ROR / EGFR bispecific antigen binding molecules

Abstract

The present invention relates to bispecific antigen binding molecules specific for both the receptor tyrosine kinase-like orphan receptor 1 (ROR1) and the epidermal growth factor receptor (EGFR), as well as related fusion proteins and conjugates. In another aspect, the invention relates to conjugated immunoglobulin-like shark variable neoantigen receptors (VNARs).

Description

Technical Field

[0001] The present invention relates to bispecific antigen binding molecules specific for both receptor tyrosine kinase-like orphan receptor 1 (ROR1) and epidermal growth factor receptor (EGFR), as well as related fusion proteins and conjugates. In another aspect, the present invention relates to conjugated immunoglobulin-like shark variable neoantigen receptors (VNARs). Background Art

[0002] Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a 937-amino acid, glycosylated, type I, single-pass transmembrane protein. The extracellular region consists of three distinct domains: an N-terminal immunoglobulin domain (Ig), followed by a cysteine-rich Fizzled domain (fz), which in turn connects to a membrane-proximal kringle domain (kr). The intracellular region of the protein contains a pseudokinase domain, followed by two Ser / Thr-rich domains interspersed with a proline-rich region. This same overall domain architecture is conserved in the closely related family member ROR2, with which it shares high sequence identity.

[0003] ROR1 is expressed during embryonic development and is prominently expressed in neural crest cells and necrotic and interdigital zones in late development. However, its expression is silenced soon after birth and is essentially absent in normal adult tissues. ROR1 expression has been observed at both the mRNA and protein levels in a variety of solid tumors and hematological malignancies, including lung, endometrial, pancreatic, ovarian, colon, head and neck, and prostate cancers, melanoma and renal cell carcinoma, breast cancer, and chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (AML). In addition, increased ROR1 expression has been reported to be associated with adverse clinical outcomes in a variety of cancer indications, including breast cancer, ovarian cancer, colorectal cancer, lung adenocarcinoma, and CLL.

[0004] Consistent with ROR1's expression pattern and association with poor clinical prognosis, the functional role of ROR1 in tumorigenesis and disease progression has been demonstrated across many different cancer indications. ROR1 promotes epithelial-mesenchymal transition and metastasis in breast cancer models and spheroid formation and tumor engraftment in ovarian cancer models. ROR1 is a transcriptional target of the NKX2-1 / TTF-1 lineage survival factor oncogene in lung adenocarcinoma. It maintains EGFR signaling and inhibits pro-apoptotic signaling, and EGF-induced interactions between ROR1 and EGFR have been observed. Co-expression of EGFR and ROR1 mRNA was identified from breast cancer gene expression database mining. ROR1 has also been shown to act as a scaffold to maintain caveolar structure and bypass signaling mechanisms that confer resistance to EGFR tyrosine kinase inhibitors. Signaling through the ROR1-HER3 complex regulates the Hippo-YAP pathway and promotes breast cancer bone metastasis, and this protein can promote Met-driven tumorigenesis. ROR1 expression is associated with chemoresistance in breast cancer through activation of the Hippo-YAP / TAZ and BMI1 pathways. It has been reported that in CLL, ROR1 hetero-oligomerizes with ROR2 in response to Wnt5a, thereby transducing signaling and enhancing proliferation and migration.

[0005] Given ROR1's functional role in cancer pathology and its general lack of expression in normal adult tissues, this oncofetal protein is an attractive target for cancer therapy. Antibodies against ROR1 have been described in WO2021097313 (4A5kipps), WO2014031174 (UC961), WO2016187220 (Five Prime), WO2010124188 (2A2), WO2012075158 (R11, R12), WO2011054007 (Oxford Bio), WO2011079902 (Bioinvent), WO2017127664, WO2017127664 (NBE Therapeutics, SCRIPPS), WO2016094847 (Emergent), and WO2017127499. The humanized mouse anti-ROR1 antibody UC961 has entered clinical trials for relapsed or refractory chronic lymphocytic leukemia. Chimeric antigen receptor T cells targeting ROR1 have also been reported (Hudecek M et al, Clin. Cancer Res., 2013, 19, 3153-64), and preclinical primate studies of UC961 and CAR-T cells targeting ROR1 showed no obvious toxicity, which is consistent with the general lack of expression of this protein in adult tissues (Choi M et al, Clinical Lymphoma, myeloma & leukemia, 2015, S167; Berger C et al, Cancer Immunol. Res., 2015, 3, 206).

[0006] The epidermal growth factor receptor (EGFR) is a member of the ErbB receptor tyrosine kinase family. It is a 170 kDa transmembrane protein composed of four extracellular domains, a transmembrane region, an intracellular tyrosine kinase domain, and a carboxy-terminal tail. While the normal function of EGFR is associated with the regulation of epithelial tissue development, it is also implicated in a variety of pathological conditions. In particular, overexpression of EGFR is associated with a variety of cancers. Therefore, it is an important drug target, and numerous therapeutic approaches have been developed. In addition to several small molecule-based EGFR inhibitors (such as gefitinib, erlotinib, afatinib, brigatinib, icotinib, and osimertinib), a number of antibodies targeting EGFR have been developed. These include the anti-EGFR antibodies cetuximab, panitumumab, zalutumumab, nimotuzumab, and matuzumab. These antibodies block the extracellular ligand-binding domain, preventing ligand binding and subsequent activation of the tyrosine kinase domain. Single domain antibodies (sdAbs) that show competitive binding with cetuximab or matuzumab have also been developed.

[0007] Single-domain binding molecules can be derived from a range of proteins from different species. The immunoglobulin isotope new antigen receptor (IgNAR) is a homodimeric heavy chain complex originally found in the serum of nurse sharks (Ginglymostoma cirratum) and other shark and ray species. IgNAR does not contain a light chain and is different from the typical immunoglobulin structure. Each molecule consists of a single variable domain (VNAR) and five constant domains (CNAR). The nomenclature in the literature refers to IgNAR as immunoglobulin isotope new antigen receptor or immunoglobulin isotope new antigen receptor, and these terms are synonymous.

[0008] There are three main defined types of shark IgNARs, termed type I, type II, and type III (Kovalena et al, Exp Opin Biol Ther 2014 14(10)1527-1539). These are classified based on the position of atypical cysteine ​​residues, which are under strong selective pressure and are therefore rarely replaced.

[0009] All three types have classical immunoglobulin-typical cysteines at positions 35 and 107, which, together with the invariant tryptophan at position 36, stabilize the standard immunoglobulin fold. There is no defined CDR2, but relatively close regions of sequence variation, TCR HV2 and HV4, are defined in framework 2 and framework 3, respectively. Type I has germline-encoded cysteine ​​residues in framework 2 and framework 4, and an even number of additional cysteines in CDR3. Crystal structure studies of type I IgNARs isolated from and in complex with lysozyme have enabled the determination of the contribution of these cysteine ​​residues. Both framework 2 and 4 cysteines form disulfide bridges with cysteines in CDR3, resulting in a tightly packed structure in which the CDR3 loops are tightly pressed against the HV2 region. To date, type I IgNARs have been found only in nurse sharks, while all other cartilaginous fish, including members of the same order, have only type II or variants of this type.

[0010] Type II IgNARs are defined as having cysteine ​​residues in both CDR1 and CDR3 that form intramolecular disulfide bonds, bringing the two regions into close proximity and forming a protruding CDR3 that favors a binding pocket or groove. Type I sequences typically have longer CDR3s than type II sequences, averaging 21 and 15 residues, respectively. This is thought to be due to the strong selective pressure of two or more cysteine ​​residues in type I CDR3s to associate with their framework 2 and 4 counterparts. Studies of somatic mutation accumulation have shown that type II CDR1 has more mutations than type I, while the sequence variation in the HV2 region of type I is greater than that of type II. This evidence is closely related to the well-defined positioning of these regions within the antigen-binding site. A third type of IgNAR, termed type III, has been discovered in newborns. Due to the germline fusion of the D1 and D2 regions (forming the CDR3) with the V gene, this member of the IgNAR family lacks diversity within the CDR3. The CDR3 of almost all known clones is 15 residues in length, with little or no sequence diversity.

[0011] Another structural type of VNAR, designated type IIb or IV, has only two typical cysteine ​​residues (in the framework 1 and framework 3b regions). To date, this type has been found primarily in dogfish sharks and has also been isolated from a semisynthetic V-NAR library derived from the wobbegong shark.

[0012] ROR1-specific antigen binding molecules, including VNARs, are described in WO2019 / 122447, the entire contents of which are incorporated herein by reference. Among other things, WO2019 / 122447 describes the sequences of B1, P3A1, and P3A1 G1 identified below.

[0013] B1

[0014] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWLVQWYDGAGTVLTVN(SEQ ID NO: 113)

[0015] P3A1

[0016] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVN (SEQ ID NO: 206)

[0017] P3A1 G1

[0018] TRVDQSPSSSLSASVGDRVTITCVLTDTSYGLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 114).

[0019] WO2019 / 122445 describes a ROR1 / EGFR bispecific binding molecule, wherein the ROR1 binding molecule is as described in WO2019 / 122447.

[0020] The conjugate of ROR1 specific antigen binding molecule (including VNAR) is described in WO2020 / 254640, the entire contents of which are incorporated herein by reference. WO2020 / 254640 describes anthracycline (PNU) derivatives suitable for drug conjugates. Specifically, there is provided a derivative of PNU159682, which lacks a C14 carbon and a connected hydroxyl functional group, and wherein an ethylenediamine (EDA) group forms a part of the joint region between the C13 carbonyl and maleimide groups of PNU159682. Alternatively, the same molecule can be described with EDA-PNU as a "warhead" so that the EDA group is not considered to be a part of the joint region. When the joint includes val-cit-PAB, the maleimide group can be replaced by any reactive group suitable for conjugation reaction. Such payload can react with the free thiol group on another molecule. When the free thiol is located on a protein, a protein-drug conjugate (PDC) can be formed.

[0021] The anthracycline derivative PNU-159682 has been described as a metabolite of nemorubicin and reportedly exhibits extremely high in vitro cell-killing potency in the picomolar to femtomolar range against an ovarian (A2780) and a breast cancer (MCF7) cell line (WO2012 / 073217A1). Derivatives of PNU-159682 have also been described in WO2016 / 102679.

[0022] Conjugation of PNU-159682 derivatives to antibodies is described in WO2009 / 099741, WO2016 / 127081 and WO2016 / 102679, Yu et al, Clin. Cancer Res 2015, 21, 3298 and Stefan et al, Mol. Cancer. Ther., 2017, 16, 879.

[0023] Auristatin E (AE) and monomethyl auristatin E (MMAE) are synthetic analogs of dolastatin, a unique class of linear pseudopeptides originally isolated from marine sources, some of which possess very potent cytotoxic activity against tumor cells. However, a disadvantage of MMAE is its relatively high systemic toxicity. To improve tumor selectivity, MMAE is particularly used in combination with enzymatically cleavable valine-citrulline linkers in ADC settings to achieve more targeted tumor therapy (see, for example, WO2005 / 081711). Following proteolytic cleavage, MMAE is preferentially released intracellularly from the corresponding ADC. Monomethyl auristatin F (MMAF) is an auristatin derivative with a C-terminal phenylalanine moiety. MMAF and its various ester and amide derivatives are disclosed in WO2005 / 081711. Other auristatin analogs with a C-terminal amide-substituted phenylalanine unit are described in WO01 / 18032. WO02 / 088172 and WO2007 / 008603 claim MMAF analogs involving modifications of the phenylalanine side chain, while WO2007 / 008848 claims MMAF analogs in which the carboxyl group of phenylalanine is modified. Auristatin conjugates linked via the C-terminus have been described in WO2009 / 117531, and other conjugates are described in WO2013 / 087716.

[0024] ROR1-specific variant antigen-binding molecules with improved properties and conjugates thereof with PNU-159682 derivatives are described in PCT / EP2021 / 086667, filed December 17, 2021, which is hereby incorporated by reference in its entirety. PCT / EP2021 / 086667 does not disclose any bispecific molecules comprising the ROR1-specific variant antigen-binding molecules and the EGFR-specific variant antigen-binding molecules of PCT / EP2021 / 086667. Summary of the Invention

[0025] The present invention generally relates to bispecific antigen-binding molecules. Specifically, the present invention relates to bispecific molecules capable of binding to ROR1 and EGFR.

[0026] According to a first aspect, the present invention provides a bispecific antigen binding molecule comprising:

[0027] (i) a receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen-binding molecule comprising the amino acid sequence represented by formula (I):

[0028] FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4(I)

[0029] in

[0030] CDR3 is selected from the group consisting of YPWGAGAPYNVQWY (SEQ ID NO: 23), YPWGAGAPYLVQWY (SEQ ID NO: 20), YPWGAGAPWNVQWY (SEQ ID NO: 24), YPSGAGAPRPVQWY (SEQ ID NO: 11), YPWGAGAPCLVQWY (SEQ ID NO: 11), NO: 12), YPWGAGAPRLVQWY (SEQ ID NO: 13), YPWGAGAPRQVQWY (SEQ ID NO: 14), YPWGAGAPRSVQWY (SEQ ID NO: 15), YPWGAGAPSLVQWY (SEQ ID NO: 16), YPWGAGAPSNVQWY (SEQ ID NO: 17), YPWGAGAPSQVQWY (SEQ ID NO: 18), YPWGAGAPSSVQWY (SEQ ID NO: 19), YPWGAGAPWQVQWY (SEQ ID NO: 21), YPWGAGAPWSVQWY (SEQ ID NO: 22), and YPWGAGAPWLVQWY (SEQ ID NO: 10);

[0031] CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of GANYGLAA (SEQ ID NO: 1), DANYGLAA (SEQ ID NO: 5), GANYDLSA (SEQ ID NO: 2), GANYGLSA (SEQ ID NO: 3), and GANYDLAA (SEQ ID NO: 4);

[0032] FW1 is the framework region;

[0033] FW2 is the framework region;

[0034] HV2 is a hypervariable sequence having an amino acid sequence selected from the group consisting of SSNQERISIS (SEQ ID NO: 6) and SSNKERISIS (SEQ ID NO: 7);

[0035] FW3a is the framework region;

[0036] HV4 is a hypervariable sequence having an amino acid sequence selected from the group consisting of NKRTM (SEQ ID NO: 8) and NKGTM (SEQ ID NO: 9);

[0037] FW3b is the framework region;

[0038] FW4 is the framework region;

[0039] wherein if CDR3 is YPWGAGAPWLVQWY (SEQ ID NO: 10), CDR1 is selected from the group consisting of DANYGLAA (SEQ ID NO: 5), GANYGLSA (SEQ ID NO: 3) and GANYDLAA (SEQ ID NO: 4); and

[0040] (ii) Epidermal growth factor receptor (EGFR)-specific antigen-binding molecules.

[0041] According to a second aspect, the present invention provides a bispecific antigen binding molecule comprising:

[0042] (i) a receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen-binding molecule comprising the amino acid sequence represented by formula (I):

[0043] FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4(I)

[0044] in

[0045] CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of GTRYGLYS (SEQ ID NO: 25), GTRYGLYSS (SEQ ID NO: 26), DTRYALYS (SEQ ID NO: 27), DTRYALYSS (SEQ ID NO: 28), GTKYGLYA (SEQ ID NO: 29), GTKYGLYAS (SEQ ID NO: 30), and DTSYGLYS (SEQ ID NO: 207);

[0046] FW1 is the framework region;

[0047] FW2 is the framework region;

[0048] HV2 is a hypervariable sequence having an amino acid sequence selected from the group consisting of SSDEERISIS (SEQ ID NO: 31), STDEERISIG (SEQ ID NO: 32), SPNKDRMIIG (SEQ ID NO: 33), STDKERIIIG (SEQ ID NO: 34), and TTDWERMSIG (SEQ ID NO: 208);

[0049] FW3a is the framework region;

[0050] HV4 is a hypervariable sequence having an amino acid sequence selected from the group consisting of NKGTK (SEQ ID NO: 35), NKGSK (SEQ ID NO: 36), NNGTK (SEQ ID NO: 37), NNRSK (SEQ ID NO: 38), and NKGAK (SEQ ID NO: 209);

[0051] FW3b is the framework region;

[0052] CDR3 is a CDR sequence having an amino acid sequence according to REARHPWLRQWY (SEQ ID NO: 39);

[0053] FW4 is the framework region; and

[0054] (ii) an epidermal growth factor receptor (EGFR)-specific antigen-binding molecule,

[0055] wherein when CDR1 is DTSYGLYS (SEQ ID NO: 207) and / or HV2 is TTDWERMSIG (SEQ ID NO: 208) and / or HV4 is NKGAK (SEQ ID NO: 209), the ROR1-specific antigen binding molecule is fused to a first fragment of the immunoglobulin Fc region, the EGFR-specific binding molecule is fused to a second fragment of the immunoglobulin Fc region, and the first fragment of the immunoglobulin Fc region and the second fragment of the immunoglobulin Fc region are engineered to dimerize.

[0056] According to a third aspect, the present invention provides a recombinant fusion protein comprising the bispecific antigen binding molecule according to the first aspect or the second aspect of the present invention.

[0057] According to a fourth aspect, the present invention provides a recombinant fusion protein dimer comprising:

[0058] (a) a first recombinant fusion protein, wherein the first recombinant fusion protein comprises a receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen binding molecule, and the receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen binding molecule comprises the amino acid sequence represented by formula (I):

[0059] FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4(I)

[0060] in

[0061] CDR3 is selected from the group consisting of YPWGAGAPYNVQWY (SEQ ID NO: 23), YPWGAGAPYLVQWY (SEQ ID NO: 20), YPWGAGAPWNVQWY (SEQ ID NO: 24), YPSGAGAPRPVQWY (SEQ ID NO: 11), YPWGAGAPCLVQWY (SEQ ID NO: 11), NO: 12), YPWGAGAPRLVQWY (SEQ ID NO: 13), YPWGAGAPRQVQWY (SEQ ID NO: 14), YPWGAGAPRSVQWY (SEQ ID NO: 15), YPWGAGAPSLVQWY (SEQ ID NO: 16), YPWGAGAPSNVQWY (SEQ ID NO: 17), YPWGAGAPSQVQWY (SEQ ID NO: 18), YPWGAGAPSSVQWY (SEQ ID NO: 19), YPWGAGAPWQVQWY (SEQ ID NO: 21), YPWGAGAPWSVQWY (SEQ ID NO: 22), and YPWGAGAPWLVQWY (SEQ ID NO: 10);

[0062] CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of GANYGLAA (SEQ ID NO: 1), DANYGLAA (SEQ ID NO: 5), GANYDLSA (SEQ ID NO: 2), GANYGLSA (SEQ ID NO: 3), and GANYDLAA (SEQ ID NO: 4);

[0063] FW1 is the framework region;

[0064] FW2 is the framework region;

[0065] HV2 is a hypervariable sequence having an amino acid sequence selected from the group consisting of SSNQERISIS (SEQ ID NO: 6) and SSNKERISIS (SEQ ID NO: 7);

[0066] FW3a is the framework region;

[0067] HV4 is a hypervariable sequence having an amino acid sequence selected from the group consisting of NKRTM (SEQ ID NO: 8) and NKGTM (SEQ ID NO: 9);

[0068] FW3b is the framework region;

[0069] FW4 is the framework region;

[0070] wherein if CDR3 is YPWGAGAPWLVQWY (SEQ ID NO: 10), CDR1 is selected from the group consisting of DANYGLAA (SEQ ID NO: 5), GANYGLSA (SEQ ID NO: 3) and GANYDLAA (SEQ ID NO: 4),

[0071] and wherein the first antigen binding molecule is fused to a first fragment of an immunoglobulin Fc region, the first fragment of the immunoglobulin Fc region being engineered to dimerize with a second fragment of the immunoglobulin Fc region;

[0072] and

[0073] (b) a second recombinant fusion protein, wherein the second recombinant fusion protein comprises an epidermal growth factor receptor (EGFR)-specific antigen binding molecule fused to a second fragment of the immunoglobulin Fc region, and the second fragment of the immunoglobulin Fc region is engineered to dimerize with the first fragment of the immunoglobulin Fc region.

[0074] According to a fifth aspect, the present invention provides a recombinant fusion protein dimer comprising:

[0075] (a) a first recombinant fusion protein, wherein the first recombinant fusion protein comprises a receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen binding molecule, and the receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen binding molecule comprises the amino acid sequence represented by formula (I):

[0076] FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4(I)

[0077] in

[0078] CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of GTRYGLYS (SEQ ID NO: 25), GTRYGLYSS (SEQ ID NO: 26), DTRYALYS (SEQ ID NO: 27), DTRYALYSS (SEQ ID NO: 28), GTKYGLYA (SEQ ID NO: 29), GTKYGLYAS (SEQ ID NO: 30), and DTSYGLYS (SEQ ID NO: 207);

[0079] FW1 is the framework region;

[0080] FW2 is the framework region;

[0081] HV2 is a hypervariable sequence having an amino acid sequence selected from the group consisting of SSDEERISIS (SEQ ID NO: 31), STDEERISIG (SEQ ID NO: 32), SPNKDRMIIG (SEQ ID NO: 33), STDKERIIIG (SEQ ID NO: 34), and TTDWERMSIG (SEQ ID NO: 208);

[0082] FW3a is the framework region;

[0083] HV4 is a hypervariable sequence having an amino acid sequence selected from the group consisting of NKGTK (SEQ ID NO: 35), NKGSK (SEQ ID NO: 36), NNGTK (SEQ ID NO: 37), NNRSK (SEQ ID NO: 38), and NKGAK (SEQ ID NO: 209);

[0084] FW3b is the framework region;

[0085] CDR3 is a CDR sequence having an amino acid sequence according to REARHPWLRQWY (SEQ ID NO: 39);

[0086] FW4 is the framework area,

[0087] and wherein the first antigen binding molecule is fused to a first fragment of an immunoglobulin Fc region, the first fragment of the immunoglobulin Fc region being engineered to dimerize with a second fragment of the immunoglobulin Fc region;

[0088] and

[0089] (b) a second recombinant fusion protein, wherein the second recombinant fusion protein comprises an epidermal growth factor receptor (EGFR)-specific antigen binding molecule fused to a second fragment of the immunoglobulin Fc region, and the second fragment of the immunoglobulin Fc region is engineered to dimerize with the first fragment of the immunoglobulin Fc region.

[0090] According to the sixth aspect, the present invention provides a ROR1-specific chimeric antigen receptor (CAR), which comprises at least one bispecific antigen binding molecule defined in the first or second aspect of the present invention, at least one recombinant fusion protein defined in the third aspect of the present invention, or at least one recombinant fusion protein dimer defined in the fourth or fifth aspect of the present invention, fused or conjugated to at least one transmembrane region and at least one intracellular domain.

[0091] The present invention also provides a cell comprising the chimeric antigen receptor according to the sixth aspect, which cell is preferably an engineered T cell.

[0092] In the seventh aspect of the present invention, a nucleic acid sequence is provided, which comprises a polynucleotide sequence encoding a bispecific antigen binding molecule, recombinant fusion protein, recombinant fusion protein dimer or chimeric antigen receptor according to the first, second, third, fourth, fifth or sixth aspect of the present invention.

[0093] Also provided are vectors comprising the nucleic acid sequence according to the seventh aspect and host cells comprising such nucleic acid.

[0094] Provided is a method for preparing the bispecific antigen-binding molecule, recombinant fusion protein, recombinant fusion protein dimer, or chimeric antigen receptor of the first, second, third, fourth, fifth, or sixth aspect, the method comprising culturing or maintaining a host cell comprising the above-mentioned polynucleotide or vector under conditions such that the host cell produces the bispecific antigen-binding molecule, recombinant fusion protein, or chimeric antigen receptor. Optionally, the method further comprises isolating the specific antigen-binding molecule, recombinant fusion protein, recombinant fusion protein dimer, or chimeric antigen receptor.

[0095] In an eighth aspect of the present invention, a pharmaceutical composition comprising the bispecific antigen binding molecules, fusion proteins, recombinant fusion protein dimers or chimeric antigen receptors of the first, second, third, fourth, fifth or sixth aspects is provided. The pharmaceutical composition may contain a variety of pharmaceutically acceptable carriers. The pharmaceutical composition of the present invention may be administered by any suitable method known in the art, including but not limited to intravenous, intramuscular, oral, intraperitoneal or topical administration. In a preferred embodiment, the pharmaceutical composition may be prepared in the form of a liquid, gel, powder, tablet, capsule or foam.

[0096] The bispecific antigen binding molecules, recombinant fusion proteins, recombinant fusion protein dimers or chimeric antigen receptors of the first, second, third, fourth, fifth or sixth aspects can be used for therapy. More specifically, the bispecific antigen binding molecules, recombinant fusion proteins, recombinant fusion protein dimers or chimeric antigen receptors of the first, second, third, fourth, fifth or sixth aspects can be used for the treatment of cancer. Preferably, cancer is a ROR1 positive cancer type and / or an EGFR positive cancer type. More preferably, cancer is selected from the group comprising: blood cancers, such as lymphomas and leukemias, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B cell acute lymphoblastic leukemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML); and solid tumors, including neuroblastoma, kidney cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, breast cancer, skin cancer, uterine cancer, prostate cancer, thyroid cancer, head and neck cancer, bladder cancer, esophageal cancer, gastric cancer or liver cancer.

[0097] Also provided herein is the use of the bispecific antigen binding molecule, recombinant fusion protein, recombinant fusion protein dimer or chimeric antigen receptor of the first, second, third, fourth, fifth or sixth aspect in the preparation of a medicament for treating a disease in a patient in need thereof.

[0098] The bispecific antigen binding molecules, recombinant fusion proteins, recombinant fusion protein dimers or chimeric antigen receptors of the first, second, third, fourth, fifth and sixth aspects or the pharmaceutical composition of the eighth aspect can be administered in a single dose. As used herein, "single dose" refers to a dosage regimen consisting of one dose. Alternatively, a multiple dose regimen can be used. Without being bound by theory, when the specific binding molecules, recombinant fusion proteins, recombinant fusion protein dimers or chimeric antigen receptors of the first, second, third, fourth, fifth and sixth aspects or the pharmaceutical composition of the eighth aspect are administered in a single dose, the advantages can be particularly evident.

[0099] In addition, according to the present invention, there is provided a method for treating a disease in a patient in need of treatment, the method comprising administering to the patient a therapeutically effective dose of the bispecific antigen binding molecule, recombinant fusion protein, recombinant fusion protein dimer or chimeric antigen receptor of the first, second, third, fourth, fifth or sixth aspect, or the pharmaceutical composition of the eighth aspect.

[0100] Preferably, the cancer is a ROR1-positive cancer type and / or an EGFR-positive cancer type. More preferably, the cancer is selected from the group consisting of blood cancers such as lymphomas and leukemias, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B-cell acute lymphoblastic leukemia (B-ALL), marginal zone lymphoma (MZL), non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML); and solid tumors including neuroblastoma, kidney cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, breast cancer, skin cancer, uterine cancer, prostate cancer, thyroid cancer, head and neck cancer, bladder cancer, esophageal cancer, stomach cancer or liver cancer.

[0101] Also provided herein is a method for determining the presence of a target analyte in a sample, comprising adding the bispecific antigen-binding molecule of the first aspect or the second aspect, or the recombinant fusion protein of the third aspect, or the recombinant fusion protein dimer of the fourth or fifth aspect having a detectable label to the sample and detecting binding of the molecule to the target analyte.

[0102] In addition, provided herein is a method for imaging a disease site in a subject, comprising administering to the subject the bispecific antigen-binding molecule of the first or second aspect having a detectable label, or the recombinant fusion protein of the third aspect having a detectable label, or the recombinant fusion protein dimer of the fourth or fifth aspect.

[0103] Also provided herein is a method of diagnosing a disease or medical condition in a subject comprising administering the bispecific antigen binding molecule of the first or second aspect, or the recombinant fusion protein of the third aspect, or the recombinant fusion protein dimer of the fourth or fifth aspect.

[0104] Also contemplated herein is a bispecific antigen binding molecule comprising an antibody, antibody fragment, or antigen binding molecule that competes with the ROR1 specific antigen binding molecule of the first or second aspect for binding to ROR1. When used in the context of an antigen binding protein (e.g., a neutralizing antigen binding protein or a neutralizing antibody), the term "competition" means competition between antigen binding proteins as determined by an assay in which the tested antigen binding protein (e.g., an antibody or a functional fragment thereof) prevents or inhibits specific binding of an antigen binding molecule as defined herein (e.g., a specific antigen binding molecule of the first aspect) to a common antigen (e.g., ROR1 in the case of the specific antigen binding molecule of the first or second aspect).

[0105] Also described herein is a kit for diagnosing a subject with a tendency to cancer or cancer, or for providing a prognosis of the subject's condition, the kit comprising a detection tool for detecting the concentration of an antigen present in a sample from a test subject, wherein the detection tool comprises the bispecific antigen binding molecule of the first or second aspect, the recombinant fusion protein of the third aspect, or the recombinant fusion protein dimer of the fourth or fifth aspect, the chimeric antigen receptor of the sixth aspect, or the nucleic acid sequence of the seventh aspect, the bispecific antigen binding molecule, recombinant fusion protein, recombinant fusion protein dimer, chimeric antigen receptor, or nucleic acid sequence are each optionally derivatized, wherein the presence of an antigen in the sample indicates that the subject has cancer. Preferably, the antigen comprises a ROR1 protein, more preferably an extracellular domain thereof. More preferably, the kit is used to identify the presence of ROR1-positive cells in a sample, or to determine its concentration in a sample. The kit may also comprise a positive control and / or a negative control and / or a detectable marker for comparative determination.

[0106] The present invention also provides a method for diagnosing a subject having cancer or a predisposition to cancer, or for providing a prognosis of the subject's condition, the method comprising detecting the concentration of an antigen present in a sample obtained from the subject, wherein the detection is achieved using the bispecific antigen binding molecule of the first or second aspect, the recombinant fusion protein of the third aspect, or the recombinant fusion protein dimer of the fourth or fifth aspect, the chimeric antigen receptor of the sixth aspect, or the nucleic acid sequence of the seventh aspect, each of the bispecific antigen binding molecule, recombinant fusion protein, recombinant fusion protein dimer, chimeric antigen receptor or nucleic acid sequence being optionally derivatized, and wherein the presence of the antigen in the sample indicates that the subject has cancer.

[0107] Also contemplated herein is a method of killing or inhibiting the growth of cells expressing ROR1 in vitro or in a patient, the method comprising administering to the cell a pharmaceutically effective amount or dosage of (i) the bispecific antigen binding molecule of the first or second aspect, the recombinant fusion protein of the third aspect, or the recombinant fusion protein dimer of the fourth or fifth aspect, the nucleic acid sequence of the seventh aspect, or the CAR or cell of the sixth aspect, or (ii) the pharmaceutical composition of the eighth aspect. Preferably, the cell expressing ROR1 is a cancer cell. More preferably, ROR1 is human ROR1.

[0108] According to a ninth aspect, the present invention provides a bispecific antigen-binding molecule comprising an amino acid sequence represented by formula (II):

[0109] X-FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4-Y(II)

[0110] in

[0111] FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 is a ROR1-specific antigen-binding molecule according to the first or second aspect,

[0112] X and Y are optional amino acid sequences,

[0113] wherein the ROR1 -specific antigen binding molecule is conjugated to a second moiety and wherein the bispecific antigen binding molecule further comprises an EGFR-specific antigen binding molecule.

[0114] According to a tenth aspect, the present invention provides a target binding molecule-drug conjugate comprising:

[0115] (a) the bispecific antigen binding molecule according to the first, second or ninth aspect, or the recombinant fusion protein of the third aspect, or the recombinant fusion protein dimer of the fourth or fifth aspect, and

[0116] (b) at least one toxin. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] Figure 1 : Design of B1 loop library: The sequence of B1 is shown, where "X" represents randomized amino acids in CDR1 and CDR3.

[0118] Figure 2 : Evaluation of the B1 VNAR loop variant (His6Myc tag) and A549 (ROR1) by flow cytometry hi ) cell surface binding of lung cancer cells.

[0119] Figure 3: Evaluation of the B1 VNAR loop variant (His6Myc tag) and A427 (ROR1 by flow cytometry 低 ) cell surface binding of lung cancer cells.

[0120] Figure 4 : Sequence and loop library design of P3A1 G1. CDR1 diversity resulted in 448 combinations, HV2 diversity resulted in 768 combinations, and HV4 diversity resulted in 24 combinations.

[0121] Figure 5 : Binding of P3A1G1 loop variants to human ROR1 was assessed by ELISA. Data are plotted as the OD signal obtained at 450 nm for each loop variant at a fixed concentration (5 ug / mL).

[0122] Figure 6 : Binding of P3A1G1 loop variants and parental P3A1G1 protein to human ROR1 was assessed by ELISA.

[0123] Figure 7 : Linker mouse IgG and linker human IgG sequences used in VNAR IgG Fc fusion proteins. Engineered hIgG1 Fc fusion proteins incorporate engineered cysteine ​​substitutions in the hIgG1 Fc sequence, such as at positions S239C or S442C (EU numbering), to enable site-specific labeling.

[0124] Figure 8 Flow cytometry was used to evaluate the interaction of B1 loop variants–hFc fusion proteins with A549 (ROR1 hi ) cell surface binding of lung cancer cells.

[0125] Figure 9 : ROR1 biparatopic VNAR-hFc fusions were analyzed by SDS-PAGE (4-12% Bis Tris gel, MOPS buffer, ±50 mM DTT). Lane 1 G3CP-P3A1 hFc (S239C + KIH) and Lane 2 G3CPG4-P3A1 hFc (S239C + KIH)

[0126] Figure 10 Flow cytometry was used to evaluate the interaction of ROR1 biparatopic VNAR-hFc fusion with A549 (ROR1 hi ) and A427(ROR1 低 ) cell surface binding of lung cancer cells.

[0127] Figure 11: Structures of PNU-linker payloads MA-PEG-vc-PAB-EDA-PNU159682 and MA-PEG-va-EDA-PNU159682.

[0128] Figure 12 : Shows the dose response of G3CP-hFc and G3CPG4-hFc PNU conjugates and the corresponding parent proteins for binding to human ROR1 assessed by ELISA.

[0129] Figure 13 : The killing efficacy of G3CP-hFc PNU and G3CPG4-hFc PNU conjugates on ROR1-positive PA-1 cell lines and ROR1-knockout PA-1 cell lines.

[0130] Figure 14 : In vivo efficacy of G3CP-hFc PNU and G3CPG4-hFc PNU conjugates in the ROR1+HBCx-28 patient-derived TNBC xenograft model. Data were plotted until the first animal in the vehicle group reached a humane tumor burden.

[0131] Figure 15 : Sequence alignment of B1, B1 G4, B1V15, G3CP, and G3CPG4. The points of variation within the CDR and HV regions are underlined. Note that B1V15 (SEQ ID NO: 115) is not a loop library variant of B1; they have identical CDR1, HV2, HV4, and CDR3 sequences.

[0132] Figure 16 : UV analysis of B1-hFc, B1G4-hFc, G3CP-hFc and G3CPG4-hFc after incubation in PBS pH 7.4 buffer at 37°C for 96 hours.

[0133] Figure 17 : After incubation in PBS pH 7.4 buffer at 37°C for 96 hours, B1-hFc, B1G4-hFc, G3CP-hFc and G3CPG4-hFc were subjected to size exclusion analysis (SEC).

[0134] Figure 18 : G3CP-hFc PNU and G3CPG4-hFc PNU conjugates kill ROR1 低 Efficacy in HEK293 cells and HEK293 cells stably transfected with human ROR1

[0135] Figure 19: In vivo efficacy of G3CP-hFc PNU and G3CPG4-hFc PNU conjugates in the ROR1+HBCx-10 patient-derived TNBC xenograft model. Data were plotted until the first animal in the vehicle group reached a humane tumor burden.

[0136] Figure 20: Evaluation of the interaction of ROR1 biparatopic VNAR-hFc drug conjugate with A549 (ROR1) by flow cytometry hi ) and A427(ROR1 低 ) cell surface binding of lung cancer cells.

[0137] Figure 21 : Efficacy of biparatopic G3CP-P3A1-hFc PNU and G3CPG4-P3A1-hFc PNU conjugates in killing ROR1-positive PA-1 cell lines and ROR1-knockout PA-1 cell lines.

[0138] Figure 22 : In vivo efficacy of biparatopic G3CP-P3A1 hFc PNU and G3CPG4-P3A1-hFc PNU conjugates in the ROR1+HBCx-28 patient-derived TNBC xenograft model.

[0139] Figure 23 : Binding of ROR1×EGFR hFc bispecific protein to human ROR1 was evaluated by ELISA.

[0140] Figure 24 : Binding of ROR1×EGFR hFc bispecific protein to human EGFR was evaluated by ELISA.

[0141] Figure 25 : Example BLI trace showing simultaneous binding of ROR1×EGFR hFc bispecific protein to ROR1 and EGFR.

[0142] Figure 26 : Stability of ROR1×EGFR hFc protein in PBS at 37°C – UV analysis.

[0143] Figure 27 Figure 2: Stability of ROR1×EGFR hFc Protein in PBS at 37°C – SEC analysis of ROR1×7D12 hFc at T0 and T96. The increase in HMW% in the protein containing B1 is highlighted.

[0144] Figure 28 Figure 2: Stability of ROR1×EGFR hFc Protein in PBS at 37°C – SEC analysis of ROR1×EGFR hFc at T0 and T96. The increase in HMW% in the protein containing B1 is highlighted.

[0145] Figure 29 : Evaluation of the interaction of ROR1×EGFR hFc MMAE conjugate with ROR1 by flow cytometry + EGFR + Cell surface binding of PA-1 cancer cells and dual receptor-negative HCC1419 cancer cells (example of ROR1 series binding).

[0146] Figure 30 : Evaluation of the interaction of ROR1×EGFR hFc MMAE conjugate with ROR1 by flow cytometry + EGFR + Cell surface binding of PA-1 cancer cells and dual receptor-negative HCC1419 cancer cells (EGFR series binding example).

[0147] Figure 31 : Evaluation of G3CP-7D12 hFc MMAE and P3A1-9G8 hFc MMAE with ROR1 by flow cytometry + EGFR + Cell surface binding of PA-1 cancer cells and dual receptor-negative HCC1419 cancer cells.

[0148] Figure 32 :Dose-response curves of G3CP-7D12 hFc MMAE in killing different cancer cell lines and normal cells.

[0149] Figure 33 :Dose-response curves of P3A1-7D12 hFc MMAE killing different cancer cell lines and normal cells.

[0150] Figure 34 : Dose-response curve of G3CPG4-7D12 hFc MMAE killing different cancer cell lines and normal cells.

[0151] Figure 35 : Cell killing by the G3CP×EGFR MMAE series in PC9 and NHEK-Ad cell lines.

[0152] Figure 36 : Cell killing by the G3CPG4×EGFR MMAE series in PC9 and NHEK-Ad cell lines.

[0153] Figure 37 : Cell killing by the P3A1×EGFR MMAE series in PC9 and NHEK-Ad cell lines.

[0154] Figure 38Summary of Western blot analysis of ROR1 and EGFR expression in TNBC (triple-negative breast cancer), NSCLC (non-small cell lung cancer), and pancreatic cancer PDX models. Fluorescence detection. A549 and Kasumi-2 cell lines serve as positive and negative controls for EGFR expression, respectively. PA-1 and PA-1ROR1-KO cell lines serve as positive and negative controls for ROR1 expression, respectively.

[0155] Figure 39 Summary of Western blots of ROR1 and EGFR expression in other PDX models (head and neck cancer, renal cancer, esophageal cancer, gastric cancer, sarcoma, and colorectal cancer). Chemiluminescence detection was used. ROR1 images were acquired with a 30-second exposure, and EGFR images were acquired with a 1-second exposure. The HeLa cell line was used as a positive control for EGFR expression. PA-1 and T47D cell lines served as positive and negative controls for ROR1 expression, respectively.

[0156] Figure 40 :ROR1×EGFR bispecific conjugates (G3CP-7D12 hFc MMAE, G3CPG4-7D12 hFc MMAE and P3A1-7D12 hFc MMAE) and ROR1 + EGFR + Cell surface binding of PA-1 cancer cells and comparison to the corresponding parental EGFR monospecific conjugate (7D12 hFc MMAE) and the corresponding parental ROR1 monospecific conjugates (G3CP hFc MMAE, G3CPG4 hFc MMAE, and P3A1 hFc MMAE). Binding was assessed by flow cytometry.

[0157] Figure 41 :ROR1×EGFR bispecific conjugates (P3A1-7D12 hFc MMAE, P3A1-9G8 hFc MMAE, P3A1-EGFR33hFc MMAE and P3A1-EGFR13 hFc MMAE) and ROR1 + EGFR + Cell surface binding of PA-1 cancer cells and comparison to the corresponding parental ROR1 monospecific conjugate (P3A1 hFc MMAE) and the corresponding parental EGFR monospecific conjugates (7D12 hFc MMAE, 9G8 hFc MMAE, EGFR33 hFc MMAE, and EGFR13 hFc MMAE). Binding was assessed by flow cytometry.

[0158] Figure 42: Internalization of ROR1×EGFR bispecific hFc proteins G3CP-7D12(585), P3A1-7D12(589) and G3CPG4-7D12(849), parental monospecific molecules targeting EGFR or ROR1, and isotype controls in ROR1+EGFR++PC9 lung adenocarcinoma cells. DETAILED DESCRIPTION

[0159] The present invention generally relates to bispecific antigen binding molecules. Specifically, the present invention provides immunoglobulin-like shark variable new antigen receptors (VNARs) and related fusion proteins, chimeric antigen receptors, conjugates and nucleic acids specific for receptor tyrosine kinase-like orphan receptor 1 (ROR1), as well as accompanying methods. ROR1-specific VNAR domains are described herein as ROR1-specific antigen binding molecules.

[0160] The novel or new antigen receptor (IgNAR) is a homodimeric protein of approximately 160 kDa found in the serum of cartilaginous fish (Greenberg AS, et al., Nature, 1995. 374(6518): p.168-173, Dooley, H., et al, Mol. Immunol, 2003. 40(1): p.25-33; Müller, MR, et al., mAbs, 2012. 4(6): p.673-685). Each molecule consists of a single N-terminal variable domain (VNAR) and five constant domains (CNAR). The IgNAR domain is a member of the immunoglobulin superfamily. VNAR is a tightly folded domain whose structure and certain sequences are similar to those of immunoglobulin and T cell receptor variable domains and cell adhesion molecules, and is referred to as VNAR by analogy with the N-variable terminal domains of classical immunoglobulins and T cell receptors. VNARs have limited sequence homology with immunoglobulins, for example, there is 25-30% similarity between VNARs and human light chain sequences.

[0161] Kovaleva M. et al Expert Opin. Biol. Ther. 2014. 14(10): p. 1527-1539 and Zielonka S. et al mAbs 2015. 7(1): p. 15-25 (which are incorporated herein by reference) provide summaries of the structural characterization and generation of VNARs.

[0162] VNARs do not appear to have evolved from a classical immunoglobulin antibody ancestor. Unique structural features of VNARs are a truncated sequence comparable to the CDR2 loop present in conventional immunoglobulin variable domains and a lack of hydrophobic VH / VL interface residues that typically allow association with the light chain domain, which is absent in IgNAR structures. Furthermore, unlike classical immunoglobulins, some VNAR subtypes contain additional cysteine ​​residues in the CDR regions that have been observed to form disulfide bridges in addition to the typical immunoglobulin superfamily bridges between cysteines in the framework 1 and 3 regions adjacent to CDR1 and CDR3 at the N-terminus.

[0163] To date, there are three defined types of shark IgNARs, termed type I, type II, and type III. These are classified based on the position of atypical cysteine ​​residues, which are under strong selective pressure and are therefore rarely replaced.

[0164] All three types have classical immunoglobulin typical cysteine ​​(as Kabat, EA et al. Sequences of proteins of immunological interest. 5th ed. 1991, Bethesda: USDept. of Health and Human Services, PHS, NIH, numbering) at positions 35 and 107, which, together with the invariant tryptophan at position 36, stabilize the standard immunoglobulin fold. There is no defined CDR2, but the sequence variation regions closer to TCR HV2 and HV4 are defined in framework 2 and framework 3, respectively. Type I has germline-encoded cysteine ​​residues in framework 2 and framework 4, and has an even number of extra cysteines in CDR3. The crystal structure study of type I IgNAR separated by lysozyme and compounded with lysozyme makes it possible to determine the contribution of these cysteine ​​residues. Framework 2 and 4 cysteines all form disulfide bridges with the cysteine ​​in CDR3, thereby forming a tightly packed structure in which the CDR3 loop is tightly pressed against the HV2 region. To date, type I IgNARs have only been found in nurse sharks, while all other cartilaginous fish, including members of the same order, possess only type II or variants of this type.

[0165] Type II IgNARs are defined as having cysteine ​​residues in both the CDR1 and CDR3 that form intramolecular disulfide bonds, bringing these two regions into close proximity and resulting in a CDR3 that favors a protruding binding pocket or groove. Type I sequences typically have longer CDR3s than type II sequences, averaging 21 and 15 residues, respectively. This is thought to be due to strong selective pressure for the two or more cysteine ​​residues in type I CDR3 to associate with their framework 2 and 4 counterparts. Studies of somatic mutation accumulation have shown that type II CDR1 has a greater number of mutations than type I, while the sequence variation in the HV2 region of type I is greater than that of type II. This evidence correlates well with the well-defined positioning of these regions within the antigen-binding site.

[0166] A third IgNAR type, termed type III, has been identified in newborns. This member of the IgNAR family lacks diversity within the CDR3 due to germline fusion of the D1 and D2 regions (forming the CDR3) with the V gene. Almost all known clones have a CDR3 length of 15 residues, with little or no sequence diversity.

[0167] Another structural type of VNAR, designated type IIb or IV, has only two typical cysteine ​​residues (in the framework 1 and framework 3b regions). To date, this type has been found primarily in dogfish sharks and has also been isolated from a semisynthetic V-NAR library derived from the wobbegong shark.

[0168] Unlike variable domains in other natural immunoglobulins, the VNAR binding surface is derived from four diversity regions: CDR1, HV2, HV4, and CDR3, connected by intervening framework sequences in the following order: FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4. The lack of a natural light chain partner combined with the absence of CDR2 makes VNAR the smallest naturally occurring binding domain in the vertebrate kingdom.

[0169] IgNARs share some incidental features with heavy chain-only immunoglobulins (HCAbs) found in camelids (camels, dromedaries, and llamas). Unlike IgNARs, HCAbs are clearly derived from the immunoglobulin family and share significant sequence homology with standard immunoglobulins. Importantly, a key difference of VNARs is that, unlike classical immunoglobulins or HCAbs, this molecule has no partner light chain at any point in its evolution. Flajnik MF et al PLoS Biol 2011.9(8):e1001120 and Zielonka S. et al mAbs 2015.7(1):p.15-25 review the similarities and differences between VNARs and the VHH single binding domains derived from immunoglobulins from camelids and their possible and distinct evolutionary origins.

[0170] Although antibodies targeting ROR1 have been reported in the literature, the high sequence identity between the extracellular domains of human, mouse, and rat ROR1, as well as between human ROR1 and ROR2 family members, means that generating high-affinity hROR1-specific binders is not straightforward. Furthermore, the large size of antibodies impairs their ability to penetrate solid tumors and renders target protein regions inaccessible due to steric factors, which is particularly problematic for cell surface proteins where oligomerization or receptor clustering is observed.

[0171] Therefore, there is a need in the art for improved anti-ROR1 binding protein agents with functional or physical characteristics or properties distinct from antibodies, as well as for the development of therapeutic and diagnostic agents for malignancies associated with ROR1 expression. The present invention provides such agents in the form of ROR1-specific antigen binding molecules as described herein.

[0172] Without being bound by theory, the ROR1-specific antigen-binding molecules described herein are believed to bind to both human ROR1 and murine ROR1. Various variants, including G3CP, G3CPG4, 1E5, 1B11, C3CP, 1G9, 1H8, G11CP, D9CP, 1B6, 1F10, F2CP, B6CP, 1E1, and P3A1, P3A1G1 NAC6.S, P3A1G1 AE3.S, P3A1G1 NAC6, P3A1G1 AE3, and P3A1G1NAG8, have been experimentally confirmed to bind to both hROR1 and mROR1. In addition, the ROR1-specific antigen-binding molecules described herein may bind to deglycosylated forms of ROR1. Furthermore, they may not bind to many linear peptides associated with anti-ROR1 antibodies described in the prior art. Therefore, the presently described ROR1 -specific antigen binding molecules are believed to bind to a different epitope in the ROR1 sequence compared to these prior art anti-ROR1 antibodies.

[0173] Binding and internalization of ROR1-specific antigen binding molecules to cancer cell lines have been demonstrated, demonstrating the potential for using such molecules in the treatment of cancer, particularly cancers expressing ROR1.

[0174] Various forms of ROR1-specific antigen-binding molecules are described, including several types of fusion proteins. Fusion proteins comprising the Fc region of an immunoglobulin, as well as homodimers and heterodimers, are described. Fusion of proteins to Fc domains can improve protein solubility and stability, significantly extend plasma half-life, and enhance overall therapeutic efficacy.

[0175] The present inventors have also created VNAR molecules conjugated to multiple moieties and payloads. Thus, the present application discloses chemically conjugated VNARs. More specifically, several conjugated forms of ROR1-specific antigen-binding molecules are provided.

[0176] The ROR1 specific antigen binding molecules described herein have been formatted in combination with EGFR specific binding molecules. The present invention therefore relates to bispecific ROR1 / EGFR antigen binding molecules.

[0177] According to a first aspect, the present invention provides a bispecific antigen binding molecule comprising:

[0178] (i) a receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen-binding molecule comprising the amino acid sequence represented by formula (I):

[0179] FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4(I)

[0180] in

[0181] CDR3 is selected from the group consisting of YPWGAGAPYNVQWY (SEQ ID NO: 23), YPWGAGAPYLVQWY (SEQ ID NO: 20), YPWGAGAPWNVQWY (SEQ ID NO: 24), YPSGAGAPRPVQWY (SEQ ID NO: 11), YPWGAGAPCLVQWY (SEQ ID NO: 11), NO: 12), YPWGAGAPRLVQWY (SEQ ID NO: 13), YPWGAGAPRQVQWY (SEQ ID NO: 14), YPWGAGAPRSVQWY (SEQ ID NO: 15), YPWGAGAPSLVQWY (SEQ ID NO: 16), YPWGAGAPSNVQWY (SEQ ID NO: 17), YPWGAGAPSQVQWY (SEQ ID NO: 18), YPWGAGAPSSVQWY (SEQ ID NO: 19), YPWGAGAPWQVQWY (SEQ ID NO: 21), YPWGAGAPWSVQWY (SEQ ID NO: 22), and YPWGAGAPWLVQWY (SEQ ID NO: 10);

[0182] CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of GANYGLAA (SEQ ID NO: 1), DANYGLAA (SEQ ID NO: 5), GANYDLSA (SEQ ID NO: 2), GANYGLSA (SEQ ID NO: 3), and GANYDLAA (SEQ ID NO: 4);

[0183] FW1 is the framework region;

[0184] FW2 is the framework region;

[0185] HV2 is a hypervariable sequence having an amino acid sequence selected from the group consisting of SSNQERISIS (SEQ ID NO: 6) and SSNKERISIS (SEQ ID NO: 7);

[0186] FW3a is the framework region;

[0187] HV4 is a hypervariable sequence having an amino acid sequence selected from the group consisting of NKRTM (SEQ ID NO: 8) and NKGTM (SEQ ID NO: 9);

[0188] FW3b is the framework region;

[0189] FW4 is the framework region;

[0190] wherein if CDR3 is YPWGAGAPWLVQWY (SEQ ID NO: 10), CDR1 is selected from the group consisting of DANYGLAA (SEQ ID NO: 5), GANYGLSA (SEQ ID NO: 3) and GANYDLAA (SEQ ID NO: 4); and

[0191] (ii) Epidermal growth factor receptor (EGFR)-specific antigen-binding molecules.

[0192] In one embodiment of the ROR1-specific antigen binding molecule:

[0193] CDR3 is selected from the group consisting of YPWGAGAPYNVQWY (SEQ ID NO: 23), YPWGAGAPYLVQWY (SEQ ID NO: 20), YPWGAGAPWNVQWY (SEQ ID NO: 24), YPSGAGAPRPVQWY (SEQ ID NO: 11), YPWGAGAPCLVQWY (SEQ ID NO: 11), NO: 12), YPWGAGAPRLVQWY (SEQ ID NO: 13), YPWGAGAPRQVQWY (SEQ ID NO: 14), YPWGAGAPRSVQWY (SEQ ID NO: 15), YPWGAGAPSLVQWY (SEQ ID NO: 16), YPWGAGAPSNVQWY (SEQ ID NO: 17), YPWGAGAPSSVQWY (SEQ ID NO: 19), YPWGAGAPWQVQWY (SEQ ID NO: 21), YPWGAGAPWSVQWY (SEQ ID The CDR sequences of the amino acid sequences in the group consisting of YPWGAGAPWLVQWY (SEQ ID NO: 22) and YPWGAGAPWLVQWY (SEQ ID NO: 10).

[0194] If CDR3 is not YPWGAGAPWLVQWY (SEQ ID NO: 10), CDR1 may be a CDR sequence having an amino acid sequence selected from the group consisting of GANYGLAA (SEQ ID NO: 1), DANYGLAA (SEQ ID NO: 5), GANYDLSA (SEQ ID NO: 2), GANYGLSA (SEQ ID NO: 3), and GANYDLAA (SEQ ID NO: 4). Therefore, a ROR1-specific antigen-binding molecule can be defined as comprising an amino acid sequence represented by formula (I):

[0195] FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4(I)

[0196] in

[0197] CDR3 is selected from the group consisting of YPWGAGAPYNVQWY (SEQ ID NO: 23), YPWGAGAPYLVQWY (SEQ ID NO: 20), YPWGAGAPWNVQWY (SEQ ID NO: 24), YPSGAGAPRPVQWY (SEQ ID NO: 11), YPWGAGAPCLVQWY (SEQ ID NO: 11), NO: 12), YPWGAGAPRLVQWY (SEQ ID NO: 13), YPWGAGAPRQVQWY (SEQ ID NO: 14), YPWGAGAPRSVQWY (SEQ ID NO: 15), YPWGAGAPSLVQWY (SEQ ID NO: 16), YPWGAGAPSNVQWY (SEQ ID NO: 17), YPWGAGAPSQVQWY (SEQ ID NO: 18), YPWGAGAPSSVQWY (SEQ ID NO: 19), YPWGAGAPWQVQWY (SEQ ID CDR sequences of amino acid sequences in the group consisting of YPWGAGAPWSVQWY (SEQ ID NO: 21) and YPWGAGAPWSVQWY (SEQ ID NO: 22);

[0198] CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of GANYGLAA (SEQ ID NO: 1), DANYGLAA (SEQ ID NO: 5), GANYDLSA (SEQ ID NO: 2), GANYGLSA (SEQ ID NO: 3), and GANYDLAA (SEQ ID NO: 4);

[0199] FW1 is the framework region;

[0200] FW2 is the framework region;

[0201] HV2 is a hypervariable sequence having an amino acid sequence selected from the group consisting of SSNQERISIS (SEQ ID NO: 6) and SSNKERISIS (SEQ ID NO: 7);

[0202] FW3a is the framework region;

[0203] HV4 is a hypervariable sequence having an amino acid sequence selected from the group consisting of NKRTM (SEQ ID NO: 8) and NKGTM (SEQ ID NO: 9);

[0204] FW3b is the framework region; and

[0205] FW4 is the framework region.

[0206] In one embodiment of the ROR1-specific antigen binding molecule:

[0207] CDR3 is a CDR sequence having an amino acid sequence selected from the group consisting of YPWGAGAPYNVQWY (SEQ ID NO: 23), YPWGAGAPYLVQWY (SEQ ID NO: 20), and YPWGAGAPWNVQWY (SEQ ID NO: 24), and / or

[0208] CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of GANYGLAA (SEQ ID NO: 1) and DANYGLAA (SEQ ID NO: 5).

[0209] In one embodiment of the ROR1-specific antigen binding molecule:

[0210] CDR3 is a CDR sequence having an amino acid sequence according to YPWGAGAPYNVQWY (SEQ ID NO: 23).

[0211] In one embodiment of the ROR1-specific antigen binding molecule:

[0212] CDR3 is a CDR sequence having an amino acid sequence according to YPWGAGAPYNVQWY (SEQ ID NO: 23);

[0213] CDR1 is a CDR sequence having an amino acid sequence according to GANYGLAA (SEQ ID NO: 1);

[0214] HV2 is a hypervariable sequence having an amino acid sequence according to SSNQERISIS (SEQ ID NO: 6); and

[0215] HV4 is a hypervariable sequence having an amino acid sequence according to NCRTM (SEQ ID NO: 8).

[0216] In one embodiment of the ROR1-specific antigen binding molecule:

[0217] CDR3 is a CDR sequence having an amino acid sequence according to YPWGAGAPYNVQWY (SEQ ID NO: 23);

[0218] CDR1 is a CDR sequence having an amino acid sequence according to DANYGLAA (SEQ ID NO: 5);

[0219] HV2 is a hypervariable sequence having an amino acid sequence according to SSN KERISIS (SEQ ID NO: 7); and

[0220] HV4 is a hypervariable sequence having an amino acid sequence according to NKGTM (SEQ ID NO: 9).

[0221] In one embodiment of the ROR1-specific antigen binding molecule:

[0222] CDR3 is a CDR sequence having an amino acid sequence according to YPWGAGAPWLVQWY (SEQ ID NO: 10);

[0223] CDR1 is a CDR sequence having an amino acid sequence according to DANYGLAA (SEQ ID NO: 5);

[0224] HV2 is a hypervariable sequence having an amino acid sequence according to SSN KERISIS (SEQ ID NO: 7); and

[0225] HV4 is a hypervariable sequence having an amino acid sequence according to NKGTM (SEQ ID NO: 9).

[0226] In a preferred embodiment, the ROR1-specific antigen binding molecule comprises an amino acid sequence selected from the group consisting of:

[0227] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVN (SEQ ID NO: 50), referred to herein as G3CP;

[0228] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIK (SEQ ID NO: 51), referred to herein as B1G4;

[0229] ASVNQTPRTATKETGESLTINCVVTGANYDLSATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPSGAGAPRPVQWYDGAGTVLTVN (SEQ ID NO: 52), referred to herein as 1E2;

[0230] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPCLVQWYDGAGTVLTVN (SEQ ID NO: 53), referred to herein as 1E5;

[0231] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPRLVQWYDGAGTVLTVN (SEQ ID NO: 54), referred to herein as 1B11;

[0232] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPRQVQWYDGAGTVLTVN (SEQ ID NO: 55), referred to herein as C3CP;

[0233] ASVNQTPRTATKETGESLTINCVVTGANYGLSATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPRSVQWYDGAGTVLTVN (SEQ ID NO: 56), referred to herein as 2G5;

[0234] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPRSVQWYDGAGTVLTVN (SEQ ID NO: 57), referred to herein as 1G12;

[0235] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSLVQWYDGAGTVLTVN (SEQ ID NO: 58), referred to herein as G5CP;

[0236] ASVNQTPRTATKETGESLTINCVVTGANYGLSATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSNVQWYDGAGTVLTVN (SEQ ID NO: 59), referred to herein as 2F4;

[0237] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSQVQWYDGAGTVLTVN (SEQ ID NO: 60), referred to herein as 1G9;

[0238] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVN (SEQ ID NO: 61), referred to herein as 1H8;

[0239] ASVNQTPRTATKETGESLTINCVVTGANYGLSATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWLVQWYDGAGTVLTVN (SEQ ID NO: 62), referred to herein as G11CP;

[0240] ASVNQTPRTATKETGESLTINCVVTGANYDLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWLVQWYDGAGTVLTVN (SEQ ID NO: 63), referred to herein as D9CP;

[0241] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWNVQWYDGAGTVLTVN (SEQ ID NO: 64), referred to herein as 1B6;

[0242] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWQVQWYDGAGTVLTVN (SEQ ID NO: 65), referred to herein as 1F10;

[0243] ASVNQTPRTATKETGESLTINCVVTGANYGLSATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWQVQWYDGAGTVLTVN (SEQ ID NO: 66), referred to herein as E6CP;

[0244] ASVNQTPRTATKETGESLTINCVVTGANYDLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWQVQWYDGAGTVLTVN (SEQ ID NO: 67), referred to herein as F2CP;

[0245] ASVNQTPRTATKETGESLTINCVVTGANYDLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWSVQWYDGAGTVLTVN (SEQ ID NO: 68), referred to herein as B6CP;

[0246] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWSVQWYDGAGTVLTVN (SEQ ID NO: 69), referred to herein as 1G1; and

[0247] ASVNQTPRTATKETGESLTINCVVTGANYGLSATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWSVQWYDGAGTVLTVN (SEQ ID NO: 70), referred to herein as A10CP;

[0248] or a functional variant having a CDR1, HV2, HV4 and CDR2 sequence according to any of the sequences therein and having a FW1, FW2, FW3a, FW3b and FW4 sequence having at least 45% combined sequence identity to the combined FW1, FW2, FW3a, FW3b and FW4 sequence of any of the sequences therein.

[0249] In a particularly preferred embodiment, the ROR1 -specific antigen binding molecule may comprise an amino acid sequence according to ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVN (SEQ ID NO: 50).

[0250] The ROR1 -specific antigen binding molecule may comprise an amino acid sequence according to ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVN (SEQ ID NO: 50), or a functional variant thereof, having a CDR1, HV2, HV4 and CDR2 sequence according to SEQ ID NO: 50 and having a FW1, FW2, FW3a, FW3b and FW4 sequence that has at least 45% combined sequence identity to the combined FW1, FW2, FW3a, FW3b and FW4 sequence of SEQ ID NO: 50.

[0251] Specific advantages associated with SEQ ID NO: 50 ("G3CP") and its functional variants include increased expression yield and hydrophilicity, as well as analytical purification and increased monomericity of these proteins in non-optimized aqueous buffer systems. Without being bound by theory, these advantages may be particularly pronounced in VNAR-hFc fusion proteins comprising the G3CP sequence or its functional variants. Thus, the G3CP sequence and its functional variants may provide improved manufacturing and / or handling characteristics. In addition, G3CP-hFc showed excellent in vivo efficacy in a patient-derived triple-negative breast cancer (TNBC) xenograft model when conjugated to a cytotoxic anthracycline (PNU) derivative. The effect of G3CP-hFc was surprisingly even improved over B1-hFc, which itself showed excellent in vivo efficacy. In addition, as shown herein, the ROR1×EGFR bispecific antibody comprising G3CP had a shorter retention time (RT) in size exclusion chromatography (SEC) than the bispecific antibody comprising B1, indicating that the hydrophilicity of the ROR1×EGFR bispecific antibody containing G3CP was improved. In addition, in PBS stability studies, ROR1×EGFR bispecific antibodies containing G3CP may have reduced turbidity and reduced formation of high molecular weight (HMW) species compared to bispecific antibodies containing B1. This suggests that the ease of analysis, purification, and monomericity of ROR1×EGFR bispecific antibodies containing G3CP is increased in non-optimized aqueous buffer systems. Therefore, the G3CP sequence and its functional variants may provide improved manufacturing and / or operating characteristics that are retained in the ROR1×EGFR bispecific format.

[0252] Without being bound by theory, in cases where ROR1 monospecific antibodies are refractory or have low specificity, ROR1×EGFR bispecific antibodies (including but not limited to antibodies comprising G3CP sequences) can also improve therapeutic efficacy. For example, the data disclosed herein use protein blotting to show not only the co-expression of ROR1 and EGFR in TNBC (this application also contains data showing that the G3CP-hFc PNU conjugate has excellent in vivo efficacy in patient-derived TNBC xenograft models), but also high expression of EGFR and ROR1 in non-small cell lung cancer models. Co-expression of ROR1 and EGFR has also been found in large cell lung cancer, head and neck cancer, esophageal cancer, kidney cancer, gastric cancer, sarcoma, pancreatic cancer and colorectal cancer models. Thus, the surprisingly improved efficacy of the G3CP-hFc PNU conjugate in a patient-derived TNBC xenograft model compared to B1-hFc PNU, combined with the in vitro data disclosed herein, suggests that the presently claimed ROR1×EGFR bispecific antibody has therapeutic potential in the potential treatment of cancers that co-express ROR1 and EGFR, such as the cancers identified herein.

[0253] In a particularly preferred embodiment, the ROR1 -specific antigen binding molecule may comprise an amino acid sequence according to ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVN (SEQ ID NO: 61).

[0254] Specific advantages of SEQ ID NO: 61 ("1H8") and its functional variants include good stability in PBS, at least in the case of the 1H8-hFc fusion and as measured by SEC (t = 0 vs 96 h), which is improved relative to B1-hFc under the same conditions. Similarly, improvements were observed for the 1H8 x EGFR bispecific relative to the B1 x EGFR bispecific from the data disclosed herein.

[0255] In a particularly preferred embodiment, the ROR1 -specific antigen binding molecule may comprise the amino acid sequence according to TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIK (SEQ ID NO: 51).

[0256] Specific advantages associated with SEQ ID NO: 51 ("B1G4") and functional variants thereof include increased expression yield and monomericity of fusion proteins (e.g., VNAR-hFc fusion proteins) comprising the B1G4 sequence or functional variants thereof in aqueous buffer systems. Thus, the B1G4 sequence and functional variants thereof can provide fusion proteins with improved manufacturing and / or handling properties.

[0257] The ROR1-specific antigen binding molecule may comprise an amino acid sequence according to TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIK (SEQ ID NO: 51) or a functional variant thereof having CDR1, HV2, HV4 and CDR2 sequences according to SEQ ID NO: 51 and having a FW1, FW2, FW3a, FW3b and FW4 sequence, said FW1, FW2, FW3a, FW3b and FW4 sequence having a combined sequence identity of at least 45% with the combined FW1, FW2, FW3a, FW3b and FW4 sequence of SEQ ID NO: 51.

[0258] In a preferred embodiment, the ROR1-specific antigen binding molecule comprises an amino acid sequence selected from the group consisting of:

[0259] TRVDQSPSSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIK (SEQ ID NO: 71), referred to as G3CP G4 in this article;

[0260] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPYNVQWYDGQGTKLEVK (SEQ ID NO: 72), referred to herein as G3CP V15;

[0261] TRVDQSPSSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIK (SEQ ID NO: 73), referred to herein as 1H8 G4;

[0262] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVK (SEQ ID NO: 74), referred to herein as 1H8 V15;

[0263] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPRQVQWYDGAGTKVEIK (SEQ ID NO: 75), referred to herein as C3CP G4; and

[0264] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPRQVQWYDGQGTKLEVK (SEQ ID NO: 76), referred to herein as C3CPV15;

[0265] or a functional variant having a CDR1, HV2, HV4 and CDR2 sequence according to any of the sequences therein and having a FW1, FW2, FW3a, FW3b and FW4 sequence having at least 45% combined sequence identity to the combined FW1, FW2, FW3a, FW3b and FW4 sequence of any of the sequences therein.

[0266] In a particularly preferred embodiment, the ROR1 -specific antigen binding molecule may comprise the amino acid sequence according to TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIK (SEQ ID NO: 71).

[0267] The ROR1-specific antigen binding molecule may comprise an amino acid sequence according to TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIK (SEQ ID NO: 71) or a functional variant thereof having CDR1, HV2, HV4 and CDR2 sequences according to SEQ ID NO: 71 and having a FW1, FW2, FW3a, FW3b and FW4 sequence, said FW1, FW2, FW3a, FW3b and FW4 sequence having a combined sequence identity of at least 45% with the combined FW1, FW2, FW3a, FW3b and FW4 sequence of SEQ ID NO: 71.

[0268] Specific advantages associated with SEQ ID NO: 71 ("G3CP G4") and functional variants thereof include increased expression yield and hydrophilicity as well as analytical purification and increased monomericity of these proteins in non-optimized aqueous buffer systems. Without being bound by theory, these advantages may be particularly pronounced in VNAR-hFc fusion proteins comprising the G3CP G4 sequence or a functional variant thereof. Thus, the G3CP G4 sequence and functional variants thereof may provide improved manufacturing and / or handling characteristics. Furthermore, G3CPG4-hFc exhibited excellent in vivo efficacy in a patient-derived triple-negative breast cancer (TNBC) xenograft model when conjugated to a cytotoxic anthracycline (PNU) derivative. The efficacy of G3CPG4-hFc was surprisingly even improved over that of B1-hFc, which exhibited excellent in vivo efficacy on its own. In addition, as shown herein, the ROR1×EGFR bispecific antibody containing G3CP G4 has a shorter retention time (RT) in size exclusion chromatography (SEC) than the bispecific antibody containing B1, indicating that the hydrophilicity of the ROR1×EGFR bispecific antibody containing G3CPG4 is improved. In addition, in PBS stability studies, the ROR1×EGFR bispecific antibody containing G3CP (DAR4) may have reduced turbidity and reduced formation of high molecular weight (HMW) species compared to the bispecific antibody containing B1 (DAR2). Based on the slight differences observed between the DAR2 and DAR4 G3CP×EGFR bispecific antibodies, this may indicate an increased ease of analysis, purification, and monomericity of the ROR1×EGFR bispecific containing G3CP G4 in a non-optimized aqueous buffer system. Therefore, the G3CP G4 sequence and its functional variants may provide improved manufacturing and / or operational characteristics that are retained in a ROR1×EGFR bispecific format.

[0269] Without being bound by theory, in cases where ROR1 monospecific antibodies are refractory or have low specificity, ROR1×EGFR bispecific antibodies (including but not limited to antibodies comprising G3CP G4 sequences) can also improve therapeutic efficacy. For example, the data disclosed herein use protein blotting to show not only the co-expression of ROR1 and EGFR in TNBC (this application also contains data showing that the G3CPG4-hFc PNU conjugate has excellent in vivo efficacy in patient-derived TNBC xenograft models), but also high expression of EGFR and ROR1 in non-small cell lung cancer models. Co-expression of ROR1 and EGFR has also been found in large cell lung cancer, head and neck cancer, esophageal cancer, kidney cancer, gastric cancer, sarcoma, pancreatic cancer and colorectal cancer models. Thus, the surprisingly improved efficacy of the G3CPG4-hFc PNU conjugate compared to B1-hFc PNU in a patient-derived TNBC xenograft model, combined with the in vitro data disclosed herein, suggests that the presently claimed ROR1×EGFR bispecific antibody has therapeutic potential for the treatment of cancers identified herein as co-expressing ROR1 and EGFR.

[0270] The sequences of G3CP and G3CPG4 have two common single amino acid changes relative to the sequence of B1. They are both within CDR3 and are the following substitutions:

[0271] 1. Y residues replace W residues, and

[0272] 2. N residues replace L residues.

[0273] Compared to G3CP, G3CPG4 has additional single amino acid changes in each of CDR1, HV2, and HV4 relative to B1 (also present in B1 G4), and the changes are to humanize the framework regions (some of which are also present in B1V15, SEQ ID NO: 115, as shown in FIG. Figure 15 Shown - B1V15 has the same CDR1, HV2, HV4 and CDR3 sequences as B1, ie it is not a loop library variant; the changes in B1V15 relative to B1 are only in the framework regions).

[0274] Without being bound by theory, any improvements over B1 exhibited by both G3CP and G3CPG4 that are not exhibited by B1 G4 or B1 V15 are believed to be due to one or both of the two mutations they share in CDR3. Thus, the advantages of G3CP and G3CPG4 are believed to arise from a CDR3 comprising the sequence YPWGAGAPYNVQWY (SEQ ID NO: 23).

[0275] Without being bound by theory, the surprising advantage associated with YPWGAGAPYNVQWY (SEQ ID NO: 23) may represent a synergistic effect of the W to Y substitution and the L to N substitution. Alternatively, the surprising advantage may be primarily derived from the W to Y substitution and thus also shared by YPWGAGAPYLVQWY (SEQ ID NO: 20). 1B6, which has an L to N mutation and a CDR3 sequence of YPWGAGAPWNVQWY (SEQ ID NO: 24), has a lower elution volume than B1, so the L to N mutation in the CDR3 does lead to improved manufacturing and / or handling properties.

[0276] The EGFR specific antigen binding molecule can be any molecule combined with EGFR. Specifically, the EGFR specific antigen binding molecule can be selected from the group comprising: immunoglobulin, immunoglobulin Fab region, Fv, single-chain Fv (scFv), double antibody, three antibodies, four antibodies, VNAR domains, single domain antibodies (sdAb), VH domains or scaffold proteins (affibodies, centyrins, darpins etc.). Preferably, the EGFR specific antigen binding molecule is a single domain antibody (sdAb).

[0277] Cetuximab is an approved monoclonal antibody therapeutic that inhibits the epidermal growth factor receptor (EGFR). Cetuximab prevents EGF and other ligands from binding to EGFR and otherwise activating EGFR (i.e., preventing the extended receptor conformation required for high-affinity ligand binding and dimerization). Cetuximab binds to a specific epitope within the EGFR domain encompassing amino acids 384-408.

[0278] 7C12 and 7D12 are camelid single-domain antibodies (nanobodies) that compete for the cetuximab epitope on EGFR (see WO2007042289A2, the entire contents of which are incorporated herein by reference). Both 7C12 and 7D12 exhibit high-affinity EGFR binding (low nM KD) [Roovers 2011 Int J Cancer 129p2013, Gainkam 2010 Mol Imaging] and block EGF binding to EGFR [Schmitz 2013 Structure 21p1214]. 7C12 and 7D12 differ by five amino acids, and 7C12 has a higher off-rate for EGFR binding [Roovers 2011 Int J Cancer 129p2013].

[0279] EGFR#33 and EGFR#13 are low-affinity variants of the 7C12 nanobody [US2016 / 0251440]. EGFR#33 differs from 7C12 by 3 amino acids and has an affinity of 240 nM as reported by SPR. EGFR#13 differs from 7C12 by 2 amino acids and has an affinity of 2.5 uM as reported by SPR.

[0280] Matuzumab is another approved monoclonal antibody therapeutic that inhibits EGFR. Matuzumab binding sterically blocks the EGFR domain rearrangements required for high-affinity ligand binding and receptor dimerization. Matuzumab binds primarily to the loop preceding the C-terminal strand of the domain III β-helix (aa 454-464 of EGFR).

[0281] 9G8 is a sdAb (nanobody) sequence that competes for the EGFR epitope of matuzumab [WO2007042289A2], but it has a unique EGFR epitope that is closer to the N-terminus of EGFR domain III and away from the domain II ligand binding site, regions that are inaccessible to conventional antibodies [Schmitz 2013Structure 21p1214].

[0282] Examples of sdAbs for use in the bispecific antigen binding molecules of the first aspect of the present invention include, but are not limited to, molecules that compete for binding with cetuximab or matuzumab. Preferably, the sdAb is selected from the group comprising:

[0283] 7D12: QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSS (SEQ ID NO: 210)

[0284] 7C12:AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTQVTVSS (SEQ ID NO: 211)

[0285] 9G8: EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSS (SEQ ID NO: 212)

[0286] 38G7: EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYVMGWFRQATGKEREFVATIAWDSGSTYYADSVKGRFTISRDNAKNTVHLQMNSLKPEDTAVYYCAASYNVYYNNYYYPISRDEYDYWGQGTQVTVSS (SEQ ID NO: 213)

[0287] EGFR#33

[0288] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSS (SEQ ID NO: 214)

[0289] EGFR#13

[0290] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS (SEQ ID NO: 215)

[0291] Preferably, the sdAb is selected from the group consisting of 7D12, EGFR#33, EGFR#13, and 9G8.

[0292] EGFR specific antigen binding molecules can be derived from cetuximab or matuzumab, or compete with cetuximab or matuzumab.For example, EGFR specific antigen binding molecules can be derived from cetuximab or matuzumab or compete with cetuximab or matuzumab immunoglobulin, immunoglobulin Fab district, Fv, single-chain Fv (scFv), double antibody, three antibodies, four antibodies, VNAR domains, single domain antibodies (sdAb), VH domains or scaffold proteins (affibodies, centyrins, darpins etc.).EGFR specific antigen binding molecules can include the CDR of cetuximab or matuzumab.EGFR specific antigen binding molecules can include VH and / or VL domains of cetuximab or matuzumab.

[0293] The EGFR-specific antigen binding molecule may include cetuximab Fab or cetuximab-based scFv.

[0294] Cetuximab Fab

[0295] Fab LC

[0296] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO: 357)

[0297] Fab HC

[0298] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQ GTLVTVSAASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV(SEQID NO:358)

[0299] Cetuximab scFv

[0300] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAGGG GSGGGGSGGGGSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK(SEQ ID NO:359)

[0301] The EGFR-specific antigen-binding molecule may include a humanized cetuximab Fab or a humanized cetuximab-based scFv, for example:

[0302] Humanized Cetuximab Fab LC

[0303] DIQMTQSPSSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO: 360)

[0304] Humanized Cetuximab Fab HC

[0305] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV(SEQID NO:361)

[0306] Humanized cetuximab scFv

[0307] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGG SGGGGSGGGGSMDIQMTQSPSSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIK(SEQ ID NO: 362)

[0308] Wherein the EGFR-specific antigen-binding molecule comprises a Fab, the EGFR-specific antigen-binding molecule will typically comprise a FabLC and a FabHC. The FabHC can be fused to a fragment of the Fc region of an immunoglobulin. Typically, the FabLC and FabHC are associated via a disulfide bond. For example, the EGFR-specific antigen-binding molecule may comprise SEQ ID NO: 360 and SEQ ID NO: 361, wherein SEQ ID NO: 361 is fused to a fragment of the Fc region of an immunoglobulin, and wherein SEQ ID NO: 360 and SEQ ID NO: 361 are associated via a disulfide bond.

[0309] The EGFR-specific antigen binding molecule may include Matuzumab Fab or Matuzumab-based scFv.

[0310] The EGFR-specific antigen-binding molecule may include humanized Matuzumab Fab or humanized Matuzumab-based scFv.

[0311] Humanized Matuzumab Fab LC

[0312] DIQMTQSPSSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKAPKLLIYDTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFFTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE(SEQ IDNO: 381)

[0313] The humanized matuzumab Fab HC may be selected from SEQ ID NO: 382 or SEQ ID NO: 383, which differ by one amino acid:

[0314] Humanized Matuzumab Fab HC "3c08"

[0315] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLEWIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDGRYFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS(SEQ ID NO: 382)

[0316] Humanized Matuzumab Fab HC "3c09"

[0317] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLEWIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYAGRYFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS(SEQ ID NO: 383)

[0318] EGFR specific antigen binding molecules can be derived from panitumumab, nimotuzumab or necitoximb, or compete with panitumumab, nimotuzumab or necitoximb. For example, EGFR specific antigen binding molecules can be immunoglobulins, immunoglobulin Fab regions, Fv, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, VNAR domains, single-domain antibodies (sdAb), VH domains, or scaffold proteins (affibodies, centyrins, darpins, etc.) derived from panitumumab, nimotuzumab or necitoximb or compete with panitumumab, nimotuzumab or necitoximb. EGFR specific antigen binding molecules can include the CDRs of panitumumab, nimotuzumab or necitoximb. EGFR specific antigen binding molecules can include the VH and / or VL domains of panitumumab, nimotuzumab or necitoximb. EGFR-specific antigen-binding molecules may include panitumumab, nimotuzumab or nesimab Fab or scFv based on panitumumab, nimotuzumab or nesimab. EGFR-specific antigen-binding molecules may include humanized panitumumab, nimotuzumab or nesimab Fab, or humanized scFv based on panitumumab, nimotuzumab or nesimab.

[0319] Panitumumab Fab HC

[0320] QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQG TMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKC(SEQ ID NO: 384)

[0321] Panitumumab Fab LC

[0322] DIQMTQSPSSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO: 385)

[0323] Nimotuzumab Fab HC

[0324] QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVP(SEQ ID NO: 386)

[0325] Nimotuzumab Fab LC

[0326] DIQMTQSPSSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQI TREVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 387)

[0327] Alternatively, nimotuzumab may refer to an affinity-matured variant, such as the variant with improved activity reported in Tundidor 2020 Sci Reports 10: 1194, which is hereby incorporated by reference in its entirety.

[0328] Neximab Fab HC

[0329] QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYW GQGTLVTVSSASTKGPSVLPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV(SEQ ID NO: 388)

[0330] Neximab Fab LC

[0331] EIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQYGSTPLTFGGGTKAEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO: 389)

[0332] Alternatively, the EGFR-specific antigen binding molecule may comprise the sequence of any EGFR-specific antigen binding molecule disclosed herein, comprising:

[0333] (i) at least 85% identical thereto, and / or

[0334] (ii) one, two or three amino acid substitutions relative thereto.

[0335] The sequence identity is at least about 85% sequence identity, and thus can be at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity. Preferably the sequence identity is at least 90% or at least 95%.

[0336] One or more amino acid substitutions can be conservative amino acid substitutions. The term "conservative amino acid substitution" as used herein refers to an amino acid substitution in which an amino acid residue is substituted by another amino acid residue with a similar side chain. Amino acids with similar side chains tend to have similar properties, and therefore it can be expected that conservative substitutions of amino acids that are important for the structure or function of a polypeptide will have less impact on polypeptide structure / function than non-conservative amino acid substitutions at the same position. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine), non-polar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, conservative amino acid substitutions can be considered as substitutions of different amino acids in the same family with specific amino acid residues. However, substitutions of epitope residues may also be non-conservative substitutions, where one amino acid is replaced by another having a side chain belonging to a different family.

[0337] EGFR-specific antigen binding molecules can be humanized. At least 85% identity and / or one, two or three amino acid substitutions relative to the sequence of the EGFR-specific antigen binding molecules disclosed herein can comprise substitutions that humanize the EGFR-specific antigen binding molecules.

[0338] Preferably, the EGFR-specific antigen binding molecule selectively interacts with the EGFR protein with an affinity constant of about 1 to 2,000 nM or 2 to 2,000 nM, preferably 1 to 200 nM, and even more preferably 1 to 20 nM. The affinity constant can be around 2 nM. The affinity constant can be measured for ROR1-specific antigen binding molecules as described elsewhere herein, for example, by surface plasmon resonance (SPR) or biolayer interferometry (BLI).

[0339] It will be understood that the ROR1-specific antigen binding molecule and the EGFR-specific antigen binding molecule can be combined in any order to form the bispecific antigen binding molecule of the first aspect, i.e., the ROR1-specific antigen binding molecule can be located at the N-terminus of the EGFR-specific antigen binding molecule or vice versa, or when the bispecific antigen binding molecule is formed by knobs-into-holes, for example, the ROR1-specific antigen binding molecule can be on one arm and the EGFR-specific antigen binding molecule can be on the other arm or vice versa.

[0340] In addition, it should be understood that higher order constructs are also contemplated herein, such as constructs composed of multiple ROR1-specific antigen binding molecules and EGFR-specific antigen binding molecules. These may take the form of multiple copies of a single primary amino acid sequence, such as ROR1 binder-EGFR binder-ROR1 binder or EGFR binder-ROR1 binder-EGFR binder.

[0341] The bispecific antigen binding molecule of the first aspect may additionally include a linker region between the ROR1-specific antigen binding molecule and the EGFR-specific antigen binding molecule. Preferred linkers include but are not limited to [G4S] x , wherein x is 1, 2, 3, 4, 5, 5, 6, 7, 8, 9, or 10. Preferred linkers include [G4S]3, [G4S]5, and G4S. A preferred linker is [G4S]3. A preferred linker is G4S. A preferred linker is [G4S]5. Other linkers may include, but are not limited to, PGVQPSPGGGGS (SEQ ID NO: 89) (Wobbe-G4S), PGVQPAPGGGGS (SEQ ID NO: 90) (Wobbe-G4SGM). It will be appreciated that different combinations of different linkers may be combined in the same construct.

[0342] The bispecific antigen binding molecules of the first aspect can also include additional domains, which can be added in the form of N-terminal or C-terminal additions, or can be placed between the ROR1 specific antigen binding molecules and the EGFR specific antigen binding molecules in the amino acid sequence of the bispecific binding molecules. Each domain of the bispecific antigen binding molecules of the first aspect can be connected by a joint region as described above. Preferred additional domains include but are not limited to immunoglobulin, immunoglobulin Fc region, immunoglobulin Fab region, single-chain Fv (scFv), double antibodies, three antibodies, four antibodies, bispecific t cell adapter (BiTE), intein, VNAR domains, single domain antibodies (sdAb), VH domains or scaffold proteins (affibodies, centyrins, darpins etc.). Particularly preferred additional domains are immunoglobulin Fc regions, preferably human Fc regions.

[0343] Combinations specifically contemplated in this application include, but are not limited to, monovalent ROR1×EGFR (Fc fusion) bispecific antibodies; bivalent ROR1×EGFR (Fc fusion) bispecific antibodies; monovalent ROR1×EGFR (non-Fc) bispecific antibodies; bivalent ROR1×EGFR (non-Fc) bispecific antibodies; and monovalent ROR1, half-life extended ROR1×EGFR (non-Fc) bispecific antibodies.

[0344] The monovalent ROR1×EGFR (Fc fusion) bispecific antibody can be in the form of a ROR1 binder-Fc-EGFR binder or an EGFR binder-Fc-ROR1 binder.

[0345] The bivalent ROR1×EGFR (Fc fusion) bispecific antibody may be in a format selected from the group consisting of:

[0346] ROR1 binder-ROR1 binder-Fc-EGFR binder

[0347] ROR1 binder-EGFR binder-Fc-ROR1 binder

[0348] EGFR binder-ROR1 binder-Fc-ROR1 binder

[0349] EGFR binder-EGFR binder-Fc-ROR1 binder

[0350] EGFR binder-ROR1 binder-Fc-EGFR binder

[0351] ROR1 binder-EGFR binder-Fc-EGFR binder

[0352] EGFR binder-Fc-ROR1 binder-ROR1 binder

[0353] ROR1 binder-Fc-EGFR binder-EGFR binder

[0354] EGFR binder-Fc-ROR1 binder-EGFR binder

[0355] EGFR binder-Fc-EGFR binder-ROR1 binder

[0356] ROR1 binder-Fc-EGFR binder-ROR1 binder, and

[0357] ROR1 binder-Fc-ROR1 binder-EGFR binder.

[0358] The monovalent ROR1×EGFR (non-Fc) bispecific antibody can be in the form of a ROR1 binder-EGFR binder or an EGFR binder-ROR1 binder.

[0359] The bivalent ROR1×EGFR (non-Fc) bispecific antibody may take a format selected from the group consisting of:

[0360] ROR1 binder-ROR1 binder-EGFR binder

[0361] ROR1 binder-EGFR binder-ROR1 binder

[0362] EGFR binder-ROR1 binder-ROR1 binder

[0363] The monovalent ROR1, half-life extended ROR1×EGFR (non-Fc) bispecific antibody can take a format selected from the group consisting of:

[0364] ROR1 binder-BA11-EGFR binder

[0365] ROR1 binder-EGFR binder-BA11

[0366] BA11-ROR1 binder-EGFR binder

[0367] BA11-EGFR binder-ROR1 binder

[0368] EGFR binder-ROR1 binder-BA11

[0369] EGFR binder-BA11-ROR1 binder

[0370] The ROR1 binder can be any ROR1-specific antigen binding molecule disclosed herein. When there are two ROR1 binders, they can be the same ROR1-specific antigen binding molecule or two different ROR1-specific antigen binding molecules.

[0371] The EGFR binder can be any EGFR-specific antigen binding molecule disclosed herein. When there are two EGFR binders, they can be the same EGFR-specific antigen binding molecule or two different EGFR-specific antigen binding molecules.

[0372] The linker between the domains is preferably but not limited to (G4S) X , wherein X is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, PGVQPSPGGGGS (SEQ ID NO: 89) (Wobbe-G4S), PGVQPAPGGGGS (SEQ ID NO: 90) (Wobbe-G4SGM), and wherein different combinations of different linkers can be combined in the same construct.

[0373] Thus, an additional C-terminal (or N-terminal) tag sequence may or may not be present.

[0374] C-terminal tags include, but are not limited to, tags containing a polyhistidine sequence to facilitate purification (e.g., His6), tags containing a c-Myc sequence (e.g., EQKLISEEDL (SEQ ID NO: 112)) to enable detection, and / or tags containing a cysteine ​​residue to enable labeling and bioconjugation using thiol-reactive payloads and probes, and combinations thereof. Preferred C-terminal tags include, but are not limited to:

[0375] QASGAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 98)

[0376] QACGAHHHHHHGAEFEQKLISEEDL(SEQ ID NO:99)

[0377] QACKAHHHHHHGAEFEQKLISEEDL(SEQ ID NO:97)

[0378] AAAHHHHHGAEFEQKLISEDL (SEQ ID NO: 100)

[0379] ACAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 101)

[0380] QASGAHHHHHH (SEQ ID NO: 102)

[0381] QACGAHHHHHH (SEQ ID NO: 103)

[0382] QACKAHHHHHH (SEQ ID NO: 104)

[0383] AAAHHHHHH (SEQ ID NO: 105)

[0384] ACAHHHHHH (SEQ ID NO: 106)

[0385] QASGA (SEQ ID NO: 107)

[0386] QACGA (SEQ ID NO: 108)

[0387] QACKA (SEQ ID NO: 109)

[0388] ACA (SEQ ID NO: 110)

[0389] SAPSA (SEQ ID NO: 111)

[0390] The domain may also be combined with an Fc domain via N-terminal fusion, C-terminal fusion, or both N-terminal and C-terminal fusion, including but not limited to:

[0391] hIgG1

[0392] EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:216)

[0393] hIgG1(S239C)

[0394] EPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:217)

[0395] hIgG1(S442C)

[0396] EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:218)

[0397] hIgG1(S239C+S442C)

[0398] EPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:219)

[0399] in:

[0400] ·P3A1 is TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVN (SEQ ID NO: 206)

[0401] ·BA11 is TRVDQSPSSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIK (SEQ ID NO: 95)

[0402] It will be clear to those skilled in the relevant art that bispecific antigen binding molecules comprising the additional domains described herein may, in certain circumstances, include additional specificities beyond ROR1 and EGFR. Such configurations are also within the scope of the present invention.

[0403] According to a second aspect, the present invention provides a bispecific antigen binding molecule comprising:

[0404] (i) a receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen-binding molecule comprising the amino acid sequence represented by formula (I):

[0405] FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4(I)

[0406] in

[0407] CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of GTRYGLYS (SEQ ID NO: 25), GTRYGLYSS (SEQ ID NO: 26), DTRYALYS (SEQ ID NO: 27), DTRYALYSS (SEQ ID NO: 28), GTKYGLYA (SEQ ID NO: 29), GTKYGLYAS (SEQ ID NO: 30), and DTSYGLYS (SEQ ID NO: 207);

[0408] FW1 is the framework region;

[0409] FW2 is the framework region;

[0410] HV2 is a hypervariable sequence having an amino acid sequence selected from the group consisting of SSDEERISIS (SEQ ID NO: 31), STDEERISIG (SEQ ID NO: 32), SPNKDRMIIG (SEQ ID NO: 33), STDKERIIIG (SEQ ID NO: 34), and TTDWERMSIG (SEQ ID NO: 208);

[0411] FW3a is the framework region;

[0412] HV4 is a hypervariable sequence having an amino acid sequence selected from the group consisting of NKGTK (SEQ ID NO: 35), NKGSK (SEQ ID NO: 36), NNGTK (SEQ ID NO: 37), NNRSK (SEQ ID NO: 38), and NKGAK (SEQ ID NO: 209);

[0413] FW3b is the framework region;

[0414] CDR3 is a CDR sequence having an amino acid sequence according to REARHPWLRQWY (SEQ ID NO: 39);

[0415] FW4 is the framework region; and

[0416] (ii) an epidermal growth factor receptor (EGFR)-specific antigen-binding molecule,

[0417] wherein when CDR1 is DTSYGLYS (SEQ ID NO: 207) and / or HV2 is TTDWERMSIG (SEQ ID NO: 208) and / or HV4 is NKGAK (SEQ ID NO: 209), the ROR1-specific antigen binding molecule is fused to a first fragment of the immunoglobulin Fc region, the EGFR-specific binding molecule is fused to a second fragment of the immunoglobulin Fc region, and the first fragment of the immunoglobulin Fc region and the second fragment of the immunoglobulin Fc region are engineered to dimerize.

[0418] The ROR1-specific antigen binding molecule can be fused to a first fragment of an immunoglobulin Fc region, the EGFR-specific binding molecule can be fused to a second fragment of an immunoglobulin Fc region, and the first fragment of the immunoglobulin Fc region and the second fragment of the immunoglobulin Fc region can be engineered to dimerize. As used herein, the terms "first fragment" and "second fragment" are interchangeable.

[0419] In a preferred embodiment, the ROR1-specific antigen binding molecule comprises an amino acid sequence selected from the group consisting of:

[0420] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVN (SEQ ID NO: 206), referred to herein as P3A1;

[0421] TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 77), referred to herein as P3A1 G1AE3;

[0422] TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 78), referred to herein as P3A1G1AE3.S;

[0423] TRVDQSPSSLSASVGDRVTITCVLTDTRYALYSTYWYRKNPGSTDEERISIGGRYSESVNKGSKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 79), referred to herein as P3A1G1NAC6;

[0424] TRVDQSPSSLSASVGDRVTITCVLTDTRYALYSSTYWYRKNPGSTDEERISIGGRYSESVNKGSKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 80), referred to herein as P3A1G1NAC6.S;

[0425] TRVDQSPSSLSASVGDRVTITCVLTGTKYGLYATYWYRKNPGSPNKDRMIIGGRYSESVNNGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 81), referred to herein as P3A1G1NAG8;

[0426] TRVDQSPSSLSASVGDRVTITCVLTGTKYGLYASTYWYRKNPGSPNKDRMIIGGRYSESVNNGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 82), referred to herein as P3A1G1NAG8.S;

[0427] TRVDQSPSSLSASVGDRVTITCVLTGTKYGLYASTYWYRKNPGSTDKERIIIGGRYSESVNNRSKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 83), referred to herein as P3A1G1AF7.S;

[0428] or a functional variant having a CDR1, HV2, HV4 and CDR2 sequence according to any of the sequences therein and having a FW1, FW2, FW3a, FW3b and FW4 sequence having at least 45% combined sequence identity to the combined FW1, FW2, FW3a, FW3b and FW4 sequence of any of the sequences therein.

[0429] Specific advantages associated with affinity matured variants of P3A1G1 and functional variants thereof include improved binding to hROR1-Fc compared to the parent P3A1G1, as shown in the Examples.

[0430]

[0431]

[0432]

[0433]

[0434]

[0435] The sequence identity associated with the molecules of the present invention can be judged at the level of individual CDRs, HVs or FWs, combined CDRs, HVs or FWs, or can be judged over the entire length of the molecule. The described CDR, HV and FW sequences can also be longer or shorter, whether by adding or deleting amino acids at the N-terminus or C-terminus of the sequence, or by inserting or deleting amino acids in the sequence.

[0436] The length of the framework region FW1 is preferably 20 to 28 amino acids, more preferably 22 to 26 amino acids, and even more preferably 23 to 25 amino acids. In certain preferred embodiments, the length of FW1 is 26 amino acids. In other preferred embodiments, the length of FW1 is 25 amino acids. In still other preferred embodiments, the length of FW1 is 24 amino acids.

[0437] In alternative definitions, the length of the CDR region CDR1 is preferably 7 to 11 amino acids, more preferably 8 to 10 amino acids in length. In certain preferred embodiments, the length of CDR1 is 9 amino acids. In other preferred embodiments, the length of CDR1 is 8 amino acids.

[0438] The framework region FW2 is preferably 6 to 14 amino acids in length, more preferably 8 to 12 amino acids in length. In certain preferred embodiments, FW2 is 12 amino acids in length. In other preferred embodiments, FW2 is 10 amino acids in length. In other preferred embodiments, FW2 is 9 amino acids in length. In other preferred embodiments, FW2 is 8 amino acids in length.

[0439] In alternative definitions, the length of the hypervariable sequence HV2 is preferably 4 to 11 amino acids, more preferably 5 to 10 amino acids in length. In certain preferred embodiments, the length of HV2 is 10 amino acids. In certain preferred embodiments, the length of HV2 is 9 amino acids. In other preferred embodiments, the length of HV2 is 6 amino acids.

[0440] The framework region FW3a is preferably 6 to 10 amino acids in length, more preferably 7 to 9 amino acids in length. In certain preferred embodiments, FW3a is 8 amino acids in length. In certain preferred embodiments, FW3a is 7 amino acids in length.

[0441] In alternative definitions, the length of the hypervariable sequence HV4 is preferably 3 to 7 amino acids, more preferably 4 to 6 amino acids. In certain preferred embodiments, the length of HV4 is 5 amino acids. In other preferred embodiments, the length of HV4 is 4 amino acids.

[0442] The length of the framework region FW3b is preferably 17 to 24 amino acids, more preferably 18 to 23 amino acids, and even more preferably 19 to 22 amino acids. In certain preferred embodiments, the length of FW3b is 21 amino acids. In other preferred embodiments, the length of FW3b is 20 amino acids.

[0443] In alternative definitions, the length of the CDR region CDR3 is preferably 8 to 21 amino acids, more preferably 9 to 20 amino acids, and even more preferably 10 to 19 amino acids. In certain preferred embodiments, the length of CDR3 is 17 amino acids. In other preferred embodiments, the length of CDR3 is 14 amino acids. In still other preferred embodiments, the length of CDR3 is 12 amino acids. In yet other preferred embodiments, the length of CDR3 is 10 amino acids.

[0444] The length of the framework region FW4 is preferably 7 to 14 amino acids, more preferably 8 to 13 amino acids, and even more preferably 9 to 12 amino acids. In certain preferred embodiments, the length of FW4 is 12 amino acids. In other preferred embodiments, the length of FW4 is 11 amino acids. In still other preferred embodiments, the length of FW4 is 10 amino acids. In yet other preferred embodiments, the length of FW4 is 9 amino acids.

[0445] In one embodiment of the ROR1-specific antigen binding molecule:

[0446] FW1 is a framework region of 20 to 28 amino acids;

[0447] FW2 is a framework region of 6 to 14 amino acids;

[0448] FW3a is a framework region of 6 to 10 amino acids;

[0449] FW3b is a framework region of 17 to 24 amino acids; and / or

[0450] FW4 is a framework region of 7 to 14 amino acids.

[0451] In one embodiment of the ROR1-specific antigen binding molecule:

[0452] FW1 has an amino acid sequence selected from the group consisting of: ASVNQTPRTATKETGESLTINCVVT(SEQ

[0453] ID NO: 40), TRVDQSPSSLSASVGDRVTITCVLT (SEQ ID NO: 41) and

[0454] ASVTQSPRSASKETGESLTITCRVT (SEQ ID NO: 42), or a functional variant of any sequence therein having at least 45% sequence identity;

[0455] FW2 has an amino acid sequence according to TYWYRKNPG (SEQ ID NO: 43), or any sequence thereof having at least 45%

[0456] functional variants of sequence identity;

[0457] FW3a has an amino acid sequence selected from the group consisting of GRYVESV (SEQ ID NO: 44) and

[0458] GRYSESV (SEQ ID NO: 45), or a functional variant of any sequence therein having at least 45% sequence identity;

[0459] FW3b has an amino acid sequence selected from the group consisting of SFSLRIKDLTVADSATYYCKA (SEQ ID NO: 84), SFTLTISSLQPEDSATYYCRA (SEQ ID NO: 46) and SFSLRISSLTVEDSATYYCKA (SEQ ID NO: 47), or a functional variant of any thereof having at least 45% sequence identity;

[0460] and / or

[0461] FW4 has an amino acid sequence selected from the group consisting of DGAGTVLTVN (SEQ ID NO: 48), DGAGTKVEIK (SEQ ID NO: 49) or DGQGTKLEVK (SEQ ID NO: 85), or a functional variant of any thereof having at least 45% sequence identity.

[0462] The ROR1-specific antigen binding molecules can be humanized. The ROR1-specific antigen binding molecules can be deimmunized. The B1 loop variants on the humanized backbone G4 and V15 described herein are humanized. Since P3A1G1 has been humanized, all loop variants of P3A1G1 are humanized. Examples of humanized sequences of the present invention include, but are not limited to:

[0463] B1G4

[0464] G3CP G4

[0465] G3CP V15

[0466] 1H8 G4

[0467] 1H8 V15

[0468] C3CP G4

[0469] C3CPV15

[0470] P3A1 G1 AE3

[0471] P3A1 G1 AE3.S

[0472] P3A1 G1 NAC6

[0473] P3A1 G1 NAC6.S

[0474] P3A1 G1 NAG8

[0475] P3A1 G1 NAG8.S

[0476] P3A1 G1 AF7.S.

[0477] A skilled artisan will appreciate that the humanized ROR1 -specific antigen binding molecules described herein can be further humanized, for example, by substituting amino acids from DPK-9 for additional FW region amino acids.

[0478] The ROR1-specific antigen binding molecules can also be conjugated to detectable labels, dyes, toxins, drugs, prodrugs, radionuclides, or biologically active molecules.

[0479] Preferably, the ROR1-specific antigen binding molecule does not bind to receptor tyrosine kinase-like orphan receptor 2 (ROR2). More preferably, the ROR1-specific antigen binding molecule binds to human ROR1 and murine ROR1 (mROR1). Even more preferably, the ROR1-specific antigen binding molecule binds to deglycosylated ROR1.

[0480] Certain ROR1-specific antigen-binding molecules of the present invention may not bind to a linear peptide sequence selected from the following:

[0481] YMESLHMQGEIENQI(SEQ ID NO:91)

[0482] CQPWNSQYPHTHTFTALRFP (SEQ ID NO: 92)

[0483] RSTIYGSRLRIRNLDTTDTGYFQ(SEQ ID NO:93)

[0484] QCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYE (SEQ ID NO: 94).

[0485] Preferably, the ROR1-specific antigen binding molecule selectively interacts with the ROR1 protein with an affinity constant of about 0.01 nM to 50 nM, preferably 0.1 nM to 30 nM, and even more preferably 0.1 nM to 10 nM. The affinity constant can be measured by biolayer interferometry (BLI). For monomers, the interaction is 1:1. For the VNAR-hFc format, the inventors used two approaches. One of them is that ROR1 is immobilized, so that when the avidity effect comes into play, the bivalent VNAR-hFc has a significant K D Another approach is to use a 1:1 format where VNAR-hFc is immobilized and ROR1 is flowed over the surface, giving a K of “true” 1:1 binding. D In general, the affinity constants used herein refer to those measured by biolayer interferometry (BLI) using a 1:1 binding format. By this method, for example, G3CP and G3CP G4 are in the range of 0.1 to 10 nM. The K values ​​of the exemplary P3A1 G1 loop variants are D The values ​​were 5.0 nM (AE3), 13.8 nM (NAC6), and 12.2 nM (NAG8).

[0486] Alternatively, the antigen binding molecule specifically binds to ROR1 with an affinity determined by SPR (e.g., under SPR conditions disclosed herein). Similarly, the antigen binding molecule can specifically bind to EGFR with an affinity determined by SPR (e.g., under SPR conditions disclosed herein). Such binding measurements can be performed using a variety of binding assays known in the art, for example, using surface plasmon resonance (SPR), such as by Biacore. TM Or use ProteOn XPR36 TM use (Sapidyne Instruments, Inc) or using bio-layer interferometry (BLI) such as the Octet system (Sartorius).

[0487] Binding capacity, specificity and affinity (K D 、k off and / or k on ) can be determined by any conventional method in the art, such as by surface plasmon resonance (SPR) or biolayer interferometry (BLI). The term "k" as used herein on ” or “k a ” refers to the association constant. The term “k d ” or “k off ” refers to the dissociation constant. The term “K D " refers to the equilibrium dissociation constant for a particular antibody-antigen interaction. Such binding measurements can be performed using a variety of binding assays known in the art, for example using surface plasmon resonance (SPR), such as by Biacore TM Or use ProteOnXPR36 TM use (Sapidyne Instruments, Inc) or BLI using the Octet system (Sartorius).

[0488] In one embodiment, surface plasmon resonance (SPR) is performed at 25° C. In another embodiment, SPR is performed at 37° C.

[0489] In one embodiment, SPR is performed at physiological pH (eg, about pH 7) or at pH 7.6 (eg, using Hepes buffered saline (also known as HBS-EP) at pH 7.6).

[0490] In one embodiment, SPR is performed at physiological salt levels (eg, 150 mM NaCl).

[0491] In one embodiment, SPR is performed at a detergent level of no more than 0.05% by volume, such as in the presence of 0.05% P20 (polysorbate 20; e.g., Tween-20 TM ) and 3 mM EDTA.

[0492] In one example, SPR is performed in a buffer (pH 7.6, 150 mM NaCl, 0.05% detergent (e.g., P20), and 3 mM EDTA) at 25° C. or 37° C. The buffer may contain 10 mM Hepes. In one example, SPR is performed in HBS-EP at 25° C. or 37° C. HBS-EP is available from Teknova Inc (California; catalog number H8022).

[0493] In one example, the affinity of the ROR1×EGFR bispecific antibody to the target antigen is determined using SPR as follows:

[0494] 1. Coupling the target antigen (e.g., ROR1 or EGFR) to a biosensor chip (e.g., GLM chip) (e.g., via primary amine coupling). Alternatively, the target antigen can be indirectly coupled to the biosensor chip via an initial anti-tag IgG capture step (e.g., an appropriate anti-Fc IgG).

[0495] 2. The test ROR1×EGFR bispecific antibody was passed over the capture surface of the chip at 1024 nM, 256 nM, 64 nM, 16 nM, 4 nM, and 0 nM (i.e., buffer alone);

[0496] 3. Determine the binding affinity of the test ROR1×EGFR bispecific antibody to the target antigen using surface plasmon resonance, for example, under the above-mentioned SPR conditions (e.g., in physiological buffer at 25°C). SPR can be performed using any standard SPR instrument such as Biacore TM Or use ProteOn XPR36 TM conduct.

[0497] Alternatively, the test ROR1×EGFR bispecific antibody can be coupled to a biosensor chip directly (e.g., primary amine coupling) or indirectly through an initial anti-tag IgG capture step (e.g., anti-hFc IgG), and the target antigen (e.g., ROR1 or EGFR) passed over the capture surface of the chip.

[0498] Regeneration of the capture surface can be performed using 10 mM glycine at pH 1.7. This will remove the captured antibody and allow the surface to be used for another interaction. Binding data can be fitted to a 1:1 model using standard techniques, for example, using ProteOn XPR36TM Analyze the models inherent in the software.

[0499] Alternatively, affinity is determined using the OctetB LI system (Sartorius) using the BLI method. ROR1 or EGFR ligands are attached to the biosensor by standard amine coupling (ARG2 biosensors) or affinity capture (e.g., using anti-human Fc capture using AHC biosensors or anti-His capture using HIS1K biosensors). The sensor is immersed in the test analyte, and the binding affinity is calculated based on the association and dissociation rates of the analyte.

[0500] In addition, the ROR1-specific antigen-binding molecule is preferably capable of mediating the killing of tumor cells expressing ROR1 or inhibiting the proliferation of cancer cells.

[0501] The ROR1-specific antigen-binding molecule can also be internalized after binding to ROR1. In other embodiments, the ROR1-specific antigen-binding molecule may not be internalized after binding to ROR1.

[0502] According to a third aspect, the present invention provides a recombinant fusion protein comprising a bispecific antigen binding molecule according to the first or second aspect of the invention.

[0503] Preferably, in the recombinant fusion protein of the third aspect, the ROR1-specific antigen binding molecule and / or the EGFR-specific antigen binding molecule are fused to one or more biologically active proteins. The specific antigen binding molecule can be fused to one or more biologically active proteins via one or more linker domains. Preferred linkers include, but are not limited to, [G4S] x , wherein x is 1, 2, 3, 4, 5, or 6. Particularly preferred linkers are G4S (SEQ ID NO: 222), [G4S]3 (SEQ ID NO: 86), and [G4S]5 (SEQ ID NO: 87). Other preferred linkers include the sequences PGVQPSP (SEQ ID NO: 88), PGVQPSPGGGGS (SEQ ID NO: 89), and PGVQPAPGGGGS (SEQ ID NO: 90). These linkers may be particularly useful when the recombinant fusion protein is expressed in different expression systems with different glycosylation patterns (e.g., CHO and insect) and in expression systems that do not glycosylate the expressed protein (e.g., E. coli). Any recombinant fusion protein sequence disclosed herein comprising a [G4S]3 linker may alternatively have any other linker sequence disclosed herein.

[0504] It should also be understood that the fusion proteins of the present invention can be constructed in any order, i.e., the ROR1-specific antigen binding molecule can be located at the N-terminus, the C-terminus, or not at any terminus (e.g., in the middle of a longer amino acid sequence). The EGFR-specific antigen binding molecule can be located at the N-terminus, the C-terminus, or not at any terminus (e.g., in the middle of a longer amino acid sequence).

[0505] Preferred biologically active proteins include, but are not limited to, immunoglobulins, immunoglobulin Fc regions, fragments of immunoglobulin Fc regions, Fc heavy chains, CH2 regions, CH3 regions, immunoglobulin Fab regions, Fab', Fv, Fv-Fc, single-chain Fv (scFv), scFv-Fc, (scFv) 2, diabodies, triabodies, tetrabodies, bispecific t cell engagers (engager), intein, VNAR domains, single domain antibodies (sdAb), VH domains or scaffold proteins (affibodies, centyrins, darpins, etc.). Particularly preferred biologically active proteins are immunoglobulin Fc regions. Other preferred fusion proteins include VNAR-VNAR and VNAR-VNAR-VNAR.

[0506] In one embodiment, the at least one biologically active protein is an immunoglobulin Fc region.

[0507] The recombinant fusion protein may comprise a ROR1-specific antigen binding molecule fused to an immunoglobulin Fc region or a fragment thereof. The immunoglobulin Fc region or a fragment thereof may be fused to the ROR1-specific antigen binding molecule via a linker. The immunoglobulin Fc region or a fragment thereof and / or a linker may be fused to the C-terminus of the ROR1-specific antigen binding molecule.

[0508] Thus, the recombinant fusion protein may comprise a sequence according to SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185 or SEQ ID NO: 223.

[0509] G3CP-hFc

[0510] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 186)

[0511] G3CPG4-hFc

[0512] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGSGGGSGGGSEGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 187)

[0513] 1H8-hFc

[0514] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:183)

[0515] 1H8 G4-hFc

[0516] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:184)

[0517] 1H8 V15-hFc

[0518] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 185)

[0519] P3A1-hFc

[0520] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 223)

[0521] The recombinant fusion protein may comprise a sequence according to SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 183 or SEQ ID NO: 223.

[0522] The recombinant fusion protein may comprise a sequence according to SEQ ID NO: 186, SEQ ID NO: 187 or SEQ ID NO: 183.

[0523] In a further embodiment, the at least one biologically active protein is an immunoglobulin Fc region further modified to comprise an S to C mutation.

[0524] The S to C mutation may be at position S239 (EU numbering). Thus, the recombinant fusion protein may comprise a sequence according to SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181 or SEQ ID NO: 182 or SEQ ID NO: 224.

[0525] G3CP-hFc(S239C)

[0526] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:178)

[0527] G3CPG4-hFc(S239C)

[0528] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:179)

[0529] 1H8-hFc(S239C)

[0530] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:180)

[0531] 1H8 G4-hFc(S239C)

[0532] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:181)

[0533] 1H8 V15-hFc(S239C)

[0534] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:182)

[0535] P3A1-hFc(S239C)

[0536] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:224)

[0537] The recombinant fusion protein may comprise a sequence according to SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 224.

[0538] The recombinant fusion protein may comprise a sequence according to SEQ ID NO: 178, SEQ ID NO: 179 or SEQ ID NO: 180.

[0539] The S to C mutation may be at position S442 (EU numbering).Thus, the recombinant fusion protein may comprise a sequence according to SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228 or SEQ ID NO: 229 or SEQ ID NO: 230.

[0540] G3CP-hFc(S442C)

[0541] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:225)

[0542] G3CPG4-hFc(S442C)

[0543] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:226)

[0544] 1H8-hFc(S442C)

[0545] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:227)

[0546] 1H8 G4-hFc(S442C)

[0547] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:228)

[0548] 1H8 V15-hFc(S442C)

[0549] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:229)

[0550] P3A1-hFc(S442C)

[0551] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:230)

[0552] The recombinant fusion protein may comprise a sequence according to SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227 or SEQ ID NO: 230.

[0553] The recombinant fusion protein may comprise a sequence according to SEQ ID NO: 225, SEQ ID NO: 226 or SEQ ID NO: 227.

[0554] The S to C mutation can occur simultaneously at positions S239 and S442 (EU numbering). Thus, the recombinant fusion protein can comprise a sequence according to SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, or SEQ ID NO: 235, or SEQ ID NO: 236.

[0555] G3CP-hFc(S239C&S442C)

[0556] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:231)

[0557] G3CPG4-hFc(S239C&S442C)

[0558] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:232)

[0559] 1H8-hFc(S239C&S442C)

[0560] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:233)

[0561] 1H8 G4-hFc(S239C&S442C)

[0562] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:234)

[0563] 1H8 V15-hFc(S239C&S442C)

[0564] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:235)

[0565] P3A1-hFc(S239C&S442C)

[0566] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:236)

[0567] The recombinant fusion protein may comprise a sequence according to SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233 or SEQ ID NO:236.

[0568] The recombinant fusion protein may comprise a sequence according to SEQ ID NO:231, SEQ ID NO:232 or SEQ ID NO:233.

[0569] The recombinant fusion protein can comprise an EGFR-specific antigen binding molecule fused to an immunoglobulin Fc region or its fragment. The immunoglobulin Fc region or its fragment can be fused to the EGFR-specific antigen binding molecule through a linker. The immunoglobulin Fc region or its fragment and / or linker can be fused to the C-terminus of the EGFR-specific antigen binding molecule.

[0570] Thus, the EGFR-specific antigen binding molecule fused to an immunoglobulin Fc region may comprise a sequence according to any one of SEQ ID NOs: 237 to 252. Thus, the EGFR-specific antigen binding molecule fused to an immunoglobulin Fc region may comprise a sequence according to any one of SEQ ID NOs: 237 to 240. Thus, the EGFR-specific antigen binding molecule fused to an immunoglobulin Fc region may comprise a sequence according to any one of SEQ ID NOs: 241 to 244. Thus, the EGFR-specific antigen binding molecule fused to an immunoglobulin Fc region may comprise a sequence according to any one of SEQ ID NOs: 245 to 248. Thus, the EGFR-specific antigen binding molecule fused to an immunoglobulin Fc region may comprise a sequence according to any one of SEQ ID NOs: 249 to 252.

[0571] 7D12-hFc

[0572] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:237)

[0573] EGFR#33-hFc

[0574] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:238)

[0575] EGFR#13-hFc

[0576] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:239)

[0577] 9G8-hFc

[0578] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:240)

[0579] 7D12-hFc(S239C)

[0580] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:241)

[0581] EGFR#33-hFc(S239C)

[0582] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:242)

[0583] EGFR#13-hFc(S239C)

[0584] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:243)

[0585] 9G8-hFc(S239C)

[0586] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:244)

[0587] 7D12-hFc(S442C)

[0588] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:245)

[0589] EGFR#33-hFc(S442C)

[0590] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:246)

[0591] EGFR#13-hFc(S442C)

[0592] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:247)

[0593] 9G8-hFc(S442C)

[0594] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:248)

[0595] 7D12-hFc(S239C&S442C)

[0596] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:249)

[0597] EGFR#33-hFc(S239C&S442C)

[0598] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:250)

[0599] EGFR#13-hFc(S239C&S442C)

[0600] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:251)

[0601] 9G8-hFc(S239C&S442C)

[0602] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:252)

[0603] The EGFR-specific antigen-binding molecule fused to the Fc region of an immunoglobulin may include cetuximab Fab or a cetuximab-based scFv. Cetuximab Fab or a cetuximab-based scFv may be any one of SEQ ID NOs: 357 to 359. Cetuximab Fab or a cetuximab-based scFv may be a humanized sequence of any one of SEQ ID NOs: 360 to 362. For example, for the humanized cetuximab sequence, the EGFR-specific antigen-binding molecule fused to the Fc region of an immunoglobulin may comprise a sequence according to any one of SEQ ID NOs: 360, 363 to 380, and 390 to 407.

[0604] Humanized Cetuximab Fab LC

[0605] DIQMTQSPSSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO: 360)

[0606] Humanized Cetuximab Fab HC hFc (S239C)

[0607] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:363)

[0608] Humanized cetuximab scFv hFc (S239C)

[0609] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:364)

[0610] Humanized Cetuximab Fab HC hFc (S239C+S442C)

[0611] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:365)

[0612] Humanized cetuximab scFv hFc (S239C+S442C)

[0613] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:366)

[0614] Humanized Cetuximab Fab HC hFc(S442C)

[0615] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:367)

[0616] Humanized cetuximab scFv hFc (S442C)

[0617] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSS GGGGSGGGGSGGGGSMDIQMTQSPSSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKV EIKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 368)

[0618] Where the EGFR-specific antigen-binding molecule comprises a Fab, the EGFR-specific antigen-binding molecule will typically comprise a FabLC and a FabHC. The FabHC can be fused to a fragment of the immunoglobulin Fc region, such as SEQ ID NOs: 363, 365, and 367. Typically, the FabLC and FabHC are associated via a disulfide bond. For example, the EGFR-specific antigen-binding molecule may comprise SEQ ID NO: 360 and SEQ ID NO: 363, wherein SEQ ID NO: 360 and SEQ ID NO: 363 are associated via a disulfide bond.

[0619] In one embodiment, the at least one biologically active protein is a fragment of an immunoglobulin Fc region selected from the group consisting of an Fc heavy chain, a CH2 region, and a CH3 region.

[0620] In one embodiment, the fragment of the immunoglobulin Fc region is an Fc heavy chain.

[0621] In one embodiment, one or more residues of the fusion protein comprise one or more amino acid substitutions suitable for conjugation.The one or more residues suitable for conjugation may be residues of a fragment of an immunoglobulin Fc region.

[0622] Any part of the fusion protein of the present invention can be engineered to be able to be conjugated. In a preferred example, when using an immunoglobulin Fc region, it can be engineered to include a cysteine ​​residue as a conjugation site. The cysteine ​​residues preferably introduced include but are not limited to S252C and S473C (Kabat numbering), which correspond to S239C and S442C in EU numbering, respectively. In some embodiments, any fusion protein disclosed herein may include an S239C point mutation. In some embodiments, any fusion protein disclosed herein may include an S442C point mutation. In some embodiments, any fusion protein disclosed herein may include an S239C and S442C point mutation. It is clearly contemplated herein that the sequence of any fusion protein disclosed herein may be modified to include an S239C and / or S442C point mutation.

[0623] According to a third aspect, a recombinant fusion comprising a plurality of VNAR domains is provided. Therefore, the recombinant fusion of the present invention can be a dimer, trimer or a more advanced multimer of VNAR. In this type of recombinant fusion, the specificity of each VNAR can be the same or different. The recombinant fusion of the present invention includes but is not limited to bispecific or trispecific molecules, in which each VNAR domain is combined with different antigens or with different epitopes (biparatope binders) on a single antigen. The term "biparatope" as used herein is intended to encompass molecules that are combined with multiple epitopes on a given antigen. This paper also contemplates molecules that are combined with three or more epitopes on a given antigen, and when using the term "biparatope", it should be understood that the possibility of a triparatope or multiparatope molecule is also encompassed.

[0624] According to a third aspect, a recombinant fusion is provided, comprising the ROR1-specific antigen binding molecule of the first aspect and a humanized VNAR domain. The humanized VNAR domain may be referred to as a soloMER and includes, but is not limited to, VNARBA11, a humanized VNAR that binds to human serum albumin with high affinity.

[0625] Examples of biparatopic and multivalent fusion proteins include, but are not limited to:

[0626] B1-G3CP

[0627] ·G3CP-BA11

[0628] BA11-G3CP

[0629] 1H8-BA11

[0630] BA11-1H8

[0631] G3CP V15-BA11

[0632] G3CP G4-BA11

[0633] B1-G3CP Cys

[0634] G3CP-BA11 Cys

[0635] BA11-G3CP Cys

[0636] 1H8-BA11 Cys

[0637] ·BA11-1H8 Cys

[0638] ·G3CP V15-BA11 Cys

[0639] G3CP G4-BA11 Cys

[0640] ·P3A1G1AE3-(L2)-G3CPG4

[0641] ·G3CPG4(L2)--P3A1G1AE3

[0642] ·P3A1G1AE3-(L2)-G3CPG4 Cys

[0643] ·G3CPG4-(L2)-P3A1G1AE3 Cys

[0644] ·P3A1-(L2)-BA11-(L2)-G3CP

[0645] ·P3A1-(L2)-G3CP-(L2)-BA11

[0646] BA11-(L2)-G3CP-(L2)-P3A1

[0647] BA11-(L2)-P3A1-(L2)-G3CP

[0648] ·P3A1-(L2)-BA11-(L2)-1H8

[0649] BA11-(L2)-P3A1-(L2)-1H8

[0650] ·P3A1-(L2)-BA11-(L2)-G3CP Cys

[0651] ·P3A1-(L2)-G3CP-(L2)-BA11 Cys

[0652] BA11-(L2)-G3CP-(L2)-P3A1 Cys

[0653] ·BA11-(L2)-P3A1-(L2)-G3CP Cys

[0654] ·P3A1-(L2)-BA11-(L2)-1H8 Cys

[0655] ·BA11-(L2)-1P3A1-(L2)-1H8 Cys

[0656] Where:

[0657] G3CP is

[0658] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVN(SEQ ID NO: 50)

[0659] 1H8 is

[0660] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVN(SEQ ID NO: 61)

[0661] G3CP G4 is

[0662] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIK(SEQ ID NO: 71)

[0663] G3CP V15 is

[0664] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPYNVQWYDGQGTKLEVK(SEQ ID NO: 72)

[0665] BA11 is TRVDQSPSSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIK (SEQ ID NO: 95)

[0666] P3A1 G1 AE3 is

[0667] TRVDQSPSSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK(SEQ ID NO: 77)

[0668] as well as

[0669] When no linker is defined, (-) corresponds to the linker Wobbe-G5S, which is PGVQPSPGGGGGS (SEQ ID NO: 96),

[0670] -(L2)- corresponds to the linker Wobbe-G4S-GM, which is PGVQPAPGGGGS (SEQ ID NO: 90).

[0671] Cys—corresponds to a Cys containing C-terminal tag—eg, QACKAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 97).

[0672] Recombinant biparatopic fusion protein dimers can also be prepared by fusing any recombinant fusion protein disclosed herein (particularly a ROR1-specific antigen binding molecule disclosed herein) to one arm of an Fc fusion and fusing an epidermal growth factor receptor (EGFR)-specific antigen binding molecule to the other arm.

[0673] In certain embodiments, the specific binding molecules or recombinant fusions of the present invention can be expressed with an N-terminal tag or a C-terminal tag to assist in purification. Examples include, but are not limited to, His6 and / or Myc. In addition, the N-terminal tag or the C-terminal tag can be further engineered to include additional cysteine ​​residues as conjugation sites. Therefore, it should be understood that the specific binding molecules or recombinant fusions mentioned in all aspects of the present invention are also intended to encompass such molecules with various N-terminal tags or C-terminal tags, which may also include additional cysteines for conjugation.

[0674] Other recombinant fusions are listed below. It should be understood that not every combination of linker and VNAR or fusion partner is listed below. However, all such combinations are clearly encompassed within the present invention.

[0675]

[0676]

[0677] The linkers between the VNAR domains are preferably, but not limited to, (G4S)5 (SEQ ID NO: 87), (G4S)3 (SEQ ID NO: 86), (G4S)7 (SEQ ID NO: 116), PGVQPSPGGGGS (SEQ ID NO: 89) (Wobbe-G4S), PGVQPAPGGGGS (SEQ ID NO: 90) (Wobbe-G4S GM), PGVQPCPGGGGGS (SEQ ID NO: 177) (WobbeCys-G5S), PGVQPCPGGGGS (SEQ ID NO: 432) (WobbeCys-G4S), and different combinations of different linkers can be combined in the same construct. The WobbeCys-G4S sequence also contains a single cysteine ​​residue to facilitate site-selective bioconjugation of the payload to the protein. In this linker, a thiol-mediated chemical coupling strategy is used. The use of this linker sequence for bioconjugation is advantageous because reoxidation and blocking of reduced cysteines are minimized, resulting in high-yield conversion of proteins to the corresponding conjugates in the bioconjugation reaction.

[0678] An additional C-terminal (or N-terminal) tag sequence may or may not be present.

[0679] C-terminal tags include, but are not limited to, tags containing a polyhistidine sequence to facilitate purification (e.g., His6), tags containing a c-Myc sequence (e.g., EQKLISEEDL (SEQ ID NO: 112)) to enable detection, and / or tags containing a cysteine ​​residue to enable labeling and bioconjugation using thiol-reactive payloads and probes, and combinations thereof. Preferred C-terminal tags include, but are not limited to:

[0680] QASGAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 98)

[0681] QACGAHHHHHHGAEFEQKLISEEDL(SEQ ID NO:99)

[0682] QACKAHHHHHHGAEFEQKLISEEDL(SEQ ID NO:97)

[0683] AAAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 100)

[0684] ACAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 101)

[0685] QASGAHHHHHH (SEQ ID NO: 102)

[0686] QACGAHHHHHH (SEQ ID NO: 103)

[0687] QACKAHHHHHH (SEQ ID NO: 104)

[0688] AAAHHHHHH (SEQ ID NO: 105)

[0689] ACAHHHHHH (SEQ ID NO: 106)

[0690] QASGA (SEQ ID NO: 107)

[0691] QACGA (SEQ ID NO: 108)

[0692] QACKA (SEQ ID NO: 109)

[0693] ACA (SEQ ID NO: 110)

[0694] SAPSA (SEQ ID NO: 111)

[0695] Where:

[0696] G3CP is

[0697] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVN (SEQ ID NO: 50)

[0698] G3CP G4 is

[0699] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIK (SEQ ID NO: 71)

[0700] BA11 is TRVDQSPSSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIK (SEQ ID NO: 95)

[0701] P3A1 G1 AE3 is

[0702] TRVDQSPSSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK(SEQ ID NO: 77)

[0703] B1G4 is

[0704] TRVDQSPSSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIK(SEQ ID NO: 51)

[0705] All combinations of VNARs and linkers are expressly contemplated herein.

[0706] Also included herein are humanized derivatives of VNARs. Any recombinant fusion protein disclosed herein involving the presence of an EGFR specific binding molecule can be modified to include an EGFR specific binding molecule. In conjunction with any recombinant fusion protein disclosed herein involving the presence of an EGFR specific binding molecule in any configuration, any EGFR specific binding molecule disclosed herein is explicitly contemplated.

[0707] According to a third aspect, a recombinant fusion is provided, comprising a ROR1-specific antigen binding molecule and a recombinant toxin. Examples of recombinant toxins include, but are not limited to, Pseudomonas exotoxin PE38 and diphtheria toxin.

[0708] According to a third aspect, a recombinant fusion is provided, which includes a ROR1-specific antigen binding molecule and a recombinant CD3 binding protein. Examples of recombinant ROR1 and CD3 binding agents include, but are not limited to:

[0709] B1G4–[WGM]–CD3

[0710] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIKPGVQPAPGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQID NO:117)

[0711] G3CP–[WGM]–CD3

[0712] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNPGVQPAPGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQID NO:118)

[0713] G3CPG4–[WGM]-CD3

[0714] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKPGVQPAPGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQID NO:119)

[0715] P3A1G1AE3–[WGM]–CD3

[0716] TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIKPGVQPAPGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ IDNO:120)

[0717] B1G4–[WGM]–BA11-[G4S]-CD3

[0718] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIKPGVQPAPGGGGSTRVDQSPSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO:121)

[0719] G3CP–[WGM]–BA11-[G4S]-CD3

[0720] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNPGVQPAPGGGGSTRVDQSPSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ IDNO:122)

[0721] G3CPG4–[WGM]–BA11-[G4S]-CD3

[0722] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKPGVQPAPGGGGSTRVDQSPSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO:123)

[0723] P3A1G1AE3–[WGM]–BA11-[G4S]-CD3

[0724] TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIKPGVQPAPGGGGSTRVDQSPSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO:124)

[0725] P3A1–[WGM]–G3CP-[G4S]-CD3

[0726] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNPGVQPAPGGGGSASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO:125)

[0727] P3A1–[WGM]–G3CPG4-[G4S]-CD3

[0728] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNPGVQPAPGGGGSTRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO:126)

[0729] P3A1G1AE3–[WGM]–G3CP-[G4S]-CD3

[0730] TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIKPGVQPAPGGGGSASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO:127)

[0731] P3A1G1AE3–[WGM]–G3CPG4-[G4S]-CD3

[0732] TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIKPGVQPAPGGGGSTRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQID NO:128)

[0733] P3A1G1AE3–[WGM]–D3-[G4S]-CD3

[0734] TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIKPGVQPAPGGGGSASVNQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKRAKSFSLRIKDLTVADSATYYCKAQSGMAISTGSGHGYNWYDGAGTVLTVNGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO:129)

[0735] Any CD3-binding sequence known in the art and its variants can be substituted into the above sequence. For example:

[0736] UCL OKT3 sequence (WO2019008379)

[0737] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTRYTMHWVRQAPGQGLEWMGYINPSRGYTNYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARYYDDHYCLDYWGQGTMVTVSSVEGGSGGSGGSGGSGGVDDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQWSSNPFTFGQGTKVEIK(SEQ ID NO:130)

[0738] Harpoon ID20 (WO2016187594)

[0739] DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGG SGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK(SEQ ID NO:131)

[0740] Fc regions can be engineered to reduce FcγR binding. Accordingly, the Fc regions disclosed herein can be engineered to reduce FcγR binding.

[0741] In one embodiment, the fragment can be a first fragment of an immunoglobulin Fc region that is engineered to dimerize with a second fragment of an immunoglobulin Fc region. For example, a ROR1-specific antigen binding molecule can be fused to the first fragment of an immunoglobulin Fc region, and an EGFR-specific binding molecule can be fused to the second fragment of an immunoglobulin Fc region.

[0742] As used herein, an immunoglobulin Fc region "engineered to dimerize" may comprise at least one amino acid substitution. Typically, the at least one amino acid substitution promotes interaction and / or association with the second fragment of the immunoglobulin Fc region and / or renders interaction and / or association with the second fragment of the immunoglobulin Fc region more energetically favorable, thereby promoting dimerization and / or rendering dimerization more energetically favorable. Such recombinant fusion proteins may have particular utility in the preparation of bispecific and / or biparatopic binders.

[0743] Methods for generating Fc-based bispecific and / or biparatopic binders by pairing two different Fc heavy chains engineered to dimerize are known in the art. These methods enable the assembly of an Fc region from two different heavy chains, each fused to a target binding domain or sequence with different binding characteristics. The target binding domains or sequences can be directed against different targets to generate multispecific binders and / or against different regions or epitopes on the same target to generate biparatopic binding proteins. Multiple binding domains or sequences can be fused to an Fc sequence to generate multispecific or multiparatopic binders or multispecific multiparatopic binders within the same protein. Methods for generating these asymmetric bispecific and / or biparatopic binders by heterodimerization of two different Fc heavy chains or fragments thereof include, but are not limited to, knob-in-hole (YT), knob-in-hole (CW-CSAV), CH3 charge pairing, Fab-arm exchange, SEED technology, BEAT technology, HA-TF, ZW1 method, Biclonic method, EW-RVT, and Triomab. See, e.g., Brinkman & Kontermann, (2017) mAbs, 9:2, 182-212; Klein et al (2012) mAbs 4:6, 653–663; Wang et al (2019) Antibodies, 8, 43; and Dietrich et al (2020) BBA - Proteins and Proteomics 1868 140250; each of which is incorporated herein by reference in its entirety.

[0744] In one embodiment, the first fragment of the immunoglobulin Fc region is engineered to dimerize with the second fragment of the immunoglobulin Fc region by a method selected from the group consisting of knob-to-hole (YT), knob-to-hole (CW-CSAV), CH3 charge pairing, Fab-arm exchange, SEED technology, BEAT technology, HA-TF, ZW1 method, Biclonic method, EW-RVT and Triomab.

[0745] The knob-to-hole (YT) may comprise a T366Y substitution in the first CH3 domain and a Y407T substitution in the second CH3 domain.

[0746] The knob-to-hole (CW-CSAV) may include one or more (preferably all) of the following substitutions in the first CH3 domain: S354C, T366W. The knob-to-hole (CW-CSAV) may include one or more (preferably all) of the following substitutions in the second CH3 domain: Y349C, T366S, L368A, Y407V. The knob-to-hole (CW-CSAV) may include a disulfide bond in the CH3 domain.

[0747] The CH3 charge pairing may comprise one or more (preferably all) of the following substitutions in the first CH3 domain: K392D, K409D. The CH3 charge pairing may comprise one or more (preferably all) of the following substitutions in the second CH3 domain: E356K, D399K.

[0748] Fab-arm exchange can include a K409R substitution in the first CH3 domain and an F405L substitution in the second CH3 domain. Fab-arm exchange and DuoBody capture the same Fc variants. Therefore, DuoBody technology can include a K409R substitution in the first CH3 domain and an F405L substitution in the second CH3 domain.

[0749] SEED technology can incorporate known substitutions and / or produce IgG / A chimeras. By designing chain exchange engineered domain (SEED) heterodimers, the complementarity of the CH3 interface that allows Fc chain heterodimer assembly was developed. These SEED CH3 domains are composed of alternating fragments derived from human IgA and IgG CH3 sequences (AG SEED CH3 and GA SEED CH3) and are used to generate so-called SEEDbodies (Davis et al (2010) PEDS 23, 4, 195-202, incorporated by reference in their entirety). Because molecular modeling showed that the interaction with FcRn in AG SEED CH3 was impaired, the residues at the CH2-CH3 junction were restored to IgG sequences. Pharmacokinetic studies confirmed that the half-life of SEEDbodies was comparable to that of other Fc fusion proteins and IgG1.

[0750] BEAT technology engineered the constant α and β domains of the human T cell receptor into the IgG1 CH3 dimer interface to drive heterodimerization (Skegro et al (2017) JBC 292 (23) 9745-9759). An additional D410Q mutation can further increase heterodimer formation in this system (Stutz & Blein 2020 JBC 295 (28) 9392-9408).

[0751] HA-TF may comprise one or more (preferably all) of the following substitutions in the first CH3 domain: S364H, F405A. HA-TF may comprise one or more (preferably all) of the following substitutions in the second CH3 domain: Y349T, T394F.

[0752] The ZW1 method may include one or more (preferably all) of the following substitutions in the first CH3 domain: T350V, L351Y, F405A, Y407V. The ZW1 method may include one or more (preferably all) of the following substitutions in the second CH3 domain: T350V, T366L, K392L, T394W.

[0753] The biclonic method may include one or more (preferably all) of the following substitutions in the first CH3 domain: 366K (+351K). The biclonic method may include one or more (preferably all) of the following substitutions in the second CH3 domain: 351D, or E or D at positions 349, 368, 349, or 349+355.

[0754] EW-RVT may include one or more (preferably all) of the following substitutions in the first CH3 domain: K360E, K409W. EW-RVT may include one or more (preferably all) of the following substitutions in the second CH3 domain: Q347R, D399V, F405T. EW-RVT may include a disulfide bond in the CH3 domain. Disulfide bridges may be supported by further incorporating Y349C into the first CH3 domain and S354C into the second CH3 domain.

[0755] Triomabs can be formed by fusing a mouse hybridoma with a rat hybridoma, resulting in a bispecific, asymmetric hybrid IgG molecule. Preferential pairing of the light chain with its corresponding heavy chain then occurs.

[0756] In one embodiment, the one or more residues of the fragment of the immunoglobulin Fc region comprise one or more amino acid substitutions suitable for heterodimerization with a second fragment of the immunoglobulin Fc region comprising one or more corresponding amino acid substitutions. In one embodiment, the one or more residues of the fragment of the immunoglobulin Fc region comprise one or more amino acid substitutions suitable for knob-in-hole (KIH) dimerization with the second fragment of the immunoglobulin Fc region comprising one or more corresponding amino acid substitutions. The one or more corresponding amino acid substitutions can be one or more corresponding amino acid substitutions suitable for knob-in-hole (KIH) dimerization with the first fragment of the immunoglobulin Fc region.

[0757] In one embodiment, the one or more amino acid substitutions are selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A and Y407V.

[0758] In one embodiment, the one or more amino acid substitutions are selected from the group consisting of T366Y and Y407T.

[0759] The recombinant fusion protein may comprise a sequence according to SEQ ID NO: 146 or SEQ ID NO: 147.

[0760] G3CP hFc(S239C+Y407T)SEQ ID NO: 146

[0761] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0762] G3CPG4 hFc(S239C+Y407T)SEQ ID NO: 147

[0763] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0764] The recombinant fusion protein may comprise a sequence according to SEQ ID NO: 194, SEQ ID NO: 195 or SEQ ID NO: 196.

[0765] 1H8 hFc(S239C+Y407T) SEQ ID NO: 194

[0766] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0767] 1H8 G4 hFc(S239C+Y407T) SEQ ID NO: 195

[0768] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0769] 1H8 v15 hFc(S239C+Y407T)SEQ ID NO: 196

[0770] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0771] The recombinant fusion protein may comprise the sequence according to SEQ ID NO: 148.

[0772] P3A1 hFc(S239C+T366Y)SEQ ID NO: 148

[0773] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0774] The recombinant fusion protein may comprise the sequence according to SEQ ID NO: 191 or SEQ ID NO: 192.

[0775] G3CP hFc(S239C+T366Y) SEQ ID NO: 191

[0776] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0777] G3CPG4 hFc(S239C+T366Y) SEQ ID NO: 192

[0778] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0779] The recombinant fusion protein may comprise a sequence according to SEQ ID NO: 197, SEQ ID NO: 198 or SEQ ID NO: 199.

[0780] 1H8 hFc(S239C+T366Y)SEQ ID NO:197

[0781] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0782] 1H8 G4 hFc(S239C+T366Y)SEQ ID NO:198

[0783] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0784] 1H8 v15 hFc(S239C+T366Y)SEQ ID NO:199

[0785] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0786] The recombinant fusion protein may comprise the sequence according to SEQ ID NO: 193.

[0787] P3A1 hFc(S239C+Y407T)SEQ ID NO: 193:

[0788] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0789] The recombinant fusion protein may comprise the sequences according to SEQ ID NOs: 550 to 561.

[0790] G3CP-hFc(S442C+T366Y)(SEQ ID NO: 550)

[0791] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGSGGGSSEGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSCLSPGK

[0792] G3CPG4 hFc(S442C+T366Y)(SEQ ID NO:551)

[0793] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTLISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGSGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSCLSPGK

[0794] 1H8-hFc(S442C+T366Y)(SEQ ID NO:552)

[0795] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0796] 1H8 G4 hFc(S442C+T366Y)(SEQ ID NO:553)

[0797] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0798] 1H8 V15 hFc(S442C+T366Y)(SEQ ID NO:554)

[0799] ASVTQSPRSASKETGESLTICRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGSGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSCLSPGK

[0800] P3A1 hFc(S442C+T366Y)(SEQ ID NO:555)

[0801] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSCLSPGK

[0802] G3CP-hFc(S442C+Y407T)(SEQ ID NO:556)

[0803] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0804] G3CPG4 hFc(S442C+Y407T)(SEQ ID NO:557)

[0805] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0806] 1H8-hFc(S442C+Y407T)(SEQ ID NO:558)

[0807] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0808] 1H8 G4 hFc(S442C+Y407T)(SEQ ID NO:559)

[0809] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0810] 1H8 V15 hFc(S442C+Y407T)(SEQ ID NO:560)

[0811] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0812] P3A1 hFc(S442C+Y407T)(SEQ ID NO: 561)

[0813] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0814] The recombinant fusion protein may comprise a sequence according to any one of SEQ ID NOs: 253 to 258.

[0815] G3CP hFc(S239C&S442C+Y407T) SEQ ID NO: 253

[0816] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0817] G3CPG4 hFc(S239C&S442C+Y407T)SEQ ID NO:254

[0818] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0819] 1H8 hFc(S239C&S442C+Y407T)SEQ ID NO:255

[0820] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0821] 1H8 G4 hFc(S239C&S442C+Y407T)SEQ ID NO:256

[0822] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0823] 1H8 v15 hFc(S239C&S442C+Y407T)SEQ ID NO:257

[0824] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0825] P3A1 hFc(S239C&S442C+Y407T) SEQ ID NO: 258

[0826] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0827] The recombinant fusion protein may comprise a sequence according to any one of SEQ ID NOs: 259 to 264.

[0828] P3A1 hFc(S239C&S442C+T366Y) SEQ ID NO: 259

[0829] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSCLSPGK

[0830] G3CP hFc(S239C&S442C+T366Y)SEQ ID NO:260

[0831] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSCLSPGK

[0832] G3CPG4 hFc(S239C&S442C+T366Y)SEQ ID NO:261

[0833] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0834] 1H8 hFc(S239C&S442C+T366Y)SEQ ID NO:262

[0835] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0836] 1H8 G4 hFc(S239C&S442C+T366Y)SEQ ID NO:263

[0837] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0838] 1H8 v15 hFc(S239C&S442C+T366Y)SEQ ID NO: 264

[0839] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0840] The recombinant fusion protein may comprise a sequence according to any one of SEQ ID NOs: 265 to 268.

[0841] 7D12-hFc(S239C+Y407T)(SEQ ID NO: 265)

[0842] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0843] EGFR#33-hFc(S239C+Y407T)(SEQ ID NO:266)

[0844] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0845] EGFR#13-hFc(S239C+Y407T)(SEQ ID NO:267)

[0846] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0847] 9G8-hFc(S239C+Y407T)(SEQ ID NO:268)

[0848] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0849] The recombinant fusion protein may comprise a sequence according to any one of SEQ ID NOs: 269 to 272.

[0850] 7D12-hFc(S442C+Y407T)(SEQ ID NO:269)

[0851] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0852] EGFR#33-hFc(S442C+Y407T)(SEQ ID NO:270)

[0853] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0854] EGFR#13-hFc(S442C+Y407T)(SEQ ID NO:271)

[0855] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0856] 9G8-hFc(S442C+Y407T)(SEQ ID NO: 272)

[0857] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0858] The recombinant fusion protein may comprise a sequence according to any one of SEQ ID NO: 273 to 276.

[0859] 7D12-hFc(S239C&S442C+Y407T)(SEQ ID NO: 273)

[0860] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0861] EGFR#33-hFc(S239C&S442C+Y407T)(SEQ ID NO:274)

[0862] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0863] EGFR#13-hFc(S239C&S442C+Y407T)(SEQ ID NO:275)

[0864] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYD A WGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0865] 9G8-hFc(S239C&S442C+Y407T)(SEQ ID NO:276)

[0866] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0867] The recombinant fusion protein may comprise a sequence according to any one of SEQ ID NOs: 277 to 280.

[0868] 7D12-hFc(S239C+T366Y)(SEQ ID NO:277)

[0869] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0870] EGFR#33-hFc(S239C+T366Y)(SEQ ID NO:278)

[0871] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0872] EGFR#13-hFc(S239C+T366Y)(SEQ ID NO:279)

[0873] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0874] 9G8-hFc(S239C+T366Y)(SEQ ID NO:280)

[0875] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0876] The recombinant fusion protein may comprise a sequence according to any one of SEQ ID NOs: 281 to 284.

[0877] 7D12-hFc(S442C+T366Y)(SEQ ID NO:281)

[0878] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0879] EGFR#33-hFc(S442C+T366Y)(SEQ ID NO:282)

[0880] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0881] EGFR#13-hFc(S442C+T366Y)(SEQ ID NO:283)

[0882] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0883] 9G8-hFc(S442C+T366Y)(SEQ ID NO: 284)

[0884] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0885] The recombinant fusion protein may comprise a sequence according to any one of SEQ ID NOs: 285 to 288.

[0886] 7D12-hFc(S239C&S442C+T366Y)(SEQ ID NO: 285)

[0887] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0888] EGFR#33-hFc(S239C&S442C+T366Y)(SEQ ID NO:286)

[0889] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0890] EGFR#13-hFc(S239C&S442C+T366Y)(SEQ ID NO:287)

[0891] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0892] 9G8-hFc(S239C&S442C+T366Y)(SEQ ID NO:288)

[0893] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0894] The recombinant fusion protein may comprise a sequence according to any one of SEQ ID NOs: 358 to 380 and 390 to 407. When the recombinant fusion protein comprises humanized cetuximab Fab HC hFc, the humanized cetuximab Fab HC hFc is typically associated with a humanized cetuximab Fab LC comprising a sequence according to SEQ ID NO: 360 via a disulfide bond.

[0895] Humanized Cetuximab Fab HC hFc (S239C+T366Y) (SEQ ID NO: 369)

[0896] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQG TLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDK THTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0897] Humanized cetuximab scFv hFc (S239C+T366Y) (SEQ ID NO: 370)

[0898] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0899] Humanized cetuximab Fab HC hFc(S239C+Y407T) (SEQ ID NO: 371)

[0900] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0901] Humanized cetuximab scFv hFc (S239C+Y407T) (SEQ ID NO: 372)

[0902] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0903] Humanized cetuximab Fab HC hFc (S239C+S442C+T366Y) (SEQ ID NO: 373)

[0904] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0905] Humanized cetuximab scFv hFc (S239C+S442C+T366Y) (SEQ ID NO: 374)

[0906] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0907] Humanized cetuximab Fab HC hFc (S239C + S442C + Y407T) (SEQ ID NO: 375)

[0908] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0909] Humanized cetuximab scFv hFc (S239C+S442C+Y407T) (SEQ ID NO: 376)

[0910] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0911] Humanized Cetuximab Fab HC hFc(S442C+T366Y) (SEQ ID NO: 377)

[0912] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0913] Humanized cetuximab scFv hFc (S442C+T366Y) (SEQ ID NO: 378)

[0914] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0915] Humanized Cetuximab Fab HC hFc (S442C + Y407T) (SEQ ID NO: 379)

[0916] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0917] Humanized cetuximab scFv hFc (S442C + Y407T) (SEQ ID NO: 380)

[0918] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVS SGGGGSGGGGSGGGGSMDIQMTQSPSSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGT KVEIKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0919] The recombinant fusion protein can be a biparatopic dimer comprising any one or any two of SEQ ID NOs: 146, 147, 194, 195, 196, 148, 191, 192, 193, 197, 198, and 199. The biparatopic dimer can comprise any one of SEQ ID NOs: 146, 147, 194, 195, 196, and 193 comprising a Y407T point mutation. The biparatopic dimer can comprise any one of SEQ ID NOs: 148, 191, 192, 197, 198, and 199 comprising a T366Y point mutation. The biparatopic dimer can comprise SEQ ID NO: 146 and SEQ ID NO: 148 or SEQ ID NO: 147 and SEQ ID NO: 148. Any recombinant fusion protein disclosed herein can be associated with any linker and payload disclosed herein. Any biparatopic dimer disclosed herein can be associated with any linker and payload disclosed herein. Conjugation can be through any one or more of the S239C or S442C residues in the biparatopic dimer.

[0920] The biparatopic dimer can be associated with a linker and payload vc-MMAE.The biparatopic dimer can include G3CP hFc(S239C+Y407T) (SEQ ID NO: 146) and P3A1 hFc(S239C+T366Y) (SEQ ID NO: 148) conjugated to vc-PAB-EDA-PNU.

[0921] Biparatopic dimers can be associated with a linker and a payload, vc-PAB-EDA-PNU. Biparatopic dimers can comprise G3CP hFc (S239C + Y407T) (SEQ ID NO: 146) and P3A1 hFc (S239C + T366Y) (SEQ ID NO: 148) conjugated to vc-PAB-EDA-PNU, or G3CPG4 hFc (S239C + Y407T) (SEQ ID NO: 147) and P3A1 hFc (S239C + T366Y) (SEQ ID NO: 148) conjugated to vc-PAB-EDA-PNU, which have been shown to be highly effective in vivo.

[0922] SEQ ID NOs: 146, 147, 194, 195, 196, 148, 191, 192, 193, 197, 198, and 199 include an S239C mutation for use in conjugation reactions. When the recombinant fusion protein is not conjugated (e.g., not conjugated to an anthracycline (PNU) derivative), the S239C mutation is not required, and position 239 can be S instead of C. Thus, in alternative embodiments, the recombinant fusion protein or biparatopic dimer can comprise a sequence according to any one of SEQ ID NOs: 146, 147, 194, 195, 196, 148, 191, 192, 193, 197, 198, and 199, but each sequence does not include an S239C mutation.

[0923] The recombinant fusion protein can be a biparatopic dimer comprising any one or any two of SEQ ID NOs: 550 to 561. The biparatopic dimer can comprise one of SEQ ID NOs: 556 to 561 comprising a Y407T point mutation. The biparatopic dimer can comprise one of SEQ ID NOs: 550 to 555 comprising a T366Y point mutation. The biparatopic dimer can comprise SEQ ID NO: 556 and SEQ ID NO: 555 or SEQ ID NO: 557 and SEQ ID NO: 555. Any recombinant fusion protein disclosed herein can be associated with any linker and payload disclosed herein. Any biparatopic dimer disclosed herein can be associated with any linker and payload disclosed herein, and conjugation can be performed through any one or more S239C or S442C residues in the biparatopic dimer.

[0924] The biparatopic dimer can be associated with a linker and a payload vc-MMAE. The biparatopic dimer can include G3CP hFc (S2442C + Y407T) (SEQ ID NO: 556) and P3A1 hFc (S442C + T366Y) (SEQ ID NO: 555) conjugated to vc-PAB-EDA-PNU.

[0925] Biparatopic dimers can be associated with a linker and a payload, vc-PAB-EDA-PNU. Biparatopic dimers can include G3CP hFc (S442C + Y407T) (SEQ ID NO: 556) and P3A1 hFc (S442C + T366Y) (SEQ ID NO: 555) conjugated to vc-PAB-EDA-PNU, or G3CPG4 hFc (S442C + Y407T) (SEQ ID NO: 557) and P3A1 hFc (S442C + T366Y) (SEQ ID NO: 555) conjugated to vc-PAB-EDA-PNU, which have been shown to be highly effective in vivo.

[0926] SEQ ID NOs: 550 to 561 include an S442C mutation for use in conjugation reactions. When the recombinant fusion protein is not conjugated (e.g., to an anthracycline (PNU) derivative), the S442C mutation is not required and position 442 can be S instead of C. Thus, in alternative embodiments, the recombinant fusion protein or biparatopic dimer can comprise a sequence according to any one of SEQ ID NOs: 550 to 561, except that each sequence does not include the S442C mutation.

[0927] The above-mentioned biparatopic dimers comprise the S442C or S239C point mutation. Also disclosed herein are biparatopic dimers corresponding to those described above comprising S442C and S239C.

[0928] The recombinant fusion protein may comprise a sequence according to SEQ ID NO: 165 or SEQ ID NO: 166.

[0929] G3CP-hFc(Y407T)SEQ ID NO: 165:

[0930] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0931] G3CP G4-hFc(Y407T) SEQ ID NO: 166

[0932] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0933] The recombinant fusion protein may comprise a sequence according to SEQ ID NO: 200, SEQ ID NO: 201 or SEQ ID NO: 202.

[0934] 1H8 hFc(Y407T) SEQ ID NO: 200

[0935] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0936] 1H8 G4 hFc(Y407T)SEQ ID NO:201

[0937] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0938] 1H8 v15 hFc(Y407T)SEQ ID NO:202

[0939] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0940] The recombinant fusion protein may comprise the sequence according to SEQ ID NO: 167.

[0941] P3A1 hFc(T366Y) SEQ ID NO: 167:

[0942] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0943] The recombinant fusion protein may comprise the sequence according to SEQ ID NO: 188 or SEQ ID NO: 189.

[0944] G3CP hFc(T366Y) SEQ ID NO: 188

[0945] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0946] G3CPG4 hFc(T366Y) SEQ ID NO: 189

[0947] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0948] The recombinant fusion protein may comprise a sequence according to SEQ ID NO: 203, SEQ ID NO: 204 or SEQ ID NO: 205.

[0949] 1H8 hFc(T366Y) SEQ ID NO: 203

[0950] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0951] 1H8 G4 hFc(T366Y)SEQ ID NO:204

[0952] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0953] 1H8 v15 hFc(T366Y)SEQ ID NO:205

[0954] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0955] The recombinant fusion protein may comprise the sequence according to SEQ ID NO: 190:

[0956] P3A1 hFc(Y407T)SEQ ID NO: 190

[0957] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0958] The recombinant fusion protein may comprise a sequence according to any one of SEQ ID NOs: 289 to 292, or a sequence corresponding to any one of SEQ ID NOs: 289 to 292 comprising S239C and / or S442C substitutions.

[0959] 7D12-hFc(Y407T)

[0960] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:289)

[0961] EGFR#33-hFc(Y407T)

[0962] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:290)

[0963] EGFR#13-hFc(Y407T)

[0964] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:291)

[0965] 9G8-hFc(Y407T)

[0966] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:292)

[0967] The recombinant fusion protein may comprise a sequence according to any one of SEQ ID NOs: 293 to 296.

[0968] 7D12-hFc(T366Y)

[0969] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:293)

[0970] EGFR#33-hFc(T366Y)

[0971] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:294)

[0972] EGFR#13-hFc(T366Y)

[0973] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYD A WGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:295)

[0974] 9G8-hFc(T366Y)

[0975] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:296)

[0976] SEQ ID NOs: 146 to 148, 165 to 167, 178 to 205, 223 to 296, and 550 to 561] each comprise a (G4S)3 linker. Corresponding sequences in which the (G4S)3 linker is replaced with a (G4S)1 linker are also expressly contemplated herein. For example, a recombinant fusion protein may comprise one or more of the following SEQ ID NOs: 297 to 356, 390 to 431, and 562 to 597.

[0977] G3CP G4S-hFc

[0978] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:408)

[0979] G3CPG4 G4S-hFc

[0980] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:409)

[0981] 1H8 G4S-hFc

[0982] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:410)

[0983] 1H8 G4 G4S-hFc

[0984] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:411)

[0985] 1H8 V15 G4S-hFc

[0986] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:412)

[0987] P3A1 G4S-hFc

[0988] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:413)

[0989] G3CP G4S-hFc(S239C)

[0990] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:414)

[0991] G3CPG4 G4S-hFc(S239C)

[0992] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:415)

[0993] 1H8 G4S-hFc(S239C)

[0994] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:416)

[0995] 1H8 G4 G4S-hFc(S239C)

[0996] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:417)

[0997] 1H8 V15 G4S-hFc(S239C)

[0998] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:418)

[0999] P3A1 G4S-hFc(S239C)

[1000] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:419)

[1001] G3CP G4S-hFc(S442C)

[1002] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSCLSPGK(SEQ ID NO: 420)

[1003] G3CPG4 G4S-hFc(S442C)

[1004] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:421)

[1005] 1H8 G4S-hFc(S442C)

[1006] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:422)

[1007] 1H8 G4 G4S-hFc(S442C)

[1008] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:423)

[1009] 1H8 V15 G4S-hFc(S442C)

[1010] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:424)

[1011] P3A1 G4S-hFc(S442C)

[1012] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:425)

[1013] G3CP G4S-hFc(S239C&S442C)

[1014] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:426)

[1015] G3CPG4 G4S-hFc(S239C&S442C)

[1016] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:427)

[1017] 1H8 G4S-hFc(S239C&S442C)

[1018] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:428)

[1019] 1H8 G4 G4S-hFc(S239C&S442C)

[1020] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:429)

[1021] 1H8 V15 G4S-hFc(S239C&S442C)

[1022] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:430)

[1023] P3A1 G4S-hFc(S239C&S442C)

[1024] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO:431)

[1025] P3A1 G4S-hFc(S239C+Y407T)SEQ ID NO:308:

[1026] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1027] G3CP G4S-hFc(S239C+Y407T)SEQ ID NO:297

[1028] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1029] G3CPG4 G4S-hFc(S239C+Y407T)SEQ ID NO:298

[1030] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1031] 1H8 G4S-hFc(S239C+Y407T)SEQ ID NO:299

[1032] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1033] 1H8 G4 G4S-hFc(S239C+Y407T)SEQ ID NO:300

[1034] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1035] 1H8 v15 G4S-hFc(S239C+Y407T)SEQ ID NO:301

[1036] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1037] P3A1 G4S-hFc(S239C+T366Y)SEQ ID NO:302

[1038] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1039] G3CP G4S-hFc(S239C+T366Y)SEQ ID NO:303

[1040] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1041] G3CPG4 G4S-hFc(S239C+T366Y)SEQ ID NO:304

[1042] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1043] 1H8 G4S-hFc(S239C+T366Y)SEQ ID NO:305

[1044] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1045] 1H8 G4 G4S-hFc(S239C+T366Y)SEQ ID NO:306

[1046] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1047] 1H8 v15 G4S-hFc(S239C+T366Y)SEQ ID NO:307

[1048] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1049] P3A1 G4S-hFc(S239C&S442C+Y407T)SEQ ID NO::309

[1050] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1051] G3CP G4S-hFc(S239C&S442C+Y407T)SEQ ID NO:310

[1052] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1053] G3CPG4 G4S-hFc(S239C&S442C+Y407T)SEQ ID NO:311

[1054] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1055] 1H8 G4S-hFc(S239C&S442C+Y407T)SEQ ID NO:312

[1056] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1057] 1H8 G4 G4S-hFc(S239C&S442C+Y407T)SEQ ID NO:313

[1058] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1059] 1H8 v15 G4S-hFc(S239C&S442C+Y407T)SEQ ID NO:314

[1060] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1061] P3A1 G4S-hFc(S239C&S442C+T366Y)SEQ ID NO:315

[1062] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1063] G3CP G4S-hFc(S239C&S442C+T366Y)SEQ ID NO:316

[1064] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1065] G3CPG4 G4S-hFc(S239C&S442C+T366Y)SEQ ID NO:317

[1066] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1067] 1H8 G4S-hFc(S239C&S442C+T366Y)SEQ ID NO:318

[1068] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1069] 1H8 G4 G4S-hFc(S239C&S442C+T366Y)SEQ ID NO:319

[1070] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1071] 1H8 v15 G4S-hFc(S239C&S442C+T366Y)SEQ ID NO:320

[1072] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1073] P3A1 G4S-hFc(S442C+Y407T)SEQ ID NO:321

[1074] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTSPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1075] G3CP G4S-hFc(S442C+Y407T)SEQ ID NO:322

[1076] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1077] G3CPG4 G4S-hFc(S442C+Y407T)SEQ ID NO:323

[1078] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1079] 1H8 G4S-hFc(S442C+Y407T)SEQ ID NO:324

[1080] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1081] 1H8 G4 G4S-hFc(S442C+Y407T)SEQ ID NO:325

[1082] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1083] 1H8 v15 G4S-hFc(S442C+Y407T)SEQ ID NO:326

[1084] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1085] P3A1 G4S-hFc(S442C+T366Y)SEQ ID NO:327

[1086] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1087] G3CP G4S-hFc(S442C+T366Y)SEQ ID NO:328

[1088] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSCLSPGK

[1089] G3CPG4 G4S-hFc(S442C+T366Y)SEQ ID NO:329

[1090] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTLISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSCLSPGK

[1091] 1H8 G4S-hFc(S442C+T366Y)SEQ ID NO:330

[1092] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1093] 1H8 G4 G4S-hFc(S442C+T366Y)SEQ ID NO:331

[1094] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1095] 1H8 v15 G4S-hFc(S442C+T366Y)SEQ ID NO:332

[1096] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1097] 7D12-G4S-hFc(S239C+Y407T)(SEQ ID NO:333

[1098] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1099] EGFR#33-G4S-hFc(S239C+Y407T)(SEQ ID NO:334)

[1100] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1101] EGFR#13-G4S-hFc(S239C+Y407T)(SEQ ID NO:335)

[1102] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1103] 9G8-G4S-hFc(S239C+Y407T)(SEQ ID NO:336)

[1104] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1105] 7D12-G4S-hFc(S442C+Y407T)(SEQ ID NO:337)

[1106] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1107] EGFR#33-G4S-hFc(S442C+Y407T)(SEQ ID NO:338)

[1108] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1109] EGFR#13-G4S-hFc(S442C+Y407T)(SEQ ID NO:339)

[1110] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1111] 9G8-G4S-hFc(S442C+Y407T)(SEQ ID NO:340)

[1112] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1113] 7D12-G4S-hFc(S239C&S442C+Y407T)(SEQ ID NO:341)

[1114] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1115] EGFR#33-G4S-hFc(S239C&S442C+Y407T)(SEQ ID NO:342)

[1116] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1117] EGFR#13-G4S-hFc(S239C&S442C+Y407T)(SEQ ID NO:343)

[1118] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1119] 9G8-G4S-hFc(S239C&S442C+Y407T)(SEQ ID NO:344)

[1120] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1121] 7D12-G4S-hFc(S239C+T366Y)(SEQ ID NO:345)

[1122] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1123] EGFR#33-G4S-hFc(S239C+T366Y)(SEQ ID NO:346)

[1124] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1125] EGFR#13-G4S-hFc(S239C+T366Y)(SEQ ID NO:347)

[1126] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1127] 9G8-G4S-hFc(S239C+T366Y)(SEQ ID NO:348)

[1128] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1129] 7D12-G4S-hFc(S442C+T366Y)(SEQ ID NO:349)

[1130] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1131] EGFR#33-G4S-hFc(S442C+T366Y)(SEQ ID NO:350)

[1132] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1133] EGFR#13-G4S-hFc(S442C+T366Y)(SEQ ID NO:351)

[1134] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1135] 9G8-G4S-hFc(S442C+T366Y)(SEQ ID NO:352)

[1136] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1137] 7D12-G4S-hFc(S239C&S442C+T366Y)(SEQ ID NO:353)

[1138] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1139] EGFR#33-G4S-hFc(S239C&S442C+T366Y)(SEQ ID NO:354)

[1140] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1141] EGFR#13-G4S-hFc(S239C&S442C+T366Y)(SEQ ID NO:355)

[1142] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1143] 9G8-G4S-hFc(S239C&S442C+T366Y)(SEQ ID NO:356)

[1144] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1145] Humanized cetuximab Fab HC-G4S-hFc(S239C) (SEQ ID NO: 390)

[1146] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1147] Humanized cetuximab scFv-G4S-hFc(S239C) (SEQ ID NO: 391)

[1148] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1149] Humanized cetuximab Fab HC-G4S-hFc(S239C+S442C) (SEQ ID NO: 392)

[1150] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1151] Humanized cetuximab scFv-G4S-hFc(S239C+S442C) (SEQ ID NO: 393)

[1152] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1153] Humanized Cetuximab Fab HC-G4S-hFc(S442C) (SEQ ID NO: 394)

[1154] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1155] Humanized cetuximab scFv-G4S-hFc(S442C) (SEQ ID NO: 395)

[1156] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1157] Humanized Cetuximab Fab HC-G4S-hFc (S239C + T366Y) (SEQ ID NO: 396)

[1158] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1159] Humanized cetuximab scFv-G4S-hFc(S239C+T366Y) (SEQ ID NO: 397)

[1160] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1161] Humanized cetuximab Fab HC-G4S-hFc (S239C + Y407T) (SEQ ID NO: 398)

[1162] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1163] Humanized cetuximab scFv-G4S-hFc(S239C+Y407T) (SEQ ID NO: 399)

[1164] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1165] Humanized cetuximab Fab HC-G4S-hFc(S239C+S442C+T366Y) (SEQ ID NO: 400)

[1166] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1167] Humanized cetuximab scFv-G4S-hFc (S239C+S442C+T366Y) (SEQ ID NO: 401)

[1168] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1169] Humanized cetuximab Fab HC-G4S-hFc (S239C + S442C + Y407T) (SEQ ID NO: 402)

[1170] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1171] Humanized cetuximab scFv-G4S-hFc (S239C+S442C+Y407T) (SEQ ID NO: 403)

[1172] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1173] Humanized cetuximab Fab HC-G4S-hFc(S442C+T366Y) (SEQ ID NO: 404)

[1174] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1175] Humanized cetuximab scFv-G4S-hFc (S442C+T366Y) (SEQ ID NO: 405)

[1176] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1177] Humanized cetuximab Fab HC-G4S-hFc (S442C + Y407T) (SEQ ID NO: 406)

[1178] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1179] Humanized cetuximab scFv-G4S-hFc (S442C+Y407T) (SEQ ID NO: 407)

[1180] EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1181] G3CP-G4S-hFc(Y407T)(SEQ ID NO:562)

[1182] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1183] G3CP G4 G4S-hFc(Y407T)(SEQ ID NO:563)

[1184] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1185] P3A1 G4S hFc(Y407T)(SEQ ID NO:564)

[1186] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1187] 1H8 G4ShFc(Y407T)(SEQ ID NO:565

[1188] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1189] 1H8 G4 G4S hFc(Y407T)(SEQ ID NO:566

[1190] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1191] 1H8 v15 G4S hFc(Y407T)(SEQ ID NO:567

[1192] ASVTQSPRSASKETGESLTICRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1193] P3A1G4S hFc(T366Y)(SEQ ID NO:568

[1194] TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1195] G3CP G4ShFc(T366Y)(SEQ ID NO:569

[1196] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1197] G3CPG4 G4S hFc(T366Y)(SEQ ID NO:570)

[1198] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1199] 1H8 G4ShFc(T366Y)(SEQ ID NO:571)

[1200] ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSPGK

[1201] 1H8 G4 G4ShFc(T366Y)(SEQ ID NO:572)

[1202] TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSPGK

[1203] 1H8 v15 G4ShFc(T366Y)(SEQ ID NO:573)

[1204] ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1205] 7D12-G4S-hFc(SEQ ID NO:574)

[1206] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1207] EGFR#33G4S-hFc(SEQ ID NO:575)

[1208] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1209] EGFR#13-G4S-hFc(SEQ ID NO:576)

[1210] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1211] 9G8-G4S-hFc(SEQ ID NO:577)

[1212] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1213] 7D12-G4S-hFc(S239C)(SEQ ID NO:578)

[1214] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1215] EGFR#33-G4S-hFc(S239C)(SEQ ID NO:579)

[1216] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1217] EGFR#13-G4S-hFc(S239C)(SEQ ID NO:580)

[1218] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1219] 9G8-G4S-hFc(S239C)(SEQ ID NO:581)

[1220] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1221] 7D12-G4S-hFc(S442C)(SEQ ID NO:582)

[1222] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1223] EGFR#33-G4S-hFc(S442C)(SEQ ID NO:583)

[1224] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1225] EGFR#13-G4S-hFc(S442C)(SEQ ID NO:584

[1226] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1227] 9G8-G4S-hFc(S442C)(SEQ ID NO:585)

[1228] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1229] 7D12-G4S-hFc(S239C&S442C)(SEQ ID NO:586)

[1230] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1231] EGFR#33-G4S-hFc(S239C&S442C)(SEQ ID NO:587)

[1232] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1233] EGFR#13-G4S-hFc(S239C&S442C)(SEQ ID NO:588)

[1234] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1235] 9G8-G4S-hFc(S239C&S442C)(SEQ ID NO:589)

[1236] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[1237] 7D12-G4S-hFc(Y407T)(SEQ ID NO:590)

[1238] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1239] EGFR#33-G4S-hFc(Y407T)(SEQ ID NO:591)

[1240] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1241] EGFR#13-G4S-hFc(Y407T)(SEQ ID NO:592)

[1242] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1243] 9G8-G4S-hFc(Y407T)(SEQ ID NO:593)

[1244] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1245] 7D12-G4S-hFc(T366Y)(SEQ ID NO:594)

[1246] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1247] EGFR#33-G4S-hFc(T366Y)(SEQ ID NO:595)

[1248] EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1249] EGFR#13-G4S-hFc(T366Y)(SEQ ID NO:596)

[1250] AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1251] 9G8-G4S-hFc(T366Y)(SEQ ID NO:597)

[1252] EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1253] In any embodiment, a specific binding molecule comprising an amino acid sequence represented by formula (I), a (G4S)3 linker and a fragment of an immunoglobulin Fc region can replace a corresponding specific binding molecule comprising an amino acid sequence represented by formula (I), a (G4S)1 linker and a fragment of an immunoglobulin Fc region, and vice versa.

[1254] According to a fourth aspect, the present invention provides a recombinant fusion protein dimer comprising:

[1255] (a) a first recombinant fusion protein, wherein the first recombinant fusion protein comprises a receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen binding molecule, and the receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen binding molecule comprises the amino acid sequence represented by formula (I):

[1256] FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4(I)

[1257] in

[1258] CDR3 is selected from the group consisting of YPWGAGAPYNVQWY (SEQ ID NO: 23), YPWGAGAPYLVQWY (SEQ ID NO: 20), YPWGAGAPWNVQWY (SEQ ID NO: 24), YPSGAGAPRPVQWY (SEQ ID NO: 11), YPWGAGAPCLVQWY (SEQ ID NO: 11), NO: 12), YPWGAGAPRLVQWY (SEQ ID NO: 13), YPWGAGAPRQVQWY (SEQ ID NO: 14), YPWGAGAPRSVQWY (SEQ ID NO: 15), YPWGAGAPSLVQWY (SEQ ID NO: 16), YPWGAGAPSNVQWY (SEQ ID NO: 17), YPWGAGAPSQVQWY (SEQ ID NO: 18), YPWGAGAPSSVQWY (SEQ ID NO: 19), YPWGAGAPWQVQWY (SEQ ID NO: 21), YPWGAGAPWSVQWY (SEQ ID NO: 22), and YPWGAGAPWLVQWY (SEQ ID NO: 10);

[1259] CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of GANYGLAA (SEQ ID NO: 1), DANYGLAA (SEQ ID NO: 5), GANYDLSA (SEQ ID NO: 2), GANYGLSA (SEQ ID NO: 3), and GANYDLAA (SEQ ID NO: 4);

[1260] FW1 is the framework region;

[1261] FW2 is the framework region;

[1262] HV2 is a hypervariable sequence having an amino acid sequence selected from the group consisting of SSNQERISIS (SEQ ID NO: 6) and SSNKERISIS (SEQ ID NO: 7);

[1263] FW3a is the framework region;

[1264] HV4 is a hypervariable sequence having an amino acid sequence selected from the group consisting of NKRTM (SEQ ID NO: 8) and NKGTM (SEQ ID NO: 9);

[1265] FW3b is the framework region;

[1266] FW4 is the framework region;

[1267] wherein if CDR3 is YPWGAGAPWLVQWY (SEQ ID NO: 10), CDR1 is selected from the group consisting of DANYGLAA (SEQ ID NO: 5), GANYGLSA (SEQ ID NO: 3) and GANYDLAA (SEQ ID NO: 4),

[1268] and wherein the first antigen binding molecule is fused to a first fragment of an immunoglobulin Fc region, the first fragment of the immunoglobulin Fc region being engineered to dimerize with a second fragment of the immunoglobulin Fc region;

[1269] ,and

[1270] (b) a second recombinant fusion protein, wherein the second recombinant fusion protein comprises an epidermal growth factor receptor (EGFR)-specific antigen binding molecule fused to a second fragment of the immunoglobulin Fc region, and the second fragment of the immunoglobulin Fc region is engineered to dimerize with the first fragment of the immunoglobulin Fc region.

[1271] In one embodiment, the second fragment of the immunoglobulin Fc region is selected from the group consisting of an Fc heavy chain, a CH2 region, and a CH3 region.

[1272] In one embodiment, the second fragment of the immunoglobulin Fc region is an Fc heavy chain.

[1273] In one embodiment, the second fragment of the immunoglobulin Fc region is engineered to dimerize with the second fragment of the immunoglobulin Fc region by a method selected from the group consisting of knob-to-hole (YT), knob-to-hole (CW-CSAV), CH3 charge pairing, Fab-arm exchange, SEED technology, BEAT technology, HA-TF, ZW1 method, Biclonic method, EW-RVT and Triomab.

[1274] One or more residues of the first fragment of the Fc region of the immunoglobulin comprise one or more amino acid substitutions suitable for knob-and-hole (KIH) dimerization with the second fragment of the Fc region of the immunoglobulin, and the second fragment of the Fc region of the immunoglobulin comprises one or more corresponding amino acid substitutions suitable for knob-and-hole (KIH) dimerization with the first fragment of the Fc region of the immunoglobulin. One or more residues of the second fragment of the Fc region of the immunoglobulin comprise one or more amino acid substitutions suitable for knob-and-hole (KIH) dimerization with the first fragment of the Fc region of the immunoglobulin, and the first fragment of the Fc region of the immunoglobulin comprises one or more corresponding amino acid substitutions suitable for knob-and-hole (KIH) dimerization with the second fragment of the Fc region of the immunoglobulin.

[1275] In one embodiment, the one or more amino acid substitutions are selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A, and Y407V. Those skilled in the art will know which amino acid substitutions represent "knobs" and which represent "holes," and therefore will know which mutations are suitable for use with corresponding mutations to promote KIH dimerization. For example, T366Y is a knob variant, and Y407T is a hole variant. When the first segment of the immunoglobulin Fc region comprises T366Y, the second segment of the immunoglobulin Fc region may comprise Y407T, and vice versa.

[1276] In one embodiment, the one or more amino acid substitutions are selected from the group consisting of T366Y and Y407T.

[1277] Any sequence of the recombinant fusion proteins disclosed herein may comprise any one or more amino acid substitutions selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A, and Y407V. Thus, SEQ ID NO: 145 (human Fc region) can be modified by incorporating any one or more amino acid substitutions selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A, and Y407V and incorporated into the recombinant fusion proteins described herein to replace the human Fc region sequence.

[1278] In one embodiment, the second specific antigen binding molecule is an immunoglobulin, an immunoglobulin Fab region, Fab', Fv, Fv-Fc, single-chain Fv (scFv), scFv-Fc, (scFv)2, a diabody, a triabody, a tetrabody, a bispecific T cell engager (BiTE), an intein, a VNAR domain, a single domain antibody (sdAb) or a VH domain.

[1279] The ROR1-specific antigen binding molecule may include any ROR1-specific antigen binding molecule disclosed herein. For example, the ROR1-specific antigen binding molecule may include G3CP, 1H8, or G3CPG4.

[1280] EGFR specific antigen binding molecules can include any EGFR specific antigen binding molecules disclosed herein.EGFR specific antigen binding molecules can, for example, include 7D12, EGFR#33, EGFR#13, 9G8, cetuximab or its derivatives, matuzumab or its derivatives, panitumumab or its derivatives, nimotuzumab or its derivatives, or neciximab or its derivatives.EGFR specific antigen binding molecules such as cetuximab, matuzumab, panitumumab, nimotuzumab or neciximab derivatives can be respectively cetuximab, matuzumab, panitumumab, nimotuzumab or neciximab humanized sequence.EGFR specific antigen binding molecules such as cetuximab, matuzumab, panitumumab, nimotuzumab or neciximab derivatives can be respectively comprising cetuximab, matuzumab, panitumumab, nimotuzumab or neciximab CDR scFv sequence. EGFR-specific antigen-binding molecules, such as derivatives of cetuximab, matuzumab, panitumumab, nimotuzumab, or necituximab, can be the Fab sequences of cetuximab, matuzumab, panitumumab, nimotuzumab, or necituximab, respectively. EGFR-specific antigen-binding molecules comprise Fab, and typically the EGFR-specific antigen-binding molecules will comprise Fab LC and Fab HC. Fab HC can be fused to a fragment of the immunoglobulin Fc region. Typically, Fab LC and Fab HC are associated via disulfide bonds. scFv, Fab LC, and / or Fab HC can be humanized. For example, the EGFR-specific antigen-binding molecule can comprise cetuximab Fab or a scFv based on cetuximab. The EGFR-specific antigen-binding molecule can comprise a humanized cetuximab Fab or a scFv based on humanized cetuximab. The EGFR-specific antigen-binding molecule can comprise 7D12, EGFR#33, EGFR#13, or 9G8.

[1281] In one embodiment:

[1282] (a) the first recombinant fusion protein comprises G3CP, 1H8 or G3CPG4, and

[1283] (b) The second recombinant fusion protein comprises 7D12, EGFR#33, EGFR#13 or 9G8.

[1284] The first recombinant fusion protein may comprise a heavy chain coupled to an Fc (optionally coupled to a [G4S] xThe first fragment of the immunoglobulin Fc region can be a first Fc heavy chain. The second fragment of the immunoglobulin Fc region can be a second Fc heavy chain. The one or more residues of the first Fc heavy chain may comprise one or more amino acid substitutions suitable for knob-and-hole (KIH) dimerization with the second Fc heavy chain, and the second Fc heavy chain comprises one or more corresponding amino acid substitutions suitable for knob-and-hole (KIH) dimerization with the first Fc heavy chain. One or more amino acid substitutions can be selected from the group consisting of T366Y and Y407T. The first Fc heavy chain may comprise S239C and / or S442C mutations. The second Fc heavy chain may comprise S239C and / or S442C mutations.

[1285] The second recombinant fusion protein may comprise a heavy chain coupled to an Fc (optionally coupled to a [G4S] x The EGFR-specific antigen-binding molecule (such as 7D12, EGFR#33, EGFR#13 or 9G8) fused to an immunoglobulin Fc region (e.g., a 5-mercaptoethanol linker). The first fragment of the immunoglobulin Fc region can be a first Fc heavy chain. The second fragment of the immunoglobulin Fc region can be a second Fc heavy chain. The one or more residues of the second Fc heavy chain may comprise one or more amino acid substitutions suitable for knob-and-hole (KIH) dimerization with the first Fc heavy chain, and the first Fc heavy chain comprises one or more corresponding amino acid substitutions suitable for knob-and-hole (KIH) dimerization with the second Fc heavy chain. One or more amino acid substitutions can be selected from the group consisting of T366Y and Y407T. The first Fc heavy chain may comprise S239C and / or S442C mutations. The second Fc heavy chain may comprise S239C and / or S442C mutations.

[1286] In one embodiment:

[1287] (a) the first recombinant fusion protein comprises G3CP-hFc, 1H8-hFc or G3CPG4-hFc, and

[1288] (b) The second recombinant fusion protein includes 7D12-hFc, EGFR#33-hFc, EGFR#13-hFc or 9G8-hFc.

[1289] In one embodiment:

[1290] (a) the first recombinant fusion protein comprises G3CP-hFc(S239C), 1H8-hFc(S239C) or G3CPG4-hFc(S239C), and

[1291] (b) The second recombinant fusion protein includes 7D12-hFc(S239C), EGFR#33-hFc(S239C), EGFR#13-hFc(S239C) or 9G8-hFc(S239C).

[1292] The recombinant fusion protein dimer may be selected from the group consisting of:

[1293] G3CP-hFc(S239C) and 7D12-hFc(S239C),

[1294] G3CP-hFc(S239C) and EGFR#33-hFc(S239C),

[1295] G3CP-hFc(S239C) and EGFR#13-hFc(S239C), and

[1296] G3CP-hFc(S239C) and 9G8-hFc(S239C);

[1297] wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, and wherein

[1298] (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution, and the hFc region fused to the EGFR-specific binding molecule comprises a Y407T substitution, or

[1299] (b) The hFc region fused to the ROR1-specific binding molecule comprises a Y407T substitution, and the hFc region fused to the EGFR-specific binding molecule comprises a T366Y substitution.

[1300] The recombinant fusion protein dimer may be selected from the group consisting of:

[1301] G3CPG4-hFc(S239C) and 7D12-hFc(S239C),

[1302] G3CPG4-hFc(S239C) and EGFR#33-hFc(S239C),

[1303] G3CPG4-hFc(S239C) and EGFR#13-hFc(S239C), and

[1304] G3CPG4-hFc(S239C) and 9G8-hFc(S239C);

[1305] wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, and wherein

[1306] (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution, and the hFc region fused to the EGFR-specific binding molecule comprises a Y407T substitution, or

[1307] (b) The hFc region fused to the ROR1-specific binding molecule comprises a Y407T substitution, and the hFc region fused to the EGFR-specific binding molecule comprises a T366Y substitution.

[1308] The recombinant fusion protein dimer may be selected from the group consisting of:

[1309] 1H8-hFc(S239C) and 7D12-hFc(S239C),

[1310] 1H8-hFc(S239C) and EGFR#33-hFc(S239C),

[1311] 1H8-hFc(S239C) and EGFR#13-hFc(S239C), and

[1312] 1H8-hFc(S239C) and 9G8-hFc(S239C);

[1313] wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, and wherein

[1314] (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution, and the hFc region fused to the EGFR-specific binding molecule comprises a Y407T substitution, or

[1315] (b) The hFc region fused to the ROR1-specific binding molecule comprises a Y407T substitution, and the hFc region fused to the EGFR-specific binding molecule comprises a T366Y substitution.

[1316] In one embodiment:

[1317] (a) the first recombinant fusion protein comprises G3CP-hFc(S442C), 1H8-hFc(S442C) or G3CPG4-hFc(S442C), and

[1318] (b) The second recombinant fusion protein includes 7D12-hFc(S442C), EGFR#33-hFc(S442C), EGFR#13-hFc(S442C) or 9G8-hFc(S442C).

[1319] The recombinant fusion protein dimer may be selected from the group consisting of:

[1320] G3CP-hFc(S442C) and 7D12-hFc(S442C),

[1321] G3CP-hFc(S442C) and EGFR#33-hFc(S442C),

[1322] G3CP-hFc(S442C) and EGFR#13-hFc(S442C), and

[1323] G3CP-hFc(S442C) and 9G8-hFc(S442C);

[1324] wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, and wherein

[1325] (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution, and the hFc region fused to the EGFR-specific binding molecule comprises a Y407T substitution, or

[1326] (b) The hFc region fused to the ROR1-specific binding molecule comprises a Y407T substitution, and the hFc region fused to the EGFR-specific binding molecule comprises a T366Y substitution.

[1327] The recombinant fusion protein dimer may be selected from the group consisting of:

[1328] G3CPG4-hFc(S442C) and 7D12-hFc(S442C),

[1329] G3CPG4-hFc(S442C) and EGFR#33-hFc(S442C),

[1330] G3CPG4-hFc(S442C) and EGFR#13-hFc(S442C), and

[1331] G3CPG4-hFc(S442C) and 9G8-hFc(S442C);

[1332] wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, and wherein

[1333] (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution, and the hFc region fused to the EGFR-specific binding molecule comprises a Y407T substitution, or

[1334] (b) The hFc region fused to the ROR1-specific binding molecule comprises a Y407T substitution, and the hFc region fused to the EGFR-specific binding molecule comprises a T366Y substitution.

[1335] The recombinant fusion protein dimer may be selected from the group consisting of:

[1336] 1H8-hFc(S442C) and 7D12-hFc(S442C),

[1337] 1H8-hFc(S442C) and EGFR#33-hFc(S442C),

[1338] 1H8-hFc(S442C) and EGFR#13-hFc(S442C), and

[1339] 1H8-hFc(S442C) and 9G8-hFc(S442C);

[1340] wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, and wherein

[1341] (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution, and the hFc region fused to the EGFR-specific binding molecule comprises a Y407T substitution, or

[1342] (b) The hFc region fused to the ROR1-specific binding molecule comprises a Y407T substitution, and the hFc region fused to the EGFR-specific binding molecule comprises a T366Y substitution.

[1343] In one embodiment:

[1344] (a) the first recombinant fusion protein comprises G3CP-hFc(S239C&S442C), 1H8-hFc(S239C&S442C) or G3CPG4-hFc(S239C&S442C), and

[1345] (b) The second recombinant fusion protein includes 7D12-hFc (S239C & S442C), EGFR#33-hFc (S239C & S442C), EGFR#13-hFc (S239C & S442C) or 9G8-hFc (S239C & S442C).

[1346] The recombinant fusion protein dimer may be selected from the group consisting of:

[1347] G3CP-hFc(S239C&S442C) and 7D12-hFc(S239C&S442C),

[1348] G3CP-hFc(S239C&S442C) and EGFR#33-hFc(S239C&S442C),

[1349] G3CP-hFc(S239C&S442C) and EGFR#13-hFc(S239C&S442C), and

[1350] G3CP-hFc (S239C & S442C) and 9G8-hFc (S239C & S442C);

[1351] wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, and wherein

[1352] (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution, and the hFc region fused to the EGFR-specific binding molecule comprises a Y407T substitution, or

[1353] (b) The hFc region fused to the ROR1-specific binding molecule comprises a Y407T substitution, and the hFc region fused to the EGFR-specific binding molecule comprises a T366Y substitution.

[1354] The recombinant fusion protein dimer may be selected from the group consisting of:

[1355] G3CPG4-hFc(S239C&S442C) and 7D12-hFc(S239C&S442C),

[1356] G3CPG4-hFc(S239C&S442C) and EGFR#33-hFc(S239C&S442C),

[1357] G3CPG4-hFc(S239C&S442C) and EGFR#13-hFc(S239C&S442C), and

[1358] G3CPG4-hFc (S239C&S442C) and 9G8-hFc (S239C&S442C);

[1359] wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, and wherein

[1360] (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution, and the hFc region fused to the EGFR-specific binding molecule comprises a Y407T substitution, or

[1361] (b) The hFc region fused to the ROR1-specific binding molecule comprises a Y407T substitution, and the hFc region fused to the EGFR-specific binding molecule comprises a T366Y substitution.

[1362] The recombinant fusion protein dimer may be selected from the group consisting of:

[1363] 1H8-hFc(S239C&S442C) and 7D12-hFc(S239C&S442C),

[1364] 1H8-hFc(S239C&S442C) and EGFR#33-hFc(S239C&S442C),

[1365] 1H8-hFc(S239C&S442C) and EGFR#13-hFc(S239C&S442C), and

[1366] 1H8-hFc(S239C&S442C) and 9G8-hFc(S239C&S442C);

[1367] wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, and wherein

[1368] (a) the hFc region fused to the ROR1-specific binding molecule comprises a T366Y substitution, and the hFc region fused to the EGFR-specific binding molecule comprises a Y407T substitution, or

[1369] (b) The hFc region fused to the ROR1-specific binding molecule comprises a Y407T substitution, and the hFc region fused to the EGFR-specific binding molecule comprises a T366Y substitution.

[1370] In an alternative embodiment, the above-mentioned recombinant fusion protein dimer may comprise any other ROR1-specific binding molecule described herein instead of G3CP, 1H8 or G3CPG4.

[1371] In an alternative embodiment, the above-mentioned recombinant fusion protein dimer may comprise any other EGFR-specific binding molecule described herein instead of 7D12, EGFR#33, EGFR#13 or 9G8.

[1372] Any recombinant fusion protein dimer disclosed herein can be associated with any linker and payload disclosed herein, and conjugation can be performed through any one or more S239C or S442C residues in the recombinant fusion protein dimer. Typically, when conjugation is performed through the S239C residue in the first hFc region of the recombinant fusion protein dimer, conjugation will also be performed through the S239C residue in the second hFc region of the recombinant fusion protein dimer. Typically, when conjugation is performed through the S442C residue in the first hFc region of the recombinant fusion protein dimer, conjugation will also be performed through the S442C residue in the second hFc region of the recombinant fusion protein dimer. Typically, when conjugation is performed through the S239C and S442C residues in the first hFc region of the recombinant fusion protein dimer, conjugation will also be performed through the S239C and S442C residues in the second hFc region of the recombinant fusion protein dimer.

[1373] According to a fifth aspect, the present invention provides a recombinant fusion protein dimer comprising:

[1374] (a) a first recombinant fusion protein, wherein the first recombinant fusion protein comprises a receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen binding molecule, and the receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen binding molecule comprises the amino acid sequence represented by formula (I):

[1375] FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4(I)

[1376] in

[1377] CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of GTRYGLYS (SEQ ID NO: 25), GTRYGLYSS (SEQ ID NO: 26), DTRYALYS (SEQ ID NO: 27), DTRYALYSS (SEQ ID NO: 28), GTKYGLYA (SEQ ID NO: 29), GTKYGLYAS (SEQ ID NO: 30), and DTSYGLYS (SEQ ID NO: 207);

[1378] FW1 is the framework region;

[1379] FW2 is the framework region;

[1380] HV2 is a hypervariable sequence having an amino acid sequence selected from the group consisting of SSDEERISIS (SEQ ID NO: 31), STDEERISIG (SEQ ID NO: 32), SPNKDRMIIG (SEQ ID NO: 33), STDKERIIIG (SEQ ID NO: 34), and TTDWERMSIG (SEQ ID NO: 208);

[1381] FW3a is the framework region;

[1382] HV4 is a hypervariable sequence having an amino acid sequence selected from the group consisting of NKGTK (SEQ ID NO: 35), NKGSK (SEQ ID NO: 36), NNGTK (SEQ ID NO: 37), NNRSK (SEQ ID NO: 38), and NKGAK (SEQ ID NO: 209);

[1383] FW3b is the framework region;

[1384] CDR3 is a CDR sequence having an amino acid sequence according to REARHPWLRQWY (SEQ ID NO: 39);

[1385] FW4 is the framework area,

[1386] and wherein the first antigen binding molecule is fused to a first fragment of an immunoglobulin Fc region, the first fragment of the immunoglobulin Fc region being engineered to dimerize with a second fragment of the immunoglobulin Fc region;

[1387] and

[1388] (b) a second recombinant fusion protein, wherein the second recombinant fusion protein comprises an epidermal growth factor receptor (EGFR)-specific antigen binding molecule fused to a second fragment of the immunoglobulin Fc region, and the second fragment of the immunoglobulin Fc region is engineered to dimerize with the first fragment of the immunoglobulin Fc region.

[1389] In one embodiment:

[1390] (a) the first recombinant fusion protein comprises P3A1, and

[1391] (b) The second recombinant fusion protein comprises 7D12, EGFR#33, EGFR#13 or 9G8.

[1392] In one embodiment:

[1393] (a) the first recombinant fusion protein comprises P3A1-hFc, and

[1394] (b) The second recombinant fusion protein comprises 7D12-hFc, EGFR#33-hFc, EGFR#13-hFc or 9G8-hFc.

[1395] In one embodiment:

[1396] (a) the first recombinant fusion protein comprises P3A1-hFc(S239C), and

[1397] (b) The second recombinant fus...

Claims

1. A recombinant fusion protein dimer, comprising: (a) a first recombinant fusion protein, wherein the first recombinant fusion protein comprises a receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen binding molecule, and the receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen binding molecule comprises an amino acid sequence represented by formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4(I) in CDR3 is selected from YPWGAGAPYNVQWY (SEQ ID NO: 23), YPWGAGAPYLVQWY (SEQ ID NO: 20), YPWGAGAPWNVQWY (SEQ ID NO: 24), YPSGAGAPRPVQWY (SEQ ID NO: 11), YPWGAGAPCLVQWY (SEQ ID NO: 11) NO: 12), YPWGAGAPRLVQWY (SEQ ID NO: 13), YPWGAGAPRQVQWY (SEQ ID NO: 14), YPWGAGAPRSVQWY (SEQ ID NO: 15), YPWGAGAPSLVQWY (SEQ ID NO: 16), YPWGAGAPSNVQWY (SEQ ID NO: 17), YPWGAGAPSQVQWY (SEQ ID NO: 18), YPWGAGAPSSVQWY (SEQ ID NO: 19), YPWGAGAPWQVQWY (SEQ ID CDR sequences of amino acid sequences in the group consisting of: YPWGAGAPWSVQWY (SEQ ID NO: 21), YPWGAGAPWSVQWY (SEQ ID NO: 22) and YPWGAGAPWLVQWY (SEQ ID NO: 10); CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of GANYGLAA (SEQ ID NO: 1), DANYGLAA (SEQ ID NO: 5), GANYDLSA (SEQ ID NO: 2), GANYGLSA (SEQ ID NO: 3), and GANYDLAA (SEQ ID NO: 4); FW1 is the framework region; FW2 is the framework region; HV2 is a hypervariable sequence having an amino acid sequence selected from the group consisting of SSNQERISIS (SEQ ID NO: 6) and SSNKERISIS (SEQ ID NO: 7); FW3a is the framework region; HV4 is a hypervariable sequence having an amino acid sequence selected from the group consisting of NKRTM (SEQ ID NO: 8) and NKGTM (SEQ ID NO: 9); FW3b is the framework region; FW4 is the framework region; wherein if CDR3 is YPWGAGAPWLVQWY (SEQ ID NO: 10), CDR1 is selected from the group consisting of DANYGLAA (SEQ ID NO: 5), GANYGLSA (SEQ ID NO: 3) and GANYDLAA (SEQ ID NO: 4), and wherein the first antigen binding molecule is fused to a first fragment of an immunoglobulin Fc region, said first fragment of the immunoglobulin Fc region being engineered to dimerize with a second fragment of the immunoglobulin Fc region; and (b) a second recombinant fusion protein, wherein the second recombinant fusion protein comprises an epidermal growth factor receptor (EGFR)-specific antigen binding molecule fused to a second fragment of an immunoglobulin Fc region, wherein the second fragment of the immunoglobulin Fc region is engineered to dimerize with the first fragment of the immunoglobulin Fc region.

2. The recombinant fusion protein dimer according to claim 1, wherein: (i) CDR1 is GANYGLAA (SEQ ID NO: 1), HV2 is SSNQERISIS (SEQ ID NO: 6), HV4 is NKRTM (SEQ ID NO: 8), and CDR3 is YPWGAGAPYNVQWY (SEQ ID NO: 23) or YPWGAGAPSSVQWY (SEQ ID NO: 19); and / or (ii) CDR1 is DANYGLAA (SEQ ID NO: 5), HV2 is SSNKERISIS (SEQ ID NO: 7), HV4 is NKGTM (SEQ ID NO: 9), and CDR3 is YPWGAGAPYNVQWY (SEQ ID NO: 23).

3. The recombinant fusion protein dimer according to claim 1, wherein: (i) FW1 is ASVNQTPRTATKETGESLTINCVVT (SEQ ID NO: 40), FW2 is TYWYRKNPG (SEQ ID NO: 43), FW3a is GRYVESV (SEQ ID NO: 44), FW3b is SFSLRIKDLTVADSATYYCKA (SEQ ID NO: 84), and FW4 is DGAGTVLTVN (SEQ ID NO: 48), and / or (ii) FW1 is TRVDQSPSSLSASVGDRVTITCVLT (SEQ ID NO:41), FW2 is TYWYRKNPG (SEQ ID NO:43), FW3a is GRYSESV (SEQ ID NO:45), FW3b is SFTLTISSLQPEDSATYYCRA (SEQ ID NO:46), and FW4 is DGAGTKVEIK (SEQ ID NO:49). 4 . The recombinant fusion protein dimer according to any one of claims 1 to 3 , wherein the ROR1-specific antigen binding molecule comprises SEQ ID NO: 50, SEQ ID NO: 61 or SEQ ID NO:

71.

5. A recombinant fusion protein dimer, comprising: (a) a first recombinant fusion protein, wherein the first recombinant fusion protein comprises a receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen binding molecule, and the receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen binding molecule comprises an amino acid sequence represented by formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4(I) in CDR1 is a CDR sequence having an amino acid sequence selected from the group consisting of GTRYGLYS (SEQ ID NO: 25), GTRYGLYSS (SEQ ID NO: 26), DTRYALYS (SEQ ID NO: 27), DTRYALYSS (SEQ ID NO: 28), GTKYGLYA (SEQ ID NO: 29), GTKYGLYAS (SEQ ID NO: 30), and DTSYGLYS (SEQ ID NO: 207); FW1 is the framework region; FW2 is the framework region; HV2 is a hypervariable sequence having an amino acid sequence selected from the group consisting of SSDEERISIS (SEQ ID NO: 31), STDEERISIG (SEQ ID NO: 32), SPNKDRMIIG (SEQ ID NO: 33), STDKERIIIG (SEQ ID NO: 34), and TTDWERMSIG (SEQ ID NO: 208); FW3a is the framework region; HV4 is a hypervariable sequence having an amino acid sequence selected from the group consisting of NKGTK (SEQ ID NO: 35), NKGSK (SEQ ID NO: 36), NNGTK (SEQ ID NO: 37), NNRSK (SEQ ID NO: 38), and NKGAK (SEQ ID NO: 209); FW3b is the framework region; CDR3 is a CDR sequence having an amino acid sequence according to REARHPWLRQWY (SEQ ID NO: 39); FW4 is the framework area. and wherein the first antigen binding molecule is fused to a first fragment of an immunoglobulin Fc region, said first fragment of the immunoglobulin Fc region being engineered to dimerize with a second fragment of the immunoglobulin Fc region; and (b) a second recombinant fusion protein, wherein the second recombinant fusion protein comprises an epidermal growth factor receptor (EGFR)-specific antigen binding molecule fused to a second fragment of an immunoglobulin Fc region, wherein the second fragment of the immunoglobulin Fc region is engineered to dimerize with the first fragment of the immunoglobulin Fc region.

6. The recombinant fusion protein dimer of claim 5, wherein CDR1 is DTSYGLYS (SEQ ID NO: 207), HV2 is TTDWERMSIG (SEQ ID NO: 208), HV4 is NKGAK (SEQ ID NO: 209), and CDR3 is REARHPWLRQWY (SEQ ID NO: 39).

7. The recombinant fusion protein dimer according to claim 5 or 6, wherein FW1 is TRVDQTPRTATKETGESLTINCVLT (SEQ ID NO: 220), FW2 is TSWFRKNPG (SEQ ID NO: 221), FW3a is GRYVESV (SEQ ID NO: 44), FW3b is SFSLRIKDLTVADSATYYCKA (SEQ ID NO: 84), and FW4 is DGAGTVLTVN (SEQ ID NO: 48). 8 . The recombinant fusion protein dimer according to any one of claims 5 to 7 , wherein the ROR1-specific antigen binding molecule comprises SEQ ID NO:

206.

9. The recombinant fusion protein dimer according to any one of claims 1 to 8, wherein the EGFR-specific antigen binding molecule comprises a single domain antibody.

10. The recombinant fusion protein dimer according to any one of claims 1 to 9, wherein the EGFR-specific antigen binding molecule comprises any one of SEQ ID NO: 210 to SEQ ID NO: 215, or an amino acid sequence having the following sequence for each sequence: (i) at least 85% identical to, and / or (ii) one, two or three amino acid substitutions relative thereto. 11 . The recombinant fusion protein dimer according to any one of claims 1 to 10 , wherein the EGFR-specific antigen binding molecule comprises any one of SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214 or SEQ ID NO:

215. 12 . The recombinant fusion protein dimer according to claim 1 , wherein the EGFR-specific antigen binding molecule comprises any one of SEQ ID NO: 210, SEQ ID NO: 212 or SEQ ID NO:

214.

13. The recombinant fusion protein dimer according to any one of claims 1 to 12, wherein the EGFR-specific antigen binding molecule is humanized. 14 . The recombinant fusion protein dimer according to claim 1 , wherein the EGFR-specific antigen binding molecule comprises any one or more of SEQ ID NO: 357 to SEQ ID NO:

362.

15. The recombinant fusion protein dimer according to any one of claims 1 to 8 and 14, wherein the EGFR-specific antigen binding molecule comprises: (i) SEQ ID NO: 357 and SEQ ID NO: 358, (ii) SEQ ID NO: 359, (iii) SEQ ID NO: 360 and SEQ ID NO: 361, or (iv) SEQ ID NO:

362.

16. A target binding molecule-drug conjugate comprising: (a) the recombinant fusion protein dimer according to any one of claims 1 to 15, and (b) at least one toxin selected from the group consisting of: ·Auristatin Anthracyclines, preferably PNU-derived anthracyclines Maytansine alkaloids · Amanitin derivatives, preferably α-amanitin derivatives ·Calichyma Tubulin inhibitors ·Duocarcin Radioisotopes - for example, alpha-emitting radionuclides such as 227Th and 225Ac Liposomes containing toxic payloads Protein toxins Taxanes ·Pyrrolidine Class and its dimer Indolebenzodiazepines Pseudodimer Spliceosome inhibitors CDK11 inhibitors Nicotinamide phosphoribosyltransferase inhibitors (NAMPTi) ·Pyridobenzodiazepine Class and its dimer Cyclopropylpyrroloindole (CPI), cyclopropylbenzidindole (CBI) or cyclopropylthienoindole (CTI) and optionally dimers thereof Irinotecan or exotecan and their derivatives.

17. The target binding molecule-drug conjugate of claim 16, wherein the toxin is auristatin, and (b) is an MMAE derivative, in, The target binding molecule-drug conjugate has the structure of formula (VI): [X] is an optional spacer selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and [L2] are optional linkers selected from the group consisting of valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), peptides, -(CH2)n-, -(CH2CH2O)n-, p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, any amino acid except glycine, and combinations thereof; and Y comprises the bispecific antigen binding molecule according to any one of claims 1 to 15.

18. The target binding molecule-drug conjugate of claim 16, wherein the toxin is auristatin, and (b) is an MMAE derivative, in, The target binding molecule-drug conjugate has the structure of formula (VIII):

19. The target binding molecule-drug conjugate according to claim 16, wherein (b) is an anthracycline (PNU) derivative, and in, The target binding molecule-drug conjugate has the structure of formula (III): wherein [X] is an optional spacer selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, one or more heteroatoms, polyethylene glycol, or a combination thereof; [L1] and [L2] are optional linkers selected from the group consisting of valine (Val), citrulline (Cit), alanine (Ala), asparagine (Asn), peptides, -(CH2)n-, -(CH2CH2O)n-, p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, any amino acid except glycine, and combinations thereof; and Y comprises the bispecific antigen binding molecule according to any one of claims 1 to 15.

20. The target binding molecule-drug conjugate according to claim 19, wherein the target binding molecule-drug conjugate has a structure selected from the following:

21. A recombinant fusion protein dimer according to any one of claims 1 to 15, or a target binding molecule-drug conjugate according to any one of claims 16 to 20, wherein one or more residues of the first fragment of the immunoglobulin Fc region include one or more amino acid substitutions suitable for knob-to-hole (KIH) dimerization with the second fragment of the immunoglobulin Fc region, and wherein one or more residues of the second fragment of the immunoglobulin Fc region include one or more amino acid substitutions suitable for knob-to-hole (KIH) dimerization with the first fragment of the immunoglobulin Fc region.

22. The recombinant fusion protein dimer or target binding molecule-drug conjugate according to any one of claims 1 to 21, wherein (i) the first fragment of the immunoglobulin Fc region comprises a T366Y substitution, and the second fragment of the immunoglobulin Fc region comprises a Y407T substitution, or (ii) the first fragment of the immunoglobulin Fc region comprises a Y407T substitution, and the second fragment of the immunoglobulin Fc region comprises a T366Y substitution.

23. The recombinant fusion protein dimer or target binding molecule-drug conjugate according to any one of claims 1 to 22, wherein the first fragment and / or the second fragment of the immunoglobulin Fc region each independently comprises S239C and / or S442C substitutions.

24. The recombinant fusion protein dimer or target binding molecule-drug conjugate according to any one of claims 1 to 23, wherein the ROR1 specific binding molecule and / or the EGFR specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker.

25. A recombinant fusion protein dimer or target binding molecule-drug conjugate according to any one of claims 1 to 24, wherein the first recombinant fusion protein comprises any one of SEQ ID NOs: 148, 167, 188, 189, 191, 192, 197 to 199, 203 to 205, 259 to 264, 302 to 307, 315 to 320, 327 to 332, 550 to 555, and 568 to 573, and the second recombinant fusion protein comprises any one of SEQ ID NOs: 265 to 276, 289 to 292, 333 to 344, 371, 372, 375, 376, 379, 380, 398, 399, 402, 403, 406, 407, and 590 to 593.

26. A recombinant fusion protein dimer or target binding molecule-drug conjugate according to any one of claims 1 to 24, wherein the first recombinant fusion protein comprises any one of SEQ ID NOs: 146, 147, 165, 166, 190, 193 to 196, 200 to 202, 253 to 258, 297 to 301, 308 to 314, 321 to 326, and 556 to 567, and the second recombinant fusion protein comprises any one of SEQ ID NOs: 277 to 288, 293 to 296, 345 to 356, 369, 370, 373, 374, 377, 378, 396, 397, 400, 401, 404, 405, and 594 to 597.

27. A recombinant fusion protein dimer or target binding molecule-drug conjugate according to any one of claims 1 to 26, wherein the recombinant fusion protein dimer or target binding molecule-drug conjugate comprises any one of SEQ ID NO: 369, 373, 377, 396, 400, 404, 371, 375, 379, 398, 402 and 406, and the recombinant fusion protein dimer or target binding molecule-drug conjugate also comprises SEQ ID NO:

360.

28. A recombinant fusion protein dimer or target binding molecule-drug conjugate according to any one of claims 1 to 25, wherein the first recombinant fusion protein comprises any one of SEQ ID NOs: 148, 191, 192, 197, 198, 199, 302, 303, 304, 305, 306 and 307, and the second recombinant fusion protein comprises any one of SEQ ID NOs: 265 to 268 and 333 to 336.

29. A recombinant fusion protein dimer or target binding molecule-drug conjugate according to any one of claims 1 to 24 and claim 27, wherein the first recombinant fusion protein comprises any one of SEQ ID NOs: 146, 147, 193, 194, 195, 196, 297, 298, 299, 300, 301 and 308, and the second recombinant fusion protein comprises any one of SEQ ID NOs: 277 to 280 and 345 to 348.

30. A recombinant fusion protein dimer or target binding molecule-drug conjugate according to any one of claims 1 to 25, wherein the first recombinant fusion protein comprises any one of SEQ ID NOs: 327 to 332 and 550 to 555, and the second recombinant fusion protein comprises any one of SEQ ID NOs: 269, 270, 271, 272, 337, 338, 339, 340, 379, 380, 406 and 407.

31. A recombinant fusion protein dimer or target binding molecule-drug conjugate according to any one of claims 1 to 24 and claim 27, wherein the first recombinant fusion protein comprises any one of SEQ ID NOs: 321 to 326 and 556 to 561, and the second recombinant fusion protein comprises any one of SEQ ID NOs: 281, 282, 283, 284, 349, 350, 351, 352, 377, 378, 404 and 405.

32. A recombinant fusion protein dimer or target binding molecule-drug conjugate according to any one of claims 1 to 25, wherein the first recombinant fusion protein comprises any one of SEQ ID NOs: 259 to 264 and 315 to 320, and the second recombinant fusion protein comprises any one of SEQ ID NOs: 273 to 276 and 341 to 344.

33. A recombinant fusion protein dimer or target binding molecule-drug conjugate according to any one of claims 1 to 24 and claim 27, wherein the first recombinant fusion protein comprises any one of SEQ ID NOs: 253 to 258 and 309 to 314, and the second recombinant fusion protein comprises any one of SEQ ID NOs: 285 to 288 and 353 to 356.

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