Activatable polypeptide complex formulations

Abstract

The present disclosure relates to methods of treating a disease or disorder comprising administering activatable anti-EGFR, anti-CD3, heteromultimeric bispecific polypeptide complexes (HBPCs) to subjects in need thereof.

Description

ACTIVATABLE POLYPEPTIDE COMPLEX FORMULATIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Provisional Application No. 63 / 643,561, filed May 7, 2024, which is hereby incorporated by reference in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS WEB

[0002] The content of the electronically submitted sequence listing (4680_003PC01_SequenceListing_ST26.xml; Size: 151,865 bytes; and Date of Creation: May 6, 2025) submitted in this application is incorporated herein by reference in its entirety.FIELD

[0003] The present disclosure relates to formulations and dosing of activatable anti-EGFR, anti-CD3, heteromultimeric bispecific polypeptide complexes (HBPCs) for the treatment of a disease or disorder.BACKGROUND

[0004] The Epidermal growth factor receptor (EGFR) signaling pathway is a driver of cell proliferation, survival, differentiation, and motility (Sigismund 2018), all of which are required for tumor growth (Rocha-Lima 2007). In cancer biology, dysregulation in EGFR signaling may occur due to gene amplification and / or protein over-expression (Sigismund 2018). Consequently, EGFR over-expression is considered a well-established prognostic marker in several cancers (Wykosky 2011), with a strong association for head and neck, ovarian, cervical, bladder, and esophageal cancer and a modest association with gastric, breast, endometrial, and colorectal cancer (Nicholson 2001). For example, in head and neck cancer EGFR expression has been shown to be an independent determinant of overall survival (OS) and disease-free survival (DFS) with high EGFR expression being associated with poorer OS and DFS (Kian 2002). Although EGFR expression in these cancers signifiespoor prognosis, its increased frequency of expression, strong oncogenic function, and ease of targeting makes it a promising focus for development of novel cancer therapeutics (Thomas 2019).

[0005] Therapeutic options that employ the dependence of cancer cells on the EGFR signaling pathway are available in the form of monoclonal antibodies (mAbs) such as cetuximab, which bind to the extracellular domain of EGFR, and small molecule inhibitors such as erlotinib or afatinib, which target the tyrosine kinase domain (Sigismund 2018; Rocha-Lima 2007). Monoclonal antibodies such as cetuximab exert their therapeutic effect by inhibiting the binding of ligands to EGFR, thereby halting the signaling cascade that leads to tumor proliferation. Traditional antibody based approaches to targeting EGFR have demonstrated limited durability of clinical response due to activating mutations in downstream signaling proteins that bypass EGFR inhibition (Van Emburgh 2016; Cai 2020). Furthermore, expression levels of EGFR in cancer tissues are not predictive of efficacy for anti-EGFR mAbs (Thomas 2019).

[0006] In addition to targeting EGFR with a mAb to directly to inhibit its activity, anti- EGFR mAbs can also be used as a delivery vehicle to aim anticancer agents at tumors in an antibody-drug conjugate (ADC) format that links the anti-EGFR mAb with a small molecule cytotoxin (Santos 2021). However, a challenge experienced with this approach has been adverse reactions resulting from premature deconjugation of the cytotoxin from the antibody, i.e., a release of the toxic payload prior to successful entry of the mAb- cytotoxin entity into an EGFR-bearing tumor cell (Santos 2021). For example, in a clinical trial of an anti-EGFR ADC in patients with recurrent glioblastoma, 50% of patients presented with thrombocytopenia (Rosenthal 2019). Additional limitations include the off- tumor, on-target effects of the mAbs, given that the antigen is also highly expressed on most epithelial cells (Maruani 2018).

[0007] Recent innovations attempted to overcome these limitations by shifting the focus from tumor antigen targeting to immune cell targeting as a way of enhancing the inherent antitumor capabilities of the immune cell (Zahavi 2020). This strategy has led to the development of bispecific T-cell engagers (bsTCE), such as blinatumomab, a CD19-CD3 BiTE® molecule (Zahavi 2020). Blinatumomab targets the tumor antigen CD 19 and the activating receptor CD3 on T cells. Binding of the T cell and tumor cell by the bsTCE leads to T-cell release of the pore-forming perforin and cytotoxic granzyme-B, resulting in killing of the target cell (Suurs 2019).

[0008] The approvals of bsTCEs such as blinatumomab have led to an influx of additional bsTCEs into oncology clinical trials (Suurs 2019). However, a repeated observation of this antibody construct is cytokine release syndrome (CRS), which is common to T-cell engaging therapies (Suurs 2019). CRS occurs as a result of on-target effects induced by binding of the bsTCE to its antigens and subsequent activation of bystander immune and non-immune cells (Shimabukuro-Vomhagen 2018). Therefore, there is a continued need for innovation in cancer therapeutics to optimize delivery of anticancer antibody-based therapeutics to the tumor while reducing systemic toxicity.BRIEF SUMMARY

[0009] Disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC is administered at a dose of about 1 pg to about 50,000 pg (0.001 mg to about 50 mg).

[0010] Also disclosed herein are methods of treating a subject afflicted with a cancer, the method comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising(i) a single-chain variable fragment (scFv) comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), wherein the VH1 and the VL1 together form a T-cell cluster of differentiation (CD3)-targeting domain that specifically binds a CD3 polypeptide, wherein the scFv comprises a set of VH1 and VL1 selected from:(A) a VH1 comprising: a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), and a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO: 5), and a VL1 comprising:a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:6), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO: 7), and a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO: 8); or(B) a VH1 comprising: a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129), and a VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 130), and a VL1 comprising: a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 131), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO: 132), and a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO: 133);(ii) a first masking moiety (MM 1),(iii) a first cleavable moiety (CM1),(iv) a second heavy chain variable domain (VH2) comprising a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15), a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17);(v) a first monomeric Fc domain (Fcl);(b) a second polypeptide comprising:(i) a second light chain variable domain (VL2), wherein the VH2 and the VL2 together form an EGFR targeting domain that specifically binds EGFR, wherein the VL2 comprises a VL CDR1 comprisingthe amino acid sequence RASQSIGTNIH (SEQ ID NO: 18), a VL CDR2 comprising the amino acid sequence YASESIS (SEQ ID NO: 19), and a VL CDR3 comprising the amino acid sequence QQNNNWPTT (SEQ ID NO:20),(ii) a second masking moiety (MM2), and(iii) a second cleavable moiety (CM2); and(c) a third polypeptide that(i) comprises a second monomeric Fc domain (Fc2), and(ii) does not comprise an immunoglobulin variable domain; wherein the HBPC is administered at a dose of about 1 pg (0.001 mg) to about 50,000 pg (50 mg).

[0011] In some aspects, the first polypeptide comprises the scFv, MM1, CM1, VH2, and Fcl in the N-terminal to C-terminal orientation. In some aspects, the scFv comprises the VH1 and VL1 in the N-terminal to C-terminal orientation.

[0012] In some aspects, the second polypeptide comprises the VL2, MM2, and CM2 in the N-terminal to C-terminal orientation.

[0013] In some aspects, the HBPC comprises the first polypeptide, second polypeptide, and third polypeptide in the N-terminal to C-terminal orientation.

[0014] In some aspects, the MM1 comprises an amino acid sequence that is selected from the group consisting of SEQ ID NOs: 1, 67, 68, 69, 70, 71, and 72.

[0015] In some aspects, the VH1 comprises a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), and a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:5), and the VL1 comprises a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:6), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NOV), and a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO:8).

[0016] In some aspects, the VH1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NOV and / or wherein the VL1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 10. In some aspects, the VH1 comprises the amino acid sequence of SEQ ID NOV and the VL1 comprises the amino acid sequence of SEQID NO: 10.

[0017] In some aspects, the MM1 comprises the amino acid sequence of SEQ ID NO: 1.

[0018] In some aspects, the VH1 comprises a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129), and a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO: 130), and the VL1 comprises a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 131), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO: 132), and a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO: 133).

[0019] In some aspects, the VH1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 134 and / or wherein the VL1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 135. In some aspects, the VH1 comprises the amino acid sequence of SEQ ID NO: 134 and the VL1 comprises the amino acid sequence of SEQ ID NO: 135.

[0020] In some aspects, the MM1 comprises the amino acid sequence of SEQ ID NO:72.

[0021] In some aspects, the MM2 comprises the amino acid sequence of SEQ ID NO: 13.

[0022] In some aspects, the CM1 and the CM2 each independently comprise a substrate for a protease selected from the group of proteases shown in Table 1.

[0023] In some aspects, the CM1 and the CM2 each independently comprise a substrate having an amino acid sequence selected from the group shown in Table 2.

[0024] In some aspects, the CM1 comprises the amino acid sequence of SEQ ID NO:73.

[0025] In some aspects, the CM2 comprises the amino acid sequence of SEQ ID NO: 14.

[0026] Also disclosed herein are methods of treating a subject afflicted with a cancer, the method comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 144; a second polypeptide comprising the amino acid sequence of SEQ ID NO:37; and a third polypeptide comprising the amino acid sequence of SEQ ID NO:32; and wherein the HBPC is administered at a dose of about 1 pg to about 50,000 pg.

[0027] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a thirdpolypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 1 pg to about 50,000 pg.

[0028] In some aspects, the HBPC is administered at a dose of about 1 pg, about 2.5 pg, about 5 pg, about 7 pg, about 10 pg, about 20 pg, about 30 pg, about 40 pg, about 50 pg, about 60 pg, about 70 pg, about 80 pg, about 90 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, about 1000 pg, about 1250 pg, about 1500 pg, about 1750 pg, about 2000 pg, about 2250 pg, about 2500 pg, about 2750 pg, about 3000 pg, about 3500 pg, about 4000 pg, about 4500 pg, about 5000 pg, about 5500 pg, about 6000 pg, about 6500 pg, about 7000 pg, about 7500 pg, about 8000 pg, about 8500 pg, about 9000 pg, about 9500 pg, about 10,000 pg, about 10,500 pg, about 11,000 pg, about 11,500 pg, about 12,000 pg, about 12,500 pg, about 13,000 pg, about 13,500 pg, about 14,000 pg, about 14,500 pg, about 15,000 pg, about 15,500 pg, about 16,000 pg, about 16,500 pg, about 17,000 pg, about 17,500 pg, about 18,000 pg, about 18,500 pg, about 19,000 pg, about 19,500 pg, 20,000 pg, about 22,500 pg, about 25,000 pg, about 27,500 pg, about30,000 pg, about 32,500 pg, about 35,000 pg, about 37,500 pg, about 40,000 pg, about42,500 pg, about 45,000 pg, about 47,500 pg, or about 50,000 pg.

[0029] In some aspects, the HBPC is administered at a dose of about 7 pg. In some aspects, the HBPC is administered at a dose of about 70 pg. In some aspects, the HBPC is administered at a dose of about 250 pg. In some aspects, the HBPC is administered at a dose of about 750 pg. In some aspects, the HBPC is administered at a dose of about 1500 pg. In some aspects, the HBPC is administered at a dose of about 3000 pg. In some aspects, the HBPC is administered at a dose of about 5000 pg. In some aspects, the HBPC is administered at a dose of about 6000 pg. In some aspects, the HBPC is administered at a dose of about 10,000 pg. In some aspects, the HBPC is administered at a dose of about 15,000 pg.

[0030] In some aspects, the HBPC is administered once every two weeks (Q2W). In some aspects, the HBPC is administered at a dose of between about 7 pg and about 70 pg once every two weeks. In some aspects, the HBPC is administered at a dose of about 7 pg once every two weeks. In some aspects, the HBPC is administered at a dose of about 70 pg once every two weeks. In some aspects, the HBPC is administered at a dose of about 250 pg once every two weeks. In some aspects, the HBPC is administered at a dose of about 750pg once every two weeks. In some aspects, the HBPC is administered at a dose of about 1500 pg once every two weeks. In some aspects, the HBPC is administered at a dose of about 3000 pg once every two weeks. In some aspects, the HBPC is administered at a dose of about 6000 pg once every two weeks. In some aspects, the HBPC is administered at a dose of about 5000 pg once every two weeks. In some aspects, the HBPC is administered at a dose of about 10,000 pg once every two weeks. In some aspects, the HBPC is administered at a dose of about 15,000 pg once every two weeks. In some aspects, the HBPC is administered at a dose of about 20,000 pg once every two weeks. In some aspects, the HBPC is administered at a dose of about 25,000 pg once every two weeks. In some aspects, the HBPC is administered at a dose of about 30,000 pg once every two weeks. In some aspects, the HBPC is administered at a dose of about 35,000 pg once every two weeks. In some aspects, the HBPC is administered at a dose of about 40,000 pg once every two weeks. In some aspects, the HBPC is administered at a dose of about 45,000 pg once every two weeks.

[0031] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31 ; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the method comprises:(i) administering at least a first priming dose of HBPC to the subject on day 1; and(ii) administering to the subject a dose of the HBPC of about 1 pg to about 50,000 pg; wherein the first priming dose of the HBPC is less than the dose of HBPC in (ii).

[0032] In some aspects, the dose in (ii) is administered on day 8.

[0033] In some aspects, the method further comprises administering a second priming dose of HBPC to the subject on day 8, and wherein the dose in (ii) is administered on day 15, wherein the second priming dose is 100% or less than the dose in (ii).

[0034] In some aspects, the first priming dose and / or the second priming dose is 50% or less than the dose in (ii).

[0035] In some aspects, the first priming dose is 25% or less than the dose in (ii).

[0036] In some aspects, a priming dose of the HBPC is administered prior to the first dose of the HBPC. In some aspects, the subject is administered: a) a priming dose of the HBPC at about 3000 pg on day 1; b) about 5000 pg on day 15; and c) about 5000 pg every two weeks thereafter. In some aspects, the subject is administered: a) a priming dose of the HBPC at about 1500 pg on day 1; b) about 5000 pg on day 8; and c) about 5000 pg every two weeks thereafter. In some aspects, the subject is administered: a) a priming dose of the HBPC at about 3000 pg on day 1; b) about 5000 pg on day 8; and c) about 5000 pg every two weeks thereafter. In some aspects, the subject is administered: a) a priming dose of the HBPC at about 3000 pg on day 1; b) about 10,000 pg on day 15; and c) about 10,000 pg every two weeks thereafter. In some aspects, the subject is administered: a) a priming dose of the HBPC at about 1500 pg on day 1; b) a second priming dose of the HBPC at about 5000 pg on day 8; c) about 10,000 pg on day 15; and d) about 10,000 pg every two weeks thereafter. In some aspects, the subject is administered: a) a priming dose of the HBPC at about 3000 pg on day 1; b) a second priming dose of the HBPC at about 5000 pg on day 8; c) about 10,000 pg on day 15; and d) about 10,000 pg every two weeks thereafter. In some aspects, the subject is administered: a) a priming dose of the HBPC at about 3000 pg on day 1; b) a second priming dose of the HBPC at about 5000 pg on day 8; c) about 15,000 pg on day 15; and d) about 15,000 pg every two weeks thereafter.

[0037] In some aspects, the subject is administered an IL-6 inhibitor prior to administration of the HBPC. In some aspects, the IL-6 inhibitor is a small molecule or an antibody. In some aspects, the IL-6 inhibitor is a steroid, such as, for example, a corticosteroid (e.g., dexamethasone, and the like). In some aspects, the IL-6 inhibitor is tocilizumab, siltuximab, or sarilumab. In some aspects, the IL-6 inhibitor is tocilizumab. In some aspects, the amount of tocilizumab that the subject is administered does not exceed 800 mg. In some aspects, the subject is administered 8 mg / kg tocilizumab prior to administration of the HBPC. In certain aspects, the IL-6 receptor inhibitor (e.g., tocilizumab, siltuximab, sarilumab, and the like) is administered up to 4 hours prior to administration of the HBPC.

[0038] In some aspects, the subject is administered: a) a priming dose of the HBPC at about 3000 pg on day 1; b) about 5000 pg on day 15; and c) about 5000 pg every two weeks thereafter, wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC. In some aspects, the subject is administered: a) a priming dose of the HBPC at about 1500 pg on day 1; b) about 5000 pg on day 8; and c) about 5000 pg every two weeksthereafter, wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC except for the priming dose. In some aspects, the subject is administered: a) a priming dose of the HBPC at about 3000 pg on day 1; b) about 5000 pg on day 8; and c) about 5000 pg every two weeks thereafter, wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC. In some aspects, the subject is administered: a) a priming dose of the HBPC at about 3000 pg on day 1; b) about 10,000 pg on day 15; and c) about 10,000 pg every two weeks thereafter, wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC. In some aspects, the subject is administered: a) a priming dose of the HBPC at about 1500 pg on day 1; b) a second priming dose of the HBPC at about 5000 pg on day 8; c) about 10,000 pg on day 15; and c) about 10,000 pg every two weeks thereafter, wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC. In some aspects, the subject is administered: a) a priming dose of the HBPC at about 3000 pg on day 1; b) a second priming dose of the HBPC at about 5000 pg on day 8; c) about 10,000 pg on day 15; and d) about 10,000 pg every two weeks thereafter, wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC. In some aspects, the subject is administered: a) a priming dose of the HBPC at about 3000 pg on day 1; b) a second priming dose of the HBPC at about 5000 pg on day 8; c) about 15,000 pg on day 15; and d) about 15,000 pg every two weeks thereafter, wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC. In some aspects, the tocilizumab is administered up to 4 hours prior to administration of the HBPC.

[0039] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 6 mg once every two weeks.

[0040] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC isadministered at a dose of about 1.5 mg on day 1, 5 mg on day 8, and 5 mg once every two weeks thereafter.

[0041] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 1.5 mg on day 1, 5 mg on day 8, 10 mg on day 15, and 10 mg once every two weeks thereafter.

[0042] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 3 mg once every two weeks.

[0043] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 5 mg once every two weeks.

[0044] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 10 mg once every two weeks.

[0045] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecificpolypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 15 mg once every two weeks.

[0046] In some aspects, the method further comprises administering a second therapeutic agent. In some aspects, the second therapeutic agent is selected from a chemotherapeutic agent, an immune-oncology agent, or a monoclonal antibody. In some aspects, the monoclonal antibody is selected from alemtuzumab, cetuximab, bevacizumab, dostarlimab, gemtuzumab, ipilimumab, nivolumab, ofatumumab, panitumumab, pembrolizumab, ranibizumab, rituximab, or trastuzumab.

[0047] In some aspects, the cancer is sensitive to treatment with immune-oncology agents. In some aspects, the cancer is an EGFR-expressing cancer. In some aspects, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), cutaneous squamous cell carcinoma, colorectal carcinoma (CRC), esophageal cancer, gastric / gastroesophageal junction (g / GEJ) cancer, breast cancer, bladder cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, and renal cell carcinoma (RCC). In some aspects, the cancer is selected from the group consisting of NSCLC, HNSCC, pancreatic cancer, CRC, esophageal cancer, and g / GEJ cancer. In certain aspects, the cancer is selected from the group consisting of pancreatic cancer, NSCLC, HNSCC, GEJ / Gastric cancer, and esophageal cancer. In other aspects, the cancer is selected from the group consisting of pancreatic cancer, NSCLC, HNSCC, and GEJ / Gastric cancer. In some aspects, the cancer comprises a tumor. In some aspects, the tumor is an EGFR-expressing cancer. In some aspects, the tumor is a solid tumor. In some aspects, the solid tumor is sensitive to treatment with immune-oncology agents.

[0048] In some aspects, the subject is administered the HBPC via intravenous infusion. In some aspects, the intravenous infusion is administered for 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, or 120 minutes. In some aspects, the intravenous infusion is administered for 30 minutes. In certain aspects, the intravenous infusion is administered for at least 60 minutes. In some aspects, the intravenous infusion is administered for 60 minutes. In some aspects, the subject is a human.BRIEF DESCRIPTION OF THE FIGURES

[0049] FIG. 1 is a schematic of an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) described herein.

[0050] FIG. 2 is a graph showing the target dose of Complex 67, number of prior regimens, and response after administration of Complex 67 in patients with colorectal cancer (CRC), esophageal cancer, gastric cancer, head and neck squamous cell carcinoma (HNSCC), nonsmall cell lung cancer (NSCLC), and pancreatic cancer. PR - partial response; SD - stable disease; PD - progressive disease.

[0051] FIG. 3 is a graph showing the fold change in IL-6 levels in patients administered various doses of Complex 67 after non-step cycle 1 or non-step cycle 2.

[0052] FIGs. 4A-4B show clinical pharmacokinetic data of Complex 67. FIG. 4A shows the circulating levels of intact CD3, intact EGFR, or intact total Complex 67 in patients administered 3 mg Complex 67. FIG. 4B shows the AUC of Complex 67 in patients administered Complex 67.

[0053] FIGs. 5A-5D show the amount and localization of CD8+ T cells in patients after administration of Complex 67. FIG. 5A shows amount and localization of CD3+ cells, CD8+ cells, CD3+ and CD8+ cells, and tumor cells in a patient with pancreatic cancer prior to administration of Complex 67. FIG. 5B shows the fold change in CD8+ cells in patients with colorectal cancer (CRC), esophageal cancer (ESGC), gastric cancer (GC), head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), and pancreatic cancer (PDAC) after administration of Complex 67. FIGs. 5C and 5D show the amount and localization of CD3+ cells, CD8+ cells, CD3+ and CD8+ cells, and tumor cells in a patient with microsatellite stable colorectal cancer (MSS CRC) prior to administration of Complex 67 (FIG. 5C) and after treatment with Complex 67 (FIG. 5D).

[0054] FIG. 6 is a graph showing the percent change in target lesions in patients with pancreatic cancer after administration of Complex 67.

[0055] FIG. 7 is a graph showing the percent change in target lesions in patients with colorectal cancer (CRC), esophageal cancer, gastric cancer, non-small cell lung cancer (NSCLC), pancreatic cancer, and head and neck squamous cell carcinoma (HNSCC) after administration of Complex 67.

[0056] FIGs. 8A-8C are scans showing the size of a target lesion and stable non-target nodal lesion in a patient with pancreatic cancer prior to administration of Complex 67 (FIG.8A), 6 weeks after administration of Complex 67 (FIG. 8B), and 12 weeks after administration of Complex 67 (FIG. 8C).DETAILED DESCRIPTION

[0057] In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.Definitions

[0058] As used herein, the term “heteromultimeric bispecific polypeptide complex” and “HBPC” are used interchangeably to refer to a set of polypeptides that together form a complex that has binding domains that are capable of binding to two different biological targets.

[0059] The term “activatable” when used in connection with the term “heteromultimeric bispecific polypeptide complex” or “HBPC” refers herein to an HBPC whose binding activity is impaired by the presence of masking moieties appended to the structure of the HBPC. The terms “activated” and “act-” are each used to refer to an activated HBPC. The terms “activated” and “unmasked,” are used interchangeably herein.

[0060] As used herein, the term “EGFR” refers to a receptor and transmembrane glycoprotein and a member of the protein kinase superfamily. Human epidermal growth factor receptor is a 170 kDa transmembrane receptor encoded by the c-erb B-l protooncogene, and exhibits intrinsic tyrosine kinase activity (Modjtahedi et al., Br. J. Cancer 73:228-235 (1996); Herbst and Shin, Cancer 94: 1593-1611 (2002)). There are also known isoforms and variants of EGFR (e.g., alternative RNA transcripts, truncated versions, polymorphisms, etc.), which are contemplated for use herein. EGFR regulates numerous cellular processes via tyrosine-kinase mediated signal transduction pathways, including, but not limited to, activation of signal transduction pathways that control cell proliferation, differentiation, cell survival, apoptosis, angiogenesis, mitogenesis, and metastasis (Atalay et al., Ann. Oncology 14: 1346-1363 (2003); Tsao and Herbst, Signal 4:4-9 (2003); Herbst and Shin, Cancer 94: 1593-1611 (2002); Modjtahedi et al., Br. J. Cancer 73:228-235 (1996)). Overexpression of EGFR is associated with numerous humancancers, including cancers of the bladder, brain, head and neck, pancreas, lung, breast, ovary, colon, prostate, and kidney. EGFR is also expressed in the cells of normal tissues at lower levels than expressed in malignant cells. Exemplary anti-EGFR antigen-binding proteins include but are not limited to human wildtype EGFR (NCBI Accession No.NG 007726.E), human wildtype EGFR Transcript Variant 1 (NCBI Accession No.NP_005219.2), human wildtype EGFR Transcript Variant 2 (NCBI Accession No.NP_958439.1), human wildtype EGFR Transcript Variant 3 (NCBI Accession No.NP_958440.1), human wildtype EGFR Transcript Variant 4 (NCBI Accession No.NP_958441.1), human wildtype EGFR Transcript Variant 5 (NCBI Accession No.NP_001333826.1), human wildtype EGFR Transcript Variant 6 (NCBI Accession No.NP_001333827.1), human wildtype EGFR Transcript Variant 7 (NCBI Accession No.NP_001333828.1), human wildtype EGFR Transcript Variant 8 (NCBI Accession No.NM 001346941.2), human wildtype EGFR Transcript Variant EGFRvIII (NCBI Accession No. NP_001333870.1), and the like.

[0061] The term “CD3” or “cluster of differentiation 3” as used herein refers to a protein complex of six chains which are subunits of the T cell receptor complex. (Janeway et al., p. 166, 9thed.) The TCR a:P heterodimer associates with CD3 subunits to complete the TCR cell-surface antigen receptor. Two CD3s chains, a CD3y chain, and a CD35 chain and a homodimer of CD3(^ chains complete the T cell receptor complex, which is involved in the recognition of peptides bound to the major histocompatibility complex class I and II and involves T cell activation. The CD3 antigen is expressed by mature T lymphocytes and by a subset of thymocytes. The CD3 -targeting domain that specifically binds a CD3 polypeptide, disclosed herein, can be from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats). The term encompasses “full- length,” unprocessed CD3 (e.g., unprocessed or unmodified CD3s or CD3 y) as well as any form of CD3 that results from processing in the cell. The term also encompasses naturally occurring variants of CD3, including, for example, splice variants or allelic variants. An anti-CD3 targeting domain described herein can specifically bind to human wildtype CD3E (NCBI Accession No. NM_000733.3).

[0062] The term “T cell,” as used herein is defined as a thymus-derived lymphocyte that participates in a variety of cell-mediated immune reactions. The term “regulatory T cell” as used herein refers to a CD4+CD25+FoxP3+T cell. “Treg” is the abbreviation used herein for a regulatory T cell.

[0063] The term “helper T cell” as used herein refers to a CD4+T cell; helper T cells recognize antigen bound to MHC Class II molecules. There are at least two types of helper T cells, Thl and Th2, which produce different cytokines. Helper T cells become CD25+when activated, but only transiently become FoxP3+.

[0064] The term “cytotoxic T cell” as used herein refers to a CD8+T cell; cytotoxic T cells recognize antigen bound to MHC Class I molecules.

[0065] The term “variable region” or “variable domain” refers to the domain of an antigen binding protein (e.g., an antibody) heavy or light chain that is involved in binding the antigen binding protein (e.g., antibody) to antigen. The variable regions or domains of the heavy chain and light chain (VH and VL, respectively) of an antigen binding protein such as an antibody can be further subdivided into regions of hypervariability (or hypervariable regions, which may be hypervariable in sequence and / or form of structurally defined loops), such as hypervariable regions (HVRs) or complementarity-determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). As used herein, the terms "hypervariable region" or "HVR" and "complementaritydetermining region" or "CDR" are used interchangeably herein. In general, there are three HVRs (HVR-H1, HVR-H2, HVR-H3) or CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region, and three HVRs (HVR-L1, HVR-L2, HVR-L3) or CDRs in (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. “Framework regions” and “FR” are known in the art to refer to the non-HVR or non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4). Within each VH and VL, three HVRs or CDRs and four FRs are typically arranged from amino-terminus to carboxy -terminus in the following order: FR1, HVR1, FR2, HVR2, FR3, HVR3, FR4 in the case of HVRs, or FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 in the case of CDRs (See also Chothia and Lesk J. Mot. Biol., 195, 901-917 (1987)). A single VH or VL domain can be sufficient to confer antigen-binding specificity. In addition, antibodies that bind a particular antigen can be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al. J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0066] The term “heavy chain variable region” (VH) as used herein refers to a region comprising heavy chain HVR-H1, FR-H2, HVR-H2, FR-H3, and HVR-H3. For example, a heavy chain variable region may comprise heavy chain CDR-H1, FR-H2, CDR-H2, FR- H3, and CDR-H3. In some aspects, a heavy chain variable region also comprises at least a portion of an FR-H1 and / or at least a portion of an FR-H4.

[0067] The term “heavy chain constant region” as used herein refers to a region comprising at least three heavy chain constant domains, CHI, CH2, and CH3. Nonlimiting exemplary heavy chain constant regions include y, 5, and a. Nonlimiting exemplary heavy chain constant regions also include a and p.

[0068] The term “light chain variable region” (VL) as used herein refers to a region comprising light chain HVR-L1, FR-L2, HVR-L2, FR-L3, and HVR-L3. In some aspects, the light chain variable region comprises light chain CDR-L1, FR-L2, CDR-L2, FR-L3, and CDR-L3. In some aspects, a light chain variable region also comprises an FR-L1 and / or an FR-L4.

[0069] The term “light chain constant region” as used herein refers to a region comprising a light chain constant domain, CL. Nonlimiting exemplary light chain constant regions include X and K.

[0070] The term “light chain” (LC) as used herein refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some aspects, a light chain comprises at least a portion of a light chain constant region. The term “full- length light chain” as used herein refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[0071] The term “antibody” refers to an immunoglobulin molecule or an immunologically active portion of an immunoglobulin (Ig) molecule, i.e., a molecule that contains an antigen binding site that specifically binds (immunoreacts with) an antigen. An “antigen-binding portion” of an antibody or polypeptide (also called an “antigen-binding fragment”) refers to one or more portions of an antibody or polypeptide that bind specifically to the target antigen. Antibodies and antigen-binding portions include, but are not limited to, polyclonal, monoclonal, chimeric, domain antibody, single chain antibodies, Fab, and F(ab')2 fragments, scFvs, Fd fragments, Fv fragments, single domain antibody (sdAb) fragments, dual-affinity re-targeting antibodies (DARTs), dual variable domain immunoglobulins; isolated complementarity determining regions (CDRs), and a combination of two or more isolated CDRs, which can optionally be joined by a synthetic linker, and a Fab expressionlibrary. A nonhuman antibody, e.g., a camelid antibody, may be humanized by recombinant methods to reduce its immunogenicity in a human.

[0072] The CDR sequences specified herein are determined in accordance with the Kabat numbering system (i.e., the “Kabat CDRs”) as described in Abhinandan, K. R. and Martin, A.C.R. (2008) "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains", Molecular Immunology, 45, 3832-3839, which is incorporated herein by reference in its entirety. The Kabat CDRs are defined as CDR-L1 : residues L24- L34; CDR-L2: residues L50-L56; CDR-L3: residues L89-L97; CDR-H1 : residues H31- H35; CDR-H2: residues H50-H65; and CDR-H3: residues H95-H102, where “L” refers to the light chain variable domain and “H” refers to the heavy chain variable domain.

[0073] “Specifically binds” or “immunospecifically binds” means that the targeting domain, antibody or antigen-binding fragment reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds at much lower affinity (Kd > 106), wherein a smaller Kd represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigenbinding site / antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the “on rate constant” (kon) and the “off rate constant” (koir) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361 : 186-87 (1993)). The ratio of koff / konenables the cancellation of all parameters not related to affinity, and is equal to the dissociation constant Kd. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). In some aspects, the antigen-targeting domain, antibody, or antigen-binding fragment that specifically binds to its corresponding antigen exhibits a Kd of less than about 10 pM, and in some aspects, less than about 100 pM with respect to the target antigen.

[0074] An immunoglobulin may derive from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4. “Isotype” refers to the antibody class or subclass (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes.

[0075] An “anti-antigen” antibody or polypeptide refers to an antibody or polypeptide that binds specifically to the antigen. For example, an anti-CD3 polypeptide binds specifically to CD3.

[0076] As used herein, the terms “MM” and “masking moiety” are used interchangeably to refer to a peptide that interferes with binding of the targeting domain to its corresponding antigen. For example, MM1 is a peptide that interferes with binding of the first targeting domain to the first target and MM2 is a peptide that interferes with binding of the second targeting domain to the second target. The extent to which a masking moiety interferes with the binding of the targeting domain to its corresponding target is quantified by its “masking efficiency.” The terms “masking efficiency” and “ME” are used interchangeably herein to refer to a ratio that is determined as follows:ME = EC50, activatable HBPC (i.e., intact / not cleaved by protease)EC50, activated HBPC

[0077] As used herein, the terms “CM” and “cleavable moiety” are used interchangeably to refer to a peptide substrate that is susceptible to cleavage by a protease that is upregulated in tumor cells. Protease-mediated cleavage of the CM results in the release of the MM from the structure of the activatable HBPC, thereby generating an “activated” (i.e., unmasked) product, where each corresponding “activated” (i.e, unmasked) first and / or second targeting domain is free to bind its respective target.

[0078] The term “isolated polynucleotide” as used herein refers to a recombinant polynucleotide or polynucleotide of synthetic origin which by virtue of its origin the “isolated polynucleotide” (1) is not associated with all or a portion of a polynucleotide in which the “isolated polynucleotide” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence. Polynucleotides in accordance with the disclosure include the nucleic acid molecules encoding the first, second, and third polypeptides.

[0079] The term “operably linked” as used herein refers to positions of components so described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0080] As discussed herein, minor variations in the amino acid sequences described herein (i.e., each reference sequence) are contemplated as being encompassed by the presentdisclosure, provided that the resulting analog sequence maintains at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% sequence identity to the reference sequence. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related with respect to the nature of their side chains. Amino acids may be divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. The hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. The hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are the amide containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family. For example, within the HBPC polypeptides and polypeptide complexes described herein, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a CDR or framework region. Whether an amino acid change results in a functional polypeptide complex can readily be determined by assaying the specific activity of the resulting molecule, i.e., the resulting analog sequence. Assays are described in detail herein. Preferred amino- and carboxy-termini of analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and / or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function. Methods to identify protein sequences that fold into a known three- dimensional structure are known. Bowie et al. Science 253 : 164 (1991). Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs andstructural conformations that may be used to define structural and functional domains in accordance with the disclosure.

[0081] A conservative amino acid substitution should not substantially change the structural characteristics of the reference sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the reference sequence, or disrupt other types of secondary structure that characterizes the reference sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature 354: 105 (1991).

[0082] Exemplary amino acid substitutions also include those which: (1) reduce susceptibility to proteolysis in regions of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide other than in the cleavable linker comprising the CM, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities to antigen, and (4) confer or modify other physicochemical or functional properties of such analogs. Such amino acid substitutions may be identified using known mutagenesis methods and / or directed molecular evolution methods using the assays described herein. See, e.g., International Publication No. WO 2001 / 032712, U.S. Pat. No. 7,432,083, U.S. Pub. No. 2004 / 0180340, and U.S. Pat. No. 6,297,053, each of which is incorporated herein by reference. Analogs may be prepared by introducing one or more mutations in a reference sequence within an activatable HBPC. For example, single or multiple amino acid substitutions may be made in the reference sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts).

[0083] As used herein, by “pharmaceutically acceptable” or “pharmacologically compatible” is meant a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to an individual or subject without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients have for example met the required standards of toxicological and manufacturing testing and / or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration.

[0084] A “patient” as used herein includes any patient who is afflicted with a cancer. The terms “subject” and “patient” are used interchangeably herein.

[0085] The terms “cancer,” “cancerous,” or “malignant” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, for example, melanoma, such as unresectable or metastatic melanoma, leukemia, lymphoma, blastoma, carcinoma and sarcoma. More particular examples of such cancers include chronic myeloid leukemia, acute lymphoblastic leukemia, Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL), squamous cell carcinoma, small-cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer, gastric cancer, germ cell tumor, pediatric sarcoma, sinonasal natural killer, multiple myeloma, acute myelogenous leukemia (AML), and chronic lymphocytic leukemia (CML).

[0086] The term “tumor” as used herein refers to any mass of tissue that results from excessive cell growth or proliferation, either benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions.

[0087] “Administering” refers to the physical introduction of a composition comprising a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion, as well as in vivo electroporation. In some aspects, the formulation is administered via a non-parenteral route, in some aspects, orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering can alsobe performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0088] Treatment” or “therapy” of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease.

[0089] As used herein, “effective treatment” refers to treatment producing a beneficial effect, e.g., amelioration of at least one symptom of a disease or disorder. A beneficial effect can take the form of an improvement over baseline, i.e., an improvement over a measurement or observation made prior to initiation of therapy according to the method. A beneficial effect can also take the form of arresting, slowing, retarding, or stabilizing of a deleterious progression of a marker of a tumor. Effective treatment may refer to alleviation of at least one symptom associated with a cancer. Such effective treatment may, e.g., reduce patient pain, reduce the size and / or number of lesions, may reduce or prevent metastasis of a tumor, and / or may slow tumor growth.

[0090] The term “effective amount” refers to an amount of an agent that provides the desired biological, therapeutic, and / or prophylactic result. That result can be reduction, amelioration, palliation, lessening, delaying, and / or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In reference to solid tumors, an effective amount comprises an amount sufficient to cause a tumor to shrink and / or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to delay other unwanted cell proliferation. In some aspects, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount can be administered in one or more administrations. The effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and may stop cancer cell infiltration into peripheral organs; (iv) inhibit, slow to some extent and may stop tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and / or recurrence of tumor; and / or (vii) relieve to some extent one or more of the symptoms associated with the cancer.

[0091] An “immune response” refers to the action of a cell of the immune system (for example, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells and neutrophils) and soluble macromoleculesproduced by any of these cells or the liver, spleen, and / or bone marrow (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and / or elimination from a vertebrate’s body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

[0092] The term "priming dose" refers to a dose of a primer agent (e.g., a dose of the HBPC) that primes a subject for administration of a therapeutically effective dose of the HBPC.

[0093] The term "target dose" refers to an amount sufficient to effect desired clinical results(i.e., achieve therapeutic efficacy). A target dose can be administered in one or more administrations. For purposes of this invention, a target dose of a HBPC is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, prevent, slow or delay the progression of the disease state (e.g., cancer).

[0094] The term "step-dose," "step-dosing," or "step-up dosing" refers to a incremental increase in the dose of the HBPC before reaching the target dose.

[0095] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the indefinite articles “a” or “an” should be understood to refer to “one or more” of any recited or enumerated component.

[0096] The term “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0097] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided.

[0098] The term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “comprising essentially of’ can mean within 1 or more than 1 standard deviation per the practice in theart. Alternatively, “about” or “comprising essentially of’ can mean a range of up to 10% or 20% (i.e., ±10% or ±20%). For example, about 3 mg can include any number between 2.7 mg and 3.3 mg (for 10%) or between 2.4 mg and 3.6 mg (for 20%). Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the application and claims, unless otherwise stated, the meaning of “about” should be assumed to be within an acceptable error range for that particular value or composition.

[0099] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.

[0100] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 5th ed., 2013, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, 2006, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0101] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the above-defined terms are more fully defined by reference to the specification in its entirety.

[0102] Schematic representations of activatable polypeptides of the present disclosure, e.g., FIG. 1, are not intended to be exclusive. Other sequence elements, such as linkers, spacers, and signal sequences, may be present before, after, or between the listed sequence elements in such schematic representations. It is also to be appreciated that a MM and a CM can be joined to a VH of an antibody or polypeptide instead of to a VL of an antibody or polypeptide, and vice versa.

[0103] Various aspects of the disclosure are described in further detail in the following subsections.Activatable Anti-EGFR, Anti-CD3 Heteromultimeric Bispecific Polypeptide Complex

[0104] The present disclosure provides methods of treating a subject afflicted with a cancer, comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide (HBPC) comprising: (a) a first polypeptide comprising (i) a singlechain variable fragment (scFv) comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), wherein the VH1 and the VL1 together form a T- cell cluster of differentiation (CD3)-targeting domain that specifically binds a CD3 polypeptide, (ii) a first masking moiety (MM1), (iii) a first cleavable moiety (CM1), (iv) a second heavy chain variable domain (VH2), and (v) a first monomeric Fc domain (Fcl); (b) a second polypeptide comprising (i) a second light chain variable domain (VL2), wherein the VH2 and the VL2 together form an EGFR targeting domain that specifically binds EGFR, (ii) a second masking moiety (MM2), and (iii) a second cleavable moiety (CM2); and (c) a third polypeptide that (i) comprises a second monomeric Fc domain (Fc2), and (ii) does not comprise an immunoglobulin variable domain, and wherein the MM1 is a peptide that interferes with binding of the CD3- targeting domain to a CD3 polypeptide and MM2 is a peptide that interferes with binding of the EGFR-targeting domain to an EGFR.

[0105] HBPCs that are suitable for use in the practice of the present disclosure include any that are known in the art, including those described in, for example, PCT Publication Nos. WO2023064927A1 and WO2023064929A1, each of which are incorporated herein by reference in their entireties.

[0106] As described herein above, among the components present in the first polypeptide of the activatable anti-EGFR, anti-CD3 HBPC is a T-cell CD3-targeting domain comprising a single-chain variable fragment (scFv) that specifically binds a CD3 polypeptide. In some aspects, the CD3 polypeptide is the epsilon chain of CD3. In some aspects, the scFv (anti-CD3 scFv) employed herein comprises a heavy chain variable domain (VH1) and a light chain variable domain (VL1).

[0107] The VH1 comprises a variable heavy chain CDR1 (VH CDR1, also referred to herein as CDRH1), a variable heavy chain CDR2 (VH CDR2, also referred to herein as CDRH2), and a variable heavy chain CDR3 (VH CDR3, also referred to herein as CDRH3), the VL1 comprises a variable light chain CDR1 (VL CDR1, also referred to herein as CDRL1), a variable light chain CDR2 (VL CDR2, also referred to herein as CDRL2), and a variable light chain CDR3 (VL CDR3, also referred to herein as CDRL3).

[0108] The activatable anti-EGFR, anti-CD3 HBPC provided herein comprises a masking moiety (MM). As used herein, the terms "masking moiety" and "MM", are used interchangeably to refer to a peptide that, when positioned proximal to a targeting domain, interferes with binding of the targeting domain to its target. In some aspects, the MM is an amino acid sequence that is coupled, or otherwise attached, to the activatable anti-EGFR, anti-CD3 HBPC and is attached to the activatable anti-EGFR, anti-CD3 HBPC such that each MM reduces the ability of the activatable anti-EGFR, anti-CD3 HBPC to specifically bind to its targets. In some aspects, MM1 prevents or decreases the ability of the activatable anti-EGFR, anti-CD3 HBPC from specifically binding to CD3. In some aspects, MM2 prevents or decreases the ability of the activatable anti-EGFR, anti-CD3 HBPC from specifically binding to EGFR. In some aspects, the MM binds specifically to the antigen targeting domain(s). Suitable MMs can be identified using any of a variety of known techniques.

[0109] For example, anti-EGFR masking moi eties that are suitable for use in the practice of the present disclosure in connection with a variety of antibody binding domains include any that are known in the art, including those described in, for example, PCT Publication Nos. WO 2013 / 163631, WO 2015 / 013671, WO 2016 / 014974, WO 2019 / 075405, and WO 2019 / 213444, each of which are incorporated herein by reference in their entireties. Anti- CD3 masking moieties that are suitable for use in the practice of the present disclosure include any of those that are known in the art, including those described in, for example, WO2013 / 163631, WO 2015 / 013671, WO 2016 / 014974, WO 2019 / 075405, and WO 2019 / 213444, each of which is incorporated herein by reference in their entireties.

[0110] In some aspects of the activatable anti-EGFR, anti-CD3 HBPC provided herein, theMM1 and / or the MM2 comprises from 5 amino acids to about 40 amino acids, or any range there between, and including both 5 amino acids and 40 amino acids. As used herein, the term “MMI” indicates a masking moiety for the CD3 targeting domain. As used herein, the term “MM2” indicates a masking moiety on the EGFR targeting domain.[OHl] In some aspects of the activatable anti-EGFR, anti-CD3 HBPC provided herein, MMI is selected from the group consisting of SEQ ID NOs: 1, 67, 68, 69, 70, 71, and 72. In some aspects, MMI comprises the amino acid sequence of SEQ ID NO: 1. In some aspects, MM2 comprises the amino acid sequence of SEQ ID NO: 13. In some aspects, MMI comprises SEQ ID NO: 1 and MM2 is SEQ ID NO: 13. In some aspects, MMI comprises SEQ ID NO:72 and MM2 comprises SEQ ID NO: 13.

[0112] In some aspects of the present disclosure, the single-chain variable fragment comprise a heavy chain variable domain (VH1) comprising: (i) a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), (ii) a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), and (iii) a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:5); and a light chain variable domain (VL1) comprising (i) a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:6), (ii) a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NOV), and (iii) a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO:8).

[0113] In some aspects of the present disclosure, a VH1 comprises the amino acid sequence of SEQ ID NO:9. In some aspects of the present disclosure, a VL1 comprises the amino acid sequence of SEQ ID NO: 10. In a specific aspect of the present disclosure, the scFv comprises the amino acid sequence of SEQ ID NO: 11 (which comprises SEQ ID NOs: 9 and 10).

[0114] In some aspects of the present disclosure, VH1 comprises an amino acid sequence that is at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOV. In some aspects of the present disclosure, VL1 comprises an amino acid sequence that is at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10.

[0115] In some aspects of the present disclosure, the first polypeptide single-chain variable fragment comprises a heavy chain variable domain (VH1) comprising: (i) a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), (ii) a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), (iii) a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:5), and comprises a heavy chain variable domain at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOV.

[0116] In some aspects of the present disclosure, VL1 comprises an amino acid sequence that comprises (i) a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NOV), (ii) a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NOV), (iii) a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO:8), wherein the amino acid sequence of VL1 is at least 90% identical, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10.

[0117] In some aspects, when the VH1 comprises: (i) a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), (ii) a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), and (iii) a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:5); and the VL1 comprises (i) a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:6), (ii) a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO:7), and (iii) a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO:8), the MM1 comprises the amino acid sequence of SEQ ID NO: 1.

[0118] In an alternative aspect, the single-chain variable fragment comprises a heavy chain variable domain (VH1) comprising: (i) a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128), (ii) a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129) and (iii) a VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 130); and a light chain variable domain (VL1) comprising (i) a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 131), (ii) a VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO: 132) (iii) a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO: 133).

[0119] In some of these aspects of the present disclosure, VH1 comprises the amino acid sequence of SEQ ID NO: 134. In certain aspects of the present disclosure, VL1 comprises the amino acid sequence of SEQ ID NO: 135. In a specific aspect of the present disclosure, the scFv comprises the amino acid sequence of SEQ ID NO: 122 (which comprises SEQ ID NOs: 134 and 135).

[0120] In some aspects of the present disclosure, VH1 comprises an amino acid sequence that is at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 134. In some aspects of the present disclosure, VL1 comprises an amino acid sequence that is at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 135.

[0121] In some aspects of the present disclosure, the first polypeptide single-chain variable fragment comprises a heavy chain variable domain (VH1) comprising: (i) a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128), (ii) a VH CDR2comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129), (iii) a VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 130), and comprises a heavy chain variable domain at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 134.

[0122] In some aspects of the present disclosure, VL1 comprises an amino acid sequence that comprises (i) a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 131), (ii) a VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO: 132), (iii) a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO: 133), wherein the amino acid sequence of VL1 is at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 135.

[0123] In some of these aspects, when the VH1 comprises (i) a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128), (ii) a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129) and (iii) a VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY(SEQ ID NO: 130); and the VL1 comprises (i) a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN(SEQ ID NO: 131), (ii) a VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO: 132), (iii) a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO: 133), the MM1 comprises the amino acid sequence of SEQ ID NO:72.

[0124] The EGFR targeting domain comprises a VH2 (disposed within the 1stpolypeptide) and a VH1 (disposed within the second polypeptide). The VH2 comprises a variable heavy chain CDR1 (VH CDR1, also referred to herein as CDRH1), a variable heavy chain CDR2 (VH CDR2, also referred to herein as CDRH2), and a variable heavy chain CDR3 (VH CDR3, also referred to herein as CDRH3), the VL2 comprises a variable light chain CDR1 (VL CDR1, also referred to herein as CDRL1), a variable light chain CDR2 (VL CDR2, also referred to herein as CDRL2), and a variable light chain CDR3 (VL CDR3, also referred to herein as CDRL3).

[0125] In some aspects of the present disclosure, the EGFR-targeting heavy chain variable domain (VH2) comprises: (i) a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15), (ii) a VH CDR2 comprising the amino acid sequenceVIWSGGNTDYNTPFTS (SEQ ID NO: 16), and (iii) a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17).

[0126] In some aspects of the present disclosure, VH2 comprises: (i) a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15), (ii) a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and (iii) a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17, wherein the amino acid sequence of VH2 that is at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:21

[0127] In some aspects of the present disclosure, VH2 comprises the amino acid sequence of SEQ ID NO:21.

[0128] In certain specific aspects of the present disclosure, the activatable HBPC comprises a first polypeptide comprising an MM1 having the amino acid sequence of SEQ ID NO: 1; a VH1 having a VH CDR1 comprising the amino acid sequence of SEQ ID NO:3, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:4, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 5; a VL1 having a VL CDR 1 having the amino acid sequence of SEQ ID NO:6, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:7, and a VL CDR3 having the amino acid sequence of SEQ ID NO:8; and a VH2 comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17. In some of these activatable HBPCs, the second polypeptide comprises an MM2 having the amino acid sequence of SEQ ID NO: 13 and a VL2 comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:20.

[0129] In another specific aspect of the present disclosure, the activatable HBPC comprises a first polypeptide comprising an MM1 having the amino acid sequence of SEQ ID NO:72; a VH1 having a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 128, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 129, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 130; a VL1 having a VL CDR 1 having the amino acid sequence of SEQ ID NO: 131, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 132, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 133; and a VH2 comprising a VH CDR1 comprising the amino acid sequence of SEQID NO: 15, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 17. In some of these activatable HBPCs, the second polypeptide comprises an MM2 having the amino acid sequence of SEQ ID NO: 13 and a VL2 comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:20.

[0130] As described above, the first polypeptide further comprises a monomeric Fc domain (Fcl). Fc domains that are known in the art are suitable for use in the activatable HBPCs of the present disclosure and are described herein below in more detail.

[0131] In some aspects of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide described herein, the Fcl comprises an amino acid sequence that is at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:23. In some aspects, the Fcl comprises the amino acid sequence of SEQ ID NO:23. In certain aspects, the Fcl comprises the amino acid sequence of SEQ ID NO:24.

[0132] In some aspects of the activatable anti-EGFR, anti-CD3 HBPC described herein, the first polypeptide further comprises a heavy chain CHI domain disposed between the VH2 and the Fcl.

[0133] In some aspects of the activatable anti-EGFR, anti-CD3 HBPC described herein, the first polypeptide further comprises an immunoglobulin hinge region disposed between the VH2 and the Fcl . In some aspects where a CHI domain is present, the immunoglobulin hinge region is disposed between the CHI domain and the Fcl domain.

[0134] In some aspects of the activatable anti-EGFR, anti-CD3 HBPC described herein, the first polypeptide comprises a structural arrangement from amino-terminus to carboxyterminus of:MMl-CMl-scFv-VH2-CHl-hinge region-Fcl, wherein each is independently a direct or indirect (e.g., via a linker) linkage.

[0135] In some aspects of the activatable anti-EGFR, anti-CD3 HBPC described herein, the first polypeptide further comprises one or more optional linkers, which are described herein below in more detail.

[0136] In some aspects of the present disclosure, the activatable anti-EGFR, anti-CD3 HBPC comprises a first polypeptide comprising an Fcl having the amino acid sequence set forth in SEQ ID NO:23 or SEQ ID NO:24. In some aspects of the present disclosure, anactivatable anti-EGFR, anti-CD3 HBPC comprises a first polypeptide comprising a hinge region having the sequence of Hinge-1 (SEQ ID NO:34) or Hinge-2 (SEQ ID NO:35).

[0137] In some aspects of the present disclosure, an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide comprises a second polypeptide comprising an EGFR-targeting light chain variable domain (VL2) that comprises a VL CDR1, VL CDR2, and VL CDR3.

[0138] In some aspects, the present disclosure provides an activatable anti-EGFR, anti- CD3 HBPC comprising a second polypeptide comprising an EGFR-targeting light chain variable domain (VL2) comprising: (i) a CDR1 comprising the amino acid sequence RASQSIGTNIH (SEQ ID NO: 18), (ii) a CDR2 comprising the amino acid sequence YASESIS (SEQ ID NO: 19), and (iii) a CDR3 comprising the amino acid sequence QQNNNWPTT (SEQ ID NO:20).

[0139] In some aspects of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide described herein, the second polypeptide comprises a VL2 having an amino acid sequence that is at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:22.

[0140] In some aspects of the present disclosure, the VL2 comprises the amino acid sequence set forth in SEQ ID NO:22.

[0141] In certain of the above-described aspects of the present disclosure, MM2 comprises the amino acid sequence of SEQ ID NO: 13.

[0142] In some aspects of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide described herein, the second polypeptide can comprise a structural arrangement from amino-terminus to carboxy -terminus of: MM2-CM2-VL2, wherein each is independently a direct or indirect (e.g., via a linker) linkage.

[0143] In some aspects of the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide described herein, the second polypeptide comprises one or more linkers. In some aspects, MM2 is joined to CM2 via a linker.

[0144] In some aspects, the second polypeptide of the activatable anti-EGFR, anti-CD3 HBPC described herein further comprises a linker comprising between about 1 and about 20 amino acids. Linkers suitable for use in the present disclosure are discussed in more detail below.

[0145] In some aspects, the second polypeptide further comprises a constant light chain domain (CL). Exemplary CLs include any of those known in the art. In some aspects, the second polypeptide comprises a CL having the amino acid sequence of SEQ ID NO:25. In certain of these aspects, the second polypeptide comprises a structural arrangement from amino-terminus to carboxy-terminus of: MM2-CM2-VL2-CL, wherein each is independently a direct or indirect (e.g., via a linker) linkage.

[0146] In some aspects, the third polypeptide of the activatable HBPC described herein comprises a monomeric Fc domain (Fc2) and does not comprise an immunoglobulin variable domain.

[0147] In some aspects, the activatable anti-EGFR, anti-CD3 HBPC disclosed herein comprises a third polypeptide comprising a structural arrangement from amino-terminus to carboxy-terminus of: hinge region-Fc2, wherein each is independently a direct or indirect (e.g., via a linker) linkage. In some aspects, the third polypeptide comprises an Fc2 having an amino acid sequence comprising SEQ ID NO:28 (optionally, with a C-terminal lysine, i.e., SEQ ID NO:29). In one aspect, the third polypeptide comprises a hinge comprising the amino acid sequence of SEQ ID NO:35 and an Fc2 comprising the amino acid sequence of SEQ ID NO:28 (optionally, with a C-terminal lysine, i.e., SEQ ID NO:29). In certain aspects, the first polypeptide comprises a hinge comprising the amino acid sequence of SEQ ID NO:34 and an Fcl comprising the amino acid sequence of SEQ ID NO:23 (optionally, with a C-terminal lysine, i.e. SEQ ID NO:24).

[0148] As provided above, in some aspects, the third polypeptide can comprise a linker, for example between a hinge region and a second Fc domain. The linker can comprise any of the linkers discussed herein.

[0149] The activatable anti-EGFR, anti-CD3 HBPCs of the disclosure are activated when the cleavable moiety is cleaved by a protease, thereby generating an activated (i.e., unmasked) anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) that is capable of binding to EGFR and CD3. By comparison, the activatable anti- EGFR, anti-CD3 HBPCs exhibit greatly reduced binding to EGFR and CD3 compared to the activated heteromultimeric bispecific polypeptide because the activatable HBPC remains masked until activated by proteases in the tumor environment. Without wishing to by bound by theory or mechanism, the typical protease activity levels in healthy tissues are likely reduced due to the presence of endogenous inhibitors and / or unfavorable protease pH conditions, while protease activity is generally up-regulated within the tumorenvironment through up-regulation of protease expression, activation of zymogen, downmodulation of inhibitor expression, or a combination of these effects (See Desnoyers et al., ScienceTranslationalMedicine.org, vol. 5, Issue 207 (October 2013), hereby incorporated by reference).

[0150] These activatable anti-EGFR, anti-CD3 HBPCs can therefore be useful in the treatment of a subject having cancer, where proteolytic activity in the tumor microenvironment is upregulated relative to normal tissue and controlled in normal tissues. The greatly reduced binding to EGFR and CD3 of the activatable HBPCs in normal tissue may allow for a reduction in the side effects associated with anti-EGFR and anti-CD3 engagement outside the tumor. In some aspects, the activatable anti-EGFR, anti-CD3 HBPC comprises a first CM and a second CM (CM1 and CM2, respectively).

[0151] In some aspects, the CM comprises a substrate for a protease that is upregulated in tumor cells. In some aspects of the HBPC disclosed herein, the CM may comprise a substrate for two or more proteases (i.e., a first protease, a second protease, a third protease, etc.) that are upregulated in tumor cells. There are reports in the literature of increased levels of proteases in a number of cancers, e.g., liquid tumors or solid tumors. See, e.g., La Rocca et al, (2004) British J. of Cancer 90(7): 1414-1421. Numerous studies have demonstrated the correlation of aberrant protease levels, e.g., uPA, legumain, MT-SP1, matrix metalloproteases (MMPs), in solid tumors. (See e.g., Murthy R V, et al. “Legumain expression in relation to clinicopathologic and biological variables in colorectal cancer,” Clin Cancer Res. 11 (2005): 2293-2299; Nielsen B S, et al. “Urokinase plasminogen activator is localized in stromal cells in ductal breast cancer,” Lab Invest 81 (2001): 1485- 1501; Look O R, et al. “In situ localization of gelatinolytic activity in the extracellular matrix of metastases of colon cancer in rat liver using quenched fluorogenic DQ-gelatin,” J Histochem Cytochem. 51 (2003): 821-829). A CM may comprise a substrate for multiple proteases, e.g. a substrate for a serine protease and a second different protease, e.g. an MMP. In some aspects, a CM may comprise a substrate for more than one serine protease, e.g., a matriptase and / or uPA. In some aspects, a CM may comprise a substrate for more than one MMP, e.g., MMP9 and MMP14.

[0152] In certain embodiments, CM1 and CM2 each independently comprise an amino acid sequence that is a substrate for a protease set forth in Table 1, below.Table 1. Exemplary Proteases

[0153] In some aspects of an activatable anti-EGFR, anti-CD3 HBPC described herein, theCM1 and / or the CM2 includes about three amino acids to about 15 amino acids. In someaspects, the CM1 and / or CM2 may comprise two or more cleavage sites. In some aspects, the two or more cleavage sites on CM1 may comprise a substrate for one protease. In some aspects, the two or more cleavage sites on CM2 may comprise a substrate for two or more proteases. In some aspects, the first protease and the second protease are the same protease. In some aspects, CM1 and CM2 comprise different substrates for the same protease. In some aspects, the CM1 and CM2 comprise the same amino acid sequence. In some aspects, the CM1 and CM2 comprise different amino acid sequences. In some aspects, CM1 comprises the amino acid sequence of SEQ ID NO:73. In some aspects, CM1 comprises the amino acid sequence of SEQ ID NO:2. In some aspects, CM2 comprises the amino acid sequence of SEQ ID NO: 14. In certain aspects, the activatable anti-EGFR, anti-CD3 HBPC described herein comprises a CM1 comprising the amino acid sequence of SEQ ID NO:2 and a CM2 comprising the amino acid sequence of SEQ ID NO: 14. In some aspects, the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complexes described herein comprise a CM1 that comprises the amino acid sequence of SEQ ID NO:73 and a CM2 the comprises the amino acid sequence of SEQ ID NO: 14.

[0154] Exemplary CMs that are suitable for use in the activatable anti-EGFR, anti-CD3 HBPC described herein include those which are known in the art. Exemplary CMs include but are not limited to those described in, for example, Table 2, and International Publication Nos.: WO 2009 / 025846, WO 2010 / 081173, WO 2015 / 013671, WO 2015 / 048329, WO 2015 / 116933, WO 2016 / 014974, and WO 2016 / 118629, each of which is incorporated herein by reference in its entirety.

[0155] In some aspects, CM1 and / or CM2 comprise an amino acid sequence set forth in Table 2 below. In certain aspects, CM1 and CM2 each independently comprise an amino acid sequence set forth in Table 2 below.Table 2. Cleavable Moieties

[0156] In some aspects of the present disclosure, when the activatable HBPC comprises (i) a heavy chain variable domain (VH1) comprising, a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), and a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:5); and a VL1 comprising a VL CDR1comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:6), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO:7), and a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO:8), the CM1 comprises the amino acid sequence of SEQ ID NO:2. In certain of these activatable HBPCs, MM1 comprises the amino acid sequence of SEQ ID NO: 1.

[0157] In some aspects of the present disclosure, when the activatable HBPC comprises (i) a heavy chain variable domain (VH1) comprising, a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), and a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:5); and a VL1 comprising a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:6), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO:7), and a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO:8), the CM1 comprises the amino acid sequence of SEQ ID NO:73. In certain of these activatable HBPCs, MM1 comprises the amino acid sequence of SEQ ID NO: 1. In some of these activatable HBPCs, MM1 comprises the amino acid sequence of SEQ ID NO: 1 and CM1 comprises the amino acid sequence of SEQ ID NO: 73.

[0158] In a specific aspect of the present disclosure, the activatable HBPC comprises:(a) a first polypeptide comprising a first heavy chain variable domain (VH1), a first light chain variable domain (VL1), and a second heavy chain variable domain (VH2), a first masking moiety (MM1), a first cleavable moiety (CM1), and a first Fc domain (Fcl), wherein the VH1 comprises:(i) a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3),(ii) a VH CDR2 comprising the amino acid sequenceRIRSKYNNYATYYADSVKD (SEQ ID NO:4), and(iii) a VH CDR3 comprising the amino acid sequenceHGNFGNSYISYWAY (SEQ ID NO: 5), wherein the VL1 comprises:(i) a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:6),(ii) a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO: 7), and(iii) a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO: 8); wherein the VH2 comprises:(i) a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15),(ii) a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and(iii) a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17);(b) a second polypeptide comprising a second light chain variable domain (VL2) and a second masking moiety (MM2) and a second cleavable moiety (CM2), wherein the VL2 comprises:(i) a VL CDR1 comprising RASQSIGTNIH (SEQ ID NO: 18),(ii) a VL CDR2 comprising YASESIS (SEQ ID NO: 19), and(iii) a VL CDR3 comprising QQNNNWPTT (SEQ ID NO:20); and(c) a third polypeptide comprising a second Fc domain (Fc2), wherein the Fcl binds to Fc2, wherein the VH1 and the VL1 together form a targeting domain that specifically binds a CD3 polypeptide, wherein the VH2 and the VL2 together form a targeting domain that specifically binds EGFR, wherein the third polypeptide does not comprise an immunoglobulin variable domain, wherein MM1 is a peptide that interferes with binding of the first targeting domain to the first target, wherein MM2 is a peptide that interferes with binding of the second targeting domain to the second target, and wherein CM1 and CM2 each independently comprise a substrate for a protease, and wherein the third polypeptide does not comprise an immunoglobulin variable domain. In certain aspects of the present disclosure the VH1 and the VL1 are disposed within an scFv. In some of these aspects, MM1 comprises SEQ IDNO: 1, CM1 comprises SEQ ID NO:73, MM2 comprises SEQ ID NO: 13, and CM2 comprises SEQ ID NO: 14. In some of these aspects, the second polypeptide further comprises a constant light domain (CL) and the third polypeptide further comprises a hinge (HR).

[0159] In some aspects, the activatable HBPC comprises:(a) a first polypeptide comprising(i) a single-chain variable fragment (scFv) comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), wherein the VH1 and the VL1 together form a T-cell cluster of differentiation (CD3)-targeting domain the specifically binds a CD3 polypeptide, wherein the scFv comprises a set of VH1 and VL1 selected from:(A) a VH1 comprising: a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), and a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO: 5), and a VL1 comprising: a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO: 6), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO:7), and a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO: 8); or(B) a VH1 comprising: a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129), and a VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 130), and a VL1 comprising: a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 131), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO: 132), and a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO: 133);(ii) a first masking moiety (MM 1),(iii) a first cleavable moiety (CM1),(iv) a second heavy chain variable domain (VH2) comprising a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15), a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17);(v) a first monomeric Fc domain (Fcl);(b) a second polypeptide comprising:(i) a second light chain variable domain (VL2), wherein the VH2 and the VL2 together form an EGFR targeting domain that specifically binds EGFR, wherein the VL2 comprises a VL CDR1 comprising the amino acid sequence RASQSIGTNIH (SEQ ID NO: 18), a VL CDR2 comprising the amino acid sequence YASESIS (SEQ ID NO: 19), and a VL CDR3 comprising the amino acid sequence QQNNNWPTT (SEQ ID NO:20),(ii) a second masking moiety (MM2), and(iii) a second cleavable moiety (CM2); and(c) a third polypeptide that(i) comprises a second monomeric Fc domain (Fc2), and(ii) does not comprise an immunoglobulin variable domain.

[0160] In some aspects, the first polypeptide comprises the scFv, MM1, CM1, VH2, and Fcl in the N-terminal to C-terminal orientation. In some aspects, the scFv comprises the VH1 and VL1 in the N-terminal to C-terminal orientation.

[0161] In some aspects, the second polypeptide comprises the VL2, MM2, and CM2 in the N-terminal to C-terminal orientation.

[0162] In some aspects, the HBPC comprises the first polypeptide, second polypeptide, and third polypeptide in the N-terminal to C-terminal orientation.

[0163] In some aspects, the MM1 comprises an amino acid sequence that is selected from the group consisting of SEQ ID NOs: 1, 67, 68, 69, 70, 71, and 72.

[0164] In some aspects, the VH1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NOV and / or wherein the VL1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 10

[0165] In some aspects, the VH1 comprises the amino acid sequence of SEQ ID NOV and the VL1 comprises the amino acid sequence of SEQ ID NO: 10.

[0166] In some aspects, the MM1 comprises the amino acid sequence of SEQ ID NO: 1.

[0167] In some aspects, the VH1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 134 and / or wherein the VL1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 135.

[0168] In some aspects, the VH1 comprises the amino acid sequence of SEQ ID NO: 134 and the VL1 comprises the amino acid sequence of SEQ ID NO: 135.

[0169] In some aspects, the MM1 comprises the amino acid sequence of SEQ ID NO:72.

[0170] In some aspects, the MM2 comprises the amino acid sequence of SEQ ID NO: 13.

[0171] In some aspects, the CM1 and the CM2 each independently comprise a substrate for a protease selected from the group of proteases shown in Table 1.

[0172] In some aspects, the CM1 and the CM2 each independently comprise a substrate having an amino acid sequence selected from the group shown in Table 2.

[0173] In some aspects, the CM1 comprises the amino acid sequence of SEQ ID NO:73.

[0174] In some aspects, the CM2 comprises the amino acid sequence of SEQ ID NO: 14.

[0175] In some aspects, the first polypeptide comprises the amino acid sequence of SEQID NO: 144, and / or the second polypeptide comprising the amino acid sequence of SEQ ID NO:37, and / or the third polypeptide comprising the amino acid sequence of SEQ ID NO:32.

[0176] In some aspects, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 144, the second polypeptide comprising the amino acid sequence of SEQ ID NO:37, and the third polypeptide comprising the amino acid sequence of SEQ ID NO:32.

[0177] In some aspects, the activatable HBPC comprises:(a) a first polypeptide comprising(i) a single-chain variable fragment (scFv) comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), wherein the VH1 and the VL1 together form a T-cell cluster of differentiation (CD3)-targeting domain the specifically binds a CD3 polypeptide, wherein the scFv comprises a VH1 and VL1 comprising: a VH1 comprising: a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), and a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:5), anda VL1 comprising: a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:6), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO:7), and a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO: 8); and(ii) a first masking moiety (MM 1),(iii) a first cleavable moiety (CM1),(iv) a second heavy chain variable domain (VH2) comprising a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15), a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17);(v) a first monomeric Fc domain (Fcl);(b) a second polypeptide comprising:(i) a second light chain variable domain (VL2), wherein the VH2 and the VL2 together form an EGFR targeting domain that specifically binds EGFR, wherein the VL2 comprises a VL CDR1 comprising the amino acid sequence RASQSIGTNIH (SEQ ID NO: 18), a VL CDR2 comprising the amino acid sequence YASESIS (SEQ ID NO: 19), and a VL CDR3 comprising the amino acid sequence QQNNNWPTT (SEQ ID NO:20),(ii) a second masking moiety (MM2), and(iii) a second cleavable moiety (CM2); and(c) a third polypeptide that(i) comprises a second monomeric Fc domain (Fc2), and(ii) does not comprise an immunoglobulin variable domain.

[0178] In some aspects, the first polypeptide comprises the scFv, MM1, CM1, VH2, and Fcl in the N-terminal to C-terminal orientation. In some aspects, the scFv comprises the VH1 and VL1 in the N-terminal to C-terminal orientation.

[0179] In some aspects, the second polypeptide comprises the VL2, MM2, and CM2 in the N-terminal to C-terminal orientation.

[0180] In some aspects, the HBPC comprises the first polypeptide, second polypeptide, and third polypeptide in the N-terminal to C-terminal orientation.

[0181] In some aspects, the MM1 comprises an amino acid sequence that is selected from the group consisting of SEQ ID NOs: 1, 67, 68, 69, 70, 71, and 72.

[0182] In some aspects, the VH1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:9 and / or wherein the VL1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 10

[0183] In some aspects, the VH1 comprises the amino acid sequence of SEQ ID NO:9 and the VL1 comprises the amino acid sequence of SEQ ID NO: 10.

[0184] In some aspects, the MM1 comprises the amino acid sequence of SEQ ID NO: 1.

[0185] In some aspects, the MM2 comprises the amino acid sequence of SEQ ID NO: 13.

[0186] In some aspects, the CM1 and the CM2 each independently comprise a substrate for a protease selected from the group of proteases shown in Table 1.

[0187] In some aspects, the CM1 and the CM2 each independently comprise a substrate having an amino acid sequence selected from the group shown in Table 2.

[0188] In some aspects, the CM1 comprises the amino acid sequence of SEQ ID NO:73.

[0189] In some aspects, the CM2 comprises the amino acid sequence of SEQ ID NO: 14.

[0190] In some aspects, the first polypeptide comprises the amino acid sequence of SEQID NO: 144, and / or the second polypeptide comprising the amino acid sequence of SEQ ID NO:37, and / or the third polypeptide comprising the amino acid sequence of SEQ ID NO:32.

[0191] In some aspects, the activatable HBPC comprises:(a) a first polypeptide comprising(i) a single-chain variable fragment (scFv) comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), wherein the VH1 and the VL1 together form a T-cell cluster of differentiation (CD3)-targeting domain the specifically binds a CD3 polypeptide, wherein the scFv comprises a VH1 and VL1 comprising: a VH1 comprising: a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129), and a VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 130), and a VL1 comprising: a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 131), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO: 132), and a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO: 133);(ii) a first masking moiety (MM 1),(iii) a first cleavable moiety (CM1),(iv) a second heavy chain variable domain (VH2) comprising a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15), a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17);(v) a first monomeric Fc domain (Fcl);(b) a second polypeptide comprising:(i) a second light chain variable domain (VL2), wherein the VH2 and the VL2 together form an EGFR targeting domain that specifically binds EGFR, wherein the VL2 comprises a VL CDR1 comprising the amino acid sequence RASQSIGTNIH (SEQ ID NO: 18), a VL CDR2 comprising the amino acid sequence YASESIS (SEQ ID NO: 19), and a VL CDR3 comprising the amino acid sequence QQNNNWPTT (SEQ ID NO:20),(ii) a second masking moiety (MM2), and(iii) a second cleavable moiety (CM2); and(c) a third polypeptide that(i) comprises a second monomeric Fc domain (Fc2), and(ii) does not comprise an immunoglobulin variable domain.

[0192] In some aspects, the first polypeptide comprises the scFv, MM1, CM1, VH2, and Fcl in the N-terminal to C-terminal orientation. In some aspects, the scFv comprises the VH1 and VL1 in the N-terminal to C-terminal orientation.

[0193] In some aspects, the second polypeptide comprises the VL2, MM2, and CM2 in the N-terminal to C-terminal orientation.

[0194] In some aspects, the HBPC comprises the first polypeptide, second polypeptide, and third polypeptide in the N-terminal to C-terminal orientation.

[0195] In some aspects, the MM1 comprises an amino acid sequence that is selected from the group consisting of SEQ ID NOs: 1, 67, 68, 69, 70, 71, and 72.

[0196] In some aspects, the VH1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 134 and / or wherein the VL1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 135.

[0197] In some aspects, the VH1 comprises the amino acid sequence of SEQ ID NO: 134 and the VL1 comprises the amino acid sequence of SEQ ID NO: 135.

[0198] In some aspects, the MM1 comprises the amino acid sequence of SEQ ID NO:72.

[0199] In some aspects, the MM2 comprises the amino acid sequence of SEQ ID NO: 13.

[0200] In some aspects, the CM1 and the CM2 each independently comprise a substrate for a protease selected from the group of proteases shown in Table 1.

[0201] In some aspects, the CM1 and the CM2 each independently comprise a substrate having an amino acid sequence selected from the group shown in Table 2.

[0202] In some aspects, the CM1 comprises the amino acid sequence of SEQ ID NO:73.

[0203] In some aspects, the CM2 comprises the amino acid sequence of SEQ ID NO: 14.

[0204] In some aspects, the activatable HBPC comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 144, and / or a second polypeptide comprising the amino acid sequence of SEQ ID NO:37, and / or a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32.

[0205] In some aspects, the activatable HBPC comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO:30, and / or a second polypeptide comprising the amino acid sequence of SEQ ID NO:31, and / or a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32.

[0206] In some aspects, the activatable HBPC comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO:30; a second polypeptide comprising the amino acid sequence of SEQ ID NO:31; and a third polypeptide comprising the amino acid sequence of SEQ ID NO:32.

[0207] In some aspects of the present disclosure, when the activatable HBPC comprises (i) a VH1 comprising a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129) and a VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 130); and (ii) a VL1 comprising a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 131), a VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO: 132), a VL CDR3 comprising the amino acid sequence of ALWYSNLWV (SEQ ID NO: 133), the CM1 comprises the amino acid sequence of SEQ ID NO: 73. In some of these activatable HBPCs, MM1 comprises the amino acid sequence of SEQ ID NO:72.

[0208] In some of the above-described activatable HBPCs, the first polypeptide further comprises a VH2 having a VH CDR1 comprising the amino acid sequence of SEQ ID NO:15, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:16, a VH CDR3comprising the amino acid sequence of SEQ ID NO: 17. In certain of these activatable HBPCs, the second polypeptide comprises a VL2 comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:20. In some of these HBPCs, the third polypeptide comprises the amino acid sequence of SEQ ID NO:28 (and no immunoglobulin variable domain).

[0209] In a specific aspect of the present disclosure, the activatable HBPC comprises:(i) a first polypeptide comprising a first heavy chain variable domain (VH1), a first light chain variable domain (VL1), and a second heavy chain variable domain (VH2), a first masking moiety (MM1), a first cleavable moiety (CM1), and a first Fc domain (Fcl), wherein the VH1 comprises:(i) a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128),(ii) a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129),(iii) a VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 130); wherein the VL1 comprises(i) a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 131),(ii) a VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO: 132), and(iii) a VL CDR3 comprising the amino acid sequence of ALWYSNLWV (SEQ ID NO: 133 wherein the VH2 comprises:(ii) a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15),(ii) a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and(iii) a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17)(b) a second polypeptide comprising a second light chain variable domain (VL2) and a second masking moiety (MM2) and a second cleavable moiety (CM2), wherein the VL2 comprises:(i) a VL CDR1 comprising RASQSIGTNIH (SEQ ID NO: 18),(ii) a VL CDR2 comprising YASESIS (SEQ ID NO: 19), and(iii) a VL CDR3 comprising QQNNNWPTT (SEQ ID NO:20); and(c) a third polypeptide comprising a second Fc domain (Fc2), wherein the Fcl binds to Fc2, wherein the VH1 and the VL1 together form a targeting domain that specifically binds a CD3 polypeptide, wherein the VH2 and the VL2 together form a targeting domain that specifically binds EGFR, wherein the third polypeptide does not comprise an immunoglobulin variable domain, wherein MM1 is a peptide that interferes with binding of the first targeting domain to the first target, wherein MM2 is a peptide that interferes with binding of the second targeting domain to the second target, and wherein CM1 and CM2 each independently comprise a substrate for a protease, and wherein the third polypeptide does not comprise an immunoglobulin variable domain. In certain aspects of the present disclosure the VH1 and the VL1 are disposed within an scFv. In some of these aspects, MM1 comprises SEQ ID NO:72, CM1 comprises SEQ ID NO:73, MM2 comprises SEQ ID NO: 13, and CM2 comprises SEQ ID NO:22. In some of these aspects, the second polypeptide further comprises a constant light domain (CL) and the third polypeptide further comprises a hinge (HR).

[0210] In some aspects of the activatable anti-EGFR, anti-CD3 HBPCs of the present disclosure, the first polypeptide comprises one or more linkers between the MM and the CM. In some aspects, MM1 is joined to CM1 via a linker. In some aspects, the first polypeptide comprises a linker between the CM1 and the VH2. In certain aspects, the first polypeptide comprises a linker between the VH2 and the Fcl. In some aspects, the first polypeptide comprises at least one linker that is disposed between a pair of elements selected from the group consisting of the MM1 and the CM1; the CM1 and the scFv; the scFv and the VH2; and the VH2 and the Fcl. Linkers suitable for use in the activatable anti-EGFR, anti-CD3 HBPCs described herein are generally ones that provide flexibilityof the activatable anti-EGFR, anti-CD3 HBPCs to facilitate the inhibition of the binding of the activatable polypeptide to the target. Such linkers are generally referred to as flexible linkers. Suitable linkers can be readily selected and can be of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0211] Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n and (GGGS)n (SEQ ID NO:41 and SEQ ID NO:40 respectfully), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycineserine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between components. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). The ordinarily skilled artisan will recognize that the polypeptides of the HBPC can be designed to include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure to provide for a desired structure.

[0212] In some aspects, the activatable anti-EGFR, anti-CD3 HBPCs comprise one or more linker sequences disposed in the first, second, and / or third polypeptides. For example in the first polypeptide, a linker is disposed between the MM1 and the CM1, between a heavy chain variable domain and a CHI domain, between a CHI domain and a hinge region if both are present, and / or between a hinge region if present and the first Fc domain. In the second polypeptide, which is described elsewhere herein, a linker can be present, for example, between the MM2 and the CM2, between the CM2 and the light chain variable domain, and / or between the light chain variable domain and a CL. In the third polypeptide, which is described elsewhere herein, a linker can be present, for example between a CHI domain and a second Fc domain, between a CHI domain and a hinge region, and or between a hinge region and a second Fc domain.

[0213] In some aspects of the activatable anti-EGFR, anti-CD3 HBPCs described herein, MM1 is linked to CM1 via linker LI. In some aspects, MM2 is linked to CM2 via linker L2. In some aspects, the amino acid sequence of LI and L2 are the same. In some aspects,the linker is selected from the group consisting of: (i) a glycine-serine-based linker selected from the group consisting of (GS)n, wherein n is an integer of at least 1, (GGS)n, wherein n is an integer of at least 1 (e.g., an integer from about 1 to about 20, or from about 1 to about 10), (GGGS)n (SEQ ID NO:40), wherein n is an integer of at least 1 (e.g., an integer from about 1 to about 20, or from about 1 to about 10), (GGGGS)n (SEQ ID NO: 126), where n is an integer of at least 1 (e.g., an integer from about 1 to about 20, or from about 1 to about 10), (GSGGS)n (SEQ ID NO:41), wherein n is an integer of at least 1 (e.g., an integer from about 1 to about 20, or from about 1 to about 10), GSSGGSGGSG (SEQ ID NO: 12), GGSG (SEQ ID NO:42), GGSGG (SEQ ID NO:43), GSGSG (SEQ ID NO:44), GSGGG (SEQ ID NO:45), GGGSG (SEQ ID NO:46), and GSSSG (SEQ ID NO:47), GGGGSGGGGSGGGGSGS (SEQ ID NO:48), GGGGSGS (SEQ ID NO:49), GGGGSGGGGSGGGGS (SEQ ID NO:50), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:51), GGGGS (SEQ ID NO:52), GGGGSGGGGS (SEQ ID NO:53), GGGS (SEQ ID NO:54), GGGSGGGS (SEQ ID NO:55), GGGSGGGSGGGS (SEQ ID NO:56), GSSGGSGGSGG (SEQ ID NO:57), GGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:58), GGGSSGGS (SEQ ID NO: 127) and GS; and (ii) a linker comprising glycine and serine, and at least one of lysine, threonine, or proline selected from the group consisting of GSTSGSGKPGSSEGST (SEQ ID NO:59), SKYGPPCPPCPAPEFLG (SEQ ID NO: 60), GGSLDPKGGGGS (SEQ ID NO:61), PKSCDKTHTCPPCPAPELLG (SEQ ID NO:62), GKSSGSGSESKS (SEQ ID NO:63), GSTSGSGKSSEGKG (SEQ ID NO:64), GSTSGSGKSSEGSGSTKG (SEQ ID NO:65), and GSTSGSGKPGSGEGSTKG (SEQ ID NO:66).

[0214] In some aspects of the present disclosure, an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide can comprise components in addition to those described above. Such components can include a spacer. The term “spacer” refers herein to an amino acid residue or a peptide incorporated at a free terminus of the first, second, and / or third polypeptide. Spacers that are suitable for use in the practice of the present disclosure include any single amino acid residue or any peptide. Suitable spacers include any of those described in, for example, International Publication Nos.: WO 2016 / 014974, WO 2019 / 075405, and WO 2019 / 213444, each of which is incorporated herein by reference in their entireties.

[0215] In some aspects, a spacer can comprise from about 1 amino acid to about 10 amino acids (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids) or any number there between. Insome aspects of an activatable anti-EGFR, anti-CD3 HBPC described herein, the spacer is N-terminally positioned relative to the MM1 and / or MM2. In some aspects, the spacer has a sequence of QGQSGS (SEQ ID NO: 116). In some aspects, the spacer has a sequence of QGQSGQG (SEQ ID NO: 117). In some aspects, the spacer has a sequence of QGQSGS (SEQ ID NO: 118). In some aspects, the spacer has a sequence of QGQSGQG (SEQ ID NO: 117).

[0216] In some aspects, the Fc domains employed as Fcl and / or Fc2 are native Fc domains (e.g., a human IgGl Fc domain or a human IgG4 Fc domain). In some aspects of the present disclosure, the Fc domains employed as Fcl and / or Fc2 are mutated forms of a native Fc amino acid sequence. The mutations may confer a desired beneficial property to the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide (and commensurately, the activated HBPC). For example, certain mutations in the FcRn binding site are known to modulate effector function (see, e.g., Petkova et al., Inti. Immunol. 18: 1759-1769, 2006; Deng et al., MAbs 4:101-109, 2012; and Olafson et al., Methods Mol. Biol. 907:537-556, 2012.) The inclusion of any known mutations in an Fc domain that can modulate effector function are suitable. For example, a N297A or N297G mutation in the Fc amino acid sequence may be employed to reduce IgG effector functions (e.g., ADCC and CDC) which may reduce target independent toxicities (see, e.g., Lund et al., Mol. Immunol. 29:35-39, 1992). The Fc domains suitable for use in the context of the present disclosure include any Fc domain known in the art, including but not limited to any known heterodimeric Fc, such as, for example, knob in holes, and the like.

[0217] In some aspects, the activatable anti-EGFR, anti-CD3 HBPC disclosed herein further comprises an immunoglobulin hinge region. Suitable hinge regions include any known hinge regions. For example, a hinge region from any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) are suitable for use in the present disclosure. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations.

[0218] In some aspects, the first polypeptide Fcl and the third polypeptide Fc2 hinge regions comprise the same sequence. In some aspects, the first and second Fc domains (Fcl and Fc2, respectively) of the activatable anti-EGFR, anti-CD3 HBPC described herein are IgGl Fc domains or IgG4 Fc domains (e.g., a human IgGl Fc domain or a human IgG4 Fc domain), or variants thereof. In some aspects, Fcl and / or Fc2 are modified variants of anative (e.g., human) IgGl Fc domain. In some aspects, Fcl and / or Fc2 are modified variants of a native (e.g., human) IgG4 Fc domain.

[0219] In some aspects of the activatable anti-EGFR, anti-CD3 HBPC described herein, the Fcl comprises an amino acid sequence that is at least 90% identical, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:23. In some aspects, the Fcl comprises the amino acid sequence of SEQ ID NO:23 (optionally with a C-terminal lysine (i.e., SEQ ID NO:24)).

[0220] In some aspects, the third polypeptide further comprises a monomeric Fc domain (Fc2) that binds to Fcl. In some aspects, Fc2 comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:28. In some aspects, the Fc2 comprises SEQ ID NO:28 (optionally with a terminal lysine (i.e., SEQ ID NO:29)).

[0221] In some aspects, the third polypeptide comprises a hinge region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 and 35.

[0222] As provided elsewhere herein, the format or structure of an activatable anti-EGFR, anti-CD3 HBPC disclosed herein can include any number of optional additional components, including linkers and spacers. By way of example only, the structures set forth below are among the contemplated aspects. However, the aspects shown below are not meant to limit the disclosure in any way.

[0223] In some aspects, the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide comprises a first polypeptide having a structure (I).First polypeptide structure (I):(Sl) - MMl - (Ll) - CMl - L2 - VH1 - L3 - VL1 - (L4) - VH2 - (L5) - (CH11) - (L6) - (Hingel) — (L7) - Fcl wherein• (SI) is an optional spacer;• (LI), (L4), (L5), (L6), and (L7) are each independently an optional linker,• L2 and L3 are linkers,• (CHI 1) is an optional CHI domain,• (Hingel) is an optional hinge region,• the Fcl is as described hereinabove.

[0224] In some aspects, the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide comprises a second polypeptide having a structure (II).Second polypeptide structure (II):(S2) - (L8) - MM2 - (L9) - CM2 - (LIO) -VL2 - (CL) wherein• (S2) is an optional spacer,• (L8), (L9), and (LIO) are each independently an optional linker,• MM2 is an anti-EGFR masking moiety, and• VL2 is as described hereinabove; and• (CL) is an optional light chain constant domain.

[0225] In some aspects, the activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide comprises a third polypeptide having a structure (III).Third polypeptide structure (III):(S3) - (CH12) - (LI 1) - (Hinge2) - (L12) - Fc2 wherein,• (S3) is an optional spacer,• (CH12) is an optional CHI domain,• (LI 1) and (LI 2) are each independently an optional linker, and• Fc2 is as described hereinabove.

[0226] Linkers, spacers, MMs, CMs, Fc domains, CHI (i.e., CH11 and CH12) domains, hinge regions, and CLs that are suitable for use in structures (I), (II), and (III) include any that are known in the art or that are described herein.

[0227] In some aspects of the present disclosure, an activatable anti-EGFR, anti-CD3 HBPC comprises first, second, and third polypeptides, wherein (1) the first polypeptide comprises the amino acid sequence of SEQ ID NO:30 (optionally with a C-terminal lysine and / or optionally without a spacer (e.g., SEQ ID NO: 120 (with a terminal lysine and without a spacer)), (2) the second polypeptide comprises the amino acid sequence of SEQ ID NO:31 (or SEQ ID NO:37 (without a spacer)), and (3) the third polypeptide comprises the amino acid sequence of SEQ ID NO:32 (optionally with a C-terminal lysine (i.e., SEQ ID NO:36) (and does not comprise an immunoglobulin variable domain). In some aspects of the present disclosure, an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide comprises a first, a second, and a third polypeptide, wherein: (1) the firstpolypeptide comprises the amino acid sequence of SEQ ID NO: 120, (2) the second polypeptide comprises the amino acid sequence of SEQ ID NO:37, and (3) the third polypeptide comprises the amino acid sequence of SEQ ID NO:32 (and does not comprise an immunoglobulin variable domain), as provided in the sequence below.

[0228] In some aspects of the present disclosure, an activatable anti-EGFR, anti-CD3 HBPC comprises first, second, and third polypeptides, wherein (1) the first polypeptide comprises the amino acid sequence of SEQ ID NO:30, (2) the second polypeptide comprises the amino acid sequence of SEQ ID NO:31, and (3) the third polypeptide consists of, or consists essentially of the amino acid sequence of SEQ ID NO:32. In some aspects of the present disclosure, an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide comprises a first, a second, and a third polypeptide, wherein: (1) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 120, (2) the second polypeptide comprises the amino acid sequence of SEQ ID NO:37, and (3) the third polypeptide consists of, or consists essentially of the amino acid sequence of SEQ ID NO:32, and does not comprise an immunoglobulin variable domain, as provided in the sequence below.

[0229] In some aspects of the present disclosure, an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide comprises a first, a second, and a third polypeptide, wherein: (1) the first polypeptide comprises the amino acid sequence of SEQ ID NO: 144, (2) the second polypeptide comprises the amino acid sequence of SEQ ID NO:37, and (3) the third polypeptide consists of, or consists essentially of the amino acid sequence of SEQ ID NO:32, and does not comprise an immunoglobulin variable domain, as provided in the sequence below.

[0230] In the first polypeptide shown below, the spacer sequence is in brackets, the mask sequence is underlined, the linkers are bolded, (the linker within the scFv is also italicized and underlined) the substrate (i.e., cleavable moiety) is italicized, and the scFv (which binds a CD3 polypeptide) is italicized and underlined.First Polypeptide[OGQSGS1VSTTCWWDPPCTPNTGSSGGSGGSGGZ5G7?5DD GGGSEFQZFE5GGGZFC> PGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDD SKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGG GGSOTWTOEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVOQKPGQAPRGLIGGTKFLAPG TPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGGGSQNQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTS RLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGST YRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:30), optionally with a C-terminal lysine and / or without spacer (e.g., SEQ ID NO: 137). In some aspects, the first polypeptide has the amino acid sequence of SEQ ID NO: 120 (without a spacer, but with a C-terminal lysine) or the amino acid sequence of SEQ ID NO: 144 (without a spacer and without a C-terminal lysine).

[0231] In the second polypeptide depicted below, the spacer sequence is in brackets, the mask sequence is underlined, the linkers are bolded, and the substrate (i.e., cleavable moiety) is italicized.Second Polypeptide [OGQSGQG1LSCEGWAMNREOCRAGGGSSGGS / 55GZZ5GA5Z GGGSQILLTQSPVIL SVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLS INSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:31), optionally without spacer (SEQ ID NO:37).

[0232] In the third polypeptide depicted below, the hinge region is bolded and underlined, and the remainder of the sequence is the Fc2.Third PolypeptideDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:32), optionally with a C-terminal lysine (SEQ ID NO:36).Therapeutic Uses and Methods

[0233] Disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC is administered at a dose (e.g., target dose) of about 1 pg to about 50,000 pg (0.001 mg to about 50 mg).

[0234] Disclosed herein is an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) for use in treating a subject afflicted with a cancer, wherein the HBPC is administered at a dose (e.g., target dose) of about 1 pg to about 50,000 pg (0.001 mg to about 50 mg).

[0235] Disclosed herein are methods of treating a subject afflicted with a cancer, the method comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising(i) a single-chain variable fragment (scFv) comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), wherein the VH1 and the VL1 together form a T-cell cluster of differentiation (CD3)-targeting domain the specifically binds a CD3 polypeptide, wherein the scFv comprises a set of VH1 and VL1 selected from:(A) a VH1 comprising: a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), and a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO: 5), and a VL1 comprising: a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO: 6), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO:7), and a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO: 8); or(B) a VH1 comprising: a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129), and a VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 130), and a VL1 comprising: a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 131), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO: 132), and a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO: 133);(ii) a first masking moiety (MM 1),(iii) a first cleavable moiety (CM1),(iv) a second heavy chain variable domain (VH2) comprising a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15), a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17);(v) a first monomeric Fc domain (Fcl);(b) a second polypeptide comprising:(i) a second light chain variable domain (VL2), wherein the VH2 and the VL2 together form an EGFR targeting domain that specifically binds EGFR, wherein the VL2 comprises a VL CDR1 comprising the amino acid sequence RASQSIGTNIH (SEQ ID NO: 18), a VL CDR2 comprising the amino acid sequence YASESIS (SEQ ID NO: 19), and a VL CDR3 comprising the amino acid sequence QQNNNWPTT (SEQ ID NO:20),(ii) a second masking moiety (MM2), and(iii) a second cleavable moiety (CM2); and(c) a third polypeptide that(i) comprises a second monomeric Fc domain (Fc2), and(ii) does not comprise an immunoglobulin variable domain; wherein the HBPC is administered at a dose (e.g., target dose) of about 1 pg (0.001 mg) to about 50,000 pg (50 mg).

[0236] Disclosed herein is an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) for use in treating are methods of treating a subject afflicted with a cancer, wherein the HBPC comprises: (a) a first polypeptide comprising(i) a single-chain variable fragment (scFv) comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), wherein the VH1 and the VL1 together form a T-cell cluster of differentiation (CD3)-targeting domain the specifically binds a CD3 polypeptide, wherein the scFv comprises a set of VH1 and VL1 selected from:(A) a VH1 comprising: a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), and a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO: 5), and a VL1 comprising: a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO: 6), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO:7), and a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO: 8); or(B) a VH1 comprising: a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129), and a VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 130), and a VL1 comprising: a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 131), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO: 132), and a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO: 133);(ii) a first masking moiety (MM 1),(iii) a first cleavable moiety (CM1),(iv) a second heavy chain variable domain (VH2) comprising a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15), a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17);(v) a first monomeric Fc domain (Fcl);(b) a second polypeptide comprising:(i) a second light chain variable domain (VL2), wherein the VH2 and the VL2 together form an EGFR targeting domain that specifically binds EGFR, wherein the VL2 comprises a VL CDR1 comprising the amino acid sequence RASQSIGTNIH (SEQ ID NO: 18), a VL CDR2 comprising the amino acid sequence YASESIS (SEQ ID NO: 19), and a VL CDR3 comprising the amino acid sequence QQNNNWPTT (SEQ ID NO:20),(ii) a second masking moiety (MM2), and(iii) a second cleavable moiety (CM2); and(c) a third polypeptide that(i) comprises a second monomeric Fc domain (Fc2), and(ii) does not comprise an immunoglobulin variable domain; wherein the HBPC is administered at a dose (e.g., target dose) of about 1 pg (0.001 mg) to about 50,000 pg (50 mg). In some aspects, the HBPC is administered at a dose (e.g., target dose) of about 7 pg (0.007 mg) to about 50,000 pg (50 mg).

[0237] In some aspects, the first polypeptide comprises the scFv, MM1, CM1, VH2, and Fcl in the N-terminal to C-terminal orientation. In some aspects, the scFv comprises the VH1 and VL1 in the N-terminal to C-terminal orientation.

[0238] In some aspects, the second polypeptide comprises the VL2, MM2, and CM2 in the N-terminal to C-terminal orientation.

[0239] In some aspects, the HBPC comprises the first polypeptide, second polypeptide, and third polypeptide in the N-terminal to C-terminal orientation.

[0240] Disclosed herein are methods of treating a subject afflicted with a cancer, the method comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising(i) a single-chain variable fragment (scFv) comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), wherein the VH1 and the VL1 together form a T-cell cluster of differentiation (CD3)-targeting domain the specifically binds a CD3 polypeptide, wherein the scFv comprises a VH1 and VL1 comprising: a VH1 comprising: a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), and a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:5), and a VL1 comprising: a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:6), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO:7), and a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO: 8);(ii) a first masking moiety (MM 1),(iii) a first cleavable moiety (CM1),(iv) a second heavy chain variable domain (VH2) comprising a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15), a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17);(v) a first monomeric Fc domain (Fcl);(b) a second polypeptide comprising:(i) a second light chain variable domain (VL2), wherein the VH2 and the VL2 together form an EGFR targeting domain that specifically binds EGFR, wherein the VL2 comprises a VL CDR1 comprising the amino acid sequence RASQSIGTNIH (SEQ ID NO: 18), a VL CDR2 comprising the amino acid sequence YASESIS (SEQ ID NO: 19), and a VL CDR3 comprising the amino acid sequence QQNNNWPTT (SEQ ID NO:20),(ii) a second masking moiety (MM2), and(iii) a second cleavable moiety (CM2); and(c) a third polypeptide that(i) comprises a second monomeric Fc domain (Fc2), and(ii) does not comprise an immunoglobulin variable domain; wherein the HBPC is administered at a dose (e.g., target dose) of about 1 pg (0.001 mg) to about 50,000 pg (50 mg). In some aspects, the HBPC is administered at a dose (e.g., target dose) of about 7 pg (0.007 mg) to about 50,000 pg (50 mg).

[0241] Disclosed herein are methods of treating a subject afflicted with a cancer, the method comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising(i) a single-chain variable fragment (scFv) comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), wherein the VH1 and the VL1 together form a T-cell cluster of differentiation (CD3)-targeting domain the specifically binds a CD3 polypeptide, wherein the scFv comprises a VH1 and VL1 comprising: a VH1 comprising: a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), and a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:5), and a VL1 comprising: a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:6), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO:7), and a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO: 8);(ii) a first cleavable moiety (CM1),(iii) a second heavy chain variable domain (VH2) comprising a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15), a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17);(iv) a first monomeric Fc domain (Fcl);(b) a second polypeptide comprising:(i) a second light chain variable domain (VL2), wherein the VH2 and the VL2 together form an EGFR targeting domain that specifically binds EGFR, wherein the VL2 comprises a VL CDR1 comprising the amino acidsequence RASQSIGTNIH (SEQ ID NO: 18), a VL CDR2 comprising the amino acid sequence YASESIS (SEQ ID NO: 19), and a VL CDR3 comprising the amino acid sequence QQNNNWPTT (SEQ ID NO:20),(ii) a second masking moiety (MM2), and(iii) a second cleavable moiety (CM2); and(c) a third polypeptide that(i) comprises a second monomeric Fc domain (Fc2), and(ii) does not comprise an immunoglobulin variable domain; wherein the HBPC is administered at a dose (e.g., target dose) of about 1 pg (0.001 mg) to about 50,000 pg (50 mg). In some aspects, the HBPC is administered at a dose (e.g., target dose) of about 7 pg (0.007 mg) to about 50,000 pg (50 mg).

[0242] Disclosed herein are methods of treating a subject afflicted with a cancer, the method comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising(i) a single-chain variable fragment (scFv) comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), wherein the VH1 and the VL1 together form a T-cell cluster of differentiation (CD3)-targeting domain the specifically binds a CD3 polypeptide, wherein the scFv comprises a VH1 and VL1 comprising: a VH1 comprising: a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), and a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:5), and a VL1 comprising: a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:6), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO:7), and a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO: 8);(ii) a first masking moiety (MM 1),(iii) a first cleavable moiety (CM1),(iv) a second heavy chain variable domain (VH2) comprising a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15), a VHCDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17);(v) a first monomeric Fc domain (Fcl);(b) a second polypeptide comprising:(i) a second light chain variable domain (VL2), wherein the VH2 and the VL2 together form an EGFR targeting domain that specifically binds EGFR, wherein the VL2 comprises a VL CDR1 comprising the amino acid sequence RASQSIGTNIH (SEQ ID NO: 18), a VL CDR2 comprising the amino acid sequence YASESIS (SEQ ID NO: 19), and a VL CDR3 comprising the amino acid sequence QQNNNWPTT (SEQ ID NO:20),(ii) a second masking moiety (MM2),(iii) a second cleavable moiety (CM2); and(c) a third polypeptide that(i) comprises a second monomeric Fc domain (Fc2), and(ii) does not comprise an immunoglobulin variable domain; wherein the HBPC is administered at a dose (e.g., target dose) of about 1 pg (0.001 mg) to about 50,000 pg (50 mg). In some aspects, the HBPC is administered at a dose (e.g., target dose) of about 7 pg (0.007 mg) to about 50,000 pg (50 mg).

[0243] Disclosed herein is an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) for use in treating a subject afflicted with a cancer, wherein the HBPC comprises:(a) a first polypeptide comprising(i) a single-chain variable fragment (scFv) comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), wherein the VH1 and the VL1 together form a T-cell cluster of differentiation (CD3)-targeting domain the specifically binds a CD3 polypeptide, wherein the scFv comprises a VH1 and VL1 comprising: a VH1 comprising: a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), and a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:5), anda VL1 comprising: a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:6), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO:7), and a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO: 8);(ii) a first cleavable moiety (CM1),(iii) a second heavy chain variable domain (VH2) comprising a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15), a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17);(iv) a first monomeric Fc domain (Fcl);(b) a second polypeptide comprising:(i) a second light chain variable domain (VL2), wherein the VH2 and the VL2 together form an EGFR targeting domain that specifically binds EGFR, wherein the VL2 comprises a VL CDR1 comprising the amino acid sequence RASQSIGTNIH (SEQ ID NO: 18), a VL CDR2 comprising the amino acid sequence YASESIS (SEQ ID NO: 19), and a VL CDR3 comprising the amino acid sequence QQNNNWPTT (SEQ ID NO:20),(ii) a second masking moiety (MM2), and(iii) a second cleavable moiety (CM2); and(c) a third polypeptide that(i) comprises a second monomeric Fc domain (Fc2), and(ii) does not comprise an immunoglobulin variable domain; wherein the HBPC is administered at a dose (e.g., target dose) of about 1 pg (0.001 mg) to about 50,000 pg (50 mg). In some aspects, the HBPC is administered at a dose (e.g., target dose) of about 7 pg (0.007 mg) to about 50,000 pg (50 mg).

[0244] In some aspects, the first polypeptide comprises the scFv, MM1, CM1, VH2, and Fcl in the N-terminal to C-terminal orientation. In some aspects, the scFv comprises the VH1 and VL1 in the N-terminal to C-terminal orientation.

[0245] In some aspects, the second polypeptide comprises the VL2, MM2, and CM2 in the N-terminal to C-terminal orientation.

[0246] In some aspects, the HBPC comprises the first polypeptide, second polypeptide, and third polypeptide in the N-terminal to C-terminal orientation.

[0247] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 144; and / or(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:37; and / or(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:32; and wherein the HBPC is administered at a dose (e.g., target dose) of about 1 pg (0.001 mg) to about 50,000 pg (50 mg). In some aspects, the HBPC is administered at a dose (e.g., target dose) of about 7 pg (0.007 mg) to about 50,000 pg (50 mg).

[0248] Also disclosed herein is an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) for use in treating a subject afflicted with a cancer, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 144; and / or(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:37; and / or(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:32; and wherein the HBPC is administered at a dose (e.g., target dose) of about 1 pg (0.001 mg) to about 50,000 pg (50 mg). In some aspects, the HBPC is administered at a dose (e.g., target dose) of about 7 pg (0.007 mg) to about 50,000 pg (50 mg).

[0249] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 144;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:37; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:32; and wherein the HBPC is administered at a dose (e.g., target dose) of about 1 pg (0.001 mg) to about 50,000 pg (50 mg). In some aspects, the HBPC is administered at a dose (e.g., target dose) of about 7 pg (0.007 mg) to about 50,000 pg (50 mg).

[0250] Also disclosed herein is an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) for use in treating a subject afflicted with a cancer, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 144;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:37; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:32; and wherein the HBPC is administered at a dose (e.g., target dose) of about 1 pg (0.001 mg) to about 50,000 pg (50 mg). In some aspects, the HBPC is administered at a dose (e.g., target dose) of about 7 pg (0.007 mg) to about 50,000 pg (50 mg).

[0251] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:30; and / or(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:31; and / or(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:32; and wherein the HBPC is administered at a dose (e.g., target dose) of about 1 pg (0.001 mg) to about 50,000 pg (50 mg). In some aspects, the HBPC is administered at a dose (e.g., target dose) of about 7 pg (0.007 mg) to about 50,000 pg (50 mg).

[0252] Also disclosed herein is an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) for use in treating a subject afflicted with a cancer, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:30; and / or(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:31; and / or(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:32; and wherein the HBPC is administered at a dose (e.g., target dose) of about 1 pg (0.001 mg) to about 50,000 pg (50 mg). In some aspects, the HBPC is administered at a dose (e.g., target dose) of about 7 pg (0.007 mg) to about 50,000 pg (50 mg).

[0253] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:30;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:31; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:32; and wherein the HBPC is administered at a dose (e.g., target dose) of about 1 pg (0.001 mg) to about 50,000 pg (50 mg). In some aspects, the HBPC is administered at a dose (e.g., target dose) of about 7 pg (0.007 mg) to about 50,000 pg (50 mg).

[0254] Also disclosed herein is an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) for use in treating a subject afflicted with a cancer, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:30;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:31; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:32; and wherein the HBPC is administered at a dose (e.g., target dose) of about 1 pg (0.001 mg) to about 50,000 pg (50 mg). In some aspects, the HBPC is administered at a dose (e.g., target dose) of about 7 pg (0.007 mg) to about 50,000 pg (50 mg).

[0255] Disclosed herein are methods of treating a subject afflicted with a cancer, the method comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising(i) a single-chain variable fragment (scFv) comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), wherein the VH1 and the VL1 together form a T-cell cluster of differentiation (CD3)-targeting domain the specifically binds a CD3 polypeptide, wherein the scFv comprises a VH1 and VL1 comprising: a VH1 comprising: a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129), and a VHCDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 130), and a VL1 comprising: a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 131), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO: 132), and a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO: 133);(ii) a first masking moiety (MM 1),(iii) a first cleavable moiety (CM1),(iv) a second heavy chain variable domain (VH2) comprising a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15), a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17);(v) a first monomeric Fc domain (Fcl);(b) a second polypeptide comprising:(i) a second light chain variable domain (VL2), wherein the VH2 and the VL2 together form an EGFR targeting domain that specifically binds EGFR, wherein the VL2 comprises a VL CDR1 comprising the amino acid sequence RASQSIGTNIH (SEQ ID NO: 18), a VL CDR2 comprising the amino acid sequence YASESIS (SEQ ID NO: 19), and a VL CDR3 comprising the amino acid sequence QQNNNWPTT (SEQ ID NO:20),(ii) a second masking moiety (MM2), and(iii) a second cleavable moiety (CM2); and(c) a third polypeptide that(i) comprises a second monomeric Fc domain (Fc2), and(ii) does not comprise an immunoglobulin variable domain; wherein the HBPC is administered at a dose (e.g., target dose) of about 1 pg (0.001 mg) to about 50,000 pg (50 mg). In some aspects, the HBPC is administered at a dose (e.g., target dose) of about 7 pg (0.007 mg) to about 50,000 pg (50 mg).

[0256] Disclosed herein is an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) for use in treating a subject afflicted with a cancer, wherein the HBPC comprises:(a) a first polypeptide comprising(i) a single-chain variable fragment (scFv) comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), wherein the VH1 and the VL1 together form a T-cell cluster of differentiation (CD3)-targeting domain the specifically binds a CD3 polypeptide, wherein the scFv comprises a VH1 and VL1 comprising: a VH1 comprising: a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129), and a VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 130), and a VL1 comprising: a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 131), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO: 132), and a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO: 133);(ii) a first masking moiety (MM 1),(iii) a first cleavable moiety (CM1),(iv) a second heavy chain variable domain (VH2) comprising a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15), a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17);(v) a first monomeric Fc domain (Fcl);(b) a second polypeptide comprising:(i) a second light chain variable domain (VL2), wherein the VH2 and the VL2 together form an EGFR targeting domain that specifically binds EGFR, wherein the VL2 comprises a VL CDR1 comprising the amino acid sequence RASQSIGTNIH (SEQ ID NO: 18), a VL CDR2 comprising the amino acid sequence YASESIS (SEQ ID NO: 19), and a VL CDR3 comprising the amino acid sequence QQNNNWPTT (SEQ ID NO:20),(ii) a second masking moiety (MM2), and(iii) a second cleavable moiety (CM2); and(c) a third polypeptide that(i) comprises a second monomeric Fc domain (Fc2), and(ii) does not comprise an immunoglobulin variable domain; wherein the HBPC is administered at a dose (e.g., target dose) of about 1 pg (0.001 mg) to about 50,000 pg (50 mg). In some aspects, the HBPC is administered at a dose (e.g., target dose) of about 7 pg (0.007 mg) to about 50,000 pg (50 mg).

[0257] In some aspects, the first polypeptide comprises the scFv, MM1, CM1, VH2, and Fcl in the N-terminal to C-terminal orientation. In some aspects, the scFv comprises the VH1 and VL1 in the N-terminal to C-terminal orientation.

[0258] In some aspects, the second polypeptide comprises the VL2, MM2, and CM2 in the N-terminal to C-terminal orientation.

[0259] In some aspects, the HBPC comprises the first polypeptide, second polypeptide, and third polypeptide in the N-terminal to C-terminal orientation.

[0260] In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 1 pg, about 2.5 pg, about 5 pg, about 7 pg, about 10 pg, about 20 pg, about 30 pg, about 40 pg, about 50 pg, about 60 pg, about 70 pg, about 80 pg, about 90 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, about 1000 pg, about 1250 pg, about 1500 pg, about 1750 pg, about 2000 pg, about 2250 pg, about 2500 pg, about 2750 pg, about 3000 pg, about 3500 pg, about 4000 pg, about 4500 pg, about 5000 pg, about 5500 pg, about 6000 pg, about 6500 pg, about 7000 pg, about 7500 pg, about8000 pg, about 8500 pg, about 9000 pg, about 9500 pg, about 10,000 pg, about 10,500 pg, about 11,000 pg, about 11,500 pg, about 12,000 pg, about 12,500 pg, about 13,000 pg, about 13,500 pg, about 14,000 pg, about 14,500 pg, about 15,000 pg, about 15,500 pg, about 16,000 pg, about 16,500 pg, about 17,000 pg, about 17,500 pg, about 18,000 pg, about 18,500 pg, about 19,000 pg, about 19,500 pg, about 20,000 pg, about 22,500 pg, about 25,000 pg, about 27,500 pg, about 30,000 pg, about 32,500 pg, about 35,000 pg, about 37,500 pg, about 40,000 pg, about 42,500 pg, about 45,000 pg, about 47,500 pg, or about 50,000 pg.

[0261] In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 0.001 mg, about 0.0025 mg, about 0.005 mg, about 0.007 mg, about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.15 mg, about 0.2 mg, about 0.25 mg, about 0.3 mg, about 0.35 mg, about 0.4 mg, about 0.45 mg, about 0.5 mg, about 0.55mg, about 0.6 mg, about 0.65 mg, about 0.7 mg, about 0.75 mg, about 0.8 mg, about 0.85 mg, about 0.9 mg, about 0.95 mg, about 1 mg, about 1.25 mg, about 1.5 mg, about 1.75 mg, about 2 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3 mg, about 3.5 mg, about 4 mg, about 4.5 mg, about 5 mg, about 5.5 mg, about 6 mg, about 6.5 mg, about 7 mg, about 7.5 mg, about 8 mg, about 8.5 mg, about 9 mg, about 9.5 mg, about 10 mg, about10.5 mg, about 11 mg, about 11.5 mg, about 12 mg, about 12.5 mg, about 13 mg, about13.5 mg, about 14 mg, about 14.5 mg, about 15 mg, about 15.5 mg, about 16 mg, about16.5 mg, about 17 mg, about 17.5 mg, about 18 mg, about 18.5 mg, about 19 mg, about19.5 mg, about 20 mg, about 22.5 mg, about 25 mg, about 27.5 mg, about 30 mg, about32.5 mg, about 35 mg, about 37.5 mg, about 40 mg, about 42.5 mg, about 45 mg, about47.5 mg, or about 50 mg.

[0262] In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 7 pg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 70 pg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 250 pg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 750 pg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 1500 pg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 3000 pg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 5000 pg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 6000 pg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 10,000 pg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 15,000 pg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 20,000 pg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 25,000 pg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 30,000 pg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 40,000 pg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 45,000 pg.

[0263] In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 0.007 mg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 0.07 mg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 0.25 mg. In some aspects, the activatable HBPC isadministered at a dose (e.g., target dose) of about 0.75 mg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 1.500 mg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 3 mg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 5 mg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 6 mg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 10 mg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 15 mg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 20 mg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 25 mg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 30 mg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 40 mg. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 45 mg.

[0264] In some aspects, the HBPC is administered once every two weeks.

[0265] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC is administered at a dose (e.g., target dose) of about 1 pg (0.001 mg) to about 50,000 pg (50 mg) once every two weeks.

[0266] In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 1 pg, about 2.5 pg, about 5 pg, about 7 pg, about 10 pg, about 20 pg, about 30 pg, about 40 pg, about 50 pg, about 60 pg, about 70 pg, about 80 pg, about 90 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, about 1000 pg, about 1250 pg, about 1500 pg, about 1750 pg, about 2000 pg, about 2250 pg, about 2500 pg, about 2750 pg, about 3000 pg, about 3500 pg, about 4000 pg, about 4500 pg, about 5000 pg, about 5500 pg, about 6000 pg, about 6500 pg, about 7000 pg, about 7500 pg, about8000 pg, about 8500 pg, about 9000 pg, about 9500 pg, about 10,000 pg, about 10,500 pg, about 11,000 pg, about 11,500 pg, about 12,000 pg, about 12,500 pg, about 13,000 pg, about 13,500 pg, about 14,000 pg, about 14,500 pg, about 15,000 pg, about 15,500 pg, about 16,000 pg, about 16,500 pg, about 17,000 pg, about 17,500 pg, about 18,000 pg, about 18,500 pg, about 19,000 pg, about 19,500 pg, about 20,000 pg, about 22,500 pg,about 25,000 pg, about 27,500 ig, about 30,000 pig, about 32,500 pig, about 35,000 pig, about 37,500 pig, about 40,000 pig, about 42,500 pig, about 45,000 pig, about 47,500 pig, or about 50,000 pig once every two weeks.

[0267] In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 0.001 mg, about 0.0025 mg, about 0.005 mg, about 0.007 mg, about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.15 mg, about 0.2 mg, about 0.25 mg, about 0.3 mg, about 0.35 mg, about 0.4 mg, about 0.45 mg, about 0.5 mg, about 0.55 mg, about 0.6 mg, about 0.65 mg, about 0.7 mg, about 0.75 mg, about 0.8 mg, about 0.85 mg, about 0.9 mg, about 0.95 mg, about 1 mg, about 1.25 mg, about 1.5 mg, about 1.75 mg, about 2 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3 mg, about 3.5 mg, about 4 mg, about 4.5 mg, about 5 mg, about 5.5 mg, about 6 mg, about 6.5 mg, about 7 mg, about 7.5 mg, about 8 mg, about 8.5 mg, about 9 mg, about 9.5 mg, about 10 mg, about10.5 mg, about 11 mg, about 11.5 mg, about 12 mg, about 12.5 mg, about 13 mg, about13.5 mg, about 14 mg, about 14.5 mg, about 15 mg, about 15.5 mg, about 16 mg, about16.5 mg, about 17 mg, about 17.5 mg, about 18 mg, about 18.5 mg, about 19 mg, about19.5 mg, about 20 mg, about 22.5 mg, about 25 mg, about 27.5 mg, about 30 mg, about32.5 mg, about 35 mg, about 37.5 mg, about 40 mg, about 42.5 mg, about 45 mg, about47.5 mg, or about 50 mg once every two weeks.

[0268] In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 7 pg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 70 pg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 250 pg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 750 pg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 1500 pg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 3000 pg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 5000 pg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 6000 pg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 10,000 pg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 15,000 pg once every two weeks. In some aspects, theactivatable HBPC is administered at a dose (e.g., target dose) of about 20,000 pg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 25,000 pg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 30,000 pg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 40,000 pg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 45,000 pg once every two weeks.

[0269] In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 0.007 mg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 0.07 mg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 0.25 mg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 0.75 mg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 1.500 mg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 3 mg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 5 mg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 6 mg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 10 mg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 15 mg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 20 mg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 25 mg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 30 mg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 40 mg once every two weeks. In some aspects, the activatable HBPC is administered at a dose (e.g., target dose) of about 45 mg once every two weeks.

[0270] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC is administered at a dose (e.g., target dose) of about 7 pg (0.007mg) once every two weeks.

[0271] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC is administered at a dose (e.g., target dose) of about 70 pg (.07 mg) once every two weeks.

[0272] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC is administered at a dose (e.g., target dose) of about 250 pg (.25 mg) once every two weeks.

[0273] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC is administered at a dose (e.g., target dose) of about 750 pg (.75 mg) once every two weeks.

[0274] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC is administered at a dose (e.g., target dose) of about 1500 pg (1.5 mg) once every two weeks.

[0275] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC is administered at a dose (e.g., target dose) of about 3000 pg (3 mg) once every two weeks.

[0276] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC is administered at a dose (e.g., target dose) of about 5000 pg (5 mg) once every two weeks.

[0277] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC is administered at a dose (e.g., target dose) of about 6000 pg (6 mg) once every two weeks.

[0278] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC is administered at a dose (e.g., target dose) of about 10,000 pg (10 mg) once every two weeks.

[0279] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC is administered at a dose (e.g., target dose) of about 15,000 pg (15 mg) once every two weeks.

[0280] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC is administered at a dose (e.g., target dose) of about 20,000 pg (20 mg) once every two weeks.

[0281] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC is administered at a dose (e.g., target dose) of about 25,000 pg (25 mg) once every two weeks.

[0282] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC is administered at a dose (e.g., target dose) of about 30,000 pg (30 mg) once every two weeks.

[0283] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC is administered at a dose (e.g., target dose) of about 40,000 pg (40 mg) once every two weeks.

[0284] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC is administered at a dose (e.g., target dose) of about 45,000 pg (45 mg) once every two weeks. In some aspects, the HBPC is administered as a step dose comprising a priming dose of HBPC and a target dose of HBPC.

[0285] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32;wherein the method comprises:(i) administering at least a first priming dose of HBPC to the subject on day 1; and(ii) administering to the subject a dose (e.g. target dose) of the HBPC of about 1 pg to about 50,000 pg; wherein the first priming dose of the HBPC is less than the dose of HBPC in (ii).

[0286] In some aspects, the dose in (ii) is administered on day 8.

[0287] In some aspects, the method further comprises administering a second priming dose of HBPC to the subject on day 8, and wherein the dose in (ii) is administered on day 15, wherein the second priming dose is 100% or less than the dose in (ii).

[0288] In some aspects, the first priming dose and / or the second priming dose is 50% or less than the dose in (ii).

[0289] In some aspects, the first priming dose is 25% or less than the dose in (ii).

[0290] Also disclosed herein is an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) for use in treating a subject afflicted with a cancer, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the subject is:(i) administered at least a first priming dose of HBPC on day 1; and(ii) administered a dose (e.g. target dose) of the HBPC of about 1 pg to about 50,000 pg; wherein the first priming dose of the HBPC is less than the dose of HBPC in (ii).

[0291] In some aspects, the dose in (ii) is administered on day 8.

[0292] In some aspects, the subject is administered a second priming dose of HBPC on day8, and wherein the dose in (ii) is administered on day 15, wherein the second priming dose is 100% or less than the dose in (ii).

[0293] In some aspects, the first priming dose and / or the second priming dose is 50% or less than the dose in (ii).

[0294] In some aspects, the first priming dose is 25% or less than the dose in (ii).

[0295] In some aspects, the HBPC is administering by step-dosing. In some aspects, the step-dosing schedule comprises administration of a priming dose and a target dose. In someaspects, step-dosing schedule comprises administration of two or more priming doses and the target dose.

[0296] In some aspects, the first priming dose of the HBPC is less than the target dose. In some aspects, the first priming dose and second priming dose of the HBPC is less than the target dose. In some aspects, all of the priming doses of the HBPC are less than the target dose.

[0297] In some aspects, the first priming dose of the HBPC is 100% or less than the target dose. In some aspects, the first priming dose and / or the second priming dose is 50% or less than the target dose. In some aspects, first priming dose is 25% or less than the target dose.

[0298] In some aspects, a first priming dose and / or second priming dose of the HBPC is administered prior to the first dose of the HBPC.

[0299] In some aspects, the first priming dose and / or second priming dose of the activatableHBPC is administered at a dose of about 1 pg, about 2.5 pg, about 5 pg, about 7 pg, about 10 pg, about 20 pg, about 30 pg, about 40 pg, about 50 pg, about 60 pg, about 70 pg, about 80 pg, about 90 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, about 1000 pg, about 1250 pg, about 1500 pg, about 1750 pg, about 2000 pg, about 2250 pg, about 2500 pg, about 2750 pg, about 3000 pg, about 3500 pg, about 4000 pg, about 4500 pg, about 5000 pg, about 5500 pg, about 6000 pg, about 6500 pg, about 7000 pg, about 7500 pg, about 8000 pg, about 8500 pg, about 9000 pg, about 9500 pg, about10,000 pg, about 10,500 pg, about 11,000 pg, about 11,500 pg, about 12,000 pg, about12.500 pg, about 13,000 pg, about 13,500 pg, about 14,000 pg, about 14,500 pg, about15,000 pg, about 15,500 pg, about 16,000 pg, about 16,500 pg, about 17,000 pg, about17.500 pg, about 18,000 pg, about 18,500 pg, about 19,000 pg, about 19,500 pg, about20,000 pg, about 22,500 pg, about 25,000 pg, about 27,500 pg, about 30,000 pg, about32.500 pg, about 35,000 pg, about 37,500 pg, about 40,000 pg, about 42,500 pg, about45,000 pg, about 47,500 pg, or about 50,000 pg.

[0300] In some aspects, the first priming dose and / or second priming dose of the activatableHBPC is administered at a dose of about 0.001 mg, about 0.0025 mg, about 0.005 mg, about 0.007 mg, about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.15 mg, about 0.2 mg, about 0.25 mg, about 0.3 mg, about 0.35 mg, about 0.4 mg, about 0.45mg, about 0.5 mg, about 0.55 mg, about 0.6 mg, about 0.65 mg, about 0.7 mg, about 0.75 mg, about 0.8 mg, about 0.85 mg, about 0.9 mg, about 0.95 mg, about 1 mg, about 1.25 mg, about 1.5 mg, about 1.75 mg, about 2 mg, about 2.25 mg, about 2.5 mg, about 2.75 mg, about 3 mg, about 3.5 mg, about 4 mg, about 4.5 mg, about 5 mg, about 5.5 mg, about6 mg, about 6.5 mg, about 7 mg, about 7.5 mg, about 8 mg, about 8.5 mg, about 9 mg, about 9.5 mg, about 10 mg, about 10.5 mg, about 11 mg, about 11.5 mg, about 12 mg, about 12.5 mg, about 13 mg, about 13.5 mg, about 14 mg, about 14.5 mg, about 15 mg, about 15.5 mg, about 16 mg, about 16.5 mg, about 17 mg, about 17.5 mg, about 18 mg, about 18.5 mg, about 19 mg, about 19.5 mg, about 20 mg, about 22.5 mg, about 25 mg, about 27.5 mg, about 30 mg, about 32.5 mg, about 35 mg, about 37.5 mg, about 40 mg, about 42.5 mg, about 45 mg, about 47.5 mg, or about 50 mg.

[0301] In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 7 pg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 70 pg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 250 pg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 750 pg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 1500 pg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 3000 pg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 5000 pg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 6000 pg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 10,000 pg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 15,000 pg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 20,000 pg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 25,000 pg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 30,000 pg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 40,000 pg.In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 45,000 pg.

[0302] In some aspects, the first priming dose and / or second priming dose of the activatableHBPC is administered at dose of about 0.007 mg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 0.07 mg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 0.25 mg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 0.75 mg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 1.500 mg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 3 mg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 5 mg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 6 mg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 10 mg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 15 mg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 20 mg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 25 mg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 30 mg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 40 mg. In some aspects, the first priming dose and / or second priming dose of the activatable HBPC is administered at a dose of about 45 mg.

[0303] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) about 5000 pg (5 mg) on day 15; and c) about 5000 pg (5 mg) every two weeks thereafter (i.e., Q2W thereafter).

[0304] Also disclosed herein are methods of treating a subject afflicted with a tumor comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) apriming dose of the HBPC at about 1500 pg (1.5 mg) on day 1; b) about 5000 pg (5 mg) on day 8; and c) about 5000 pg (5 mg) on day 15 and every two weeks thereafter (i.e., Q2W thereafter).

[0305] Also disclosed herein are methods of treating a subject afflicted with a tumor comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) about 5000 pg (5 mg) on day 8; and c) about 5000 pg (5 mg) on day 15 and every two weeks thereafter (i.e., Q2W thereafter).

[0306] Also disclosed herein are methods of treating a subject afflicted with a tumor comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) about 10,000 pg (10 mg) on day 15; and c) about 10,000 pg (10 mg) every two weeks thereafter (i.e., Q2W thereafter).

[0307] Also disclosed herein are methods of treating a subject afflicted with a tumor comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 1500 pg (1.5 mg) on day 1; b) a second priming dose of the HBPC at about 5000 pg (5 mg) on day 8; c) about 10,000 pg (10 mg) on day 15; and d) about 10,000 pg (10 mg) every two weeks thereafter (i.e., Q2W thereafter).

[0308] Also disclosed herein are methods of treating a subject afflicted with a tumor comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) a second priming dose of the HBPC at about 5000 pg (5 mg) on day 8; c) about 10,000 pg (10 mg) on day 15; and d) about 10,000 pg (10 mg) every two weeks thereafter (i.e., Q2W thereafter).

[0309] Also disclosed herein are methods of treating a subject afflicted with a tumor comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) a second priming dose of the HBPC at about 5000 pg (5 mg) on day 8; c) about 15,000 pg (15 mg) on day 15; and d) about 15,000 pg (15 mg) every two weeks thereafter (i.e., Q2W thereafter).

[0310] Also disclosed herein are methods of treating a subject afflicted with a tumor comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) a second priming dose of the HBPC at about 5000 pg (5 mg) on day 8; c) about 20,000 pg (20 mg) on day 15; and d) about 15,000 pg (15 mg) every two weeks thereafter (i.e., Q2W thereafter).

[0311] Also disclosed herein are methods of treating a subject afflicted with a tumor comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) a second priming dose of the HBPC at about 5000 pg (5 mg) on day 8; c) about 15,000 pg (15 mg) on day 15; and d) about 20,000 pg (20 mg) every two weeks thereafter (i.e., Q2W thereafter).

[0312] Also disclosed herein are methods of treating a subject afflicted with a tumor comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) a second priming dose of the HBPC at about 5000 pg (5 mg) on day 8; c) about 20,000 pg (20 mg) on day 15; and d) about 20,000 pg (20 mg) every two weeks thereafter (i.e., Q2W thereafter).

[0313] Also disclosed herein are methods of treating a subject afflicted with a tumor comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) a second priming dose of the HBPC at about 5000 pg (5 mg) on day 8; c) about 25,000 pg (25 mg) on day 15; and d) about 25,000 pg (25 mg) every two weeks thereafter (i.e., Q2W thereafter).

[0314] Also disclosed herein are methods of treating a subject afflicted with a tumor comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) a second priming dose of the HBPC at about 5000 pg (5 mg) on day 8; c) about 30,000 pg (30 mg) on day 15; and d) about 30,000 pg (30 mg) every two weeks thereafter (i.e., Q2W thereafter).

[0315] Also disclosed herein are methods of treating a subject afflicted with a tumor comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) apriming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) a second priming dose of the HBPC at about 5000 pg (5 mg) on day 8; c) about 40,000 pg (40 mg) on day 15; and d) about 40,000 pg (40 mg) every two weeks thereafter (i.e., Q2W thereafter).

[0316] Also disclosed herein are methods of treating a subject afflicted with a tumor comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) a second priming dose of the HBPC at about 5000 pg (5 mg) on day 8; c) about 45,000 pg (45 mg) on day 15; and d) about 45,000 pg (45 mg) every two weeks thereafter (i.e., Q2W thereafter).

[0317] In some aspects, the subject is administered an IL-6 inhibitor prior to administration of the HBPC. In some aspects, the IL-6 inhibitor is a small molecule, an antibody, or a steroid. In some aspects, the IL-6 inhibitor targets IL-6 or the IL-6 receptor. In some aspects, the IL-6 inhibitor that targets IL-6 is siltuximab. In some aspects, the IL-6 inhibitor that targets the IL-6 receptor is tocilizumab or sarilumab. In some aspects, the IL-6 inhibitor is siltuximab, tocilizumab, or sarilumab. In some aspects, the IL-6 inhibitor is tocilizumab. In some aspects, the amount of tocilizumab that the subject is administered does not exceed 800 mg. In some aspects, the subject is administered 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, or 12 mg / kg tocilizumab prior to administration of the HBPC. In some aspects, the subject is administered 8 mg / kg tocilizumab prior to administration of the HBPC. In some aspects, the tocilizumab is administered up to 4 hours prior to administration of the HBPC. In some aspects, the IL-6 inhibitor is a corticosteroid, such as, for example, dexamethasone, and the like.

[0318] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) about 5000 pg (5 mg) on day 15; and c) about 5000 pg (5 mg) every two weeks thereafter (i.e., Q2W thereafter), wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC.

[0319] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 1500 pg (1.5 mg) on day 1; b) about 5000 pg (5 mg)on day 8; and c) about 5000 ig (5 mg) every two weeks thereafter (i.e., Q2W thereafter), wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC except for the priming dose.

[0320] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) about 5000 pg (5 mg) on day 8; and c) about 5000 pg (5 mg) every two weeks thereafter (i.e., Q2W thereafter), wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC.

[0321] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) about 10,000 pg (10 mg) on day 15; and c) about 10,000 pg (10 mg) every two weeks thereafter (i.e., Q2W thereafter), wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC.

[0322] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 1500 pg (1.5 mg) on day 1; b) a second priming dose of the HBPC at about 5000 pg (5 mg) on day 8; c) about 10,000 pg (10 mg) on day 15; and d) about 10,000 pg (10 mg) every two weeks thereafter (i.e., Q2W thereafter), wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC.

[0323] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) a second priming dose of the HBPC at about 5000 pg (5 mg) on day 8; c) about 10,000 pg (10 mg) on day 15; and d) about 10,000 pg (10 mg) every two weeks thereafter (i.e., Q2W thereafter), wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC.

[0324] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) apriming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) a second priming dose of the HBPC at about 5000 pg (5 mg) on day 8; c) about 15,000 pg (15 mg) on day 15; and d) about 15,000 pg (15 mg) every two weeks thereafter (i.e., Q2W thereafter), wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC.

[0325] Also disclosed herein are methods of treating a subject afflicted with a tumor comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) a second priming dose of the HBPC at about 5000 pg (5 mg) on day 8; c) 20,000 pg (20 mg) on day 15; and d) 15,000 pg (15 mg) every two weeks thereafter (i.e., Q2W thereafter), wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC.

[0326] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) a second priming dose of the HBPC at about 5000 pg (5 mg) on day 8; c) about 15,000 pg (15 mg) on day 15; and d) about 20,000 pg (20 mg) every two weeks thereafter (i.e., Q2W thereafter), wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC.

[0327] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) a second priming dose of the HBPC at about 5000 pg (5 mg) on day 8; c) about 20,000 pg (20 mg) on day 15; and d) about 20,000 pg (20 mg) every two weeks thereafter (i.e., Q2W thereafter), wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC.

[0328] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) a second priming dose of the HBPC at about 5000 pg (5 mg) on day 8; c) about 25,000 pg (25 mg) on day 15; and d) about 25,000 pg (25 mg) every two weeks thereafter (i.e., Q2W thereafter), wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC.

[0329] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) a second priming dose of the HBPC at about 5000 pg (5 mg) on day 8; c) about 30,000 pg (30 mg) on day 15; and d) about 30,000 pg (30 mg) every two weeks thereafter (i.e., Q2W thereafter), wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC.

[0330] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) a second priming dose of the HBPC at about 5000 pg (5 mg) on day 8; c) about 40,000 pg (40 mg) on day 15; and d) about 40,000 pg (40 mg) every two weeks thereafter (i.e., Q2W thereafter), wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC.

[0331] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the subject is administered a) a priming dose of the HBPC at about 3000 pg (3 mg) on day 1; b) a second priming dose of the HBPC at about 5000 pg (5 mg) on day 8; c) about 45,000 pg (45 mg) on day 15; and d) about 45,000 pg (45 mg) every two weeks thereafter (i.e., Q2W thereafter), wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC.

[0332] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 7 pg once every two weeks.

[0333] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a thirdpolypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 70 pg once every two weeks.

[0334] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 250 pg once every two weeks.

[0335] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 750 pg once every two weeks.

[0336] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 1.5 mg once every two weeks.

[0337] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 3 mg once every two weeks.

[0338] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a firstpolypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 5 mg once every two weeks.

[0339] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 6 mg once every two weeks.

[0340] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 10 mg once every two weeks.

[0341] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 15 mg once every two weeks.

[0342] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 3 mg on DI, 5 mg on DI 5, and 5 mg once every two weeks thereafter. Also disclosed herein are methods of treating a subject afflicted with a cancercomprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 3 mg on DI, 5 mg on DI 5, and 5 mg once every two weeks thereafter, wherein the subject is administered 8 mg / kg tocilizumab prior to all HBPC doses.

[0343] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 1.5 mg on day 1, 5 mg on day 8, and 5 mg once every two weeks thereafter.

[0344] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 1.5 mg on day 1, 5 mg on day 8, and 5 mg once every two weeks thereafter, wherein the subject is administered 8 mg / kg tocilizumab prior to all HBPC doses except on day 1.

[0345] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 3 mg on day 1, 5 mg on day 8, and 5 mg once every two weeks thereafter.

[0346] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 3 mg on day 1, 5 mg on day 8, and 5 mg once every two weeks thereafter, wherein the subject is administered 8 mg / kg tocilizumab prior to all HBPC doses.

[0347] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 3 mg on day 1, 10 mg on day 15, and 10 mg once every two weeks thereafter.

[0348] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 3 mg on day 1, 10 mg on day 15, and 10 mg once every two weeks thereafter, wherein the subject is administered 8 mg / kg tocilizumab prior to all HBPC doses.

[0349] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC isadministered at a dose of about 1.5 mg on day 1, 5 mg on day 8, 10 mg on day 15, and 10 mg once every two weeks thereafter.

[0350] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 1.5 mg on day 1, 5 mg on day 8, 10 mg on day 15, and 10 mg once every two weeks thereafter, wherein the subject is administered 8 mg / kg tocilizumab prior to all HBPC doses expect on day 1.

[0351] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 3 mg on day 1, 5 mg on day 8, 10 mg on day 15, and 10 mg once every two weeks thereafter.

[0352] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 1.5 mg on day 1, 5 mg on day 8, 10 mg on day 15, and 10 mg once every two weeks thereafter, wherein the subject is administered 8 mg / kg tocilizumab prior to all HBPC doses.

[0353] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a thirdpolypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 3 mg on day 1, 5 mg on day 8, 15 mg on day 15, and 15 mg once every two weeks thereafter.

[0354] Also disclosed herein are methods of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 3 mg on day 1, 5 mg on day 8, 15 mg on day 15, and 15 mg once every two weeks thereafter, wherein the subject is administered 8 mg / kg tocilizumab prior to all HBPC.

[0355] In some aspects, the method further comprises administering a second therapeutic agent (e.g., combination therapy). In some aspects, the second therapeutic agent is selected from a chemotherapeutic agent, an immune-oncology agent, or a monoclonal antibody. In some aspects, the monoclonal antibody is selected from alemtuzumab, cetuximab, bevacizumab, dostarlimab, gemtuzumab, ipilimumab, nivolumab, ofatumumab, panitumumab, pembrolizumab, ranibizumab, rituximab, or trastuzumab

[0356] In some aspects, the cancer is sensitive to treatment with immune-oncology agents. In some aspects, the cancer is an EGFR-expressing cancer.

[0357] In some aspects, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), cutaneous squamous cell carcinoma, colorectal carcinoma (CRC), esophageal, gastric / gastroesophageal junction (g / GEJ) cancer, breast cancer, bladder cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, and renal cell carcinoma (RCC). In some aspects, the cancer is selected from the group consisting of NSCLC, HNSCC, pancreatic cancer, CRC, and g / GEJ cancer. In certain aspects, the cancer is selected from the group consisting of pancreatic cancer, NSCLC, HNSCC, GEJ / Gastric cancer, and esophageal cancer. In certain aspects, the cancer is NSCLC. In certain other aspects, the cancer is HNSCC. In some aspects, the cancer is pancreatic cancer. In still further aspects, the cancer is CRC. Sometimes the cancer is g / GEJ cancer. In some aspects, the cancer is esophageal cancer.

[0358] In some aspects, the cancer comprises a tumor. In some aspects, the tumor is a solid tumor. In some aspects, the solid tumor is sensitive to treatment with immune-oncology agents. In certain aspects, the tumor is an EGFR-expressing cancer.

[0359] In some aspects, the tumor is selected from the group consisting of non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), cutaneous squamous cell carcinoma, colorectal carcinoma (CRC), gastric / gastroesophageal junction (g / GEJ) cancer, esophageal cancer, breast cancer, bladder cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, and renal cell carcinoma (RCC). In some aspects, the tumor is selected from the group consisting of NSCLC, HNSCC, pancreatic cancer, CRC, esophageal cancer, and g / GEJ cancer. In certain aspects, the tumor is selected from the group consisting of pancreatic cancer, NSCLC, HNSCC, GEJ / Gastric cancer, and esophageal cancer. In certain aspects, the tumor (or cancer) is NSCLC. In certain other aspects, it is HNSCC. In some aspects, the tumor (or cancer) is pancreatic cancer. In still further aspects, the tumor (or cancer) is CRC. Sometimes the tumor (or cancer) is g / GEJ cancer. In certain aspects, the tumor (or cancer) is esophageal cancer.

[0360] In some aspects, the activatable HBPCs of the disclosures are administered via intravenous (IV) infusion. In some aspects, the activatable HBPCs of the disclosure are administered by IV infusion for 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, or 120 minutes. In some aspects, the activatable HBPCs of the disclosure are administered by IV infusion for 30 minutes. In some aspects, the activatable HBPCs of the disclosure are administered by IV infusion for at least 60 minutes. In some aspects, the activatable HBPCs of the disclosure are administered by IV infusion for 60 minutes.

[0361] In some aspects, the subject is a human.Polynucleotides

[0362] In some aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding the first, second, and / or third polypeptide of an activatable anti-EGFR, anti-CD3 HBPC of the present disclosure (correspondingly referred to herein as the "first polynucleotide" the "second polynucleotide", and the "third polynucleotide"), respectively). Suitable polynucleotides include any that encode any of the first, second, and / or third polypeptides described herein, or portion thereof. An illustrative set ofpolynucleotide sequences encoding a first, second, and third polypeptide is provided herein below.

[0363] Polynucleotides of the present disclosure may be sequence optimized for optimal production from the host organism selected for expression, e.g., by codon / RNA optimization, replacement with heterologous signal sequences, and elimination of mRNA instability elements. Methods to generate optimized nucleic acids encoding an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide or antigen-binding fragment thereof (e.g., heavy chain, light chain, VH domain, or VL domain) for recombinant expression by introducing codon changes (e.g., a codon change that encodes the same amino acid due to the degeneracy of the genetic code) and / or eliminating inhibitory regions in the mRNA can be carried out by adapting the optimization methods described in, e.g., U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, accordingly.Nucleic AcidsPolynucleotide Encoding a First Polypeptide (SEQ ID NO: 112)CAAGGACAATCTGGCTCTGTGTCCACCACCTGTTGGTGGGACCCTCCATGCACAC CTAATACCGGCAGCTCTGGTGGCTCTGGCGGAAGCGGAGGACTGTCTGGCAGAT CCGATGATCACGGCGGAGGATCTGAGGTGCAGCTGGTTGAATCTGGTGGCGGAC TGGTTCAGCCTGGCGGATCTCTGAAACTGAGCTGTGCCGCCAGCGGCTTCACCTT CAACAAATACGCCATGAACTGGGTCCGACAGGCCCCTGGCAAAGGCCTTGAATG GGTCGCCAGAATCAGAAGCAAGTACAACAACTATGCCACCTACTACGCCGACAG CGTGAAGGACAGATTCACCATCAGCCGGGACGACAGCAAGAACACCGCCTACCT GCAGATGAACAACCTGAAAACCGAGGACACCGCCGTGTACTACTGTGTGCGGCA CGGCAACTTCGGCAACAGCTACATCAGCTACTGGGCCTATTGGGGCCAGGGCAC ACTGGTCACAGTTTCTAGTGGCGGAGGCGGATCTGGCGGCGGTGGAAGTGGCGG CGGAGGTTCTCAAACAGTGGTCACCCAAGAGCCTAGCCTGACCGTTTCTCCTGGC GGAACCGTGACACTGACATGCGGATCTTCTACAGGCGCCGTGACCAGCGGCAAC TACCCTAATTGGGTGCAGCAGAAGCCAGGCCAGGCTCCTAGAGGACTGATCGGC GGCACAAAGTTTCTGGCTCCCGGAACACCAGCCAGATTCAGCGGTTCTCTGCTCG GAGGAAAGGCCGCTCTGACACTTTCTGGCGTGCAGCCTGAGGATGAGGCCGAGT ACTATTGCGTGCTGTGGTACAGCAACAGATGGGTGTTCGGCGGAGGCACCAAGC TGACAGTTCTTGGAGGTGGCGGTAGCCAGGTCCAGCTGAAACAATCTGGACCCG GACTCGTGCAGCCAAGCCAGAGCCTGTCTATCACCTGTACCGTGTCCGGCTTCAGCCTGACCAATTACGGCGTGCACTGGGTTCGACAATCTCCCGGCAAGGGACTCGA ATGGCTGGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCATTCAC CAGCAGACTGAGCATCAACAAGGACAACAGCAAGTCCCAGGTGTTCTTCAAGAT GAACTCCCTGCAGAGCCAGGATACCGCCATCTATTACTGCGCTCGGGCCCTGACC TACTATGACTACGAGTTTGCCTACTGGGGACAGGGAACCCTCGTGACAGTGTCTG CTGCTAGCACAAAGGGCCCTAGCGTTTTCCCACTGGCTCCCAGCAGCAAGTCTAC ATCCGGTGGAACAGCCGCTCTGGGCTGCCTGGTCAAGGATTACTTTCCCGAGCCA GTGACCGTGTCCTGGAATAGCGGAGCACTGACATCTGGCGTGCACACATTTCCAG CCGTGCTGCAGTCTAGCGGCCTGTACTCTCTGTCCAGCGTTGTGACAGTGCCCAG CAGCTCTCTGGGCACCCAGACCTACATCTGCAATGTGAACCACAAGCCTAGCAA CACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGATAAGACACACACCT GTCCTCCATGTCCTGCTCCAGAGCTGCTCGGAGGCCCTTCCGTGTTTCTGTTCCCT CCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTG GTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTCGAC GGCGTGGAAGTGCACAATGCCAAGACCAAGCCTTGCGAGGAACAGTACGGCAGC ACCTACAGATGCGTGTCCGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGC AAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAA ACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAACCCCAGGTGTACACACTGCCT CCAAGCCGGAAAGAGATGACCAAGAATCAGGTGTCCCTGACCTGCCTGGTCAAG GGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGACAGCCCGAG AACAACTACAAGACAACCCCTCCTGTGCTGAAGTCCGACGGCTCATTCTTCCTGT ACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCT GCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCT GAGCCCCGGCAAA

[0364] The terminal lysine may not be present in the purified protein regardless of being present or absent in the gene. In a variation of this illustrative polynucleotide, the codon encoding the C-terminal lysine may be absent (i.e., SEQ ID NO: 139).Polynucleotide Encoding a Second Polypeptide (SEQ ID NO: 113)CAAGGCCAGTCTGGCCAAGGTCTTAGTTGTGAAGGTTGGGCGATGAATAGAGAA CAATGTCGAGCCGGAGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGT CCGGCAGATCCGACCAGCACGGCGGAGGATCCCAAATCCTGCTGACACAGTCTC CTGTCATACTGAGTGTCTCCCCCGGCGAGAGAGTCTCTTTCTCATGTCGGGCCAGTCAGTCTATTGGGACTAACATACACTGGTACCAGCAACGCACCAACGGAAGCCC GCGCCTGCTGATTAAATATGCGAGCGAAAGCATTAGCGGCATTCCGAGCCGCTTT AGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGC GAAGATATTGCGGATTATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTG GCGCGGGCACCAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCA TCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCT GCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGC CCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAG CACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACA CAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAA GAGCTTCAACAGGGGAGAGTGT

[0365] The second polypeptide is also encoded by the polynucleotide having the sequence of SEQ ID NO: 115.Polynucleotide Encoding a Third Polypeptide (SEQ ID NO: 114)GATAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAACTGCTCGGCGGACCTT CCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCC TGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTT CAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACAAAGCCCTGCGA GGAACAGTACGGCAGCACCTACAGATGCGTGTCCGTGCTGACAGTGCTGCACCA GGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCC TGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAACCCCA GGTGTACACACTGCCTCCAAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCT GACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGC AATGGACAGCCCGAGAACAACTACGACACCACACCTCCAGTGCTGGACAGCGAC GGCTCATTCTTCCTGTACAGCGACCTGACCGTGGACAAGAGCAGATGGCAGCAG GGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACC CAGAAGTCCCTGAGCCTGTCTCCTGGCAAA

[0366] In a variation of this illustrative polynucleotide, the codon encoding the C-terminal lysine may be absent (i.e., SEQ ID NO: 141).

[0367] A further illustrative activatable HBPC of the present disclosure is described in Example 1 that comprises a first polypeptide having the amino acid sequence of SEQ IDNO:30 (encoded by a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 139 or 112 (the terminal lysine is not present in the purified protein regardless of being present or absent in the gene); a second polypeptide having the amino acid sequence of SEQ ID NO:31 (encoded by the polynucleotide sequence of SEQ ID NO: 113 or SEQ ID NO: 115; and a third polypeptide having the amino acid sequence of SEQ ID NO:32 (encoded by the polynucleotide sequence of SEQ ID NO: 114 (the terminal lysine is not present in the purified protein regardless of being present or absent in the gene) or SEQ ID NO: 141.

[0368] In another illustrative activatable HBPC of the present disclosure, described in Example 1, the activatable HBPC comprises a first polypeptide having the amino acid sequence of SEQ ID NO:38 (encoded by a polynucleotide sequence comprising the polynucleotide sequence of SEQ ID NO: 142 or 143 (the terminal lysine is not present in the purified protein regardless of being present or absent in the gene); a second polypeptide having the amino acid sequence of SEQ ID NO:31 (encoded by the polynucleotide sequence of SEQ ID NO: 113 or SEQ ID NO: 115; and a third polypeptide having the amino acid sequence of SEQ ID NO:32 (encoded by the polynucleotide sequence of SEQ ID NO: 114 (the terminal lysine is not present in the purified protein regardless of being present or absent in the gene). or SEQ ID NO: 141 (the terminal lysine is not present in the purified protein regardless of being present or absent in the gene).

[0369] In some aspects, the activatable HBPC comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 144, and / or a second polypeptide having the amino acid sequence of SEQ ID NO: 37, and / or a third polypeptide having the amino acid sequence of SEQ ID NO: 32. In some aspects, the activatable HBPC comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 144, a second polypeptide having the amino acid sequence of SEQ ID NO: 37, and a third polypeptide having the amino acid sequence of SEQ ID NO: 32.

[0370] In some aspects, the activatable HBPC comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 30, and / or a second polypeptide having the amino acid sequence of SEQ ID NO: 31, and / or a third polypeptide having the amino acid sequence of SEQ ID NO: 32. In some aspects, the activatable HBPC comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 30, a second polypeptide having the amino acid sequence of SEQ ID NO: 31, and a third polypeptide having the amino acid sequence of SEQ ID NO: 32.

[0371] A polynucleotide encoding a polypeptide or antigen-binding fragment thereof described herein or a domain thereof can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods), synthesized using techniques that are well known in the art, and the like. Polynucleotides encoding the first, second, and third polypeptides can be cloned into one or more vectors for expression in host cells and for further cloning, for example, to generate chimeric and humanized antibodies or antigen-binding fragments thereof.

[0372] Polynucleotides provided herein can be an RNA or a DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and DNA can be double-stranded or single-stranded. If single stranded, DNA can be the coding strand or non-coding (anti-sense) strand. In some aspects, the polynucleotide is a cDNA or a DNA lacking one more endogenous introns. In some aspects, a polynucleotide is a non-naturally occurring polynucleotide. In some aspects, a polynucleotide is recombinantly produced. In some aspects, the polynucleotides are isolated. In some aspects, the polynucleotides are substantially pure. In some aspects, a polynucleotide is purified from natural components.

[0373] In some aspects, the polynucleotides described herein encode an activatable anti- EGFR, anti-CD3 HBPCs or antigen-binding fragment thereof and comprises the heavy chain (VH) and light chain (VL) and CDRs provided herein.Vectors, Host Cells, and Methods of Production

[0374] Provided herein are one or more vectors comprising polynucleotides encoding the first, second, and / or third polypeptides of the present disclosure (corresponding to a first polynucleotide, a second polynucleotide, and a third polynucleotide, respectively). In some aspects, such vectors may be used to recombinantly produce the polypeptides of the HBPC from a host cell, as described in more detail hereinbelow. In some aspects, the vector comprises the first, the second, and / or the third polynucleotide operably linked to one or more promoter sequences. In certain aspects, the present disclosure provides a plurality of vectors that collectively comprise the polynucleotides encoding the first, second, and third polypeptides (i.e., the first, second, and third polynucleotides), where the plurality comprises at least one vector that comprises no more than two, or no more than one of the first, the second, and the third polynucleotides. In these aspects, the first, the second, andthe third polynucleotide sequences in the plurality of vectors are usually operably linked to one or more promoter sequences.

[0375] Also provided herein are recombinant host cells comprising any of the abovedescribed polynucleotides and / or vectors for recombinantly expressing the polynucleotides encoding the polypeptides of the activatable anti-EGFR, anti-CD3 HBPC of the present disclosure. A variety of host-expression vector systems can be utilized to express the polypeptides described herein (see, e.g., U.S. Patent No. 5,807,715). Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody or antigen-binding fragment thereof described herein in situ. Exemplary host cells that are suitable for use as a recombinant expression host for the above-described polynucleotides include mammalian cell systems (e.g, COS (e.g, COS1 or COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, Rl. l, B-W, L-M, BSC1, BSC40, YB / 20, BMT10 cells, and the like). Vectors employed in the construction of a recombinant mammalian host cell may comprise a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or from a mammalian virus (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In some aspects, the recombinant host cell is a CHO cell or a NSO cell.

[0376] In some aspects, recombinant expression of a polypeptide described herein, e.g., a first, second, and / or third polypeptide, involves construction of an expression vector containing a polynucleotide that encodes the activatable anti-EGFR, anti-CD3 HBPC. Vector(s) comprising polynucleotides encoding the HBPC can be readily generated by recombinant DNA technology using techniques well known in the art. Methods which are well known to those skilled in the art can be used to construct expression vectors containing one or more polynucleotides encoding that polypeptides described herein, e.g., a first, second, and / or third polypeptide, as well as appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors comprising a nucleotide sequence operably linked to a promoter. Such vectors can, for example, include the nucleotide sequence encoding the constant region of a polypeptide described herein, e.g., a first, second, and / or third polypeptide (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036; and U.S. Patent No. 5,122,464), andvariable domains of the polypeptide can be cloned into such a vector for expression of the entire VH, the entire VL, or both the entire VH and VL.

[0377] An expression vector can be transferred to a cell (e.g., host cell) by conventional techniques and the resulting cells can then be cultured by conventional techniques to produce the HBPC described herein (e.g., the CDRs, the VH1, VH2, VL1, and VL2 of an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide provided herein). Thus, provided herein are host cells containing a polynucleotide encoding the HBPC described herein, operably linked to a promoter for expression of such sequences in the host cell. In some aspects, a host cell contains a vector comprising a polynucleotide encoding the HBPC described herein, or a domain thereof. In some aspects, a host cell contains three different vectors, a first vector comprising a first polynucleotide encoding a first polypeptide described herein, a second vector comprising a second polynucleotide encoding a second polypeptide described herein, and a third vector comprising a third polynucleotide encoding a third polypeptide described herein.

[0378] In some aspects, provided herein is a population of vectors that collectively comprise polynucleotides encoding the first, second, and third polypeptide, where each vector comprises only one or two of the polynucleotides encoding the first, second, or third polypeptides. In certain aspects, a single vector is provided herein that comprises the polynucleotides encoding the first, second, and third polypeptides (i.e., the first, second, and third polynucleotides, respectively).

[0379] In some aspects, the present disclosure provides methods of producing an activatable HBPC comprising: (a) culturing a host cell comprising one or more polynucleotides encoding the polypeptides of the present disclosure (e.g., a first polynucleotide, a second polynucleotide, and / or a third polynucleotide, as well as vector(s) comprising the aforementioned polynucleotides) in a liquid culture medium under conditions sufficient to produce the activatable HBPC; and (b) recovering the activatable HBPC.

[0380] In a particular aspect, provided herein are methods for producing an activatable anti-EGFR, anti-CD3 HBPC, comprising expressing such a polypeptide thereof in a host cell. More specifically, provided herein is a method of producing an activatable HBPC comprising: (a) culturing a host cell comprising one or more polynucleotides encoding the polypeptides of the present disclosure in a liquid culture medium under conditions sufficient to produce the HBPC; and (b) recovering the activatable HBPC.

[0381] In another aspect, the method further comprises purifying a bioharvest (cell-free expression product) of activatable HBPC or other in-process composition comprising subjecting an aqueous composition comprising activatable HBPC to a unit operation such as, for example, affinity chromatography, size exclusion chromatography, ion exchange chromatography, ceramic hydroxyapatite chromatography, and the like. In certain aspects, the unit operation is ceramic hydroxyapatite chromatography.Compositions

[0382] In some aspects, the activatable HBPCs of the present disclosure or HBPC thereof can be utilized in a pharmaceutical composition useful for any of the therapeutic applications disclosed herein. In certain aspects, the pharmaceutical composition comprises a therapeutically effective amount of one or more activatable HBPC, together with pharmaceutically acceptable diluent or carrier. In other aspects, the pharmaceutical composition comprises a therapeutically effective amount of one or more activatable HBPC, a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant. Acceptable formulation materials are nontoxic to recipients at the dosages and concentrations employed. The pharmaceutical compositions can be formulated as liquid, frozen or lyophilized compositions.

[0383] In some aspects, the activatable HBPCs of the present disclosure or HBPC thereof formulated as a lyophilized cake in a vial. In some aspects, the activatable HBPCs of the present disclosure or HBPC thereof is formulated at 1 mg / vial, 2.5 mg / vial, 5 mg / vial, 7.5 mg / vial, 10 mg / vial, 12.5 mg / vial, 15 mg / vial, 17.5 mg / vial, 20 mg / vial, 22.5 mg / vial, 25 mg / vial, 27.5 mg / vial, or 30 mg / vial. In some aspects, the activatable HBPCs of the present disclosure or HBPC thereof is formulated at 25 mg / vial.

[0384] In some aspects, the activatable HBPCs of the present disclosure or HBPC thereof is formulated as a lyophilized cake. In some aspects, the lyophilized HBPCs of the present disclosure are reconstituted with a pharmaceutically acceptable diluent or carrier. In some aspects, the lyophilized HBPCs of the present disclosure are reconstituted with water. In some aspects, the lyophilized HBPCs of the present disclosure are reconstituted with sterile water. In some aspects, the lyophilized HBPCs of the present disclosure are reconstituted with sterile water for injection. In some aspects, the lyophilized HBPCs of the present disclosure are reconstituted with a pharmaceutically acceptable diluent or carrier to a concentration of 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 1 mg / mL, 1.5 mg / mL,2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 7.5 mg / mL, 8 mg / mL, 8.5 mg / mL, 9 mg / mL, 9.5 mg / mL, or 10 mg / mL. In some aspects, the lyophilized HBPCs of the present disclosure are reconstituted with a pharmaceutically acceptable diluent or carrier to a concentration of 5 mg / mL.

[0385] In some aspects, the reconstituted HBPCs are diluted with a pharmaceutically acceptable diluent or carrier. In some aspects, the reconstituted HBPCs are diluted in saline. In some aspects, the reconstituted HBPCs are diluted in 0.9% saline (e.g., a USP bag).

[0386] In certain aspects, the pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids; antimicrobials; antioxidants; buffers; bulking agents; chelating agents; complexing agents; fillers; carbohydrates such as monosaccharides or disaccharides; proteins; coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers; low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives; solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols; suspending agents; surfactants or wetting agents; stability enhancing agents; tonicity enhancing agents; delivery vehicles; and / or pharmaceutical adjuvants. Additional details and options for suitable agents that can be incorporated into pharmaceutical compositions are provided in, for example, Remington's Pharmaceutical Sciences, 22ndEdition, (Loyd V. Allen, ed.) Pharmaceutical Press (2013); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7thed., Lippencott Williams and Wilkins (2004); and Kibbe et al., Handbook of Pharmaceutical Excipients, 3rded., Pharmaceutical Press (2000).

[0387] The components of the pharmaceutical composition are selected depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington's Pharmaceutical Sciences, 22ndEdition, (Loyd V. Allen, ed.) Pharmaceutical Press (2013). The compositions are selected to influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the antigen binding proteins disclosed. The primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier can be water for injection or physiological saline solution. In certain aspects, antigen binding proteincompositions can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of a lyophilized cake or an aqueous solution. Further, in certain aspects, the antigen binding protein can be formulated as a lyophilizate using appropriate excipients. Some compositions include a buffer or a pH adjusting agent. Representative buffers include, but are not limited to: organic acid salts (such as salts of citric acid, acetic acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, or phthalic acid); Tris; phosphate buffers; and, in some instances, an amino acid as described below. In certain aspects, the composition further comprises glutamic acid, sucrose, and Polysorbate 80.

[0388] In certain aspects, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8. Some compositions have a pH from about 5-6, 6-7, or 7-8. In other aspects, the pH is from 5.5-6.5, 6.5-7.5, or 7.5-8.5.

[0389] A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, and rectal administration.

[0390] In some aspects, the pharmaceutical composition is formulated to be compatible with intravenous infusion. In some aspects, an IV bag is coated with an IV stabilizing solution (IVSS) prior to the addition of the diluted HBPCs. As used herein, the term "intravenous stabilizing solution" and "IVSS" are used interchangeably herein to refer to a solution that, when used to treat an intravenous (IV) bag, functions to prevent the adhesion of protein to the interior surfaces of IV bags and IV tubing. In some aspects, the IVSS comprises citric acid, lysine hydrochloride, polysorbate 80, water for injection (WFI) and an acid, base, or buffer. In certain aspects, the IVSS has a pH of from about 6.5 to about7.5. In certain aspects, the IVSS has a pH of about 7. IVSS solutions suitable for use in the methods described herein include those described in PCT publication number WO 2018 / 204907, which is incorporated herein by reference in its entirety. In a specific aspect, the IVSS solution comprises citric acid monohydrate (5.25 mg / ml) or citric acid, anhydrous (4.80 mg / ml), L-lysine hydrochloride (228.31 mg / ml), polysorbate 80 (1 mg / ml), sodium hydroxide to adjust pH to 7.0, and WFI. In some aspects, the IVSS solution on a per vial basis comprises 52.5 mg citric acid monohydrate or 4.80 mg citric acid, anhydrous, 2283.1 mg L-lysine hydrochloride, 10 mg polysorbate 80, sodium hydroxide to adjust pH to 7.0,and WFI (q.s. to 10 ml), collectively, per vial. In certain aspects, the IVSS solution is added to the IV bag to a final v / v of 3% prior to addition of the HBPC to the IV solution.

[0391] Formulation components suitable for parenteral administration (e.g., intravenous, subcutaneous, intraocular, intraperitoneal, intramuscular) include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[0392] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0393] Further guidance on appropriate formulations depending upon the form of delivery is provided, for example, in Remington's Pharmaceutical Sciences, 22ndEdition, (Loyd V. Allen, ed.) Pharmaceutical Press (2013).

[0394] Pharmaceutical formulations can be sterile. Sterilization can be accomplished by any suitable method, e.g., filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution. As demonstrated in Examples 7 and 8, the activatable HBPCs described herein appear relatively aggregation-resistant even at relatively high concentrations. Thus, in another aspect, provided herein are compositions comprising any of the activatable HBPCs described herein, and water, wherein the activatable HBPC is present at a concentration of at least 1 mg / mL and wherein the composition comprises at least about 95% monomeric activatable HBPC, or at least about 96% monomeric activatable HBPC, or at least about 97% monomeric activatable HBPC, or at least about 98% monomeric activatable HBPC, or at least about 99% monomeric activatable HBPC. As used herein, the term "monomeric activatable HBPC" refers to the activatable HBPC in non-aggregated form. In certain of these aspects the composition comprises at least about 2 mg / ml and at least about 95% monomeric activatable HBPC, or at least about 96% monomeric activatable HBPC, or at least about 97% monomeric activatable HBPC, or atleast about 98% monomeric activatable HBPC, or at least about 99% monomeric activatable HBPC. In some aspects, the composition comprises at least about 3 mg / ml and at least about 95% monomeric activatable HBPC, or at least about 96% monomeric activatable HBPC, or at least about 97% monomeric activatable HBPC, or at least about 98% monomeric activatable HBPC, or at least about 99% monomeric activatable HBPC. In some aspects, the composition comprises at least about 4 mg / ml and at least about 95% monomeric activatable HBPC, or at least about 96% monomeric activatable HBPC, or at least about 97% monomeric activatable HBPC, or at least about 98% monomeric activatable HBPC, or at least about 99% monomeric activatable HBPC. The percentage of monomeric activatable HBPC can be readily determined by, for example, size exclusion (SE)-HPLC, as illustrated in Example 7, where percent monomeric activatable HBPC is determined as the percentage peak area corresponding to monomeric activatable HBPC on the basis of total peak area.Kits

[0395] Provided herein are kits comprising one or more an activatable anti-EGFR, anti- CD3 HBPC described herein or an HBPC thereof, as, wherein the kits are diagnostic or for treatment. In certain aspects, provided herein is a pack or kit comprising one or more containers filled with one or more of the ingredients of the compositions described herein, such as one or more an activatable anti-EGFR, anti-CD3 HBPCs provided herein or an antigen-binding fragment thereof, optional an instructing for use. In some aspects, the kits contain a composition described herein and any diagnostic, prophylactic or therapeutic agent, such as those described herein. In certain aspects, the kit further comprises an IVSS solution (e.g., vial of IVSS solution).Embodiments

[0396] Embodiment 1 : A method treating a subject afflicted with a cancer, the method comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising (i) a single-chain variable fragment (scFv) comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), wherein the VH1 and the VL1 together form a T-cell cluster of differentiation (CD3)-targeting domainthat specifically binds a CD3 polypeptide, wherein the scFv comprises a set of VH1 and VL1 selected from:(A) a VH1 comprising a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), and a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO: 5), and a VL1 comprising a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO: 6), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO: 7), and a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO:8); or(B) a VH1 comprising a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129), and a VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 130), and a VL1 comprising a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 131), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO: 132), and a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO: 133);(ii) a first masking moiety (MM 1),(iii) a first cleavable moiety (CM1),(iv) a second heavy chain variable domain (VH2) comprising a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15), a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17);(v) a first monomeric Fc domain (Fcl);(b) a second polypeptide comprising(i) a second light chain variable domain (VL2), wherein the VH2 and the VL2 together form an EGFR targeting domain that specifically binds EGFR, wherein the VL2 comprises a VL CDR1 comprising the amino acid sequence RASQSIGTNIH (SEQ ID NO: 18), a VL CDR2 comprising the amino acid sequence YASESIS (SEQ ID NO: 19), and a VL CDR3 comprising the amino acid sequence QQNNNWPTT (SEQ ID NO:20),(ii) a second masking moiety (MM2), and(iii) a second cleavable moiety (CM2); and(c) a third polypeptide that(i) comprises a second monomeric Fc domain (Fc2), and(ii) does not comprise an immunoglobulin variable domain; and wherein the HBPC is administered at a dose of about 1 pg to about 50,000 pg.

[0397] Embodiment 2: The method of Embodiment 1, wherein the first polypeptide comprises the scFv, MM1, CM1, VH2, and Fcl in the N-terminal to C-terminal orientation.

[0398] Embodiment 3: The method of Embodiment 1 or 2, wherein the second polypeptide comprises the VL2, MM2, and CM2 in the N-terminal to C-terminal orientation.

[0399] Embodiment 4: The method of any one of Embodiments 1-3, wherein the HBPC comprises the first polypeptide, second polypeptide, and third polypeptide in the N- terminal to C-terminal orientation.

[0400] Embodiment 5: The method of any one of Embodiments 1-4, wherein the MM1 comprises an amino acid sequence that is selected from the group consisting of SEQ ID NOs: 1, 67, 68, 69, 70, 71, and 72.

[0401] Embodiment 6: The method of any one of Embodiments 1-5, wherein the VH1 comprises a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), and a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:5), and wherein the VL1 comprises a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:6), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO:7), and a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO:8).

[0402] Embodiment 7: The method of any one of Embodiments 1-6, wherein the VH1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NOV and / or wherein the VL1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 10.

[0403] Embodiment 8: The method of any of Embodiments 1-7, wherein the VH1 comprises the amino acid sequence of SEQ ID NOV and the VL1 comprises the amino acid sequence of SEQ ID NO: 10.

[0404] Embodiment 9: The method of any of Embodiments 6-8, wherein the MM1 comprises the amino acid sequence of SEQ ID NO: 1.

[0405] Embodiment 10: The method of Embodiment 1, wherein the VH1 comprises a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129), and a VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 130), and wherein the VL1 comprises a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 131), a VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO: 132), and a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO: 133) .

[0406] Embodiment 11 : The method of any of Embodiments 1-5, and 10, wherein the VH1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 134 and / or wherein the VL1 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 135.

[0407] Embodiment 12: The method of any of Embodiments 1-5 and 10-11, wherein the VH1 comprises the amino acid sequence of SEQ ID NO: 134 and the VL1 comprises the amino acid sequence of SEQ ID NO: 135.

[0408] Embodiment 13: The method of any of Embodiments 1-5 and 10-12 wherein the MM1 comprises the amino acid sequence of SEQ ID NO:72.

[0409] Embodiment 14: The method of any of Embodiments 1-13, wherein the MM2 comprises the amino acid sequence of SEQ ID NO: 13.

[0410] Embodiment 15: The method of any of Embodiments 1-14, wherein the CM1 and the CM2 each independently comprise a substrate for a protease selected from the group of proteases shown in Table 1.

[0411] Embodiment 16: The method of any of Embodiments 1-14, wherein the CM1 and the CM2 each independently comprise a substrate having an amino acid sequence selected from the group shown in Table 2.

[0412] Embodiment 17: The method of any of Embodiments 1-11, wherein the CM1 comprises the amino acid sequence of SEQ ID NO:73.

[0413] Embodiment 18: The method of any of Embodiments 1-10, wherein the CM2 comprises the amino acid sequence of SEQ ID NO: 14.

[0414] Embodiment 19: A method of treating a subject afflicted with a cancer, the method comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 144; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 37; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:32; and wherein the HBPC is administered at a dose of about 1 pg to about 50,000 pg.

[0415] Embodiment 20: A method of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide- I l l - comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 1 pg to about 50,000 pg.

[0416] Embodiment 21 : The method of any of the preceding Embodiments, wherein theHBPC is administered at a dose of about 1 pg, about 2.5 pg, about 5 pg, about 7 pg, about 10 pg, about 20 pg, about 30 pg, about 40 pg, about 50 pg, about 60 pg, about 70 pg, about 80 pg, about 90 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, about 1000 pg, about 1250 pg, about 1500 pg, about 1750 pg, about 2000 pg, about 2250 pg, about 2500 pg, about 2750 pg, about 3000 pg, about 3500 pg, about 4000 pg, about 4500 pg, about 5000 pg, about 5500 pg, about 6000 pg, about 6500 pg, about 7000 pg, about 7500 pg, about 8000 pg, about 8500 pg, about 9000 pg, about 9500 pg, about10,000 pg, about 10,500 pg, about 11,000 pg, about 11,500 pg, about 12,000 pg, about12,500 pg, about 13,000 pg, about 13,500 pg, about 14,000 pg, about 14,500 pg, about15,000 pg, about 15,500 pg, about 16,000 pg, about 16,500 pg, about 17,000 pg, about17,500 pg, about 18,000 pg, about 18,500 pg, about 19,000 pg, about 19,500 pg, 20,000 pg, about 22,500 pg, about 25,000 pg, about 27,500 pg, about 30,000 pg, about 32,500 pg, about 35,000 pg, about 37,500 pg, about 40,000 pg, about 42,500 pg, about 45,000 pg, about 47,500 pg, or about 50,000 pg.

[0417] Embodiment 22: The method of any of the preceding Embodiments, wherein the HBPC is administered at a dose of about 7 pg.

[0418] Embodiment 23 : The method of any of the preceding Embodiments, wherein the HBPC is administered at a dose of about 70 pg.

[0419] Embodiment 24: The method of any of the preceding Embodiments, wherein the HBPC is administered at a dose of about 250 pg.

[0420] Embodiment 25: The method of any of the preceding Embodiments, wherein the HBPC is administered at a dose of about 750 pg.

[0421] Embodiment 26: The method of any of the preceding Embodiments, wherein the HBPC is administered at a dose of about 1500 pg.

[0422] Embodiment 27: The method of any of the preceding Embodiments, wherein the HBPC is administered at a dose of about 3000 pg.

[0423] Embodiment 28: The method of any of the preceding Embodiments, wherein the HBPC is administered at a dose of about 5000 pg.

[0424] Embodiment 29: The method of any of the preceding Embodiments, wherein the HBPC is administered at a dose of about 6000 pg.

[0425] Embodiment 30: The method of any of the preceding Embodiments, wherein the HBPC is administered at a dose of about 10,000 pg.

[0426] Embodiment 31 : The method of any of the preceding Embodiments, wherein the HBPC is administered at a dose of about 15,000 pg.

[0427] Embodiment 32: The method of any of the preceding Embodiments, wherein the HBPC is administered once every two weeks.

[0428] Embodiment 33: The method of Embodiment 32, wherein the HBPC is administered at a dose of between about 7 pg and about 70 pg once every two weeks.

[0429] Embodiment 34: The method of Embodiment 32 or Embodiment 33, wherein theHBPC is administered at a dose of about 7 pg once every two weeks.

[0430] Embodiment 35: The method of Embodiment 32 or Embodiment 33, wherein theHBPC is administered at a dose of about 70 pg once every two weeks.

[0431] Embodiment 36: The method of Embodiment 32, wherein the HBPC is administered at a dose of about 250 pg once every two weeks.

[0432] Embodiment 37: The method of Embodiment 32, wherein the HBPC is administered at a dose of about 750 pg once every two weeks.

[0433] Embodiment 38: The method of Embodiment 32, wherein the HBPC is administered at a dose of about 1500 pg once every two weeks.

[0434] Embodiment 39: The method of Embodiment 32, wherein the HBPC is administered at a dose of about 3000 pg once every two weeks.

[0435] Embodiment 40: The method of Embodiment 32, wherein the HBPC is administered at a dose of about 6000 pg once every two weeks.

[0436] Embodiment 41 : The method of Embodiment 32, wherein the HBPC is administered at a dose of about 5000 pg once every two weeks.

[0437] Embodiment 42: The method of Embodiment 32, wherein the HBPC is administered at a dose of about 10,000 pg once every two weeks.

[0438] Embodiment 43: The method of Embodiment 32, wherein the HBPC is administered at a dose of about 15,000 pg once every two weeks.

[0439] Embodiment 44: The method of any of the preceding Embodiments, wherein a priming dose of the HBPC is administered prior to the first dose of the HBPC.

[0440] Embodiment 45: A method of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the method comprises: (i) administering at least a first priming dose of HBPC to the subject on day 1; and (ii) administering to the subject a dose of the HBPC of about 1 pg to about 50,000 pg; wherein the first priming dose of the HBPC is less than the dose of HBPC in (ii).

[0441] Embodiment 46: The method of Embodiment 45, wherein the dose in (ii) is administered on day 8.

[0442] Embodiment 47: The method of Embodiments 45 or 46, wherein the method further comprises administering a second priming dose of HBPC to the subject on day 8, and wherein the dose in (ii) is administered on day 15, wherein the second priming dose is 100% or less than the dose in (ii).

[0443] Embodiment 48: The method of any one of Embodiments 45-47, wherein the first priming dose and / or the second priming dose is 50% or less than the dose in (ii).

[0444] Embodiment 49: The method of any one of Embodiments 45-48, wherein the first priming dose is 25% or less than the dose in (ii).

[0445] Embodiment 50: The method of Embodiment 44, wherein the subject is administered: (a) a priming dose of the HBPC at about 3000 pg on day 1; (b) about 5000 pg on day 15; and (c) about 5000 pg every two weeks thereafter.

[0446] Embodiment 51 : The method of Embodiment 44, wherein the subject is administered: (a) a priming dose of the HBPC at about 1500 pg on day 1; (b) about 5000 pg on day 8; and (c) about 5000 pg every two weeks thereafter.

[0447] Embodiment 52: The method of Embodiment 44, wherein the subject is administered: (a) a priming dose of the HBPC at about 3000 pg on day 1; (b) about 5000 pg on day 8; and (c) about 5000 pg every two weeks thereafter.

[0448] Embodiment 53: The method of Embodiment 44, wherein the subject is administered: (a) a priming dose of the HBPC at about 3000 pg on day 1; (b) about 10,000 pg on day 15; and (c) about 10,000 pg every two weeks thereafter.

[0449] Embodiment 54: The method of Embodiment 44, wherein the subject is administered: (a) a priming dose of the HBPC at about 1500 pg on day 1; (b) a secondpriming dose of the HBPC at about 5000 pg on day 8; (c) about 10,000 pg on day 15; and (d) about 10,000 pg every two weeks thereafter.

[0450] Embodiment 55: The method of Embodiment 44, wherein the subject is administered: (a) a priming dose of the HBPC at about 3000 pg on day 1; (b) a second priming dose of the HBPC at about 5000 pg on day 8; (c) about 10,000 pg on day 15; and (d) about 10,000 pg every two weeks thereafter.

[0451] Embodiment 56: The method of Embodiment 44, wherein the subject is administered: (a) a priming dose of the HBPC at about 3000 pg on day 1; (b) a second priming dose of the HBPC at about 5000 pg on day 8; (c) about 15,000 pg on day 15; and (d) about 15,000 pg every two weeks thereafter.

[0452] Embodiment 57: The method of any of the preceding Embodiments, wherein the subject is administered an IL-6 inhibitor prior to administration of the HBPC.

[0453] Embodiment 58: The method of Embodiment 57, wherein the IL-6 inhibitor is a small molecule or an antibody.

[0454] Embodiment 59: The method of Embodiment 57, wherein the IL-6 inhibitor is a corticosteroid.

[0455] Embodiment 60: The method of Embodiment 59, wherein the corticosteroid is dexamethasone.

[0456] Embodiment 61 : The method of Embodiment 57 or Embodiment 58, wherein the IL-6 inhibitor is tocilizumab, siltuximab, or sarilumab.

[0457] Embodiment 62: The method of any one of Embodiment 57, 58, and 61, wherein the IL-6 inhibitor is tocilizumab.

[0458] Embodiment 63: The method of any one of Embodiments 61 and 62, wherein the amount of tocilizumab that the subject is administered does not exceed 800 mg.

[0459] Embodiment 64: The method of any one of Embodiments 61-63, wherein the subject is administered 8 mg / kg tocilizumab prior to administration of the HBPC.

[0460] Embodiment 65: The method of any one of Embodiments 61-64, wherein the subject is administered: (a) a priming dose of the HBPC at about 3000 pg on day 1; (b) about 5000 pg on day 15; and (c) about 5000 pg every two weeks thereafter, wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC.

[0461] Embodiment 66: The method of any one of Embodiments 61-64, wherein the subject is administered: (a) a priming dose of the HBPC at about 1500 pg on day 1; (b) about 5000 pg on day 8; and (c) about 5000 pg every two weeks thereafter, wherein thesubject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC except for the priming dose.

[0462] Embodiment 67: The method of any one of Embodiments 61-64, wherein the subject is administered: (a) a priming dose of the HBPC at about 3000 pg on day 1; (b) about 5000 pg on day 8; and (c) about 5000 pg every two weeks thereafter, wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC.

[0463] Embodiment 68: The method of any one of Embodiments 61-64, wherein the subject is administered: (a) a priming dose of the HBPC at about 3000 pg on day 1; (b) about 10,000 pg on day 15; and(c) about 10,000 pg every two weeks thereafter, wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC.

[0464] Embodiment 69: The method of any one of Embodiments 61-64, wherein the subject is administered: (a) a priming dose of the HBPC at about 1500 pg on day 1; (b) a second priming dose of the HBPC at about 5000 pg on day 8; (c) about 10,000 pg on day 15; and (d) about 10,000 pg every two weeks thereafter, wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC.

[0465] Embodiment 70: The method of any one of Embodiments 61-64, wherein the subject is administered: (a) a priming dose of the HBPC at about 3000 pg on day 1; (b) a second priming dose of the HBPC at about 5000 pg on day 8; (c) about 10,000 pg on day 15; and (d) about 10,000 pg every two weeks thereafter, wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC.

[0466] Embodiment 71 : The method of any one of Embodiments 61-64, wherein the subject is administered: (a) a priming dose of the HBPC at about 3000 pg on day 1; (b) a second priming dose of the HBPC at about 5000 pg on day 8; (c) about 15,000 pg on day 15; and (d) about 15,000 pg every two weeks thereafter, wherein the subject is administered 8 mg / kg tocilizumab prior to all doses of the HBPC.

[0467] Embodiment 72: The method of any one of Embodiments 61-71, wherein the tocilizumab is administered up to 4 hours prior to administration of the HBPC.

[0468] Embodiment 73: A method of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 6 mg once every two weeks.

[0469] Embodiment 74: A method of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 1.5 mg on day 1, 5 mg on day 8, and 5 mg once every two weeks thereafter.

[0470] Embodiment 75: A method of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 1.5 mg on day 1, 5 mg on day 8, 10 mg on day 15, and 10 mg once every two weeks thereafter.

[0471] Embodiment 76: A method of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 3 mg once every two weeks.

[0472] Embodiment 77: The method of any one of the preceding Embodiments, wherein the method further comprises administering a second therapeutic agent.

[0473] Embodiment 78: The method of Embodiment 77, wherein the second therapeutic agent is selected from a chemotherapeutic agent, an immune-oncology agent, or a monoclonal antibody.

[0474] Embodiment 79: The method of Embodiment 78, wherein the monoclonal antibody is selected from alemtuzumab, cetuximab, bevacizumab, dostarlimab, gemtuzumab, ipilimumab, nivolumab, ofatumumab, panitumumab, pembrolizumab, ranibizumab, rituximab, or trastuzumab.

[0475] Embodiment 80: The method of any of the preceding Embodiments, wherein the cancer is sensitive to treatment with immune-oncology agents.

[0476] Embodiment 81 : The method of any of the preceding Embodiments, wherein the cancer is an EGFR-expressing cancer.

[0477] Embodiment 82: The method of any of the preceding Embodiments, wherein the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), cutaneous squamous cell carcinoma, colorectal carcinoma (CRC), gastric / gastroesophageal junction (g / GEJ) cancer, esophageal cancer, breast cancer, bladder cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, and renal cell carcinoma (RCC).

[0478] Embodiment 83: The method of Embodiment 82, wherein the cancer is selected from the group consisting of NSCLC, HNSCC, pancreatic cancer, CRC, and g / GEJ cancer.

[0479] Embodiment 84: The method of Embodiment 82, wherein the cancer is selected from the group consisting of pancreatic cancer, NSCLC, HNSCC, g / GEJ, and esophageal cancer.

[0480] Embodiment 85: The method any of the preceding Embodiments, wherein the cancer comprises a tumor.

[0481] Embodiment 86: The method of Embodiment 85, wherein the tumor is an EGFR- expressing cancer.

[0482] Embodiment 87: The method of Embodiment 85 or 86, wherein the tumor is a solid tumor.

[0483] Embodiment 88: The method of any one of Embodiments 85-87, wherein the solid tumor is sensitive to treatment with immune-oncology agents.

[0484] Embodiment 89: The method of any of the preceding Embodiments, wherein the subject is administered the HBPC via intravenous infusion.

[0485] Embodiment 90: The method of Embodiment 89, wherein the intravenous infusion is administered for 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, or 120 minutes.

[0486] Embodiment 91 : The method of Embodiments 89 or Embodiment 90, wherein the intravenous infusion is administered for 30 minutes.

[0487] Embodiment 92: The method of Embodiment 89 or Embodiment 90, wherein the intravenous infusion is administered for 60 minutes.

[0488] Embodiment 93 : The method of any of the preceding Embodiments, wherein the subject is a human.

[0489] Embodiment 94: A method of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 5 mg once every two weeks.

[0490] Embodiment 95: A method of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 10 mg once every two weeks.

[0491] Embodiment 96: A method of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 15 mg once every two weeks.EXAMPLES

[0492] The examples in this Examples Section are offered by way of illustration, and not by way of limitation.Example 1: Construction and Expression of Activatable Anti-EGFR, Anti-CD3 Hetero nuiltimeric Bispecific Polypeptides

[0493] In this example, two illustrative activatable anti-EGFR, anti-CD3 HBPCs, Complex-57 and Complex-67, were previously prepared having the structure shown in Figure 1. With reference to Figure 1, each of the activatable anti-EGFR, anti-CD3 HBPCs were constructed in three polypeptides as described below:(a) a first polypeptide including a CD3 masking moiety (MM1) 100, a first cleavable moiety (CM1) 101, an anti-CD3 scFv 102 (including VH1 and VL1 sequences connected via a linker), an anti-EGFR heavy chain variable domain (VH2) (top) and a CHI domain (bottom), together indicated as 103, which is linked, via a hinge region 109, to a first Fc domain (Fcl) 104; and(b) a second polypeptide including a EGFR masking moiety (MM2) 105, a second cleavable moiety (CM2) 106, and an anti-EGFR light chain variable domain (VL2) (top) and constant light domain (CL) (bottom), together indicated as 107; and(c) a third polypeptide including a hinge region 110 and a second Fc domain (Fc2) 108. As seen in Figure 1, the first and second Fc domains bind each other, and the anti- EGFR heavy and light chain variable domains form an EGFR targeting domain that binds specifically to EGFR. Complex-57 and Complex-67 included the same anti-EGFR targeting domain but included different anti-CD3 scFvs. The components of Complex-67 are listed in Table 3A-3C, and the components of Complex-57 are listed in Tables 4A-4C.Table 3A. Complex-67 First Polypeptide Components* Corresponding polynucleotide sequence is SEQ ID NO: 112 (the terminal lysine is not present in the purified protein regardless of being present or absent in the gene) or SEQ ID NO: 139.++Contains an N-terminal spacer, SEQ ID NO:33.AFcl is located at the C-terminus of a CHI (SEQ ID NO:26)-Hinge (SEQ ID NO:34) sequence.Table 3B. Complex-67 Second Polypeptide Components* Corresponding polynucleotide sequence is SEQNO: 113 or a ternatively SEQ ID NO: 115.++Contains an N-terminal spacer, SEQ ID NO: 117.Table 3C. Complex-67 Third Polypeptide ComponentsCorresponding polynucleotide sequence is SEQ ID NO: 114 (the terminal lysine is not present in the purified protein regardless of being present or absent in the gene) or SEQ ID NO: 141.++Contains a hinge (SEQ ID NO:35) located at the N-terminus of Fc2.Table 4A. Complex-57 First PolypeptideCorresponding polynucleotide sequence is SEQ ID NO: 143 (the terminal lysine is not present in the purified protein regardless of being present or absent in the gene) or SEQ ID NO: 142.Contains an N-terminal spacer (SEQ ID NO:117).AFcl is located at the C-terminus of a CHI (SEQ ID NO:26)-Hinge (SEQ ID NO:34) sequence.Table 4B. Complex-57 Second Polypeptide++Contains an N-terminal spacer, SEQ ID NO: 117.Table 4C. Complex-57 Third Polypeptide ComponentsCorresponding polynucleotide sequence is SEQ ID NO: 114 (the terminal lysine is not present in the purified protein regardless of being present in the gene) or SEQ ID NO: 141.++Contains a hinge (SEQ ID NO:35) located at the N-terminus of Fc2.Example 2. Phase 1 / lb, Open-label, Dosing-finding, First-in-human Study to Evaluate the Safety and Antitumor activity of an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (EIBPC)2.1 Study Design

[0494] A phase 1 / lb study will be performed to study the safety and tolerability of an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) comprising a first polypeptide having the sequence of SEQ ID NO: 30, a second polypeptide having the sequence of SEQ ID NO: 31, and a third polypeptide having the sequence of SEQ ID NO: 32.

[0495] The HBPC is supplied as 25 mg / vial white-to-off-white lyophilized cake in Type I 20 mL clear glass vial for reconstitution with 5 mL water for injection (WFI), 5 mg / mL. The HBPC will be further diluted in 0.9% United States Pharmacopeia (USP), NaCl (normal saline) for administration by intravenous (IV) infusion.

[0496] The final diluted drug product will be made using an IV stabilizing solution (IVSS). The IVSS is used to coat the IV bag prior to addition of the reconstituted HBPC to preventadhesion of protein to IV bags and IV tubing. The IVSS is supplied as vials containing 10 ml of 4.8 mg / ml anhydrous citric acid, 228.31 mg / ml L-Lysine monohydrochloride, 1.0 mg / ml polysorbate 80, NaOH q.s. to pH target 7.0, and WFI q.s. to 10 ml. The IVSS is added to the IV bag prior to addition of the reconstituted HBPC, at a total concentration of 3% v / v. The final administration set will incorporate a 0.2 micron in-line filter.

[0497] To qualify for the study, patients are required to have active metastatic or locally advanced, unresectable tumor. Fresh tumor tissue biopsy must be performed during screening period, unless it is medically contraindicated, in which case recent archival tumor sample (obtained within 6 months) must be provided in a form of slides (20 slides) or paraffin block for exploratory analyses by immunohistochemistry (IHC).

[0498] Patients will be enrolled into escalating dose cohorts in which the HBPC will be administered intravenously once every two weeks (1 cycle). The dose-escalation part begins with the enrollment of a single patient into the lowest planned dose level (minimum anticipated biologic effect level [MABEL]). The dose-limiting toxicity (DLT) period for all cohorts is 21 days after the first dose of the HBPC. Dose escalation in single patient cohorts will proceed until the rules for expansion of dose escalation cohorts in accordance with a 3+3 dose escalation design are met. Dose escalation will continue using a 3+3 design until the maximum tolerated dose (MTD) or maximum administered dose (MAD) is reached. The MTD is defined as the highest dose at which less than 33% in a group of at least 6 DLT-evaluable patients experienced a DLT. DLT-evaluable is defined as a patient who experiences a DLT or who received at least 50% of the planned dose during the DLT period during the dose escalation part of the study.

[0499] The recommended phase 2 dose (RP2D) is the dose at or below the MTD that is pharmacologically optimal based on pharmacokinetics (PK), pharmacodynamics, target engagement, and antitumor activity and demonstrates acceptable safety and tolerability as determined by the Safety Review Committee (SRC). Additional patients with tumor types selected by sponsor may be enrolled at the MTD or lower dose levels to better understand safety and efficacy and to optimize recommended Phase 2 dose (RP2D) selection. Treatment-emergent adverse events (TEAEs), including those that would meet the protocol-defined DLT criteria, occurring in patients enrolled at these lower dose levels will be monitored as part of the ongoing safety oversight of the study and will be considered with all other data by the SRC when making decisions regarding future dosing in the study or the selection of the RP2D.

[0500] The initial infusion duration will be set at 60 minutes and may be altered based on emerging safety data. If the patient does not experience an infusion-related reaction (IRR) or if there are no signs of cytokine release syndrome (CRS) observed, the infusion duration may be reduced to 30 minutes for subsequent infusions. However, if the patient experiences an IRR or shows signs of CRS, then the infusion should be increased back up to 60 minutes. The SRC may modify the infusion rate should IRRs with unacceptable severity or frequency occur at a dose level, or if it is deemed necessary to improve safe administration of the HBPCs. Pre-medications with antihistamines and / or corticosteroids may be administered as needed based on emerging safety data.

[0501] An SRC, consisting of Investigators and Sponsor personnel, will review emerging safety data to determine the dose level and schedule for the HBPC for each cohort, including altering the schedule (if necessary) to include priming doses of HBPC to improve tolerability. When patients are enrolled in groups of 3-6 patients, enrollment of the first and second patients at a given dose level will be staggered by 16 days, to occur outside the period of maximum risk of CRS. Simultaneous screening of subjects is allowed including during the dose-escalation part of the study.

[0502] When at least 2 patients in the 3-6 patient cohort have completed the DLT observation period and the third patient has been observed for least 10 days without safety concerns the SRC may permit intra-patient dose escalation to a higher dose level (including the priming schedule if implemented) if the patient did not experience a clinically significant drug-related Grade > 3 AE or DLT in any prior cycle. Should the patient experience a DLT at the increased dose level, their dose will be reduced to the lower dose they have received prior to escalation. Dosing of cohorts 1-8 is shown in Table 5.Table 5. Dosing Schedule for Cohorts 1-8

[0503] If emerging safety data document Grade 2 or higher CRS (in any patient in one dose level, the HBPC may be administered in a priming dosing manner (two initial lower doses at a percentage of the full dose, followed by escalation to full treatment dose) to improve tolerability and reduce the risk of CRS. If the HBPC is administered via a priming dosing schedule, the DLT period will be extended from the first dose of the HBPC to 21 days after the third dose (when the targeted dose is attained) of the HBPC. The target dose for priming dosing administration shall be the highest dose level explored at that time with the conventional dose schedule. The priming dosing schedule may consist of every-7-day (Q7D) administration of the HBPC for Cycle 1, with the goal of reaching the target dose by Cycle 2 Day 1, at which time the full target dose will be administered every 14 days (Q14D) henceforth. An example of a possible Priming Dose Schedule is outlined in Table 6 (eg, dose of 25%, 50%, and 100% of target dose on Cycle 1 Day 1, Cycle 1 Day 8, and Cycle 2 Day 1, respectively; the SRC may also opt for an alternative priming dose schedule). Patients whose dosing is delayed for 4 weeks or longer after receiving a priming dose will resume treatment at the lowest priming dose.Table 6. Example Priming Dose Schedule

[0504] A prophylactic dose of tocilizumab may be administered at 8mg / kg up to 4 hours prior to administration of the HBPC. The dose of tocilizumab is not to exceed 800 mg. The dosing schedule of cohorts 9-12, including administration of tocilizumab, is shown in Table 7.Table 7. Dosing Criteria of Cohorts 9-122.2 Inclusion Criteria

[0505] To be eligible for the study, patients must meet all the following criteria prior to participation:Disease Characteristics

[0506] 1) Histologically confirmed diagnosis of metastatic or locally advanced unresectable solid tumor. Patients must have received, completed, or been intolerant of prior standard therapy appropriate for their tumor type and stage of disease, or in the opinion of the Investigator, would be unlikely to tolerate or derive clinically meaningful benefit from appropriate standard-of-care therapy.

[0507] 2) Agrees to provide fresh tumor tissue from tumor lesion that has not been previously irradiated for exploratory biomarker evaluation (if the tumor lesion wasirradiated with subsequent recurrence of disease, this lesion may be acceptable for biopsy and submission; please consult the Medical Monitor). Procedures more invasive than a core biopsy should not be utilized. Note: patients for whom the biopsy is medically contraindicated could be enrolled if a recent (within 6 months of screening) archival tumor tissue (20 slides or a tissue block are requested) can be submitted.

[0508] 3) Eligibility malignancies: Patients must have a tumor with histology known to be associated with EGFR expression and likely sensitive to immune-oncology agents, limited to non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), cutaneous squamous cell carcinoma, colorectal carcinoma (CRC) and gastric / gastroesophageal junction (g / GEJ) cancer.

[0509] 4) Measurable disease per RECIST vl. l. Dose escalation patients with evaluable(nonmeasurable) disease may be permitted to enroll with Medical Monitor approval.Baseline Characteristics

[0510] 5) > 18 years of age

[0511] 6) Eastern Cooperative Oncology Group (ECOG) performance status of 0-1 within14 days of Cycle 1, Day 1 (C1D1)Laboratory Values

[0512] Screening laboratory values must meet the following criteria within 14 days prior to ClDl :

[0513] Hematologic

[0514] 7) Absolute neutrophil count (ANC) > 1500 / / / L without granulocyte colonystimulating factor support within 14 days prior to C1D1 (if a patient has a documented history of benign ethnic neutropenia, the patient is eligible with a screening ANC > 1000 / pL without granulocyte colony-stimulating factor support within 14 days prior to C1D1)

[0515] 8) Platelet count > 75 * Kh / / / L; must not have been transfused within 14 days prior to ClDl

[0516] 9) Hemoglobin > 9.0 g / dL, maintained without transfusion within 14 days

[0517] Renal

[0518] 10) Calculated creatinine clearance > 45 mL / min calculated per the Cockcroft-GaultEquation.

[0519] Hepatic

[0520] 11) Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) < 3.0 x institution's upper limit of normal (ULN)

[0521] 12) Total bilirubin < 1.5 x institutional ULN (total bilirubin must be < 3 * institution's ULN in patients with Gilbert's syndrome)Contraception use

[0522] 13) Patients of childbearing potential or those with partners of childbearing potential must agree to use a highly effective method of birth control from signing the ICF, while receiving study treatment, and for a period of 30 days after the last dose of the HBPC. Male patients must refrain from donating sperm during the aforementioned period. Childbearing potential is defined as one who has experienced menarche and who is not permanently sterile or postmenopausal; postmenopausal is defined as 12 consecutive months with no menses without an alternative medical cause.2.3 Exclusion Criteria

[0523] Patients will be excluded if they meet any of the following criteria prior to participation:

[0524] 1) History of malignancy (unique from that which is being evaluated in this study) that is active within the previous 2 years except for localized cancers that are not related to the current cancer being treated, that are considered to have been cured, and in the opinion of the Investigator, present a low risk for recurrence, including but not limited to histories of basal or squamous cell skin cancer, superficial bladder cancer, or carcinoma in situ of the prostate or cervix or other cancer, which in the opinion of the Medical Monitor, may be eligible

[0525] 2) History of primary brain or spinal cord cancer

[0526] 3) Patients with known brain and / or leptomeningeal metastases

[0527] 4) Disease refractory (ie, which has been non-responsive) to prior immunooncology treatment, defined as not receiving clinical benefit as assessed by treating provider after receiving at least 2 doses of immuno-oncology agent

[0528] 5) Unresolved prior cancer therapy-related acute toxicity Grade > 1. Alopecia and other nonacute toxicities are not exclusionary.

[0529] 6) A patient with a screening electrocardiogram (ECG; performed in triplicate) demonstrating a mean QTcF value > 480 msec (auto-calculated); a screening echocardiogram or multi-gated acquisition (MUGA) scan with left ventricular ejection fraction (LVEF) < 50%.

[0530] 7) Serious concurrent illness including, but not limited to, the following: a) Active hepatitis B virus (HBV; defined as having a positive hepatitis B surface antigen [HBsAg] test at screening) or hepatitis C virus (HCV; defined as detectable HCV RNA) b) Active human immunodeficiency virus infection (as determined by viral load) or cytomegalovirus (CMV) infection c) Active infection requiring intravenous systemic therapy within 2 weeks prior to C1D1; active COVID-19 infection (defined as unresolved symptomatic disease with the exception of taste and smell), d) Significant cardiac disease such as recent myocardial infarction (< 6 months prior to C1D1), unstable angina pectoris, uncontrolled congestive heart failure (New York Heart Association Class > II), uncontrolled hypertension, Grade > 3 as measured by the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v5, uncontrolled cardiac arrhythmias, severe aortic stenosis, Grade > 3 cardiac toxicity following prior cytotoxic chemotherapy, or Grade > 2 myocarditis, e) History of multiple sclerosis or other demyelinating disease, Eaton-Lambert syndrome (paraneoplastic syndrome), history of hemorrhagic or ischemic stroke within the last 6 months, or alcoholic liver disease f) Nonhealing wound(s) or ulcer(s) except for ulcerative lesions caused by the underlying neoplasm g) Severe pulmonary disease, including prior lung disease secondary to EGFR therapy, interstitial lung disease, uncontrolled asthma, chronic obstructive pulmonary disease, or evidence of oxygen saturation < 92% on room air by pulse oximetry during screening examination h) Severe gastrointestinal disease including prior inflammatory bowel disease or uncontrolled diarrhea i) History of severe (Grade 4) cutaneous reactions to anti-EGFR therapy j) History of encephalitis, meningitis, organic brain disease (eg, Parkinson's disease) or uncontrolled seizures (those which continue despite medication) within 1 year prior to the first dose of study drug k) History of allogeneic tissue / solid organ transplant, stem cell transplant, or bone marrow transplant 1) History of autoimmune disease requiring maintenance immunosuppressive therapy including treatment with antitumor necrosis factor (TNF) alpha therapies, systemic corticosteroids (prednisone dose >10mg per day or equivalent), or other immune suppressive drugs within the 2 weeks prior to first dose of study drug. Note: The following conditions are not exclusionary: patients with vitiligo, resolved childhood atopic dermatitis or asthma, controlled type 1 diabetes mellitus, arthritis that is managed without systemic therapy beyond oral acetaminophen and nonsteroidal anti-inflammatory drugs, hypothyroidism, or hyperthyroidism that is clinically euthyroid at screening. Patients who require a brief course of steroids (up to 2 days in the week before enrollment) are not excluded, m) Active or chronic corneal disorder, including but not limited to the following: Sjogren’s syndrome, Fuchs’ corneal dystrophy (requiring treatment), history of corneal transplantation, active herpetic keratitis, prior refractive surgery (within 3 months of Cycle 1 Day 1), or checkpoint-inhibitor associated uveitis.Prior or concomitant therapies

[0531] 8) Receipt of systemic anticancer treatment, including anti-EGFR or investigational agent(s) within 14 days prior to C1D1

[0532] 9) Receipt of radiotherapy within 14 days prior to C1D1. Patients also must have recovered from acute effects of radiation.

[0533] 10) History of severe allergic or anaphylactic reactions to previous monoclonal antibody therapy or known hypersensitivity to any component of the HBPC

[0534] 11) Major surgery (requiring general anesthesia) within 3 months prior to dosing.Patients who have undergone major surgery within this period may be enrolled after consultation with the Medical Monitor.

[0535] 12) Receipt of live vaccine within 30 days prior to the planned first dose, including but not limited to measles, mumps, rubella, chicken pox / zoster, yellow fever, rabies, Bacillus Calmette-Guerin, and typhoid vaccines. Intranasal influenza vaccines contain live attenuated virus and are therefore prohibited. COVID-19 vaccination within 7 days of HBPC infusion prior to Cycle 1 Day 1 and Cycle 2 Day 1 is not permitted, as it may interfere with safety assessments.Other exclusions

[0536] 13) Current participation in an ongoing clinical study involving treatment with medications, radiation, or surgery.

[0537] 14) Pregnant or breastfeeding

[0538] 15) Situations or conditions that would limit compliance with study requirementsExample 3. Results of the Phase 1 / lb, Open-label, Dosing-finding, First-in-human Study to Evaluate the Safety and Antitumor activity of an activatable anti-EGFR, anti- CD3 heteromultimeric bispecific polypeptide complex (EIBPC)

[0539] Patients were treated in the Phase 1 / lb, Open-label, Dosing-finding, First-in-human Study to Evaluate the Safety and Antitumor activity of the activatable anti-EGFR, anti- CD3 heteromultimeric bispecific polypeptide complex (HBPC), Complex-67. The patients had locally advanced / metastatic cancer associated with EGFR expression, received or were intolerant to standard therapies, ECOG 0-1, measurable disease per RECIST 1.1, and adequate organ function. Complex 67 was administered at various dose levels (DLs) / schedules. Study objectives include assessment of safety / tolerability, antitumor activity, PK and determination of maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). As of April 16, 2024, 35 patients with a median of 4 prior lines of therapy had been treated with Complex 67 at 0.007 to 10 mg (FIG. 2).

[0540] Of these patients 18 had been diagnosed with colorectal cancer (CRC), 3 with nonsmall cell lung cancer (NSCLC), 2 with Gastric cancer, 1 with Esophageal cancer, 3 with head and neck squamous cell carcinoma (HNSCC), 7 with pancreatic cancer, and 1 with another cancer. Nineteen (19) patients were dosed with non-step doses 7pg-6mg on a Q2W schedule. Sixteen (16) patients were subsequently enrolled into step-dosing cohorts with target doses in the range of from 5 mg to 10 mg. The baseline characteristics of the patients is shown in Table 8.Table 8. Baseline Patient Characteristics

[0541] Dose limiting toxicities (DLTs) were observed in two patients treated at an initial dose of 6 mg Q2W (one with Grade 3 rash, a second with Grade 3 tenosynovitis). Subsequent dose escalation with step-dosing allowed escalation to a target dose of 10 mg without additional DLTs.

[0542] Reported treatment-emergent adverse events (TEAEs)in >15% were arthralgia (13, 37%; grade 3 in 2 (6%)), rash maculo-papular (12, 34%; grade 3 in 2 (6%)), fatigue (7, 20%; no grade 3), nausea (6, 17%; grade 3 in 1 (3%)), vomiting (6, 17%; no grade 3), and decreased appetite (6, 17%; no grade 3).

[0543] Treatment-Related adverse events are show in Tables 9 and 10 in non-step dosing and step-dosing patients, respectively. Arthralgia severity, incidence appear to be mitigated by use of step-dosing and tocilizumab prophylaxis. Rash adverse events appear to be manageable with topic and / or oral steroids and dose days. Adverse events were generally manageable as outpatient. There was no mandatory hospitalization for administration and monitoring at cleared dose levels.Table 9. Treatment Related adverse events (AEs) in Non-Step Dosing Patientsa Includes the preferred terms rash maculopapular, dermatitis acneiform, rash pustular, and skin exfoliationTable 10. Treatment Related AEs in Step Dosing Patientsa Includes the preferred terms rash maculo-papular, dermatitis acneiform, rash pustular, and skin exfoliation b Not protocol-defined DLTs

[0544] IL-6 levels were found to increase with increasing amounts of Complex 67 in nonstep dosing (FIG. 3). Musculoskeletal adverse events (AEs) emerged in dose escalation and were associated with dose dependent IL-6 elevation. IL-6 is associated with musculoskeletal pathology in autoimmune conditions and the toxicities elicited by immune-oncology agents. Use of tocilizumab has demonstrated potential to ameliorate immune-mediated musculoskeletal pathologies from immune-checkpoint inhibitor (ICI) etiologies. IL-6 inhibition with tocilizumab has not been reported to impact TCE efficacy.

[0545] Grade 1 cytokine release syndrome (CRS) was observed in 5 (14%) patients treated at the highest dose of 6 mg (n=5), with no CRS of any grade with step-dosing. Per protocol treatment at cleared dose levels did not require inpatient administration and monitoring. Eight patients dosed >0.75 mg had measurable tumor reduction, including 2 of 6 efficacy- evaluable patients (33%) with pancreatic cancer with confirmed partial responses perRECIST 1.1. All 6 patients had disease control (objective response or stable disease). For the patients with confirmed partial response, one patient (6 mg target dose) had an 83% tumor reduction and the second patient (5 mg target dose) had a 51% tumor reduction and remains on study treatment. In addition, a third pancreatic cancer patient has maintained stable disease with 3.5 months of study treatment. A description of the treatment and results for these patients is described in more detail hereinbelow.

[0546] Preliminary PK showed predominantly intact Complex 67 in circulation after administration of 3 mg Complex 67 (FIG. 4A). The preliminary estimate of half-life is between 2.8 and 5.3 days. Total Complex 67 exposure (Cmax and AUC) increased linearly with increasing dose (FIG. 4B). There was no apparent change in clearance with dose and no apparent target mediated drug disposition (TMDD).

[0547] A pre-treatment biopsy of a pancreatic cancer patient showed high levels of CD8+ cells in the tumor microenvironment (FIG. 5A). After treatment, patients had reduced CD8+ cells in the periphery, showing a preliminary correlation with response (FIG.5B). T cell margination was found in MSS CRC patients after treatment with Complex 67 (FIGs. 5C-5D). CD8+ infiltration post-Complex 67 treatment supports pursuit of future combination strategies.

[0548] The HBPC was generally safe and tolerable, with minimal CRS, and encouraging preliminary efficacy in heavily pretreated patients with advanced solid tumors, including pancreatic cancer.

[0549] Complex 67 masking was maintained in circulation. No CRS was observ...

Claims

WHAT IS CLAIMED IS:

1. A method of treating a subj ect afflicted with a cancer, the method comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising(i) a single-chain variable fragment (scFv) comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), wherein the VH1 and the VL1 together form a T-cell cluster of differentiation (CD3)-targeting domain that specifically binds a CD3 polypeptide, wherein the scFv comprises a set of VH1 and VL1 selected from:(A) a VH1 comprising a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), and a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO: 5), and a VL1 comprising a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:6), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO: 7), and a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO: 8); or(B) a VH1 comprising a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129), anda VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 130), and a VL1 comprising a VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 131), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO: 132), and a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO: 133);(ii) a first masking moiety (MM 1),(iii) a first cleavable moiety (CM1),(iv) a second heavy chain variable domain (VH2) comprising a VH CDR1 comprising the amino acid sequence NYGVH (SEQ ID NO: 15), a VH CDR2 comprising the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 16), and a VH CDR3 comprising the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 17);(v) a first monomeric Fc domain (Fcl);(b) a second polypeptide comprising(i) a second light chain variable domain (VL2), wherein the VH2 and the VL2 together form an EGFR targeting domain that specifically binds EGFR, wherein the VL2 comprises a VL CDR1 comprising the amino acid sequence RASQSIGTNIH (SEQ ID NO: 18), a VL CDR2 comprising the amino acid sequence YASESIS (SEQ ID NO: 19), and a VL CDR3 comprising the amino acid sequence QQNNNWPTT (SEQ ID NO:20),(ii) a second masking moiety (MM2), and(iii) a second cleavable moiety (CM2); and(c) a third polypeptide that(i) comprises a second monomeric Fc domain (Fc2), and(ii) does not comprise an immunoglobulin variable domain; andwherein the HBPC is administered at a dose of about 1 jug to about 50,000 jug.

2. The method of claim 1, wherein the VH1 comprises a VH CDR1 comprising the amino acid sequence KYAMN (SEQ ID NO:3), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:4), and a VH CDR3 comprising the amino acid sequence HGNFGNSYISYWAY (SEQ ID NO: 5), and wherein the VL1 comprises a VL CDR1 comprising the amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:6), a VL CDR2 comprising the amino acid sequence GTKFLAP (SEQ ID NO: 7), and a VL CDR3 comprising the amino acid sequence VLWYSNRWV (SEQ ID NO:8).

3. The method of claim 1 or 2, wherein the VH1 comprises the amino acid sequence of SEQ ID NOV and the VL1 comprises the amino acid sequence of SEQ ID NO: 10.

4. The method of any of claims 1-3, wherein the MM1 comprises the amino acid sequence of SEQ ID NO: 1.

5. The method of claim 1, wherein the VH1 comprises a VH CDR1 comprising the amino acid sequence TYAMN (SEQ ID NO: 128), a VH CDR2 comprising the amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO: 129), and a VH CDR3 comprising the amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 130), and wherein the VL1 comprisesa VL CDR1 comprising the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 131), a VL CDR2 comprising the amino acid sequence GTNKRAP (SEQ ID NO: 132), and a VL CDR3 comprising the amino acid sequence ALWYSNLWV (SEQ ID NO: 133) .

6. The method of claim 1 or 5, wherein the VH1 comprises the amino acid sequence of SEQ ID NO: 134 and the VL1 comprises the amino acid sequence of SEQ ID NO: 135.

7. The method of any of claims 1 and 5-6, wherein the MM1 comprises the amino acid sequence of SEQ ID NO:72.

8. The method of any of claims 1-7, wherein the MM2 comprises the amino acid sequence of SEQ ID NO: 13.

9. The method of any of claims 1-5, wherein the CM1 comprises the amino acid sequence of SEQ ID NO: 73.

10. The method of any of claims 1-5, wherein the CM2 comprises the amino acid sequence of SEQ ID NO: 14.

11. A method of treating a subj ect afflicted with a cancer, the method comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 144;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 37; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:32; and wherein the HBPC is administered at a dose of about 1 pg to about 50,000 pg.

12. A method of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31 ; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 1 pg to about 50,000 pg.

13. The method of any of the preceding claims, wherein the HBPC is administered at a dose of about 1 pg, about 2.5 pg, about 5 pg, about 7 pg, about 10 pg, about 20 pg, about 30 pg, about 40 pg, about 50 pg, about 60 pg, about 70 pg, about 80 pg, about 90 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, about 1000 pg, about 1250 pg, about 1500 pg, about 1750 pg, about 2000 pg, about 2250 pg, about 2500 pg, about 2750 pg, about 3000 pg, about 3500 pg, about 4000 pg, about 4500 pg, about 5000 pg, about 5500 pg, about 6000 pg, about 6500 pg, about 7000 pg, about 7500 pg, about8000 pg, about 8500 pg, about 9000 pg, about 9500 pg, about 10,000 pg, about 10,500 pg, about 11,000 pg, about 11,500 pg, about 12,000 pg, about 12,500 pg, about 13,000 pg, about 13,500 pg, about 14,000 pg, about 14,500 pg, about 15,000 pg, about 15,500 pg, about 16,000 pg, about 16,500 pg, about 17,000 pg, about 17,500 pg, about 18,000 pg, about 18,500 pg, about 19,000 pg, about 19,500 pg, 20,000 pg, about 22,500 pg, about25,000 pg, about 27,500 pg, about 30,000 pg, about 32,500 pg, about 35,000 pg, about 37,500 pg, about 40,000 pg, about 42,500 pg, about 45,000 pg, about 47,500 pg, or about50,000 pg.

14. The method of any of the preceding claims, wherein the HBPC is administered at a dose of about 7 pg, 70 pg, 250 pg, 750 pg, 1500 pg, 3000 pg, 5000 pg, 6000 pg, 10,000 pg, or 15,000 pg.

15. The method of any of the preceding claims, wherein the HBPC is administered once every two weeks.

16. The method of claim 15, wherein the HBPC is administered at a dose of between about 7 pg and about 70 pg once every two weeks.

17. The method of claim 15 or claim 16, wherein the HBPC is administered at a dose of about 7 pg once every two weeks.

18. The method of claim 15 or claim 16, wherein the HBPC is administered at a dose of about 70 pg once every two weeks, about 250 pg once every two weeks, about 750 pg once every two weeks, of about 1500 pg once every two weeks, about 3000 pg once every two weeks, about 6000 pg once every two weeks, about 5000 pg once every two weeks, about 10,000 pg once every two weeks, about 15,000 pg once every two weeks.

19. The method of any of the preceding claims, wherein a priming dose of the HBPC is administered prior to the first dose of the HBPC.

20. A method of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31 ; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the method comprises:(i) administering at least a first priming dose of HBPC to the subject on day 1; and(ii) administering to the subject a dose of the HBPC of about 1 pg to about 50,000 pg; wherein the first priming dose of the HBPC is less than the dose of HBPC in (ii).

21. The method of claim 20, wherein the dose in (ii) is administered on day 8.

22. The method of claim 20 or 21, wherein the method further comprises administering a second priming dose of HBPC to the subject on day 8, and wherein the dose in (ii) isadministered on day 15, wherein the second priming dose is 100% or less than the dose in (ii).

23. The method of any one of claims 20-22, wherein the first priming dose and / or the second priming dose is 50% or less than the dose in (ii).

24. The method of any one of claims 20-23, wherein the first priming dose is 25% or less than the dose in (ii).

25. The method of claim 19, wherein the subject is administered: a) a priming dose of the HBPC at about 3000 pg on day 1; b) about 5000 pg on day 15; and c) about 5000 pg every two weeks thereafter.

26. The method of claim 19, wherein the subject is administered: a) a priming dose of the HBPC at about 1500 pg on day 1; b) about 5000 pg on day 8; and c) about 5000 pg every two weeks thereafter.

27. The method of claim 19, wherein the subject is administered: a) a priming dose of the HBPC at about 3000 pg on day 1; b) about 5000 pg on day 8; and c) about 5000 pg every two weeks thereafter.

28. The method of claim 19, wherein the subject is administered: a) a priming dose of the HBPC at about 3000 pg on day 1; b) about 10,000 pg on day 15; and c) about 10,000 pg every two weeks thereafter.

29. The method of claim 19, wherein the subject is administered: a) a priming dose of the HBPC at about 1500 pg on day 1; b) a second priming dose of the HBPC at about 5000 pg on day 8; c) about 10,000 pg on day 15; andd) about 10,000 ig every two weeks thereafter.

30. The method of claim 19, wherein the subject is administered: a) a priming dose of the HBPC at about 3000 pg on day 1; b) a second priming dose of the HBPC at about 5000 pg on day 8; c) about 10,000 pg on day 15; and d) about 10,000 pg every two weeks thereafter.

31. The method of claim 19, wherein the subject is administered: a) a priming dose of the HBPC at about 3000 pg on day 1; b) a second priming dose of the HBPC at about 5000 pg on day 8; c) about 15,000 pg on day 15; and d) about 15,000 pg every two weeks thereafter.

32. The method of any of the preceding claims, wherein the subject is administered an IL-6 inhibitor prior to administration of the HBPC.

33. The method of claim 32, wherein the IL-6 inhibitor is a small molecule or an antibody.

34. The method of claim 32, wherein the IL-6 inhibitor is a corticosteroid.

35. The method of claim 34, wherein the corticosteroid is dexamethasone.

36. The method of claim 32 or claim 33, wherein the IL-6 inhibitor is tocilizumab, siltuximab, or sarilumab.

37. A method of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31 ; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32;wherein the HBPC is administered at a dose of about 6 mg once every two weeks.

38. A method of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31 ; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 1.5 mg on day 1, 5 mg on day8, and 5 mg once every two weeks thereafter.

39. A method of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31 ; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 1.5 mg on day 1, 5 mg on day8, 10 mg on day 15, and 10 mg once every two weeks thereafter.

40. A method of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31 ; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 3 mg once every two weeks.

41. The method of any one of the preceding claims, wherein the method further comprises administering a second therapeutic agent.

42. The method of claim 41, wherein the second therapeutic agent is selected from a chemotherapeutic agent, an immune-oncology agent, or a monoclonal antibody.

43. The method of claim 42, wherein the monoclonal antibody is selected from alemtuzumab, cetuximab, bevacizumab, dostarlimab, gemtuzumab, ipilimumab, nivolumab, ofatumumab, panitumumab, pembrolizumab, ranibizumab, rituximab, or trastuzumab.

44. The method of any of the preceding claims, wherein the cancer is sensitive to treatment with immune-oncology agents.

45. The method of any of the preceding claims, wherein the cancer is an EGFR-expressing cancer.

46. The method of any of the preceding claims, wherein the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), cutaneous squamous cell carcinoma, colorectal carcinoma (CRC), gastric / gastroesophageal junction (g / GEJ) cancer, esophageal cancer, breast cancer, bladder cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, and renal cell carcinoma (RCC).

47. The method of claim 46, wherein the cancer is selected from the group consisting of NSCLC, HNSCC, pancreatic cancer, CRC, and g / GEJ cancer.

48. The method of claim 46, wherein the cancer is selected from the group consisting of pancreatic cancer, NSCLC, HNSCC, g / GEJ, and esophageal cancer.

49. The method any of the preceding claims, wherein the cancer comprises a tumor.

50. The method of claim 49, wherein the tumor is an EGFR-expressing cancer.

51. The method of claim 49 or 50, wherein the tumor is a solid tumor.

52. The method of claim 51, wherein the solid tumor is sensitive to treatment with immune- oncology agents.

53. The method of any of the preceding claims, wherein the subject is administered the HBPC via intravenous infusion.

54. The method of claim 53, wherein the intravenous infusion is administered for 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, or 120 minutes.

55. The method of claims 53 or claim 54, wherein the intravenous infusion is administered for 30 minutes.

56. The method of claims 53 or claim 54, wherein the intravenous infusion is administered for 60 minutes.

57. The method of any of the preceding claims, wherein the subject is a human.

58. A method of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31 ; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 5 mg once every two weeks.

59. A method of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31 ; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 10 mg once every two weeks.

60. A method of treating a subject afflicted with a cancer comprising administering an activatable anti-EGFR, anti-CD3 heteromultimeric bispecific polypeptide complex (HBPC) to the subject, wherein the HBPC comprises:(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 30;(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 31 ; and(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 32; wherein the HBPC is administered at a dose of about 15 mg once every two weeks.

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