Bispecific binding molecule

Abstract

The present invention relates to a bispecific binding molecule that binds to CD3 and CA19-9. The present invention further relates to such a bispecific binding molecule for use in treating cancer.

Description

[0001] BISPECIFIC BINDING MOLECULE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a bispecific binding molecule that binds to CD3 and CA19-9. The present invention further relates to such a bispecific binding molecule for use in treating cancer.

[0004] BACKGROUND TO THE INVENTION

[0005] Carbohydrate antigen 19-9 (CA19-9) also known as sialyl-LewisA, is a tetrasaccharide molecule that is commonly attached to glycans on the surface of cells. CA19-9 has increased expression in cancer tissue, including in pancreatic cancer but also in other types of cancer. CA19-9 has therefore been proposed as a diagnostic and therapeutic target for such cancers.

[0006] CD3 (cluster of differentiation 3) is a protein complex expressed on T cells. The CD3 complex is composed of a CD3y chain, a CD35 chain, and two CD3E chains. These chains associate with the T cell receptor (TCR) a and chains and two CD3 chains. Antigen binding by the TOR is translated into an intracellular activation signal by the CD3 subunits via a phosphorylation cascade. The phosphorylation causes the activation of transcription factors such as NFAT and NFkB, which leads to increased expression of cytokines and effector proteins such as granzymes and perforin and ultimately enables the T cell to perform its effector functions, i.e. killing of the target cell in case of a cytotoxic T cell.

[0007] Due to their ability to bind and activate T cells, antibodies binding to CD3 are useful in T cell engager molecules. T cell engager molecules are antigen binding proteins comprising at least two antigen binding sites, wherein one binds to T cells, usually via CD3, and the other binds to a tumor cell via a tumor-specific molecule, thereby engaging the T cell and recruiting it to the tumor. Desired properties of T cell engagers are an efficient cell killing activity, a manageable side effect profile, a long shelf life, good solubility, stability, ease of formulation and production scalability. There remains a need in the art for effective anti-CD3 antibodies.

[0008] Although various approaches have been investigated in the art, there is still a need for novel, improved immunotherapeutic candidates (such as bispecific T cell engager molecules) targeting both CA19-9 and CD3.SUMMARY OF THE INVENTION

[0009] The present invention provides a bispecific binding molecule comprising a CD3-binding moiety and a first Carbohydrate antigen 19-9 (CA19-9)-binding moiety.

[0010] In some embodiments, the bispecific binding molecule is a bispecific antigen-binding molecule.

[0011] In some embodiments, the bispecific binding molecule comprises or is a bispecific antibody.

[0012] In some embodiments, the CD3-binding moiety comprises a CD3 heavy chain variable (CD3-VH) domain. In some embodiments, the CD3-binding moiety comprises a CD3 light chain variable (CD3-VL) domain.

[0013] In some embodiments, the CD3-binding moiety comprises a CD3 heavy chain variable (CD3-VH) domain and / or CD3 light chain variable (CD3-VL) domain.

[0014] In some embodiments, the CD3-binding moiety comprises a scFv, wherein the scFv comprises:

[0015] (i) a CD3 heavy chain variable (CD3-VH) domain;

[0016] (ii) a linker; and

[0017] (iii) a CD3 light chain variable (CD3-VL) domain.

[0018] In some embodiments, the first CA19-9-binding moiety comprises a CA19-9 heavy chain variable (CA19-9-VH) domain.

[0019] In some embodiments, the first CA19-9-binding moiety comprises a CA19-9 light chain variable (CA19-9-VL) domain.

[0020] In some embodiments, the first CA19-9-binding moiety comprises a CA19-9 heavy chain variable (CA19-9-VH) domain and / or a CA19-9 light chain variable (CA19-9-VL) domain.

[0021] In some embodiments, the first CA19-9-binding moiety comprises a CA19-9-binding Fab, wherein the Fab comprises:

[0022] (i) a heavy chain comprising a CA19-9 heavy chain variable (CA19-9-VH) domain and a CA19-9 heavy chain constant (CA19-9-CH1) domain; and

[0023] (ii) a light chain comprising a CA19-9 light chain variable (CA19-9-VL) domain and a CA19-9 light chain constant (CA19-9-CL) domain.In some embodiments, the bispecific binding molecule further comprises a second CA19-9-binding moiety.

[0024] In some embodiments, the second CA19-9-binding moiety comprises a CA19-9 heavy chain variable (CA19-9-VH) domain.

[0025] In some embodiments, the second CA19-9-binding moiety comprises a CA19-9 light chain variable (CA19-9-VL) domain.

[0026] In some embodiments, the second CA19-9-binding moiety comprises a CA19-9 heavy chain variable (CA19-9-VH) domain and a CA19-9 light chain variable (CA19-9-VL) domain.

[0027] In some embodiments, the second CA19-9-binding moiety comprises a CA19-9-binding Fab, wherein the Fab comprises:

[0028] (i) a heavy chain comprising a CA19-9 heavy chain variable (CA19-9-VH) domain and a CA19-9 heavy chain constant (CA19-9-CH1) domain; and

[0029] (ii) a light chain comprising a CA19-9 light chain variable (CA19-9-VL) domain and a CA19-9 light chain constant (CA19-9-CL) domain.

[0030] In some embodiments, the first CA19-9-binding moiety and the second CA19-9-binding moiety are the same.

[0031] In some embodiments, the first CA19-9-binding moiety is linked to the CD3-binding moiety.

[0032] In some embodiments, the C-terminus of the first CA19-9-binding moiety, preferably the C-terminus of the CA19-9-CH1 domain, is linked to the N-terminus of the CD3-VH domain.

[0033] In some embodiments, the C-terminus of the first CA19-9-binding moiety, preferably the C-terminus of the CA19-9-CH1 domain, is linked to the N-terminus of the CD3-VL domain.

[0034] In some embodiments, the bispecific binding molecule further comprises an Fc region comprising a first subunit and a second subunit.

[0035] In some embodiments, the CD3-binding moiety is linked to the Fc region.

[0036] In some embodiments, the second CA19-9-binding moiety is linked to the Fc region.In some embodiments, the C-terminus of the CD3-VH domain is linked to the first subunit of the Fc region.

[0037] In some embodiments, the C-terminus of the CD3-VL domain is linked to the first subunit of the Fc region.

[0038] In some embodiments, the C-terminus of the second CA19-9-binding moiety, preferably the C-terminus of the CA19-9-CH1 domain, is linked to the second subunit of the Fc region.

[0039] In some embodiments, the C-terminus of the CD3-VH domain is linked to a hinge region of the first subunit of the Fc region.

[0040] In some embodiments, the C-terminus of the CD3-VL domain is linked to a hinge region of the first subunit of the Fc region.

[0041] In some embodiments, the C-terminus of the second CA19-9-binding moiety, preferably the C-terminus of the CA19-9-CH1 domain, is linked to a hinge region of the second subunit of the Fc region.

[0042] In some embodiments, the first subunit of the Fc region and the second subunit of the Fc region each comprise a CH2 domain.

[0043] In some embodiments, the first subunit of the Fc region and the second subunit of the Fc region each comprise a CH3 domain.

[0044] In some embodiments, the first subunit of the Fc region and the second subunit of the Fc region each comprise a CH2 domain and a CH3 domain.

[0045] In some embodiments of the bispecific binding binding molecule according to the invention: (i) the CD3-binding moiety comprises an scFv, wherein the scFv comprises:

[0046] (i) a CD3 heavy chain variable (CD3-VH) domain;

[0047] (ii) a linker; and

[0048] (iii) a CD3 light chain variable (CD3-VL) domain;

[0049] wherein the N-terminal to C-terminal orientation of the scFv is:

[0050] CD3-VH domain - linker - CD3-VL domain;

[0051] (ii) the bispecific binding molecule further comprises a second CA19-9-binding moiety, wherein the first CA19-9-binding moiety and the second CA19-9-binding moiety are the same, whereinthe first CA19-9-binding moiety and the second CA19-9-binding moiety each comprise a CA19-9-binding Fab, wherein each Fab comprises:

[0052] (i) a heavy chain comprising a CA19-9 heavy chain variable (CA19-9-VH) domain and a CA19-9 heavy chain constant (CA19-9-CH1) domain; and

[0053] (ii) a light chain comprising a CA19-9 light chain variable (CA19-9-VL) domain and a CA19-9 light chain constant (CA19-9-CL) domain;

[0054] (iii) the bispecific binding molecule further comprises an Fc region comprising a first subunit and a second subunit; wherein the first subunit of the Fc region and the second subunit of the Fc region each comprise a CH2 domain and a CH3 domain, optionally wherein the CH3 domain of one subunit of the Fc region comprises one or more mutations selected from the list consisting of: Y349C, T366S, L368A, and Y407V; and the CH3 domain of the other subunit of the Fc region comprises one or more mutations selected from the list consisting of: S354C and T366W;

[0055] (iv) the C-terminus of CA19-9-CH1 domain of the first CA19-9-binding moiety is linked to the N-terminus of the CD3-VH domain;

[0056] (v) the C-terminus of the CD3-VL domain is linked to the N-terminus of the CH2 domain of the first subunit of the Fc region, preferably to the N-terminus of a hinge region of the CH2 domain of the first subunit of the Fc region; and

[0057] (vi) the C-terminus of the CA19-9-CH1 domain of the second CA19-9-binding moiety is linked to the N-terminus of the CH2 domain of the second subunit of the Fc region, preferably to the N-terminus of a hinge region of the CH2 domain of the second subunit of the Fc region.

[0058] In some embodiments, the CD3-binding moiety comprises heavy chain complementarity determining regions (HCDRs) 1-3, and / or light chain complementarity determining regions (LCDRs) 1-3, wherein (according to Kabat numbering)

[0059] (i)

[0060] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 1, HCDR2 comprises the amino acid sequence according to SEQ ID NO: 3, and HCDR3 comprises the amino acid sequence according to SEQ ID NO: 5,

[0061] and / or

[0062] (ii)

[0063] LCDR1 comprises the amino acid sequence according to SEQ ID NO: 7, LCDR2 comprises the amino acid sequence according to SEQ ID NO: 9, and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 11;optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0064] In some embodiments, the CD3-binding moiety comprises heavy chain complementarity determining regions (HCDRs) 1-3, and / or light chain complementarity determining regions (LCDRs) 1-3, wherein (according to IMGT numbering)

[0065] (i)

[0066] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 2, HCDR2 comprises the amino acid sequence according to SEQ ID NO: 4, and HCDR3 comprises the amino acid sequence according to SEQ ID NO: 6,

[0067] and / or

[0068] (ii)

[0069] LCDR1 comprises the amino acid sequence according to SEQ ID NO: 8, LCDR2 comprises the amino acid sequence according to SEQ ID NO: 10, and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 12; optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0070] In some embodiments, the HCDR1, HCDR2 and HCDR3 sequences comprise or consist of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, respectively.

[0071] In some embodiments, the HCDR1, HCDR2 and HCDR3 sequences comprise or consist of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, respectively.

[0072] In some embodiments, the LCDR1, LCDR2 and LCDR3 sequences comprise or consist of SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11, respectively.

[0073] In some embodiments, the LCDR1, LCDR2 and LCDR3 sequences comprise or consist of SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12, respectively.

[0074] In some embodiments, the CD3-binding moiety comprises a CD3-VH domain sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to any one of SEQ ID NO: 14, SEQ ID NO: 22, and SEQ ID NO: 30.In some embodiments, the CD3-VH domain comprises or consists of a sequence as set forth in any one of SEQ ID NO: 14, SEQ ID NO: 22, and SEQ ID NO: 30.

[0075] In some embodiments, the CD3-binding moiety comprises a CD3-VL domain sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or preferably 100% identity to any one of SEQ ID NO: 16, SEQ ID NO: 24, and SEQ ID NO: 32.

[0076] In some embodiments, the CD3-VL domain comprises or consists of a sequence as set forth in any one of SEQ ID NO: 16, SEQ ID NO: 24, and SEQ ID NO: 32.

[0077] In some embodiments, the CD3-binding moiety is or comprises a scFv having the format VH-L1-VL, wherein L1 and L2 are peptide linkers.

[0078] In some embodiments, the CD3-binding moiety is or comprises a scFv having the format VL-L2-VH, wherein L1 and L2 are peptide linkers.

[0079] In some embodiments, the amino acids of L1 and L2 are selected from glycines and serines.

[0080] In some embodiments, L1 and L2 independently have a length of 10 to 30 amino acids.

[0081] In some embodiments, L1 is a (G4S)4 linker.

[0082] In some embodiments, L2 is a (G4S)s linker.

[0083] In some embodiments, L1 comprises a sequence as set forth in SEQ ID NO: 80, or a variant having at least 80% identity thereto.

[0084] In some embodiments, L2 comprises a sequence as set forth in SEQ ID NO: 81, or a variant having at least 80% identity thereto.

[0085] In some embodiments, the scFv comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or preferably 100% identity to any one of SEQ ID NO: 18, SEQ ID NO: 26, and SEQ ID NO: 34.

[0086] In some embodiments, the scFv comprises or consists of a sequence as set forth in any one of SEQ ID NO: 18, SEQ ID NO: 26, and SEQ ID NO: 34In some embodiments, the CD3 is human CD3. In some embodiments, the CD3 is CD3E.

[0087] In some embodiments, the, or each, CA19-9-binding moiety comprises heavy chain complementarity determining regions (HCDRs) 1-3, and / or light chain complementarity determining regions (LCDRs) 1-3, wherein (according to Kabat numbering):

[0088] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 37, HCDR2 comprises the amino acid sequence according to SEQ ID NO: 39, and HCDR3 comprises the amino acid sequence according to SEQ ID NO: 41,

[0089] and / or

[0090] LCDR1 comprises the amino acid sequence according to SEQ ID NO: 43, LCDR2 comprises the amino acid sequence according to SEQ ID NO: 45, and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 47; optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0091] In some embodiments, the, or each, CA19-9-binding moiety comprises heavy chain complementarity determining regions (HCDRs) 1-3, and / or light chain complementarity determining regions (LCDRs) 1-3, wherein (according to IMGT numbering):

[0092] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 38, HCDR2 comprises the amino acid sequence according to SEQ ID NO: 40, and HCDR3 comprises the amino acid sequence according to SEQ ID NO: 42,

[0093] and / or

[0094] LCDR1 comprises the amino acid sequence according to SEQ ID NO: 44, LCDR2 comprises the amino acid sequence according to SEQ ID NO: 46, and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 48; optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0095] In some embodiments, the CA19-9-binding moiety comprises a CA19-9-VH domain comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 50.In some embodiments, the CA19-9-VH domain comprises or consists of a sequence as set forth in SEQ ID NO: 50.

[0096] In some embodiments, the CA19-9-binding moiety comprises a CA19-9-VL domain comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or preferably 100% identity to SEQ ID NO: 52.

[0097] In some embodiments, the CA19-9-VL domain comprises or consists of a sequence as set forth in SEQ ID NO: 52.

[0098] In some embodiments, the CA19-9-binding moiety comprises a heavy chain comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or preferably 100% identity to SEQ ID NO: 85.

[0099] In some embodiments, the CA19-9-binding moiety comprises a heavy chain comprising or consisting of a sequence as set forth in SEQ ID NO: 85.

[0100] In some embodiments, the CA19-9-binding moiety comprises a light chain comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or preferably 100% identity to SEQ ID NO: 78.

[0101] In some embodiments, the CA19-9-binding moiety comprises a light chain comprising or consisting of a sequence as set forth in SEQ ID NO: 78.

[0102] In some embodiments, the bispecific binding molecule comprises:

[0103] (i) a first polypeptide comprising the amino acid sequence according to SEQ ID NO: 66 or a variant having at least 80% sequence identity thereto;

[0104] (ii) a second polypeptide comprising the amino acid sequence according to SEQ ID NO: 64 or a variant having at least 80% sequence identity thereto; and

[0105] (iii) a third polypeptide comprising the amino acid sequence according to SEQ ID NO: 78 or a variant having at least 80% sequence identity thereto.

[0106] In some embodiments, the bispecific binding molecule comprises:

[0107] (i) a first polypeptide comprising the amino acid sequence according to SEQ ID NO: 70 or a variant having at least 80% sequence identity thereto;

[0108] (ii) a second polypeptide comprising the amino acid sequence according to SEQ ID NO: 64 or a variant having at least 80% sequence identity thereto; and(iii) a third polypeptide comprising the amino acid sequence according to SEQ ID NO: 78 or a variant having at least 80% sequence identity thereto.

[0109] In some embodiments, the bispecific binding molecule comprises:

[0110] (i) a first polypeptide comprising the amino acid sequence according to SEQ ID NO: 74 or a variant having at least 80% sequence identity thereto;

[0111] (ii) a second polypeptide comprising the amino acid sequence according to SEQ ID NO: 64 or a variant having at least 80% sequence identity thereto; and

[0112] (iii) a third polypeptide comprising the amino acid sequence according to SEQ ID NO: 78 or a variant having at least 80% sequence identity thereto.

[0113] The present invention provides one or more polynucleotide(s) comprising a nucleic acid sequence encoding the bispecific binding molecule according to the invention.

[0114] In some embodiments, the nucleic acid sequence is selected from the list consisting of: a DNA sequence, a cDNA sequence, an RNA sequence, and an mRNA sequence.

[0115] In some embodiments, the nucleic acid sequence is an RNA sequence.

[0116] In some embodiments, the RNA sequence is an mRNA sequence.

[0117] In some embodiments, the RNA is modRNA (N1-methyl-pseudouridine).

[0118] In some embodiments, the RNA sequence comprises a 5'-cap.

[0119] In some embodiments, the 5'-cap is CC413.

[0120] In some embodiments, the 5'-cap is m27’3'oGppp(mi2'°)ApG, which comprises the following structure:

[0121]

[0122] OH OH

[0123] In some embodiments, the RNA sequence comprises a poly adenine sequence.

[0124] In some embodiments, the poly adenine sequence comprises the nucleic acid sequence according to SEQ ID NO: 84 or a variant having 80% sequence identity thereto.

[0125] In some embodiments, the RNA sequence comprises a 5’IITR.

[0126] In some embodiments, the 5’IITR comprises the nucleic acid sequence according to SEQ ID NO: 82 or a variant having 80% sequence identity thereto.

[0127] In some embodiments, the RNA sequence comprises a 3’IITR

[0128] In some embodiments, the 3’IITR comprises the nucleic acid sequence according to SEQ ID NO: 83 or a variant having 80% sequence identity thereto.

[0129] In some embodiments, a plurality of polynucleotides comprises:

[0130] (i) a first polynucleotide comprising a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[0131] CA19-9-VH domain - CA19-9-CH1 domain - optional linker- CD3-VH domain - L1 - CD3-VL domain - optional linker - CH2 domain - CH3 domain;

[0132] (ii) a second polynucleotide comprising a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[0133] CA19-9-VH domain - CA19-9-CH1 domain - CH2 domain - CH3 domain; and / or

[0134] (iii) a third polynucleotide comprising a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[0135] CA19-9-VL domain - CA19-9-CL domain.In some embodiments:

[0136] (i) the first polynucleotide comprises a nucleic acid sequence comprising in 5’-3’ orientation:

[0137] 5’-Cap - 5’UTR - CA19-9-VH domain - CA19-9-CH1 domain - optional linker - CD3-VH domain - linker L1 - CD3-VL domain - optional linker - CH2 domain - CH3 domain - 3’UTR - Poly(A)

[0138] (ii) the second polynucleotide comprises a nucleic acid sequence comprising in 5’-3’ orientation:

[0139] 5’-Cap - 5’IITR - CA19-9-VH domain - CA19-9-CH1 domain - CH2 domain - CH3 domain - 3’UTR - Poly(A); and / or

[0140] (iii) the third polynucleotide comprises a nucleic acid sequence comprising in 5’-3’ orientation:

[0141] 5’-Cap - 5’UTR - CA19-9-VL domain - CA19-9-CL domain - 3’UTR - Poly(A).

[0142] In some embodiments, the first polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 65 or a variant having at least 80% sequence identity to SEQ ID NO: 65.

[0143] In some embodiments, the first polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 69 or a variant having at least 80% sequence identity to SEQ ID NO: 69.

[0144] In some embodiments, the first polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 73 or a variant having at least 80% sequence identity to SEQ ID NO: 73.

[0145] In some embodiments, the second polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 63 or a variant having at least 80% sequence identity to SEQ ID NO: 63.

[0146] In some embodiments, the third polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 77 or a variant having at least 80% sequence identity to SEQ ID NO: 77.

[0147] In some embodiments:

[0148] (i) the first polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 65 or a variant having at least 80% sequence identity to SEQ ID NO: 65;

[0149] (ii) the second polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 63 or a variant having at least 80% sequence identity to SEQ ID NO: 63; and(iii) the third polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 77 or a variant having at least 80% sequence identity to SEQ ID NO: 77.

[0150] In some embodiments:

[0151] (i) the first polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 69 or a variant having at least 80% sequence identity to SEQ ID NO: 69; or

[0152] (ii) the second polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 63 or a variant having at least 80% sequence identity to SEQ ID NO: 63; and (iii) the third polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 77 or a variant having at least 80% sequence identity to SEQ ID NO: 77.

[0153] In some embodiments:

[0154] (i) the first polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 73 or a variant having at least 80% sequence identity to SEQ ID NO: 73;

[0155] (ii) the second polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 63 or a variant having at least 80% sequence identity to SEQ ID NO: 63; and (iii) the third polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 77 or a variant having at least 80% sequence identity to SEQ ID NO: 77.

[0156] The present invention provides a vector which comprises one or more polynucleotide(s) according to the invention.

[0157] The present invention provides a composition comprising

[0158] (i) the bispecific binding molecule according to the invention;

[0159] (ii) the one or more polynucleotide(s) according to the invention; or

[0160] (iii) the vector according to the invention.

[0161] The present invention provides a pharmaceutical composition comprising:

[0162] (i)

[0163] (a) the bispecific binding molecule according to the invention; or (b) the one or more polynucleotide(s) according to the invention; or

[0164] (c) the vector according to the invention; or

[0165] (d) the composition according to the invention;

[0166] and

[0167] (ii) a pharmaceutically acceptable carrier, diluent or excipient,

[0168] optionally wherein the pharmaceutical composition further comprises(iii) a vesicle, a nanoparticle, a lipid nanoparticle (LNP), a liposome or a polymeric mixture wherein the multi-specific binding molecule, the one or more polynucleotide(s), the vector, or the composition is encapsulated within the vesicle, nanoparticle, LNP, liposome or polymeric mixture.

[0169] In some embodiments, (iii) is an LNP.

[0170] In some embodiments, the pharmaceutical composition is formulated for parenteral administration.

[0171] In some embodiments, the pharmaceutical composition is formulated for intravenous or intraarterial administration.

[0172] The present invention provides a method for treating and / or preventing a disease which comprises the step of administering the bispecific binding molecule according to the invention; or the one or more polynucleotide(s) according to the invention; or the vector according to the invention; or the composition according to the invention, or the pharmaceutical composition according to the invention, to a subject.

[0173] The present invention provides use of the bispecific binding molecule according to the invention; or the one or more polynucleotide(s) according to the invention; or the vector according to the invention; or the composition according to the invention, or the pharmaceutical composition according to the invention, in the manufacture of a medicament for treating and / or preventing a disease.

[0174] The present invention provides the bispecific binding molecule according to the invention; or the one or more polynucleotide(s) according to the invention; or the vector according to the invention; or the composition according to the invention, or the pharmaceutical composition according to the invention, for use in the treatment or prevention of a disease.

[0175] In some embodiments, the disease is cancer.

[0176] In some embodiments, the cancer is pancreatic cancer.

[0177] In some embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

[0178] In some embodiments, the cancer is cholangiocarcinoma.In some embodiments, the cancer is gastrointestinal cancer.

[0179] In some embodiments, the gastrointestinal cancer is selected from the listing consisting of: colorectal cancer, oesophageal cancer, gastric cancer, and hepatocellular cancer.

[0180] BRIEF DESCRIPTION OF THE FIGURES

[0181] Figure 1 shows a modular scheme illustrating the structural design of the 2+1 Fab(2)-scFv-Fc RiboMab. The 2+1 Fab(2)-scFv-Fc constructs consist of three different polypeptide chains. One heavy chain comprises a human CA19-9-binding VH with a corresponding human CH1 domain and a human lgG1 Fc part with hole mutations and the Fc-silencing LALA mutations. The other heavy chain is generated from the same CA19-9-binding VH with a corresponding human CH1 domain, a C-terminally fused human CD3-binding scFv that is linked via short glycine serine linker to both the CA19-9-binding moiety and a human lgG1 Fc part with knob mutations and the Fc-silencing LALA mutations N-terminally. The light chain comprises a human CA19-9-binding VL with a corresponding human CL domain.

[0182] Figure 2 shows productivity data of 2+1 CA19-9 x scFv-anti-CD3 RiboMabs that were produced and secreted into the cell culture supernatant after transient in vitro RNA transfection of Expi293F™ cells. Expi293F cells were transiently transfected with three different anti-CA19-9xCD3 Ribomab-coding RNA mixes via ribofectamine. Resulting antibody titers were determined via Gyros ELISA. The results are a mean of two technical replicates. The 2+1 CA19-9xscFv-anti-CD3 RiboMabs were produced well and secreted into the cell culture supernatant. All supernatant samples were unconcentrated (1x).

[0183] Figure 3 shows Western blot (WB)-based analysis of individual chain assemblies under nonreduced and reduced conditions after transient production of 2+1 CA19-9 x scFv-anti-CD3 RiboMabs. The Western blot shows several protein species present in the cell culture supernatant after transient transfection. All three individual chains of the 2+1 CA19-9xCD3 RiboMabs are present, and the fully assembled antibody is the dominant species in all samples.

[0184] Figure 4 shows T-cell dependent cellular cytotoxicity assay data of 2+1 CA19-9 x scFv-anti-CD3 RiboMabs when co-cultured with PBMCs and CA 19-9-positive cells. The graphs show the increase in specific cell lysis as it relates to increasing concentrations of two exemplary anti-CA19-9xCD3 RiboMabs in cell culture supernatant. The error bars are calculated from thestandard deviation of three individual test well replicates. The co-culture assay was conducted with CA19-9-positive COLO 205 cells (solid lines) or CA 19-9-negative MDA-MB-231 cells (dotted lines).

[0185] Figure 5 shows blotted data from FACS binding assays of an exemplary 2+1 CA19-9 x scFv-anti-CD3 RiboMab binding to CA19-9-high expressing COLO 205 cells.

[0186] Figure 6 shows blotted data from FACS binding assays of an exemplary 2+1 CA19-9 x scFv-anti-CD3 RiboMab binding to CA19-9-mediocre expressing BxPC-3 cells.

[0187] Figure 7 shows data from T cell activation assays of an exemplary 2+1 CA19-9 x scFv-anti-CD3 RiboMab that efficiently activates T cells when CA19-9-high expressing COLO 205 or CA19-9-mediocre expressing BxPC-3 target cells are present.DETAILED DESCRIPTION OF THE INVENTION

[0188] BINDING MOLECULE

[0189] The present invention provides a bispecific binding molecule comprising a CD3-binding moiety and a first Carbohydrate antigen 19-9 (CA19-9)-binding moiety.

[0190] In some embodiment, the bispecific binding molecule is a bispecific antigen-binding molecule comprising a CD3-binding moiety and a first Carbohydrate antigen 19-9 (CA19-9)-binding moiety.

[0191] In some embodiments, the bispecific binding molecule comprises one or more amino acid sequence(s) as shown in Table 1.

[0192] In some embodiments, the bispecific binding molecule comprises a bispecific antibody.

[0193] A “binding moiety” as used herein (e.g. a CD3-binding moiety or a CA19-9 binding moiety) may comprise an antigen-binding domain, which is well-known term in the art and typically means a protein or polypeptide comprising at least one complementarity determining region(s) (CDR(s)).

[0194] It will be understood that an binding domain is a domain of a molecule that interacts with a target (e.g., a target antigen) such that a stable association is formed. An example of a molecule comprising an binding domain is an antibody (or antibody-like molecule) that binds to its cognate antigen. Such binding interaction will be known in the art. The binding interaction may be non-covalent, reversible covalent or irreversible covalent.

[0195] In some embodiments, a binding moiety as used herein (e.g. a CD3-binding moiety and / or a CA19-9 binding moiety) comprises one or more complementarity determining regions (CDRs).

[0196] It will be understood that a mutation in any of the CDRs described herein may encompass a deletion of an amino acid, an insertion of an amino acid, or a substitution of an amino acid. It will also be understood that such a mutation may not prevent the binding moiety according to the invention from binding to its target. In other words, a CD3-binding moiety according to the invention comprising a mutation in one or more CDRs described herein may suitably maintain the capacity (e.g. affinity) to bind to CD3. A CA19-9-binding moiety according to the invention comprising a mutation in one or more CDRs described herein may suitably maintain the capacity (e.g. affinity) to bind to CA19-9. In some embodiments, the mutation suitably maintains the same capacity (e.g. affinity) to bind to CD3 or CA19-9 (respectively) as the parentbinding molecule. The term “parent binding molecule” in this context refers to the binding moiety without the mutation(s) in question.

[0197] It will be understood that the bispecific binding molecule according to the invention is capable of binding to (i) one or more CA19-9 antigen(s) associated with (e.g. presented on the surface of) tumour cells and (ii) CD3 associated with (e.g. presented on the surface of) T cells. Thus, the bispecific binding molecule according to the invention is capable of recruiting T cells to the site of the tumour cells.

[0198] In some embodiments, the CD3-binding moiety of the bispecific binding molecule is capable of binding to T cells. In some embodiments, the CD3-binding moiety of the bispecific binding molecule is capable of recruiting T cells.

[0199] In some embodiments, the T cells are cytotoxic T cells. In some embodiments, the T cells are capable of killing target cells. In some embodiments, the T cells are capable of killing target tumour or cancer cells.

[0200] Accordingly, it will also be understood that the bispecific binding molecule according to the invention may be defined as a T cell engager molecule.

[0201] In some embodiments, the bispecific binding molecule further comprises a second CA19-9-binding moiety.

[0202] It will be understood that the bispecific binding molecule according to the invention may therefore comprise a CD3 binding moiety, a first CA19-9-binding moiety, and a second CA19-9-binding moiety.

[0203] In some embodiments, the bispecific binding molecule comprises:

[0204] (i) a first CA19-9-binding moiety;

[0205] (ii) a second CA19-9-binding moiety; and

[0206] (iii) a CD3-binding moiety.

[0207] It will be understood that a bispecific binding molecule comprising (i), (ii) and (iii) as described above may be defined as a (CA19-9-binding moiety)2-CD3 binding moiety format.

[0208] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9 heavy chain variable (CA19-9-VH) domain.In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9 light chain variable (CA19-9-VL) domain.

[0209] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9-VH domain and a CA19-9-VL domain.

[0210] The terms “heavy chain variable domain” or “VH” may refer to the portion of the heavy chain of a binding molecule, antigen-binding molecule, or antibody, that typically contains three CDRs, where each CDR is positioned between stretches of amino acids known as framework regions that form a scaffold to support the CDRs.

[0211] The terms “light chain variable domain” or “VL” may refer to the fragment of the light chain of a binding molecule, antigen-binding molecule, or antibody, that typically contains three CDRs, where each CDR is positioned between stretches of amino acids known as framework regions that form a scaffold to support the CDRs.

[0212] The terms “complementarity determining region” or “CDR” refer to a highly variable loop in the variable region which interact with a cognate antigen and is largely responsible for determining the ability of a binding molecule (e.g., an antibody) to bind to an antigen and for determining binding affinity. The CDRs within a variable region are typically numbered from the amino to the carboxy terminus, with CDR1 being closest to the amino terminus of the variable region and CDR3 being closest to the carboxy terminus of the variable region.

[0213] It will also be understood that each CDR is positioned between stretches of amino acids known as framework regions that form a scaffold to support the CDRs.

[0214] In some embodiments, the bispecific binding molecule comprises:

[0215] (i) a first CA19-9-binding moiety comprising:

[0216] (i) a CA19-9-VH domain; and

[0217] (ii) a CA19-9-VL domain;

[0218] (ii) a second CA19-9-binding moiety comprising:

[0219] (i) a CA19-9-VH domain; and

[0220] (ii) a CA19-9-VL domain; and

[0221] (iii) a CD3-binding moiety.In some embodiments, the first CA19-9-binding moiety comprises a CA19-9-binding fragment antigen-binding region (Fab).

[0222] It will be understood that a Fab typically comprises: (i) a heavy chain comprising one heavy constant (CH1) domain and one heavy variable (VH) domain; and (ii) a light chain comprising one light constant domain (CL) and one light variable (VL) domain.

[0223] It will be understood that the term “Fab” also encompasses fragments thereof.

[0224] In some embodiments, the second CA19-9-binding moiety comprises a CA19-9-binding Fab domain.

[0225] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a Fab comprising:

[0226] (i) a heavy chain comprising a CA19-9 heavy chain variable (CA19-9-VH) domain and a CA19-9 heavy chain constant (CA19-9-CH1) domain; and

[0227] (ii) a light chain comprising a CA19-9 light chain variable (CA19-9-VL) domain and a CA19-9 light chain constant (CA19-9-CL) domain.

[0228] In some embodiments, the first CA19-9-binding moiety comprises a CA19-9-binding Fab domain and the second CA19-9-binding moiety comprises a CA19-9-binding Fab domain. In some embodiments, each CA19-9-binding moiety comprises a CA19-9-binding Fab domain.

[0229] In some embodiments, the bispecific binding molecule comprises:

[0230] (i) a first CA19-9-binding moiety comprises a Fab comprising:

[0231] (i) a heavy chain comprising a CA19-9-VH domain and a CA19-9-CH1 domain; and (ii) a light chain comprising a CA19-9-VL domain and a CA19-9-CL domain;

[0232] (ii) a second CA19-9-binding moiety comprises a Fab comprising:

[0233] (i) a heavy chain comprising a CA19-9-VH domain and a CA19-9-CH1 domain; and (ii) a light chain comprising a CA19-9-VL domain and a CA19-9-CL domain; and (iii) a CD3-binding moiety.

[0234] In some embodiments, the first CA19-9-binding moiety and the second CA19-9-binding moiety are the same.

[0235] In some embodiments, the CD3-binding moiety comprises a CD3 heavy chain variable (CD3-VH) domain and CD3 light chain variable (CD3-VL) domain.In some embodiments, the bispecific binding molecule comprises:

[0236] (i) a first CA19-9-binding moiety;

[0237] (ii) a second CA19-9-binding moiety; and

[0238] (iii) a CD3-binding moiety comprising:

[0239] (i) a CD3-VH domain; and

[0240] (ii) a CD3-VL domain.

[0241] In some embodiments, the bispecific binding molecule comprises:

[0242] (i) a first CA19-9-binding moiety comprising:

[0243] (i) a CA19-9-VH domain; and

[0244] (ii) a CA19-9-VL domain;

[0245] (ii) a second CA19-9-binding moiety comprising:

[0246] (i) a CA19-9-VH domain; and

[0247] (ii) a CA19-9-VL domain; and

[0248] (iii) a CD3-binding moiety comprising:

[0249] (i) a CD3-VH domain; and

[0250] (ii) a CD3-VL domain.

[0251] In some embodiments, the bispecific binding molecule comprises:

[0252] (i) a first CA19-9-binding moiety comprising a CA19-9-binding Fab domain comprising:

[0253] (i) a heavy chain comprising a CA19-9-VH domain and a CA19-9-CH1 domain; and (ii) a light chain comprising a CA19-9-VL domain and a CA19-9-CL domain;

[0254] (ii) a second CA19-9-binding moiety comprising a CA19-9-binding Fab domain comprising:

[0255] (i) a heavy chain comprising a CA19-9-VH domain and a CA19-9-CH1 domain; and (ii) a light chain comprising a CA19-9-VL domain and a CA19-9-CL domain; and (iii) a CD3-binding moiety comprising:

[0256] (i) a CD3-VH domain; and

[0257] (ii) a CD3-VL domain.

[0258] In some embodiments, the CD3-binding moiety comprises a single-chain variable fragment (scFv) domain.

[0259] It will be understood that a scFv typically comprises: (i) a heavy variable (VH) domain; (ii) a linker; and (iii) a light variable (VL) domain.It will be understood that a VH domain and a VL domain of a scFv may together form a variable region.

[0260] It will be understood that the term “Fab” also encompasses fragments thereof.

[0261] In some embodiments, the CD3-binding moiety comprises a scFv comprising:

[0262] (i) a CD3-VH domain;

[0263] (ii) a linker; and

[0264] (iii) a CD3-VL domain.

[0265] In some embodiments, the bispecific binding molecule comprises:

[0266] (i) a first CA19-9-binding moiety comprising a CA19-9-binding Fab domain comprising:

[0267] (i) a heavy chain comprising a CA19-9-VH domain and a CA19-9-CH1 domain; and (ii) a light chain comprising a CA19-9-VL domain and a CA19-9-CL domain;

[0268] (ii) a second CA19-9-binding moiety comprising a CA19-9-binding Fab domain comprising:

[0269] (i) a heavy chain comprising a CA19-9-VH domain and a CA19-9-CH1 domain; and (ii) a light chain comprising a CA19-9-VL domain and a CA19-9-CL domain; and (iii) a CD3-binding moiety comprising a scFv comprising:

[0270] (i) a CD3-VH domain;

[0271] (ii) a linker; and

[0272] (iii) a CD3-VL domain.

[0273] It will be understood that a bispecific binding molecule comprising (i), (ii) and (iii) as described above may be defined as a (Fab)2-scFv format.

[0274] In some embodiments, the bispecific binding molecule further comprises an Fc region comprising a first subunit and a second subunit.

[0275] In some embodiments, the bispecific binding molecule comprises:

[0276] (i) a first CA19-9-binding moiety as described herein;

[0277] (ii) a second CA19-9-binding moiety as described herein;

[0278] (iii) a CD3-binding moiety as described herein; and

[0279] (iv) an Fc region comprising a first subunit and a second subunit.

[0280] In some embodiments, the bispecific binding molecule comprises:

[0281] (i) a first CA19-9-binding moiety comprising a CA19-9-binding Fab domain comprising:

[0282] (i) a heavy chain comprising a CA19-9-VH domain and a CA19-9-CH1 domain; and(ii) a light chain comprising a CA19-9-VL domain and a CA19-9-CL domain;

[0283] (ii) a second CA19-9-binding moiety comprising a CA19-9-binding Fab domain comprising:

[0284] (i) a heavy chain comprising a CA19-9-VH domain and a CA19-9-CH1 domain; and (ii) a light chain comprising a CA19-9-VL domain and a CA19-9-CL domain; and (iii) a CD3-binding moiety comprising a scFv comprising:

[0285] (i) a CD3-VH domain;

[0286] (ii) a linker; and

[0287] (iii) a CD3-VL domain;

[0288] and

[0289] (iv) an Fc region comprising a first subunit and a second subunit.

[0290] In some embodiments, the bispecific binding molecule comprises:

[0291] (i) a first CA19-9-binding moiety comprising a CA19-9-binding Fab domain comprising:

[0292] (i) a heavy chain comprising a CA19-9-VH domain and a CA19-9-CH1 domain; and (ii) a light chain comprising a CA19-9-VL domain and a CA19-9-CL domain;

[0293] (ii) a second CA19-9-binding moiety comprising a CA19-9-binding Fab domain comprising:

[0294] (i) a heavy chain comprising a CA19-9-VH domain and a CA19-9-CH1 domain; and (ii) a light chain comprising a CA19-9-VL domain and a CA19-9-CL domain; and (iii) a CD3-binding moiety comprising a scFv comprising:

[0295] (i) a CD3-VH domain;

[0296] (ii) a linker; and

[0297] (iii) a CD3-VL domain;

[0298] and

[0299] (iv) an Fc region comprising

[0300] (a) a first subunit comprising a CH2 domain and a CH3 domain; and

[0301] (b) a second subunit comprising a CH2 domain and a CH3 domain.

[0302] It will be understood that the first CA19-9-binding moiety, the second CA19-9-binding moiety, the CD3-binding moiety and / or the Fc region may be arranged in any format.

[0303] In some embodiments, the first CA19-9-binding moiety is linked to the CD3-binding moiety.

[0304] In some embodiments, the C-terminus of the first CA19-9-binding moiety is linked to the N-terminus of the CD3-binding moiety

[0305] It will be understood that any suitable linker molecule can be used to link the first CA19-9-binding moiety to the CD3-binding moiety.In some embodiments, the linker that links the first CA19-9-binding moiety to the CD3-binding moiety is a peptide linker. In some embodiments, the linker comprises one or more serine amino acid(s). In some embodiments, the linker comprises one or more glycine amino acid(s). In some embodiments, the linker comprises one or more serine amino acid(s) and one or more glycine amino acid(s).

[0306] In some embodiments, the linker that links the first CA19-9-binding moiety to the CD3-binding moiety has a length of 10 to 30 amino acids. In some embodiments, the linker has a length of 10 to 30 amino acids and comprises one or more serine amino acid(s) and one or more glycine amino acid(s).

[0307] In some embodiments, the linker that links the first CA19-9-binding moiety to the CD3-binding moiety is a (G4S)2 linker.

[0308] In some embodiments, the linker that links the first CA19-9-binding moiety to the CD3-binding moiety comprises an amino acid sequence according to SEQ ID NO: 79 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 79. In some embodiments, the linker consists of an amino acid sequence according to SEQ ID NO: 79.

[0309] In some embodiments, the C-terminus of the first CA19-9-binding moiety is linked to the N-terminus of the CD3-VH domain

[0310] In some embodiments, the C-terminus of the first CA19-9-binding moiety is linked to the N-terminus of the CD3-VL domain.

[0311] In some embodiments, the C-terminus of the CA19-9-binding Fab domain of the first CA19-9-binding moiety is linked to the N-terminus of the CD3-VH domain

[0312] In some embodiments, the C-terminus of the CA19-9-binding Fab domain of the first CA19-9-binding moiety is linked to the N-terminus of the CD3-VL domain.

[0313] In some embodiments, the C-terminus of the CA19-9-CH1 domain of the CA19-9-binding Fab domain of the first CA19-9-binding moiety is linked to the N-terminus of the CD3-VH domainIn some embodiments, the C-terminus of the CA19-9-CH1 domain of the CA19-9-binding Fab domain of the first CA19-9-binding moiety is linked to the N-terminus of the CD3-VL domain.

[0314] In some embodiments, the CD3-binding moiety is linked to the Fc region.

[0315] It will be understood that any suitable linker molecule can be used to link the CD3-binding moiety to the Fc region.

[0316] In some embodiments, the linker that links the CD3-binding moiety to the Fc region is a peptide linker. In some embodiments, the linker comprises one or more serine amino acid(s). In some embodiments, the linker comprises one or more glycine amino acid(s). In some embodiments, the linker comprises one or more serine amino acid(s) and one or more glycine amino acid(s).

[0317] In some embodiments, the linker that links the CD3-binding moiety to the Fc region has a length of 10 to 30 amino acids. In some embodiments, the linker has a length of 10 to 30 amino acids and comprises one or more serine amino acid(s) and one or more glycine amino acid(s).

[0318] In some embodiments, the linker that links the CD3-binding moiety to the Fc region is a (G4S)2 linker.

[0319] In some embodiments, the linker that links the CD3-binding moiety to the Fc region comprises an amino acid sequence according to SEQ ID NO: 79 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 79. In some embodiments, the linker consists of an amino acid sequence according to SEQ ID NO: 79.

[0320] In some embodiments, the C-terminus of the CD3-binding moiety is linked to the N-terminus of the first subunit of the Fc region.

[0321] In some embodiments, the C-terminus of the CD3-binding moiety is linked to a hinge region which is linked to the first subunit of the Fc region. In some embodiments, the C-terminus of the CD3-binding moiety comprises part of a hinge region, wherein the hinge region also comprises part of the first subunit of the Fc region.

[0322] In some embodiments, the C-terminus of the CD3-binding moiety is linked to the CH2 domain of the first subunit of the Fc region.In some embodiments, the C-terminus of the CD3-binding moiety is linked to a hinge region which is linked to the CH2 domain of the first subunit of the Fc region. In some embodiments, the C-terminus of the CD3-binding moiety comprises part of a hinge region, wherein the hinge region also comprises part of the CH2 domain of the first subunit of the Fc region.

[0323] In some embodiments, the C-terminus of the CD3-binding moiety is linked to the N-terminus of a CH2 domain of the first subunit of the Fc region.

[0324] In some embodiments, the C-terminus of the CD3-binding moiety is linked to a hinge region which is linked to the N-terminus of a CH2 domain of the first subunit of the Fc region. In some embodiments, the C-terminus of the CD3-binding moiety comprises part of a hinge region, wherein the hinge region also comprises part of the N-terminus of a CH2 domain of the first subunit of the Fc region.

[0325] In some embodiments, the C-terminus of the CD3-VL domain is linked to the first subunit of the Fc region.

[0326] In some embodiments, the C-terminus of the CD3-VL domain is linked to a hinge region which is linked to the first subunit of the Fc region. In some embodiments, the C-terminus of the CD3-VL domain comprises part of a hinge region, wherein the hinge region also comprises part of the first subunit of the Fc region..

[0327] In some embodiments, the C-terminus of the CD3-VL domain is linked to the CH2 domain of the first subunit of the Fc region.

[0328] In some embodiments, the C-terminus of the CD3-VL domain is linked to a hinge region which is linked to the CH2 domain of the first subunit of the Fc region. In some embodiments, the C-terminus of the CD3-VL domain comprises part of a hinge region, wherein the hinge region also comprises part of the CH2 domain of the first subunit of the Fc region.

[0329] In some embodiments, the C-terminus of the CD3-VL domain is linked to the N-terminus of a CH2 domain of the first subunit of the Fc region.

[0330] In some embodiments, the C-terminus of the CD3-VL domain is linked to a hinge region which is linked to the N-terminus of a CH2 domain of the first subunit of the Fc region. In some embodiments, the C-terminus of the CD3-VL domain comprises part of a hinge region, whereinthe hinge region also comprises part of the N-terminus of the CH2 domain of the first subunit of the Fc region.

[0331] In some embodiments, the C-terminus of the CD3-VH domain is linked to the first subunit of the Fc region.

[0332] In some embodiments, the C-terminus of the CD3-VH domain is linked to a hinge region which is linked to the first subunit of the Fc region. In some embodiments, the C-terminus of the CD3-VH domain comprises part of a hinge region, wherein the hinge region also comprises part of first subunit of the Fc region.

[0333] In some embodiments, the C-terminus of the CD3-VH domain is linked to the CH2 domain of the first subunit of the Fc region.

[0334] In some embodiments, the C-terminus of the CD3-VH domain is linked to a hinge region which is linked to the CH2 domain of the first subunit of the Fc region. In some embodiments, the C-terminus of the CD3-VH domain comprises part of a hinge region, wherein the hinge region also comprises part of the first subunit of the Fc region.

[0335] In some embodiments, the C-terminus of the CD3-VH domain is linked to the N-terminus of a CH2 domain of the first subunit of the Fc region.

[0336] In some embodiments, the C-terminus of the CD3-VH domain is linked to a hinge region which is linked to the N-terminus of a CH2 domain of the first subunit of the Fc region. In some embodiments, the C-terminus of the CD3-VH domain comprises part of a hinge region, wherein the hinge region also comprises part of the N-terminus of the CH2 domain of the first subunit of the Fc region.

[0337] In some embodiments, the second CA19-9-binding moiety is linked to the Fc region.

[0338] In some embodiments, the C-terminus of the second CA19-9-binding moiety is linked to the N-terminus of the second subunit of the Fc region.

[0339] In some embodiments, the C-terminus of the second CA19-9-binding moiety is linked to a hinge region which is linked to the second subunit of the Fc region. In some embodiments, the C-terminus of the second CA19-9-binding moiety comprises part of a hinge region, wherein the hinge region also comprises part of the second subunit of the Fc region.In some embodiments, the C-terminus of the second CA19-9-binding moiety is linked to the CH2 domain of the second subunit of the Fc region.

[0340] In some embodiments, the C-terminus of the second CA19-9-binding moiety is linked to a hinge region which is linked to the CH2 domain of the second subunit of the Fc region. In some embodiments, the C-terminus of the second CA19-9-binding moiety comprises part of a hinge region, wherein the hinge region also comprises part of the CH2 domain of the second subunit of the Fc region.

[0341] In some embodiments, the C-terminus of the second CA19-9-binding moiety is linked to the N-terminus of a CH2 domain of the second subunit of the Fc region.

[0342] In some embodiments, the C-terminus of the second CA19-9-binding moiety is linked to a hinge region which is linked to the N-terminus of a CH2 domain of the second subunit of the Fc region. In some embodiments, the C-terminus of the second CA19-9-binding moiety comprises part of a hinge region, wherein the hinge region also comprises part of the N-terminus of the CH2 domain of the second subunit of the Fc region.

[0343] In some embodiments, the C-terminus of the CA19-9-CH1 domain of the second CA19-9-binding moiety is linked to the second subunit of the Fc region.

[0344] In some embodiments, the C-terminus of the CA19-9-CH1 domain of the second CA19-9-binding moiety is linked to a hinge region which is linked to the second subunit of the Fc region. In some embodiments, the C-terminus of the CA19-9-CH1 domain of the second CA19-9-binding moiety comprises part of a hinge region, wherein the hinge region also comprises part of the second subunit of the Fc region.

[0345] In some embodiments, the C-terminus of the CA19-9-CH1 domain of the second CA19-9-binding moiety is linked to the CH2 domain of the second subunit of the Fc region.

[0346] In some embodiments, the C-terminus of the CA19-9-CH1 domain of the second CA19-9-binding moiety is linked to a hinge region which is linked to the CH2 domain of the second subunit of the Fc region. In some embodiments, the C-terminus of the CA19-9-CH1 domain of the second CA19-9-binding moiety comprises part of a hinge region, wherein the hinge region also comprises part of the CH2 domain of the second subunit of the Fc region.In some embodiments, the C-terminus of the CA19-9-CH1 domain of the second CA19-9-binding moiety is linked to the N-terminus of a CH2 domain of the second subunit of the Fc region.

[0347] In some embodiments, the C-terminus of the CA19-9-CH1 domain of the second CA19-9-binding moiety is linked to a hinge region which is linked to the N-terminus of a CH2 domain of the second subunit of the Fc region. In some embodiments, the C-terminus of the CA19-9-CH1 domain of the second CA19-9-binding moiety comprises part of a hinge region, wherein the hinge region also comprises part of the N-terminus of the CH2 domain of the second subunit of the Fc region.

[0348] In some embodiments, the bispecific binding molecule may comprise a structure as depicted in Figure 1.

[0349] In some embodiments, the bispecific binding molecule comprises:

[0350] (i) a CD3-binding moiety comprising a scFv, wherein the Fab comprises:

[0351] (i) a CD3-VH domain;

[0352] (ii) a linker; and

[0353] (iii) a CD3-VL domain;

[0354] (ii) a first CA19-9-binding moiety and a second CA19-9-binding moiety, wherein the first CA19-9-binding moiety and the second CA19-9-binding moiety are the same, wherein the first CA19-9-binding moiety and the second CA19-9-binding moiety each independently comprise a CA19-9-binding Fab domain, wherein each Fab comprises:

[0355] (i) a heavy chain comprising a CA19-9-VH domain and a CA19-9-CH1 domain; and (ii) a light chain comprising a CA19-9-VL domain and a CA19-9-CL domain;

[0356] (iii) an Fc region comprising a first subunit and a second subunit; wherein the first subunit of the Fc region and the second subunit of the Fc region each independently comprise a CH2 domain and a CH3 domain, and wherein the CH2 domain comprises a hinge region, optionally wherein the CH3 domain of one subunit of the Fc region comprises one or more mutations selected from the list consisting of: Y349C, T366S, L368A, and Y407V; and the CH3 domain of the other subunit of the Fc region comprises one or more mutations selected from the list consisting of: S354C and T366W;

[0357] (iv) the C-terminus of the CA19-9-CH1 domain of the first CA19-9-binding moiety is linked to the N-terminus of the CD3-VH domain;

[0358] (v) the C-terminus of the CD3-VL domain is linked to the N-terminus of the hinge region of the CH2 domain of the first subunit of the Fc region; and(vi) the C-terminus of the CA19-9-CH1 domain of the second CA19-9-binding moiety is linked to the N-terminus of the hinge region of the CH2 domain of the second subunit of the Fc region.

[0359] In some embodiments, the bispecific binding molecule comprises:

[0360] (i) a CD3-binding moiety comprising a scFv, wherein the Fab comprises:

[0361] (i) a CD3-VH domain;

[0362] (ii) a linker; and

[0363] (iii) a CD3-VL domain;

[0364] (ii) a first CA19-9-binding moiety and a second CA19-9-binding moiety, wherein the first CA19-9-binding moiety and the second CA19-9-binding moiety are the same, wherein the first CA19-9-binding moiety and the second CA19-9-binding moiety each independently comprise a CA19-9-binding Fab domain, wherein each Fab comprises:

[0365] (i) a heavy chain comprising a CA19-9-VH domain and a CA19-9-CH1 domain; and (ii) a light chain comprising a CA19-9-VL domain and a CA19-9-CL domain;

[0366] (iii) an Fc region comprising a first subunit and a second subunit; wherein the first subunit of the Fc region and the second subunit of the Fc region each independently comprise a CH2 domain and a CH3 domain, optionally wherein the CH3 domain of one subunit of the Fc region comprises one or more mutations selected from the list consisting of: Y349C, T366S, L368A, and Y407V; and the CH3 domain of the other subunit of the Fc region comprises one or more mutations selected from the list consisting of: S354C and T366W;

[0367] (iv) the C-terminus of the CA19-9-CH1 domain of the first CA19-9-binding moiety is linked to the N-terminus of the CD3-VH domain;

[0368] (v) the C-terminus of the CD3-VL domain is linked to the N-terminus of a hinge region, wherein the hinge region is linked to, or also comprises part of, the N-terminus of the CH2 domain of the first subunit of the Fc region; and

[0369] (vi) the C-terminus of the CA19-9-CH1 domain of the second CA19-9-binding moiety is linked to, or comprises part of, a hinge region, wherein the hinge region is linked to, or also comprises part of, the N-terminus of the CH2 domain of the second subunit of the Fc region.

[0370] In some embodiments, the bispecific binding molecule comprises:

[0371] (i) a CD3-binding moiety comprising a scFv, wherein the Fab comprises:

[0372] (i) a CD3-VL domain;

[0373] (ii) a linker; and

[0374] (iii) a CD3-VL domain;

[0375] (ii) a first CA19-9-binding moiety and a second CA19-9-binding moiety, wherein the first CA19-9-binding moiety and the second CA19-9-binding moiety are the same, wherein the first CA19-9-binding moiety and the second CA19-9-binding moiety each independently comprise a CA19-9-binding Fab domain, wherein each Fab comprises:

[0376] (i) a heavy chain comprising a CA19-9-VH domain and a CA19-9-CH1 domain; and (ii) a light chain comprising a CA19-9-VL domain and a CA19-9-CL domain;

[0377] (iii) an Fc region comprising a first subunit and a second subunit; wherein the first subunit of the Fc region and the second subunit of the Fc region each independently comprise a CH2 domain and a CH3 domain, and wherein the CH2 domain comprises a hinge region, optionally wherein the CH3 domain of one subunit of the Fc region comprises one or more mutations selected from the list consisting of: Y349C, T366S, L368A, and Y407V; and the CH3 domain of the other subunit of the Fc region comprises one or more mutations selected from the list consisting of: S354C and T366W;

[0378] (iv) the C-terminus of the CA19-9-CH1 domain of the first CA19-9-binding moiety is linked to the N-terminus of the CD3-VL domain;

[0379] (v) the C-terminus of the CD3-VH domain is linked to the N-terminus of the hinge region of the CH2 domain of the first subunit of the Fc region; and

[0380] (vi) the C-terminus of the CA19-9-CH1 domain of the second CA19-9-binding moiety is linked to the N-terminus of the hinge region of the CH2 domain of the second subunit of the Fc region.

[0381] In some embodiments, the bispecific binding molecule comprises:

[0382] (i) a CD3-binding moiety comprising a scFv, wherein the Fab comprises:

[0383] (i) a CD3-VL domain;

[0384] (ii) a linker; and

[0385] (iii) a CD3-VL domain;

[0386] (ii) a first CA19-9-binding moiety and a second CA19-9-binding moiety, wherein the first CA19-9-binding moiety and the second CA19-9-binding moiety are the same, wherein the first CA19-9-binding moiety and the second CA19-9-binding moiety each independently comprise a CA19-9-binding Fab domain, wherein each Fab comprises:

[0387] (i) a heavy chain comprising a CA19-9-VH domain and a CA19-9-CH1 domain; and (ii) a light chain comprising a CA19-9-VL domain and a CA19-9-CL domain;

[0388] (iii) an Fc region comprising a first subunit and a second subunit; wherein the first subunit of the Fc region and the second subunit of the Fc region each independently comprise a CH2 domain and a CH3 domain, optionally wherein the CH3 domain of one subunit of the Fc region comprises one or more mutations selected from the list consisting of: Y349C, T366S, L368A, and Y407V; and the CH3 domain of the other subunit of the Fc region comprises one or more mutations selected from the list consisting of: S354C and T366W;

[0389] (iv) the C-terminus of the CA19-9-CH1 domain of the first CA19-9-binding moiety is linked to the N-terminus of the CD3-VL domain;(v) the C-terminus of the CD3-VH domain is linked to the N-terminus of a hinge region, wherein the hinge region is part of the N-terminus of the CH2 domain of the first subunit of the Fc region and part of the C-terminus of the CD3-VH domain of the first CA19-9-binding moiety; and (vi) the C-terminus of the CA19-9-CH1 domain of the second CA19-9-binding moiety is linked to a hinge region, wherein the hinge region is part of the N-terminus of the CH2 domain of the second subunit of the Fc region and part of the C-terminus of the CA19-9-CH1 domain of the second CA19-9-binding moiety.

[0390] In some embodiments, the bispecific binding molecule comprises:

[0391] (i) a CD3-binding moiety comprising a scFv, wherein the Fab comprises:

[0392] (i) a CD3-VL domain;

[0393] (ii) a linker; and

[0394] (iii) a CD3-VL domain;

[0395] (ii) a first CA19-9-binding moiety and a second CA19-9-binding moiety, wherein the first CA19-9-binding moiety and the second CA19-9-binding moiety are the same, wherein the first CA19-9-binding moiety and the second CA19-9-binding moiety each independently comprise a CA19-9-binding Fab domain, wherein each Fab comprises:

[0396] (i) a heavy chain comprising a CA19-9-VH domain and a CA19-9-CH1 domain; and (ii) a light chain comprising a CA19-9-VL domain and a CA19-9-CL domain;

[0397] (iii) an Fc region comprising a first subunit and a second subunit; wherein the first subunit of the Fc region and the second subunit of the Fc region each independently comprise a CH2 domain and a CH3 domain, optionally wherein the CH3 domain of one subunit of the Fc region comprises one or more mutations selected from the list consisting of: Y349C, T366S, L368A, and Y407V; and the CH3 domain of the other subunit of the Fc region comprises one or more mutations selected from the list consisting of: S354C and T366W;

[0398] (iv) the C-terminus of the CA19-9-CH1 domain of the first CA19-9-binding moiety is linked to the N-terminus of the CD3-VL domain;

[0399] (v) the C-terminus of the CD3-VH domain is linked to the N-terminus of a hinge region, wherein the hinge region is linked to, or also comprises part of, the N-terminus of the CH2 domain of the first subunit of the Fc region; and

[0400] (vi) the C-terminus of the CA19-9-CH1 domain of the second CA19-9-binding moiety is linked to, or comprises part of, a hinge region, wherein the hinge region is linked to, or also comprises part of, the N-terminus of the CH2 domain of the second subunit of the Fc region.

[0401] In some embodiments, the CH3 domain of one subunit of the Fc region comprises one or more mutations selected from the list consisting of: Y349C, T366S, L368A, and Y407V; and the CH3domain of the other subunit of the Fc region comprises one or more mutations selected from the list consisting of: S354C and T366W.

[0402] In some embodiments, the CH2 domain of one subunit of the Fc region comprises one or more LALA mutations. In some embodiments, the CH2 domain of both subunits of the Fc region comprises one or more LALA mutations.

[0403] In some embodiments, the CH2 domain of one subunit of the Fc region comprises L234A and L235A mutations. In some embodiments, the CH2 domain of both subunits of the Fc region comprises L234A and L235A mutations.

[0404] Suitable assays and techniques for measuring / quantifying binding activity of the bispecific binding molecule according to the invention may include, but are not limited to, ELISA, surface plasmon resonance (SPR), bio-layer interferometry (BLI), quartz crystal microbalance (QCM), bioluminescence assays and flow cytometry. Other suitable techniques will be known in the art.

[0405] In particular, it will be understood that the ability of a binding molecule to bind to its cognate target may be assessed by flow cytometry, where the effectiveness of a binding molecule to bind to a cognate target expressed by a population of cells can be measured in a flow cytometry assay.

[0406] Moreover, it will be understood that EC50 is a measure of the concentration of a binding molecule that induces a specific response that is 50% between the maximum response and the baseline response. As such, EC50 can be used to assess the ability of a binding molecule to bind to a target.

[0407] In some embodiments, the bispecific binding molecule comprises one or more sequences as shown in Table 1.

[0408] CA19-9-bindinq moiety

[0409] As used herein, a “CA19-9-binding moiety” comprises a binding domain that is capable of binding to CA19-9. In some embodiments, the, or each, CA19-9-binding moiety independently comprises an antigen-binding domain, e.g. a CA19-9-binding Fab domain.

[0410] It will be understood that the term “CA19-9-binding moiety” also encompasses fragments thereof.In some embodiments, the first CA19-9-binding moiety and the second CA19-9-binding moiety are the same.

[0411] In some embodiments, the, or each, CA19-9-binding moiety independently comprises one, two, three, four, five or six CDRs.

[0412] In some embodiments, the, or each, CA19-9-binding moiety independently comprises six CDRs.

[0413] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a heavy chain variable (CA19-9-VH) domain.

[0414] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a light chain variable (CA19-9-VL) domain.

[0415] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9-VH domain and CA19-9-VL domain.

[0416] It will be understood that the CA19-9-VH domain and / or CA19-9-VL domain are capable of binding to CA19-9. It will also be understood that the CA19-9-VH domain and CA19-9-VL domain may form the antigen-binding domain of the, or each, CA19-9-binding moiety as described herein.

[0417] In some embodiments, the CA19-9-VH domain comprises one or more complementarity determining regions (CDRs). In some embodiments, the CA19-9-VH domain comprises one, two or three CDRs. In some embodiments, the CA19-9-VH domain comprises three CDRs. It will be understood that CDRs of the CA19-9-VH domain may be termed HCDRs. It will also be understood that each of the three CDRs of the CA19-9-VH domain may be termed HCDR1, HCDR2 and HCDR3 respectively.

[0418] In some embodiments, the CA19-9-VL domain comprises one or more CDRs. In some embodiments, the CA19-9-VL domain comprises one, two or three CDRs. In some embodiments, the CA19-9-VL domain comprises three CDRs. It will be understood that CDRs of the CA19-9-VL domain may be termed LCDRs. It will also be understood that each of the three CDRs of the CA19-9-VL domain may be termed LCDR1, LCDR2 and LCDR3 respectively.In some embodiments, the, or each, CA19-9-binding moiety further comprises framework regions. In some embodiments, the framework regions are a scaffold (e.g. amino acid sequences) that support the CDRs such that the, or each, CA19-9-binding moiety specifically binds to CA19-9 via the CDRs.

[0419] The CDRs may be determined according to the Kabat definition and / or the IMGT definition, both of which are well-known in the art.

[0420] The CDRs may be determined according to the Kabat definition and / or the IMGT definition, both of which are well-known in the art (e.g. see Edelman et al., Proc Natl Acad Sci U S A.

[0421] 1969 May;63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242; and Marie-Paule Lefranc, Veronique Giudicelli, Chantal Ginestoux, Julia Bodmer, Werner Muller, Ronald Bontrop, Marc Lemaitre, Ansar Malik, Valerie Barbie, Denys Chaume, IMGT, the international ImMunoGeneTics database, Nucleic Acids Research, Volume 27, Issue 1, 1 January 1999, Pages 209-212).

[0422] It will be understood that a variant of a CA19-9-binding moiety as described herein may have an equivalent function to a full length CA19-9-binding moiety amino acid sequence as described herein and may suitably maintain the capacity to bind to CA19-9.

[0423] It will be understood that a variant of a CA19-9-VH domain as described herein may have an equivalent function to a full length CA19-9-VH domain amino acid sequence as described herein and may suitably maintain the capacity to bind to CA19-9.

[0424] It will be understood that a variant of a CA19-9-VL domain as described herein may have an equivalent function to a full length CA19-9-VL domain amino acid sequence as described herein and may suitably maintain the capacity to bind to CA19-9.

[0425] In some embodiments, the, or each, CA19-9-binding moiety specifically binds to CA19-9.

[0426] In some embodiments, it will be understood herein that “specifically binds to” refers to the antibody-like binding of the, or each, CA19-9-binding moiety to its target: CA19-9. In some embodiments, “specifically binds to” means that the, or each, CA19-9-binding moiety binds to an epitope on CA19-9.In some embodiments, the, or each, CA19-9-binding moiety is capable of recruiting immune cells.

[0427] All CDRs specified herein, i.e. each CDR of the CA19-9-binding variable domains of the antigen binding proteins described herein, i.e. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 defined below, can be variant CDRs. The amino acid sequence of a variant CDR can be varied by one or more, preferably one, two or three amino acid deletions, insertions and / or substitutions from the sequence of the corresponding non-variant CDR. In some embodiments, the variant CDR comprises not more than 3, preferably not more than two, more preferably not more than one conservative substitution. In some embodiments, the variant CDR has an amino acid sequence that is highly homologous to the sequence of the corresponding non-variant CDR. The variant CDRs, when in the context of the antigen binding proteins of the invention have at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% of the binding affinity to CA19-9 compared to the non-variant CDR.

[0428] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9-VH domain; wherein the CA19-9-VH domain comprises a HCDR3 sequence, wherein the HCDR3 sequence comprises or consists of SEQ ID NO: 41 (Kabat).

[0429] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9-VH domain; wherein the CA19-9-VH domain comprises a HCDR3 sequence, wherein the HCDR3 sequence comprises or consists of SEQ ID NO: 42 (IMGT).

[0430] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9-VH domain; wherein the CA19-9-VH domain comprises a HCDR1 sequence, wherein the HCDR1 sequence comprises or consists of SEQ ID NO: 37 (Kabat).

[0431] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9-VH domain; wherein the CA19-9-VH domain comprises a HCDR1 sequence, wherein the HCDR1 sequence comprises or consists of SEQ ID NO: 38 (IMGT).

[0432] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9-VH domain; wherein the CA19-9-VH domain comprises a HCDR2 sequence, wherein the HCDR2 sequence comprises or consists of SEQ ID NO: 39 (Kabat).In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9-VH domain; wherein the CA19-9-VH domain comprises a HCDR2 sequence, wherein the HCDR2 sequence comprises or consists of SEQ ID NO: 40 (IMGT).

[0433] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9-VL domain; wherein the CA19-9-VL domain comprises a LCDR3 sequence, wherein the LCDR3 sequence comprises or consists of SEQ ID NO: 47 (Kabat).

[0434] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9-VL domain; wherein the CA19-9-VL domain comprises a LCDR3 sequence, wherein the LCDR3 sequence comprises or consists of SEQ ID NO: 48 (IMGT).

[0435] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9-VL domain; wherein the CA19-9-VL domain comprises a LCDR1 sequence, wherein the LCDR1 sequence comprises or consists of SEQ ID NO: 43 (Kabat).

[0436] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9-VL domain; wherein the CA19-9-VL domain comprises a LCDR1 sequence, wherein the LCDR1 sequence comprises or consists of SEQ ID NO: 44 (IMGT).

[0437] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9-VL domain; wherein the CA19-9-VL domain comprises a LCDR2 sequence, wherein the LCDR2 sequence comprises or consists of SEQ ID NO: 45 (Kabat).

[0438] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9-VL domain; wherein the CA19-9-VL domain comprises a LCDR2 sequence, wherein the LCDR2 sequence comprises or consists of SEQ ID NO: 46 (IMGT).

[0439] In some embodiments, one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0440] In some embodiments, the HCDR1 sequence comprises one, two or three amino acid mutations relative to the recited sequences. In some embodiments, the HCDR2 sequence comprises one, two or three amino acid mutations relative to the recited sequences. In some embodiments, the HCDR3 sequence comprises one, two or three amino acid mutations relative to the recited sequences.In some embodiments, the LCDR1 sequence comprises one, two or three amino acid mutations relative to the recited sequences. In some embodiments, the LCDR2 sequence comprises one, two or three amino acid mutations relative to the recited sequences. In some embodiments, the LCDR3 sequence comprises one, two or three amino acid mutations relative to the recited sequences.

[0441] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9-VH domain; wherein the CA19-9-VH domain comprises HCDRs 1-3, wherein (according to Kabat numbering):

[0442] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 37, HCDR2 comprises the amino acid sequence according to SEQ ID NO: 39, and HCDR3 comprises the amino acid sequence according to SEQ ID NO: 41, optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0443] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9-VL domain; wherein the CA19-9-VL domain comprises LCDRs 1-3, wherein (according to Kabat numbering):

[0444] LCDR1 comprises the amino acid sequence according to SEQ ID NO: 43, LCDR2 comprises the amino acid sequence according to SEQ ID NO: 45, and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 47; optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0445] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9-VH domain and a CA19-9-VL domain; wherein the CA19-9-VH domain comprises HCDRs 1-3, and the CA19-9-VL domain comprises LCDRs 1-3, wherein (according to Kabat numbering):

[0446] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 37, HCDR2 comprises the amino acid sequence according to SEQ ID NO: 39, HCDR3 comprises the amino acid sequence according to SEQ ID NO: 41, LCDR1 comprises the amino acid sequence according to SEQ ID NO: 43, LCDR2 comprises the amino acid sequence according to SEQ ID NO: 45, and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 47; optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9-VH domain; wherein the CA19-9-VH domain comprises HCDRs 1-3, wherein (according to IMGT numbering):

[0447] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 38, HCDR2 comprises the amino acid sequence according to SEQ ID NO: 40, and HCDR3 comprises the amino acid sequence according to SEQ ID NO: 42, optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0448] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9-VL domain; wherein the CA19-9-VL domain comprises LCDRs 1-3, wherein (according to IMGT numbering):

[0449] LCDR1 comprises the amino acid sequence according to SEQ ID NO: 44, LCDR2 comprises the amino acid sequence according to SEQ ID NO: 46, and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 48; optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0450] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a CA19-9-VH domain and a CA19-9-VL domain; wherein the CA19-9-VH domain comprises HCDRs 1-3, and the CA19-9-VL domain comprises LCDRs 1-3, wherein (according to IMGT numbering):

[0451] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 38, HCDR2 comprises the amino acid sequence according to SEQ ID NO: 40, HCDR3 comprises the amino acid sequence according to SEQ ID NO: 42, LCDR1 comprises the amino acid sequence according to SEQ ID NO: 44, LCDR2 comprises the amino acid sequence according to SEQ ID NO: 46, and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 48; optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0452] In some embodiments, the HCDR1, HCDR2 and HCDR3 sequences comprise SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 41, respectively.

[0453] In some embodiments, the HCDR1, HCDR2 and HCDR3 sequences consist of SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 41, respectively.In some embodiments, the HCDR1, HCDR2 and HCDR3 sequences comprise SEQ ID NO: 38, SEQ ID NO: 40, and SEQ ID NO: 42, respectively.

[0454] In some embodiments, the HCDR1, HCDR2 and HCDR3 sequences consist of SEQ ID NO: 38, SEQ ID NO: 40, and SEQ ID NO: 42, respectively.

[0455] In some embodiments, the LCDR1, LCDR2 and LCDR3 sequences comprise SEQ ID NO: 43, SEQ ID NO: 45, and SEQ ID NO: 47, respectively.

[0456] In some embodiments, the LCDR1 , LCDR2 and LCDR3 sequences consist of SEQ ID NO: 43, SEQ ID NO: 45, and SEQ ID NO: 47, respectively.

[0457] In some embodiments, the LCDR1, LCDR2 and LCDR3 sequences comprise SEQ ID NO: 44, 46, and SEQ ID NO: 48, respectively.

[0458] In some embodiments, the LCDR1, LCDR2 and LCDR3 sequences consist of SEQ ID NO: 44, 46, and SEQ ID NO: 48, respectively.

[0459] In the VH domain and VL domain the CDR sequences are interspersed with FR sequences. Like the CDR sequences, the FR sequences differ depending on whether they are defined according to Kabat or IMGT. Preferably, CDR sequences defined according to Kabat are combined with FR sequences defined according to Kabat and CDR sequences defined according to IMGT are combined with FR sequences according to IMGT.

[0460] In some embodiments, the CA19-9-VH domain comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 50.

[0461] In some embodiments, the CA19-9-VH domain comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 50.

[0462] In some embodiments, the CA19-9-VH domain comprises of a sequence as set forth in SEQ ID NO: 50.

[0463] In some embodiments, the CA19-9-VH domain consists of a sequence as set forth in SEQ ID NO: 50.It will be understood that the variants of the CA19-9-VH domains described herein comprise the HCDR1, HCDR2 and HCDR3 sequences described herein (i.e. SEQ ID NO: 37, SEQ ID NO: 39, and SEQ ID NO: 41, respectively defined according to Kabat; or SEQ ID NO: 38, SEQ ID NO: 40, and SEQ ID NO: 42, respectively defined according to IMGT), optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0464] In some embodiments, the CA19-9-VL domain comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 52.

[0465] In some embodiments, the CA19-9-VL domain comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 52.

[0466] In some embodiments, the CA19-9-VL domain comprises a sequence as set forth in SEQ ID NO: 52.

[0467] In some embodiments, the CA19-9-VL domain consists of a sequence as set forth in SEQ ID NO: 52.

[0468] It will be understood that the variants of the CA19-9-VL domains described herein comprise the LCDR1, LCDR2 and LCDR3 sequences described herein (i.e. SEQ ID NO: 43, SEQ ID NO: 45, and SEQ ID NO: 47, respectively defined according to Kabat; or SEQ ID NO: 44, 46, and SEQ ID NO: 48, respectively defined according to IMGT), optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0469] In some embodiments, the, or each, CA19-9-binding moiety independently comprises:

[0470] (i) a VH domain comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 50; and

[0471] (ii) a VL domain comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 52.In some embodiments, the, or each, CA19-9-binding moiety independently comprises:

[0472] (i) a VH domain consisting of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 50; and

[0473] (ii) a VL domain consisting of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 52.

[0474] In some embodiments, the, or each, CA19-9-binding moiety independently comprises:

[0475] (i) a VH domain comprising a sequence as set forth in SEQ ID NO: 50; and

[0476] (ii) a VL domain comprising a sequence as set forth in SEQ ID NO: 52.

[0477] In some embodiments, the, or each, CA19-9-binding moiety independently comprises:

[0478] (i) a VH domain consisting of a sequence as set forth in SEQ ID NO: 50; and

[0479] (ii) a VL domain consisting of a sequence as set forth in SEQ ID NO: 52.

[0480] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a fragment antigen-binding region (Fab). In some embodiments, the, or each, CA19-9-binding moiety independently consists of a Fab. In some embodiments, the, or each, CA19-9-binding moiety independently is a Fab.

[0481] It will be understood that a Fab typically comprises: (i) a heavy chain comprising one heavy constant (CH) domain and one heavy variable (VH) domain; and (ii) a light chain comprising one light constant domain (CL) and one light variable (VL) domain.

[0482] It will be understood that the Fab may comprise any of the CA19-9 binding CDRs, VH domains and / or VL domains as described herein.

[0483] In some embodiments, the heavy chain constant domain is selected from the list consisting of: a constant heavy 1 (CH1) domain, a constant heavy 2 (CH2) domain, and a constant heavy 3 (CH3) domain.

[0484] In some embodiments, the light chain constant domain is selected from the list consisting of: a light chain kappa constant domain and a light chain lambda constant domain.

[0485] In some embodiments, the, or each, CA19-9-binding moiety independently comprises a Fab comprising:(i) a heavy chain comprising a CA19-9-VH domain as described herein and a CA19-9 heavy chain constant (CA19-9-CH1) domain; and

[0486] (ii) a light chain comprising a CA19-9-VL domain as described herein and a CA19-9 light chain constant (CA19-9-CL) domain.

[0487] In some embodiments, the heavy chain comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 85.

[0488] In some embodiments, the heavy chain comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 85.

[0489] In some embodiments, the heavy chain comprises a sequence as set forth in SEQ ID NO: 85.

[0490] In some embodiments, the heavy chain consists of a sequence as set forth in SEQ ID NO: 85.

[0491] In some embodiments, the light chain comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 78.

[0492] In some embodiments, the light chain comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 78.

[0493] In some embodiments, the light chain comprises a sequence as set forth in SEQ ID NO: 78.

[0494] In some embodiments, the light chain consists of a sequence as set forth in SEQ ID NO: 78.

[0495] In some embodiments, the, or each, CA19-9-binding moiety independently comprises:

[0496] (i) a heavy chain comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 85; and

[0497] (ii) a light chain comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 78.In some embodiments, the, or each, CA19-9-binding moiety independently comprises:

[0498] (i) a heavy chain consisting of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 85; and

[0499] (ii) a light chain consisting of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 78.

[0500] In some embodiments, the, or each, CA19-9-binding moiety independently comprises:

[0501] (i) a heavy chain comprising a sequence as set forth in SEQ ID NO: 85; and

[0502] (ii) a light chain comprising a sequence as set forth in SEQ ID NO: 78.

[0503] In some embodiments, the, or each, CA19-9-binding moiety independently comprises:

[0504] (i) a heavy chain consisting of a sequence as set forth in SEQ ID NO: 85; and

[0505] (ii) a light chain consisting of a sequence as set forth in SEQ ID NO: 78.

[0506] The, or each, CA19-9-binding moiety according to the invention independently may have a high affinity for CA19-9. In particular, the, or each, CA19-9-binding moiety of the invention independently may have the ability to bind to a recombinant human CA19-9 with an ECso of less than 75 ng / ml, less than 50 ng / ml, less than 40 ng / ml, less than 30 ng / ml, less than 25 ng / ml, less than 20 ng / ml, less than 15 ng / ml, less than 10 ng / ml or less than 5 ng / ml, such as 4-75 ng / ml, 4-50 ng / ml, 4-40 ng / ml, 4-30 ng / ml, 4-25 ng / ml, 4-20 ng / ml, 4-15 ng / ml, 4-10 ng / ml or 4-5 ng / ml.

[0507] The, or each, CA19-9-binding moiety of the invention also independently may have a high affinity for cells expressing CA19-9. In particular, the, or each, CA19-9-binding moiety of the invention independently may bind to cells genetically modified to express human CA19-9 protein on the cell surface with an ECso of less than 12 ng / ml, less than 11 ng / ml, less than 10 ng / ml, less than 9 ng / ml or less than 8 ng / ml, such as 1-12 ng / ml, 1-11 ng / ml, 1-10 ng / ml, 1-9 ng / ml, 1-8 ng / ml, 3-12 ng / ml, 3-11 ng / ml, 3-10 ng / ml, 3-9 ng / ml, 3-8 ng / ml, 6-12 ng / ml, 6-11 ng / ml, 6-10 ng / ml, 6-9 ng / ml or 6-8 ng / ml.

[0508] It will be understood that a CA19-9-binding moiety having % identity to the SEQ ID NO of any CA19-9-binding moieties defined herein may have an equivalent function to the CA19-9-binding moiety having the sequence set forth in the SEQ ID NO defined herein and may suitably maintain the capacity to bind to CA19-9, e.g. the same capacity as the CA19-9-binding moiety having the sequence set forth in the SEQ ID NO defined herein.In some embodiments, the, or each, CA19-9-binding moiety or a fragment thereof is independently non-human.

[0509] In some embodiments, the, or each, CA19-9-binding moiety or a fragment thereof is independently humanised.

[0510] In some embodiments, the, or each, CA19-9-binding moiety or a fragment thereof is independently chimeric.

[0511] In some embodiments, the, or each, CA19-9-binding moiety or a fragment thereof is independently fully human.

[0512] Techniques for obtaining antibodies and fragments thereof are well-known in the art. By way of example, it is well-known that antibodies may be obtained by standard techniques comprising immunizing an animal with a target antigen and isolating antibodies from serum.

[0513] It will be understood that a fragment of an antibody comprises an amino acid sequence that is shorter than the full-length sequence of an antibody, but retains the biological activity (e.g., binding specificity and / or affinity) of the full-length antibody.

[0514] The term "humanised antibody" typically refers to a genetically engineered antibody generated in a non-human animal that has been engineered to reduce immunogenicity when used in a human whilst retaining antigen specificity. The humanised antibody typically contains human antibody constant domains and non-human variable domains that are modified to contain a high level of sequence homology to human variable domains.

[0515] Suitable assays and techniques for measuring / quantifying binding activity of CA19-9-binding moiety according to the invention may include, but are not limited to, ELISA, surface plasmon resonance (SPR), bio-layer interferometry (BLI), quartz crystal microbalance (QCM), bioluminescence assays and flow cytometry. Other suitable techniques will be known in the art.

[0516] In particular, it will be understood that the ability of a binding molecule to bind to its cognate target may be assessed by flow cytometry, where the effectiveness of a binding molecule to bind to a cognate target expressed by a population of cells can be measured in a flow cytometry assay.Moreover, it will be understood that EC50 is a measure of the concentration of a binding molecule that induces a specific response that is 50% between the maximum response and the baseline response. As such, EC50 can be used to assess the ability of a binding molecule to bind to a target.

[0517] CD3-binding moiety

[0518] As used herein, a “CD3-binding moiety” comprises a binding domain, e.g. an antigen-binding domain, that is capable of binding to CD3.

[0519] It will be understood that the term “CD3-binding moiety” also encompasses fragments thereof.

[0520] In some embodiments, CD3 is human CD3. In some embodiments, CD3 is CD3E.

[0521] In some embodiments, the CD3-binding moiety comprises one or more complementarity determining regions (CDRs).

[0522] In some embodiments, the CD3-binding moiety comprises one, two, three, four, five or six CDRs.

[0523] In some embodiments, the CD3-binding moiety comprises six CDRs.

[0524] In some embodiments, the CD3-binding moiety comprises a heavy chain variable (CD3-VH) domain.

[0525] In some embodiments, the CD3-binding moiety comprises a light chain variable (CD3-VL) domain.

[0526] In some embodiments, the CD3-binding moiety comprises a CD3-VH domain and CD3-VL domain.

[0527] It will be understood that the CD3-VH domain and / or CD3-VL domain are capable of binding to CD3. It will also be understood that the CD3-VH domain and CD3-VL domain may form the antigen-binding domain of the CD3-binding moiety as described herein.

[0528] In some embodiments, the CD3-VH domain comprises one or more complementarity determining regions (CDRs). In some embodiments, the CD3-VH domain comprises one, twoor three CDRs. In some embodiments, the CD3-VH domain comprises three CDRs. It will be understood that CDRs of the CD3-VH domain may be termed HCDRs. It will also be understood that each of the three CDRs of the CD3-VH domain may be termed HCDR1, HCDR2 and HCDR3 respectively.

[0529] In some embodiments, the CD3-VL domain comprises one or more CDRs. In some embodiments, the CD3-VL domain comprises one, two or three CDRs. In some embodiments, the CD3-VL domain comprises three CDRs. It will be understood that CDRs of the CD3-VL domain may be termed LCDRs. It will also be understood that each of the three CDRs of the CD3-VL domain may be termed LCDR1, LCDR2 and LCDR3 respectively.

[0530] In some embodiments, the CD3-binding moiety further comprises framework regions. In some embodiments, the framework regions are a scaffold (e.g. amino acid sequences) that support the CDRs such that the CD3-binding moiety specifically binds to CD3 via the CDRs.

[0531] The CDRs may be determined according to the Kabat definition and / or the IMGT definition, both of which are well-known in the art.

[0532] It will be understood that a variant of a CD3-binding moiety as described herein may have an equivalent function to a full length CD3-binding moiety amino acid sequence as described herein and may suitably maintain the capacity to bind to CD3.

[0533] It will be understood that a variant of a CD3-VH domain as described herein may have an equivalent function to a full length CD3-VH domain amino acid sequence as described herein and may suitably maintain the capacity to bind to CD3.

[0534] It will be understood that a variant of a CD3-VL domain as described herein may have an equivalent function to a full length CD3-VL domain amino acid sequence as described herein and may suitably maintain the capacity to bind to CD3.

[0535] In some embodiments, the CD3-binding moiety specifically binds to CD3.

[0536] In some embodiments, it will be understood herein that “specifically binds to” refers to the antibody-like binding of the CD3-binding moiety to its target: CD3. In some embodiments, “specifically binds to” means that the CD3-binding moiety binds to an epitope on CD3.

[0537] In some embodiments, the CD3-binding moiety is capable of recruiting immune cells.All CDRs specified herein, i.e. each CDR of the CD3-binding variable domains of the antigen binding proteins described herein, i.e. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 defined below, can be variant CDRs. The amino acid sequence of a variant CDR can be varied by one or more, preferably one, two or three amino acid deletions, insertions and / or substitutions from the sequence of the corresponding non-variant CDR. In some embodiments, the variant CDR comprises not more than 3, preferably not more than two, more preferably not more than one conservative substitution. In some embodiments, the variant CDR has an amino acid sequence that is highly homologous to the sequence of the corresponding non-variant CDR. The variant CDRs, when in the context of the antigen binding proteins of the invention have at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% of the binding affinity to CD3 compared to the non-variant CDR.

[0538] In some embodiments, the CD3-binding moiety comprises a CD3-VH domain; wherein the CD3-VH domain comprises a HCDR3 sequence, wherein the HCDR3 sequence comprises or consists of SEQ ID NO: 5 (Kabat).

[0539] In some embodiments, the CD3-binding moiety comprises a CD3-VH domain; wherein the CD3-VH domain comprises a HCDR3 sequence, wherein the HCDR3 sequence comprises or consists of SEQ ID NO: 6 (IMGT).

[0540] In some embodiments, the CD3-binding moiety comprises a CD3-VH domain; wherein the CD3-VH domain comprises a HCDR1 sequence, wherein the HCDR1 sequence comprises or consists of SEQ ID NO: 1 (Kabat).

[0541] In some embodiments, the CD3-binding moiety comprises a CD3-VH domain; wherein the CD3-VH domain comprises a HCDR1 sequence, wherein the HCDR1 sequence comprises or consists of SEQ ID NO: 2 (IMGT).

[0542] In some embodiments, the CD3-binding moiety comprises a CD3-VH domain; wherein the CD3-VH domain comprises a HCDR2 sequence, wherein the HCDR2 sequence comprises or consists of SEQ ID NO: 3 (Kabat).

[0543] In some embodiments, the CD3-binding moiety comprises a CD3-VH domain; wherein the CD3-VH domain comprises a HCDR2 sequence, wherein the HCDR2 sequence comprises or consists of SEQ ID NO: 4 (IMGT).In some embodiments, the CD3-binding moiety comprises a CD3-VL domain; wherein the CD3-VL domain comprises a LCDR3 sequence, wherein the LCDR3 sequence comprises or consists of SEQ ID NO: 11 (Kabat).

[0544] In some embodiments, the CD3-binding moiety comprises a CD3-VL domain; wherein the CD3-VL domain comprises a LCDR3 sequence, wherein the LCDR3 sequence comprises or consists of SEQ ID NO: 12 (IMGT).

[0545] In some embodiments, the CD3-binding moiety comprises a CD3-VL domain; wherein the CD3-VL domain comprises a LCDR1 sequence, wherein the LCDR1 sequence comprises or consists of SEQ ID NO: 7 (Kabat).

[0546] In some embodiments, the CD3-binding moiety comprises a CD3-VL domain; wherein the CD3-VL domain comprises a LCDR1 sequence, wherein the LCDR1 sequence comprises or consists of SEQ ID NO: 8 (IMGT).

[0547] In some embodiments, the CD3-binding moiety comprises a CD3-VL domain; wherein the CD3-VL domain comprises a LCDR2 sequence, wherein the LCDR2 sequence comprises or consists of SEQ ID NO: 9 (Kabat).

[0548] In some embodiments, the CD3-binding moiety comprises a CD3-VL domain; wherein the CD3-VL domain comprises a LCDR2 sequence, wherein the LCDR2 sequence comprises or consists of 10 (IMGT).

[0549] In some embodiments, one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0550] In some embodiments, the HCDR1 sequence comprises one, two or three amino acid mutations relative to the recited sequences. In some embodiments, the HCDR2 sequence comprises one, two or three amino acid mutations relative to the recited sequences. In some embodiments, the HCDR3 sequence comprises one, two or three amino acid mutations relative to the recited sequences.

[0551] In some embodiments, the LCDR1 sequence comprises one, two or three amino acid mutations relative to the recited sequences. In some embodiments, the LCDR2 sequence comprises one, two or three amino acid mutations relative to the recited sequences. In someembodiments, the LCDR3 sequence comprises one, two or three amino acid mutations relative to the recited sequences.

[0552] In some embodiments, the CD3-binding moiety comprises a CD3-VH domain; wherein the CD3-VH domain comprises HCDRs 1-3, wherein (according to Kabat numbering):

[0553] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 1, HCDR2 comprises the amino acid sequence according to SEQ ID NO: 3, and HCDR3 comprises the amino acid sequence according to SEQ ID NO: 5, optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0554] In some embodiments, the CD3-binding moiety comprises a CD3-VL domain; wherein the CD3-VL domain comprises LCDRs 1-3, wherein (according to Kabat numbering):

[0555] LCDR1 comprises the amino acid sequence according to SEQ ID NO: 7, LCDR2 comprises the amino acid sequence according to SEQ ID NO: 9, and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 11; optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0556] In some embodiments, the CD3-binding moiety comprises a CD3-VH domain and a CD3-VL domain; wherein the CD3-VH domain comprises HCDRs 1-3, and the CD3-VL domain comprises LCDRs 1-3, wherein (according to Kabat numbering):

[0557] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 1, HCDR2 comprises the amino acid sequence according to SEQ ID NO: 3, HCDR3 comprises the amino acid sequence according to SEQ ID NO: 5, LCDR1 comprises the amino acid sequence according to SEQ ID NO: 7, LCDR2 comprises the amino acid sequence according to SEQ ID NO: 9, and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 11; optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0558] In some embodiments, the CD3-binding moiety comprises a CD3-VH domain; wherein the CD3-VH domain comprises HCDRs 1-3, wherein (according to IMGT numbering):

[0559] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 2, HCDR2 comprises the amino acid sequence according to SEQ ID NO: 4, and HCDR3 comprises the amino acid sequence according to SEQ ID NO: 6,optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0560] In some embodiments, the CD3-binding moiety comprises a CD3-VL domain; wherein the CD3-VL domain comprises LCDRs 1-3, wherein (according to IMGT numbering):

[0561] LCDR1 comprises the amino acid sequence according to SEQ ID NO: 8,

[0562] LCDR2 comprises the amino acid sequence according to SEQ ID NO: 10, and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 12; optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0563] In some embodiments, the CD3-binding moiety comprises a CD3-VH domain and a CD3-VL domain; wherein the CD3-VH domain comprises HCDRs 1-3, and the CD3-VL domain comprises LCDRs 1-3, wherein (according to IMGT numbering):

[0564] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 2, HCDR2 comprises the amino acid sequence according to SEQ ID NO: 4, HCDR3 comprises the amino acid sequence according to SEQ ID NO: 6, LCDR1 comprises the amino acid sequence according to SEQ ID NO: 8,

[0565] LCDR2 comprises the amino acid sequence according to SEQ ID NO: 10, and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 12; optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0566] In some embodiments, the HCDR1, HCDR2 and HCDR3 sequences comprise SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, respectively.

[0567] In some embodiments, the HCDR1, HCDR2 and HCDR3 sequences consist of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, respectively.

[0568] In some embodiments, the HCDR1, HCDR2 and HCDR3 sequences comprise SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, respectively.

[0569] In some embodiments, the HCDR1, HCDR2 and HCDR3 sequences consist of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, respectively.

[0570] In some embodiments, the LCDR1, LCDR2 and LCDR3 sequences comprise SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11, respectively.In some embodiments, the LCDR1, LCDR2 and LCDR3 sequences consist of SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11, respectively.

[0571] In some embodiments, the LCDR1, LCDR2 and LCDR3 sequences comprise SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12, respectively.

[0572] In some embodiments, the LCDR1, LCDR2 and LCDR3 sequences consist of SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12, respectively.

[0573] In the VH domain and VL domain the CDR sequences are interspersed with FR sequences. Like the CDR sequences, the FR sequences differ depending on whether they are defined according to Kabat or IMGT. Preferably, CDR sequences defined according to Kabat are combined with FR sequences defined according to Kabat and CDR sequences defined according to IMGT are combined with FR sequences according to IMGT.

[0574] In some embodiments, the CD3-VH domain comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to any one of SEQ ID NO: 14, SEQ ID NO: 22, and SEQ ID NO: 30.

[0575] In some embodiments, the CD3-VH domain comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 14.

[0576] In some embodiments, the CD3-VH domain comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 22.

[0577] In some embodiments, the CD3-VH domain comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 30.

[0578] In some embodiments, the CD3-VH domain comprises of a sequence as set forth in any one of SEQ ID NO: 14, SEQ ID NO: 22, and SEQ ID NO: 30.

[0579] In some embodiments, the CD3-VH domain comprises of a sequence as set forth in SEQ ID NO: 14. In some embodiments, the CD3-VH domain comprises of a sequence as set forth inSEQ ID NO: 22. In some embodiments, the CD3-VH domain comprises of a sequence as set forth in SEQ ID NO: 30.

[0580] In some embodiments, the CD3-VH domain consists of a sequence as set forth in any one of SEQ ID NO: 14, SEQ ID NO: 22, and SEQ ID NO: 30.

[0581] In some embodiments, the CD3-VH domain consists of a sequence as set forth in SEQ ID NO: 14. In some embodiments, the CD3-VH domain consists of a sequence as set forth in SEQ ID NO: 22. In some embodiments, the CD3-VH domain consists of a sequence as set forth in SEQ ID NO: 30.

[0582] It will be understood that the variants of the CD3-VH domains described herein comprise the HCDR1 , HCDR2 and HCDR3 sequences described herein (i.e. SEQ ID NO: 1 , SEQ ID NO: 3, and SEQ ID NO: 5, respectively defined according to Kabat; or SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, respectively defined according to IMGT), optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0583] In some embodiments, the CD3-VL domain comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to any one of SEQ ID NO: 16, SEQ ID NO: 24, and SEQ ID NO: 32.

[0584] In some embodiments, the CD3-VL domain comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 16.

[0585] In some embodiments, the CD3-VL domain comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 24.

[0586] In some embodiments, the CD3-VL domain comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 32.

[0587] In some embodiments, the CD3-VL domain comprises a sequence as set forth in any one of SEQ ID NO: 16, SEQ ID NO: 24, and SEQ ID NO: 32.In some embodiments, the CD3-VL domain comprises a sequence as set forth in SEQ ID NO: 16. In some embodiments, the CD3-VL domain comprises a sequence as set forth in SEQ ID NO: 24. In some embodiments, the CD3-VL domain comprises a sequence as set forth in SEQ ID NO: 32.

[0588] In some embodiments, the CD3-VL domain consists of a sequence as set forth in any one of SEQ ID NO: 16, SEQ ID NO: 24, and SEQ ID NO: 32.

[0589] In some embodiments, the CD3-VL domain consists of a sequence as set forth in SEQ ID NO: 16. In some embodiments, the CD3-VL domain consists of a sequence as set forth in SEQ ID NO: 24. In some embodiments, the CD3-VL domain consists of a sequence as set forth in SEQ ID NO: 32.

[0590] It will be understood that the variants of the CD3-VL domains described herein comprise the LCDR1, LCDR2 and LCDR3 sequences described herein (i.e. SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11, respectively defined according to Kabat; or SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12, respectively defined according to IMGT), optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0591] In some embodiments, the CD3-binding moiety comprises:

[0592] (i) a VH domain comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 14; and

[0593] (ii) a VL domain comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 16.

[0594] In some embodiments, the CD3-binding moiety comprises:

[0595] (i) a VH domain consisting of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 14; and

[0596] (ii) a VL domain consisting of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 16.In some embodiments, the CD3-binding moiety comprises:

[0597] (i) a VH domain comprising a sequence as set forth in SEQ ID NO: 14; and

[0598] (ii) a VL domain comprising a sequence as set forth in SEQ ID NO: 16.

[0599] In some embodiments, the CD3-binding moiety comprises:

[0600] (i) a VH domain consisting of a sequence as set forth in SEQ ID NO: 14; and

[0601] (ii) a VL domain consisting of a sequence as set forth in SEQ ID NO: 16.

[0602] In some embodiments, the CD3-binding moiety comprises:

[0603] (i) a VH domain comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 22; and

[0604] (ii) a VL domain comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 24.

[0605] In some embodiments, the CD3-binding moiety comprises:

[0606] (i) a VH domain consisting of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 22; and

[0607] (ii) a VL domain consisting of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 24.

[0608] In some embodiments, the CD3-binding moiety comprises:

[0609] (i) a VH domain comprising a sequence as set forth in SEQ ID NO: 22; and

[0610] (ii) a VL domain comprising a sequence as set forth in SEQ ID NO: 24.

[0611] In some embodiments, the CD3-binding moiety comprises:

[0612] (i) a VH domain consisting of a sequence as set forth in SEQ ID NO: 22; and

[0613] (ii) a VL domain consisting of a sequence as set forth in SEQ ID NO: 24.

[0614] In some embodiments, the CD3-binding moiety comprises:

[0615] (i) a VH domain comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 30; and(ii) a VL domain comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 32.

[0616] In some embodiments, the CD3-binding moiety comprises:

[0617] (i) a VH domain consisting of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 30; and

[0618] (ii) a VL domain consisting of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 32.

[0619] In some embodiments, the CD3-binding moiety comprises:

[0620] (i) a VH domain comprising a sequence as set forth in SEQ ID NO: 30; and

[0621] (ii) a VL domain comprising a sequence as set forth in SEQ ID NO: 32.

[0622] In some embodiments, the CD3-binding moiety comprises:

[0623] (i) a VH domain consisting of a sequence as set forth in SEQ ID NO: 30; and

[0624] (ii) a VL domain consisting of a sequence as set forth in SEQ ID NO: 32.

[0625] In some embodiments, the CD3-binding moiety comprises a single-chain variable fragment (scFv) domain. In some embodiments, the CD3-binding moiety consists of a scFv. In some embodiments, the CD3-binding moiety is a scFv.

[0626] It will be understood that a scFv typically comprises: (i) a heavy variable (VH) domain; (ii) a linker; and (iii) a light variable (VL) domain.

[0627] It will be understood that the scFv may comprise any of the CD3 binding CDRs, VH domains and / or VL domains as described herein.

[0628] In some embodiments, the CD3-binding moiety comprises a scFv comprising:

[0629] (i) a CD3-VH domain;

[0630] (ii) a linker; and

[0631] (iii) a CD3-VL domain.

[0632] In some embodiments, the orientation of the scFv is

[0633] CD3-VH domain - linker (L1) - CD3-VL domainIn some embodiments, the orientation of the scFv is

[0634] CD3-VL domain - linker (L2) - CD3-VH domain

[0635] In some embodiments, the linker is a peptide linker. In some embodiments, the linker comprises one or more serine amino acid(s). In some embodiments, the linker comprises one or more glycine amino acid(s). In some embodiments, the linker comprises one or more serine amino acid(s) and one or more glycine amino acid(s).

[0636] In some embodiments, the linker has a length of 10 to 30 amino acids. In some embodiments, the linker has a length of 10 to 30 amino acids and comprises one or more serine amino acid(s) and one or more glycine amino acid(s).

[0637] In some embodiments, L1 is a (G4S)4 linker.

[0638] In some embodiments, L2 is a (G4S)s linker.

[0639] In some embodiments, the L1 comprises an amino acid sequence according to SEQ ID NO: 80 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 80. In some embodiments, the L1 consists of an amino acid sequence according to SEQ ID NO: 80.

[0640] In some embodiments, the L2 comprises an amino acid sequence according to SEQ ID NO: 81 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 81. In some embodiments, the L2 consists of an amino acid sequence according to SEQ ID NO: 81.

[0641] In some embodiments, the scFv comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to any one of SEQ ID NO: 18, SEQ ID NO: 26, and SEQ ID NO: 34.

[0642] In some embodiments, the scFv comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 18.In some embodiments, the scFv comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 26.

[0643] In some embodiments, the scFv comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 34.

[0644] In some embodiments, the scFv comprises a sequence as set forth in any one of SEQ ID NO: 18, SEQ ID NO: 26, and SEQ ID NO: 34.

[0645] In some embodiments, the scFv comprises a sequence as set forth in SEQ ID NO: 18.

[0646] In some embodiments, the scFv comprises a sequence as set forth in SEQ ID NO: 26.

[0647] In some embodiments, the scFv comprises a sequence as set forth in SEQ ID NO: 34.

[0648] In some embodiments, the scFv consists of a sequence as set forth in any one of SEQ ID NO: 18, SEQ ID NO: 26, and SEQ ID NO: 34.

[0649] In some embodiments, the scFv consists of a sequence as set forth in SEQ ID NO: 18. In some embodiments, the scFv consists of a sequence as set forth in SEQ ID NO: 26. In some embodiments, the scFv consists of a sequence as set forth in SEQ ID NO: 34.

[0650] In some embodiments, the scFv comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to any one of SEQ ID NO: 20, SEQ ID NO: 28, and SEQ ID NO: 36.

[0651] In some embodiments, the scFv comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 20.

[0652] In some embodiments, the scFv comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 28.

[0653] In some embodiments, the scFv comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 36.In some embodiments, the scFv comprises a sequence as set forth in any one of SEQ ID NO: 20, SEQ ID NO: 28, and SEQ ID NO: 36.

[0654] In some embodiments, the scFv comprises a sequence as set forth in SEQ ID NO: 20.

[0655] In some embodiments, the scFv comprises a sequence as set forth in SEQ ID NO: 28.

[0656] In some embodiments, the scFv comprises a sequence as set forth in SEQ ID NO: 36.

[0657] In some embodiments, the scFv consists of a sequence as set forth in any one of SEQ ID NO: 20, SEQ ID NO: 28, and SEQ ID NO: 36.

[0658] In some embodiments, the scFv consists of a sequence as set forth in SEQ ID NO: 20. In some embodiments, the scFv consists of a sequence as set forth in SEQ ID NO: 28. In some embodiments, the scFv consists of a sequence as set forth in SEQ ID NO: 36.

[0659] The CD3-binding moiety according to the invention may have a high affinity for CD3. In particular, the CD3-binding moiety of the invention may have the ability to bind to a recombinant human CD3E / 5 heterodimer with an ECso of less than 75 ng / ml, less than 50 ng / ml, less than 40 ng / ml, less than 30 ng / ml, less than 25 ng / ml, less than 20 ng / ml, less than 15 ng / ml, less than 10 ng / ml or less than 5 ng / ml, such as 4-75 ng / ml, 4-50 ng / ml, 4-40 ng / ml, 4-30 ng / ml, 4-25 ng / ml, 4-20 ng / ml, 4-15 ng / ml, 4-10 ng / ml or 4-5 ng / ml. In some embodiments, the ECso for CD3E / 5 is 6-15 ng / ml.

[0660] Furthermore, the CD3-binding moiety of the invention may have the ability to bind to a recombinant human CD3E / Y heterodimer with an EC50 of less than 20 ng / ml, less than 18 ng / ml, less than 16 ng / ml, less than 14 ng / ml, less than 12 ng / ml, less than 10 ng / ml, less than 8 ng / ml, less than 7 ng / ml or less than 6 ng / ml, such as 4-20 ng / ml, 4-18 ng / ml, 4-16 ng / ml, 4-14 ng / ml, 4-12 ng / ml, 4-10 ng / ml, 4-8 ng / ml, 4-7 ng / ml or 4-6 ng / ml. In some embodiments, the EC50 for CD3E / Y is 5-14 ng / ml.

[0661] The CD3-binding moiety of the invention may have a high affinity for cells expressing CD3. In particular, the CD3-binding moiety of the invention may bind to cells genetically modified to express human CD3 protein on the cell surface with an EC50 of less than 15 ng / ml, of less than 14 ng / ml, of less than 13 ng / ml, of less than 12 ng / ml, less than 11 ng / ml, less than 10 ng / ml, less than 9 ng / ml or less than 8 ng / ml, such as 10-15 ng / ml, 1-15 ng / ml, 1-14 ng / ml, 1-13 ng / ml, 1-12 ng / ml, 1-11 ng / ml, 1-10 ng / ml, 1-9 ng / ml, 1-8 ng / ml, 3-12 ng / ml, 3-11 ng / ml, 3-10 ng / ml, 3-9 ng / ml, 3-8 ng / ml, 6-12 ng / ml, 6-11 ng / ml, 6-10 ng / ml, 6-9 ng / ml or 6-8 ng / ml. In some embodiments, the EC50 for is 10-15 ng / ml.

[0662] In some embodiments, the CD3-binding moiety binds to CD3E. In some embodiments, the CD3E is human CD3E. Surprisingly, the inventors have shown that the CD3-binding moiety of the invention is capable of binding to cells expressing only CD3 and not the TCR. In other words, the antigen binding proteins of the invention are capable of binding to CD3 independently of the TCR.

[0663] In some embodiments, the CD3-binding moiety or a fragment thereof is non-human.

[0664] In some embodiments, the CD3-binding moiety or a fragment thereof is humanised.

[0665] In some embodiments, the CD3-binding moiety or a fragment thereof is chimeric.

[0666] In some embodiments, the CD3-binding moiety or a fragment thereof is fully human.

[0667] Techniques for obtaining antibodies and fragments thereof are well-known in the art. By way of example, it is well-known that antibodies may be obtained by standard techniques comprising immunizing an animal with a target antigen and isolating antibodies from serum.

[0668] It will be understood that a fragment of an antibody comprises an amino acid sequence that is shorter than the full-length sequence of an antibody, but retains the biological activity (e.g., binding specificity and / or affinity) of the full-length antibody.

[0669] The term "humanised antibody" typically refers to a genetically engineered antibody generated in a non-human animal that has been engineered to reduce immunogenicity when used in a human whilst retaining antigen specificity. The humanised antibody typically contains human antibody constant domains and non-human variable domains that are modified to contain a high level of sequence homology to human variable domains.

[0670] Suitable assays and techniques for measuring / quantifying binding activity of CD3-binding moiety according to the invention may include, but are not limited to, ELISA, surface plasmon resonance (SPR), bio-layer interferometry (BLI), quartz crystal microbalance (QCM), bioluminescence assays and flow cytometry. Other suitable techniques will be known in the art.In particular, it will be understood that the ability of a binding molecule to bind to its cognate target may be assessed by flow cytometry, where the effectiveness of a binding molecule to bind to a cognate target expressed by a population of cells can be measured in a flow cytometry assay.

[0671] Moreover, it will be understood that EC50 is a measure of the concentration of a binding molecule that induces a specific response that is 50% between the maximum response and the baseline response. As such, EC50 can be used to assess the ability of a binding molecule to bind to a target.

[0672] Fc region

[0673] In some embodiments, the bispecific binding molecule further comprises an Fc (fragment crystallisable) region.

[0674] In some embodiments, the Fc region comprises a first subunit and a second subunit.

[0675] In some embodiments, each subunit of the Fc region independently comprises one or more immunoglobulin constant domain(s).

[0676] In some embodiments, each subunit of the Fc region independently comprises a heavy chain constant domain. In some embodiments, the heavy chain constant domain is a heavy chain IgG constant domain. In some embodiments, the heavy chain IgG constant domain is a heavy chain IgG 1 constant domain. In some embodiments, the heavy chain IgG constant domain is a heavy chain lgG2 constant domain. In some embodiments, the heavy chain IgG constant domain is a heavy chain lgG3 constant domain. In some embodiments, the heavy chain IgG constant domain is a heavy chain lgG4 constant domain.

[0677] In some embodiments, each subunit of the Fc region independently comprises a light chain constant domain. In some embodiments, the light chain constant domain is a light chain kappa constant domain. In some embodiments, the light chain constant domain is a light chain lambda constant domain.

[0678] In some embodiments, the, or each, immunoglobulin constant domain is independently selected from the list consisting of: a constant heavy 1 (CH1) domain, a constant heavy 2 (CH2) domain, and a constant heavy 3 (CH3) domain.In some embodiments, the first subunit of the Fc region comprises a CH2 domain. In some embodiments, the second subunit of the Fc region comprises a CH2 domain. In some embodiments, the first subunit of the Fc region and the second subunit of the Fc region each independently comprises a CH2 domain.

[0679] In some embodiments, the first subunit of the Fc region comprises a CH3 domain. In some embodiments, the second subunit of the Fc region comprises a CH3 domain. In some embodiments, the first subunit of the Fc region and the second subunit of the Fc region each independently comprises a CH3 domain.

[0680] In some embodiments, the first subunit of the Fc region comprises a CH2 domain and a CH3 domain. In some embodiments, the second subunit of the Fc region comprises a CH2 domain and a CH3 domain. In some embodiments, the first subunit of the Fc region and the second subunit of the Fc region each independently comprises a CH2 domain and a CH3 domain.

[0681] In some embodiments, a hinge region is part of the Fc region. In some embodiments, a hinge region is separate to the Fc region. In some embodiments, a hinge region comprises part of the Fc region.

[0682] In some embodiments, a hinge region is part of the CH2 domain. In some embodiments, a hinge region is separate to the CH2 domain. In some embodiments, a hinge region comprises part of the CH2 domain.

[0683] In some embodiments, the Fc region is linked to a hinge region. In some embodiments, the Fc region comprises a hinge region.

[0684] In some embodiments, the Fc region comprises a full hinge region. In some embodiments, the Fc region comprises a part of a hinge region.

[0685] It will be understood that a hinge region can be present between a CH1 domain and a CH2 domain. It will also be understood that part of a CH1 domain and part of a CH2 domain can together form the hinge region.

[0686] In some embodiments, the Fc region comprises (i) a hinge region, (ii) a CH2 domain, and (iii) a CH3 domain.In some embodiments, the Fc region comprises (i) part of a hinge region, (ii) a CH2 domain, and (iii) a CH3 domain.

[0687] In some embodiments, the Fc region comprises a CH2 domain and a CH3 domain, wherein a part of the CH2 domain is a part of a hinge region. In some embodiments, the Fc region comprises a CH2 domain and a CH3 domain, wherein an N-terminal part of the CH2 domain is a part of a hinge region.

[0688] In some embodiments, the hinge region is derived from lgG1 or lgG3. In some embodiments, the hinge region is derived from lgG1. In some embodiments, the hinge region is derived from lgG3.

[0689] In some embodiments, the hinge region is derived from an lgG1 hinge region or an lgG3 hinge region. In some embodiments, the hinge region is derived from an lgG1 hinge region. In some embodiments, the hinge region is derived from an lgG3 hinge region.

[0690] In some embodiments, the hinge region is an lgG1 hinge region or an lgG3 hinge region. In some embodiments, the hinge region is an I gG 1 hinge region. In some embodiments, the hinge region is an lgG3 hinge region.

[0691] In some embodiments, the first subunit of the Fc region and the second subunit of the Fc region each independently comprise a CH2 domain and a CH3 domain, wherein the Fc region is linked to a hinge region.

[0692] In some embodiments, the first subunit of the Fc region and the second subunit of the Fc region each independently comprise a CH2 domain and a CH3 domain, wherein the CH2 domain is linked to a hinge region.

[0693] In some embodiments, the first subunit of the Fc region and the second subunit of the Fc region each independently comprise a CH2 domain and a CH3 domain, wherein the CH2 domain comprises part of a hinge region.

[0694] It will be understood that the Fc region may interact with Fc receptors presented on the surface of a cell. The Fc receptors may be Fc gamma receptors.In some embodiments, the Fc region binds to one or more or all of the Fc receptors. In some embodiments, the Fc receptors comprise one or more or all of FcyRI (CD64), FcyRlla (CD32A), FcyRllb (CD32B), FcyRIII (CD16), C1q and FcRn.

[0695] In some embodiments, the Fc region is capable of interacting with Fc receptors and / or be able to recruit immune cells.

[0696] In some embodiments, the Fc region binds to one or more Fc receptors. In some embodiments, the Fc region binds to one or more Fc gamma receptors.

[0697] In some embodiments, the Fc region is capable of binding to immune cells. In some embodiments, the Fc domain is capable of recruiting immune cells.

[0698] In some embodiments, the immune cells are cytotoxic immune cells. In some embodiments, the immune cells are capable of killing target cells. In some embodiments, the cytotoxic immune cells are selected from the list consisting of: NK cells, macrophages, neutrophils and eosinophils.

[0699] In some embodiments, the Fc region interacts with proteins of the complement system. The proteins of the complement system may include C1q.

[0700] In some embodiments, the Fc region maintains the same Fc functionality as an antibody comprising the same Fc region. In some embodiments, Fc-mediated effector functions include antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular phagocytosis (ADCP).

[0701] However, in some embodiments the Fc region is silenced to reduce, negate or abolish one or more Fc receptor binding and / or functionalities. In some embodiments, the Fc region of the bispecific binding molecule according to the invention is modified to negate one or more Fc receptor functionalities. In some embodiments, the Fc region of the bispecific binding molecule according to the invention is silenced in respect of one or more or all of FcyRI (CD64), FcyRlla (CD32A), FcyRllb (CD32B), FcyRIII (CD16) and C1q functionality.

[0702] In some embodiments, the Fc region comprises one or more STR mutations.

[0703] In some embodiments, the Fc region comprises L234S, L235T, and G236R (STR; according to Ell numbering mutations.In some embodiments, the CH2 domain comprises one or more STR mutations.

[0704] In some embodiments, the CH2 domain comprises L234S, L235T, and G236R (STR; according to Ell numbering) mutations.

[0705] In some embodiments, the Fc region comprises one or more LALA mutations.

[0706] In some embodiments, the Fc region comprises L234A and L235A (according to Ell numbering) mutations.

[0707] In some embodiments, the CH2 domain comprises one or more LALA mutations.

[0708] In some embodiments, the CH2 domain comprises L234A and L235A (according to EU numbering) mutations.

[0709] It will be understood that the first subunit of the Fc region may be present on a first polypeptide.

[0710] It will also be understood that the second subunit of the Fc region may be present on a second polypeptide.

[0711] It will be understood that within the scope of the invention the first subunit and the second subunit may be the same or different, such that the subunits may independently possess any combination of the features defined herein.

[0712] In some embodiments, the first subunit and / or the second subunit independently comprise one or more modifications that enhance the formation of the bispecific binding molecule according to the invention. In some embodiments, the modifications comprise knobs-into-holes modifications.

[0713] In some embodiments, the first and second subunits comprise modifications that enhance the formation of a dimer, comprising one monomer of the first subunit of the Fc region as described herein and one monomer of the second subunit of the Fc region as described herein.

[0714] It will be understood that dimerisation of the first and second subunit of the Fc region as described herein may result in dimerisation of the first and second polypeptide as described herein, thus forming the bispecific binding molecule according to the invention.In some embodiments, the dimer is a heterodimer. In some embodiments, the dimer is a homodimer.

[0715] Challenges exist in producing bispecific antibody agents from a single composition (e.g., a composition comprising polyribonucleotides sufficient to encode multiple antibody agents), particularly because the random pairing of different antibody heavy and light chains can yield undesired antibody species. Due to the presence of mispaired byproducts, and significantly reduced production yields, sophisticated purification procedures are required to isolate the desired antibody agent in those situations (see e.g. Morrison, S.L., Nature Biotech. 25, 1233-1234, 2007, which is herein incorporated by reference). In general, the same problem of mispaired byproducts remains if recombinant expression techniques are used.

[0716] In some embodiments, the modifications can be those modifications which are widely known in the art of antibody technology for the purpose of (hetero)dimerisation between antibody chains (e.g. “knobs-into-holes” (KiH) modifications). In some embodiments, the modifications are present in the CH domains, e.g. the CH3 domains, of the first and second subunits.

[0717] In some embodiments, one or more of the CH domains of the first subunit of the Fc region as described herein and / or one or more of the CH domains of the second subunit of the Fc region as described herein comprise a modification that enhances the formation of a dimer comprising the first and second subunits of the Fc region.

[0718] In some embodiments wherein the first and second subunits of the Fc region each comprise more than one constant immunoglobulin domain, e.g. a CH2 domain and a CH3 domain, one or more or all of the CH domains may comprise such modifications.

[0719] In some embodiments, the modification is in the CH2 domain of the first and second subunits.

[0720] In some embodiments, the modification is in the CH3 domain of the first and second subunits.

[0721] As used herein, the term “enhances the formation” includes promoting dimerisation of the first and second subunits of the Fc region as described herein. Thus, in some embodiments, the modifications promote dimerisation of the first and second subunits of the Fc region as described herein, which in turn may promote dimerisation of the first and second polypeptide as described herein, thus forming the bispecific binding molecule according to the invention.In some embodiments, the modifications promote heterodimerisation of the first and second subunits of the Fc region as described herein. By heterodimerisation it is meant that the first subunit of the Fc region is different to the second subunit of the Fc region, and the first subunit of the Fc region will form a dimer with the second subunit of the Fc region, but the first subunit of the Fc region will not form (or is unlikely to form) a dimer with the first subunit of the Fc region, and the second subunit of the Fc region will not form (or is unlikely to form) a dimer with the second subunit of the Fc region.

[0722] It will be understood that alternative modifications may promote homodimerisation of the first and second subunits of the Fc region. By homodimerisation it is meant that the first subunit of the Fc region is the same as the second subunit of the Fc region, and the first subunit of the Fc region will form a dimer with the second subunit of the Fc region.

[0723] Various means of promoting dimerisation of two domains (e.g. homo- or hetero-dimerisation) are known in the art. In some embodiments, the two domains may comprise a modification that enhances formation of dimer, e.g. a modification that increases the affinity of the two domains, promotes and / or enables the formation of one or more disulphide bonds, reduces steric hindrance to the dimerisation, promotes electrostatic and / or hydrophilic / hydrophobic interactions between the two domains, or any combination thereof.

[0724] In some embodiments, one or more of the CH domains of the first subunit of the Fc region and / or one or more of the CH domains of the second subunit of the Fc region comprise a modification that enhances the affinity between the first and second subunits of the Fc region. In some embodiments, the modification enhances the affinity between the CH domains of the first and second subunits of the Fc region.

[0725] In some embodiments, one or more of the CH domains of the first subunit of the Fc region and / or one or more of the CH domains of the second subunit of the Fc region comprise a modification that reduces steric hindrance to the formation of the first and second polypeptide as described herein, thus forming the bispecific binding molecule according to the invention.

[0726] In some embodiments, the modification reduces steric hindrance between the CH domains of the first and second subunits of the Fc region.

[0727] In some embodiments, one or more of the CH domains of the first subunit of the Fc region and one or more of the CH domains of the second subunit of the Fc region comprise a ‘knobs-into-holes’ modification.In some embodiments, one or more of the CH domains of the first subunit of the Fc region and one or more of the CH domains of the second subunit of the Fc region are associated via a ‘knobs-into-holes’ modification.

[0728] In some embodiments, one or more of the CH domains of the first binding molecule and / or one or more of the CH domains of the second binding molecule comprise a modification that promotes electrostatic interactions between the first and second subunits of the Fc region. In some embodiments, the modification promotes electrostatic interactions between the CH domains of the first and second subunits of the Fc region. In some embodiments, the electrostatic interactions that are promoted are favourable to the formation of a dimer (e.g. a heterodimer) comprising the first and second subunit of the Fc region.

[0729] In some embodiments, one or more of the CH domains of the first subunit of the Fc region and / or one or more of the CH domains of the second subunit of the Fc region comprise a modification that promotes hydrophilic / hydrophobic interactions between the first and second subunits of the Fc region. In some embodiments, the modification promotes hydrophilic / hydrophobic interactions between the CH domains of the first and second subunits of the Fc region. In some embodiments, the hydrophilic / hydrophobic interactions that are promoted are favourable to the formation of a dimer comprising the first and second subunits of the Fc region.

[0730] In some embodiments, a CH domain of the first subunit of the Fc region forms a disulphide bond with a CH domain of the second subunit of the Fc region. In some embodiments, a CH domain of the first subunit of the Fc region forms a disulphide bond with a CH domain of the second subunit of the Fc region, and in addition one or more of the CH domains of the first subunit of the Fc region and / or one or more of the CH domains of the second subunit of the Fc region comprise a modification that enhances the affinity between the first and second subunits of the Fc region.

[0731] Any suitable technology for promoting the dimerisation of CH domains, i.e. of at least one of the CH domains of the first subunit of the Fc region and at least one of the CH domains of the second subunit of the Fc region, may be employed in the practice of the invention. The modification that enhances the formation of a dimer between the CH domains may be KiH technology (Ridgeway et al. , Protein Engineering, Design and Selection, 1996, 9: 617-621 and Merchant et al., Nat Biotechnol, 1998, 16: 677-681). The KiH technology is based on an engineered pair of CH domains that heterodimerises (CH heterodimer) in which asymmetrichydrophobic mutations are introduced between the homodimeric CH domain. KiH involves introducing mutations that create a protuberance (“knob”) in the interface of the first CH domain and a corresponding cavity (“hole”) in the interface of the second CH domain, such that the protuberance can be positioned in the cavity to promote heterodimer assembly and hinder homodimer formation. KiH variants therefore thermodynamically favour the formation of heterodimers rather than homodimers.

[0732] In some embodiments, one or more of the CH domains of the first subunit of the Fc region and one or more of the CH domains of the second subunit of the Fc region are associated via a KiH modification.

[0733] It will be understood that the CH2 domain of the first subunit of the Fc region and the CH2 domain of the second subunit of the Fc region may each comprise a ‘knobs-into-holes’ modification.

[0734] It will be understood that the CH3 domain of the first subunit of the Fc region and the CH3 domain of the second subunit of the Fc region may each comprise a ‘knobs-into-holes’ modification.

[0735] Any suitable ‘knobs-into-holes’ modifications known in the art may be employed in the practice of the present invention.

[0736] In some embodiments, the first subunit of the Fc region and the second subunit of the Fc region comprise a ‘knobs-into-holes’ modification.

[0737] In some embodiments, the CH2 domain of the first subunit of the Fc region and the CH2 domain of the second subunit of the Fc region comprise a ‘knobs-into-holes’ modification.

[0738] In some embodiments, the CH3 domain of the first subunit of the Fc region and the CH3 domain of the second subunit of the Fc region comprise a ‘knobs-into-holes’ modification.

[0739] It will be understood that the “Knob” modifications may comprise one or more mutations selected from the list consisting of: S354C, and T366W.

[0740] It will be understood that the “Hole” modifications may comprise one or more mutations selected from the list consisting of: Y349C,T366S,L368A, andY407V.In some embodiments, one subunit of the Fc region comprises one or more mutations selected from the list consisting of: Y349C, T366S, L368A, and Y407V (according to Ell numbering). In some embodiments, the subunit comprises each of the following mutations: Y349C, T366S, L368A, and Y407V (according to Ell numbering). Such a combination of mutations is referred to herein as “cah”.

[0741] In some embodiments, one subunit of the Fc region comprises one or more mutations selected from the list consisting of: S354C and T366W (according to Ell numbering). In some embodiments, the subunit comprises each of the following mutations: S354C and T366W (according to Ell numbering). Such a combination of CH3 mutations is referred to herein as “cak”.

[0742] In some embodiments, the CH3 domain of one subunit of the Fc region comprises one or more mutations selected from the list consisting of: Y349C, T366S, L368A, and Y407V (according to Ell numbering). In some embodiments, the CH3 domain comprises each of the following mutations: Y349C, T366S, L368A, and Y407V (according to Ell numbering). Such a combination of mutations is referred to herein as “cah”.

[0743] In some embodiments, the CH3 domain of the one subunit of the Fc region comprises one or more mutations selected from the list consisting of: S354C and T366W (according to Ell numbering). In some embodiments, the CH3 domain comprises each of the following mutations: S354C and T366W (according to Ell numbering). Such a combination of CH3 mutations is referred to herein as “cak”.

[0744] In some embodiments, the CH3 domain of the first subunit of the Fc region comprises one or more mutations selected from the list consisting of: Y349C, T366S, L368A, and Y407V (according to Ell numbering). In some embodiments, the CH3 domain comprises each of the following mutations: Y349C, T366S, L368A, and Y407V (according to Ell numbering). Such a combination of mutations is referred to herein as “cah”.

[0745] In some embodiments, the CH3 domain of the second subunit of the Fc region comprises one or more mutations selected from the list consisting of: S354C and T366W (according to Ell numbering). In some embodiments, the CH3 domain comprises each of the following mutations: S354C and T366W (according to Ell numbering). Such a combination of CH3 mutations is referred to herein as “cak”.In some embodiments, the CH3 domain of the second subunit of the Fc region comprises one or more mutations selected from the list consisting of: Y349C, T366S, L368A, and Y407V (according to Ell numbering). In some embodiments, the CH3 domain comprises each of the following mutations: Y349C, T366S, L368A, and Y407V (according to Ell numbering). Such a combination of mutations is referred to herein as “cah”.

[0746] In some embodiments, the CH3 domain of the first subunit of the Fc region comprises one or more mutations selected from the list consisting of: S354C and T366W (according to Ell numbering). In some embodiments, the CH3 domain comprises each of the following mutations: S354C and T366W (according to Ell numbering). Such a combination of CH3 mutations is referred to herein as “cak”.

[0747] In some embodiments, the first subunit of the Fc region comprises knob mutations and Fc-silencing LALA mutations.

[0748] In some embodiments, the second subunit of the Fc region comprises hole mutations and Fc-silencing LALA mutations.

[0749] In some embodiments, the first subunit of the Fc region comprises knob mutations and Fc-silencing LALA mutations, and the second subunit of the Fc region comprises hole mutations and Fc-silencing LALA mutations.

[0750] In some embodiments, the first subunit of the Fc region comprises hole mutations and Fc-silencing LALA mutations.

[0751] In some embodiments, the second subunit of the Fc region comprises knob mutations and Fc-silencing LALA mutations.

[0752] In some embodiments, the first subunit of the Fc region comprises hole mutations and Fc-silencing LALA mutations, and the second subunit of the Fc region comprises knob mutations and Fc-silencing LALA mutations.Full-length molecule

[0753] In some embodiments, the bispecific binding molecule comprises:

[0754] (i) a first polypeptide comprising the following from N to C terminus:

[0755] CA19-9-VH domain - CA19-9-CH1 domain - optional linker - CD3-VH domain - linker - CD3-VL domain - optional linker - first subunit of the Fc region as described herein;

[0756] (ii) a second polypeptide comprising the following from N to C terminus:

[0757] CA19-9-VH domain - CA19-9-CH1 domain - second subunit of the Fc region as described herein; and

[0758] (iii) a third polypeptide comprising the following from N to C terminus:

[0759] CA19-9-VL domain - CA19-9-CL domain.

[0760] In some embodiments, the bispecific binding molecule comprises:

[0761] (i) a first polypeptide comprising the following from N to C terminus:

[0762] CA19-9-VH domain - CA19-9-CH1 domain - optional linker- CD3-VL domain - linker - CD3-VH domain - optional linker - first subunit of the Fc region as described herein;

[0763] (ii) a second polypeptide comprising the following from N to C terminus:

[0764] CA19-9-VH domain - CA19-9-CH1 domain - second subunit of the Fc region as described herein; and

[0765] (iii) a third polypeptide comprising the following from N to C terminus:

[0766] CA19-9-VL domain - CA19-9-CL domain.

[0767] It will be understood that the hinge region may be formed from part of the CH1 domain and part of the Fc region described herein.

[0768] It will be also understood that the hinge region may be considered as separate to the CH1 domain and the Fc region described herein.

[0769] Accordingly, in some embodiments, the bispecific binding molecule comprises:

[0770] (i) a first polypeptide comprising the following from N to C terminus:

[0771] CA19-9-VH domain - CA19-9-CH1 domain - optional linker - CD3-VH domain - linker - CD3-VL domain - optional linker - hinge region - first subunit of the Fc region as described herein;

[0772] (ii) a second polypeptide comprising the following from N to C terminus:CA19-9-VH domain - CA19-9-CH1 domain - hinge region - second subunit of the Fc region as described herein; and

[0773] (iii) a third polypeptide comprising the following from N to C terminus:

[0774] CA19-9-VL domain - CA19-9-CL domain.

[0775] Accordingly, in some embodiments, the bispecific binding molecule comprises:

[0776] (i) a first polypeptide comprising the following from N to C terminus:

[0777] CA19-9-VH domain - CA19-9-CH1 domain - optional linker- CD3-VL domain - linker - CD3-VH domain - optional linker - hinge region - first subunit of the Fc region as described herein;

[0778] (ii) a second polypeptide comprising the following from N to C terminus:

[0779] CA19-9-VH domain - CA19-9-CH1 domain - hinge region - second subunit of the Fc region as described herein; and

[0780] (iii) a third polypeptide comprising the following from N to C terminus:

[0781] CA19-9-VL domain - CA19-9-CL domain.

[0782] In some embodiments, the first subunit of the Fc region comprises a CH2 domain as described herein and a CH3 domain as described herein.

[0783] In some embodiments, the second subunit of the Fc region comprises a CH2 domain as described herein and a CH3 domain as described herein.

[0784] Accordingly, in some embodiments, the bispecific binding molecule comprises:

[0785] (i) a first polypeptide comprising the following from N to C terminus:

[0786] CA19-9-VH domain - CA19-9-CH1 domain - optional linker - CD3-VH domain - linker - CD3-VL domain - optional linker - CH2 domain - CH3 domain; (ii) a second polypeptide comprising the following from N to C terminus:

[0787] CA19-9-VH domain - CA19-9-CH1 domain - CH2 domain - CH3 domain; and (iii) a third polypeptide comprising the following from N to C terminus:

[0788] CA19-9-VL domain - CA19-9-CL domain.

[0789] Accordingly, in some embodiments, the bispecific binding molecule comprises:

[0790] (i) a first polypeptide comprising the following from N to C terminus:

[0791] CA19-9-VH domain - CA19-9-CH1 domain - optional linker- CD3-VL domain - linker - CD3-VH domain - optional linker - CH2 domain - CH3 domain; (ii) a second polypeptide comprising the following from N to C terminus:

[0792] CA19-9-VH domain - CA19-9-CH1 domain - CH2 domain - CH3 domain; and(iii) a third polypeptide comprising the following from N to C terminus:

[0793] CA19-9-VL domain - CA19-9-CL domain.

[0794] It will be understood that the hinge region may be formed from part of the CH1 domain and part of the CH2 domain described herein.

[0795] It will be also understood that the hinge region may be considered as separate to the CH1 domain and the CH2 domain described herein.

[0796] Accordingly, in some embodiments, the bispecific binding molecule comprises:

[0797] (i) a first polypeptide comprising the following from N to C terminus:

[0798] CA19-9-VH domain - CA19-9-CH1 domain - optional linker - CD3-VH domain - linker - CD3-VL domain - optional linker - hinge region - CH2 domain - CH3 domain;

[0799] (ii) a second polypeptide comprising the following from N to C terminus:

[0800] CA19-9-VH domain -CA19-9-CH1 domain-hinge region-CH2 domain-CH3 domain; and

[0801] (iii) a third polypeptide comprising the following from N to C terminus:

[0802] CA19-9-VL domain - CA19-9-CL domain.

[0803] Accordingly, in some embodiments, the bispecific binding molecule comprises:

[0804] (i) a first polypeptide comprising the following from N to C terminus:

[0805] CA19-9-VH domain - CA19-9-CH1 domain - optional linker- CD3-VL domain - linker- CD3-VH domain - optional linker- hinge region - CH2 domain - CH3 domain;

[0806] (ii) a second polypeptide comprising the following from N to C terminus:

[0807] CA19-9-VH domain -CA19-9-CH1 domain-hinge region-CH2 domain-CH3 domain; and

[0808] (iii) a third polypeptide comprising the following from N to C terminus:

[0809] CA19-9-VL domain - CA19-9-CL domain.

[0810] In some embodiments, the first polypeptide comprises knob mutations and Fc-silencing LALA mutations.

[0811] In some embodiments, the second polypeptide comprises a hole mutations and Fc-silencing LALA mutations.In some embodiments, the first polypeptide comprises knob mutations and Fc-silencing LALA mutations, and the second polypeptide comprises a hole mutations and Fc-silencing LALA mutations.

[0812] In some embodiments, the first polypeptide comprises a hole mutations and Fc-silencing LALA mutations.

[0813] In some embodiments, the second polypeptide comprises knob mutations and Fc-silencing LALA mutations.

[0814] In some embodiments, the first polypeptide comprises a hole mutations and Fc-silencing LALA mutations, and the second polypeptide comprises knob mutations and Fc-silencing LALA mutations.

[0815] It will be understood that the bispecific binding molecule described herein may comprise two copies of the third polypeptide according to part (iii) described above. Indeed, the third polypeptide is the CA19-9 light chain (i.e. the polypeptide comprising the CA19-9-VL domain and the CA19-9-CL domain). As such, it will be understood that two copies of the third polypeptide contribute to the 2+1 Fab(2)-scFv-Fc bispecific binding molecule structure described herein (i.e. as depicted in Figure 1).

[0816] In some embodiments, the first polypeptide comprises the amino acid sequence according to SEQ ID NO: 66 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 66. In some embodiments, the first polypeptide consists of an amino acid sequence according to SEQ ID NO: 66.

[0817] In some embodiments, the first polypeptide comprises the amino acid sequence according to SEQ ID NO: 70 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70. In some embodiments, the first polypeptide consists of an amino acid sequence according to SEQ ID NO: 70.

[0818] In some embodiments, the first polypeptide comprises the amino acid sequence according to SEQ ID NO: 74 or a variant having at least 80% sequence identity thereto. In someembodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74. In some embodiments, the first polypeptide consists of an amino acid sequence according to SEQ ID NO: 74.

[0819] In some embodiments, the first polypeptide comprises the amino acid sequence according to SEQ ID NO: 68 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 68. In some embodiments, the first polypeptide consists of an amino acid sequence according to SEQ ID NO: 68.

[0820] In some embodiments, the first polypeptide comprises the amino acid sequence according to SEQ ID NO: 72 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72. In some embodiments, the first polypeptide consists of an amino acid sequence according to SEQ ID NO: 72.

[0821] In some embodiments, the first polypeptide comprises the amino acid sequence according to SEQ ID NO: 76 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76. In some embodiments, the first polypeptide consists of an amino acid sequence according to SEQ ID NO: 76.

[0822] In some embodiments, the second polypeptide comprises the amino acid sequence according to SEQ ID NO: 64 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 64. In some embodiments, the second polypeptide consists of an amino acid sequence according to SEQ ID NO: 64.

[0823] In some embodiments, the third polypeptide comprises a third polypeptide comprising the amino acid sequence according to SEQ ID NO: 78 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity toSEQ ID NO: 78. In some embodiments, the third polypeptide consists of an amino acid sequence according to SEQ ID NO: 78.

[0824] In some embodiments, the bispecific binding molecule comprises:

[0825] (i) a first polypeptide comprising the amino acid sequence according to SEQ ID NO: 66 or a variant having at least 80% sequence identity thereto;

[0826] (ii) a second polypeptide comprising the amino acid sequence according to SEQ ID NO: 64 or a variant having at least 80% sequence identity thereto; and

[0827] (iii) a third polypeptide comprising the amino acid sequence according to SEQ ID NO: 78 or a variant having at least 80% sequence identity thereto.

[0828] In some embodiments, each variant independently has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0829] In some embodiments, the bispecific binding molecule comprises:

[0830] (i) a first polypeptide comprising the amino acid sequence according to SEQ ID NO: 66;

[0831] (ii) a second polypeptide comprising the amino acid sequence according to SEQ ID NO: 64; and

[0832] (iii) a third polypeptide comprising the amino acid sequence according to SEQ ID NO: 78.

[0833] In some embodiments, the bispecific binding molecule comprises:

[0834] (i) a first polypeptide comprising the amino acid sequence according to SEQ ID NO: 70 or a variant having at least 80% sequence identity thereto;

[0835] (ii) a second polypeptide comprising the amino acid sequence according to SEQ ID NO: 64 or a variant having at least 80% sequence identity thereto; and

[0836] (iii) a third polypeptide comprising the amino acid sequence according to SEQ ID NO: 78 or a variant having at least 80% sequence identity thereto.

[0837] In some embodiments, each variant independently has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0838] In some embodiments, the bispecific binding molecule comprises:

[0839] (i) a first polypeptide comprising the amino acid sequence according to SEQ ID NO: 70;(ii) a second polypeptide comprising the amino acid sequence according to SEQ ID NO: 64; and

[0840] (iii) a third polypeptide comprising the amino acid sequence according to SEQ ID NO: 78.

[0841] In some embodiments, the bispecific binding molecule comprises:

[0842] (i) a first polypeptide comprising the amino acid sequence according to SEQ ID NO: 74 or a variant having at least 80% sequence identity thereto;

[0843] (ii) a second polypeptide comprising the amino acid sequence according to SEQ ID NO: 64 or a variant having at least 80% sequence identity thereto; and

[0844] (iii) a third polypeptide comprising the amino acid sequence according to SEQ ID NO: 78 or a variant having at least 80% sequence identity thereto.

[0845] In some embodiments, each variant independently has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0846] In some embodiments, the bispecific binding molecule comprises:

[0847] (i) a first polypeptide comprising the amino acid sequence according to SEQ ID NO: 74;

[0848] (ii) a second polypeptide comprising the amino acid sequence according to SEQ ID NO: 64; and

[0849] (iii) a third polypeptide comprising the amino acid sequence according to SEQ ID NO: 78.

[0850] In some embodiments, the bispecific binding molecule comprises:

[0851] (i) a first polypeptide comprising the amino acid sequence according to SEQ ID NO: 68 or a variant having at least 80% sequence identity thereto;

[0852] (ii) a second polypeptide comprising the amino acid sequence according to SEQ ID NO: 64 or a variant having at least 80% sequence identity thereto; and

[0853] (iii) a third polypeptide comprising the amino acid sequence according to SEQ ID NO: 78 or a variant having at least 80% sequence identity thereto.

[0854] In some embodiments, each variant independently has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0855] In some embodiments, the bispecific binding molecule comprises:(i) a first polypeptide comprising the amino acid sequence according to SEQ ID NO: 68;

[0856] (ii) a second polypeptide comprising the amino acid sequence according to SEQ ID NO: 64; and

[0857] (iii) a third polypeptide comprising the amino acid sequence according to SEQ ID NO: 78.

[0858] In some embodiments, the bispecific binding molecule comprises:

[0859] (i) a first polypeptide comprising the amino acid sequence according to SEQ ID NO: 72 or a variant having at least 80% sequence identity thereto;

[0860] (ii) a second polypeptide comprising the amino acid sequence according to SEQ ID NO: 64 or a variant having at least 80% sequence identity thereto; and

[0861] (iii) a third polypeptide comprising the amino acid sequence according to SEQ ID NO: 78 or a variant having at least 80% sequence identity thereto.

[0862] In some embodiments, each variant independently has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0863] In some embodiments, the bispecific binding molecule comprises:

[0864] (i) a first polypeptide comprising the amino acid sequence according to SEQ ID NO: 72;

[0865] (ii) a second polypeptide comprising the amino acid sequence according to SEQ ID NO: 64; and

[0866] (iii) a third polypeptide comprising the amino acid sequence according to SEQ ID NO: 78.

[0867] In some embodiments, the bispecific binding molecule comprises:

[0868] (i) a first polypeptide comprising the amino acid sequence according to SEQ ID NO: 76 or a variant having at least 80% sequence identity thereto;

[0869] (ii) a second polypeptide comprising the amino acid sequence according to SEQ ID NO: 64 or a variant having at least 80% sequence identity thereto; and

[0870] (iii) a third polypeptide comprising the amino acid sequence according to SEQ ID NO: 78 or a variant having at least 80% sequence identity thereto.

[0871] In some embodiments, each variant independently has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.In some embodiments, the bispecific binding molecule comprises:

[0872] (i) a first polypeptide comprising the amino acid sequence according to SEQ ID NO: 76;

[0873] (ii) a second polypeptide comprising the amino acid sequence according to SEQ ID NO: 64; and

[0874] (iii) a third polypeptide comprising the amino acid sequence according to SEQ ID NO: 78.

[0875] POLYNUCLEOTIDE

[0876] The present invention provides a polynucleotide comprising a nucleic acid sequence encoding the bispecific binding molecule according to the invention.

[0877] In some embodiments, the polynucleotide comprises a nucleic acid sequence(s) as shown in Table 1.

[0878] The present invention provides one or more polynucleotide(s) comprising a nucleic acid sequence encoding the bispecific binding molecule according to the invention.

[0879] In some embodiments, the one or more polynucleotide(s) comprises one or more nucleic acid sequence(s) as shown in Table 1.

[0880] As used herein, the terms “nucleotide sequence”, and “nucleic acid sequence” are intended to be synonymous with each other. The nucleic acid sequence may be a DNA or an RNA sequence. The nucleic acid sequence may be single-stranded or may be double-stranded. The nucleic acid sequence may be, for example, genomic, recombinant, cDNA or mRNA. The nucleic acid sequence may comprise synthetic nucleotides and / or modified nucleotides. These synthetic nucleotides and / or modified nucleotides may enhance the in vivo activity and / or stability of the polynucleotide.

[0881] In some embodiments, the, or each, nucleic acid sequence is independently a DNA sequence.

[0882] In some embodiments, the, or each, nucleic acid sequence is independently a cDNA sequence.

[0883] In some embodiments, the, or each, nucleic acid sequence is independently an RNA sequence.In some embodiments, the, or each, nucleic acid sequence is independently an mRNA sequence.

[0884] In some embodiments, the, or each RNA sequence is independently in vitro transcribed RNA. Processes for producing in vitro transcribed RNA are known in the art. For example, CleanCap 413 ((m7(3'OMeG)(5')ppp(5')(2'OMeA)pG)-capped RNA can be produced following the process as disclosed, e.g., in Kreiter, S. et al. Cancer Immunol. Immunother. 56, 1577-87 (2007) and WO 2021214204A1, each of which is incorporated herein by reference in its entirety. Methyl pseudo-uridine can be used in the in vitro transcription reaction and incorporated into the produced RNA.

[0885] In some embodiments, the, or each RNA sequence is independently modRNA (N1-methyl-pseudouridine).

[0886] In some embodiments, the modRNA contains N1-methyl-pseudouridine instead of uridine. In some embodiments, an additional purification step may be applied for modRNA to reduce double stranded RNA contaminants generated during the in vitro transcription reaction.

[0887] In some embodiments, the nucleic acid sequence(s) contain one or more structural elements optimized for maximal efficacy of the RNA or mRNA with respect to stability and translational efficiency, wherein the structural elements are selected from the list consisting of: a 5'-cap, a 5'-UTR, a 3'-UTR, and a poly(A)-tail.

[0888] In some embodiments, the nucleic acid sequence(s) comprise a 5'-cap, a 5'-UTR, a 3'-UTR, and a poly(A)-tail.

[0889] In some embodiments, the, or each RNA sequence independently comprises a 5'-cap.

[0890] In some embodiments, the 5'-cap is CC413. It will be understood that CC413 may be defined as CleanCap® Reagent AG (3' OMe) - (N-7413), which has the molecular formula: C33H45N15O24P4 (free acid).

[0891] In some embodiments, the 5'-cap is m27’3'oGppp(mi2'°)ApG, which comprises the following structure:

[0892]

[0893] OH OH

[0894] In some embodiments, the, or each RNA sequence independently comprises a poly adenine sequence (poly(A)-tail).

[0895] In some embodiments, the poly(A) tail is about 10 to about 250 nucleotides in length.

[0896] In some embodiments, the poly(A) tail is about 50 to about 200 nucleotides in length.

[0897] In some embodiments, the poly(A) tail is about 50 to about 100 nucleotides in length.

[0898] In some embodiments, the poly(A) tail is about 100 to about 200 nucleotides in length.

[0899] In some embodiments, the poly(A) tail is about 100 to about 150 nucleotides in length.

[0900] In some embodiments, the poly(A) tail is about 100 to about 120 nucleotides in length.

[0901] In some embodiments, the poly(A) tail is poly A30LA70.

[0902] In some embodiments, the poly(A) tail comprises the nucleic acid sequence according to SEQ ID NO: 84. In some embodiments, the poly(A) tail comprises a variant having at least 80% sequence identity to SEQ ID NO: 84. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 84. In some embodiments, the poly(A) tail consists of the nucleic acid sequence according to SEQ ID NO: 84.

[0903] In some embodiments, the, or each RNA sequence independently comprises a 5’IITR.

[0904] In some embodiments, the 5’IITR comprises the nucleic acid sequence according to SEQ ID NO: 82. In some embodiments, the 5’IITR comprises a variant having at least 80% sequenceidentity to SEQ ID NO: 82. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 82. In some embodiments, the 5’IITR consists of the nucleic acid sequence according to SEQ ID NO: 82.

[0905] In some embodiments, the, or each RNA sequence independently comprises a 3’IITR

[0906] In some embodiments, the 3’IITR comprises the nucleic acid sequence according to SEQ ID NO: 83. In some embodiments, the 3’IITR comprises a variant having at least 80% sequence identity to SEQ ID NO: 83. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 83. In some embodiments, the 3’IITR consists of the nucleic acid sequence according to SEQ ID NO: 83.

[0907] In some embodiments, the nucleic acid sequence(s) comprises a Kozak sequence.

[0908] In some embodiments, the nucleic acid sequence(s) comprises a signal peptide sequence (e.g., husec2).

[0909] In some embodiments, one polynucleotide comprises a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[0910] CA19-9-VH domain - CA19-9-CH1 domain - optional linker - CD3-VH domain - linker - CD3-VL domain - optional linker - CH2 domain - CH3 domain

[0911] In some embodiments, one polynucleotide comprises a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[0912] CA19-9-VH domain - CA19-9-CH1 domain - optional linker- CD3-VL domain - linker - CD3-VH domain - optional linker - CH2 domain - CH3 domain

[0913] In some embodiments, one polynucleotide comprises a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[0914] CA19-9-VH domain - CA19-9-CH1 domain - CH2 domain - CH3 domain

[0915] In some embodiments, one polynucleotide comprises a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:CA19-9-VH domain - CA19-9-CH1 domain - optional linker - CD3-VH domain - linker - CD3-VL domain - optional linker - hinge region - CH2 domain - CH3 domain

[0916] In some embodiments, one polynucleotide comprises a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[0917] CA19-9-VH domain - CA19-9-CH1 domain - optional linker- CD3-VL domain - linker- CD3-VH domain - optional linker- hinge region - CH2 domain - CH3 domain

[0918] In some embodiments, one polynucleotide comprises a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[0919] CA19-9-VH domain -CA19-9-CH1 domain-hinge region-CH2 domain-CH3 domain

[0920] In some embodiments, one polynucleotide comprises a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[0921] CA19-9-VL domain - CA19-9-CL domain

[0922] It will be understood that the CA19-9-VH domain, CA19-9-CH1 domain, CA19-9-VL domain, CA19-9-CL domain, CD3-VH domain, linker, CD3-VL domain, hinge region, CH2 domain, and CH3 domain may be defined as described herein.

[0923] In some embodiments, one polynucleotide comprises a nucleic acid sequence comprising in 5’-3’ orientation:

[0924] 5’-cap - 5’IITR - CA19-9-VH domain - CA19-9-CH1 domain - optional linker - CD3- VH domain - linker - CD3-VL domain - optional linker - CH2 domain - CH3 domain - 3’IITR - Poly(A)

[0925] In some embodiments, one polynucleotide comprises a nucleic acid sequence comprising in 5’-3’ orientation:

[0926] 5’-cap - 5’IITR - CA19-9-VH domain - CA19-9-CH1 domain - optional linker - CD3- VL domain - linker - CD3-VH domain - optional linker - CH2 domain - CH3 domain - 3’IITR - Poly(A)

[0927] In some embodiments, one polynucleotide comprises a nucleic acid sequence comprising in 5’-3’ orientation:5’-cap - 5’UTR - CA19-9-VH domain - CA19-9-CH1 domain - CH2 domain - CH3 domain - 3’UTR - Poly(A)

[0928] In some embodiments, one polynucleotide comprises a nucleic acid sequence comprising in 5’-3’ orientation:

[0929] 5’-cap - 5’UTR - CA19-9-VH domain - CA19-9-CH1 domain - optional linker - CD3- VH domain - linker - CD3-VL domain - optional linker - hinge region - CH2 domain - CH3 domain - 3’UTR - Poly(A)

[0930] In some embodiments, one polynucleotide comprises a nucleic acid sequence comprising in 5’-3’ orientation:

[0931] 5’-cap - 5’IITR - CA19-9-VH domain - CA19-9-CH1 domain - optional linker - CD3- VL domain - linker - CD3-VH domain - optional linker - hinge region - CH2 domain - CH3 domain - 3’UTR - Poly(A)

[0932] In some embodiments, one polynucleotide comprises a nucleic acid sequence comprising in 5’-3’ orientation:

[0933] 5’-cap - 5’UTR - CA19-9-VH domain - CA19-9-CH1 domain - hinge region - CH2 domain - CH3 domain - 3’UTR - Poly(A)

[0934] In some embodiments, one polynucleotide comprises a nucleic acid sequence comprising in 5’-3’ orientation:

[0935] 5’-cap - 5’UTR - CA19-9-VL domain - CA19-9-CL domain - 3’UTR - Poly(A)

[0936] It will be understood that the CA19-9-VH domain, CA19-9-CH1 domain, CA19-9-VL domain, CA19-9-CL domain, CD3-VH domain, linker, CD3-VL domain, hinge region, CH2 domain, and CH3 domain may be defined as described herein.

[0937] It will be understood that the 5’-cap, the 5’UTR, the 3’UTR and the Poly(A) tail may be defined as described herein.

[0938] The present invention provides a plurality of polynucleotides encoding the bispecific binding molecule according to the invention, wherein the plurality of polynucleotides independently comprise a nucleic acid sequence encoding part of the bispecific binding molecule according to the invention.In other words, the bispecific binding molecule according to the invention may be encoded by a plurality of polynucleotides, such that expression of the plurality of polynucleotides results in polypeptides that form the bispecific binding molecule according to the invention.

[0939] In some embodiments, a plurality of polynucleotides may comprise:

[0940] (i) a first polynucleotide comprising a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[0941] CA19-9-VH domain - CA19-9-CH1 domain - optional linker - CD3-VH domain - linker - CD3-VL domain - optional linker - CH2 domain - CH3 domain; (ii) a second polynucleotide comprising a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[0942] CA19-9-VH domain - CA19-9-CH1 domain - CH2 domain - CH3 domain; and (iii) a third polynucleotide comprising a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[0943] CA19-9-VL domain - CA19-9-CL domain.

[0944] In some embodiments, a plurality of polynucleotides may comprise:

[0945] (i) a first polynucleotide comprising a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[0946] CA19-9-VH domain - CA19-9-CH1 domain - optional linker- CD3-VL domain - linker - CD3-VH domain - optional linker - CH2 domain - CH3 domain; (ii) a second polynucleotide comprising a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[0947] CA19-9-VH domain - CA19-9-CH1 domain - CH2 domain - CH3 domain; and (iii) a third polynucleotide comprising a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[0948] CA19-9-VL domain - CA19-9-CL domain.

[0949] In some embodiments, a plurality of polynucleotides may comprise:

[0950] (i) a first polynucleotide comprising a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[0951] CA19-9-VH domain - CA19-9-CH1 domain - optional linker - CD3-VH domain - linker - CD3-VL domain - optional linker - hinge region - CH2 domain - CH3 domain;

[0952] (ii) a second polynucleotide comprising a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:CA19-9-VH domain -CA19-9-CH1 domain-hinge region-CH2 domain-CH3 domain; and

[0953] (iii) a third polynucleotide comprising a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[0954] CA19-9-VL domain - CA19-9-CL domain.

[0955] In some embodiments, a plurality of polynucleotides may comprise:

[0956] (i) a first polynucleotide comprising a nucleic acid sequence encoding following polypeptide chain from N to C terminus:

[0957] CA19-9-VH domain - CA19-9-CH1 domain - optional linker- CD3-VL domain - linker- CD3-VH domain - optional linker- hinge region - CH2 domain - CH3 domain;

[0958] (ii) a second polynucleotide comprising a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[0959] CA19-9-VH domain -CA19-9-CH1 domain-hinge region-CH2 domain-CH3 domain; and

[0960] (iii) a third polynucleotide comprising a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[0961] CA19-9-VL domain - CA19-9-CL domain.

[0962] It will be understood that the CA19-9-VH domain, CA19-9-CH1 domain, CA19-9-VL domain, CA19-9-CL domain, CD3-VH domain, linker, CD3-VL domain, hinge region, CH2 domain, and CH3 domain may be defined as described herein.

[0963] In some embodiments, the plurality of polynucleotides may comprise one or more copies of the third polynucleotide according to part (iii) above.

[0964] In some embodiments, the plurality of polynucleotides may comprise two copies of the third polynucleotide according to part (iii) above.

[0965] In some embodiments, a plurality of polynucleotides may comprise:

[0966] (i) a first polynucleotide comprising a nucleic acid sequence comprising in 5’-3’ orientation:

[0967] 5’-cap - 5’IITR - CA19-9-VH domain - CA19-9-CH1 domain - optional linker — CD3-VH domain - linker - CD3-VL domain - optional linker - CH2 domain - CH3 domain - 3’UTR - Poly(A)(ii) a second polynucleotide comprising a nucleic acid sequence comprising in 5’-3’ orientation:

[0968] 5’-cap - 5’UTR - CA19-9-VH domain - CA19-9-CH1 domain - CH2 domain - CH3 domain - 3’UTR - Poly(A)

[0969] (iii) a third polynucleotide comprising a nucleic acid sequence comprising in 5’-3’ orientation:

[0970] 5’-cap - 5’UTR - CA19-9-VL domain - CA19-9-CL domain - 3’UTR - Poly(A)

[0971] In some embodiments, a plurality of polynucleotides may comprise:

[0972] (i) a first polynucleotide comprising a nucleic acid sequence comprising in 5’-3’ orientation:

[0973] 5’-cap - 5’UTR - CA19-9-VH domain - CA19-9-CH1 domain - optional linker — CD3-VL domain - linker - CD3-VH domain - optional linker - CH2 domain - CH3 domain - 3’UTR - Poly(A)

[0974] (ii) a second polynucleotide comprising a nucleic acid sequence comprising in 5’-3’ orientation:

[0975] 5’-cap - 5’UTR - CA19-9-VH domain - CA19-9-CH1 domain - CH2 domain - CH3 domain - 3’UTR - Poly(A)

[0976] (iii) a third polynucleotide comprising a nucleic acid sequence comprising in 5’-3’ orientation:

[0977] 5’-cap - 5’UTR - CA19-9-VL domain - CA19-9-CL domain - 3’UTR - Poly(A)

[0978] In some embodiments, a plurality of polynucleotides may comprise:

[0979] (i) a first polynucleotide comprising a nucleic acid sequence comprising in 5’-3’ orientation:

[0980] 5’-cap - 5’UTR - CA19-9-VH domain - CA19-9-CH1 domain - optional linker — CD3-VH domain - linker - CD3-VL domain - optional linker - hinge region - CH2 domain - CH3 domain - 3’UTR - Poly(A)

[0981] (ii) a second polynucleotide comprising a nucleic acid sequence comprising in 5’-3’ orientation:

[0982] 5’-cap - 5’UTR - CA19-9-VH domain - CA19-9-CH1 domain - hinge region - CH2 domain - CH3 domain - 3’UTR - Poly(A)

[0983] (iii) a third polynucleotide comprising a nucleic acid sequence comprising in 5’-3’ orientation:

[0984] 5’-cap - 5’UTR - CA19-9-VL domain - CA19-9-CL domain - 3’UTR - Poly(A)In some embodiments, a plurality of polynucleotides may comprise:

[0985] (i) a first polynucleotide comprising a nucleic acid sequence comprising in 5’-3’ orientation:

[0986] 5’-cap - 5’UTR - CA19-9-VH domain - CA19-9-CH1 domain - optional linker — CD3-VL domain - linker - CD3-VH domain - optional linker - hinge region - CH2 domain - CH3 domain - 3’UTR - Poly(A)

[0987] (ii) a second polynucleotide comprising a nucleic acid sequence comprising in 5’-3’ orientation:

[0988] 5’-cap - 5’IITR - CA19-9-VH domain - CA19-9-CH1 domain - hinge region - CH2 domain - CH3 domain - 3’IITR - Poly(A)

[0989] (iii) a third polynucleotide comprising a nucleic acid sequence comprising in 5’-3’ orientation:

[0990] 5’-cap - 5’UTR - CA19-9-VL domain - CA19-9-CL domain - 3’UTR - Poly(A)

[0991] It will be understood that the CA19-9-VH domain, CA19-9-CH1 domain, CA19-9-VL domain, CA19-9-CL domain, CD3-VH domain, linker, CD3-VL domain, hinge region, CH2 domain, and CH3 domain may be defined as described herein.

[0992] It will be understood that the 5’-cap, the 5’IITR, the 3’UTR and the Poly(A) tail may be defined as described above.

[0993] In some embodiments, the plurality of polynucleotides may comprise one or more copies of the third polynucleotide according to part (iii) above.

[0994] In some embodiments, the plurality of polynucleotides may comprise two copies of the third polynucleotide according to part (iii) above.

[0995] In some embodiments, the first polynucleotide comprises the nucleic acid sequence according to SEQ ID NO: 65.

[0996] In some embodiments, the first polynucleotide comprises a variant having at least 80% sequence identity to SEQ ID NO: 65. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 65.

[0997] In some embodiments, the first polynucleotide consists of the nucleic acid sequence according to SEQ ID NO: 65.In some embodiments, the first polynucleotide comprises the nucleic acid sequence according to SEQ ID NO: 69.

[0998] In some embodiments, the first polynucleotide comprises a variant having at least 80% sequence identity to SEQ ID NO: 69. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69.

[0999] In some embodiments, the first polynucleotide consists of the nucleic acid sequence according to SEQ ID NO: 69.

[1000] In some embodiments, the first polynucleotide comprises the nucleic acid sequence according to SEQ ID NO: 73.

[1001] In some embodiments, the first polynucleotide comprises a variant having at least 80% sequence identity to SEQ ID NO: 73. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73.

[1002] In some embodiments, the first polynucleotide consists of the nucleic acid sequence according to SEQ ID NO: 73.

[1003] In some embodiments, the first polynucleotide comprises the nucleic acid sequence according to SEQ ID NO: 67.

[1004] In some embodiments, the first polynucleotide comprises a variant having at least 80% sequence identity to SEQ ID NO: 67. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67.

[1005] In some embodiments, the first polynucleotide consists of the nucleic acid sequence according to SEQ ID NO: 67.

[1006] In some embodiments, the first polynucleotide comprises the nucleic acid sequence according to SEQ ID NO: 71.In some embodiments, the first polynucleotide comprises a variant having at least 80% sequence identity to SEQ ID NO: 71. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 71.

[1007] In some embodiments, the first polynucleotide consists of the nucleic acid sequence according to SEQ ID NO: 71.

[1008] In some embodiments, the first polynucleotide comprises the nucleic acid sequence according to SEQ ID NO: 75.

[1009] In some embodiments, the first polynucleotide comprises a variant having at least 80% sequence identity to SEQ ID NO: 75. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 75.

[1010] In some embodiments, the first polynucleotide consists of the nucleic acid sequence according to SEQ ID NO: 75.

[1011] In some embodiments, the second polynucleotide comprises the nucleic acid sequence according to SEQ ID NO: 63.

[1012] In some embodiments, the second polynucleotide comprises a variant having at least 80% sequence identity to SEQ ID NO: 63. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 63.

[1013] In some embodiments, the second polynucleotide consists of the nucleic acid sequence according to SEQ ID NO: 63.

[1014] In some embodiments, the third polynucleotide comprises the nucleic acid sequence according to SEQ ID NO: 77.

[1015] In some embodiments, the third polynucleotide comprises a variant having at least 80% sequence identity to SEQ ID NO: 77. In some embodiments, the variant has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 77.In some embodiments, the third polynucleotide consists of the nucleic acid sequence according to SEQ ID NO: 77.

[1016] In some embodiments, the plurality of polynucleotides comprises:

[1017] (i) the first polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 65 or a variant having at least 80% sequence identity to SEQ ID NO: 65;

[1018] (ii) the second polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 63 or a variant having at least 80% sequence identity to SEQ ID NO: 63; and (iii) the third polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 77 or a variant having at least 80% sequence identity to SEQ ID NO: 77.

[1019] In some embodiments, each variant independently has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[1020] In some embodiments, the plurality of polynucleotides comprises:

[1021] (i) the first polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 65;

[1022] (ii) the second polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 63; and

[1023] (iii) the third polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 77.

[1024] In some embodiments, the plurality of polynucleotides comprises:

[1025] (i) the first polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 69 or a variant having at least 80% sequence identity to SEQ ID NO: 69;

[1026] (ii) the second polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 63 or a variant having at least 80% sequence identity to SEQ ID NO: 63; and (iii) the third polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 77 or a variant having at least 80% sequence identity to SEQ ID NO: 77.

[1027] In some embodiments, each variant independently has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[1028] In some embodiments, the plurality of polynucleotides comprises:

[1029] (i) the first polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 69;(ii) the second polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 63; and

[1030] (iii) the third polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 77.

[1031] In some embodiments, the plurality of polynucleotides comprises:

[1032] (i) the first polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 73 or a variant having at least 80% sequence identity to SEQ ID NO: 73;

[1033] (ii) the second polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 63 or a variant having at least 80% sequence identity to SEQ ID NO: 63; and (iii) the third polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 77 or a variant having at least 80% sequence identity to SEQ ID NO: 77.

[1034] In some embodiments, each variant independently has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[1035] In some embodiments, the plurality of polynucleotides comprises:

[1036] (i) the first polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 73;

[1037] (ii) the second polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 63; and

[1038] (iii) the third polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 77.

[1039] In some embodiments, the plurality of polynucleotides comprises:

[1040] (i) the first polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 67 or a variant having at least 80% sequence identity to SEQ ID NO: 67;

[1041] (ii) the second polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 63 or a variant having at least 80% sequence identity to SEQ ID NO: 63; and (iii) the third polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 77 or a variant having at least 80% sequence identity to SEQ ID NO: 77.

[1042] In some embodiments, each variant independently has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.In some embodiments, the plurality of polynucleotides comprises:

[1043] (i) the first polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 67;

[1044] (ii) the second polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 63; and

[1045] (iii) the third polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 77.

[1046] In some embodiments, the plurality of polynucleotides comprises:

[1047] (i) the first polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 71 or a variant having at least 80% sequence identity to SEQ ID NO: 71;

[1048] (ii) the second polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 63 or a variant having at least 80% sequence identity to SEQ ID NO: 63; and (iii) the third polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 77 or a variant having at least 80% sequence identity to SEQ ID NO: 77.

[1049] In some embodiments, each variant independently has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[1050] In some embodiments, the plurality of polynucleotides comprises:

[1051] (i) the first polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 71;

[1052] (ii) the second polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 63; and

[1053] (iii) the third polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 77.

[1054] In some embodiments, the plurality of polynucleotides comprises:

[1055] (i) the first polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 75 or a variant having at least 80% sequence identity to SEQ ID NO: 75;

[1056] (ii) the second polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 63 or a variant having at least 80% sequence identity to SEQ ID NO: 63; and (iii) the third polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 77 or a variant having at least 80% sequence identity to SEQ ID NO: 77.

[1057] In some embodiments, each variant independently has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.In some embodiments, the plurality of polynucleotides comprises:

[1058] (i) the first polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 75;

[1059] (ii) the second polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 63; and

[1060] (iii) the third polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 77.

[1061] In some embodiments, the ratios as calculated by mass of the first polynucleotide : second polynucleotide : third polynucleotide is: 1-2 : 1-2 : 1.

[1062] In some embodiments, the ratios as calculated by mass of the first polynucleotide : second polynucleotide : third polynucleotide is: 1.25-1.75 : 1.125-1.5 : 1.

[1063] In some embodiments, the ratios as calculated by mass of the first polynucleotide : second polynucleotide : third polynucleotide is: 1.5 : 1.5 : 1.

[1064] Due to the redundancy of the genetic code, variations in nucleic acid sequences are possible that encode for the same polypeptide. These sequences are encompassed by the present invention. Therefore, multiple polynucleotides are envisaged, each with a different nucleic acid sequence but which encodes a polypeptide according to the invention or a further polypeptide as described herein. It is known in the art how to design and produce such nucleic acid sequences.

[1065] In some embodiments, the nucleic acid sequence of the polynucleotide is codon optimised for production in the host cell of choice.

[1066] In some embodiments, the nucleic acid sequence of the polynucleotide is operably linked to a sequence such as a control sequence, e.g., a promoter sequence, an enhancer sequence or regulatory sequence, which controls transcription and / or translation. The polynucleotide may be in the form of an expression cassette.

[1067] The polynucleotide may be suitable for expression in prokaryotic cells or in eukaryotic cells, such as mammalian cells.Any promoter may be used in the polynucleotide, such as a strong promoter that is functional in prokaryotic cells or in eukaryotic cells. Suitable promoters will be known in the art. The promoter may be a constitutive promoter. The promoter may be a tissue specific promoter.

[1068] VECTOR

[1069] The present invention provides a vector comprising the polynucleotide according to the invention.

[1070] In some embodiments, the vector comprises a polynucleotide comprising a nucleic acid sequence which encodes the bispecific binding molecule according to the invention.

[1071] The present invention provides a vector comprising one or more polynucleotide(s) according to the invention.

[1072] In some embodiments, the vector comprises one or more polynucleotide(s) comprising one or more nucleic acid sequence(s) which encodes the bispecific binding molecule according to the invention.

[1073] In some embodiments, the vector comprises one or more polynucleotide(s) comprising one or more nucleic acid sequence(s) which encodes the bispecific antigen-binding molecule according to the invention.

[1074] In some embodiments, the vector comprises one or more polynucleotide(s) comprising one or more nucleic acid sequence(s) which encodes the bispecific antibody according to the invention.

[1075] The present invention provides a vector comprising the plurality of polynucleotides according to the invention.

[1076] In some embodiments, the vector comprises a plurality of polynucleotides comprising a plurality of nucleic acid sequences which together encodes the bispecific binding molecule according to the invention.

[1077] In some embodiments, the vector comprises a plurality of polynucleotides comprising a plurality of nucleic acid sequences which together encodes the bispecific antigen-binding molecule according to the invention.In some embodiments, the vector comprises a plurality of polynucleotides comprising a plurality of nucleic acid sequences which together encodes the bispecific antibody according to the invention.

[1078] The vector may be used to introduce a polynucleotide or polynucleotides according to the invention into a cell so that the cell expresses and / or produces the bispecific binding molecule according to the invention.

[1079] As used herein, the term “vector” may be considered interchangeable with the term “expression vector” and “expression construct”. The vector may be any vector that is suitable for introducing and / or expressing a nucleic acid sequence in a cell. The vector may comprise regulatory sequences, enhancer sequences and / or promoter sequences that promote expression of a nucleic acid sequence in a cell.

[1080] The vector according to the invention may be any agent capable of delivering a polynucleotide according to the invention to a cell.

[1081] The vector according to the invention may be any agent capable of expressing a polynucleotide or polynucleotides according to the invention in a cell.

[1082] Examples of suitable vectors include but are not limited to plasmids, cosmids, phages, viruses or artificial chromosomes.

[1083] In some embodiments, the vector is a plasmid or a viral vector. In some embodiments, the vector is a retroviral vector or a lentiviral vector.

[1084] Examples of appropriate DNA plasmids can be found in WO2021214204A1, which is hereby incorporated by reference in its entirety.

[1085] The vector may be capable of transfecting or transducing a cell.

[1086] COMPOSITION

[1087] The present invention provides a composition comprising the bispecific binding molecule according to the invention.

[1088] The present invention provides a composition comprising the bispecific antigen-binding molecule according to the invention.The present invention provides a composition comprising the bispecific antibody according to the invention.

[1089] The present invention provides a composition comprising the polynucleotide according to the invention.

[1090] The present invention provides a composition comprising the polynucleotides according to the invention.

[1091] In some embodiments, the composition comprises the plurality of polynucleotides as described herein.

[1092] The present invention provides a composition comprising the vector according to the invention.

[1093] PHARMACEUTICAL COMPOSITION

[1094] The present invention provides a pharmaceutical composition comprising the bispecific binding molecule according to the invention; together with a pharmaceutically acceptable carrier, diluent or excipient.

[1095] The present invention provides a pharmaceutical composition comprising the bispecific antigen-binding molecule according to the invention; together with a pharmaceutically acceptable carrier, diluent or excipient.

[1096] The present invention provides a pharmaceutical composition comprising the bispecific antibody according to the invention; together with a pharmaceutically acceptable carrier, diluent or excipient.

[1097] The present invention also provides a pharmaceutical composition comprising the polynucleotide according to the invention; together with a pharmaceutically acceptable carrier, diluent or excipient.

[1098] The present invention also provides a pharmaceutical composition comprising the polynucleotides according to the invention; together with a pharmaceutically acceptable carrier, diluent or excipient.The present invention also provides a pharmaceutical composition comprising the vector according to the invention; together with a pharmaceutically acceptable carrier, diluent or excipient.

[1099] The present invention also provides a pharmaceutical composition comprising the composition according to the invention; together with a pharmaceutically acceptable carrier, diluent or excipient.

[1100] In some embodiments, the pharmaceutical composition described herein further comprises one or more selected from this list consisting of: a pharmaceutically acceptable adjuvant, salt, active polypeptide, buffer, compound, component and active agent.

[1101] In some embodiments, the pharmaceutical composition further comprises a vesicle, a nanoparticle, a lipid nanoparticle (LNP), a liposome or a polymeric mixture.

[1102] It will be understood that the vesicle, nanoparticle, LNP, liposome or polymeric mixture may enable delivery of the bispecific binding molecule according to the invention, the polynucleotide according to the invention, the polynucleotide(s) according to the invention, the vector according to the invention, or the composition according to the invention, to a cell.

[1103] In some embodiments, the pharmaceutical composition further comprises a vesicle. In some embodiments, the bispecific binding molecule, the polynucleotide, the polynucleotides, the vector, or the composition according to the invention is encapsulated within the vesicle.

[1104] In some embodiments, the pharmaceutical composition further comprises a nanoparticle. In some embodiments, the bispecific binding molecule, the polynucleotide, the polynucleotides, the vector, or the composition according to the invention is encapsulated within the nanoparticle.

[1105] In some embodiments, the pharmaceutical composition further comprises a lipid nanoparticle (LNP). In some embodiments, the bispecific binding molecule, the polynucleotide, the polynucleotides, the vector, or the composition according to the invention is encapsulated within the LNP.

[1106] In some embodiments, the pharmaceutical composition further comprises a liposome. In some embodiments, the bispecific binding molecule, the polynucleotide, the polynucleotides, the vector, or the composition according to the invention is encapsulated within the liposome.In some embodiments, the pharmaceutical composition further comprises a polymeric mixture. In some embodiments, the bispecific binding molecule, the polynucleotide, the polynucleotides, the vector, or the composition according to the invention is encapsulated within the polymeric mixture.

[1107] Compositions typically should be sterile and stable under the conditions of manufacture and storage. The composition according to the invention may be produced using current good manufacturing practices (CGMP).

[1108] In some embodiments, the pharmaceutical composition is sterile.

[1109] In some embodiments, the pharmaceutical composition is sterile in aqueous buffer suitable for intravenous (IV) administration.

[1110] In some embodiments, the pharmaceutical composition may comprise an aqueous diluent or solvent. In some embodiments, the aqueous diluent or solvent may be a phosphate buffered saline solution, such as a sterile phosphate buffered saline solution.

[1111] The term “carrier”, as used herein, may refer to a diluent, adjuvant, excipient, or vehicle. Such carriers can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil. A sterile saline solution is a preferred carrier.

[1112] The term "nanoparticle" relates to a nano-sized particle, wherein all three external dimensions of the particle are in the nanoscale, i.e., at least about 1 nm and below about 1,000 nm. Preferably, the size of a particle is its diameter.

[1113] Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[1114] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition of the invention can be formulated as neutral or salt forms. Salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxylgroups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[1115] In some embodiments, the salt comprises a metal cation, such as a sodium salt or a potassium salt.

[1116] The pharmaceutical composition may be formulated to be suitable for administration to a patient in order to prevent and / or treat disease. Pharmaceutical compositions can be formulated for administration by different routes, for example, for oral, parenteral, topical, inhalative, intravenous, intramuscular, rectal, sublingual, transdermal, subcutaneous, intratumoral application routes, according to their chemical and physical properties.

[1117] In some embodiments, the pharmaceutical composition is in a form suitable for intravenous infusion. In some embodiments, the pharmaceutical composition is administered intravenously.

[1118] In some embodiments, the pharmaceutical composition is formulated for parenteral administration.

[1119] In some embodiments, the pharmaceutical composition is formulated for intravenous or intraarterial administration.

[1120] DISEASE

[1121] The present invention provides the bispecific binding molecule according to the invention, the bispecific antigen-binding molecule of the invention, the bispecific antibody of the invention, the polynucleotide according to the invention, the polynucleotides according to the invention, the vector according to the invention, the composition according to the invention, or the pharmaceutical composition according to the invention, for use in treating and / or preventing a disease.

[1122] The present invention provides the bispecific binding molecule according to the invention for use in treating and / or preventing a disease.

[1123] The present invention provides the bispecific antigen-binding molecule according to the invention for use in treating and / or preventing a disease.The present invention provides the bispecific antibody according to the invention for use in treating and / or preventing a disease.

[1124] The present invention provides the polynucleotide according to the invention for use in treating and / or preventing a disease.

[1125] The present invention provides the polynucleotides according to the invention for use in treating and / or preventing a disease.

[1126] The present invention provides the vector according to the invention for use in treating and / or preventing a disease.

[1127] The present invention provides the composition according to the invention for use in treating and / or preventing a disease.

[1128] The present invention provides the pharmaceutical composition according to the invention for use in treating and / or preventing a disease.

[1129] In some embodiments, the use is for treating a disease. In some embodiments, the use is for preventing a disease.

[1130] The present invention provides a method for treating and / or preventing a disease which comprises the step of administering the bispecific binding molecule according to the invention, the bispecific antigen-binding molecule of the invention, the bispecific antibody of the invention, the polynucleotide according to the invention, the polynucleotides according to the invention, the vector according to the invention, the composition according to the invention, or the pharmaceutical composition according to the invention, to a subject.

[1131] In some embodiments, the method is for treating a disease. In some embodiments, the method is for preventing a disease.

[1132] The present invention provides a use of the bispecific binding molecule according to the invention, the bispecific antigen-binding molecule of the invention, the bispecific antibody of the invention, the polynucleotide according to the invention, the polynucleotides according to the invention, the vector according to the invention, the composition according to the invention, or the pharmaceutical composition according to the invention, in the manufacture of a medicament for treating and / or preventing a disease.In some embodiments, the medicament is for treating a disease. In some embodiments, the medicament is for preventing a disease.

[1133] In some embodiments, the disease is cancer.

[1134] In some embodiments, the disease is a tumour.

[1135] In some embodiments, the disease is associated with inflammation.

[1136] In some embodiments, the cancer is a tumour or cancer expressing CA19-9.

[1137] In some embodiments, the tumour expresses CA19-9.

[1138] In some embodiments, the expression of CA19-9 is increased compared to the expression of CA19-9 by a non-cancerous tissue. It will be understood that non-cancerous tissue typically has low membranous expression, or no detectable membranous expression, of CA19-9.

[1139] In some embodiments, the cancer is selected from the listing consisting of: pancreatic cancer, cholangiocarcinoma, and gastrointestinal cancer.

[1140] In some embodiments, the cancer is pancreatic cancer. In some embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

[1141] In some embodiments, the cancer is cholangiocarcinoma. In some embodiments, the cancer is bile duct cancer.

[1142] In some embodiments, the cancer is gastrointestinal cancer. In some embodiments, the gastrointestinal cancer is selected from the listing consisting of: colorectal cancer, oesophageal cancer, gastric cancer, and hepatocellular cancer.

[1143] It will be understood that the terms “tumor” and “tumours” can be used interchangeably.

[1144] In some embodiments, the bispecific binding molecule according to the invention, the bispecific antigen-binding molecule of the invention, the bispecific antibody of the invention, the polynucleotide according to the invention, the polynucleotides according to the invention, the vector according to the invention, the composition according to the invention, or thepharmaceutical composition according to the invention, is administered to a subject systemically.

[1145] In some embodiments, the bispecific binding molecule according to the invention, the bispecific antigen-binding molecule of the invention, the bispecific antibody of the invention, the polynucleotide according to the invention, the polynucleotides according to the invention, the vector according to the invention, the composition according to the invention, or the pharmaceutical composition according to the invention, is administered intravenously (IV) to a subject.

[1146] The term "disease" and "disorder" are used interchangeably herein, referring to an abnormal condition, especially an abnormal medical condition such as an illness or injury, wherein a cell, a tissue, an organ, or an individual is not able to efficiently fulfil its function anymore. Typically, but not necessarily, a disease is associated with specific symptoms or signs indicating the presence of such disease. The presence of such symptoms or signs may thus, be indicative for a cell, a tissue, an organ, or an individual suffering from a disease. An alteration of these symptoms or signs may be indicative for the progression of such a disease.

[1147] A progression of a disease is typically characterised by an increase or decrease of such symptoms or signs which may indicate a "worsening" or "bettering" of the disease. The "worsening" of a disease is characterised by a decreasing ability of a cell, tissue, organ or individual / patient to fulfil its function efficiently, whereas the "bettering" of a disease is typically characterised by an increase in the ability of a cell, tissue, an organ or an individual / patient to fulfil its function efficiently.

[1148] The terms “treat”, “treatment” and “treating” refers to lessening, reducing or improving at least one symptom associated with an existing disease or condition and / or to slow down, reduce or block the progression of the disease or condition and / or to delay or prevent the onset of symptoms (such as further symptoms) of the disease or condition.

[1149] The terms “prevent”, “prevention” and “preventing” refers to preventing the onset of symptoms of a disease or condition, and as such encompasses prophylactic treatment.

[1150] The medical uses and methods of treatment described herein may be used in combination with additional treatments and / or medicaments.SUBJECT

[1151] In some embodiments, the subject is a mammal.

[1152] In some embodiments, the subject is a human.

[1153] In some embodiments, the subject is a non-human mammal, including for example, a dog, a cat, a horse, a cow, a sheep or a pig.

[1154] As used herein, the terms “patient” and “subject” may be used interchangeably.

[1155] In some embodiments, the subject is a patient. In some embodiments, the subject is a human patient.

[1156] In some embodiments, the subject is a human male. In some embodiments, the subject is a human female.

[1157] IDENTITY

[1158] The terms “identity” and “% sequence identity” as used herein, may refer to the proportion of amino acids (expressed in percent) of an amino acid sequence in a peptide or protein, which across the amino acid sequence, are identical to a reference sequence.

[1159] The percentage of identity is thus calculated by counting the number of aligned amino acids that are identical (a Match) between two sequences (in the amino acids sequence of the peptide or protein of the invention and in the reference sequence), dividing that number by the total number of amino acids in the aligned region and multiplying by 100.

[1160] Therefore, Percentage of Identity = (Matches divided by Length of the aligned region) multiplied by 100.

[1161] Insertions and deletions are not allowed in the calculation the percentage of identity of an amino acid sequence.

[1162] Any method of calculating percentage identity is permitted within the scope of the invention.GENERAL TERMS AND DEFINITIONS

[1163] This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure.

[1164] The term “polypeptide” is used in the conventional sense to mean a series of amino acids, typically L-amino acids, connected one to the other, typically by peptide bonds between the a-amino and carboxyl groups of adjacent amino acids.

[1165] The term “polypeptide” is used interchangeably with the terms “amino acid sequence”, “peptide” and / or “protein”.

[1166] The term “residues” is used to refer to amino acids in an amino acid sequence.

[1167] Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[1168] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

[1169] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[1170] The terms "comprising", "comprises" and "comprised of' as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of' also include the terms "consisting of' or “consisting essentially of”.As used herein, the term “variant” is synonymous with the term “mutant” and refers to an amino acid sequence or a nucleic acid sequence that differs in comparison to the corresponding wildtype sequence. The term “wild-type” is used to mean a protein comprising an amino acid sequence or a polynucleotide comprising an amino acid sequence, which is identical to the native protein or native polynucleotide (e.g. gene) respectively. The variant may have an equivalent function to the amino acid sequences or nucleic acid sequences described herein, but may include one or more amino acid or nucleic acid (respectively) substitutions, insertions or deletions. Amino acid substitutions, insertions and / or deletions may be considered as mutations. Nucleic acid substitutions, insertions and / or deletions may be considered as mutations.

[1171] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.SEQUENCES

[1172] Table 1 - List of sequences

[1173] SEQ name DNA / Sequence

[1174] ID AAS /

[1175] NO: RNA

[1176] 1 CD3 VH-CDR1 (Kabat) AAS NNYYMC

[1177] 2 CD3 VH-CDR1 (IMGT) AAS GFSFSNNYY

[1178] 3 CD3 VH-CDR2 (Kabat) AAS CIYTNNIGSTWYASWAKG

[1179] 4 CD3 VH-CDR2 (IMGT) AAS IYTNNIGST

[1180] 5 CD3 VH-CDR3 (Kabat) AAS GIYSDSSDFIGNL

[1181] 6 CD3 VH-CDR3 (IMGT) AAS ARGIYSDSSDFIGNL

[1182] 7 CD3 VL-CDR1 (Kabat) AAS QASQTIYNYLA

[1183] 8 CD3 VL-CDR1 (IMGT) AAS QTIYNY

[1184] 9 CD3 VL-CDR2 (Kabat) AAS KASTLAS

[1185] 10 CD3 VL-CDR2 (IMGT) AAS KAS

[1186] 11 CD3 VL-CDR3 (Kabat) AAS QQYNTIINVDRT

[1187] 12 CD3 VL-CDR3 (IMGT) AAS QQYNTIINVDRT

[1188] 13 CD3 CLONE 1 variable RNA GAAGUUCAGCUGGUGGAAUCUGGCGGCGGACUGGUUAAGC heavy chain (VH) CUGGUGGAUCUCUGGGCCUGAGCUGUACCGCCAGCGGCU UCAGCUUCAGCAACAACUACUACAUGUGCUGGGUUCGCAG AGCCCCUGGCAAAUGUCUUGAGUGGGUCGGAUGCAUCUAC ACCAACAACAUCGGCAGCACUUGGUACGCCAGCUGGGCCA AGGGCAGAUUCACCAUCAGCAGGGACGACAGCAAGAACAC CGUGUACCUGCAGAUGAACAGCCUGAAAACCGAGGACACC GCCGUGUACUACUGCGCCAGAGGCAUCUACAGCGACAGCA GCGACUUCAUCGGCAACCUGUGGGGACAGGGCACACUGGU CACAGUUUCUAGC

[1189] 14 CD3 CLONE 1 variable AAS EVQLVESGGGLVKPGGSLGLSCTASGFSFSNNYYMCWVRRAP heavy chain (VH) GKCLEWVGCIYTNNIGSTWYASWAKGRFTISRDDSKNTVYLQM NSLKTEDTAVYYCARGIYSDSSDFIGNLWGQGTLVTVSS

[1190] 15 CD3 CLONE 1 variable RNA GAAAUCGUGAUGACACAGAGCCCCGCCACACUGAGUGUGU heavy chain (VL) CUCCAGGCGAAAAGGCCACACUGUCCUGUCAGGCCAGCCA GACCAUCUACAACUACCUGGCCUGGUAUCAGCAGAAGCCC GGCCAACCUCCUCGGCUGCUGAUCUACAAAGCCAGCACAC UGGCCAGCGGCAUCCCUGCCAGAUUUUCUGGAAGCGGCAG CGGCACCGAGUUCACCCUGACAAUCAGCAACCUGCAGAGC GAGGACUUUGCCGUGUAUUAUUGCCAGCAGUACAACACCA UCAUCAACGUGGACCGGACCUUCGGCUGUGGCACCAAGGU GGAAAUCAAG

[1191] 16 CD3 CLONE 1 variable AAS EIVMTQSPATLSVSPGEKATLSCQASQTIYNYLAWYQQKPGQP heavy chain (VL) PRLLIYKASTLASGIPARFSGSGSGTEFTLTISNLQSEDFAVYYC

[1192]

[1193] QQYNTIINVDRTFGCGTKVEIKscFv-CD3 CLONE 1 (VH- RNA GAAGUUCAGCUGGUGGAAUCUGGCGGCGGACUGGUUAAGC (G4S)4-VL), full sequence CUGGUGGAUCUCUGGGCCUGAGCUGUACCGCCAGCGGCU UCAGCUUCAGCAACAACUACUACAUGUGCUGGGUUCGCAG AGCCCCUGGCAAAUGUCUUGAGUGGGUCGGAUGCAUCUAC ACCAACAACAUCGGCAGCACUUGGUACGCCAGCUGGGCCA AGGGCAGAUUCACCAUCAGCAGGGACGACAGCAAGAACAC CGUGUACCUGCAGAUGAACAGCCUGAAAACCGAGGACACC GCCGUGUACUACUGCGCCAGAGGCAUCUACAGCGACAGCA GCGACUUCAUCGGCAACCUGUGGGGACAGGGCACACUGGU CACAGUUUCUAGCGGAGGCGGAGGUUCAGGUGGCGGAGG AAGUGGCGGCGGAGGAUCUGGCGGAGGUGGAAGCGAAAU CGUGAUGACACAGAGCCCCGCCACACUGAGUGUGUCUCCA GGCGAAAAGGCCACACUGUCCUGUCAGGCCAGCCAGACCA UCUACAACUACCUGGCCUGGUAUCAGCAGAAGCCCGGCCA ACCUCCUCGGCUGCUGAUCUACAAAGCCAGCACACUGGCC AGCGGCAUCCCUGCCAGAUUUUCUGGAAGCGGCAGCGGCA CCGAGUUCACCCUGACAAUCAGCAACCUGCAGAGCGAGGA CUUUGCCGUGUAUUAUUGCCAGCAGUACAACACCAUCAUCA ACGUGGACCGGACCUUCGGCUGUGGCACCAAGGUGGAAAU CAAG

[1194] scFv-CD3 CLONE 1 (VH- AAS EVQLVESGGGLVKPGGSLGLSCTASGFSFSNNYYMCWVRRAP (G4S)4-VL), full sequence GKCLEWVGCIYTNNIGSTWYASWAKGRFTISRDDSKNTVYLQM NSLKTEDTAVYYCARGIYSDSSDFIGNLWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGEKATLSCQAS QTIYNYLAWYQQKPGQPPRLLIYKASTLASGIPARFSGSGSGTE FTLTISNLQSEDFAVYYCQQYNTIINVDRTFGCGTKVEIK

[1195] scFv-CD3 CLONE 1 (VL- RNA GAGAUUGUGAUGACACAGAGCCCCGCCACACUGUCCGUUU (G4S)5-VH), full sequence CUCCAGGCGAAAAAGCCACACUGAGCUGCCAGGCCAGCCA GACCAUCUACAAUUACCUGGCCUGGUAUCAGCAGAAGCCC GGCCAACCUCCUCGGCUGCUGAUCUACAAAGCCAGCACAC UGGCCAGCGGCAUCCCUGCCAGAUUUUCUGGAAGCGGCAG CGGCACCGAGUUCACCCUGACAAUCAGCAACCUGCAGAGC GAGGACUUCGCCGUGUACUACUGCCAGCAGUACAACACCA UCAUCAACGUGGACCGGACCUUCGGCUGUGGCACCAAGGU UGAAAUCAAAGGCGGCGGAGGAAGCGGAGGCGGAGGUUCU GGUGGUGGUGGCUCUGGUGGCGGAGGAUCUGGCGGAGGC GGUUCUGAAGUUCAGCUGGUGGAAUCUGGCGGCGGACUG GUUAAGCCUGGUGGAUCUCUGGGCCUGAGCUGUACCGCCA GCGGCUUCAGCUUCAGCAACAACUACUACAUGUGCUGGGU UCGCAGAGCCCCUGGCAAAUGUCUUGAGUGGGUCGGAUGC AUCUACACCAACAACAUCGGCAGCACUUGGUACGCCAGCU GGGCCAAGGGCAGAUUCACCAUCAGCAGGGACGACAGCAA GAACACCGUGUACCUGCAGAUGAACAGCCUGAAAACCGAG GACACAGCCGUGUAUUAUUGCGCCAGAGGCAUCUACAGCG ACAGCAGCGACUUCAUCGGCAACCUGUGGGGACAGGGCAC ACUGGUCACAGUGUCAUCU

[1196] scFv-CD3 CLONE 1 (VL- AAS EIVMTQSPATLSVSPGEKATLSCQASQTIYNYLAWYQQKPGQP (G4S)5-VH), full sequence PRLLIYKASTLASGIPARFSGSGSGTEFTLTISNLQSEDFAVYYC QQYNTIINVDRTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGS GGGGSEVQLVESGGGLVKPGGSLGLSCTASGFSFSNNYYMC WVRRAPGKCLEWVGCIYTNNIGSTWYASWAKGRFTISRDDSKN TVYLQMNSLKTEDTAVYYCARGIYSDSSDFIGNLWGQGTLVTVS S CD3 CLONE 2 variable RNA CAAGUUCAGCUGGUUCAAUCAGGCGGCGGACUGGUUCAAC heavy chain (VH) CUGGCGGACCUCUGAGACUGAGCUGUACCGCCAGCGGCUU CAGCUUCAGCAACAACUACUACAUGUGCUGGGUCCGACAG GCCCCUGGCAAGUGUCUUGAAUGGGUGUCCUGCAUCUACA CCAACAACAUCGGCAGCACUUGGUACGCCAGCUGGGCCAA

[1197]

[1198] GGGCAGAUUCACCAUCAGCCGGGACAACAGCAAGAACACCCUGUACCUGCAGAUGAACAGCCUGAGAGCCGAGGAUACCG CCGUGUACUAUUGUGCCAGAGGCAUCUACAGCGACAGCAG CGACUUCAUCGGCAACCUGUGGGGACAGGGCACACUGGUC ACAGUUUCUAGC CD3 CLONE 2 variable AAS QVQLVQSGGGLVQPGGPLRLSCTASGFSFSNNYYMCWVRQA heavy chain (VH) PGKCLEWVSCIYTNNIGSTWYASWAKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCARGIYSDSSDFIGNLWGQGTLVTVSS CD3 CLONE 2 variable RNA GCCAUUCAGAUGACACAGAGCCCUAGCAGCCUGAGCGCCU heavy chain (VL) CUGUGGGAGACAGAGUGACCAUUACCUGUCAGGCCAGCCA GACCAUCUACAACUACCUGGCCUGGUAUCAGCAGAAACCC GGCAAGGUGCCAGAGCUGCUGAUCUACAAGGCCAGCACAC UGGCUAGCGGCGUGCCCAGUAGAUUCAUGGGCACAGGCUU CGGCACCGACUUCACCCUGACCAUCGAUAGCCUGCAGCCU GAAGAUGCCGCCACCUACUACUGCCAGCAGUACAACACCAU CAUCAACGUGGACCGGACCUUCGGCUGUGGAACCAAGGUG GAAAUCAAA CD3 CLONE 2 variable AAS AIQMTQSPSSLSASVGDRVTITCQASQTIYNYLAWYQQKPGKVP heavy chain (VL) ELLIYKASTLASGVPSRFMGTGFGTDFTLTIDSLQPEDAATYYCQ QYNTIINVDRTFGCGTKVEIK

[1199] scFv-CD3 CLONE 2(VH- RNA CAAGUUCAGCUGGUUCAAUCAGGCGGCGGACUGGUUCAAC (G4S)4-VL), full sequence CUGGCGGACCUCUGAGACUGAGCUGUACCGCCAGCGGCUU CAGCUUCAGCAACAACUACUACAUGUGCUGGGUCCGACAG GCCCCUGGCAAGUGUCUUGAAUGGGUGUCCUGCAUCUACA CCAACAACAUCGGCAGCACUUGGUACGCCAGCUGGGCCAA GGGCAGAUUCACCAUCAGCCGGGACAACAGCAAGAACACC CUGUACCUGCAGAUGAACAGCCUGAGAGCCGAGGAUACCG CCGUGUACUAUUGUGCCAGAGGCAUCUACAGCGACAGCAG CGACUUCAUCGGCAACCUGUGGGGACAGGGCACACUGGUC ACAGUUUCUAGCGGUGGCGGAGGAAGCGGAGGCGGAGGU UCAGGUGGUGGUGGAAGCGGUGGUGGCGGAUCUGCCAUU CAGAUGACACAGAGCCCUAGCAGCCUGAGCGCCUCUGUGG GAGACAGAGUGACCAUUACCUGUCAGGCCAGCCAGACCAU CUACAACUACCUGGCCUGGUAUCAGCAGAAACCCGGCAAG GUGCCAGAGCUGCUGAUCUACAAGGCCAGCACACUGGCUA GCGGCGUGCCCAGUAGAUUCAUGGGCACAGGCUUCGGCAC CGACUUCACCCUGACCAUCGAUAGCCUGCAGCCUGAAGAU GCCGCCACCUACUACUGCCAGCAGUACAACACCAUCAUCAA CGUGGACCGGACCUUCGGCUGUGGAACCAAGGUGGAAAUC AAA

[1200] scFv-CD3 CLONE 2(VH- AAS QVQLVQSGGGLVQPGGPLRLSCTASGFSFSNNYYMCWVRQA (G4S)4-VL), full sequence PGKCLEWVSCIYTNNIGSTWYASWAKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCARGIYSDSSDFIGNLWGQGTLVTVSSGGG GSGGGGSGGGGSGGGGSAIQMTQSPSSLSASVGDRVTITCQA SQTIYNYLAWYQQKPGKVPELLIYKASTLASGVPSRFMGTGFGT DFTLTIDSLQPEDAATYYCQQYNTIINVDRTFGCGTKVEIK

[1201] scFv-CD3 CLONE 2(VL- RNA GCCAUUCAGAUGACACAGAGCCCUAGCAGCCUGAGCGCCU (G4S)5-VH), full sequence CUGUGGGAGACAGAGUGACCAUUACCUGUCAGGCCAGCCA GACCAUCUACAACUACCUGGCCUGGUAUCAGCAGAAACCC GGCAAGGUGCCAGAGCUGCUGAUCUACAAGGCCAGCACAC UGGCUAGCGGCGUGCCCAGUAGAUUCAUGGGCACAGGCUU CGGCACCGACUUCACCCUGACCAUCGAUAGCCUGCAGCCU GAAGAUGCCGCCACCUACUACUGCCAGCAGUACAACACCAU CAUCAACGUGGACCGGACCUUCGGCUGUGGAACCAAGGUG GAAAUCAAAGGUGGCGGAGGAAGCGGAGGCGGCGGUUCAG GCGGUGGUGGUAGCGGUGGUGGCGGCUCAGGCGGCGGAG GUUCUCAAGUUCAGCUGGUUCAAUCAGGCGGCGGACUGGU UCAACCUGGCGGACCUCUGAGACUGAGCUGUACCGCCAGC GGCUUCAGCUUCAGCAACAACUACUACAUGUGCUGGGUCC

[1202]

[1203] GACAGGCCCCUGGCAAGUGUCUUGAAUGGGUGUCCUGCAUCUACACCAACAACAUCGGCAGCACUUGGUACGCCAGCUGG GCCAAGGGCAGAUUCACCAUCAGCCGGGACAACAGCAAGA ACACCCUGUACCUGCAGAUGAACAGCCUGAGAGCCGAGGA UACCGCCGUGUACUAUUGUGCCAGAGGCAUCUACAGCGAC AGCAGCGACUUCAUCGGCAACCUGUGGGGACAGGGCACAC UGGUCACAGUUUCUAGC

[1204] scFv-CD3 CLONE 2(VL- AAS AIQMTQSPSSLSASVGDRVTITCQASQTIYNYLAWYQQKPGKVP (G4S)5-VH), full sequence ELLIYKASTLASGVPSRFMGTGFGTDFTLTIDSLQPEDAATYYCQ QYNTIINVDRTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSG GGGSQVQLVQSGGGLVQPGGPLRLSCTASGFSFSNNYYMCW VRQAPGKCLEWVSCIYTNNIGSTWYASWAKGRFTISRDNSKNT LYLQMNSLRAEDTAVYYCARGIYSDSSDFIGNLWGQGTLVTVSS CD3 CLONE 3 variable RNA GAAGUUCAGCUGGUUGAAUCAGGCGGCGGACUGGUUAAGC heavy chain (VH) CUGGCGGAUCUCUGGGCCUGUCUUGUACAGCCAGCGGCU UCAGCUUCAGCAACAACUACUACAUGUGCUGGGUUCGCAG AGCCCCUGGCAAGUGUCUGGAAUGGGUCGGAUGCAUCUAC ACCAACAACAUCGGCAGCACUUGGUACGCCAGCUGGGCCA AGGGCAGAUUCACCAUCAGCAGGGACGACAGCAAGAACAC CGUGUACCUGCAGAUGAACAGCCUGAAAACCGAGGAUACC GCCGUGUACUACUGCGCCAGAGGCAUCUACAGCGACAGCA GCGACUUCAUCGGCAACCUGUGGGGACAGGGCACACUGGU CACAGUUUCUAGC CD3 CLONE 3 variable AAS EVQLVESGGGLVKPGGSLGLSCTASGFSFSNNYYMCWVRRAP heavy chain (VH) GKCLEWVGCIYTNNIGSTWYASWAKGRFTISRDDSKNTVYLQM NSLKTEDTAVYYCARGIYSDSSDFIGNLWGQGTLVTVSS CD3 CLONE 3 variable RNA GAUAUUCAGAUGACACAGAGCCCUAGCAGCCUGAGCGCCU heavy chain (VL) CUGUGGGAGACAGAGUGACCAUUACCUGUCAGGCCAGCCA GACCAUCUACAACUACCUGGCCUGGUAUCAGCAGAAGCCC GGCAAGCAGCCUAAGCUGCUGAUCUACAAGGCCAGCACAC UGGCCAGCGGAGUGCCCAGUAGAUUCAAAGGCUCUGGCAG CGGCACCGACUUCACCCUGACCAUAUCUAGCCUGCAGCCA GAGGACGUGGCCACCUACUACUGCCAGCAGUACAACACCA UCAUCAACGUGGACCGGACCUUCGGCUGUGGCACAGAGGU UGUGGUCAAA CD3 CLONE 3 variable AAS DIQMTQSPSSLSASVGDRVTITCQASQTIYNYLAWYQQKPGKQ heavy chain (VL) PKLLIYKASTLASGVPSRFKGSGSGTDFTLTISSLQPEDVATYYC QQYNTIINVDRTFGCGTEWVK

[1205] scFv-CD3 CLONE 3(VH- RNA GAAGUUCAGCUGGUUGAAUCAGGCGGCGGACUGGUUAAGC (G4S)4-VL), full sequence CUGGCGGAUCUCUGGGCCUGUCUUGUACAGCCAGCGGCU UCAGCUUCAGCAACAACUACUACAUGUGCUGGGUUCGCAG AGCCCCUGGCAAGUGUCUGGAAUGGGUCGGAUGCAUCUAC ACCAACAACAUCGGCAGCACUUGGUACGCCAGCUGGGCCA AGGGCAGAUUCACCAUCAGCAGGGACGACAGCAAGAACAC CGUGUACCUGCAGAUGAACAGCCUGAAAACCGAGGAUACC GCCGUGUACUACUGCGCCAGAGGCAUCUACAGCGACAGCA GCGACUUCAUCGGCAACCUGUGGGGACAGGGCACACUGGU CACAGUUUCUAGCGGUGGCGGAGGAAGCGGAGGCGGAGG UUCAGGUGGUGGUGGAAGCGGUGGUGGCGGCAGCGAUAU UCAGAUGACACAGAGCCCUAGCAGCCUGAGCGCCUCUGUG GGAGACAGAGUGACCAUUACCUGUCAGGCCAGCCAGACCA UCUACAACUACCUGGCCUGGUAUCAGCAGAAGCCCGGCAA GCAGCCUAAGCUGCUGAUCUACAAGGCCAGCACACUGGCC AGCGGAGUGCCCAGUAGAUUCAAAGGCUCUGGCAGCGGCA CCGACUUCACCCUGACCAUAUCUAGCCUGCAGCCAGAGGA CGUGGCCACCUACUACUGCCAGCAGUACAACACCAUCAUCA ACGUGGACCGGACCUUCGGCUGUGGCACAGAGGUUGUGG

[1206]

[1207] UCAAAscFv-CD3 CLONE 3(VH- AAS EVQLVESGGGLVKPGGSLGLSCTASGFSFSNNYYMCWVRRAP (G4S)4-VL), full sequence GKCLEWVGCIYTNNIGSTWYASWAKGRFTISRDDSKNTVYLQM NSLKTEDTAVYYCARGIYSDSSDFIGNLWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQAS QTIYNYLAWYQQKPGKQPKLLIYKASTLASGVPSRFKGSGSGTD FTLTISSLQPEDVATYYCQQYNTIINVDRTFGCGTEVWK

[1208] scFv-CD3 CLONE 3(VL- RNA GAUAUUCAGAUGACACAGAGCCCUAGCAGCCUGAGCGCCU (G4S)5-VH), full sequence CUGUGGGAGACAGAGUGACCAUUACCUGUCAGGCCAGCCA GACCAUCUACAACUACCUGGCCUGGUAUCAGCAGAAGCCC GGCAAGCAGCCUAAGCUGCUGAUCUACAAGGCCAGCACAC UGGCCAGCGGAGUGCCCAGUAGAUUCAAAGGCUCUGGCAG CGGCACCGACUUCACCCUGACCAUAUCUAGCCUGCAGCCA GAGGACGUGGCCACCUACUACUGCCAGCAGUACAACACCA UCAUCAACGUGGACCGGACCUUCGGCUGUGGCACAGAGGU UGUGGUCAAAGGUGGCGGAGGAAGCGGAGGCGGCGGUUC AGGCGGUGGUGGUAGCGGUGGUGGCGGCUCAGGCGGCGG AGGUUCUGAAGUUCAGCUGGUUGAAUCAGGCGGCGGACUG GUUAAGCCUGGCGGAUCUCUGGGCCUGUCUUGUACAGCCA GCGGCUUCAGCUUCAGCAACAACUACUACAUGUGCUGGGU UCGCAGAGCCCCUGGCAAGUGUCUGGAAUGGGUCGGAUGC AUCUACACCAACAACAUCGGCAGCACUUGGUACGCCAGCU GGGCCAAGGGCAGAUUCACCAUCAGCAGGGACGACAGCAA GAACACCGUGUACCUGCAGAUGAACAGCCUGAAAACCGAG GAUACCGCCGUGUACUACUGCGCCAGAGGCAUCUACAGCG ACAGCAGCGACUUCAUCGGCAACCUGUGGGGACAGGGCAC ACUGGUCACAGUUUCUAGC

[1209] scFv-CD3 CLONE 3(VL- AAS DIQMTQSPSSLSASVGDRVTITCQASQTIYNYLAWYQQKPGKQ (G4S)5-VH), full sequence PKLLIYKASTLASGVPSRFKGSGSGTDFTLTISSLQPEDVATYYC QQYNTIINVDRTFGCGTEWVKGGGGSGGGGSGGGGSGGGG SGGGGSEVQLVESGGGLVKPGGSLGLSCTASGFSFSNNYYMC WVRRAPGKCLEWVGCIYTNNIGSTWYASWAKGRFTISRDDSKN TVYLQMNSLKTEDTAVYYCARGIYSDSSDFIGNLWGQGTLVTVS S CA19-9 VH-CDR1 (Kabat) AAS AYAMH

[1210] CA19-9 VH-CDR1 (IMGT) AAS GFTFEAYA

[1211] CA19-9 VH-CDR2 (Kabat) AAS SINWNSGRIAYADSVKG

[1212] CA19-9 VH-CDR2 (IMGT) AAS INWNSGRI

[1213] CA19-9 VH-CDR3 (Kabat) AAS DIRRFSTGGAEFEY

[1214] CA19-9 VH-CDR3 (IMGT) AAS AKDIRRFSTGGAEFEY

[1215] CA19-9 VL-CDR1 (Kabat) AAS SGSSSNIGSNFVY

[1216] CA19-9 VL-CDR1 (IMGT) AAS SSNIGSNF

[1217] CA19-9 VL-CDR2 (Kabat) AAS RNNQRPS

[1218] CA19-9 VL-CDR2 (IMGT) AAS RN

[1219] CA19-9 VL-CDR3 (Kabat) AAS AAWDDSLGGHYV

[1220] CA19-9 VL-CDR3 (IMGT) AAS AAWDDSLGGHYV

[1221]

[1222] CA19-9 variable RNA CAGGUUCAGCUUGUGGAAUCUGGCGGCGGAUCUGUGCAG heavy chain (VH) CCUGGCAGAUCUCUGAGACUGAGCUGUGAAGCCUCCGGCU UCACCUUUGAGGCCUACGCCAUGCACUGGGUCCGACAGCC ACCUGGCAAAGGCCUUGAAUGGGUGUCCAGCAUCAACUGG AACAGCGGCAGAAUCGCCUACGCCGACUCUGUGAAGGGCA GAUUCACCAUCAGCCGGGACAACGCCAGAAACAGCCUGUA CCUGCAGAUGAACAGCCUGCGGCUGGAAGAUACCGCCUUC UACUACUGCGCCAAGGACAUCAGACGGUUCAGCACAGGCG GAGCCGAGUUUGAGUAUUGGGGCCAGGGAACCCUGGUCAC CGUGUCUAGC CA19-9 variable AAS QVQLVESGGGSVQPGRSLRLSCEASGFTFEAYAMHWVRQPPG heavy chain (VH) KGLEWVSSINWNSGRIAYADSVKGRFTISRDNARNSLYLQMNS LRLEDTAFYYCAKDIRRFSTGGAEFEYWGQGTLVTVSS CA19-9 variable RNA CAGUCUGUUCUGACACAGCCUCCUAGCGCCUCUGGCACAC light chain (VL) CUGGACAGAGAGUGACCAUCAGCUGUAGCGGCAGCAGCAG CAACAUCGGCAGCAACUUCGUGUACUGGUAUCAGCAGCUG CCCGGCACAGCCCCUAAACUGCUGAUCUACCGGAACAACCA GCGGCCUAGCGGCGUGCCAGAUAGAUUUUCUGGCAGCAGA AGCGGCACCUCUGCCAGCCUGGCUAUCUCUGGCCUGAGAU CUGAGGACGAGGCCGACUACUAUUGCGCCGCCUGGGAUGA UUCUCUCGGCGGCCACUAUGUGUUUGGCACCGGCACCAAA GUGACCGUGCUG CA19-9 variable AAS QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNFVYWYQQLPGT light chain (VL) APKLLIYRNNQRPSGVPDRFSGSRSGTSASLAISGLRSEDEADY YCAAWDDSLGGHYVFGTGTKVTVL

[1223] Signal peptide (SP) RNA AUGGACUGGAUCUGGCGCAUCCUGUUUCUUGUGGGAGCC GCCACAGGCGCCCAUUCU

[1224] Signal peptide (SP) AAS MDWIWRILFLVGAATGAHS

[1225] CH1-3 hole(hlgGI) RNA GCUUCUACAAAGGGCCCCAGCGUGUUCCCUCUGGCUCCUA GCAGCAAGAGCACAAGCGGAGGAACAGCCGCUCUGGGCUG UCUGGUCAAGGACUACUUUCCCGAGCCUGUGACCGUGUCC UGGAAUUCUGGCGCUCUGACAAGCGGCGUGCACACCUUUC CAGCUGUGCUGCAAAGCAGCGGCCUGUACUCUCUGAGCAG CGUGGUCACAGUGCCAAGCUCUAGCCUGGGCACCCAGACC UACAUCUGCAAUGUGAACCACAAGCCUAGCAACACCAAGGU GGACAAGAAGGUGGAACCCAAGAGCUGCGACAAGACCCAC ACCUGUCCUCCAUGUCCUGCUCCAGAAGCCGCCGGAGGAC CUUCCGUGUUUCUGUUCCCUCCAAAGCCUAAGGACACCCU GAUGAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUGGUG GAUGUGUCUCACGAGGACCCCGAAGUGAAGUUCAAUUGGU ACGUGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCC UAGAGAGGAACAGUACAACAGCACCUACAGAGUGGUGUCC GUGCUGACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAG AGUACAAGUGCAAGGUGUCCAACAAGGCCCUGCCUGCUCC UAUCGAGAAAACCAUCAGCAAGGCCAAGGGCCAGCCUCGC GAACCUCAAGUCUGCACACUUCCACCAAGCCGGGACGAGC UGACCAAGAAUCAGGUGUCACUCAGCUGCGCCGUGAAGGG CUUCUACCCUUCUGAUAUCGCCGUGGAAUGGGAGAGCAAC GGCCAGCCUGAGAACAACUACAAGACAACCCCUCCUGUGC UGGACAGCGACGGCUCAUUCUUCCUGGUGUCCAAGCUGAC AGUGGACAAGUCCAGAUGGCAGCAGGGCAACGUGUUCAGC UGCAGCGUGAUGCACGAGGCCCUGCACAAUCACUACACCC AGAAGUCCCUGUCUCUGAGCCCCGGCAAGUGAUGA CH1-3 hole(hlgGI) AAS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK

[1226]

[1227] PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK

[1228] Ckappa (hlgG) RNA AGAACAGUGGCCGCUCCUAGCGUGUUCAUCUUCCCACCUU CCGACGAGCAGCUGAAGUCUGGCACAGCCAGCGUUGUGUG CCUGCUGAACAACUUCUACCCCAGAGAAGCCAAGGUGCAG UGGAAGGUGGACAACGCCCUGCAGAGCGGCAAUAGCCAAG AGAGCGUGACCGAGCAGGACAGCAAGGACUCUACCUACAG CCUGAGCAGCACCCUGACACUGAGCAAGGCCGAUUACGAG AAGCACAAAGUGUACGCCUGCGAAGUGACCCACCAGGGCC UUUCUAGCCCUGUGACCAAGAGCUUCAACCGGGGCGAGUG CUGAUGA

[1229] Ckappa (hlgG) AAS RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC CH1 (hlgG1) RNA GCUUCUACAAAGGGCCCCAGCGUGUUCCCUCUGGCUCCUA GCAGCAAGUCUACAAGCGGAGGAACAGCCGCUCUGGGCUG CCUGGUCAAGGAUUACUUUCCCGAGCCUGUGACCGUGUCC UGGAAUUCUGGCGCUCUGACAAGCGGCGUGCACACCUUUC CAGCUGUGCUGCAAAGCAGCGGCCUGUACUCUCUGAGCAG CGUGGUCACAGUGCCAAGCUCUAGCCUGGGCACCCAGACC UACAUCUGCAAUGUGAACCACAAGCCUAGCAACACCAAGGU GGACAAGAAGGUGGAACCCAAGAGCUGUGAU CH1 (hlgG1) AAS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCD

[1230] Hinge-CH2-3 knob (hlgG1) RNA AAAACCCACACCUGUCCUCCAUGUCCUGCUCCGGAAGCUG CUGGUGGCCCUUCCGUGUUUCUGUUCCCUCCAAAGCCUAA GGACACCCUGAUGAUCAGCAGGACCCCUGAAGUGACCUGC GUGGUGGUCGAUGUGUCUCACGAGGACCCAGAAGUGAAGU UCAAUUGGUACGUGGACGGCGUGGAAGUGCACAACGCCAA GACCAAGCCUAGAGAGGAACAGUACAACAGCACCUACAGAG UGGUGUCCGUGCUGACCGUGCUGCACCAGGAUUGGCUGAA CGGCAAAGAGUACAAGUGCAAGGUGUCCAACAAGGCCCUG CCUGCUCCUAUCGAGAAAACCAUCAGCAAGGCCAAGGGCCA GCCUAGGGAACCCCAGGUUUACACCCUUCCACCUUGCCGG GAUGAGCUGACCAAGAACCAGGUGUCCCUGUGGUGUCUCG UGAAGGGCUUCUACCCCUCCGAUAUCGCCGUGGAAUGGGA GAGCAAUGGCCAGCCUGAGAACAACUACAAGACAACCCCUC CUGUGCUGGACAGCGACGGCUCAUUCUUCCUGUACUCCAA GCUGACAGUGGAUAAGUCCCGGUGGCAGCAGGGCAACGUG UUCAGCUGUUCUGUGAUGCACGAGGCCCUGCACAACCACU ACACCCAGAAAAGCCUGUCUCUGAGCCCCGGCAAGUGAUG A

[1231] Hinge-CH2-3 knob (hlgG1) AAS KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCWVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK

[1232] 5’-UTR-husec2-CA19- RNA AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGA 9(VH)-CH1-3 hole (hlgG1)- GAACCCGCCACCAUGGACUGGAUCUGGCGCAUCCUGUUUC 3’-UTR-polyA, full heavy UUGUGGGAGCCGCCACAGGCGCCCAUUCUCAGGUUCAGCU chain sequence UGUGGAAUCUGGCGGCGGAUCUGUGCAGCCUGGCAGAUCU CUGAGACUGAGCUGUGAAGCCUCCGGCUUCACCUUUGAGG CCUACGCCAUGCACUGGGUCCGACAGCCACCUGGCAAAGG CCUUGAAUGGGUGUCCAGCAUCAACUGGAACAGCGGCAGA

[1233]

[1234] AUCGCCUACGCCGACUCUGUGAAGGGCAGAUUCACCAUCAGCCGGGACAACGCCAGAAACAGCCUGUACCUGCAGAUGAA CAGCCUGCGGCUGGAAGAUACCGCCUUCUACUACUGCGCC AAGGACAUCAGACGGUUCAGCACAGGCGGAGCCGAGUUUG AGUAUUGGGGCCAGGGAACCCUGGUCACCGUGUCUAGCGC UUCUACAAAGGGCCCCAGCGUGUUCCCUCUGGCUCCUAGC AGCAAGAGCACAAGCGGAGGAACAGCCGCUCUGGGCUGUC UGGUCAAGGACUACUUUCCCGAGCCUGUGACCGUGUCCUG GAAUUCUGGCGCUCUGACAAGCGGCGUGCACACCUUUCCA GCUGUGCUGCAAAGCAGCGGCCUGUACUCUCUGAGCAGCG UGGUCACAGUGCCAAGCUCUAGCCUGGGCACCCAGACCUA CAUCUGCAAUGUGAACCACAAGCCUAGCAACACCAAGGUGG ACAAGAAGGUGGAACCCAAGAGCUGCGACAAGACCCACACC UGUCCUCCAUGUCCUGCUCCAGAAGCCGCCGGAGGACCUU CCGUGUUUCUGUUCCCUCCAAAGCCUAAGGACACCCUGAU GAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUGGUGGAU GUGUCUCACGAGGACCCCGAAGUGAAGUUCAAUUGGUACG UGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAG AGAGGAACAGUACAACAGCACCUACAGAGUGGUGUCCGUG CUGACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAGAGU ACAAGUGCAAGGUGUCCAACAAGGCCCUGCCUGCUCCUAU CGAGAAAACCAUCAGCAAGGCCAAGGGCCAGCCUCGCGAA CCUCAAGUCUGCACACUUCCACCAAGCCGGGACGAGCUGA CCAAGAAUCAGGUGUCACUCAGCUGCGCCGUGAAGGGCUU CUACCCUUCUGAUAUCGCCGUGGAAUGGGAGAGCAACGGC CAGCCUGAGAACAACUACAAGACAACCCCUCCUGUGCUGGA CAGCGACGGCUCAUUCUUCCUGGUGUCCAAGCUGACAGUG GACAAGUCCAGAUGGCAGCAGGGCAACGUGUUCAGCUGCA GCGUGAUGCACGAGGCCCUGCACAAUCACUACACCCAGAA GUCCCUGUCUCUGAGCCCCGGCAAGUGAUGAGGAUCCGAU CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCG UCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGG UAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUA GUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAA CGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAU UAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUA ACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGA GCUAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAU GACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[1235] CA19-9(VH)-CH1-3 hole AAS QVQLVESGGGSVQPGRSLRLSCEASGFTFEAYAMHWVRQPPG (hlgG1), full heavy chain KGLEWVSSINWNSGRIAYADSVKGRFTISRDNARNSLYLQMNSL sequence RLEDTAFYYCAKDIRRFSTGGAEFEYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK

[1236] 5’-UTR-husec2-CA19- RNA AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGA 9(VH)-CH1-(G4S)2-scFv- GAACCCGCCACCAUGGACUGGAUCUGGCGCAUCCUGUUUC CD3 CLONE 1(VH-(G4S)4- UUGUGGGAGCCGCCACAGGCGCCCAUUCUCAGGUUCAGCU VL)-(G4S)2-Hinge-CH2-3 UGUGGAAUCUGGCGGCGGAUCUGUGCAGCCUGGCAGAUCU knob (hlgG1)- 3’-UTR- CUGAGACUGAGCUGUGAAGCCUCCGGCUUCACCUUUGAGG polyA, full heavy chain CCUACGCCAUGCACUGGGUCCGACAGCCACCUGGCAAAGG sequence CCUUGAAUGGGUGUCCAGCAUCAACUGGAACAGCGGCAGA

[1237] AUCGCCUACGCCGACUCUGUGAAGGGCAGAUUCACCAUCA

[1238]

[1239] GCCGGGACAACGCCAGAAACAGCCUGUACCUGCAGAUGAACAGCCUGCGGCUGGAAGAUACCGCCUUCUACUACUGCGCC AAGGACAUCAGACGGUUCAGCACAGGCGGAGCCGAGUUUG AGUAUUGGGGCCAGGGAACCCUGGUCACCGUGUCUAGCGC UUCUACAAAGGGCCCCAGCGUGUUCCCUCUGGCUCCUAGC AGCAAGUCUACAAGCGGAGGAACAGCCGCUCUGGGCUGCC UGGUCAAGGAUUACUUUCCCGAGCCUGUGACCGUGUCCUG GAAUUCUGGCGCUCUGACAAGCGGCGUGCACACCUUUCCA GCUGUGCUGCAAAGCAGCGGCCUGUACUCUCUGAGCAGCG UGGUCACAGUGCCAAGCUCUAGCCUGGGCACCCAGACCUA CAUCUGCAAUGUGAACCACAAGCCUAGCAACACCAAGGUGG ACAAGAAGGUGGAACCCAAGAGCUGUGAUGGCGGCGGAGG AUCUGGCGGAGGCGGAUCUGAAGUUCAGCUGGUGGAAUCU GGCGGCGGACUGGUUAAGCCUGGUGGAUCUCUGGGCCUG AGCUGUACCGCCAGCGGCUUCAGCUUCAGCAACAACUACU ACAUGUGCUGGGUUCGCAGAGCCCCUGGCAAAUGUCUUGA GUGGGUCGGAUGCAUCUACACCAACAACAUCGGCAGCACU UGGUACGCCAGCUGGGCCAAGGGCAGAUUCACCAUCAGCA GGGACGACAGCAAGAACACCGUGUACCUGCAGAUGAACAG CCUGAAAACCGAGGACACCGCCGUGUACUACUGCGCCAGA GGCAUCUACAGCGACAGCAGCGACUUCAUCGGCAACCUGU GGGGACAGGGCACACUGGUCACAGUUUCUAGCGGAGGCGG AGGUUCAGGUGGCGGAGGAAGUGGCGGCGGAGGAUCUGG CGGAGGUGGAAGCGAAAUCGUGAUGACACAGAGCCCCGCC ACACUGAGUGUGUCUCCAGGCGAAAAGGCCACACUGUCCU GUCAGGCCAGCCAGACCAUCUACAACUACCUGGCCUGGUA UCAGCAGAAGCCCGGCCAACCUCCUCGGCUGCUGAUCUAC AAAGCCAGCACACUGGCCAGCGGCAUCCCUGCCAGAUUUU CUGGAAGCGGCAGCGGCACCGAGUUCACCCUGACAAUCAG CAACCUGCAGAGCGAGGACUUUGCCGUGUAUUAUUGCCAG CAGUACAACACCAUCAUCAACGUGGACCGGACCUUCGGCU GUGGCACCAAGGUGGAAAUCAAGGGCGGUGGCGGUUCAGG UGGCGGAGGCUCUAAAACCCACACCUGUCCUCCAUGUCCU GCUCCGGAAGCUGCUGGUGGCCCUUCCGUGUUUCUGUUC CCUCCAAAGCCUAAGGACACCCUGAUGAUCAGCAGGACCCC UGAAGUGACCUGCGUGGUGGUCGAUGUGUCUCACGAGGAC CCAGAAGUGAAGUUCAAUUGGUACGUGGACGGCGUGGAAG UGCACAACGCCAAGACCAAGCCUAGAGAGGAACAGUACAAC AGCACCUACAGAGUGGUGUCCGUGCUGACCGUGCUGCACC AGGAUUGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUC CAACAAGGCCCUGCCUGCUCCUAUCGAGAAAACCAUCAGCA AGGCCAAGGGCCAGCCUAGGGAACCCCAGGUUUACACCCU UCCACCUUGCCGGGAUGAGCUGACCAAGAACCAGGUGUCC CUGUGGUGUCUCGUGAAGGGCUUCUACCCCUCCGAUAUCG CCGUGGAAUGGGAGAGCAAUGGCCAGCCUGAGAACAACUA CAAGACAACCCCUCCUGUGCUGGACAGCGACGGCUCAUUC UUCCUGUACUCCAAGCUGACAGUGGAUAAGUCCCGGUGGC AGCAGGGCAACGUGUUCAGCUGUUCUGUGAUGCACGAGGC CCUGCACAACCACUACACCCAGAAAAGCCUGUCUCUGAGCC CCGGCAAGUGAUGAGGAUCCGAUCUGGUACUGCAUGCACG CAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGU CUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCAC CUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCA AGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACA CCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAAC GAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAU UUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAA CA19-9(VH)-CH1-(G4S)2- AAS QVQLVESGGGSVQPGRSLRLSCEASGFTFEAYAMHWVRQPPG

[1240]

[1241] scFv-CD3 CLONE 1(VH- KGLEWVSSINWNSGRIAYADSVKGRFTISRDNARNSLYLQMNSL(G4S)4-VL)-(G4S)2-Hinge- RLEDTAFYYCAKDIRRFSTGGAEFEYWGQGTLVTVSSASTKGP CH2-3 knob (hlgG1), full SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG heavy chain sequence VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDGGGGSGGGGSEVQLVESGGGLVKPGGSLGLS CTASGFSFSNNYYMCWVRRAPGKCLEWVGCIYTNNIGSTWYA SWAKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCARGIYSDSS DFIGNLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVM TQSPATLSVSPGEKATLSCQASQTIYNYLAWYQQKPGQPPRLLI YKASTLASGIPARFSGSGSGTEFTLTISNLQSEDFAVYYCQQYN TIINVDRTFGCGTKVEIKGGGGSGGGGSKTHTCPPCPAPEAAG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1242] 5’-UTR-husec2-CA19- RNA AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGA 9(VH)-CH1-(G4S)2-scFv- GAACCCGCCACCAUGGACUGGAUCUGGCGCAUCCUGUUUC CD3 CLONE 1(VL-(G4S)5- UUGUGGGAGCCGCCACAGGCGCCCAUUCUCAGGUUCAGCU VH)-(G4S)2-Hinge-CH2-3 UGUGGAAUCUGGCGGCGGAUCUGUGCAGCCUGGCAGAUCU knob (hlgG1)- 3’-UTR- CUGAGACUGAGCUGUGAAGCCUCCGGCUUCACCUUUGAGG polyA, full heavy chain CCUACGCCAUGCACUGGGUCCGACAGCCACCUGGCAAAGG sequence CCUUGAAUGGGUGUCCAGCAUCAACUGGAACAGCGGCAGA AUCGCCUACGCCGACUCUGUGAAGGGCAGAUUCACCAUCA GCCGGGACAACGCCAGAAACAGCCUGUACCUGCAGAUGAA CAGCCUGCGGCUGGAAGAUACCGCCUUCUACUACUGCGCC AAGGACAUCAGACGGUUCAGCACAGGCGGAGCCGAGUUUG AGUAUUGGGGCCAGGGAACCCUGGUCACCGUGUCUAGCGC UUCUACAAAGGGCCCCAGCGUGUUCCCUCUGGCUCCUAGC AGCAAGUCUACAAGCGGAGGAACAGCCGCUCUGGGCUGCC UGGUCAAGGAUUACUUUCCCGAGCCUGUGACCGUGUCCUG GAAUUCUGGCGCUCUGACAAGCGGCGUGCACACCUUUCCA GCUGUGCUGCAAAGCAGCGGCCUGUACUCUCUGAGCAGCG UGGUCACAGUGCCAAGCUCUAGCCUGGGCACCCAGACCUA CAUCUGCAAUGUGAACCACAAGCCUAGCAACACCAAGGUGG ACAAGAAGGUGGAACCCAAGAGCUGUGAUGGCGGCGGAGG AUCUGGCGGAGGCGGAUCUGAGAUUGUGAUGACACAGAGC CCCGCCACACUGUCCGUUUCUCCAGGCGAAAAAGCCACAC UGAGCUGCCAGGCCAGCCAGACCAUCUACAAUUACCUGGC CUGGUAUCAGCAGAAGCCCGGCCAACCUCCUCGGCUGCUG AUCUACAAAGCCAGCACACUGGCCAGCGGCAUCCCUGCCA GAUUUUCUGGAAGCGGCAGCGGCACCGAGUUCACCCUGAC AAUCAGCAACCUGCAGAGCGAGGACUUCGCCGUGUACUAC UGCCAGCAGUACAACACCAUCAUCAACGUGGACCGGACCUU CGGCUGUGGCACCAAGGUUGAAAUCAAAGGCGGCGGAGGA AGCGGAGGCGGAGGUUCUGGUGGUGGUGGCUCUGGUGGC GGAGGAUCUGGCGGAGGCGGUUCUGAAGUUCAGCUGGUG GAAUCUGGCGGCGGACUGGUUAAGCCUGGUGGAUCUCUG GGCCUGAGCUGUACCGCCAGCGGCUUCAGCUUCAGCAACA ACUACUACAUGUGCUGGGUUCGCAGAGCCCCUGGCAAAUG UCUUGAGUGGGUCGGAUGCAUCUACACCAACAACAUCGGC AGCACUUGGUACGCCAGCUGGGCCAAGGGCAGAUUCACCA UCAGCAGGGACGACAGCAAGAACACCGUGUACCUGCAGAU GAACAGCCUGAAAACCGAGGACACAGCCGUGUAUUAUUGC GCCAGAGGCAUCUACAGCGACAGCAGCGACUUCAUCGGCA ACCUGUGGGGACAGGGCACACUGGUCACAGUGUCAUCUGG CGGUGGCGGUUCAGGUGGCGGAGGCUCUAAAACCCACACC UGUCCUCCAUGUCCUGCUCCGGAAGCUGCUGGUGGCCCUU CCGUGUUUCUGUUCCCUCCAAAGCCUAAGGACACCCUGAU GAUCAGCAGGACCCCUGAAGUGACCUGCGUGGUGGUCGAU GUGUCUCACGAGGACCCAGAAGUGAAGUUCAAUUGGUACG

[1243]

[1244] UGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAGAGAGGAACAGUACAACAGCACCUACAGAGUGGUGUCCGUG CUGACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAGAGU ACAAGUGCAAGGUGUCCAACAAGGCCCUGCCUGCUCCUAU CGAGAAAACCAUCAGCAAGGCCAAGGGCCAGCCUAGGGAA CCCCAGGUUUACACCCUUCCACCUUGCCGGGAUGAGCUGA CCAAGAACCAGGUGUCCCUGUGGUGUCUCGUGAAGGGCUU CUACCCCUCCGAUAUCGCCGUGGAAUGGGAGAGCAAUGGC CAGCCUGAGAACAACUACAAGACAACCCCUCCUGUGCUGGA CAGCGACGGCUCAUUCUUCCUGUACUCCAAGCUGACAGUG GAUAAGUCCCGGUGGCAGCAGGGCAACGUGUUCAGCUGUU CUGUGAUGCACGAGGCCCUGCACAACCACUACACCCAGAAA AGCCUGUCUCUGAGCCCCGGCAAGUGAUGAGGAUCCGAUC UGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGU CCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGU AUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUA GUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAA CGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAU UAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUA ACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGA GCUAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAU GACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA CA19-9(VH)-CH1-(G4S)2- AAS QVQLVESGGGSVQPGRSLRLSCEASGFTFEAYAMHWVRQPPG scFv-CD3 CLONE 1(VL- KGLEWVSSINWNSGRIAYADSVKGRFTISRDNARNSLYLQMNSL (G4S)5-VH)-(G4S)2-Hinge- RLEDTAFYYCAKDIRRFSTGGAEFEYWGQGTLVTVSSASTKGP CH2-3 knob (hlgG1), full SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG heavy chain sequence VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDGGGGSGGGGSEIVMTQSPATLSVSPGEKATLS CQASQTIYNYLAWYQQKPGQPPRLLIYKASTLASGIPARFSGSG SGTEFTLTISNLQSEDFAVYYCQQYNTIINVDRTFGCGTKVEIKG GGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGG SLGLSCTASGFSFSNNYYMCWVRRAPGKCLEWVGCIYTNNIGS TWYASWAKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCARGI YSDSSDFIGNLWGQGTLVTVSSGGGGSGGGGSKTHTCPPCPA PEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKN QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1245] 5’-UTR-husec2-CA19- RNA AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGA 9(VH)-CH1-(G4S)2-scFv- GAACCCGCCACCAUGGACUGGAUCUGGCGCAUCCUGUUUC CD3 CLONE 2(VH-(G4S)4- UUGUGGGAGCCGCCACAGGCGCCCAUUCUCAGGUUCAGCU VL)-(G4S)2-Hinge-CH2-3 UGUGGAAUCUGGCGGCGGAUCUGUGCAGCCUGGCAGAUCU knob (hlgG1)- 3’-UTR- CUGAGACUGAGCUGUGAAGCCUCCGGCUUCACCUUUGAGG polyA, full heavy chain CCUACGCCAUGCACUGGGUCCGACAGCCACCUGGCAAAGG sequence CCUUGAAUGGGUGUCCAGCAUCAACUGGAACAGCGGCAGA AUCGCCUACGCCGACUCUGUGAAGGGCAGAUUCACCAUCA GCCGGGACAACGCCAGAAACAGCCUGUACCUGCAGAUGAA CAGCCUGCGGCUGGAAGAUACCGCCUUCUACUACUGCGCC AAGGACAUCAGACGGUUCAGCACAGGCGGAGCCGAGUUUG AGUAUUGGGGCCAGGGAACCCUGGUCACCGUGUCUAGCGC UUCUACAAAGGGCCCCAGCGUGUUCCCUCUGGCUCCUAGC AGCAAGUCUACAAGCGGAGGAACAGCCGCUCUGGGCUGCC UGGUCAAGGAUUACUUUCCCGAGCCUGUGACCGUGUCCUG GAAUUCUGGCGCUCUGACAAGCGGCGUGCACACCUUUCCA GCUGUGCUGCAAAGCAGCGGCCUGUACUCUCUGAGCAGCG UGGUCACAGUGCCAAGCUCUAGCCUGGGCACCCAGACCUA CAUCUGCAAUGUGAACCACAAGCCUAGCAACACCAAGGUGG ACAAGAAGGUGGAACCCAAGAGCUGUGAUGGCGGCGGAGG

[1246]

[1247] AUCUGGCGGAGGCGGAUCUCAAGUUCAGCUGGUUCAAUCAGGCGGCGGACUGGUUCAACCUGGCGGACCUCUGAGACUGA GCUGUACCGCCAGCGGCUUCAGCUUCAGCAACAACUACUA CAUGUGCUGGGUCCGACAGGCCCCUGGCAAGUGUCUUGAA UGGGUGUCCUGCAUCUACACCAACAACAUCGGCAGCACUU GGUACGCCAGCUGGGCCAAGGGCAGAUUCACCAUCAGCCG GGACAACAGCAAGAACACCCUGUACCUGCAGAUGAACAGCC UGAGAGCCGAGGAUACCGCCGUGUACUAUUGUGCCAGAGG CAUCUACAGCGACAGCAGCGACUUCAUCGGCAACCUGUGG GGACAGGGCACACUGGUCACAGUUUCUAGCGGUGGCGGAG GAAGCGGAGGCGGAGGUUCAGGUGGUGGUGGAAGCGGUG GUGGCGGAUCUGCCAUUCAGAUGACACAGAGCCCUAGCAG CCUGAGCGCCUCUGUGGGAGACAGAGUGACCAUUACCUGU CAGGCCAGCCAGACCAUCUACAACUACCUGGCCUGGUAUC AGCAGAAACCCGGCAAGGUGCCAGAGCUGCUGAUCUACAA GGCCAGCACACUGGCUAGCGGCGUGCCCAGUAGAUUCAUG GGCACAGGCUUCGGCACCGACUUCACCCUGACCAUCGAUA GCCUGCAGCCUGAAGAUGCCGCCACCUACUACUGCCAGCA GUACAACACCAUCAUCAACGUGGACCGGACCUUCGGCUGU GGAACCAAGGUGGAAAUCAAAGGCGGUGGCGGUUCAGGUG GCGGAGGCUCUAAAACCCACACCUGUCCUCCAUGUCCUGC UCCGGAAGCUGCUGGUGGCCCUUCCGUGUUUCUGUUCCCU CCAAAGCCUAAGGACACCCUGAUGAUCAGCAGGACCCCUGA AGUGACCUGCGUGGUGGUCGAUGUGUCUCACGAGGACCCA GAAGUGAAGUUCAAUUGGUACGUGGACGGCGUGGAAGUGC ACAACGCCAAGACCAAGCCUAGAGAGGAACAGUACAACAGC ACCUACAGAGUGGUGUCCGUGCUGACCGUGCUGCACCAGG AUUGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUCCAA CAAGGCCCUGCCUGCUCCUAUCGAGAAAACCAUCAGCAAG GCCAAGGGCCAGCCUAGGGAACCCCAGGUUUACACCCUUC CACCUUGCCGGGAUGAGCUGACCAAGAACCAGGUGUCCCU GUGGUGUCUCGUGAAGGGCUUCUACCCCUCCGAUAUCGCC GUGGAAUGGGAGAGCAAUGGCCAGCCUGAGAACAACUACA AGACAACCCCUCCUGUGCUGGACAGCGACGGCUCAUUCUU CCUGUACUCCAAGCUGACAGUGGAUAAGUCCCGGUGGCAG CAGGGCAACGUGUUCAGCUGUUCUGUGAUGCACGAGGCCC UGCACAACCACUACACCCAGAAAAGCCUGUCUCUGAGCCCC GGCAAGUGAUGAGGAUCCGAUCUGGUACUGCAUGCACGCA AUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCU CCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCU GCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAG CACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACC CCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGA AAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUU CGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAA CA19-9(VH)-CH1-(G4S)2- AAS QVQLVESGGGSVQPGRSLRLSCEASGFTFEAYAMHWVRQPPG scFv-CD3 CLONE 2(VH- KGLEWVSSINWNSGRIAYADSVKGRFTISRDNARNSLYLQMNSL (G4S)4-VL)-(G4S)2-Hinge- RLEDTAFYYCAKDIRRFSTGGAEFEYWGQGTLVTVSSASTKGP CH2-3 knob (hlgG1), full SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG heavy chain sequence VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDGGGGSGGGGSQVQLVQSGGGLVQPGGPLRL SCTASGFSFSNNYYMCWVRQAPGKCLEWVSCIYTNNIGSTWY ASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGIYSDS SDFIGNLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSAIQ MTQSPSSLSASVGDRVTITCQASQTIYNYLAWYQQKPGKVPELL IYKASTLASGVPSRFMGTGFGTDFTLTIDSLQPEDAATYYCQQY NTIINVDRTFGCGTKVEIKGGGGSGGGGSKTHTCPPCPAPEAA GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWY

[1248]

[1249] VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVS LWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1250] 5’-UTR-husec2-CA19- RNA AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGA 9(VH)-CH1-(G4S)2-scFv- GAACCCGCCACCAUGGACUGGAUCUGGCGCAUCCUGUUUC CD3 CLONE 2(VL-(G4S)5- UUGUGGGAGCCGCCACAGGCGCCCAUUCUCAGGUUCAGCU VH)-(G4S)2-Hinge-CH2-3 UGUGGAAUCUGGCGGCGGAUCUGUGCAGCCUGGCAGAUCU knob (hlgG1)- 3’-UTR- CUGAGACUGAGCUGUGAAGCCUCCGGCUUCACCUUUGAGG polyA, full heavy chain CCUACGCCAUGCACUGGGUCCGACAGCCACCUGGCAAAGG sequence CCUUGAAUGGGUGUCCAGCAUCAACUGGAACAGCGGCAGA AUCGCCUACGCCGACUCUGUGAAGGGCAGAUUCACCAUCA GCCGGGACAACGCCAGAAACAGCCUGUACCUGCAGAUGAA CAGCCUGCGGCUGGAAGAUACCGCCUUCUACUACUGCGCC AAGGACAUCAGACGGUUCAGCACAGGCGGAGCCGAGUUUG AGUAUUGGGGCCAGGGAACCCUGGUCACCGUGUCUAGCGC UUCUACAAAGGGCCCCAGCGUGUUCCCUCUGGCUCCUAGC AGCAAGUCUACAAGCGGAGGAACAGCCGCUCUGGGCUGCC UGGUCAAGGAUUACUUUCCCGAGCCUGUGACCGUGUCCUG GAAUUCUGGCGCUCUGACAAGCGGCGUGCACACCUUUCCA GCUGUGCUGCAAAGCAGCGGCCUGUACUCUCUGAGCAGCG UGGUCACAGUGCCAAGCUCUAGCCUGGGCACCCAGACCUA CAUCUGCAAUGUGAACCACAAGCCUAGCAACACCAAGGUGG ACAAGAAGGUGGAACCCAAGAGCUGUGAUGGCGGCGGAGG AUCUGGCGGAGGCGGAUCUGCCAUUCAGAUGACACAGAGC CCUAGCAGCCUGAGCGCCUCUGUGGGAGACAGAGUGACCA UUACCUGUCAGGCCAGCCAGACCAUCUACAACUACCUGGC CUGGUAUCAGCAGAAACCCGGCAAGGUGCCAGAGCUGCUG AUCUACAAGGCCAGCACACUGGCUAGCGGCGUGCCCAGUA GAUUCAUGGGCACAGGCUUCGGCACCGACUUCACCCUGAC CAUCGAUAGCCUGCAGCCUGAAGAUGCCGCCACCUACUAC UGCCAGCAGUACAACACCAUCAUCAACGUGGACCGGACCUU CGGCUGUGGAACCAAGGUGGAAAUCAAAGGUGGCGGAGGA AGCGGAGGCGGCGGUUCAGGCGGUGGUGGUAGCGGUGGU GGCGGCUCAGGCGGCGGAGGUUCUCAAGUUCAGCUGGUU CAAUCAGGCGGCGGACUGGUUCAACCUGGCGGACCUCUGA GACUGAGCUGUACCGCCAGCGGCUUCAGCUUCAGCAACAA CUACUACAUGUGCUGGGUCCGACAGGCCCCUGGCAAGUGU CUUGAAUGGGUGUCCUGCAUCUACACCAACAACAUCGGCA GCACUUGGUACGCCAGCUGGGCCAAGGGCAGAUUCACCAU CAGCCGGGACAACAGCAAGAACACCCUGUACCUGCAGAUGA ACAGCCUGAGAGCCGAGGAUACCGCCGUGUACUAUUGUGC CAGAGGCAUCUACAGCGACAGCAGCGACUUCAUCGGCAAC CUGUGGGGACAGGGCACACUGGUCACAGUUUCUAGCGGCG GUGGCGGUUCAGGUGGCGGAGGCUCUAAAACCCACACCUG UCCUCCAUGUCCUGCUCCGGAAGCUGCUGGUGGCCCUUCC GUGUUUCUGUUCCCUCCAAAGCCUAAGGACACCCUGAUGA UCAGCAGGACCCCUGAAGUGACCUGCGUGGUGGUCGAUGU GUCUCACGAGGACCCAGAAGUGAAGUUCAAUUGGUACGUG GACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAGAG AGGAACAGUACAACAGCACCUACAGAGUGGUGUCCGUGCU GACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAGAGUAC AAGUGCAAGGUGUCCAACAAGGCCCUGCCUGCUCCUAUCG AGAAAACCAUCAGCAAGGCCAAGGGCCAGCCUAGGGAACCC CAGGUUUACACCCUUCCACCUUGCCGGGAUGAGCUGACCA AGAACCAGGUGUCCCUGUGGUGUCUCGUGAAGGGCUUCUA CCCCUCCGAUAUCGCCGUGGAAUGGGAGAGCAAUGGCCAG CCUGAGAACAACUACAAGACAACCCCUCCUGUGCUGGACAG CGACGGCUCAUUCUUCCUGUACUCCAAGCUGACAGUGGAU AAGUCCCGGUGGCAGCAGGGCAACGUGUUCAGCUGUUCUG UGAUGCACGAGGCCCUGCACAACCACUACACCCAGAAAAGC

[1251]

[1252] CUGUCUCUGAGCCCCGGCAAGUGAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCU GGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUG CUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUU CCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGC UUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAAC CUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCC CAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUA GCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAA CA19-9(VH)-CH1-(G4S)2- AAS QVQLVESGGGSVQPGRSLRLSCEASGFTFEAYAMHWVRQPPG scFv-CD3 CLONE 2(VL- KGLEWVSSINWNSGRIAYADSVKGRFTISRDNARNSLYLQMNSL (G4S)5-VH)-(G4S)2-Hinge- RLEDTAFYYCAKDIRRFSTGGAEFEYWGQGTLVTVSSASTKGP CH2-3 knob (hlgG1), full SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG heavy chain sequence VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDGGGGSGGGGSAIQMTQSPSSLSASVGDRVTIT CQASQTIYNYLAWYQQKPGKVPELLIYKASTLASGVPSRFMGTG FGTDFTLTIDSLQPEDAATYYCQQYNTIINVDRTFGCGTKVEIKG GGGSGGGGSGGGGSGGGGSGGGGSQVQLVQSGGGLVQPG GPLRLSCTASGFSFSNNYYMCWVRQAPGKCLEWVSCIYTNNIG STWYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARG IYSDSSDFIGNLWGQGTLVTVSSGGGGSGGGGSKTHTCPPCPA PEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKN QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1253] 5’-UTR-husec2-CA19- RNA AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGA 9(VH)-CH1-(G4S)2-scFv- GAACCCGCCACCAUGGACUGGAUCUGGCGCAUCCUGUUUC CD3 CLONE 3(VH-(G4S)4- UUGUGGGAGCCGCCACAGGCGCCCAUUCUCAGGUUCAGCU VL)-(G4S)2-Hinge-CH2-3 UGUGGAAUCUGGCGGCGGAUCUGUGCAGCCUGGCAGAUCU knob (hlgG1)- 3’-UTR- CUGAGACUGAGCUGUGAAGCCUCCGGCUUCACCUUUGAGG polyA, full heavy chain CCUACGCCAUGCACUGGGUCCGACAGCCACCUGGCAAAGG sequence CCUUGAAUGGGUGUCCAGCAUCAACUGGAACAGCGGCAGA AUCGCCUACGCCGACUCUGUGAAGGGCAGAUUCACCAUCA GCCGGGACAACGCCAGAAACAGCCUGUACCUGCAGAUGAA CAGCCUGCGGCUGGAAGAUACCGCCUUCUACUACUGCGCC AAGGACAUCAGACGGUUCAGCACAGGCGGAGCCGAGUUUG AGUAUUGGGGCCAGGGAACCCUGGUCACCGUGUCUAGCGC UUCUACAAAGGGCCCCAGCGUGUUCCCUCUGGCUCCUAGC AGCAAGUCUACAAGCGGAGGAACAGCCGCUCUGGGCUGCC UGGUCAAGGAUUACUUUCCCGAGCCUGUGACCGUGUCCUG GAAUUCUGGCGCUCUGACAAGCGGCGUGCACACCUUUCCA GCUGUGCUGCAAAGCAGCGGCCUGUACUCUCUGAGCAGCG UGGUCACAGUGCCAAGCUCUAGCCUGGGCACCCAGACCUA CAUCUGCAAUGUGAACCACAAGCCUAGCAACACCAAGGUGG ACAAGAAGGUGGAACCCAAGAGCUGUGAUGGCGGCGGAGG AUCUGGCGGAGGCGGAUCUGAAGUUCAGCUGGUUGAAUCA GGCGGCGGACUGGUUAAGCCUGGCGGAUCUCUGGGCCUG UCUUGUACAGCCAGCGGCUUCAGCUUCAGCAACAACUACUA CAUGUGCUGGGUUCGCAGAGCCCCUGGCAAGUGUCUGGAA UGGGUCGGAUGCAUCUACACCAACAACAUCGGCAGCACUU GGUACGCCAGCUGGGCCAAGGGCAGAUUCACCAUCAGCAG GGACGACAGCAAGAACACCGUGUACCUGCAGAUGAACAGC CUGAAAACCGAGGAUACCGCCGUGUACUACUGCGCCAGAG GCAUCUACAGCGACAGCAGCGACUUCAUCGGCAACCUGUG GGGACAGGGCACACUGGUCACAGUUUCUAGCGGUGGCGGA GGAAGCGGAGGCGGAGGUUCAGGUGGUGGUGGAAGCGGU GGUGGCGGCAGCGAUAUUCAGAUGACACAGAGCCCUAGCA

[1254]

[1255] GCCUGAGCGCCUCUGUGGGAGACAGAGUGACCAUUACCUGUCAGGCCAGCCAGACCAUCUACAACUACCUGGCCUGGUAU CAGCAGAAGCCCGGCAAGCAGCCUAAGCUGCUGAUCUACA AGGCCAGCACACUGGCCAGCGGAGUGCCCAGUAGAUUCAA AGGCUCUGGCAGCGGCACCGACUUCACCCUGACCAUAUCU AGCCUGCAGCCAGAGGACGUGGCCACCUACUACUGCCAGC AGUACAACACCAUCAUCAACGUGGACCGGACCUUCGGCUG UGGCACAGAGGUUGUGGUCAAAGGUGGUGGCGGAUCUGG UGGCGGAGGCAGCAAAACACACACCUGUCCUCCAUGUCCU GCUCCGGAAGCUGCUGGUGGCCCUUCCGUGUUUCUGUUC CCUCCAAAGCCUAAGGACACCCUGAUGAUCAGCAGGACCCC UGAAGUGACCUGCGUGGUGGUCGAUGUGUCUCACGAGGAC CCAGAAGUGAAGUUCAAUUGGUACGUGGACGGCGUGGAAG UGCACAACGCCAAGACCAAGCCUAGAGAGGAACAGUACAAC AGCACCUACAGAGUGGUGUCCGUGCUGACCGUGCUGCACC AGGAUUGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUC CAACAAGGCCCUGCCUGCUCCUAUCGAGAAAACCAUCAGCA AGGCCAAGGGCCAGCCUAGGGAACCCCAGGUUUACACCCU UCCACCUUGCCGGGAUGAGCUGACCAAGAACCAGGUGUCC CUGUGGUGUCUCGUGAAGGGCUUCUACCCCUCCGAUAUCG CCGUGGAAUGGGAGAGCAAUGGCCAGCCUGAGAACAACUA CAAGACAACCCCUCCUGUGCUGGACAGCGACGGCUCAUUC UUCCUGUACUCCAAGCUGACAGUGGAUAAGUCCCGGUGGC AGCAGGGCAACGUGUUCAGCUGUUCUGUGAUGCACGAGGC CCUGCACAACCACUACACCCAGAAAAGCCUGUCUCUGAGCC CCGGCAAGUGAUGAGGAUCCGAUCUGGUACUGCAUGCACG CAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGU CUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCAC CUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCA AGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACA CCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAAC GAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAU UUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAA CA19-9(VH)-CH1-(G4S)2- AAS QVQLVESGGGSVQPGRSLRLSCEASGFTFEAYAMHWVRQPPG scFv-CD3 CLONE 3(VH- KGLEWVSSINWNSGRIAYADSVKGRFTISRDNARNSLYLQMNSL (G4S)4-VL)-(G4S)2-Hinge- RLEDTAFYYCAKDIRRFSTGGAEFEYWGQGTLVTVSSASTKGP CH2-3 knob (hlgG1), full SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG heavy chain sequence VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDGGGGSGGGGSEVQLVESGGGLVKPGGSLGLS CTASGFSFSNNYYMCWVRRAPGKCLEWVGCIYTNNIGSTWYA SWAKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCARGIYSDSS DFIGNLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQM TQSPSSLSASVGDRVTITCQASQTIYNYLAWYQQKPGKQPKLLI YKASTLASGVPSRFKGSGSGTDFTLTISSLQPEDVATYYCQQYN TIINVDRTFGCGTEWVKGGGGSGGGGSKTHTCPPCPAPEAAG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1256] 5’-UTR-husec2-CA19- RNA AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGA 9(VH)-CH1-(G4S)2-scFv- GAACCCGCCACCAUGGACUGGAUCUGGCGCAUCCUGUUUC CD3 CLONE 3(VL-(G4S)5- UUGUGGGAGCCGCCACAGGCGCCCAUUCUCAGGUUCAGCU VH)-(G4S)2-Hinge-CH2-3 UGUGGAAUCUGGCGGCGGAUCUGUGCAGCCUGGCAGAUCU knob (hlgG1)- 3’-UTR- CUGAGACUGAGCUGUGAAGCCUCCGGCUUCACCUUUGAGG polyA, full heavy chain CCUACGCCAUGCACUGGGUCCGACAGCCACCUGGCAAAGG sequence CCUUGAAUGGGUGUCCAGCAUCAACUGGAACAGCGGCAGA

[1257]

[1258] AUCGCCUACGCCGACUCUGUGAAGGGCAGAUUCACCAUCAGCCGGGACAACGCCAGAAACAGCCUGUACCUGCAGAUGAA CAGCCUGCGGCUGGAAGAUACCGCCUUCUACUACUGCGCC AAGGACAUCAGACGGUUCAGCACAGGCGGAGCCGAGUUUG AGUAUUGGGGCCAGGGAACCCUGGUCACCGUGUCUAGCGC UUCUACAAAGGGCCCCAGCGUGUUCCCUCUGGCUCCUAGC AGCAAGUCUACAAGCGGAGGAACAGCCGCUCUGGGCUGCC UGGUCAAGGAUUACUUUCCCGAGCCUGUGACCGUGUCCUG GAAUUCUGGCGCUCUGACAAGCGGCGUGCACACCUUUCCA GCUGUGCUGCAAAGCAGCGGCCUGUACUCUCUGAGCAGCG UGGUCACAGUGCCAAGCUCUAGCCUGGGCACCCAGACCUA CAUCUGCAAUGUGAACCACAAGCCUAGCAACACCAAGGUGG ACAAGAAGGUGGAACCCAAGAGCUGUGAUGGCGGCGGAGG AUCUGGCGGAGGCGGAUCUGAUAUUCAGAUGACACAGAGC CCUAGCAGCCUGAGCGCCUCUGUGGGAGACAGAGUGACCA UUACCUGUCAGGCCAGCCAGACCAUCUACAACUACCUGGC CUGGUAUCAGCAGAAGCCCGGCAAGCAGCCUAAGCUGCUG AUCUACAAGGCCAGCACACUGGCCAGCGGAGUGCCCAGUA GAUUCAAAGGCUCUGGCAGCGGCACCGACUUCACCCUGAC CAUAUCUAGCCUGCAGCCAGAGGACGUGGCCACCUACUAC UGCCAGCAGUACAACACCAUCAUCAACGUGGACCGGACCUU CGGCUGUGGCACAGAGGUUGUGGUCAAAGGUGGCGGAGG AAGCGGAGGCGGCGGUUCAGGCGGUGGUGGUAGCGGUGG UGGCGGCUCAGGCGGCGGAGGUUCUGAAGUUCAGCUGGU UGAAUCAGGCGGCGGACUGGUUAAGCCUGGCGGAUCUCUG GGCCUGUCUUGUACAGCCAGCGGCUUCAGCUUCAGCAACA ACUACUACAUGUGCUGGGUUCGCAGAGCCCCUGGCAAGUG UCUGGAAUGGGUCGGAUGCAUCUACACCAACAACAUCGGC AGCACUUGGUACGCCAGCUGGGCCAAGGGCAGAUUCACCA UCAGCAGGGACGACAGCAAGAACACCGUGUACCUGCAGAU GAACAGCCUGAAAACCGAGGAUACCGCCGUGUACUACUGC GCCAGAGGCAUCUACAGCGACAGCAGCGACUUCAUCGGCA ACCUGUGGGGACAGGGCACACUGGUCACAGUUUCUAGCGG CGGUGGCGGUUCAGGUGGCGGAGGCUCUAAAACCCACACC UGUCCUCCAUGUCCUGCUCCGGAAGCUGCUGGUGGCCCUU CCGUGUUUCUGUUCCCUCCAAAGCCUAAGGACACCCUGAU GAUCAGCAGGACCCCUGAAGUGACCUGCGUGGUGGUCGAU GUGUCUCACGAGGACCCAGAAGUGAAGUUCAAUUGGUACG UGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAG AGAGGAACAGUACAACAGCACCUACAGAGUGGUGUCCGUG CUGACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAGAGU ACAAGUGCAAGGUGUCCAACAAGGCCCUGCCUGCUCCUAU CGAGAAAACCAUCAGCAAGGCCAAGGGCCAGCCUAGGGAA CCCCAGGUUUACACCCUUCCACCUUGCCGGGAUGAGCUGA CCAAGAACCAGGUGUCCCUGUGGUGUCUCGUGAAGGGCUU CUACCCCUCCGAUAUCGCCGUGGAAUGGGAGAGCAAUGGC CAGCCUGAGAACAACUACAAGACAACCCCUCCUGUGCUGGA CAGCGACGGCUCAUUCUUCCUGUACUCCAAGCUGACAGUG GAUAAGUCCCGGUGGCAGCAGGGCAACGUGUUCAGCUGUU CUGUGAUGCACGAGGCCCUGCACAACCACUACACCCAGAAA AGCCUGUCUCUGAGCCCCGGCAAGUGAUGAGGAUCCGAUC UGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGU CCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGU AUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUA GUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAA CGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAU UAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUA ACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGA GCUAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAU GACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[1259]

[1260] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACA19-9(VH)-CH1-(G4S)2- AAS QVQLVESGGGSVQPGRSLRLSCEASGFTFEAYAMHWVRQPPG scFv-CD3 CLONE 3(VL- KGLEWVSSINWNSGRIAYADSVKGRFTISRDNARNSLYLQMNSL (G4S)5-VH)-(G4S)2-Hinge- RLEDTAFYYCAKDIRRFSTGGAEFEYWGQGTLVTVSSASTKGP CH2-3 knob (hlgG1), full SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG heavy chain sequence VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDGGGGSGGGGSDIQMTQSPSSLSASVGDRVTIT CQASQTIYNYLAWYQQKPGKQPKLLIYKASTLASGVPSRFKGSG SGTDFTLTISSLQPEDVATYYCQQYNTIINVDRTFGCGTEVWKG GGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGG SLGLSCTASGFSFSNNYYMCWVRRAPGKCLEWVGCIYTNNIGS TWYASWAKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCARGI YSDSSDFIGNLWGQGTLVTVSSGGGGSGGGGSKTHTCPPCPA PEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKN QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1261] 5’-UTR-husec2-CA19- RNA AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGA 9(VL)-Ckappa (hlgG1)- 3’- GAACCCGCCACCAUGGACUGGAUCUGGCGCAUCCUGUUUC UTR-polyA, full heavy UUGUGGGAGCCGCCACAGGCGCCCAUUCUCAGUCUGUUCU chain sequence GACACAGCCUCCUAGCGCCUCUGGCACACCUGGACAGAGA GUGACCAUCAGCUGUAGCGGCAGCAGCAGCAACAUCGGCA GCAACUUCGUGUACUGGUAUCAGCAGCUGCCCGGCACAGC CCCUAAACUGCUGAUCUACCGGAACAACCAGCGGCCUAGC GGCGUGCCAGAUAGAUUUUCUGGCAGCAGAAGCGGCACCU CUGCCAGCCUGGCUAUCUCUGGCCUGAGAUCUGAGGACGA GGCCGACUACUAUUGCGCCGCCUGGGAUGAUUCUCUCGGC GGCCACUAUGUGUUUGGCACCGGCACCAAAGUGACCGUGC UGAGAACAGUGGCCGCUCCUAGCGUGUUCAUCUUCCCACC UUCCGACGAGCAGCUGAAGUCUGGCACAGCCAGCGUUGUG UGCCUGCUGAACAACUUCUACCCCAGAGAAGCCAAGGUGC AGUGGAAGGUGGACAACGCCCUGCAGAGCGGCAAUAGCCA AGAGAGCGUGACCGAGCAGGACAGCAAGGACUCUACCUAC AGCCUGAGCAGCACCCUGACACUGAGCAAGGCCGAUUACG AGAAGCACAAAGUGUACGCCUGCGAAGUGACCCACCAGGG CCUUUCUAGCCCUGUGACCAAGAGCUUCAACCGGGGCGAG UGCUGAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUG CUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCC CCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCC CCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCAC GCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCC ACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAG UUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGU GCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAA CA19-9(VL)-Ckappa AAS QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNFVYWYQQLPGT (hlgG1), full light chain APKLLIYRNNQRPSGVPDRFSGSRSGTSASLAISGLRSEDEADY sequence YCAAWDDSLGGHYVFGTGTKVTVLRTVAAPSVFIFPPSDEQLKS GTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE

[1262] C

[1263] (G4S)2AAS GGGGSGGGGS

[1264] (G4S)4AAS GGGGSGGGGSGGGGSGGGGS

[1265] (G4S)5AAS GGGGSGGGGSGGGGSGGGGSGGGGS

[1266] 5’-UTR RNA AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGA

[1267]

[1268] GAACCCGCCACC3’-UTR RNA CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCG UCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGG UAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUA GUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAA CGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAU UAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUA ACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACC

[1269] A30L70 RNA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAA CA19-9(VH)-CH1 (hlgG1), AAS QVQLVESGGGSVQPGRSLRLSCEASGFTFEAYAMHWVRQPPG full heavy Fab chain KGLEWVSSINWNSGRIAYADSVKGRFTISRDNARNSLYLQMNSL sequence RLEDTAFYYCAKDIRRFSTGGAEFEYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK

[1270]

[1271] VDKKVEPKSCDNUMBERED PARAGRAPHS

[1272] The present invention will now be described by way of the following paragraphs:

[1273] 1. A bispecific binding molecule, preferably an antigen-binding molecule, more preferably a bispecific antibody, comprising a CD3-binding moiety and a first Carbohydrate antigen 19-9 (CA19-9)-binding moiety.

[1274] 2. The bispecific binding molecule according to paragraph 1, wherein the CD3-binding moiety comprises a CD3 heavy chain variable (CD3-VH) domain and / or CD3 light chain variable (CD3-VL) domain.

[1275] 3. The bispecific binding molecule according to paragraph 1 or 2, wherein the CD3-binding moiety comprises a scFv, wherein the scFv comprises:

[1276] (i) a CD3 heavy chain variable (CD3-VH) domain;

[1277] (ii) a linker; and

[1278] (iii) a CD3 light chain variable (CD3-VL) domain.

[1279] 4. The bispecific binding molecule according to any of paragraphs 1 to 3, wherein the first CA19-9-binding moiety comprises a CA19-9 heavy chain variable (CA19-9-VH) domain and a CA19-9 light chain variable (CA19-9-VL) domain.

[1280] 5. The bispecific binding molecule according to any of paragraphs 1 to 4, wherein the first CA19-9-binding moiety comprises a CA19-9-binding Fab, wherein the Fab comprises:

[1281] (i) a heavy chain comprising a CA19-9 heavy chain variable (CA19-9-VH) domain and a CA19-9 heavy chain constant (CA19-9-CH1) domain; and

[1282] (ii) a light chain comprising a CA19-9 light chain variable (CA19-9-VL) domain and a CA19-9 light chain constant (CA19-9-CL) domain.

[1283] 6. The bispecific binding molecule according to any of paragraphs 1 to 5, wherein the bispecific binding molecule further comprises a second CA19-9-binding moiety.

[1284] 7. The bispecific binding molecule according to paragraph 6, wherein the second CA19-9-binding moiety comprises a CA19-9 heavy chain variable (CA19-9-VH) domain and a CA19-9 light chain variable (CA19-9-VL) domain.

[1285] 8. The bispecific binding molecule according to claim 6, wherein the second CA19-9-binding moiety comprises a CA19-9-binding Fab, wherein the Fab comprises:(i) a heavy chain comprising a CA19-9 heavy chain variable (CA19-9-VH) domain and a CA19-9 heavy chain constant (CA19-9-CH1) domain; and

[1286] (ii) a light chain comprising a CA19-9 light chain variable (CA19-9-VL) domain and a CA19-9 light chain constant (CA19-9-CL) domain.

[1287] 9. The bispecific binding molecule according to any of paragraphs 6 to 8, wherein the first CA19-9-binding moiety and the second CA19-9-binding moiety are the same.

[1288] 10. The bispecific binding molecule according to any of paragraphs 1 to 9, wherein the first CA19-9-binding moiety is linked to the CD3-binding moiety.

[1289] 11. The bispecific binding molecule according to paragraph 10, wherein:

[1290] (i) the C-terminus of the first CA19-9-binding moiety, preferably the C-terminus of the CA19-9-CH1 domain, is linked to the N-terminus of the CD3-VH domain; or

[1291] (ii) the C-terminus of the first CA19-9-binding moiety, preferably the C-terminus of the CA19-9-CH1 domain, is linked to the N-terminus of the CD3-VL domain.

[1292] 12. The bispecific binding molecule according to any of paragraphs 1-11, wherein the bispecific binding molecule further comprises an Fc region comprising a first subunit and a second subunit.

[1293] 13. The bispecific binding molecule according to paragraph 12, wherein the CD3-binding moiety is linked to the Fc region.

[1294] 14. The bispecific binding molecule according to paragraph 12 or paragraph 13, wherein the second CA19-9-binding moiety is linked to the Fc region.

[1295] 15. The bispecific binding molecule according to any of paragraphs 12-14, wherein:

[1296] (i) the C-terminus of the CD3-VH domain is linked to the first subunit of the Fc region; or (ii) the C-terminus of the CD3-VL domain is linked to the first subunit of the Fc region.

[1297] 16. The bispecific binding molecule according to any of paragraphs 12-15, wherein the C-terminus of the second CA19-9-binding moiety, preferably the C-terminus of the CA19-9-CH1 domain, is linked to the second subunit of the Fc region.

[1298] 17. The bispecific binding molecule according to paragraph 15 or paragraph 16, wherein:(i) the C-terminus of the CD3-VH domain is linked to a hinge region of the first subunit of the Fc region; or

[1299] (ii) the C-terminus of the CD3-VL domain is linked to a hinge region of the first subunit of the Fc region.

[1300] 18. The bispecific binding molecule according to paragraph 16 or paragraph 17, wherein the C-terminus of the second CA19-9-binding moiety, preferably the C-terminus of the CA19-9-CH1 domain, is linked to a hinge region of the second subunit of the Fc region.

[1301] 19. The bispecific binding molecule according to any of paragraphs 12-18, wherein the first subunit of the Fc region and the second subunit of the Fc region each comprise a CH2 domain and / or a CH3 domain.

[1302] 20. The bispecific binding molecule according to any of paragraphs 1-19, wherein:

[1303] (i) the CD3-binding moiety comprises an scFv, wherein the scFv comprises:

[1304] (i) a CD3 heavy chain variable (CD3-VH) domain;

[1305] (ii) a linker; and

[1306] (iii) a CD3 light chain variable (CD3-VL) domain;

[1307] wherein the N-terminal to C-terminal orientation of the scFv is:

[1308] CD3-VH domain - linker - CD3-VL domain;

[1309] (ii) the bispecific binding molecule further comprises a second CA19-9-binding moiety, wherein the first CA19-9-binding moiety and the second CA19-9-binding moiety are the same, wherein the first CA19-9-binding moiety and the second CA19-9-binding moiety each comprise a CA19-9-binding Fab, wherein each Fab comprises:

[1310] (i) a heavy chain comprising a CA19-9 heavy chain variable (CA19-9-VH) domain and a CA19-9 heavy chain constant (CA19-9-CH1) domain; and

[1311] (ii) a light chain comprising a CA19-9 light chain variable (CA19-9-VL) domain and a CA19-9 light chain constant (CA19-9-CL) domain;

[1312] (iii) the bispecific binding molecule further comprises an Fc region comprising a first subunit and a second subunit; wherein the first subunit of the Fc region and the second subunit of the Fc region each comprise a CH2 domain and a CH3 domain,; optionally wherein the CH3 domain of one subunit of the Fc region comprises one or more mutations selected from the list consisting of: Y349C, T366S, L368A, and Y407V; and the CH3 domain of the other subunit of the Fc region comprises one or more mutations selected from the list consisting of: S354C and T366W;

[1313] (iv) the C-terminus of CA19-9-CH1 domain of the first CA19-9-binding moiety is linked to the N-terminus of the CD3-VH domain;(v) the C-terminus of the CD3-VL domain is linked to the N-terminus of the CH2 domain of the first subunit of the Fc region, preferably to the N-terminus of a hinge region of the CH2 domain of the first subunit of the Fc region; and

[1314] (vi) the C-terminus of the CA19-9-CH1 domain of the second CA19-9-binding moiety is linked to the N-terminus of the CH2 domain of the second subunit of the Fc region, preferably to the N-terminus of a hinge region of the CH2 domain of the second subunit of the Fc region.

[1315] 21. The bispecific binding molecule according to any of paragraphs 1-20, wherein the CD3-binding moiety comprises heavy chain complementarity determining regions (HCDRs) 1-3, and / or light chain complementarity determining regions (LCDRs) 1-3, wherein (according to Kabat numbering)

[1316] (i)

[1317] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 1, HCDR2 comprises the amino acid sequence according to SEQ ID NO: 3, and HCDR3 comprises the amino acid sequence according to SEQ ID NO: 5,

[1318] and / or

[1319] (ii)

[1320] LCDR1 comprises the amino acid sequence according to SEQ ID NO: 7, LCDR2 comprises the amino acid sequence according to SEQ ID NO: 9, and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 11; optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[1321] 22. The bispecific binding molecule according to any of paragraphs 1-20, wherein the CD3-binding moiety comprises heavy chain complementarity determining regions (HCDRs) 1-3, and / or light chain complementarity determining regions (LCDRs) 1-3, wherein (according to IMGT numbering)

[1322] (i)

[1323] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 2, HCDR2 comprises the amino acid sequence according to SEQ ID NO: 4, and HCDR3 comprises the amino acid sequence according to SEQ ID NO: 6,

[1324] and / or

[1325] (ii)

[1326] LCDR1 comprises the amino acid sequence according to SEQ ID NO: 8,LCDR2 comprises the amino acid sequence according to SEQ ID NO: 10, and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 12; optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[1327] 23. The bispecific binding molecule according to paragraph 21 or paragraph 22, wherein the HCDR1 , HCDR2 and HCDR3 sequences comprise or consist of

[1328] (i) SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, respectively; or

[1329] (ii) SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, respectively.

[1330] 24. The bispecific binding molecule according to any of paragraphs 21 to 23, wherein the LCDR1, LCDR2 and LCDR3 sequences comprise or consist of

[1331] (i) SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 11, respectively; or

[1332] (ii) SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12, respectively.

[1333] 25. The bispecific binding molecule according to any of paragraphs 21 to 24, wherein the CD3-binding moiety comprises a CD3-VH domain sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to any one of SEQ ID NO: 14, SEQ ID NO: 22, and SEQ ID NO: 30.

[1334] 26. The bispecific binding molecule according to paragraph 25, wherein the CD3-VH domain comprises or consists of a sequence as set forth in any one of SEQ ID NO: 14, SEQ ID NO: 22, and SEQ ID NO: 30.

[1335] 27. The bispecific binding molecule according to any of paragraphs 21 to 26, wherein the CD3-binding moiety comprises a CD3-VL domain sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or preferably 100% identity to any one of SEQ ID NO: 16, SEQ ID NO: 24, and SEQ ID NO: 32

[1336] 28. The bispecific binding molecule according to paragraph 27, wherein the CD3-VL domain comprises or consists of a sequence as set forth in any one of SEQ ID NO: 16, SEQ ID NO: 24, and SEQ ID NO: 32.

[1337] 29. The bispecific binding molecule according to any of paragraphs 1-28, wherein the CD3-binding moiety is or comprises a scFv having the format VH-L1-VL or VL-L2-VH, wherein L1 and L2 are peptide linkers.30. The bispecific binding molecule according to paragraph 29, wherein the amino acids of L1 and L2 are selected from glycines and serines.

[1338] 31. The bispecific binding molecule according to paragraph 29 or paragraph 30, wherein L1 and L2 independently have a length of 10 to 30 amino acids.

[1339] 32. The bispecific binding molecule according to any of paragraphs 29 to 31 , wherein L1 is a (G4S)4 linker.

[1340] 33. The bispecific binding molecule according to any of paragraphs 29 to 31 , wherein L2 is a (G4S)s linker.

[1341] 34. The bispecific binding molecule according to any of paragraphs 29 to 32, wherein L1 comprises a sequence as set forth in SEQ ID NO: 80, or a variant having at least 80% identity thereto.

[1342] 35. The bispecific binding molecule according to any of paragraphs 29 to 31 or 33, wherein L2 comprises a sequence as set forth in SEQ ID NO: 81, or a variant having at least 80% identity thereto.

[1343] 36. The bispecific binding molecule according to any of paragraphs 29-34, wherein the scFv comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or preferably 100% identity to any one of SEQ ID NO: 18, SEQ ID NO: 26, and SEQ ID NO: 34.

[1344] 37. The bispecific binding molecule according to paragraph 36, wherein the scFv comprises or consists of a sequence as set forth in any one of SEQ ID NO: 18, SEQ ID NO: 26, and SEQ ID NO: 34

[1345] 38. The bispecific binding molecule according to any of paragraphs 1-37, wherein CD3 is human CD3, optionally wherein CD3 is CD3E.

[1346] 39. The bispecific binding molecule according to any of paragraphs 1-38, wherein the, or each, CA19-9-binding moiety comprises heavy chain complementarity determining regions (HCDRs) 1-3, and / or light chain complementarity determining regions (LCDRs) 1-3, wherein (according to Kabat numbering):

[1347] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 37,HCDR2 comprises the amino acid sequence according to SEQ ID NO: 39, and HCDR3 comprises the amino acid sequence according to SEQ ID NO: 41,

[1348] and / or

[1349] LCDR1 comprises the amino acid sequence according to SEQ ID NO: 43, LCDR2 comprises the amino acid sequence according to SEQ ID NO: 45, and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 47; optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[1350] 40. The bispecific binding molecule according to any of paragraphs 1-38, wherein the, or each, CA19-9-binding moiety comprises heavy chain complementarity determining regions (HCDRs) 1-3, and / or light chain complementarity determining regions (LCDRs) 1-3, wherein (according to IMGT numbering):

[1351] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 38, HCDR2 comprises the amino acid sequence according to SEQ ID NO: 40, and HCDR3 comprises the amino acid sequence according to SEQ ID NO: 42,

[1352] and / or

[1353] LCDR1 comprises the amino acid sequence according to SEQ ID NO: 44, LCDR2 comprises the amino acid sequence according to SEQ ID NO: 46, and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 48; optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[1354] 41. The bispecific binding molecule according to any of paragraphs 1-40, wherein the CA19-9-binding moiety comprises a CA19-9-VH domain comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 50.

[1355] 42. The bispecific binding molecule according to any of paragraphs 5-41, wherein the CA19-9-VH domain comprises or consists of a sequence as set forth in SEQ ID NO: 50.

[1356] 43. The bispecific binding molecule according to any of paragraphs 1-42, wherein the CA19-9-binding moiety comprises a CA19-9-VL domain comprising a sequence having at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or preferably 100% identity to SEQ ID NO: 52.

[1357] 44. The bispecific binding molecule according to any of paragraphs 5-43, wherein the CA19-9-VL domain comprises or consists of a sequence as set forth in SEQ ID NO: 52.

[1358] 45. The bispecific binding molecule according to any of paragraphs 1-44, wherein the CA19-9-binding moiety comprises a heavy chain comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or preferably 100% identity to SEQ ID NO: 85.

[1359] 46. The bispecific binding molecule according to any of paragraphs 1-45, wherein the CA19-9-binding moiety comprises a heavy chain comprising or consisting of a sequence as set forth in SEQ ID NO: 85.

[1360] 47. The bispecific binding molecule according to any of paragraphs 1-46, wherein the CA19-9-binding moiety comprises a light chain comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or preferably 100% identity to SEQ ID NO: 78.

[1361] 48. The bispecific binding molecule according to any of paragraphs 1-47, wherein the CA19-9-binding moiety comprises a light chain comprising or consisting of a sequence as set forth in SEQ ID NO: 78.

[1362] 49. The bispecific binding molecule according to any of paragraphs 1-48, wherein the bispecific binding molecule comprises:

[1363] (i) a first polypeptide comprising the amino acid sequence according to SEQ ID NO: 66 or a variant having at least 80% sequence identity thereto;

[1364] (ii) a second polypeptide comprising the amino acid sequence according to SEQ ID NO: 64 or a variant having at least 80% sequence identity thereto; and

[1365] (iii) a third polypeptide comprising the amino acid sequence according to SEQ ID NO: 78 or a variant having at least 80% sequence identity thereto.

[1366] 50. The bispecific binding molecule according to any of paragraphs 1-48, wherein the bispecific binding molecule comprises:

[1367] (i) a first polypeptide comprising the amino acid sequence according to SEQ ID NO: 70 or a variant having at least 80% sequence identity thereto;(ii) a second polypeptide comprising the amino acid sequence according to SEQ ID NO: 64 or a variant having at least 80% sequence identity thereto; and

[1368] (iii) a third polypeptide comprising the amino acid sequence according to SEQ ID NO: 78 or a variant having at least 80% sequence identity thereto.

[1369] 51. The bispecific binding molecule according to any of paragraphs 1-48, wherein the bispecific binding molecule comprises:

[1370] (i) a first polypeptide comprising the amino acid sequence according to SEQ ID NO: 74 or a variant having at least 80% sequence identity thereto;

[1371] (ii) a second polypeptide comprising the amino acid sequence according to SEQ ID NO: 64 or a variant having at least 80% sequence identity thereto; and

[1372] (iii) a third polypeptide comprising the amino acid sequence according to SEQ ID NO: 78 or a variant having at least 80% sequence identity thereto.

[1373] 52. One or more polynucleotide(s) comprising a nucleic acid sequence encoding the bispecific binding molecule according to any of paragraphs 1-51.

[1374] 53. The one or more polynucleotide(s) according to paragraph 52, wherein the nucleic acid sequence is selected from the list consisting of: a DNA sequence, a cDNA sequence, an RNA sequence, and an mRNA sequence.

[1375] 54. The one or more polynucleotide(s) according to paragraph 53, wherein the nucleic acid sequence is an RNA sequence.

[1376] 55. The one or more polynucleotide(s) according to paragraph 54, wherein the RNA sequence is an mRNA sequence.

[1377] 56. The one or more polynucleotide(s) according to paragraph 55, wherein the RNA is modRNA (N 1 -methyl-pseudouridine).

[1378] 57. The one or more polynucleotide(s) according to any one of paragraphs 52-56, wherein the RNA sequence comprises a 5'-cap, optionally wherein the 5'-cap is CC413, further optionally wherein the 5'-cap is m27’3'oGppp(mi2'°)ApG, which comprises the following structure:

[1379]

[1380] OH OH

[1381] 58. The one or more(s) polynucleotide according to any one of paragraphs 52-57, wherein the RNA sequence comprises a poly adenine sequence, optionally wherein the poly adenine sequence comprises the nucleic acid sequence according to SEQ ID NO: 84 or a variant having 80% sequence identity thereto.

[1382] 59. The one or more polynucleotide(s) according to any one of paragraphs 52-58, wherein the RNA sequence comprises a 5’IITR; optionally wherein the 5’IITR comprises the nucleic acid sequence according to SEQ ID NO: 82 or a variant having 80% sequence identity thereto.

[1383] 60. The one or more polynucleotide(s) according to any one of paragraphs 52-59, wherein the RNA sequence comprises a 3’IITR; optionally wherein the 3’IITR comprises the nucleic acid sequence according to SEQ ID NO: 83 or a variant having 80% sequence identity thereto.

[1384] 61. The one or more polynucleotide(s) according to any one of paragraphs 52-60 comprising:

[1385] (i) a first polynucleotide comprising a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[1386] CA19-9-VH domain - CA19-9-CH1 domain - optional linker- CD3-VH domain - L1 - CD3-VL domain - optional linker - CH2 domain - CH3 domain;

[1387] (ii) a second polynucleotide comprising a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[1388] CA19-9-VH domain - CA19-9-CH1 domain - CH2 domain - CH3 domain; and / or

[1389] (iii) a third polynucleotide comprising a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:

[1390] CA19-9-VL domain - CA19-9-CL domain.The one or more polynucleotide(s) according to any of paragraphs 52-61 , wherein: (i) a first polynucleotide comprises a nucleic acid sequence comprising in 5’-3’ orientation:

[1391] 5’-cap - 5’UTR - CA19-9-VH domain - CA19-9-CH1 domain - optional linker - CD3-VH domain - linker L1 - CD3-VL domain - optional linker - CH2 domain - CH3 domain - 3’UTR - Poly(A)

[1392] (ii) a second polynucleotide comprises a nucleic acid sequence comprising in 5’-3’ orientation:

[1393] 5’-cap - 5’IITR - CA19-9-VH domain - CA19-9-CH1 domain - CH2 domain - CH3 domain - 3’UTR - Poly(A); and / or

[1394] (iii) a third polynucleotide comprises a nucleic acid sequence comprising in 5’-3’ orientation:

[1395] 5’-cap - 5’UTR - CA19-9-VL domain - CA19-9-CL domain - 3’UTR - Poly(A).

[1396] The one or more polynucleotide(s) according to any of paragraphs 52-62, wherein: (i) a first polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 65 or a variant having at least 80% sequence identity to SEQ ID NO: 65;

[1397] (ii) a second polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 63 or a variant having at least 80% sequence identity to SEQ ID NO: 63; and / or (iii) a third polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 77 or a variant having at least 80% sequence identity to SEQ ID NO: 77.

[1398] The one or more polynucleotide(s) according to any of paragraphs 52-62, wherein: (i) a first polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 69 or a variant having at least 80% sequence identity to SEQ ID NO: 69; or

[1399] (ii) a second polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 63 or a variant having at least 80% sequence identity to SEQ ID NO: 63; and / or (iii) a third polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 77 or a variant having at least 80% sequence identity to SEQ ID NO: 77.

[1400] The one or more polynucleotide(s) according to any of paragraphs 52-62, wherein: (i) the first polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 73 or a variant having at least 80% sequence identity to SEQ ID NO: 73;

[1401] (ii) the second polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 63 or a variant having at least 80% sequence identity to SEQ ID NO: 63; and / or (iii) the third polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 77 or a variant having at least 80% sequence identity to SEQ ID NO: 77.66. A vector which comprises one or more polynucleotide(s) according to any of paragraphs 52-65.

[1402] 67. A composition comprising

[1403] (i) the bispecific binding molecule according to any of paragraphs 1-51;

[1404] (ii) the one or more polynucleotide(s) according to any of paragraphs 52-65; or

[1405] (iii) the vector according to paragraph 66.

[1406] 68. A pharmaceutical composition comprising:

[1407] (i)

[1408] (a) the bispecific binding molecule according to any of paragraphs 1-51; or (b) the one or more polynucleotide(s) according to any of paragraphs 52-65; or (c) the vector according to paragraph 66; or

[1409] (d) the composition according to paragraph 67;

[1410] and

[1411] (ii) a pharmaceutically acceptable carrier, diluent or excipient,

[1412] optionally wherein the pharmaceutical composition further comprises

[1413] (iii) a vesicle, a nanoparticle, a lipid nanoparticle (LNP), a liposome or a polymeric mixture wherein the bispecific -binding molecule, the one or more polynucleotide(s), the vector, or the composition is encapsulated within the vesicle, nanoparticle, LNP, liposome or polymeric mixture.

[1414] 69. The pharmaceutical composition according to paragraph 68, wherein (iii) is an LNP.

[1415] 70. The pharmaceutical composition according to paragraph 68 or paragraph 69, which is formulated for parenteral administration.

[1416] 71. The pharmaceutical composition according to any of paragraphs 68 to 70, which is formulated for intravenous or intraarterial administration.

[1417] 72. A method for treating and / or preventing a disease which comprises the step of administering the bispecific binding molecule according to any of paragraphs 1-51 ; or the one or more polynucleotide(s) according to any of paragraphs 52-65; or the vector according to paragraph 66; or the composition according to paragraph 67, or the pharmaceutical composition according to any of paragraphs 68 to 71 , to a subject.73. Use of the bispecific binding molecule according to any of paragraphs 1-51 ; or the one or more polynucleotide(s) according to any of paragraphs 52-65; or the vector according to paragraph 66; or the composition according to paragraph 67, or the pharmaceutical composition according to any of paragraphs 68 to 71 , in the manufacture of a medicament for treating and / or preventing a disease.

[1418] 74. The bispecific binding molecule according to any of paragraphs 1-51; or the one or more polynucleotide(s) according to any of paragraphs 52-65; or the vector according to paragraph 66; or the composition according to paragraph 67, or the pharmaceutical composition according to any of paragraphs 68 to 71 , for use in the treatment or prevention of a disease.

[1419] 75. The method according to claim 72, the use according to claim 73, or the bispecific binding molecule, the one or more polynucleotide(s), the vector, the composition, or the pharmaceutical composition for use according to claim 74, wherein the disease is cancer.

[1420] 76. The method, the use according, or the bispecific binding molecule, the one or more polynucleotide(s), the vector, the composition, or the pharmaceutical composition for use according to claim 75, wherein the cancer is selected from the list consisting of:

[1421] (i) pancreatic cancer, optionally wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC), (ii) cholangiocarcinoma; and

[1422] (iii) gastrointestinal cancer, optionally wherein the gastrointestinal cancer is selected from the listing consisting of: colorectal cancer, oesophageal cancer, gastric cancer, and hepatocellular cancer.EXAMPLES

[1423] Example 1 : Generation of polyribonucleotides encoding bispecific antibody agents

[1424] The present Example demonstrates the generation of polyribonucleotide seguences encoding bispecific anti-CA19-9 and anti-CD3 antibody agents. Specifically, ribonucleic acid seguences encoding the heavy and light chain variable domains of a CA19-9-directed CA19-9 clone and CD3-directed CD3 clones (or variants thereof) were combined with other immunoglobulin domains to form polyribonucleotides that encode the CA19-9 Clone and bispecific CA19-9 Clone-CD3 clones (or variants thereof) antibody agent immunoglobulin chains, respectively. The respective polydeoxyribonucleotides were cloned into a DNA plasmid. The polyribonucleotides were designed to encode a bispecific, asymmetric 2+1 Fab(2)-scFv-Fc format (see FIG. 1). Exemplary antibody agent configurations and component immunoglobulin chains are shown in Table 1.

[1425] Methods of the present example include:

[1426] Cloning of exemplary antibody agent-encoding DNA template vectors:

[1427] DNA seguences encoding antibody fragments composed of a) variable heavy and / or variable light chain domains, b) linker seguences (where applicable) and c) constant domains of the human immunoglobulin 1 isotype were synthesized either internally or externally (Thermo Fischer Scientific). These DNA fragments were cloned into a DNA plasmid appropriate to use for RNA expression according to standard procedures well known by the person skilled in the art. In short, the parental plasmid was digested with restriction enzymes. The resulting product was analyzed via agarose gel, purified and its concentration measured by UV spectroscopy. The antibody encoding light chain and heavy chain seguences were mixed and cloned into the plasmid by transforming competent Escherichia coli cells via heat shock and subseguent in vivo assembly (Garcia-Nafria, “IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly,” Scientific Reports volume 6, Article number: 27459 (2016), which is incorporated herein by reference in its entirety). Single bacterial clones were seguence verified by Sanger seguencing. Seguencing results were aligned to the reference seguence

[1428] Plasmid DNA preparation of exemplary antibody agent-encoding DNA template vectors: Plasmid DNA was prepared by selecting E. coli clones for inoculation in Luria-Bertani (LB) medium containing kanamycin. The cultures were grown overnight at 37 °C and ~ 150 to 200 rpm. Following cell harvest, purification was done using the QIAGEN Plasmid Plus Maxi or Giga Kit according to the manufacturer’s instructions. Finally, the concentration wasdetermined by UV spectroscopy. DNA was stored in certified RNase- and DNase-free reaction tubes.

[1429] Linearization and DNA purification of exemplary antibody agent-encoding DNA template vectors:

[1430] Linearization of plasmid DNA was performed using appropriate restriction enzymes, followed by purification of the linearized DNA template using magnetic beads (Dynabeads™ MyOne™ Carboxylic Acid) according to the manufacturer’s protocol. The DNA concentration was measured by UV spectroscopy.

[1431] In vitro transcription of exemplary antibody agent-encoding DNA template vectors:

[1432] CleanCap 413 ((m7(3'OMeG)(5')ppp(5')(2'OMeA)pG)-capped RNA was produced following the process as disclosed, e.g., in Kreiter, S. et al. Cancer Immunol. Immunother. 56, 1577-87 (2007) and WO 2021214204A1, each of which is incorporated herein by reference in its entirety. Methyl pseudo-uridine was used in the in vitro transcription reaction and incorporated into the produced RNA. Purification of the resulting RNA was performed to isolate singlestranded RNA, followed by concentration measurement by UV spectroscopy. The RNA integrity was determined by microfluidic-based electrophoresis.

[1433] Results of the present example:

[1434] Multiple polyribonucleotides encoding two exemplary 2+1 Fab(2)-scFv-Fc antibody agents (Fig.

[1435] 1) were successfully cloned (Table 2).

[1436] Table 2: Exemplary plasmids encoding bispecific anti-CA19-9 and anti-CD3 antibody agents' immunoglobulin chains

[1437] Antibody agent Format Immunoglobulin-encoding plasmid inserts

[1438] CA19-9 Clone(Fab)-HC(hole)

[1439] CA19-9 Clone x CA19-9 Clone(Fab)-LC

[1440] scFv-CD3-Clone 2 2+1 Fab(2)-scFv-Fc

[1441] (VH-VL) CA19-9 Clone(Fab)- CD3-Clone 2(scFv-VH-(G4S)4-VL)

[1442] HC(knob)

[1443] CA19-9 Clone(Fab)-HC(hole)

[1444] CA19-9 Clone x CA19-9 Clone(Fab)-LC

[1445] scFv-CD3-Clone 3 2+1 Fab(2)-scFv-Fc

[1446] (VH-VL) CA19-9 Clone(Fab)- CD3-Clone 3(scFv-VH-(G4S)4-VL)

[1447] HC(knob)

[1448]

[1449] The 2+1 Fab(2)-scFv-Fc RiboMab consists of three different polypeptide chains. One heavy chain comprises a human CA19-9-binding heavy chain Fab domain and a human IgG 1 Fc part with hole mutations and Fc-silencing mutations. The other heavy chain is generated from thesame CA19-9-binding heavy chain Fab domain, a human CD3E binding humanized scFv that is C-terminally fused via short glycine serine linkers and an Fc-part with corresponding knob mutations and Fc-silencing mutations. The third chain comprises a human CA19-9-binding light chain Fab domain.

[1450] Example 2: In vitro validation of exemplary bispecific antibody agents delivered to cells using RNA constructs: Expression analysis

[1451] Exemplary polyribonucleotide constructs used in this example are described in Example 1. The present Example demonstrates that such polyribonucleotides can be designed so that transient in vitro antibody agent production of the one or more polyribonucleotides can be achieved. Hence, the expression and protein integrities of the antibody agents were tested. In this Example, Expi293F™ suspension cells were transfected with polyribonucleotides encoding the respective antibody agent. The respective antibody agent concentrations in the supernatant were determined by GYROS ELISA. Reducing and non-reducing denaturing Western blot analyses were also performed to check the ratio of antibody agents produced versus high molecular weight (HMW) aggregates and free chains.

[1452] Methods of the present example include:

[1453] In vitro Production of exemplary antibody agents via transient mRNA transfection:

[1454] RiboMabs were generated by mRNA transfection of Expi293F™ suspension cells with the polyribonucleotides encoding the antibody agent chains. Expi293F™ are cultivated in culture flasks at 140 rpm, at 37 °C and 5% CO2 in an incubator. 24 hours prior to the transfection, the cells were seeded at 2.5 x 106cells per mL. On the day of the transfection, viability and cell density were checked and - in case of > 95% viability - the cells were seeded at 5 x 106cells per mL. Per mL cell suspension 1 pg RNA was diluted in 100 pL serum-free OptiMEM medium (Gibco). 2 pL mRNA RiboJuice Boost Reagent (Merck) and 2 pL RiboJuice mRNA Reagent (Merck) were added to the diluted RNA in immediate succession and the resulting solutions each gently mixed in between. An incubation time of at least 3 min but not more than 5 min was executed at room temperature after which the RNA-mix is added dropwise to the cells. The transfected cells were then incubated in CF50 culture tubes (TPP) at 180 rpm, 37 °C and 5% CO2 for four days. The cells were harvested by centrifugation at 300 x g for 4 min and the resulting supernatant was further cleared from remaining cells by centrifugation at 3000 x g for another 10 min. The collected supernatant was either concentrated with via centrifugation in a 50 kDa MWCO concentrator (Amicon) or directly stored at 4 °C until further analysis.Gyros ELISA for exemplary antibody agent quantitation:

[1455] Selected antibody agents expressed from polyribonucleotides delivered to a cell (referred to herein as “RiboMabs”) were quantified in serum using a Gyros xPand™ XPA1055 immunoassay system (Gyros Protein Technologies AB, Uppsala, Sweden). A sandwich immunoassay format was performed to determine the intact (fully assembled) RiboMabs content in cell culture supernatant samples from Expi293F™ cells transfected with mRNA. The RiboMabs were captured by a biotinylated anti-human IgG Fc antibody (Reagent A, ready-to-use solution from Gyrolab huIgG Kit, Gyros Protein Technologies AB). An Alexa Fluor 647 labeled F(ab')2 fragment of anti-human IgG (Reagent B, ready-to-use solution both from Generic PK Kit-Low Titer or Generic TK Kit-High Titer, Gyros Protein Technologies AB) was used for detection of the antibody agents. The assay was processed for low titer in a Gyrolab Bioaffy 1000 HC CD (Gyros Protein Technologies AB) or for high titer in a Gyrolab Bioaffy 20 HC CD (Gyros Protein Technologies AB). All antibody agents, reference proteins and reagents were centrifuged for 4 minutes at 12,000xg to sediment any aggregates. RiboMab molecules in cell culture supernatant were diluted 5 to 10-fold in Reagent E buffer (Gyrolab huIgG Kit, Gyros Protein Technologies AB). The CD columns were washed with Reagent C and D (Gyrolab PK / TK Kit, Gyrolab huIgG Kit, Gyros Protein Technologies AB). The Gyrolab huIgG Standard (Gyros Protein Technologies AB) and (in-house generated) served as reference proteins. For RiboMab quantitation, all materials prepared for the Gyros ELISA assay were loaded onto a 96-well plate (Gyros Protein Technologies AB) according to the Gyrolab loading list. Data were generated for low antibody titers with the huIgG Low Titer method and for high antibody titers with the huIgG High Titer v2 method, respectively. A standard curve was prepared with a previously purified batch of a recombinant reference protein of the same molecular composition (1693 / 1694 / 1497p; BioNTech SE) and the antibody concentration in the cell culture supernatant was calculated using the GyroLab Evaluator software.

[1456] Western blot Analysis of exemplary antibody agents in Cell Culture Supernatants:

[1457] To evaluate RiboMab integrity, the supernatant of cells transfected with mRNAs was analyzed via Western blot according to standard procedures well known by the person skilled in the art. Briefly, cell culture supernatant samples were first mixed with varying volumes of cell culture supernatant from mock-transfected Expi293F™cells to obtain equal amounts of protein (10 ng) as well as 4x Laemmli buffer and heated to 95°C for 5 minutes under non-reducing and reducing conditions. The purified anti-CA19-9xCD3 Fab-scFv-Fc protein served as reference proteins for gauging the RiboMab quality. As negative control, cell culture supernatant from Mock-transfected Expi293F™ cells was used. The samples were separated via polyacrylamide gel electrophoresis using a 4-15% Criterion™ TGX Stain-Free™ Gel (Bio-Rad Laboratories, Dreieich, Germany) followed by transfer of the separated bands onto a blotting membrane(Bio-Rad). For Western blotting, the membrane was incubated with HRP-conjugated goat antihuman kappa LC at a 1:500 dilution (ID510, Thermo Fisher Scientific) in combination with HRP-conjugated goat anti-human IgG-Fcy fragment (ID074, (Jackson ImmunoResearch Europe Ltd) at a 1:500 dilution as detection antibodies. The membrane was visualized with Clarity Western ECL Reagent (Bio-Rad) on a Vilber Fusion FX imaging device (Vilber, Collegien, France) for 0.2 seconds and the data analyzed with Image Lab Software (Bio-Rad).

[1458] Results of the present example:

[1459] The results from the Gyros ELISA in this Example (Fig. 2) show that both exemplary antibody agents, CA19-9 Clone x scFv-CD3-Clone 2 (VH-VL) and CA19-9 Clone x scFv-CD3-Clone 3 (VH-VL), were successfully produced at a similar level.

[1460] The results from the Western blot analysis are shown in Fig. 3. The non-reducing, denaturing Western blot of the exemplary antibody agents CA19-9 Clone x scFv-CD3-Clone 2(VH-VL) and CA19-9 Clone x sc...

Claims

CLAIMS1. A bispecific binding molecule comprising a CD3-binding moiety and a first Carbohydrate antigen 19-9 (CA19-9)-binding moiety.

2. The bispecific binding molecule according to claim 1, wherein the CD3-binding moiety comprises:(i) a CD3 heavy chain variable (CD3-VH) domain and / or CD3 light chain variable (CD3-VL) domain; or(ii) a scFv, wherein the scFv comprises:(i) a CD3 heavy chain variable (CD3-VH) domain;(ii) a linker; and(iii) a CD3 light chain variable (CD3-VL) domain.

3. The bispecific binding molecule according to claim 1 or claim 2, wherein the first CA19-9-binding moiety comprises(i) a CA19-9 heavy chain variable (CA19-9-VH) domain and a CA19-9 light chain variable (CA19-9-VL) domain; or(ii) a CA19-9-binding Fab, wherein the Fab comprises:(i) a heavy chain comprising a CA19-9 heavy chain variable (CA19-9-VH) domain and a CA19-9 heavy chain constant (CA19-9-CH1) domain; and(ii) a light chain comprising a CA19-9 light chain variable (CA19-9-VL) domain and a CA19-9 light chain constant (CA19-9-CL) domain.

4. The bispecific binding molecule according to any of claims 1-3, wherein the bispecific binding molecule further comprises a second CA19-9-binding moiety, optionally wherein the second CA19-9-binding moiety comprises:(i) a CA19-9 heavy chain variable (CA19-9-VH) domain and a CA19-9 light chain variable (CA19-9-VL) domain; or(ii) a CA19-9-binding Fab, wherein the Fab comprises:(i) a heavy chain comprising a CA19-9 heavy chain variable (CA19-9-VH) domain and a CA19-9 heavy chain constant (CA19-9-CH1) domain; and(ii) a light chain comprising a CA19-9 light chain variable (CA19-9-VL) domain and a CA19-9 light chain constant (CA19-9-CL) domain.

5. The bispecific binding molecule according to any of claims 1-4, wherein the bispecific binding molecule further comprises an Fc region comprising a first subunit and a second subunit;optionally wherein the first subunit of the Fc region and the second subunit of the Fc region each comprise a CH2 domain and / or a CH3 domain.

6. The bispecific binding molecule according to claim 5, wherein(i) the first CA19-9-binding moiety is linked to the CD3-binding moiety; optionally wherein (a) the C-terminus of the first CA19-9-binding moiety is linked to the N-terminus of the CD3-VH domain; or(b) the C-terminus of the first CA19-9-binding moiety is linked to the N-terminus of the CD3-VL domain; and / or(ii) the CD3-binding moiety is linked to the Fc region; optionally wherein:(a) the C-terminus of the CD3-VH domain is linked to the first subunit of the Fc region; or(b) the C-terminus of the CD3-VL domain is linked to the first subunit of the Fc region; and / or(iii) the second CA19-9-binding moiety is linked to the Fc region; optionally wherein the C-terminus of the second CA19-9-binding moiety is linked to the second subunit of the Fc region.

7. The bispecific binding molecule according to any of claims 1 to 5, wherein:(i) the CD3-binding moiety comprises an scFv, wherein the scFv comprises:(i) a CD3 heavy chain variable (CD3-VH) domain;(ii) a linker (L1); and(iii) a CD3 light chain variable (CD3-VL) domain;wherein the N-terminal to C-terminal orientation of the scFv is:CD3-VH domain - L1 - CD3-VL domain;(ii) the bispecific binding molecule further comprises a second CA19-9-binding moiety, wherein the first CA19-9-binding moiety and the second CA19-9-binding moiety are the same, wherein the first CA19-9-binding moiety and the second CA19-9-binding moiety each comprise a CA19-9-binding Fab, wherein each Fab comprises:(i) a heavy chain comprising a CA19-9 heavy chain variable (CA19-9-VH) domain and a CA19-9 heavy chain constant (CA19-9-CH1) domain; and(ii) a light chain comprising a CA19-9 light chain variable (CA19-9-VL) domain and a CA19-9 light chain constant (CA19-9-CL) domain;(iii) the bispecific binding molecule further comprises an Fc region comprising a first subunit and a second subunit; wherein the first subunit of the Fc region and the second subunit of theFc region each comprise a CH2 domain and a CH3 domain, optionally wherein the CH3 domain of one subunit of the Fc region comprises one or more mutations selected from the list consisting of: Y349C, T366S, L368A, and Y407V; and the CH3 domain of the other subunit of the Fc region comprises one or more mutations selected from the list consisting of: S354C and T366W;(iv) the C-terminus of CA19-9-CH1 domain of the first CA19-9-binding moiety is linked to the N-terminus of the CD3-VH domain;(v) the C-terminus of the CD3-VL domain is linked to the N-terminus of the CH2 domain of the first subunit of the Fc region, preferably to the N-terminus of a hinge region of the CH2 domain of the first subunit of the Fc region; and(vi) the C-terminus of the CA19-9-CH1 domain of the second CA19-9-binding moiety is linked to the N-terminus of the CH2 domain of the second subunit of the Fc region, preferably to the N-terminus of a hinge region of the CH2 domain of the second subunit of the Fc region.

8. The bispecific binding molecule according to any of claims 1-7, wherein the CD3-binding moiety comprises heavy chain complementarity determining regions (HCDRs) 1-3, and / or the VL domain comprises light chain complementarity determining regions (LCDRs) 1-3, wherein(i) (according to Kabat numbering)HCDR1 comprises the amino acid sequence according to SEQ ID NO: 1, HCDR2 comprises the amino acid sequence according to SEQ ID NO: 3, and HCDR3 comprises the amino acid sequence according to SEQ ID NO: 5,and / orLCDR1 comprises the amino acid sequence according to SEQ ID NO: 7, LCDR2 comprises the amino acid sequence according to SEQ ID NO: 9, and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 11; or(ii) (according to IMGT numbering)HCDR1 comprises the amino acid sequence according to SEQ ID NO: 2, HCDR2 comprises the amino acid sequence according to SEQ ID NO: 4, and HCDR3 comprises the amino acid sequence according to SEQ ID NO: 6,and / orLCDR1 comprises the amino acid sequence according to SEQ ID NO: 8,LCDR2 comprises the amino acid sequence according to SEQ ID NO: 10, and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 12; optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences;optionally wherein(iii)(a) the CD3-binding moiety comprises a CD3-VH domain sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to any one of SEQ ID NO: 14, SEQ ID NO: 22, and SEQ ID NO: 30; and / or(b) the CD3-binding moiety comprises a CD3-VL domain sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to any one of SEQ ID NO: 16, SEQ ID NO: 24, and SEQ ID NO: 32;or(iv) the CD3-binding moiety is or comprises a scFv, wherein the scFv comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or preferably 100% identity to any one of SEQ ID NO: 18, SEQ ID NO: 26, and SEQ ID NO: 34.

9. The bispecific binding molecule according to any of claims 1-8, wherein the, or each, CA19-9-binding moiety comprises heavy chain complementarity determining regions (HCDRs) 1-3, and / or light chain complementarity determining regions (LCDRs) 1-3, wherein(i) (according to Kabat numbering)HCDR1 comprises the amino acid sequence according to SEQ ID NO: 37, HCDR2 comprises the amino acid sequence according to SEQ ID NO: 39, and HCDR3 comprises the amino acid sequence according to SEQ ID NO: 41,and / orLCDR1 comprises the amino acid sequence according to SEQ ID NO: 43, LCDR2 comprises the amino acid sequence according to SEQ ID NO: 45, and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 47; optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences; or(ii) (according to IMGT numbering)HCDR1 comprises the amino acid sequence according to SEQ ID NO: 38, HCDR2 comprises the amino acid sequence according to SEQ ID NO: 40, and HCDR3 comprises the amino acid sequence according to SEQ ID NO: 42,and / orLCDR1 comprises the amino acid sequence according to SEQ ID NO: 44, LCDR2 comprises the amino acid sequence according to SEQ ID NO: 46, and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 48; optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences;optionally wherein(iii)(a) the, or each, CA19-9-binding moiety comprises a CA19-9-VH domain comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 50; and / or (b) the, or each, CA19-9-binding moiety comprises a CA19-9-VL domain comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 52;or(iv)(a) the, or each, CA19-9-binding moiety comprises a heavy chain comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or preferably 100% identity to SEQ ID NO: 85; and / or(b) the, or each, CA19-9-binding moiety comprises a light chain comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or preferably 100% identity to SEQ ID NO: 78.

10. One or more polynucleotide(s) comprising a nucleic acid sequence encoding the bispecific binding molecule according to any of claims 1-9.

11. The one or more polynucleotide(s) according to claim 10, wherein the nucleic acid sequence is selected from the list consisting of: a DNA sequence, a cDNA sequence, an RNA sequence, and an mRNA sequence; optionally wherein the nucleic acid sequence is an RNAsequence; optionally wherein the RNA sequence is an mRNA sequence; optionally wherein the RNA is modRNA (N1-methyl-pseudouridine); optionally wherein(i) the RNA sequence comprises a 5'-cap, further optionally wherein(a) the 5'-cap is CC413; or(b) wherein the 5'-cap is m27’3'oGppp(mi2'°)ApG, which comprises the following structure:OH OH(ii) the RNA sequence comprises a poly adenine sequence, optionally wherein the poly adenine sequence comprises the nucleic acid sequence according to SEQ ID NO: 84 or a variant having at least 80% sequence identity thereto;(iii) the RNA sequence comprises a 5’IITR; optionally wherein the 5’IITR comprises the nucleic acid sequence according to SEQ ID NO: 82 or a variant having at least 80% sequence identity thereto.(iv) the RNA sequence comprises a 3’IITR; optionally wherein the 3’IITR comprises the nucleic acid sequence according to SEQ ID NO: 83 or a variant having at least 80% sequence identity thereto.

12. The one or more polynucleotide(s) according to claim 10 or claim 11 comprising:(i) a first polynucleotide comprising a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:CA19-9-VH domain - CA19-9-CH1 domain - optional linker - CD3-VH domain - L1 - CD3-VL domain - optional linker - CH2 domain - CH3 domain;(ii) a second polynucleotide comprising a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:CA19-9-VH domain - CA19-9-CH1 domain - CH2 domain - CH3 domain; and / or(iii) a third polynucleotide comprising a nucleic acid sequence encoding the following polypeptide chain from N to C terminus:CA19-9-VL domain - CA19-9-CL domainoptionally wherein:(iv) the first polynucleotide comprises a nucleic acid sequence comprising in 5’-3’ orientation:5’-cap - 5’IITR - CA19-9-VH domain - CA19-9-CH1 domain - optional linker - CD3-VH domain - L1 - CD3-VL domain - optional linker - CH2 domain - CH3 domain - 3’IITR - Poly(A);(v) the second polynucleotide comprises a nucleic acid sequence comprising in 5’-3’ orientation:5’-cap - 5’IITR - CA19-9-VH domain - CA19-9-CH1 domain - CH2 domain - CH3 domain - 3’IITR - Poly(A); and / or(vi) the third polynucleotide comprises a nucleic acid sequence comprising in 5’-3’ orientation:5’-cap - 5’IITR - CA19-9-VL domain - CA19-9-CL domain - 3’UTR - Poly(A); further optionally wherein:(vii) the first polynucleotide comprises:(a) a nucleic acid sequence according to SEQ ID NO: 65 or a variant having at least 80% sequence identity to SEQ ID NO: 65;(b) a nucleic acid sequence according to SEQ ID NO: 69 or a variant having at least 80% sequence identity to SEQ ID NO: 69; or(c) a nucleic acid sequence according to SEQ ID NO: 73 or a variant having at least 80% sequence identity to SEQ ID NO: 73;(viii) the second polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 63 or a variant having at least 80% sequence identity to SEQ ID NO: 63; and / or (ix) the third polynucleotide comprises a nucleic acid sequence according to SEQ ID NO: 77 or a variant having at least 80% sequence identity to SEQ ID NO: 77.

13. A vector comprising one or more polynucleotide(s) according to any of claims 10-12.

14. A composition comprising:(i)(a) the bispecific binding molecule according to any of claims 1-9; or(b) the one or more polynucleotide(s) according to any of claims 10-12; or (c) the vector according to claim 13;optionally wherein the composition is a pharmaceutical composition comprising:(ii) a pharmaceutically acceptable carrier, diluent or excipient,optionally wherein the pharmaceutical composition further comprises(iii) a vesicle, a nanoparticle, a lipid nanoparticle (LNP), a liposome or a polymeric mixture wherein the bispecific binding molecule, the one or more polynucleotide(s), or the composition is encapsulated within the vesicle, nanoparticle, LNP, liposome or polymeric mixture.

15. The bispecific binding molecule according to any of claims 1-9; or the one or more polynucleotide(s) according to any of claims 10-12; or the composition according to claim claim 13, or the composition according to claim 14, for use in the treatment or prevention of a disease; optionally wherein the disease is cancer; further optionally wherein the cancer is selected from the list consisting of:(i) pancreatic cancer, optionally wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC), (ii) cholangiocarcinoma; and(iii) gastrointestinal cancer, optionally wherein the gastrointestinal cancer is selected from the listing consisting of: colorectal cancer, oesophageal cancer, gastric cancer, and hepatocellular cancer.

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