WO2020247871A2 - Compositions and methods relating to tumor activated t cell engagers - Google Patents

Compositions and methods relating to tumor activated t cell engagers Download PDF

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Publication number
WO2020247871A2
WO2020247871A2 PCT/US2020/036493 US2020036493W WO2020247871A2 WO 2020247871 A2 WO2020247871 A2 WO 2020247871A2 US 2020036493 W US2020036493 W US 2020036493W WO 2020247871 A2 WO2020247871 A2 WO 2020247871A2
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Prior art keywords
polypeptide
polypeptide complex
fab
complex
seq
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English (en)
French (fr)
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WO2020247871A3 (en
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David Campbell
Ramesh Bhatt
Thomas R. DIRAIMONDO
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Janux Therapeutics Inc
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Janux Therapeutics Inc
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Priority to JP2021572583A priority Critical patent/JP2022535924A/ja
Priority to US17/616,281 priority patent/US20230220109A1/en
Priority to KR1020227000224A priority patent/KR20220052898A/ko
Priority to CN202080056153.1A priority patent/CN114423499A/zh
Priority to EP20819457.1A priority patent/EP3980131A4/en
Publication of WO2020247871A2 publication Critical patent/WO2020247871A2/en
Publication of WO2020247871A3 publication Critical patent/WO2020247871A3/en
Anticipated expiration legal-status Critical
Priority to US18/785,900 priority patent/US20240376226A1/en
Priority to JP2025050785A priority patent/JP2025111446A/ja
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • Protein-based therapies comprising T cell engagers have proven effective as treatments for a variety of diseases. As with any therapeutic class, there is a need to improve toxicity and side effects of such treatments, along with improving the half-life of the therapeutic molecules.
  • Modified T cell engagers can be used for selective destruction of an individual cell or cell type such as cancer cells of a tumor. Such modified T cell engagers induce an immune response against the tumor to clear the tumor. However, current therapies using modified T cell engagers can be toxic and inefficacious. Further, such modified T-cell engagers can have poor pharmacokinetic properties (PK). Provided herein are modified T-cell engagers that reduce toxicity in healthy tissue and thus improving safety while having improved PK properties and efficacy in eliminating the tumor. In some embodiments, the modified T-cell engagers described herein are linked to a peptide that blocks interactions of the T-cell engager with its target in healthy tissue thereby reducing target mediated drug disposition (TMDD). The modified T-cell engagers as described herein are also linked to half-life extending molecule, such as single-domain antibody, which improves the PK profile of the modified T-cell engager as compared to an unmodified T-cell engager.
  • TMDD target mediated drug disposition
  • polypeptide complexes comprising a structural arrangement according to a configuration:
  • the polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide (Pi) at an N- tenninus of the scFv with a linking moiety (Li) that is a substrate for a tumor specific protease, wherein Pi impairs binding of the scFv to an effector cell antigen, and Pi is further linked to a half-life extending molecule; and an antigen recognizing molecule that binds to a tumor cell antigen, wherein the antigen recognizing molecule comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the antigen recognizing molecule is linked to the scFv, and the antigen recognizing molecule is further linked to P2 and L2, wherein P2 comprises a peptide that impairs binding of the antigen recognizing molecule to the tumor cell antigen; and L2 comprises
  • the antigen recognizing molecule is a Fab or a Fab'.
  • the heavy chain variable domain is linked to an N-terminus of the Fab heavy chain polypeptide and L2 is connected to an N-terminus of the Fab light chain polypeptide.
  • the heavy chain variable domain is linked to an N-terminus of the Fab light chain polypeptide and L2 is connected to an N-terminus of the Fab heavy chain polypeptide.
  • the light chain variable domain is linked to an N-terminus of the Fab heavy chain polypeptide and L2 is connected to an N- terminus of the Fab light chain polypeptide.
  • the light chain variable domain is linked to an N-terminus of the Fab light chain polypeptide and L2 is connected to an N-terminus of the Fab heavy chain polypeptide.
  • the polypeptide complex has a molecular weight of less than about 110 kDa.
  • the heavy chain variable domain, light chain variable domain, Fab heavy chain polypeptide, Fab light chain polypeptide, and half-life extending molecule have a combined molecular weight of less than about 100 kDa.
  • the tumor cell antigen comprises epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), or mesothelin.
  • the effector cell antigen comprises cluster of differentiation 3 (CD3).
  • the scFv comprises complementary determining regions (CDRs) selected from the group consisting of muromonab- CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, FI 11-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b,
  • CDRs complementary determining regions
  • the scFv comprises complementary determining regions (CDR)s of SP34.
  • the scFv comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68.
  • Pi impairs binding of the scFv to the effector cell antigen by binding to the scFv through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H- bonding interactions. In some instances, Pi impairs binding of the scFv to the effector cell antigen by binding to the scFv at or near an antigen binding site.
  • Pi comprises an amino acid sequence of at least 10 amino acids in length and no more than 20 amino acids in length. In some instances, Pi has less than 70% sequence identity to an amino acid sequence of the effector cell antigen. In some instances, Pi has less than 70% sequence identity to an amino acid sequence of CD3. In some instances, Pi comprises an amino acid sequence according to SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28.
  • Li comprises a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, matrix metalloprotease cleavable amino acid sequence, or a legumain cleavable amino acid sequence.
  • Li has a formula comprising (G2S) n , (GS) n , (GSGGS) n (SEQ ID NO: 30), (GGGS) thread (SEQ ID NO: 31) , (GGGGS) context (SEQ ID NO: 32) , or(GSSGGS)terrorism (SEQ ID NO: 33) , wherein n is an integer of at least 1.
  • Li comprises an amino acid sequence according to SEQ ID NOs: 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 55. In some instances, Li comprises an amino acid sequence according to SEQ ID NO: 42.
  • the half-life extending molecule comprises a linking moiety (L3) that connects the half-life extending molecule to Pi.
  • L3 has a formula selected from the group consisting of (G2S) n , (GS) n , (GSGGS) n (SEQ ID NO: 30), (GGGS) n (SEQ ID NO: 31) , (GGGGS) n (SEQ ID NO: 32) , and (GSSGGS) n (SEQ ID NO: 33) , wherein n is an integer of at least 1.
  • L3 comprises an amino acid sequence according to SEQ ID NO: 51.
  • the half-life extending molecule comprises an antibody.
  • the antibody comprises a single domain antibody, a single chain variable fragment, or a Fab.
  • the single domain antibody binds to albumin.
  • the single domain antibody comprises 10G or 10GE. In some instances, the single domain antibody comprises 10G, and the single domain antibody comprises an amino acid sequence according to SEQ ID NO: 52.
  • P2 impairs binding of the antigen recognizing molecule to the tumor cell antigen by binding to the antigen recognizing molecule through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions. In some instances, P2 impairs binding of the antigen recognizing molecule to the tumor cell antigen by binding to the antigen recognizing molecule at or near an antigen binding site. In some instances, P2 comprises an amino acid sequence of at least 10 amino acids in length and no more than 20 amino acids in length.
  • the tumor cell antigen comprises epidermal growth factor receptor (EGFR).
  • EGFR epidermal growth factor receptor
  • P2 comprises an amino acid sequence according to SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 7.
  • the Fab light chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 56 or SEQ ID NO: 57.
  • the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60.
  • the tumor cell antigen comprises human epidermal growth factor receptor 2 (HER2).
  • P2 comprises an amino acid sequence according to SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17.
  • the Fab light chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 61.
  • the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 62 or SEQ ID NO: 63.
  • L2 comprises a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, matrix metalloprotease cleavable amino acid sequence, or a legumain cleavable amino acid sequence.
  • L2 has a formula comprising (G 2 S) connect, (GS) come, (GSGGS) friendship (SEQ ID NO: 30), (GGGS) thread (SEQ ID NO: 31) , (GGGGS) thread (SEQ ID NO: 32) , O G (GSSGGS) n (SEQ ID NO: 33) , wherein n is an integer of at least 1.
  • L2 comprises an amino acid sequence according to SEQ ID NOs: 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 55.
  • L2 comprises the amino acid sequence according to SEQ ID NO: 42.
  • compositions comprising: (i) the polypeptide complex as described herein; and (ii) a pharmaceutically acceptable excipient.
  • nucleic acid molecules encoding the polypeptide or polypeptide complex as described herein.
  • FIGS. 1A-1G illustrates preparation and biotinylation of cetuximab and trastuzumab. Antibodies were biotinylated prior to phage panning using he EZ4ink Sulfo-NHS-LC-LC-Biotin reagent and evaluated for their ability to bind cognate antigen.
  • FIG. 1A illustrates naked Ab-1, Ab-3 binding to EGFR-biotin using 3nM, InM, 0.3nM, O.lnM, 0.03nM, and OnM Ab-1, Ab-3 in solution.
  • FIG. 1A illustrates naked Ab-1, Ab-3 binding to EGFR-biotin using 3nM, InM, 0.3nM, O.lnM, 0.03nM, and OnM Ab-1, Ab-3 in solution.
  • FIG. 1A illustrates naked Ab-1, Ab-3 binding to EGFR-biotin using 3nM, InM, 0.3nM, O.lnM, 0.03nM, and OnM Ab-1,
  • FIG. 1C illustrates naked Ab-6, Ab-7 binding to HER2 -biotin using 25nM, 12.5nM, 6.25nM, 3.125nM, 1.56nM, 0.78nM, 0.39nM and OnM Ab-6, Ab-7 in solution.
  • FIG ID illustrates HER2 binding to biotinylated Ab-6, Ab-7 using 70nM HER2 in solution
  • FIG. IE illustrates HER2 binding to biotinylated Ab-6, Ab-7 using OnM HER2 in solution
  • FIG. IF illustrates naked Ab-9, Ab-10 binding to CD3-biotin using 3nM,
  • FIG. 1G illustrates CD3 binding to biotinylated Ab-9, Ab-10 using 200nM and OnM CD3 in solution.
  • FIGS. 2A-2C illustrate peptide panning using phage display enables discovery of antibody inhibitory peptides.
  • Peptides were displayed via p3 or p8 phage protein fusion and biopanned against Trastuzumab (Ab-6, Ab-7).
  • FIG. 2A depicts a panning process involving standard bind, wash, elute, and amplify cycles. The eluted phage after multiple rounds of panning were used to infect bacteria, plated on agar, individual colonies picked and amplified, followed by binding assessments and sequencing.
  • Figure discloses SEQ ID NOS 112-113, respectively, in order of appearance.
  • FIG. 2B illustrates binding of clonal phagemid to plate captured Ab-6, Ab-7 characterized by EFISA. Biotinylated antibody was captured on neutravidin coated plates followed by incubation with phage. Bound phage was detected using an anti-ml3 HRP antibody conjugate. Phage binding to neutravidin captured biotinylated antibody was compared to phage binding to neutravidin alone.
  • FIG. 2C illustrates clonal phage binders of Ab-6, Ab-7 that did not bind neutravidin evaluated for their ability to bind in the presence and absence of the cognate antigen. Inhibition of phage binding using pre -incubation of soluble HER2 at 6nM or 20nM was used as an indicator that clonal phage bound within or near the antibody binding sites responsible for HER2 recognition.
  • FIGS. 3A-3K illustrate kinetic binding of Trastuzumab (Ab-6, Ab-7) to example peptides or HER2 via BLI.
  • FIG. 3A illustrates kinetic binding of Trastuzumab (Ab-6, Ab-7) to Peptide-8.
  • FIG. 3B illustrates kinetic binding of Trastuzumab (Ab-6, Ab-7) to Peptide-9.
  • FIG. 3C illustrates kinetic binding of
  • FIG. 3D illustrates kinetic binding of Trastuzumab (Ab-6, Ab-7) to Peptide-11.
  • FIG. 3E illustrates kinetic binding of Trastuzumab (Ab-6, Ab-7) to Peptide-12.
  • FIG. 3F illustrates kinetic binding of Trastuzumab (Ab-6, Ab-7) to Peptide-13.
  • FIG. 3G illustrates kinetic binding of Trastuzumab (Ab-6, Ab-7) to Peptide-14.
  • FIG. 3H illustrates kinetic binding of Trastuzumab (Ab-6, Ab-7) to Peptide-15.
  • FIG. 31 illustrates kinetic binding of Trastuzumab (Ab-6, Ab-7) to Peptide-16.
  • FIG. 3J illustrates kinetic binding of Trastuzumab (Ab-6, Ab-7) to Peptide-17.
  • FIG. 3K illustrates kinetic binding of Trastuzumab (Ab-6, Ab-7) to Her2.
  • FIGS. 4A-4H illustrate kinetic binding of Cetuximab (Ab-1, Ab-3) to example peptides or EGFR via BLI.
  • FIG. 4A illustrates kinetic binding of Cetuximab (Ab-1, Ab-3) to Peptide-1.
  • FIG. 4B illustrates kinetic binding of Cetuximab (Ab-1, Ab-3) to Peptide-2.
  • FIG. 4C illustrates kinetic binding of Cetuximab (Ab-1, Ab-3) to Peptide-3.
  • FIG. 4D illustrates kinetic binding of Cetuximab (Ab-1, Ab-3) to Peptide-4.
  • FIG. 4E illustrates kinetic binding of Cetuximab (Ab-1, Ab-3) to Peptide-5.
  • FIG. 4F illustrates kinetic binding of Cetuximab (Ab-1, Ab-3) to Peptide-6.
  • FIG. 4G illustrates kinetic binding of Cetuximab (Ab-1, Ab-3) to Peptide-7.
  • FIG. 4H illustrates a blank.
  • FIGS. 5A-5L illustrates kinetic binding of SP34 (Ab-9, Ab-10) to example peptides or CD3 via BLI.
  • FIG. 5A illustrates kinetic binding of SP34 (Ab-9, Ab-10) to Peptide-19.
  • FIG. 5B illustrates kinetic binding of SP34 (Ab-9, Ab-10) to Peptide-20.
  • FIG. 5C illustrates kinetic binding of SP34 (Ab-9, Ab-10) to Peptide-21.
  • FIG. 5D illustrates kinetic binding of SP34 (Ab-9, Ab-10) to Peptide-22.
  • FIG. 5E illustrates kinetic binding of SP34 (Ab-9, Ab-10) to Peptide-23.
  • FIG. 5F illustrates kinetic binding of SP34 (Ab-9, Ab- 10) to Peptide-24.
  • FIG. 5G illustrates kinetic binding of SP34 (Ab-9, Ab-10) to Peptide-26.
  • FIG. 5H illustrates kinetic binding of SP34 (Ab-9, Ab-10) to Peptide-26.
  • FIG. 51 illustrates kinetic binding of SP34 (Ab-9, Ab-10) to Peptide-27.
  • FIG. 5J illustrates kinetic binding of SP34 (Ab-9, Ab-10) to Peptide-28.
  • FIG. 5K illustrates kinetic binding of SP34 (Ab-9, Ab-10) to Peptide-18.
  • FIG. 5L illustrates a blank.
  • FIGS. 6A-6C illustrate equilibrium binding via ELISA.
  • FIG. 6A illustrates equilibrium binding of Trastuzumab (Ab-6, Ab-7) to example peptides via ELISA.
  • FIG. 6B illustrates equilibrium binding of Cetuximab (Ab-1, Ab-3) to example peptides via ELISA.
  • FIG. 6C illustrates equilibrium binding of SP34 (Ab-9, Ab-10) to example peptides via ELISA.
  • FIGS. 7A-7F illustrate that lOOuM peptides inhibit kinetic binding of 2nM Ab-6, Ab-7 to HER2 via BLI.
  • FIG. 7A illustrates kinetic binding of 2nM Ab-6, Ab-7 to HER2 via BLI in the absence of a peptide.
  • FIG. 7B illustrates that lOOuM Peptide-8 inhibit kinetic binding of 2nM Ab-6, Ab-7 to HER2 via BLI.
  • FIG. 7C illustrates that lOOuM Peptide-9 inhibit kinetic binding of 2nM Ab-6, Ab-7 to HER2 via BLI.
  • FIG. 7D illustrates that lOOuM Peptide- 10 inhibit kinetic binding of 2nM Ab-6, Ab-7 to HER2 via BLI.
  • FIG. 7A illustrates kinetic binding of 2nM Ab-6, Ab-7 to HER2 via BLI in the absence of a peptide.
  • FIG. 7B illustrates that lOOuM Peptide-8 inhibit kinetic binding of
  • FIG. 7E illustrates that lOOuM Peptide- 11 inhibit kinetic binding of 2nM Ab-6, Ab-7 to HER2 via BLI.
  • FIG. 7F illustrates that lOOuM Peptide-12 inhibit kinetic binding of 2nM Ab-6, Ab-7 to HER2 via BLI.
  • FIGS. 8A-8I illustrate that lOOuM peptides inhibit kinetic binding of 2nM Ab-1, Ab-3 to EGFR via BLI.
  • FIG. 8A illustrates kinetic binding of 2nM Ab-1, Ab-3 to EGFR via BLI in the absence of a peptide.
  • FIG. 8B illustrates that lOOuM Peptide-1 inhibit kinetic binding of 2nM Ab-1, Ab-3 to EGFR via BLI.
  • FIG. 8C illustrates that lOOuM Peptide-2 inhibit kinetic binding of 2nM Ab-1, Ab-3 to EGFR via BLI.
  • FIG. 8D illustrates that lOOuM Peptide-3 inhibit kinetic binding of 2nM Ab-1, Ab-3 to EGFR via BLI.
  • FIG. 8A illustrates kinetic binding of 2nM Ab-1, Ab-3 to EGFR via BLI in the absence of a peptide.
  • FIG. 8B illustrates that lOOuM Peptide-1 inhibit kinetic binding of 2
  • FIG. 8E illustrates that lOOuM Peptide-4 inhibit kinetic binding of 2nM Ab-1, Ab-3 to EGFR via BLI.
  • FIG. 8F illustrates that lOOuM Peptide-5 inhibit kinetic binding of 2nM Ab-1, Ab-3 to EGFR via BLI.
  • FIG. 8G illustrates that lOOuM Peptide-6 inhibit kinetic binding of 2nM Ab-1, Ab-3 to EGFR via BLI.
  • FIG. 8H illustrates that lOOuM Peptide-7 inhibit kinetic binding of 2nM Ab-1, Ab-3 to EGFR via BLI.
  • FIG. 81 illustrates a blank.
  • FIGS. 9A-9L illustrate that lOOuM peptides inhibit kinetic binding of 2nM Ab-9, Ab-10 to CD3 via BLI.
  • FIG. 9A illustrates kinetic binding of 2nM Ab-9, Ab-10 to CD3 via BLI in the absence of a peptide.
  • FIG. 9B illustrates that lOOuM Peptide-18 inhibit kinetic binding of 2nM Ab-9, Ab-10 to CD3 via BLI.
  • FIG. 9C illustrates that lOOuM Peptide- 19 inhibit kinetic binding of 2nM Ab-9, Ab-10 to CD3 via BLI.
  • FIG. 9D illustrates that lOOuM Peptide-20 inhibit kinetic binding of 2nM Ab-9, Ab-10 to CD3 via BLI.
  • FIG. 9E illustrates that lOOuM Peptide-21 inhibit kinetic binding of 2nM Ab-9, Ab-10 to CD3 via BLI.
  • FIG. 9F illustrates that lOOuM Peptide-22 inhibit kinetic binding of 2nM Ab-9, Ab-10 to CD3 via BLI.
  • FIG. 9G illustrates that lOOuM Peptide-23 inhibit kinetic binding of 2nM Ab-9, Ab-10 to CD3 via BLI.
  • FIG. 9H illustrates that lOOuM Peptide-24 inhibit kinetic binding of 2nM Ab-9, Ab-10 to CD3 via BLI.
  • FIG. 91 illustrates that lOOuM Peptide-25 inhibit kinetic binding of 2nM Ab-9, Ab-10 to CD3 via BLI.
  • FIG. 9J illustrates that lOOuM Peptide-26 inhibit kinetic binding of 2nM Ab-9, Ab-10 to CD3 via BLI.
  • FIG. 9K illustrates that lOOuM Peptide-26 inhibit kinetic binding of 2nM Ab-9, Ab-10 to CD3 via BLI.
  • FIG. 9L illustrates that lOOuM Peptide-28 inhibit kinetic binding of 2nM Ab-9, Ab-10 to CD3 via BLI.
  • FIGS. 10A-10C illustrate dose dependent inhibition.
  • FIG. 10A illustrates that peptides inhibit O.lnM Ab-6, Ab-7 binding HER2 in a dose dependent manner by ELISA.
  • FIG. 10B illustrates that peptides inhibit 0.2nM Ab-1, Ab-3 binding EGFR in a dose dependent manner by ELISA.
  • FIG. IOC illustrates that peptides inhibit InM Ab-9, Ab-10 binding CD3 in a dose dependent manner by ELISA.
  • FIG. 11 depicts the tumor specific activity and cross over PK concepts within tumor activated T cell engager molecules or polypeptide complexes.
  • FIG. 12 depicts general dual mask and single mask polypeptide complex designs.
  • FIGS. 13A-13C illustrate analysis of non-masked polypeptide complex, PC-1.
  • FIG. 13A illustrates SDS-PAGE of non-masked polypeptide complex, PC-1.
  • FIG. 13B illustrates SEC-FPLC of non-masked polypeptide complex, PC-1.
  • FIG. 13C illustrates liquid chromatography-mass spectrometry (LC-MS) analysis of non-masked polypeptide complex, PC-1.
  • FIGS. 14A-14C illustrate analysis of masked polypeptide complex, PC-2.
  • FIG. 14A illustrates SDS-PAGE of masked polypeptide complex, PC-2.
  • FIG. 14B illustrates SEC-FPLC of masked polypeptide complex, PC-2.
  • FIG. 14C illustrates liquid chromatography-mass spectrometry (LC-MS) analysis of masked polypeptide complex, PC-2.
  • FIGS. 15A-15C illustrate analysis of masked polypeptide complex, PC-3.
  • FIG. 15A illustrates SDS-PAGE of masked polypeptide complex, PC-3.
  • FIG. 15B illustrates SEC-FPLC of masked polypeptide complex, PC-3.
  • FIG. 15C illustrates liquid chromatography-mass spectrometry (LC-MS) analysis of masked polypeptide complex, PC-3.
  • LC-MS liquid chromatography-mass spectrometry
  • FIGS. 16A-16C illustrate analysis of masked polypeptide complex, PC-4.
  • FIG. 16A illustrates SDS-PAGE of masked polypeptide complex, PC-4.
  • FIG. 16B illustrates SEC-FPLC of masked polypeptide complex, PC-4.
  • FIG. 16C illustrates liquid chromatography-mass spectrometry (LC-MS) analysis of masked polypeptide complex, PC-4.
  • LC-MS liquid chromatography-mass spectrometry
  • FIGS. 17A-17B illustrate analysis of masked polypeptide complex, PC-5.
  • FIG. 17A illustrates SDS-PAGE of masked polypeptide complex, PC-5.
  • FIG. 17B illustrates SEC-FPLC of masked polypeptide complex, PC-5.
  • FIGS. 18A-18C illustrate analysis of masked polypeptide complex, PC-6.
  • FIG. 18A illustrates SDS-PAGE of masked polypeptide complex, PC-6.
  • FIG. 18B illustrates SEC-FPLC of masked polypeptide complex, PC-6.
  • FIG. 18C illustrates liquid chromatography-mass spectrometry (LC-MS) analysis of masked polypeptide complex, PC-6.
  • LC-MS liquid chromatography-mass spectrometry
  • FIGS. 19A-19B illustrate analysis of masked polypeptide complex, PC-7.
  • FIG. 19A illustrates SDS-PAGE of masked polypeptide complex, PC-7.
  • FIG. 19B illustrates SEC-FPLC of masked polypeptide complex, PC-7.
  • FIGS. 20A-20B illustrate analysis of non-masked polypeptide complex, PC-8.
  • FIG. 20A illustrates SDS-PAGE of non-masked polypeptide complex, PC-8.
  • FIG. 20B illustrates SEC-FPLC of non-masked polypeptide complex, PC-8.
  • FIGS. 21A-21B illustrate analysis of masked polypeptide complex, PC-9.
  • FIG. 21A illustrates SDS-PAGE of masked polypeptide complex, PC-9.
  • FIG. 21B illustrates SEC-FPLC of masked polypeptide complex, PC-9.
  • FIGS. 22A-22B illustrate analysis of masked polypeptide complex, PC-10.
  • FIG. 22A illustrates SDS-PAGE of masked polypeptide complex, PC-10.
  • FIG. 22B illustrates SEC-FPLC of masked polypeptide complex, PC- 10.
  • FIGS. 23A-23B illustrate analysis of non-masked polypeptide complex, PC-11.
  • FIG. 23A illustrates SDS-PAGE of non-masked polypeptide complex, PC-11.
  • FIG. 23B illustrates SEC-FPLC of non-masked polypeptide complex, PC-11.
  • FIGS. 24A-24B illustrate analysis of masked polypeptide complex, PC- 12.
  • FIG. 24A illustrates SDS-PAGE of masked polypeptide complex, PC-12.
  • FIG. 24B illustrates SEC-FPLC of masked polypeptide complex, PC- 12.
  • FIG. 25 illustrates polypeptide complex binding albumin from different species by ELISA.
  • HSA human serum albumin
  • CSA cynomolgus serum albumin
  • MSA mouse serum albumin
  • BSA bovine serum albumin
  • FIGS. 26A-26D illustrate polypeptide complex kinetic binding to HER2 by BLI.
  • FIG. 26A illustrates binding of non-masked polypeptide complex PC-11 to HER2.
  • FIG. 26B illustrates binding of masked polypeptide complex PC- 12 to HER2.
  • FIG. 26C illustrate binding of masked polypeptide complex PC- 12 treated with MTSP1 to HER2
  • FIG. 26D illustrates a blank.
  • FIGS. 27A-27H illustrate polypeptide complex kinetic binding to EGFR in the presence of bovine serum albumin (BSA) buffer by BLI.
  • FIG. 27A illustrates kinetic binding of PC-1 to EGFR in the presence of BSA buffer.
  • FIG. 27B illustrates kinetic binding of PC-1 treated with uPa to EGFR in the presence of BSA buffer.
  • FIG. 27C illustrates kinetic binding of PC-2 to EGFR in the presence of BSA buffer.
  • FIG. 27D illustrates kinetic binding of PC-2 treated with uPato EGFR in the presence of BSA buffer.
  • FIG. 27E illustrates kinetic binding of PC-3 to EGFR in the presence of BSA buffer.
  • FIG. 27F illustrates kinetic binding of PC-3 treated with uPa to EGFR in the presence of BSA buffer.
  • FIG. 27G illustrates kinetic binding of PC-10 to EGFR in the presence of BSA buffer.
  • FIG. 27H illustrates kinetic binding of PC-6 treated with uPa to EGFR in the presence of BSA buffer.
  • FIGS. 28A-28R illustrate polypeptide complex kinetic binding to EGFR in the presence of human serum albumin (HSA) buffer by BLI.
  • FIG. 28A illustrates kinetic binding of PC-1 to EGFR in the presence of HSA buffer.
  • FIG. 28B illustrates kinetic binding of PC- 1 treated with uPa to EGFR in the presence of HSA buffer.
  • FIG. 28C illustrates kinetic binding of PC-2 to EGFR in the presence of HSA buffer.
  • FIG. 28D illustrates kinetic binding of PC-2 treated with uPa to EGFR in the presence of HSA buffer.
  • FIG. 28E illustrates kinetic binding of PC-3 to EGFR in the presence of HSA buffer.
  • FIG. 28F illustrates kinetic binding of PC-3 treated with uPa to EGFR in the presence of HSA buffer.
  • FIG. 28G illustrates kinetic binding of PC-4 to EGFR in the presence of HSA buffer.
  • FIG. 28H illustrates kinetic binding of PC-4 treated with MTSP1 to EGFR in the presence of HSA buffer.
  • FIG. 281 illustrates kinetic binding of PC-5 to EGFR in the presence of HSA buffer.
  • FIG. 28 J illustrates kinetic binding of PC-5 treated with MTSP1 to EGFR in the presence of HSA buffer.
  • FIG. 28K illustrates kinetic binding of PC-7 to EGFR in the presence of HSA buffer.
  • FIG. 28L illustrates kinetic binding of PC-7 treated with MTSP1 to EGFR in the presence of HSA buffer.
  • FIG. 28M illustrates kinetic binding of PC-8 to EGFR in the presence of HSA buffer.
  • FIG. 28N illustrates kinetic binding of PC-8 treated with MTSP 1 to EGFR in the presence of HSA buffer.
  • FIG. 280 illustrates kinetic binding of PC-9 to EGFR in the presence of HSA buffer.
  • FIG. 28P illustrates kinetic binding of PC-9 treated with MTSP1 to EGFR in the presence of HSA buffer.
  • FIG. 28Q illustrates kinetic binding of PC-10 to EGFR in the presence of HSA buffer.
  • FIG. 28R illustrates kinetic binding of PC- lOtreated with MTSP1 to EGFR in the presence of HSA buffer.
  • FIGS. 29A-29L illustrate polypeptide complex kinetic binding to CD3 in the presence of bovine serum albumin (BSA) buffer by BLI.
  • FIG. 29A illustrates kinetic binding of PC-1 to CD3 in the presence of BSA buffer.
  • FIG. 29B illustrates kinetic binding of PC-1 treated with uPa to CD3 in the presence of BSA buffer.
  • FIG. 29C illustrates kinetic binding of PC-2 to CD3 in the presence of BSA buffer.
  • FIG. 29D illustrates kinetic binding of PC-2 treated with uPato CD3 in the presence of BSA buffer.
  • FIG. 29E illustrates kinetic binding of PC-3 to CD3 in the presence of BSA buffer.
  • FIG. 29A illustrates kinetic binding of PC-1 to CD3 in the presence of BSA buffer.
  • FIG. 29B illustrates kinetic binding of PC-1 treated with uPa to CD3 in the presence of BSA buffer.
  • FIG. 29C illustrates kinetic binding of PC-2 to CD3 in the presence of BSA buffer
  • FIG. 29F illustrates kinetic binding of PC-3 treated with uPa to CD3 in the presence of BSA buffer.
  • FIG. 29G illustrates kinetic binding of PC-10 to CD3 in the presence of BSA buffer .
  • FIG. 29H illustrates kinetic binding of PC-10 treated with MTSP1 to CD3 in the presence of BSA buffer.
  • FIG. 291 illustrates kinetic binding of PC-12 to CD3 in the presence of BSA buffer .
  • FIG. 29J illustrates kinetic binding of PC-12 treated with MTSP1 to CD3 in the presence of BSA buffer.
  • FIG. 29K illustrates kinetic binding of PC-11 to CD3 in the presence of BSA buffer .
  • FIG. 29L illustrates kinetic binding of PC-6 to CD3 in the presence of BSA buffer.
  • FIGS. 30A-30R illustrate polypeptide complex kinetic binding to CD3 in the presence of human serum albumin (HSA) buffer by BLI.
  • FIG. 30A illustrates kinetic binding of PC-1 to CD3 in the presence of HSA buffer.
  • FIG. 30B illustrates kinetic binding of PC-1 treated with uPato CD3 in the presence of HSA buffer.
  • FIG. 30C illustrates kinetic binding of PC-2 to CD3 in the presence of HSA buffer.
  • FIG. 30D illustrates kinetic binding of PC-2 treated with uPato CD3 in the presence of HSA buffer.
  • FIG. 30E illustrates kinetic binding of PC-3 to CD3 in the presence of HSA buffer.
  • FIG. 30F illustrates kinetic binding of PC-3 treated with uPa to CD3 in the presence of HSA buffer.
  • FIG. 30G illustrates kinetic binding of PC-4 to CD3 in the presence of HSA buffer.
  • FIG. 30H illustrates kinetic binding of PC-4 treated with MTSP 1 to CD3 in the presence of HSA buffer.
  • FIG. 301 illustrates kinetic binding of PC-5 to CD3 in the presence of HSA buffer.
  • FIG. 30J illustrates kinetic binding of PC-5 treated with MTSP1 to CD3 in the presence of HSA buffer.
  • FIG. 30K illustrates kinetic binding of PC-7 to CD3 in the presence of HSA buffer.
  • FIG. 30L illustrates kinetic binding of PC-7 treated with MTSP1 to CD3 in the presence of HSA buffer.
  • FIG. 30M illustrates kinetic binding of PC-8 to CD3 in the presence of HSA buffer.
  • FIG. 30N illustrates kinetic binding of PC-8 treated with MTSP1 to CD3 in the presence of HSA buffer.
  • FIG. 300 illustrates kinetic binding of PC-9 to CD3 in the presence of HSA buffer.
  • FIG. 30P illustrates kinetic binding of PC-9 treated with MTSP1 to CD3 in the presence of HSA buffer.
  • FIG. 30Q illustrates kinetic binding of PC-6 to CD3 in the presence of HSA buffer.
  • FIG. 30R illustrates a blank.
  • FIG. 31 illustrates polypeptide complex equilibrium binding HER2 in buffer containing bovine (BSA) or human albumin (HSA) by ELISA.
  • FIGS. 32A-32C illustrates polypeptide complex equilibrium binding EGFR in buffer containing BSA or HSA by ELISA, before or after a protease treatment.
  • FIG. 32A illustrates polypeptide complex equilibrium binding EGFR in buffer containing HSA, before or after uPa treatment.
  • FIG. 32B illustrates polypeptide complex equilibrium binding EGFR in buffer containing BSA, before or after uPa treatment.
  • FIG. 32C illustrates polypeptide complex equilibrium binding EGFR in buffer containing BSA or HSA, before or after MTSP1 treatment.
  • FIGS. 33A-33D illustrates polypeptide complex equilibrium binding CD3 in buffer containing BSA or HSA by ELISA, before or after a protease treatment.
  • FIG. 33A illustrate polypeptide complex equilibrium binding CD3 in buffer containing HSA by ELISA, before or after uPa treatment.
  • FIG. 33B illustrate polypeptide complex equilibrium binding CD3 in buffer containing BSA by ELISA, before or after MTSP 1 treatment.
  • FIG. 33C illustrate polypeptide complex equilibrium binding CD3 in buffer containing HSA by ELISA, before or after MTSP1 treatment.
  • FIG. 33C illustrate polypeptide complex equilibrium binding CD3 in buffer containing BSA by ELISA, before or after MTSP1 treatment.
  • FIG. 34 illustrates cellular CD3, polypeptide complex, and EGFR tetramer ternary complex formation on the surface of human T cells by flow cytometry.
  • FIG. 35 illustrate cellular EGFR, polypeptide complex, and CD3 tetramer ternary complex formation on the surface of HCT116 cells by flow cytometry.
  • FIG. 36 illustrates polypeptide complex mediated cytotoxicity against tumor target cells, HCC1569 by LDH-Glo assay.
  • FIG. 37 illustrates polypeptide complex mediated t cell activation against tumor target cells, HCC1569 by IFNy ELISA.
  • FIG. 38 illustrates polypeptide complex mediated HCC1569 tumor cell killing using real time cell analyzer (RTCA).
  • FIGS. 39A-39C illustrate polypeptide complex mediated cytotoxicity against tumor target cells, HCT116, by LDH-Glo assay.
  • FIG. 39A illustrates polypeptide complex (PC-1, PC-2, PC-3) mediated cytotoxicity against tumor target cells, HCT116, by LDH-Glo assay before or after uPa treatment.
  • FIG. 39B illustrates polypeptide complex (PC-4) mediated cytotoxicity against tumor target cells, HCT116, by LDH- Glo assay before or after MTSP1 treatment.
  • FIG. 39C illustrates polypeptide complex (PC-10) mediated cytotoxicity against tumor target cells, HCT116, by LDH-Glo assay before or after MTSP1 treatment.
  • FIGS. 40A-40C illustrate polypeptide complex mediated T cell activation against tumor target cells, HCT116 by IFNy ELISA.
  • FIG. 40A illustrates polypeptide complex (PC-1, PC-2, PC-3) mediated T cell activation against tumor target cells, HCT116 by IFNy ELISA before or after uPa treatment.
  • FIG. 40B illustrates polypeptide complex (PC-4) mediated T cell activation against tumor target cells, HCT116 by IFNy ELISA before or after MTSP1 treatment.
  • FIG. 40C illustrates polypeptide complex (PC- 10) mediated T cell activation against tumor target cells, HCT116 by IENg ELISA before or after MTSP1 treatment.
  • FIG. 41 illustrate polypeptide complex (PC-8, PC-4) mediated HCT116 tumor cell killing using real time cell analyzer (RTCA).
  • RTCA real time cell analyzer
  • FIG. 42 illustrates PC-8 and PC-4 mouse pharmacokinetics.
  • FIG. 43 illustrates HCT116 growth kinetics in tumor bearing NCG mice.
  • FIG. 44 illustrates mouse bodyweight over time.
  • FIG. 45 depicts polypeptide complexes used in Example 5.
  • FIG. 46 illustrates TRACT plasma concentrations (nM) in cynomolgus monkey.
  • FIGS. 47A-47F illustrate pro-inflammatory cytokine release in cynomolgus monkey plasma after dosing polypeptide complex molecules.
  • FIG. 47A illustrates plasma IFNy concentration (pg/mL).
  • FIG. 47B illustrates plasma TNFa concentration (pg/mL).
  • FIG. 47C illustrates plasma IL-6 concentration (pg/mL).
  • FIG. 47D illustrates plasma IL-5 concentration (pg/mL).
  • FIG. 47E illustrates plasma IL-4 concentration (pg/mL).
  • FIG. 47F illustrates plasma IL-2 concentration (pg/mLO.
  • FIGS. 48A-48C illustrates PBMC populations from cynomolgus monkeys after polypeptide complex dosing.
  • FIG. 48A illustrates percent of PBMCs which were CD3+.
  • FIG. 48B illustrates % of CD3+ cells which were CD69+.
  • FIG. 48C illustrates % of CD3+ cells which were Ki-67+.
  • FIGS. 49A-49D illustrate clinical chemistry and hematology after polypeptide complex dosing in cynomolgus monkeys.
  • FIG. 49A illustrates lymphocyte (LYM) concentration over time after polypeptide complex dosing.
  • FIG. 49B illustrates aspartate aminotransferase (AST) concentration overtime after polypeptide complex dosing.
  • FIG. 49C illustrates albumin (ALB) concentration over time after polypeptide complex dosing.
  • FIG. 49D illustrates alamine aminotransferase (ALT) concentration over time after polypeptide complex dosing.
  • FIGS. 50A-50P illustrate exemplary schemas for polypeptide complexes described herein.
  • “Fragment” as used herein refers to a peptide or a polypeptide that comprises less than the full length amino acid sequence.
  • Antigen-binding site refers to the region of a polypeptide that interacts with an antigen.
  • the antigen binding site includes amino acid residues that interact directly with an antigen and those amino acid residues that are within proximity to the antigen but that may not interact directly with the antigen.
  • polypeptides or polypeptide complexes comprising a half-life extending molecule.
  • the polypeptides or polypeptide complexes comprise an antibody or an antibody fragment.
  • the polypeptides or polypeptide complexes bind to a tumor cell antigen.
  • the polypeptides or polypeptide complexes bind to an effector cell antigen.
  • the polypeptide or polypeptide complexes described herein have an optimal molecular weight for enhanced tissue penetration and distribution.
  • the polypeptide or polypeptide complexes have a molecular weight of about 80 kDa. In some embodiments, the polypeptide or polypeptide complexes have a molecular weight of about 90 kDa. In some embodiments, the polypeptide or polypeptide complexes have a molecular weight of about 100 kDa. In some embodiments, the polypeptide or polypeptide complexes have a molecular weight of about 110 kDa. In some embodiments, the polypeptide or polypeptide complexes have a molecular weight of about 120 kDa. In some embodiments, the polypeptide or polypeptide complexes have a molecular weight of about 130 kDa. In some embodiments,
  • the polypeptide or polypeptide complexes have a molecular weight of less than about 80 kDa. In some embodiments, the polypeptide or polypeptide complexes have a molecular weight of less than about 90 kDa. In some embodiments, the polypeptide or polypeptide complexes have a molecular weight of less than about 100 kDa. In some embodiments, the polypeptide or polypeptide complexes have a molecular weight of less than about 110 kDa. In some embodiments, the polypeptide or polypeptide complexes have a molecular weight of less than about 120 kDa. In some embodiments, the polypeptide or polypeptide complexes have a molecular weight of less than about 130 kDa.
  • polypeptides or polypeptide complexes according to Formula I are polypeptides or polypeptide complexes according to Formula I:
  • Ai comprises a first antigen recognizing molecule that binds to a first target antigen
  • Pi comprises a peptide that binds to Ai
  • Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • A2 comprises a second antigen recognizing molecule that binds to a second target antigen.
  • polypeptides or polypeptide complexes according to Formula I are polypeptides or polypeptide complexes according to Formula I:
  • Ai is a first antigen recognizing molecule that binds to a first target antigen
  • Pi is a peptide that binds to Ai
  • Li is a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi is a half-life extending molecule
  • A is a second antigen recognizing molecule that binds to a second target antigen.
  • polypeptides or polypeptide complexes comprising Formula I:
  • Ai comprises a first antigen recognizing molecule that binds to a first target antigen
  • Pi comprises a peptide that binds to Ai
  • Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • A comprises a second antigen recognizing molecule that binds to a second target antigen.
  • polypeptides or polypeptide complexes comprising Formula F
  • Ai is a first antigen recognizing molecule that binds to a first target antigen
  • Pi is a peptide that binds to Ai
  • Li is a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi is a half-life extending molecule
  • A is a second antigen recognizing molecule that binds to a second target antigen.
  • the first target antigen comprises a tumor cell antigen and the second target antigen comprises an effector cell antigen.
  • the first target antigen comprises an effector cell antigen and the second target antigen comprises a tumor cell antigen.
  • the polypeptide or polypeptide complex of formula I binds to a target cell when Li is cleaved by the tumor specific protease. In some embodiments, the polypeptide of formula I binds to an effector cell when Li is cleaved by the tumor specific protease.
  • Ai comprises a first antigen recognizing molecule that binds to a first target antigen
  • Pi comprises a peptide that binds to Ai
  • Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • A comprises a second antigen recognizing molecule that binds to a second target antigen
  • P comprises a peptide that binds to A
  • L comprises a linking moiety that connects A to P and is a substrate for a tumor specific protease.
  • Ai is a first antigen recognizing molecule that binds to a first target antigen
  • Pi is a peptide that binds to Ai
  • Li is a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi is a half-life extending molecule
  • A2 is a second antigen recognizing molecule that binds to a second target antigen
  • P2 is a peptide that binds to A2
  • L2 is a linking moiety that connects A2to P2 and is a substrate for a tumor specific protease.
  • polypeptides or polypeptide complexes comprising Formula la:
  • Ai comprises a first antigen recognizing molecule that binds to a first target antigen
  • Pi comprises a peptide that binds to Ai
  • Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • A2 comprises a second antigen recognizing molecule that binds to a second target antigen
  • P2 comprises a peptide that binds to A2
  • L2 comprises a linking moiety that connects A2to P2 and is a substrate for a tumor specific protease.
  • polypeptides or polypeptide complexes comprising Formula la:
  • Ai is a first antigen recognizing molecule that binds to a first target antigen
  • Pi is a peptide that binds to Ai
  • Li is a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi is a half-life extending molecule
  • A2 is a second antigen recognizing molecule that binds to a second target antigen
  • P2 is a peptide that binds to A2
  • L2 is a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease.
  • the polypeptide or polypeptide complex comprises a modified amino acid, a non-natural amino acid, a modified non-natural amino acid, or a combination thereof.
  • the modified amino acid or modified non-natural amino acid comprises a post-translational modification.
  • polypeptides or polypeptide complexes according to Formula II are polypeptides or polypeptide complexes according to Formula II:
  • Li a comprises a tumor specific protease-cleaved linking moiety that when uncleaved connects Pi a to an antigen recognizing molecule that binds to a target antigen and; Pi a comprises a peptide that binds to the antigen recognizing molecule when Li a is uncleaved; and Hi a comprises a half-life extending molecule.
  • polypeptides or polypeptide complexes according to Formula II are polypeptides or polypeptide complexes according to Formula II:
  • Li a is a tumor specific protease-cleaved linking moiety that when uncleaved connects Pi a to an antigen recognizing molecule that binds to a target antigen and; Pi a is a peptide that binds to the antigen recognizing molecule when Li a is uncleaved; and Hi a is a half-life extending molecule.
  • polypeptides or polypeptide complexes comprising Formula P:
  • Li a comprises a tumor specific protease-cleaved linking moiety that when uncleaved connects Pi a to an antigen recognizing molecule that binds to a target antigen and; Pi a comprises a peptide that binds to the antigen recognizing molecule when Li a is uncleaved; and Hi a comprises a half-life extending molecule.
  • Li a is a tumor specific protease-cleaved linking moiety that when uncleaved connects Pi a to an antigen recognizing molecule that binds to a target antigen and; Pi a is a peptide that binds to the antigen recognizing molecule when Li a is uncleaved; and Hi a is a half-life extending molecule.
  • the antigen recognizing molecule comprises an antibody or antibody fragment.
  • the target antigen is an anti-CD3 effector cell antigen.
  • the first target antigen comprises an effector cell antigen and the second target antigen comprises a tumor cell antigen.
  • the effector cell antigen comprises CD3.
  • the tumor cell antigen comprises EGFR, HER2, mesothelin, or CEACAM5.
  • Ai comprises an antibody or antibody fragment. In some embodiments, Ai comprises an antibody or antibody fragment that is human or humanized. In some embodiments, Li is bound to N-terminus of the antibody or antibody fragment. In some embodiments, Li is bound to N-terminus of the antibody or antibody fragment and A is bound to the other N-terminus of the antibody or antibody fragment. In some embodiments, A is bound to C-terminus of the antibody or antibody fragment. In some embodiments, Li is bound to C-terminus of the antibody or antibody fragment. In some embodiments, A is bound to N-terminus of the antibody or antibody fragment. In some embodiments, the antibody or antibody fragment comprises a single chain variable fragment, a single domain antibody, or a Fab fragment.
  • Ai is the single chain variable fragment (scFv).
  • the scFv comprises a scFv heavy chain polypeptide and a scFv light chain polypeptide.
  • Ai is the single domain antibody.
  • Ai is a Fab fragment.
  • Ai comprises an anti-CD3e single chain variable fragment.
  • Ai comprises an anti-CD3e single chain variable fragment that has a K D binding of 1 mM or less to CD3 on CD3 expressing cells.
  • Ai comprises a variable light chain and variable heavy chain each of which is capable of specifically binding to human CD3.
  • Ai comprises complementary determining regions (CDRs) selected from the group consisting of muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, FI 11-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865vl2, 15865vl6, and 15865vl9.
  • CDRs complementary determining regions
  • the polypeptide or polypeptide complex of formula I binds to an effector cell when Li is cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex of formula I binds to an effector cell when Li is cleaved by the tumor specific protease and Aibinds to the effector cell. In some embodiments, the effector cell is a T cell. In some embodiments, Ai binds to a polypeptide that is part of a TCR-CD3 complex on the effector cell. In some embodiments, the polypeptide that is part of the TCR-CD3 complex is human CD3s. In some embodiments, the effector cell antigen comprises CD3, and the scFv comprises an amino acid sequence according to SEQ ID NOs: 64, 65, or 66.
  • the first target antigen comprises a tumor cell antigen and the second target antigen comprises an effector cell antigen.
  • the tumor cell antigen comprises EGFR, HER2, mesothelin, or CEACAM5.
  • the effector cell antigen comprises CD3.
  • Ai comprises an antibody or antibody fragment. In some embodiments, Ai comprises an antibody or antibody fragment that is human or humanized. In some embodiments, Li is bound to N-terminus of the antibody or antibody fragment. In some embodiments, A is bound to C-terminus of the antibody or antibody fragment. In some embodiments, Li is bound to C-terminus of the antibody or antibody fragment. In some embodiments, A is bound to N-terminus of the antibody or antibody fragment. In some embodiments, the antibody or antibody fragment thereof comprises a single chain variable fragment, a single domain antibody, or a Fab.
  • the antibody or antibody fragment thereof comprises a single chain variable fragment (scFv), a heavy chain variable domain (VH domain), a light chain variable domain (VL domain), a variable domain (VHH) of a camelid derived single domain antibody.
  • the antibody or antibody fragment thereof is humanized or human.
  • Ai is the Fab.
  • the Fab comprises (a) a Fab light chain polypeptide and (b) a Fab heavy chain polypeptide wherein the Fab light chain polypeptide of Ai is bound to a C-terminus of the single chain variable fragment (scFv) of A .
  • the Fab heavy chain polypeptide of Ai is bound to a C-terminus of the single chain variable fragment (scFv) A .
  • the Fab light chain polypeptide of Ai is bound to a N-terminus of the single chain variable fragment (scFv) of A .
  • the Fab heavy chain polypeptide of Ai is bound to a N- terminus of the single chain variable fragment (scFv) A . In some embodiments, the Fab heavy chain polypeptide of Ai is bound to the scFv heavy chain polypeptide of A and Li is bound to the Fab light chain polypeptide of Ai. In some embodiments, the Fab light chain polypeptide of Ai is bound to the scFv heavy chain polypeptide of A2 and Li is bound to the Fab heavy chain polypeptide of Ai. In some embodiments, the Fab heavy chain polypeptide of Ai is bound to the scFv light chain polypeptide of A2 and Li is bound to the Fab light chain polypeptide of Ai.
  • the Fab light chain polypeptide of Ai is bound to the scFv light chain polypeptide of A2 and Li is bound to the Fab heavy chain polypeptide of Ai.
  • A2 further comprises P2 and L2, wherein P2 comprises a peptide that binds to A2; and L2 comprises a linking moiety that connects A2to P2 and is a substrate for a tumor specific protease.
  • the Fab heavy chain polypeptide of Ai is bound to the scFv heavy chain polypeptide of A2 and Li is bound to the Fab light chain polypeptide of Ai and L2 is bound to the scFv light chain polypeptide of A2.
  • the Fab heavy chain polypeptide of Ai is bound to the scFv heavy chain polypeptide of A2 and Li is bound to the Fab light chain polypeptide of Ai and L2 is bound to the scFv light chain polypeptide of A2, and the polypeptide complex comprises amino acid sequence according to SEQ ID NO: 72 and SEQ ID NO: 71.
  • the Fab light chain polypeptide of Ai is bound to the scFv heavy chain polypeptide of A2 and Li is bound to the Fab heavy chain polypeptide of Ai and L2 is bound to the scFv light chain polypeptide of A2.
  • the Fab heavy chain polypeptide of Ai is bound to the scFv light chain polypeptide of A2 and Li is bound to the Fab light chain polypeptide of Ai and L2 is bound to the scFv heavy chain polypeptide of A2.
  • the Fab light chain polypeptide of Ai is bound to the scFv light chain polypeptide of A2 and Li is bound to the Fab heavy chain polypeptide of Ai and L2 is bound to the scFv heavy chain polypeptide of A2.
  • the antibody or antibody fragment thereof comprises an epidermal growth factor receptor (EGFR) binding domain. In some embodiments, the antibody or antibody fragment thereof comprises a mesothelin binding domain. In some embodiments, the antibody or antibody fragment thereof comprises a carcinoembryonic antigen-related cell adhesion molecule CEACAM5 binding domain. In some embodiments, the antibody or antibody fragment thereof comprises a HER2 binding domain. In some embodiments, the tumor cell antigen comprises EGFR, and the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NOs: 56 or 57.
  • the tumor cell antigen comprises EGFR, and the Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NOs: 59 or 60. In some embodiments, the tumor cell antigen comprises HER2, and the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 61. In some embodiments, the tumor cell antigen comprises HER2 and the Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NOs: 62 or 63.
  • the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen as compared to the binding affinity for the tumor cell antigen of a polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 5X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 8X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 10X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 15X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 20X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 25X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 3 OX higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 35X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 40X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 45X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 5 OX higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 55X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 60X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 65X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 70X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 75X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 80X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 85X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 90X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 95X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 100X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 120X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 1000X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen as compared to the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 5X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 8X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 10X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 15X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 20X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 25X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 3 OX higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 35X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 40X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 45X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 5 OX higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 55X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 60X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 65X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 70X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 75X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 80X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 85X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 90X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 95X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 100X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 120X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 1000X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has an increased EC 50 in an IENg release T-cell activation assay as compared to the EC 50 in an IFNy release T-cell activation assay of a polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in an IENg release T-cell activation assay that is at least 10X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • the polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 20X higher than the EC 50 in an IENg release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 30X higher than the EC 50 in an IENg release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • the polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 40X higher than the EC 50 in an IENg release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 50X higher than the EC 50 in an IENg release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • the polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 60X higher than the EC 50 in an IENg release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 70X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • the polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 80X higher than the EC 50 in an IENg release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 90X higher than the EC 50 in an IENg release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • the polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 100X higher than the EC 50 in an IENg release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 1000X higher than the EC 50 in an IENg release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • the polypeptide or polypeptide complex has an increased EC 50 in an IENg release T-cell activation assay as compared to the EC 50 in an IFNy release T-cell activation assay of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 10X higher than the EC 50 in an IFNy release T-cell activation assay of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 20X higher than the EC 50 in an IFNy release T-cell activation assay of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 30X higher than the EC 50 in an IFNy release T-cell activation assay of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 40X higher than the EC 50 in an IFNy release T-cell activation assay of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 50X higher than the EC 50 in an IFNy release T-cell activation assay of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 60X higher than the EC 50 in an IFNy release T-cell activation assay of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 70X higher than the EC 50 in an IFNy release T-cell activation assay of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 80X higher than the EC 50 in an IFNy release T-cell activation assay of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 90X higher than the EC 50 in an IFNy release T-cell activation assay of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 100X higher than the EC 50 in an IFNy release T-cell activation assay of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 1000X higher than the EC 50 in an IENg release T-cell activation assay of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay as compared to the EC 50 in a T-cell cytolysis assay of a polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 10X higher than the EC 50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 20X higher than the EC 50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 30X higher than the EC 50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 40X higher than the EC 50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 5 OX higher than the EC 50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 60X higher than the EC 50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 70X higher than the EC 50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 80X higher than the EC 50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 90X higher than the EC 50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 100X higher than the EC 50 in a T-cell cytolysis assay of a polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 1000X higher than the EC 50 in a T-cell cytolysis assay of a polypeptide or polypeptide complex that does not have Pi or Li.
  • the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay as compared to the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 10X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 20X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 30X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 40X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 50X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 60X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 70X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 80X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 90X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 100X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 1,000X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease.
  • the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 has weaker binding affinity for the tumor cell antigen as compared to the binding affinity for the tumor cell antigen of a polypeptide or polypeptide complex of formula la that does not have Pi, Li, P2, or L2.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 10X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 5 OX higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 75X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 100X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 - A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 120X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 200X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 300X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 400X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 500X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 600X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 - L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 700X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 800X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 900X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 1000X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least IO,OOOC higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen as compared to the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 - L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 10X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 5 OX higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 75X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 - A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 100X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 120X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 200X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 300X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 400X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 - A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 500X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 600X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 700X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 800X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 900X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 - A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 1000X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least IO,OOOC higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay as compared to the EC 50 in an IENg release T-cell activation assay of a polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least 10X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 - L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least 50X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IENg release T-cell activation assay that is at least 75X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IENg release T-cell activation assay that is at least 100X higher than the EC 50 in an IENg release T-cell activation assay of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least 200X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IENg release T-cell activation assay that is at least 300X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least 400X higher than the EC 50 in an IENg release T-cell activation assay of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least 500X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 - L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least 600X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IENg release T-cell activation assay that is at least 700X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the polypeptide or polypeptide complex of formula la that does not Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IENg release T-cell activation assay that is at least 800X higher than the EC 50 in an IENg release T-cell activation assay of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least 900X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IENg release T-cell activation assay that is at least 1000X higher than the EC 50 in an IENg release T-cell activation assay of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least IO,OOOC higher than the EC 50 in an IFNy release T-cell activation assay of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P2, or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay as compared to the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 10X higher than the EC 50 in a T- cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 - A 2 -A 1 -L 1 -P 1 -H 1 (formula la) has an increased EC 50 in a T-cell cytolysis assay that is at least 50X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 75X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 100X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 200X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 - Hi has an increased EC 50 in a T-cell cytolysis assay that is at least 300X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 400X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 500X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 600X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 - Hi has an increased EC 50 in a T-cell cytolysis assay that is at least 700X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 800X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 900X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 1000X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 - Hi has an increased EC 50 in a T-cell cytolysis assay that is at least IO,OOOC higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay as compared to the EC 50 in a T-cell cytolysis assay of a polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 10X higher than the EC 50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 5 OX higher than the EC 50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 75X higher than the EC 50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 75X higher than the EC 50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex of formula la that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 100X higher than the EC 50 in a T-cell cytolysis assay of a polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 200X higher than the EC 50 in a T-cell cytolysis assay of a polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 300X higher than the EC 50 in a T-cell cytolysis assay of a polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 - A 1 -L 1 -P 1 -H 1 (formula la) has an increased EC 50 in a T-cell cytolysis assay that is at least 400X higher than the EC 50 in a T-cell cytolysis assay of a polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 500X higher than the EC 50 in a T-cell cytolysis assay of a polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 600X higher than the EC 50 in a T-cell cytolysis assay of a polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 700X higher than the EC 50 in a T-cell cytolysis assay of a polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 - A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 800X higher than the EC 50 in a T-cell cytolysis assay of a polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 900X higher than the EC 50 in a T-cell cytolysis assay of a polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 1000X higher than the EC 50 in a T-cell cytolysis assay of a polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least IO,OOOC higher than the EC 50 in a T-cell cytolysis assay of a polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay as compared to the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 10X higher than the EC 50 in a T- cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 - L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 50X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 75X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 100X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 200X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 - L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 300X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 400X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 500X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 600X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 - L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 700X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 800X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 900X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 1000X higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L have been cleaved by the tumor specific proteases.
  • the polypeptide or polypeptide complex P 2 - L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least IO,OOOC higher than the EC 50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex of formula la in which Li and L have been cleaved by the tumor specific proteases.
  • A2 comprises an antibody or antibody fragment.
  • the antibody or antibody fragment thereof comprises a single chain variable fragment, a single domain antibody, or a Fab.
  • the antibody or antibody fragment thereof comprises a single chain variable fragment (scFv), a heavy chain variable domain (VH domain), a light chain variable domain (VL domain), a variable domain (VHH) of a camelid derived single domain antibody.
  • the antibody or antibody fragment thereof is humanized or human.
  • A2 is the Fab.
  • the Fab comprises (a) a Fab light chain polypeptide and (b) a Fab heavy chain polypeptide.
  • the antibody or antibody fragment thereof comprises an epidermal growth factor receptor (EGFR) binding domain. In some embodiments, the antibody or antibody fragment thereof comprises a mesothelin binding domain. In some embodiments, the antibody or antibody fragment thereof comprises a carcinoembryonic antigen-related cell adhesion molecule CEACAM5 binding domain. In some embodiments, the antibody or antibody fragment thereof comprises a carcinoembryonic antigen-related cell adhesion molecule HER2 binding domain. In some embodiments, the tumor cell antigen comprises EGFR, and the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NOs: 56 or 57.
  • the tumor cell antigen comprises EGFR, and the Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NOs: 59 or 60. In some embodiments, the tumor cell antigen comprises HER2, and the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 61. In some embodiments, the tumor cell antigen comprises HER2 and the Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NOs: 62 or 63.
  • the Fab light chain polypeptide of A2 is bound to a C-terminus of the single chain variable fragment (scFv) of Ai. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to a C-terminus of the single chain variable fragment (scFv) Ai. In some embodiments, the Fab light chain polypeptide of A2 is bound to a N-terminus of the single chain variable fragment (scFv) of Ai. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to a N-terminus of the single chain variable fragment (scFv) Ai.
  • the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai, and the polypeptide complex comprises amino acid sequences of SEQ ID NO: 57 and SEQ ID NO: 76. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and the polypeptide complex comprises amino acid sequences of SEQ ID NO: 57 and SEQ ID NO: 78.
  • the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and the polypeptide complex comprises amino acid sequences of SEQ ID NO: 57 and SEQ ID NO: 73.
  • the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai.
  • the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of Ai.
  • the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of Ai . and the polypeptide complex comprises amino acid sequences of SEQ ID NO: 57 and SEQ ID NO: 74.
  • the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of Ai.
  • A2 further comprises P2 and L2, wherein P2 comprises a peptide that binds to A2; and L2 comprises a linking moiety that connects A2to P2 and is a substrate for a tumor specific protease.
  • the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and L2 is bound to the Fab light chain polypeptide of A2.
  • the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and L2 is bound to the Fab light chain polypeptide of A2 and the polypeptide complex comprises amino acid sequences of SEQ ID NO: 70 and SEQ ID NO: 73.
  • the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and L2 is bound to the Fab light chain polypeptide of A2 and the polypeptide complex comprises amino acid sequences of SEQ ID NO: 80 and SEQ ID NO: 81.
  • the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and L2 is bound to the Fab heavy chain polypeptide of A2. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of Ai and L2 is bound to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of Ai and L2 is bound to the Fab heavy chain polypeptide of A2.
  • A2 comprises an anti-CD3e single chain variable fragment. In some embodiments, A2 comprises an anti-CD3e single chain variable fragment that has a K D binding of 1 mM or less to CD3 on CD3 expressing cells. In some embodiments, A2 comprises a variable light chain and variable heavy chain each of which is capable of specifically binding to human CD3.
  • A2 comprises complementary determining regions (CDRs) selected from the group consisting of muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, FI 11-409, CLB-T3.4.2, TR-66, WT32, SPv- T3b, 11D8, XIII- 141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865vl2, 15865vl6, and 15865vl9.
  • CDRs complementary determining regions
  • the polypeptide or polypeptide complex of formula I binds to an effector cell.
  • the effector cell is a T cell.
  • A2 binds to a polypeptide that is part of a TCR-CD3 complex on the effector cell.
  • the polypeptide that is part of the TCR-CD3 complex is human CD3s.
  • the effector cell antigen comprises CD3, and the scFv comprises an amino acid sequence according to SEQ ID NOs: 66, 67, or 68.
  • Pi impairs binding of Ai to the first target antigen.
  • Pi is bound to Ai through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof.
  • Pi is bound to Ai at or near an antigen binding site.
  • Pi becomes unbound from Ai when Li is cleaved by the tumor specific protease thereby exposing Ai to the first target antigen.
  • Pi has less than 70% sequence identity to the first target antigen.
  • Pi has less than 75% sequence identity to the first target antigen.
  • Pi has less than 80% sequence identity to the first target antigen.
  • Pi has less than 85% sequence identity to the first target antigen. In some embodiments, Pi has less than 90% sequence identity to the first target antigen. In some embodiments, Pi has less than 95% sequence identity to the first target antigen. In some embodiments, Pi has less than 98% sequence identity to the first target antigen. In some embodiments, Pi has less than 99% sequence identity to the first target antigen. In some embodiments, Pi comprises a de novo amino acid sequence that shares less than 10% sequence identity to the first target antigen.
  • P2 impairs binding of A2to the second target antigen.
  • P2 is bound to A2 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof.
  • P2 is bound to A2 at or near an antigen binding site.
  • P2 becomes unbound from A2 when L2 is cleaved by the tumor specific protease thereby exposing A2 to the second target antigen.
  • P2 has less than 70% sequence identity to the second target antigen. In some embodiments, P2 has less than 75% sequence identity to the second target antigen.
  • P2 has less than 80% sequence identity to the second target antigen. In some embodiments, P2 has less than 85% sequence identity to the second target antigen. In some embodiments, P2 has less than 90% sequence identity to the second target antigen. In some embodiments, P2 has less than 95% sequence identity to the second target antigen. In some embodiments, P2 has less than 98% sequence identity to the second target antigen. In some embodiments, P2 has less than 99% sequence identity to the second target antigen. In some embodiments, P2 comprises a de novo amino acid sequence that shares less than 10% sequence identity to the second target antigen.
  • the antigen recognizing molecule comprises an antibody or antibody fragment.
  • the target antigen is an anti-CD3 effector cell antigen.
  • the target antigen is a tumor cell antigen.
  • the tumor cell antigen is EGFR, HER2, mesothelin, or CEACAM5.
  • Pi a has less than 70% sequence identity to the target antigen. In some embodiments, Pi a has less than 75% sequence identity to the target antigen. In some embodiments, Pi a has less than 80% sequence identity to the target antigen.
  • Pi a has less than 85% sequence identity to the target antigen. In some embodiments, Pi a has less than 90% sequence identity to the target antigen. In some embodiments, Pi a has less than 95% sequence identity to the target antigen. In some embodiments, Pi a has less than 98% sequence identity to the target antigen. In some embodiments, Pi a has less than 99% sequence identity to the target antigen. In some embodiments, Pi a comprises a de novo amino acid sequence that shares less than 10% sequence identity to the second target antigen.
  • Pi, P2, or Pi a comprises a peptide sequence of at least 5 amino acids in length. In some embodiments, Pi, P2, or Pi a comprises a peptide sequence of at least 6 amino acids in length. In some embodiments, Pi, P2, or Pi a comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, Pi, P2, or Pi a comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, Pi, P2, or Pi a comprises a peptide sequence of at least 16 amino acids in length. In some embodiments, Pi, P2, or Pi a comprises a peptide sequence of no more than 40 amino acids in length.
  • Pi, P2, or Pi a comprises at least two cysteine amino acid residues. In some embodiments, Pi, P2, or Pi a comprises a cyclic peptide or a linear peptide. In some embodiments, Pi, P2, or Pi a comprises a cyclic peptide. In some embodiments, Pi, P2, or Pi a comprises a linear peptide. In some embodiments, the tumor cell antigen comprises EGFR, and the Pi or P2 comprises Peptide-1, Peptide-2, Peptide-3, Peptide-4, Peptide-5, Peptide-6, or Peptide-7.
  • the tumor cell antigen comprises EGFR
  • the Pi or P2 comprises an amino acid sequence selected from the group consisting of GGDWCRSLMSYTDLCP (SEQ ID NO: 1), GGTSCADAHLIAPSCS (SEQ ID NO: 2), GGN CQWDRVEHTY ACS (SEQ ID NO: 3), GGWV SCHDGSHMTCFH (SEQ ID NO: 4),
  • GGMN CLNRLWVEY CLV (SEQ ID NO: 5), GGY CGQDNTWVREGCF (SEQ ID NO: 6) and
  • the tumor cell antigen comprises HER2, and the Pi or P2 comprises Peptide-8, Peptide-9, Peptide-10, Peptide-11, Peptide-12, Peptide-13, Peptide-14, Peptide-15, Peptide-16 or Peptide-17.
  • the tumor cell antigen comprises HER2, and the Pi or P2 comprises an amino acid sequence selected from the group consisting of GGPLCSDLDHITRLCD (SEQ ID NO: 8), GGIDCASLDHYTESCY (SEQ ID NO: 9),
  • GGNPV CTLGDPYEC SH SEQ ID NO: 10
  • GGTFCQLNADPYECQS SEQ ID NO: 11
  • GGGY CELIGDYVV CSP (SEQ ID NO: 12), GGLCDRW GWID APY CH (SEQ ID NO: 13),
  • GGTGCTEGHWHWGTCS (SEQ ID NO: 14)
  • GGNICMDY SWRSGCAV (SEQ ID NO: 15)
  • the effector cell antigen comprises CD3, and the Pi or P2 comprises Peptide-18, Peptide-19, Peptide-20, Peptide-21, Peptide-22, Peptide-23, Peptide-24, Peptide-25, Peptide-26, Peptide-27, Peptide-28, or Peptide-29.
  • the effector cell antigen comprises CD3, and the Pi or P2 comprises an amino acid sequence selected from the group consisting of QGQSGQGYLWGCEWNCGGITT (SEQ ID NO: 18), GGD S V CADPEVPICEI (SEQ ID NO: 19), GGMSDCGDPGVEICTH (SEQ ID NO: 20), GGIQCHDPDLPSPCYI (SEQ ID NO: 21), GGEW CLFDPD VPT CQD (SEQ ID NO: 22),
  • GGLGCNDIDPGEQCIV SEQ ID NO: 23
  • GGLECFDPEIPEAF Cl SEQ ID NO: 24
  • GGQGCGTIADPEPHCW (SEQ ID NO: 25), GGNCHDPDIPAYVLCS (SEQ ID NO: 26),
  • GGLCPINDWEPQDICW SEQ ID NO: 27
  • GGLCMIGDWLPGD V CL SEQ ID NO: 28
  • Pi, P2, or Pi a or Pi, P2, and Pi a comprise a modified amino acid or non-natural amino acid, or a modified non-natural amino acid, or a combination thereof.
  • the modified amino acid or a modified non-natural amino acid comprises a post-translational modification.
  • Pi, P2, or Pi a or Pi, P2, and Pi a comprise a modification including, but not limited to acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of
  • pyroglutamate formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Modifications are made anywhere to Pi, P2, or Pi a or Pi, P2, and Pi a including the peptide backbone, the amino acid side chains, and the terminus.
  • Pi, P2, or Pi a does not comprise albumin or an albumin fragment. In some embodiments, Pi, P2, or Pi a does not comprise an albumin binding domain.
  • Linking Moiety Li, L 2 , L 3 , and L 3a
  • Li, L 2 , L 3 , or L 3a is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, Li, L 2 , L 3 , or L 3a is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, Li, L 2 , L 3 , or L 3a is a peptide sequence having at least 10 amino acids. In some embodiments, Li, L 2 , L 3 , or L 3a is a peptide sequence having at least 18 amino acids. In some embodiments, Li, L 2 , L 3 , or L 3a is a peptide sequence having at least 26 amino acids.
  • Li, L 2 , L 3 , or L 3a has a formula comprising (G 2 S) n , wherein n is an integer from 1 to 3 (SEQ ID NO: 29). In some embodiments, Li, L 2 , L 3 , or L 3a has a formula comprising (G 2 S) n , wherein n is an integer of at least 1.
  • Li, L 2 , L 3 , or L 3a has a formula selected from the group consisting of (G 2 S) n , (GS) n , (GSGGS) worship (SEQ ID NO: 30), (GGGS) thread (SEQ ID NO: 31) , (GGGGS) context (SEQ ID NO: 32) , and (GSSGGS)fact (SEQ ID NO: 33) , wherein n is an integer of at least 1.
  • the tumor specific protease is selected from the group consisting of metalloprotease, serine protease, cysteine protease, threonine protease, and aspartic protease.
  • Li, L 2 , L 3 , or L 3a comprises a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, a legumain cleavable amino acid sequence, or a matrix metalloprotease cleavable amino acid sequence.
  • Li, L 2 , L 3 , or L3 a is Linker-1, Linker-2, Linker-3, Linker-4, Linker-5, Linker- 6, Linker-7, Linker-8, Linker-9, Linker-10, Linker-11, Linker-12, Linker-13, Linker-14, Linker-15, Linker- 16, Linker-17, Linker-18, Linker-19, or Linker 20.
  • LAGRSDNH (SEQ ID NO: 37), ISSGLASGRSDNH (SEQ ID NO: 38), ISSGLLAGRSDNH (SEQ ID NO: 39), LSGRSDNH (SEQ ID NO: 40), ISSGLLSGRSDNP (SEQ ID NO: 41), IS SGLLSGRSDNH (SEQ ID NO: 42), LSGRSDNHSPLGLAGS (SEQ ID NO: 43), SPLGLAGSLSGRSDNH (SEQ ID NO: 44), SPLGLSGRSDNH (SEQ ID NO: 45), LAGRSDNHSPLGLAGS (SEQ ID NO: 46),
  • LSGRSDNHVPLSLKMG (SEQ ID NO: 47), LSGRSDNHVPLSLSMG (SEQ ID NO: 48),
  • Li or L2 comprises an amino acid sequence ASGRSDNH (SEQ ID NO: 36), LAGRSDNH (SEQ ID NO: 37),
  • L3 or L 3a comprises an amino acid sequence GGGGSGGGS (SEQ ID NO: 51).
  • Li is bound to N-terminus of Ai.
  • Li is bound to C- tenninus of Ai.
  • L2 is bound to N-terminus of A2.
  • L2 is bound to C-terminus of A2.
  • Pi becomes unbound from Ai when Li is cleaved by the tumor specific protease thereby exposing Ai to the first target antigen.
  • P2 becomes unbound from A2 when L2 is cleaved by the tumor specific protease thereby exposing A2 to the second target antigen.
  • Li, L 2 , L 3 , or L3 or Li, L 2 , L 3 , and L 3a comprise a modified amino acid or non-natural amino acid, or a modified non-natural amino acid, or a combination thereof.
  • the modified amino acid or a modified non-natural amino acid comprises a post-translational modification.
  • Li, L 2 , L 3 , or L3 a or Li, L 2 , L 3 , and L 3a comprise a modification including, but not limited, to acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer
  • Hi does not block Ai binding to the first target antigen.
  • Hi comprises a linking moiety (L 3 ) that connects Hi to Pi. In some embodiments, Hi a does not block antigen recognizing molecule binding to the target antigen. In some embodiments, Hi a comprises a linking moiety (L 3 ) that connects Hi a to Pi a . In some embodiments, half-life extending molecule (Hi or Hi a ) does not have binding affinity to antigen recognizing molecule. In some embodiments, half-life extending molecule (Hi or Hi a ) does not have binding affinity to the target antigen. In some embodiments, half-life extending molecule (Hi or Hi a ) does not shield antigen recognizing molecule from the target antigen. In some embodiments, half-life extending molecule (Hi or Hi a ) is not directly linked to antigen recognizing molecule.
  • Hi or Hi a comprise an amino acid sequence that has repetitive sequence motifs. In some embodiments, Hi or Hi a comprises an amino acid sequence that has highly ordered secondary structure. “Highly ordered secondary structure,” as used in this context, means that at least about 50%, or about 70%, or about 80%, or about 90%, of amino acid residues of Hi or Hi a contribute to secondary structure, as measured or determined by means, including, but not limited to, spectrophotometry (e.g. by circular dichroism spectroscopy in the“far-UV” spectral region (190-250 nm), and computer programs or algorithms, such as the Chou-Fasman algorithm and the Gamier-Osguthorpe-Robson (“GOR”) algorithm.
  • spectrophotometry e.g. by circular dichroism spectroscopy in the“far-UV” spectral region (190-250 nm
  • computer programs or algorithms such as the Chou-Fasman algorithm and the Gamier-Osguthorpe-Robson (“GOR”) algorithm.
  • Hi or Hi a comprises a polymer.
  • the polymer is polyethylene glycol (PEG).
  • Hi or Hi a comprises albumin.
  • Hi or Hi a comprises an Fc domain.
  • the albumin is serum albumin.
  • the albumin is human serum albumin.
  • Hi or Hi a comprises a polypeptide, a ligand, or a small molecule.
  • the polypeptide, the ligand or the small molecule binds serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35/CR1.
  • the serum protein comprises a thyroxine-binding protein, a transthyretin, a 1-acid
  • the circulating immunoglobulin molecule comprises IgGl, IgG2, IgG3, IgG4, slgA, IgM or IgD.
  • the serum protein is albumin.
  • the polypeptide is an antibody.
  • the antibody comprises a single domain antibody, a single chain variable fragment or a Fab.
  • the single domain antibody comprises a single domain antibody that binds to albumin.
  • the antibody is a human or humanized antibody. In some embodiments, the antibody is selected from the group consisting of 645gHlgFl,
  • the single domain antibody is 10G, and the single domain antibody comprises an amino acid sequence
  • Hi or Hi a or Hi and Hi a comprise a modified amino acid or non-natural amino acid, or a modified non-natural amino acid, or a combination thereof.
  • the modified amino acid or a modified non-natural amino acid comprises a post-translational modification.
  • Hi or Hi a or Hi and Hi a comprise a modification including, but not limited to acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubi
  • Hi comprises a linking moiety (L3) that connects Hi to Pi .
  • L3 is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L3 is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L3 is a peptide sequence having at least 10 amino acids. In some embodiments, L3 is a peptide sequence having at least 18 amino acids. In some embodiments, L3 is a peptide sequence having at least 26 amino acids.
  • L3 has a formula selected from the group consisting of (G2S) n , (GS) n , (GSGGS) context (SEQ ID NO: 30), (GGGS) thread (SEQ ID NO: 31) , (GGGGS) thread (SEQ ID NO: 32) , and (GSSGGS)fact (SEQ ID NO: 33) , wherein n is an integer of at least 1.
  • L3 comprises an amino acid sequence GGGGSGGGS (SEQ ID NO: 51).
  • Hi a comprises a linking moiety (L3 a ) that connects Hi a to Pi a.
  • L3 a is a peptide sequence having at least 5 to no more than 50 amino acids.
  • L3 a is a peptide sequence having at least 10 to no more than 30 amino acids.
  • L3 a is a peptide sequence having at least 10 amino acids.
  • L3 a is a peptide sequence having at least 18 amino acids.
  • L3 a is a peptide sequence having at least 26 amino acids.
  • L3 a has a formula selected from the group consisting of (G2S) n , (GS) context, (GSGGS) context (SEQ ID NO: 30), (GGGS) thread (SEQ ID NO: 31) , (GGGGS) repeat (SEQ ID NO: 32) , and (GSSGGS) n (SEQ ID NO: 33) , wherein n is an integer of at least 1.
  • L3 comprises an amino acid sequence GGGGSGGGS (SEQ ID NO: 51).
  • polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 1 :
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the light chain variable domain of the scFv with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab heavy chain polypeptide is linked to a C terminus of the heavy chain variable domain of the scFv, and wherein the Fab is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding of the Fab to the tumor
  • polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 2:
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide that impairs binding of the scFv to an effector cell antigen and the peptide is linked to the light chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide chain and a Fab heavy chain polypeptide chain, and wherein the Fab heavy chain polypeptide chain is linked to a C terminus of the heavy chain variable domain of the scFv.
  • scFv single chain variable fragment
  • polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 3:
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the light chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab light chain polypeptide is linked to a C terminus of the heavy chain variable domain of the scFv, and wherein the Fab is linked to P 2 and L 2 , wherein P 2 comprises a peptide that impairs binding to the tumor cell antigen; and L
  • polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 4:
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is further linked to a peptide that impairs binding of the scFv to an effector cell antigen and the peptide is linked to a N-terminus of the light chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab light chain polypeptide is linked to a C terminus of the heavy chain variable domain of the scFv.
  • scFv single chain variable fragment
  • polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 5:
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab heavy chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding to the tumor cell antigen;
  • polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 6:
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide that impairs binding of the scFv to an effector cell antigen and the peptide is linked to the heavy chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide chain and a Fab heavy chain polypeptide chain, and wherein the Fab heavy chain polypeptide chain is linked to a C terminus of the light chain variable domain of the scFv.
  • scFv single chain variable fragment
  • polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 7:
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab light chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding to the tumor cell antigen;
  • polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 8:
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide that impairs binding of the scFv to an effector cell antigen and the peptide is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab light chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv.
  • scFv single chain variable fragment
  • polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 9:
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide (Pi) that impairs binding of the Fab to the tumor cell antigen and Pi is linked to a N terminus of the Fab light chain polypeptide with a linking moiety (Li) that is a substrate for a tumor specific protease, and the Pi is further linked to a half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain of the scFv is linked to an N terminus of the Fab heavy chain polypeptide, wherein the scFv is linked to P and L , wherein P comprises a peptide that impairs binding of
  • polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 10:
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide that impairs binding of the Fab to the tumor cell antigen and the peptide is linked to a N terminus of the Fab light chain polypeptide with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain of the scFv is linked to an N terminus of the Fab heavy chain polypeptide.
  • scFv single chain variable fragment
  • polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 11 :
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide (Pi) that impairs binding of the Fab to the tumor cell antigen and Pi is linked to a N terminus of the Fab heavy chain polypeptide with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi
  • scFv single chain variable fragment
  • the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain of the scFv is linked to an N terminus of the Fab light chain polypeptide, wherein the scFv further is linked to P and L , wherein P comprises a peptide that impairs binding of the scFv to the effector cell antigen, and L comprises a linking moiety that connects the light chain variable domain of the scFv to P and is a substrate for a tumor specific protease.
  • P comprises a peptide that impairs binding of the scFv to the effector cell antigen
  • L comprises a linking moiety that connects the light chain variable domain of the scFv to P and is a substrate for a tumor specific protease.
  • polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 12:
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide that impairs binding of the Fab to the tumor cell antigen and the peptide is linked to a N terminus of the Fab heavy chain polypeptide with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain of the scFv is linked to an N terminus of the Fab light chain polypeptide.
  • scFv single chain variable fragment
  • polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 13:
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide (Pi) that impairs binding of the Fab to the tumor cell antigen and Pi is linked to a N terminus of the Fab light chain polypeptide with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi
  • scFv single chain variable fragment
  • the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain of the scFv is linked to an N terminus of the Fab heavy chain polypeptide, wherein the scFv is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding of the scFv to the effector cell antigen, and L2 comprises a linking moiety that connects the heavy chain variable domain of the scFv to P2 and is a substrate for a tumor specific protease.
  • scFv single chain variable fragment
  • polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 14:
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide that impairs binding of the Fab to the tumor cell antigen and the peptide is linked to a N terminus of the Fab light chain polypeptide with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain of the scFv is linked to an N terminus of the Fab heavy chain polypeptide.
  • scFv single chain variable fragment
  • polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 15:
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a (Pi) that impairs binding of the Fab to the tumor cell antigen and Pi is linked to a N terminus of the Fab heavy chain polypeptide with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain of the scFv is linked to an N terminus of the Fab light chain polypeptide, wherein the scFv is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding of the s
  • polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 16:
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide that impairs binding of the Fab to the tumor cell antigen and the peptide is linked to a N terminus of the Fab heavy chain polypeptide with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a single chain variable fragment
  • scFv that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain of the scFv is linked to an N terminus of the Fab light chain polypeptide.
  • polypeptides or polypeptide complexes as disclosed herein.
  • the polypeptides or polypeptide complexes comprise an antibody or an antibody fragment.
  • the polypeptides or polypeptide complexes comprise a Fab and a single chain variable fragment (scFv).
  • nucleic acid molecules encoding polypeptides or polypeptide complexes according to Formula I:
  • Ai comprises a first antigen recognizing molecule that binds to a first target antigen
  • Pi comprises a peptide that binds to Ai
  • Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • A2 comprises a second antigen recognizing molecule that binds to a second target antigen.
  • nucleic acid molecules encoding polypeptides or polypeptide complexes according to Formula I:
  • Ai is a first antigen recognizing molecule that binds to a first target antigen
  • Pi is a peptide that binds to Ai
  • Li is a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • A2 is a second antigen recognizing molecule that binds to a second target antigen.
  • Ai comprises a first antigen recognizing molecule that binds to a first target antigen
  • Pi comprises a peptide that binds to Ai
  • Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • A2 comprises a second antigen recognizing molecule that binds to a second target antigen.
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising Formula I:
  • Ai is a first antigen recognizing molecule that binds to a first target antigen
  • Pi is a peptide that binds to Ai
  • Li is a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi is s a half-life extending molecule
  • A2 is a second antigen recognizing molecule that binds to a second target antigen.
  • nucleic acid molecules encoding polypeptides or polypeptide complexes according to Formula la:
  • Ai comprises a first antigen recognizing molecule that binds to a first target antigen
  • Pi comprises a peptide that binds to Ai
  • Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • A2 comprises a second antigen recognizing molecule that binds to a second target antigen
  • P2 comprises a peptide that binds to A2
  • L2 comprises a linking moiety that connects A2to P2 and is a substrate for a tumor specific protease.
  • nucleic acid molecules encoding polypeptides or polypeptide complexes according to Formula la:
  • Ai is a first antigen recognizing molecule that binds to a first target antigen
  • Pi is a peptide that binds to Ai
  • Li is a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi is a half-life extending molecule
  • A2 is a second antigen recognizing molecule that binds to a second target antigen
  • P2 is a peptide that binds to A2
  • L2 is a linking moiety that connects A2to P2 and is a substrate for a tumor specific protease.
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising Formula la:
  • Ai comprises a first antigen recognizing molecule that binds to a first target antigen
  • Pi comprises a peptide that binds to Ai
  • Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • a 2 comprises a second antigen recognizing molecule that binds to a second target antigen
  • P 2 comprises a peptide that binds to A 2
  • L 2 comprises a linking moiety that connects A 2 to P 2 and is a substrate for a tumor specific protease.
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising Formula la:
  • Ai is a first antigen recognizing molecule that binds to a first target antigen
  • Pi is a peptide that binds to Ai
  • Li is a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi is a half-life extending molecule
  • a 2 is a second antigen recognizing molecule that binds to a second target antigen
  • P 2 is a peptide that binds to A 2
  • L 2 is a linking moiety that connects A 2 to P 2 and is a substrate for a tumor specific protease.
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 1 :
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the light chain variable domain of the scFv with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab heavy chain polypeptide is linked to a C terminus of the heavy chain variable domain of the scFv, and wherein the Fab is linked to P 2 and L 2 , wherein P 2 comprises a peptide that impairs binding of the Fab to
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 2:
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide that impairs binding of the scFv to an effector cell antigen and the peptide is linked to the light chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide chain and a Fab heavy chain polypeptide chain, and wherein the Fab heavy chain polypeptide chain is linked to a C terminus of the heavy chain variable domain of the scFv.
  • scFv single chain variable fragment
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 3 :
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the light chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab light chain polypeptide is linked to a C terminus of the heavy chain variable domain of the scFv, and wherein the Fab is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding to the tumor cell antigen; and L2 comprises
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 4:
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is further linked to a peptide that impairs binding of the scFv to an effector cell antigen and the peptide is linked to a N-terminus of the light chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab light chain polypeptide is linked to a C terminus of the heavy chain variable domain of the scFv.
  • scFv single chain variable fragment
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 5 :
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab heavy chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding to the tumor cell antigen;
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 6:
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide that impairs binding of the scFv to an effector cell antigen and the peptide is linked to the heavy chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide chain and a Fab heavy chain polypeptide chain, and wherein the Fab heavy chain polypeptide chain is linked to a C terminus of the light chain variable domain of the scFv.
  • scFv single chain variable fragment
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 7 :
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab light chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding to the tumor cell antigen;
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 8:
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide that impairs binding of the scFv to an effector cell antigen and the peptide is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab light chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv.
  • scFv single chain variable fragment
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 9:
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide (Pi) that impairs binding of the Fab to the tumor cell antigen and Pi is linked to a N terminus of the Fab light chain polypeptide with a linking moiety (Li) that is a substrate for a tumor specific protease, and the Pi is further linked to a half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain of the scFv is linked to an N terminus of the Fab heavy chain polypeptide, wherein the scFv is linked to P and L , wherein P comprises a peptide that impairs binding of
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 10:
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide that impairs binding of the Fab to the tumor cell antigen and the peptide is linked to a N terminus of the Fab light chain polypeptide with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain of the scFv is linked to an N terminus of the Fab heavy chain polypeptide.
  • scFv single chain variable fragment
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 11 :
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide (Pi) that impairs binding of the Fab to the tumor cell antigen and Pi is linked to a N terminus of the Fab heavy chain polypeptide with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi
  • scFv single chain variable fragment
  • the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain of the scFv is linked to an N terminus of the Fab light chain polypeptide, wherein the scFv further is linked to P and L , wherein P comprises a peptide that impairs binding of the scFv to the effector cell antigen, and L comprises a linking moiety that connects the light chain variable domain of the scFv to P and is a substrate for a tumor specific protease.
  • P comprises a peptide that impairs binding of the scFv to the effector cell antigen
  • L comprises a linking moiety that connects the light chain variable domain of the scFv to P and is a substrate for a tumor specific protease.
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 12:
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide that impairs binding of the Fab to the tumor cell antigen and the peptide is linked to a N terminus of the Fab heavy chain polypeptide with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain of the scFv is linked to an N terminus of the Fab light chain polypeptide.
  • scFv single chain variable fragment
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 13:
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide (Pi) that impairs binding of the Fab to the tumor cell antigen and Pi is linked to a N terminus of the Fab light chain polypeptide with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi
  • scFv single chain variable fragment
  • the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain of the scFv is linked to an N terminus of the Fab heavy chain polypeptide, wherein the scFv is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding of the scFv to the effector cell antigen, and L2 comprises a linking moiety that connects the heavy chain variable domain of the scFv to P2 and is a substrate for a tumor specific protease.
  • scFv single chain variable fragment
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 14:
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide that impairs binding of the Fab to the tumor cell antigen and the peptide is linked to a N terminus of the Fab light chain polypeptide with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain of the scFv is linked to an N terminus of the Fab heavy chain polypeptide.
  • scFv single chain variable fragment
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 15:
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a (Pi) that impairs binding of the Fab to the tumor cell antigen and Pi is linked to a N terminus of the Fab heavy chain polypeptide with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain of the scFv is linked to an N terminus of the Fab light chain polypeptide, wherein the scFv is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding of the s
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 16:
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide that impairs binding of the Fab to the tumor cell antigen and the peptide is linked to a N terminus of the Fab heavy chain polypeptide with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a single chain variable fragment
  • scFv that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain of the scFv is linked to an N terminus of the Fab light chain polypeptide.
  • compositions comprising: (a) the polypeptides or polypeptide complexes as disclosed herein; and (b) a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes according to Formula I:
  • Ai comprises a first antigen recognizing molecule that binds to a first target antigen
  • Pi comprises a peptide that binds to Ai
  • Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • A2 comprises a second antigen recognizing molecule that binds to a second target antigen
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes according to Formula I:
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising Formula I:
  • Ai comprises a first antigen recognizing molecule that binds to a first target antigen
  • Pi comprises a peptide that binds to Ai
  • Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • A2 comprises a second antigen recognizing molecule that binds to a second target antigen
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising Formula I:
  • Ai is a first antigen recognizing molecule that binds to a first target antigen
  • Pi is a peptide that binds to Ai
  • Li is a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi is a half-life extending molecule
  • A2 is a second antigen recognizing molecule that binds to a second target antigen
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes according to Formula la:
  • Ai comprises a first antigen recognizing molecule that binds to a first target antigen
  • Pi comprises a peptide that binds to Ai
  • Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • A2 comprises a second antigen recognizing molecule that binds to a second target antigen
  • P2 comprises a peptide that binds to A2
  • L2 comprises a linking moiety that connects A2to P2 and is a substrate for a tumor specific protease
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes according to Formula la:
  • Ai is a first antigen recognizing molecule that binds to a first target antigen
  • Pi is a peptide that binds to Ai
  • Li is a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi is a half-life extending molecule
  • A2 is a second antigen recognizing molecule that binds to a second target antigen
  • P2 is a peptide that binds to A2
  • L2 is a linking moiety that connects A2to P2 and is a substrate for a tumor specific protease
  • (b) a pharmaceutically acceptable excipient is a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising Formula la: P 2 -L 2 -A 2 -A 1 -L 1 -P l -Hi
  • Ai comprises a first antigen recognizing molecule that binds to a first target antigen
  • Pi comprises a peptide that binds to Ai
  • Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • a 2 comprises a second antigen recognizing molecule that binds to a second target antigen
  • P 2 comprises a peptide that binds to A 2
  • L 2 comprises a linking moiety that connects A 2 to P 2 and is a substrate for a tumor specific protease
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising Formula la:
  • Ai is a first antigen recognizing molecule that binds to a first target antigen
  • Pi is a peptide that binds to Ai
  • Li is a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi is a half-life extending molecule
  • a 2 is a second antigen recognizing molecule that binds to a second target antigen
  • P 2 is a peptide that binds to A 2
  • L 2 is a linking moiety that connects A 2 to P 2 and is a substrate for a tumor specific protease
  • (b) a pharmaceutically acceptable excipient is a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 1 :
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the light chain variable domain of the scFv with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab heavy chain polypeptide is linked to a C terminus of the heavy chain variable domain of the scFv, and wherein the Fab is linked to P 2 and L 2 , wherein P 2 comprises a peptide that impairs binding of the Fab to
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide that impairs binding of the scFv to an effector cell antigen and the peptide is linked to the light chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide chain and a Fab heavy chain polypeptide chain, and wherein the Fab heavy chain polypeptide chain is linked to a C terminus of the heavy chain variable domain of the scFv; and (b) a pharmaceutically acceptable excipient.
  • scFv single chain variable fragment
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 3 :
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the light chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab light chain polypeptide is linked to a C terminus of the heavy chain variable domain of the scFv, and wherein the Fab is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding to the tumor cell antigen; and L2 comprises
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is further linked to a peptide that impairs binding of the scFv to an effector cell antigen and the peptide is linked to a N-terminus of the light chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab light chain polypeptide is linked to a C terminus of the heavy chain variable domain of the scFv; and (b) a pharmaceutically acceptable excipient.
  • scFv single chain variable fragment
  • the scFv is further linked to a
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 5 :
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab heavy chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding to the tumor cell antigen;
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide that impairs binding of the scFv to an effector cell antigen and the peptide is linked to the heavy chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide chain and a Fab heavy chain polypeptide chain, and wherein the Fab heavy chain polypeptide chain is linked to a C terminus of the light chain variable domain of the scFv; and (b) a pharmaceutically acceptable excipient.
  • scFv single chain variable fragment
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 7 :
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab light chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding to the tumor cell antigen;
  • the polypeptide or polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide that impairs binding of the scFv to an effector cell antigen and the peptide is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab light chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv; and (b) a pharmaceutically acceptable excipient.
  • scFv single chain variable fragment
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 9:
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide (Pi) that impairs binding of the Fab to the tumor cell antigen and Pi is linked to a N terminus of the Fab light chain polypeptide with a linking moiety (Li) that is a substrate for a tumor specific protease, and the Pi is further linked to a half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain of the scFv is linked to an N terminus of the Fab heavy chain polypeptide, wherein the scFv is linked to P and L , wherein P comprises a peptide that impairs binding of
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 10:
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide that impairs binding of the Fab to the tumor cell antigen and the peptide is linked to a N terminus of the Fab light chain polypeptide with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain of the scFv is linked to an N terminus of the Fab heavy chain polypeptide; and (b) a pharmaceutically acceptable excipient.
  • scFv single chain variable fragment
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 11 :
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide (Pi) that impairs binding of the Fab to the tumor cell antigen and Pi is linked to a N terminus of the Fab heavy chain polypeptide with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain of the scFv is linked to an N terminus of the Fab light chain polypeptide, wherein the scFv further is linked to P and L , wherein P comprises a peptide that impairs binding of
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 12:
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide that impairs binding of the Fab to the tumor cell antigen and the peptide is linked to a N terminus of the Fab heavy chain polypeptide with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain of the scFv is linked to an N terminus of the Fab light chain polypeptide; and (b) a pharmaceutically acceptable excipient.
  • scFv single chain variable fragment
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 13:
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide (Pi) that impairs binding of the Fab to the tumor cell antigen and Pi is linked to a N terminus of the Fab light chain polypeptide with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain of the scFv is linked to an N terminus of the Fab heavy chain polypeptide, wherein the scFv is linked to P and L , wherein P comprises a peptide that impairs binding of the
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide that impairs binding of the Fab to the tumor cell antigen and the peptide is linked to a N terminus of the Fab light chain polypeptide with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain of the scFv is linked to an N terminus of the Fab heavy chain polypeptide; and (b) a pharmaceutically acceptable excipient.
  • scFv single chain variable fragment
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 15:
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a (Pi) that impairs binding of the Fab to the tumor cell antigen and Pi is linked to a N terminus of the Fab heavy chain polypeptide with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain of the scFv is linked to an N terminus of the Fab light chain polypeptide, wherein the scFv is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding of the s
  • the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide that impairs binding of the Fab to the tumor cell antigen and the peptide is linked to a N terminus of the Fab heavy chain polypeptide with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a single chain variable fragment
  • scFv that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain of the scFv is linked to an N terminus of the Fab light chain polypeptide; and (b) a pharmaceutically acceptable excipient.
  • the polypeptide or polypeptide complex further comprises a detectable label, a therapeutic agent, or a pharmacokinetic modifying moiety.
  • the detectable label comprises a fluorescent label, a radiolabel, an enzyme, a nucleic acid probe, or a contrast agent.
  • the polypeptide or polypeptide complex as disclosed herein may be provided in a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients.
  • pharmaceutically acceptable carrier includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the patient to whom it is administered.
  • suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
  • Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose.
  • the compositions are sterile.
  • compositions may also contain adjuvants such as preservative, emulsifying agents and dispersing agents.
  • the pharmaceutical composition may be in any suitable form, (depending upon the desired method of administration). It may be provided in unit dosage form, may be provided in a sealed container and may be provided as part of a kit. Such a kit may include instructions for use. It may include a plurality of said unit dosage forms.
  • compositions may be adapted for administration by any appropriate route, including a parenteral (e.g., subcutaneous, intramuscular, or intravenous) route.
  • parenteral e.g., subcutaneous, intramuscular, or intravenous
  • Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.
  • Dosages of the substances of the present disclosure can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used.
  • Table 1 provides the amino acid sequences of constructs described herein.
  • Polypeptides or polypeptide complexes comprise a sequence set forth in Table 1.
  • the sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
  • the sequence comprises at least or about 95% identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81.
  • the sequence comprises at least or about 97% identity to SEQ ID Nos: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
  • sequence comprises at least or about 99% identity to SEQ ID NOs:
  • the sequence comprises at least or about 100% identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81.
  • the sequence comprises at least a portion having at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, or more than 400 amino acids of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
  • sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • sequence identity typically includes comparing two nucleotide or amino acid sequences and the determining their percent identity. Sequence comparisons, such as for the purpose of assessing identities, may be performed by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needle aligner available at www.ebi.ac.uk/Tools/psa/emboss_needle/, optionally with default settings), the BLAST algorithm (see, e.g., the BLAST alignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi, optionally with default settings), and the Smith-Waterman algorithm (see, e.g., the EMBOSS Water aligner available at www.ebi.ac.uk/Tools/psa/emboss_water/, optionally with default settings).
  • the Needleman-Wunsch algorithm see, e.g., the EMBOSS Needle aligner available at
  • Optimal alignment may be assessed using any suitable parameters of a chosen algorithm, including default parameters.
  • the “percent identity”, also referred to as“percent homology”, between two sequences may be calculated as the number of exact matches between two optimally aligned sequences divided by the length of the reference sequence and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul, et ah, J. Mol. Biol.
  • the BLAST program defines identity as the number of identical aligned symbols (i.e., nucleotides or amino acids), divided by the total number of symbols in the shorter of the two sequences. The program may be used to determine percent identity over the entire length of the sequences being compared. Default parameters are provided to optimize searches with short query sequences, for example, with the blastp program.
  • the program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Lederhen, Computers and Chemistry 17: 149-163 (1993).
  • High sequence identity generally includes ranges of sequence identity of approximately 80% to 100% and integer values there between.
  • Embodiment 1 comprises a polypeptide or polypeptide complex according to Formula I:
  • Ai comprises a first antigen recognizing molecule that binds to a first target antigen
  • Pi comprises a peptide that binds to Ai
  • Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • A comprises a second antigen recognizing molecule that binds to a second target antigen.
  • Embodiment 2 comprises a polypeptide or polypeptide complex of embodiment 1, wherein the first target antigen comprises an effector cell antigen and the second target antigen comprises a tumor cell antigen.
  • Embodiment 3 comprises a polypeptide or polypeptide complex of any one of embodiments 1-2, wherein the effector cell antigen comprises CD3.
  • Embodiment 4 comprises a polypeptide or polypeptide complex of any one of embodiments 1-3, wherein the tumor cell antigen comprises EGFR, HER2, mesothelin, or CEACAM5.
  • Embodiment 5 comprises a polypeptide or polypeptide complex of any one of embodiments 1-4, wherein Ai comprises an antibody or antibody fragment.
  • Embodiment 6 comprises a polypeptide or polypeptide complex of any one of embodiments 1-5, wherein Ai comprises an antibody or antibody fragment that is human or humanized.
  • Embodiment 7 comprises a polypeptide or polypeptide complex of any one of embodiments 1-6, wherein Li is bound to N-terminus of the antibody or antibody fragment.
  • Embodiment 8 comprises a polypeptide or polypeptide complex of any one of embodiments 1-7, wherein A is bound to C-terminus of the antibody or antibody fragment.
  • Embodiment 9 comprises a polypeptide or polypeptide complex of any one of embodiments 1-8, wherein Li is bound to C-terminus of the antibody or antibody fragment.
  • Embodiment 10 comprises a polypeptide or polypeptide complex of any one of embodiments 1-9, wherein A is bound to N-terminus of the antibody or antibody fragment.
  • Embodiment 11 comprises a polypeptide or polypeptide complex of any one of embodiments 1-10, wherein the antibody or antibody fragment comprises a single chain variable fragment, a single domain antibody, or a Fab fragment.
  • Embodiment 12 comprises a polypeptide or polypeptide complex of any one of embodiments 1-11, wherein Ai is the single chain variable fragment (scFv).
  • Embodiment 13 comprises a polypeptide or polypeptide complex of any one of embodiments 1-12, wherein the scFv comprises a scFv heavy chain polypeptide and a scFv light chain polypeptide.
  • Embodiment 14 comprises a polypeptide or polypeptide complex of any one of embodiments 1-13, wherein Ai is the single domain antibody.
  • Embodiment 15 comprises a polypeptide or polypeptide complex of any one of embodiments 1-14, Ai is a single chain variable fragment (scFv), a heavy chain variable domain (VH domain), a light chain variable domain (VL domain), or a variable domain (VHH) of a camelid derived single domain antibody.
  • scFv single chain variable fragment
  • VH domain heavy chain variable domain
  • VL domain light chain variable domain
  • VHH variable domain
  • Embodiment 16 comprises a polypeptide or polypeptide complex of any one of embodiments 1-15, wherein Ai comprises an anti-CD3e single chain variable fragment.
  • Embodiment 17 comprises a polypeptide or polypeptide complex of any one of embodiments 1-16, wherein Ai comprises an anti-CD3e single chain variable fragment that has a K D binding of 1 mM or less to CD3 on CD3 expressing cells.
  • Embodiment 18 comprises a polypeptide or polypeptide complex of any one of embodiments 1-17, wherein Ai comprises a variable light chain and variable heavy chain each of which is capable of specifically binding to human CD3.
  • Embodiment 19 comprises a polypeptide or polypeptide complex of any one of embodiments 1-18, wherein Ai comprises complementary determining regions (CDRs) selected from the group consisting of muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, FI 11-409, CLB-T3.4.2, TR-66, WT32, SPv- T3b, 11D8, XIII- 141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865vl2, 15865vl6, and 15865vl9.
  • Embodiment 20 comprises a polypeptide or polypeptide complex of any one of embodiments 1-19, wherein the polypeptide or polypeptide complex of formula I binds to an effector cell when Li is cleaved by the tumor specific protease.
  • Embodiment 21 comprises a polypeptide or polypeptide complex of any one of embodiments 1-20, wherein the polypeptide or polypeptide complex of formula I binds to an effector cell when Li is cleaved by the tumor specific protease and Aibinds to the effector cell.
  • Embodiment 22 comprises a polypeptide or polypeptide complex of any one of embodiments 1-21, wherein the effector cell is a T cell.
  • Embodiment 23 comprises a polypeptide or polypeptide complex of any one of embodiments 1-22, wherein Ai binds to a polypeptide that is part of a TCR-CD3 complex on the effector cell.
  • Embodiment 24 comprises a polypeptide or polypeptide complex of any one of embodiments 1-23, wherein the polypeptide that is part of the TCR-CD3 complex is human CD3s.
  • Embodiment 25 comprises a polypeptide or polypeptide complex of any one of embodiments 1-24, wherein the effector cell antigen comprises CD3, and the scFv comprises an amino acid sequence according to SEQ ID NOs: 66, 67, or 68.
  • Embodiment 26 comprises a polypeptide or polypeptide complex of any one of embodiments 1-25, wherein A2 comprises an antibody or antibody fragment.
  • Embodiment 27 comprises a polypeptide or polypeptide complex of any one of embodiments 1-26, wherein the antibody or antibody fragment thereof comprises a single chain variable fragment, a single domain antibody, or a Fab.
  • Embodiment 28 comprises a polypeptide or polypeptide complex of any one of embodiments 1-27, wherein the antibody or antibody fragment thereof comprises a single chain variable fragment (scFv), a heavy chain variable domain (VH domain), a light chain variable domain (VL domain), a variable domain (VHH) of a camelid derived single domain antibody.
  • scFv single chain variable fragment
  • VH domain heavy chain variable domain
  • VL domain light chain variable domain
  • VHH variable domain
  • Embodiment 29 comprises a polypeptide or polypeptide complex of any one of embodiments 1-28, wherein the antibody or antibody fragment thereof is humanized or human.
  • Embodiment 30 comprises a polypeptide or polypeptide complex of any one of embodiments 1-29, wherein A is the Fab.
  • Embodiment 31 comprises a polypeptide or polypeptide complex of any one of embodiments 1-30, wherein the Fab comprises (a) a Fab light chain polypeptide and (b) a Fab heavy chain polypeptide.
  • Embodiment 32 comprises a polypeptide or polypeptide complex of any one of embodiments 1-31, wherein the antibody or antibody fragment thereof comprises an epidermal growth factor receptor (EGFR) binding domain.
  • EGFR epidermal growth factor receptor
  • Embodiment 33 comprises a polypeptide or polypeptide complex of any one of embodiments 1-32, wherein the antibody or antibody fragment thereof comprises a mesothelin binding domain.
  • Embodiment 34 comprises a polypeptide or polypeptide complex of any one of embodiments 1-33, wherein the antibody or antibody fragment thereof comprises a carcinoembryonic antigen-related cell adhesion molecule CEACAM5 binding domain.
  • Embodiment 35 comprises a polypeptide or polypeptide complex of any one of embodiments 1-34, wherein the tumor cell antigen comprises EGFR, and the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NOs: 56 or 57.
  • Embodiment 36 comprises a polypeptide or polypeptide complex of any one of embodiments 1-35, wherein the tumor cell antigen comprises EGFR, and the Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NOs: 59 or 60.
  • Embodiment 37 comprises a polypeptide or polypeptide complex of any one of embodiments 1-36, wherein the tumor cell antigen comprises HER2, and the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 61.
  • Embodiment 38 comprises a polypeptide or polypeptide complex of any one of embodiments 1-37, wherein the tumor cell antigen comprises HER2 and the Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NOs: 62 or 63.
  • Embodiment 39 comprises a polypeptide or polypeptide complex of any one of embodiments 1-38, wherein the Fab light chain polypeptide of A is bound to a C-terminus of the single chain variable fragment (scFv) of Ai.
  • scFv single chain variable fragment
  • Embodiment 40 comprises a polypeptide or polypeptide complex of any one of embodiments 1-39, wherein the Fab heavy chain polypeptide of A is bound to a C-terminus of the single chain variable fragment (scFv) Ai.
  • Embodiment 41 comprises a polypeptide or polypeptide complex of any one of embodiments 1-40, wherein the Fab light chain polypeptide of A2 is bound to a N-terminus of the single chain variable fragment (scFv) of Ai.
  • Embodiment 42 comprises a polypeptide or polypeptide complex of any one of embodiments 1-41, wherein the Fab heavy chain polypeptide of A2 is bound to a N-terminus of the single chain variable fragment (scFv) Ai.
  • scFv single chain variable fragment
  • Embodiment 43 comprises a polypeptide or polypeptide complex of any one of embodiments 1-42, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai.
  • Embodiment 44 comprises a polypeptide or polypeptide complex of any one of embodiments 1-43, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai. and the polypeptide complex comprises amino acid sequences of SEQ ID NO: 57 and SEQ ID NO: 76.
  • Embodiment 45 comprises a polypeptide or polypeptide complex of any one of embodiments 1-44, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai. and the polypeptide complex comprises amino acid sequences of SEQ ID NO: 57 and SEQ ID NO: 78.
  • Embodiment 46 comprises a polypeptide or polypeptide complex of any one of embodiments 1-45, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai. and the polypeptide complex comprises amino acid sequences of SEQ ID NO: 57 and SEQ ID NO: 73.
  • Embodiment 47 comprises a polypeptide or polypeptide complex of any one of embodiments 1-46, wherein the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai.
  • Embodiment 48 comprises a polypeptide or polypeptide complex of any one of embodiments 1-47, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of Ai.
  • Embodiment 49 comprises a polypeptide or polypeptide complex of any one of embodiments 1-48, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of A 1 . and the polypeptide complex comprises amino acid sequences of SEQ ID NO: 57 and SEQ ID NO: 74.
  • Embodiment 50 comprises a polypeptide or polypeptide complex of any one of embodiments 1-49, wherein the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of Ai.
  • Embodiment 51 comprises a polypeptide or polypeptide complex of any one of embodiments 1-50, wherein A2 further comprises P2 and L2, wherein P2 comprises a peptide that binds to A2; and L2 comprises a linking moiety that connects A2to P2 and is a substrate for a tumor specific protease.
  • Embodiment 52 comprises the polypeptide or polypeptide complex of any one of embodiments 1- 51, wherein the polypeptide or polypeptide complex is according to Formula la
  • Embodiment 53 comprises a polypeptide or polypeptide complex of any one of embodiments 1-52, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and L2 is bound to the Fab light chain polypeptide of A2.
  • Embodiment 54 comprises a polypeptide or polypeptide complex of any one of embodiments 1-53, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and F2 is bound to the Fab light chain polypeptide of A2 and the polypeptide complex comprises amino acid sequences of SEQ ID NO : 70 and SEQ ID NO : 73.
  • Embodiment 55 comprises a polypeptide or polypeptide complex of any one of embodiments 1-54, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and L2 is bound to the Fab light chain polypeptide of A2 and the polypeptide complex comprises amino acid sequences of SEQ ID NO: 80 and SEQ ID NO: 81.
  • Embodiment 56 comprises a polypeptide or polypeptide complex of any one of embodiments 1-55, wherein the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and L2 is bound to the Fab heavy chain polypeptide of A2.
  • Embodiment 57 comprises a polypeptide or polypeptide complex of any one of embodiments 1-56, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of Ai and F2 is bound to the Fab light chain polypeptide of A2.
  • Embodiment 58 comprises a polypeptide or polypeptide complex of any one of embodiments 1-57, wherein the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of Ai and F2 is bound to the Fab heavy chain polypeptide of A2.
  • Embodiment 59 comprises a polypeptide or polypeptide complex of any one of embodiments 1-58, wherein the first target antigen comprises a tumor cell antigen and the second target antigen comprises an effector cell antigen
  • Embodiment 60 comprises a polypeptide or polypeptide complex of any one of embodiments 1-59, wherein the tumor cell antigen comprises EGFR, HER2, mesothelin, or CEACAM5.
  • Embodiment 61 comprises a polypeptide or polypeptide complex of any one of embodiments 1-60, wherein the effector cell antigen comprises CD3.
  • Embodiment 62 comprises a polypeptide or polypeptide complex of any one of embodiments 1-61, wherein Ai comprises an antibody or antibody fragment.
  • Embodiment 63 comprises a polypeptide or polypeptide complex of any one of embodiments 1-62, wherein Ai comprises an antibody or antibody fragment that is human or humanized.
  • Embodiment 64 comprises a polypeptide or polypeptide complex of any one of embodiments 1-63, wherein Li is bound to N-terminus of the antibody or antibody fragment.
  • Embodiment 65 comprises a polypeptide or polypeptide complex of any one of embodiments 1-64, wherein A2 is bound to C-terminus of the antibody or antibody fragment.
  • Embodiment 66 comprises a polypeptide or polypeptide complex of any one of embodiments 1-65, wherein Li is bound to C-terminus of the antibody or antibody fragment.
  • Embodiment 67 comprises a polypeptide or polypeptide complex of any one of embodiments 1-66, wherein A2 is bound to N-terminus of the antibody or antibody fragment.
  • Embodiment 68 comprises a polypeptide or polypeptide complex of any one of embodiments 1-67, wherein the antibody or antibody fragment thereof comprises a single chain variable fragment, a single domain antibody, or a Fab.
  • Embodiment 69 comprises a polypeptide or polypeptide complex of any one of embodiments 1-68, wherein the antibody or antibody fragment thereof comprises a single chain variable fragment (scFv), a heavy chain variable domain (VH domain), a light chain variable domain (VL domain), a variable domain (VHH) of a camelid derived single domain antibody.
  • scFv single chain variable fragment
  • VH domain heavy chain variable domain
  • VL domain light chain variable domain
  • VHH variable domain
  • Embodiment 70 comprises a polypeptide or polypeptide complex of any one of embodiments 1-69, wherein the antibody or antibody fragment thereof is humanized or human.
  • Embodiment 71 comprises a polypeptide or polypeptide complex of any one of embodiments 1-70, wherein Ai is the Fab.
  • Embodiment 72 comprises a polypeptide or polypeptide complex of any one of embodiments 1-71, wherein the Fab comprises (a) a Fab light chain polypeptide and (b) a Fab heavy chain polypeptide.
  • Embodiment 73 comprises a polypeptide or polypeptide complex of any one of embodiments 1-72, wherein the antibody or antibody fragment thereof comprises an epidermal growth factor receptor (EGFR) binding domain.
  • EGFR epidermal growth factor receptor
  • Embodiment 74 comprises a polypeptide or polypeptide complex of any one of embodiments 1-73, wherein the antibody or antibody fragment thereof comprises a mesothelin binding domain.
  • Embodiment 75 comprises a polypeptide or polypeptide complex of any one of embodiments 1-74, wherein the antibody or antibody fragment thereof comprises a carcinoembryonic antigen-related cell adhesion molecule CEACAM5 binding domain.
  • Embodiment 76 comprises a polypeptide or polypeptide complex of any one of embodiments 1-75, wherein the tumor cell antigen comprises EGFR, and the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NOs: 56 or 57.
  • Embodiment 77 comprises a polypeptide or polypeptide complex of any one of embodiments 1-76, wherein the tumor cell antigen comprises EGFR, and the Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NOs: 59 or 60.
  • Embodiment 78 comprises a polypeptide or polypeptide complex of any one of embodiments 1-77, wherein the tumor cell antigen comprises HER2, and the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 61.
  • Embodiment 79 comprises a polypeptide or polypeptide complex of any one of embodiments 1-78, wherein the tumor cell antigen comprises HER2 and the Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NOs: 62 or 63.
  • Embodiment 80 comprises a polypeptide or polypeptide complex of any one of embodiments 1-79, wherein A comprises an antibody or antibody fragment.
  • Embodiment 81 comprises a polypeptide or polypeptide complex of any one of embodiments 1-80, wherein A comprises an antibody or antibody fragment that is human or humanized.
  • Embodiment 82 comprises a polypeptide or polypeptide complex of any one of embodiments 1-81, wherein the antibody or antibody fragment comprises a single chain variable fragment, a single domain antibody, or a Fab fragment.
  • Embodiment 83 comprises a polypeptide or polypeptide complex of any one of embodiments 1-82, wherein A IS the single chain variable fragment (scFv).
  • Embodiment 84 comprises a polypeptide or polypeptide complex of any one of embodiments 1-83, wherein the scFv comprises a scFv heavy chain polypeptide and a scFv light chain polypeptide.
  • Embodiment 85 comprises a polypeptide or polypeptide complex of any one of embodiments 1-84, wherein A is the single domain antibody.
  • Embodiment 86 comprises a polypeptide or polypeptide complex of any one of embodiments 1-85, wherein the single domain antibody comprises a single chain variable fragment (scFv), a heavy chain variable domain (VH domain), a light chain variable domain (VL domain), or a variable domain (VHH) of a camelid derived single domain antibody.
  • the single domain antibody comprises a single chain variable fragment (scFv), a heavy chain variable domain (VH domain), a light chain variable domain (VL domain), or a variable domain (VHH) of a camelid derived single domain antibody.
  • Embodiment 87 comprises a polypeptide or polypeptide complex of any one of embodiments 1-86, wherein A comprises an anti-CD3e single chain variable fragment.
  • Embodiment 88 comprises a polypeptide or polypeptide complex of any one of embodiments 1-87, wherein A comprises an anti-CD3e single chain variable fragment that has a K D binding of 1 mM or less to CD3 on CD3 expressing cells.
  • Embodiment 89 comprises a polypeptide or polypeptide complex of any one of embodiments 1-88, wherein A comprises a variable light chain and variable heavy chain each of which is capable of specifically binding to human CD3.
  • Embodiment 90 comprises a polypeptide or polypeptide complex of any one of embodiments 1-89, wherein A comprises complementary determining regions (CDRs) selected from the group consisting of muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, FI 11-409, CLB-T3.4.2, TR-66, WT32, SPv- T3b, 11D8, XIII- 141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865vl2, 15865vl6, and 15865vl9.
  • CDRs
  • Embodiment 91 comprises a polypeptide or polypeptide complex of any one of embodiments 1-90, wherein the polypeptide or polypeptide complex of formula I binds to an effector cell.
  • Embodiment 92 comprises a polypeptide or polypeptide complex of any one of embodiments 1-91, wherein the effector cell is a T cell.
  • Embodiment 93 comprises a polypeptide or polypeptide complex of any one of embodiments 1-92, wherein A binds to a polypeptide that is part of a TCR-CD3 complex on the effector cell.
  • Embodiment 94 comprises a polypeptide or polypeptide complex of any one of embodiments 1-93, wherein the polypeptide that is part of the TCR-CD3 complex is human CD3s.
  • Embodiment 95 comprises a polypeptide or polypeptide complex of any one of embodiments 1-94, wherein the effector cell antigen comprises CD3, and the scFv comprises an amino acid sequence according to SEQ ID NOs: 66, 67, or 68.
  • Embodiment 96 comprises a polypeptide or polypeptide complex of any one of embodiments 1-95, wherein the Fab light chain polypeptide of Ai is bound to a C-terminus of the single chain variable fragment (scFv) of A2.
  • Embodiment 97 comprises a polypeptide or polypeptide complex of any one of embodiments 1-96, wherein the Fab heavy chain polypeptide of Ai is bound to a C-terminus of the single chain variable fragment (scFv) A2.
  • Embodiment 98 comprises a polypeptide or polypeptide complex of any one of embodiments 1-97, wherein the Fab light chain polypeptide of Ai is bound to a N-terminus of the single chain variable fragment (scFv) of A2.
  • Embodiment 99 comprises a polypeptide or polypeptide complex of any one of embodiments 1-98, wherein the Fab heavy chain polypeptide of Ai is bound to a N-terminus of the single chain variable fragment (scFv) A2.
  • Embodiment 100 comprises a polypeptide or polypeptide complex of any one of embodiments 1-99, wherein the Fab heavy chain polypeptide of Ai is bound to the scFv heavy chain polypeptide of A2 and Li is bound to the Fab light chain polypeptide of Ai.
  • Embodiment 101 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 102 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 103 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 104 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • A2 further comprises P2 and L2, wherein P2 comprises a peptide that binds to A2; and L2 comprises a linking moiety that connects A2to P2 and is a substrate for a tumor specific protease.
  • Embodiment 105 comprises the polypeptide or polypeptide complex of any one of embodiments 1-
  • polypeptide or polypeptide complex is according to Formula la
  • Embodiment 106 comprises a polypeptide or polypeptide complex of any one of embodiments 1- 105, wherein the Fab heavy chain polypeptide of Ai is bound to the scFv heavy chain polypeptide of A2 and Li is bound to the Fab light chain polypeptide of Ai and L is bound to the scFv light chain polypeptide of A 2 .
  • Embodiment 107 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the polypeptide complex comprises amino acid sequences of SEQ ID NO: 72 and SEQ ID NO: 71.
  • Embodiment 108 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 109 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 110 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 11 1 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen as compared to the binding affinity for the tumor cell antigen of a polypeptide or polypeptide complex that does not have Pi or Li.
  • Embodiment 112 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 1 OX higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • Embodiment 113 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 100X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have Pi or Li.
  • Embodiment 114 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen as compared to the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • Embodiment 115 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 1 OX higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • Embodiment 116 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 100X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • Embodiment 117 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 118 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Pi is bound to Ai through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof.
  • Embodiment 119 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 120 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 121 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • P2 is bound to A2 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof.
  • Embodiment 122 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 123 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 124 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Pi or P2 comprises a peptide sequence of at least 10 amino acids in length.
  • Embodiment 125 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Pi or P2 comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length.
  • Embodiment 126 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Pi or P2 comprises a peptide sequence of at least 16 amino acids in length.
  • Embodiment 127 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Pi or P2 comprises a peptide sequence of no more than 40 amino acids in length.
  • Embodiment 128 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Pi or P2 comprises at least two cysteine amino acid residues.
  • Embodiment 129 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Pi or P2 comprises a cyclic peptide or a linear peptide.
  • Embodiment 130 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 131 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Pi or P2 comprises a linear peptide
  • Embodiment 132 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Pi comprises at least two cysteine amino acid residues.
  • Embodiment 133 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the tumor cell antigen comprises EGFR
  • the Pi or P2 comprises an amino acid sequence selected from the group consisting of GGDWCRSLMSYTDLCP (SEQ ID NO: 1), GGTSCADAHLIAPSCS (SEQ ID NO: 2), GGNCQWDRVEHTYACS (SEQ ID NO: 3), GGWV SCHDGSHMTCFH (SEQ ID NO: 4), GGMN CLNRLWVEY CLV (SEQ ID NO: 5), GGY CGQDNTWVREGCF (SEQ ID NO: 6) and QGQSGQLSCEGWAMNREQCRA (SEQ ID NO: 7).
  • Embodiment 134 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the tumor cell antigen comprises HER2
  • the Pi or P2 comprises an amino acid sequence selected from the group consisting of GGPLCSDLDHITRLCD (SEQ ID NO: 8), GGIDCASLDHYTESCY (SEQ ID NO: 9), GGNPV CTLGDPYECSH (SEQ ID NO: 10), GGTFCQLNADPYECQS (SEQ ID NO:
  • GGTGCTEGHWHWGTCS (SEQ ID NO: 14)
  • GGNICMDY SWRSGCAV (SEQ ID NO: 15)
  • GGHSCTFGDWSLGTCA SEQ ID NO: 16
  • GGFICTLGNWWDGSCE SEQ ID NO: 17
  • Embodiment 135 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the effector cell antigen comprises CD3, and the Pi or P2 comprises an amino acid sequence selected from the group consisting of QGQSGQGYLWGCEWNCGGITT (SEQ ID NO: 18),
  • GGD S V CADPEVPICEI (SEQ ID NO: 19), GGMSDCGDPGVEICTH (SEQ ID NO: 20),
  • GGIQCHDPDLPSPCYI SEQ ID NO: 21
  • GGEW CLFDPD VPT CQD SEQ ID NO: 22
  • GGLGCNDIDPGEQCIV SEQ ID NO: 23
  • GGLECFDPEIPEAF Cl SEQ ID NO: 24
  • GGQGCGTIADPEPHCW (SEQ ID NO: 25), GGNCHDPDIPAYVLCS (SEQ ID NO: 26),
  • GGLCPINDWEPQDICW SEQ ID NO: 27
  • GGLCMIGDWLPGD V CL SEQ ID NO: 28
  • Embodiment 136 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 137 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 138 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 139 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 140 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Li or L2 is a peptide sequence having at least 5 to no more than 50 amino acids.
  • Embodiment 141 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 142 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Li or L2 IS a peptide sequence having at least 10 amino acids.
  • Embodiment 143 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Li or L2 IS a peptide sequence having at least 18 amino acids.
  • Embodiment 144 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Li or L2 IS a peptide sequence having at least 26 amino acids.
  • Embodiment 145 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Li or L2 has a formula comprising (G2S) n , wherein n is an integer from 1 to 3 (SEQ ID NO:
  • Embodiment 146 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Li has a formula selected from the group consisting of (G2S) n , (GS) n , (GSGGS) n (SEQ ID NO:
  • Embodiment 147 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 148 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 149 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • tumor specific protease is selected from the group consisting of metalloprotease, serine protease, cysteine protease, threonine protease, and aspartic protease.
  • Embodiment 150 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Li or L2 comprises a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, matrix metalloprotease cleavable amino acid sequence, or a legumain cleavable amino acid sequence.
  • Embodiment 151 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Li or L2 comprises an amino acid sequence selected from the group consisting of
  • GGGGSLSGRSDNHGSSGT SEQ ID NO: 34
  • GGGGSSGGSGGSGLSGRSDNHGSSGT SEQ ID NO: 35
  • ASGRSDNH SEQ ID NO: 36
  • LAGRSDNH SEQ ID NO: 37
  • ISSGLASGRSDNH SEQ ID NO: 38
  • IS SGLLAGRSDNH SEQ ID NO: 39
  • LSGRSDNH SEQ ID NO: 40
  • ISSGLLSGRSDNP SEQ ID NO: 41
  • ISSGLLSGRSDNH SEQ ID NO: 42
  • LSGRSDNHSPLGLAGS SEQ ID NO: 43
  • SPLGLAGSLSGRSDNH SEQ ID NO: 44
  • SPLGLSGRSDNH SEQ ID NO: 45
  • Embodiment 152 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Li or L2 comprises an amino acid sequence ASGRSDNH (SEQ ID NO: 36), LAGRSDNH (SEQ ID NO: 37), IS SGLA SGRSDNH (SEQ ID NO: 38), and IS SGLLAGRSDNH (SEQ ID NO: 39).
  • Embodiment 153 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Hi comprises a polymer
  • Embodiment 154 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • polymer is polyethylene glycol (PEG).
  • Embodiment 155 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Hi comprises albumin
  • Embodiment 156 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Hi comprises an Fc domain
  • Embodiment 157 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • albumin is serum albumin
  • Embodiment 158 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • albumin is human serum albumin.
  • Embodiment 159 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Hi comprises a polypeptide, a ligand, or a small molecule.
  • Embodiment 160 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • polypeptide, the ligand or the small molecule binds serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35/CR1.
  • Embodiment 161 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the serum protein comprises a thyroxine -binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an albumin.
  • Embodiment 162 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the circulating immunoglobulin molecule comprises IgGl, IgG2, IgG3, IgG4, slgA, IgM or IgD.
  • Embodiment 163 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • serum protein is albumin
  • Embodiment 164 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • polypeptide 163 wherein the polypeptide is an antibody.
  • Embodiment 165 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the antibody comprises a single domain antibody, a single chain variable fragment, or a Fab.
  • Embodiment 166 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the single domain antibody comprises a single domain antibody that binds to albumin wherein the single domain antibody is a human or humanized antibody.
  • Embodiment 166 comprises a polypeptide or polypeptide complex of any one of embodiments 1- 165, wherein the single domain antibody is 645gHlgLl.
  • Embodiment 168 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 169 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 170 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the single domain antibody is A10m3 or a fragment thereof.
  • Embodiment 171 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 172 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 173 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the single domain antibody is Alb-1, Alb-8, or Alb-23.
  • Embodiment 174 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 175 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the single domain antibody is 10G, and the single domain antibody comprises an amino acid sequence
  • Embodiment 176 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 177 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • polypeptide or polypeptide complex comprises a modified amino acid, a non-natural amino acid, a modified non-natural amino acid, or a combination thereof.
  • Embodiment 178 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • modified amino acid or modified non-natural amino acid comprises a post-translational modification
  • Embodiment 179 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Hi comprises a linking moiety (L3) that connects Hi to Pi .
  • Embodiment 180 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • L3 is a peptide sequence having at least 5 to no more than 50 amino acids.
  • Embodiment 181 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • L3 is a peptide sequence having at least 10 to no more than 30 amino acids.
  • Embodiment 182 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • L3 is a peptide sequence having at least 10 amino acids.
  • Embodiment 183 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 184 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • L is a peptide sequence having at least 26 amino acids.
  • Embodiment 185 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • L has a formula selected from the group consisting of (G S) n , (GS) n , (GSGGS) n (SEQ ID NO: 30), (GGGS) thread (SEQ ID NO: 31) , (GGGGS) context (SEQ ID NO: 32) , and (GSSGGS)structure (SEQ ID NO: 33) , wherein n is an integer of at least .
  • Embodiment 186 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • L comprises an amino acid sequence according to GGGGSGGGS (SEQ ID NO: 51).
  • Embodiment 187 comprises a pharmaceutical composition comprising: the polypeptide or polypeptide complex of any one of embodiments 1-186; and a pharmaceutically acceptable excipient.
  • Embodiment 188 comprises an isolated recombinant nucleic acid molecule encoding the polypeptide or polypeptide complex of any one of embodiments 1-187.
  • Embodiment 189 comprises a polypeptide or polypeptide complex according to Formula II:
  • Li a comprises a tumor specific protease-cleaved linking moiety that when uncleaved connects Pi a to an antigen recognizing molecule that binds to a target antigen and; Pi a comprises a peptide that binds to the antigen recognizing molecule when Li a is uncleaved; and Hi a comprises a half-life extending molecule.
  • Embodiment 190 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 191 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the antigen recognizing molecule comprises an antibody or antibody fragment.
  • Embodiment 192 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the target antigen is an anti-CD3 effector cell antigen.
  • Embodiment 193 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the target antigen is a tumor cell antigen.
  • Embodiment 194 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • tumor cell antigen is EGFR, HER2, mesothelin, or CEACAM5.
  • Embodiment 195 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 196 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Pi a comprises a peptide sequence of at least 10 amino acids in length.
  • Embodiment 197 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 198 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Pi a comprises a peptide sequence of at least 16 amino acids in length.
  • Embodiment 199 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Pi a comprises a peptide sequence of no more than 40 amino acids in length.
  • Embodiment 200 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Pi a comprises at least two cysteine amino acid residues.
  • Embodiment 201 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Pi a comprises a cyclic peptide or a linear peptide.
  • Embodiment 202 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Pi a comprises a cyclic peptide
  • Embodiment 203 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Pi a comprises a linear peptide
  • Embodiment 204 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the target antigen comprises EGFR
  • the Pi a comprises an amino acid sequence selected from the group consisting of GGDWCRSLMSYTDLCP (SEQ ID NO: 1), GGTSCADAHLIAPSCS (SEQ ID NO: 2), GGNCQWDRVEHTYACS (SEQ ID NO: 3), GGWVSCHDGSHMTCFH (SEQ ID NO: 4), GGMN CLNRLWVEY CLV (SEQ ID NO: 5), GGY CGQDNTWVREGCF (SEQ ID NO: 6) and
  • Embodiment 205 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the target comprises HER2
  • the Pi a comprises an amino acid sequence selected from the group consisting of GGPLCSDLDHITRLCD (SEQ ID NO: 8), GGIDCASLDHYTESCY (SEQ ID NO: 9), GGNPV CTLGDPYEC SH (SEQ ID NO: 10), GGTFCQLNADPYECQS (SEQ ID NO: 11),
  • GGGY CELIGDYVV CSP (SEQ ID NO: 12), GGLCDRW GWID APY CH (SEQ ID NO: 13),
  • GGTGCTEGHWHWGTCS (SEQ ID NO: 14)
  • GGNICMDY SWRSGCAV (SEQ ID NO: 15)
  • GGHSCTFGDWSLGTCA SEQ ID NO: 16
  • GGFICTLGNWWDGSCE SEQ ID NO: 17
  • Embodiment 206 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the target comprises CD3, and the Pi a comprises an amino acid sequence selected from the group consisting of QGQSGQGYLWGCEWNCGGITT (SEQ ID NO: 18), GGDSVCADPEVPICEI (SEQ ID NO: 19), GGMSDCGDPGVEICTH (SEQ ID NO: 20), GGIQCHDPDLPSPCYI (SEQ ID NO: 21), GGEWCLFDPDVPTCQD (SEQ ID NO: 22), GGLGCNDIDPGEQCIV (SEQ ID NO: 23),
  • GGLECFDPEIPEAF Cl (SEQ ID NO: 24), GGQGCGTIADPEPHCW (SEQ ID NO: 25),
  • GGNCHDPDIPAYVLCS (SEQ ID NO: 26), GGLCPINDWEPQDICW (SEQ ID NO: 27), and
  • Embodiment 207 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Hi a comprises a polymer
  • Embodiment 208 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 209 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Hi a comprises albumin
  • Embodiment 210 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Hi a comprises an Fc domain.
  • Embodiment 211 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • albumin is serum albumin.
  • Embodiment 212 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • albumin is human serum albumin.
  • Embodiment 213 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Hi a comprises a polypeptide, a ligand, or a small molecule.
  • Embodiment 214 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • polypeptide, the ligand or the small molecule binds a serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35/CR1.
  • Embodiment 215 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the serum protein comprises a thyroxine -binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an albumin.
  • Embodiment 216 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the circulating immunoglobulin molecule comprises IgGl, IgG2, IgG3, IgG4, slgA, IgM or IgD.
  • Embodiment 217 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • serum protein is albumin
  • Embodiment 218 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • polypeptide is an antibody
  • Embodiment 219 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the antibody comprises a single domain antibody, a single chain variable fragment or a Fab.
  • Embodiment 220 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the antibody comprises a single domain antibody that binds to albumin.
  • Embodiment 221 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the antibody is a human or humanized antibody.
  • Embodiment 222 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 223 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 224 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 225 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 226 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 227 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 228 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 229 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 230 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • the single domain antibody is 10G, and the single domain antibody comprises an amino acid sequence
  • Embodiment 231 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Embodiment 232 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • Hi a comprises a linking moiety (L a ) that connects Hi a to Pi a.
  • Embodiment 233 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • L a is a peptide sequence having at least 5 to no more than 50 amino acids.
  • Embodiment 234 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • L a is a peptide sequence having at least 10 to no more than 30 amino acids.
  • Embodiment 235 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • L a is a peptide sequence having at least 10 amino acids.
  • Embodiment 236 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • L a is a peptide sequence having at least 18 amino acids.
  • Embodiment 237 comprises a polypeptide or polypeptide complex of any one of embodiments 1-
  • L a is a peptide sequence having at least 26 amino acids.
  • Embodiment 238 comprises a polypeptide or polypeptide complex of any one of embodiments 1-

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US20230220105A1 (en) 2023-07-13
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US20240376226A1 (en) 2024-11-14
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