ROR1 / EGFR bispecific antigen-binding molecule

Abstract

The present invention relates to bispecific antigen-binding molecules having specificity 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 a further aspect, the present invention relates to conjugated immunoglobulin-like shark variable new antigen receptors (VNARs).

Description

Technical Field

[0001] The present invention relates to bispecific antigen-binding molecules that are 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 a further aspect, the present invention relates to conjugated immunoglobulin-like shark variable new antigen 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 frizzled domain (fz), and then a membrane-proximal kringle domain (kr) to which it is bound. The intracellular region of the protein contains a pseudokinase domain, followed by two Ser / Thr-rich domains interspersed with proline-rich regions, and this entire domain architecture is conserved in the closely related family member ROR2 and shares high sequence identity with it.

[0003] ROR1 is expressed during embryonic development and is prominently expressed in neural crest cells and in late developmental necrosis and interdigital webs. However, this expression is rapidly silenced after birth and is mostly absent in normal adult tissues. ROR1 expression has been observed at both the mRNA and protein levels across a wide range of solid tumors and hematological malignancies, such as lung, endometrial, pancreatic, ovarian, colorectal, head and neck, and prostate cancers, melanoma and renal cell carcinoma, breast cancer, and chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (AML). Furthermore, increased ROR1 expression has been reported to correlate with poor clinical outcomes for a number of cancer indications, such as breast cancer, ovarian cancer, colorectal cancer, lung adenocarcinoma, and CLL.

[0004] Consistent with its expression pattern and association with poor clinical prognosis, the functional role of ROR1 in tumorigenesis and disease progression has been demonstrated for a number of 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, which sustains EGFR signaling, suppresses apoptosis-promoting signaling, and an EGF-induced interaction between ROR1 and EGFR has been observed. Mining of breast cancer gene expression databases has revealed co-expression of EGFR and ROR1 mRNA. ROR1 has also been shown to act as a scaffold for maintaining caveolae structures and bypass signaling mechanisms that confer resistance to EGFR tyrosine kinase inhibitors. Signaling through the ROR1-HER3 complex modulates the Hippo-YAP pathway and promotes breast cancer bone metastasis, and this protein may promote Met-driven tumorigenesis. ROR1 expression is associated with chemotherapy resistance in breast cancer through activation of the Hippo-YAP / TAZ and BMI1 pathways. In contrast, in CLL, ROR1 has been reported to hetero-oligomerize with ROR2 in response to Wnt5a to transmit signaling and improve proliferation and migration.

[0005] Considering the functional role of ROR1 in cancer pathology and the general lack of expression in normal adult tissues, this cancer - fetal protein is an attractive target for cancer therapy. Antibodies against ROR1 are described in WO 2021097313 pamphlet (4A5 kipps), WO 2014031174 pamphlet (UC961), WO 2016187220 pamphlet (Five Prime), WO 2010124188 pamphlet (2A2), WO 2012075158 pamphlet (R11, R12), WO 2011054007 pamphlet (Oxford Bio), WO 2011079902 pamphlet (Bioinvent), WO 2017127664 pamphlet, WO 2017127664 pamphlet (NBE Therapeutics, SCRIPPS), WO 2016094847 pamphlet (Emergent), WO 2017127499 pamphlet). 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). Pre - clinical primate studies using 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) belongs to the ErbB family of receptor tyrosine kinases. EGFR is a 170 kDa transmembrane protein consisting of four extracellular domains, a transmembrane region, an intracellular tyrosine kinase domain, and a carboxy-terminal tail. The normal function of EGFR is associated with controlling the development of epithelial tissues, but it is also associated with many pathological conditions. In particular, overexpression of EGFR is associated with many cancers. Therefore, EGFR is an important drug target, and many therapeutic approaches have been applied. In addition to many small molecule-based EGFR inhibitors such as gefitinib, erlotinib, afatinib, brigatinib, icotinib, and osimertinib, numerous antibodies against EGFR have also been developed. Anti-EGFR antibodies, cetuximab, panitumumab, zalutumumab, nimotuzumab, and matuzumab. These antibodies block the extracellular ligand-binding domain and inhibit 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 series of proteins from distinct species. Immunoglobulin new antigen receptor (IgNAR) is a homodimeric heavy chain complex first found in the serum of nurse sharks (Ginglymostoma cirratum) and other sharks and rays. IgNAR does not contain a light chain and is distinct from the typical immunoglobulin structure. Each molecule consists of one single variable domain (VNAR) and five constant domains (CNAR). The nomenclature in the literature refers to IgNAR as immunoglobulin new antigen receptor or immunoglobulin new antigen receptor, and these terms are synonyms.

[0008] There are three main defined types of shark IgNAR known as types I, II, and III (Kovalena et al, Exp Opin Biol Ther 2014 14(10)1527-1539). These are classified based on the position of non-canonical cysteine residues that are under strong selective pressure and are therefore rarely replaced.

[0009] All three types have the classical immunoglobulin canonical cysteines at positions 35 and 107 that stabilize the standard immunoglobulin fold, together with the invariant tryptophan at position 36. The defined CDR2 itself is absent, but regions of sequence variation that more closely correspond to TCR HV2 and HV4 are defined in frameworks 2 and 3, respectively. Type I has germline-encoded cysteine residues in frameworks 2 and 4 and an even number of additional cysteines within CDR3. Crystal structure studies of type I IgNAR isolated from and complexed with lysozyme have allowed determination of the contribution of those cysteine residues. Both the framework 2 and 4 cysteines form disulfide bridges with cysteines in CDR3, forming a tightly packed structure in which the CDR3 loop dips down towards the HV2 region and is held tightly. So far, type I IgNAR has been identified only in the spiny dogfish, and all other elasmobranchs, including members of the same order, have only type II or a variation of this type.

[0010] Type II IgNAR is defined as having cysteine residues in CDR1 and CDR3 that form an intramolecular disulfide bond holding those two regions in proximity, resulting in a protruding CDR3 that promotes a binding pocket or groove. Type I sequences typically have a longer CDR3 than Type II, with an average of 21 and 15 residues respectively. This is thought to be due to strong selection pressure for two or more cysteine residues in Type I CDR3 to associate with their framework 2 and 4 counterparts. Studies on the accumulation of somatic mutations have shown that there are a large number of mutations in CDR1 of Type II compared to Type I, while the HV2 region of Type I shows greater sequence variation than Type II. This evidence correlates well with the positioning of those regions determined within the antigen-binding site. A third IgNAR type, known as Type III, has been identified in neonates. This member of the IgNAR family lacks diversity within CDR3 due to germline fusion of the D1 and D2 regions (which form CDR3) with the V gene. Almost all known clones have a CDR3 length of 15 residues and little or no sequence diversity.

[0011] Another structural form of VNAR, designated (IIb or IV) type, has only two canonical cysteine residues (in the framework 1 and framework 3b regions). So far, this type has been mainly found in dogfish and has also been isolated from a semi-synthetic V-NAR library derived from angelshark.

[0012] ROR1-specific antigen-binding molecules, such as VNAR, are described in WO 2019 / 122447 pamphlet, which is incorporated herein by reference in its entirety. In particular, WO 2019 / 122447 pamphlet describes the sequences of B1, P3A1 and P3A1 G1 specified below.

[0013] B1 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWLVQWYDGAGTVLTVN (SEQ ID NO: 113)

[0014] P3A1 TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVN (SEQ ID NO: 206)

[0015] P3A1 G1 TRVDQSPSSLSASVGDRVTITCVLTDTSYGLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 114)

[0016] WO 2019 / 122445 pamphlet describes an ROR1 / EGFR bispecific binding molecule, and the ROR1 binding molecule is as described in WO 2019 / 122447 pamphlet.

[0017] ROR1-specific antigen-binding molecules, such as conjugates of VNARs, are described in WO 2020 / 254640, which is hereby incorporated by reference in its entirety. WO 2020 / 254640 describes anthracycline (PNU) derivatives suitable for use in drug conjugates. Specifically, derivatives of PNU159682 are provided that lack the C14 carbon and attached hydroxyl functionality and in which the ethylenediamino (EDA) group forms part of the linker region between the C13 carbonyl of PNU159682 and the maleimide group. Alternatively, the same molecule may be described as EDA-PNU as the "warhead" such that the EDA group is not considered part of the linker region. When the linker contains val-cit-PAB, the maleimide group may be replaced with any reactive group suitable for the conjugation reaction. Such a payload may react with free thiol groups on another molecule. If the free thiol is present on a protein, a protein-drug conjugate (PDC) may be formed.

[0018] The anthracycline derivative PNU-159682 has been described as a metabolite of nemorubicin and has been reported to show extremely high potency for in vitro cell killing in the pico- to femtomolar range using one ovarian (A2780) and one breast cancer (MCF7) cell line (WO 2012 / 073217A1). Derivatives of PNU-159682 are also described in WO 2016 / 102679.

[0019] Conjugation of PNU-159682 derivatives to antibodies is described in WO 2009 / 099741, WO 2016 / 127081, WO 2016 / 102679, Yu et al, Clin. Cancer Res 2015, 21, 3298 and Stefan et al, Mol. Cancer. Ther., 2017, 16, 879.

[0020] Auristatin E (AE) and monomethyl auristatin E (MMAE) are synthetic analogs of dolastatin, a particular group of linear pseudopeptides originally isolated from marine organisms. Some of them have very potent cytotoxic activity against tumor cells. However, MMAE has the drawback of relatively high systemic toxicity. To improve tumor selectivity, MMAE is used in ADC settings for more targeted tumor therapy, particularly in combination with an enzymatically cleavable valine-citrulline linker (see, for example, WO 2005 / 081711). After proteolytic cleavage, MMAE is preferably released intracellularly from the corresponding ADC. Monomethyl auristatin F (MMAF) is an auristatin derivative having a C-terminal phenylalanine moiety. MMAF as well as various esters and their amide derivatives are disclosed in WO 2005 / 081711. Further auristatin analogs having a phenylalanine unit with an amide substitution at the C-terminus are described in WO 01 / 18032. WO 02 / 088172, WO 2007 / 008603, and WO 2007 / 008848 claim MMAF analogs related to side chain modification of phenylalanine, and MMAF analogs in which the carboxyl group of phenylalanine is modified. Auristatin conjugates linked via the C-terminus are described in WO 2009 / 117531, and further conjugates are described in WO 2013 / 087716.

[0021] ROR1-specific variant antigen-binding molecules having improved properties and conjugates thereof to derivatives of PNU-159682 are described in PCT / European Patent Application Publication No. 2021 / 086667, filed on December 17, 2021, which is incorporated herein by reference in its entirety. PCT / European Patent Application Publication No. 2021 / 086667 does not disclose any bispecific substances, including the ROR1-specific variant antigen-binding molecule and the EGFR-specific variant antigen-binding molecule of PCT / European Patent Application Publication No. 2021 / 086667.

Summary of the Invention

Means for Solving the Problems

[0022] The present invention generally relates to bispecific antigen-binding molecules. Specifically, the present invention relates to bispecific molecules having the ability to bind to both ROR1 and EGFR.

[0023] According to a first aspect, the present invention is a bispecific antigen-binding molecule, (i) Formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) (wherein, 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), YPWGAGAPWNVQWY (SEQ ID NO: 24), YPSGAGAPRPVQWY (SEQ ID NO: 11), YPWGAGAPCLVQWY (SEQ ID 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); 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 a framework region; FW2 is a 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 a 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 a framework region; FW4 is a framework region; When 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) A receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen-binding molecule comprising an amino acid sequence represented by; and (ii) An epidermal growth factor receptor (EGFR)-specific antigen-binding molecule A bispecific antigen-binding molecule comprising is provided.

[0024] According to a second aspect, the present invention is a bispecific antigen-binding molecule, (i) Formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) (Wherein, 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 a framework region; FW2 is a 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 a 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 a framework region; CDR3 is a CDR sequence having an amino acid sequence by REARHPWLRQWY (SEQ ID NO: 39); FW4 is a framework region) A receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen-binding molecule comprising the amino acid sequence represented by; and (ii) An epidermal growth factor receptor (EGFR)-specific antigen-binding molecule comprising, 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 the first fragment of the immunoglobulin Fc region, the EGFR-specific binding molecule is fused to the 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, A bispecific antigen-binding molecule is provided.

[0025] According to a third aspect, the present invention provides a recombinant fusion protein comprising the bispecific antigen-binding molecule according to the first or second aspect of the present invention.

[0026] According to a fourth aspect, the present invention is a recombinant fusion protein dimer, (a) A first recombinant fusion protein; The first recombinant fusion protein comprises a receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen-binding molecule comprising the amino acid sequence represented by formula (I); Formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) (wherein, 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), YPWGAGAPWNVQWY (SEQ ID NO: 24), YPSGAGAPRPVQWY (SEQ ID NO: 11), YPWGAGAPCLVQWY (SEQ ID 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); 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 a framework region; FW2 is a 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 a 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 a framework region; FW4 is a framework region; When 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 The first antigen-binding molecule is fused to a first fragment of an immunoglobulin Fc region that has been engineered to dimerize with a second fragment of the immunoglobulin Fc region; and (b) a second recombinant fusion protein; 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 that has been engineered to dimerize with a first fragment of the immunoglobulin Fc region; A recombinant fusion protein dimer comprising:

[0027] According to a fifth aspect, the present invention provides a recombinant fusion protein dimer, (a) a first recombinant fusion protein; The first recombinant fusion protein comprises a receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen-binding molecule comprising an amino acid sequence represented by formula (I); Formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) (wherein, 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 a framework region; FW2 is a 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 a 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 a framework region; CDR3 is a CDR sequence having an amino acid sequence by REARHPWLRQWY (SEQ ID NO: 39); FW4 is a framework region); and The first antigen-binding molecule is fused to a first fragment of the immunoglobulin Fc region that has been engineered to dimerize with a second fragment of the immunoglobulin Fc region; and (b) A second recombinant fusion protein; 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 that has been engineered to dimerize with a first fragment of the immunoglobulin Fc region; A recombinant fusion protein dimer comprising is provided.

[0028] According to a sixth aspect, the present invention provides an ROR1-specific chimeric antigen receptor (CAR) comprising at least one bispecific antigen-binding molecule defined by the first or second aspect of the present invention, at least one recombinant fusion protein defined by the third aspect of the present invention, or at least one recombinant fusion protein dimer defined by the fourth or fifth aspect of the present invention, which is fused or conjugated to at least one transmembrane region and at least one intracellular domain.

[0029] The present invention also provides a cell comprising the chimeric antigen receptor according to the sixth aspect, preferably an engineered T cell.

[0030] In a seventh aspect of the present invention, there is provided a nucleic acid sequence comprising a polynucleotide sequence encoding a bispecific antigen-binding molecule, a recombinant fusion protein, a recombinant fusion protein dimer or a chimeric antigen receptor according to the first, second, third, fourth, fifth or sixth aspect of the present invention.

[0031] There are also provided vectors containing the nucleic acid sequence according to the seventh aspect and host cells containing such nucleic acids.

[0032] A method for preparing a bispecific antigen-binding molecule, a recombinant fusion protein, a recombinant fusion protein dimer or a chimeric antigen receptor according to the first, second, third, fourth, fifth or sixth aspect, comprising culturing or maintaining a host cell containing the polynucleotide or vector under conditions such that the host cell produces a bispecific antigen-binding molecule, a recombinant fusion protein or a chimeric antigen receptor, and optionally further comprising isolating the specific antigen-binding molecule, recombinant fusion protein, recombinant fusion protein dimer or chimeric antigen receptor.

[0033] In an eighth aspect of the present invention, there is provided a pharmaceutical composition comprising a bispecific antigen-binding molecule, a fusion protein, a recombinant fusion protein dimer or a chimeric antigen receptor according to the first, second, third, fourth, fifth or sixth aspect. The pharmaceutical composition may contain various pharmaceutically acceptable carriers. The pharmaceutical composition of the present invention may be for administration by any suitable method known in the art, by way of example and not limitation, intravenous, intramuscular, oral, intraperitoneal or topical administration. In a preferred embodiment, the pharmaceutical composition may be prepared in the form of a solution, gel, powder, tablet, capsule or foam.

[0034] The bispecific antigen-binding molecules, recombinant fusion proteins, recombinant fusion protein dimers or chimeric antigen receptors of the 1st, 2nd, 3rd, 4th, 5th or 6th aspect may be for use in a method of treatment. More specifically, the bispecific antigen-binding molecules, recombinant fusion proteins, recombinant fusion protein dimers or chimeric antigen receptors of the 1st, 2nd, 3rd, 4th, 5th or 6th aspect may be for use in the treatment of cancer. Preferably, the cancer is an ROR1-positive cancer type and / or an EGFR-positive cancer type. More preferably, the cancer is selected from the group comprising blood cancers such as lymphoma and leukemia, 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, renal 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.

[0035] Also provided herein is the use of a bispecific antigen-binding molecule, recombinant fusion protein, recombinant fusion protein dimer or chimeric antigen receptor of the 1st, 2nd, 3rd, 4th, 5th or 6th aspect in the manufacture of a medicament for use in treating a disease in a patient in need thereof.

[0036] The bispecific antigen-binding molecules, recombinant fusion proteins, recombinant fusion protein dimers or chimeric antigen receptors of the 1st, 2nd, 3rd, 4th, 5th, 6th aspect or the pharmaceutical composition of the 8th aspect may be administered in a single dose. As used herein, "single dose" refers to a dosing regimen consisting of one dose. Alternatively, a multiple-dose regimen may be used. Without being bound by theory, the advantages of the specific binding molecules, recombinant fusion proteins, recombinant fusion protein dimers or chimeric antigen receptors of the 1st, 2nd, 3rd, 4th, 5th, 6th aspect or the pharmaceutical composition of the 8th aspect may be particularly apparent when administered in a single dose.

[0037] Furthermore, according to the present invention, there is provided a method of treating a disease in a patient in need thereof, the method comprising administering to the patient a therapeutically effective dose of a bispecific antigen-binding molecule of the first, second, third, fourth, fifth or sixth aspect, a recombinant fusion protein, a recombinant fusion protein dimer or a chimeric antigen receptor, or a pharmaceutical composition of the eighth aspect.

[0038] Preferably, the cancer is an 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 lymphoma and leukemia, 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, renal 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.

[0039] Also provided herein is a method of assaying for the presence of a target analyte in a sample, the method comprising adding to the sample a bispecific antigen-binding molecule of the first or second aspect, or a recombinant fusion protein of the third aspect, or a recombinant fusion protein dimer of the fourth or fifth aspect, which is detectably labeled, and detecting the binding of the molecule to the target analyte.

[0040] Further provided herein is a method of imaging a disease site in a subject, the method comprising administering to the subject a bispecific antigen-binding molecule of the first or second aspect, which is detectably labeled, or a recombinant fusion protein of the third aspect, which is detectably labeled, or a recombinant fusion protein dimer of the fourth or fifth aspect.

[0041] Also provided herein are methods of diagnosing a disease or medical condition in a subject, the methods comprising administering a bispecific antigen-binding molecule of the first or second aspect, or a recombinant fusion protein of the third aspect, or a recombinant fusion protein dimer of the fourth or fifth aspect.

[0042] Also contemplated herein are bispecific antigen-binding molecules comprising an antibody, antibody fragment, or antigen-binding molecule that competes for binding to ROR1 with an ROR1-specific antigen-binding molecule of the first or second aspect. The term "competes," as used in connection with an antigen-binding protein (e.g., a neutralizing antigen-binding protein or neutralizing antibody), means that the antigen-binding protein under test (e.g., an antibody or a functional fragment thereof) competes with an antigen-binding molecule as defined herein (e.g., a specific antigen-binding molecule of the first aspect) for binding to a common antigen (e.g., ROR1 in the case of a specific antigen-binding molecule of the first or second aspect), as determined by an assay in which the test antigen-binding protein interferes with or inhibits the specific binding of the antigen-binding molecule to the common antigen.

[0043] Also described herein are kits for diagnosing a subject having or at risk of having cancer, or for providing a prognostic diagnosis of a subject's medical condition, the kits comprising means for detecting the concentration of an antigen present in a sample from the test subject, the means for detecting comprising a bispecific antigen-binding molecule of the first or second aspect, a recombinant fusion protein of the third aspect, or a recombinant fusion protein dimer of the fourth or fifth aspect, each optionally derivatized, a chimeric antigen receptor of the sixth aspect, or a nucleic acid sequence of the seventh aspect, wherein the presence of the antigen in the sample suggests that the subject has cancer. Preferably, the antigen is the ROR1 protein, more preferably comprising its extracellular domain. More preferably, the kit is used to identify the presence or absence of ROR1-positive cells in the sample or to determine its concentration in the sample. The kit may also include a positive control and / or a negative control to which the assay is compared and / or a label that can be detected.

[0044] The present invention provides a method for diagnosing a subject suffering from cancer or having a susceptibility thereto, or for providing a prognostic diagnosis of the disease state of a subject, which method comprises detecting the concentration of an antigen present in a sample obtained from the subject, and the detection is achieved using a bispecific antigen-binding molecule of the first or second aspect, each optionally derivatized, a recombinant fusion protein of the third aspect, or a recombinant fusion protein dimer of the fourth or fifth aspect, a chimeric antigen receptor of the sixth aspect, or a nucleic acid sequence of the seventh aspect, and the presence of the antigen in the sample suggests that the subject is suffering from cancer.

[0045] Also contemplated herein is a method of killing or inhibiting the growth of cells expressing ROR1 in vitro or in a patient, which method comprises administering to the cells a pharmaceutically effective amount or dose of (i) a bispecific antigen-binding molecule of the first or second aspect, a recombinant fusion protein of the third aspect, or a recombinant fusion protein dimer of the fourth or fifth aspect, a nucleic acid sequence of the seventh aspect, or a CAR or cell according to the sixth aspect, or (ii) a pharmaceutical composition of the eighth aspect. Preferably, the cells expressing ROR1 are cancer cells. More preferably, ROR1 is human ROR1.

[0046] According to a ninth aspect, the present invention provides a bispecific antigen-binding molecule comprising an amino acid sequence represented by formula (II): X-FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4-Y (II) (wherein FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 is an ROR1-specific antigen-binding molecule according to the first or second aspect, X and Y are optional amino acid sequences) and wherein the ROR1-specific antigen-binding molecule is conjugated to a second moiety and further comprises an EGFR-specific antigen-binding molecule.

[0047] According to a tenth aspect, the present invention provides (a) A bispecific antigen-binding molecule according to the first, second or ninth aspect, or a recombinant fusion protein according to the third aspect, or a recombinant fusion protein dimer according to the fourth or fifth aspect, and (b) at least one toxin to provide a target-binding molecule-drug conjugate.

Brief Description of the Drawings

[0048]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27

Figure 28

Figure 29

Figure 30

Figure 31

Figure 32

Figure 33

Figure 34

Figure 35

Figure 36

Figure 37

Figure 38

Figure 39

Figure 40

Figure 41

Figure 42

Mode for Carrying Out the Invention

[0049] The present invention generally relates to bispecific antigen-binding molecules. Specifically, the present invention provides immunoglobulin-like shark variable new antigen receptors (VNARs) specific for receptor tyrosine kinase-like orphan receptor 1 (ROR1), as well as related fusion proteins, chimeric antigen receptors, conjugates, and nucleic acids, and methods associated therewith. The ROR1-specific VNAR domain is described herein as an ROR1-specific antigen-binding molecule.

[0050] The novel or new antigen receptor (IgNAR) is an approximately 160 kDa homodimeric protein found in the serum of cartilaginous fish (Greenberg A.S., et al., Nature, 1995. 374(6518): p. 168-173, Dooley, H., et al, Mol. Immunol, 2003. 40(1): p. 25-33; Mueller, M.R., 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 that has structural similarity and some sequence similarity with immunoglobulin and T cell receptor variable domains and cell adhesion molecules, and is named VNAR due to its similarity with the N-variable terminal domains of classical immunoglobulins and T cell receptors. VNAR shares limited sequence homology with immunoglobulins, for example, 25-30% similarity between VNAR and human light chain sequences.

[0051] 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 provide an overview of the structural characterization and generation of VNAR, which are incorporated herein by reference.

[0052] VNAR is not thought to have evolved from the ancestors of classical immunoglobulin antibodies. The distinct structural features of VNAR are the truncation of a sequence equivalent to the CDR2 loop present in conventional immunoglobulin variable domains and the absence of hydrophobic VH / VL interface residues that normally enable association with the light chain domain and that are not present in the IgNAR structure. Furthermore, unlike classical immunoglobulins, some VNAR subtypes contain special cysteine residues in the CDR regions, and it has been observed that they form disulfide bridges in addition to the canonical immunoglobulin superfamily cross-links between cysteines in the framework 1 and 3 regions adjacent to the N-terminus in CDR1 and 3.

[0053] To date, there are three defined types of shark IgNAR known as types I, II, and III. These are classified based on the positions of non-canonical cysteine residues that are under strong selective pressure and thus are rarely replaced.

[0054] All three types have the classical immunoglobulin canonical cysteines at positions 35 and 107 (numbering as in Kabat, E. A. et al. Sequences of proteins of immunological interest. 5th ed. 1991, Bethesda: US Dept. of Health and Human Services, PHS, NIH) that stabilize the standard immunoglobulin fold, along with the invariant tryptophan at position 36. The defined CDR2 itself is absent, but regions of sequence variation that more closely correspond to TCR HV2 and HV4 are defined in frameworks 2 and 3, respectively. Type I has germline-encoded cysteine residues in frameworks 2 and 4 and an even number of additional cysteines within CDR3. Crystal structure studies of type I IgNAR isolated from and complexed with lysozyme have allowed determination of the contribution of those cysteine residues. Both the framework 2 and 4 cysteines form disulfide bridges with cysteines in CDR3, forming a tightly packed structure in which the CDR3 loop dips down towards the HV2 region and is held tightly. To date, type I IgNAR has only been identified in the thresher shark, and all other elasmobranchs, including members of the same order, have only type II or variations of this type.

[0055] Type II IgNAR is defined as having cysteine residues in CDR1 and CDR3 that form an intramolecular disulfide bond holding these two regions in proximity, resulting in a protruding CDR3 that promotes a binding pocket or groove. Type I sequences typically have a longer CDR3 than Type II, with an average of 21 and 15 residues respectively. This is thought to be due to strong selective pressure for two or more cysteine residues in Type I CDR3 to associate with their framework 2 and 4 counterparts. Studies on the accumulation of somatic mutations have shown that there are a large number of mutations in CDR1 of Type II compared to Type I, while the HV2 region of Type I shows greater sequence variation than Type II. This evidence correlates well with the positioning of those regions determined within the antigen-binding site.

[0056] A third IgNAR type, known as Type III, has been identified in neonates. This member of the IgNAR family lacks diversity within CDR3 due to germline fusion of the D1 and D2 regions (which form CDR3) with the V gene. Almost all known clones have a 15-residue CDR3 length and little or no sequence diversity.

[0057] Another structural form of VNAR, designated (IIb or IV) type, has only two canonical cysteine residues (in the framework 1 and framework 3b regions). To date, this type has been mainly found in dogfish and has also been isolated from a semi-synthetic V-NAR library derived from nurse sharks.

[0058] The VNAR binding surface, unlike the variable domains in other natural immunoglobulins, is derived from four diversity regions: CDR1, HV2, HV4, and CDR3, which are linked by intervening framework sequences in the order FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4. The combination of the absence of a natural light chain partner and the absence of CDR2 results in VNAR being the smallest naturally occurring binding domain in the vertebrate kingdom.

[0059] IgNAR shares some incidental features with heavy chain antibodies (HCAb) found in camels (camels, dromedaries and llamas). Unlike IgNAR, HCAb is clearly derived from the immunoglobulin family and shares significant sequence homology with standard immunoglobulins. Importantly, one important distinction of VNAR is that the molecule, unlike classical immunoglobulins or HCAb, never had a partner light chain at any point in its evolution. Flajnik M.F. et al PLoS Biol 2011.9(8):e1001120 and Zielonka S. et al mAbs 2015.7(1):p.15-25 have shown views on the similarities and differences between VNAR and immunoglobulin-derived VHH single binding domains from camels and their presumably distinct evolutionary origins.

[0060] Antibodies against ROR1 have been reported in the literature, but the high sequence identity between the extracellular domains of human, mouse and rat ROR1 and between human ROR1 and ROR2 family members means that it is not easy to generate high-affinity hROR1-specific binding agents. Furthermore, the large size of antibodies impairs their ability to penetrate solid tumors, making regions of the target protein inaccessible due to steric factors, which can be particularly serious for cell surface proteins where oligomerization or receptor clustering is observed.

[0061] As a result, there is a need in the art for the development of improved anti-ROR1 binding protein agents having functional or physical characteristics or properties different from antibodies, as well as 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 described herein.

[0062] Although not bound by theory, the presently described ROR1-specific antigen-binding molecules are thought to bind to both human and mouse ROR1. A number of variants, such as 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 P3A1G1 NAG8, have been experimentally confirmed to bind to both hROR1 and mROR1. Further, the ROR1-specific antigen-binding molecules described herein may bind to the deglycosylated form of ROR1. Further, these cannot bind to a number of linear peptides associated with anti-ROR1 antibodies described in the prior art. Thus, the presently described ROR1-specific antigen-binding molecules are thought to bind to distinct epitopes in the ROR1 sequence as compared to their prior art anti-ROR1 antibodies.

[0063] Binding and internalization of ROR1-specific antigen-binding molecules to cancer cell lines has been demonstrated. This supports the potential for the use of such molecules in the treatment of cancer, specifically cancers that express ROR1.

[0064] Various forms of ROR1-specific antigen-binding molecules are described, including several types of fusion proteins. Also described are fusion proteins containing the immunoglobulin Fc region as well as both homo- and hetero-dimers. Fusion of a protein to the Fc domain can improve protein solubility and stability, significantly increase plasma half-life, and improve overall therapeutic efficacy.

[0065] The inventors have also created VNAR molecules conjugated to various moieties and payloads. Accordingly, the present application also discloses chemically conjugated VNARs. More specifically, ROR1-specific antigen-binding molecules in several conjugate formats are provided.

[0066] The ROR1-specific antigen-binding molecule described in this specification is formulated in combination with an EGFR-specific binding molecule. Accordingly, the present invention relates to an ROR1 / EGFR bispecific antigen-binding molecule.

[0067] According to a first aspect, the present invention is a bispecific antigen-binding molecule, (i) Formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) (wherein, 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), YPWGAGAPWNVQWY (SEQ ID NO: 24), YPSGAGAPRPVQWY (SEQ ID NO: 11), YPWGAGAPCLVQWY (SEQ ID 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); 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 a framework region; FW2 is a 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 a 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 a framework region; FW4 is a framework region; When 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) A receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen-binding molecule comprising an amino acid sequence represented by; and (ii) An epidermal growth factor receptor (EGFR)-specific antigen-binding molecule Provided is a bispecific antigen-binding molecule comprising.

[0068] In one embodiment of the ROR1-specific antigen-binding molecule, 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), YPWGAGAPWNVQWY (SEQ ID NO: 24), YPSGAGAPRPVQWY (SEQ ID NO: 11), YPWGAGAPCLVQWY (SEQ ID 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 NO: 22), and YPWGAGAPWLVQWY (SEQ ID NO: 10).

[0069] When the CDR3 is not YPWGAGAPWLVQWY (SEQ ID NO: 10), the CDR1 can 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). Accordingly, the ROR1-specific antigen-binding molecule has the formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) (wherein, the 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), YPWGAGAPWNVQWY (SEQ ID NO: 24), YPSGAGAPRPVQWY (SEQ ID NO: 11), YPWGAGAPCLVQWY (SEQ ID 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), and YPWGAGAPWSVQWY (SEQ ID NO: 22); the 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 a framework region; FW2 is a 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 a 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 a framework region; FW4 is a framework region) can be defined as including the amino acid sequence represented by.

[0070] In one embodiment of the ROR1-specific antigen-binding molecule, 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 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).

[0071] In one embodiment of the ROR1-specific antigen-binding molecule, CDR3 is a CDR sequence having the amino acid sequence of YPWGAGAPYNVQWY (SEQ ID NO: 23).

[0072] In one embodiment of the ROR1-specific antigen-binding molecule, CDR3 is a CDR sequence having the amino acid sequence of YPWGAGAPYNVQWY (SEQ ID NO: 23); CDR1 is a CDR sequence having the amino acid sequence of GANYGLAA (SEQ ID NO: 1); HV2 is a hypervariable sequence having the amino acid sequence of SSNQERISIS (SEQ ID NO: 6); and HV4 is a hypervariable sequence having the amino acid sequence of NKRTM (SEQ ID NO: 8).

[0073] In one embodiment of the ROR1-specific antigen-binding molecule, CDR3 is a CDR sequence having the amino acid sequence of YPWGAGAPYNVQWY (SEQ ID NO: 23); CDR1 is a CDR sequence having the amino acid sequence by DANYGLAA (SEQ ID NO: 5); HV2 is a hypervariable sequence having the amino acid sequence by SSNKERISIS (SEQ ID NO: 7); and HV4 is a hypervariable sequence having the amino acid sequence by NKGTM (SEQ ID NO: 9).

[0074] In one embodiment of the ROR1-specific antigen-binding molecule, CDR3 is a CDR sequence having the amino acid sequence by YPWGAGAPWLVQWY (SEQ ID NO: 10); CDR1 is a CDR sequence having the amino acid sequence by DANYGLAA (SEQ ID NO: 5); HV2 is a hypervariable sequence having the amino acid sequence by SSNKERISIS (SEQ ID NO: 7); and HV4 is a hypervariable sequence having the amino acid sequence by NKGTM (SEQ ID NO: 9).

[0075] In a preferred embodiment, the ROR1-specific antigen-binding molecule is referred to herein as G3CP ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVN (SEQ ID NO: 50);

[0076] referred to herein as B1G4 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIK (SEQ ID NO: 51);

[0077] referred to herein as 1E2 ASVNQTPRTATKETGESLTINCVVTGANYDLSATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPSGAGAPRPVQWYDGAGTVLTVN (SEQ ID NO: 52);

[0078] referred to as 1E5 in this specification ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPCLVQWYDGAGTVLTVN (SEQ ID NO: 53);

[0079] referred to as 1B11 in this specification ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPRLVQWYDGAGTVLTVN (SEQ ID NO: 54);

[0080] referred to as C3CP in this specification ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPRQVQWYDGAGTVLTVN (SEQ ID NO: 55);

[0081] referred to as 2G5 in this specification ASVNQTPRTATKETGESLTINCVVTGANYGLSATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPRSVQWYDGAGTVLTVN (SEQ ID NO: 56);

[0082] referred to as 1G12 in this specification ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPRSVQWYDGAGTVLTVN (SEQ ID NO: 57);

[0083] referred to herein as G5CP ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSLVQWYDGAGTVLTVN (SEQ ID NO: 58);

[0084] referred to herein as 2F4 ASVNQTPRTATKETGESLTINCVVTGANYGLSATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSNVQWYDGAGTVLTVN (SEQ ID NO: 59);

[0085] referred to herein as 1G9 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSQVQWYDGAGTVLTVN(SEQ ID NO: 60);

[0086] referred to herein as 1H8 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVN(SEQ ID NO: 61);

[0087] referred to herein as G11CP ASVNQTPRTATKETGESLTINCVVTGANYGLSATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWLVQWYDGAGTVLTVN (SEQ ID NO: 62);

[0088] referred to herein as D9CP ASVNQTPRTATKETGESLTINCVVTGANYDLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWLVQWYDGAGTVLTVN (SEQ ID NO: 63);

[0089] referred to herein as 1B6 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWNVQWYDGAGTVLTVN (SEQ ID NO: 64);

[0090] referred to herein as 1F10 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWQVQWYDGAGTVLTVN (SEQ ID NO: 65);

[0091] referred to herein as E6CP ASVNQTPRTATKETGESLTINCVVTGANYGLSATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWQVQWYDGAGTVLTVN (SEQ ID NO: 66);

[0092] referred to herein as F2CP ASVNQTPRTATKETGESLTINCVVTGANYDLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWQVQWYDGAGTVLTVN (SEQ ID NO: 67);

[0093] In this specification, it is referred to as B6CP ASVNQTPRTATKETGESLTINCVVTGANYDLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWSVQWYDGAGTVLTVN (SEQ ID NO: 68);

[0094] In this specification, it is referred to as 1G1 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWSVQWYDGAGTVLTVN (SEQ ID NO: 69); and

[0095] In this specification, it is referred to as A10CP ASVNQTPRTATKETGESLTINCVVTGANYGLSATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPWSVQWYDGAGTVLTVN (SEQ ID NO: 70) An amino acid sequence selected from the group consisting of or CDR1, HV2, HV4, and CDR2 sequences by any of them, and having at least 45% combined sequence identity with any combination of FW1, FW2, FW3a, FW3b, and FW4 sequences of any of them, including functional variants having FW1, FW2, FW3a, FW3b, and FW4 sequences.

[0096] In a particularly preferred embodiment, the ROR1-specific antigen-binding molecule is It may contain the amino acid sequence according to ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVN (SEQ ID NO: 50). It may contain the amino acid sequence according to .

[0097] The ROR1-specific antigen-binding molecule may include the amino acid sequence according to ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVN (SEQ ID NO: 50). or its functional variant having the CDR1, HV2, HV4 and CDR2 sequences according to SEQ ID NO: 50 and having at least 45% combined sequence identity with the combined FW1, FW2, FW3a, FW3b and FW4 sequences of SEQ ID NO: 50.

[0098] Specific advantages associated with SEQ ID NO: 50 ("G3CP") and its functional variants include increased expression yield and hydrophilicity in suboptimal aqueous buffer systems for these proteins, as well as increased ease of analysis, purification, and monomericity. Without being bound by theory, these advantages may be particularly evident in VNAR-hFc fusion proteins containing the G3CP sequence or its functional variants. Thus, the G3CP sequence and its functional variants may provide improved manufacturing and / or handling characteristics. Furthermore, G3CP-hFc exhibits excellent in vivo efficacy in a patient-derived xenograft model of triple-negative breast cancer (TNBC) when conjugated to a cytotoxic anthracycline (PNU) derivative. Surprisingly, the effect of G3CP-hFc is also improved relative to B1-hFc, which itself exhibits excellent in vivo efficacy. Additionally, as shown herein, ROR1xEGFR bispecificity containing G3CP has a shorter retention time (RT) in size-exclusion chromatography (SEC) compared to B1 containing bispecificity, indicating improved hydrophilicity of ROR1xEGFR bispecificity containing G3CP. Further, in a PBS stability test, ROR1xEGFR bispecificity containing G3CP may reduce turbidity and the formation of high molecular weight (HMW) species compared to B1 containing bispecificity. This indicates increased ease of analysis, purification, and monomericity of ROR1xEGFR bispecificity containing G3CP in suboptimal aqueous buffer systems. Thus, the G3CP sequence and its functional variants may provide improved manufacturing and / or handling characteristics retained in ROR1xEGFR bispecific formulations.

[0099] Although not bound by theory, ROR1xEGFR bispecificity (including but not limited to G3CP sequences) may improve efficacy even when ROR1 monospecificity is refractory or low in specificity. For example, data showing co-expression of ROR1 and EGFR in TNBC (this application also includes data showing excellent in vivo efficacy of G3CP-hFc PNU conjugate in a patient-derived xenograft model of TNBC), as well as data showing high expression of EGFR along with ROR1 expression in a non-small cell lung cancer model by Western blotting are disclosed herein. Co-expression of ROR1 and EGFR has also been confirmed in models of large cell lung cancer, head and neck cancer, esophageal cancer, renal cancer, gastric cancer, sarcoma, pancreatic cancer, and colorectal cancer. In a patient-derived xenograft model of TNBC, the surprisingly improved effect of the G3CP-hFc PNU conjugate compared to B1-hFc PNU, in conjunction with the in vitro data disclosed herein, demonstrates the therapeutic efficacy of the currently claimed ROR1xEGFR bispecificity in the potential treatment of cancers co-expressing ROR1 and EGFR as confirmed herein.

[0100] In a particularly preferred embodiment, the ROR1-specific antigen-binding molecule may comprise the amino acid sequence according to ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVN (SEQ ID NO: 61).

[0101] Specific advantages associated with SEQ ID NO: 61 (“1H8”) and its functional variants include good stability in PBS, which is improved compared to B1-hFc under the same conditions when measured by SEC (t = 0 vs 96 h), at least in the 1H8-hFc fusion. Similarly, an improvement in 1H8xEGFR bispecificity compared to B1xEGFR bispecificity has been observed in the data disclosed herein.

[0102] In a particularly preferred embodiment, the ROR1-specific antigen-binding molecule is TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIK (SEQ ID NO: 51) may include the amino acid sequence according to

[0103] Specific advantages associated with SEQ ID NO: 51 ("B1G4") and its functional variants include increased expression yields and monomericity in aqueous buffer systems for fusion proteins, such as VNAR-hFc fusion proteins, containing the B1G4 sequence or its functional variants. Thus, the B1G4 sequence and its functional variants may provide fusion proteins with improved manufacturing and / or handling characteristics.

[0104] The ROR1-specific antigen-binding molecule is TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIK (SEQ ID NO: 51) has the amino acid sequence according to or the CDR1, HV2, HV4 and CDR2 sequences according to SEQ ID NO: 51, and may include its functional variants having FW1, FW2, FW3a, FW3b and FW4 sequences with at least 45% combinatorial sequence identity to the combinatorial FW1, FW2, FW3a, FW3b and FW4 sequences of SEQ ID NO: 51.

[0105] In a preferred embodiment, the ROR1-specific antigen-binding molecule is referred to herein as G3CP G4 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIK (SEQ ID NO: 71);

[0106] referred to herein as G3CP V15 ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPYNVQWYDGQGTKLEVK (SEQ ID NO: 72);

[0107] referred to herein as 1H8 G4 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIK (SEQ ID NO: 73);

[0108] referred to herein as 1H8 V15 ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVK (SEQ ID NO: 74);

[0109] referred to herein as C3CP G4 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPRQVQWYDGAGTKVEIK (SEQ ID NO: 75); and

[0110] referred to herein as C3CPV15 ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPRQVQWYDGQGTKLEVK (SEQ ID NO: 76) comprises a functional variant having a CDR1, HV2, HV4 and CDR2 sequence selected from the group consisting of amino acid sequences or any of them, and having a FW1, FW2, FW3a, FW3b and FW4 sequence with at least 45% combinatorial sequence identity to any combination of their FW1, FW2, FW3a, FW3b and FW4 sequences.

[0111] In a particularly preferred embodiment, the ROR1-specific antigen-binding molecule may comprise the amino acid sequence according to TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIK (SEQ ID NO: 71)

[0112] The ROR1-specific antigen-binding molecule may comprise the amino acid sequence according to TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIK (SEQ ID NO: 71) or may comprise a functional variant thereof having a CDR1, HV2, HV4 and CDR2 sequence according to SEQ ID NO: 71 and having a FW1, FW2, FW3a, FW3b and FW4 sequence with at least 45% combinatorial sequence identity to the combinatorial FW1, FW2, FW3a, FW3b and FW4 sequences of SEQ ID NO: 71.

[0113] ​​Specific advantages associated with SEQ ID NO: 71 (「G3CP G4」) and its functional variants include increased expression yield and hydrophilicity in sub-optimal aqueous buffer systems for these proteins, as well as increased ease of analysis, purification and monomericity. Without being bound by theory, these advantages may be particularly evident in VNAR-hFc fusion proteins comprising a G3CP G4 sequence or a functional variant thereof. Thus, the G3CP G4 sequence and its functional variants may provide improved manufacturing and / or handling characteristics. Furthermore, G3CPG4-hFc exhibits excellent in vivo efficacy in a patient-derived xenograft model of triple negative breast cancer (TNBC) when conjugated to a cytotoxic anthracycline (PNU) derivative. Surprisingly, the effect of G3CPG4-hFc is also improved over B1-hFc, which itself exhibits excellent in vivo efficacy. Furthermore, as shown herein, ROR1xEGFR bispecificity comprising G3CP G4 has a shorter retention time (RT) in size exclusion chromatography (SEC) compared to B1 comprising bispecificity, indicating improved hydrophilicity of ROR1xEGFR bispecificity comprising G3CP G4. Furthermore, in a PBS stability test, ROR1xEGFR bispecificity comprising G3CP (DAR4) may reduce turbidity and the formation of high molecular weight (HMW) species compared to B1 (DAR2) comprising bispecificity. Given that little difference is observed between the G3CPxEGFR bispecificities of DAR2 and DAR4, this may indicate increased ease of analysis, purification and monomericity of ROR1xEGFR bispecificity comprising G3CP G4 in sub-optimal aqueous buffer systems. Thus, the G3CP G4 sequence and its functional variants may provide improved manufacturing and / or handling characteristics retained in ROR1xEGFR bispecific formulations.

[0114] Although not bound by theory, ROR1xEGFR bispecificity (including but not limited to the G3CP G4 sequences) can improve efficacy even when ROR1 monospecificity is refractory or of low specificity. For example, data showing co-expression of ROR1 and EGFR in TNBC (this application also includes data showing excellent in vivo efficacy of the G3CPG4-hFc PNU conjugate in a patient-derived xenograft model of TNBC), as well as data showing high expression of EGFR along with ROR1 expression in a non-small cell lung cancer model by Western blotting are disclosed herein. Co-expression of ROR1 and EGFR has also been confirmed in models of large cell lung cancer, head and neck cancer, esophageal cancer, renal cancer, gastric cancer, sarcoma, pancreatic cancer, and colorectal cancer. In a patient-derived xenograft model of TNBC, the surprisingly improved effect of the G3CPG4-hFc PNU conjugate compared to B1-hFc PNU, together with the in vitro data disclosed herein, demonstrates the therapeutic effectiveness of the currently claimed ROR1xEGFR bispecificity in the potential treatment of cancers that co-express ROR1 and EGFR as confirmed herein.

[0115] The sequences of G3CP and G3CPG4 commonly have two single amino acid changes relative to the sequence of B1. These are both present within CDR3, 1. at the Y residue of the W residue, and 2. at the N residue of the L residue substitutions.

[0116] Compared to G3CP, G3CPG4 has additional single amino acid changes (also occurring in B1 G4) in each of CDR1, HV2, and HV4 relative to B1, as well as changes that humanize the framework region (a portion of which also occurs in B1V15, SEQ ID NO: 115 as shown in FIG. 15, and B1V15 has the same CDR1, HV2, HV4, and CDR3 sequences as B1, i.e., this is not a loop library variant; the changes in B1V15 relative to B1 are present only in the framework region).

[0117] Although not bound by theory, any improvement to B1 shown by both G3CP and G3CPG4, but not shown by B1 G4 or B1 V15, is thought to result from one or both of two mutations they share in CDR3. Thus, the advantages of G3CP and G3CPG4 are thought to derive from a CDR3 containing the sequence YPWGAGAPYNVQWY (SEQ ID NO: 23).

[0118] Although not bound by theory, the surprising advantages associated with YPWGAGAPYNVQWY (SEQ ID NO: 23) may represent the synergistic effect of both the W to Y and L to N substitutions. Alternatively, the surprising advantages may primarily derive from the W to Y substitution and are thus shared by YPWGAGAPYLVQWY (SEQ ID NO: 20). 1B6 has the L to N mutation and the CDR3 sequence of YPWGAGAPWNVQWY (SEQ ID NO: 24) has a lower elution volume than B1, and thus the L to N mutation in CDR3 results in improved manufacturing and / or handling characteristics.

[0119] The EGFR-specific antigen-binding molecule can be any molecule that binds to EGFR. In particular, the EGFR-specific antigen-binding molecule can be selected from the group consisting of immunoglobulins, immunoglobulin Fab regions, Fv, single-chain Fv (scFv), diabodies, triabodies, tetra-bodies, VNAR domains, single-domain antibodies (sdAb), VH domains, or scaffold proteins (such as affibody, centyrin, darpin, etc.). Preferably, the EGFR-specific antigen-binding molecule is a single-domain antibody (sdAb).

[0120] Cetuximab is an approved monoclonal antibody drug that inhibits the epidermal growth factor receptor (EGFR). Cetuximab inhibits the binding of EGF and other ligands to EGFR while inhibiting the activation of EGFR (i.e., inhibiting the extended receptor conformation required for high-affinity ligand binding and dimerization). Cetuximab binds to a specific epitope within the EGFR domain containing amino acids 384 - 408.

[0121] 7C12 and 7D12 are single domain antibodies (nanobodies) of the camelid family that compete with the epitope of cetuximab on EGFR (see WO 2007042289 A2, which is incorporated herein by reference in its entirety). Both 7C12 and 7D12 exhibit high affinity EGFR binding (low nM KD) (Roovers 2011 Int J Cancer 129 p2013, Gainkam 2010 Mol Imaging) and inhibit EGF binding to EGFR (Schmitz 2013 Structure 21 p1214). 7C12 and 7D12 differ by 5 amino acids, and 7C12 has a high off-rate for EGFR binding (Roovers 2011 Int J Cancer 129 p2013).

[0122] EGFR#33 and EGFR#13 are low affinity variants of the 7C12 nanobody (U.S. Patent Application Publication No. 2016 / 0251440). EGFR#33 differs from 7C12 by 3 amino acids, and the affinity by SPR is reported to be 240 nM. EGFR#13 differs from 7C12 by 2 amino acids, and the affinity by SPR is reported to be 2.5 μM.

[0123] Matuzumab is another approved monoclonal antibody drug that inhibits EGFR. The binding of matuzumab sterically inhibits the rearrangement of the EGFR domain required for high affinity ligand binding and receptor dimerization. Matuzumab binds mainly to the loop (amino acids 454 - 464 of EGFR) in front of the most C-terminal strand of the β-helix of domain III.

[0124] 9G8 is an sdAb (nanobody) sequence that competes with the EGFR epitope of matuzumab (WO 2007042289 A2), but has another epitope of EGFR in a region closer to the N-terminus of domain III of EGFR and further away from the ligand binding site of domain II, which is inaccessible to conventional antibodies (Schmitz 2013 Structure 21 p1214).

[0125] Examples of sdAbs used in the bispecific antigen-binding molecules of the first aspect of the present invention include, but are not limited to, molecules that compete in binding to cetuximab or matuzumab. Preferably, the sdAb is 7D12: QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSS (SEQ ID NO: 210);

[0126] 7C12: AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTQVTVSS (SEQ ID NO: 211);

[0127] 9G8: EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSS (SEQ ID NO: 212);

[0128] 38G7: EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYVMGWFRQATGKEREFVATIAWDSGSTYYADSVKGRFTISRDNAKNTVHLQMNSLKPEDTAVYYCAASYNVYYNNYYYPISRDEYDYWGQGTQVTVSS (SEQ ID NO: 213);

[0129] EGFR#33 EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSS (SEQ ID NO: 214); and

[0130] EGFR#13 AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSS (SEQ ID NO: 215) selected from the group consisting of.

[0131] Preferably, the sdAb is selected from the group consisting of 7D12, EGFR#33, EGFR#13, and 9GB.

[0132] The EGFR-specific antigen-binding molecule may be derived from cetuximab or matuzumab, or may compete with cetuximab or matuzumab. For example, the EGFR-specific antigen-binding molecule may be an immunoglobulin, an immunoglobulin Fab region, Fv, single-chain Fv (scFv), diabody, triabody, tetrabody, VNAR domain, single-domain antibody (sdAb), VH domain, or scaffold protein (such as affibody, centyrin, darpin, etc.) that is derived from cetuximab or matuzumab or competes with cetuximab or matuzumab. The EGFR-specific antigen-binding molecule may contain the CDRs of cetuximab or matuzumab. The EGFR-specific antigen-binding molecule may contain the VH and / or VL domains of cetuximab or matuzumab.

[0133] The EGFR-specific antigen-binding molecule may contain cetuximab Fab or a cetuximab-based scFv.

[0134] Cetuximab Fab Fab LC DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 357)

[0135] Fab HC QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 358)

[0136] Cetuximab scFv QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 359)

[0137] The EGFR-specific antigen-binding molecule can include, for example, humanized cetuximab Fab or a cetuximab-based scFv.

[0138] Humanized cetuximab Fab LC DIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence No. 360)

[0139] Humanized cetuximab Fab HC EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV (Sequence No. 361)

[0140] Humanized cetuximab scFv EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIK (Sequence No. 362)

[0141] Here, the EGFR-specific antigen-binding molecule contains a Fab and usually contains both a Fab LC and a Fab HC. The Fab HC may be fused to a fragment of the immunoglobulin Fc region. Usually, the Fab LC and Fab HC are linked via a disulfide bond. For example, the EGFR-specific antigen-binding molecule contains SEQ ID NO: 360 and SEQ ID NO: 361, SEQ ID NO: 361 is fused to a fragment of the immunoglobulin Fc region, and SEQ ID NO: 360 and SEQ ID NO: 361 are linked via a disulfide bond.

[0142] The EGFR-specific antigen-binding molecule may contain a matuzumab Fab or a matuzumab-based scFv.

[0143] The EGFR-specific antigen-binding molecule may contain a humanized matuzumab Fab or a humanized matuzumab-based scFv.

[0144] Humanized matuzumab Fab LC DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKAPKLLIYDTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE (SEQ ID NO: 381)

[0145] The humanized matuzumab Fab HC may be selected from SEQ ID NO: 382 or SEQ ID NO: 383. These differ by 1 amino acid.

[0146] Humanized matuzumab Fab HC "3c08" QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLEWIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDGRYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS (SEQ ID NO: 382)

[0147] Humanized Mab Fab HC "3c09" QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLEWIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYAGRYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS (SEQ ID NO: 383)

[0148] The EGFR-specific antigen-binding molecule may be derived from panitumumab, nimotuzumab, or necitumumab, or may compete with panitumumab, nimotuzumab, or necitumumab. For example, the EGFR-specific antigen-binding molecule may be an immunoglobulin, immunoglobulin Fab region, Fv, single-chain Fv (scFv), diabody, triabody, tetrabody, VNAR domain, single-domain antibody (sdAb), VH domain, or scaffold protein (such as affibody, centyrin, darpin, etc.) derived from panitumumab, nimotuzumab, or necitumumab, or competing with panitumumab, nimotuzumab, or necitumumab. The EGFR-specific antigen-binding molecule may contain the CDRs of panitumumab, nimotuzumab, or necitumumab. The EGFR-specific antigen-binding molecule may contain the VH and / or VL domains of panitumumab, nimotuzumab, or necitumumab. The EGFR-specific antigen-binding molecule may contain the Fab of panitumumab, nimotuzumab, or necitumumab, or a panitumumab, nimotuzumab, or necitumumab-based scFv. The EGFR-specific antigen-binding molecule may contain a humanized Fab of panitumumab, nimotuzumab, or necitumumab, or a humanized panitumumab, nimotuzumab, or necitumumab-based scFv.

[0149] Panitumumab Fab HC QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKC (SEQ ID NO: 384)

[0150] Panitumumab Fab LC DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 385)

[0151] Nimotuzumab Fab HC QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVP (SEQ ID NO: 386)

[0152] Nimotuzumab Fab LC DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITREVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 387)

[0153] Nimotuzumab may refer to affinity - matured variants (such as those reported in Tundidor 2020 Sci Reports 10:1194, which are incorporated herein by reference in their entirety and have improved activity).

[0154] Necitumumab Fab HC QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSSASTKGPSVLPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV (SEQ ID NO: 388)

[0155] Necitumumab Fab LC EIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQYGSTPLTFGGGTKAEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 389)

[0156] Alternatively, the EGFR-specific antigen-binding molecule may comprise (i) at least 85% identity thereto, and / or (ii) one, two, or three amino acid substitutions thereto and may include the sequence of any EGFR-specific antigen-binding molecule disclosed herein.

[0157] The sequence identity is at least about 85% sequence identity, and thus may 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%.

[0158] One or more amino acid substitutions can be conservative amino acid substitutions. As used herein, the term "conservative amino acid substitution" refers to an amino acid substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Since amino acids with similar side chains often have similar properties, conservative substitutions of amino acids that are important for the structure or function of a polypeptide are expected to have less impact on the structure / function of the polypeptide than non-conservative amino acid substitutions at the same position. Families of amino acid residues with similar side chains have been identified in the art and include, for example, basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), polar uncharged 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). Thus, a conservative amino acid substitution can be considered a substitution in which one amino acid residue is replaced with a different amino acid from the same family. However, substitution of an epitope residue can also be a non-conservative substitution in which one amino acid is replaced with an amino acid having a side chain belonging to a different family.

[0159] The EGFR-specific antigen-binding molecule may be humanized. At least 85% identity and / or one, two, or three amino acid substitutions to the sequence of the EGFR-specific antigen-binding molecule disclosed herein may include substitutions for humanizing the EGFR-specific antigen-binding molecule.

[0160] 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, more preferably 1 to 20 nM. The affinity constant can be about 2 nM. The affinity constant can be measured as described elsewhere herein (for the ROR1-specific antigen-binding molecule, for example, by surface plasmon resonance (SPR) or biolayer interferometry (BLI)).

[0161] It is understood that the ROR1-specific antigen-binding molecule and the EGFR-specific antigen-binding molecule can be combined in any order, thereby forming a bispecific antigen-binding molecule of the first aspect. That is, the ROR1-specific antigen-binding molecule may be on the N-terminal side of the EGFR-specific antigen-binding molecule, or vice versa. Alternatively, when the bispecific antigen-binding molecule is formed, for example, by knob-into-hole, the ROR1-specific antigen-binding molecule may be in one arm while the EGFR-specific antigen-binding molecule may be in the other arm, or vice versa.

[0162] Furthermore, it is understood that higher-order constructs, such as constructs composed of multiple ROR1-specific antigen-binding molecules and EGFR-specific antigen-binding molecules, are also contemplated herein. These may take the form of multiple units in a single primary amino acid sequence, such as, for example, ROR1 binder-EGFR binder-ROR1 binder, or EGFR binder-ROR1 binder-EGFR binder.

[0163] The bispecific antigen-binding molecule of the first aspect may further include a linker region between the ROR1-specific antigen-binding molecule and the EGFR-specific antigen-binding molecule. A preferred linker is [G4S] X(X is 1, 2, 3, 4, 5, 5, 6, 7, 8, 9, or 10), but is not limited thereto. 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 include, but are not limited to, PGVQPSPGGGGS (SEQ ID NO: 89) (Wobbe-G4S), PGVQPAPGGGGS (SEQ ID NO: 90) (Wobbe-G4S GM), etc. It is understood that different combinations of different linkers can be combined within the same construct.

[0164] The bispecific antigen-binding molecule of the first aspect may further comprise an additional domain, which may take the form of an N-terminal or C-terminal addition, or may be disposed between the ROR1-specific antigen-binding molecule and the EGFR-specific antigen-binding molecule in the amino acid sequence of the bispecific binding molecule. Each domain of the bispecific antigen-binding molecule of the first aspect may be connected via the linker region described above. Preferred additional domains include, but are not limited to, immunoglobulin, immunoglobulin Fc region, immunoglobulin Fab region, single-chain Fv (scFv), diabody, triabody, tetrabody, bispecific T cell engager (BiTE), intein, VNAR domain, single-domain antibody (sdAb), VH domain, or scaffold protein (such as affibody, centyrin, darpin, etc.). A particularly preferred additional domain is the immunoglobulin Fc region, preferably the human Fc region.

[0165] Combinations specifically contemplated herein include, but are not limited to, monovalent ROR1xEGFR (Fc fusion) bispecific; bivalent ROR1xEGFR (Fc fusion) bispecific; monovalent ROR1xEGFR (non-Fc) bispecific; bivalent ROR1xEGFR (non-Fc) bispecific; monovalent ROR1, half-life extended ROR1xEGFR (non-Fc) bispecific.

[0166] The monovalent ROR1xEGFR (Fc-fusion) bispecificity can take the form of a ROR1 binder-Fc-EGFR binder or an EGFR binder-Fc-ROR1 binder.

[0167] The divalent ROR1xEGFR (Fc-fusion) bispecificity is ROR1 binder-ROR1 binder-Fc-EGFR binder ROR1 binder-EGFR binder-Fc-ROR1 binder EGFR binder-ROR1 binder-Fc-ROR1 binder EGFR binder-EGFR binder-Fc-ROR1 binder EGFR binder-ROR1 binder-Fc-EGFR binder ROR1 binder-EGFR binder-Fc-EGFR binder EGFR binder-Fc-ROR1 binder-ROR1 binder ROR1 binder-Fc-EGFR binder-EGFR binder EGFR binder-Fc-ROR1 binder-EGFR binder EGFR binder-Fc-EGFR binder-ROR1 binder ROR1 binder-Fc-EGFR binder-ROR1 binder, and ROR1 binder-Fc-ROR1 binder-EGFR binder can take a form selected from the group consisting of.

[0168] The monovalent ROR1xEGFR (non-Fc) bispecificity can take the form of a ROR1 binder-EGFR binder or an EGFR binder-ROR1 binder.

[0169] The divalent ROR1xEGFR (non-Fc) bispecificity is ROR1 binder-ROR1 binder-EGFR binder ROR1 binder-EGFR binder-ROR1 binder EGFR binder - ROR1 binder - ROR1 binder It may take a form selected from the group consisting of.

[0170] The monovalent ROR1, half - life extended ROR1xEGFR (non - Fc) bispecificity is ROR1 binder - BA11 - EGFR binder ROR1 binder - EGFR binder - BA11 BA11 - ROR1 binder - EGFR binder BA11 - EGFR binder - ROR1 binder EGFR binder - ROR1 binder - BA11 EGFR binder - BA11 - ROR1 binder It may take a form selected from the group consisting of.

[0171] The ROR1 binder can be any ROR1 - specific antigen - binding molecule disclosed herein. If two ROR1 binders are present, they may be the same ROR1 - specific antigen - binding molecule or two different ROR1 - specific antigen - binding molecules.

[0172] The EGFR binder can be any EGFR - specific antigen - binding molecule disclosed herein. If two EGFR binders are present, they may be the same EGFR - specific antigen - binding molecule or two different EGFR - specific antigen - binding molecules.

[0173] The linker between domains is preferably, but not limited to, (G4S) X (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 - G4S GM), and different combinations of different linkers can be combined within the same construct.

[0174] Thus, an additional C-terminal (or N-terminal) tag sequence may or may not be present. Examples of C-terminal tags include, but are not limited to, tags containing a poly-histidine sequence (such as His6) for facilitating purification, tags containing a c-Myc sequence (such as EQKLISEEDL (SEQ ID NO: 112)) for enabling detection, and / or tags containing a cysteine residue for enabling labeling and bioconjugation using a thiol-reactive payload and probe, and combinations thereof. Preferred C-terminal tags include QASGAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 98) QACGAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 99) QACKAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 97) AAAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 100) ACAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 101) QASGAHHHHHH (SEQ ID NO: 102) QACGAHHHHHH (SEQ ID NO: 103) QACKAHHHHHH (SEQ ID NO: 104) AAAHHHHHH (SEQ ID NO: 105) ACAHHHHHH (SEQ ID NO: 106) QASGA (SEQ ID NO: 107) QACGA (SEQ ID NO: 108) QACKA (SEQ ID NO: 109) ACA (SEQ ID NO: 110) SAPSA (SEQ ID NO: 111) but are not limited thereto.

[0175] The domain can also be bound to the Fc domain via N-terminal fusion, C-terminal fusion, or both N-terminal and C-terminal fusion. For example, but not limited thereto, the following are included.

[0176] hIgG1 EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 216)

[0177] hIgG1(S239C) EPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 217)

[0178] hIgG1(S442C) EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 218)

[0179] hIgG1(S239C + S442C) EPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 219)

[0180] Here: P3A1 is TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVN (SEQ ID NO: 206) and

[0181] BA11 is TRVDQSPSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIK (SEQ ID NO: 95) and

[0182] According to a second aspect, the invention provides a bispecific antigen-binding molecule, (i) Formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) (wherein 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 a framework region; FW2 is a 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 a 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 a framework region; CDR3 is a CDR sequence having an amino acid sequence by REARHPWLRQWY (SEQ ID NO: 39); FW4 is a framework region) A receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen-binding molecule comprising an amino acid sequence represented by, and (ii) an epidermal growth factor receptor (EGFR)-specific antigen-binding molecule comprising, 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, A bispecific antigen-binding molecule is provided. As used herein, the terms "first fragment" and "second fragment" are interchangeable.

[0183] In a preferred embodiment, the ROR1-specific antigen-binding molecule is referred to herein as P3A1 TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVN (SEQ ID NO: 206);

[0184] Referred to as P3A1 G1 AE3 in this specification TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 77);

[0185] Referred to as P3A1 G1 AE3.S in this specification TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 78);

[0186] Referred to as P3A1 G1 NAC6 in this specification TRVDQSPSSLSASVGDRVTITCVLTDTRYALYSTYWYRKNPGSTDEERISIGGRYSESVNKGSKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 79);

[0187] Referred to as P3A1 G1 NAC6.S in this specification TRVDQSPSSLSASVGDRVTITCVLTDTRYALYSSTYWYRKNPGSTDEERISIGGRYSESVNKGSKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 80);

[0188] Referred to as P3A1 G1 NAG8 in this specification TRVDQSPSSLSASVGDRVTITCVLTGTKYGLYATYWYRKNPGSPNKDRMIIGGRYSESVNNGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 81);

[0189] In the present specification, it is referred to as P3A1 G1 NAG8.S TRVDQSPSSLSASVGDRVTITCVLTGTKYGLYASTYWYRKNPGSPNKDRMIIGGRYSESVNNGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 82);

[0190] In the present specification, it is referred to as P3A1 G1 AF7.S TRVDQSPSSLSASVGDRVTITCVLTGTKYGLYASTYWYRKNPGSTDKERIIIGGRYSESVNNRSKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 83) An amino acid sequence selected from the group consisting of or having CDR1, HV2, HV4 and CDR2 sequences by any of them, and having FW1, FW2, FW3a, FW3b and FW4 sequences with at least 45% combined sequence identity with any combination of FW1, FW2, FW3a, FW3b and FW4 sequences of any of them, including functional variants.

[0191] As a specific advantage related to the affinity maturation variant of P3A1G1 and its functional variant, as described by the examples, an improvement in the binding to hROR1-Fc compared to the parental P3A1G1 can be mentioned.

[0192] The ROR1-specific antigen-binding molecule may contain the CDR and HV sequences of the clones shown in Table 1 below. In the first and / or second preferred embodiments of the present invention, the ROR1-specific antigen-binding molecule has any combination sequence of the clones shown in Table 1 below.

[0193]

Table 1

[0194] The ROR1-specific antigen-binding molecule may comprise the CDR and HV sequences of the clones shown in Table 2 below. In the first and / or second preferred embodiments of the present invention, the ROR1-specific antigen-binding molecule has a combined sequence of any of the clones shown in Table 2 below.

[0195]

Table 2

[0196] All possible combinations and permutations of the framework regions, complementarity-determining regions, and hypervariable regions listed herein are expressly contemplated herein.

[0197] The sequence identity referred to in connection with the molecules of the present invention can be determined at the level of individual CDRs, HVs or FWs, combined CDRs, HVs or FWs, or it can be determined over the length of the entire molecule. The CDR, HV, and FW sequences described may be longer or shorter, whether due to addition or deletion of amino acids at the N or C terminus of the sequence, or insertion or deletion of amino acids in the sequence.

[0198] The framework region FW1 is preferably 20 - 28 amino acids in length, more preferably 22 - 26 amino acids in length, even more preferably 23 - 25 amino acids in length. In certain preferred embodiments, FW1 is 26 amino acids in length. In other preferred embodiments, FW1 is 25 amino acids in length. In other even more preferred embodiments, FW1 is 24 amino acids in length.

[0199] In an alternative definition, the CDR region CDR1 is preferably 7 to 11 amino acids in length, more preferably 8 to 10 amino acids in length. In certain preferred embodiments, CDR1 is 9 amino acids in length. In other preferred embodiments, CDR1 is 8 amino acids in length.

[0200] 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.

[0201] In an alternative definition, the hypervariable array HV2 is preferably 4 to 11 amino acids in length, more preferably 5 to 10 amino acids in length. In certain preferred embodiments, HV2 is 10 amino acids in length. In certain preferred embodiments, HV2 is 9 amino acids in length. In other preferred embodiments, HV2 is 6 amino acids in length.

[0202] 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.

[0203] In an alternative definition, the hypervariable array HV4 is preferably 3 to 7 amino acids in length, more preferably 4 to 6 amino acids in length. In certain preferred embodiments, HV4 is 5 amino acids in length. In other preferred embodiments, HV4 is 4 amino acids in length.

[0204] The framework region FW3b is preferably 17 to 24 amino acids in length, more preferably 18 to 23 amino acids in length, even more preferably 19 to 22 amino acids in length. In certain preferred embodiments, FW3b is 21 amino acids in length. In other preferred embodiments, FW3b is 20 amino acids in length.

[0205] In an alternative definition, the CDR region CDR3 is preferably 8 to 21 amino acids in length, more preferably 9 to 20 amino acids in length, even more preferably 10 to 19 amino acids in length. In certain preferred embodiments, CDR3 is 17 amino acids in length. In other preferred embodiments, CDR3 is 14 amino acids in length. In still other preferred embodiments, CDR3 is 12 amino acids in length. In yet other even more preferred embodiments, CDR3 is 10 amino acids in length.

[0206] The framework region FW4 is preferably 7 to 14 amino acids in length, more preferably 8 to 13 amino acids in length, even more preferably 9 to 12 amino acids in length. In certain preferred embodiments, FW4 is 12 amino acids in length. In other preferred embodiments, FW4 is 11 amino acids in length. In yet other even more preferred embodiments, FW4 is 10 amino acids in length. In still other preferred embodiments, FW4 is 9 amino acids in length.

[0207] In one embodiment of the ROR1-specific antigen-binding molecule, FW1 is a framework region of 20 to 28 amino acids; FW2 is a framework region of 6 to 14 amino acids; FW3a is a framework region of 6 to 10 amino acids; FW3b is a framework region of 17 to 24 amino acids; and / or FW4 is a framework region of 7 to 14 amino acids.

[0208] In one embodiment of the ROR1-specific antigen-binding molecule, FW1 has an amino acid sequence selected from the group consisting of ASVNQTPRTATKETGESLTINCVVT (SEQ ID NO: 40), TRVDQSPSSLSASVGDRVTITCVLT (SEQ ID NO: 41), and ASVTQSPRSASKETGESLTITCRVT (SEQ ID NO: 42) or a functional variant thereof having at least 45% sequence identity; FW2 has an amino acid sequence according to TYWYRKNPG (SEQ ID NO: 43) or any of its functional variants having at least 45% sequence identity; FW3a has an amino acid sequence selected from the group consisting of GRYVESV (SEQ ID NO: 44) and GRYSESV (SEQ ID NO: 45) or any of their functional variants having at least 45% sequence identity; 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 any of their functional variants having at least 45% sequence identity; and / or 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 any of their functional variants having at least 45% sequence identity.

[0209] The ROR1-specific antigen-binding molecule can be humanized. The ROR1-specific antigen-binding molecule can be deimmunized. The B1 loop variants on the humanized frameworks G4 and V15 described herein are humanized. If P3A1G1 is already humanized, all loop variants of P3A1G1 are humanized. Examples of the humanized sequences of the present invention include, but are not limited to, B1G4 G3CP G4 G3CP V15 1H8 G4 1H8 V15 C3CP G4 C3CPV15 P3A1 G1 AE3 P3A1 G1 AE3.S P3A1 G1 NAC6 P3A1 G1 NAC6.S P3A1 G1 NAG8 P3A1 G1 NAG8.S P3A1 G1 AF7.S include the following.

[0210] Those skilled in the art will recognize that the humanized ROR1-specific antigen-binding molecule described in this specification can be further humanized, for example, by substituting additional FW region amino acids with the amino acids of DPK-9.

[0211] The ROR1-specific antigen-binding molecule can be conjugated to a detectable label, dye, toxin, drug, prodrug, radionuclide, or biologically active molecule.

[0212] 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 both human ROR1 and mouse ROR1 (mROR1). Even more preferably, the ROR1-specific antigen-binding molecule binds to deglycosylated ROR1.

[0213] A certain ROR1-specific antigen-binding molecule of the present invention YMESLHMQGEIENQI (SEQ ID NO: 91) CQPWNSQYPHTHTFTALRFP (SEQ ID NO: 92) RSTIYGSRLRIRNLDTTDTGYFQ (SEQ ID NO: 93) QCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYE (SEQ ID NO: 94) cannot bind to a linear peptide sequence selected from

[0214] Preferably, the ROR1-specific antigen-binding molecule selectively interacts with the ROR1 protein with an affinity constant of approximately 0.01 to 50 nM, preferably 0.1 to 30 nM, more preferably 0.1 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 is to immobilize ROR1, and thus, due to the avidity effect, the bivalent VNAR-hFc binds with an apparent K D The other approach is a 1:1 format, where VNAR-hFc is immobilized and ROR1 is flowed over its surface, thus giving the K D for "true" 1:1 binding. Typically, as used herein, the affinity constant refers to that 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. Among the P3A1 G1 loop variants, examples have K D values of 5.0 nM (AE3), 13.8 nM (NAC6), and 12.2 nM (NAG8).

[0215] Optionally, the antigen-binding molecule specifically binds to ROR1 with an affinity determined by SPR (e.g., under the SPR conditions disclosed herein). Similarly, the antigen-binding molecule specifically binds to EGFR with an affinity determined by SPR (e.g., under the SPR conditions disclosed herein). Such binding measurements can be performed using various binding assays well known in the art, such as surface plasmon resonance (SPR) using Biacore™, or ProteOn XPR36™ (using Bio-Rad®), KinExA® (Sapidyne Instruments, Inc), or biolayer interferometry (BLI) such as the Octet system (Sartorius).

[0216] The binding ability, specificity, and affinity (KD , k off and / or k on ) can be determined by any routine means in the art, such as surface plasmon resonance (SPR) or biolayer interferometry (BLI). As used herein, "k on " or "k a " refers to the binding constant. As used herein, "k d " or "k off " refers to the dissociation constant. As used herein, the term "K D " is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction. Such binding measurements can be performed using various binding assays well known in the art, such as surface plasmon resonance (SPR) using Biacore™, or ProteOn XPR36™ (using Bio-Rad®), using KinExA® (Sapidyne Instruments, Inc), or using BLI with an Octet system (Sartorius).

[0217] In one embodiment, the surface plasmon resonance (SPR) is performed at 25°C. In another embodiment, the SPR is performed at 37°C.

[0218] In one embodiment, the SPR is performed at physiological pH, such as about pH 7 or pH 7.6 (e.g., using Hepes buffered saline at pH 7.6 (also referred to as HBS-EP)).

[0219] In one embodiment, the SPR is performed at physiological salt concentration, such as 150 mM NaCl.

[0220] In one embodiment, the SPR is performed in the presence of a surfactant level of only 0.05% by volume, such as 0.05% P20 (polysorbate 20; e.g., Tween-20®) and 3 mM EDTA.

[0221] In one example, SPR is performed at 25°C or 37°C in a buffer of pH 7.6, 150 mM NaCl, 0.05% surfactant (e.g., P20), and 3 mM EDTA. The buffer may contain 10 mM Hepes. In one example, SPR is performed at 25°C or 37°C in HBS-EP. HBS-EP is available from Teknova Inc (California; catalog number H8022).

[0222] In one example, the affinity of the test ROR1xEGFR bispecificity for the target antigen is determined using SPR as follows. 1. Bind the target antigen (e.g., ROR1 or EGFR) to a biosensor chip (e.g., GLM chip) by a primary amine coupling method or the like. Alternatively, the target antigen can also be indirectly bound to the biosensor chip via a primary anti-tag IgG capture step (e.g., appropriate anti-Fc IgG). 2. Pass the test ROR1xEGFR bispecificity at 0 nM (i.e., buffer only), 1024 nM, 256 nM, 64 nM, 16 nM, 4 nM across the capture surface of the chip. 3. Use surface plasmon resonance to determine the binding affinity of the test ROR1xEGFR bispecificity for the target antigen, for example, under the above-mentioned SPR conditions (e.g., in physiological buffer, 25°C). SPR can be performed using any standard SPR apparatus, for example, using Biacore™ or ProteOn XPR36™.

[0223] Alternatively, the test ROR1xEGFR bispecificity can be directly (e.g., by primary amine coupling) or indirectly via a primary anti-tag IgG capture step (e.g., anti-hFc IgG) bound to the biosensor chip, and the target antigen (e.g., ROR1 or EGFR) can be passed across the capture surface of the chip.

[0224] The capture surface can be regenerated with 10 mM glycine (pH 1.7). This removes the captured antibody, allowing the surface to be used for another interaction. Binding data can be fit to the native 1:1 model using standard techniques, for example, the model native to the ProteOn XPR36™ analysis software can be used.

[0225] Alternatively, the BLI method may be used to determine affinity using an Octet BLI system (Sartorius). The ROR1 or EGFR ligand is conjugated to the biosensor by standard amine coupling (ARG2 biosensor) or affinity capture (e.g., anti-human Fc capture with an AHC biosensor or anti-His capture with a HIS1K biosensor). The sensor is immersed in the test analyte, and the binding affinity is calculated from the binding and dissociation rates of the analyte.

[0226] Furthermore, the ROR1-specific antigen-binding molecule preferably can mediate the killing of ROR1-expressing tumor cells or inhibit cancer cell proliferation.

[0227] The ROR1-specific antigen-binding molecule can also be endocytosed upon binding to ROR1. In other embodiments, the ROR1-specific antigen-binding molecule cannot be endocytosed upon binding to ROR1.

[0228] According to a third aspect, the invention provides a recombinant fusion protein comprising a bispecific antigen-binding molecule of the first or second aspect of the invention.

[0229] Preferably, in the recombinant fusion protein of the third aspect, the ROR1-specific antigen-binding molecule and / or the EGFR-specific antigen-binding molecule is 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). Specific preferred linkers include 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 can be particularly useful when the recombinant fusion protein is expressed in expression systems with different glycosylation patterns, such as CHO and insect, as well as in expression systems that do not glycosylate the expressed protein (e.g., Escherichia coli (E. coli)). Any recombinant fusion protein sequence disclosed herein that includes the [G4S]3 linker can alternatively have any other linker sequence disclosed herein.

[0230] The fusion proteins of the present invention can be constructed in any order, i.e., it is also recognized that the ROR1-specific antigen-binding molecule can be present at the N-terminus, C-terminus, or not present at either terminus (e.g., present within a longer amino acid sequence). The EGFR-specific antigen-binding molecule can be present at the N-terminus, C-terminus, or not present at either terminus (e.g., present within a longer amino acid sequence).

[0231] 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, inteins, VNAR domains, single-domain antibodies (sdAb), VH domains, or scaffold proteins (such as affibody, centyrin, darpin, etc.). Another preferred biologically active protein is the immunoglobulin Fc region. Other preferred fusion proteins include VNAR-VNAR and VNAR-VNAR-VNAR.

[0232] In one embodiment, at least one biologically active protein is an immunoglobulin Fc region.

[0233] This recombinant fusion protein may contain an 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 the linker may be fused to the C-terminus of the ROR1-specific antigen-binding molecule.

[0234] Thus, the recombinant fusion protein may contain 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.

[0235] G3CP-hFc ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 186)

[0236] G3CPG4-hFc TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 187)

[0237] 1H8-hFc ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 183)

[0238] 1H8 G4-hFc TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 184)

[0239] 1H8 V15-hFc ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 185)

[0240] P3A1-hFc TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 223)

[0241] 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.

[0242] The recombinant fusion protein may comprise a sequence according to SEQ ID NO: 186, SEQ ID NO: 187 or SEQ ID NO: 183.

[0243] In a further embodiment, the at least one biologically active protein is an immunoglobulin Fc region that has been further modified to include a mutation from S to C.

[0244] The mutation from S to C may be at the position of 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, SEQ ID NO: 182 or SEQ ID NO: 224.

[0245] G3CP-hFc(S239C) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 178)

[0246] G3CPG4-hFc(S239C) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 179)

[0247] 1H8-hFc(S239C) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 180)

[0248] 1H8 G4-hFc(S239C) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 181)

[0249] 1H8 V15-hFc(S239C) ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 182)

[0250] P3A1-hFc(S239C) TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 224)

[0251] The recombinant fusion protein may contain a sequence according to SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 224.

[0252] The recombinant fusion protein may contain a sequence according to SEQ ID NO: 178, SEQ ID NO: 179, or SEQ ID NO: 180.

[0253] The mutation from S to C can be at the position of S442 (EU numbering). Thus, the recombinant fusion protein may contain the 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.

[0254] G3CP-hFc(S442C) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 225)

[0255] G3CPG4-hFc(S442C) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 226)

[0256] 1H8-hFc(S442C) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 227)

[0257] 1H8 G4-hFc(S442C) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 228)

[0258] 1H8 V15-hFc(S442C) ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 229)

[0259] P3A1-hFc(S442C) TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 230)

[0260] The recombinant fusion protein may contain a sequence according to SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227 or SEQ ID NO: 230.

[0261] The recombinant fusion protein may contain a sequence according to SEQ ID NO: 225, SEQ ID NO: 226, or SEQ ID NO: 227.

[0262] The mutation from S to C can be at positions S239 and S442 (EU numbering). Thus, the recombinant fusion protein may contain 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.

[0263] G3CP-hFc(S239C&S442C) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 231)

[0264] G3CPG4-hFc(S239C&S442C) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 232)

[0265] 1H8-hFc(S239C&S442C) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 233)

[0266] 1H8 G4-hFc(S239C&S442C) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(Sequence number 234)

[0267] 1H8 V15-hFc(S239C&S442C) ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(Sequence number 235)

[0268] P3A1-hFc(S239C&S442C) TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 236)

[0269] 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.

[0270] The recombinant fusion protein may comprise a sequence according to SEQ ID NO: 231, SEQ ID NO: 232, or SEQ ID NO: 233.

[0271] The recombinant fusion protein may comprise an EGFR-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 EGFR-specific antigen-binding molecule via a linker. The immunoglobulin Fc region or a fragment thereof and / or the linker may be fused to the C-terminus of the EGFR-specific antigen-binding molecule.

[0272] Therefore, the EGFR-specific antigen-binding molecule fused to the immunoglobulin Fc region may contain a sequence according to any one of SEQ ID NO: 237 to SEQ ID NO: 252. Therefore, the EGFR-specific antigen-binding molecule fused to the immunoglobulin Fc region may contain a sequence according to any one of SEQ ID NO: 237 to SEQ ID NO: 240. Therefore, the EGFR-specific antigen-binding molecule fused to the immunoglobulin Fc region may contain a sequence according to any one of SEQ ID NO: 241 to SEQ ID NO: 244. Therefore, the EGFR-specific antigen-binding molecule fused to the immunoglobulin Fc region may contain a sequence according to any one of SEQ ID NO: 245 to SEQ ID NO: 248. Therefore, the EGFR-specific antigen-binding molecule fused to the immunoglobulin Fc region may contain a sequence according to any one of SEQ ID NO: 249 to SEQ ID NO: 252.

[0273] 7D12-hFc QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 237)

[0274] EGFR#33-hFc EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 238)

[0275] EGFR#13-hFc AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 239)

[0276] 9G8-hFc EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 240)

[0277] 7D12-hFc(S239C) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 241)

[0278] EGFR#33-hFc(S239C) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 242)

[0279] EGFR#13-hFc(S239C) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 243)

[0280] 9G8-hFc(S239C) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 244)

[0281] 7D12-hFc(S442C) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 245)

[0282] EGFR#33-hFc(S442C) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 246)

[0283] EGFR#13-hFc(S442C) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 247)

[0284] 9G8-hFc(S442C) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 248)

[0285] 7D12-hFc(S239C&S442C) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 249)

[0286] EGFR#33-hFc(S239C&S442C) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 250)

[0287] EGFR#13-hFc(S239C&S442C) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 251)

[0288] 9G8-hFc(S239C&S442C) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 252)

[0289] The EGFR-specific antigen-binding molecule fused to the immunoglobulin Fc region may contain cetuximab Fab or a cetuximab-based scFv. The cetuximab Fab or cetuximab-based scFv may be any one of SEQ ID NOs: 357 to 359. The cetuximab Fab or cetuximab-based scFv may be a humanized sequence of any one of SEQ ID NOs: 360 to 362. Thus, for example, as a humanized cetuximab sequence, the EGFR-specific antigen-binding molecule fused to the immunoglobulin Fc region may contain a sequence by any one of SEQ ID NO: 360 and SEQ ID NOs: 363 to 380 and SEQ ID NOs: 390 to 407.

[0290] Humanized cetuximab Fab LC DIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 360)

[0291] Humanized cetuximab Fab HC hFc (S239C) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 363)

[0292] Humanized cetuximab scFv hFc (S239C) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 364)

[0293] Humanized cetuximab Fab HC hFc (S239C+S442C) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 365)

[0294] Humanized cetuximab scFv hFc (S239C + S442C) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 366)

[0295] Humanized Sertuximab Fab HC hFc (S442C) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 367)

[0296] Humanized Cetuximab scFv hFc (S442C) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 368)

[0297] Here, the EGFR-specific antigen-binding molecule contains a Fab, and usually, the EGFR-specific antigen-binding molecule contains both a Fab LC and a Fab HC. The Fab HC may be fused to a fragment of an immunoglobulin Fc region such as SEQ ID NOs: 363, 365, and 367. Usually, the Fab LC and the Fab HC are linked via a disulfide bond. For example, the EGFR-specific antigen-binding molecule contains SEQ ID NO: 360 and SEQ ID NO: 363, and SEQ ID NO: 360 and SEQ ID NO: 363 are linked via a disulfide bond.

[0298] 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.

[0299] In one embodiment, the fragment of the immunoglobulin Fc region is an Fc heavy chain.

[0300] In one embodiment, one or more residues of the fusion protein comprise one or more amino acid substitutions suitable for conjugation. One or more residues suitable for conjugation can be residues of a fragment of the immunoglobulin Fc region.

[0301] Any portion of the fusion protein of the present invention can be engineered to enable conjugation. In a preferred example where the immunoglobulin Fc region is used, this can be engineered to contain a cysteine residue as a conjugation site. Preferred introduced cysteine residues 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 of the fusion proteins disclosed herein can include the S239C point mutation. In some embodiments, any of the fusion proteins disclosed herein can include the S442C point mutation. In some embodiments, any of the fusion proteins disclosed herein can include both the S239C and S442C point mutations. It is explicitly contemplated herein that any sequence of the fusion proteins disclosed herein can be modified to include the S239C and / or S442C point mutations.

[0302] According to a third aspect, a recombinant fusion comprising a plurality of VNAR domains is provided. Thus, the recombinant fusions of the invention can be dimers, trimers or higher order multimers of VNARs. In such recombinant fusions, the specificity of each VNAR can be the same or different. Examples of the recombinant fusions of the invention include, but are not limited to, bispecific or trispecific molecules in which each VNAR domain binds to a different antigen or to different epitopes on a single antigen (biparatopic binders). As used herein, the term "biparatopic" is intended to encompass molecules that bind to multiple epitopes on a given antigen. Molecules that bind to three or more epitopes on a given antigen are also contemplated herein, and it should be understood that when the term "biparatopic" is used, the possibility of triparatopic or multiparatopic molecules is also contemplated.

[0303] According to a third aspect, a recombinant fusion comprising the ROR1-specific antigen-binding molecule of the first aspect and a humanized VNAR domain is also provided. The humanized VNAR domain can be referred to as soloMER, and examples thereof include, but are not limited to, VNAR BA11, a humanized VNAR that binds to human serum albumin with high affinity.

[0304] Examples of biparatopic and multivalent fusion proteins include, but are not limited to, · B1-G3CP · G3CP-BA11 · BA11-G3CP · 1H8-BA11 · BA11-1H8 · G3CP V15-BA11 · G3CP G4-BA11 · B1-G3CP Cys · G3CP-BA11 Cys · BA11-G3CP Cys · 1H8-BA11 Cys · BA11-1H8 Cys · G3CP V15-BA11 Cys ·G3CP G4-BA11 Cys ·P3A1G1AE3-(L2)-G3CPG4 ·G3CPG4(L2)--P3A1G1AE3 ·P3A1G1AE3-(L2)-G3CPG4 Cys ·G3CPG4-(L2)-P3A1G1AE3 Cys ·P3A1-(L2)-BA11-(L2)-G3CP ·P3A1-(L2)-G3CP-(L2)-BA11 ·BA11-(L2)-G3CP-(L2)-P3A1 ·BA11-(L2)-P3A1-(L2)-G3CP ·P3A1-(L2)-BA11-(L2)-1H8 ·BA11-(L2)-P3A1-(L2)-1H8 ·P3A1-(L2)-BA11-(L2)-G3CP Cys ·P3A1-(L2)-G3CP-(L2)-BA11 Cys ·BA11-(L2)-G3CP-(L2)-P3A1 Cys ·BA11-(L2)-P3A1-(L2)-G3CP Cys ·P3A1-(L2)-BA11-(L2)-1H8 Cys ·BA11-(L2)-1P3A1-(L2)-1H8 Cys include

[0305] G3CP is ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVN (SEQ ID NO: 50) and

[0306] 1H8 is ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVN (SEQ ID NO: 61) and

[0307] G3CP G4 is TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIK (SEQ ID NO: 71) and

[0308] G3CP V15 is ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPYNVQWYDGQGTKLEVK (SEQ ID NO: 72) and

[0309] BA11 is TRVDQSPSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIK (SEQ ID NO: 95) and

[0310] P3A1 G1 AE3 is TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 77) and

[0311] When no linker is defined, (-) corresponds to linker Wobbe-G5S, which in turn is PGVQPSPGGGGGS (SEQ ID NO: 96), -(L2)- corresponds to linker Wobbe-G4S-GM, which in turn is PGVQPAPGGGGS (SEQ ID NO: 90), Cys- corresponds to a Cys-containing C-terminal tag, such as QACKAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 97).

[0312] Recombinant biparatopic fusion protein dimers can also be prepared by fusing any of the recombinant fusion proteins disclosed herein, particularly the ROR1-specific antigen-binding molecules disclosed herein, on one arm of an Fc fusion and an epidermal growth factor receptor (EGFR)-specific antigen-binding molecule on the other.

[0313] In certain embodiments, the specific binding molecules or recombinant fusions of the invention can be expressed with N- or C-terminal tags to assist in purification. Examples include, but are not limited to, His6 and / or Myc. Additionally, the N- or C-terminal tags can be further engineered to contain additional cysteine residues to function as conjugation points. Accordingly, references to specific binding molecules or recombinant fusions in all aspects of the invention are intended to encompass such molecules with various N- or C-terminal tags, and it is recognized that those tags can also include additional cysteines for conjugation.

[0314] Additional recombinant fusions are listed below. It is recognized that not all combinations of linkers and VNARs or fusion partners are listed below. However, all such combinations are expressly encompassed by the invention.

[0315] monovalent-BA11 fusion BA11-G3CP G3CP-BA11 BA11-G3CPG4 G3CPG4-BA1 P3A1G1 AE3-BA11 BA11-P3A1G1 AE3 divalent-BA11 fusion P3A1G1 AE3-P3A1G1 AE3-BA11 BA11-P3A1G1 AE3-P3A1G1 AE3 P3A1G1 AE3-BA11-P3A1G1 AE3 G3CP-G3CP-BA11 G3CP-BA11-G3CP BA11-G3CP-G3CP G3CPG4-G3CPG4-BA11 G3CPG4-BA11-G3CPG4 BA11-G3CPG4-G3CPG4 B1G4-B1G4-BA11 B1G4-BA11-B1G4 BA11-B1G4-B1G4 biparatopic dimer G3CP-P3A1G1 AE3 P3A1G1 AE3-G3CP G3CPG4-P3A1G1 AE3 P3A1G1 AE3-G3CPG4 dimeric biparatopic BA11 fusion G3CP-P3A1G1 AE3-BA11 P3A1G1 AE3-G3CP-BA11 G3CP-BA11-P3A1G1 AE3 P3A1G1 AE3-BA11-G3CP G3CPG4-P3A1G1 AE3-BA11 P3A1G1 AE3-G3CPG4-BA11 G3CPG4-BA11-P3A1G1 AE3 P3A1G1 AE3-BA11-G3CPG4 B1G4-P3A1G1 AE3-BA11 P3A1G1 AE3-B1G4-BA11 B1G4-BA11-P3A1G1 AE3 P3A1G1 AE3-BA11-B1G4

[0316] The linkers between 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 within 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 using a thiol-mediated chemical coupling strategy. The use of this linker sequence for bioconjugation is advantageous because reoxidation and capping of reduced cysteine are minimal, resulting in a high yield conversion of the protein to the corresponding conjugate in the bioconjugation reaction.

[0317] Additional C-terminal (or N-terminal) tag sequences may or may not be present. C-terminal tags include, but are not limited to, tags containing a poly-histidine sequence (e.g., His6) to facilitate purification, tags containing a c-Myc sequence (e.g., EQKLISEEDL (SEQ ID NO: 112)) to enable detection, and / or tags containing cysteine residues that enable labeling and bioconjugation using thiol-reactive payloads and probes, and combinations thereof. Preferred C-terminal tags include, but are not limited to, QASGAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 98) QACGAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 99) QACKAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 97) AAAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 100) ACAHHHHHHGAEFEQKLISEEDL (SEQ ID NO: 101) QASGAHHHHHH (SEQ ID NO: 102) QACGAHHHHHH (SEQ ID NO: 103) QACKAHHHHHH (SEQ ID NO: 104) AAAHHHHHH (SEQ ID NO: 105) ACAHHHHHH (SEQ ID NO: 106) QASGA (SEQ ID NO: 107) QACGA (SEQ ID NO: 108) QACKA (SEQ ID NO: 109) ACA (SEQ ID NO: 110) SAPSA (SEQ ID NO: 111) include

[0318] G3CP is ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVN (SEQ ID NO: 50) and

[0319] G3CP G4 is TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIK (SEQ ID NO: 71) and

[0320] BA11 is TRVDQSPSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIK (SEQ ID NO: 95) and

[0321] P3A1 G1 AE3 is TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIK (SEQ ID NO: 77) and

[0322] B1G4 is TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIK (SEQ ID NO: 51) and is

[0323] All combinations of VNAR and linker are explicitly included herein. Humanized derivatives of VNAR are also included herein. Any recombinant fusion protein disclosed herein that exhibits the presence of an EGFR-specific binding molecule can be modified to contain an EGFR-specific binding molecule. It is expressly intended that any EGFR-specific binding molecule disclosed herein can be bound to any recombinant fusion protein disclosed herein that exhibits the presence of an EGFR-specific binding molecule in any configuration.

[0324] According to a third aspect, there is also provided a recombinant fusion comprising an 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.

[0325] According to a third aspect, there is also provided a recombinant fusion comprising an 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,

[0326] B1G4-[WGM]-CD3 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIKPGVQPAPGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO: 117)

[0327] G3CP-[WGM]-CD3 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNPGVQPAPGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO: 118)

[0328] G3CPG4-[WGM]-CD3 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKPGVQPAPGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO: 119)

[0329] P3A1G1AE3-[WGM]-CD3 TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIKPGVQPAPGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO: 120)

[0330] B1G4-[WGM]-BA11-[G4S]-CD3 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPWLVQWYDGAGTKVEIKPGVQPAPGGGGSTRVDQSPSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO: 121)

[0331] G3CP-[WGM]-BA11-[G4S]-CD3 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNPGVQPAPGGGGSTRVDQSPSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO: 122)

[0332] G3CPG4-[WGM]-BA11-[G4S]-CD3 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKPGVQPAPGGGGSTRVDQSPSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO: 123)

[0333] P3A1G1AE3-[WGM]-BA11-[G4S]-CD3 TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIKPGVQPAPGGGGSTRVDQSPSSLSASVGDRVTITCVLTDTSYPLYSTYWYRKNPGSSNKEQISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAMSTNIWTGDGAGTKVEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO: 124)

[0334] P3A1-[WGM]-G3CP-[G4S]-CD3 TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNPGVQPAPGGGGSASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO: 125)

[0335] P3A1-[WGM]-G3CPG4-[G4S]-CD3 TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNPGVQPAPGGGGSTRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO: 126)

[0336] P3A1G1AE3-[WGM]-G3CP-[G4S]-CD3 TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIKPGVQPAPGGGGSASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO: 127)

[0337] P3A1G1AE3-[WGM]-G3CPG4-[G4S]-CD3 TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIKPGVQPAPGGGGSTRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO: 128)

[0338] P3A1G1AE3-[WGM]-D3-[G4S]-CD3 TRVDQSPSSLSASVGDRVTITCVLTGTRYGLYSTYWYRKNPGSSDEERISISGRYSESVNKGTKSFTLTISSLQPEDSATYYCRAREARHPWLRQWYDGAGTKVEIKPGVQPAPGGGGSASVNQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKRAKSFSLRIKDLTVADSATYYCKAQSGMAISTGSGHGYNWYDGAGTVLTVNGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSHHHHHH(SEQ ID NO:129) include those below.

[0339] Any CD3 binding sequence known in the art and its variants may be substituted as described above. For example,

[0340] UCL OKT3 sequence (WO 2019 / 008379 pamphlet) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTRYTMHWVRQAPGQGLEWMGYINPSRGYTNYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARYYDDHYCLDYWGQGTMVTVSSVEGGSGGSGGSGGSGGVDDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQWSSNPFTFGQGTKVEIK(SEQ ID NO:130)

[0341] Harpoon ID20 (Pamphlet of International Publication No. 2016 / 187594) DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 131) is as follows.

[0342] The Fc region can be engineered to reduce FcγR binding. Thus, the Fc regions disclosed herein can be engineered to reduce FcγR binding.

[0343] In one embodiment, the fragment may be a first fragment of an immunoglobulin Fc region engineered to dimerize with a second fragment of the immunoglobulin Fc region. For example, the ROR1-specific antigen-binding molecule may be fused to the first fragment of the immunoglobulin Fc region, and the EGFR-specific binding molecule may be fused to the second fragment of the immunoglobulin Fc region.

[0344] As used herein, an immunoglobulin Fc region that is "engineered to dimerize" may contain 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 is more energetically favorable, and thus promotes dimerization and / or makes dimerization more energetically favorable. Such recombinant fusion proteins may have particular utility in the preparation of bispecific and / or biparatopic binders.

[0345] Methods for generating Fc-based bispecific and / or biparatopic binders via the pairing of two distinct Fc heavy chains that are engineered to dimerize are known in the art. These methods enable the assembly of Fc regions from two different heavy chains, each fused to a sequence having a target binding domain or different binding characteristics. The target binding domains or sequences can be directed to different targets to generate multispecific binders and / or to different regions or epitopes on the same target to generate biparatopic binding proteins. Multiple binding domains or sequences can be fused to the Fc region to create multispecific or multiparatopic binders or both multispecific multiparatopic binders within the same protein. Methods for generating these asymmetric bispecific and / or biparatopic binders via the heterodimerization of two different Fc heavy chains or fragments thereof include, but are not limited to, knob-into-hole (Y-T), knob-into-hole (CW-CSAV), CH3 charge pairing, Fab-arm exchange, SEED technology, BEAT technology, HA-TF, ZW1 approach, Biclonic approach, EW-RVT, and Triomab. See, for example, 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.

[0346] In one embodiment, the first fragment of the immunoglobulin Fc region is engineered to dimerize with a second fragment of the immunoglobulin Fc region by a method selected from the group consisting of knob-into-hole (Y-T), knob-into-hole (CW-CSAV), CH3 charge pairing, Fab-arm exchange, SEED technology, BEAT technology, HA-TF, ZW1 approach, Biclonic approach, EW-RVT, and Triomab.

[0347] Knob-into-Hole (Y-T) may include a T366Y substitution in the first CH3 domain and a Y407T substitution in the second CH3 domain.

[0348] Knob-into-Hole (CW-CSAV) may include one or more (preferably all) of the following substitutions in the first CH3 domain: S354C, T366W. Knob-into-Hole (CW-CSAV) may include one or more (preferably all) of the following substitutions in the second CH3 domain: Y349C, T366S, L368A, Y407V. Knob-into-Hole (CW-CSAV) may include a disulfide bond in CH3.

[0349] CH3 charge pair formation may include one or more (preferably all) of the following substitutions in the first CH3 domain: K392D, K409D. CH3 charge pair formation may include one or more (preferably all) of the following substitutions in the second CH3 domain: E356K, D399K.

[0350] Fab arm exchange may 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 changes. Thus, DuoBody technology may include a K409R substitution in the first CH3 domain and an F405L substitution in the second CH3 domain.

[0351] The SEED technology can incorporate known substitutions and / or result in IgG / A chimeras. Complementarity in the CH3 interface that enables heterodimeric assembly of the Fc chains was exploited to engineer chain-exchange engineered domains (SEED) heterodimers. These SEED CH3 domains are composed of alternating segments (AG SEED CH3 and GA SEED CH3) derived from human IgA and IgG CH3 sequences and were used to generate so-called SEED bodies, incorporated herein by reference in its entirety: Davis et al (2010) PEDS 23,4,195-202. Since molecular modeling suggested that interaction with FcRn was impaired in AG SEED CH3, residues at the CH2-CH3 junction were reverted to the IgG sequence. Pharmacokinetic studies have confirmed that the half-life of the SEED body is comparable to that of other Fc fusion proteins and IgG1.

[0352] The 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).

[0353] HA-TF may include one or more (preferably all) of the following substitutions in the first CH3 domain: S364H, F405A. HA-TF may include one or more (preferably all) of the following substitutions in the second CH3 domain: Y349T, T394F.

[0354] The ZW1 approach may include one or more (preferably all) of the following substitutions in the first CH3 domain: T350V, L351Y, F405A, Y407V. The ZW1 approach may include one or more (preferably all) of the following substitutions in the second CH3 domain: T350V, T366L, K392L, T394W.

[0355] The biclonic approach may include one or more (preferably all) of the following substitutions in the first CH3 domain: 366K(+351K). The biclonic approach may include one or more (preferably all) of the following substitutions in the second CH3 domain: 351D or one or more of E or D at 349, 368, 349 or 349+355.

[0356] 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. The disulfide bridge may be supported by further incorporation of Y349C into the first CH3 domain and S354C into the second CH3 domain.

[0357] Triomab can be formed by fusing a mouse hybridoma with a rat hybridoma, resulting in the production of a bispecific asymmetric hybrid IgG molecule. Then, preferential pairing of the light chain with its corresponding heavy chain can occur.

[0358] In one embodiment, one or more residues of a fragment of the immunoglobulin Fc region include one or more amino acid substitutions suitable for heterodimerization with a second fragment of the immunoglobulin Fc region that includes one or more corresponding amino acid substitutions. In one embodiment, one or more residues of a fragment of the immunoglobulin Fc region include one or more amino acid substitutions suitable for knob-in-hole (KIH) dimerization with a second fragment of the immunoglobulin Fc region that includes 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.

[0359] 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.

[0360] In one embodiment, one or more amino acid substitutions are selected from the group consisting of T366Y and Y407T.

[0361] The recombinant fusion protein may comprise the sequence according to SEQ ID NO: 146 or SEQ ID NO: 147.

[0362] G3CP hFc(S239C+Y407T) SEQ ID NO: 146 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0363] G3CPG4 hFc(S239C+Y407T) SEQ ID NO: 147 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0364] The recombinant fusion protein may contain the sequence according to SEQ ID NO: 194, SEQ ID NO: 195 or SEQ ID NO: 196.

[0365] 1H8 hFc(S239C+Y407T) SEQ ID NO: 194 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0366] 1H8 G4 hFc(S239C+Y407T) SEQ ID NO: 195 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0367] 1H8 v15 hFc(S239C+Y407T) Sequence number 196 ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0368] The recombinant fusion protein may contain the sequence according to SEQ ID NO: 148.

[0369] P3A1 hFc(S239C+T366Y) Sequence number 148 TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0370] The recombinant fusion protein comprises the sequence according to SEQ ID NO: 191 or SEQ ID NO: 192.

[0371] G3CP hFc(S239C+T366Y) SEQ ID NO: 191 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0372] G3CPG4 hFc(S239C+T366Y) SEQ ID NO: 192 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0373] The recombinant fusion protein may contain the sequence according to SEQ ID NO: 197, SEQ ID NO: 198, or SEQ ID NO: 199.

[0374] 1H8 hFc(S239C+T366Y) SEQ ID NO: 197 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0375] 1H8 G4 hFc(S239C+T366Y) SEQ ID NO: 198 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0376] 1H8 v15 hFc(S239C+T366Y) SEQ ID NO:199 ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0377] The recombinant fusion protein may comprise the sequence according to SEQ ID NO:193.

[0378] P3A1 hFc(S239C+Y407T) SEQ ID NO:193: TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0379] The recombinant fusion protein may comprise the sequence according to SEQ ID NO: 550 to SEQ ID NO: 561.

[0380] G3CP-hFc(S442C+T366Y) (SEQ ID NO: 550) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0381] G3CPG4 hFc(S442C+T366Y) (SEQ ID NO: 551) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0382] 1H8-hFc(S442C+T366Y)(SEQ ID NO: 552) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0383] 1H8 G4 hFc(S442C+T366Y)(SEQ ID NO: 553) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0384] 1H8 V15 hFc(S442C+T366Y) (SEQ ID NO: 554) ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0385] P3A1 hFc(S442C+T366Y) (SEQ ID NO: 555) TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0386] G3CP-hFc(S442C+Y407T)(SEQ ID NO: 556) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0387] G3CPG4 hFc(S442C+Y407T)(SEQ ID NO: 557) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0388] 1H8-hFc(S442C+Y407T) (Accession No. 558) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0389] 1H8 G4 hFc(S442C+Y407T) (Accession No. 559) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0390] 1H8 V15 hFc(S442C+Y407T) (SEQ ID NO: 560) ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0391] P3A1 hFc(S442C+Y407T) (SEQ ID NO: 561) TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0392] The recombinant fusion protein may contain a sequence according to any one of SEQ ID NO: 253 to SEQ ID NO: 258.

[0393] G3CP hFc(S239C&S442C+Y407T) SEQ ID NO: 253 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0394] G3CPG4 hFc(S239C&S442C+Y407T) SEQ ID NO: 254 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0395] 1H8 hFc(S239C&S442C+Y407T) Sequence number 255 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0396] 1H8 G4 hFc(S239C&S442C+Y407T) Sequence number 256 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0397] 1H8 v15 hFc(S239C&S442C+Y407T) Sequence No. 257 ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0398] P3A1 hFc(S239C&S442C+Y407T) Sequence No. 258 TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0399] The recombinant fusion protein may contain a sequence according to any one of SEQ ID NO: 259 to SEQ ID NO: 264.

[0400] P3A1 hFc(S239C&S442C+T366Y) SEQ ID NO: 259 TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0401] G3CP hFc(S239C&S442C+T366Y) SEQ ID NO: 260 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0402] G3CPG4 hFc(S239C&S442C+T366Y) Sequence number 261 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0403] 1H8 hFc(S239C&S442C+T366Y) Sequence number 262 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0404] 1H8 G4 hFc(S239C&S442C+T366Y) Sequence number 263 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0405] 1H8 v15 hFc(S239C&S442C+T366Y) Sequence number 264 ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0406] The recombinant fusion protein may contain a sequence according to any one of SEQ ID NO: 265 to SEQ ID NO: 268.

[0407] 7D12-hFc(S239C+Y407T) (SEQ ID NO: 265) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0408] EGFR#33-hFc(S239C+Y407T) (SEQ ID NO: 266) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0409] EGFR#13-hFc(S239C+Y407T)(SEQ ID NO: 267) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0410] 9G8-hFc(S239C+Y407T)(SEQ ID NO: 268) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0411] The recombinant fusion protein may contain a sequence according to any one of SEQ ID NO: 269 to SEQ ID NO: 272.

[0412] 7D12-hFc(S442C+Y407T) (SEQ ID NO: 269) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0413] EGFR#33-hFc(S442C+Y407T) (SEQ ID NO: 270) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0414] EGFR#13-hFc(S442C+Y407T)(SEQ ID NO: 271) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0415] 9G8-hFc(S442C+Y407T)(SEQ ID NO: 272) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0416] The recombinant fusion protein may contain a sequence according to any one of SEQ ID NO: 273 to SEQ ID NO: 276.

[0417] 7D12-hFc(S239C&S442C+Y407T) (SEQ ID NO: 273) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0418] EGFR#33-hFc(S239C&S442C+Y407T) (SEQ ID NO: 274) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0419] EGFR#13-hFc(S239C&S442C+Y407T)(SEQ ID NO: 275) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0420] 9G8-hFc(S239C&S442C+Y407T)(SEQ ID NO: 276) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0421] The recombinant fusion protein may contain a sequence according to any one of SEQ ID NO: 277 to SEQ ID NO: 280.

[0422] 7D12-hFc(S239C+T366Y) (SEQ ID NO: 277) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0423] EGFR#33-hFc(S239C+T366Y) (SEQ ID NO: 278) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0424] EGFR#13-hFc(S239C+T366Y)(SEQ ID NO: 279) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0425] 9G8-hFc(S239C+T366Y)(SEQ ID NO: 280) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0426] The recombinant fusion protein may contain a sequence according to any one of SEQ ID NO: 281 to SEQ ID NO: 284.

[0427] 7D12-hFc(S442C+T366Y) (SEQ ID NO: 281) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0428] EGFR#33-hFc(S442C+T366Y) (SEQ ID NO: 282) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0429] EGFR#13-hFc(S442C+T366Y)(SEQ ID NO: 283) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0430] 9G8-hFc(S442C+T366Y)(SEQ ID NO: 284) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0431] The recombinant fusion protein may contain a sequence according to any one of SEQ ID NO: 285 to SEQ ID NO: 288.

[0432] 7D12-hFc(S239C&S442C+T366Y) (SEQ ID NO: 285) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0433] EGFR#33-hFc(S239C&S442C+T366Y) (SEQ ID NO: 286) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0434] EGFR#13-hFc(S239C&S442C+T366Y) (SEQ ID NO: 287) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0435] 9G8-hFc(S239C&S442C+T366Y) (SEQ ID NO: 288) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0436] The recombinant fusion protein may contain a sequence according to any one of SEQ ID NO: 358 to SEQ ID NO: 380 and SEQ ID NO: 390 to SEQ ID NO: 407. When the recombinant fusion protein contains humanized cetuximab Fab HC hFc, the humanized cetuximab Fab HC hFc is usually linked via a disulfide bond to a humanized cetuximab Fab LC containing the sequence according to SEQ ID NO: 360.

[0437] Humanized cetuximab Fab HC hFc (S239C+T366Y) (SEQ ID NO: 369) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0438] Humanized Cetuximab scFv hFc (S239C+T366Y) (SEQ ID NO: 370) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0439] Humanized Cetuximab Fab HC hFc (S239C + Y407T) (SEQ ID NO: 371) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0440] Humanized Cetuximab scFv hFc (S239C+Y407T) (SEQ ID NO: 372) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0441] Humanized Cetuximab Fab HC hFc (S239C+S442C+T366Y) (SEQ ID NO: 373) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0442] Humanized Cetuximab scFv hFc (S239C+S442C+T366Y) (SEQ ID NO: 374) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0443] Humanized Cetuximab Fab HC hFc (S239C + S442C + Y407T) (SEQ ID NO: 375) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0444] Humanized cetuximab scFv hFc (S239C+S442C+Y407T) (SEQ ID NO: 376) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0445] Humanized Cetuximab Fab HC hFc (S442C + T366Y) (SEQ ID NO: 377) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0446] Humanized cetuximab scFv hFc (S442C + T366Y) (SEQ ID NO: 378) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0447] Humanized Cetuximab Fab HC hFc (S442C + Y407T) (SEQ ID NO: 379) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0448] Humanized cetuximab scFv hFc (S442C+Y407T) (SEQ ID NO: 380) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0449] 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 one of SEQ ID NOs: 146, 147, 194, 195, 196, and 193 containing the Y407T point mutation. The biparatopic dimer can comprise one of SEQ ID NOs: 148, 191, 192, 197, 198, and 199 containing the 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 of the recombinant fusion proteins disclosed herein may be conjugated to any of the linkers and payloads disclosed herein, and any of the biparatopic dimers disclosed herein may be conjugated to any of the linkers and payloads disclosed herein. The conjugation can be by any one or more S239C or S442C residues in the biparatopic dimer.

[0450] The bivalent paratopic dimer may be conjugated to a linker and payload vc-MMAE. The bivalent paratopic dimer may comprise G3CP hFc(S239C+Y407T) (SEQ ID NO: 146) and P3A1 hFc(S239C+T366Y) (SEQ ID NO: 148) conjugated to vc-PAB-EDA-PNU.

[0451] The bivalent paratopic dimer may be conjugated to a linker and payload vc-PAB-EDA-PNU. The bivalent paratopic dimer may 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 has been shown to be more potent in vivo.

[0452] SEQ ID NOs: 146, 147, 194, 195, 196, 148, 191, 192, 193, 197, 198, and 199 contain the S239C mutation for use in conjugation reactions. When the recombinant fusion protein is not conjugated (e.g., to an anthracycline (PNU) derivative), the S239C mutation is not required and position 239 may be S instead of C. Thus, in alternative embodiments, the recombinant fusion protein or bivalent paratopic dimer may comprise a sequence according to any one of SEQ ID NOs: 146, 147, 194, 195, 196, 148, 191, 192, 193, 197, 198, and 199, except that each sequence does not contain the S239C mutation.

[0453] The recombinant fusion protein can be a biparatopic dimer comprising any one or any two of SEQ ID NO: 550 to SEQ ID NO: 561. The biparatopic dimer may comprise one of SEQ ID NO: 556 to SEQ ID NO: 561, including the Y407T point mutation. The biparatopic dimer may comprise one of SEQ ID NO: 550 to SEQ ID NO: 555, including the T366Y point mutation. The biparatopic dimer may 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 may be conjugated to any linker and payload disclosed herein. Any biparatopic dimer disclosed herein may be conjugated to any linker and payload disclosed herein. Conjugation can be effected by any one or more S239C or S442C residues in the biparatopic dimer.

[0454] The biparatopic dimer may be conjugated to a linker and the payload vc-MMAE. The biparatopic dimer may comprise G3CP hFc(S2442C+Y407T) (SEQ ID NO: 556) and P3A1 hFc(S442C+T366Y) (SEQ ID NO: 555) conjugated to vc-PAB-EDA-PNU.

[0455] The biparatopic dimer may be conjugated to a linker and the payload vc-PAB-EDA-PNU. The biparatopic dimer may comprise 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 has been shown to be more potent in vivo.

[0456] Array numbers 550 to 561 contain the S442C mutation for use in the conjugation reaction. If the recombinant fusion protein is not conjugated (e.g., with an anthracycline (PNU) derivative), the S442C mutation is not necessary and position 442 may be S instead of C. Thus, in another embodiment, the recombinant fusion protein or biparatopic dimer may contain the sequence by any one of array numbers 550 to 561. However, each sequence does not contain the S442C mutation.

[0457] The above-mentioned biparatopic dimer contains either the point mutation of S442C or S239C. Also disclosed herein is a biparatopic dimer containing both S442C and S239C corresponding to the above-mentioned biparatopic dimer.

[0458] The recombinant fusion protein may contain the sequence by array number 165 or array number 166.

[0459] G3CP-hFc(Y407T) Array number 165: ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0460] G3CP G4-hFc(Y407T) Array number 166: TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0461] The recombinant fusion protein may contain the sequence according to SEQ ID NO: 200, SEQ ID NO: 201 or SEQ ID NO: 202.

[0462] 1H8 hFc(Y407T) SEQ ID NO: 200 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0463] 1H8 G4 hFc(Y407T) SEQ ID NO: 201 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0464] 1H8 v15 hFc(Y407T) Sequence number 202 ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0465] The recombinant fusion protein may contain the sequence according to SEQ ID NO: 167.

[0466] P3A1 hFc(T366Y) Sequence number 167: TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0467] The recombinant fusion protein may contain the sequence according to SEQ ID NO: 188 or SEQ ID NO: 189.

[0468] G3CP hFc(T366Y) SEQ ID NO: 188 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0469] G3CPG4 hFc(T366Y) SEQ ID NO: 189 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0470] The recombinant fusion protein may contain a sequence according to SEQ ID NO: 203, SEQ ID NO: 204 or SEQ ID NO: 205.

[0471] 1H8 hFc(T366Y) SEQ ID NO: 203 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0472] 1H8 G4 hFc(T366Y) SEQ ID NO: 204 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0473] 1H8 v15 hFc(T366Y) Sequence number 205 ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0474] The recombinant fusion protein may contain the sequence according to SEQ ID NO: 190.

[0475] P3A1 hFc(Y407T) Sequence number 190 TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0476] The recombinant fusion protein may comprise a sequence according to any one of SEQ ID NO: 289 to SEQ ID NO: 292, or a sequence according to any one of SEQ ID NO: 289 to SEQ ID NO: 292 comprising S239C and / or S442C substitutions.

[0477] 7D12-hFc(Y407T) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 289)

[0478] EGFR#33-hFc(Y407T) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 290)

[0479] EGFR#13-hFc(Y407T) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 291)

[0480] 9G8-hFc(Y407T) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 292)

[0481] The recombinant fusion protein may contain a sequence according to any one of SEQ ID NOs: 293 to 296.

[0482] 7D12-hFc(T366Y) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 293)

[0483] EGFR#33-hFc(T366Y) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 294)

[0484] EGFR#13-hFc(T366Y) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 295)

[0485] 9G8-hFc(T366Y) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 296)

[0486] Each of SEQ ID NOs: 146 to 148, 165 to 167, 178 to 205, 223 to 296, and 550 to 561 contains a (G4S)3 linker. Also explicitly contemplated herein are the corresponding sequences in which the (G4S)3 linker has been replaced with a (G4S)1 linker. For example, the recombinant fusion protein may contain one or more of SEQ ID NOs: 297 to 356, 390 to 431, and 562 to 597 below.

[0487] G3CP G4S-hFc ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 408)

[0488] G3CPG4 G4S-hFc TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 409)

[0489] 1H8 G4S-hFc ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 410)

[0490] 1H8 G4 G4S-hFc TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 411)

[0491] 1H8 V15 G4S-hFc ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 412)

[0492] P3A1 G4S-hFc TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 413)

[0493] G3CP G4S-hFc(S239C) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 414)

[0494] G3CPG4 G4S-hFc(S239C) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 415)

[0495] 1H8 G4S-hFc(S239C) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 416)

[0496] 1H8 G4 G4S-hFc(S239C) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 417)

[0497] 1H8 V15 G4S-hFc(S239C) ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 418)

[0498] P3A1 G4S-hFc(S239C) TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 419)

[0499] G3CP G4S-hFc(S442C) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 420)

[0500] G3CPG4 G4S-hFc(S442C) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 421)

[0501] 1H8 G4S-hFc(S442C) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 422)

[0502] 1H8 G4 G4S-hFc(S442C) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 423)

[0503] 1H8 V15 G4S-hFc(S442C) ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 424)

[0504] P3A1 G4S-hFc(S442C) TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 425)

[0505] G3CP G4S-hFc(S239C&S442C) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 426)

[0506] G3CPG4 G4S-hFc(S239C&S442C) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 427)

[0507] 1H8 G4S-hFc(S239C&S442C) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 428)

[0508] 1H8 G4 G4S-hFc(S239C&S442C) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK(SEQ ID NO: 429)

[0509] 1H8 V15 G4S-hFc(S239C&S442C) ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 430)

[0510] P3A1 G4S-hFc(S239C&S442C) TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK (SEQ ID NO: 431)

[0511] P3A1 G4S-hFc(S239C+Y407T) SEQ ID NO: 308: TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0512] G3CP G4S-hFc(S239C+Y407T) SEQ ID NO: 297 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0513] G3CPG4 G4S-hFc(S239C+Y407T) SEQ ID NO: 298 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0514] 1H8 G4S-hFc(S239C+Y407T) Sequence number 299 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0515] 1H8 G4 G4S-hFc(S239C+Y407T) Sequence number 300 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0516] 1H8 v15 G4S-hFc(S239C+Y407T) Sequence number 301 ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0517] P3A1 G4S-hFc(S239C+T366Y) Sequence number 302 TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0518] G3CP G4S-hFc(S239C+T366Y) Sequence number 303 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0519] G3CPG4 G4S-hFc(S239C+T366Y) Sequence number 304 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0520] 1H8 G4S-hFc(S239C+T366Y) Sequence number 305 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0521] 1H8 G4 G4S-hFc(S239C+T366Y) Sequence number 306 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0522] 1H8 v15 G4S-hFc(S239C+T366Y) Sequence number: 307 ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0523] P3A1 G4S-hFc(S239C&S442C+Y407T) Sequence number: 309 TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0524] G3CP G4S-hFc(S239C&S442C+Y407T) Sequence number 310 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0525] G3CPG4 G4S-hFc(S239C&S442C+Y407T) Sequence number 311 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0526] 1H8 G4S-hFc(S239C&S442C+Y407T) Sequence number 312 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0527] 1H8 G4 G4S-hFc(S239C&S442C+Y407T) Sequence number 313 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0528] 1H8 v15 G4S-hFc(S239C&S442C+Y407T) Sequence number 314 ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0529] P3A1 G4S-hFc(S239C&S442C+T366Y) Sequence number 315 TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0530] G3CP G4S-hFc(S239C&S442C+T366Y) Sequence number 316 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0531] G3CPG4 G4S-hFc(S239C&S442C+T366Y) Sequence number 317 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0532] 1H8 G4S-hFc(S239C&S442C+T366Y) Sequence number 318 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0533] 1H8 G4 G4S-hFc(S239C&S442C+T366Y) Sequence number 319 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0534] 1H8 v15 G4S-hFc(S239C&S442C+T366Y) Sequence number 320 ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0535] P3A1 G4S-hFc(S442C+Y407T) Sequence number 321 TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTSPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0536] G3CP G4S-hFc(S442C+Y407T) Sequence number 322 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0537] G3CPG4 G4S-hFc(S442C+Y407T) Sequence number 323 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0538] 1H8 G4S-hFc(S442C +Y407T) Sequence number 324 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0539] 1H8 G4 G4S-hFc(S442C +Y407T) Sequence number 325 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0540] 1H8 v15 G4S-hFc(S442C+Y407T) Sequence number 326 ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0541] P3A1 G4S-hFc(S442C +T366Y) Sequence number 327 TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0542] G3CP G4S-hFc(S442C+T366Y) Sequence number 328 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0543] G3CPG4 G4S-hFc(S442C+T366Y) Sequence number 329 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0544] 1H8 G4S-hFc(S442C+T366Y) Sequence number 330 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0545] 1H8 G4 G4S-hFc(S442C+T366Y) Sequence number 331 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0546] 1H8 v15 G4S-hFc(S442C+T366Y) Sequence number 332 ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0547] 7D12-G4S-hFc(S239C+Y407T)(Sequence number 333 QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0548] EGFR#33-G4S-hFc(S239C+Y407T)(SEQ ID NO: 334) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0549] EGFR#13-G4S-hFc(S239C+Y407T)(SEQ ID NO: 335) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0550] 9G8 - G4S - hFc(S239C + Y407T) (SEQ ID NO: 336) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0551] 7D12 - G4S - hFc(S442C + Y407T) (SEQ ID NO: 337) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0552] EGFR#33-G4S-hFc(S442C+Y407T)(SEQ ID NO: 338) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0553] EGFR#13-G4S-hFc(S442C+Y407T)(SEQ ID NO: 339) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0554] 9G8 - G4S - hFc(S442C+Y407T) (SEQ ID NO: 340) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0555] 7D12 - G4S - hFc(S239C&S442C+Y407T) (SEQ ID NO: 341) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0556] EGFR#33-G4S-hFc(S239C&S442C+Y407T)(SEQ ID NO: 342) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0557] EGFR#13-G4S-hFc(S239C&S442C+Y407T)(SEQ ID NO: 343) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0558] 9G8 - G4S - hFc(S239C&S442C+Y407T) (SEQ ID NO: 344) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0559] 7D12 - G4S - hFc(S239C+T366Y) (SEQ ID NO: 345) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0560] EGFR#33-G4S-hFc(S239C+T366Y)(SEQ ID NO: 346) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0561] EGFR#13-G4S-hFc(S239C+T366Y)(SEQ ID NO: 347) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0562] 9G8 - G4S - hFc(S239C+T366Y) (SEQ ID NO: 348) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0563] 7D12 - G4S - hFc(S442C+T366Y) (SEQ ID NO: 349) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0564] EGFR#33-G4S-hFc(S442C+T366Y)(SEQ ID NO: 350) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0565] EGFR#13-G4S-hFc(S442C+T366Y)(SEQ ID NO: 351) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0566] 9G8 - G4S - hFc(S442C+T366Y) (SEQ ID NO: 352) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0567] 7D12 - G4S - hFc(S239C&S442C+T366Y) (SEQ ID NO: 353) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0568] EGFR#33-G4S-hFc(S239C&S442C+T366Y)(SEQ ID NO: 354) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0569] EGFR#13-G4S-hFc(S239C&S442C+T366Y)(SEQ ID NO: 355) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0570] 9G8 - G4S - hFc(S239C&S442C+T366Y) (SEQ ID NO: 356) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0571] Humanized Cetuximab Fab HC - G4S - hFc(S239C) (SEQ ID NO: 390) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0572] Humanized Cetuximab scFv-G4S-hFc(S239C) (SEQ ID NO: 391) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0573] Humanized Cetuximab Fab HC-G4S-hFc(S239C+S442C) (SEQ ID NO: 392) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0574] Humanized cetuximab scFv-G4S-hFc (S239C+S442C) (SEQ ID NO: 393) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0575] Humanized cetuximab Fab HC-G4S-hFc(S442C) (SEQ ID NO: 394) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0576] Humanized Cetuximab scFv-G4S-hFc(S442C) (SEQ ID NO: 395) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0577] Humanized cetuximab Fab HC-G4S-hFc (S239C+T366Y) (SEQ ID NO: 396) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0578] Humanized cetuximab scFv-G4S-hFc (S239C+T366Y) (SEQ ID NO: 397) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0579] Humanized Cetuximab Fab HC-G4S-hFc(S239C+Y407T) (SEQ ID NO: 398) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0580] Humanized cetuximab scFv-G4S-hFc (S239C+Y407T) (SEQ ID NO: 399) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0581] Humanized cetuximab Fab HC-G4S-hFc (S239C+S442C+T366Y) (SEQ ID NO: 400) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0582] Humanized cetuximab scFv-G4S-hFc (S239C+S442C+T366Y) (SEQ ID NO: 401) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0583] Humanized Cetuximab Fab HC-G4S-hFc (S239C + S442C + Y407T) (SEQ ID NO: 402) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0584] Humanized cetuximab scFv-G4S-hFc (S239C+S442C+Y407T) (SEQ ID NO: 403) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0585] Humanized Cetuximab Fab HC-G4S-hFc(S442C+T366Y) (SEQ ID NO: 404) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0586] Humanized Cetuximab scFv-G4S-hFc (S442C+T366Y) (SEQ ID NO: 405) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0587] Humanized cetuximab Fab HC-G4S-hFc(S442C+Y407T) (SEQ ID NO: 406) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0588] Humanized cetuximab scFv-G4S-hFc (S442C+Y407T) (SEQ ID NO: 407) EVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGLEWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLIKYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTFGQGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0589] G3CP-G4S-hFc(Y407T) (Accession No. 562) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPYNVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0590] G3CP G4 G4S-hFc(Y407T) (Accession No. 563) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0591] P3A1 G4S hFc(Y407T) (Accession No. 564) TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0592] 1H8 G4ShFc(Y407T) (Accession No. 565 ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0593] 1H8 G4 G4S hFc(Y407T) (Sequence number 566 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0594] 1H8 v15 G4S hFc(Y407T) (Sequence number 567 ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0595] P3A1G4S hFc(T366Y) (Accession No. 568 TRVDQTPRTATKETGESLTINCVLTDTSYGLYSTSWFRKNPGTTDWERMSIGGRYVESVNKGAKSFSLRIKDLTVADSATYYCKAREARHPWLRQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0596] G3CP G4ShFc(T366Y) (Accession No. 569 TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0597] G3CPG4 G4S hFc(T366Y) (SEQ ID NO: 570) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPYNVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0598] 1H8 G4ShFc(T366Y) (SEQ ID NO: 571) ASVNQTPRTATKETGESLTINCVVTGANYGLAATYWYRKNPGSSNQERISISGRYVESVNKRTMSFSLRIKDLTVADSATYYCKAYPWGAGAPSSVQWYDGAGTVLTVNGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0599] 1H8 G4 G4ShFc(T366Y) (Accession No. 572) TRVDQSPSSLSASVGDRVTITCVLTDANYGLAATYWYRKNPGSSNKERISISGRYSESVNKGTMSFTLTISSLQPEDSATYYCRAYPWGAGAPSSVQWYDGAGTKVEIKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0600] 1H8 v15 G4ShFc(T366Y) (Accession No. 573) ASVTQSPRSASKETGESLTITCRVTGANYGLAATYWYRKNPGSSNQERISISGRYSESVNKRTMSFSLRISSLTVEDSATYYCKAYPWGAGAPSSVQWYDGQGTKLEVKGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0601] 7D12-G4S-hFc (Accession No. 574) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0602] EGFR#33G4S-hFc (Accession No. 575) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0603] EGFR#13 - G4S - hFc (SEQ ID NO: 576) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0604] 9G8 - G4S - hFc (SEQ ID NO: 577) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0605] 7D12 - G4S - hFc(S239C) (SEQ ID NO: 578) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0606] EGFR#33 - G4S - hFc(S239C) (SEQ ID NO: 579) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0607] EGFR#13-G4S-hFc(S239C) (SEQ ID NO: 580) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0608] 9G8-G4S-hFc(S239C) (SEQ ID NO: 581) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0609] 7D12 - G4S - hFc(S442C) (SEQ ID NO: 582) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0610] EGFR#33 - G4S - hFc(S442C) (SEQ ID NO: 583) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0611] EGFR#13-G4S-hFc(S442C)(SEQ ID NO: 584 AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0612] 9G8-G4S-hFc(S442C)(SEQ ID NO: 585) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0613] 7D12 - G4S - hFc(S239C&S442C) (SEQ ID NO: 586) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0614] EGFR#33 - G4S - hFc(S239C&S442C) (SEQ ID NO: 587) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0615] EGFR#13-G4S-hFc(S239C&S442C)(SEQ ID NO: 588) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0616] 9G8-G4S-hFc(S239C&S442C)(SEQ ID NO: 589) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLCLSPGK

[0617] 7D12 - G4S - hFc(Y407T) (SEQ ID NO: 590) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0618] EGFR#33 - G4S - hFc(Y407T) (SEQ ID NO: 591) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0619] EGFR#13-G4S-hFc(Y407T) (SEQ ID NO: 592) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0620] 9G8-G4S-hFc(Y407T) (SEQ ID NO: 593) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0621] 7D12-G4S-hFc(T366Y) (SEQ ID NO: 594) QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0622] EGFR#33-G4S-hFc(T366Y) (SEQ ID NO: 595) EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDYWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0623] EGFR#13-G4S-hFc(T366Y) (SEQ ID NO: 596) AVQLVESGGGSVQAGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSTWYGTLYEYDAWGQGTLVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0624] 9G8-G4S-hFc(T366Y) (SEQ ID NO: 597) EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEYDYWGQGTQVTVSSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0625] In any embodiment, the specific binding molecule comprising the amino acid sequence represented by formula (I), the (G4S)3 linker, and the fragment of the immunoglobulin Fc region may be replaced with the corresponding specific binding molecule comprising the amino acid sequence represented by formula (I), the (G4S)1 linker, and the fragment of the immunoglobulin Fc region, and vice versa.

[0626] According to a fourth aspect, the present invention is a recombinant fusion protein dimer, comprising: (a) a first recombinant fusion protein; The first recombinant fusion protein comprises a receptor tyrosine kinase-like orphan receptor 1 (ROR1) specific antigen-binding molecule comprising the amino acid sequence represented by formula (I); Formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) (wherein, 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), YPWGAGAPWNVQWY (SEQ ID NO: 24), YPSGAGAPRPVQWY (SEQ ID NO: 11), YPWGAGAPCLVQWY (SEQ ID 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); 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 a framework region; FW2 is a 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 a 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 a framework region; FW4 is a framework region; When 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 The first antigen-binding molecule is fused to a first fragment of an immunoglobulin Fc region that has been engineered to dimerize with a second fragment of the immunoglobulin Fc region; and (b) a second recombinant fusion protein; 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 that has been engineered to dimerize with a first fragment of the immunoglobulin Fc region; Provided is a recombinant fusion protein dimer comprising:

[0627] 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.

[0628] In one embodiment, the second fragment of the immunoglobulin Fc region is an Fc heavy chain.

[0629] In one embodiment, the second fragment of the immunoglobulin Fc region has been engineered to dimerize with the second fragment of the immunoglobulin Fc region by a method selected from the group consisting of knob-into-hole (Y-T), knob-into-hole (CW-CSAV), CH3 charge pair formation, Fab arm exchange, SEED technology, BEAT technology, HA-TF, ZW1 approach, Biclonic approach, EW-RVT, and Triomab.

[0630] One or more residues of the first fragment of the immunoglobulin Fc region comprise one or more corresponding amino acid substitutions suitable for knob-into-hole (KIH) dimerization with the second fragment of the immunoglobulin Fc region. One or more residues of the second fragment of the immunoglobulin Fc region comprise one or more corresponding amino acid substitutions suitable for knob-into-hole (KIH) dimerization with the first fragment of the immunoglobulin Fc region.

[0631] In one embodiment, 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 understand which amino acid substitutions represent "knobs" and which represent "holes", and thus understand which mutations are suitable for KIH dimerization with corresponding mutations. For example, T366Y is a knob variant and Y407T is a hole variant. When the first fragment of the immunoglobulin Fc region contains T366Y, the second fragment of the immunoglobulin Fc region may contain Y407T, and vice versa.

[0632] In one embodiment, one or more amino acid substitutions are selected from the group consisting of T366Y and Y407T.

[0633] Any sequence of the recombinant fusion proteins disclosed herein may contain 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 incorporation of any one or more amino acid substitutions selected from the group consisting of T366Y, Y407T, S354C, T366W, Y349C, T366S, L368A, and Y407V and can be incorporated into the recombinant fusion proteins described herein in place of the human Fc region sequence.

[0634] 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, diabody, triabody, tetrabody, bispecific T cell engager (BiTE), intein, VNAR domain, single-domain antibody (sdAb), or VH domain.

[0635] The ROR1-specific antigen-binding molecule can include any ROR1-specific antigen-binding molecule disclosed herein. The ROR1-specific antigen-binding molecule can include, for example, G3CP, 1H8, or G3CPG4.

[0636] The EGFR-specific antigen-binding molecule can include any EGFR-specific antigen-binding molecule disclosed herein. The EGFR-specific antigen-binding molecule can include, for example, 7D12, EGFR#33, EGFR#13, 9G8, cetuximab or its derivative, matuzumab or its derivative, panitumumab or its derivative, nimotuzumab or its derivative, or necitumumab or its derivative. Derivatives of EGFR-specific antigen-binding molecules such as cetuximab, matuzumab, panitumumab, nimotuzumab, or necitumumab can be the respective humanized sequences of cetuximab, matuzumab, panitumumab, nimotuzumab, or necitumumab. Derivatives of EGFR-specific antigen-binding molecules such as cetuximab, matuzumab, panitumumab, nimotuzumab, or necitumumab can be scFv sequences containing the respective CDRs of cetuximab, matuzumab, panitumumab, nimotuzumab, or necitumumab. Derivatives of EGFR-specific antigen-binding molecules such as cetuximab, matuzumab, panitumumab, nimotuzumab, or necitumumab can be the respective Fab sequences of cetuximab, matuzumab, panitumumab, nimotuzumab, or necitumumab. Here, the EGFR-specific antigen-binding molecule includes Fab and usually can include both Fab LC and Fab HC. Fab HC may be fused with a fragment of the immunoglobulin Fc region. Usually, Fab LC and Fab HC are bound via disulfide bonds. scFv, Fab LC, and / or Fab HC can be humanized. For example, the EGFR-specific antigen-binding molecule can include cetuximab Fab or a cetuximab-based scFv. The EGFR-specific antigen-binding molecule can include humanized cetuximab Fab or a humanized cetuximab-based scFv. The EGFR-specific antigen-binding molecule can include 7D12, EGFR#33, EGFR#13, or 9G8.

[0637] In one embodiment, (a) The first recombinant fusion protein comprises G3CP, 1H8, or G3CPG4, and (b) The second recombinant fusion protein comprises 7D12, EGFR#33, EGFR#13, or 9G8.

[0638] The first recombinant fusion protein may optionally comprise an ROR1-specific antigen-binding molecule such as G3CP, 1H8, or G3CPG4 fused to an Fc heavy chain via a [G4S] X linker. The first fragment of the immunoglobulin Fc region can be the first Fc heavy chain. The second fragment of the immunoglobulin Fc region can be the second Fc heavy chain. One or more residues of the first Fc heavy chain can comprise one or more amino acid substitutions suitable for knob-in-hole (KIH) dimerization with a second Fc heavy chain that comprises one or more corresponding amino acid substitutions suitable for knob-in-hole (KIH) dimerization with the first Fc heavy chain. The one or more amino acid substitutions can be selected from the group consisting of T366Y and Y407T. The first Fc heavy chain can comprise the S239C and / or S442C mutation. The second Fc heavy chain can comprise the S239C and / or S442C mutation.

[0639] The second recombinant fusion protein may optionally comprise an EGFR-specific antigen-binding molecule such as 7D12, EGFR#33, EGFR#13, or 9G8 fused to an Fc heavy chain via a [G4S] X linker. The first fragment of the immunoglobulin Fc region can be the first Fc heavy chain. The second fragment of the immunoglobulin Fc region can be the second Fc heavy chain. One or more residues of the second Fc heavy chain can comprise one or more amino acid substitutions suitable for knob-in-hole (KIH) dimerization with a first Fc heavy chain that comprises one or more corresponding amino acid substitutions suitable for knob-in-hole (KIH) dimerization with the second Fc heavy chain. The one or more amino acid substitutions can be selected from the group consisting of T366Y and Y407T. The first Fc heavy chain can comprise the S239C and / or S442C mutation. The second Fc heavy chain can comprise the S239C and / or S442C mutation.

[0640] In one embodiment, (a) The first recombinant fusion protein comprises G3CP-hFc, 1H8-hFc, or G3CPG4-hFc, and (b) The second recombinant fusion protein comprises 7D12-hFc, EGFR#33-hFc, EGFR#13-hFc, or 9G8-hFc.

[0641] In one embodiment, (a) The first recombinant fusion protein comprises G3CP-hFc(S239C), 1H8-hFc(S239C) or G3CPG4-hFc(S239C), and (b) The second recombinant fusion protein comprises 7D12-hFc(S239C), EGFR#33-hFc(S239C), EGFR#13-hFc(S239C) or 9G8-hFc(S239C).

[0642] The recombinant fusion protein dimer may be G3CP-hFc(S239C) and 7D12-hFc(S239C), G3CP-hFc(S239C) and EGFR#33-hFc(S239C), G3CP-hFc(S239C) and EGFR#13-hFc(S239C), and G3CP-hFc(S239C) and 9G8-hFc(S239C) selected from the group consisting of, wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, (a) the hFc region fused to the ROR1-specific binding molecule contains a T366Y substitution and the hFc region fused to the EGFR-specific binding molecule contains a Y407T substitution, or (b) the hFc region fused to the ROR1-specific binding molecule contains a Y407T substitution and the hFc region fused to the EGFR-specific binding molecule contains a T366Y substitution.

[0643] The recombinant fusion protein dimer may be G3CPG4-hFc(S239C) and 7D12-hFc(S239C), G3CPG4-hFc(S239C) and EGFR#33-hFc(S239C), G3CPG4-hFc(S239C) and EGFR#13-hFc(S239C), and G3CPG4-hFc(S239C) and 9G8-hFc(S239C) may be selected from the group consisting of, wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, (a) the hFc region fused to the ROR1-specific binding molecule contains a T366Y substitution and the hFc region fused to the EGFR-specific binding molecule contains a Y407T substitution, or (b) the hFc region fused to the ROR1-specific binding molecule contains a Y407T substitution and the hFc region fused to the EGFR-specific binding molecule contains a T366Y substitution.

[0644] The recombinant fusion protein dimer is, 1H8-hFc(S239C) and 7D12-hFc(S239C), 1H8-hFc(S239C) and EGFR#33-hFc(S239C), 1H8-hFc(S239C) and EGFR#13-hFc(S239C), and 1H8-hFc(S239C) and 9G8-hFc(S239C) may be selected from the group consisting of, wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, (a) the hFc region fused to the ROR1-specific binding molecule contains a T366Y substitution and the hFc region fused to the EGFR-specific binding molecule contains a Y407T substitution, or (b) the hFc region fused to the ROR1-specific binding molecule contains a Y407T substitution and the hFc region fused to the EGFR-specific binding molecule contains a T366Y substitution.

[0645] In one embodiment, (a) The first recombinant fusion protein comprises G3CP-hFc(S442C), 1H8-hFc(S442C), or G3CPG4-hFc(S442C), and (b) The second recombinant fusion protein comprises 7D12-hFc(S442C), EGFR#33-hFc(S442C), EGFR#13-hFc(S442C), or 9G8-hFc(S442C).

[0646] The recombinant fusion protein dimer may be selected from the group consisting of G3CP-hFc(S442C) and 7D12-hFc(S442C), G3CP-hFc(S442C) and EGFR#33-hFc(S442C), G3CP-hFc(S442C) and EGFR#13-hFc(S442C), and G3CP-hFc(S442C) and 9G8-hFc(S442C), wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, and (a) the hFc region fused to the ROR1-specific binding molecule contains a T366Y substitution, and the hFc region fused to the EGFR-specific binding molecule contains a Y407T substitution, or (b) the hFc region fused to the ROR1-specific binding molecule contains a Y407T substitution, and the hFc region fused to the EGFR-specific binding molecule contains a T366Y substitution.

[0647] The recombinant fusion protein dimer may be selected from the group consisting of G3CPG4-hFc(S442C) and 7D12-hFc(S442C), G3CPG4-hFc(S442C) and EGFR#33-hFc(S442C), G3CPG4-hFc(S442C) and EGFR#13-hFc(S442C), and G3CPG4-hFc(S442C) and 9G8-hFc(S442C), wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, Here, the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, (a) the hFc region fused to the ROR1-specific binding molecule contains a T366Y substitution and the hFc region fused to the EGFR-specific binding molecule contains a Y407T substitution, or (b) the hFc region fused to the ROR1-specific binding molecule contains a Y407T substitution and the hFc region fused to the EGFR-specific binding molecule contains a T366Y substitution.

[0648] The recombinant fusion protein dimer may be 1H8-hFc(S442C) and 7D12-hFc(S442C), 1H8-hFc(S442C) and EGFR#33-hFc(S442C), 1H8-hFc(S442C) and EGFR#13-hFc(S442C), and 1H8-hFc(S442C) and 9G8-hFc(S442C) selected from the group consisting of, where the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, (a) the hFc region fused to the ROR1-specific binding molecule contains a T366Y substitution and the hFc region fused to the EGFR-specific binding molecule contains a Y407T substitution, or (b) the hFc region fused to the ROR1-specific binding molecule contains a Y407T substitution and the hFc region fused to the EGFR-specific binding molecule contains a T366Y substitution.

[0649] In one embodiment, (a) the first recombinant fusion protein comprises G3CP-hFc(S239C&S442C), 1H8-hFc(S239C&S442C) or G3CPG4-hFc(S239C&S442C), and (b) The second recombinant fusion protein comprises 7D12-hFc(S239C&S442C), EGFR#33-hFc(S239C&S442C), EGFR#13-hFc(S239C&S442C), or 9G8-hFc(S239C&S442C).

[0650] The recombinant fusion protein dimer is G3CP-hFc(S239C&S442C) and 7D12-hFc(S239C&S442C), G3CP-hFc(S239C&S442C) and EGFR#33-hFc(S239C&S442C), G3CP-hFc(S239C&S442C) and EGFR#13-hFc(S239C&S442C), and G3CP-hFc(S239C&S442C) and 9G8-hFc(S239C&S442C) may be selected from the group consisting of, wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, (a) the hFc region fused to the ROR1-specific binding molecule contains a T366Y substitution and the hFc region fused to the EGFR-specific binding molecule contains a Y407T substitution, or (b) the hFc region fused to the ROR1-specific binding molecule contains a Y407T substitution and the hFc region fused to the EGFR-specific binding molecule contains a T366Y substitution.

[0651] The recombinant fusion protein dimer is G3CPG4-hFc(S239C&S442C) and 7D12-hFc(S239C&S442C), G3CPG4-hFc(S239C&S442C) and EGFR#33-hFc(S239C&S442C), G3CPG4-hFc(S239C&S442C) and EGFR#13-hFc(S239C&S442C), and G3CPG4-hFc(S239C&S442C) and 9G8-hFc(S239C&S442C) may be selected from the group consisting of, Here, the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, (a) the hFc region fused to the ROR1-specific binding molecule contains a T366Y substitution, and the hFc region fused to the EGFR-specific binding molecule contains a Y407T substitution, or (b) the hFc region fused to the ROR1-specific binding molecule contains a Y407T substitution, and the hFc region fused to the EGFR-specific binding molecule contains a T366Y substitution.

[0652] The recombinant fusion protein dimer is 1H8-hFc(S239C&S442C) and 7D12-hFc(S239C&S442C), 1H8-hFc(S239C&S442C) and EGFR#33-hFc(S239C&S442C), 1H8-hFc(S239C&S442C) and EGFR#13-hFc(S239C&S442C), and 1H8-hFc(S239C&S442C) and 9G8-hFc(S239C&S442C) may be selected from the group consisting of, Here, the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, (a) the hFc region fused to the ROR1-specific binding molecule contains a T366Y substitution, and the hFc region fused to the EGFR-specific binding molecule contains a Y407T substitution, or (b) the hFc region fused to the ROR1-specific binding molecule contains a Y407T substitution, and the hFc region fused to the EGFR-specific binding molecule contains a T366Y substitution.

[0653] In another method, the recombinant fusion protein dimer described above may contain any other ROR1-specific binding molecule described herein, instead of G3CP, 1H8, or G3CPG4.

[0654] In another method, the recombinant fusion protein dimer described above may include any other EGFR-specific binding molecule described herein instead of 7D12, EGFR#33, EGFR#13, or 9G8.

[0655] Any recombinant fusion protein dimer disclosed herein may be conjugated to any linker and drug disclosed herein, and the conjugation may be effected by any one or more S239C or S442C residues in the recombinant fusion protein dimer. Usually, when the conjugation is by the S239C residue of the first hFc region of the recombinant fusion protein dimer, the conjugation is also effected by the S239C residue of the second hFc region of the recombinant fusion protein dimer. Usually, when the conjugation is by the S442C residue of the first hFc region of the recombinant fusion protein dimer, the conjugation is also effected by the S442C residue of the second hFc region of the recombinant fusion protein dimer. Usually, when the conjugation is by the S239C and S442C residues of the first hFc region of the recombinant fusion protein dimer, the conjugation is also effected by the S239C and S442C residues of the second hFc region of the recombinant fusion protein dimer.

[0656] According to a fifth aspect, the present invention provides a recombinant fusion protein dimer, (a) a first recombinant fusion protein; The first recombinant fusion protein includes a receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen-binding molecule comprising an amino acid sequence represented by formula (I); Formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) (wherein, 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 a framework region; FW2 is a 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 a 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 a framework region; CDR3 is a CDR sequence having an amino acid sequence by REARHPWLRQWY (SEQ ID NO: 39); FW4 is a framework region); and The first antigen-binding molecule is fused to a first fragment of an immunoglobulin Fc region that has been engineered to dimerize with a second fragment of the immunoglobulin Fc region; and (b) A second recombinant fusion protein; 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 that has been engineered to dimerize with a first fragment of the immunoglobulin Fc region; Provided is a recombinant fusion protein dimer comprising.

[0657] In one embodiment, (a) The first recombinant fusion protein comprises P3A1, and (b) The second recombinant fusion protein comprises 7D12, EGFR#33, EGFR#13 or 9G8.

[0658] In one embodiment, (a) The first recombinant fusion protein comprises P3A1-hFc, and (b) The second recombinant fusion protein comprises 7D12-hFc, EGFR#33-hFc, EGFR#13-hFc or 9G8-hFc.

[0659] In one embodiment, (a) The first recombinant fusion protein comprises P3A1-hFc(S239C), and (b) The second recombinant fusion protein comprises 7D12-hFc(S239C), EGFR#33-hFc(S239C), EGFR#13-hFc(S239C) or 9G8-hFc(S239C).

[0660] The recombinant fusion protein dimer may be P3A1-hFc(S239C) and 7D12-hFc(S239C), P3A1-hFc(S239C) and EGFR#33-hFc(S239C), P3A1-hFc(S239C) and EGFR#13-hFc(S239C), and P3A1-hFc(S239C) and 9G8-hFc(S239C) selected from the group consisting of, wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, (a) the hFc region fused to the ROR1-specific binding molecule contains a T366Y substitution and the hFc region fused to the EGFR-specific binding molecule contains a Y407T substitution, or (b) the hFc region fused to the ROR1-specific binding molecule contains a Y407T substitution and the hFc region fused to the EGFR-specific binding molecule contains a T366Y substitution.

[0661] In one embodiment, (a) The first recombinant fusion protein comprises P3A1-hFc(S442C), and (b) The second recombinant fusion protein comprises 7D12-hFc(S442C), EGFR#33-hFc(S442C), EGFR#13-hFc(S442C) or 9G8-hFc(S442C).

[0662] The recombinant fusion protein dimer may be P3A1-hFc(S442C) and 7D12-hFc(S442C), P3A1-hFc(S442C) and EGFR#33-hFc(S442C), P3A1-hFc(S442C) and EGFR#13-hFc(S442C), and P3A1-hFc(S442C) and 9G8-hFc(S442C) selected from the group consisting of, wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, (a) the hFc region fused to the ROR1-specific binding molecule contains a T366Y substitution and the hFc region fused to the EGFR-specific binding molecule contains a Y407T substitution, or (b) the hFc region fused to the ROR1-specific binding molecule contains a Y407T substitution and the hFc region fused to the EGFR-specific binding molecule contains a T366Y substitution.

[0663] In one embodiment, (a) The first recombinant fusion protein comprises P3A1-hFc(S239C&S442C), and (b) The second recombinant fusion protein comprises 7D12-hFc(S239C&S442C), EGFR#33-hFc(S239C&S442C), EGFR#13-hFc(S239C&S442C) or 9G8-hFc(S239C&S442C).

[0664] The recombinant fusion protein dimer is P3A1-hFc(S239C&S442C) and 7D12-hFc(S239C&S442C), P3A1-hFc(S239C&S442C) and EGFR#33-hFc(S239C&S442C), P3A1-hFc(S239C&S442C) and EGFR#13-hFc(S239C&S442C), and P3A1-hFc(S239C&S442C) and 9G8-hFc(S239C&S442C) may be selected from the group consisting of, wherein the ROR1-specific binding molecule is fused to the hFc region via a [G4S]3 linker or a G4S linker, (a) the hFc region fused to the ROR1-specific binding molecule contains a T366Y substitution and the hFc region fused to the EGFR-specific binding molecule contains a Y407T substitution, or (b) the hFc region fused to the ROR1-specific binding molecule contains a Y407T substitution and the hFc region fused to the EGFR-specific binding molecule contains a T366Y substitution.

[0665] In another method, the recombinant fusion protein dimer described above may contain any other ROR1-specific binding molecule described herein instead of P3A1.

[0666] In another method, the recombinant fusion protein dimer described above may contain any other EGFR-specific binding molecule described herein instead of 7D12, EGFR#33, EGFR#13, or 9G8.

[0667] In one embodiment of the recombinant fusion protein dimer, 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.

[0668] In one embodiment of the recombinant fusion protein dimer, 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.

[0669] In one embodiment of the recombinant fusion protein dimer, the recombinant fusion protein dimer or the target-binding molecule-drug conjugate comprises any one of SEQ ID NOs: 369, 373, 377, 396, 400, 404, 371, 375, 379, 398, 402, and 406, and the recombinant fusion protein dimer or the target-binding molecule-drug conjugate further comprises SEQ ID NO: 360.

[0670] In one embodiment of the recombinant fusion protein dimer, 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.

[0671] In one embodiment of the recombinant fusion protein dimer, 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.

[0672] In one embodiment of the recombinant fusion protein dimer, 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.

[0673] In one embodiment of the recombinant fusion protein dimer, 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.

[0674] In one embodiment of the recombinant fusion prote...

Claims

1. A recombinant fusion protein dimer, comprising: (a) a first recombinant fusion protein; The first recombinant fusion protein comprises a receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen-binding molecule comprising an amino acid sequence represented by formula (I); Formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) (wherein CDR3 is a CDR sequence having an amino acid sequence selected from the group consisting of YPWGAGAPYN VQW Y (SEQ ID NO: 23), YPWGAGAPYLVQW Y (SEQ ID NO: 20), YPWGAGAPWN VQW Y (SEQ ID NO: 24), YPSGAGAPRPVQW Y (SEQ ID NO: 11), YPWGAGAPCLVQW Y (SEQ ID NO: 12), YPWGAGAPRLVQW Y (SEQ ID NO: 13), YPWGAGAPRQVQW Y (SEQ ID NO: 14), YPWGAGAPRSVQW Y (SEQ ID NO: 15), YPWGAGAPSLVQW Y (SEQ ID NO: 16), YPWGAGAPSNVQW Y (SEQ ID NO: 17), YPWGAGAPSQVQW Y (SEQ ID NO: 18), YPWGAGAPSSVQW Y (SEQ ID NO: 19), YPWGAGAPWQVQW Y (SEQ ID NO: 21), YPWGAGAPWSVQW Y (SEQ ID NO: 22) and YPWGAGAPWL VQW Y (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), GANYDLS A (SEQ ID NO: 2), GANYGLS A (SEQ ID NO: 3) and GANYDLA A (SEQ ID NO: 4); FW1 is a framework region; FW2 is a 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 a framework region; HV4 is a hypervariable sequence having an amino acid sequence selected from the group consisting of NKRTM (SEQ ID NO: 8) and NKG TM (SEQ ID NO: 9); FW3b is a framework region; FW4 is a framework region; When CDR3 is YPWGAGAPWL VQW Y (SEQ ID NO: 10), CDR1 is selected from the group consisting of DANYGLAA (SEQ ID NO: 5), GANYGLS A (SEQ ID NO: 3) and GANYDLA A (SEQ ID NO: 4)); and The first antigen-binding molecule is fused to a first fragment of an immunoglobulin Fc region that has been engineered to dimerize with a second fragment of the immunoglobulin Fc region; and (b) a second recombinant fusion protein; 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 that has been engineered to dimerize with the first fragment of the immunoglobulin Fc region; A recombinant fusion protein dimer comprising: **Claim 2** (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), The recombinant fusion protein dimer according to claim 1. **Claim 3** (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 DGAGTVLT VN (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 SFTLTISSSLQPEDSATYYCRA (SEQ ID NO: 46); and FW4 is DGAGTKVEIK (SEQ ID NO: 49), The recombinant fusion protein dimer according to claim 1. **Claim 4** The ROR1-specific antigen-binding molecule comprises SEQ ID NO: 50, SEQ ID NO: 61, or SEQ ID NO: 71, The recombinant fusion protein dimer according to any one of claims 1 to 3. **Claim 5** A recombinant fusion protein dimer, comprising (a) a first recombinant fusion protein; The first recombinant fusion protein comprises a receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific antigen-binding molecule comprising an amino acid sequence represented by formula (I); formula (I): FW1-CDR1-FW2-HV2-FW3a-HV4-FW3b-CDR3-FW4 (I) (wherein 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), DTRIALYS (SEQ ID NO: 27), DTRIALYSS (SEQ ID NO: 28), GTKYGLYA (SEQ ID NO: 29), GTKYGLYAS (SEQ ID NO: 30), and DTSYGLYS (SEQ ID NO: 207); FW1 is a framework region; FW2 is a framework region; HV2 is a hypervariable sequence having an amino acid sequence selected from the group consisting of SSDEERISIS (SEQ ID NO: 31), STDDEERISIG (SEQ ID NO: 32), SPNKDRMIIIG (SEQ ID NO: 33), STDKERIIIG (SEQ ID NO: 34), and TTDEWERMSIG (SEQ ID NO: 208); FW3a is a framework region; HV4 is a hypervariable sequence having an amino acid sequence selected from the group consisting of NKG TK (SEQ ID NO: 35), NKG SK (SEQ ID NO: 36), NNG TK (SEQ ID NO: 37), NNR SK (SEQ ID NO: 38), and NKGAK (SEQ ID NO: 209); FW3b is a framework region; CDR3 is a CDR sequence having an amino acid sequence according to REARHPWLQRQW (SEQ ID NO: 39); FW4 is a framework region; and The first antigen-binding molecule is fused to a first fragment of an immunoglobulin Fc region that has been engineered to dimerize with a second fragment of the immunoglobulin Fc region; and (b) a second recombinant fusion protein; 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 that has been engineered to dimerize with the first fragment of the immunoglobulin Fc region; A recombinant fusion protein dimer comprising

6. CDR1 is DTSYGLYS (SEQ ID NO: 207); HV2 is TTDEWERMSIG (SEQ ID NO: 208); HV4 is NKGAK (SEQ ID NO: 209); and CDR3 is REARHPWLRQWYY (SEQ ID NO: 39). The recombinant fusion protein dimer according to claim 5.

7. (i) 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 DGAGTVLT VN (SEQ ID NO: 48) The recombinant fusion protein dimer according to claim 5 or claim 6.

8. The ROR1-specific antigen-binding molecule contains SEQ ID NO:

206. The recombinant fusion protein dimer according to any one of claims 5 to 7.

9. The EGFR-specific antigen-binding molecule contains a single-domain antibody. The recombinant fusion protein dimer according to any one of claims 1 to 8.

10. The EGFR-specific antigen-binding molecule is any one of SEQ ID NOs: 210 to 215, or for each sequence, (i) at least 85% identity thereto, and / or (ii) one, two, or three amino acid substitutions thereto An amino acid sequence having containing, The recombinant fusion protein dimer according to any one of claims 1 to 9.

11. The EGFR-specific antigen-binding molecule contains any one of SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, or SEQ ID NO:

215. The recombinant fusion protein dimer according to any one of claims 1 to 10.

12. The EGFR-specific antigen-binding molecule contains any one of SEQ ID NO: 210, SEQ ID NO: 212, or SEQ ID NO:

214. The recombinant fusion protein dimer according to any one of claims 1 to 11.

13. The EGFR-specific antigen-binding molecule is humanized. The recombinant fusion protein dimer according to any one of claims 1 to 12.

14. The EGFR-specific antigen-binding molecule contains one or more of SEQ ID NOs: 357 to 362. The recombinant fusion protein dimer according to any one of claims 1 to 8.

15. The EGFR-specific antigen-binding molecule is (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 containing, The recombinant fusion protein dimer according to any one of claims 1 to 8 and 14.

16. (a) a 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, - anthracycline, preferably PNU-derived anthracycline - maytansinoid, - amatoxin derivative, preferably α-amanitin derivative, - calicheamicin, - tubulysin, - duocarmycin, - radioisotopes such as alpha-emitting radionuclides like 227Th and 225Ac labels, - liposomes containing a toxic payload, - protein toxins, - taxane, - pyrrolobenzodiazepine and its dimer, - indolinobenzodiazepine pseudodimer, - spliceosome inhibitor, - CDK11 inhibitor, - nicotinamide phosphoribosyltransferase inhibitor (NAMPTi) - pyridinobenzodiazepine and its dimer, - cyclopropapyrroloindole (CPI), cyclopropabenzindole (CBI), cyclopropathiienoindole (CTI) and optionally their dimers, - irinotecan or exatecan and their derivatives A target-binding molecule-drug conjugate comprising.

17. The toxin is auristatin, and (b) is an MMAE derivative, The target-binding molecule-drug conjugate has the formula (VI): 【Chemical 1】 (wherein [X] is an optional spacer selected from the group consisting of a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, 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, -(CH ２ ) ｎ -, -(CH ２ CH ２ O) ｎ -, p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, glycine, and any amino acid and combinations thereof other than; and Y comprises a bispecific antigen-binding molecule according to any one of claims 1 to 15) having the structure of The target-binding molecule-drug conjugate according to claim 16.

18. The toxin is auristatin, and (b) is an MMAE derivative, The target-binding molecule-drug conjugate has the formula (VIII): 【Chemical 2】 having the structure of The target-binding molecule-drug conjugate according to claim 16.

19. (b) is an anthracycline (PNU) derivative, and The target-binding molecule-drug conjugate has the formula (III): 【Chemical 3】 (wherein, [X] is an optional spacer selected from the group consisting of a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, 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, -(CH ２ ) ｎ -, -(CH ２ CH ２ O) ｎ -, p-aminobenzyloxycarbonyl (PAB), Val-Cit-PAB, Val-Ala-PAB, Ala-Ala-Asn-PAB, Val-Ala, Asn-Ala, glycine, and any combination thereof; and Y comprises the bispecific antigen-binding molecule according to any one of claims 1 to 15) having the structure of The target-binding molecule-drug conjugate according to claim 16.

20. The target-binding molecule-drug conjugate is 【Chemical 4】 [Chemical Formula 5] 【Chemical Formula 6】 【Chemical Formula 7】 having a structure selected from The target-binding molecule-drug conjugate according to claim 19.

21. One or more residues of the first 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, One or more residues of the second fragment of the immunoglobulin Fc region comprise one or more amino acid substitutions suitable for knob-in-hole (KIH) dimerization with the first fragment of the immunoglobulin Fc region, The recombinant fusion protein dimer according to any one of claims 1 to 15, or The target-binding molecule-drug conjugate according to any one of claims 16 to 20.

22. (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, The recombinant fusion protein dimer or target-binding molecule-drug conjugate according to any one of claims 1 to 21.

23. The first and / or second fragment of the immunoglobulin Fc region individually comprises an S239C and / or S442C substitution, The recombinant fusion protein dimer or target-binding molecule-drug conjugate according to any one of claims 1 to 22.

24. An ROR1-specific binding molecule and / or an EGFR-specific binding molecule is fused to the hFc region via a ４ S ３ linker or a ４ GS linker. The recombinant fusion protein dimer or target-binding molecule-drug conjugate according to any one of claims 1 to 23.

25. 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, 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. The recombinant fusion protein dimer or target-binding molecule-drug conjugate according to any one of claims 1 to 24. **Claim 26** 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. 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. The recombinant fusion protein dimer or target-binding molecule-drug conjugate according to any one of claims 1 to 24. **Claim 27** The recombinant fusion protein dimer or target-binding molecule-drug conjugate has SEQ ID NO comprises any one of 369, 373, 377, 396, 400, 404, 371, 375, 379, 398, 402, and 406. The recombinant fusion protein dimer or target-binding molecule-drug conjugate further comprises SEQ ID NO:

360. The recombinant fusion protein dimer or target-binding molecule-drug conjugate according to any one of claims 1 to 26. **Claim 28** 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. The second recombinant fusion protein comprises any one of SEQ ID NOs: 265 to 268 and 333 to 336. The recombinant fusion protein dimer or target-binding molecule-drug conjugate according to any one of claims 1 to 25. **Claim 29** 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. The second recombinant fusion protein comprises any one of SEQ ID NOs: 277 to 280 and 345 to 348. The recombinant fusion protein dimer or target-binding molecule-drug conjugate according to any one of claims 1 to 24 and claim 27.

30. The first recombinant fusion protein comprises any one of SEQ ID NOs: 327 to 332 and 550 to 555, 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, The recombinant fusion protein dimer or target-binding molecule-drug conjugate according to any one of claims 1 to 25.

31. The first recombinant fusion protein comprises any one of SEQ ID NOs: 321 to 326 and 556 to 561, 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, The recombinant fusion protein dimer or target-binding molecule-drug conjugate according to any one of claims 1 to 24 and claim 27.

32. The first recombinant fusion protein comprises any one of SEQ ID NOs: 259 to 264 and 315 to 320, The second recombinant fusion protein comprises any one of SEQ ID NOs: 273 to 276 and 341 to 344, The recombinant fusion protein dimer or target-binding molecule-drug conjugate according to any one of claims 1 to 25.

33. The first recombinant fusion protein comprises any one of SEQ ID NOs: 253 to 258 and 309 to 314, The second recombinant fusion protein comprises any one of SEQ ID NOs: 285 to 288 and 353 to 356, The recombinant fusion protein dimer or target-binding molecule-drug conjugate according to any one of claims 1 to 24 and claim 27.

Images