating the subject with a therapy for an infusion-related reaction before or after administration. In some embodiments, the method further comprises treating the subject with a therapy for an infusion-related reaction before administration. In some embodiments, the method further comprises treating the subject with a therapy for an infusion-related reaction after administration. In some embodiments, the method further comprises treating the subject with a therapy for an infusion-related reaction before each dose of the isolated recombinant polypeptide complex is administered. In some embodiments, the method further comprises treating the subject with a therapy for an infusion-related reaction after administering one dose of the isolated recombinant polypeptide complex. In some embodiments, the method further comprises treating the subject with a therapy for an infusion-related reaction after administering one dose of the isolated recombinant polypeptide complex and before administering a second dose of the isolated recombinant polypeptide complex. In some embodiments, the therapy for an infusion-related reaction comprises acetaminophen, paracetamol, or an antihistamine. In some embodiments, the therapy for an infusion-related reaction comprises acetaminophen or paracetamol and an antihistamine. In some embodiments, the therapy for infusion-related reactions includes a therapy for treating fever, chills, rash, hives, dyspnea, hypotension or nausea. In some embodiments, the method further includes treating the subject with a therapy for cytokine release syndrome (CRS) before or after administration. In some embodiments, the therapy for CRS includes administering glucocorticoids, intravenous pre-hydration (pre-hydration) or suspending antihypertensive drugs before the first dose in the method. In some embodiments, the therapy for CRS includes administering glucocorticoids, intravenous pre-hydration or suspending antihypertensive drugs after the first dose in the method. In some embodiments, the therapy for CRS includes administering glucocorticoids, intravenous pre-hydration or suspending antihypertensive drugs before a dose in the method. In some embodiments, the therapy for CRS includes administering glucocorticoids, intravenous pre-hydration or suspending antihypertensive drugs before a dose in the method. In some embodiments, the therapy for CRS includes administering glucocorticoids, intravenous pre-hydration or suspending antihypertensive drugs after ... a fever, tachycardia, hypotension, hypoxia, fatigue, nausea, headache, dyspnea, chills, myalgia / arthralgia or anorexia. In some embodiments, the method further comprises treating the subject with a therapy for tumor lysis syndrome (TLS) before or after administration. In some embodiments, the therapy for TLS comprises intravenous rehydration, hypouric acid agents, or correction of acidosis before or after administration. In some embodiments, the therapy for TLS comprises therapy for hyperuricemia, hyperkalemia, hyperphosphatemia, and hypocalcemia.

[0190] In some embodiments, after administration, the separated recombinant polypeptide complex is cleaved by a protease to generate an enzyme product of the separated recombinant polypeptide complex. In some embodiments, after administration, the separated recombinant polypeptide complex is cleaved by a tumor-specific protease to generate an enzyme product of the separated recombinant polypeptide complex. In some embodiments, the tumor-specific protease comprises two or more proteases. In some embodiments, the separated recombinant polypeptide complex is cleaved by a first protease of the two or more proteases to generate a first metabolite of the separated recombinant polypeptide complex. In some embodiments, the separated recombinant polypeptide complex is cleaved by a second protease of the two or more proteases to generate a second metabolite of the separated recombinant polypeptide complex. 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 human membrane serine protease (MTSP1). In some embodiments, the matrix metalloprotease comprises human matrix metalloprotease 9 (MMP9).

[0191] In some embodiments, the enzyme product of the separated recombinant polypeptide includes a first metabolite. In some embodiments, the enzyme product of the separated recombinant polypeptide includes a second metabolite. In some embodiments, the enzyme product of the separated recombinant polypeptide includes a first metabolite and a second metabolite.

[0192] In some embodiments, the first metabolite comprises: a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6, or both. In some embodiments, the first metabolite comprises a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the first metabolite comprises a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, and the first amino acid sequence is 229 amino acids in length. In some embodiments, the first metabolite comprises a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the first metabolite comprises a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the second amino acid sequence is 492 amino acids in length.

[0193] In some embodiments, the first metabolite comprises a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, and a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the first metabolite comprises: a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, and the first amino acid sequence is 229 amino acids in length; and a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the second amino acid sequence is 492 amino acids in length.

[0194] In some embodiments, the first metabolite comprises a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, and a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the first metabolite comprises: a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, and the first amino acid sequence is 229 amino acids in length; and a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2, and the second amino acid sequence is 653 amino acids in length.

[0195] In some embodiments, the first metabolite comprises a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1, and a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the first metabolite comprises: a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1, and the first amino acid sequence is 256 amino acids in length; and a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the second amino acid sequence is 492 amino acids in length.

[0196] In some embodiments, the first metabolite comprises a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the first metabolite comprises a first amino acid sequence comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, the first metabolite comprises a second amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the first metabolite comprises a second amino acid sequence comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the first metabolite comprises a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first metabolite comprises a first amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first metabolite comprises a second amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the first metabolite comprises a second amino acid sequence comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the first amino acid sequence of the first metabolite is 256 amino acids in length. In some embodiments, the first amino acid sequence of the first metabolite is less than 256 amino acids in length. In some embodiments, the second amino acid sequence of the first metabolite is 653 amino acids in length.In some embodiments, the second amino acid sequence of the first metabolite is less than 653 amino acids in length. In some embodiments, the first amino acid sequence of the first metabolite is 229 amino acids in length. In some embodiments, the first amino acid sequence of the first metabolite is less than 229 amino acids in length. In some embodiments, the second amino acid sequence of the first metabolite is 492 amino acids in length. In some embodiments, the second amino acid sequence of the first metabolite is less than 492 amino acids in length.

[0197] In some embodiments, the first metabolite comprises: a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4, and a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: NO:6 amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to a second amino acid sequence. In some embodiments, the first metabolite comprises a first amino acid sequence comprising the amino acid sequence of SEQ ID NO:4 and a second amino acid sequence comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first metabolite comprises a second amino acid sequence comprising the amino acid sequence of SEQ ID NO:2. In some embodiments, the first amino acid sequence of the first metabolite is 256 amino acids in length. In some embodiments, the first amino acid sequence of the first metabolite has less than 256 amino acids in length. In some embodiments, the second amino acid sequence of the first metabolite is 653 amino acids in length. In some embodiments, the second amino acid sequence of the first metabolite is less than 653 amino acids in length. In some embodiments, the first amino acid sequence of the first metabolite is 229 amino acids in length. In some embodiments, the first amino acid sequence of the first metabolite is less than 229 amino acids in length. In some embodiments, the second amino acid sequence of the first metabolite is 492 amino acids in length. In some embodiments, the second amino acid sequence of the first metabolite is less than 492 amino acids in length.

[0198] In some embodiments, the first metabolite comprises: a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4, and a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: NO:2 amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to a second amino acid sequence. In some embodiments, the first metabolite comprises a first amino acid sequence comprising the amino acid sequence of SEQ ID NO:4 and a second amino acid sequence comprising the amino acid sequence of SEQ ID NO:2. In some embodiments, the first metabolite comprises a second amino acid sequence comprising the amino acid sequence of SEQ ID NO:2. In some embodiments, the first amino acid sequence of the first metabolite is 256 amino acids in length. In some embodiments, the first amino acid sequence of the first metabolite has less than 256 amino acids in length. In some embodiments, the second amino acid sequence of the first metabolite is 653 amino acids in length. In some embodiments, the second amino acid sequence of the first metabolite is less than 653 amino acids in length. In some embodiments, the first amino acid sequence of the first metabolite is 229 amino acids in length. In some embodiments, the first amino acid sequence of the first metabolite is less than 229 amino acids in length. In some embodiments, the second amino acid sequence of the first metabolite is 492 amino acids in length. In some embodiments, the second amino acid sequence of the first metabolite is less than 492 amino acids in length.

[0199] In some embodiments, the first metabolite comprises: a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1, and a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. NO:6 amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to a second amino acid sequence. In some embodiments, the first metabolite comprises a first amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1 and a second amino acid sequence comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the first metabolite comprises a second amino acid sequence comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the first amino acid sequence of the first metabolite is 256 amino acids in length. In some embodiments, the first amino acid sequence of the first metabolite has less than 256 amino acids in length. In some embodiments, the second amino acid sequence of the first metabolite is 653 amino acids in length. In some embodiments, the second amino acid sequence of the first metabolite is less than 653 amino acids in length. In some embodiments, the first amino acid sequence of the first metabolite is 229 amino acids in length. In some embodiments, the first amino acid sequence of the first metabolite is less than 229 amino acids in length. In some embodiments, the second amino acid sequence of the first metabolite is 492 amino acids in length. In some embodiments, the second amino acid sequence of the first metabolite is less than 492 amino acids in length.

[0200] In some embodiments, the second metabolite comprises: a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 3, a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 5, or both.

[0201] In some embodiments, the second metabolite comprises a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the second metabolite comprises a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 3, and the first amino acid sequence is 221 amino acids in length.

[0202] In some embodiments, the second metabolite comprises a second 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 a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the second amino acid sequence is 484 amino acids in length.

[0203] In some embodiments, the second metabolite comprises: a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 3, and the first amino acid sequence is 221 amino acids in length; and a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the second amino acid sequence is 484 amino acids in length.

[0204] In some embodiments, the second metabolite comprises a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 3, and a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the second metabolite comprises: a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 3, and the first amino acid sequence is 221 amino acids in length; and a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2, and the second amino acid sequence is 653 amino acids in length.

[0205] In some embodiments, the second metabolite comprises a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1, and a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:5.

[0206] In some embodiments, the second metabolite comprises: a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1, and the first amino acid sequence is 256 amino acids in length; and a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the second amino acid sequence is 484 amino acids in length.

[0207] In some embodiments, the second metabolite comprises a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the second metabolite comprises a first amino acid sequence comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the second metabolite comprises a second amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the second metabolite comprises a second amino acid sequence comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the second metabolite comprises a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the second metabolite comprises a first amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the second metabolite comprises a second amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the second metabolite comprises a second amino acid sequence comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the first amino acid sequence of the second metabolite is 256 amino acids in length. In some embodiments, the first amino acid sequence of the second metabolite is less than 256 amino acids in length. In some embodiments, the second amino acid sequence of the second metabolite is 653 amino acids in length.In some embodiments, the second amino acid sequence of the first metabolite is less than 653 amino acids in length. In some embodiments, the first amino acid sequence of the second metabolite is 221 amino acids in length. In some embodiments, the first amino acid sequence of the second metabolite is less than 221 amino acids in length. In some embodiments, the second amino acid sequence of the second metabolite is 484 amino acids in length. In some embodiments, the second amino acid sequence of the second metabolite is less than 484 amino acids in length.

[0208] In some embodiments, the second metabolite comprises: a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3, and a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: NO:5 amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to a second amino acid sequence. In some embodiments, the second metabolite comprises a first amino acid sequence comprising the amino acid sequence of SEQ ID NO:3 and a second amino acid sequence comprising the amino acid sequence of SEQ ID NO:5. In some embodiments, the first amino acid sequence of the second metabolite is 256 amino acids in length. In some embodiments, the first amino acid sequence of the second metabolite has less than 256 amino acids in length. In some embodiments, the second amino acid sequence of the second metabolite is 653 amino acids in length. In some embodiments, the second amino acid sequence of the first metabolite is less than 653 amino acids in length. In some embodiments, the first amino acid sequence of the second metabolite is 221 amino acids in length. In some embodiments, the first amino acid sequence of the second metabolite is less than 221 amino acids in length. In some embodiments, the second amino acid sequence of the second metabolite is 484 amino acids in length. In some embodiments, the second amino acid sequence of the second metabolite is less than 484 amino acids in length.

[0209] In some embodiments, the second metabolite comprises: a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3; and a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3; NO:2 amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to a second amino acid sequence. In some embodiments, the second metabolite comprises a first amino acid sequence comprising the amino acid sequence of SEQ ID NO:3 and a second amino acid sequence comprising the amino acid sequence of SEQ ID NO:2. In some embodiments, the first amino acid sequence of the second metabolite is 256 amino acids in length. In some embodiments, the first amino acid sequence of the second metabolite has less than 256 amino acids in length. In some embodiments, the second amino acid sequence of the second metabolite is 653 amino acids in length. In some embodiments, the second amino acid sequence of the first metabolite is less than 653 amino acids in length. In some embodiments, the first amino acid sequence of the second metabolite is 221 amino acids in length. In some embodiments, the first amino acid sequence of the second metabolite is less than 221 amino acids in length. In some embodiments, the second amino acid sequence of the second metabolite is 484 amino acids in length. In some embodiments, the second amino acid sequence of the second metabolite is less than 484 amino acids in length.

[0210] In some embodiments, the second metabolite comprises: a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1; and a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1; NO:5 amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to a second amino acid sequence. In some embodiments, the second metabolite comprises a first amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1 and a second amino acid sequence comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the first amino acid sequence of the second metabolite is 256 amino acids in length. In some embodiments, the first amino acid sequence of the second metabolite has less than 256 amino acids in length. In some embodiments, the second amino acid sequence of the second metabolite is 653 amino acids in length. In some embodiments, the second amino acid sequence of the first metabolite is less than 653 amino acids in length. In some embodiments, the first amino acid sequence of the second metabolite is 221 amino acids in length. In some embodiments, the first amino acid sequence of the second metabolite is less than 221 amino acids in length. In some embodiments, the second amino acid sequence of the second metabolite is 484 amino acids in length. In some embodiments, the second amino acid sequence of the second metabolite is less than 484 amino acids in length.

[0211] In some embodiments, the enzyme product of the isolated recombinant polypeptide complex comprises: a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3; and In some embodiments, the enzyme product of the isolated recombinant polypeptide complex comprises a first amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:3 and a second amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:6. In some embodiments, the enzyme product of the isolated recombinant polypeptide complex comprises: a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 3, and the first amino acid sequence is 221 amino acids in length; and a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6, and the second amino acid sequence is 492 amino acids in length. In some embodiments, the second metabolite comprises a first amino acid sequence comprising the amino acid sequence of SEQ ID NO: 3 and a second amino acid sequence comprising the amino acid sequence of SEQ ID NO: 6.

[0212] In some embodiments, the enzyme product of the isolated recombinant polypeptide complex comprises: a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4, and a first amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4. In some embodiments, the enzyme product of the isolated recombinant polypeptide complex comprises a first amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:4 and a second amino acid sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:5. In some embodiments, the enzyme product of the isolated recombinant polypeptide complex comprises: a first amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, and the first amino acid sequence is 229 amino acids in length; and a second amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 5, and the second amino acid sequence is 484 amino acids in length. In some embodiments, the second metabolite comprises a first amino acid sequence comprising the amino acid sequence of SEQ ID NO: 4 and a second amino acid sequence comprising the amino acid sequence of SEQ ID NO: 5.

[0213] In some embodiments, the first amino acid sequence of a first metabolite is 256 amino acids in length. In some embodiments, the first amino acid sequence of a first metabolite is less than 256 amino acids in length. In some embodiments, the second amino acid sequence of a first metabolite is 653 amino acids in length. In some embodiments, the second amino acid sequence of a first metabolite is less than 653 amino acids in length. In some embodiments, the first amino acid sequence of a first metabolite is 229 amino acids in length. In some embodiments, the first amino acid sequence of a first metabolite is less than 229 amino acids in length. In some embodiments, the second amino acid sequence of a first metabolite is 492 amino acids in length. In some embodiments, the second amino acid sequence of a first metabolite is less than 492 amino acids in length.

[0214] In some embodiments, the first amino acid sequence of the second metabolite is 256 amino acids in length. In some embodiments, the first amino acid sequence of the second metabolite is less than 256 amino acids in length. In some embodiments, the second amino acid sequence of the second metabolite is 653 amino acids in length. In some embodiments, the second amino acid sequence of the first metabolite is less than 653 amino acids in length. In some embodiments, the first amino acid sequence of the second metabolite is 221 amino acids in length. In some embodiments, the first amino acid sequence of the second metabolite is less than 221 amino acids in length. In some embodiments, the second amino acid sequence of the second metabolite is 484 amino acids in length. In some embodiments, the second amino acid sequence of the second metabolite is less than 484 amino acids in length.

[0215] In some embodiments, the isolated recombinant polypeptide complex exhibits a dose-dependent cancer cell killing activity, wherein an increase in the dose of the isolated recombinant polypeptide complex results in an increase in the cancer cell killing activity. In some embodiments, the isolated recombinant polypeptide complex exhibits a dose-dependent cancer cell killing activity. In some embodiments, an increase in the dose of the isolated recombinant polypeptide complex results in an increase in the cancer cell killing activity. In some embodiments, the isolated recombinant polypeptide complex exhibits a cancer cell killing activity that is dependent on the amount of the enzyme product, wherein an increase in the amount of the enzyme product results in an increase in the cancer cell killing activity. In some embodiments, the isolated recombinant polypeptide complex exhibits a cancer cell killing activity that is dependent on the amount of the enzyme product. In some embodiments, an increase in the amount of the enzyme product results in an increase in the cancer cell killing activity. In some embodiments, the isolated recombinant polypeptide complex exhibits a cancer cell killing activity that is correlated with the expression level of EGFR on cancer cells, wherein an increase in the expression level of EGFR on cancer cells results in an increase in the cancer cell killing activity. In some embodiments, the isolated recombinant polypeptide complex exhibits a cancer cell killing activity that is correlated with the expression level of EGFR on cancer cells. In some embodiments, an increase in the expression level of EGFR on cancer cells results in an increase in the cancer cell killing activity. In some embodiments, the binding affinity of the enzyme product to EGFR is more than 300 times the binding affinity of the isolated recombinant polypeptide complex to EGFR. In some embodiments, the binding affinity of the enzyme product to cluster of differentiation 3 (CD3) is more than 1000 times the binding affinity of the isolated recombinant polypeptide complex to CD3.

[0216] In some embodiments, the isolated recombinant polypeptide complex binds to:

[0217] (a) Human EGFR, at a half-maximal effective concentration (EC) of approximately 109 nM50 );

[0218] (b) Human CD3, at an EC of approximately 89 nM 50 ;

[0219] (c) Human albumin, at an EC of approximately 0.1 nM 50 ;

[0220] (d) Cynomolgus monkey EGFR, with an EC of approximately 126 nM 50 ;

[0221] (e) Cynomolgus monkey CD3, at an EC of approximately 87 nM 50 and / or

[0222] (f) Cynomolgus monkey albumin, at an EC of approximately 0.3 nM 50 .

[0223] In some embodiments, the isolated recombinant polypeptide complexes are bound to mouse or rat EGFR, CD3, or albumin-binding ECs. 50 ECs that bind to human or cynomolgus monkey EGFR, CD3, or albumin 50 More than 1000 times.

[0224] In some embodiments, the first metabolite of the isolated recombinant polypeptide complex binds to:

[0225] (a) Human EGFR, at an EC of approximately 0.28 nM 50 ;

[0226] (b) Human CD3, at an EC of approximately 0.08 nM 50 ;

[0227] (c) Cynomolgus monkey EGFR, at an EC of approximately 0.27 nM 50 and / or

[0228] (d) Cynomolgus monkey CD3, at an EC of approximately 0.08 nM 50 .

[0229] In some embodiments, the first metabolite of the isolated recombinant polypeptide complex:

[0230] (a) does not bind to albumin derived from human, cynomolgus monkey, mouse, or rat;

[0231] (b) Binds to mouse EGFR with an EC of approximately 29 nM 50 ;

[0232] (c) EC binding to mouse CD3 50 EC that binds to human or cynomolgus monkey CD3 50 More than 1000 times;

[0233] (d) Binds to rat EGFR with an EC of approximately 26 nM 50 and / or

[0234] (e) Binds to rat CD3 with an EC of approximately 542 nM 50 .

[0235] In some embodiments, the second metabolite of the isolated recombinant polypeptide complex binds to:

[0236] (a) Human EGFR, at an EC of approximately 0.27 nM 50 ;

[0237] (b) Human CD3, at an EC of approximately 0.09 nM 50 ;

[0238] (c) Cynomolgus monkey EGFR, at an EC of approximately 0.26 nM 50 and / or

[0239] (d) Cynomolgus monkey CD3, at an EC of approximately 0.09 nM 50 .

[0240] In some embodiments, the second metabolite of the isolated recombinant polypeptide complex:

[0241] (a) does not bind to albumin derived from human, cynomolgus monkey, mouse, or rat;

[0242] (b) Binds to mouse EGFR with an EC of approximately 20 nM 50 ;

[0243] (c) EC binding to mouse CD3 50 EC that binds to human or cynomolgus monkey CD3 50 More than 1000 times;

[0244] (d) Binds to rat EGFR with an EC of approximately 18 nM 50 and / or

[0245] (e) Binds to rat CD3 with an EC of approximately 415 nM 50 .

[0246] In some embodiments, the isolated recombinant polypeptide complex exhibits cancer cell killing activity in an in vitro assay that is at least 100-fold weaker than the enzyme product of the isolated recombinant polypeptide complex. In some embodiments, the isolated recombinant polypeptide complex exhibits cancer cell killing activity in an in vitro assay that is at least 100-fold weaker than the EC of the enzyme product of the isolated recombinant polypeptide complex. 50Induce the release of cytokines from immune cells. In some embodiments, cancer cell killing activity is measured in the presence of cancer cells and immune cells. In some embodiments, cytokine release is measured in the presence of cancer cells and immune cells. In some embodiments, immune cells are human peripheral blood mononuclear cells (PBMCs). In some embodiments, cytokines include IFNγ, TNF, or IL-6. In some embodiments, cytokines include IFNγ. In some embodiments, cytokines include TNF. In some embodiments, cytokines include IL-6. In some embodiments, cytokines include IFNγ, TNF, and IL-6. In some embodiments, the subject is a human.

[0247] Antibody production

[0248] In some embodiments, the polypeptides described herein (e.g., antibodies and binding fragments thereof) are produced using any method known in the art for synthesizing polypeptides (e.g., antibodies), particularly by chemical synthesis or recombinant expression, and preferably by recombinant expression technology.

[0249] In some cases, the antibody or its binding fragment is expressed recombinantly and the nucleic acid encoding the antibody or its binding fragment is assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves synthesizing overlapping oligonucleotides comprising portions of the sequence encoding the antibody, annealing and ligating those oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0250] Alternatively, nucleic acid molecules encoding antibodies are optionally generated from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell that expresses immunoglobulins) by PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of the sequence or by cloning using oligonucleotide probes specific for a particular gene sequence.

[0251] In some cases, antibodies or binding proteins thereof are optionally generated by immunizing an animal (such as a mouse) to generate polyclonal antibodies, or more preferably, by generating monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497), or as described by Kozbor et al. (1983, Immunology Today 4:72), or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, clones encoding at least the Fab portion of an antibody are optionally obtained by screening a Fab expression library for Fab fragment clones that bind to a specific antigen (e.g., as described in Huse et al., 1989, Science 246: 1275-1281), or by screening antibody libraries (see, e.g., Clackson et al., 1991, Nature 352: 624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94: 4937).

[0252] In some embodiments, a technique developed for producing "chimeric antibodies" is used by splicing together the genes for a mouse antibody molecule of appropriate antigen specificity with the genes for a human antibody molecule of appropriate biological activity (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454). Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having variable regions derived from a murine monoclonal antibody and human immunoglobulin constant regions.

[0253] In some embodiments, the technology described for producing single-chain antibodies (U.S. Patent No. 4,694,778; Bird, 1988, Science 242: 423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; and Ward et al., 1989, Nature 334: 544-54) is adjusted to produce single-chain antibodies. Single-chain antibodies are formed by connecting the heavy chain and light chain fragments of the Fv region by an amino acid bridge, thereby producing a single-chain polypeptide. The technology for assembling functional Fv fragments in Escherichia coli (E. coli) (Skerra et al., 1988, Science 242: 1038-1041) is also optionally used.

[0254] In some embodiments, an expression vector comprising the nucleotide sequence of an antibody or a fragment thereof or the nucleotide sequence of an antibody or a fragment thereof is transported into a host cell by conventional techniques (e.g., electroporation, lipofection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In specific embodiments, the expression of the antibody is regulated by a constitutive, inducible, or tissue-specific promoter.

[0255] 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 vehicles for producing and subsequently purifying antibody coding sequences, but also represent cells that express antibodies or binding fragments thereof in situ when transformed or transfected with appropriate nucleotide coding sequences. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and Bacillus subtilis (B. subtilis)) transformed with recombinant bacterial phage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody or binding fragment coding sequences; yeast (e.g., Pichia pastoris) transformed with recombinant yeast expression vectors containing antibody or binding fragment coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing antibody or binding fragment coding sequences; infected heavy metalloproteinases (HMTs) or other similar vectors. The antibody or its binding fragment encoding sequence can be transformed into a plant cell system using a viral expression vector (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or a recombinant plasmid expression vector (e.g., Ti plasmid) containing the antibody or its binding fragment encoding sequence; or a mammalian cell system (e.g., COS, CHO, BH, HEK293, 293T, 3T3 cells) containing a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0256] In order to produce recombinant protein for a long time and in high yield, stable expression is preferred. In some cases, the cell line of through engineering approaches stably expressed antibody is optionally used. Host cell is not to use the expression vector containing viral replication origin, but is transformed with DNA and the selectable marker controlled by suitable expression control elements (for example, promoter, enhancer, sequence, transcription terminator, polyadenylation site etc.). After introducing foreign DNA, the cell of through engineering approaches is allowed to grow 1-2 days in enrichment medium, and then switch to selective medium. The selectable marker in the recombinant plasmid gives the resistance to selection, and allows cell to stably integrate plasmid into its chromosome and grow to form colony, and then clone and expand into cell line. This method can be advantageously used for the cell line of through engineering approaches expressed antibody or its binding fragment.

[0257] In some cases, a number of selection systems are used, including, but not limited to, blasticidin, bleomycin, herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes in tk cells, hgprt cells, or aprt cells, respectively. In addition, antimetabolite resistance has been used as the basis for selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy Pharmacy 12:576-581). 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May 1993, TIB TECH 11(5):155-215) and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).Methods well known in the 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 in Chapters 12 and 13 of Dracopoli et al. (eds.), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY; Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1).

[0258] In some cases, the expression level of the isolated recombinant polypeptide complex is 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)). When the marker in the vector system expressing the isolated recombinant polypeptide complex is amplifiable, an increase in the inhibitor level in the host cell culture will increase the copy number of the marker gene. Since the amplified region is related to the nucleotide sequence of the isolated recombinant polypeptide complex, the production of the isolated recombinant polypeptide complex will also be increased (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0259] In some cases, the isolated recombinant polypeptide complex is purified using any method known in the art, for example, by chromatography (e.g., ion exchange, affinity, particularly for a specific antigen following protein A, and fractionated column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying the protein.

[0260] expression vector

[0261] In some embodiments, the vector includes any suitable vector derived from a eukaryotic or prokaryotic source. In some cases, the vector is obtained from bacteria (e.g., Escherichia coli), insects, yeast (e.g., Pichia pastoris), algae, or mammalian sources. Exemplary bacterial vectors include pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

[0262] Exemplary insect vectors include pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBac M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393M11, pVL1393 M12, a FLAG vector (such as pPolh-FLAG1 or pPolh-MAT 2), or a MAT vector (such as pPolh-MAT1 or pPolh-MAT2).

[0263] In some cases, the yeast carrier includes pDEST TM 14 carriers, pDEST TM 15 carriers, pDEST TM 17 carriers, pDEST TM 24 carriers, pYES-DEST52 vector, pBAD-DEST49 Destination vector, pAO815 Pichia pastoris vector, pFLD1 Pichia pastoris vector, pGAPZA, pGAPZB & pGAPZC Pichia pastoris vector, pPIC3.5K Pichia pastoris vector, pPIC6 A, pPIC6 B & pPIC6 C Pichia pastoris vector, pPIC9K Pichia pastoris vector, pTEF1 / Zeo, pYES2 yeast vector, pYES2 / CT yeast vector, pYES2 / NT A, pYES2 / NT B & pYES2 / NT C yeast vector, or pYES3 / CT yeast vector.

[0264] Exemplary algal vectors include pChlamy-4 vectors or MCS vectors.

[0265] Examples of mammalian vectors include transient expression vectors or stable expression vectors. Mammalian transient expression vectors may include pcDNA, pcDNA3.1 + , pcDNA 3.1, pRK5, p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a, pFLAG-CMV 6b, pFLAG-CMV 6c, pFLAG-CMV 5.1, pFLAG-CMV 5a, pFLAG-CMV 5b, pFLAG-CMV 5c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3 or pBICEP-CMV 4. Mammalian stable expression vectors can include pFLAG-CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2.

[0266] In some cases, the cell-free system is a mixture of cytoplasmic and / or nuclear components from cells and is used for in vitro nucleic acid synthesis. In some cases, the cell-free system utilizes prokaryotic cell components or eukaryotic cell components. Sometimes, nucleic acid synthesis is obtained in a cell-free system based on, for example, Drosophila cells, Xenopus eggs, or HeLa cells. Exemplary cell-free systems include, but are not limited to, E. coli S30 extract systems, E. coli T7 S30 systems, or

[0267] host cells

[0268] In some embodiments, the host cell includes any suitable cell, such as a cell of natural origin or a genetically modified cell. In some cases, the host cell is a production host cell. In some cases, the host cell is a eukaryotic cell. In other cases, 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 bacteria or gram-negative bacteria. Sometimes gram-negative bacteria are anaerobic, rod-shaped, or both.

[0269] In some cases, the gram-positive bacteria include Actinomycetes, Firmicutes, or Soft-walled bacteria. In some cases, the gram-negative bacteria include Aquificae, Deinococcus-Thermus, Fibrobacteres-Chlorobi / Bacteroidetes (FCB group), Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes-Verrucomicrobia / Chlamydiae (PVC group), Proteobacteria, Spirochaetes, or Synergistetes. Other bacteria may be Acidobacteria, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Dictyoglomi, Thermodesulfobacteria, or Thermotogae. The bacterial cell may be Escherichia coli, Clostridium botulinum, or Coli bacilli.

[0270] Exemplary prokaryotic host cells include, but are not limited to, BL21, Mach1 TM DH10B TM 、TOP10、DH5α、DH10Bac TM 、OmniMax TM 、MegaXTM DH12S TM , INV110, TOP10F', INVαF, TOP10 / P3, ccdB Survival, PIR1, PIR2, Stbl2 TM 、Stbl3 TM or Stbl4 TM .

[0271] In some cases, animal cells include cells from vertebrates or from invertebrates. In some cases, animal cells include cells from marine invertebrates, fish, insects, amphibians, reptiles, or mammals. In some cases, fungal cells include yeast cells, such as brewer's yeast, baker's yeast, or wine yeast.

[0272] In some cases, yeast includes ascomycetes, such as yeast, mold, filamentous fungi, basidiomycetes or zygomycetes. In some cases, yeast includes ascomycetes or basidiomycetes. In some cases, ascomycetes include Saccharomycotina (true yeast, for example Saccharomyces cerevisiae (baker's yeast)) or Taphrinomycotina (for example Schizosaccharomycetes (fission yeast)). In some cases, Basidiomycetes include Agaricomycotina (for example Tremellomycetes) or Pucciniomycotina (for example Microbotryomycetes).

[0273] Exemplary yeasts or filamentous fungi include, for example, the genera Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula, Kluyveromyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidi, Aspergillus, Fusarium, or Trichoderma.Exemplary yeasts or filamentous fungi include, e.g., species such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, Candida boidini, Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida viswanathii, Candidalusitaniae, Rhodotorula mucilaginosa, Pichia metanolica, Pichia angusta, Pichia pastoris, Pichia anomala, and Pichia spp. anomala), Hansenula polymorpha, Kluyveromyces lactis, Zygosaccharomyces rouxii, Yarrowia lipolytica, Trichosporon pullulans, Rhodosporidium toru-Aspergillus niger, Aspergillus nidulans, Aspergillus awamori, Aspergillus oryzae, Trichoderma reesei, Yarrowia lipolytica, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii, Zygosaccharomyces bailii), Cryptococcus neoformans, Cryptococcus gattii, or Saccharomyces boulardii.

[0274] Exemplary yeast host cells include, but are not limited to, Pichia pastoris strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33; and Saccharomyces cerevisiae strains such as INVSc1.

[0275] In some cases, additional animal cells include cells obtained from molluscs, arthropods, annelids, or sponges. In some cases, additional animal cells are mammalian cells, such as from primates, apes, horses, cattle, pigs, dogs, cats, or rodents. In some cases, rodents include mice, rats, hamsters, gerbils, hamsters, chinchillas, squirrels, or guinea pigs.

[0276] Exemplary mammalian host cells include, but are not limited to, 293A cell line, 293FT cell line, 293F cells, 293H cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, FUT8 KO CHOK1, ExpiCHO-S cells, Expi293F cells, TM cells, Flp-In TM T-REx TM 293 cell line, Flp-In TM -293 cell line, Flp-In TM -3T3 cell line, Flp-In TM -BHK cell line, Flp-In TM -CHO cell lines, 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 MSR cell line, GS-CHO cell line, HepaRG TM cells, T-REx TM Jurkat cell line, Per.C6 cells, T-REx TM -293 cell line, T-REx TM -CHO cell lines and T-REx TM -HeLa cell line.

[0277] In some cases, the mammalian host cell is a stable cell line, or a cell line that has integrated the genetic material of interest into its own genome and has the ability to express the product of the genetic material after multiple generations of cell division. In some cases, the mammalian host cell is a transient cell line, or a cell line that has not integrated the genetic material of interest into its own genome and does not have the ability to express the product of the genetic material after multiple generations of cell division.

[0278] Exemplary insect host cells include, but are not limited to, Drosophila S2 cells, Sf9 cells, Sf21 cells, HighFive TM Cells and cell.

[0279] In some cases, the plant cell comprises a cell from algae.Exemplary insect cell lines include, but are not limited to, strains from Chlamydomonas reinhardtii 137c or Synechococcuse longatus PPC 7942.

[0280] Products

[0281] In another aspect of the invention, a product is provided that contains materials that can be used to treat, prevent and / or diagnose the above-mentioned conditions. The product includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be made of a variety of materials (e.g., glass or plastic). The container holds a composition that is effective for treating, preventing and / or diagnosing the condition, either alone or in combination with another composition, and can have a sterile import (e.g., the container can be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody that specifically binds to EGFR and CD3.

[0282] The label or package insert indicates that the composition is used to treat the selected condition. In addition, the article of manufacture may include (a) a first container containing a composition contained therein, wherein the composition comprises a bispecific antibody of the invention; and (b) a second container containing a composition contained therein, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent. The article of manufacture in this embodiment of the invention may also include a package insert indicating that the composition can be used to treat a specific condition.

[0283] Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0284] Although preferred embodiments of the present invention have been shown and described herein, it will be understood by those skilled in the art that these embodiments are provided by way of example only. Many variations, changes, and substitutions will now occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in practicing the present invention. The appended claims are intended to define the scope of the present invention and are intended to encompass methods and structures within the scope of these claims and their equivalents.

[0285] Certain terms

[0286] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the claimed subject matter belongs. It should be understood that the above general description and the following detailed description are exemplary and explanatory only and do not limit any subject matter claimed. In this application, unless otherwise expressly stated, the use of the singular also includes the plural. It must be noted that, as used in the specification and the appended claims, the singular forms "a", "an", and "the / said" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "including" as well as other forms such as "comprising", "containing", and "having" are not restrictive.

[0287] As used herein, ranges and amounts can be expressed as "about" a particular value or range. About also includes an exact amount. Thus, "about 5 μL" means "about 5 μL" and "5 μL." In general, the term "about" includes amounts that might be expected to be within experimental error.

[0288] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0289] "Antibodies" and "immunoglobulins" (IG) are glycoproteins with the same structural characteristics. The two terms are used synonymously. In some cases, the antigen specificity of an immunoglobulin is known.

[0290] The term "antibody" is used in the broadest sense and covers fully assembled antibodies, antibody fragments that can bind to an antigen (e.g., Fab, F(ab')2, Fv, single-chain antibodies (scFv), diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, etc.), and recombinant peptides comprising the above antibodies.

[0291] As used herein, the terms "individual," "subject," and "patient" refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is non-human. None of these terms require or are limited to situations characterized by monitoring (e.g., continuous or intermittent monitoring) by a healthcare worker (e.g., a physician, registered nurse, licensed practical nurse, physician's assistant, caregiver, or hospice worker).

[0292] As used herein, the term "amino acid sequence identity percentage (%)" about sequence is defined as comparing the sequence and introducing a gap to reach maximum sequence identity percentage, and after any conservative substitution is not considered as a part for sequence identity, the percentage of the amino acid residue identical with the amino acid residue in the specific sequence in the candidate sequence. The comparison for determining the amino acid sequence identity percentage as purpose can be realized by the whole bag of tricks that those skilled in the art understand, for example, using the available computer software of the public, such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the comparison, are included in and realize maximum comparison required any algorithm on the entire length of the comparative sequence.

[0293] In the case where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A compared to, with, or relative to the amino acid sequence of a given amino acid sequence B (which can also be expressed as a given amino acid sequence A having or including a certain % amino acid sequence identity compared to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 multiplied by the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program alignment of A and B, and where Y is the total number of amino acid residues in B. It will be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless expressly stated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program, as described in the preceding paragraph.

[0294] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is non-human. None of these terms require or are limited to situations characterized by monitoring (e.g., continuous or intermittent monitoring) by a healthcare worker (e.g., a physician, registered nurse, licensed practical nurse, physician's assistant, orderlies, or hospice workers).

[0295] Example

[0296] Example 1: Production and purification process

[0297] summary

[0298] The production process is divided into upstream (cell culture), harvest, and downstream (purification). The upstream process consists of upstream fed-batch expression in Chinese hamster ovary (CHO)-K1 cell culture, followed by the harvest step. The downstream process includes multiple purification steps, followed by final formulation and batch filtration.

[0299] The production and purification of recombinant antibodies follow Figure 3-4 The steps shown are shown in Figure 2. The upstream process starts with thawing a single vial of the master cell bank, followed by cell culture expansion and fed-batch production of recombinant antibodies in the harvested cell culture fluid (HCCF). Basal medium 1 consisting of CD CHO culture medium containing glutamine, hypoxanthine and thymidine is inoculated with one vial of the master cell bank and expanded to a specified viable cell density in a shake flask. The culture is further expanded in 20 liters (L) and 50L single-use bioreactors, which are equipped with basal medium 2 consisting of ActiPro culture medium containing glutamine, hypoxanthine and thymidine, and then transferred to a 250L single-use bioreactor for fed-batch production. The cell culture is carried out for approximately 12 days, at which point it is harvested by depth filtration.

[0300] The downstream process first captures the recombinant antibody from the harvested cell culture fluid (HCCF) by protein A affinity chromatography, followed by viral inactivation, neutralization, and depth filtration steps at low pH. Host cell impurities are removed by a moderate polishing step using a multimodal anion exchange chromatography resin. The recombinant antibody is recovered in the flow-through and further refined by step elution with a hydrophobic interaction chromatography resin. A viral nanofiltration step is performed to remove any potential adventitious agents. The product is then ultrafiltered and diafiltered using histidine buffer pH 5.3, followed by the addition of sucrose and polysorbate 20 to improve batch stability. The formulated product is filtered and aseptically dispensed into single-use sterile polycarbonate bottles, and the bulk drug substance is stored at -70°C ± 10°C.

[0301] Upstream cell culture process

[0302] Thawing of vials and cell culture in shake flasks

[0303] The manufacturing process begins by thawing a vial of master cell bank in a 37.0°C ± 1°C water bath and aseptically transferring the cells to a shake flask. The cells are then resuspended in fresh Basal Medium 1, consisting of CD CHO medium containing glutamine, hypoxanthine, and thymidine. Under controlled environmental conditions, the cell culture is further subcultured into shake flasks of increasing size at a defined viable cell density (VCD) and viability by adding fresh Basal Medium 1. A backup inoculum is generated at the N-3 stage and maintained for up to three passages.

[0304] Cell expansion in a 20L bioreactor

[0305] After two rounds of expansion in shake flasks, and when the cell culture reached the desired VCD and viability was above 90.0%, the cells were cultured with basal medium 2 consisting of ActiPro medium containing glutamine, hypoxanthine, and thymidine at a concentration of approximately 0.35 × 10 6 The cell culture was transferred into a 20 L disposable bioreactor at a cell density of 10 cells / mL.

[0306] Cell expansion in a 50L bioreactor

[0307] Cells from the 20 L bioreactor were cultured at a rate of approximately 0.45 × 10 6 The target VCD of cells / mL and a viability greater than 90% were inoculated into a 50 L single-use bioreactor filled with basal medium 2. The temperature and agitation speed were set to approximately 36.5° C. and 150 rpm, respectively. pH and dissolved oxygen (DO) were controlled by CO and air / oxygen sparging. The cell culture was continued to expand to the desired VCD with a viability greater than 90%.

[0308] Cell culture in a 250L bioreactor

[0309] Cell culture was performed in a 250 L single-use bioreactor. The temperature was set at approximately 36.5°C during inoculation and the culture temperature was set at approximately (10.00-16.00) × 10 6 cells / mL or around day 5, the temperature was shifted to 32.0°C. pH and dissolved oxygen were monitored and controlled at approximately 6.90% and 40.0%, respectively, and the 250 L bioreactor was operated with agitation at 100 rpm and air / oxygen and CO sparging. Cell culture was continued with regular feed additions and glucose supplementation. The culture was harvested on day 12 or when the viability dropped below 85%, whichever came first.

[0310] Cell harvesting and clarification

[0311] When harvest criteria were met, cell cultures were harvested and clarified by depth filtration using a 2-stage double layer regenerated cellulose filter to remove cells and cell debris.

[0312] Downstream purification process

[0313] Affinity chromatography

[0314] The principle of this chromatography step is to capture the target protein recombinant antibody by affinity binding using MabSelect PrismA Protein A resin or equivalent, while allowing impurities to be removed by flow-through of the packed column.

[0315] The column was rinsed, sterilized, and equilibrated with 50 mM Tris-HAc, 150 mM NaCl, pH 7.4 buffer. The clarified cell culture fluid was loaded onto the column, which was then washed with high / low salt and pH to remove impurities. The bound recombinant antibody was eluted with 30 mM sodium acetate-acetic acid (NaAc-HAc), pH 4.3 at the same flow rate.

[0316] Low pH virus inactivation and neutralization

[0317] The pH of the Protein A eluate was adjusted to pH 3.6 ± 0.1 with 1 molar (M) acetic acid (HAc) and maintained at 18-26°C while stirring to achieve robust viral inactivation. After 1-2 hours under these conditions, the solution was neutralized with Tris-base and maintained at ambient temperature before a medium depth filtration step.

[0318] Medium depth filtration

[0319] The mid-depth filtration step removes precipitates that can form during the low-pH viral inactivation and neutralization process steps. Prior to loading the neutralized product pool, the regenerated cellulose depth filter is equilibrated with buffer. After loading, the filter is rinsed with equilibration buffer, and the combined filtrates are further filtered through 0.5 / 0.2 μm into a sterile mixing and storage bag.

[0320] Multimodal anion exchange chromatography

[0321] The multimodal anion exchange chromatography step was performed in flowthrough mode using Capto Adhere resin or equivalent. The pH and conductivity of the filtrate from the intermediate depth filtration step were adjusted, and loading was performed at a maximum linear flow rate of 300 cm / h and a minimum residence time of 5 minutes. After loading was complete, the column was washed, and the eluate was filtered 0.5 / 0.2 μm during collection.

[0322] Hydrophobic interaction chromatography

[0323] Hydrophobic interaction (Butyl Sepharose 4FF or equivalent) chromatography in bind-elute mode is used as a polishing step to further remove impurities. During preparation, the conductivity of the multimodal anion exchange eluate is adjusted and then 0.5 / 0.2 μm filtered into a storage bag. The Butyl Sepharose 4FF column is rinsed, sterilized, and equilibrated before loading the adjusted pool. After loading is complete, the column is washed with equilibration / wash buffer. The bound recombinant antibody is eluted from the column and the eluate is filtered 0.5 / 0.2 μm during collection.

[0324] Virus filtration

[0325] The virus filtration step removes potential viral particles and consists of a 0.5 / 0.2 μm prefilter, a 20 nanometer (nm) virus-retaining filter, and a 0.5 / 0.2 μm filter in series. During loading, the pressure differential across the prefilter is maintained at ≤2 bar, the pressure differential across the virus-retaining nanofilter is maintained between 0.7 and 1.0 bar, and the pressure differential across the final 0.5 / 0.2 μm filter is maintained at ≤2 bar. After loading is complete, the filters are rinsed with wash buffer. The final combined filtrates are mixed before proceeding to the next step.

[0326] Ultrafiltration and diafiltration

[0327] Ultrafiltration and diafiltration are used to adjust in-process drug substance protein concentration and exchange buffers prior to final batch formulation.

[0328] Equilibrate an ultrafiltration / diafiltration unit with a 30 kilodalton (kDa) molecular weight cutoff filter cartridge until the defined pH and conductivity are reached. The filtrate from the previous step is pumped across the membrane surface and first concentrated, then diafiltered with histidine buffer until the pH and conductivity criteria are met. After circulating at a low flow rate, the DF pool is recovered and then rinsed with histidine buffer, pH 5.3.

[0329] Excipient addition, bulk formulation and filling

[0330] During batch formulation, sucrose and polysorbate 20 were added to the in-process material from stock solutions to final concentrations of 8% (w / v) and 0.01% (w / v), respectively, to improve product stability. The product was then diluted to the target concentration range using histidine buffer, pH 5.3. The formulated product was filtered and aseptically dispensed into individual, single-use, sterile polycarbonate bottles.

[0331] Example 2: Formulation Development

[0332] Initial screening

[0333] Formulation development activities are carried out to identify robust formulations that stabilize recombinant antibodies in liquid dosage forms that can be frozen or stored refrigerated. For this reason, initial formulation development studies were carried out at a protein concentration of 2 mg / mL to determine pH range (4.5, 5.0, 5.5, 6.0, 6.5, or 7.0), stabilizer type (8% (w / v) sucrose or 150 mM sodium chloride), buffer type (10 mM glutamate, 10 mM acetate, 10 mM histidine, 10 mM phosphate buffer), and surfactant was kept constant at 0.01% (w / v) polysorbate 20 (PS20). 12 formulations were prepared in total, and thermal stability, appearance (visible particles, color, and clarity), pH, protein concentration, size exclusion chromatography (SEC), capillary electrophoresis (reduction and non-reduction), and imaging capillary isoelectric focusing (iCIEF) were analyzed by differential scanning calorimetry (DSC). A summary of the formulation matrix is ​​shown in Table 2.

[0334] The samples were filled into 2R vials and subjected to three freeze / thaw (F / T) cycles (-70°C to room temperature), agitation (300 rpm at 25°C for 2 days), and 4 weeks at 33°C. The formulations were then evaluated for appearance, aggregation, and impurities.

[0335] Table 2: Formulation Design Matrix

[0336]

[0337] Thermal stability (DSC), appearance (visible particles, color, clarity), pH, protein concentration, SEC, capillary electrophoresis (reducing and non-reducing), and iCIEF were performed.

[0338] For SEC, notable changes included a significant decrease in purity observed for Formulations 6–12 after one week at 33°C, and a significant decrease in the purity of the main peak after two days of stirring for Formulation 7. For iCIEF, Formulations 6–12 showed severe degradation after one week at 33°C.

[0339] Based on the data generated from the initial screening experiments, the formulation matrices selected for further development in secondary screening studies were 10 mM acetate pH 5.0, 10 mM histidine pH 5.5, and 10 mM histidine pH 6.0.

[0340] Secondary screening

[0341] Secondary screening studies were conducted to further optimize the stabilizer, polysorbate 20 concentration, and pH so that the final formulation of the drug product could be selected. The study design for the secondary screening experiments is summarized in Table 3.

[0342] Table 3: Secondary Screening Study Design

[0343]

[0344] The samples were filled into 2R vials and subjected to three or five cycles of F / T (-70°C to room temperature), agitation (300 rpm at 25°C for 3 days), and storage for 3 weeks at 33°C. In addition to the test panels used to evaluate the samples in the initial screening study, the formulations were tested for osmotic pressure and subvisible particulate matter.

[0345] Samples from the secondary screening study were subjected to appearance (visible particles, color, clarity), pH, protein concentration, SEC, particulate matter (subvisible particles), capillary electrophoresis (reducing and non-reducing), osmolarity, and iCIEF.

[0346] No significant changes were observed after three weeks of incubation at 33°C, after three days of agitation, or for F / T of all formulations for pH, protein concentration, iCIEF, capillary electrophoresis (reduced and non-reduced), and subvisible particle results. The appearance of all samples was a clear, colorless solution with no visible particles.

[0347] SEC data showed that sucrose and lower concentrations of surfactant were most effective in stabilizing the protein.

[0348] To minimize the risk of aggregation, a relatively low pH (5.0 to 5.5) was chosen to increase the surface charge of the protein.

[0349] Based on the data generated from the secondary screening experiments, the formulation matrix selected for formulation validation was 2 mg / mL recombinant antibody, 10 mM histidine, 8% (w / v) sucrose, 0.01% (w / v) PS20, pH 5.3.

[0350] Formulation validation

[0351] Formulations selected from the secondary screening study (2 mg / mL recombinant antibody, 10 mM L-histidine, 8% (w / v) sucrose, 0.01% (w / v) PS20, pH 5.3) were evaluated in validation studies to assess the stability of the molecule in its final formulation.

[0352] For formulation validation studies, the final formulations of the recombinant antibodies were subjected to one and three F / T cycles (-20 ± 5 ° C / room temperature), stored at -20 ± 5 ° C for 12 weeks, stored at 2-8 ° C for 12 weeks, and stored at 25 ± 2 ° C for 4 weeks for stability studies. The frozen samples (-20 ± 5 ° C and F / T) were initially frozen at -40 ± 5 ° C and then transferred to -20 ± 5 ° C for storage. An overview of the validation studies is shown in Table 4.

[0353] Table 4: Overview of formulation validation studies

[0354]

[0355] NT – Not tested

[0356] No substantial changes were observed in the formulation validation studies for appearance (visible particles, color, clarity), pH, protein concentration, SEC, iCIEF, reduced CE-SDS, non-reduced caliper-SDS, and ELISA binding-EGFR (cleavage).

[0357] For all conditions, particle counts for particles ≥ 2 μm increased. However, particle counts for particles ≥ 2 μm were all < 130 particles / mL and were obtained using HIAC, so the increase in particle counts may be due to methodological and instrumental variations. These increases in particle counts are not considered practically significant.

[0358] Glass transition (Tg')

[0359] The glass transition temperature (Tg') was tested at the beginning of the formulation validation study. Tg' was measured to be -34.2°C, which is lower than the expected long-term storage temperature. The recombinant antibody can be frozen at -40±5°C before being transferred to -20±5°C.

[0360] Preparation validation conclusion

[0361] Based on data generated from formulation validation experiments, the formulation matrix was selected to be 2 mg / mL recombinant antibody, 10 mM L-histidine, 8% (w / v) sucrose, 0.01% (w / v) PS20, pH 5.3.

[0362] Example 3: Confirmation of disulfide bonds by LC-MS / MS

[0363] Disulfide bonds are important in protein folding and play a crucial role in both protein structure and function. The number and location of disulfide bonds are important properties for ensuring the safety and efficacy of biopharmaceuticals.

[0364] There are 20 cysteine ​​residues in PC-1, such as Figure 2 As shown in , it is cross-linked by 1 interchain disulfide bond and 9 intrachain disulfide bonds between the heavy chain and the light chain. The heavy chain contains six domains, and the light chain contains three domains. Non-reducing Lys-C / trypsin sequential digestion using PNGase F is expected to produce 10 disulfide bond-related peptides (DS1 to DS10). Since the theoretical mass of peptide DS8 is 11380.3093Da, it indicates that the peptide may be highly hydrophobic and difficult to be ionized by MS spectroscopy. In order to further illustrate the cysteine ​​connection, Lys-C / trypsin sequential digestion was applied. DS8 was digested into smaller peptides. Liquid chromatography and tandem mass spectrometry (LC-MS / MS) results are provided in Table 5.

[0365] Table 5: Liquid chromatography and tandem mass spectrometry (LC-MS / MS) results

[0366]

[0367]

[0368]

[0369] Example 4: Secondary structure determination by far-UV CD

[0370] Far-UV circular dichroism (Far-UV CD) spectroscopy of proteins can reveal characteristic secondary structures, such as α-helices, β-sheets, and random coils. Prior to measurement, protein samples were diluted with ultrapure water to a concentration of 0.1 mg / mL. Data were collected and analyzed using a JASCO / J-815CD spectrometer and Spectra Manager software.

[0371] Figure 5 The CD spectrum of PC-1 in the far UV region (190-260 nm) is shown. β-sheets and random coils are the main secondary structural components.

[0372] Example 5: Tertiary structure determination by near UV-CD

[0373] Protein near-UV CD spectroscopy provides information about the tertiary structure of proteins. The CD spectral pattern in the 250-350 nm region is determined by the absorption, dipole orientation, and surrounding properties of phenylalanine (250-270 nm), tyrosine (270-290 nm), and tryptophan (290-305 nm), respectively. For measurement, protein samples were diluted to 1.0 mg / mL with 10 mM histidine, 8% (w / v) sucrose, and 0.01% (w / v) PS20 pH 5.3. Data collection and analysis were performed using a JASCO / J-815 CD spectrometer and Spectra Manager software.

[0374] Figure 6 The CD spectrum of PC-1 in the near UV region (250-350 nm) is shown. Spectral similarity / structural identity analysis was performed by calculating the correlation coefficient of the two spectra using the "Quick Compare" tool of OPUS Spectroscopy software.

[0375] Example 6: Thermal stability analysis by DSC

[0376] Differential Scanning Calorimetry (DSC) is an experimental technique for characterizing thermal transitions in materials. It is widely used in protein characterization to study the folding / unfolding transition of proteins under programmed temperature. m) of the melting onset (T 起始 ) and midpoint (T m ) temperature is often used as an indicator of thermal stability, and the DSC thermogram reveals thermal transition features.

[0377] PC-1 was analyzed using Malvern's MicroCal DSC. Prior to analysis, the protein sample was diluted to 1 mg / mL with 10 mM histidine, 8% (w / v) sucrose, 0.01% (w / v) PS20 pH 5.3. 400 microliters (μL) of the corresponding formulation buffer was added to a 96-well plate as a reference, and 400 μL of the protein sample was added. The sample was heated from 10°C to 95°C at a heating rate of 90°C / h in a capillary DSC system. The DSC data were analyzed and fitted using MicroCal PEAQ-DSC software 1.51. The results are shown in Figure 2. Figure 7 As shown. For PC-1, T 起始 The tm1 temperature was determined to be 62.5°C, and the midpoint temperature of both transitions (Tm2) was determined to be 73.8°C.

[0378] Example 7: Molar mass and size analysis by SEC-MALS

[0379] Size exclusion chromatography coupled with a multi-angle light scattering (SEC-MALS) detector separates proteins based on their size, and the molecular weight of the separated components is then measured by the MALS detector. Smaller proteins elute from the SEC column later, while larger proteins elute at earlier retention times and result in the separation of proteins based on their size differences. The separated components, including monomers, high molecular weight species (HMWS), and low molecular weight species (LMWS), are quantified by a UV detector. The absolute molar mass and size of the molecules in the solution are calculated using the intensity and angular dependence of the scattered light signal from the MALS detector. Static multi-angle light scattering characterizes the absolute molecular weight of proteins based on the principle of static light scattering, expressed as Zimm's equation. For proteins larger than 10 nm, the laser scattering intensity is directly proportional to the molecular weight and protein concentration. Therefore, the absolute molecular weight can be determined based on the relationship between the scattered light intensity and the angle P(θ), protein size R g The molecular weights of those proteins were calculated based on the relationship between the protein concentration c.

[0380] The SEC-MALS chromatogram of PC-1 is shown in Figures 8A-8D The molecular weight of the main peak (peak 1) was 90.2 kDa (RT = 3.111 min), corresponding to a monomer.

[0381] Example 8: Protein size analysis by dynamic light scattering

[0382] Dynamic light scattering (DLS) analysis is used to measure particle size by illuminating the particles with a laser and analyzing the intensity fluctuations of the scattered light. DLS analysis of PC-1 samples was performed using a Malvern dynamic light scattering instrument, Zen 3600. Protein samples in formulation buffer were transferred to DLS disposable cuvettes. Each sample was sampled once and tested three times. The data was automatically analyzed by Zetasizer Nano software and displayed as particle size (Z-average particle size) and polydispersity index (PDI). These results indicate that there were no significant differences in particle size and distribution among PC-1 samples.

[0383] The DLS results are shown in Figures 9A-9B and Table 6. The Z-average particle size of PC-1v2 was 14.8 nm, and the Z-average particle size of PC-1v1 was 14.6 nm. The polydispersity index (PDI) of PC1-v2 was 0.03, and the PDI of PC1-v1 was 0.02.

[0384] Table 6: DLS results

[0385]

[0386] (b)

[0387] #1 The Z-average represents the diameter of the protein particles.

[0388] #2 PDI indicates the uniformity of protein particles; the smaller the PDI, the more uniform the protein particles.

[0389] Example 9: Activity against tumors harboring resistance mutations

[0390] The isolated recombinant antibodies were evaluated in a functional in vitro tumor cell killing assay using EGFR-positive tumor cell lines harboring resistance mutations: HEK293, HeLa, A459, A549 EGFR knockout, H1650, HCT116, HT29, H1975, PC-9, FaDu, mutant PC-9, mutant H1975, Cal27, and A431, as shown in Table 7. The peptide complexes were evaluated in a functional in vitro tumor cell killing assay using EGFR-positive tumor cell lines. Tumor cell killing was measured using an xCelligence real-time cell analyzer from Agilent, which relies on sensor impedance measurement (cell index), which increases as tumor cells adhere, spread, and expand on the sensor surface. Similarly, impedance decreases when tumor cells are killed. 10,000 tumor cells were added per well and allowed to adhere overnight to a 96-well E-Plate. The next day, peptide complexes titrated in human serum-supplemented medium and 30,000 CD8+ T cells were added to the wells. The cell index was measured every 10 minutes for an additional 72 hours. The cell index multiplied by the number of hours (tumor cell growth kinetics) was then plotted against the concentration of the peptide complex, and the concentration required to reduce tumor growth by 50% (IC50) was calculated using Graphpad Prism software.

[0391] Table 7: Activity against tumors harboring resistance mutations

[0392]

[0393] Example 10: Clinical effects of polypeptide complex 1 (PC-1)

[0394] This example illustrates the clinical effects of the polypeptide complex 1 (PC-1) disclosed herein.

[0395] Summarize

[0396] 1.1PC-1

[0397] like Figure 1 As shown in , PC-1 is a tumor-activated T cell engager (TRACTr) that comprises a humanized trispecific protein containing epidermal growth factor receptor (EGFR) and cluster of differentiation 3 (CD3) binding domains, an albumin binding domain that extends circulation half-life, and two separate peptide masks. The peptide masks are fused to the molecule via a tumor protease-cleavable linker. One peptide mask inhibits EGFR engagement on target cells, while the other peptide mask inhibits CD3 engagement on T cells.

[0398] 1.2 Pharmacology

[0399] Nonclinical pharmacology in vitro studies of PC-1 examined the following:

[0400] 1. PC-1 binds to EGFR, CD3, and albumin antigens in mouse, rat, cynomolgus monkey, and human

[0401] 2. Stability of PC-1 in sera from healthy human donors and patients with colorectal cancer (CRC), head and neck squamous cell carcinoma (SCCHN), and non-small cell lung cancer (NSCLC), and cynomolgus monkeys

[0402] 3. PC-1's ability to induce T cell-mediated anti-tumor cytotoxic activity

[0403] 4. PC-1's ability to induce cytokine production

[0404] Results of binding studies concluded that PC-1 binds to human and cynomolgus monkey EGFR, CD3, and albumin with low nanomolar affinity, while exhibiting minimal binding to mouse or rat antigens. Cleavage-dependent unmasking of PC-1 results in subnanomolar affinity for human and cynomolgus monkey EGFR and CD3. Serum stability studies demonstrated that PC-1 is more susceptible to cleavage in cynomolgus monkey serum than in human serum or serum from CRC, SCCHN, and NSCLC patients. The cleavage rates of both masks were approximately 1% / day in healthy human serum and 2% in serum from CRC, SCCHN, and NSCLC patients, while the cleavage rates of PC-1 in cynomolgus monkey serum were 11.5% for the EGFR mask and 6.6% for the CD3 mask.

[0405] Cytotoxicity studies have shown that PC-1-induced tumor cell killing is cleavage-dependent and dose-dependent. Compared to the cleaved forms (PC-1 serine protease [SP]-cleavage and PC-1 matrix metalloproteinase [MMP]-cleavage) and unmasked PC-1-T cell engager (TCE), PC-1 exhibits much lower potency. Activity, which correlates with the density of EGFR expression on the surface of target tumor cells, is lowest in the A549 cell line (~8,000 to 12,000x relative to SP-cleaved PC-1). The estimated reduction in tumor killing in HCT116 and CAL27 cell lines is ~5,500x and ~450x, respectively. The activity of PC-1, i.e., the ability to induce T cell-mediated tumor cell killing, depends on the expression level of EGFR on the surface of target tumor cells. None of the test articles induced cytotoxicity against the control EGFR-deficient A549 cell line. In in vitro cytokine release assays, cleaved (PC-1-SP and PC-1-MMP cleavage) and unmasked (PC-1-TCE) test articles demonstrated potent dose-dependent induction of interferon gamma (IFNγ), tumor necrosis factor (TNF), and interleukin (IL)-6 release in the presence of EGFR-positive cell lines HCT116 and A549, while dose-dependent production of IFNγ and TNF, but not IL-6, was observed in the presence of EGFR-positive Cal27 cell line. Masked PC-1 showed reduced ability to induce cytokine release compared to PC-1-TCE in the presence of all cell lines.

[0406] An investigational variant of PC-1 with a histidine tag (PC-1-His tag) exhibited dose-dependent and cleavage-dependent antitumor activity in a mouse model of CRC. Preclinical evidence for this mechanism supports the requirement for protease activity in human tumors to achieve cleavage and antitumor activity of PC-1 in vivo. The nonclinical toxicity profile of PC-1 was studied in nonhuman primates. Based on clinical observations and detailed weekly examinations of animals after administration of ≤0.6 mg / kg / dose of PC-1, there were no effects on the central nervous system (CNS) or respiratory system. At PC-1 doses ≤0.6 mg / kg / week, PC-1 had no effect on qualitative electrocardiogram (ECG) parameters or on the QRS or heart rate-corrected QT interval. At doses ≥0.2 mg / kg / week, there was an increased incidence of animals with minimal increase in heart rate and associated shortening of mean RR, PR, and QT intervals 1 to 3 hours after the end of infusion (EOI) on days 1 and 22. These changes were not observed after the 4-week recovery period and were likely secondary to cytokine release and were not considered to be direct physiological effects of PC-1.

[0407] 1.3 Pharmacokinetics

[0408] The pharmacokinetic (PK) properties of PC-1 were evaluated in cynomolgus monkeys following single-dose and repeated-dose studies. The PK characteristics of PC-1 were studied in cynomolgus monkeys as part of nonclinical PK and toxicokinetic (TK) studies. A total of 3 studies were conducted: two single-dose non-good laboratory practice (GLP) PK studies in cynomolgus monkeys (one study at doses of 0.1, 0.3, and 1 mg / kg, and the other study at doses of 0.05, 0.2, and 0.6 mg / kg), and a repeated-dose GLP toxicology study in cynomolgus monkeys examining weekly doses of PC-1 (4 weeks; a total of 5 doses) at 0.05, 0.2, and 0.6 mg / kg / dose. When present, anti-drug antibodies (ADA) affect the PK characteristics of PC-1 in cynomolgus monkeys. In the first single-dose non-GLP PK study, PC-1 was administered to cynomolgus monkeys at doses of 0.1, 0.3, and 1 mg / kg via a single intravenous (IV) bolus. PC-1 concentrations increased with increasing doses. Mean Cmax increased in a greater than dose-proportional manner between doses of 0.1 and 0.3 mg / kg, and in a dose-proportional manner between 0.3 and 1 mg / kg. Mean AUC 0-216h The effect was greater than dose-proportional between doses of 0.1 and 0.3 mg / kg and less than dose-proportional between doses of 0.3 and 1 mg / kg. The mean t 1 / 2 In the range of 88.8 to 101 hours. Animals sacrificed after 24 hours had the highest observed Cmax and AUC0-24h in this group. PC-1-TCE was not detectable in any sample. In a second single-dose non-GLP PK study, PC-1 was administered to cynomolgus monkeys at doses of 0.05, 0.2, and 0.6 mg / kg by a single 30-minute IV infusion. The mean Cmax values ​​of PC-1 increased in an approximately dose-proportional manner. The mean AUC0-168h increased less than dose-proportionally from 0.05 to 0.2 mg / kg and increased in an approximately dose-proportional manner from 0.2 to 0.6 mg / kg. At 0.05, 0.2, and 0.6 mg / kg, the mean PC-1t1 / 2 The values ​​were 81.3, 68.2 and 96.5 hours, respectively. PC-1-TCE was not detectable in any sample. After five weekly doses of PC-1 administered IV at 0.05, 0.2 and 0.6 mg / kg / dose to cynomolgus monkeys over 30 minutes, the mean Cmax, mean AUC0-24hr and mean AUC0-24hr on day 1 post-dose were 0-168hrHowever, due to the presence of ADA in 3 of 6 animals at the 0.05 mg / kg dose, 6 of 6 animals at the 0.2 mg / kg dose, and 10 of 10 animals at the 0.6 mg / kg dose, the mean Cmax, mean AUC, and 0-24hr and / or mean AUC 0-168hr The increase was less than dose-proportional. No accumulation was observed after multiple doses, and Groups 2, 3, and 4 could not be fully evaluated due to the presence of confirmed ADA. No significant accumulation was observed in ADA-negative animals at 0.05 mg / kg / dose (Group 2) and at 0.2 mg / kg / dose (Group 3). One of the ADA-positive animals in Group 2 (2503) and two of the ADA-positive animals in Group 3 remained significantly exposed after the 22nd and / or 29th day dose, slightly lower than the day 1 dose, as measured by Cmax, AUC 0-24h and AUC 0-168h In addition, five of the ADA-positive animals in Group 4 maintained some exposure after the Day 22 dose, as measured by AUC0-24h, AUC0-168h, and Cmax, however, the exposure was significantly lower compared to the Day 1 dose. PC-1-TCE was not detectable in most samples, with the exception of one sample that had a measurable concentration close to the detection limit of the assay.

[0409] Pharmacokinetic drug interaction studies have not been conducted on PC-1. Typically, molecules such as PC-1 are not metabolized by cytochrome P450 (CYP) enzymes and are not transported by P-glycoprotein (Pgp) or related adenosine triphosphate-binding cassette membrane transporters. Cytokines produced by activated lymphocytes may affect Pgp levels and CYP enzyme activity (Harvey and Morgan, 2014). The clinical relevance of PC-1 in causing immunomodulation and potential cytokine production that may affect Pgp and CYP is unknown, but clinically relevant drug-drug interaction effects are considered highly unlikely. Therefore, based on guidance and scientific evidence (FDA August 2020; Huang et al., 2010; Seitz and Zhou, 2007), no PK drug-drug interaction studies have been conducted on PC-1.

[0410] 1.4 Toxicology

[0411] In a 4-week repeat dose toxicity study in monkeys, temporary clinical signs considered to be secondary to cytokine release were observed after administration of PC-1 at 0.6 mg / kg / dose on day 1. PC-1-related clinical chemistry and hematological changes associated with acute phase responses were observed at ≥0.05 mg / kg / dose. An increase in IL-10 at ≥0.2 mg / kg / dose and an increase in IL-6 and IFNγ at 0.6 mg / kg / dose were observed after PC-1 on day 1. Most changes noted during the study were observed after the first dose and returned to pre-dose levels before the next dose. After a 4-week recovery period, any remaining minor changes were fully reversible. The no observed adverse effect level (NOAEL) was considered to be 0.6 mg / kg / dose, the highest dose tested. Concentration-dependent or dose-dependent cytokine release (i.e., IL-6, IFNγ, and / or TNF) induced by PC-1 was observed in the presence of tumor cells in vitro and in normal monkeys without tumors in vivo. In vivo, cytokine release was primarily observed after the first dose and correlated with clinical signs and clinical chemistry changes indicative of cytokine release syndrome.

[0412] 1.5 Phase 1 Clinical Study Design

[0413] The study is a first-in-human (FIH), Phase 1 / 1b, open-label, multicenter dose escalation and dose expansion study to evaluate the safety, tolerability, PK, pharmacodynamics (PD), and preliminary anti-tumor activity of PC-1 in adult subjects with histologically confirmed advanced or metastatic CRC, NSCLC, renal cell carcinoma (RCC), and SCCHN. The study will be conducted in 3 parts: dose escalation (Part 1), approximately 40 to 50 subjects; cohort backfill expansion (Part 2), up to approximately 40 subjects, enrolled at 4 dose levels; dose expansion (Part 3), up to approximately 40 subjects, enrolled at the recommended Phase 2 dose (RP2D). The dose escalation (Part 1) will evaluate the safety, tolerability, PK, PD, and preliminary efficacy of PC-1 administered by IV infusion. The cohort backfill (Part 2) will allow further characterization of the safety and activity of the dose levels. The dose expansion (Part 3) will determine additional safety, tolerability, PK, PD, and preliminary clinical activity data for PC-1. The dose and schedule will be determined by the Safety Review Committee after reviewing all available safety, PK, PD, and preliminary efficacy data. Depending on the data, randomization may be integrated into two different RP2D doses or two different treatment intervals.

[0414] cancer

[0415] The following is a non-limiting list of cancers that can be treated with PC-1.

[0416] CRC: colorectal cancer, rectal adenocarcinoma, colon adenocarcinoma.

[0417] SCCHN: Squamous cell carcinoma with a primary tumor site in the oral cavity, oropharynx, hypopharynx, or larynx.

[0418] RCC: clear cell or papillary cell renal cell carcinoma (non-chromophobe, hereditary cancer syndrome, or other types).

[0419] NSCLC: non-small cell lung cancer; squamous cell carcinoma and adenocarcinoma.

[0420] Breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, brain cancer (glioblastoma multiforme)

[0421] 2.2 PC-1 design, structure, and mechanism of action

[0422] PC-1 is TRACTr, a humanized trispecific protein comprising EGFR and cluster of differentiation 3 (CD3) binding domains, an albumin binding domain that extends circulating half-life, and two separate peptide masks. The peptide masks are fused to the molecule via a tumor protease-cleavable linker. One peptide mask inhibits EGFR engagement on target cells, and the other peptide mask inhibits CD3 engagement on T cells ( Figure 1 ). TRACTr target engagement requires proteolysis of its two cleavable linkers by proteases present in the tumor microenvironment (TME). Once the cleavage sequence is proteolytically cleaved, the EGFR mask and tandem CD3 mask plus albumin binding domain are released, allowing for optimized EGFR and CD3 target engagement. This tumor-restricted binding and subsequent T cell activation achieved by the EGFR x CD3 bispecific components of PC-1 promotes T cell-mediated killing of EGFR-expressing cancer cells. Furthermore, the absence of the albumin binding domain ensures that any cleaved PC-1 that migrates from the tumor will be rapidly cleared from the hemostasis, minimizing its accumulation in healthy tissues, which can lead to long-term safety risks. PC-1 is being developed for the treatment of advanced or metastatic tumors known to overexpress EGFR, including adult metastatic CRC, NSCLC, SCCHN, and RCC.

[0423] TRACTr's conditional masking and half-life extension are protease cleavage-dependent. Published work has described the upregulation of many proteases in tumors, including MMPs and SPs, relative to healthy tissues. In addition, several protease-activated biological agents and imaging agents have been clinically validated in a wide spectrum of tumor types. By design, the TRACTr molecule is highly sensitive to tumor-selective proteases. Once TRACTr reaches the TME, the protease cuts the specific substrate (an SP substrate and an MMP substrate) within the cleavable linker, releasing the CD3 mask and albumin binding domain. The result of protease cleavage is that TRACTr is converted into its active form, the TCE.

[0424] It is worth noting that the TCE form of PC-1 (PC-1-TCE) has a very short serum half-life, so that the cleaved form of PC-1 that escapes the TME is expected to be cleared from the body before they can produce significant extra-tumor toxicity. Preclinical data indicate that TRACTr can drive a potent anti-tumor response by being demasked by proteases at the tumor site, while producing 1 / 25 systemic IL-6 (the main marker of cytokine release syndrome [CRS]) at 10x high dose levels relative to unmasked TCE. We expect that the TRACTr approach will enable us to demonstrate different safety and efficacy characteristics in patients with metastatic and advanced NSCLC, SCCHN and CRC. Therefore, a FIH, Phase 1, multicenter, open-label study is planned to determine the safety, PK, RP2D and preliminary anti-tumor activity of PC-1 administered as a single agent in adult subjects with metastatic or advanced NSCLC, SCCHN, CRC and RCC.

[0425] 2.3 Basic Principles of PC-1

[0426] Therefore, there is an unmet need to optimize the use of T cell-mediated cytotoxicity to target tumor cells. Products that can be selectively activated within the TME may have significant advantages in developing favorable risk-benefit profiles. TRACTr-based approaches are designed to provide a more focused way to activate T cells within the tumor, minimize systemic activation, achieve higher dosing, and thereby improve anti-tumor efficacy. The TRACTr molecule (PC-1) is designed to improve the therapeutic profile of EGFR-targeted TCEs in patients with tumors known to overexpress EGFR, including metastatic CRC, NCSLC, SCCHN, and RCC. PC-1 consists of a core bispecific TCE that recognizes EGFR and CD3 on T cells, modified by the addition of a tumor protease-cleavable linker connected to a peptide mask that specifically inhibits (1) the CD3 binding domain and (2) the EGFR binding domain of PC-1. Figure 1). CD3 maskers are designed to limit activity outside the TME by inhibiting CD3 binding in peripheral blood, thereby helping to alleviate the extensive T cell activation that causes CRS. Similarly, EGFR maskers are designed to limit on-target off-tumor EGFR binding and related toxicities. In addition, PC-1 also exhibits an extended half-life in plasma by incorporating an albumin binding domain fused to a CD3 masker. The conditional masking and half-life extension of TRACTr are protease cleavage dependent. The TRACTr molecule is designed to be highly sensitive to tumor-selective proteases. Once TRACTr reaches the TME, two types of proteases can cut the joint (which contains SP and MMP substrate sequences), releasing the CD3 masker and the albumin binding domain as well as the EGFR masker. The result of protease cleavage is that TRACTr is converted into its active form, TCE.

[0427] 3 Physical, chemical and pharmaceutical properties and

[0428] preparation

[0429] 3.1 Drug substances

[0430] PC-1 is a 97.1 kDa humanized trispecific glycosylated protein composed of:

[0431] Anti-EGFR antigen-binding fragment (Fab)

[0432] Anti-CD3 single-chain variable fragment (scFv)

[0433] Anti-albumin single domain antibody (SDA / sdAb)

[0434] A peptide mask that inhibits anti-EGFR Fab binding to EGFR and is incorporated into the molecule via a tumor protease-cleavable amino acid linker

[0435] A second peptide mask that inhibits the binding of the anti-CD3 scFv to CD3 and is incorporated into the molecule via a tumor protease-cleavable amino acid linker

[0436] The light chain of the anti-CD3 scFv was fused to the N-terminal heavy chain of the anti-EGFR Fab via a short flexible linker. The EGFR inhibitory peptide mask was fused to the amino terminus of the anti-EGFR Fab light chain via a protease cleavable linker. The tandem albumin-binding sdAb and the CD3 inhibitory peptide mask were fused to the amino terminus of the anti-CD3 scFv via a tumor protease cleavable linker. The albumin-binding SDA was linked to the amino terminus of the CD3 inhibitory peptide mask via a short flexible linker. Figure 1 ).

[0437] The molecular formula of PC-1 is C 4181 H6437 N 1147 O 1339 S 26 The estimated average molecular weight of unglycosylated PC-1 is 95,027 Da. PC-1TRACTr consists of two protein chains connected by a single intermolecular disulfide bond between the light chain (LC) and heavy chain (HC) of TRACTr, and nine disulfide bonds within the molecule. Each peptide mask contains a single internal disulfide bond. The LC and HC arrangements of PC-1TRACTr are provided in Figure 11 middle.

[0438] 3.2 Drug products

[0439] PC-1 solution for injection (e.g., IV infusion (referred to as PC-1 drug product [DP])) will be provided for clinical studies as a sterile aqueous solution formulated at a nominal concentration of 2 mg / mL. This solution is formulated in 10 mM histidine, 8% (w / v) sucrose, and 0.01% (w / v) polysorbate 20 and has a pH of approximately 5.3. The formulation of PC-1 DP is summarized in Table 8. Abbreviations: cGMP = Current Good Manufacturing Practice; BP = British Pharmacopoeia; Ch.P = Chinese Pharmacopoeia; JP = Japanese Pharmacopoeia; Ph Eur = European Pharmacopoeia; QS = Quantitative Sufficient; USP / NF = US Pharmacopoeia / National Formulary.

[0440] Table 8. Composition of PC-1 drug product per vial

[0441]

[0442] cGMP = current good manufacturing practice; BP = British Pharmacopoeia; Ch.P = Chinese Pharmacopoeia; JP = Japanese Pharmacopoeia; PhEur = European Pharmacopoeia; QS = quantitatively sufficient; USP / NF = US Pharmacopoeia / National Formulary.

[0443] PC-1DP includes drug substance filled at a target volume of 1.23 mL to achieve an extractable volume of ≥1.0 mL in a single-dose 2R, Type 1 borosilicate glass vial, and is sealed with a polypropylene nested cap containing an embedded elastomeric stopper.

[0444] The physical and chemical properties of PC-1 are summarized in Table 9.

[0445] Table 9. Physical and chemical properties of PC-1 drug product

[0446] characteristic describe Appearance (color, transparency) Colorless 1.0NTU <![CDATA[Molecular weight 1 > 97.1 kDa Melting point Tm determined by DSC 73.8℃ Osmotic pressure 276mOsmol / kg Solution pH 5.3 Isoelectric point 9.1 <![CDATA[Potency determined by ELISA binding assay after cutting the CD3 mask 2 > 102% relative efficacy <![CDATA[Potency determined by ELISA binding assay after cleavage of the EGFR masking agent 2 > 103% relative efficacy Extinction coefficient <![CDATA[1.77(mg / mL) -1 cm -1 ]]>

[0447] CD3=cluster of differentiation 3; DSC=differential scanning calorimetry;

[0448] ELISA = enzyme-linked immunosorbent assay; NTU = nephelometric unit; Tm = melting temperature.

[0449] 1 Molecular weight measured by the intact mass of the most abundant sugar species (G2FS1)

[0450] 2 The relative effectiveness of one batch compared to the second batch

[0451] 3.3 Storage and handling

[0452] PC-1 DP vials will be stored and shipped frozen (at -20 ± 5°C). The vial contents will be diluted into an infusion solution that will be selected based on the results of compatibility studies at the time of use.

[0453] Nonclinical studies

[0454] 4.1 Introduction

[0455] Nonclinical studies of PC-1DP are designed to support a Phase 1 clinical program in subjects diagnosed with advanced or metastatic colorectal cancer (CRC), non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), and squamous cell carcinoma of the head and neck (SCCHN). Nonclinical pharmacology studies are conducted in in vitro, ex vivo, and in vivo model systems. In vitro studies examine the following:

[0456] PC-1 binds to EGFR, CD3, and albumin antigens in mouse, rat, cynomolgus monkey, and human;

[0457] The stability of PC-1 in sera from healthy human donors, CRC, SCCHN, and NSCLC patients, and cynomolgus monkeys;

[0458] The ability of PC-1 to induce T cell-mediated anti-tumor cytotoxic activity; and

[0459] • The ability of PC-1 to induce cytokine production.

[0460] Nonclinical pharmacology showed that:

[0461] • PC-1 exhibits cleavage-dependent binding to the human and cynomolgus monkey antigens EGFR, CD3, and albumin.

[0462] Masking the EGFR-binding and CD3-binding domains of PC-1 reduces the affinity of the molecule for EGFR and CD3 antigens. The affinity of PC-1 for human EGFR and CD3 is reduced by ...

Claims

1. An isolated recombinant polypeptide complex comprising a first chain having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1 and a second chain having an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2, wherein the isolated recombinant polypeptide complex comprises at least one of the following characteristics: (a) at least one N-glycan moiety; (b) at least one disulfide bond; (c) when the isolated recombinant polypeptide complex is formulated at a concentration of about 1.0 mg / mL in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) polysorbate 20 (PS20), pH about 5.3, the melting onset temperature (T 起始 ) and a transition midpoint temperature (T m1 ), wherein the T 起始 and the T m1 Measured using differential scanning calorimetry (DSC); (d) a deep UV circular dichroism at wavelengths between 210 nm and 230 nm when the isolated recombinant polypeptide complex is formulated in water at a concentration of about 0.1 mg / mL; or (e) The isolated recombinant polypeptide complex has a depressed near-UV circular dichroism at wavelengths between 275 nm and 290 nm when formulated in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) PS20, pH about 5.3, at a concentration of about 1.0 mg / mL.

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

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

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

5. The isolated recombinant polypeptide complex of claim 1, wherein the polypeptide complex comprises at least five of the characteristics. The isolated recombinant polypeptide complex of claim 1 , wherein the first chain comprises at least 75% sequence identity to SEQ ID NO:

1.

7. The isolated recombinant polypeptide complex of claim 1, wherein the first chain comprises at least 80% sequence identity to SEQ ID NO:

1.

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

1.

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

1.

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

1.

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

1.

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

1.

13. The isolated recombinant polypeptide complex of claim 1, wherein the second chain comprises at least 75% sequence identity to SEQ ID NO:

2.

14. The isolated recombinant polypeptide complex of claim 1, wherein the second chain comprises at least 80% sequence identity to SEQ ID NO:

2.

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

2.

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

2.

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

2.

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

2.

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

2.

20. The isolated recombinant polypeptide complex of claim 1, wherein the at least one N-glycan moiety is located on the first chain.

21. The isolated recombinant polypeptide complex of claim 1, wherein the at least one N-glycan moiety is located on the second chain.

22. The isolated recombinant polypeptide complex of claim 1, wherein the at least one N-glycan moiety comprises G2F, G2FS1, or G2FS2.

23. The isolated recombinant polypeptide complex of claim 1, wherein the at least one N-glycan moiety comprises G2F.

24. The isolated recombinant polypeptide complex of claim 1, wherein the at least one N-glycan moiety comprises G2FS1.

25. The isolated recombinant polypeptide complex of claim 1, wherein the at least one N-glycan moiety comprises G2FS2.

26. The isolated recombinant polypeptide complex of claim 1, wherein at least one asparagine deamidation moiety is located at asparagine 83 of SEQ ID NO:

1.

27. The isolated recombinant polypeptide complex of claim 1, wherein the at least one N-glycan moiety is located at asparagine 519 of SEQ ID NO:

2.

28. The isolated recombinant polypeptide complex of claim 1, wherein the isolated recombinant polypeptide complex further comprises O-xylosylation, asparagine deamidation, or succinimide formation.

29. The isolated recombinant polypeptide complex according to claim 26, wherein the succinimide formation is located at asparagine 83 of SEQ ID NO:

1.

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

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

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

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

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

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

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

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

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

39. The isolated recombinant polypeptide complex of claim 1, wherein the pair of cysteine ​​residues comprises cysteine ​​4 and cysteine ​​15 of SEQ ID NO:

1.

40. The isolated recombinant polypeptide complex of claim 1, wherein the pair of cysteine ​​residues comprises cysteine ​​65 and cysteine ​​130 of SEQ ID NO:

1.

41. The isolated recombinant polypeptide complex of claim 1, wherein the pair of cysteine ​​residues comprises cysteine ​​176 and cysteine ​​236 of SEQ ID NO:

1.

42. The isolated recombinant polypeptide complex of claim 1, wherein the pair of cysteine ​​residues comprises cysteine ​​256 of SEQ ID NO: 1 and cysteine ​​653 of SEQ ID NO:

2.

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

2.

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

2.

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

2.

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

2.

47. The isolated recombinant polypeptide complex of claim 1, wherein the pair of cysteine ​​residues comprises cysteine ​​453 and cysteine ​​526 of SEQ ID NO:

2.

48. The isolated recombinant polypeptide complex of claim 1, wherein the pair of cysteine ​​residues comprises cysteine ​​577 and cysteine ​​633 of SEQ ID NO:

2.

49. The isolated recombinant polypeptide complex of claim 1, wherein the polypeptide complex comprises one interchain disulfide bond and nine intrachain disulfide bonds between the first chain and the second chain.

50. The isolated recombinant polypeptide complex of claim 1, wherein the polypeptide complex comprises 1 interchain disulfide bond between the first chain and the second chain, and the second chain comprises 6 intrachain disulfide bonds, and the first chain comprises 3 intrachain disulfide bonds.

51. The isolated recombinant polypeptide complex of claim 1, having a T between about 61°C and about 64.5°C. 起始 .

52. The isolated recombinant polypeptide complex of claim 1, having a T between about 62°C and about 64°C. 起始 .

53. The isolated recombinant polypeptide complex of claim 1, having a T of about 62.5°C. 起始 .

54. The isolated recombinant polypeptide complex of claim 1, having a T of about 63.2°C. 起始 .

55. The isolated recombinant polypeptide complex of claim 1, having a T between about 71°C and about 75°C. m1 .

56. The isolated recombinant polypeptide complex of claim 1, having a T between about 72.5°C and about 74.5°C. m1 .

57. The isolated recombinant polypeptide complex of claim 1, having a T of about 73.8°C. m1 .

58. The isolated recombinant polypeptide complex of claim 1, having a T of about 74.0°C. m1 .

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

60. The isolated recombinant polypeptide complex of claim 1, having a far-UV circular dichroism dip at a wavelength between 215 nm and 225 nm.

61. The isolated recombinant polypeptide complex of claim 1, having a far-UV circular dichroism dip at a wavelength between 215 nm and 220 nm.

62. The isolated recombinant polypeptide complex of claim 1, having a near-UV circular dichroism dip at a wavelength between 280 nm and 290 nm.

63. The isolated recombinant polypeptide complex of claim 1, having a near-UV circular dichroism dip at a wavelength between 280 nm and 285 nm.

64. The isolated recombinant polypeptide complex of claim 1, having a near-UV circular dichroism peak at a wavelength between 270 nm and 275 nm.

65. The isolated recombinant polypeptide complex of claim 1, having a near-UV circular dichroism peak at a wavelength between 285 nm and 290 nm.

66. The isolated recombinant polypeptide complex of claim 1 , wherein the first chain comprises the amino acid sequence according to SEQ ID NO: 1, and the second chain comprises the amino acid sequence according to SEQ ID NO: 2, and the at least one N-glycan moiety comprises G2F, G2FS1, or G2FS2, and the recombinant polypeptide complex comprises disulfide bonds formed by the following pairs of cysteine ​​residues: Cysteine ​​4 and Cysteine ​​15 of SEQ ID NO: 1, Cysteine ​​65 and Cysteine ​​130 of SEQ ID NO: 1, Cysteine ​​176 and Cysteine ​​236 of SEQ ID NO: 1, Cysteine ​​256 of SEQ ID NO: 1 and Cysteine ​​653 of SEQ ID NO: 2, Cysteine ​​138 and Cysteine ​​148 of SEQ ID NO: 2, Cysteine ​​22 and Cysteine ​​96 of SEQ ID NO: 2, Cysteine ​​199 and Cysteine ​​275 of SEQ ID NO: 2, Cysteine ​​339 and Cysteine ​​407 of SEQ ID NO: 2, Cysteine ​​339 and Cysteine ​​408 of SEQ ID NO: 2 NO:2 cysteine ​​453 and cysteine ​​526 and cysteine ​​577 and cysteine ​​633 of SEQ ID NO:2, and when the isolated recombinant polypeptide complex is formulated at a concentration of about 1.0 mg / mL in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) PS20 pH about 5.3, the T 起始 Between about 60°C and about 65°C and the transition midpoint temperature (T m1 ) is between about 70°C and about 75°C, wherein the T 起始 and the T m1 The isolated recombinant polypeptide complex was measured using differential scanning calorimetry (DSC), and the far-UV circular dichroism at wavelengths between 210 nm and 230 nm was depressed when the isolated recombinant polypeptide complex was formulated at a concentration of about 0.1 mg / mL in water, and the near-UV circular dichroism at wavelengths between 275 nm and 290 nm was depressed when the isolated recombinant polypeptide complex was formulated at a concentration of about 1.0 mg / mL in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) PS20, pH about 5.

3.

67. An isolated recombinant polypeptide complex comprising a first chain having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1 and a second chain having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2, wherein the isolated recombinant polypeptide complex comprises the following features: (a) at least one N-glycan moiety; (b) at least one disulfide bond; (c) when the isolated recombinant polypeptide complex is formulated at a concentration of about 1.0 mg / mL in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) PS20 pH about 5.3, the melting onset temperature (T 起始 ) and a transition midpoint temperature (T m1 ), wherein the T 起始 and the T m1 Measured using differential scanning calorimetry (DSC); (d) a deep UV circular dichroism at wavelengths between 210 nm and 230 nm when the isolated recombinant polypeptide complex is formulated in water at a concentration of about 0.1 mg / mL; or (e) The isolated recombinant polypeptide complex has a depressed near-UV circular dichroism at wavelengths between 275 nm and 290 nm when formulated in about 10 mM histidine, about 8% (w / v) sucrose, about 0.01% (w / v) PS20, pH about 5.3, at a concentration of about 1.0 mg / mL.

68. A pharmaceutical composition comprising: (a) the isolated recombinant polypeptide complex according to any one of claims 1 to 67; and (b) a pharmaceutically acceptable carrier.

69. The pharmaceutical composition of claim 68, wherein the pharmaceutically acceptable carrier comprises a buffer, a stabilizer, a tonicity agent, a surfactant, or a combination thereof.

70. The pharmaceutical composition of claim 68, wherein the buffer comprises an amino acid or a derivative thereof.

71. The pharmaceutical composition of claim 70, wherein the amino acid or derivative thereof comprises L-histidine, L-histidine hydrochloride monohydrate, or a combination thereof.

72. The pharmaceutical composition of claim 68, wherein the surfactant is polysorbate 20.

73. The pharmaceutical composition of claim 68, wherein the stabilizer is sucrose.

74. The pharmaceutical composition of claim 68, having a pH below 6.0.

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