Chimeric polypeptide assemblies and their preparation and use

Abstract

This invention relates to chimeric peptide assemblies and methods for their preparation and use, specifically to bispecific chimeric peptide assembly compositions comprising a filling portion connected to a binding domain via a cleavable release segment. When the cleavable release segment is cleaved, it enables effector T cells to simultaneously bind to a targeted tumor or cancer cell, resulting in cell lysis of the tumor or cancer cell. The invention also provides compositions and methods for preparing and using the said cleavable chimeric peptide assembly compositions.

Description

[0001] This application is a divisional application of Chinese invention patent application filed on August 26, 2016, with Chinese application number 201680050442.4 and invention title "Chimeric polypeptide assembly and preparation and use method thereof". Technical Field

[0002] This invention relates to a bispecific chimeric polypeptide assembly composition comprising a filling portion connected to a binding domain via a cleavable release segment, wherein cleavage of the cleavable release segment enables effector T cells to simultaneously bind to a targeted tumor or cancer cell and induce cell lysis of the tumor or cancer cell. The invention also provides compositions and methods for preparing and using the said cleavable chimeric polypeptide assembly composition. Background Technology

[0003] Many approved cancer treatments are cytotoxic drugs that kill both normal and tumor cells. The therapeutic benefit of these cytotoxic drugs depends on the fact that tumor cells are more sensitive than normal cells, allowing for the use of doses that do not cause unacceptable side effects to achieve a clinical response. However, essentially all of these nonspecific drugs cause some (if not serious) damage to normal tissue, which often limits the applicability of the treatment.

[0004] Bispecific antibodies offer a different approach to cytotoxic drugs because they direct immune effector cells to kill cancer cells. Bispecific antibodies combine the benefits of the different binding specificities derived from two monoclonal antibodies into a single composition, enabling coverage methods or combinations that are impossible with monospecific antibodies. This approach relies on one arm of the bispecific antibody binding to a tumor-associated antigen or marker, while the other arm, after binding to CD3 molecules on T cells, triggers its cytotoxic activity by releasing effector molecules such as TNF-α, IFN-γ, interleukins 2, 4, and 10, perforin, and granzymes. Advances in antibody engineering have led to the development of several bispecific antibody forms and compositions for redirecting effector cells to tumor targets, including bispecific T cell conjugates (BiTEs®) such as blinatumomab. BiTEs work by recruiting and activating a polyclonal population of T cells at the tumor site, and do so without requiring co-stimulation or conventional MHC recognition. However, some patients still have a dual problem, experiencing severe side effects known as “cytokine storm” or “cytokine release syndrome” mediated by the release of cytokines such as TNF-α and IFN-γ (Lee DW et al., Current concepts in the diagnosis and management of cytokine release syndrome. Blood. 2014 124(2):188-195), and in fact, BiTE compositions have a very short half-life, thus requiring continuous infusion for 4-8 weeks in order to maintain BiTE for a sufficient time to achieve therapeutic effect within the therapeutic window. Summary of the Invention

[0005] There remains a considerable need for alternative therapies that offer the pharmacological advantages of this bispecific antibody form, but with increased safety, reduced side effects, increased selectivity, and / or enhanced pharmacokinetic properties, such as requiring less frequent dosing or administration via a single injection.

[0006] This invention discloses chimeric peptide assemblies for the treatment or prevention of diseases including, but not limited to, cancer, autoimmune diseases, and inflammatory diseases. In a first aspect, this disclosure provides cleavable chimeric peptide assemblies. These cleavable chimeric peptide assembly compositions address unmet needs and are superior to conventional bispecific antibody formulations in use in one or more aspects, including enhanced terminal half-life, targeted delivery, and reduced toxicity to healthy tissues.

[0007] Thematic polypeptide assemblies typically comprise a first part, a second part, and a third part, wherein: the first part comprises (i) a first binding domain having binding specificity to a target cell marker; and (ii) a second binding domain having binding specificity to an effector cell antigen; the second part comprises a peptide release segment (RS) capable of being cleaved by one or more mammalian proteases; and the third part comprises a filler portion; wherein the filler portion is capable of being released from the first part by the action of the mammalian proteases on the second part.

[0008] The various components in a chimeric peptide assembly can be configured in various different orders. In one embodiment, the chimeric peptide assembly is configured from N-terminus to C-terminus, wherein a first portion is linked to a second portion, which in turn is linked to a third portion. In another embodiment, the chimeric peptide assembly is configured from N-terminus to C-terminus, wherein a third portion is linked to a second portion, which in turn is linked to a first portion. In one embodiment, the chimeric peptide assembly is a fusion protein. In another embodiment, the second and third portions are fusion proteins, and the first portion is conjugated to the second portion. In one embodiment of a chemically conjugated peptide assembly composition, the C-terminus of the first portion peptide can be conjugated to the N-terminus of the second portion peptide by cysteine ​​or other suitable amino acids suitable for cross-linking (by reagents such as maleamide or other cross-linking agents known in the art). In another embodiment, the first and second portions are monomeric fusion proteins, and the third portion is chemically conjugated to the second portion.

[0009] Optionally, the chimeric polypeptide assembly composition may include an additional filling portion connected to the composition via a second release segment attached to opposite ends of the composition, thereby closing the first and second portions.

[0010] The first and second binding domains are typically antibody fragments derived from monoclonal antibodies. In one embodiment, the first and second binding domains of the first portion of the chimeric peptide assembly composition are scFv or configured asa biantibodies. In other embodiments, the first and second binding domains of the first portion of the chimeric peptide assembly composition are selected from Fv, Fab, Fab', Fab'-SH, F(ab')2, linear antibodies, single-domain antibodies, non-antibody scaffolds, and single-domain camel antibodies. In other embodiments, the first and second binding domains of the first portion of the chimeric peptide assembly composition are selected from peptides, non-antibody scaffolds such as anticalins, adnectin, fynomers, affiliates, affibodies, centyrins, and DARPins. In other embodiments, the binding domain of the tumor cell target is a variable domain of a T cell receptor, which has been engineered to bind to the MHC of a peptide fragment carrying a protein overexpressed by tumor cells.

[0011] In one embodiment of the chimeric polypeptide assembly, a first binding domain of the first portion has binding affinity for a target cell marker. Target cells include any eukaryotic cell type, such as cells derived from the ectoderm, mesoderm, or endoderm. Of particular interest are tumor cells and markers expressed by tumor cells. Tumor cells may originate from a selection of stromal cells, fibroblasts, myofibroblasts, glial cells, epithelial cells, adipocytes, lymphocytes, vascular cells, smooth muscle cells, mesenchymal cells, breast tissue cells, prostate cells, kidney cells, brain cells, colon cells, ovarian cells, uterine cells, bladder cells, skin cells, gastric cells, urogenital tract cells, cervical cells, uterine cells, small intestinal cells, hepatocytes, pancreatic cells, gallbladder cells, bile duct cells, esophageal cells, salivary gland cells, lung cells, and thyroid cells.In some cases, tumor-specific markers include α4 integrin, Ang2, B7-H3, B7-H6, CEACAM5, cMET, CTLA4, FOLR1, EpCAM, CCR5, CD19, HER2, HER2neu, HER3, HER4, HER1 (EGFR), PD-L1, PSMA, CEA, MUC1 (mucin), MUC-2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC16, βhCG, Lewis-Y, CD20, CD33, CD38, CD30, CD56 (NCAM), CD133, ganglioside GD3; 9-O-acetyl-GD3, GM2, and Globo. H, Fucosyl GM1, GD2, Carbonic anhydrase IX, CD44v6, Shh, Wue-1, Plasma cell antigen 1, Melanoma chondroitin sulfate proteoglycan (MCSP), CCR8, Prostate 6-transmembrane epithelial antigen (STEAP), Mesothelin, A33 antigen, Prostate stem cell antigen (PSCA), Ly-6, Desmosome core protein 4, Fetal acetylcholine receptor (fnAChR), CD25, Cancer antigen 19-9 (CA19-9), Cancer antigen 125 (CA-125), Type II Müllerian inhibitory substance receptor (MISIIR), Sialized Tn antigen (sTN), Fibroblast activation antigen (FAP), Endothelial sialic acid protein (CD248), Epidermal growth factor receptor variant III (EGFRvIII), tumor-associated antigen L6 (TAL6), SAS, CD63, TAG72, Thomsen-Friedenreich antigen (TF-antigen), insulin-like growth factor I receptor (IGF-IR), Cora antigen, CD7, CD22, CD70, CD79a, CD79b, G250, MT-MMP, F19 antigen, CA19-9, CA-125, alpha-fetoprotein (AFP), VEGFR1, VEGFR2, DLK1, SP17, ROR1, and EphA2. In another embodiment of the chimeric polypeptide assembly, the first binding domain of the first portion has binding affinity for target cell markers that serve as inflammatory markers.

[0012] In one embodiment, the first binding domain of the first portion of the chimeric polypeptide assembly composition comprises VH and VL regions having specific binding affinity for tumor-specific markers or target cell antigens. In one of the foregoing embodiments, the first binding domains VH and VL are derived from monoclonal antibody VH and VL selected from the paired sequences listed in Table 2. The VH and VL regions of the first and second binding domains may be configured in different orders regarding the N-terminal to C-terminal sequence. In one embodiment, the first binding domains VH and VL regions are arranged in the VH-VL order. In another embodiment, the first binding domains VH and VL regions are arranged in the VL-VH order. In other cases, the first binding domain comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 regions, wherein each of these regions is derived from a monoclonal antibody sequence selected from the sequences listed in Table 2. Various configurations of the VH and VL regions, and the CDRs contained therein, generally maintain the desired binding specificity to the intended target cell marker.

[0013] In other embodiments of the chimeric polypeptide assembly, the second binding domain of the first portion has binding affinity for effector cells. Effector cells may be immune cells, including but not limited to plasma cells, T cells, B cells, cytokine-induced killer cells (CIK cells), mast cells, dendritic cells, regulatory T cells (RegT cells), helper T cells, myeloid cells, and NK cells. The second binding domain typically exhibits binding specificity for antigens expressed on effector cells. In some embodiments, the antigen is expressed on the cell surface of the effector cell. In another embodiment, the second binding domain has binding specificity for effector cell antigens expressed on T cells. Non-limiting exemplary effector cell antigens include: CD3, CD4, CD8, αβ TCR, CD25, CD45RO, CD69, CD127, and CD196 (CCR6). Of particular interest is the use of a second binding domain with an scFv conformation having regions derived from monoclonal antibodies that specifically bind to human CD3. In one embodiment, the second binding domains VH and VL are derived from monoclonal antibodies VH and VL selected from the sequences listed in Table 1. In another embodiment, the second binding domain comprises VH and VL regions derived from a monoclonal antibody capable of binding human CD3ε.

[0014] The VH and VL regions of the scFv binding domain can be arranged in different configurations without affecting the utility of the resulting composition. In one embodiment, the second binding domain scFv comprises VH and VL regions arranged in a VH-VL or VL-VH order in the N-terminal to C-terminal direction. The binding domain can also be created from the CDR region. In one embodiment, the second binding domain comprises CDR-H1, CDR-H2, CDR-L3, CDR-L1, CDR-L2, and CDR-L3 regions, each of which is derived from the monoclonal antibody listed in Table 1. In the aforementioned embodiments of this paragraph, the VH and VL regions, as well as the first and second binding domains, are linked by a flexible polypeptide linker selected from the sequences listed in Tables 8 and 9. In another embodiment, the first portion of the chimeric polypeptide assembly composition has a sequence having at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity with a sequence selected from the sequences in Table 13.

[0015] One advantage of the described chimeric polypeptide assembly is that it is assembled in prodrug form, wherein the complete composition can be activated near a target tissue or cellular environment in which a mammalian protease is present, releasing the first-part binding domain at the site where its activity is most desired. For example, the first-part binding domain, when present in the complete assembly, has a low binding affinity due to the shielding effect of the filling portion. Upon release by cleavage of RS by a mammalian protease preferentially expressed in a target tissue, such as tumor tissue, the first-part binding domain becomes "activated" without being shielded by the filling portion. In another embodiment, the invention provides a chimeric polypeptide assembly in which a mammalian protease capable of cleaving RS is preferentially expressed in inflamed tissue. In one embodiment, the chimeric polypeptide assembly comprises RS, wherein RS comprises an amino acid sequence selected from the sequences listed in Table 4. If desired, RS comprises an amino acid sequence selected from the following sequences: LSGRSDNHSPLGLAGS, SPLGLAGSLSGRSDNH, SPLGLSGRSDNH, LAGRSDNHSPLGLAGS, LSGRSDNHVPLSLKMG, SPLGLAGS, GPLLARG, LSGRSDNH, VPLSLTMG, VPLSLKMG, VPLSLSMG, EPLELVAG, EPLELRAG, EPAALMAG, EPASLMAG, RIGSLRTA, RIQFLRTA, EPFHLMAG, VPLSLFMG, EPLELPAG, and EPLELAAG. When desired, the release segment of the chimeric polypeptide assembly composition comprises the amino acid sequence LSGRSDNHSPLGLAGS. In RS embodiments, RS comprises an amino acid sequence capable of being cleaved by one or more proteases selected from those listed in Table 3.

[0016] On the other hand, the third portion of the chimeric polypeptide assembly composition includes a filler portion. Exemplary filler portions include, but are not limited to: extended recombinant polypeptide (XTEN), albumin-binding domain, albumin, IgG-binding domain, polypeptides composed of proline, serine, and alanine; fatty acids, ELP biopolymers, Fc domains, polyethylene glycol (PEG), PLGA, and hydroxyethyl starch. In one embodiment, the filler portion is an XTEN sequence. If desired, the XTEN of the third portion comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with sequences selected from those listed in Table 5.

[0017] On the other hand, the subject-specific chimeric polypeptide assembly exhibits the ability to bind and connect effector cells and target cells, thereby forming an immune synapse that allows effector cells to mediate their biological effects in a target-cell-specific manner. For example, the subject-specific chimeric polypeptide assembly has the ability to: (1) specifically bind to target cell markers such as tumor-specific markers, and (2) specifically bind to antigens expressed on effector cells (e.g., antigens expressed by T cells). The simultaneous binding of T cells and tumor cells mediates the killing, destruction, and / or lysis of tumor cells. In one embodiment, after the second portion is cleaved by one or more mammalian proteases and the first portion is released, the first portion is able to simultaneously bind to both the T cell carrying the human CD3 antigen and the tumor cell carrying the tumor-specific marker in an in vitro assay. In an exemplary design feature of the composition of the present invention, after cleaving the second portion RS to release the first and third portions from the chimeric polypeptide assembly, the released first portion has a molecular weight at least 2, 3, 4, or 5 times lower than the third portion, and has a molecular weight at least 20%, 30%, 40%, 50%, or 60% lower than the intact chimeric polypeptide composition. In one embodiment, after cleaving the second portion RS, the first portion released from the chimeric polypeptide assembly exhibits increased binding affinity for effector T cells carrying CD3 antigens and / or tumor cell markers compared to the chimeric binding assembly with the second portion uncleaved. The binding affinity of the released first portion for T cells carrying human CD3 antigens and / or tumor cell markers is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times compared to the binding affinity of the chimeric polypeptide assembly with the RS uncleaved for T cells carrying human CD3 antigens or tumor cell markers. In another embodiment, after cleaving the second portion RS and releasing the first portion from the chimeric polypeptide assembly, in an in vitro assay comprising a population of T cells and tumor cells, the first portion simultaneously binds to both T cells and tumor cells, generating cytotoxic activity against the tumor cells. In another embodiment, in an in vitro assay, the released first portion of the chimeric polypeptide assembly achieves a greater number of tumor cell lysis amounts compared to the intact chimeric binding assembly. For example, in an in vitro assay, the released first portion of the chimeric polypeptide assembly achieves at least 10-fold, or at least 30-fold, or at least 100-fold, or at least 300-fold, or at least 1000-fold cell lysis compared to the intact chimeric binding assembly.In one embodiment, the cytotoxic activity and / or cell lysis of tumor cells are mediated by target-specific activation of T cells, wherein the amount of T cell activation achieved by the first portion of the released chimeric polypeptide assembly is at least 10-fold, or at least 30-fold, or at least 100-fold, or at least 300-fold, or at least 1000-fold compared to the intact chimeric-binding assembly. Since the RS of the chimeric-binding assembly may undergo partial cleavage during in vitro cytotoxicity assays, to determine the maximum relative difference in cytotoxicity, the RS of the assembly can be replaced with an incleavable peptide and compared with a sample containing the released first portion. If desired, in vitro assays may be selected from the following: cell membrane integrity assay, mixed cell culture assay, FACS-based propidium iodide assay, trypan blue influx assay, photoluminescent enzyme release assay, radioactive 51Cr release assay, fluorescent europium release assay, Calcein AM release assay, photoluminescent MTT assay, XTT assay, WST-1 assay, Alamar blue assay, radioactive 3H-Thd incorporation assay, clonogenic assay to measure cell division activity, fluorescent rhodamine 123 assay to measure mitochondrial transmembrane gradient, apoptosis monitoring assay by FACS-based phosphatidylserine exposure, TUNEL assay based on ELISA, sandwich ELISA, caspase activity assay, cell-based LDH release assay and cell morphology assay, or any combination thereof, or performed by the methods described in the examples below.

[0018] On the other hand, the present invention provides a chimeric polypeptide assembly composition comprising: a first portion, wherein the first portion comprises i) a second binding domain having binding specificity to effector cell antigens; and ii) a first binding domain having binding specificity to tumor-specific markers or target cell antigens; a second portion, wherein the second portion comprises a first release segment (RS) cleavable by a mammalian protease; a third portion comprising a first filling portion, wherein the filling portion is releaseable from the first portion by the action of the mammalian protease on the second portion; a fourth portion comprising a release segment (RS) that may be the same as or different from the second portion RS; and a fifth portion comprising the second filling portion, which may be the same as or different from the third portion, wherein the filling portion is releaseable from the first portion by the action of the mammalian protease on the fourth portion. In one of the foregoing embodiments, the second release segment of the chimeric polypeptide assembly composition comprises an amino acid sequence selected from the sequences listed in Table 4. In another embodiment described above, the second release segment of the chimeric peptide assembly composition comprises an amino acid sequence selected from the following sequences: LSGRSDNHSPLGLAGS, SPLGLAGSLSGRSDNH, SPLGLSGRSDNH, LAGRSDNHSPLGLAGS, LSGRSDNHVPLSLKMG, SPLGLAGS, GPLLARG, LSGRSDNH, VPLSLTMG, VPLSLKMG, VPLSLSMG, EPLELVAG, EPLELRAG, EPAALMAG, EPASLMAG, RIGSLRTA, RIQFLRTA, EPFHLMAG, VPLSLFMG, EPLELPAG, and EPLELAAG. In another embodiment described above, the second release segment of the chimeric peptide assembly composition comprises an amino acid sequence capable of being cleaved by a protease selected from the proteases listed in Table 3. In another embodiment described above, the filling portion of the fifth part of the composition is selected from: XTEN; an albumin-binding domain; albumin; an IgG-binding domain; a polypeptide of at least 350 amino acid residues consisting of proline, serine, and alanine; a fatty acid; and an Fc domain. In another embodiment described above, the filler portion of the fifth part of the composition is XTEN. In yet another embodiment described above, the filler portion of the composition is an XTEN comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the sequences listed in Table 5 at optimal alignment.In one embodiment, the present invention provides a chimeric polypeptide assembly composition comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequences of the no-signal peptide shown in Table 10 at optimal alignment. In another embodiment, the present invention provides a chimeric polypeptide assembly composition comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequences listed in Table 12 at optimal alignment. In yet another embodiment, the present invention provides a chimeric polypeptide assembly composition comprising, as shown... Figure 36 or Figure 37 The listed amino acid sequences have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In another embodiment, the present invention provides a chimeric polypeptide assembly comprising amino acid sequences selected from... Figure 36 or Figure 37 The amino acid sequence composition of the listed polypeptide sequences.

[0019] In an exemplary feature of the chimeric polypeptide assembly composition, its ability to achieve cell lysis of target cells after release of the first portion binding domain (compared to the intact composition) is proportionally greater than the increased binding affinity of the released first portion for target cell markers. In one embodiment of this feature, compared to in vitro assays using K... d Compared to the logarithmic representation of binding affinity, EC 50 The relative cytotoxicity, expressed as an integer, is at least about 2:1, or at least 10:1, or at least 50:1, or at least 100:1, or at least 300:1, or at least 500:1, or at least 1000:1. In another embodiment, cytotoxicity (e.g., in EC50) 50 The binding affinity (e.g., denoted as K) between the first portion of the chimeric polypeptide assembly released in vitro (integer representation) and the molecule. d The proportion of the logarithm is at least about 2 times, at least about 3 times, at least about 5 times, at least about 10 times, at least about 30 times, at least about 50 times, at least about 100 times higher.

[0020] In some embodiments, the comparison of the following items includes: a) relative cytotoxicity, which is measured as a ratio between the following cytotoxicities: (i) the cytotoxicity of the released first portion to target tumor cells in an in vitro assay comprising T cells and tumor cells carrying target cell markers, and (ii) the cytotoxicity of the composition comprising a corresponding first portion and a corresponding third portion of the chimeric polypeptide assembly, the two portions being linked by an incleavable peptide of 1 to about 10 amino acids; and b) relative binding affinity to effector cell antigens, which is measured as a ratio between the following binding affinities: (i) the binding affinity of the released first portion to the effector cell antigen, and (ii) The composition exhibits binding affinity for an effector cell antigen, comprising a corresponding first portion and a corresponding third portion of a chimeric polypeptide assembly linked by an incleavable peptide of 1 to 10 amino acids, wherein the ratio between relative cytotoxicity and relative binding affinity is greater than at least 3:1, 10:1, or greater than at least 30:1, or greater than at least 50:1, or greater than at least 100:1, or greater than at least 300:1, or greater than at least 500:1, or greater than at least 1000:1. In one of the foregoing embodiments, the incleavable peptide has a sequence of multiple units of glycine-serine, serine-glycine, or any dipeptide, and the effector cell antigen is CD3. In one embodiment, the comparison of the following items includes: a) relative cytotoxicity, which is measured as a ratio between the following cytotoxicities: (i) the cytotoxicity of the released first portion to target tumor cells in an in vitro assay comprising T cells and tumor cells carrying target cell markers; and (ii) the cytotoxicity of the composition comprising a corresponding first portion and a corresponding third portion of the chimeric polypeptide assembly, the two portions being linked by an incleavable peptide of 1 to about 10 amino acids; and b) relative binding affinity to the target cell marker, which is measured as a ratio between the following binding affinities: (i) the binding affinity of the released first portion to the target cell marker; and (ii) the binding affinity of the released first portion to the target cell marker. The composition exhibits binding affinity for a target cell marker, comprising a corresponding first portion and a corresponding third portion of a chimeric polypeptide assembly linked by an incleavable peptide of 1 to 10 amino acids, wherein the ratio between relative cytotoxicity and relative binding affinity is greater than at least 3:1, 10:1, or greater than at least 30:1, or greater than at least 50:1, or greater than at least 100:1, or greater than at least 300:1, or greater than at least 500:1, or greater than at least 1000:1. In one of the foregoing embodiments, the incleavable peptide has a sequence of multiple units of glycine-serine, serine-glycine, or any dipeptide, and the effector cell antigen is CD3.In another embodiment, the comparison of the following items includes: a) relative cytotoxicity, which is measured as a ratio between the following cytotoxicities: (i) the cytotoxicity of the released first portion to target tumor cells in an in vitro assay comprising T cells and tumor cells carrying target cell markers, and (ii) the cytotoxicity of the composition comprising a corresponding first portion and a corresponding third portion of the chimeric polypeptide assembly linked by an incleavable peptide of 1 to 10 amino acids; and b) relative effector cell antigen binding affinity, which is measured as a ratio between the following binding affinities: (i) the binding affinity of the released first portion to effector cell antigens, and (ii) the binding affinity of the composition comprising a corresponding first portion and a corresponding third portion of the chimeric polypeptide assembly linked by an incleavable peptide of 1 to 10 amino acids; and c) relative binding affinity to target cell markers, which is measured as a ratio between the following binding affinities: (i) the binding affinity of the released first portion to the target cell markers, and (ii) The composition exhibits binding affinity for a target cell marker, comprising a corresponding first portion and a corresponding third portion of a chimeric polypeptide assembly linked by an incleavable peptide of 1 to 10 amino acids, wherein the ratio of relative cytotoxicity to relative effector cell antigen binding affinity (multiplied by the relative binding affinity to the target cell marker) is greater than at least 3:1, 10:1, or greater than at least 30:1, or greater than at least 50:1, or greater than at least 100:1, or greater than at least 300:1, or greater than at least 500:1, or greater than at least 1000:1. In one of the foregoing embodiments, the incleavable peptide has a sequence of multiple units of glycine-serine, serine-glycine, or any dipeptide, and the effector cell antigen is CD3.

[0021] In one embodiment, the present invention provides a chimeric polypeptide assembly composition wherein, in an in vitro cytotoxicity assay comprising T cells and tumor cells carrying target cell markers, the chimeric polypeptide assembly composition releases a first portion of EC... 50 The value is ≤5000 pg / ml, even more preferably ≤1000 pg / ml, even more preferably ≤500 pg / ml, even more preferably ≤350 pg / ml, even more preferably ≤250 pg / ml, even more preferably <100 pg / ml, even more preferably ≤50 pg / ml, even more preferably <10 pg / ml, and most preferably ≤5 pg / ml. In one embodiment, in an in vitro assay, the EC of the first portion of the released chimeric polypeptide assembly composition is... 50 The EC value of the complete chimeric polypeptide assembly composition is higher than that of the complete chimeric polypeptide assembly composition. 50The value is less than 10 times, or at least 20 times, or at least 30 times, or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times, or at least 80 times, or at least 90 times, or at least 100 times, or at least 120 times.

[0022] In some cases, the first binding domain of the released first portion exhibits a greater binding affinity for tumor-specific markers than the second binding domain of the released first portion for CD3 antigen. In one embodiment, the binding affinity of the first binding domain of the released first portion to target cells, such as K+, is determined in vitro. d The constant measured is at least an order of magnitude higher than the binding affinity of the second binding domain for the CD3 antigen. In some other embodiments, the binding affinity of the first binding domain of the released first portion to target cells, such as by K0 in an in vitro binding assay, is... d The constant is measured in 10 -5 Up to 10 -9 Between M, K of the second associative domain d In 10 -5 Up to 10 -9 Between M and M. Binding affinity can be determined by standard cell-based assays, ELISA, the assays described in the examples herein, or other in vitro assays known in the art.

[0023] On the other hand, the present invention relates to the enhanced performance of the chimeric polypeptide assembly when administered to a subject. In particular, it is envisioned that a complete chimeric polypeptide assembly composition comprising the release segment exhibits less cytotoxicity and / or reduced ability to induce pro-inflammatory cytokine production compared to a released first fraction. In one embodiment, the present invention provides a chimeric polypeptide assembly composition wherein, when or after administration of the composition comprising the chimeric polypeptide assembly to a subject, a second fraction of the assembly is cleaved near a tumor expressing a protease capable of cleaving RS. After the second fraction is cleaved by the mammalian protease and the first fraction is released from the assembly, the first fraction is capable of simultaneously binding to T cells carrying the human CD3 antigen and tumor cells carrying tumor-specific markers in the subject. In one embodiment, the simultaneous binding of the released first fraction to T cells carrying the CD3 antigen and tumor cells carrying tumor cell markers leads to the release of T cell-derived effector molecules. In the foregoing, the effector molecules are selected from one or more effector molecules selected from TNF-α, IFN-γ, interleukin-2, perforin, and granzyme, or other T cell effector molecules known in the art. As a result of the simultaneous binding of effector cells and target cells, an immune synapse is generated, which enables the lysis of target cells by T cells and effector molecules.

[0024] On the other hand, the present invention relates to chimeric peptide assembly compositions having increased terminal half-life and other properties conferred by filling portions such as XTEN. In one embodiment, the invention provides a chimeric peptide assembly composition wherein the complete composition exhibits a half-life at or after administration to a subject that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times longer than the half-life of the first portion not connected to the second and third portions at or after administration to the subject at a comparable dose. In another embodiment, at or after administration of the chimeric peptide assembly to a subject and cleavage of the second portion and release of the first and third portions from the chimeric peptide assembly, the half-life of the first portion in the subject is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times shorter than the half-life of the complete chimeric peptide assembly. In a related embodiment, at or after a single administration of the chimeric peptide composition to a subject, the plasma C of the released first portion is... max The concentration shall not exceed about 0.01 ng / ml, or about 0.1 ng / ml, or about 1 ng / ml, or about 10 ng / ml, or about 100 ng / ml. In another related embodiment, at or after a single administration of the complete chimeric peptide composition to a subject, the first portion of plasma C is released. max The concentration of the complete chimeric polypeptide assembly in the plasma C of the same subject was higher than that of the complete chimeric polypeptide assembly. max The concentration is at least 3-fold lower, or at least 10-fold lower, or at least 30-fold lower, or at least 100-fold lower. Pharmacokinetic parameters can be measured using plasma samples from subjects who have been administered the subject chimeric polypeptide assembly, using methods described herein or conventional methods known in the art. In another embodiment, at or after administration to a subject, the intact chimeric polypeptide assembly exhibits reduced exudation from the circulatory system in the subject compared to its RS-cleaved chimeric polypeptide assembly, releasing the first and third portions. In the aforementioned embodiments described in this paragraph, the subject can be a mouse, rat, monkey, dog, or human.

[0025] On the other hand, the present invention relates to pharmaceutical compositions of chimeric polypeptide assemblies. In one embodiment, the invention provides a pharmaceutical composition comprising any chimeric polypeptide assembly disclosed herein, one or more pharmaceutically suitable excipients, and optionally one or more carriers or stabilizers. In another embodiment, the pharmaceutical composition is formulated for intradermal, subcutaneous, intravenous, intraarterial, intraperitoneal, intrathecal, intrasheathal, or intramuscular administration. In yet another embodiment, the pharmaceutical composition is in liquid form. In related embodiments, the liquid form of the pharmaceutical composition is supplied in a pre-filled syringe for a single injection. In several other embodiments, the pharmaceutical composition is formulated as a lyophilized powder reconstituted prior to administration.

[0026] On the other hand, the present invention relates to methods and uses of the chimeric polypeptide assembly or a pharmaceutical composition comprising the chimeric polypeptide assembly. In one embodiment, the present invention provides a chimeric polypeptide assembly or a pharmaceutical composition comprising the chimeric polypeptide assembly for preparing a medicament for treating a disease in a subject. In a related embodiment, the medicament is used for a disease selected from: cancer, Hodgkin's lymphoma and non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, ER / PR+ breast cancer, Her2+ breast cancer, triple-negative breast cancer, colon cancer, colon cancer with malignant ascites, mucinous tumor, prostate cancer, head and neck cancer, skin cancer, melanoma, genitourinary tract cancer, ovarian cancer, and other cancers with malignant ascites. Watery ovarian cancer, peritoneal cancer metastasis, serous uterine cancer, endometrial cancer, cervical cancer, colorectal cancer, uterine cancer, mesothelioma in the peritoneum, kidney cancer, Wilms' tumor, lung cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, stomach cancer, small intestine cancer, liver cancer, hepatocellular carcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, thyroid cancer, epithelial cancer, male cell tumor, adenocarcinoma, sarcoma, and chronic lymphocytic leukemia of B cell origin.

[0027] On the other hand, the present invention relates to chimeric polypeptide assemblies or pharmaceutical compositions comprising such chimeric polypeptide assemblies used in methods for treating a disease in a subject, wherein the method comprises administering the chimeric polypeptide assembly or the pharmaceutical composition to a subject suffering from the disease, including but not limited to cancer. If desired, the method comprises administering to the subject in need a therapeutically effective dose of a pharmaceutical composition comprising the chimeric polypeptide assembly and one or more excipients. In one embodiment of the treatment method, the disease is selected from: cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, T-cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, colon cancer, prostate cancer, head and neck cancer, any form of skin cancer, melanoma, genitourinary tract cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal cancer metastasis, serous uterine cancer, endometrial cancer, cervical cancer, colorectal cancer, intraepithelial malignant tumor with malignant ascites, uterine cancer, and mesothelioma in the peritoneum. The treatment is indicated for the following cancers: renal cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, gastric cancer, small intestinal cancer, liver cancer, hepatocellular carcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, bile duct cancer, salivary gland cancer, thyroid cancer, epithelial carcinoma, adenocarcinoma, sarcoma of any origin, primary hematologic malignancies including acute or chronic lymphocytic leukemia, acute or chronic myeloid leukemia, myeloproliferative neoplasms or myelodysplastic disorders, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, or Goodpasture syndrome. In another embodiment of this treatment, the pharmaceutical composition is administered to the subject at one or more therapeutically effective doses twice weekly, once weekly, every two weeks, every three weeks, or once monthly. In another embodiment of this treatment, the pharmaceutical composition is administered to the subject at one or more therapeutically effective doses for at least two weeks, or at least one month, or at least two months, or at least three months, or at least four months, or at least five months, or at least six months. In another embodiment of this treatment method, the dose is administered intradermally, subcutaneously, intravenously, intraarterially, intraperitoneally, intrathecally, or intramuscularly. In yet another embodiment of this treatment method, the dose is administered, under tolerable conditions, as a bolus dose or by infusion over 5 minutes to 96 hours to achieve maximum safety and efficacy.In another embodiment of the treatment method, the dose is selected from: at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.02 mg / kg, at least about 0.04 mg / kg, at least about 0.08 mg / kg, at least about 0.1 mg / kg, at least about 0.12 mg / kg, at least about 0.14 mg / kg, at least about 0.16 mg / kg, at least about 0.18 mg / kg, at least about 0.20 mg / kg, at least about 0.22 mg / kg, at least about 0.24 mg / kg, at least about 0.26 mg / kg, at least about 0.27 mg / kg, at least about 0.28 mg / kg, at least 0.3 mg / kg, at least 0.4 mg / kg, at least about 0.5 mg / kg, at least about 0.6 mg / kg, at least about 0.7 mg / kg, at least about 0.8 mg / kg, at least about 0.9 mg / kg, at least about 1.0 mg / kg, at least about 1.5 mg / kg. mg / kg or at least about 2.0 mg / kg. In another embodiment of this treatment method, the initial dose is selected from: at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.02 mg / kg, at least about 0.04 mg / kg, at least about 0.08 mg / kg, at least about 0.1 mg / kg, and subsequent doses are selected from: at least about 0.1 mg / kg, at least about 0.12 mg / kg, at least about 0.14 mg / kg, at least about 0.16 mg / kg, at least about 0.18 mg / kg, at least about 0.20 mg / kg, at least about 0.22 mg / kg, at least about 0.24 mg / kg, at least about 0.26 mg / kg, at least about 0.27 mg / kg, at least about 0.28 mg / kg, at least 0.3 mg / kg, at least 0.4 mg / kg, at least about 0.5 mg / kg, at least about 0.6 mg / kg, at least about 0.7 mg / kg, at least about 0.8 mg / kg, at least about 0.9 mg / kg. mg / kg, at least about 1.0 mg / kg, at least about 1.5 mg / kg, or at least about 2.0 mg / kg. In another embodiment of this treatment method, administration of a therapeutically effective dose of the pharmaceutical composition to a subject results in a plasma concentration of the chimeric polypeptide assembly in the subject of at least about 0.1 ng / mL to at least about 2 ng / mL or higher, lasting for at least about 3 days, at least about 7 days, at least about 10 days, at least about 14 days, or at least about 21 days.

[0028] In another embodiment, the present invention provides a pharmaceutical composition for use in a method of treating a disease, the method comprising administering the pharmaceutical composition to a subject suffering from the disease according to a treatment regimen comprising one or more consecutive doses of a therapeutically effective dose. In one embodiment of the pharmaceutical composition used in the method of treating a disease, the disease is selected from: cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, T-cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, colon cancer, prostate cancer, head and neck cancer, any form of skin cancer, melanoma, genitourinary tract cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal cancer metastasis, serous uterine carcinoma, endometrial cancer, cervical cancer, colorectal cancer, intraepithelial malignant tumor with malignant ascites, uterine cancer, and peritoneal cancer. Mesothelioma, renal cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, gastric cancer, small intestinal cancer, liver cancer, hepatocellular carcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, bile duct cancer, salivary gland cancer, thyroid cancer, epithelial carcinoma, adenocarcinoma, sarcoma of any origin, primary hematologic malignancies including acute or chronic lymphocytic leukemia, acute or chronic myeloid leukemia, myeloproliferative neoplasms or myelodysplastic disorders, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, or Goodpasture syndrome. In another embodiment of the pharmaceutical composition used in the method of treating the disease, the treatment regimen is part of a specific treatment cycle, wherein the specified treatment cycle includes administration of the pharmaceutical composition twice a week, once a week, once every ten days, once every two weeks, once every three weeks, or once a month per treatment cycle. In one embodiment, the treatment regimen results in an improvement in a disease-related clinical parameter or endpoint in the subject, wherein the clinical parameter or endpoint is selected from one or any combination of the following: tumor shrinkage as a complete, partial, or incomplete response; time to progression; time to treatment failure; biomarker response; progression-free survival; disease-free survival; time to recurrence; time to metastasis; overall survival; improved quality of life; and symptom improvement. In the aforementioned embodiments of the method, the subject is selected from mice, rats, monkeys, and humans.

[0029] On the other hand, the present invention relates to kits comprising the pharmaceutical composition. In one embodiment, the present invention provides a kit comprising a pharmaceutical composition of any embodiment disclosed herein, a container, and a label or packaging instructions on or associated with the container. In another embodiment, the present invention provides a kit comprising a pre-filled syringe containing a pharmaceutical composition of any embodiment disclosed herein, and a label or packaging insert on or associated with the syringe.

[0030] On the other hand, the present invention relates to the different features and effects of intact chimeric peptide assembly compositions and cleaved chimeric peptide assembly compositions. In one embodiment, the present invention provides a chimeric peptide assembly of any embodiment disclosed herein, wherein, when said assembly is in contact with effector cells and target cells, the intact chimeric peptide assembly is at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 1000-fold lower than a corresponding first portion of said assembly not connected to said assembly in terms of the likelihood of generating Th1 cytokines from effector cells.In one embodiment, the production of Th1 cytokines is determined in an in vitro assay comprising PBMCs or CD3+ T cells and target cells with tumor-specific marker antigens selected from: α4 integrin, Ang2, B7-H3, B7-H6, CEACAM5, cMET, CTLA4, FOLR1, EpCAM, CCR5, CD19, HER2, HER2 neu, HER3, HER4, HER1 (EGFR), PD-L1, PSMA, CEA, MUC1 (mucin), MUC-2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC16, βhCG, Lewis-Y, CD20, CD33, CD38, CD30, CD56. (NCAM), CD133, ganglioside GD3, 9-O-acetyl-GD3, GM2, GloboH, fucose GM1, GD2, carbonic anhydrase IX, CD44v6, Shh, Wue-1, plasma cell antigen 1, melanoma chondroitin sulfate proteoglycan (MCSP), CCR8, prostate 6-transmembrane epithelial antigen (STEAP), mesothelin, A33 antigen, prostate stem cell antigen (PSCA), Ly-6, desmosome core protein 4, fetal acetylcholine receptor (fnAChR), CD25, cancer antigen 19-9 (CA19-9), cancer antigen 125 (CA-125), type II Müllerian inhibitory substance receptor (MISIIR), sialylated Tn antigen (sTN), fibroblast activation antigen (FAP), endothelial sialic acid protein (CD248), epidermal growth factor receptor variant III (EGFRvIII), tumor-associated antigen L6 (TAL6), SAS, CD63, TAG72, Thomsen-Friedenreich antigen (TF-antigen), insulin-like growth factor I receptor (IGF-IR), Cora antigen, CD7, CD22, CD70, CD79a, CD79b, G250, MT-MMP, F19 antigen, CA19-9, CA-125, alpha-fetoprotein (AFP), VEGFR1, VEGFR2, DLK1, SP17, ROR1, and EphA2. In the aforementioned embodiments, the Th1 cytokines are selected from IL-2, TNF-α, and IFN-γ.In another embodiment, the production of Th1 cytokines is measured using a blood or fluid sample from a subject who has been administered the chimeric polypeptide assembly, compared to a subject who has been administered a corresponding first portion unlinked to the chimeric polypeptide assembly. The result shows that the complete chimeric polypeptide assembly is at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 1000-fold lower in likelihood of achieving Th1 cytokine production. In the foregoing embodiments, the subjects are selected from mice, rats, monkeys, and humans.

[0031] In other cases, the chimeric polypeptide assembly of any embodiment disclosed herein exhibits the following characteristic: the complete chimeric polypeptide assembly is at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold lower than the first portion of the chimeric polypeptide assembly when administered to a subject at a comparable dose.

[0032] On the other hand, the present invention relates to nucleic acids encoding the compositions of the present invention. In one embodiment, the present invention provides an isolated nucleic acid comprising a polynucleotide sequence selected from: (a) a polynucleotide encoding a chimeric polypeptide assembly of any embodiment disclosed herein, or (b) a complement of the polynucleotide of (a). In another embodiment, the present invention provides an isolated nucleic acid comprising a polynucleotide sequence having at least 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the polynucleotide sequences listed in Table 10 or Table 14. In another embodiment, the present invention provides an expression vector comprising the polynucleotide sequence of the foregoing embodiments and a recombinant regulatory sequence operatively linked to the polynucleotide sequence. In another embodiment, the present invention provides an isolated host cell comprising the above-described expression vector. In one embodiment, the host cell is *Escherichia coli*.

[0033] On the other hand, the present invention relates to T-cell binding compositions and nucleic acids encoding them. In one embodiment, the present invention provides a monomeric fusion protein comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from those listed in Table 7, wherein the monomeric fusion protein exhibits binding affinity for the CD3 antigen of T cells. In another embodiment, the present invention provides an isolated nucleic acid comprising a polynucleotide sequence selected from the following: (a) a polynucleotide encoding the aforementioned fusion protein of the T-cell binding composition; (b) a polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a polynucleotide sequence selected from those listed in Table 7; or (c) a complement of the polynucleotide of (a) or (b).

[0034] In another embodiment, the present invention provides a method for using a nucleic acid encoding a fusion protein of the aforementioned T-cell binding composition in a method for preparing a polynucleotide sequence encoding a chimeric polypeptide assembly of any of the chimeric polypeptide assembly embodiments disclosed herein, the method comprising operatively linking a polynucleotide sequence encoding a target cell marker having binding affinity to a target cell marker disclosed herein or selected from the targets listed in Table 2, which conforms to a reading frame of the polynucleotide encoding the aforementioned disclosed T-cell binding composition fusion protein. In another embodiment, the present invention provides an expression vector comprising the aforementioned polynucleotide sequence and a recombinant regulatory sequence operatively linked to the polynucleotide sequence. The present invention also provides an isolated host cell containing the expression vector, wherein the host cell is *Escherichia coli*.

[0035] In another aspect, the present invention relates to methods for generating chimeric polypeptide assemblies of the embodiments disclosed herein. In one embodiment, the present invention provides a method for generating chimeric polypeptide assemblies of the embodiments disclosed herein, the method comprising transforming host cells with an expression vector encoding the chimeric polypeptide assembly, culturing the host cells under conditions allowing the chimeric polypeptide assembly to be expressed in the transformed host cells, and isolating the chimeric polypeptide assembly into a soluble fusion protein. In some embodiments, at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99% of the first and second binding domains of the expressed fusion protein are correctly folded.

[0036] In other instances, the present invention provides a method for inducing target cell death. This method typically involves contacting the target cells with a chimeric polypeptide assembly of an embodiment disclosed herein and effector cells. In one embodiment, this contact produces effects in the target cells including, but not limited to, loss of membrane integrity, condensation, nuclear fragmentation, induction of intrinsic apoptosis pathways, induction of extrinsic apoptosis pathways, apoptosis, cell lysis, and cell death.

[0037] Cytotoxicity leading to cell death (e.g., necrosis or apoptosis) can be determined by any suitable method, including but not limited to: cell counting before and after treatment, or measuring the levels of markers associated with live or dead cells. The degree of cell death can be determined by any suitable method. In some embodiments, the degree of cell death is determined relative to the starting conditions. For example, an individual may have a known starting amount of target cells, such as a known-sized starting cell mass or tumor, or a known concentration of circulating target cells. In another instance, the degree of cell death induced by one composition can be compared to the degree of cell death induced by another composition (e.g., a chimeric polypeptide assembly attached to a filling portion and a chimeric polypeptide assembly not attached to a filling portion). In this case, the degree of cell death can be expressed as the ratio of surviving cells after treatment to the starting cell population. In some embodiments, the degree of cell death can be determined by appropriate cell death assays. In one embodiment, the degree of cell death can be determined by measuring tumor size over time. Various cell death assays are available, and a variety of detection methods can be utilized. Examples of such detection methods include, but are not limited to, the use of cell staining, microscopy, flow cytometry, cell sorting, and combinations thereof. Further non-limiting examples of cell death assays described in WO2011131472A1 and US20130052160 are cited by reference.

[0038] In one of the foregoing embodiments, the method is used in a cell-based in vitro assay comprising a mixed population of target cells and effector cells, and an effective amount of a chimeric polypeptide assembly having binding affinity for antigens on both the target cells and effector cells. In this assay, the target cells express tumor-specific marker antigens, including but not limited to: α4 integrin, Ang2, B7-H3, B7-H6, CEACAM5, cMET, CTLA4, FOLR1, EpCAM, CCR5, CD19, HER2, HER2 neu, HER3, HER4, HER1 (EGFR), PD-L1, PSMA, CEA, MUC1 (mucin), MUC-2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC16 βhCG, Lewis-Y, CD20, CD33, CD38, CD30, CD56 (NCAM), CD133, ganglioside GD3, 9-O-acetyl-GD3, GM2, Globo H, Fucosyl GM1, GD2, Carbonic anhydrase IX, CD44v6, Shh, Wue-1, Plasma cell antigen 1, Melanoma chondroitin sulfate proteoglycan (MCSP), CCR8, Prostate 6-transmembrane epithelial antigen (STEAP), Mesothelin, A33 antigen, Prostate stem cell antigen (PSCA), Ly-6, Desmosome core protein 4, Fetal acetylcholine receptor (fnAChR), CD25, Cancer antigen 19-9 (CA19-9), Cancer antigen 125 (CA-125), Type II Müllerian inhibitory substance receptor (MISIIR), Sialized Tn antigen (sTN), Fibroblast activation antigen (FAP), Endothelial sialic acid protein (CD248), Epidermal growth factor receptor variant III (EGFRvIII), Tumor-associated antigen L6 The chimeric polypeptide assembly includes (TAL6), SAS, CD63, TAG72, Thomsen-Friedenreich antigen (TF-antigen), insulin-like growth factor I receptor (IGF-IR), Cora antigen, CD7, CD22, CD70, CD79a, CD79b, G250, MT-MMP, F19 antigen, CA19-9, CA-125, alpha-fetoprotein (AFP), VEGFR1, VEGFR2, DLK1, SP17, ROR1, and EphA2, and the effector cells are T cells, wherein the effector cell antigen is CD3. In some other embodiments, the method of inducing target cell death is used in a subject with cancer containing a target cell population, wherein the method includes administering a therapeutically effective amount of the chimeric polypeptide assembly to the subject.In one of the foregoing embodiments, the method includes administering a chimeric polypeptide assembly as one or more consecutively administered therapeutically effective doses of a pharmaceutical composition, the pharmaceutical composition comprising the chimeric polypeptide assembly and one or more excipients. In another of the foregoing embodiments, the method includes determining the amount of pharmaceutical composition required to achieve a therapeutic effect in a subject with cancer and administering that amount as one or more consecutive doses to the subject. In methods for inducing target cell death in a subject, the target cells are cancer cells, wherein the cancer may be: carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, ER / PR+ breast cancer, Her2+ breast cancer, triple-negative breast cancer, colon cancer, colon cancer with malignant ascites, mucinous tumor, prostate cancer, head and neck cancer, skin cancer, melanoma, genitourinary tract cancer, ovarian cancer, Ovarian cancer with malignant ascites, metastatic peritoneal cancer, serous uterine carcinoma, endometrial cancer, cervical cancer, colorectal cancer, uterine cancer, mesothelioma in the peritoneum, renal cancer, Wilms' tumor, lung cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, stomach cancer, small intestinal cancer, liver cancer, hepatocellular carcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, thyroid cancer, epithelial carcinoma, androgenetic adenocarcinoma, sarcoma, and B-cell-derived chronic lymphocytic leukemia. By using the method of the present invention in subjects with cancer, the method leads to improvements in clinical parameters or endpoints, wherein said clinical parameters or endpoints may be: overall survival, symptom endpoint, disease-free survival, objective response rate, complete response, duration of response, progression-free survival, time to progression, time to treatment failure, tumor measurement, tumor size, tumor response rate, time to metastasis, and biomarker concentration. In other cases, compared to administration of a composition comprising the first portion of a chimeric polypeptide assembly but excluding the second and third portions to comparable subjects at a comparable dose of mmol / kg, the use of the method of the present invention in subjects with cancer results in a reduction in the frequency, duration, or severity of diagnosis-related side effects in the subjects, said side effects being one or more of the following: elevated plasma IL-2 levels, elevated plasma TNF-α levels, elevated plasma IFN-γ levels, sepsis, febrile neutropenia, neurotoxicity, seizures, encephalopathy, cytokine release syndrome, speech disorders, balance disorders, fever, headache, confusion, hypotension, neutropenia, nausea, impaired consciousness, disorientation, and increased liver enzymes.

[0039] In another embodiment, the present invention provides a method for delivering a therapeutic agent to tumor cells containing a tumor-specific marker, the method comprising administering to target cells a chimeric polypeptide assembly of any embodiment disclosed herein, wherein the therapeutic agent is delivered to the target cells via a first binding domain that specifically binds to a first portion of the tumor-specific marker. In the foregoing, the tumor-specific marker is selected from: α4 integrin, Ang2, B7-H3, B7-H6, CEACAM5, cMET, CTLA4, FOLR1, EpCAM, CCR5, CD19, HER2, HER2 neu, HER3, HER4, HER1 (EGFR), PD-L1, PSMA, CEA, MUC1 (mucin), MUC-2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC16 βhCG, Lewis-Y, CD20, CD33, CD38, CD30, CD56 (NCAM), CD133, ganglioside GD3, 9-O-acetyl-GD3, GM2, GloboH, fucose GM1, GD2, carbonic anhydrase IX, CD44v6, Shh, Wue-1, plasma cell antigen 1, melanoma chondroitin sulfate proteoglycan (MCSP), CCR8, prostate 6-transmembrane epithelial antigen (STEAP), mesothelin, A33 antigen, prostate stem cell antigen (PSCA), Ly-6, desmosome core protein 4, fetal acetylcholine receptor (fnAChR), CD25, cancer antigen 19-9 (CA19-9), cancer antigen 125 (CA-125), type II Müllerian inhibitory substance receptor (MISIIR), sialylated Tn antigen (sTN), fibroblast activation antigen (FAP), endothelial sialic acid protein (CD248), epidermal growth factor receptor variant III (EGFRvIII), tumor-associated antigen L6 (TAL6), SAS, CD63, TAG72, Thomsen-Friedenreich antigen (TF-antigen), insulin-like growth factor I receptor (IGF-IR), Cora antigen, CD7, CD22, CD70, CD79a, CD79b, G250, MT-MMP, F19 antigen, CA19-9, CA-125, alpha-fetoprotein (AFP), VEGFR1, VEGFR2, DLK1, SP17, ROR1, and EphA2.If desired, the tumor-specific markers are selected from: α4 integrin, Ang2, CEACAM5, CD19, CD20, CD33, CD38, cMET, CTLA4, EpCAM, EphA2, FOLR1, HER1 (EGFR), HER2, HER3, HER1 (EGFR) / HER3, HER2 / 3, mesothelin, MUC1, PD-L1, PSMA, TAG-72, VEGFR1, and VEGFR2. In one embodiment of the method, the chimeric polypeptide assembly comprises an amino acid sequence having at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 100% sequence identity with a polypeptide sequence selected from the sequences listed in Table 12. In another embodiment of the method, the chimeric polypeptide assembly comprises an amino acid sequence selected from the... Figure 36 or Figure 37 The listed polypeptide sequences have amino acid sequences with at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 100% sequence identity. In one embodiment of the foregoing method, the tumor cells reside in a tumor of a subject, wherein the subject may be a mouse, rat, monkey, dog, or human.

[0040] On the other hand, the present invention relates to the physical properties of chimeric peptide assembly compositions and the performance produced when administered to a subject. Regarding embodiments of the chimeric peptide assemblies disclosed herein, neither the second nor the third portion of the chimeric peptide assembly has a specific binding affinity for the first portion. For each embodiment of the chimeric peptide assembly disclosed herein, the first portion constitutes less than 50% of the molecular weight of the complete chimeric peptide assembly. In another embodiment, when the apparent molecular weight factor is evaluated by size exclusion chromatography, the first portion of the chimeric peptide assembly of the disclosed embodiments constitutes less than 30%, or less than 40%, or less than 50% of the apparent molecular weight factor of the chimeric peptide assembly. Furthermore, when the hydrodynamic radius is evaluated by size exclusion chromatography, when the second portion is cleaved and the first and third portions are released from the chimeric peptide assembly of any embodiment disclosed herein, the hydrodynamic radius of the released first portion is approximately 30%, or approximately 40%, or approximately 50% smaller than the hydrodynamic radius of the complete chimeric peptide assembly. In one embodiment, the present invention provides a chimeric peptide assembly wherein, upon determination of the hydrodynamic radius by size exclusion chromatography, after cleaving the second portion and releasing the first and third portions from the chimeric peptide assembly, the hydrodynamic radius of the released first portion is less than about 5 nm, or less than about 4 nm, or less than about 3 nm. Therefore, the released first portion has a higher ability to penetrate tumor tissue compared to the intact chimeric peptide assembly. In another embodiment, upon determination of the hydrodynamic radius by size exclusion chromatography, the intact chimeric peptide assembly disclosed herein has a hydrodynamic radius greater than about 8 nm, or greater than about 9 nm, or greater than about 10 nm. Therefore, the intact chimeric peptide assembly administered to a subject with a tumor has a weaker ability to leak from the blood vessels of the subject's normal tissue than it has a weaker ability to leak from the blood vessels of a tumor.

[0041] In particular, it is envisioned that the embodiments of the chimeric polypeptide assembly composition can exhibit one or more, or any combination of, the properties disclosed herein. More specifically, it is envisioned that the therapeutic methods can exhibit one or more, or any combination of, the properties disclosed herein. Incorporation

[0042] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually cited and incorporated herein by reference. Attached Figure Description

[0043] The features and advantages of the invention can be further explained by referring to the following detailed description of illustrative embodiments and the accompanying drawings.

[0044] Figure 1 The various schematic diagrams used in the accompanying drawings are depicted, along with descriptions of what they represent.

[0045] Figure 2 The figure depicts the ProTIA composition (also described herein as a chimeric polypeptide assembly) in its uncleaved "Pro" form and its cleavage state after action by tumor-associated proteases. Some non-limiting properties of the two compositional forms are also illustrated.

[0046] Figures 3A-3B exist Figure 3A The uncut "Pro" form is shown in the image. Figure 3B The diagram illustrates the cleavage forms, with the uncleaved forms depicted near effector cells and tumor-associated cells, each possessing cell surface antigens; however, due to the steric hindrance and shielding effect of the filling portion on the targeting (or binding) domain, Figure 3A The uncut form in the middle cannot bind to two types of cells simultaneously, while Figure 3B The cleavage form with a release target domain allows both types of cells to bind simultaneously and allows effector cells to activate immune responses against target tumor-associated cells.

[0047] Figure 4 shows a schematic diagram of two configurations of the ProTIA composition, illustrating that the release segment and filling portion can be attached to the effector cell binding portion or the tumor antigen binding portion.

[0048] Figures 5A-5B A schematic diagram of two configurations of the ProTIA composition is shown, in which two release segments and two filler portions are connected to the binding portion. Figure 5A In this case, one RS and filling portion are connected to the effector cell binding portion, and another RS ​​and filling portion are connected to the tumor antigen binding portion, and the composition will be in the scFv configuration. Figure 5B In this case, both the RS and the filling portion are attached to the effector cell binding portion (on the left) or the tumor antigen binding portion (on the right), and the binding portion will be in a biantibody configuration (thus allowing the composition to be produced in a recombinant form).

[0049] Figure 6A- Figure 6D A schematic diagram of two configurations of the ProTIA composition is shown, wherein the filler portion is an XTEN peptide, and RS and the filler portion are linked to the effector cell binding portion (on the left) or RS, and the filler portion is linked to the tumor antigen binding portion (on the right).

[0050] Figures 7A-7BA schematic diagram of two configurations of the ProTIA composition is shown, in which two release segments and two XTENs are connected to the binding portion. Figure 7A In this case, one RS and one XTEN are connected to the effector cell binding site and another RS, and the filling site is connected to the tumor antigen binding site, and the composition will be in the scFv configuration. Figure 7B In this case, both RS and XTEN are linked to either the effector cell binding site (on the right) or the tumor antigen binding site (on the left), and the binding site will be in a dual antibody configuration (thus allowing the composition to be produced in a recombinant form).

[0051] Figures 8A-8C A schematic diagram of two configurations of the ProTIA composition is shown, in which the RS and filler portion are connected to either the effector cell binding portion (on the left) or the tumor antigen binding portion (on the right). Figure 8A The combined part is described as XTEN. Figure 8B The binding portion is depicted as albumin. Figure 8C The junction is depicted as an Fc segment.

[0052] Figures 9A-9C A schematic diagram of the ProTIA composition configuration is shown, in which two release segments and two filler portions are connected to the binding portion. Figure 9A Three configurations are depicted, in which the two S and XTEN are connected to the effector cell binding part and the tumor antigen binding part (left side), connected to the tumor antigen binding part (center), or connected to the effector cell binding part (right side). Figure 9B Four configurations were depicted, in which RS and XTEN are connected to the effector cell binding site, and RS and albumin are connected to the tumor antigen binding site (on the upper left), RS and XTEN are connected to the tumor antigen binding site, RS and albumin are connected to the effector cell binding site (on the upper right), both RS and XTEN and both RS and albumin are connected to the tumor antigen binding site (on the lower left), and both RS and XTEN and both RS and albumin are connected to the effector cell binding site (on the lower right). Figure 9C Four configurations were depicted, in which RS and XTEN are connected to the effector cell binding site, and RS and Fc are connected to the tumor antigen binding site (on the upper left), RS and XTEN are connected to the tumor antigen binding site, RS and Fc are connected to the effector cell binding site (on the upper right), both RS and XTEN and both RS and Fc are connected to the tumor antigen binding site (on the lower left), and both RS and XTEN and both RS and Fc are connected to the effector cell binding site (on the lower right).

[0053] Figure 10The diagram illustrates ProTIA near tumor tissue (left) and normal tissue (right). In tumor tissue, the more permeable vascular system allows ProTIA to leak into tissues where its tumor-associated protease can act on RS, cleaving and releasing its binding moiety. This, in turn, allows for the simultaneous binding of both effector cells and tumor-associated cells. In normal tissue, the exudate is either blocked by a denser vascular system, or, in the case of ProTIA leakage, ProTIA remains in its "Pro" form and is able to bind effector cells, but without tumor cells, or, if present, with insufficient protease to release the binding moiety, resulting in a net effect without the formation of immune synapses.

[0054] Figure 11 A schematic diagram of the scFv configuration of the effector cell binding portion and the tumor antigen binding portion is shown, each having VH / VL pairs bound by linkers in a tandem configuration.

[0055] Figure 12 A schematic diagram of a dual antibody configuration with an effector cell binding portion and a tumor antigen binding portion is shown, each having a VH / VL pair linked by a linker.

[0056] Figure 13A A schematic diagram of the general structural design is shown. Figure 13B and 13C A schematic diagram of the ProTIA composition is shown, in which the effector cell binding portion and the tumor antigen binding portion are in the scFv configuration ( Figure 13B [Having a variable heavy chain (VH) and a variable light chain (VL) linked by an intramolecular long linker (L) or an intermolecular short linker (1)] and a biantibody configuration ( Figure 13C [VH and VL domains connected by long linkers (L) or short intermolecular linkers (1)] are in various arrangements.

[0057] Figures 14A-14C The purification of uncut AC1278 from fermentation medium is shown as described in Example 2. Figure 14A An exemplary SDS-PAGE of AC1278 captured from fermentation medium IMAC is shown; Figure 14B The SDS-PAGE analysis of fractions in the HIC purification step is shown; Figure 14C The SDS-PAGE analysis of fractions in the ImpRes-Q purification step is shown.

[0058] Figures 15A-15B Batch release analysis of uncut AC1278 is shown, as described in Example 2. Figure 15ABatch release analysis of uncut AC1278 (shown as solid line) against XTEN length standard (shown as dashed line) by SEC chromatography is shown; Figure 15B The batch release SDS-PAGE of uncut AC1278 is shown.

[0059] Figures 16A-16B The preparation of cut ProTIA-A using uncut AC1278 is shown as described in Example 2. Figure 16A The SDS-PAGE analysis of the MMP-9 digestion reaction mixture is shown. Figure 16B The SDS-PAGE analysis of the IMAC-purified MMP-9 digest mixture to remove the cleaved XTEN fragment is shown.

[0060] Figures 17A-17B Batch release analysis of the cut AC1278 is shown, as described in Example 2. Figure 17A The batch release analysis of cleaved AC1278 (shown as solid line) against globular protein standards (shown as dashed line) by SEC chromatography is shown. Figure 17B The image shows a batch release SDS-PAGE of a cut AC1278.

[0061] Figures 18A-18C The purification of uncut AC1476 from fermentation medium is shown as described in Example 3. Figure 18A An exemplary SDS-PAGE of AC1476 captured from fermentation medium IMAC is shown; Figure 18B The SDS-PAGE analysis of fractions in the HIC purification step is shown; Figure 18C The SDS-PAGE analysis of fractions in the ImpRes-Q purification step is shown.

[0062] Figures 19A-19C Batch release analysis of uncut AC1476 is shown, as described in Example 3. Figure 19A Batch release analysis of uncut AC1476 (shown as solid line) against XTEN length standard (shown as dashed line) by SEC chromatography is shown; Figure 19B The image shows a batch-release SDS-PAGE of uncut AC1476 stained with Coomassie stain; Figure 19C The batch release SDS-PAGE of uncut AC1476 stained with silver is shown.

[0063] Figures 20A-20B Additional batch release analysis of uncut AC1476 is shown, as described in Example 3. Figure 20A The batch release ESI-MS of uncutterd AC1476 is shown; Figure 20BBatch release cation exchange chromatography of uncutterd AC1476 is shown.

[0064] Figures 21A-21B The preparation of cut ProTIA-A using uncut AC1476 is shown as described in Example 3. Figure 21A The SDS-PAGE analysis of the MMP-9 digestion reaction mixture is shown. Figure 21B SDS-PAGE analysis of the anion exchange fraction of the MMP-9 digest mixture is shown to remove uncut substrate and cleaved XTEN segments.

[0065] Figures 22A-22C Batch release analysis of the cut AC1476 is shown, as described in Example 3. Figure 22A The SEC shows the batch release analysis of cleaved AC1476 (indicated by solid lines) against globular protein standards (indicated by dashed lines); Figure 22B The batch release SDS-PAGE of Coomassie-stained cut AC1476 is shown. Figure 22C Batch release SDS-PAGE of cut AC1476 with silver staining is shown.

[0066] Figures 23A-23B Additional batch release analysis of the cut AC1476 is shown, as described in Example 3. Figure 23A The batch release ESI-MS of the cut AC1476 is shown; Figure 23B Batch release cation exchange chromatography of cleaved AC1476 is shown.

[0067] Figure 24 The binding of protease-treated and untreated anti-EpCAM x anti-CD3 ProTIA to its ligands is shown as described in Example 4.

[0068] Figure 25 The results of experiments measuring the in vitro activity of protease-treated and untreated anti-EpCAM x anti-CD3 ProTIA are described as in Example 6.

[0069] Figure 26 The results of experiments determining the in vitro specificity of anti-EpCAM x anti-CD3 ProTIA are described as in Example 6.

[0070] Figure 27 Results of experiments measuring the in vitro activity of protease-treated, protease-untreated, and protease-incompatible anti-EpCAMx anti-CD3 ProTIA are described as in Example 6.

[0071] Figure 28The results of experiments measuring the PK of anti-EpCAM x anti-CD3 ProTIA in protease-treated and untreated samples are described as in Example 9.

[0072] Figures 29A-29B A schematic diagram of the alternating N-terminal to C-terminal configuration of the T-cell binding composition is shown. Figure 29A The configuration of the effector cell binding portion (ECBM), followed by the release site segment (RS) and XTEN is shown. Figure 29B The configuration shown is XTEN followed by RS segment and then ECBM.

[0073] Figure 30 Results of experiments measuring the in vitro activity of protease-treated, protease-untreated, and protease-incompatible anti-EpCAM x anti-CD3 ProTIA in SK-OV-3 are described as in Example 6.

[0074] Figure 31 Tumor volume results from experiments measuring the antitumor effects of protease-treated and untreated anti-EpCAM x anti-CD3 ProTIA were described, as in Example 10.

[0075] Figure 32 The results of experiments determining the antitumor effects of protease-treated and untreated anti-EpCAM x anti-CD3 ProTIA were described as weight-based, as set forth in Example 10.

[0076] Figures 33A-33B The results of experiments that determined the cytokine curves of anti-EpCAM x anti-CD3 ProTIA with and without protease treatment were described in Example 12. Figure 33A The results of the test for detecting IL-2 are shown, while Figure 33B The results of IL-4 detection are shown.

[0077] Figures 34A-34B The results of experiments depicting the cytokine curves for determining anti-EpCAM×anti-CD3 ProTIA in protease-treated and untreated samples were described in Example 12. Figure 34A The results of the test for detecting IL-6 are shown, while Figure 34B The results of IL-10 detection are shown.

[0078] Figures 35A-35B The results of experiments depicting the cytokine curves for determining anti-EpCAM×anti-CD3 ProTIA in protease-treated and untreated samples were described in Example 12. Figure 35A The results of the experiment for detecting IFN-γ are shown, while Figure 35B The results of TNF-α detection are shown.

[0079] Figure 36 The amino acid sequence of AC1476 aEpCAM-aCD3 ProTIA.

[0080] Figure 37 The amino acid sequence of AC1489 aEpCAM-aCD3 ProTIA.

[0081] Figure 38 The HCT-116 tumor volume results for the experiments that determined the antitumor effects of anti-EpCAM x anti-CD3 ProTIA, protease-treated anti-EpCAM x anti-CD3 ProTIA, and non-cleavable anti-EpCAM x anti-CD3 ProTIA are described as in Example 13.

[0082] Figure 39 The experimental results for determining the anti-HCT-116 tumor efficacy of anti-EpCAM×anti-CD3 ProTIA, protease-treated anti-EpCAM×anti-CD3 ProTIA, and non-cleavable anti-EpCAM×anti-CD3 ProTIA are described as in Example 13.

[0083] Figure 40 Experimental results were described in SK-OV-3 to determine the in vitro activity of protease-treated, protease-untreated, and protease-incompatible anti-EpCAM x anti-CD3 ProTIA using purified human CD3-positive T cells, as described in Example 14.

[0084] Figure 41 Experimental results were described in OVCAR-3 to determine the in vitro activity of protease-treated, protease-untreated, and protease-insoluble anti-EpCAM x anti-CD3 ProTIA using purified human CD3-positive T cells, as described in Example 14.

[0085] Figures 42A-42B Experimental results depicting CD69 activation in CD8 and CD4 cells in PBMC and SK-OV-3 cell co-cultures using protease-treated, untreated, and protease-insoluble anti-EpCAM×anti-CD3 ProTIA were described as follows: Figure 42A The activation of CD69 on CD8 cells was described, and Figure 42B The activation of CD69 on CD4 cells was described.

[0086] Figures 43A-43BExperimental results depicting the activation of CD69 and CD25 on CD8 and CD4 cells in PBMC and SK-OV-3 cell co-cultures using protease-treated, untreated, and protease-insoluble anti-EpCAM×anti-CD3 ProTIA were described as follows: Figure 43A The activation of CD69 and CD25 on CD8 cells was described, and Figure 43B The activation of CD69 and CD25 on CD4 cells was described.

[0087] Figures 44A-44B Experimental results depicting CD69 activation on CD8 and CD4 cells in co-cultures of purified CD3+ cells and SK-OV-3 cells using protease-treated, untreated, and protease-insoluble anti-EpCAM×anti-CD3 ProTIA were described as follows: Figure 44A The activation of CD69 on CD8 cells was described, and Figure 44B The activation of CD69 on CD4 cells was described.

[0088] Figures 45A-45B Experimental results depicting the activation of both CD69 and CD25 on CD8 and CD4 cells in purified CD3+ cells and SK-OV-3 cell co-cultures using protease-treated, untreated, and protease-insoluble anti-EpCAM x anti-CD3 ProTIA were described as described in Example 8. Figure 45A The activation effects of both CD69 and CD25 on CD8 cells were described, and Figure 45B The activation effects of CD69 and CD25 on CD4 cells were described.

[0089] Figures 46A-46B Experimental results depicting CD69 activation on CD8 and CD4 cells in purified CD3+ cells and OVCAR3 cell co-cultures using protease-treated, untreated, and protease-insoluble anti-EpCAM×anti-CD3 ProTIA were described as follows: Figure 46A The activation of CD69 on CD8 cells was described, and Figure 46B The activation of CD69 on CD4 cells was described.

[0090] Figures 47A-47BExperimental results depicting the activation of both CD69 and CD25 on CD8 and CD4 cells in co-cultures of purified CD3+ cells and OVCAR3 cells using protease-treated, untreated, and protease-insoluble anti-EpCAM x anti-CD3 ProTIA were described as described in Example 8. Figure 47A The activation effects of CD69 and CD25 on CD8 cells were described, and Figure 47B The activation effects of CD69 and CD25 on CD4 cells were described.

[0091] Figures 48A-48B Experimental results depicting CD69 activation in CD8 and CD4 cells in co-cultures of PBMC and OVCAR3 cells using protease-treated, untreated, and protease-insoluble anti-EpCAM x anti-CD3 ProTIA were described as follows: Figure 48A The activation of malignant CD69 on CD8 cells was described, and Figure 48B The activation of CD69 on CD4 cells was described.

[0092] Figures 49A-49B The results of experiments measuring the activation of both CD69 and granzyme B in CD8 and CD4 cells in PBMC and OVCAR3 cell co-cultures using protease-treated, untreated, and protease-insoluble anti-EpCAM×anti-CD3 ProTIA are described as described in Example 8. Figure 49A The activation of CD69 and granzyme B in CD8 cells was described, and Figure 49B The activation effects of both CD69 and granzyme B in CD4 cells were described.

[0093] Figures 50A-50B Experimental results were described using protease-treated, untreated, and protease-insoluble anti-EpCAM×anti-CD3 ProTIA to measure the release of cytokines IL-2 and IL-4 in purified CD3+ cells and SK-OV-3 cell co-cultures, as described in Example 15. Figure 50A The concentration of released IL-2 was depicted, while Figure 50B The concentration of released IL-4 was depicted.

[0094] Figures 51A-51B Experimental results were described using anti-EpCAM×anti-CD3 ProTIA, which was prepared by protease, unprepared by protease, and prepared by protease-insoluble protease, to measure the release of cytokines IL-6 and IL-10 in co-cultures of purified CD3+ cells and SK-OV-3 cells, as described in Example 15. Figure 51A The concentration of released IL-6 was depicted, while Figure 51B The concentration of released IL-10 was depicted.

[0095] Figures 52A-52B Experimental results were described using protease-treated, untreated, and protease-insoluble anti-EpCAM×anti-CD3 ProTIA to measure the release of cytokines TNF-α and IFN-γ in purified CD3+ cells and SK-OV-3 cell co-cultures, as described in Example 15. Figure 52A The concentration of released TNF-α was depicted, while Figure 52B The concentration of released IFN-γ was depicted.

[0096] Figure 53 Binding curves of protease-treated, untreated, and non-cleavable anti-EpCAM x anti-CD3ProTIA for CD3εδ ligands are shown as described in Example 16.

[0097] Figure 54 The binding specificity of protease-treated anti-EpCAM x anti-CD3 ProTIA to the rhEpCAM ligand is shown as described in Example 17.

[0098] Figure 55 The SW480 tumor volume results for the experiments determining the antitumor effects of anti-EpCAM x antiCD3 ProTIA, protease-treated anti-EpCAM x antiCD3 ProTIA, and non-cleavable anti-EpCAM x antiCD3 ProTIA are described as in Exercise 18.

[0099] Figure 56 The results of experiments determining the anti-SW480 tumor activity of anti-EpCAM x anti-CD3 ProTIA, protease-treated anti-EpCAM x anti-CD3 ProTIA, and non-cleavable anti-EpCAM x anti-CD3 ProTIA are described as in Example 18.

[0100] Figure 57 Experimental results for determining the in vitro activity of protease-treated, protease-untreated, and protease-insoluble anti-EpCAM x anti-CD3 ProTIA in SKOV3 of human PBMCs are described as in Example 23.

[0101] Figure 58 Experimental results were described using human PBMCs to determine the in vitro activity of protease-treated, protease-untreated, and protease-insoluble anti-EpCAM x anti-CD3 ProTIA in OVCAR3, as described in Example 23.

[0102] Figure 59 Experimental results were described using human PBMCs to determine the in vitro activity of protease-treated, protease-untreated, and protease-insoluble anti-EpCAM x anti-CD3 ProTIA in HCT116, as described in Example 23.

[0103] Figure 60 Experimental results were described using human PBMCs to determine the in vitro activity of protease-treated, protease-untreated, and protease-incompatible anti-EpCAM x anti-CD3 ProTIA in SW480, as described in Example 23. Detailed Implementation

[0104] Before describing the embodiments of the invention, it should be understood that these embodiments are provided by way of example only, and the invention can be implemented using various alternatives to the embodiments described herein. Those skilled in the art will now be able to conceive of many changes, variations, and substitutions without departing from the invention.

[0105] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials to those described herein may be used to practice or test the invention, suitable methods and materials are described below. In case of conflict, this patent specification, including the definitions, shall prevail. Furthermore, materials, methods, and examples are illustrative only and not intended to be limiting. Many changes, variations, and substitutions will now occur to those skilled in the art without departing from the invention. definition

[0106] Unless otherwise specified, the following terms shall have their own meanings in the context of this application.

[0107] Except where a specific upper limit is specified herein, the terms “a,” “an,” and “the” as used throughout the specification and claims are used to mean “at least one,” “at least first,” “one or more,” or “a plurality of” the components or steps mentioned. Thus, as used herein, “cleavage sequence” means “at least a first cleavage sequence,” but includes multiple cleavage sequences. Based on this disclosure, the operational limitations and parameters of combinations, such as the amount of any single reagent, will be known to those skilled in the art.

[0108] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acids of any length. This polymer may be linear or branched, may contain modified amino acids, and may have non-amino acids inserted between them. These terms also include amino acid polymers modified, for example, by disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation (e.g., conjugation with a labeled component).

[0109] The term "monomer" applied to polypeptides refers to the state of a polypeptide as a single, continuous amino acid sequence that is substantially not associated with one or more additional polypeptides of the same or different sequence. The monomeric state of a polypeptide can be determined by size exclusion chromatography as a single protein entity with the same molecular weight.

[0110] As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including but not limited to D or L optical isomers, as well as amino acid analogs and peptide mimics. Amino acids may be designated using standard single-letter or three-letter codes.

[0111] The term “natural L-amino acid” or “L-amino acid” refers to the L-optical isomers of the following amino acids: glycine (G), proline (P), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), cysteine ​​(C), phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R), glutamine (Q), asparagine (N), glutamic acid (E), aspartic acid (D), serine (S), and threonine (T).

[0112] The term “non-naturally occurring” as used herein, applied to sequences, refers to a polypeptide or polynucleotide sequence that has no counterpart in a wild-type or naturally occurring sequence found in mammals, is not complementary to a wild-type or naturally occurring sequence found in mammals, or does not share a high degree of homology with a wild-type or naturally occurring sequence found in mammals. For example, when properly aligned, a non-naturally occurring polypeptide or fragment may share no more than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, or even lower amino acid sequence identity with a natural sequence.

[0113] The terms "hydrophilic" and "hydrophobic" refer to the degree of affinity of a substance for water. Hydrophilic substances have a strong affinity for water, readily dissolve in water, mix with water, or are wetted by water, while hydrophobic substances have virtually no affinity for water, readily repel water and do not absorb water, and are not readily soluble in water, mix with water, or are wetted by water. Amino acids can be characterized by their hydrophobicity. Many scales have been developed. One example is the scale developed by Levitt, M ​​et al., J Mol Biol (1976) 104:59, which is listed in Hopp, TP et al., Proc Natl Acad Sci USA (1981) 78:3824. Examples of "hydrophilic amino acids" are arginine, lysine, threonine, alanine, asparagine, and glutamine. Of particular interest are the hydrophilic amino acids aspartic acid, glutamic acid, as well as serine and glycine. Examples of "hydrophobic amino acids" are tryptophan, tyrosine, phenylalanine, methionine, leucine, isoleucine, and valine.

[0114] “Fragment,” when used for bioactive proteins (not antibodies), is a truncated form of a bioactive protein that retains at least a portion of its therapeutic and / or biological activity. “Variant,” when used for bioactive proteins, is a protein that has sequence homology with the native bioactive protein and retains at least a portion of its therapeutic and / or biological activity. For example, a variant protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with a reference bioactive protein. As used herein, the term “bioactive protein variant” includes proteins that are intentionally modified, for example, through site-directed mutagenesis, coding gene synthesis, insertion, or unintentionally modified through mutation and retain their activity.

[0115] The term "sequence variant" refers to a polypeptide that has been modified compared to its native or original sequence by the insertion, deletion, or substitution of one or more amino acids. Insertions may be located at one or both ends of the protein and / or may be located within an internal region of the amino acid sequence. A non-limiting example is the substitution of amino acids in XTEN with different amino acids. In deletion variants, one or more amino acid residues of the polypeptide described herein are removed. Thus, deletion variants encompass all segments of the polypeptide sequence described herein. In substitution variants, one or more amino acid residues of the polypeptide are removed and replaced with other residues. In one respect, substitutions are inherently conserved, and this type of conserved substitution is well known in the art.

[0116] The term "part" refers to a component of a larger composition or a component intended to be incorporated into a larger composition, such as a protein moiety that is linked as a continuous or discontinuous sequence to a larger polypeptide. A part of a larger composition can impart a desired function. For example, a filler moiety can impart increased molecular weight and / or half-life to the resulting larger composition associated with that filler moiety.

[0117] The term "release segment" or "RS" refers to a cleavage sequence in a composition that can be recognized and cleaved by one or more proteases, thereby releasing one or more portions or fractions from the composition. As used herein, "mammalian protease" means that which is commonly present in the body fluids, cells, and tissues of mammals and is visible at higher levels in certain target tissues or cells, for example, in diseased tissues of mammals (e.g., tumors). RS sequences can be engineered to be cleaved in vitro by a variety of mammalian proteases present in or near the subject's target tissue or introduced therein. Other equivalent proteases (endogenous or exogenous) capable of recognizing defined cleavage sites can be used. In particular, the RS sequence can be adapted and customized depending on the protease used and can be incorporated into linker amino acids for linkage with adjacent peptides.

[0118] The term "within," when referring to a first polypeptide linked to a second polypeptide, includes the linking or fusion of additional components that link the N-terminus of the first or second polypeptide to the C-terminus of the second or first polypeptide, respectively, and also includes the insertion of the first polypeptide into the sequence of the second polypeptide. For example, when the RS component is linked "within" a chimeric polypeptide assembly, the RS may be linked to the N-terminus or C-terminus of the payload polypeptide, or may be inserted between any two amino acids of the XTEN polypeptide.

[0119] The term "activity" as used in the context of the compositions provided herein means any action or effect known in the art to the effector component of the composition, including but not limited to receptor binding, antagonist activity, agonist activity, cellular or physiological response, cell lysis, cell death, or effect, whether measured by in vitro, ex vivo, or in vivo analysis or by clinical efficacy.

[0120] As used in this article, "effective cells" include any eukaryotic cell capable of conferring effects on target cells. For example, effector cells can induce loss of membrane integrity, condensation, nuclear fragmentation, apoptosis, lysis, and / or death of target cells. In another instance, effector cells can induce division, growth, differentiation, or otherwise alter signal transduction in target cells. Non-limiting examples of effector cells include: plasma cells, T cells, CD4 cells, CD8 cells, B cells, cytokine-induced killer cells (CIK cells), chief cells, dendritic cells, regulatory T cells (RegT cells), helper T cells, myeloid cells, macrophages, and NK cells.

[0121] "Effective cell antigen" refers to molecules expressed by effector cells, including but not limited to cell surface molecules such as proteins, glycoproteins, or lipoproteins. Exemplary effector cell antigens include proteins of the CD3 complex or T cell receptor (TCR), CD4, CD8, CD25, CD38, CD69, CD45RO, CD57, CD95, CD107, and CD154, as well as effector molecules such as cytokines associated with and bound to effector cells, cytokines expressed within effector cells, or cytokines expressed and released by effector cells. Effector cell antigens can be used as binding counterparts to the binding domains of the subject chimeric polypeptide assembly. Non-limiting examples of effector cell antigens that the subject composition can bind include antigens on the cell surface, such as CD3, CD4, CD8, CD25, CD38, CD69, CD45RO, CD57, CD95, CD107, and CD154, and Th1 cytokines selected from IL2, IL10, IL12, IFNγ, and TNFα.

[0122] As used herein, the term "ELISA" refers to an enzyme-linked immunosorbent assay as described herein or known in the art.

[0123] "Host cell" includes a single cell or cell culture that may be, or has been, a recipient of a subject vector for which a foreign nucleic acid has been introduced, as described herein. Host cell includes the progeny of a single host cell. Due to natural, accidental, or intentional mutations, the progeny may not necessarily be identical to the original parent cell (morphologically or genomically in terms of total DNA complementarity). Host cell includes cells transfected in vivo using the vector of the present invention.

[0124] When used to describe the various polypeptides disclosed herein, "isolated" means a polypeptide that has been identified, isolated, and / or recovered from components in its native environment or from more complex mixtures (e.g., during protein purification). Dopant components in its native environment are materials that typically interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other protein or non-protein solutes. It will be apparent to those skilled in the art that non-naturally occurring polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof do not need to be "isolated" to distinguish them from their naturally occurring counterparts. Furthermore, "concentrated," "isolated," or "diluted" polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof can be distinguished from their naturally occurring counterparts because the concentration or number of molecules per volume is generally greater than that of their naturally occurring counterparts. Generally, polypeptides prepared recombinantly and expressed in host cells are considered "isolated."

[0125] "Isolated nucleic acid" refers to a nucleic acid molecule identified and isolated from at least one doped nucleic acid molecule that normally accompanies the natural source of the polypeptide-encoded nucleic acid. For example, the isolated polypeptide-encoded nucleic acid differs from its naturally occurring form or environment. Therefore, the isolated polypeptide-encoded nucleic acid molecule is distinct from the specific polypeptide-encoded nucleic acid molecule present in natural cells. However, the isolated polypeptide-encoded nucleic acid molecule includes polypeptide-encoded nucleic acid molecules contained in cells that normally express the polypeptide, where, for example, the nucleic acid molecule is located intrachromosomally or extrachromosomally, unlike the nucleic acid molecule in natural cells.

[0126] A "chimeric" protein or polypeptide contains at least one fusion polypeptide comprising at least one region at a sequence location different from its naturally occurring location. These regions may be normally present in different proteins and aggregated together in the fusion polypeptide; or they may be normally present in the same protein but in a new arrangement within the fusion polypeptide. Chimeric proteins can be produced, for example, by chemical synthesis or by generating and translating polynucleotides in which peptide regions are encoded in a desired relationship.

[0127] The terms “fusion” and “fusion” are used interchangeably in this document and refer to the linking of two or more peptide or polypeptide sequences together through recombination. A “fusion protein” or “chimeric protein” contains a first amino acid sequence linked to a second amino acid sequence that is not naturally linked in nature.

[0128] "XTENized" is used to refer to a peptide or polypeptide that has been modified by linking or fusing one or more XTEN peptides (described below) to the peptide or polypeptide, whether by recombination or chemical crosslinking.

[0129] "Operably linked" means that the linked DNA sequences are contiguous and within the reading frame. "In-frame fusion" refers to two or more open reading frames (ORFs) combining in a manner that maintains the correct reading frame of the original ORF to form a longer, continuous ORF. For example, if a promoter or enhancer affects the transcription of a polypeptide sequence, then that promoter or enhancer is operatively linked to the coding sequence of the polypeptide. Therefore, the resulting recombinant fusion protein is a single protein containing two or more segments corresponding to the polypeptide encoded by the original ORF (segments that would not normally be linked in this way in nature).

[0130] The terms “crosslinking,” “combination,” “link,” “linking,” and “connection” are used interchangeably herein and refer to the covalent connection of two different molecules through a chemical reaction. As is known in the art, crosslinking can occur in one or more chemical reactions.

[0131] As used in this article, the term "combined couple" refers to individual components that can be linked or linked in a combination reaction.

[0132] The term "conjugate" (used as a noun) refers to a heterogeneous molecule formed by the covalent connection of conjugate partners to each other, such as a binding domain covalently connected to a release segment.

[0133] The terms "crosslinker" and "crosslinking agent" are used interchangeably and, in their broadest context, refer to a chemical entity used to covalently link two or more entities. When defining an entity herein, for example, a crosslinker links two, three, four, or more peptides, or links peptides to XTEN. Those skilled in the art will understand that a crosslinker can refer to the covalently bonded reaction product remaining after the reactants have been crosslinked. A crosslinker may also contain one or more reactants that have not yet reacted but are capable of reacting with another entity.

[0134] In the context of peptides, a "linear sequence" or "sequence" is the amino acid sequence of a peptide from the amino terminus to the carboxyl terminus (N-terminus to C-terminus), where adjacent residues in the sequence are continuous in the primary structure of the peptide. A "partial sequence" is a linear sequence of a peptide that is known to contain additional residues in one or two directions.

[0135] "Heterologous" means derived from an entity whose genotype differs from that of the entity being compared. For example, a glycine-rich sequence taken from its natural coding sequence and operatively linked to a coding sequence different from the natural sequence is a heterologous glycine-rich sequence. The term "heterologous" when applied to polynucleotides and polypeptides means that the polynucleotide or polypeptide is derived from an entity whose genotype differs from that of the entity being compared.

[0136] The terms “polynucleotide,” “nucleic acid,” “nucleotide,” and “oligonucleotide” are used interchangeably. They refer to nucleotides of any length, including singular and plural nucleic acids, which are deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci (multiple loci) identified by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be made before or after polymer assembly. Non-nucleotide components can be inserted into the nucleotide sequence. Polynucleotides can be further modified after polymerization, for example, by conjugation with labeled components.

[0137] The term "polynucleotide complement" refers to a polynucleotide molecule that has a complementary base sequence and is reversed compared to a reference sequence, so that the complement can hybridize with the reference sequence with complete fidelity.

[0138] When applied to a polynucleotide, “recombinant” means that the polynucleotide is the product of various combinations of recombination steps, which may include cloning, restriction digestion and / or ligation steps, as well as other procedures that result in the expression of the recombinant protein in the host cell.

[0139] The terms "gene" and "gene segment" are used interchangeably in this document. They refer to polynucleotides containing at least one open reading frame (ORF) capable of encoding a specific protein after transcription and translation. A gene or gene segment can be genomic DNA or cDNA, provided that the polynucleotide contains at least one ORF, which may cover the entire coding region or a segment thereof. A "fusion gene" is a gene consisting of at least two heterologous polynucleotides linked together.

[0140] As used herein, a “coding region” or “coding sequence” is a portion of a polynucleotide consisting of codons that can be translated into amino acids. While “stop codons” (TAG, TGA, or TAA) are not typically translated into amino acids, they can be considered part of a coding region; however, any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of a coding region. The boundaries of a coding region are typically defined by the 5' start codon at the N-terminus of the resulting polypeptide and the 3' translation stop codon at the C-terminus of the resulting polypeptide. Two or more coding regions of the present invention may be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. Subsequently, a single vector may contain only a single coding region, or two or more coding regions, e.g., a single vector may encode binding domain A and binding domain B, as described below, respectively. Furthermore, the vectors, polynucleotides, or nucleic acids of the present invention may encode heterologous coding regions fused to or not fused with the nucleic acid encoding the binding domains of the present invention. Heterogeneous coding regions include, but are not limited to, specific elements or motifs, such as secretory signal peptides or heterogeneous functional domains.

[0141] The term "downstream" refers to the nucleotide sequence located at the 3' of the reference nucleotide sequence. In some embodiments, the downstream nucleotide sequence involves the sequence following the transcription start site. For example, the translation start codon of a gene is located downstream of the transcription start site.

[0142] The term "upstream" refers to the nucleotide sequence located at the 5' end of a reference nucleotide sequence. In some embodiments, the upstream nucleotide sequence involves a sequence located 5' to the side of the coding region or transcription start site. For example, most promoters are located upstream of the transcription start site.

[0143] "Homology" or "homology" refers to the sequence similarity or interchangeability between two or more polynucleotide sequences or two or more polypeptide sequences. When using programs such as BestFit to determine sequence identity, similarity, or homology between two different amino acid sequences, default settings can be used, or an appropriate scoring matrix such as blosum45 or blosum80 can be selected to optimize the identity, similarity, or homology score. Preferably, homologous polynucleotides are those sequences that hybridize under stringent conditions as defined herein and have at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably 97%, more preferably 98%, and even more preferably 99% sequence identity compared to those sequences. Homologous polypeptides, when optimally aligned on sequences of comparable length, preferably have at least 70%, preferably at least 80%, and even more preferably at least 90%, and even more preferably at least 95-99% identical sequence identity.

[0144] In polynucleotides, "ligation" refers to the process of forming phosphodiester bonds between two nucleic acid fragments or genes, thus linking them together. For DNA fragments or genes to be joined, their ends must be compatible. In some cases, the ends are compatible after digestion with endonucleases. However, it may be necessary to first convert the staggered ends, which are usually produced after endonuclease digestion, into blunt ends to make them suitable for ligation.

[0145] The term "stringent conditions" or "stringent hybridization conditions" refers to conditions under which a polynucleotide will hybridize to its target sequence to a much greater extent than other sequences (e.g., at least twice the background). Generally, the stringency of hybridization is expressed in part by the temperature and salt concentration at which the washing steps are performed. Typically, stringent conditions are those where the salt concentration is less than about 1.5 M Na ions at pH 7.0 to 8.3, typically about 0.01 to 1.0 M Na ion concentration (or other salt), and the temperature is at least about 30°C for short polynucleotides (e.g., 10 to 50 nucleotides) and at least about 60°C for long polynucleotides (e.g., greater than 50 nucleotides)—for example, "stringent conditions" could include hybridization at 37°C in 50% formamide, 1 M NaCl, and 1% SDS, followed by three washes at 60°C to 65°C for 15 minutes each in 0.1 × SSC / 1% SDS. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C can be used. SSC concentrations can range from approximately 0.1 to 2 × SSC, with SDS present at approximately 0.1%. This washing temperature is typically chosen to be approximately 5°C to 20°C lower than the thermodynamic melting point of the specific sequence at a given ionic strength and pH. Tm is the temperature at which 50% of the target sequence hybridizes with a perfectly matched probe (at a given ionic strength and pH). Equations for calculating Tm and nucleic acid hybridization conditions are well known and can be found in Sambrook, J. Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, 2001. Typically, blocking agents are used to block nonspecific hybridization. Such blocking agents include, for example, approximately 100–200 µg / ml of cleaved and denatured salmon sperm DNA. In specific cases, such as for RNA:DNA hybridization, organic solvents, such as formamide at a concentration of approximately 35–50% v / v, may also be used. Useful modifications to these washing conditions will be apparent to those skilled in the art.

[0146] The terms “percentage identity,” “sequence identity percentage,” and “% identity” when applied to polynucleotide sequences refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a normalization algorithm. Such an algorithm can insert gaps in the compared sequences in a normalized and reproducible manner to optimize the alignment between the two sequences, thus enabling a more meaningful comparison. Percentage identity can be measured over the entire length of a given polynucleotide sequence, or over a shorter length, such as a fragment length derived from a larger given polynucleotide sequence, for example, a fragment of at least 45, 60, 90, 120, 150, 210, or 450 consecutive residues. These lengths are merely exemplary, and it should be understood that any fragment length supported by the sequences shown herein in tables, figures, or sequence listings can be used to describe the length at which percentage identity can be measured. The percentage of sequence identity is calculated as follows: Two best-aligned sequences are compared within a comparison window. The number of matching positions (where the same residues appear in both polypeptide sequences) is determined. The number of matching positions is divided by the total number of positions within the comparison window (i.e., the window size), and the result is multiplied by 100 to obtain the percentage of sequence identity. When comparing sequences of different lengths, the shortest sequence defines the length of the comparison window. Conservative substitutions are not considered when calculating sequence identity.

[0147] The "percentage (%) sequence identity" of the polypeptide sequence identified herein is defined as the percentage of amino acid residues in the sequence that are identical to those in a second comparable length of a reference polypeptide sequence or a portion thereof, after alignment of the sequence and, where necessary, the introduction of vacancies to achieve maximum percentage sequence identity and without treating any conserved substitutions as part of sequence identity to result in optimal alignment. Alignments intended to determine percentage amino acid sequence identity can be performed in various ways within the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve optimal alignment over the full length of the sequences being compared. Percentage identity can be measured over the entire determined length of the polypeptide sequence, or it can be measured over shorter lengths, such as fragments derived from larger determined polypeptide sequences, said fragments being, for example, fragments of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 consecutive residues. These lengths are merely illustrative, and it should be understood that any segment length supported by the sequences shown in the tables, figures, or sequence lists herein can be used to describe the length of measurable percentage identity.

[0148] In the context of polynucleotide sequences, "reproducibility" refers to the degree of internal homology within a sequence, such as the frequency of identical nucleotide sequences of a given length. For example, reproducibility can be determined by analyzing the frequency of identical sequences.

[0149] As used herein, the term "expression" refers to the process by which polynucleotides produce gene products, such as RNA or polypeptides. This includes, but is not limited to, the transcription of polynucleotides into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product, and the translation of mRNA into polypeptides. Expression produces "gene products." As used herein, gene products can be nucleic acids, such as messenger RNA produced through gene transcription or polypeptides translated from transcripts. Gene products described herein further include nucleic acids with post-transcriptional modifications, such as polyadenylation or splicing, or polypeptides with post-translational modifications, such as methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage.

[0150] The terms "vector" and "expression vector" are used interchangeably and refer to a nucleic acid molecule that preferably self-replicates in a suitable host, transferring an inserted nucleic acid molecule into a host cell and / or between host cells. The term includes vectors whose primary function is to insert DNA or RNA into a cell, replication vectors whose primary function is to replicate DNA or RNA, and expression vectors whose function is to transcribe and / or translate DNA or RNA. It also includes vectors that provide more than one of the above functions. An "expression vector" is a polynucleotide that, when introduced into a suitable host cell, can be transcribed and translated into a polypeptide. An "expression system" generally refers to a suitable host cell containing an expression vector that can be used to produce a desired expression product.

[0151] When applied to peptides, "serum degradation resistance" refers to the peptide's ability to resist degradation in blood or its components, typically associated with proteases in serum or plasma. Serum degradation resistance can be measured by mixing the protein with human (or, where appropriate, mouse, rat, dog, or monkey) serum or plasma, typically at approximately 37°C, for a specific number of days (e.g., 0.25, 0.5, 1, 2, 4, 8, or 16 days). Samples at these time points can be analyzed using Western blotting, and the protein can be detected using antibodies. Antibodies can target tags on the protein. If the protein shows a single band on the Western blot, where the protein size is the same as the injected protein size, degradation has not occurred. In this exemplary method, the time point at which 50% of the protein is degraded, determined by Western blotting or equivalent techniques, is the serum degradation half-life or "serum half-life" of the protein.

[0152] The term "t" 1 / 2The terms “half-life”, “terminal half-life”, “elimination half-life”, and “cyclic half-life” are used interchangeably in this paper, and when used in this paper, they refer to the calculation ln(2) / K. el Terminal half-life. K el The terminal elimination rate constant is calculated by linear regression of the terminal linear portion of the log concentration versus time curve. Half-life generally refers to the time required for half of the administered substance stored in a living organism to be metabolized or eliminated by normal biological processes. When the clearance curve of a given peptide is constructed as a function of time, the curve is typically biphasic, with a rapid α phase and a longer β phase. The typical β-phase half-life of human antibodies in humans is 21 days. Half-life can be measured using timed samples from any bodily fluid, but is ultimately measured in plasma samples.

[0153] The term "molecular weight" generally refers to the sum of the atomic masses of the constituent atoms in a molecule. Molecular weight can be theoretically determined by adding the atomic masses of the constituent atoms in the molecule. When applied in the context of peptides, molecular weight is calculated by adding the molecular weights of each type of amino acid in the composition based on the amino acid composition, or by comparing with molecular weight standards in an SDS electrophoresis gel. The calculated molecular weight of a molecule may differ from the molecule's "apparent molecular weight," which typically refers to the molecular weight of a molecule determined by one or more analytical techniques. "Apparent molecular weight factor" and "apparent molecular weight" are related terms, and when applied in the context of peptides, the term refers to a measure of the relative increase or decrease in the apparent molecular weight exhibited by a particular amino acid or peptide sequence. For example, apparent molecular weight can be determined using size exclusion chromatography (SEC) or similar methods by comparison with globular protein standards, as measured in "apparent kD" units. The apparent molecular weight factor is the ratio between the apparent molecular weight and the "molecular weight," which is calculated by adding based on the amino acid composition as described above, or by estimating by comparing with molecular weight standards in an SDS electrophoresis gel. The determination of apparent molecular weight and apparent molecular weight factor is described in U.S. Patent No. 8,673,860.

[0154] The term "hydrodynamic radius" or "Stokes radius" is the effective radius (Rh, in nm) of a molecule in solution, measured by assuming it is a bulk that moves through the solution and is resisted by the solution's viscosity. In embodiments of the invention, the hydrodynamic radius measurement of XTEN peptides is associated with the "apparent molecular weight factor," a more intuitive measure. The "hydrodynamic radius" of a protein affects its diffusion rate in aqueous solutions and its ability to migrate in macromolecular gels. The hydrodynamic radius of a protein is determined by its molecular weight and its structure, including shape and density. Methods for determining the hydrodynamic radius are well known in the art, for example, by using size exclusion chromatography (SEC), as described in U.S. Patent Nos. 6,406,632 and 7,294,513. Most proteins have a globular structure, which is the most compact three-dimensional structure a protein can have at the minimum hydrodynamic radius. Some proteins have random and open unstructured or 'linear' conformations, and therefore have much larger hydrodynamic radii compared to typical globular proteins of similar molecular weight.

[0155] The "diffusion coefficient" refers to the order of magnitude of the molar flux through a surface per unit out-of-plane concentration gradient. In the transport of dilute substances, the flux caused by diffusion is derived from Fick's first law, and it depends solely on a single property of the interaction between the solute and the solvent: the diffusion coefficient.

[0156] “Physiological conditions” refers to a set of conditions in a live host and in vitro conditions that mimic those of a live subject, including temperature, salt concentration, and pH. Many physiologically relevant conditions have been established for in vitro analysis. Generally, physiological buffers contain physiological concentrations of salt and are adjusted to a neutral pH, ranging from about 6.5 to about 7.8, preferably from about 7.0 to about 7.5. Many physiological buffers are listed in Sambrook et al. (2001). Physiologically relevant temperatures are from about 25°C to about 38°C, preferably from about 35°C to about 37°C.

[0157] The term “binding domain” as used in this article is specifically intended to include a class of antibodies or antibody fragments that have a specific binding affinity to target antigens or ligands such as cell surface receptors or antigens or glycoproteins, oligonucleotides, enzyme substrates, antigenic determinants, or binding sites that may be present in or on the surface of target tissues or cells.

[0158] The term "antibody" is used in its broadest sense herein and includes a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity. Full-length antibodies may be, for example, monoclonal, recombinant, chimeric, immune-depleted, humanized, and human antibodies.

[0159] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that the individual antibodies comprising this population are identical and / or bind to the same epitopes, except for possible variant antibodies, such as those containing naturally occurring mutations or those occurring during the production of the monoclonal antibody formulation. These variants are typically present in small quantities. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies used according to the invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci. Such methods and other exemplary methods for preparing monoclonal antibodies are known in the art or described herein.

[0160] "Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody and binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to: Fv, Fab, Fab′, Fab′-SH, F(ab′)2, biantibodies, linear antibodies, single-domain antibodies, single-domain camel antibodies, single-chain antibody molecules (scFv), and multispecific antibodies formed from antibody fragments.

[0161] "scFv" or "single-chain variable fragment" is used interchangeably in this document and refers to an antibody fragment containing two copies of a heavy chain variable region ("VH") and a light chain variable region ("VL"), or either the VH or VL chain, linked together by a short, flexible peptide linker. scFv is not actually a fragment of an antibody, but rather a fusion protein of the heavy chain variable region (VH) and the light chain variable region (VL) of an immunoglobulin, and can be readily expressed in its functional form in *E. coli*.

[0162] The terms “antigen,” “target antigen,” and “immunogen” are used interchangeably in this document and refer to the structural or binding determinants to which antibodies, antibody fragments, or antibody fragment-based molecules bind or are specifically bound.

[0163] The term "epitope" refers to a specific site on an antigen molecule to which an antibody, antibody fragment, or binding domain binds. An epitope is a ligand for an antibody or antibody fragment.

[0164] As used in this article, "CD3" or "differentiation group 3" refers to the T cell surface antigen CD3 complex, which contains all known CD3 subunits, such as CD3ε, CD3δ, CD3γ, CD3ζ, CD3α, and CD3β, either alone or in independent combinations. The extracellular domains of CD3ε, γ, and δ contain immunoglobulin-like domains and are therefore considered part of the immunoglobulin superfamily.

[0165] The terms “specific binding” or “specifically binding” or “binding specificity” are used interchangeably herein and refer to a high binding affinity of the binding domain for its corresponding target. Typically, specific binding measured by one or more of the assays disclosed herein will have a binding affinity of less than about 10. -6 The dissociation constant of M or K d .

[0166] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, "binding affinity" as used herein refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be determined by the dissociation constant (K). d The term "higher binding affinity" as used in this paper refers to a lower K0. d Value; for example, 1 x 10 -9 M is 1 x 10 -8 M has a greater binding affinity.

[0167] The terms "inhibition constant" and "Ki" are used interchangeably. It refers to the dissociation constant of the enzyme-inhibitor complex or the reciprocal of the inhibitor's affinity for the enzyme.

[0168] "Dissociation constant" or "K" d "Can be used interchangeably" refers to the affinity between the ligand "L" and the protein "P"; that is, how tightly the ligand binds to a specific protein. Formula K can be used to express this affinity. d The calculation is performed using [L][P] / [LP], where [P], [L], and [LP] represent the molar concentrations of the protein, ligand, and complex, respectively. The term "k" is used in this paper. on "k" refers to the binding rate constant for the association of an antibody with an antigen to form an antibody / antigen complex, as is known in the art. The term "k" as used herein... off "Intended to represent the dissociation rate constant of an antibody from an antibody / antigen complex, as is known in the art."

[0169] As used herein, the term "antagonist" includes any molecule that partially or completely blocks, inhibits, or neutralizes the biological activity of the natural polypeptides disclosed herein. Methods for identifying polypeptide antagonists may include contacting a natural polypeptide with a candidate antagonist molecule and measuring detectable changes in one or more biological activities typically associated with the natural polypeptide. In the context of this invention, antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecule that reduces the effect of a biologically active protein.

[0170] "Target cell markers" refer to molecules expressed by target cells, including but not limited to cell surface receptors, antigens, glycoproteins, oligonucleotides, enzyme substrates, antigenic determinants, or binding sites that may be present on or in the surface of target tissues or target cells that can act as ligands for antibodies.

[0171] "Target tissue" refers to tissue that is a cause of or part of a disease condition, such as, but not limited to, cancer or inflammation. Sources of diseased target tissue include body organs, tumors, cancer cells, or populations of cancer cells or cell populations that form the matrix or are associated with cancer cell populations, bone, skin, and cells that produce cytokines or factors that contribute to the disease condition.

[0172] "Definitive-component medium" refers to a medium containing the nutrients and hormones necessary for cell survival and / or growth in a culture, such that the components of the medium are known. Traditionally, specific-component mediums have been prepared by adding nutrients and growth factors necessary for growth and / or survival. Typically, a specific-component medium provides at least one component from one or more of the following categories: a) all essential amino acids, typically a basic combination of twenty amino acids plus cysteine; b) an energy source, typically in the form of carbohydrates such as glucose; c) low concentrations of required vitamins and / or other organic compounds; d) free fatty acids; and e) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements typically required in very low concentrations, typically in the micromolar range. Specific-component mediums may also optionally be supplemented with one or more components from any of the following categories: a) one or more mitogenic agents; b) salts and buffers, such as calcium, magnesium, and phosphates; c) nucleosides and bases, such as, for example, adenosine and thymidine, hypoxanthine; and d) proteins and tissue hydrolysates.

[0173] The term "agonist" is used in the broadest sense and includes any molecule that mimics the biological activity of the natural polypeptides disclosed herein. Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments or amino acid sequence variants of natural polypeptides, peptides, small organic molecules, etc. Methods for identifying agonists of natural polypeptides may include contacting the natural polypeptide with a candidate agonist molecule and measuring detectable changes in one or more biological activities typically associated with the natural polypeptide.

[0174] The terms “treatment,” “management,” “mitigation,” or “improvement” as used herein are used interchangeably. These terms refer to methods for obtaining beneficial or desired outcomes, including but not limited to therapeutic and / or preventive benefits. A therapeutic benefit is the eradication or improvement of the underlying condition being treated. Additionally, a therapeutic benefit can be achieved by eradicating or improving one or more physical symptoms or improving one or more clinical parameters associated with the underlying condition, such that improvement is observed in a subject, even though the subject may still have the underlying disease. Regarding preventive benefits, the composition may be administered to subjects at risk of developing a specific disease or to subjects reporting physical symptoms of one or more diseases, even if the disease may not yet be diagnosed.

[0175] As used herein, "therapeutic effect" or "therapeutic benefit" refers to physiological effects, including but not limited to the mitigation, relief, or prevention of disease in humans or other animals, or improvement of one or more clinical parameters associated with an underlying condition, or, in addition, enhancement of the physical or mental state of humans or animals caused by the ability of administering the polypeptides of the present invention to induce the production of antibodies against antigenic epitopes of biologically active proteins. For preventative benefits, the composition may be administered to subjects at risk of developing a specific disease, a recurrence of a previous disease, a condition or symptoms of a disease, or subjects who have reported one or more physiological symptoms of a disease, although a diagnosis of the disease may not yet have been made.

[0176] As used herein, the terms "therapeutic effective amount" and "therapeutic dose" refer to an amount of a drug or bioactive protein, alone or as part of a polypeptide composition, that, when administered to a subject in a single or repeated dose, is capable of having any detectable beneficial effect on any symptom, aspect, measurement parameter, or characteristic of a disease state or condition. Such effect is not necessarily absolutely beneficial. The determination of a therapeutically effective amount is within the competence of a skilled person, particularly based on the detailed disclosure provided herein.

[0177] As used herein, the term "therapeuticly effective and non-toxic dose" refers to a tolerable dose of a composition as defined herein, which is sufficiently high in subjects to induce tumor or cancer cell depletion, tumor elimination, tumor shrinkage, or disease stabilization without or with minimal major toxic effects. Such therapeutically effective and non-toxic doses can be determined through dose escalation studies as described in the art and should be below doses that could induce serious adverse side effects.

[0178] As used herein, the term "dosing regimen" refers to a regimen in which multiple doses (i.e., at least two or more) of a composition are administered consecutively, wherein the doses are given in a therapeutically effective amount to produce a sustained beneficial effect on any symptom, aspect, measurement parameter, endpoint, or characteristic of a disease state or condition.

[0179] The terms "cancer" and "carcinomatous" refer to or describe a physiological condition in mammals typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to: carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, T-cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, colon cancer, prostate cancer, head and neck cancer, any form of skin cancer, melanoma, genitourinary tract cancer, ovarian cancer, ovarian cancer with malignant ascites, metastatic peritoneal cancer, serous uterine carcinoma, endometrial cancer, cervical cancer, colorectal cancer, intraepithelial malignant tumors with malignant ascites, uterine cancer, mesothelioma in the peritoneum, kidney cancer, lung cancer, etc. Small cell lung cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, stomach cancer, small intestinal cancer, liver cancer, hepatocellular carcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, bile duct cancer, salivary gland cancer, thyroid cancer, epithelial cancer, adenocarcinoma, sarcoma of any origin, primary hematologic malignancies including acute or chronic lymphocytic leukemia, acute or chronic myeloid leukemia, myeloproliferative neoplasms or myelodysplastic disorders, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, or Goodpasture syndrome.

[0180] The term "tumor-specific marker" as used in this article refers to antigens found on or in cancer cells that may be found in higher numbers in or on cancer cells than in normal cells or tissues, but this is not always the case.

[0181] “Target cells” refers to cells that have ligands of antibodies or antibody fragments of the subject composition and that are associated with or cause a disease or pathological state, including cancer cells, tumor cells, and inflammatory cells. Ligands of target cells are referred to herein as “target cell markers” or “target cell antigens” and include, but are not limited to, cell surface receptors or antigens, cytokines, MHC proteins, and exogenously presented cytoplasmic proteins or peptides. As used herein, “target cells” will not include effector cells. I) General technology

[0182] Unless otherwise stated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA are employed in the practice of this invention, which are within the scope of the art. See J. et al., "Molecular Cloning: A Laboratory Manual", 3rd edition, Cold Spring Harbor Laboratory Press, 2001; "Current protocols in molecular biology", edited by FMAusubel et al., 1987; "Methods in Enzymology" series, Academic Press, San Diego, CA.; "PCR 2: a practical approach", edited by MJ MacPherson, BD Hames and GR Taylor, Oxford University Press, 1995; "Antibodies, a laboratory manual" Harlow, E. and Lane, D., eds., Cold Spring Harbor Laboratory, 1988; "Goodman & Gilman's The Pharmacological Basis of Therapeutics", 11th ed., McGraw-Hill, 2005; and Freshney, RI, "Culture of Animal Cells: A Manual of Basic Technique", 4th ed., John Wiley & Sons, Somerset, NJ, In 2000, all of these contents were incorporated into this article in their entirety through citation.

[0183] Host cells can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco Modified Eagle Medium (DMEM, Sigma) are suitable for culturing eukaryotic cells. In addition, animal cells can be grown in serum-deficient media supplemented with hormones, growth factors, or any other factors necessary for the survival and / or growth of a specific cell type. Definitely defined media supporting cell survival maintain viability, morphology, metabolic capacity, and potential cell differentiation capacity, while those promoting cell growth provide all the chemical substances required for cell proliferation or hyperplasia. General parameters for controlling mammalian cell survival and in vitro growth are established in the art. Physicochemical parameters that can be controlled in different cell culture systems include, for example, pH, pO2, temperature, and molar osmotic pressure concentration. The nutritional needs of cells are typically met in standard culture medium formulations developed to provide optimal conditions. Nutrients can be categorized into several classes: amino acids and their derivatives, carbohydrates, sugars, fatty acids, complex lipids, nucleic acid derivatives, and vitamins. In addition to nutrients necessary to maintain cellular metabolism, most cells require one or more hormones from at least one of the following groups: steroids, prostaglandins, growth factors, pituitary hormones, and peptide hormones, to proliferate in serum-free media (Sato, GH et al., “Growth of Cells in Hormonally Defined Media”, Cold Spring Harbor Press, NY, 1982). Besides hormones, cells may require transport proteins such as transferrin (plasma iron transporter), ceruloplasmin (copper transporter), and high-density lipoprotein (lipid carrier) for in vitro survival and growth. The optimal combination of hormones or transport proteins will vary depending on the cell type. Most of these hormones or transport proteins are exogenously added, or, in rare cases, mutant cell lines that do not require specific factors have been discovered. Those skilled in the art will know of other factors required to maintain cell culture without extensive experimentation.

[0184] Growth media for prokaryotic host cell growth include nutrient liquid media (liquid nutrient media) or LB medium (Luria Bertani). Suitable media include defined-component and undefined-component media. Typically, the media contains carbon sources required for bacterial growth, such as glucose, water, and salts. The media may also contain amino acid and nitrogen sources, such as beef or yeast extract (in undefined-component media) or known amounts of amino acids (in defined-component media). In some embodiments, the growth medium is LB liquid medium, such as LB Miller liquid medium or LB Lennox liquid medium. LB liquid medium contains peptone (an enzymatic digestion product of casein), yeast extract, and sodium chloride. In some embodiments, selective media containing antibiotics are used. In such media, only desired cells with antibiotic resistance will grow. II) Chimeric polypeptide assembly composition

[0185] This invention relates in part to chimeric polypeptide assembly compositions (also known as "ProTIA") that can be used to treat, improve or prevent diseases including but not limited to cancer, autoimmune or inflammatory diseases.

[0186] In a first aspect, the present invention provides a chimeric polypeptide assembly that generally comprises a first portion, a second portion, and a third portion, wherein: the first portion comprises (i) a first binding domain having binding specificity to a target cell marker; and (ii) a second binding domain having binding specificity to an effector cell antigen; the second portion comprises a peptide release segment (RS) capable of being cleaved by one or more mammalian proteases; and the third portion comprises a filler portion; wherein the filler portion is capable of being released from the first portion by the action of the mammalian proteases on the second portion. Unbound by theory, the exemplary polypeptide assemblies of this disclosure exhibit one or more of the following features: 1) the assembly comprises at least two binding domains capable of simultaneously binding to effector cells and target cells; 2) the filling portion of the assembly i) shields the binding domains and reduces the binding affinity to the target antigen by, for example, steric hindrance when the composition is intact; ii) provides an increased half-life of the composition when administered to a subject; and / or iii) reduces the leakage of the composition from blood vessels in normal tissues and organs compared to diseased tissues (e.g., tumors), resulting in increased safety compared to bispecific cytotoxic antibody therapeutics currently used or evaluated in clinical trials; and 3) when the assembly is near diseased tissues such as tumors or inflamed tissues, it can be cleaved by one or more mammalian proteases, thereby releasing the binding domains of the first portion, such that the binding domains can bind to target cell markers and effector cell antigens with higher affinity compared to a state where the binding domains are not cleaved from the assembly. Because the therapeutic portion (e.g., a first portion capable of bridging target cells and effector cells) is released at the site of the lesion, the assembly of the present invention can be advantageously used as a "prodrug" in which the protease is preferentially expressed compared to normal tissue. The assembly of this subject matter addresses the problems including BiTE... ® This addresses several serious drawbacks of existing bispecific antibodies, including BiTE. The subject assembly typically retains the properties of bispecific antibodies such as BiTE. ® The known therapeutic benefits of tumor contraction are achieved while mitigating the inherent side effects of conventional bispecific antibodies. In one embodiment, the present invention provides a chimeric polypeptide assembly composition wherein a first portion comprises two binding domains in single-chain form, wherein the first binding domain has binding specificity for tumor-specific markers or antigens of target cells, and the second binding domain has binding specificity for effector cell antigens, such as receptors on effector cells or ligands within effector cells, such that the composition is bispecific.

[0187] In some embodiments, the subject composition is designed such that the action of a protease cleaves a release segment (RS) of the subject composition, thereby releasing a binding domain and a filler portion from the composition. Upon release from the composition, a first binding domain, specific for binding to tumor-specific markers or target cell antigens, and a second binding domain, specific for binding to effector cell antigens, are simultaneously able to bind to target cells with a greater binding affinity than the intact composition, linking effector cells to target cells and forming an immune synapse. As a result, at a very low effector-to-target (E:T) ratio, target cells are acted upon by effector molecules released by effector cells into the cell-to-cell immune synapse, causing damage including, but not limited to, perforin-mediated lysis, granzyme B-induced cell death and / or apoptosis of target cells. In some embodiments, the first portion of the composition released is designed to have binding specificity, enabling it to simultaneously bind to preselected surface antigens on effector cytotoxic T lymphocytes and tumor cells in the subject, thereby achieving selective, targeted, and localized effects of the immune synapse and released cytokines and effector molecules against the target tumor. This results in damage or destruction of tumor cells, producing antitumor activity and therapeutic benefit for the subject. In some other embodiments, the effector cells bound by the first portion of the released cells are selected from plasma cells, B cells, cytokine-induced killer cells (CIK cells), mast cells, dendritic cells, regulatory T cells (RegT cells), helper T cells, myeloid cells, and NK cells.

[0188] On the other hand, the present invention provides a chimeric polypeptide assembly composition comprising a first part, a second part, a third part, a fourth part, and a fifth part, wherein the first part comprises a first and a second binding domain (described in more detail below), the second part comprises a release segment (RS), the third part comprises a filler portion (described in more detail below), the fourth part comprises a release segment (RS) that may be the same as or different from the second part RS, and the fifth part comprises a filler portion that may be the same as or different from the third part filler portion; the composition is substantially in a prodrug form until it is subjected to protease action.

[0189] The composition fulfills a long-standing need for a bispecific therapeutic agent with higher selectivity, a longer half-life, and less toxicity and side effects once cleaved by a protease associated with the unhealthy target tissue or tissue caused by the disease. Thus, the composition exhibits an improved therapeutic index compared to known bispecific antibody compositions in the art. Such a composition can be used to treat certain diseases, including but not limited to cancer. 1. Associative domain

[0190] The object of this invention is to provide a chimeric polypeptide assembly composition comprising a first portion, the first portion comprising at least a first binding domain having binding specificity to a target cell marker (e.g., a tumor-specific marker) and a second binding domain having binding specificity to an effector cell antigen. In some embodiments, the binding domain is attached as a single strand exhibiting bispecific binding specificity to the target cell marker and the effector cell antigen.

[0191] On the other hand, an object of the present invention is to provide a cleavable chimeric polypeptide assembly composition designed to have a configuration in which a first binding domain is connected to a filler portion via a short peptide release segment containing a cleavage sequence. In this exemplary configuration, the binding domain is shielded by a proximal filler portion component to reduce or eliminate nonspecific interactions and to bind to non-pathological tissues or cells that are not the intended target of the composition, thereby reducing undesirable toxicity or side effects. Furthermore, when the release segment is cleaved by a protease (described in more detail below) preferentially expressed in pathological tissues, the shielded filler portion is released at the target site (e.g., pathological tissue). The released first portion then regains its ability to bind corresponding ligands more freely or more preferentially, including target cell markers and effector cell markers. Without wishing to be bound by any particular theory, the subject-specific chimeric polypeptide assembly offers multiple advantages as a therapeutic agent compared to the side effects experienced when or after administration of a composition having only a first-part bispecific binding domain at comparable doses of mmol / kg, in terms of reduced administration frequency, increased duration of therapeutic effect, and reduced severity of diagnosis-related side effects in subjects. Non-limiting examples of side effects that can be avoided or reduced by using the compositions of the present invention include undesirable increases in plasma levels of IL-2, TNF-α, IFN-γ, liver enzymes, and / or the incidence of sepsis, febrile neutropenia, neurotoxicity, seizures, encephalopathy, cytokine release syndrome, speech disorders, balance disorders, fever, headache, confusion, hypotension, neutropenia, nausea, impaired consciousness, and disorientation.

[0192] This invention contemplates the use of single-chain binding domains in the subject compositions, such as, but not limited to, Fv, Fab, Fab′, Fab′-SH, F(ab′)2, linear antibodies, single-domain antibodies, single-domain camel antibodies, single-chain antibody molecules (scFv), and biantibodies capable of binding ligands or receptors associated with effector cells and antigens of diseased tissues or cells of cancer, tumors, or other malignant tissues. In some other embodiments, the first and second binding domains of the first portion of the chimeric polypeptide assembly composition may be non-antibody scaffolds, such as anticalins, adnectins, fynomers, affiliates, affibodies, centyrins, and DARPins. In some other embodiments, the binding domain of the tumor cell target is a variable domain of a T-cell receptor that has been engineered to bind MHC fragments carrying proteins overexpressed by tumor cells. The compositions of this invention are designed to provide a broad therapeutic window by considering the location of the target tissue protease, the presence of the same protease in healthy tissue that is not targeted, and the presence of the target ligand in healthy tissue but more of the ligand in unhealthy target tissue. "Therapeutic window" refers to the maximum difference between the minimum effective dose and the maximum tolerated dose of a given therapeutic composition. To help obtain a wide therapeutic window, the binding domain of the first portion of the composition is shielded by proximity to the filler portion (e.g., XTEN), such that the binding affinity of the intact composition for one or both ligands is reduced compared to the composition cleaved by mammalian proteases, thereby releasing the first portion from the shielding effect of the filler portion.

[0193] It has been established that, regarding the single-chain binding domain, an Fv is the smallest antibody fragment containing complete antigen recognition and binding sites; it consists of a dimer of a non-covalently associated heavy chain variable domain (VH) and a light chain variable domain (VL). Within each VH and VL chain are three complementarity-determining regions (CDRs), which interact to determine the antigen-binding site on the surface of the VH-VL dimer; the six CDRs of the binding domain provide antigen-binding specificity to the antibody or single-chain binding domain. In some cases, scFvs are created, where each has 3, 4, or 5 CDRs within each binding domain. The framework sequences flanking the CDRs have a largely conserved tertiary structure in transspecies natural immunoglobulins, and framework residues (FRs) serve to maintain the CDRs in their proper orientation. Binding function does not require a constant domain, but a constant domain can contribute to stabilizing the VH-VL interaction. The binding site domains of the polypeptides of the present invention can be a pair of VH-VL, VH-VH, or VL-VL domains of the same or different immunoglobulins; however, it is generally preferred to prepare single-chain binding domains using the respective VH and VL chains from the parent antibody. The order of the VH and VL domains in the polypeptide chain is not limiting to the present invention; the given order of binding domains can generally be reversed without any loss of function, but it is understood that the VH and VL domains are arranged to allow the antigen binding site to fold correctly. Therefore, the single-chain binding domains of the bispecific scFv embodiments of the subject composition can be in the order of (VL-VH)1-(VL-VH)2, or (VL-VH)1-(VH-VL)2, or (VH-VL)1-(VL-VH)2, or (VH-VL)1-(VH-VL)2, where “1” and “2” represent the first and second binding domains, respectively, wherein the paired binding domains are linked by a polypeptide linker as described below.

[0194] Therefore, in the exemplary bispecific single-chain antibodies disclosed herein, the binding domains can be arranged such that the first binding domain is located at the C-terminus of the second binding domain. The V chain arrangement can be VH (target cell surface antigen)-VL (target cell surface antigen)-VL (effective cell antigen)-VH (effective cell antigen), VH (target cell surface antigen)-VL (target cell surface antigen)-VH (effective cell antigen)-VL (effective cell antigen), VL (target cell surface antigen)-VH (target cell surface antigen)-VL (effective cell antigen)-VH (effective cell antigen) or VL (target cell surface antigen)-VH (target cell surface antigen)-VL (effective cell antigen). For arrangements where the second binding domain is located at the N-terminus of the first binding domain, the following sequences are possible: VH (effective cell antigen)-VL (effective cell antigen)-VL (target cell surface antigen)-VH (target cell surface antigen), VH (effective cell antigen)-VL (effective cell antigen)-VH (target cell surface antigen)-VL (target cell surface antigen), VL (effective cell antigen)-VH (effective cell antigen)-VL (target cell surface antigen)-VH (target cell surface antigen), or VL (effective cell antigen)-VH (effective cell antigen)-VH (target cell surface antigen)-VL (target cell surface antigen). As used herein, "N-terminus" or "C-terminus" and their grammatical variations indicate relative positions within the primary amino acid sequence, rather than absolute N-terminus or C-terminus of the bispecific single-chain antibody. Therefore, as a non-limiting example, "located at the C-terminus of the second binding domain" means that the first binding domain is located on the carboxyl side of the second binding domain within the bispecific single-chain antibody, and does not exclude the possibility of additional sequences, such as His-tags or other compounds, such as radioisotopes, being located at the C-terminus of the bispecific single-chain antibody.

[0195] In one embodiment, the chimeric polypeptide assembly composition comprises a first portion containing a first binding domain and a second binding domain, wherein each of the binding domains is an scFv, and wherein each scFv contains a VL and a VH. In another embodiment, the chimeric polypeptide assembly composition comprises a first portion containing a first binding domain and a second binding domain, wherein the binding domains are a biantibody configuration, and wherein each domain contains a VL domain and a VH. In the foregoing embodiments, the first binding domain has binding specificity for tumor-specific markers or antigens of target cells, and the second binding domain has binding specificity for effector cell antigens. In one of the foregoing embodiments, the effector cell antigen is expressed on or within effector cells. In one embodiment, the effector cell antigen is expressed on T cells such as CD4+, CD8+, or natural killer (NK) cells. In another embodiment, the effector cell antigen is expressed on B cells, mast cells, dendritic cells, or myeloid cells. In one embodiment, the effector cell antigen is CD3—a group 3 antigen of cytotoxic T cells. In some of the foregoing embodiments, the first binding domain exhibits binding specificity for tumor-specific markers associated with tumor cells. In one embodiment, the binding domain has a binding affinity for tumor-specific markers, wherein the tumor cells may include, but are not limited to, cells derived from: stromal cell tumors, fibroblast tumors, myofibroblast tumors, glial cell tumors, epithelial cell tumors, adipocyte tumors, immune cell tumors, vascular cell tumors, and smooth muscle cell tumors.In one embodiment, the tumor-specific marker or target cell antigen is selected from: α4 integrin, Ang2, B7-H3, B7-H6, CEACAM5, cMET, CTLA4, FOLR1, EpCAM, CCR5, CD19, HER2, HER2 neu, HER3, HER4, HER1 (EGFR), PD-L1, PSMA, CEA, MUC1 (mucin), MUC-2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC16, βhCG, Lewis-Y, CD20, CD33, CD38, CD30, CD56 (NCAM), CD133, ganglioside GD3, 9-O-acetyl-GD3, GM2, Globo H, Fucosyl GM1, GD2, Carbonic anhydrase IX, CD44v6, Shh, Wue-1, Plasma cell antigen 1, Melanoma chondroitin sulfate proteoglycan (MCSP), CCR8, Prostate 6-transmembrane epithelial antigen (STEAP), Mesothelin, A33 antigen, Prostate stem cell antigen (PSCA), Ly-6, Desmosome core protein 4, Fetal acetylcholine receptor (fnAChR), CD25, Cancer antigen 19-9 (CA19-9), Cancer antigen 125 (CA-125), Type II Müllerian inhibitory substance receptor (MISIIR), Sialized Tn antigen (sTN), Fibroblast activation antigen (FAP), Endothelial sialic acid protein (CD248), Epidermal growth factor receptor variant III (EGFRvIII), Tumor-associated antigen L6 (TAL6), SAS, CD63, TAG72, Thomsen-Friedenreich antigen (TF-antigen), insulin-like growth factor I receptor (IGF-IR), Cora antigen, CD7, CD22, CD70, CD79a, CD79b, G250, MT-MMP, F19 antigen, CA19-9, CA-125, alpha-fetoprotein (AFP), VEGFR1, VEGFR2, DLK1, SP17, ROR1, and EphA2. In one embodiment, the first binding domain showing binding affinity to CD70 is its native ligand CD27, rather than the antibody fragment. In another embodiment, the first binding domain showing binding affinity to B7-H6 is its native ligand Nkp30, rather than the antibody fragment.

[0196] Conceivedly, the scFv embodiment of the subject composition of the present invention comprises a first binding domain and a second binding domain, wherein the VL and VH domains are derived from monoclonal antibodies having binding specificity against tumor-specific markers or target cell antigens and effector cell antigens, respectively. In other cases, the first and second binding domains each comprise six CDRs derived from monoclonal antibodies having binding specificity against target cell markers such as tumor-specific markers and effector cell antigens, respectively. In some other embodiments, the first and second binding domains of the first portion of the subject composition have 3, 4, or 5 CHRs within each binding domain. In some other embodiments, embodiments of the present invention comprise a first binding domain and a second binding domain, wherein each comprises a CDR-H1 region, a CDR-H2 region, a CDR-H3 region, a CDR-L1 region, a CDR-L2 region, and a CDR-L3 region, wherein each of these regions is derived from a monoclonal antibody capable of binding to tumor-specific markers or target cell antigens and effector cell antigens, respectively. In one embodiment, the present invention provides a chimeric polypeptide assembly composition wherein the second binding domain comprises VH and VL regions derived from a monoclonal antibody capable of binding human CD3. In another embodiment, the present invention provides a chimeric polypeptide assembly composition wherein the scFv second binding domain comprises VH and VL regions, wherein each VH and VL region exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98% or 99% identity or complete identicalness to paired VL and VH sequences selected from anti-CD3 antibodies in Table 1. Alternatively, embodiments of the second domain of the present invention comprise CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 regions, wherein each of these regions is derived from a monoclonal antibody selected from the antibodies listed in Table 1. In the foregoing embodiments, the VH and / or VL domains may be configured as scFv, a biantibody, a single-domain antibody, or a single-domain camel antibody.

[0197] In some other embodiments, the second domain of the subject composition is derived from an anti-CD3 antibody selected from the antibodies listed in Table 1. In one of the foregoing embodiments, the second domain of the subject composition comprises paired VL and VH region sequences of an anti-CD3 antibody selected from the antibodies listed in Table 1. In another embodiment, the present invention provides a chimeric polypeptide assembly composition wherein the second binding domain comprises VH and VL regions, wherein each VH and VL region exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity or complete identity with the paired VL and VH sequences of the huUCHT1 anti-CD3 antibody in Table 1. In the foregoing embodiments, the VH and / or VL domains may be configured as scFv, part of a biantibody, a single-domain antibody, or a single-domain camel antibody.

[0198] In some other embodiments, the scFv of the first binding domain of the composition is derived from an antitumor cell antibody selected from the antibodies listed in Table 2. In another embodiment, the present invention provides a chimeric polypeptide assembly composition wherein the first binding domain comprises VH and VL regions, wherein each VH and VL region exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity or complete identity with a paired VL and VH sequence of an antitumor cell antibody selected from Table 2. In one of the foregoing embodiments, the first binding domain of the composition comprises a paired VL and VH region sequence of an antitumor cell antibody disclosed herein. In the foregoing embodiments, the VH and / or VL domains may be configured as an scFv, part of a biantibody, a single-domain antibody, or a single-domain camel antibody.

[0199] In another embodiment, the first portion of the chimeric polypeptide assembly composition has a sequence that is at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identical to a sequence selected from the sequences in Table 13.

[0200] In another embodiment, the chimeric polypeptide assembly composition comprises a first portion containing a first binding domain and a second binding domain, wherein the binding domain is a biantibody configuration, and each binding domain comprises a VL domain and a VH domain. In one embodiment, the biantibody embodiment of the present invention comprises a first binding domain and a second binding domain, wherein the VL and VH domains are derived from monoclonal antibodies having binding specificity to tumor-specific markers or target cell antigens and effector cell antigens, respectively. In another embodiment, the biantibody embodiment of the present invention comprises a first binding domain and a second binding domain, wherein each comprises a CDR-H1 region, a CDR-H2 region, a CDR-H3 region, a CDR-L1 region, a CDR-L2 region, and a CDR-L3 region, wherein each of the regions is derived from a monoclonal antibody capable of binding to tumor-specific markers or target cell antigens and effector cell antigens, respectively. It is conceivable that the biantibody embodiment of the present invention comprises a first binding domain and a second binding domain, wherein the VL and VH domains are derived from monoclonal antibodies having binding specificity to tumor-specific markers or target cell antigens and effector cell antigens, respectively. On the other hand, the biantibody embodiment of the present invention includes a first binding domain and a second binding domain, each comprising a CDR-H1 region, a CDR-H2 region, a CDR-H3 region, a CDR-L1 region, a CDR-L2 region, and a CDR-L3 region, wherein each of said regions is derived from a monoclonal antibody capable of binding tumor-specific markers or target cell antigens and effector cell antigens, respectively. In one embodiment, the present invention provides a chimeric polypeptide assembly composition wherein the second binding domain of the biantibody comprises paired VH and VL regions derived from a monoclonal antibody capable of binding human CD3. In another embodiment, the present invention provides a chimeric polypeptide assembly composition wherein the second binding domain of the biantibody comprises VH and VL regions, wherein each VH and VL region exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity or complete identicalness to paired VL and VH sequences selected from anti-CD3 antibodies in Table 1. In another embodiment, the present invention provides a chimeric polypeptide assembly composition wherein the second binding domain of the biantibody comprises VH and VL regions, wherein each VH and VL region exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity or complete identity with the VL and VH sequences selected from the huUCHT1 antibodies of Table 1. In some other embodiments, the second binding domain of the biantibody in the composition is derived from the anti-CD3 antibody described herein.In another embodiment, the present invention provides a chimeric polypeptide assembly composition wherein the first binding domain of the biantibody comprises VH and VL regions, wherein each VH and VL region exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity or complete identity with the VL and VH sequences selected from the antitumor cell antibodies listed in Table 2. In some other embodiments, the first binding domain of the biantibody in the composition is derived from the antitumor cell antibodies described herein.

[0201] Therapeutic monoclonal antibodies that can be derived for the VL and VH and CDR domains of the subject compositions are known in the art. Such therapeutic antibodies include, but are not limited to: rituximab, IDEC / Genentech / Roche (see, for example, U.S. Patent No. 5,736,137), a chimeric anti-CD20 antibody for the treatment of many lymphomas, leukemias, and some autoimmune diseases; ofamumumab, an anti-CD20 antibody developed by GlaxoSmithKline and approved for chronic lymphocytic leukemia, and being developed for follicular non-Hodgkin lymphoma, diffuse large B-cell lymphoma, rheumatoid arthritis, and relapsing-remitting multiple sclerosis; rukammumab (HCD122), an anti-CD40 antibody developed by Novartis for non-Hodgkin lymphoma or Hodgkin lymphoma (see, for example, U.S. Patent No. 6,899,879); AME-133, developed by Applied Molecular... An antibody developed by Evolution that binds to CD20-expressing cells to treat non-Hodgkin's lymphoma; vetuzumab (hA20), developed by Immunomedics, Antibodies developed by Inc. that bind to CD20-expressing cells to treat immune thrombocytopenic purpura; HumaLYM developed by Intracel for the treatment of low-grade malignant B-cell lymphoma; and oligrinzab—an anti-CD20 monoclonal antibody developed by Genentech for the treatment of rheumatoid arthritis (see, for example, U.S. Patent Application 20090155257); trastuzumab (see, for example, U.S. Patent No. 5,677,171), a humanized anti-Her2 / neu antibody developed by Genentech approved for the treatment of breast cancer; pertuzumab, an anti-HER2 dimerization inhibitor antibody developed by Genentech for the treatment of prostate, breast, and ovarian cancer (see, for example, U.S. Patent No. 4,753,894); cetuximab, an anti-EGFR antibody developed by Imclone and BMS for the treatment of KRAS wild-type metastatic colorectal and head and neck cancer expressing epidermal growth factor receptor (EGFR) (see, for example, U.S. Patent No. 4,943,533; PCT WO 96 / 40210); Panitumumab, a fully human monoclonal antibody specifically targeting the epidermal growth factor receptor (also known as EGF receptor, EGFR, ErbB-1, and HER1) currently marketed by Amgen for the treatment of metastatic colorectal cancer (see U.S. Patent No. 6,235,883); Zalumab, a fully human monoclonal antibody against the epidermal growth factor receptor (EGFR) developed by Genmab for the treatment of squamous cell carcinoma of the head and neck (see, for example, U.S. Patent No. 7,247,301);Nimotuzumab, a chimeric antibody against EGFR developed by Biocon, YM Biosciences, Cuba, and Oncosciences, Europe for the treatment of squamous cell carcinoma of the head and neck, nasopharyngeal carcinoma, and glioma (see, for example, US Patent No. 5,891,996; US Patent No. 6,506,883); alenzusuzumab, a humanized monoclonal antibody against CD52 marketed by Bayer Schering Pharma for the treatment of chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma (CTCL), and T-cell lymphoma; moromumab-CD3, an anti-CD3 antibody developed by Ortho Biotech / Johnson & Johnson as an immunosuppressive biologic to reduce acute rejection in organ transplant patients; and tiemotuzumab, developed by IDEC / Schering... AG developed an anti-CD20 monoclonal antibody for the treatment of certain forms of B-cell non-Hodgkin's lymphoma; Giemumab ozomicin, developed by Celltech / Wyeth, is an anti-CD33 (p67 protein) antibody linked to the radioactive isotope-conjugated cytotoxic chelator tiuxetan for the treatment of acute myeloid leukemia; ABX-CBL, an anti-CD147 antibody developed by Abgenix; ABX-IL8, an anti-IL8 antibody developed by Abgenix; ABX-MA1, an anti-MUC18 antibody developed by Abgenix; Pemtumomab (R1549, 90Y-muHMFG1), an anti-MUC1 antibody developed by Antisoma; Therex (R1550), an anti-MUC1 antibody developed by Antisoma; AngioMab (AS1405) developed by Antisoma; HuBC-1 developed by Antisoma; Thioplatin (AS1407) developed by Antisoma; ANTEGREN Natalizumab, an anti-α-4-β-1 (VLA4) and α-4-β-7 antibody developed by Biogen; VLA-1 mAb, an anti-VLA-1 integrin antibody developed by Biogen; LTBR mAb, an anti-lymphotoxin β receptor (LTBR) antibody developed by Biogen; CAT-152, an anti-TGF-β2 antibody developed by Cambridge Antibody Technology; J695, an anti-IL-12 antibody developed by Cambridge Antibody Technology and Abbott; CAT-192, an anti-TGFβ1 antibody developed by Cambridge Antibody Technology and Genzyme.CAT-213, an anti-Eotaxin1 antibody developed by Cambridge Antibody Technology; LYMPHOSTAT-B, an anti-Blys antibody developed by Cambridge Antibody Technology and Human Genome Sciences Inc.; TRAIL-R1mAb, an anti-TRAIL-R1 antibody developed by Cambridge Antibody Technology and Human Genome Sciences Inc.; HERCEPTIN, an anti-HER receptor family antibody developed by Genentech; anti-tissue factor (ATF), an anti-tissue factor antibody developed by Genentech; XOLAIR (omalizumab), an anti-IgE antibody developed by Genentech; MLN-02 antibody (formerly known as LDP-02) developed by Genentech and Millennium Pharmaceuticals; HUMAX CD4®, an anti-CD4 antibody developed by Genmab; tocilizuma, an anti-IL6R antibody developed by Chugai; HUMAX-IL15, an anti-IL15 antibody developed by Genmab and Amgen; HUMAX-Inflam, developed by Genmab and Medarex; HUMAX-Cancer, an anti-heparinase I antibody developed by Genmab, Medarex, and Oxford GlycoSciences; HUMAX-Lymphoma, developed by Genmab and Amgen; HUMAX-TAC, developed by Genmab; IDEC-131, an anti-CD40L antibody developed by IDEC Pharmaceuticals; IDEC-151 (crixaximab), an anti-CD4 antibody developed by IDEC Pharmaceuticals; IDEC-114, an anti-CD80 antibody developed by IDEC Pharmaceuticals; IDEC-152, developed by IDEC... Anti-CD23 developed by Pharmaceuticals; anti-KDR antibody developed by Imclone; DC101, anti-flk-1 antibody developed by Imclone; anti-VE cadherin antibody developed by Imclone; CEA-CIDE (labezizumab), anti-carcinoembryonic antigen (CEA) antibody developed by Immunomedics; Yervoy (ipilimumab), anti-CTLA4 antibody for the treatment of melanoma developed by Bristol-Myers Squibb.Lumphocide® (epazolizumab), an anti-CD22 antibody developed by Immunomedics; AFP-Cide developed by Immunomedics; MyelomaCide developed by Immunomedics; LkoCides developed by Immunomedics; ProstaCide developed by Immunomedics; MDX-010, an anti-CTLA4 antibody developed by Medarex; MDX-060, an anti-CD30 antibody developed by Medarex; MDX-070 developed by Medarex; MDX-018 developed by Medarex; OSIDEM (IDM-1), an anti-HER2 antibody developed by Medarex and Immuno-Designed Molecules; HUMAX®-CD4, an anti-CD4 antibody developed by Medarex and Genmab; HuMax-IL15, an anti-IL15 antibody developed by Medarex and Genmab; and an anti-intercellular adhesion molecule-1 developed by MorphoSys. (ICAM-1)(CD54) antibody, MOR201; trimelimumab, an anti-CTLA-4 antibody developed by Pfizer; vexizumab, an anti-CD3 antibody developed by Protein Design Labs; an anti-α5β1 integrin antibody developed by Protein Design Labs; an anti-IL-12 antibody developed by Protein Design Labs; ING-1, an anti-Ep-CAM antibody developed by Xoma; and MLN01, an anti-β2 integrin antibody developed by Xoma; all antibody references cited above in this paragraph are expressly incorporated herein by reference. The sequences of the above antibodies are available from publicly available databases, patents, or references. Furthermore, Table 1 lists non-limiting examples of monoclonal antibodies and VH and VL sequences from anti-CD3 antibodies, while Table 2 provides non-limiting examples of monoclonal antibodies against cancer, tumor, or target cell markers, and their VH and VL sequences. Table 1: Anti-CD3 Monoclonal Antibodies and Sequences *The underlined sequence, if it exists, is a CDR within VL and VH. Table 2: Anti-target cell monoclonal antibodies and their sequences *The underlined and bolded sequence, if present, is a CDR within VL and VH.

[0202] Methods for measuring the binding affinity and / or other biological activities of the compositions of this invention may be those disclosed herein or methods generally known in the art. For example, denoted as K d The binding affinity of a binding pair (e.g., antibody and antigen) can be determined using a variety of appropriate assays, including but not limited to radioactive binding assays, non-radioactive binding assays such as fluorescence resonance energy transfer and surface plasmon resonance (SPR, Biacore), and enzyme-linked immunosorbent assays (ELISA), kinetic repulsion assays (KinExA®), or as described in the examples. For example, an increase or decrease in the binding affinity of a chimeric polypeptide assembly that has been cleaved to remove the filler portion compared to a chimeric polypeptide assembly with the filler portion attached can be determined by measuring the binding affinity of the chimeric polypeptide assembly to its target binding partner with and without the filler portion.

[0203] The half-life of the subject chimeric assembly can be measured using a variety of suitable methods. For example, the half-life of a substance can be determined by administering the substance to a subject and periodically sampling biological samples (e.g., biological fluids, such as blood or plasma or ascites) to determine the concentration and / or amount of the substance in the samples over time. The concentration of the substance in the biological samples can be determined using a variety of suitable methods, including enzyme-linked immunosorbent assay (ELISA), immunoblotting, and chromatographic techniques, including high-performance liquid chromatography and rapid protein liquid chromatography. In some cases, the substance can be labeled with a detectable tag, such as a radioactive tag or a fluorescent tag, which can be used to determine the concentration of the substance in a sample (e.g., a blood sample or plasma sample). Various pharmacokinetic parameters are then determined based on the results, which can be performed using software packages such as SoftMaxPro or by manual calculations known in the art.

[0204] Furthermore, the physicochemical properties of the chimeric polypeptide assembly composition can be measured to determine solubility, structure, and stability retention. Determining the subject composition allows for the determination of the binding characteristics of the binding domain to the ligand, including the binding dissociation constant (K). d K on and K off The half-life of the ligand-receptor complex dissociation, and the activity of the binding domain in inhibiting the biological activity of the isolated ligand compared to the free ligand (IC50). 50 Value). Term "IC" 50 "EC" refers to the concentration required to inhibit half of the maximal biological response of a ligand agonist, and is typically determined by a competitive binding assay. 50 "" refers to the concentration required to achieve half of the maximum biological response of an active substance, and is typically determined by ELISA or cell-based assays, including the methods of the embodiments described herein. (i) Anti-CD3 binding domain

[0205] In some embodiments, the present invention provides a chimeric polypeptide assembly composition comprising a binding domain of a first portion having binding affinity for T cells. In one embodiment, the binding domain of a second portion comprises VL and VH sequences derived from monoclonal antibodies against a CD3 antigen. In another embodiment, the binding domain comprises VL and VH sequences derived from monoclonal antibodies against CD3ε and CD3δ. Monoclonal antibodies against CD3neu are known in the art. Exemplary, non-limiting examples of VL and VH sequences of monoclonal antibodies against CD3 are listed in Table 1. In one embodiment, the present invention provides a chimeric polypeptide assembly comprising a binding domain having binding affinity for CD3, the binding domain comprising the anti-CD3VL and VH sequences listed in Table 1. In another embodiment, the present invention provides a chimeric polypeptide assembly comprising a binding domain of a first portion having binding affinity for CD3ε, the binding domain comprising the anti-CD3ε VL and VH sequences listed in Table 1. In another embodiment, the present invention provides a chimeric peptide assembly composition wherein the scFv second binding domain of the first portion comprises VH and VL regions, wherein each VH and VL region exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity or complete identity with the paired VL and VH sequences of the huUCHT1 anti-CD3 antibody in Table 1. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a binding domain having binding affinity for CD3, the binding domain comprising a CDR-L1 region, a CDR-L2 region, a CDR-L3 region, a CDR-H1 region, a CDR-H2 region, and a CDR-H3 region, wherein each region is derived from the respective anti-CD3 VL and VH sequences listed in Table 1. In another embodiment, the present invention provides a chimeric polypeptide assembly composition comprising a binding domain having binding affinity for CD3, the binding domain comprising a CDR-L1 region, a CDR-L2 region, a CDR-L3 region, a CDR-H region, a CDR-H2 region, and a CDR-H3 region, wherein the CDR sequence is RASQDIRNYLN, YTSRLES, QQGNTLPWT, GYSFTGYTMN, LINPYKGVST, and SGYYGDSDWYFDV.

[0206] The CD3 complex is a group of cell surface molecules that are associated with the T cell antigen receptor (TCR) and play a role in the cell surface expression of the TCR, as well as in the signal transduction cascade that occurs when a peptide:MHC ligand binds to the TCR. Normally, when an antigen binds to the T cell receptor, CD3 sends a signal across the cell membrane to the cytoplasm within the T cell. This leads to T cell activation, and the T cell rapidly divides to produce sensitized new T cells to attack the specific antigen exposed by the TCR. The CD3 complex consists of the CD3ε molecule and four other membrane-bound peptides (CD3-γ, CD3-δ, CD3-ζ, and CD3-β). In humans, CD3-ε is encoded by the CD3E gene on chromosome 11. The intracellular domain of each CD3 chain contains an immune receptor tyrosine-based activation motif (ITAM), which serves as the nucleation site for intracellular signal transduction mechanisms following T cell receptor binding.

[0207] Many therapeutic strategies modulate T-cell immunity by targeting TCR signaling, particularly anti-human CD3 monoclonal antibodies (mAbs) which are widely used in clinical immunosuppressive regimens. The CD3-specific mouse mAb OKT3 was the first approved human mAb (Sgro, C. Side-effects of a monoclonal antibody, muromonab CD3 / orthocloneOKT3: bibliographic review. Toxicology 105:23-29, 1995) and is widely used clinically as an immunosuppressant in transplantation (Chatenoud, Clin. Transplant 7: 422-430, (1993); Chatenooud, Nat. Rev. Immunol. 3: 123-132 (2003); Kumar, Transplant. Proc. 30:1351-1352 (1998)), type 1 diabetes, and psoriasis. Importantly, anti-CD3 mAbs can induce partial T cell signaling and clonal non-responsiveness (Smith, JA, Nonmitogenic anti-CD3 monoclonal antibodies Deliver a Partial T Cell Receptor Signal and Induce Clonal Anergy J. Exp. Med. 185:1413-1422 (1997)). OKT3 has been described in the literature as a T cell mitogen and an effective T cell killer (Wong, JT. The mechanism of anti-CD3 monoclonal antibodies. Mediation of cytolysis by inter-T cell bridging. Transplantation 50: 683-689 (1990)). In particular, Wong's research showed that target killing can be achieved by bridging CD3 T cells and target cells, and that FcR-mediated ADCC and complement fixation are unnecessary for bivalent anti-CD3 MAB to lyse target cells.

[0208] OKT3 exhibits time-dependent mitogenic and T-cell cytotoxic activity; it leads to cytokine release after early T-cell activation, and upon further administration, OKT3 subsequently blocks all known T-cell functions. Due to this subsequent T-cell function blockade, OKT3 is widely used in treatment regimens as an immunosuppressant to reduce or even eliminate allogeneic tissue rejection. Other antibodies specific to the CD3 molecule are disclosed in Tunnacliffe, Int. Immunol. 1 (1989), 546-50. WO2005 / 118635 and WO2007 / 033230 describe anti-human monoclonal CD3ε antibodies. U.S. Patent 5,821,337 describes the VL and VH sequences of mouse anti-CD3 monoclonal Ab UCHT1 (muxCD3, Shalaby et al., J. Exp. Med. 175, 217-225 (1992)) and a humanized variant of the antibody (huUCHT1). U.S. Patent Application 20120034228 discloses a binding domain capable of binding to epitopes of human and non-chimpanzee primate CD3ε chains. (ii) Anti-EpCAM binding domain

[0209] In some embodiments, the present invention provides a chimeric polypeptide assembly composition comprising a binding domain having binding affinity for the tumor-specific marker EpCAM. In one embodiment, the binding domain comprises VL and VH regions derived from a monoclonal antibody against EpCAM. Monoclonal antibodies against EpCAM are known in the art. Exemplary, non-limiting examples of EpCAM monoclonal antibodies and their VL and VH sequences are listed in Table 2. In one embodiment, the present invention provides a chimeric polypeptide assembly composition comprising a binding domain having binding affinity for the tumor-specific marker EpCAM, the binding domain comprising the anti-EpCAM VL and VH sequences listed in Table 2. In another embodiment, the present invention provides a chimeric polypeptide assembly composition wherein a first binding domain of a first portion comprises VH and VL regions, wherein each VH and VL region exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity or complete identicalness to the paired VL and VH sequences of the 4D5MUCB anti-EpCAM antibody of Table 2. In another embodiment, the present invention provides a chimeric polypeptide assembly composition comprising a binding domain having binding affinity for a tumor-specific marker, the binding domain comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from the respective VL and VH sequences listed in Table 2.

[0210] Epithelial cell adhesion molecule (EpCAM, also known as the 17-1A antigen) is a 40-kDa membrane-integrated glycoprotein consisting of 314 amino acids expressed in some epithelial cells and in many human tumors (see Balzar, The biology of the 17-1A antigen (Ep-CAM), J. Mol. Med. 1999, 77: 699-712). EpCAM was initially discovered using the mouse monoclonal antibody 17-1A / epsilonumab, produced by immunizing mice with colon cancer cells (Goettlinger, Int J Cancer. 1986; 38, 47-53 and Simon, Proc. Natl. Acad. Sci. USA. 1990; 87, 2755-2759). Due to their epithelial origin, tumor cells from most cancers express EpCAM on their surface (more than normal healthy cells), including most primary, metastatic, and disseminated non-small cell lung cancer cells (Passlick, B., et al., The 17-1A antigen is expressed on primary, metastatic, and disseminated non-small cell lung carcinoma cells. Int. J. cancer 87(4):548–552, 2000), gastric and gastroesophageal junction adenocarcinomas (Martin, IG., 17-1A antigen in gastric and gastro-oesophageal junction adenocarciomas: a potential immunotherapeutic target? J Clin Pathol 1999; 52:701–704), and breast and colorectal cancers (Packeisen J, et al., Dentection of surface antigen 17-1S in breast and colorectal cancer. Hybridoma. 1999). 18(1):37-40), therefore, this is an attractive target for immunotherapy. In fact, increased expression of EpCAM is associated with increased epithelial proliferation; in breast cancer, overexpression of EpCAM on tumor cells is a predictor of survival (Gastl, Lancet. 2000,356,1981-1982).Due to their epithelial origin, tumor cells from most cancers still express EpCAM on their surface, and bispecific solitomab single-chain antibody compositions targeting EpCAM on tumor cells and also containing a CD3 binding region have been proposed for use against primary uterine and ovarian CS cell lines (Ferrari F, et al., Solitomab, an EpCAM / CD3 bispecific antibody construct (BiTE®), is highly active against primary uterine and ovarian carcino cancer celllines in vitro. J Exp Clin Cancer Res. 2015 34:123).

[0211] Monoclonal antibodies against EpCAM are known in the art. EpCAM monoclonal antibodies ING-1, 3622W94, adenomyumab, and ezetimibe have been described as having been tested in human patients (Münz, M. Side-by-side analysis of five clinically tested anti-EpCAM monoclonal antibody cancer Cell International, 10:44-56, 2010). Bispecific antibodies against EpCAM and CD3 have also been described, including the construction of two distinct bispecific antibodies by fusing a hybridoma that produces a monoclonal antibody against EpCAM with either of two hybridomas, OKT3 and 9.3 (Möller, SA, Reisfeld, RA, Bispecific-monoclonal-antibody-directed lysis of ovarian tumor cells by activated human Tlymphocytes. Cancer Immunol. Immunother. 33:210-216, 1991). Other examples of bispecific antibodies against EpCAM include BiUII (anti-CD3 (rat) x anti-EpCAM (mouse)) (Zeidler, J. Immunol., 1999, 163: 1247-1252), scFv CD3 / 17-1A bispecific antibody (Mack, M. A smallbispecific antibody composition expressed as a functional single-chain molecule with high tumor cell cytotoxicity. Proc. Natl. Acad. Sci., 1995, 92: 7021-7025), and a partially humanized bispecific antibody with anti-CD3 and anti-EpCAM specificity (Helfrich, W. Construction and characterization of a bispecific antibody for retargeting T cells to human tumors. Int. J. Cancer, 1998, 76: 232-239).

[0212] In one embodiment, what is provided herein is a bispecific chimeric polypeptide assembly composition having a first portion having an EpCAM-specific binding domain and a CD3-specific binding domain. The technical aspect to be solved is to provide means and methods for producing improved compositions that exhibit properties of being well-tolerated and more convenient (with lower dosing frequency) for the effective treatment and / or improvement of oncological diseases. The solution to the technical problem is achieved through the embodiments disclosed herein and characterized in the claims.

[0213] Therefore, in some embodiments, the present invention relates to a chimeric polypeptide assembly composition, wherein the composition comprises a first portion containing a bispecific single-chain antibody composition, the first portion comprising at least two binding domains, wherein one of the domains binds an effector cell antigen, such as a CD3 antigen, and a second domain binds an EpCAM antigen, wherein the binding domains comprise EpCAM-specific VL and VH and human CD3 antigen-specific VL and VH. Preferably, in this embodiment, the EpCAM-specific binding domain has a density greater than 10. -7 Up to 10 −10 M of K d The value, as determined in an in vitro binding assay. In one of the foregoing embodiments, the binding domain is in the form of scFv. In another of the foregoing embodiments, the binding domain is in the form of a single-chain biantibody. (iii) CCR5 binding domain

[0214] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CCR5, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against CCR5. Monoclonal antibodies against CCR5 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CCR5, comprising anti-CCR5 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (iv) Anti-CD19 binding domain

[0215] In some embodiments, the present invention provides a chimeric polypeptide assembly composition comprising a first binding domain having binding affinity for the tumor-specific marker CD19, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against CD19. Monoclonal antibodies against CD19 are known in the art. Exemplary, non-limiting examples of VL and VH sequences are listed in Table 2. In one embodiment, the present invention provides a chimeric polypeptide assembly composition comprising a first binding domain having binding affinity for the tumor-specific marker CD19, comprising the anti-CD19 VL and VH sequences listed in Table 2. In another embodiment, the present invention provides a chimeric peptide assembly composition wherein the scFv second binding domain comprises VH and VL regions, wherein each VH and VL region exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity or complete identity with the paired VL and VH sequences of the MT103 anti-CD19 antibody in Table 2. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, wherein each is derived from the respective VL and VH sequences listed in Table 2. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (v) Anti-HER-2 binding domain

[0216] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker HER-2, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against HER-2. Monoclonal antibodies against HER-2 are known in the art. Exemplary non-limiting examples of VL and VH sequences are listed in Table 2. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker HER-2, comprising the anti-HER-2 VL and VH sequences listed in Table 2. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from the respective VL and VH sequences listed in Table 2. Preferably, in an embodiment, the combination has a value higher than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (vi) Anti-HER-3 binding domain

[0217] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker HER-3, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against HER-3. Monoclonal antibodies against HER-3 are known in the art. Exemplary, non-limiting examples of VL and VH sequences are listed in Table 2. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker HER-3, comprising the anti-HER-3 VL and VH sequences listed in Table 2. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from the respective VL and VH sequences listed in Table 2. Preferably, in an embodiment, the combination has a value higher than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (vii) Anti-HER-4 binding domain

[0218] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker HER-4, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against HER-4. Monoclonal antibodies against HER-4 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker HER-4, comprising anti-HER-4 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (viii) Anti-EGFR binding domain

[0219] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first binding domain having binding affinity for the tumor-specific marker EGFR, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against EGFR. Monoclonal antibodies against EGFR are known in the art. Exemplary, non-limiting examples of VL and VH sequences are listed in Table 2. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first binding domain having binding affinity for the tumor-specific marker EGFR, comprising the anti-EGFR VL and VH sequences listed in Table 2. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from the respective VL and VH sequences listed in Table 2. Preferably, in an embodiment, the combination has a value higher than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (ix) Anti-PSMA binding domain

[0220] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker PSMA, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against PSMA. Monoclonal antibodies against PSMA are known in the art. Exemplary, non-limiting examples of VL and VH sequences are listed in Table 2. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker PSMA, comprising the anti-PSMA VL and VH sequences listed in Table 2. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from the respective VL and VH sequences listed in Table 2. In another embodiment, the present invention provides a chimeric polypeptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from the respective VL and VH sequences listed in Table 2. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (x) Anti-CEA binding domain

[0221] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CEA, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against CEA. Monoclonal antibodies against CEA are known in the art. Exemplary, non-limiting examples of VL and VH sequences are listed in Table 2. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CEA, comprising the anti-CEA VL and VH sequences listed in Table 2. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from the respective VL and VH sequences listed in Table 2. Preferably, in an embodiment, the combination has a value higher than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xi) Anti-MUC1 binding domain

[0222] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker MUC1, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against MUC1. Monoclonal antibodies against MUC1 are known in the art. Exemplary, non-limiting examples of VL and VH sequences are listed in Table 2. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker MUC1, comprising the anti-MUC1 VL and VH sequences listed in Table 2. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from the respective VL and VH sequences listed in Table 2. Preferably, in an embodiment, the combination has a value higher than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xii) Anti-MUC2 binding domain

[0223] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker MUC2, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against MUC2. Monoclonal antibodies against MUC2 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker MUC2, comprising anti-MUC2 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xiii) Anti-MUC3 binding domain

[0224] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first binding domain having binding affinity for the tumor-specific marker MUC3, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH derived from a monoclonal antibody against MUC3. Monoclonal antibodies against MUC3 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first binding domain having binding affinity for the tumor-specific marker MUC3, comprising anti-MUC3 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xiv) Anti-MUC4 binding domain

[0225] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker MUC4, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against MUC4. Monoclonal antibodies against MUC4 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker MUC4, comprising anti-MUC4 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xv) Anti-MUC5AC binding domain

[0226] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker MUC5AC, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against MUC5AC. Monoclonal antibodies against MUC5AC are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker MUC5AC, comprising anti-MUC5AC VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xvi) Anti-MUC5B binding domain

[0227] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker MUC5B, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against MUC5B. Monoclonal antibodies against MUC5B are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker MUC5B, comprising anti-MUC5B VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xvii) Anti-MUC7 binding domain

[0228] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker MUC7, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against MUC7. Monoclonal antibodies against MUC7 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker MUC7, comprising anti-MUC7 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xviii) Anti-βhCG binding domain

[0229] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker βhCG, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against βhCG. Monoclonal antibodies against βhCG are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker βhCG, comprising anti-βhCG VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xix) Anti-Lewis-Y binding domain

[0230] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker Lewis-Y, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises monoclonal antibodies VL and VH derived from Lewis-Y. Monoclonal antibodies against Lewis-Y are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker Lewis-Y, comprising the anti-Lewis-Y VL and VH sequences listed in Table 2. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from its respective VL and VH sequences. In another embodiment, the present invention provides a chimeric polypeptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10−10 M of K d Values, such as those determined in in vitro binding assays. (xx) Anti-CD20 binding domain

[0231] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CD20, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against CD20. Monoclonal antibodies against CD20 are known in the art. Exemplary, non-limiting examples of VL and VH sequences are listed in Table 2. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CD20, comprising the anti-CD20 VL and VH sequences listed in Table 2. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from the respective VL and VH sequences listed in Table 2. Preferably, in an embodiment, the combination has a value higher than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xxi) Anti-CD33 binding domain

[0232] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CD33, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against CD33. Monoclonal antibodies against CD33 are known in the art. Exemplary, non-limiting examples of VL and VH sequences are listed in Table 2. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CD33, comprising the anti-CD33 VL and VH sequences listed in Table 2. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from the respective VL and VH sequences listed in Table 2. Preferably, in an embodiment, the combination has a value higher than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xxii) Anti-CD30 binding domain

[0233] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CD30, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against CD30. Monoclonal antibodies against CD30 are known in the art. Exemplary, non-limiting examples of VL and VH sequences are listed in Table 2. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CD30, comprising the anti-CD30 VL and VH sequences listed in Table 2. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from the respective VL and VH sequences listed in Table 2. Preferably, in an embodiment, the combination has a value higher than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xxiii) Antiganglioside GD3 binding domain

[0234] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker ganglioside GD3, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH of a monoclonal antibody derived from ganglioside GD3. Monoclonal antibodies against ganglioside GD3 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker ganglioside GD3, comprising anti-ganglioside GD3 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xxiv) Anti-9-O-acetyl-GD3 binding domain

[0235] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first binding domain having binding affinity for the tumor-specific marker 9-O-acetyl-GD3, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against 9-O-acetyl-GD3. Monoclonal antibodies against 9-O-acetyl-GD3 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first binding domain having binding affinity for the tumor-specific marker 9-O-acetyl-GD3, comprising anti-9-O-acetyl-GD3 VL and VH sequences. In another embodiment, the present invention provides a chimeric polypeptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xxv) Anti-Globo H binding domain

[0236] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker Globo H, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against Globo H. Monoclonal antibodies against Globo H are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker Globo H, comprising anti-Globo H VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xxvi) Anti-fucosylation GM1 binding domain

[0237] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker fucosylation GM1, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against fucosylation GM1. Monoclonal antibodies against fucosylation GM1 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker fucosylation GM1, comprising anti-fucosylation GM1 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xxvii) GD2 binding domain

[0238] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker GD2, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against GD2. Monoclonal antibodies against GD2 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker GD2, comprising anti-GD2 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xxviii) Anticarbonic anhydrase IX binding domain

[0239] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker carbonic anhydrase IX, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against carbonic anhydrase IX. Monoclonal antibodies against carbonic anhydrase IX are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker carbonic anhydrase IX, comprising anti-carbonic anhydrase IX VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xxix) Anti-CD44v6 binding domain

[0240] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CD44v6, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against CD44v6. Monoclonal antibodies against CD44v6 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CD44v6, comprising anti-CD44v6 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xxx) Anti-sound hedgehog binding domain

[0241] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific biomarker *Sound Hedgehog*, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against *Sound Hedgehog*. Monoclonal antibodies against *Sound Hedgehog* are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific biomarker *Sound Hedgehog*, comprising anti-*Sound Hedgehog* VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific biomarker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xxxi) Wue-1 binding domain

[0242] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker Wue-1, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against Wue-1. Monoclonal antibodies against Wue-1 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker Wue-1, comprising anti-Wue-1 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xxxii) Anti-plasma cell antigen 1 binding domain

[0243] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker plasma cell antigen 1, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against plasma cell antigen 1. Monoclonal antibodies against plasma cell antigen 1 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker plasma cell antigen 1, comprising anti-plasma cell antigen 1 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xxxiii) Anti-melanoma chondroitin sulfate proteoglycan binding domain

[0244] In some embodiments, the present invention provides a chimeric polypeptide assembly composition comprising a first binding domain having binding affinity for the tumor-specific marker melanoma chondroitin sulfate proteoglycan, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody targeting melanoma chondroitin sulfate proteoglycan. Monoclonal antibodies targeting melanoma chondroitin sulfate proteoglycan are known in the art. In one embodiment, the present invention provides a chimeric polypeptide assembly composition comprising a first binding domain having binding affinity for the tumor-specific marker melanoma chondroitin sulfate proteoglycan, comprising anti-melanoma chondroitin sulfate proteoglycan VL and VH sequences. In another embodiment, the present invention provides a chimeric polypeptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xxxiv) CCR8 binding domain

[0245] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CCR8, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against CCR8. Monoclonal antibodies against CCR8 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CCR8, comprising anti-CCR8 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xxxv) Anti-STEAP binding domain

[0246] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker prostate 6-transmembrane epithelial antigen (STEAP), and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against STEAP. Monoclonal antibodies against STEAP are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker STEAP, comprising anti-STEAP VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xxxvi) Anti-mesothelin binding domain

[0247] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker mesothelin, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against mesothelin. Monoclonal antibodies against mesothelin are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker mesothelin, comprising anti-mesothelin VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xxxvii) Anti-A33 antigen binding domain

[0248] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker A33 antigen, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against the A33 antigen. Monoclonal antibodies against the A33 antigen are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker A33 antigen, comprising anti-A33 antigen VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xxxviii) Anti-PSCA binding domain

[0249] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker prostate stem cell antigen (PSCA), and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against PSCA. Monoclonal antibodies against PSCA are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker PSCA, comprising anti-PSCA VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xxxix) anti-Ly-6 binding domain

[0250] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker Ly-6, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against LY-6. Monoclonal antibodies against LY-6 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker LY-6, comprising anti-LY-6 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xl) Anti-SAS binding domain

[0251] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker SAS, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against SAS. Monoclonal antibodies against SAS are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker SAS, comprising anti-SAS VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xli) Anti-desmosome core protein 4 binding domain

[0252] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker desmosome core 4, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against desmosome core 4. Monoclonal antibodies against desmosome core 4 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker desmosome core 4, comprising anti-desmosome core 4 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xlii) Anti-fetal acetylcholine receptor binding domain

[0253] In some embodiments, the present invention provides a chimeric polypeptide assembly composition comprising a first binding domain having binding affinity for the tumor-specific marker fetal acetylcholine receptor, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against the fetal acetylcholine receptor. Monoclonal antibodies against the fetal acetylcholine receptor are known in the art. In one embodiment, the present invention provides a chimeric polypeptide assembly composition comprising a first binding domain having binding affinity for the tumor-specific marker fetal acetylcholine receptor, comprising anti-fetal acetylcholine receptor VL and VH sequences. In another embodiment, the present invention provides a chimeric polypeptide assembly composition comprising a first binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xliii) Anti-CD25 binding domain

[0254] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CD25, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against CD25. Monoclonal antibodies against CD25 are known in the art; for example, dacrolimus. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CD25, comprising anti-CD25 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xliv) Anticancer antigen 19-9 binding domain

[0255] In some embodiments, the present invention provides a chimeric polypeptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker cancer antigen 19-9 (CA 19-9), and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against CA 19-9. Monoclonal antibodies against CA 19-9 are known in the art. In one embodiment, the present invention provides a chimeric polypeptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CA 19-9, comprising anticancer antigen 19-9 VL and VH sequences. In another embodiment, the present invention provides a chimeric polypeptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from its respective VL and VH sequences. Preferably, in an embodiment, the combination has a value higher than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xlv) CA-125 binding domain

[0256] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker cancer antigen 125 (CA-125), and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against CA-125. Monoclonal antibodies against CA-125 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CA-125, comprising anti-CA-125 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xlvi) Anti-MISIIR binding domain

[0257] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker Müllerian inhibitory substance receptor type II (MISIIR), and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against MISIIR. Monoclonal antibodies against MISIIR are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker MISIIR, comprising anti-MISIIR VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xlvii) Anti-sialylated Tn antigen-binding domain

[0258] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker sialylated Tn antigen, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against sialylated Tn antigen. Monoclonal antibodies against sialylated Tn antigen are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker sialylated Tn antigen, comprising anti-sialylated Tn antigen VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xlviii) Anti-FAP binding domain

[0259] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker fibroblast activation antigen (FAP), and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against FAP. Monoclonal antibodies against FAP are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker FAP, comprising anti-FAP VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (xlix) Anti-CD248 binding domain

[0260] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker endothelial sialic acid protein (CD248), and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against CD248. Monoclonal antibodies against CD248 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CD248, comprising anti-CD248 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (l) Anti-EGFRvIII binding domain

[0261] In some embodiments, the present invention provides a chimeric polypeptide assembly composition comprising a first binding domain having binding affinity for the tumor-specific marker epidermal growth factor receptor variant III (EGFRvIII), and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against EGFRvIII. Monoclonal antibodies against EGFRvIII are known in the art. Exemplary, non-limiting examples of VL and VH sequences are listed in Table 2. In one embodiment, the present invention provides a chimeric polypeptide assembly composition comprising a first binding domain having binding affinity for the tumor-specific marker EGFRvIII, comprising the anti-EGFRvIII VL and VH sequences listed in Table 2. In another embodiment, the present invention provides a chimeric polypeptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from the respective VL and VH sequences listed in Table 2. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (li) Anti-TAL6 binding domain

[0262] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker tumor-associated antigen L6 (TAL6), and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against TAL6. Monoclonal antibodies against TAL6 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker TAL6, comprising anti-TAL6 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (lii) Anti-SAS binding domain

[0263] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker tumor-associated antigen (SAS), and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against SAS. Monoclonal antibodies against SAS are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker SAS, comprising anti-SAS VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (liii) Anti-CD63 binding domain

[0264] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker tumor-associated antigen CD63, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against CD63. Monoclonal antibodies against CD63 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CD63, comprising anti-CD63 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (liv) Anti-TAG72 binding domain

[0265] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first binding domain having binding affinity for the tumor-specific marker tumor-associated antigen TAG72, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against TAG72. Monoclonal antibodies against TAG72 are known in the art. Exemplary, non-limiting examples of VL and VH sequences are listed in Table 2. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first binding domain having binding affinity for the tumor-specific marker TAG72, comprising the anti-TAG72 VL and VH sequences listed in Table 2. In another embodiment, the present invention provides a chimeric polypeptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from the respective VL and VH sequences listed in Table 2. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (lv) Anti-TF antigen binding domain

[0266] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker Thomsen-Friedenreich antigen (TF-antigen), and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against the TF-antigen. Monoclonal antibodies against the TF-antigen are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker TF-antigen, comprising anti-TF-antigen VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (lvi) Anti-IGF-IR binding domain

[0267] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker tumor-associated antigen insulin-like growth factor I receptor (IGF-IR), and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against IGF-IR. Monoclonal antibodies against IGF-IR are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker IGF-IR, comprising anti-IGF-IR VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (lvii) Anti-Cora antigen binding domain

[0268] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker Cora antigen, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against Cora antigen. Monoclonal antibodies against Cora antigen are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker Cora antigen, comprising anti-Cora antigen VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (lviii) Anti-CD7 binding domain

[0269] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having affinity for the tumor-specific marker tumor-associated antigen CD7, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against CD7. Monoclonal antibodies against CD7 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CD7, comprising anti-CD7 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (lix) Anti-CD22 binding domain

[0270] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker tumor-associated antigen CD22, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against CD22. Monoclonal antibodies against CD22 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CD22, comprising anti-CD22 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (lx) Anti-CD79a binding domain

[0271] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker tumor-associated antigen CD79a, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against CD79a. Monoclonal antibodies against CD79a are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CD79a, comprising anti-CD79a VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (lxi) Anti-CD79b binding domain

[0272] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker tumor-associated antigen CD79b, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against CD79b. Monoclonal antibodies against CD79b are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker CD79b, comprising anti-CD79b VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (lxii) G250 binding domain

[0273] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker tumor-associated antigen G250, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against G250. Monoclonal antibodies against G250 are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker G250, comprising anti-G250 VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (lxiii) Anti-MT-MMP binding domain

[0274] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker tumor-associated antigen MT-MMP, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against MT-MMP. Monoclonal antibodies against MT-MMP are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker MT-MMP, comprising anti-MT-MMP VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (lxiv) Anti-F19 antigen binding domain

[0275] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker tumor-associated antigen F19, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against the F19 antigen. Monoclonal antibodies against the F19 antigen are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker F19 antigen, comprising anti-F19 antigen VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays. (lxv) Anti-EphA2 receptor binding domain

[0276] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker tumor-associated antigen EphA2 receptor, and a second binding domain binding effector cell antigens, such as CD3 antigen. In one embodiment, the binding domain comprises VL and VH sequences derived from a monoclonal antibody against the EphA2 receptor. Monoclonal antibodies against the EphA2 receptor are known in the art. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for the tumor-specific marker EphA2 receptor, comprising anti-EphA2 receptor VL and VH sequences. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a first partial binding domain having binding affinity for a tumor-specific marker, comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from a respective VL and VH sequence. Preferably, in an embodiment, the binding has a binding affinity greater than 10. -7 Up to 10 −10 M of K d Values, such as those determined in in vitro binding assays.

[0277] It is particularly envisioned that the chimeric polypeptide assembly composition may comprise any of the aforementioned domains or sequence variants, provided that the variant exhibits binding specificity to the antigen. In one embodiment, the sequence variant is generated by substituting different amino acids in the VL or VH sequence. In deletion variants, one or more amino acid residues in the VL or VH sequence described herein are removed. Thus, deletion variants comprise all fragments of the binding domain polypeptide sequence. In substitution variants, one or more amino acid residues of the VL or VH (or CDR) polypeptide are removed and replaced with substituted residues. On the one hand, the substitutions are inherently conserved, and this type of conserved substitution is well known in the art. Furthermore, it is particularly envisioned that compositions comprising the first and second binding domains disclosed herein can be used in any of the methods disclosed herein. 2. Release section

[0278] On the other hand, the present invention relates to a chimeric polypeptide assembly composition incorporating a release segment (RS) peptide sequence cleavable by one or more mammalian proteases, wherein when the RS is exposed to a protease (or multiple proteases), the RS is cleaved and a bispecific binding domain is released from the composition. Upon release of the bispecific binding domain and the shielding filler portion of the chimeric polypeptide assembly, due to the loss of the shielding effect of the filler portion, the binding domain regains its full ability to simultaneously bind to effector T cells as well as cancer, tumor, or target cells, thereby leading to damage or lysis of the cancer, tumor, or target cells.

[0279] In some embodiments, the present invention provides a chimeric polypeptide assembly composition comprising a single fusion protein comprising a bifunctional binding domain portion, a binding portion such as XTEN, and an introduced peptide RS, which is a substrate of one or more proteases associated with a target tissue, wherein the RS is recombinantly linked to the end of a filler portion and the RS is recombinantly linked to a first portion comprising first and second binding domains; thus, the RS is located between the first portion and the XTEN or other filler portion.

[0280] In embodiments, the present invention provides a chimeric polypeptide assembly composition comprising one or more RSs, wherein the RSs are substrates of a protease associated with a diseased target tissue in a subject; non-limiting examples of target tissues are cancer, tumors, or tissues or organs involved in proliferative disorders or inflammatory diseases. The object of the present invention is to provide RSs specifically configured for use in chimeric polypeptide assembly compositions comprising bispecific binding domains, such that when the composition comprising the RSs approaches a protease associated with the target tissue, the binding domain is released from the composition. The chimeric polypeptide assembly composition is designed such that the component thereby released, comprising the binding domain, has an enhanced ability to expel from and attach to or penetrate the target tissue, whether through a reduced molecular weight of the resulting fragment or through reduced steric hindrance resulting from a cleaved filler portion (e.g., XTEN).

[0281] The matrix in human cancers consists of the extracellular matrix and various types of non-cancerous cells such as leukocytes, endothelial cells, fibroblasts, and myofibroblasts. Tumor-associated matrix actively supports tumor growth by stimulating angiogenesis and also supports the proliferation and invasion of coexisting cancer cells. Matrix fibroblasts, often referred to as cancer-associated fibroblasts (CAFs), play a particularly important role in tumor progression due to their ability to dynamically alter the composition of the extracellular matrix (ECM), thereby promoting tumor cell invasion and subsequent metastatic colonization. In particular, it is known in the art that proteases are important components contributing to malignant progression, including tumor angiogenesis, invasion, extracellular matrix remodeling, and metastasis, where proteases function as part of a broad, multidirectional network of proteolytic interactions. Since malignancies require the acquisition of a vascular system to infiltrate surrounding normal tissues and spread to distant sites, tumors primarily rely on increased expression of extracellular endopeptidases from various enzyme classes, such as metalloproteinases (MMPs), as well as serine, threonine, cysteine, and aspartic proteases. However, the role of proteases is not limited to tissue invasion and angiogenesis. These enzymes also play major roles in growth factor activation, cell adhesion, cell survival, and immune surveillance. For example, MMPs can influence tumor cell behavior due to their ability to cleave growth factors, cell surface receptors, cell adhesion molecules, or chemokines. Overall, tumor-associated proteases demonstrate significant power in the phenotypic evolution of cancer.

[0282] Given substantial evidence of differential expression of many of these proteases between normal and tumor tissues, it is particularly envisioned that this differential expression could be used as a means of activating the subject composition near the tumor. In this regard, serine and metalloproteinases are particularly promising candidates for targeted, differential drug delivery of the subject composition, due to their elevated activity in the extracellular tumor environment and their ability to selectively and specifically cleave short peptide sequences of RS, resulting in a high level of activity of the subject composition at the tumor site and a low level of the intact chimeric polypeptide assembly composition in normal healthy tissue. As a result of the selective delivery of the chimeric polypeptide assembly, the required activity or dose of these drugs is correspondingly reduced, and toxicity to normal tissues (including the liver, heart, and bone marrow) is decreased, thereby significantly improving the therapeutic index of the chimeric polypeptide assembly composition. In particular, the disclosed compositions are envisioned to possess the beneficial properties of this prodrug concept, since in their uncut state they exhibit reduced binding affinity to their respective ligands and reduced exudation in normal, healthy tissues, but after cutting they are able to exudate and penetrate tumors better and have higher binding affinity to their respective ligands; all of these contribute to enhancing the therapeutic index and side effects of the subject composition.

[0283] In some embodiments, the present invention comprises a chimeric polypeptide assembly composition containing RS, wherein when the composition is cleaved by a tissue-associated protease, a fragment comprising a first-part binding domain is released, wherein the fragment is capable of penetrating the target tissue, such as a tumor, to a concentration at least 2, 3, 4, or 5 times that of the uncleaned composition. In other embodiments, the present invention comprises a chimeric polypeptide assembly composition containing RS, wherein when the composition is cleaved by a tissue-associated protease, a fragment comprising a first-part binding domain is released, wherein the fragment comprising the first part is capable of penetrating the tissue at a rate at least 2, 3, 4, or 5 times that of a composition without RS. In one embodiment, the present invention comprises a chimeric polypeptide assembly composition containing RS, wherein when the composition is cleaved by a tissue-associated protease, a fragment comprising a first partial binding domain is released, the cleaved first partial fragment having a resulting molecular weight that is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% smaller than that of the intact chimeric polypeptide assembly composition uncleaved by the protease. In another embodiment, the present invention comprises a chimeric polypeptide assembly composition containing RS, wherein when the composition is cleaved by a tissue-associated protease, a fragment comprising a first partial binding domain is released, the cleaved first partial fragment having a resulting hydrodynamic radius that is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% smaller than that of the intact chimeric polypeptide assembly uncleaved by the protease. In particular, it is envisioned that in embodiments of the subject chimeric polypeptide assembly, cleavage by a tissue-associated protease produces a fragment containing a first-part binding domain, which is capable of more effectively penetrating tissues such as tumors. This is because the fragment is smaller in size relative to the intact composition, resulting in pharmacological effects known in the art for the binding domain of the intracellular assembly, which may include membrane damage, induction of apoptosis, cell lysis or death of target cells. It is also envisioned that the RS of the chimeric polypeptide assembly composition is designed to target a specific tissue with a composition containing a specific protease known to be produced by the target tissue or cell. In one embodiment, the RS contains an amino acid sequence as a substrate of a protease associated with cancerous tissue. In another embodiment, the RS contains an amino acid sequence as a substrate of a protease associated with cancerous tumors. In another embodiment, the RS contains an amino acid sequence as a substrate of a protease associated with cancers such as leukemia. In yet another embodiment, the RS contains an amino acid sequence as a substrate of a protease associated with proliferative disorders.In another embodiment, the RS of the chimeric polypeptide assembly composition comprises an amino acid sequence that serves as a substrate for a protease associated with inflammatory diseases.

[0284] In some embodiments, RS is a substrate of at least one protease selected from metalloproteinases, cysteine ​​proteases, aspartic proteases, and serine proteases. In another embodiment, RS is a substrate of one or more proteases selected from the group consisting of: transmembrane peptidase, neutral lysozyme (CD10), PSMA, BMP-1, unlinking protein and metalloproteinase (ADAM), ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 (TACE), ADAM19, ADAM28 (MDC-L), ADAM with platelet-reactive protein motif (ADAMTS), ADAMTS1, ADAMTS4, ADAMTS5, MMP-1 (collagenase 1), MMP-2 (gelatinase A), MMP-3 (macrolysin 1), MMP-7 (macrolysin 1), MMP-8 (collagenase 2), MMP-9 (gelatinase B), MMP-10 (macrolysin 2), MMP-11 (macrolysin 3), MMP-12 (macrophage elastase), MMP-13 (collagenase 3), MMP-14 (MT1-MMP), MMP-15 (MT2-MMP), MMP-19, MMP-23 (CA-MMP), MMP-24 (MT5-MMP), MMP-26 (Matrix factor 2), MMP-27 (CMMP), Legumin, Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin X, Cathepsin D, Cathepsin E, Secretase, Urokinase (uPA), Tissue-type plasminogen activating factor (tPA), Plasmin, Thrombin, Prostate-specific antigen (PSA, KLK3), Human neutrophil elastase (HNE), Elastase, Trypsin, Type II transmembrane serine protease (TTSP), DESC1, Hepsin (HPN), proteolytic enzymes, proteolytic enzyme-2, TMPRSS2, TMPRSS3, TMPRSS4 (CAP2), fibroblast activating protein (FAP), kallikrein-related peptidases (KLK family), KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and KLK14. In some embodiments, RS is a substrate of ADAM17. In some embodiments, RS is a substrate of BMP-1. In some embodiments, RS is a substrate of cathepsins. In some embodiments, RS is a substrate of cysteine ​​proteases. In some embodiments, RS is a substrate of HtrA1. In some embodiments, RS is a substrate of pod protein. In some embodiments, RS is a substrate of MT-SP1. In some embodiments, RS is a substrate of metalloproteinases.In some embodiments, RS is a substrate of neutrophil elastase. In some embodiments, RS is a substrate of thrombin. In some embodiments, RS is a substrate of type II transmembrane serine protease (TTSP). In some embodiments, RS is a substrate of TMPRSS3. In some embodiments, RS is a substrate of TMPRSS4. In some embodiments, RS is a substrate of uPA. In one embodiment, RS of the chimeric polypeptide assembly composition is a substrate of at least two proteases selected from MMP-2, MMP-9, uPA, and protein lyase. In another embodiment, RS of the chimeric polypeptide assembly composition is a substrate of MMP-2, MMP-9, uPA, and protein lyase.

[0285] In one embodiment, the RS of the chimeric polypeptide assembly composition comprises an amino acid sequence that is a substrate of an extracellular protease (including but not limited to the proteases in Table 3) secreted by the target tissue. In another embodiment, the RS of the chimeric polypeptide assembly composition comprises an amino acid sequence that is a substrate of an intracellular protease (including but not limited to the proteases in Table 3).

[0286] In some embodiments, the present invention provides an RS composition intended for use in a subject chimeric peptide assembly composition, comprising at least a first cleavage sequence selected from the sequences listed in Table 4. In some embodiments, the RS sequence of the subject composition is selected from: LSGRSDNHSPLGLAGS, SPLGLAGSLSGRSDNH, SPLGLSGRSDNH, LAGRSDNHSPLGLAGS, LSGRSDNHVPLSLKMG, SPLGLAGS, GPLLARG, LSGRSDNH, VPLSLTMG, VPLSLKMG, VPLSLSMG, EPLELVAG, EPLELRAG, EPAALMAG, EPASLMAG, RIGSLRTA, RIQFLRTA, EPFHLMAG, VPLSLFMG, EPLELPAG, and EPLELAAG. If desired, the RS sequence of the subject chimeric peptide assembly is LSGRSDNHSPLGLAGS. In one embodiment, the RS of the chimeric peptide assembly composition comprises the sequence of BSRS1 in Table 4. In another embodiment, the RS of the chimeric peptide assembly composition consists of the sequence of BSRS1 in Table 4.

[0287] In another embodiment, the RS of the cleavage conjugate composition comprises a first cleavage sequence and a second cleavage sequence different from the first cleavage sequence, wherein each sequence is selected from the sequences shown in Table 4, and the first and second cleavage sequences are linked to each other by one to six amino acids selected from glycine, serine, alanine, and threonine. In another embodiment, the RS of the cleavage conjugate composition comprises a first cleavage sequence, a second cleavage sequence different from the first cleavage sequence, and a third cleavage sequence, wherein each sequence is selected from the sequences shown in Table 4, and the first, second, and third cleavage sequences are linked to each other by one to six amino acids selected from glycine, serine, alanine, and threonine. In other embodiments, the present invention provides chimeric polypeptide assemblies comprising one, two, or three RSs. Specifically, it is intended that multiple RSs of a chimeric polypeptide assembly can be linked to form a universal sequence that can be cleaved by a variety of proteases. Considering that such compositions would be more readily cleaved by lesion target tissues expressing multiple proteases, the resulting fragments carrying binding domains would more easily penetrate target tissues and exert the pharmacological effects of the binding domains. Table 3: Proteases in target tissues Table 4: Sequence of Release Segments (RS) Indicates the cutting site Specific amino acid abbreviations: Cit: Citrulline; Cha: β-Cyclohexylalanine; Hof: Homophenylalanine; Nva: Aminooctanoic acid; Dpa: D-phenylalanine; NLE: Leucine; SMC: S-methylcysteine; MnL: Methylleucine; Mel: Melphalan. * A list of multiple amino acids before, between, or after a slash indicates the alternative amino acids that can be substituted at that position; a "-" indicates that any amino acid can replace the corresponding amino acid shown in the middle column. **x is any L-amino acid other than proline. Hy is any hydrophilic L-amino acid γ indicates that the bond is linked by a γ-carboxyl group. 3. Filling part

[0288] On the other hand, the present invention relates to chimeric peptide assembly compositions comprising at least a first filler portion. In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a filler portion. Non-limiting examples of filler portions include extended recombinant peptides (XTEN, as described below); albumin-binding domains; albumin; IgG-binding domains; peptides of at least 350 amino acid residues consisting of proline, serine, and alanine; fatty acids; elastin-like proteins (ELPs) (a single subunit or structural unit of an ELP is derived from a five-amino acid motif found in the human protein elastin, which is repeated multiple times to form an ELP biopolymer, as described in WO2016081884), Fc domains, polyethylene glycol (PEG), PLGA, and hydroxyethyl starch. In another embodiment, the filler portion comprises two distinct filler portions selected from: XTEN; albumin-binding domains; albumin; IgG-binding domains; peptides consisting of proline, serine, and alanine; fatty acids; Fc domains; polyethylene glycol (PEG), PLGA, and hydroxyethyl starch, wherein the two filler portions are connected to each other and in turn to a release segment of the composition. In one preferred embodiment, the filler portion of the subject composition is one or more XTEN molecules. In another preferred embodiment, the chimeric polypeptide assembly composition comprises a filler sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an XTEN sequence of comparable length selected from the sequences listed in Table 5. In an embodiment, the XTEN polypeptide is recombinantly linked to a second release region (RS) of the composition.

[0289] Unconstrained by theory, the introduction of filler portions into the design of the subject composition is incorporated to impart certain important properties; ii) providing a chimeric peptide assembly composition having a filler portion that, when the composition is intact, shields the binding domain and reduces the binding affinity for target antigens and effector cell antigens; iii) providing a chimeric peptide assembly composition having a filler portion that, when administered to a subject, provides an enhanced half-life; iv) providing a chimeric peptide assembly composition having a filler portion that reduces exudation in normal tissues and organs, but still allows a degree of exudation in lesions (e.g., tumors) with larger pore sizes in the vascular system, but can be released from the composition by the action of certain mammalian proteases, thereby allowing the binding domains of the composition to more easily penetrate lesions and simultaneously bind target antigens on effector cells and tumor cells. To meet these needs, the present invention provides a chimeric peptide assembly composition in which the filler portion provides increased mass and hydrodynamic radius to the resulting composition. In a preferred embodiment, the filling portion is an XTEN polypeptide, which provides certain advantages in the design of the compositions of the present invention, not only providing increased mass and hydrodynamic radius, but also its flexible, unstructured characteristics providing shielding of the binding domain of the first portion of the composition, thereby reducing the likelihood of binding to antigens in normal tissues or in the vascular system of normal tissues that do not express or express reduced levels of target antigens and / or effector cell antigens, and enhancing the solubility and proper folding of scFv. (i) XTEN

[0290] In some embodiments, the present invention provides a chimeric polypeptide assembly composition comprising one or more recombinant XTEN molecules linked to the composition.

[0291] As used herein, “XTEN” refers to a polypeptide with a non-naturally occurring, substantially non-repetitive sequence that, under physiological conditions, has little or no secondary or tertiary structure, and the additional properties described in the following paragraphs. XTENs typically have at least about 100 to at least about 1000 or more amino acids, more preferably at least about 200 to at least about 900 amino acids, and more preferably at least about 400 to about 866 amino acids, most or all of which are small hydrophilic amino acids. “XTEN” as used herein explicitly excludes complete antibodies or antibody fragments (e.g., single-chain antibodies and Fc fragments). XTEN polypeptides have the function of acting as fusion couplers because they perform a variety of functions and, when linked to compositions, for example, those comprising a bispecific binding domain of the first part of the chimeric polypeptide assembly composition described herein, impart certain desired properties. The resulting compositions have enhanced properties compared to the corresponding binding domain not linked to XTENs, such as enhanced pharmacokinetic, physicochemical, pharmacological, and improved toxicological and pharmaceutical properties, thereby making them suitable for the treatment of certain conditions for which binding domains are known to be used in the art.

[0292] The unstructured characteristics and physicochemical properties of XTENs are partly due to the disproportionately limited overall amino acid composition to 4-6 types of hydrophilic amino acids, the quantifiable, substantially non-reproducible design of amino acid linkages, and the resulting length and / or configuration of the XTEN polypeptide. Among the advantageous features common to XTENs but not to natural polypeptides, the properties of XTENs disclosed herein are independent of the absolute primary amino acid sequence, as demonstrated by the various exemplary sequences in Table 5, which possess similar properties across different length ranges and impart enhanced properties to the compositions to which they are linked, many of which are demonstrated in the examples. In fact, particularly considering that the compositions of the invention are not limited to those specifically listed in Table 5, but rather that embodiments include sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequences in Table 5, as these sequences exhibit the properties of XTENs described below. It has been determined that such XTENs have properties more like non-protein hydrophilic polymers (e.g., polyethylene glycol or “PEG”) than proteins. The XTEN of the present invention exhibits one or more of the following advantageous properties: defined and uniform length (for a given sequence), conformational flexibility, reduced or absent secondary structure, high random coil formation, high water solubility, high protease resistance, low immunogenicity, low binding to mammalian receptors, defined degree of charge and enhanced hydrodynamic (or Stokes) radius; similar to certain hydrophilic polymers (e.g., polyethylene glycol), making them particularly suitable for use as conjugate partners.

[0293] The XTEN component of the subject fusion protein is designed to behave like a denatured peptide sequence under physiological conditions, despite the elongation of the polymer length. "Denatured" describes the state of a peptide in solution characterized by a large degree of conformational freedom in its peptide backbone. Most peptides and proteins adopt a denatured conformation in the presence of high concentrations of denaturing agents or at high temperatures. Peptides in a denatured conformation have, for example, a characteristic circular dichroism (CD) spectrum and are characterized by a lack of long-range interactions, as determined by NMR. In this document, "denatured conformation" and "unstructured conformation" are used as synonyms. In some embodiments, the present invention provides chimeric peptide assembly compositions comprising an XTEN sequence that, under physiological conditions, resembles a denatured sequence substantially lacking secondary structure. "Substantially lacking" as used in this context means that at least about 80%, or about 90%, or about 95%, or about 97%, or at least about 99% of the XTEN amino acid residues of the XTEN sequence do not contribute to the secondary structure, as measured or determined by the methods described herein (including algorithms or spectrophotometry).

[0294] Various established methods and assays are known in the art for determining and confirming the physicochemical properties of the subject XTEN. These properties include, but are not limited to, secondary or tertiary structure, solubility, protein aggregation, stability, absolute and apparent molecular weight, purity and homogeneity, chain-breaking properties, contamination, and water content. Methods for measuring these properties include analytical centrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion chromatography (SEC), HPLC-reversed phase, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, HPLC-ion exchange, HPLC-size exclusion, IR, NMR, Raman spectroscopy, refractive index measurement, and UV / Vis spectroscopy. In particular, secondary structure can be measur...

Claims

1. A chimeric polypeptide assembly comprising a first part, a second part, and a third part, wherein: a. The first part includes i. A first binding domain with binding specificity to target cell markers; as well as ii. A second binding domain with binding specificity to effector cell antigens; b. The second portion comprises a peptide release region (RS) capable of being cleaved by one or more mammalian proteases; and c. The third part includes a filling portion; The filling portion is capable of being released from the first portion by the action of the mammalian protease on the second portion.

2. A pharmaceutical composition comprising the chimeric polypeptide assembly according to claim 1 and one or more pharmaceutically suitable excipients.

3. A method of treating a disease in a subject, comprising administering to a subject in need a therapeutically effective dose of a chimeric polypeptide assembly of any one of claims 1-2 or a pharmaceutical composition comprising said chimeric polypeptide assembly.

4. The method according to claim 3, wherein the disease is selected from cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, T-cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, colon cancer, prostate cancer, head and neck cancer, any form of skin cancer, melanoma, genitourinary tract cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal cancer metastasis, serous uterine carcinoma, endometrial cancer, cervical cancer, colorectal cancer, intraperitoneal malignant tumor with malignant ascites, uterine cancer, and mesothelioma in the peritoneum. Kidney cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, stomach cancer, esophageal cancer, gastric cancer, small intestinal cancer, liver cancer, hepatocellular carcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, bile duct cancer, salivary gland cancer, thyroid cancer, epithelial cancer, adenocarcinoma, sarcoma of any origin, primary hematologic malignancies including acute or chronic lymphocytic leukemia, acute or chronic myeloid leukemia, myeloproliferative neoplasms or myelodysplastic disorders, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis or Goodpasture syndrome.

5. The pharmaceutical composition of claim 2 or the chimeric polypeptide assembly of any one of claims 1, in a method of treating a disease, the method comprising optionally administering the pharmaceutical composition or the chimeric polypeptide assembly to a subject suffering from the disease according to a treatment regimen comprising using one or more consecutive doses of a therapeutically effective dose.

6. A kit comprising the pharmaceutical composition of claim 2, a container, and a label or packaging insert on or associated with the container.

7. A chimeric polypeptide assembly comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequences listed in Table 10 or Table 12.

8. An isolated nucleic acid comprising a polynucleotide sequence selected from: (a) a polynucleotide encoding the chimeric polypeptide assembly of claim 1, or (b) a complement of the polynucleotide in (a).

9. An expression vector comprising the polynucleotide sequence of claim 8 and a recombinant regulatory sequence operatively linked to the polynucleotide sequence.

10. An isolated host cell comprising the expression vector of claim 9.

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