Antibodies targeting EGFR and CD3 and uses thereof

Abstract

Disclosed herein are antibodies that selectively bind to EGFR and CD3, wherein the anti-EGFR domain is in the form of a Fab or Fab'antibody linked to a single chain variable fragment (scFv) that binds to CD3. Bispecific antibody forms of Fab or Fab'linked to scFv provide potency and safety advantages over other bispecific antibody forms, Provided herein are antibodies that selectively bind to EGFR and CD3, pharmaceutical compositions thereof, and methods of producing such antibodies.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 424,314, filed on November 10, 2022, and U.S. Provisional Application No. 63 / 488,696, filed on March 6, 2023, both of which are incorporated herein by reference in their entireties.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy was created on October 20, 2023, is named 52426-749_601_SL.xml and is 30,073 bytes in size. Summary of the Invention

[0005] Disclosed herein is an isolated polypeptide complex according to the formula:

[0006] ALB

[0007] (Formula I)

[0008] Wherein A comprises a single-chain variable fragment (scFv) that binds to CD3, wherein the scFv comprises a scFv light chain variable domain and a scFv heavy chain variable domain, the scFv heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv heavy chain variable domain comprise HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3. NO:3; B comprises an antigen-binding fragment (Fab) or Fab' that binds to EGFR, wherein the Fab or Fab' comprises a Fab light chain polypeptide comprising a Fab light chain variable domain and a Fab heavy chain polypeptide comprising a Fab heavy chain variable domain, wherein the Fab heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the Fab heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19, and HC-CDR3: SEQ ID NO: 20, and wherein the Fab heavy chain polypeptide comprises an N-glycosylation site having an amino acid sequence of N-X1-X2, wherein X1 is any amino acid except P and X2 is T or S; and L comprises a linker connecting A to B.

[0009] In some embodiments, the Fab light chain variable domain comprises a complementarity determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the Fab light chain variable domain comprise LC-CDR1: SEQ ID NO: 21, LC-CDR2: SEQ ID NO: 22, and LC-CDR3: SEQ ID NO: 23, and the CDRs comprise 0-2 amino acid modifications in at least one of LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, the Fab light chain polypeptide comprises a mutation that eliminates pyroglutamic acid formation at the N-terminus of the Fab light chain polypeptide relative to the non-mutated form of the Fab light chain polypeptide. In some embodiments, the mutation is a Q to X3 mutation at residue number 1 at the N-terminus of the Fab light chain polypeptide, and X3 is an amino acid selected from the group consisting of: A, R, N, D, C, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X3 is selected from D and E. In some embodiments, X3 is D.

[0010] In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 26. In some embodiments, the Fab light chain polypeptide comprises a D at residue number 1 from the N-terminus of the Fab light chain polypeptide. In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the Fab light chain polypeptide comprises a Q at residue number 1 from the N-terminus of the Fab light chain polypeptide. In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 24.

[0011] In some embodiments, X2 of the N-glycosylation site is T. In some embodiments, X1 of the N-glycosylation site is D. In some embodiments, the Fab heavy chain polypeptide comprising the N-glycosylation site comprises the amino acid sequence QSNDTAIY (SEQ ID NO: 33). In some embodiments, the N of the N-glycosylation site is located at residue number 88 of the Fab heavy chain polypeptide. In some embodiments, the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 25.

[0012] In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 24 and the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide consists of the amino acid sequence of SEQ ID NO: 24 and the Fab heavy chain polypeptide consists of the amino acid sequence of SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 26 and the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide consists of the amino acid sequence of SEQ ID NO: 26 and the Fab heavy chain polypeptide consists of the amino acid sequence of SEQ ID NO: 25.

[0013] In some embodiments, the scFv light chain variable domain comprises a complementarity determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv light chain variable domain comprise LC-CDR1: SEQ ID NO: 6, LC-CDR2: SEQ ID NO: 7, and LC-CDR3: SEQ ID NO: 8. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 13. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of SEQ ID NO: 13. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 12. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of SEQ ID NO: 12. In some embodiments, the scFv comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 14. In some embodiments, the scFv comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 14. In some embodiments, the scFv comprises an amino acid sequence of at least 225 consecutive amino acid residues of SEQ ID NO: 14. In some embodiments, the scFv comprises an amino acid sequence of SEQ ID NO: 14.

[0014] In some embodiments, a linker connects the C-terminus of A to the N-terminus of B. In some embodiments, a linker connects the C-terminus of A to the N-terminus of a Fab light chain polypeptide. In some embodiments, a linker connects the C-terminus of A to the N-terminus of a Fab heavy chain polypeptide. In some embodiments, a linker connects the Fab heavy chain polypeptide to the C-terminus of a scFv light chain variable domain. In some embodiments, a linker connects the N-terminus of A to the C-terminus of B. In some embodiments, a linker connects the N-terminus of A to the C-terminus of a Fab heavy chain polypeptide. In some embodiments, a linker connects the N-terminus of A to the C-terminus of a Fab light chain polypeptide. In some embodiments, the linker is at least 5 amino acids in length. In some embodiments, the linker is no more than 10 amino acids in length. In some embodiments, the linker is no more than 30 amino acids in length. In some embodiments, the linker is at least 5 amino acids in length and no more than 30 amino acids in length. In some embodiments, the linker is 5 amino acids in length. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 28 (GGGGSGGGGSGGGGS), SEQ ID NO: 29 (GGGGS), or SEQ ID NO: 30 (GGGGSGGGS). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 29 (GGGGS).

[0015] In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and the Fab light chain polypeptide comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and the Fab light chain polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and the Fab light chain polypeptide comprises an amino acid sequence of at least 200 contiguous amino acid residues of SEQ ID NO: 24 or SEQ ID NO: 26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 24 or SEQ ID NO: 26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO: 32.

[0016] In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 24 and an amino acid sequence of SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises the amino acid sequences of SEQ ID NO: 24 and SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex consists of the amino acid sequences of SEQ ID NO: 24 and SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26 and an amino acid sequence of SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises the amino acid sequences of SEQ ID NO: 26 and SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex consists of the amino acid sequences of SEQ ID NO: 26 and SEQ ID NO: 32.

[0017] Disclosed herein is an isolated polypeptide complex according to the formula:

[0018] ALB

[0019] (Formula I)

[0020] Wherein A comprises a single-chain variable fragment (scFv) that binds to CD3, wherein the scFv comprises an scFv light chain variable domain and an scFv heavy chain variable domain; B comprises an antigen-binding fragment (Fab) or Fab' that binds to EGFR, wherein the Fab or Fab' comprises a Fab light chain polypeptide comprising a Fab light chain variable domain and a Fab heavy chain polypeptide comprising a Fab heavy chain variable domain, wherein the Fab heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2 and HC-CDR3, wherein the HC-CDR1, HC-CDR2 and HC-CDR3 of the Fab heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19 and HC-CDR3: SEQ ID NO: 20. NO:20, and wherein the Fab heavy chain polypeptide comprises an N-glycosylation site having an amino acid sequence of N-X1-X2, wherein X1 is any amino acid except P and X2 is T or S; and L comprises a linker connecting A to B, wherein the linker connects the Fab heavy chain polypeptide to the scFv light chain variable domain.

[0021] In some embodiments, the Fab light chain variable domain comprises a complementarity determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the Fab light chain variable domain comprise LC-CDR1: SEQ ID NO: 21, LC-CDR2: SEQ ID NO: 22, and LC-CDR3: SEQ ID NO: 23, and wherein the CDR comprises 0-2 amino acid modifications in at least one of LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, the Fab light chain polypeptide comprises a mutation that eliminates pyroglutamic acid formation at the N-terminus of the Fab light chain polypeptide relative to the non-mutated form of the Fab light chain polypeptide. In some embodiments, the mutation is a Q to X3 mutation at residue number 1 at the N-terminus of the Fab light chain polypeptide, and X3 is an amino acid selected from the group consisting of A, R, N, D, C, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X3 is selected from D and E. In some embodiments, X3 is D. In some embodiments, the Fab light chain polypeptide comprises a D at residue number 1 from the N-terminus of the Fab light chain polypeptide. In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 26. In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the Fab light chain polypeptide comprises a Q at residue number 1 from the N-terminus of the Fab light chain polypeptide. In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 24.

[0022] In some embodiments, X2 of the N-glycosylation site is T. In some embodiments, X1 of the N-glycosylation site is D. In some embodiments, the Fab heavy chain polypeptide comprising the N-glycosylation site comprises the amino acid sequence of QSNDTAIY (SEQ ID NO: 33). In some embodiments, the N of the N-glycosylation site is located at residue number 88 of the Fab heavy chain polypeptide. In some embodiments, the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 25.

[0023] In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 24 and the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide consists of the amino acid sequence of SEQ ID NO: 24 and the Fab heavy chain polypeptide consists of the amino acid sequence of SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 26 and the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide consists of the amino acid sequence of SEQ ID NO: 26 and the Fab heavy chain polypeptide consists of the amino acid sequence of SEQ ID NO: 25.

[0024] In some embodiments, the scFv heavy chain variable domain comprises the complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv heavy chain variable domain comprise HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3. In some embodiments, the scFv light chain variable domain comprises the complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv light chain variable domain comprise LC-CDR1: SEQ ID NO: 6, LC-CDR2: SEQ ID NO: 7, and LC-CDR3: SEQ ID NO: 8. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 100 contiguous amino acid residues of SEQ ID NO: 13. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of SEQ ID NO: 13. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 100 contiguous amino acid residues of SEQ ID NO: 12. In some embodiments, the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the scFv comprises an amino acid sequence of at least 200 contiguous amino acid residues of SEQ ID NO: 14. In some embodiments, the scFv comprises an amino acid sequence of at least 225 contiguous amino acid residues of SEQ ID NO: 14. In some embodiments, the scFv comprises an amino acid sequence of SEQ ID NO: 14.

[0025] In some embodiments, a linker connects the C-terminus of A to the N-terminus of B. In some embodiments, a linker connects the C-terminus of A to the N-terminus of a Fab heavy chain polypeptide. In some embodiments, a linker connects the Fab heavy chain polypeptide to the C-terminus of a scFv light chain variable domain. In some embodiments, a linker connects the N-terminus of a Fab heavy chain polypeptide to the C-terminus of a scFv light chain variable domain. In some embodiments, a linker connects the N-terminus of A to the C-terminus of B. In some embodiments, the linker is at least 5 amino acids in length. In some embodiments, the linker is no more than 10 amino acids in length. In some embodiments, the linker is no more than 30 amino acids in length. In some embodiments, the linker is at least 5 amino acids in length and no more than 30 amino acids in length. In some embodiments, the linker is 5 amino acids in length. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 28 (GGGGSGGGGSGGGGS), SEQ ID NO: 29 (GGGGS), or SEQ ID NO: 30 (GGGGSGGGS). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 29 (GGGGS).

[0026] In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and the Fab light chain polypeptide comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and the Fab light chain polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and the Fab light chain polypeptide comprises an amino acid sequence of at least 200 contiguous amino acid residues of SEQ ID NO: 24 or SEQ ID NO: 26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 24 or SEQ ID NO: 26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO: 32.

[0027] In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 24 and an amino acid sequence of SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises the amino acid sequences of SEQ ID NO: 24 and SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex consists of the amino acid sequences of SEQ ID NO: 24 and SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26 and an amino acid sequence of SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises the amino acid sequences of SEQ ID NO: 26 and SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex consists of the amino acid sequences of SEQ ID NO: 26 and SEQ ID NO: 32.

[0028] Additionally disclosed herein are pharmaceutical compositions comprising: (i) an isolated polypeptide complex according to any embodiment herein, and (ii) a pharmaceutically acceptable excipient.

[0029] Additionally disclosed herein are isolated recombinant nucleic acid molecules encoding the isolated polypeptide complex according to any embodiment herein.

[0030] Further disclosed herein are methods of treating cancer in a subject comprising administering to the subject the isolated polypeptide complex of any embodiment herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description and accompanying drawings which illustrate illustrative embodiments in which the principles of the invention are utilized, in which:

[0032] Figure 1 An exemplary configuration of a polypeptide complex that selectively binds to EGFR and CD3 is shown;

[0033] Figure 2 Shown is the binding of Ab-1, Ab-2, and Ab-3 to EGFR measured by ELISA;

[0034] Figure 3 shows the binding of Ab-1, Ab-2, and Ab-3 to CD3 as measured by ELISA; and

[0035] Figure 4Shown is the killing of HCT116 tumor cells mediated by Ab-1, Ab-2, and Ab-3 in the presence of CD8+ T cells. DETAILED DESCRIPTION

[0036] Certain definitions

[0037] The terms used herein are for the purpose of describing the particular situation only and are not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof are used in the detailed description and / or claims, such terms are intended to connote open-endedness in a manner similar to the term "comprising."

[0038] The term "antibody" is used in the broadest sense and encompasses fully assembled antibodies, antibody fragments that can bind antigen, such as Fab, F(ab')2, Fv, single-chain antibodies (scFv), diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, etc.

[0039] The term "complementarity determining region" or "CDR" refers to a segment of the variable region of an antibody that is structurally complementary to the epitope to which the antibody binds and is more variable than the rest of the variable region. For this reason, CDRs are sometimes referred to as hypervariable regions. The variable region comprises three CDRs. CDR peptides can be obtained by constructing genes encoding the CDRs of the antibody of interest. For example, such genes are prepared by synthesizing the variable regions from RNA of antibody-producing cells using the polymerase chain reaction. See, e.g., Larrick et al., Methods: A Companion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (Eds.), pp. 166-179 (Cambridge University Press 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al. (Eds.), pp. 137-185 (Wiley-Liss, Inc. 1995).

[0040] The term "Fab" refers to a protein containing the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments in that a few residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation for Fab' herein, in which the cysteine residues of the constant domains carry free thiol groups. Fab' fragments are produced by reducing the heavy chain disulfide bridges of the F(ab')2 fragment. Other chemical couplings of antibody fragments are also known.

[0041] "Single-chain variable fragment (scFv)" is a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an antibody, connected to a short linker peptide of 10 to about 25 amino acids. The linker is usually rich in glycine for flexibility, and serine or threonine for solubility, and can connect the N-terminus of VH to the C-terminus of VL, and vice versa. The protein retains the specificity of the original antibody, despite the removal of the constant region and the introduction of a linker. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. In addition, the antibody fragment comprises a single-chain polypeptide having the characteristics of a VH domain, i.e., capable of being assembled with a VL domain; or a single-chain polypeptide having the characteristics of a VL domain, i.e., capable of being assembled with a VH domain to reach a functional antigen binding site, thereby providing the antigen binding properties of a full-length antibody.

[0042] As used herein, the term " amino acid sequence identity percentage (%) " relevant to sequence is defined as after the alignment sequence and introducing room (if necessary) to obtain maximum percentage sequence identity, the percentage of the amino acid residue identical with the amino acid residue in the particular sequence in the candidate sequence, and does not consider any conservative replacement as a part for sequence identity.Comparison for determining amino acid sequence identity percentage can be realized in the various ways within the scope of the art, for example, use publicly available computer software, such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software implementation.Those skilled in the art can determine to be used to measure the appropriate parameters for comparison, are included in and realize maximum comparison required any algorithm on the full-length sequence compared.

[0043] Where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A relative to, with, or against a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or comprising a specific % amino acid sequence identity relative to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues recorded as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be understood that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0044] The terms "complementarity determining region" and "CDR", which are synonymous with "hypervariable region" or "HVR", are known in the art to refer to non-contiguous amino acid sequences within the variable region of an antibody that confer antigen specificity and / or binding affinity. In general, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. "Framework region" and "FR" are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region.The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography", J. Mol. Biol. 262, 732-745" ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains", Dev. Comp Immunol, 2003 Jan;27(1):55-77 ("IMGT" numbering scheme); Honegger A and Plückthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool", J Mol Biol, 2001 Jun 8;309(3):657-70, ("Aho" numbering scheme); and Whitelegg NR and Rees AR, "WAM: an improved algorithm for modelling antibodies on the WEB", Protein Eng. 2000 Dec;13(12):819-24 ("AbM" numbering scheme). In certain embodiments, the CDRs of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or a combination thereof.

[0045] The boundaries of a given CDR or FR can vary according to the scheme used to identify them. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. The numbering of both the Kabat scheme and the Chothia scheme is based on the most common antibody region sequence length, wherein insertion is regulated by inserting letters such as "30a", and deletion occurs in some antibodies. These two schemes place certain insertions and deletions ("indels") at different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures, and is similar to the Chothia numbering scheme in many respects.

[0046] Isolated peptide complex

[0047] Multispecific antibodies combine the benefits of different binding specificities from two or more antibodies into a single composition. Multispecific antibodies for redirecting T cells to cancer have shown promise in both preclinical and clinical studies. This approach relies on one antigen-interacting portion of the antibody binding to a tumor-associated antigen or marker, while the second antigen-interacting portion can bind to an effector cell antigen such as CD3 on T cells, which then triggers cytotoxic activity.

[0048] One such tumor-associated antigen is EGFR. Epidermal growth factor receptor (EGFR), also known as ERBB1, receptor tyrosine protein kinase ErbB-1, or proto-oncogene C ErbB-1, is a transmembrane glycoprotein that is a member of the protein kinase superfamily. EGFR is a cell surface protein that binds to epidermal growth factor, thereby inducing receptor dimerization and tyrosine autophosphorylation, leading to cell proliferation. Amplification and mutation of EGFR have been shown to be driving events in many cancer types.

[0049] Disclosed herein are antibodies that selectively bind to EGFR and CD3, wherein the anti-EGFR domain is in the form of a Fab or Fab' antibody linked to a single-chain variable fragment (scFv) that binds to CD3. The bispecific antibody format of Fab or Fab' linked to scFv offers efficacy and safety advantages over other bispecific antibody formats.

[0050] Disclosed herein is an isolated polypeptide complex according to the formula:

[0051] ALB

[0052] (Formula I)

[0053] Wherein A comprises a single-chain variable fragment (scFv) that binds to CD3, wherein the scFv comprises a scFv light chain variable domain and a scFv heavy chain variable domain, the scFv heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv heavy chain variable domain comprise HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3. NO: 3 (see Table 1); B comprises an antigen-binding fragment (Fab) or Fab' that binds to EGFR, wherein the Fab or Fab' comprises a Fab light chain polypeptide comprising a Fab light chain variable domain and a Fab heavy chain polypeptide comprising a Fab heavy chain variable domain, wherein the Fab heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the Fab heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19, and HC-CDR3: SEQ ID NO: 20 (see Table 4), and the Fab heavy chain polypeptide comprises an N-glycosylation site having an amino acid sequence of N-X1-X2, wherein X1 is any amino acid except P and X2 is T or S; and L comprises a linker connecting A to B.

[0054] Table 1. Anti-CD3 scFv heavy chain variable domain complementarity determining regions (CDRs) (as based on the IMGT CDR numbering system).

[0055]

[0056] Table 2. Anti-CD3 scFv light chain variable domain complementarity determining regions (CDRs) (as based on the IMGT CDR numbering system).

[0057]

[0058] Table 3. Anti-CD3 scFv light chain variable domain, heavy chain variable domain sequence and full length sequence

[0059]

[0060]

[0061] Table 4. Anti-EGFR Fab heavy chain variable domain complementarity determining regions (CDRs) (as based on the IMGT CDR numbering system).

[0062]

[0063] Table 5. Anti-EGFR Fab light chain variable domain complementarity determining regions (CDRs) (as based on the IMGT CDR numbering system).

[0064]

[0065] Antigen-binding fragment (Fab or Fab') that binds to EGFR

[0066] In some embodiments, the Fab light chain variable domain comprises a complementary determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the Fab light chain variable domain comprise LC-CDR1: SEQ ID NO: 21, LC-CDR2: SEQ ID NO: 22, and LC-CDR3: SEQ ID NO: 23 (see Table 5), and wherein the CDR comprises 0-2 amino acid modifications in at least one of LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, the Fab light chain variable domain comprises a complementary determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the Fab light chain variable domain comprise LC-CDR1: SEQ ID NO: 21, LC-CDR2: SEQ ID NO: 22, and LC-CDR3: SEQ ID NO: 23.

[0067] In some embodiments, the Fab light chain polypeptide comprises a mutation that eliminates pyroglutamate formation at the N-terminus of the Fab light chain polypeptide relative to the non-mutated form of the Fab light chain polypeptide. In some embodiments, the mutation is a Q to X3 mutation at residue number 1 at the N-terminus of the Fab light chain polypeptide, and wherein X3 is an amino acid selected from the group consisting of A, R, N, D, C, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X3 is A. In some embodiments, X3 is R. In some embodiments, X3 is N. In some embodiments, X3 is C. In some embodiments, X3 is E. In some embodiments, X3 is G. In some embodiments, X3 is H. In some embodiments, X3 is I. In some embodiments, X3 is L. In some embodiments, X3 is K. In some embodiments, X3 is M. In some embodiments, X3 is F. In some embodiments, X3 is P. In some embodiments, X3 is S. In some embodiments, X3 is T. In some embodiments, X3 is W. In some embodiments, X3 is Y. In some embodiments, X3 is V. In some embodiments, X3 is selected from D and E. In some embodiments, X3 is D.

[0068] In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 26 (see Table 6). In some embodiments, the Fab light chain polypeptide comprises a D at residue number 1 at the N-terminus of the Fab light chain polypeptide. In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the Fab light chain polypeptide comprises a Q at residue number 1 at the N-terminus of the Fab light chain polypeptide. In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 24.

[0069] Table 6. Anti-EGFR Fab light chain polypeptide and Fab heavy chain polypeptide sequences

[0070]

[0071] In some embodiments, X2 of the N-glycosylation site is T. In some embodiments, X1 of the N-glycosylation site is D. In some embodiments, X1 of the N-glycosylation site is A. In some embodiments, X1 of the N-glycosylation site is R. In some embodiments, X1 of the N-glycosylation site is N. In some embodiments, X1 of the N-glycosylation site is C. In some embodiments, X1 of the N-glycosylation site is E. In some embodiments, X1 of the N-glycosylation site is Q. In some embodiments, X1 of the N-glycosylation site is G. In some embodiments, X1 of the N-glycosylation site is H. In some embodiments, X1 of the N-glycosylation site is I. In some embodiments, X1 of the N-glycosylation site is L. In some embodiments, X1 of the N-glycosylation site is K. In some embodiments, X1 of the N-glycosylation site is M. In some embodiments, X1 of the N-glycosylation site is F. In some embodiments, X1 of the N-glycosylation site is S. In some embodiments, X1 of the N-glycosylation site is T. In some embodiments, X1 of the N-glycosylation site is W. In some embodiments, X1 of the N-glycosylation site is Y. In some embodiments, X1 of the N-glycosylation site is V. In some embodiments, the N-glycosylation site has the amino acid sequence NDT. In some embodiments, the Fab heavy chain polypeptide comprising the N-glycosylation site comprises the amino acid sequence QSNDTAIY (SEQ ID NO: 33). In some embodiments, the N of the N-glycosylation site is located at residue number 88 of the Fab heavy chain polypeptide.

[0072] In some embodiments, the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 25 (see Table 6).

[0073] In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 24, and the Fab heavy chain polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 24, and the Fab heavy chain polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 24, and the Fab heavy chain polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 200 contiguous amino acid residues of SEQ ID NO: 24, and the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 contiguous amino acid residues of SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 24, and the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide consists of the amino acid sequence of SEQ ID NO:24 and the Fab heavy chain polypeptide consists of the amino acid sequence of SEQ ID NO:25.

[0074] In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 26, and the Fab heavy chain polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 26, and the Fab heavy chain polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 26, and the Fab heavy chain polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 200 contiguous amino acid residues of SEQ ID NO: 26, and the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 contiguous amino acid residues of SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 26, and the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide consists of the amino acid sequence of SEQ ID NO:26 and the Fab heavy chain polypeptide consists of the amino acid sequence of SEQ ID NO:25.

[0075] Single-chain variable fragment (scFv) that binds to CD3

[0076] In some embodiments, the scFv light chain variable domain comprises a complementarity determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv light chain variable domain comprise LC-CDR1: SEQ ID NO: 6, LC-CDR2: SEQ ID NO: 7, and LC-CDR3: SEQ ID NO: 8, and the CDRs comprise 0-2 amino acid modifications in at least one of LC-CDR1, LC-CDR2, or LC-CDR3 (see Table 2). In some embodiments, the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv light chain variable domain comprise LC-CDR1: SEQ ID NO: 6, LC-CDR2: SEQ ID NO: 7, and LC-CDR3: SEQ ID NO: 8.

[0077] In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 13 (see Table 3). In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 100 contiguous amino acid residues of SEQ ID NO: 13. In some embodiments, the scFv heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the scFv heavy chain variable domain consists of the amino acid sequence of SEQ ID NO: 13.

[0078] In some embodiments, the scFv light chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 12 (see Table 3). In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 100 contiguous amino acid residues of SEQ ID NO: 12. In some embodiments, the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the scFv light chain variable domain consists of the amino acid sequence of SEQ ID NO: 12.

[0079] In some embodiments, the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 14 (see Table 3). In some embodiments, the scFv comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 14. In some embodiments, the scFv comprises an amino acid sequence of at least 225 consecutive amino acid residues of SEQ ID NO: 14. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the scFv consists of the amino acid sequence of SEQ ID NO: 14.

[0080] Connectors and connections

[0081] In some embodiments, a linker connects the C-terminus of A to the N-terminus of B. In some embodiments, a linker connects the C-terminus of A to the N-terminus of a Fab light chain polypeptide. In some embodiments, a linker connects the C-terminus of A to the N-terminus of a Fab heavy chain polypeptide. In some embodiments, a linker connects the C-terminus of a Fab heavy chain polypeptide to the C-terminus of a scFv light chain variable domain. In some embodiments, a linker connects the N-terminus of a Fab heavy chain polypeptide to the C-terminus of a scFv light chain variable domain.

[0082] In some embodiments, the linker connects the N-terminus of A to the C-terminus of B. In some embodiments, the linker connects the N-terminus of A to the C-terminus of the Fab heavy chain polypeptide. In some embodiments, the linker connects the N-terminus of A to the C-terminus of the Fab light chain polypeptide.

[0083] In some embodiments, the length of the linker is at least 5 amino acids. In some embodiments, the length of the linker is no more than 10 amino acids. In some embodiments, the length of the linker is no more than 30 amino acids. In some embodiments, the length of the linker is at least 5 amino acids and no more than 30 amino acids. In some embodiments, the length of the linker is 5 amino acids. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 28 (GGGGSGGGGSGGGGS), SEQ ID NO: 29 (GGGGS) or SEQ ID NO: 30 (GGGGSGGGS) (see Table 7). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 29 (GGGGS).

[0084] Table 7. Linker sequences

[0085]

[0086] In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and the Fab light chain polypeptide comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:32.

[0087] In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and the Fab light chain polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:32.

[0088] In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and the Fab light chain polypeptide comprises an amino acid sequence of at least 200 contiguous amino acid residues of SEQ ID NO:24 or SEQ ID NO:26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:32.

[0089] In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and the Fab light chain polypeptide comprises the amino acid sequence according to SEQ ID NO:24 or SEQ ID NO:26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:32.

[0090] Peptide complex sequence

[0091] In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 24 and an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 24 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 24 and an amino acid sequence that is at least 92% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 24 and an amino acid sequence that is at least 95% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 24 and an amino acid sequence that is at least 96% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 24 and an amino acid sequence that is at least 97% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 24 and an amino acid sequence that is at least 98% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 24 and an amino acid sequence that is at least 99% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises the amino acid sequences of SEQ ID NO: 24 and SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex consists of the amino acid sequences of SEQ ID NO: 24 and SEQ ID NO: 32.

[0092] In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26 and an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 26 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 26 and an amino acid sequence that is at least 92% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 26 and an amino acid sequence that is at least 95% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 26 and an amino acid sequence that is at least 97% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 26 and an amino acid sequence that is at least 98% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 26 and an amino acid sequence that is at least 99% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises the amino acid sequences of SEQ ID NO: 26 and SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex consists of the amino acid sequences of SEQ ID NO: 26 and SEQ ID NO: 32.

[0093] In some embodiments, the isolated polypeptide complex comprises a modified amino acid or non-natural amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or modified non-natural amino acid comprises a post-translational modification. In some embodiments, the isolated polypeptide complex comprises modifications including, but not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. Modifications can be made anywhere on the isolated polypeptide complex, including the peptide backbone, amino acid side chains, and termini.

[0094] Disclosed herein is an isolated polypeptide complex according to the formula:

[0095] ALB

[0096] (Formula I)

[0097] Wherein A comprises a single-chain variable fragment (scFv) that binds to CD3, wherein the scFv comprises an scFv light chain variable domain and an scFv heavy chain variable domain; B comprises an antigen-binding fragment (Fab) or Fab' that binds to EGFR, wherein the Fab or Fab' comprises a Fab light chain polypeptide comprising a Fab light chain variable domain and a Fab heavy chain polypeptide comprising a Fab heavy chain variable domain, wherein the Fab heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2 and HC-CDR3, wherein the HC-CDR1, HC-CDR2 and HC-CDR3 of the Fab heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19 and HC-CDR3: SEQ ID NO: 20. NO:20, and wherein the Fab heavy chain polypeptide comprises an N-glycosylation site having an amino acid sequence of N-X1-X2, wherein X1 is any amino acid except P and X2 is T or S; and L comprises a linker connecting A to B, wherein the linker connects the Fab heavy chain polypeptide to the scFv light chain variable domain.

[0098] Antigen-binding fragment (Fab or Fab') that binds to EGFR

[0099] In some embodiments, the Fab light chain variable domain comprises a complementary determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the Fab light chain variable domain comprise LC-CDR1: SEQ ID NO: 21, LC-CDR2: SEQ ID NO: 22, and LC-CDR3: SEQ ID NO: 23, and the CDRs comprise 0-2 amino acid modifications in at least one of LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, the Fab light chain variable domain comprises a complementary determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the Fab light chain variable domain comprise LC-CDR1: SEQ ID NO: 21, LC-CDR2: SEQ ID NO: 22, and LC-CDR3: SEQ ID NO: 23.

[0100] In some embodiments, the Fab light chain polypeptide comprises a mutation that eliminates pyroglutamate formation at the N-terminus of the Fab light chain polypeptide relative to the non-mutated form of the Fab light chain polypeptide. In some embodiments, the mutation is a Q to X3 mutation at residue number 1 at the N-terminus of the Fab light chain polypeptide, and wherein X3 is an amino acid selected from the group consisting of A, R, N, D, C, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X3 is A. In some embodiments, X3 is R. In some embodiments, X3 is N. In some embodiments, X3 is C. In some embodiments, X3 is E. In some embodiments, X3 is G. In some embodiments, X3 is H. In some embodiments, X3 is I. In some embodiments, X3 is L. In some embodiments, X3 is K. In some embodiments, X3 is M. In some embodiments, X3 is F. In some embodiments, X3 is P. In some embodiments, X3 is S. In some embodiments, X3 is T. In some embodiments, X3 is W. In some embodiments, X3 is Y. In some embodiments, X3 is V. In some embodiments, X3 is selected from D and E. In some embodiments, X3 is D.

[0101] In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 26. In some embodiments, the Fab light chain polypeptide comprises a D at residue number 1 from the N-terminus of the Fab light chain polypeptide. In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the Fab light chain polypeptide comprises a Q at residue number 1 from the N-terminus of the Fab light chain polypeptide. In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 24.

[0102] In some embodiments, X2 of the N-glycosylation site is T. In some embodiments, X1 of the N-glycosylation site is D. In some embodiments, X1 of the N-glycosylation site is A. In some embodiments, X1 of the N-glycosylation site is R. In some embodiments, X1 of the N-glycosylation site is N. In some embodiments, X1 of the N-glycosylation site is C. In some embodiments, X1 of the N-glycosylation site is E. In some embodiments, X1 of the N-glycosylation site is Q. In some embodiments, X1 of the N-glycosylation site is G. In some embodiments, X1 of the N-glycosylation site is H. In some embodiments, X1 of the N-glycosylation site is I. In some embodiments, X1 of the N-glycosylation site is L. In some embodiments, X1 of the N-glycosylation site is K. In some embodiments, X1 of the N-glycosylation site is M. In some embodiments, X1 of the N-glycosylation site is F. In some embodiments, X1 of the N-glycosylation site is S. In some embodiments, X1 of the N-glycosylation site is T. In some embodiments, X1 of the N-glycosylation site is W. In some embodiments, X1 of the N-glycosylation site is Y. In some embodiments, X1 of the N-glycosylation site is V. In some embodiments, the N-glycosylation site has the amino acid sequence NDT. In some embodiments, the Fab heavy chain polypeptide comprising the N-glycosylation site comprises the amino acid sequence QSNDTAIY (SEQ ID NO: 33). In some embodiments, the N of the N-glycosylation site is located at residue number 88 of the Fab heavy chain polypeptide.

[0103] In some embodiments, the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO:25.

[0104] In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 24, and the Fab heavy chain polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 24, and the Fab heavy chain polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 24, and the Fab heavy chain polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 200 contiguous amino acid residues of SEQ ID NO: 24, and the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 contiguous amino acid residues of SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 24, and the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide consists of the amino acid sequence of SEQ ID NO:24 and the Fab heavy chain polypeptide consists of the amino acid sequence of SEQ ID NO:25.

[0105] In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 26, and the Fab heavy chain polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 26, and the Fab heavy chain polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 26, and the Fab heavy chain polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 200 contiguous amino acid residues of SEQ ID NO: 26, and the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 contiguous amino acid residues of SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 26, and the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the Fab light chain polypeptide consists of the amino acid sequence of SEQ ID NO:26 and the Fab heavy chain polypeptide consists of the amino acid sequence of SEQ ID NO:25.

[0106] Single-chain variable fragment (scFv) that binds to CD3

[0107] In some embodiments, the scFv heavy chain variable domain comprises a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv heavy chain variable domain comprise HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3, and the CDRs comprise 0-2 amino acid modifications in at least one of HC-CDR1, HC-CDR2, or HC-CDR3. In some embodiments, the HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv heavy chain variable domain comprise HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3.

[0108] In some embodiments, the scFv light chain variable domain comprises a complementarity determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv light chain variable domain comprise LC-CDR1: SEQ ID NO: 6, LC-CDR2: SEQ ID NO: 7, and LC-CDR3: SEQ ID NO: 8, and the CDRs comprise 0-2 amino acid modifications in at least one of LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv light chain variable domain comprise LC-CDR1: SEQ ID NO: 6, LC-CDR2: SEQ ID NO: 7, and LC-CDR3: SEQ ID NO: 8.

[0109] In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 100 contiguous amino acid residues of SEQ ID NO: 13. In some embodiments, the scFv heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the scFv heavy chain variable domain consists of the amino acid sequence of SEQ ID NO: 13.

[0110] In some embodiments, the scFv light chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 100 contiguous amino acid residues of SEQ ID NO: 12. In some embodiments, the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the scFv light chain variable domain consists of the amino acid sequence of SEQ ID NO: 12.

[0111] In some embodiments, the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the scFv comprises an amino acid sequence of at least 200 contiguous amino acid residues of SEQ ID NO: 14. In some embodiments, the scFv comprises an amino acid sequence of at least 225 contiguous amino acid residues of SEQ ID NO: 14. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the scFv consists of the amino acid sequence of SEQ ID NO: 14.

[0112] Connectors and connections

[0113] In some embodiments, a linker connects the C-terminus of A to the N-terminus of B. In some embodiments, a linker connects the C-terminus of A to the N-terminus of a Fab heavy chain polypeptide. In some embodiments, a linker connects the C-terminus of a Fab heavy chain polypeptide to the C-terminus of a scFv light chain variable domain. In some embodiments, a linker connects the N-terminus of a Fab heavy chain polypeptide to the C-terminus of a scFv light chain variable domain.

[0114] In some embodiments, a linker connects the N-terminus of A to the C-terminus of B. In some embodiments, a linker connects the N-terminus of A to the C-terminus of a Fab heavy chain polypeptide. In some embodiments, a linker connects the Fab heavy chain polypeptide to the N-terminus of a scFv light chain variable domain. In some embodiments, a linker connects the C-terminus of a Fab heavy chain polypeptide to the N-terminus of a scFv light chain variable domain.

[0115] In some embodiments, the length of the linker is at least 5 amino acids. In some embodiments, the length of the linker is no more than 10 amino acids. In some embodiments, the length of the linker is no more than 30 amino acids. In some embodiments, the length of the linker is at least 5 amino acids and no more than 30 amino acids. In some embodiments, the length of the linker is 5 amino acids. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 28 (GGGGSGGGGSGGGGS), SEQ ID NO: 29 (GGGGS) or SEQ ID NO: 30 (GGGGSGGGS). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 29 (GGGGS).

[0116] In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and the Fab light chain polypeptide comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:32.

[0117] In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and the Fab light chain polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:32.

[0118] In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and the Fab light chain polypeptide comprises an amino acid sequence of at least 200 contiguous amino acid residues of SEQ ID NO:24 or SEQ ID NO:26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:32.

[0119] In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and the Fab light chain polypeptide comprises the amino acid sequence according to SEQ ID NO:24 or SEQ ID NO:26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:32.

[0120] Peptide complex sequence

[0121] In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 24 and an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 24 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 24 and an amino acid sequence that is at least 92% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 24 and an amino acid sequence that is at least 95% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 24 and an amino acid sequence that is at least 96% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 24 and an amino acid sequence that is at least 97% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 24 and an amino acid sequence that is at least 98% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 24 and an amino acid sequence that is at least 99% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises the amino acid sequences of SEQ ID NO: 24 and SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex consists of the amino acid sequences of SEQ ID NO: 24 and SEQ ID NO: 32.

[0122] In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26 and an amino acid sequence that is 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 26 and an amino acid sequence that is at least 90% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 26 and an amino acid sequence that is at least 92% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 26 and an amino acid sequence that is at least 95% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 26 and an amino acid sequence that is at least 97% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 26 and an amino acid sequence that is at least 98% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 26 and an amino acid sequence that is at least 99% identical to SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex comprises the amino acid sequences of SEQ ID NO: 26 and SEQ ID NO: 32. In some embodiments, the isolated polypeptide complex consists of the amino acid sequences of SEQ ID NO: 26 and SEQ ID NO: 32.

[0123] In some embodiments, the isolated polypeptide complex comprises a modified amino acid or non-natural amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or modified non-natural amino acid comprises a post-translational modification. In some embodiments, the isolated polypeptide complex comprises modifications including, but not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. Modifications can be made anywhere on the isolated polypeptide complex, including the peptide backbone, amino acid side chains, and termini.

[0124] Pharmaceutical composition

[0125] In some embodiments, disclosed herein are pharmaceutical compositions comprising: (i) an isolated polypeptide or polypeptide complex according to any embodiment disclosed herein; and (ii) a pharmaceutically acceptable excipient.

[0126] In some embodiments, disclosed herein are pharmaceutical compositions comprising an isolated polypeptide complex according to the following formula:

[0127] ALB

[0128] (Formula I)

[0129] Wherein A comprises a single-chain variable fragment (scFv) that binds to CD3, wherein the scFv comprises a scFv light chain variable domain and a scFv heavy chain variable domain, the scFv heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv heavy chain variable domain comprise HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3. NO:3; B comprises an antigen-binding fragment (Fab) or Fab' that binds to EGFR, wherein the Fab or Fab' comprises a Fab light chain polypeptide comprising a Fab light chain variable domain and a Fab heavy chain polypeptide comprising a Fab heavy chain variable domain, wherein the Fab heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the Fab heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19, and HC-CDR3: SEQ ID NO: 20, and wherein the Fab heavy chain polypeptide comprises an N-glycosylation site having an amino acid sequence of N-X1-X2, wherein X1 is any amino acid except P and X2 is T or S; and L comprises a linker connecting A to B.

[0130] In some embodiments, disclosed herein are pharmaceutical compositions comprising an isolated polypeptide complex according to the following formula:

[0131] ALB

[0132] (Formula I)

[0133] Wherein A comprises a single-chain variable fragment (scFv) that binds to CD3, wherein the scFv comprises an scFv light chain variable domain and an scFv heavy chain variable domain; B comprises an antigen-binding fragment (Fab) or Fab' that binds to EGFR, wherein the Fab or Fab' comprises a Fab light chain polypeptide comprising a Fab light chain variable domain and a Fab heavy chain polypeptide comprising a Fab heavy chain variable domain, wherein the Fab heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2 and HC-CDR3, wherein the HC-CDR1, HC-CDR2 and HC-CDR3 of the Fab heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19 and HC-CDR3: SEQ ID NO: 20. NO:20, and wherein the Fab heavy chain polypeptide comprises an N-glycosylation site having an amino acid sequence of N-X1-X2, wherein X1 is any amino acid except P and X2 is T or S; and L comprises a linker connecting A to B, wherein the linker connects the Fab heavy chain polypeptide to the scFv light chain variable domain.

[0134] In some embodiments, disclosed herein are pharmaceutical compositions comprising an isolated polypeptide complex comprising the amino acid sequence of SEQ ID NO:24 and the amino acid sequence of SEQ ID NO:32.

[0135] In some embodiments, disclosed herein are pharmaceutical compositions comprising an isolated polypeptide complex comprising the amino acid sequence of SEQ ID NO:26 and the amino acid sequence of SEQ ID NO:32.

[0136] In some embodiments, the polypeptide or polypeptide complex further comprises a detectable label, a therapeutic agent, or a pharmacokinetic modifying moiety. In some embodiments, the detectable label comprises a fluorescent label, a radiolabel, an enzyme, a nucleic acid probe, or a contrast agent.

[0137] For administration to a subject, a polypeptide or polypeptide complex as disclosed herein can be provided in the form of a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the component and is non-toxic to the patient to which it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions (such as oil / water emulsions), various types of wetting agents, sterile solutions, and the like. Such carriers can be formulated by conventional methods and can be administered to a subject at a suitable dose. Preferably, the composition is sterile. These compositions may also contain adjuvants, such as preservatives, emulsifiers, and dispersants. Protection against the action of microorganisms can be ensured by including various antibacterial and antifungal agents.

[0138] The pharmaceutical composition can be in any suitable form (depending on the desired method of administration). It can be provided in unit dosage form, can be provided in a sealed container, and can be provided as part of a kit. Such a kit can include instructions for use. It can include a plurality of such unit dosage forms.

[0139] The pharmaceutical composition can be suitable for administration by any suitable route, including parenteral (e.g., subcutaneous, intramuscular or intravenous) routes. Such compositions can be prepared by any method known in the pharmaceutical art, for example, by mixing the active ingredient with a carrier or excipient under aseptic conditions.

[0140] The dosage of the substances of the present disclosure can vary between wide limits, depending on the disease or disorder to be treated, the age and condition of the individual to be treated, etc., and the physician will ultimately determine the appropriate dosage to use.

[0141] Isolated recombinant nucleic acid molecules

[0142] Disclosed herein are isolated recombinant nucleic acid molecules encoding the isolated polypeptide complexes as disclosed herein. In some embodiments, described herein are isolated recombinant nucleic acid molecules encoding polypeptides comprising antibodies that selectively bind to CD3 and EGFR.

[0143] In some embodiments, disclosed herein are isolated recombinant nucleic acid molecules encoding an isolated polypeptide complex according to the following formula:

[0144] ALB

[0145] (Formula I)

[0146] Wherein A comprises a single-chain variable fragment (scFv) that binds to CD3, wherein the scFv comprises a scFv light chain variable domain and a scFv heavy chain variable domain, the scFv heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv heavy chain variable domain comprise HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3. NO:3; B comprises an antigen-binding fragment (Fab) or Fab' that binds to EGFR, wherein the Fab or Fab' comprises a Fab light chain polypeptide comprising a Fab light chain variable domain and a Fab heavy chain polypeptide comprising a Fab heavy chain variable domain, wherein the Fab heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the Fab heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19, and HC-CDR3: SEQ ID NO: 20, and wherein the Fab heavy chain polypeptide comprises an N-glycosylation site having an amino acid sequence of N-X1-X2, wherein X1 is any amino acid except P and X2 is T or S; and L comprises a linker connecting A to B.

[0147] In some embodiments, disclosed herein are isolated recombinant nucleic acid molecules encoding an isolated polypeptide complex according to the following formula:

[0148] ALB

[0149] (Formula I)

[0150] Wherein A comprises a single-chain variable fragment (scFv) that binds to CD3, wherein the scFv comprises an scFv light chain variable domain and an scFv heavy chain variable domain; B comprises an antigen-binding fragment (Fab) or Fab' that binds to EGFR, wherein the Fab or Fab' comprises a Fab light chain polypeptide comprising a Fab light chain variable domain and a Fab heavy chain polypeptide comprising a Fab heavy chain variable domain, wherein the Fab heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2 and HC-CDR3, wherein the HC-CDR1, HC-CDR2 and HC-CDR3 of the Fab heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19 and HC-CDR3: SEQ ID NO: 20. NO:20, and wherein the Fab heavy chain polypeptide comprises an N-glycosylation site having an amino acid sequence of N-X1-X2, wherein X1 is any amino acid except P and X2 is T or S; and L comprises a linker connecting A to B, wherein the linker connects the Fab heavy chain polypeptide to the scFv light chain variable domain.

[0151] In some embodiments, disclosed herein are isolated recombinant nucleic acid molecules encoding an isolated polypeptide complex comprising the amino acid sequence of SEQ ID NO:24 and the amino acid sequence of SEQ ID NO:32.

[0152] In some embodiments, disclosed herein are isolated recombinant nucleic acid molecules encoding an isolated polypeptide complex comprising the amino acid sequence of SEQ ID NO:26 and the amino acid sequence of SEQ ID NO:32.

[0153] Treatment

[0154] In some embodiments, disclosed herein are methods for treating cancer in a subject in need thereof, the methods comprising administering to the subject an isolated polypeptide complex of any embodiment disclosed herein. In some embodiments, the cancer has cells that express EGFR. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods for treating renal cell carcinoma (RCC), colorectal cancer (CRC), squamous cell carcinoma of the head and neck (SCCHN), non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, colon / rectal cancer, head and neck cancer, esophagogastric cancer, liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, or pancreatic cancer. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods for treating subjects that are resistant to treatment with EGFR inhibitors. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods for treating subjects that have KRAS mutations. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods for treating subjects that are resistant to treatment with EGFR inhibitors and have KRAS mutations.

[0155] In some embodiments, disclosed herein are methods of treating cancer in a subject in need thereof, the method comprising administering to the subject an isolated polypeptide complex according to the formula: ALB (Formula I), wherein A comprises a single-chain variable fragment (scFv) that binds to CD3, wherein the scFv comprises a scFv light chain variable domain and a scFv heavy chain variable domain, the scFv heavy chain variable domain comprising complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv heavy chain variable domain comprise HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3. NO:3; B comprises an antigen-binding fragment (Fab) or Fab' that binds to EGFR, wherein the Fab or Fab' comprises a Fab light chain polypeptide comprising a Fab light chain variable domain and a Fab heavy chain polypeptide comprising a Fab heavy chain variable domain, wherein the Fab heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the Fab heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19, and HC-CDR3: SEQ ID NO: 20, and wherein the Fab heavy chain polypeptide comprises an N-glycosylation site having an amino acid sequence of N-X1-X2, wherein X1 is any amino acid except P and X2 is T or S; and L comprises a linker connecting A to B.

[0156] In some embodiments, disclosed herein are methods of treating cancer in a subject in need thereof, the method comprising administering to the subject an isolated polypeptide complex according to the following formula: ALB (Formula I), wherein A comprises a single-chain variable fragment (scFv) that binds to CD3, wherein the scFv comprises a scFv light chain variable domain and a scFv heavy chain variable domain; and B comprises an antigen-binding fragment (Fab) or Fab' that binds to EGFR, wherein the Fab or Fab' comprises a Fab light chain polypeptide comprising a Fab light chain variable domain and a Fab heavy chain polypeptide comprising a Fab heavy chain variable domain, wherein the Fab heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the Fab heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19, and HC-CDR3: SEQ ID NO: 20. NO:20, and wherein the Fab heavy chain polypeptide comprises an N-glycosylation site having an amino acid sequence of N-X1-X2, wherein X1 is any amino acid except P and X2 is T or S; and L comprises a linker connecting A to B, wherein the linker connects the Fab heavy chain polypeptide to the scFv light chain variable domain.

[0157] Generation of antibodies that bind to EGFR and CD3

[0158] In some embodiments, the polypeptides described herein are produced using any method known in the art for synthesizing polypeptides (e.g., antibodies), particularly by chemical synthesis or by recombinant expression, and preferably by recombinant expression techniques.

[0159] In some cases, the antibody or its binding fragment is expressed recombinantly and nucleic acids encoding the antibody or its binding fragment are assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves synthesizing overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating those oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0160] Alternatively, nucleic acid molecules encoding antibodies are optionally generated from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell that expresses immunoglobulins) by PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for a particular gene sequence.

[0161] In some cases, antibodies or binding partners thereof are optionally produced by immunizing an animal (e.g., a mouse) to produce polyclonal antibodies, or more preferably, by producing monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497), or as described by Kozbor et al. (1983, Immunology Today 4:72), or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, clones encoding at least the Fab portion of an antibody are optionally obtained by screening a Fab expression library for clones that bind to the specific antigen (e.g., as described in Huse et al. 1989, Science 246: 1275-1281) or by screening antibody libraries (see, e.g., Clackson et al., 1991, Nature 352: 624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94: 4937).

[0162] In some embodiments, a technique developed for producing "chimeric antibodies" is used by splicing together genes from a mouse antibody molecule with appropriate antigenic specificity and genes from a human antibody molecule with appropriate biological activity (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454). Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having variable regions derived from mouse monoclonal antibodies and human immunoglobulin constant regions.

[0163] In some embodiments, the techniques described for producing single-chain antibodies (U.S. Patent No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are suitable for producing single-chain antibodies. Single-chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge to obtain a single-chain polypeptide. Techniques for assembling functional Fv fragments in E. coli (Skerra et al., 1988, Science 242:1038-1041) can also be optionally used.

[0164] In some embodiments, an expression vector comprising the nucleotide sequence of the antibody or the nucleotide sequence of the antibody is transferred to a host cell by conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In specific embodiments, the expression of the antibody is regulated by a constitutive, inducible, or tissue-specific promoter.

[0165] In some embodiments, a variety of host expression vector systems are utilized to express the antibodies or binding fragments thereof described herein. Such host expression systems represent vehicles for the coding sequences of antibodies that are produced and subsequently purified, but also represent cells that express the antibodies or binding fragments thereof in situ when transformed or transfected with appropriate nucleotide coding sequences. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and Bacillus subtilis (B. subtilis)) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody or binding fragment coding sequences; yeast (e.g., Pichia pastoris (Saccharomyces cerevisiae)) transformed with recombinant yeast expression vectors containing antibody or binding fragment coding sequences. Pichia); an insect cell system infected with a recombinant viral expression vector (e.g., baculovirus) containing the antibody or its binding fragment coding sequence; a plant cell system infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing the antibody or its binding fragment coding sequence; or a mammalian cell system (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) comprising a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or a promoter from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0166] For the long-term high yield production of recombinant protein, stable expression is preferred.In some cases, the cell line of stably expressing antibody is optionally engineered.Do not use the expression vector containing viral replication origin, but transform host cell with the DNA controlled by suitable expression control element (for example, promotor, enhancer, sequence, transcription terminator, polyadenylation site etc.) and selective marker.After introducing foreign DNA, the engineered cell allows to grow in enrichment medium and continues 1-2 days, is then transferred in the selective medium. The selective marker in the recombinant plasmid gives the resistance to selection, and allows the cell to stably integrate the plasmid in their chromosome, and grow to form a stove, and this stove is cloned and expanded in the cell line again.This method can be advantageously used for the cell line of engineered expression antibody or its binding fragment.

[0167] In some cases, a number of selection systems are used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes for tk cells, hgprt cells, or aprt cells, respectively. In addition, antimetabolite resistance has been used as the basis for selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 78: 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May 1993, TIB TECH 11(5):155-215) and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods well known in the art of recombinant DNA technology that can be used are described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds.), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY; Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1).

[0168] In some cases, the expression level of the antibody is increased by vector amplification (for review, see Bebbington and Hentschel, the use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Volume 3 (Academic Press, New York, 1987)). When the marker in the vector system expressing the antibody is amplifiable, an increase in the level of inhibitor present in the culture of the host cell will increase the copy number of the marker gene. Since the amplified region is associated with the nucleotide sequence of the antibody, the production of the antibody will also increase (Crouse et al., 1983, Mol. Cell Biol. 3: 257).

[0169] In some cases, any method known in the art for purifying antibodies is used, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen followed by protein A, and size column chromatography), centrifugation, differential solubilization, or by any other standard technique for purifying proteins.

[0170] expression vector

[0171] In some embodiments, the vector includes any suitable vector derived from a eukaryotic or prokaryotic source. In some cases, the vector is obtained from bacteria (e.g., Escherichia coli), insects, yeast (e.g., Pichia pastoris), algae, or mammalian sources. Exemplary bacterial vectors include pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

[0172] Exemplary insect vectors include pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393M11, pVL1393 M12, FLAG vectors such as pPolh-FLAG1 or pPolh-MAT2, or MAT vectors such as pPolh-MAT1 or pPolh-MAT2.

[0173] In some cases, the yeast carrier comprises pDEST TM 14 carriers, pDEST TM 15 carriers, pDEST TM 17 carriers, pDEST TM 24 carriers, pYES-DEST52 vector, pBAD-DEST49 Destination vector, pAO815 Pichia vector, pFLD1 Pichia pastoris vector, pGAPZA, B, and C Pichia pastoris vector, pPIC3.5K Pichia vector, pPIC6 A, B, and C Pichia vector, pPIC9K Pichia vector, pTEF1 / Zeo, pYES2 yeast vector, pYES2 / CT yeast vector, pYES2 / NT A, B, and C yeast vector, or pYES3 / CT yeast vector.

[0174] Exemplary algal vectors include pChlamy-4 vectors or MCS vectors.

[0175] Examples of mammalian vectors include transient expression vectors or stable expression vectors. Mammalian transient expression vectors can include pRK5, p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a, b, c, pFLAG-CMV 5.1, pFLAG-CMV 5a, b, c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4. Mammalian stable expression vectors can include pFLAG-CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2.

[0176] In some cases, the cell-free system is a mixture of cytoplasmic and / or nuclear components from cells and is used for in vitro nucleic acid synthesis. In some cases, the cell-free system utilizes prokaryotic cell components or eukaryotic cell components. Sometimes, nucleic acid synthesis is obtained in a cell-free system based on, for example, Drosophila cells, Xenopus eggs, or HeLa cells. Exemplary cell-free systems include, but are not limited to, the E. coli S30 extraction system, the E. coli T7 S30 system, or the

[0177] host cells

[0178] In some embodiments, the host cell includes any suitable cell, such as a naturally derived cell or a genetically modified cell. In some cases, the host cell is a production host cell. In some cases, the host cell is a eukaryotic cell. In other cases, the host cell is a prokaryotic cell. In some cases, the eukaryotic cell includes a fungus (e.g., a yeast cell), an animal cell, or a plant cell. In some cases, the prokaryotic cell is a bacterial cell. Examples of bacterial cells include gram-positive bacteria or gram-negative bacteria. Sometimes gram-negative bacteria are anaerobic, rod-shaped, or both.

[0179] In some cases, gram-positive bacteria include Actinobacteria, Firmicutes, or Tenericutes. In some cases, gram-negative bacteria include Aquificae, Deinococcus-Thermus, Fibrobacteres-Chlorobi / Bacteroidetes (FCB group), Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes-Verrucomicrobia / Chlamydiae (PVC group), Proteobacteria, Spirochaetes, or Synergistetes. The other bacteria may be of the phylum Acidobacteria, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Dictyoglomi, Thermodesulfobacteria, or Thermotogae. The bacterial cell may be Escherichia coli, Clostridium botulinum, or Coli bacilli.

[0180] Exemplary prokaryotic host cells include, but are not limited to, BL21, Mach1 TM DH10B TM 、TOP10、DH5α、DH10Bac TM 、OmniMax TM 、MegaX TM DH12S TM , INV110, TOP10F', INVαF, TOP10 / P3, ccdB Survival, PIR1, PIR2, Stbl2 TM 、Stbl3 TM or Stbl4 TM .

[0181] In some cases, animal cells include cells from vertebrates or invertebrates. In some cases, animal cells include cells from marine invertebrates, fish, insects, amphibians, reptiles, or mammals. In some cases, fungal cells include yeast cells, such as brewer's yeast, baker's yeast, or wine yeast.

[0182] In some cases, yeast includes ascomycetes, such as yeast, mold, filamentous fungi, basidiomycetes or zygomycetes. In some cases, yeast includes ascomycota (Ascomycota) or Basidiomycota (Basidiomycota). In some cases, ascomycota includes Saccharomyces (true yeast, such as Saccharomyces cerevisiae (baker's yeast)) or Exomycota (such as Schizosaccharomycetes (fission yeast)). In some cases, Basidiomycota includes Agaricomycota (for example, Tremellomycetes (Tremellomycetes)) or Pucciniomycotina (for example, Microbotryomycetes (Microbotryomycetes)).

[0183] Exemplary yeast or filamentous fungi include, for example, the genera Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula, Kluyveromyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidium, Aspergillus, Fusarium, or Trichoderma.

[0184] Exemplary yeasts or filamentous fungi include, e.g., species such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, Candida boidini, Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida viswanathii, Candida lusitaniae, Rhodotorula mucilaginosa, Pichia metanolica, Pichia angusta, Pichia pastoris, Pichia anomala, and Pichia spp. anomala), Hansenula polymorpha, Kluyveromyces lactis, Zygosaccharomyces rouxii, Yarrowia lipolytica, Trichosporon pullulans, Rhodosporidium toru-Aspergillus niger, Aspergillus nidulans, Aspergillus awamori, Aspergillus oryzae, Trichoderma reesei, Yarrowia lipolytica, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii, Zygosaccharomyces bailii), Cryptococcus neoformans, Cryptococcus gattii, or Saccharomyces boulardii.

[0185] Exemplary yeast host cells include, but are not limited to, Pichia pastoris strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33; and Saccharomyces cerevisiae yeast strains such as INVSc1.

[0186] In some cases, additional animal cells include cells obtained from molluscs, arthropods, annelids, or sponges. In some cases, additional animal cells are mammalian cells, such as cells from primates, apes, equines, bovines, porcines, canines, felines, or rodents. In some cases, rodents include mice, rats, hamsters, gerbils, hamsters, chinchillas, squirrels, or guinea pigs.

[0187] Exemplary mammalian host cells include, but are not limited to, 293A cell line, 293FT cell line, 293F cells, 293H cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, FUT8 KO CHOK1, Expi293F TM cells, Flp-In TM T-REx TM 293 cell line, Flp-In TM -293 cell line, Flp-In TM -3T3 cell line, Flp-In TM -BHK cell line, Flp-In TM -CHO cell lines, Flp-In TM -CV-1 cell line, Flp-In TM -Jurkat cell line, FreeStyle TM 293-F cells, FreeStyle TM CHO-S cells, GripTite TM 293MSR cell line, GS-CHO cell line, HepaRG TM cells, T-REx TM Jurkat cell line, Per.C6 cells, T-REx TM -293 cell line, T-REx TM -CHO cell lines and T-REx TM -HeLa cell line.

[0188] In some cases, the mammalian host cell is a stable cell line, or a cell line that has incorporated the genetic material of interest into its own genome and has the ability to express the products of that genetic material after many generations of cell division. In some cases, the mammalian host cell is a transient cell line, or a cell line that has not incorporated the genetic material of interest into its own genome and has no ability to express the products of that genetic material after many generations of cell division.

[0189] Exemplary insect host cells include, but are not limited to, Drosophila S2 cells, Sf9 cells, Sf21 cells, HighFive TM Cells and cell.

[0190] In some cases, the plant cell comprises a cell from algae.Exemplary insect cell lines include, but are not limited to, strains from Chlamydomonas reinhardtii 137c or Synechococcuse longatus PPC 7942.

[0191] Products

[0192] In another aspect of the invention, a product is provided that contains materials for treating, preventing and / or diagnosing the above-mentioned conditions. The product includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from a variety of materials (such as glass or plastic). The container is filled with a composition that can be effectively used to treat, prevent and / or diagnose the condition by itself or in combination with another composition; and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is a bispecific antibody as defined above, comprising a first antigen binding site that specifically binds to CD3 and a second antigen binding site that specifically binds to EGFR.

[0193] The label or package insert indicates that the composition is used to treat the selected condition. In addition, the article of manufacture may include (a) a first container containing a composition comprising a bispecific antibody of the invention; and (b) a second container containing a composition comprising an additional cytotoxic agent or other therapeutic agent. The article of manufacture in this embodiment of the invention may further include a package insert indicating that the composition can be used to treat a specific condition.

[0194] Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0195] Implementation Plan

[0196] Embodiment 1 includes an isolated polypeptide complex according to the following formula: ALB (Formula I), wherein A comprises a single-chain variable fragment (scFv) that binds to CD3, wherein the scFv comprises a scFv light chain variable domain and a scFv heavy chain variable domain, the scFv heavy chain variable domain comprising complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv heavy chain variable domain comprise HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3. NO:3; B comprises an antigen-binding fragment (Fab) or Fab' that binds to EGFR, wherein the Fab or Fab' comprises a Fab light chain polypeptide comprising a Fab light chain variable domain and a Fab heavy chain polypeptide comprising a Fab heavy chain variable domain, wherein the Fab heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the Fab heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19, and HC-CDR3: SEQ ID NO: 20, and wherein the Fab heavy chain polypeptide comprises an N-glycosylation site having an amino acid sequence of N-X1-X2, wherein X1 is any amino acid except P and X2 is T or S; and L comprises a linker connecting A to B.

[0197] Embodiment 2 includes the isolated polypeptide complex according to embodiment 1, wherein the Fab light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2 and LC-CDR3, wherein the LC-CDR1, LC-CDR2 and LC-CDR3 of the Fab light chain variable domain comprise LC-CDR1: SEQ ID NO: 21, LC-CDR2: SEQ ID NO: 22 and LC-CDR3: SEQ ID NO: 23, and wherein the CDRs comprise 0-2 amino acid modifications in at least one of LC-CDR1, LC-CDR2 or LC-CDR3.

[0198] Embodiment 3 includes the isolated polypeptide complex of embodiment 1 or 2, wherein the Fab light chain polypeptide comprises a mutation that eliminates pyroglutamate formation at the N-terminus of the Fab light chain polypeptide relative to a non-mutated form of the Fab light chain polypeptide.

[0199] Embodiment 4 includes the isolated polypeptide complex of embodiment 3, wherein the mutation is a Q to X3 mutation at residue number 1 of the N-terminus of the Fab light chain polypeptide, and wherein X3 is an amino acid selected from the group consisting of: A, R, N, D, C, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V.

[0200] Embodiment 5 includes the isolated polypeptide complex according to embodiment 4, wherein X3 is selected from D and E.

[0201] Embodiment 6 includes the isolated polypeptide complex according to embodiment 5, wherein X3 is D.

[0202] Embodiment 7 includes the isolated polypeptide complex of embodiment 1 or 2, wherein the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:26.

[0203] Embodiment 8 includes the isolated polypeptide complex of any one of embodiments 1-6, wherein the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO:26.

[0204] Embodiment 9 includes the isolated polypeptide complex of embodiment 1 or 2, wherein the Fab light chain polypeptide comprises a Q at residue number 1 of the N-terminus of the Fab light chain polypeptide.

[0205] Embodiment 10 includes the isolated polypeptide complex of any one of embodiments 1, 2, or 9, wherein the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO:24.

[0206] Embodiment 11 includes the isolated polypeptide complex according to any of the preceding embodiments, wherein X2 is T.

[0207] Embodiment 12 includes the isolated polypeptide complex according to any of the preceding embodiments, wherein X1 is D.

[0208] Embodiment 13 comprises the isolated polypeptide complex according to any of the preceding embodiments, wherein the Fab heavy chain polypeptide comprising an N-glycosylation site comprises the amino acid sequence QSNDTAIY (SEQ ID NO: 33).

[0209] Embodiment 14 includes the isolated polypeptide complex according to any of the preceding embodiments, wherein the N of the N-glycosylation site is located at residue number 88 of the Fab heavy chain polypeptide.

[0210] Embodiment 15 includes the isolated polypeptide complex according to any of the preceding embodiments, wherein the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO:25.

[0211] Embodiment 16 includes the isolated polypeptide complex of embodiment 1 or 2, wherein the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO:24 and the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO:25.

[0212] Embodiment 17 includes the isolated polypeptide complex of embodiment 1 or 2, wherein the Fab light chain polypeptide consists of the amino acid sequence of SEQ ID NO:24 and the Fab heavy chain polypeptide consists of the amino acid sequence of SEQ ID NO:25.

[0213] Embodiment 18 includes the isolated polypeptide complex of any one of embodiments 1-6, wherein the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO:26 and the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO:25.

[0214] Embodiment 19 includes the isolated polypeptide complex of any one of embodiments 1-6, wherein the Fab light chain polypeptide consists of the amino acid sequence of SEQ ID NO:26 and the Fab heavy chain polypeptide consists of the amino acid sequence of SEQ ID NO:25.

[0215] Embodiment 20 includes an isolated polypeptide complex according to any of the preceding embodiments, wherein the scFv light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv light chain variable domain comprise LC-CDR1: SEQ ID NO: 6, LC-CDR2: SEQ ID NO: 7, and LC-CDR3: SEQ ID NO: 8.

[0216] Embodiment 21 includes the isolated polypeptide complex of any of the preceding embodiments, wherein the scFv heavy chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:13.

[0217] Embodiment 22 includes the isolated polypeptide complex of any one of embodiments 1-20, wherein the scFv heavy chain variable domain comprises an amino acid sequence of at least 100 contiguous amino acid residues of SEQ ID NO:13.

[0218] Embodiment 23 includes the isolated polypeptide complex of any of the preceding embodiments, wherein the scFv heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:13.

[0219] Embodiment 24 includes the isolated polypeptide complex of any of the preceding embodiments, wherein the scFv light chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:12.

[0220] Embodiment 25 includes the isolated polypeptide complex of any one of embodiments 1-23, wherein the scFv light chain variable domain comprises an amino acid sequence of at least 100 contiguous amino acid residues of SEQ ID NO:12.

[0221] Embodiment 26 includes the isolated polypeptide complex of any of the preceding embodiments, wherein the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:12.

[0222] Embodiment 27 includes the isolated polypeptide complex of any of the preceding embodiments, wherein the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:14.

[0223] Embodiment 28 includes the isolated polypeptide complex of any one of embodiments 1-26, wherein the scFv comprises an amino acid sequence of at least 200 contiguous amino acid residues of SEQ ID NO:14.

[0224] Embodiment 29 includes the isolated polypeptide complex of any one of embodiments 1-26, wherein the scFv comprises an amino acid sequence of at least 225 contiguous amino acid residues of SEQ ID NO:14.

[0225] Embodiment 30 includes the isolated polypeptide complex of any of the preceding embodiments, wherein the scFv comprises the amino acid sequence of SEQ ID NO:14.

[0226] Embodiment 31 includes the isolated polypeptide complex according to any of the preceding embodiments, wherein a linker connects the C-terminus of A to the N-terminus of B.

[0227] Embodiment 32 includes the isolated polypeptide complex according to any of the preceding embodiments, wherein a linker connects the C-terminus of A to the N-terminus of the Fab light chain polypeptide.

[0228] Embodiment 33 includes the isolated polypeptide complex of any one of embodiments 1-31, wherein a linker connects the C-terminus of A to the N-terminus of the Fab heavy chain polypeptide.

[0229] Embodiment 34 includes the isolated polypeptide complex of any one of embodiments 1-31 and 33, wherein a linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain.

[0230] Embodiment 35 includes the isolated polypeptide complex of any one of embodiments 1-30, wherein a linker connects the N-terminus of A to the C-terminus of B.

[0231] Embodiment 36 includes the isolated polypeptide complex of any one of embodiments 1-30 and 35, wherein the linker connects the N-terminus of A to the C-terminus of the Fab heavy chain polypeptide.

[0232] Embodiment 37 includes the isolated polypeptide complex of any one of embodiments 1-30 and 35, wherein the linker connects the N-terminus of A to the C-terminus of the Fab light chain polypeptide.

[0233] Embodiment 38 includes the isolated polypeptide complex according to any of the preceding embodiments, wherein the linker is at least 5 amino acids in length.

[0234] Embodiment 39 includes the isolated polypeptide complex according to any of the preceding embodiments, wherein the linker is no more than 10 amino acids in length.

[0235] Embodiment 40 includes the isolated polypeptide complex according to any of the preceding embodiments, wherein the linker is no more than 30 amino acids in length.

[0236] Embodiment 41 includes the isolated polypeptide complex according to any of the preceding embodiments, wherein the linker is at least 5 amino acids and no more than 30 amino acids in length.

[0237] Embodiment 42 includes the isolated polypeptide complex according to any of the preceding embodiments, wherein the linker is 5 amino acids in length.

[0238] Embodiment 43 includes the isolated polypeptide complex of any one of embodiments 1-37, wherein the linker comprises the amino acid sequence of SEQ ID NO: 28 (GGGGSGGGGSGGGGS), SEQ ID NO: 29 (GGGGS), or SEQ ID NO: 30 (GGGGSGGGS).

[0239] Embodiment 44 includes the isolated polypeptide complex of embodiment 43, wherein the linker comprises the amino acid sequence of SEQ ID NO: 29 (GGGGS).

[0240] Embodiment 45 includes the isolated polypeptide complex of any one of embodiments 1-31, 33-34, and 38-44, wherein the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and wherein the Fab light chain polypeptide comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:32.

[0241] Embodiment 46 includes the isolated polypeptide complex of any one of embodiments 1-31, 33-34, and 38-44, wherein the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and wherein the Fab light chain polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:32.

[0242] Embodiment 47 includes the isolated polypeptide complex of any one of embodiments 1-31, 33-34, and 38-44, wherein the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and wherein the Fab light chain polypeptide comprises an amino acid sequence of at least 200 contiguous amino acid residues of SEQ ID NO:24 or SEQ ID NO:26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:32.

[0243] Embodiment 48 includes the isolated polypeptide complex of any one of embodiments 1-31, 33-34, and 38-44, wherein the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and wherein the Fab light chain polypeptide comprises the amino acid sequence according to SEQ ID NO:24 or SEQ ID NO:26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:32.

[0244] Embodiment 49 includes the isolated polypeptide complex of any one of embodiments 1-2, 9-17, and 20-48, wherein the isolated polypeptide complex comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:24 and an amino acid sequence of SEQ ID NO:32.

[0245] Embodiment 50 includes the isolated polypeptide complex of any one of embodiments 1-2, 9-17, and 20-48, wherein the isolated polypeptide complex comprises the amino acid sequences of SEQ ID NO:24 and SEQ ID NO:32.

[0246] Embodiment 51 includes the isolated polypeptide complex of any one of embodiments 1-2, 9-17, and 20-48, wherein the isolated polypeptide complex consists of the amino acid sequences of SEQ ID NO:24 and SEQ ID NO:32.

[0247] Embodiment 52 includes the isolated polypeptide complex of any one of embodiments 1-8, 11-15, and 18-48, wherein the isolated polypeptide complex comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:26 and an amino acid sequence of SEQ ID NO:32.

[0248] Embodiment 53 includes the isolated polypeptide complex of any one of embodiments 1-8, 11-15, and 18-48, wherein the isolated polypeptide complex comprises the amino acid sequences of SEQ ID NO:26 and SEQ ID NO:32.

[0249] Embodiment 54 includes the isolated polypeptide complex of any one of embodiments 1-8, 11-15, and 18-48, wherein the isolated polypeptide complex consists of the amino acid sequences of SEQ ID NO:26 and SEQ ID NO:32.

[0250] Embodiment 55 includes an isolated polypeptide complex according to the following formula: ALB (Formula I), wherein A comprises a single-chain variable fragment (scFv) that binds to CD3, wherein the scFv comprises a scFv light chain variable domain and a scFv heavy chain variable domain; and B comprises an antigen binding fragment (Fab) or Fab' that binds to EGFR, wherein the Fab or Fab' comprises a Fab light chain polypeptide comprising a Fab light chain variable domain and a Fab heavy chain polypeptide comprising a Fab heavy chain variable domain, wherein the Fab heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the Fab heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19, and HC-CDR3: SEQ ID NO: 20. ID NO: 20, and wherein the Fab heavy chain polypeptide comprises an N-glycosylation site having an amino acid sequence of N-X1-X2, wherein X1 is any amino acid except P and X2 is T or S; and L comprises a linker connecting A to B, wherein the linker connects the Fab heavy chain polypeptide to the scFv light chain variable domain.

[0251] Embodiment 56 includes the isolated polypeptide complex of embodiment 55, wherein the Fab light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the Fab light chain variable domain comprise LC-CDR1: SEQ ID NO: 21, LC-CDR2: SEQ ID NO: 22, and LC-CDR3: SEQ ID NO: 23, and wherein the CDRs comprise 0-2 amino acid modifications in at least one of LC-CDR1, LC-CDR2, or LC-CDR3.

[0252] Embodiment 57 includes the isolated polypeptide complex of embodiment 55 or 56, wherein the Fab light chain polypeptide comprises a mutation that eliminates pyroglutamate formation at the N-terminus of the Fab light chain polypeptide relative to a non-mutated form of the Fab light chain polypeptide.

[0253] Embodiment 58 includes the isolated polypeptide complex of embodiment 57, wherein the mutation is a Q to X3 mutation at residue number 1 of the N-terminus of the Fab light chain polypeptide, and wherein X3 is an amino acid selected from the group consisting of: A, R, N, D, C, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V.

[0254] Embodiment 59 includes the isolated polypeptide complex of embodiment 58, wherein X3 is selected from D and E.

[0255] Embodiment 60 includes the isolated polypeptide complex of embodiment 58, wherein X3 is D.

[0256] Embodiment 61 includes the isolated polypeptide complex of any one of embodiments 55-60, wherein the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:26.

[0257] Embodiment 62 includes the isolated polypeptide complex of any one of embodiments 55-61, wherein the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO:26.

[0258] Embodiment 63 includes the isolated polypeptide complex of embodiment 55 or 56, wherein the Fab light chain polypeptide comprises a Q at residue number 1 of the N-terminus of the Fab light chain polypeptide.

[0259] Embodiment 64 includes the isolated polypeptide complex of any one of embodiments 55-56 and 63, wherein the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO:24.

[0260] Embodiment 65 includes the isolated polypeptide complex of any one of embodiments 55-64, wherein X2 is T.

[0261] Embodiment 66 includes the isolated polypeptide complex of any one of embodiments 55-65, wherein X1 is D.

[0262] Embodiment 67 comprises the isolated polypeptide complex of any one of embodiments 55-66, wherein the Fab heavy chain polypeptide comprising an N-glycosylation site comprises the amino acid sequence of QSNDTAIY (SEQ ID NO: 33).

[0263] Embodiment 68 includes the isolated polypeptide complex of any one of embodiments 55-67, wherein the N of the N-glycosylation site is at residue number 88 of the Fab heavy chain polypeptide.

[0264] Embodiment 69 includes the isolated polypeptide complex of any one of embodiments 55-68, wherein the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO:25.

[0265] Embodiment 70 includes the isolated polypeptide complex of any one of embodiments 55-56, 61, and 63-69, wherein the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO:24 and the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO:25.

[0266] Embodiment 71 includes the isolated polypeptide complex of any one of embodiments 55-56, 61, and 63-70, wherein the Fab light chain polypeptide consists of the amino acid sequence of SEQ ID NO:24 and the Fab heavy chain polypeptide consists of the amino acid sequence of SEQ ID NO:25.

[0267] Embodiment 72 includes the isolated polypeptide complex of any one of embodiments 55-62 and 65-69, wherein the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO:26 and the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO:25.

[0268] Embodiment 73 includes the isolated polypeptide complex of any one of embodiments 55-62 and 65-69, wherein the Fab light chain polypeptide consists of the amino acid sequence of SEQ ID NO:26 and the Fab heavy chain polypeptide consists of the amino acid sequence of SEQ ID NO:25.

[0269] Embodiment 74 includes the isolated polypeptide complex of any one of embodiments 55-73, wherein the scFv heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv heavy chain variable domain comprise HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3.

[0270] Embodiment 75 includes an isolated polypeptide complex according to any one of embodiments 55-74, wherein the scFv light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv light chain variable domain comprise LC-CDR1: SEQ ID NO: 6, LC-CDR2: SEQ ID NO: 7, and LC-CDR3: SEQ ID NO: 8.

[0271] Embodiment 76 includes the isolated polypeptide complex of any one of embodiments 55-75, wherein the scFv heavy chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:13.

[0272] Embodiment 77 includes the isolated polypeptide complex of any one of embodiments 55-75, wherein the scFv heavy chain variable domain comprises an amino acid sequence of at least 100 contiguous amino acid residues of SEQ ID NO:13.

[0273] Embodiment 78 includes the isolated polypeptide complex of any one of embodiments 55-77, wherein the scFv heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:13.

[0274] Embodiment 79 includes the isolated polypeptide complex of any one of embodiments 55-78, wherein the scFv light chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:12.

[0275] Embodiment 80 includes the isolated polypeptide complex of any one of embodiments 55-78, wherein the scFv light chain variable domain comprises an amino acid sequence of at least 100 contiguous amino acid residues of SEQ ID NO:12.

[0276] Embodiment 81 includes the isolated polypeptide complex of any one of embodiments 55-80, wherein the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:12.

[0277] Embodiment 82 includes the isolated polypeptide complex of any one of embodiments 55-81, wherein the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:14.

[0278] Embodiment 83 includes the isolated polypeptide complex of any one of embodiments 55-81, wherein the scFv comprises an amino acid sequence of at least 200 contiguous amino acid residues of SEQ ID NO:14.

[0279] Embodiment 84 includes the isolated polypeptide complex of any one of embodiments 55-81, wherein the scFv comprises an amino acid sequence of at least 225 contiguous amino acid residues of SEQ ID NO:14.

[0280] Embodiment 85 includes the isolated polypeptide complex of any one of embodiments 55-84, wherein the scFv comprises the amino acid sequence of SEQ ID NO:14.

[0281] Embodiment 86 includes the isolated polypeptide complex of any one of embodiments 55-85, wherein a linker connects the C-terminus of A to the N-terminus of B.

[0282] Embodiment 87 includes the isolated polypeptide complex of any one of embodiments 55-86, wherein the linker connects the C-terminus of A to the N-terminus of the Fab heavy chain polypeptide.

[0283] Embodiment 88 includes the isolated polypeptide complex of any one of embodiments 55-87, wherein a linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain.

[0284] Embodiment 89 includes the isolated polypeptide complex of any one of embodiments 55-88, wherein the linker connects the N-terminus of the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain.

[0285] Embodiment 90 includes the isolated polypeptide complex of any one of embodiments 55-85, wherein a linker connects the N-terminus of A to the C-terminus of B.

[0286] Embodiment 91 comprises the isolated polypeptide complex of any one of embodiments 55-90, wherein the linker is at least 5 amino acids in length.

[0287] Embodiment 92 includes the isolated polypeptide complex of any one of embodiments 55-91, wherein the linker is no more than 10 amino acids in length.

[0288] Embodiment 93 includes the isolated polypeptide complex of any one of embodiments 55-91, wherein the linker is no more than 30 amino acids in length.

[0289] Embodiment 94 includes the isolated polypeptide complex of any one of embodiments 55-91, wherein the linker is at least 5 amino acids and no more than 30 amino acids in length.

[0290] Embodiment 95 includes the isolated polypeptide complex of any one of embodiments 55-90, wherein the linker is 5 amino acids in length.

[0291] Embodiment 96 includes the isolated polypeptide complex of any one of embodiments 55-90, wherein the linker comprises the amino acid sequence of SEQ ID NO:28 (GGGGSGGGGSGGGGS), SEQ ID NO:29 (GGGGS), or SEQ ID NO:30 (GGGGSGGGS).

[0292] Embodiment 97 includes the isolated polypeptide complex of embodiment 96, wherein the linker comprises the amino acid sequence of SEQ ID NO: 29 (GGGGS).

[0293] Embodiment 98 includes the isolated polypeptide complex of any one of embodiments 55-89 and 91-97, wherein the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and wherein the Fab light chain polypeptide comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:32.

[0294] Embodiment 99 includes the isolated polypeptide complex of any one of embodiments 55-89 and 91-97, wherein the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and wherein the Fab light chain polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:32.

[0295] Embodiment 100 includes the isolated polypeptide complex of any one of embodiments 55-89 and 91-97, wherein the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and wherein the Fab light chain polypeptide comprises an amino acid sequence of at least 200 contiguous amino acid residues of SEQ ID NO:24 or SEQ ID NO:26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:32.

[0296] Embodiment 101 includes the isolated polypeptide complex of any one of embodiments 55-89 and 91-97, wherein the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and wherein the Fab light chain polypeptide comprises the amino acid sequence according to SEQ ID NO:24 or SEQ ID NO:26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:32.

[0297] Embodiment 102 includes the isolated polypeptide complex of any one of embodiments 55-56, 61, 63-71, and 74-101, wherein the isolated polypeptide complex comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:24 and an amino acid sequence of SEQ ID NO:32.

[0298] Embodiment 103 includes the isolated polypeptide complex of any one of embodiments 55-56, 61, 63-71, and 74-101, wherein the isolated polypeptide complex comprises the amino acid sequences of SEQ ID NO:24 and SEQ ID NO:32.

[0299] Embodiment 104 includes the isolated polypeptide complex of any one of embodiments 55-56, 61, 63-71, and 74-101, wherein the isolated polypeptide complex consists of the amino acid sequence of SEQ ID NO:24 and SEQ ID NO:32.

[0300] Embodiment 105 includes the isolated polypeptide complex of any one of embodiments 55-62, 65-69, and 72-101, wherein the isolated polypeptide complex comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:26 and an amino acid sequence of SEQ ID NO:32.

[0301] Embodiment 106 includes the isolated polypeptide complex of any one of embodiments 55-62, 65-69, and 72-101, wherein the isolated polypeptide complex comprises the amino acid sequences of SEQ ID NO:26 and SEQ ID NO:32.

[0302] Embodiment 107 includes the isolated polypeptide complex of any one of embodiments 55-62, 65-69, and 72-101, wherein the isolated polypeptide complex consists of the amino acid sequence of SEQ ID NO:26 and SEQ ID NO:32.

[0303] Embodiment 108 includes a pharmaceutical composition comprising: (i) the isolated polypeptide complex according to any one of embodiments 1-107, and (ii) a pharmaceutically acceptable excipient.

[0304] Embodiment 109 includes an isolated recombinant nucleic acid molecule encoding the isolated polypeptide complex according to any one of embodiments 1-107.

[0305] Embodiment 110 includes a method of treating cancer in a subject in need thereof, the method comprising administering to the subject the isolated polypeptide complex according to any one of embodiments 1-107.

[0306] Example

[0307] Example 1: Binding of EGFR polypeptide complex to EGFR and CD3e

[0308] EGFR polypeptide complexes Ab-1, Ab-2, and Ab-3 were evaluated for EGFR and CD3ε binding.

[0309] Ab-1, Ab-2 and Ab-3 comprise the sequences listed in Table 8.

[0310] Table 8. Antibody sequences that bind to EGFR and CD3e

[0311]

[0312]

[0313] The binding of Ab-1, Ab-2, and Ab-3 to EGFR and CD3 was evaluated using enzyme-linked immunosorbent assay (ELISA). Biotinylated EGFR or biotinylated CD3 was captured on a neutravidin-coated plate. Peptide complex molecules diluted in buffer were added to the antigen-coated plate. Bound peptide complexes were detected using a standard horseradish peroxidase-conjugated secondary antibody. Graphpad Prism software was used to calculate the 50% maximum signal (EC 50 ) The concentration of the polypeptide complex required. Figure 2 See the binding data for Ab-1, Ab-2, and Ab-3 to EGFR in Figure 3 Binding data for Ab-1, Ab-2, and Ab-3 binding to CD3 can be seen in . Figure 2-3 The EC values for EGFR and CD3 binding of all three polypeptides are shown in Table 9. 50 .

[0314] Table 9. ECs associated with EGFR and CD3 binding 50

[0315]

[0316] Example 2: In vitro efficacy of EGFR polypeptide complexes

[0317] The polypeptide complexes were evaluated in a functional in vitro tumor cell killing assay using the EGFR-positive tumor cell line HCT116. Tumor cell killing was measured using the xCelligence real-time cell analyzer from Agilent, which relies on sensor impedance measurements (cell index), which increase as tumor cells adhere, spread, and proliferate on the sensor surface. Likewise, as tumor cells are killed, the impedance decreases. 10,000 tumor cells were added to each well and allowed to adhere overnight on a 96-well E plate. The next day, the polypeptide complexes titrated in culture medium supplemented with human serum and 30,000 CD8+ T cells were added to the wells. Cell index measurements were taken every 10 minutes for an additional 72 hours. The cell index was then multiplied by the number of hours (tumor cell growth kinetics) versus the concentration of the polypeptide complexes. Graphpad Prism software was used to calculate the concentration required to reduce tumor growth by 50% (IC 50 ). The tumor cell killing graphs of Ab-1, Ab-2 and Ab-3 are shown in Figure 4 In, and IC 50 Provided on Figure 4 and Table 10.

[0318] Table 10. IC of HCT116 tumor cell killing 50

[0319] peptides <![CDATA[IC 50 (pM)]]> Ab-1 0.4 Ab-2 0.4 Ab-3 0.3

[0320] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific embodiments provided in this specification. Although the present invention has been described with reference to the above description, the description and illustration of the embodiments herein are not intended to be interpreted in a restrictive sense. Without departing from the present invention, those skilled in the art will now appreciate various modifications, changes and substitutions. In addition, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations or relative proportions set forth herein, which depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be used to practice the present invention. Therefore, it is envisioned that the present invention should also encompass any such alternatives, modifications, variations or equivalents. The following claims are intended to define the scope of the present invention, and thus encompass methods and structures within the scope of these claims and their equivalents.

Claims

1. An isolated polypeptide complex according to the formula: ALB (Formula I) in A comprises a single-chain variable fragment (scFv) that binds to CD3, wherein the scFv comprises a scFv light chain variable domain and a scFv heavy chain variable domain, the scFv heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the scFv heavy chain variable domain comprise HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3; B comprises an antigen-binding fragment (Fab) or Fab′ that binds to EGFR, wherein the Fab or Fab′ comprises a Fab light chain polypeptide comprising a Fab light chain variable domain and a Fab heavy chain polypeptide comprising a Fab heavy chain variable domain, the Fab heavy chain variable domain comprising complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the Fab heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19, and HC-CDR3: SEQ ID NO: 20, and wherein the Fab heavy chain polypeptide comprises an N-glycosylation site having an amino acid sequence of N-X1-X2, wherein X1 is any amino acid except P and X2 is T or S; and L contains the linker that connects A to B.

2. The isolated polypeptide complex according to claim 1, wherein the Fab light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2 and LC-CDR3, wherein the LC-CDR1, the LC-CDR2 and the LC-CDR3 of the Fab light chain variable domain comprise LC-CDR1: SEQ ID NO: 21, LC-CDR2: SEQ ID NO: 22 and LC-CDR3: SEQ ID NO: 23, and wherein the CDR comprises 0-2 amino acid modifications in at least one of the LC-CDR1, LC-CDR2 or LC-CDR3.

3. The isolated polypeptide complex of claim 1, wherein the Fab light chain polypeptide comprises a mutation that eliminates pyroglutamate formation at the N-terminus of the Fab light chain polypeptide relative to a non-mutated form of the Fab light chain polypeptide.

4. The isolated polypeptide complex of claim 3, wherein the mutation is a Q to X3 mutation at residue number 1 of the N-terminus of the Fab light chain polypeptide, and wherein X3 is an amino acid selected from the group consisting of: A, R, N, D, C, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V. The isolated polypeptide complex according to claim 4 , wherein X 3 is selected from the group consisting of D and E. The isolated polypeptide complex according to claim 5 , wherein X 3 is D.

7. The isolated polypeptide complex according to claim 1, wherein the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO:

26.

8. The isolated polypeptide complex of claim 1, wherein the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO:

26.

9. The isolated polypeptide complex of claim 1, wherein the Fab light chain polypeptide comprises a Q at residue number 1 of the N-terminus of the Fab light chain polypeptide.

10. The isolated polypeptide complex of claim 1, wherein the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO:

24. The isolated polypeptide complex according to claim 1 , wherein X 2 is T. The isolated polypeptide complex according to claim 11 , wherein X 1 is D.

13. The isolated polypeptide complex of claim 1, wherein the Fab heavy chain polypeptide comprising the N-glycosylation site comprises the amino acid sequence QSNDTAIY (SEQ ID NO: 33).

14. The isolated polypeptide complex according to claim 1, wherein the N of the N-glycosylation site is located at residue number 88 of the Fab heavy chain polypeptide.

15. The isolated polypeptide complex of claim 1, wherein the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO:

25.

16. The isolated polypeptide complex of claim 1, wherein the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 24 and the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO:

25.

17. The isolated polypeptide complex of claim 1, wherein the Fab light chain polypeptide consists of the amino acid sequence of SEQ ID NO: 24 and the Fab heavy chain polypeptide consists of the amino acid sequence of SEQ ID NO:

25.

18. The isolated polypeptide complex of claim 1, wherein the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 26 and the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO:

25.

19. The isolated polypeptide complex of claim 1, wherein the Fab light chain polypeptide consists of the amino acid sequence of SEQ ID NO: 26 and the Fab heavy chain polypeptide consists of the amino acid sequence of SEQ ID NO:

25.

20. The isolated polypeptide complex of claim 1, wherein the scFv light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the scFv light chain variable domain comprise LC-CDR1: SEQ ID NO: 6, LC-CDR2: SEQ ID NO: 7, and LC-CDR3: SEQ ID NO:

8.

21. The isolated polypeptide complex of claim 1, wherein the scFv heavy chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

13.

22. The isolated polypeptide complex of claim 1, wherein the scFv heavy chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO:

13.

23. The isolated polypeptide complex of claim 1, wherein the scFv heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:

13.

24. The isolated polypeptide complex of claim 1, wherein the scFv light chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

12.

25. The isolated polypeptide complex of claim 1, wherein the scFv light chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO:

12.

26. The isolated polypeptide complex of claim 1, wherein the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:

12.

27. The isolated polypeptide complex of claim 1, wherein the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

14.

28. The isolated polypeptide complex of claim 1, wherein the scFv comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO:

14.

29. The isolated polypeptide complex of claim 1, wherein the scFv comprises an amino acid sequence of at least 225 consecutive amino acid residues of SEQ ID NO:

14.

30. The isolated polypeptide complex of claim 1, wherein the scFv comprises the amino acid sequence of SEQ ID NO:

14.

31. The isolated polypeptide complex of claim 1, wherein the linker connects the C-terminus of A to the N-terminus of B.

32. The isolated polypeptide complex of claim 1, wherein the linker connects the C-terminus of A to the N-terminus of the Fab light chain polypeptide.

33. The isolated polypeptide complex of claim 1, wherein the linker connects the C-terminus of A to the N-terminus of the Fab heavy chain polypeptide.

34. The isolated polypeptide complex of claim 1, wherein the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain.

35. The isolated polypeptide complex according to claim 1, wherein the linker connects the N-terminus of A to the C-terminus of B.

36. The isolated polypeptide complex of claim 1, wherein the linker connects the N-terminus of A to the C-terminus of the Fab heavy chain polypeptide.

37. The isolated polypeptide complex of claim 1, wherein the linker connects the N-terminus of A to the C-terminus of the Fab light chain polypeptide.

38. The isolated polypeptide complex of claim 1, wherein the linker is at least 5 amino acids in length.

39. The isolated polypeptide complex of claim 1, wherein the linker is no more than 10 amino acids in length.

40. The isolated polypeptide complex of claim 1, wherein the linker is no more than 30 amino acids in length.

41. The isolated polypeptide complex of claim 1, wherein the linker is at least 5 amino acids and no more than 30 amino acids in length.

42. The isolated polypeptide complex of claim 1, wherein the linker is 5 amino acids in length.

43. The isolated polypeptide complex of claim 1, wherein the linker comprises the amino acid sequence of SEQ ID NO: 28 (GGGGSGGGGSGGGGS), SEQ ID NO: 29 (GGGGS), or SEQ ID NO: 30 (GGGGSGGGS).

44. The isolated polypeptide complex of claim 1, wherein the linker comprises the amino acid sequence of SEQ ID NO: 29 (GGGGS).

45. The isolated polypeptide complex of claim 1 , wherein the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and wherein the Fab light chain polypeptide comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:

32.

46. The isolated polypeptide complex of claim 1 , wherein the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and wherein the Fab light chain polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:

32.

47. The isolated polypeptide complex of claim 1, wherein the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and wherein the Fab light chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 24 or SEQ ID NO: 26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:

32.

48. The isolated polypeptide complex of claim 1 , wherein the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and wherein the Fab light chain polypeptide comprises the amino acid sequence according to SEQ ID NO: 24 or SEQ ID NO: 26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:

32.

49. The isolated polypeptide complex of claim 1, wherein the isolated polypeptide complex comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:24 and an amino acid sequence of SEQ ID NO:

32.

50. The isolated polypeptide complex of claim 1, wherein the isolated polypeptide complex comprises the amino acid sequences of SEQ ID NO: 24 and SEQ ID NO:

32.

51. The isolated polypeptide complex of claim 1, wherein the isolated polypeptide complex consists of the amino acid sequences of SEQ ID NO: 24 and SEQ ID NO:

32.

52. The isolated polypeptide complex of claim 1, wherein the isolated polypeptide complex comprises an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:26 and an amino acid sequence of SEQ ID NO:

32.

53. The isolated polypeptide complex of claim 1, wherein the isolated polypeptide complex comprises the amino acid sequences of SEQ ID NO: 26 and SEQ ID NO:

32.

54. The isolated polypeptide complex of claim 1, wherein the isolated polypeptide complex consists of the amino acid sequences of SEQ ID NO: 26 and SEQ ID NO:

32.

55. An isolated polypeptide complex according to the formula: ALB (Formula I) in A comprises a single-chain variable fragment (scFv) that binds to CD3, wherein the scFv comprises an scFv light chain variable domain and an scFv heavy chain variable domain; B comprises an antigen-binding fragment (Fab) or Fab′ that binds to EGFR, wherein the Fab or Fab′ comprises a Fab light chain polypeptide comprising a Fab light chain variable domain and a Fab heavy chain polypeptide comprising a Fab heavy chain variable domain, the Fab heavy chain variable domain comprising complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the Fab heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19, and HC-CDR3: SEQ ID NO: 20, and wherein the Fab heavy chain polypeptide comprises an N-glycosylation site having an amino acid sequence of N-X1-X2, wherein X1 is any amino acid except P and X2 is T or S; and L comprises a linker connecting A to B, wherein the linker connects the Fab heavy chain polypeptide to the scFv light chain variable domain.

56. The isolated polypeptide complex of claim 55, wherein the Fab light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the Fab light chain variable domain comprise LC-CDR1: SEQ ID NO: 21, LC-CDR2: SEQ ID NO: 22, and LC-CDR3: SEQ ID NO: 23, and wherein the CDR comprises 0-2 amino acid modifications in at least one of the LC-CDR1, LC-CDR2, or LC-CDR3.

57. The isolated polypeptide complex of claim 55, wherein the Fab light chain polypeptide comprises a mutation that eliminates pyroglutamate formation at the N-terminus of the Fab light chain polypeptide relative to a non-mutated form of the Fab light chain polypeptide.

58. The isolated polypeptide complex of claim 57, wherein the mutation is a Q to X3 mutation at residue number 1 of the N-terminus of the Fab light chain polypeptide, and wherein X3 is an amino acid selected from the group consisting of: A, R, N, D, C, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V.

59. The isolated polypeptide complex according to claim 58, wherein X3 is selected from D and E.

60. The isolated polypeptide complex according to claim 58, wherein X3 is D.

61. The isolated polypeptide complex of claim 55, wherein the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO:

26.

62. The isolated polypeptide complex of claim 55, wherein the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO:

26.

63. The isolated polypeptide complex of claim 55, wherein the Fab light chain polypeptide comprises a Q at residue number 1 of the N-terminus of the Fab light chain polypeptide.

64. The isolated polypeptide complex of claim 55, wherein the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO:

24.

65. The isolated polypeptide complex of claim 55, wherein X2 is T.

66. The isolated polypeptide complex according to claim 65, wherein X1 is D.

67. The isolated polypeptide complex of claim 55, wherein the Fab heavy chain polypeptide comprising the N-glycosylation site comprises the amino acid sequence of QSNDTAIY (SEQ ID NO: 33).

68. The isolated polypeptide complex of claim 55, wherein the N of the N-glycosylation site is located at residue number 88 of the Fab heavy chain polypeptide.

69. The isolated polypeptide complex of claim 55, wherein the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO:

25.

70. The isolated polypeptide complex of claim 55, wherein the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 24 and the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO:

25.

71. The isolated polypeptide complex of claim 55, wherein the Fab light chain polypeptide consists of the amino acid sequence of SEQ ID NO: 24 and the Fab heavy chain polypeptide consists of the amino acid sequence of SEQ ID NO:

25.

72. The isolated polypeptide complex of claim 55, wherein the Fab light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 26 and the Fab heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO:

25.

73. The isolated polypeptide complex of claim 55, wherein the Fab light chain polypeptide consists of the amino acid sequence of SEQ ID NO: 26 and the Fab heavy chain polypeptide consists of the amino acid sequence of SEQ ID NO:

25.

74. The isolated polypeptide complex of claim 55, wherein the scFv heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the scFv heavy chain variable domain comprise HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO:

3.

75. The isolated polypeptide complex of claim 55, wherein the scFv light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the scFv light chain variable domain comprise LC-CDR1: SEQ ID NO: 6, LC-CDR2: SEQ ID NO: 7, and LC-CDR3: SEQ ID NO:

8.

76. The isolated polypeptide complex of claim 55, wherein the scFv heavy chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

13.

77. The isolated polypeptide complex of claim 55, wherein the scFv heavy chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO:

13.

78. The isolated polypeptide complex of claim 55, wherein the scFv heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:

13.

79. The isolated polypeptide complex of claim 55, wherein the scFv light chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

12.

80. The isolated polypeptide complex of claim 55, wherein the scFv light chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO:

12.

81. The isolated polypeptide complex of claim 55, wherein the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:

12.

82. The isolated polypeptide complex of claim 55, wherein the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

14.

83. The isolated polypeptide complex of claim 55, wherein the scFv comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO:

14.

84. The isolated polypeptide complex of claim 55, wherein the scFv comprises an amino acid sequence of at least 225 consecutive amino acid residues of SEQ ID NO:

14.

85. The isolated polypeptide complex of claim 55, wherein the scFv comprises the amino acid sequence of SEQ ID NO:

14.

86. The isolated polypeptide complex of claim 55, wherein the linker connects the C-terminus of A to the N-terminus of B.

87. The isolated polypeptide complex of claim 55, wherein the linker connects the C-terminus of A to the N-terminus of the Fab heavy chain polypeptide.

88. The isolated polypeptide complex of claim 55, wherein the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain.

89. The isolated polypeptide complex of claim 55, wherein the linker connects the N-terminus of the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain.

90. The isolated polypeptide complex of claim 55, wherein the linker connects the N-terminus of A to the C-terminus of B.

91. The isolated polypeptide complex of claim 55, wherein the linker is at least 5 amino acids in length.

92. The isolated polypeptide complex of claim 55, wherein the linker is no more than 10 amino acids in length.

93. The isolated polypeptide complex of claim 55, wherein the linker is no more than 30 amino acids in length.

94. The isolated polypeptide complex of claim 55, wherein the linker is at least 5 amino acids and no more than 30 amino acids in length.

95. The isolated polypeptide complex of claim 55, wherein the linker is 5 amino acids in length.

96. The isolated polypeptide complex of claim 55, wherein the linker comprises the amino acid sequence of SEQ ID NO: 28 (GGGGSGGGGSGGGGS), SEQ ID NO: 29 (GGGGS), or SEQ ID NO: 30 (GGGGSGGGS).

97. The isolated polypeptide complex of claim 55, wherein the linker comprises the amino acid sequence of SEQ ID NO: 29 (GGGGS).

98. The isolated polypeptide complex of claim 55, wherein the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and wherein the Fab light chain polypeptide comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:

32.

99. The isolated polypeptide complex of claim 55, wherein the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and wherein the Fab light chain polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:

32.

100. The isolated polypeptide complex of claim 55, wherein the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and wherein the Fab light chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 24 or SEQ ID NO: 26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:

32.

101. The isolated polypeptide complex of claim 55, wherein the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain, and wherein the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 24 or SEQ ID NO: 26, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence of SEQ ID NO:

32.

102. A pharmaceutical composition comprising: (i) the isolated polypeptide complex according to claim 1; and (ii) a pharmaceutically acceptable excipient.

103. An isolated recombinant nucleic acid molecule encoding the isolated polypeptide complex of claim 1.

104. A method of treating cancer in a subject in need thereof, comprising administering to the subject the isolated polypeptide complex of claim 1.

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