OA17758A

OA17758A - Production of T cell retargeting heterodimeric immunoglobulins.

Info

Publication number: OA17758A
Application number: OA1201600163
Authority: OA
Inventors: Stanislas Blein; Romain OLLIER; Samuel Hou; Darko Skegro
Original assignee: Glenmark Pharmaceuticals S.A.
Priority date: 2013-11-04
Filing date: 2014-11-04
Publication date: 2017-11-13

Abstract

The present invention describes novel hetero-dimeric immunoglobulins or fragments thereof which bind to CD3 and a disease associated antigen. These hetero-dimeric immunoglobulins have been engineered to promote hetero-dimer formation during expression and can be purified to a high degree using a Protein A differential purification technique.

Description

Production of T cell retargeting hetero-dimeric immunoglobulins

Field of the Invention

The présent invention relates to hetero-dimeric immunoglobulins that target both a component of the human CD3 antigen and a disease associated antigen and methods of making the same.

Background of the invention

T cell redirected killing is a désirable mode of action in many therapeutic areas. Various bispecific antibody formats hâve been shown to médiate T cell redirection both in pre-clinical and clinical investigations (May C et al., (2012) Biochem Pharmacol, 84(9): 1105-12; Frankel SR & Baeuerle PA, (2013) Curr Opin Chem Biol, 17(3): 385-92). Ail T cell retargeting bispecific antibodies or fragments thereof are engineered to hâve at least two antigen binding sites wherein one site binds a surface antigen on a target cell and the other site binds a T cell surface antigen. Amongst T cell surface antigens, the human CD3 epsilon subunit from the TCR protein complex has been the most targeted to redirect T cell killing.

Many bispecific antibody formats hâve been used to redirect T cell killing, these mainly include tandem of scFv fragments and diabody based formats with only few examples of Fcbased bispecific antibody formats reported (Moore PA et al., (2011) Blood, 117(17): 4542-51; May C et al., (2012) supra-, Frankel SR & Baeuerle PA, (2013) supra). Bispecific formats that will encompass a human Fc région will hâve longer circulation half-lives which may resuit in enhanced efficacy and/or less frequent dosing regimens. Among possible Fc-based bispecific formats, one preferred format to redirect T cell killing is the so-called heavy chain hetero-dimer format. This format is of particular interest as it does not allows aggregation of multiple copies of human CD3 molécules at the T cell surface thereby preventing any T cell inactivation (Klein C et al., (2012) MAbs, 4(6): 653-63).

The first described method to engineer heavy chain hetero-dimers is a method known as the “knob-into-hole” method (PCT Publication No: WO199627011 ; Merchant AM et al., (1998) Nat Biotechnol, 16(7): 677-81). Recently a chemical method known as the FAB-arm exchange method wherein two antibodies are combined into one bispecific antibody via réduction and in vitro reshuffling of half-immunoglobulins has been reported (PCT

Publication Nos: W02008119353 (Schuurman J et al.) and W02013060867 (Gramer M et al.); Labrijn AF et al., (2013) Proc Natl Acad Sci USA, 110(13): 5145-50).

Both methods and dérivatives thereof are currently inadéquate to produce Fc-based bispecific antibody formats in mammalian cell hosts. When expressing “knob-into-hole” heavy chain hetero-dimers in mammalian cell hosts, bispecific antibody recovery is impaired by the presence of homo-dimers (Jackman J et al., (2010) J Biol Chem, 285(27): 20850-9; Klein C et al., supra). The FAB-arm exchange method and dérivatives thereof suffers from the same drawback with the added problem of having first to produce the two “monospecific” antibodies separately.

When developing bispecific antibodies that redirect T cell killing via the engagement of a CD3 subunit, it is essential that no homo-dimers spécifie for the CD3 subunit are présent in the final drug product. In the case of targeting the CD3 epsilon subunit, traces of anti-human CD3 epsilon antibody species (monospecific and bivalent for the human CD3 epsilon antigen) may trigger transient T cell activation and cytokine release before leading to T cell apoptosis thereby interfering with the goal of a controlled and spécifie T cell activation. Production of stable and safe Fc-based bispecific antibodies that efficiently redirect T cell killing remains a challenge to the pharmaceutical industry with respect to purity and yields.

Accordingly there remains a need for a technology to efficiently produce anti-human CD3 based heavy chain hetero-dimers free of anti-human CD3 homo-dimers wherein the secreted bispecific antibody product is readily isolated from the cell culture supematant from a recombinant mammalian host cell line.

Techniques to purify heavy chain hetero-dimers over homo-dimers based on a differential affinity for a reagent hâve been described. The first example of known differential affinity purification technique involved the use of two different heavy chains from two different animal species, wherein one of which does not bind the affinity reagent Protein A (Lindhofer H et al., (1995) J Immunol, 155(1): 219-225). The same authors also described the use of two different heavy chains originating from two different human immunoglobulin isotypes (IGHG1 and IGHG3), one of which does not bind the affinity reagent Protein A (IGHG3; see US6,551,592 Lindhofer H et al.). More recently, a variation of this technique was reported by Davis S et al. (PCT Publication No: W02010151792) and made use of the two amino acid substitutions H435R and Y436F described by Jendeberg (1997) (Jendeberg L. et al. (1997) J

Immunol Methods, 201(1): 25-34) to abrogate the affinity for the reagent Protein A in one of the hetero-dimer heavy chains.

The preferred known differential Protein A affinity purification technique of the présent invention corresponds to a technique wherein ail three species i.e. the two homo-dimeric species and the hetero-dimer of interest differ in their total number of Protein A binding sites by at least one site and wherein one of the two homo-dimeric species has no Protein A binding site and therefore does not bind Protein A (as shown in FIG.l).

Drug stability is an important aspect of successful pharmaceutical development and VH3 based immunoglobulins or fragments thereof are of major importance to the biological drug industry. Therapeutic antibodies based on the VH3 subclass hâve been extensively developed as these frameworks bind Protein A and facilitate the testing of antibody fragments before their formatting into immunoglobulins; for example, many synthetic antibody phage display libraries used for antibody discovery are based on the VH3 subclass. In addition VH3 based antibodies are often selected for their good expression and stability over other known heavy chain variable domain subclasses.

Although a VH3 domain has only one Protein A binding site with a weaker affinity when compared to a Fc région which has two sites with a stronger affinity (Roben PW et al., (1995) J Immunol, 154(12): 6437-45), there is enough affinity to interfère with the known differential Protein A affinity purification techniques. When dealing with the purification of hetero-dimers of heavy chains wherein the heavy chain engineered in its Fc région to hâve no binding for Protein A encompasses one VH3 based antigen binding site, then Protein A binding is restored via the VH3 domain and the preferred technology described in FIG. 1 and above is no longer useful (FIG. 2A). In this instance, abrogating Protein A binding in the VH3 based antigen binding site provides a straightforward solution and allows to keep the initial architecture of the desired hetero-dimer (FIG. 2B). Alternatively, the heavy chain heterodimer can be re-engineered to hâve the VH3 based antigen binding site located on the heavy chain that binds Protein A in its Fc région (FIG. 2C; note that a VH3 domain has a weaker affinity for Protein A compared to a Fc monomer hence the hetero-dimer of interest still elutes at a separate pH value from the other homo-dimeric species, typically at pH 4, while the homo-dimeric species that binds Protein A now encompasses two additional Protein A binding sites and elutes at a pH value < 3).

More importantly, when dealing with the purification of hetero-dimers of heavy chains wherein both heavy chains encompass a VH3 based antigen binding site, then the relocation strategy described above may only be partially helpful (FIG. 2D and FIG. 15B). Protein A based differential purification is only enabled when Protein A binding in at least one (FIG. 2E) or both (FIG. 2F) VH3 based antigen binding sites is abrogated .

Accordingly, there remains a need to abrogate Protein A binding within VH3 domains when undertaking the production of hetero-dimers of heavy chains encompassing this variable domain subclass.

Summary of the Invention

The présent invention provides new anti-human CD3 bispecific antibodies comprising a second binding arm which can recognise and bind to a disease associated antigen.

In the context of the présent invention a disease associated antigen means any antigen or epitope associated with a pathological state such as an oncogenic marker or a marker of some other metabolic or immunological dysfunction. In addition a disease marker my also relate to an infectious disease such as a pathogenic virus or bacteria.

In accordance with the présent invention the two binding arms of the anti-human CD3 bispecific antibody each comprise an immunoglobulin constant région and wherein the first arm or polypeptide binds to protein A and the second arm or polypeptide does not bind to protein A.

According to the présent invention the binding of the first polypeptide to protein A and the lack of binding of the second polypeptide to protein A, is not intended to mean that the second polypeptide may not hâve some residual binding to protein A and it is instead intended that the second polypeptide binds less well to protein A in comparison to the first arm.

According to the présent invention the first and second polypeptides of the hetero-dimeric immunoglobulin or fragment thereof, comprise an engineered immunoglobulin constant région with a modified CH3 région having a protein-protein interface that favours heterodimer formation over homo-dimer formation. In a preferred embodiment, the présent invention provides a hetero-dimeric immunoglobulin or fragment thereof wherein the first and second polypeptides comprise an engineered immunoglobulin constant région with a modified CH3 domain having a protein-protein interface, wherein the protein-protein interface of the first polypeptide comprises an amino acid substitution at a position selected from the group consisting of: 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88 and 90 (IMGT® numbering), and wherein the protein-protein interface of the second polypeptide comprises an amino acid substitution at a position selected from the group consisting of: 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 84.4, 85.1, 86, 88 and 90 (IMGT® numbering).

Preferably wherein the protein-protein interface of the second polypeptide comprises an amino acid substitution at position 84.4 and at least one further substitution at a position selected from the group consisting of: 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88 and 90 (IMGT® numbering).

In a further embodiment, the présent invention provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the first and second polypeptides comprise an engineered immunoglobulin constant région with a modified CH3 domain having a protein-protein interface, wherein the protein-protein interface of the first polypeptide comprises an amino acid substitution at position 88 and at a position selected from the group consisting of: 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86 and 90 (IMGT® numbering), and wherein the protein-protein interface of the second polypeptide comprises an amino acid substitution at position 85.1 and/or 86 and at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 84.4, 88 and 90 (IMGT® numbering).

According to a further aspect of the présent invention the epitope binding région of the first polypeptide binds the CD3 protein complex and the epitope binding région of the second polypeptide binds a disease associated antigen or wherein the epitope binding région of the first polypeptide binds a disease associated antigen and the epitope binding région of the second polypeptide binds the CD3 protein complex; and wherein the epitope binding région that binds the CD3 protein complex comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 194, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 195 and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 196, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 197, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 198 and a light chain CDR3 comprising the amino acid sequences of: SEQ ID NO: 199; or wherein the epitope binding région that binds the CD3 protein complex comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 200, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 201 and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 202, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 203, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 204 and a light chain CDR3 comprising the amino acid sequences of: SEQ ID NO: 205; or wherein the epitope binding région that binds the CD3 protein complex comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 352, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 353 and aheavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 354, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 355, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 356 and a light chain CDR3 comprising the amino acid sequences ofSEQIDNO: 357.

Use of these new anti-human CD3 bispecific antibodies is not limited to but includes treatments of various human cancers and autoimmune and inflammatory diseases. The spécifie destruction of cancer cells over healthy cells and tissues represents a primary objective in oncology. Therapeutics that could safely redirect T cell killing against tumour associated cell surface antigens may offer improved clinical effïcacy. Known areas of clinical unmet needs in oncology include but are not limited to breast cancer, metastatic breast cancer, ovarian cancer, pancreatic cancer, lung cancer, lymphomas and multiple myeloma. Elimination of disease-causing T cells could be more bénéficiai than inhibiting T cell différentiation in treating autoimmune and inflammatory diseases such as psoriasis, multiple sclerosis and diabètes.

A preferred set of disease associated antigens corne from the gene products CD33, TROP2, CD105, GD2, GD3, CEA, VEGFR1, VEGFR2, NCAM, CD133, CD123, ADAM17, MCSP, PSCA, FOLR1, CD 19, CD20, CD38, EpCAM, HER2, EGFR, PSMA, IgE, Integrin a4bl, CCR5, LewisY, FAP, MUC-1, Wue-1, MSP, EGFRvIII, P glycoprotein, AFP, ALK, BAGE proteins, CD30, CD40, CTLA4, ErbB3, ErbB4, Mesothelin, 0X40, CA125, CAIX, CD66e, cMet, EphA2, HGF/SF , MUC1 , Phosphatidylserine, TAG-72 , TPBG, β-catenin, brc-abl, BRCA1, BORIS, CA9, caspase-8, CDK4, Cyclin-Bl, CYP1B1, ETV6-AML, Fra-1, FOLR1, GAGE-1, GAGE-2, GloboH, glypican-3, GM3, gplOO, HLA/B-raf, HLA/k-ras, HLA/MAGEA3, hTERT, LMP2, MAGE1, MAGE2, MAGE3, MAGE4, MAGE6, MAGE12, MART-1, ML-IAP, Muc2, Muc3, Muc4, Muc5, Mucl6, MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, pl5, p53, PAP, PAX3 PAX5, PCTA-1, PLAC1, PRLR, PRAME, RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, Steap-1, Steap-2, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TRP-1, TRP-2, tyrosinase, uroplakin-3.

A hetero-dimeric immunoglobulin or fragment thereof according to the invention, wherein the epitope binding région that binds a disease associated antigen comprises heavy chain CDR1, CDR2 and CDR3 amino acid sequences and light chain CDR1, CDR2 and CDR3 amino acid sequences, respectively, selected from the group consisting of:

i) SEQ ID NOs: 206-211;

ii) SEQ ID NOs: 212-217;

iii) SEQ ID NOs: 218-223;

iv) SEQ ID NOs: 224-229;

v) SEQ ID NOs: 230-235;

vi) SEQ ID NOs: 236-241;

vii) SEQ ID NOs: 242 - 247;

viii) SEQ ID NOs: 248 - 253;

ix) SEQ ID NOs: 254-259;

x) SEQ ID NOs: 260-265;

xi) SEQ ID NOs: 266 - 271; and xii) SEQ ID NOs: 272 - 277;

In accordance with a further aspect of the présent invention the constant région of the second polypeptide of the hetero-dimeric immunoglobulin or fragment thereof, comprises an IgG3

CH3 région.

In accordance with a further aspect of the présent invention the constant région of the second polypeptide of the hetero-dimeric immunoglobulin or fragment thereof, comprises a CH3 région other than that from IgG, and the non-IgG3 CH3 région comprises at least one substitution so as to decrease/abolish protein A binding.

According to a further aspect of the présent invention the epitope binding région of second polypeptide of the hetero-dimeric immunoglobulin or fragment thereof comprises a VH3 région comprising at least one modification that reduces protein A binding.

The inventors hâve shown that VH3 based antigen binding sites can be readily produced and purified with a high degree of purity in a single Protein A chromatography step. These antibodies may exhibit higher efficacy over current thérapies in addition to their ease of production.

The présent invention also provides a method to produce anti-human CD3 bispecific heavy chain hetero-dimers having at least one VH3 based antigen binding site from a recombinant mammalian host cell line wherein the bispecific antibody product is readily isolated after a single Protein A chromatography step with a high degree of purity.

In particular the modified VH3 région comprises an amino acid substitution selected from the group consisting of: 57, 65, 81, 82a and combination 19/57/59 (Kabat numbering) and even more preferably wherein the modified VH3 région comprises an amino acid substitution selected from the group consisting of: 57A, 57E, 65S, 81E, 82aS and combination 19G/57A/59A (Kabat numbering).

According to a further aspect of the présent invention the hetero-dimeric immunoglobulin or fragment thereof, may comprise further substitutions wherein the heavy chain variable framework région comprises an amino acid substitution selected from the group consisting of:

I34M, V48I, A49G, R58N/Y, I69L, A71T and T73K (Kabat numbering) and the light chain variable framework région comprises an amino acid substitution selected from the group consisting of: M4L, V33M, A34N, L46R, L47W, T51A, R66G, F71Y and P96F (Kabat numbering); or wherein the heavy chain variable framework région comprises the amino acid substitutions I34M, A49G and A71T (Kabat numbering) and the light chain variable framework région comprises the amino acid substitutions M4L, L46R, L47W and F71Y (Kabat numbering).

In a further embodiment, the epitope binding région that binds to the CD3 protein complex comprises a heavy chain variable framework région that is the product of or derived from the human VH3 subclass. Preferably the heavy chain variable framework région is the product of or derived from human IGHV3-23. More preferably, the heavy chain variable framework région is the product of or derived from human IGHV3-23*04 (SEQ ID NO: 22). The heavy chain variable framework région comprises at least one amino acid modification from the corresponding framework région of the heavy chain variable région of the corresponding murine antibody comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 60.

In a preferred embodiment, the epitope binding région of the first polypeptide that binds to the CD3 protein complex comprises a light chain variable framework région that is the product of or derived from the human VK1 subclass or the human VK3 subclass. Preferably the light chain variable framework région is the product of or derived from human VK1-39 or VK3-20. More preferably the light chain variable framework région is the product of or derived from human IGKV1-39*O1 (SEQ ID NO: 23) or IGKV3-20*01 (SEQ ID NO: 24). The light chain variable framework région comprises at least one amino acid modification from the corresponding framework région of the light chain variable région of the corresponding murine antibody comprising the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 61.

In a preferred embodiment, the epitope binding région that binds to the CD3 protein complex comprises a humanized heavy chain variable domain having the back mutations selected from the group consisting of: I34M, V48I, A49G, R58N/Y, I69L, A71T and T73K (Kabat numbering) and a humanized light chain variable domain having the back mutations selected from the group consisting of: M4L, V33M, A34N, L46R, L47W, R66G, F71Y and P96F (Kabat numbering). More preferably, the epitope binding région that binds to the CD3 protein complex comprises a humanized heavy chain variable domain having the back mutations

I34M, A49G and A71T (Kabat numbering) and a humanized light chain variable domain having the back mutations M4L, L46R, L47W and F71Y (Kabat numbering).

According to a further aspect of the présent invention the epitope binding région that binds the CD3 protein complex of the hetero-dimeric immunoglobulin or fragment thereof, comprises a heavy chain variable région comprising the amino acid sequence of SEQ ID NO: 48, and a light chain variable région comprising the amino acid sequence of SEQ ID NO: 51; or wherein the epitope binding région that binds the CD3 protein complex comprises a heavy chain variable région comprising the amino acid sequence of SEQ ID NO: 49, and a light chain variable région comprising the amino acid sequence of SEQ ID NO: 51; or wherein the epitope binding région that binds the CD3 protein complex comprises a heavy chain variable région comprising the amino acid sequence of SEQ ID NO: 358, and a light chain variable région comprising the amino acid sequence of SEQ ID NO: 51; or wherein the epitope binding région that binds the CD3 protein complex comprises a heavy chain variable région comprising the amino acid sequence of SEQ ID NO: 101, and a light chain variable région comprising the amino acid sequence of SEQ ID NO: 105; or wherein the epitope binding région that binds the CD3 protein complex comprises a heavy chain variable région comprising the amino acid sequence of SEQ ID NO: 103, and a light chain variable région comprising the amino acid sequence of SEQ ID NO: 106; or wherein the epitope binding région that binds the CD3 protein complex comprises a heavy chain variable région comprising the amino acid sequence of SEQ ID NO: 104, and a light chain variable région comprising the amino acid sequence of SEQ ID NO: 106.

The CD3 protein complex comprises a number of subunits, for example, delta, epsilon and gamma. In a preferred embodiment, the epitope binding région that binds to the CD3 protein complex binds to the CD3 epsilon subunit.

An epitope binding région as described herein includes the combination of one or more heavy chain variable domains and one or more complementary light chain variable domains which together form a binding site which permits the spécifie binding of the hetero-dimeric immunoglobulin or fragment thereof to one or more epitopes. In an embodiment of the présent invention, the epitope binding région of the first poly peptide comprises a FAB and the epitope binding région of the second polypeptide comprises a scFv. Altematively, the epitope binding région of the first poly peptide comprises a scFv and the epitope binding région of the second polypeptide comprises a FAB.

In one embodiment, the epitope binding région that binds a disease associated antigen binds to HER2. The epitope binding région comprises a heavy chain variable ffamework région that is the product of or derived from the human VH3 subclass, preferably human VH3-23, more preferably human IGHV3-23*04 (SEQ ID NO: 22), and a light chain variable ffamework région that is the product of or derived from the human VK1 subclass, preferably human VK1-39, more preferably human IGKV1-39*O1 (SEQ ID NO: 23).

In a preferred embodiment, the epitope binding région that binds the disease associated antigen HER2 comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 20 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 21. In a further preferred embodiment, the epitope binding région that binds HER2 may comprise a heavy chain variable domain and a light chain variable domain joined by a G4S linker forming a scFv fragment comprising the amino acid sequence of SEQ ID NO: 107. Preferably, the variable domain of the scFv fragment comprises a modification to abrogate binding to Protein A, wherein the amino acid substitution is 65S (Kabat numbering) and wherein the scFv fragment comprises the amino acid sequence of SEQ ID NO: 109 or wherein the amino acid substitution is 82aS (Kabat numbering) and wherein the scFv fragment comprises the amino acid sequence of SEQ ID NO: 111.

In particular wherein said Herceptin binding arm comprises a heavy chain variable région encoded by SEQ ID NO: 20 and a light chain variable région encoded by SEQ ID NO: 21.

In another embodiment, the epitope binding région that binds a disease associated antigen binds to CD38. The epitope binding région comprises a heavy chain variable ffamework région that is the product of or derived from the human VH3 subclass, preferably human VH3-23, more preferably human IGHV3-23*04 (SEQ ID NO: 22). The heavy chain variable ffamework région comprises at least one amino acid modification from the corresponding ffamework région of the heavy chain variable région of the corresponding murine antibody comprising the amino acid sequence of SEQ ID NO: 112 or 114 or 122. The epitope binding région further comprises a light chain variable framework région that is the product of or derived from the human VK1 subclass, preferably human VK1-39, more preferably human IGKVl-39*01 (SEQ ID NO: 23). The light chain variable framework région comprises at least one amino acid modification from the corresponding framework région of the light chain variable région of the corresponding murine antibody comprising the amino acid sequence of SEQ IDNO: 113 or 115 or 123.

In particular the CD38 binding polypeptide comprises variable heavy chain domain and variable light chain domain pair encoded by SEQ ID NOs: 116/117, 129/130, 133/134 and 135/136.

In one embodiment, the epitope binding région that binds a disease associated antigen binds to 0X40. The epitope binding région comprises a heavy chain variable framework région that is the product of or derived from the human VH3 subclass, preferably human VH3-23, more preferably human IGHV3-23*04 (SEQ ID NO: 22). The heavy chain variable framework région comprises at least one amino acid modification from the corresponding framework région of the heavy chain variable région of the corresponding murine antibody comprising the amino acid sequence of SEQ ID NO: 139. The epitope binding région further comprises a light chain variable framework région that is the product of or derived from the human VK1 subclass, preferably human VK1-39, more preferably human IGKV1-39*O1 (SEQ ID NO: 23). The light chain variable framework région comprises at least one amino acid modification from the corresponding framework région of the light chain variable région of the corresponding murine antibody comprising the amino acid sequence of SEQ ID NO: 140.

Most preferably, the humanized heavy chain variable domain comprises a modification to abrogate binding to Protein A comprising the substitution G65S or the substitution N82aS (Kabat numbering).

In particular the 0X40 binding polypeptide comprises variable heavy chain domain and variable light chain domain pair encoded by SEQ ID NOs: 141/142, 278/280 and 279/281.

In one embodiment, the epitope binding région that binds a disease associated antigen binds to CD19. The epitope binding région comprises a heavy chain variable framework région that is the product of or derived from the human VH3 subclass, preferably human VH3-23, more preferably human IGHV3-23*04 (SEQ ID NO: 22) and most preferably comprises the amino acid sequence of SEQ ID NO: 296. The epitope binding région further comprises a light chain variable framework région that is the product of or derived from the human VK1 subclass, preferably human VK1-39, more preferably human IGKV1-39*O1 (SEQ ID NO: 23) and most preferably comprises the amino acid sequence of SEQ ID NO: 297. In a preferred embodiment, the heavy chain variable domain comprises a modification to abrogate binding to Protein A comprising the substitution G65S or the substitution N82aS (Kabat numbering).

In particular the CD 19 binding polypeptide comprises variable heavy chain domain and variable light chain domain pair encoded by SEQ ID NOs: 296/297.

In one embodiment, the epitope binding région that binds a disease associated antigen binds to CD20. The epitope binding région comprises a heavy chain variable framework région that is the product of or derived from the human VH3 subclass, preferably human VH3-23, more preferably human IGHV3-23*04 (SEQ ID NO: 22). The heavy chain variable framework région comprises at least one amino acid modification from the corresponding framework région of the heavy chain variable région of the corresponding murine antibody comprising the amino acid sequence of SEQ ID NO: 143. The epitope binding région further comprises a light chain variable framework région that is the product of or derived from the human VK1 subclass, preferably human VK1-39, more preferably human IGKVl-39*01 (SEQ ID NO: 23). The light chain variable framework région comprises at least one amino acid modification from the corresponding framework région of the light chain variable région of the corresponding murine antibody comprising the amino acid sequence of SEQ ID NO: 144.

In particular the EGFR binding polypeptide comprises variable heavy chain domain and variable light chain domain pair encoded by SEQ ID NOs: 143/144, 282/284, 283/285.

In one embodiment, the epitope binding région that binds a disease associated antigen binds to EGFR. The epitope binding région comprises a heavy chain variable framework région that is the product of or derived from the human VH3 subclass, preferably human VH3-23, more preferably human IGHV3-23*04 (SEQ ID NO: 22). The heavy chain variable framework région comprises at least one amino acid modification from the corresponding framework région of the heavy chain variable région of the corresponding murine antibody comprising the amino acid sequence of SEQ ID NO: 145. The epitope binding région further comprises a light chain variable framework région that is the product of or derived from the human VK1 subclass, preferably human VK1-39, more preferably human IGKV1-39*O1 (SEQ ID NO: 23). The light chain variable framework région comprises at least one amino acid modification from the corresponding framework région of the light chain variable région of the corresponding murine antibody comprising the amino acid sequence of SEQ ID NO: 146.

In particular the CD20 binding polypeptide comprises variable heavy chain domain and variable light chain domain pair encoded by SEQ ID NOs: 145/146, 286/288, 287/289, 290/291, 292/294.

In one embodiment, the epitope binding région that binds a disease associated antigen binds to IgE. The epitope binding région comprises a heavy chain variable framework région that is the product of or derived from the human VH3 subclass, preferably human VH3-23, more preferably human IGHV3-23*04 (SEQ ID NO: 22). The heavy chain variable framework région comprises at least one amino acid modification from the corresponding framework région of the heavy chain variable région of the corresponding humanized antibody comprising the amino acid sequence of SEQ ID NO: 298 or the corresponding murine antibody comprising the amino acid sequence of SEQ ID NO: 304. The epitope binding région further comprises a light chain variable framework région that is the product of or derived from the human VK1 subclass, preferably human VK1-39, more preferably human IGKV1-39*O1 (SEQ ID NO: 23). The light chain variable framework région comprises at least one amino acid modification from the corresponding framework région of the light chain variable région of the corresponding humanized antibody comprising the amino acid sequence of SEQ ID NO: 299 or the corresponding murine antibody comprising the amino acid sequence of SEQ ID NO: 305.

Most preferably, the heavy chain variable domain comprises a modification to abrogate binding to Protein A comprising the substitution G65S or the substitution N82aS (Kabat numbering).

In particular the IgE binding polypeptide comprises variable heavy chain domain and variable light chain domain pair encoded by SEQ ID NOs:, 298/299, 300/302, 301/303, 304/305, 306/308, 307/309.

Anti-CD3 antibodies hâve been found to trigger toxicity by both direct and indirect mechanisms. Indirect mechanisms are mediated by the Fc région of the CD3 antibody which acts with the Fc receptor expressing immune cells and lead to transient T cell activation and cytokine release. Therefore in order to improve the safety of the hetero-dimeric immunoglobulins or fragment thereof as described herein, the immunoglobulin constant région of the first and/or second polypeptide has reduced or no binding for effector immune cells and/or complément Clq. Preferably, the immunoglobulin constant région is engineered to abrogate Fc receptor binding in the lower hinge région. More preferably the immunoglobulin constant région of the first and/or second polypeptide comprises the substitution(s) L234A and/or L235A (EU numbering). Most preferably, the immunoglobulin constant région of the first and/or second polypeptide comprises the substitutions L234A and L235A (EU numbering).

In another aspect, the disclosure of the présent invention also describes a hetero-dimeric immunoglobulin or fragment thereof wherein the epitope binding région binds to the CD3 epsilon subunit of the CD3 protein complex and comprises a FAB having a FAB thermostability superior to the FAB thermo-stability of the OKT3 chimera comprising a heavy chain variable domain of amino acid sequence of SEQ ID NO: 25 and a light chain variable domain of amino acid sequence of SEQ ID NO: 26, as measured by Differential Scanning Calorimetry (DSC) as described in FIG. 9.

In further aspect, the présent invention provides a hetero-dimeric immunoglobulin or fragment thereof as described herein wherein one epitope binding région binds to the CD3 epsilon subunit of the CD3 protein complex and the other epitope binding région that binds a disease associated antigen, binds HER2. The potency of such a hetero-dimeric immunoglobulin or fragment thereof to redirect T-cell killing can be measured in an in vitro assay using a flow cytometry method (RDL-FACS) or a colorimétrie based method (RDLMTS) on cell lines expressing HER2 such as JIMT-1, BT-474 and MDA-MB-231, as described in the Examples.

In one embodiment the hetero-dimeric immunoglobulin or fragment thereof that binds to CD3 epsilon and HER2 kills JIMT-1 cells with a potency of 21 pM or less. Alternatively, the hetero-dimeric immunoglobulin or fragment thereof also kills BT-474 cells with a potency of 2 pM or less. In addition, the hetero-dimeric immunoglobulin or fragment thereof also kills MDA-MB-231 cells with a potency of 0.2 nM or less. The cytotoxicity of ail cell lines was measured in a RDL assay performed with human PBMCs at an effectoritarget cell ratio of 10:1 over 48h. Furthermore, this hetero-dimeric immunoglobulin or fragment thereof shows a potent anti-tumour effect wherein tested in vivo in a JIMT-l/PBMC xenograft model. Preferably the hetero-dimeric immunoglobulin or fragment thereof kills JIMT-1 cells at 0.05 mg/kg in a JIMT-1 cell xenograft.

In a preferred embodiment, the présent invention provides hetero-dimeric immunoglobulin or fragment thereof binding to:

i) the CD3 protein complex and HER2, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 159 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 47 and binds CD3 epsilon, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 160 and binds HER2;

ii) the CD3 protein complex and HER2, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 161 and is assembled with a cognate light chain of amino acid sequence of SEQ ID NO: 3 and binds HER2, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 162 and binds CD3 epsilon;

iii) the CD3 protein complex and HER2, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 163 and is assembled with a light chain of amino acid sequence of

SEQ JD NO: 47 and binds CD3 epsilon, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 164 and binds HER2;

iv) the CD3 protein complex and HER2, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 165 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 166 and binds CD3 epsilon, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 167 and binds HER2;

v) the CD3 protein complex and HER2, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 168 and is assembled with a light chain of amino acid sequence of SEQ JD NO: 89 and binds CD3 epsilon, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 167 and binds HER2;

vi) the CD3 protein complex and CD38, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 169 and is assembled with a cognate light chain of amino acid sequence of SEQ ID NO: 119 and binds CD38, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 162 and binds CD3 epsilon;

vii) the CD3 protein complex and CD38, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 170 and is assembled with a cognate light chain of amino acid sequence of SEQ ID NO: 138 and binds CD38, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 171 and binds CD3 epsilon;

viii) the CD3 protein complex and CD38, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 176 and is assembled with a cognate light chain of amino acid sequence of SEQ ID NO: 119 and binds CD38, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 177 and binds CD3 epsilon;

ix) the CD3 protein complex and CD38, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 178 and is assembled with a cognate light chain of amino acid sequence of SEQ ID NO: 128 and binds CD38, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 179 and binds CD3 epsilon;

x) the CD3 protein complex and 0X40 wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 172 and is assembled with a cognate light chain of amino acid sequence of SEQ ID NO: 173 and binds 0X40, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 162 and binds CD3 epsilon;

xi) the CD3 protein complex and EGFR wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 174 and is assembled with a cognate light chain of amino acid sequence of SEQ ID NO: 175 and binds EGFR, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 171 and binds CD3 epsilon;

xii) the CD3 protein complex and CD20, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 180 and is assembled with a cognate light chain of amino acid sequence of SEQ ID NO: 181 and binds CD20, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 177 and binds CD3 epsilon.

In a further embodiment, the présent invention provides hetero-dimeric immunoglobulin or fragment thereof binding to:

the CD3 protein complex and HER2, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 310 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 3 and binds HER2, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 311 and binds CD3 epsilon;

the CD3 protein complex and CD38, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 312 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 132 and binds CD38, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 311 and binds CD3 epsilon;

the CD3 protein complex and CD38, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 313 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 138 and binds CD38, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 311 and binds CD3 epsilon;

the CD3 protein complex and 0X40, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 314 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 315 and binds 0X40, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 311 and binds CD3 epsilon;

the CD3 protein complex and 0X40, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 316 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 317 and binds 0X40, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 311 and binds CD3 epsilon;

the CD3 protein complex and CD20, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 318 and is assembled with a light chain of amino acid sequence of

SEQ ID NO: 319 and binds CD20, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 311 and binds CD3 epsilon;

the CD3 protein complex and CD20, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 320 and is assembled with a light chain of amino acid sequence of

SEQ ID NO: 321 and binds CD20, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 311 and binds CD3 epsilon;

the CD3 protein complex and EGFR, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 322 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 323 and binds EGFR, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 311 and binds CD3 epsilon;

the CD3 protein complex and EGFR, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 324 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 325 and binds EGFR, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 311 and binds CD3 epsilon;

the CD3 protein complex and EGFR, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 326 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 327 and binds EGFR, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 311 and binds CD3 epsilon;

the CD3 protein complex and EGFR, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 328 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 329 and binds EGFR, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 311 and binds CD3 epsilon;

the CD3 protein complex and CD 19, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 330 and is assembled with a light chain of amino acid sequence of SEQ JD NO: 331 and binds CD19, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 311 and binds CD3 epsilon;

the CD3 protein complex and IgE, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 332 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 333 and binds IgE, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 311 and binds CD3 epsilon;

the CD3 protein complex and IgE, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 334 and is assembled with a light chain of amino acid sequence of SEQ ID

NO: 335 and binds IgE, and wherein the second polypeptide has an amino acid sequence of

SEQ ID NO: 311 and binds CD3 epsilon;

the CD3 protein complex and IgE, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 336 and is assembled with a light chain of amino acid sequence of SEQ ID

NO: 337 and binds IgE, and wherein the second polypeptide has an amino acid sequence of

SEQ ID NO: 311 and binds CD3 epsilon;

the CD3 protein complex and IgE, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 338 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 339 and binds IgE, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 311 and binds CD3 epsilon;

the CD3 protein complex and 0X40, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 340 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 173 and binds 0X40, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 311 and binds CD3 epsilon;

the CD3 protein complex and CD20, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 341 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 181 and binds CD20, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 311 and binds CD3 epsilon;

the CD3 protein complex and EGFR, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 342 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 175 and binds EGFR, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 311 and binds CD3 epsilon;

the CD3 protein complex and EGFR, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 343 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 344 and binds EGFR, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 311 and binds CD3 epsilon;

the CD3 protein complex and IgE, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 345 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 346 and binds IgE, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 311 and binds CD3 epsilon;

the CD3 protein complex and IgE, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 347 and is assembled with a light chain of amino acid sequence of SEQ ID

NO: 348 and binds IgE, and wherein the second polypeptide has an amino acid sequence of

SEQ JD NO: 311 and binds CD3 epsilon.

In accordance with a further aspect of the présent invention the hetero-dimeric immunoglobulin or fragment thereof wherein said CD3 binding polypeptide comprises at least one or a combination of a heavy and light chain variable régions selected from the group: SEQ ID NOs: 48/51, 49/51,101/105,103/106,104/106, 358/51 and wherein said disease associated antigen binding polypeptide comprises at least one or a combination of a heavy and light chain variable régions selected from the group: SEQ ID NOs: 20/21,116/117, 129/130, 133/134, 135/136, 139/140,141/142, 278/280, 279/281, 143/144, 282/284, 283/285, 296/297, 145/146, 286/288, 287/289, 290/291, 292/294, 293/295, 298/299, 300/302, 301/303, 304/305, 306/308, 307/309.

As discussed above for bispecific antibody génération, there is a need to efficiently produce anti-human CD3 based heavy chain hetero-dimers free of anti-human CD3 homo-dimers wherein the secreted bispecific antibody product is readily isolated from the cell culture supematant from a recombinant mammalian host cell line. To this effect, a Protein A based differential purification technique can be used to isolate hetero-dimeric immunoglobulins or fragments thereof encompassing the variable domain subclass of VH3, wherein the Protein A binding site in at least one but preferably both VH3 based epitope binding régions is abrogated. Therefore, in another aspect, the présent invention provides an in vitro method for the production of a hetero-dimeric immunoglobulin or fragment thereof as described herein, comprising the following steps:

ia) preparing a DNA vector encoding a heavy chain of the first polypeptide and a DNA vector encoding a heavy chain of the second polypeptide wherein one or both DNA vectors or a third DNA vector optionally encode a common light chain or a light chain that assembles with a heavy chain of the first or second polypeptide; or ib) preparing one DNA vector encoding heavy chains of the first and second polypeptides wherein the DNA vector optionally encodes a common light chain or a light chain that assembles with a heavy chain of the first or second polypeptide; and wherein said DNA vectors are suitable for transient or stable expression in a mammalian host cell;

ii) transfecting or co-transfecting the DNA vector(s) from (i) in a mammalian host cell line;

iii) culturing the transfected cell line or stably selected clone therefrom and harvesting the cell culture supematant;

iv) contacting the cell culture supematant on a Protein A affïnity chromatography resin;

v) eluting and collecting the hetero-dimeric immunoglobulin of interest.

Preferably the hetero-dimeric immunoglobulin or fragment thereof found in the purified material from step (v) is at least 95% pure. More preferably the hetero-dimeric immunoglobulin or fragment thereof found in the purified material from step (v) is at least 96% pure. Even more preferably the hetero-dimeric immunoglobulin or fragment thereof found in the purified material from step (v) is at least 97%. Purity of the hetero-dimeric immunoglobulin or fragment thereof found in the purified material can be measured by capillary electrophoresis.

In accordance with a further aspect of the présent invention there is provided a polypeptide comprising at least one CDRs from the groups: SEQ ID NOs: 224-229, 230-235 and 352-357; or combinations of heavy chain variable domain and light chain variable domain pairs selected from the group: SEQ IDNOs: 122/123, 124/125, 129/130, 135/136, 133/134 104 /106; and heavy and light chain sequence pair selected from the group: 126/127 or 128, 131/132, 137/138,359/360.

Brief Description of the Figures

FIG. 1: Schematic diagram of the preferred differential affïnity purification technique using Protein A. None of the heavy chains encompass a VH3 based antigen binding site. Legend: [(A+)] means a functional Protein A binding site and [(A-)] means a nonfunctional Protein A binding site. pH of elution is indicated.

FIG. 2A-F: Schematic diagrams illustrating the problems faced when purifying hetero-dimers of heavy chains encompassing one or more VH3 domains using differential protein A chromatography. Examples of solutions based on mutating the Protein A binding site within at least one VH3 domain of the hetero-dimer are shown. FIG. 2A: Problem faced when the hetero-dimer of heavy chains encompasses a VH3 domain within the heavy chain that does not bind Protein A in its Fc région. FIG. 2B: Solution to the purification problem described in FIG.2A, the heavy chain of the hetero-dimer that does not bind Protein A in its Fc région encompasses a VH3 domain which has been mutated to abrogate its Protein A binding site.

FIG. 2C: Alternative solution to the problem described in FIG. 2A, the hetero-dimer encompasses only one VH3 domain and the hetero-dimer is engineered to hâve its VH3 domain located on the heavy chain that binds Protein A in its Fc région (VH3 domain relocation strategy as a solution). FIG. 2D: Problem faced when both heavy chains of the hetero-dimer encompass a VH3 domain. FIG. 2E: Solution to the purification problem described in FIG.2D, the heavy chain of the hetero-dimer that does not bind Protein A in its Fc région encompasses a VH3 domain which has been mutated to abrogate its Protein A binding site. FIG. 2F: Alternative solution to the purification problem described in FIG.2D, each VH3 domain has its Protein A binding site abrogated. Boxed species indicated that these species co-elute during the differential Protein A chromatography process. pH values A and B differ by about one pH unit and allow efficient séparation of the species that binds Protein A. Typically pH values for pH A and pH B are 4 and 3, respectively. Legend for ail figures: [(A+)] means a functional Protein A binding site and [(A-)] means a nonfunctional Protein A binding site.

FIG. 3: Protein A gradient mode chromatography traces for Fc 133 (HiTrap™ MabSelect SuRe™ Protein A column). Plots of absorbance at 280 nm vs. total volume of mobile phase are shown as solid line. Plots of mobile phase pH and percentage of eluent buffer (B) présent in mobile phase are shown as dashed and dotted-dashed lines, respectively.

FIG. 4A-C: Protein A gradient mode chromatography traces. Plots of absorbance at 280 nm vs. total volume of mobile phase are shown as solid line. Plots of mobile phase pH and percentage of eluent buffer (B) présent in mobile phase are shown as dashed and dotteddashed lines, respectively. FIG. 4A: Anti-HER2 FAB-Fc 133 (HiTrap™ MabSelect SuRe™ Protein A column). FIG. 4B: Anti-HER2 scFv-Fc 133 (HiTrap™ MabSelect SuRe™ Protein A column). FIG. 4C: Anti-HER2 FAB (HiTrap™ MabSelect SuRe™ Protein A column and HiTrap™ MabSelect™ Protein A column).

FIG. 5: Représentative amino acid sequences for each of the seven known human VH framework subclasses. Sequences were aligned according to the Kabat numbering. Positions in the human VH3-23 framework subclass that interact with the domain D of Protein A are shown in bold.

FIG. 6A-I: Protein A gradient mode chromatography traces (HiTrap™ MabSelect™ Protein

A column). Plots of absorbance at 280 nm vs. total volume of mobile phase are shown as solid line. Plots of mobile phase pH and percentage of eluent buffer (B) présent in mobile phase are shown as dashed and dotted-dashed fines, respectively. FIG. 6A: Anti-HER2 FAB. FIG. 6B:

Anti-HER2 FAB T57A. FIG. 6C: Anti-HER2 FAB T57E. FIG. 6D: Anti-HER2 FAB G65S.

FIG. 6E: Anti-HER2 FAB R66Q. FIG. 6F: Anti-HER2 FAB T68V. FIG. 6G: Anti-HER2 FAB Q81E. FIG. 6H: Anti-HER2 FAB N82aS. FIG. 61: Anti-HER2 FAB R19G/T57A/Y59A.

FIG. 7: Equilibrium dissociation constants (KD) of selected anti-HER2 FAB variants for the HER2 antigen.

FIG. 8A-D: Protein A gradient mode chromatography traces (HiTrap™ MabSelect SuRe™ Protein A column). Plots of absorbance at 280 nm vs. total volume of mobile phase are shown as solid line. Plots of mobile phase pH and percentage of eluent buffer (B) présent in mobile phase are shown as dashed and dotted-dashed fines, respectively. FIG. 8A: Anti-HER2 scFv(G65S)-Fc 133. FIG. 8B: Anti-HER2 scFv(N82aS)-Fc 133. FIG. 8C: Anti-HER2 FAB(G65S)-Fc 133. FIG. 8D: Anti-HER2 FAB(N82aS)-Fc 133.

FIG. 9A-F: These figures ail relate to OKT3 humanization on stable human frameworks.

FIG. 9A-C: Summary of humanized candidates formatted as human IgGl antibodies. HPBALL staining relative to the chimeric OKT3 antibody: (-) indicates no binding, (+) weaker binding, (++) moderate binding and (+++) similar binding. FIG. 9D: DSC profiles of selected antibodies of candidates. FIG. 9E: Summary of humanized candidates formatted as scFv-Fc fusions. HPB-ALL staining relative to the chimeric OKT3 antibody: (-) indicates no binding, (+) weaker binding, (++) moderate binding and (+++) similar binding. FIG. 9F: DSC profiles of selected scFv-Fc candidates.

FIG. 10A-B: These figures ail relate to SP34 humanization on stable human frameworks. FIG. 10A: Summary of humanized candidates formatted as human IgGl antibodies. FIG. 10B: Summary of humanized candidates formatted as scFv-Fc fusion proteins (Fc of human IgGl isotype). SPR data relative to the chimeric SP34 antibody for human and cynomolgus monkey CD3 epsilon l-26_Fc fusion proteins: (-) indicates no binding, (+) weaker binding, (++) moderate binding, strong but not similar binding (+++), and (++++) similar binding.

FIG. 11A-J: These figures ail relate to anti-human CD38 antibodies.

FIG. 11 A: Antibody-antigen interaction measured by SPR between the chimeric HB-7 antibody and the human CD38 antigen. A CM5 sensor chip was covalently coupled with protein G and 200 RUs of chimeric HB-7 antibody were captured. Human CD38 protein (human CD38 extracellular domain with a poly-histidine tag) was injected at 125, 31, 7.8, 3.9, 1.9, 1 and 0.5 nM at a flow rate of 30 μΐ/min in HBS-P. FIG. 11B: Antibody-antigen interaction measured by SPR between the humanized HB-7 best-fit antibody and the human CD38 antigen. A CM5 sensor chip was covalently coupled with protein G and 200 RUs of humanized HB-7 best-fit antibody were captured. Human CD38 protein (human CD38 extracellular domain with a poly-histidine tag) was injected at 50, 25,12.5, 6.25 and 0.39 nM at a flow rate of 30 μΐ/min in HBS-P. FIG. 11C: Antibody-antigen interaction measured by SPR between the humanized 9G7 best-fit antibody and the human CD38 antigen. A CM5 sensor chip was covalently coupled with protein G and 200 RUs of humanized 9G7 best-fit antibody were captured. Human CD38 protein (human CD38 extracellular domain with a poly-histidine tag) was injected at 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19, and 0.1 nM at a flow rate of 30 μΐ/min in HBS-P. FIG. 11D: Antibody-antigen interaction measured by SPR between the humanized 9G7 best-ffamework antibody and the human CD38 antigen. A CM5 sensor chip was covalently coupled with protein G and 200 RUs of humanized 9G7 bestframework antibody were captured. Human CD38 protein (human CD38 extracellular domain with a poly-histidine tag) was injected at 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19, and 0.1 nM at a flow rate of 30pl/min in HBS-P. FIG. 11E: Antibody-antigen interaction measured by SPR between the human 767 antibody and the human CD38 antigen. A CM5 sensor chip was covalently coupled with protein G and 200 RUs of human 767 antibody were captured. Human CD38 protein (human CD38 extracellular domain with a poly-histidine tag) was injected at 500, 250, 125, 62.5, 31.25, and 15.6 nM at a flow rate of 30 μΐ/min in HBS-P. Affinity was obtained from a plot of the equilibrium response (Req) vs. analyte concentration (C) according to the following équation: Req=KA*C*Rmax/(KA*C*n+l), concentration at 50% saturation is KD. Ail SPR data are expressed as number of response units (abbreviated RU; Y axis) vs. time (X axis). FIG. 11F: DSC profiles of chimeric HB-7 and humanized HB7 best-fit antibodies. FIG. 11G: DSC profiles of chimeric 9G7 and humanized 9G7 best-fit antibodies. FIG. 11H: DSC profiles of humanized 9G7 best-fiamework antibody. FIG. 111:

DSC profiles of human clone 767 antibody. FIG. 11J: summary table for the 9G7 humanized antibodies.

FIG. 12A-C: Schematic diagram of the BEAT HER2/CD3 antibodies in alternative formats. FIG.12A: BEAT HER2/CD3-1 (format A) and BEAT HER2/CD3-2 (format B) antibodies. FIG.12B: BEAT HER2/CD3-3 (format C) and BEAT HER2/CD3(SP34) (fonnat D) antibodies. FIG.12C: BEAT HER2/CD3(SP34-Kappal) (fonnat E) antibody. Legend: [(A4-)] means functional Protein A binding site. [(A-)] means nonfunctional Protein A binding site.

FIG. 13: Protein A purification profile of BEAT HER2/CD3-1 antibody (Absorbance trace at 280 nm). Column: 1ml MabSelect SuRe. Flow rate: 1 ml/min. Running buffer: 0.2 M NafEPCU pH 6. Elution buffer No 1: 20 mM Na Acetate pH 4 (20 ml). Elution buffer No 2: 0.1 M Glycine pH 3 (20ml). Neutralization: 1/10 vol. of IM Tris pH 8.

FIG. 14: Capillary Electrophoresis profile of BEAT HER2/CD3-1 antibody préparations.

FIG. 15A: SDS-PAGE analysis of N82aS substituted BEAT HER2/CD3-1 antibody. FIG. 15B: SDS-PAGE analysis ofN82aS non substituted BEAT HER2/CD3-1 antibody variant. Legend: [(A4-)] means a functional Protein A binding site and [(A-)] means a nonfunctional Protein A binding site. pH of elution is indicated.

FIG. 16A: Antibody-antigen interaction measured by SPR between the BEAT HER2/CD3-1 antibody and the human CD3 epsilon antigen. A CM5 sensor chip was covalently coupled with 7400 RUs of the human CD3 gamma-epsilon-Fc fusion protein. BEAT HER2/CD3-1 antibody was injected at 5000, 2500, 1250, 625, 312.5 and 156.25 nM at a flow rate of 10 μΐ/min in HBS-P. Data are expressed as number of response units (abbreviated RU; Y axis) vs. time (X axis). Affmity was obtained from a plot of the equilibrium response (Req) vs. analyte concentration (C) according to the following équation: Req=KA*C*Rmax/(KA*C*n+l), concentration at 50% saturation is KD. FIG. 16B: Antibody-antigen interaction measured by SPR between the BEAT HER2/CD3-1 antibody and the human HER2 antigen. A CM5 sensor chip was covalently coupled protein G and 150 RUs of BEAT HER2/CD3-1 antibody were captured. HER2-his was injected at 1000, 333,

111, 37, 12, 4.1, 1.4, 0.5 and 0.15 nM at a flow rate of 30μ1/ιηίη inHBS-P. Data are expressed as number of response units (abbreviated RU; Y axis) vs. time (X axis). FIG. 16C: DSC profiles of BEAT HER2/CD3-1 and -2 antibodies shown in profiles A and B, respectively.

FIG. 17A-G: Examples of T cell redirected killing by the BEAT HER2/CD3 antibodies. Readout: RDL-MTS method. Effector cells: human PBMCs. Effector cells-to-targeted cells ratio of 10:1. Means of three donors with 48h incubation. Antibody concentrations are shown in nM. FIG. 17A: BEAT HER2/CD3-1 and BEAT HER2/CD3-2 antibodies, target cells: BT474. FIG. 17B: BEAT HER2/CD3-1 and BEAT HER2/CD3-2 antibodies, target cells: JIMT-

1. FIG 17C: BEAT HER2/CD3-1 and BEAT HER2/CD3-2 antibodies, target cells: MDAMB-231. FIG. 17D: BEAT HER2/CD3(SP34) antibody, target cells: NCI-N87. FIG. 17E: BEAT HER2/CD3(SP34) antibody, target cells: HT-1080. FIG. 17F: BEAT HER2/CD3(SP34-Kappal) antibody, target cells: NCI-N87. FIG. 17G: BEAT HER2/CD3(SP34-Kappal) antibody, target cells: HT-1080.

FIG. 18A-C: JIMT-1 xenografts with human PBMC supplémentation. FIG. 18A: Human o

PBMCs do not interfère with tumor growth. FIG. 18B-C: Tumor volumes (mm ) for BEAT HER2/CD3-1 treated and non-treated mice, four human PBMC donors, cohorts of five mice.

FIG. 19: Schematic diagram of the BEAT CD38-HB7bestfit/CD3 (format A) and BEAT CD38-767/CD3 (format B) antibodies. [(A+)] means functional Protein A binding site. [(A-)] means nonfunctional Protein A binding site.

FIG. 20A: Antibody-antigen interaction measured by SPR between the BEAT CD38HB7bestfit/CD3 antibody and the human CD38 antigen. A CM5 sensor chip was covalently coupled with protein G and 200 RUs of BEAT CD38-HB7bestfit/CD3 antibody were captured. Human CD38 protein (poly-histidine tagged protein) was injected at 50, 25, 12.5, 6.25 and 0.39 nM at a flow rate of 30pl/min in HBS-P. Data are expressed as number of response units (abbreviated RU; Y axis) vs. time (X axis). FIG. 20B: BEAT CD38HB7bestfit/CD3 antibody DSC profile.

r

FIG. 21: Example of T cell redirected killing by the BEAT CD38-HB7bestfit/CD3 antibody.

Readout: RDL-FACS method. Effector cells: purified human T cells. Effector cells-totargeted cells ratio of 10:1. Mean of two donors with 48h incubation. Target cells: RPMI

8226. Antibody concentration is shown in nM.

FIG. 22: Example of T cell redirected killing by the BEAT CD38-767/CD3(SP34) antibody. Readout: RDL-FACS method. Effector cells: human PBMCs. Effector cells-to-targeted cells ratio of 10:1. Mean of three donors with 24h incubation. Target cells: Daudi. Antibody concentration is shown in nM.

FIG. 23: Schematic diagram of the BEAT OX40/CD3antibody. Legend: [(A+)] means functional Protein A binding site. [(A-)] means nonfunctional Protein A binding site.

FIG. 24: Example of T cell redirected killing by the BEAT OX40/CD3 antibody. Readout:

RDL-MTS method. Effector cells: Human PBMCs. Effector cells-to-targeted cells ratio of 20:1. Mean of three donors with 48h incubation. Target cells: recombinant stable CHO[OX40] cells. Antibody concentration is shown in riM.

FIG. 25: Schematic diagram ofthe BEAT EGFR/CD3 antibody. Legend: [(A+)] means functional Protein A binding site. [(A-)] means nonfunctional Protein A binding site.

FIG. 26: Example of T cell redirected killing by the BEAT EGFR/CD3antibody. Readout: RDL-MTS method. Effector cells: Human PBMCs. Effector cells-to-targeted cells ratio of 10:1. Mean of four donors with 48h incubation. Target cells: HT-29 cells. Antibody concentration is shown in nM.

FIG. 27: Schematic diagram of the BEAT CD38-HB7bestfit/CD3(SP34) (format A) and BEAT CD38-9G7bestfit/CD3(SP34-Kappa2) (format B) antibodies. [(A+)] means functional Protein A binding site.

FIG. 28: Example of T cell redirected killing by the BEAT CD38-HB7bestfit/CD3(SP34) antibody. Readout: RDL-FACS method. Effector cells: Human PBMCs. Effector cells-to17758 targeted cells ratio of 10:1. Mean of three donors with 24h incubation. Target cells: Daudi cells. Antibody concentration is shown in nM.

FIG. 29: Antibody-antigen interaction measured by SPR between the BEAT CD389G7bestfit/CD3(SP34-Kappa2) antibody and the human CD3 epsilon l-26_Fc fusion protein. A CM5 sensor chip was covalently coupled with 500 RUs of the human CD3 epsilon l-26_Fc fusion protein. BEAT CD38-9G7bestfit/CD3(SP34-Kappa2) antibody was injected at 50, 25, 12.5, 6.2, 3.1, 0.8 and 0.4 nM at a flow rate of 30 μΐ/min in HBS-P. Data are expressed as number of response units (abbreviated RU; Y axis) vs. time (X axis).

FIG. 30: Example of T cell redirected killing by the BEAT CD38/CD3(SP34-Kappa2) antibody. Readout: RDL-FACS method. Effector cells: Human PBMCs. Effector cells-totargeted cells ratio of 10:1. Mean of three donors with 24h incubation. Target cells: Daudi cells. Antibody concentration is shown in nM.

FIG. 31: Schematic diagram ofthe BEAT CD20/CD3(SP34) antibody. [(A+)] means functional Protein A binding site.

FIG. 32: Example of T cell redirected killing by the BEAT CD20/CD3(SP34) antibody. Readout: RDL-FACS method. Effector cells: Human PBMCs. Effector cells-to-targeted cells ratio of 10:1. Means of three donors with 24h incubation. Target cells: Daudi cells. Antibody concentration is shown in nM.

Detailed description of the invention

The présent invention relates generally to novel hetero-dimeric immunoglobulins that bind to the CD3 protein complex and a disease associated antigen. Furthermore, these hetero-dimeric immunoglobulins hâve reduced or eliminated binding to protein A and therefore can be purified to a very high degree of purity using affinity chromatography.

For the purposes of interpreting this spécification, the following définitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

The tenus “polypeptide” and “protein” refer to a polymer of amino acid residues wherein amino acids are combined via peptide bonds to form a chain of amino acids that hâve been linked together via déhydration synthesis. Polypeptides and proteins can be synthesized through chemical synthesis or recombinant expression and are not limited to a minimum amino acid length.

In accordance with the invention, the group of polypeptides comprises “proteins” as long as the proteins consist of a single polypeptide chain. Polypeptides may further form multimers such as dimers, trimers and higher oligomers, i.e. consisting of more than one polypeptide moiecule. Polypeptide molécules forming such dimers, trimers etc. may be identical or nonidentical. The corresponding higher order structures of such multimers are, consequently, termed homo- or hetero-dimers, homo- or hetero-trimers etc. An example for a heteromultimer is an antibody moiecule, which, in its naturally occurring form, consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms “polypeptide” and “protein” also refer to naturally modified polypeptides/proteins wherein the modification is effected e.g. by post-translational modifications like glycosylation, acétylation, phosphorylation and the like. Such modifications are well known in the art. Furthermore, for purposes of the présent invention, a “polypeptide” refers to a protein which includes modifications, such as délétions, additions and substitutions (which can be conservative in nature) to the native sequence. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidentai, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

The term “CD3 complex” as used herein refers to the protein complex known as the CD3 (cluster of différentiation 3) T-cell co-receptor (Wucherpfennig KW et al., (2010) Cold Spring Harb Perspect Biol, 2(4): a005140). The CD3 protein complex is composed of four distinct chains. In mammals, the complex contains a CD3y chain, a CD3ô chain, and two CD3s chains. These chains associate with a moiecule known as the T-cell receptor (TCR) and the ζ-chain to generate an activation signal in T lymphocytes (van der Merwe PA & Dushek O (2011) Nat Rev Immunol, 11(1): 47-55). The TCR, ζ-chain, and CD3 molécules together comprise the TCR complex. The CD3y, CD3b, and CD3s chains are highly related cellsurface proteins of the immunoglobulin superfamily containing a single extracellular immunoglobulin domain. The intracellular tails of the CD3 molécules contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM for short, which is essential for the signalling capacity of the TCR. Since CD3 is required for Tcell activation, drugs (often monoclonal antibodies) that target CD3 hâve and are being investigated as immunosuppressant thérapies.

The tenu “disease associated antigen” as used herein refers to molécules that are involved in a disease process. Examples of disease associated antigens are found in a broad range of therapeutic areas such as inflammation, cancer and autoimmune diseases. In oncology, disease associated antigens are molécules that can broadly be used for the screening and/or monitoring and/or therapeutic targeting of cancers within a patient population, for example EpCAM antigen in prostate cancer. Tumour antigens can be produced directly by the tumour or by non-tumour cells as a response to the presence of a tumour and preferred tumour antigens are cell-surface molécules. Inflammatory disease associated antigens are known, which include but are not limited to, pro-inflammatory cytokines such as TNF-α and IL-1. Autoimmune disease associated antigens are also known; examples of these include but are not limited to antibodies against double-stranded DNA in systemic lupus erythematosus and amyloid beta peptide in Alzheimers disease.

The term “immunoglobulin” as referred to herein can be used interchangeably with the term “antibody”. Immunoglobulin includes full-length antibodies and any antigen binding fragment or single chains thereof. Immunoglobulins can be homo-dimeric or hetero-dimeric. Immunoglobulins and specifically naturally occurring antibodies are glycoproteins which exist as one or more copies of a Y-shaped unit, composed of four polypeptide chains. Each “Y” shape contains two identical copies of a heavy (H) chain and two identical copies of a light (L) chain, named as such by their relative molecular weights. Each light chain pairs with a heavy chain and each heavy chain pairs with another heavy chain. Covalent interchain disulfide bonds and non-covalent interactions link the chains together. Immunoglobulins and specifically naturally occurring antibodies contain variable régions, which are the two copies of the antigen binding site. Papain, a proteolytic enzyme splits the “Y” shape into three separate molécules, two so called “Fab” or “FAB” fragments (Fab = fragment antigen binding) and one so called “Fc” fragment or “Fc région” (Fc = fragment crystallizable). A Fab fragment consists of the entire light chain and part of the heavy chain. The heavy chain contains one variable région (VH) and either three or four constant régions (CH1, CH2, CH3 and CH4, depending on the antibody class or isotype). The région between the CH1 and CH2 régions is called the hinge région and pennits flexibility between the two Fab arms of the Yshaped antibody molécule, allowing them to open and close to accommodate binding to two antigenic déterminants separated by a fixed distance. The “hinge région” as referred to herein is a sequence région of 6-62 amino acids in length, only présent in IgA, IgD and IgG, which encompasses the cysteine residues that bridge the two heavy chains. The heavy chains of IgA, IgD and IgG each hâve four régions, i.e. one variable région (VH) and three constant régions (CH1-3). IgE and IgM hâve one variable and four constant régions (CH1-4) on the heavy chain. The constant régions of the immunoglobulins may médiate the binding to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the complément system classical pathway. Each light chain is usually linked to a heavy chain by one covalent disulfide bond. Each light chain contains one variable région (VL) and one light chain constant région. The light chain constant région is a kappa light chain constant région designated herein as IGKC or is a lambda light chain constant région designated herein as IGLC. IGKC is used herein equivalently to Ck or CK and has the same meaning. IGLC is used herein equivalently to CÀ or CL and has the same meaning. The tenn “an IGLC région” as used herein refer to ail lambda light chain constant régions e.g. to ail lambda light chain constant régions selected from the group consisting of IGLC1, IGLC2, IGLC3, IGLC6 and IGLC7. The VH and VL régions can be further subdivided into régions of hypervariability, termed complementarity determining régions (CDR), interspersed with régions that are more conserved, termed framework régions (FR or FW). Each VH and VL is composed of three CDRs and four FRs, arranged from amino- terminus to carboxy-terminus in the following order: FRI, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable régions of the heavy and light chains contain an epitope- binding région that interacts with an antigen. Engineered immunoglobulins can encompass different epitope binding région formats such as scFv, FAB or dAb fragments. These fragments are usually assembled in an antibody-like structure by genetic fusion to a IgG Fc région. Engineered immunoglobulins can be constructed as homo or hetero-dimers with or without the use of hetero-dimerization enhancing techniques, and can hâve mono- or bispecific binding properties.

The term “full length antibody” as used herein includes the structure that constitutes the natural biological form of an antibody, including variable and constant régions. For example, in most mammals, including humans and mice, the full length antibody of the IgG class is a tetramer and consists of two identical pairs of two immunoglobulin chains, each pair having one light and one heavy chain, each light chain comprising immunoglobulin régions VL and a light chain constant région, and each heavy chain comprising immunoglobulin régions VH, CH1 (Cyl), CH2 (Ογ2), CH3 (Cy3) and CH4 (Cy4), depending on the antibody class or isotype). In some mammals, for example in camels and llamas, IgG antibodies may consist of only two heavy chains, each heavy chain comprising a variable région attached to the Fc région.

Antibodies are grouped into classes, also referred to as isotypes, as determined genetically by the constant région. Human constant light chains are classified as kappa (CK) and lambda (CX) light chains. Heavy chains are classified as mu (μ), delta (δ), gamma (y), alpha (a), or epsilon (ε) and define the antibody's isotype as IgM, IgD, IgG, IgA and IgE, respectively. Thus, “isotype” as used herein is meant any of the classes and/or subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant régions. The known human immunoglobulin isotypes are IGHG1 (IgGl), IGHG2 (IgG2), IGHG3 (IgG3), IGHG4 (IgG4), IGHA1 (IgAl), IGHA2 (IgA2), IGHM (IgM), IGHD (IgD) and IGHE (IgE). The so-called human immunoglobulin pseudo-gamma IGHGP gene represents an additional human immunoglobulin heavy constant région gene which has been sequenced but does not encode a protein due to an altered switch région (Bensmana M et al., (1988) Nucleic Acids Res, 16(7): 3108). In spite of having an altered switch région, the human immunoglobulin pseudo-gamma IGHGP gene has open reading frames for ail heavy constant régions (CH1-CH3) and hinge. Ail open reading frames for its heavy constant régions encode protein régions which align well with ail human immunoglobulin constant régions with the predicted structural features. This additional pseudo-gamma isotype is referred herein as IgGP or IGHGP. Other pseudo immunoglobulin genes hâve been reported such as the human immunoglobulin heavy constant région epsilon PI and P2 pseudo-genes (IGHEP1 and IGHEP2). The IgG class is the most commonly used for therapeutic purposes. In humans this class comprises subclasses IgGl, IgG2, IgG3 and IgG4. In mice this class comprises subclasses IgGl, IgG2a, IgG2b, IgG2c and IgG3.

The terni “Immunoglobulin fragments” as used herein include, but is not limited to, (i) a région including for example a CH1, a CH2 or a CH3 région, (ii) the Fab fragment consisting of VL, VH, CL or CK and CH1 régions, including Fab' and Fab'-SH, (ii) the Fd fragment consisting of the VH and CH1 régions, (iii) the dAb fragment (Ward ES et al., (1989) Nature, 341(6242): 544-6) which consists of a single variable région (iv) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (v) single chain Fv fragments (scFv), wherein a VH région and a VL région are linked by a peptide linker which allows the two régions to associate to form an antigen binding site (Bird RE et al., (1988) Science, 242(4877): 423-6; Huston JS et al., (1988) Proc Natl Acad Sci USA, 85(16): 5879-83), (vi) “diabodies” or “triabodies”, multivalent or multispecific fragments constructed by gene fusion (Holliger P et al., (1993) Proc Natl Acad Sci USA, 90(14): 6444-8; Tomlinson I & Holliger P, (2000) Methods Enzymol, 326:461-79), (vii) scFv, diabody or région antibody fused to an Fc région and (viii) scFv fused to the same or a different antibody.

The term “variable région” refers to the régions or domains that médiates antigen-binding and defines specifïcity of a particular antibody for a particular antigen. In naturally occurring antibodies, the antigen-binding site consists of two variable régions that define specifïcity: one located in the heavy chain, referred herein as heavy chain variable région (VH) and the other located in the light chain, referred herein as light chain variable région (VL). In humans, the heavy chain variable région (VH) can be divided into seven subgroups or subclasses: VH1, VH2, VH3, VH4, VH5, VH6 and VH7. In some cases, specifïcity may exclusively résidé in only one of the two régions as in single-domain antibodies from heavychain antibodies found in camelids. The V régions are usually about 110 amino acids long and consist of relatively invariant stretches of amino acid sequence called framework régions (FRs or “non-CDR régions”) of 15-30 amino acids separated by shorter régions of extreme variability called “hypervariable régions” that are 7-17 amino acids long. The variable domains of native heavy and light chains comprise four FRs, largely adopting a beta-sheet configuration, connected by three hypervariable régions, which form loops. The hypervariable régions in each chain are held together in close proximity by FRs and, with the hypervariable régions from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat EA et al., supra.). The term “hypervariable région” as used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable région generally comprises amino acid residues from a “complementary determining région” or “CDR”, the latter being of highest sequence variability and/or involved in antigen récognition. For ail variable régions numbering is according to Kabat (Kabat EA et al., supra}.

A number of CDR définitions are in use and are encompassed herein. The Kabat définition is based on sequence variability and is the most commonly used (Kabat EA et al., supra}. Chothia refers instead to the location of the structural loops (Chothia & Lesk J. (1987) Mol Biol, 196: 901-917). The AbM définition is a compromise between the Kabat and the Chothia définitions and is used by Oxford Molecular's AbM antibody modelling software (Martin ACR et al., (1989) Proc Natl Acad Sci USA 86:9268-9272; Martin ACR et al., (1991) Methods Enzymol, 203: 121-153; Pedersen JT et al., (1992) Immunomethods, 1: 126-136; Rees AR et al., (1996) In Sternberg M.J.E. (ed.), Protein Structure Prédiction. Oxford University Press, Oxford, 141-172). The contact définition has been recently introduced (MacCallum RM et al., (1996) J Mol Biol, 262: 732-745) and is based on an analysis of the available complex structures available in the Protein Databank. The définition of the CDR by IMGT®, the international ImMunoGeneTics information system® (http://www.imgt.org) is based on the IMGT numbering for ail immunoglobulin and T cell receptor V-REGIONs of ail species (IMGT®, the international ImMunoGeneTics information system®; Lefranc MP et al., (1999) Nucleic Acids Res, 27(1): 209-12; Ruiz M et al., (2000) Nucleic Acids Res, 28(1): 219-21; Lefranc MP (2001) Nucleic Acids Res, 29(1): 207-9; Lefranc MP (2003) Nucleic Acids Res, 31(1): 307-10; Lefranc MP et al., (2005) Dev Comp Immunol, 29(3): 185-203; Kaas Q et al., (2007) Briefings in Functional Genomics & Proteomics, 6(4): 253-64). Ail Complementarity Determining Régions (CDRs) as referred to in the présent invention, are defined preferably as follows (numbering according to Kabat EA et al., supra):

LCDR1: 24-34, LCDR2: 50-56, LCDR3: 89-98, HCDR1: 26-35, HCDR2: 50-65, HCDR3: 95-102.

The “non-CDR régions” of the variable domain are known as framework régions (FR). The “non-CDR régions” of the VL région as used herein comprise the amino acid sequences: 1-23 (FRI), 35-49 (FR2), 57-88 (FR3) and 99-107 (FR4). The “non-CDR régions” ofthe VH région as used herein comprise the amino acid sequences: 1-25 (FRI), 36-49 (FR2), 66-94 (FR3) and 103-113 (FR4).

The CDRs of the présent invention may comprise extended CDRs which are based on the aforementioned définitions and hâve variable domain residues as foliows: LCDR1 : 24-36, LCDR2: 46-56, LCDR3:89-97, HCDR1: 26-35, HCDR2:47-65, HCDR3: 93-102. These extended CDRs are numbered as well according to Kabat et al., supra. The “non-extended CDR région” of the VL région as used herein comprise the amino acid sequences: 1-23 (FRI), 37-45 (FR2), 57-88 (FR3) and 98- approximately 107 (FR4). The “non-extended CDR région” of the VH région as used herein comprise the amino acid sequences: 1-25 (FRI), 3746 (FR2), 66-92 (FR3) and 103- approximately 113 (FR4).

The tenn “Fab” or “FAB” or “Fab région” or “FAB région” as used herein includes the polypeptides that comprise the VH, CH1, VL and light chain constant immunoglobulin régions. Fab may refer to this région in isolation, or this région in the context of a foil length antibody or antibody fragment.

The term “Fc” or “Fc région”, as used herein includes the polypeptide comprising the constant région of an antibody heavy chain excluding the first constant région immunoglobulin région. Thus Fc refers to the last two constant région immunoglobulin régions of IgA, IgD and IgG or the last three constant région immunoglobulin régions of IgE and IgM, and the flexible hinge N-terminal to these régions. For IgA and IgM, Fc may include the J chain. For IgG, Fc comprises immunoglobulin régions Cgamma2 and Cgamma3 (Cy2 and Cy3) and the hinge between Cgammal (Cyl) and Cgamma2 (Cy2). Although the boundaries of the Fc région may vary, the human IgG heavy chain Fc région is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index. Fc may refer to this région in isolation or this région in the context of an Fc polypeptide, for example an antibody.

The term “immunoglobulin constant région” as used herein refers to immunoglobulin or antibody heavy chain constant régions from human or animal species and encompasses ail isotypes. Preferably, immunoglobulin constant régions are of human origin and are selected from the group consisting of, but not limited to: IGHG1 CH1, IGHG2 CH1, IGHG3 CH1, IGHG4 CH1, IGHA1 CH1, IGHA2 CH1, IGHE CH1, IGHEP1 CH1, IGHM CH1, IGHD CH1, IGHGP CH1, IGHG1 CH2, IGHG2 CH2, IGHG3 CH2, IGHG4 CH2, IGHA1 CH2, IGHA2 CH2, IGHE CH2, IGHEP1 CH2, IGHM CH2, IGHD CH2, IGHGP CH2, IGHG1

CH3, IGHG2 CH3, IGHG3 CH3, IGHG4 CH3, IGHA1 CH3, IGHA2 CH3, IGHE CH3, IGHEP1 CH3, IGHM CH3, IGHD CH3, IGHGP CH3, IGHE CH4 and IGHM CH4. Prefered “immunoglobulin constant régions” are selected from the group consisting of human IGHE CH2, IGHM CH2, IGHG1 CH3, IGHG2 CH3, IGHG3 CH3, IGHG4 CH3, IGHA1 CH3, IGHA2 CH3, IGHE CH3, IGHM CH3, IGHD CH3 and IGHGP CH3. More prefered “immunoglobulin constant régions” are selected from the group consisting of human IGHG1 CH3, IGHG2 CH3, IGHG3 CH3, IGHG4 CH3, IGHA1 CH3, IGHA2 CH3, IGHE CH3, IGHM CH3, IGHD CH3 and IGHGP CH3.

The term “epitope binding région” includes a polypeptide or a fragment thereof having minimal amino acid sequence to permit the spécifie binding of the immunoglobulin molécule to one or more epitopes. Naturally occurring antibodies hâve two epitope binding régions which are also known as antigen binding or combining sites or paratopes. Epitope binding régions in naturally occurring antibodies are confined within the CDR régions of the VH and/or VL domains wherein the amino acid mediating epitope binding are found. In addition to naturally occurring antibodies, artificial VH domains or VL domains or fragments thereof and combinations thereof can be engineered to provide epitope binding régions (Holt LJ et al., (2003) Trends Biotechnol, 21(11): 484-490; Polonelli L étal., (2008) PLoS ONE, 3(6): e2371). Examples of non-immunoglobulin based epitope binding régions can be found in artificial protein domains used as “scaffold” for engineering epitope binding régions (Binz HK et al., (2005) Nat Biotechnol, 23(10): 1257-1268) or peptide mimetics (Murali R & Greene MI (2012) Pharmaceuticals, 5(2): 209-235). Preferably the term 'epitope binding région' includes the combination of one or more heavy chain variable domains and one or more complementary light chain variable domains which together forms a binding site which permits the spécifie binding of the immunoglobulin molécule to one or more epitopes. Examples of an epitope binding région as exemplified in the présent invention include scFv and F AB.

As used herein, the term “epitope” includes a fragment of a polypeptide or protein or a nonprotein molécule having antigenic or immunogenic activity in an animal, preferably in a mammal and most preferably in a human. An epitope having immunogenic activity is a fragment of a polypeptide or protein that elicits an antibody response in an animal. An epitope having antigenic activity is a fragment of a polypeptide or protein to which an antibody or polypeptide specifically binds as determined by any method well-known to one of skill in the art, for example by immunoassays. Antigenic epitopes need not necessarily be immunogenic. Preferably, the term “epitope” as used herein refers to a polypeptide sequence of at least about 3 to 5, preferably about 5 to 10 or 15 and not more than about 1,000 amino acids (or any integer there between), which define a sequence that by itself or as part of a larger sequence, binds to an antibody generated in response to such sequence. There is no critical upper limit to the length of the fragment, which may comprise neariy the full-length of the protein sequence, or even a fusion protein comprising one or more epitopes. An epitope for use in the subject invention is not limited to a polypeptide having the exact sequence of the portion of the parent protein from which it is derived. Thus the term “epitope” encompasses sequences identical to the native sequence, as well as modifications to the native sequence, such as délétions, additions and substitutions (generally conservative in nature).The epitopes of protein antigens are divided into two categories, conformational epitopes and linear epitopes, based on their structure and interaction with the epitope binding site (Goldsby R et al., (2003) “Antigens (Chapter 3)” Immunology (Fifth édition ed.), New York: W. H. Freeman and Company, pp. 57-75, ISBN 0-7167-4947-5). A conformational epitope is composed of discontinuous sections of the antigen's amino acid sequence. These epitopes interact with the paratope based on the 3-D surface features and shape or tertiary structure of the antigen. Most epitopes are conformational. By contrast, linear epitopes interact with the paratope based on their primary structure. A linear epitope is formed by a continuous sequence of amino acids from the antigen.

The tenu “hetero-dimeric immunoglobulin” or “hetero-dimeric fragment” or “hetero-dimer” or “hetero-dimer of heavy chains” as used herein includes an immunoglobulin molécule or part of comprising at least a first and a second polypeptide, like a first and a second région, wherein the second polypeptide differs in amino acid sequence from the first polypeptide. Preferably, a hetero-dimeric immunoglobulin comprises two polypeptide chains, wherein the first chain has at least one non-identical région to the second chain, and wherein both chains assemble, i.e. interact through their non-identical régions. More preferably the hetero-dimeric immunoglobulin, has binding specificity for at least two different ligands, antigens or binding sites, i.e. is bispecific. Hetero-dimeric immunoglobulin as used herein includes but is not limited to full length bispecific antibodies, bispecifc Fab, bispecifc F(ab’)2, bispecific scFv fused to an Fc région, diabody fused to an Fc région and domain antibody fused to an Fc région.

The term “homo-dimeric immunoglobulin” or “homo-dimeric fragment” or “homo-dimer” or “homo-dimer of heavy chains” as used herein includes an immunoglobulin molécule or part of comprising at least a first and a second polypeptide, like a first and a second région, wherein the second polypeptide is identical in amino acid sequence to the first polypeptide. Preferably, a homo-dimeric immunoglobulin comprises two polypeptide chains, wherein the first chain has at least one identical région to the second chain, and wherein both chains assemble, i.e. interact through their identical régions. Preferably, a homo-dimeric immunoglobulin fragment comprises at least two régions, wherein the first région is identical to the second région, and wherein both régions assemble, i.e. interact through their proteinprotein interfaces.

For ail immunoglobulin constant régions included in the présent invention, numbering can be according to the IMGT® (IMGT®; supra).

For ail human CH1, CH2, CH3 immunoglobulin heavy chain constant régions selected from the group consisting of IGHG1, IGHG2, IGHG3 and IGHG4, numbering can be according to the “EU numbering System” (Edelman GM et al., (1969) ProcNatl Acad Sci USA, 63(1): 7885). A complété correspondence for the human CH1, hinge, CH2 and CH3 constant régions of IGHG1 can be found at the IMGT database (IMGT®; supra).

For the human kappa immunoglobulin light chain constant région (IGKC), numbering can be according to the “EU numbering System” (Edelman GM et al., supra). A complété correspondence for the human CK région can be found at IMGT database (IMGT®; supra).

For the human lambda immunoglobulin light chain constant régions (IGLC1, IGLC2, IGLC3, IGLC6 and IGLC7), numbering can be according to the “Kabat numbering system” (Kabat EA et al., supra). A complété correspondence for human IGLC régions can be found at the IMGT database (IMGT®; supra).

The human IGHG1 immunoglobulin heavy chain constant régions as referred to herein hâve the following région boundaries: CH1 région (EU numbering: 118-215), Hinge γΐ région (EU numbering: 216-230), CH2 région (EU numbering: 231-340) and CH3 région (EU numbering: 341-447). The human CK région referred herein spans residues 108 to 214 (EU numbering). The human IGLC1, IGLC2, IGLC3, IGLC6 and IGLC7 régions referred herein span residues 108-215 (Kabat numbering).

The terms “amino acid” or “amino acid residue” as used herein includes natural amino acids as well as non-natural amino acids. Preferably natural amino acids are included.

The tenu “modification” or “amino acid modification” herein includes an amino acid substitution, insertion and/or délétion in a polypeptide sequence. The terms “substitution” or “amino acid substitution” or “amino acid residue substitution” as used herein refers to a substitution of a first amino acid residue in an amino acid sequence with a second amino acid residue, whereas the first amino acid residue is different from the second amino acid residue

i.e. the substituted amino acid residue is different from the amino acid which has been substituted. For example, the substitution R94K refers to a variant polypeptide, in which the arginine at position 94 is replaced with a lysine. For example 94K indicates the substitution of position 94 with a lysine. For the purposes herein, multiple substitutions are typically separated by a slash or a comma. For example, “R94K/L78V” or “R94K, L78V” refers to a double variant comprising the substitutions R94K and L78V. By “amino acid insertion” or “insertion” as used herein is meant the addition of an amino acid at a particular position in a parent polypeptide sequence. For example, insert -94 désignâtes an insertion at position 94. By “amino acid délétion” or “délétion” as used herein is meant the removal of an amino acid at a particular position in a parent polypeptide sequence. For example, R94- désignâtes the délétion of arginine at position 94.

In certain embodiments, the terms “decrease”, “reduce”, or “réduction” in binding to Protein A refers to an overall decrease of at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, or 99% up to 100% (élimination) in the binding of a modified immunoglobulin or fragment thereof to Protein A detected by standard art known methods such as those described herein, as compared to a parental i.e. unmodified immunoglobulin or wild-type IgG or an IgG having the wild-type human IgG Fc région. In certain embodiments these terms altematively may refer to an overall decrease of 10-fold (i.e. 1 log), 100-fold (2 logs), 1,000fold (or 3 logs), 10,000-fold (or 4 logs), or 100,000-fold (or 5 logs).

The terms “eliminate”, “abrogate”, “élimination” or “abrogation” of binding to Protein A refers to an overall decrease of 100% in the binding of a modified immunoglobulin or fragment thereof to Protein A i.e. a complété loss of the binding of a modified immunoglobulin or fragment thereof to Protein A, detected by standard art known methods such as those described herein, as compared to a parental i.e. unmodified immunoglobulin or wild-type IgG or an IgG having the wild-type human IgG Fc région.

Similarly, the terms “decrease”, “reduce”, or “réduction” in binding to an affinity reagent refers to an overall decrease of at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, or 99% up to 100% (élimination) in the binding of a modified immunoglobulin or fragment thereof to the affinity reagent detected by standard art known methods such as those described herein, as compared to a parental, i.e. unmodified immunoglobulin or wild-type IgG or an IgG having the wild-type human IgG Fc région. In certain embodiments these terms altematively may refer to an overall decrease of 10-fold (i.e. 1 log), 100-fold (2 logs), 1,000fold (or 3 logs), 10,000-fold (or 4 logs), or 100,000-fold (or 5 logs).

The terms “eliminate” , “abrogate”, “élimination” or “abrogation” of binding to an affinity reagent refers to an overall decrease of 100% in the binding of a modified immunoglobulin or fragment thereof to the affinity reagent i.e. a complété loss of the binding of a modified immunoglobulin or fragment thereof to the affinity reagent detected by standard art known methods such as those described herein, as compared to a parental, i.e. unmodified immunoglobulin or wild-type IgG or an IgG having the wild-type human IgG Fc région.

“Bispecific antibodies” are monoclonal antibodies that hâve binding specificities for at least two different antigens. In certain embodiments, the bispecific antibodies are bispecific antibodies with one or more amino acid modifications in the VH région relative to the parental antibody. In certain embodiments, bispecific antibodies may be human or humanized antibodies. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express a target antigen. These antibodies possess a target-antigen-binding arm and an arm which binds a cytotoxic agent, such as, e.g., saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten. Bispecific antibodies can be prepared as full length antibodies or antibody fragments. Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains hâve different specificities (Milstein and Cuello, (1983) Nature, 305: 537-40). Because of the random assortaient of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of different antibody molécules, of which only one has the correct bispecific structure. The purification of the correct molécule, which is usually done by affinity chromatography steps, is rather cumbersome and the product yields are low. Similar procedures are disclosed in WO1993/08829 and in Traunecker et al., (1991) EMBO J, 10: 3655-9. According to a different approach, antibody variable régions with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant région sequences. The fusion, for example, is with an immunoglobulin heavy chain constant région, comprising at least part of the hinge, CH2 and CH3 régions. In certain embodiments, the first heavy-chain constant région (CH1), containing the site necessary for light chain binding, is présent in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and are co-transfected into a suitable host organism. This provides for flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or ail three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.

Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies hâve, for example, been proposed to target immune system cells to unwanted cells (US4,676,980) and for treatment of HIV infection (W01991/00360, WO1992/00373 and EP03089). Heteroconjugate antibodies may be made using any convenient cross-linking method. Suitable cross-linking agents are well known in the art (see US4,676,980), along with a number of cross-linking techniques. Antibodies with more than two valencies are also contemplated. For example, trispecific antibodies can be prepared (see Tutt A et al. (1991) J.

Immunol. 147: 60-9).

In some embodiments the présent disclosure provides a bispecific hetero-dimeric immunoglobulin or fragment thereof or a bispecific full-length antibody which binds to CD3 and a disease associated antigens selected from within the groups of: tumor antigens, cytokines, vascular growth factors and lympho-angiogenic growth factors. Preferably the bispecific hetero-dimeric immunoglobulin or fragment thereof or the bispecific antibody binds to CD3 and a disease associated antigen selected from the group consisting of: CD33, TROP2, CD105, GD2, GD3, CEA, VEGFR1, VEGFR2, NCAM, CD133, CD123, ADAM17, MCSP, PSCA, FOLR1, CD19, CD20, CD38, EpCAM, HER2, HER3, EGFR, PSMA, IgE, Integrin a4bl, CCR5, LewisY, FAP, MUC-1, Wue-1, MSP, EGFRvIII, P glycoprotein, AFP, ALK, BAGE proteins, CD30, CD40, CTLA4, ErbB3, ErbB4, Mesothelin, 0X40, CA125, CAJX, CD66e, cMet, EphA2, HGF/SF , MUC1 , Phosphatidylserine, TAG-72 , TPBG, βcatenin, brc-abl, BRCA1, BORIS, CA9, caspase-8, CDK4, Cyclin-Bl, CYP1B1, ETV6AML, Fra-1, FOLR1, GAGE-1, GAGE-2, GloboH, glypican-3, GM3, gplOO, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, LMP2, MAGE1, MAGE2, MAGE3, MAGE4, MAGE6, MAGE12, MART-1, ML-IAP, Muc2, Muc3, Muc4, Muc5, Mucl6, MUM1, NA17, NY-BR1, NY-BR62, NY-BR-85, NY-ESO1, pl5, p53, PAP, PAX3 PAX5, PCTA-1, PLAC1, PRLR, PRAME, RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, Steap-1, Steap-2, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TRP-1, TRP-2, tyrosinase, uroplakin-3, PSMA . Preferably the bispecific hetero-dimeric immunoglobulin or fragment thereof or the bispecific antibody binds to CD3 and HER2 or CD3 and CD38 or CD3 and 0X40.

Protein A: Protein A is a cell wall component produced by several strains of Staphylococcus aureus which consists of a single polypeptide chain. The Protein A gene product consists of five homologous repeats attached in a tandem fashion to the pathogen’s cell wall. The five domains are approximately 58 amino acids in length and denoted ED ABC, each exhibiting immunoglobulin binding activity (Tashiro M & Montelione GT (1995) Curr. Opin. Struct. Biol., 5(4): 471-481). The five homologous immunoglobulin binding domains fold into a three-helix bundle. Each domain is able to bind proteins from many mammalian species, most notably IgGs (Hober S et al., (2007) J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 848(1): 40-47). Protein A binds the heavy chain of most immunoglobulins within the Fc région but also within the Fab région in the case of the human VH3 family (Jansson B et al, (1998) FEMS Immunol. Med. Microbiol., 20(1): 69-78). Protein A binds IgG from various species including human, mouse, rabbit and guinea pig but does not bind human IgG3 (Hober S et al., (2007) supra). The inability of human IgG3 to bind Protein A can be explained by the H435R and Y436F substitutions in the human IgG3 Fc région (EU numbering, Jendeberg et al., (1997) J. Immunol. Methods, 201(1): 25-34). Besides IgG, Protein A also interacts with IgM and IgA.

Amongst human VH subclasses, VH3 is the only subclass to bind Protein A (Graille M et al., (2000) Proc. Natl. Acad. Sci. USA 97(10): 5399-5404), and ail five domains of Protein A are known to bind this variable domain subclass (Jansson B et al, (1998) FEMS Immunol. Med. Microbiol., 20(1): 69-78. VH3 based immunoglobulins or fragments thereof are of major importance to the biotechnology industry. VH3 based molécules hâve been extensively developed since their ability to bind Protein A facilitâtes their functional pre-screening, and as such many synthetic or donor based phage display libraries or transgenic animal technologies used for antibody discovery are based on the VH3 subclass. In addition VH3 based molécules are often selected for their good expression and stability over other known heavy chain variable domain subclasses.

The capacity of Protein A to bind antibodies with such high affinity is the driving motivation for its industrial scale use in biologie pharmaceuticals. Protein A used for production of antibodies in bio-pharmaceuticals is usually produced recombinantly in E. coli and functions essentially the same as native Protein A (Liu HF et al., (2010) MAbs, 2(5): 480-499).

Most commonly, recombinant Protein A is bound to a stationary phase chromatography resin for purification of antibodies. Optimal binding occurs at pH8.2, although binding is also good at neutral or physiological conditions (pH 7.0-7.6). Elution is usually achieved through pH shift towards acidic pH (glycine-HCl, pH2.5-3.0). This effectively dissociâtes most proteinprotein and antibody-antigen binding interactions without permanently affecting protein structure. Nevertheless, some antibodies and proteins are damaged by low pH and it is best to neutralize immediately after recovery by addition of 1/1 Oth volume of alkaline buffer such as 1 M Tris-HCl, pH 8.0 to minimize the duration of time in the low-pH condition.

There are various commercially available Protein A chromatography resins. The main différences between these media are the support matrix type, Protein A ligand modification, pore size and particle size. The différences in these factors give rise to différences in compressibility, chemical and physical robustness, diffusion résistance and binding capacity of the adsorbents (Hober S et al., (2007), supra). Examples of Protein A chromatography resins include but are not limited to the MabSelect SuRe™ Protein A resin and MabSelect™ Protein A resin from GE Healthcare as used in examples.

The term “chromatography” refers to protein liquid chromatography and includes fast protein liquid chromatography (FPLC) which is a form of liquid chromatography that is often used to analyze or purify mixtures of proteins. As in other forms of chromatography, séparation is possible because the different components of a mixture hâve different affïnities for two materials, a moving fluid (the mobile phase) which passes through a porous solid (the stationary phase). In FPLC, the mobile phase is an aqueous solution, or “buffer”. The buffer flow rate can be operated under gravity flow or controlled by a positive-displacement pump which is normally kept at a constant rate, while the composition of the buffer can be varied by drawing fluids in different proportions from two or more extemal réservoirs. The stationary phase is a resin composed of beads, usually of cross-linked agarose, packed into a cylindrical glass or plastic column. FPLC resins are available in a wide range of bead sizes and surface ligands depending on the application.

The process of “affmity chromatography” involves the use of an affinity reagent as ligands which are cross-linked to the stationary phase and that hâve binding affinity to spécifie molécules or a class of molécules. Ligands can be bio-molecules, like protein ligands or can be synthetic molécules. Both types of ligand tend to hâve good specifïcity. The most commonly used protein ligand in production is the affinity reagent Protein A. In affinity chromatography when the solution (for example a crude cell supematant containing a protein of interest) is loaded onto to the column the target protein is usually adsorbed while allowing contaminants (other proteins, lipids, carbohydrates, DNA, pigments, etc.) to pass through the column. The adsorbent itself is normally packed in a chromatography column; though the adsorption stage can be performed by using the adsorbent as a stirred slurry in batch binding mode. The next stage after adsorption is the wash stage, in which the adsorbent is washed to remove residual contaminants. The bound protein is then eluted in a semi-pure or pure form.

Elution is normally achieved by changing the buffer or sait composition so that the protein can no longer interact with the immobilized ligand and is released. In some instances the protein of interest may not bind the affinity resin and affinity chromatography is directed at binding unwanted contaminants and the unbound fraction is therefore collected to isolate the protein of interest. Affinity chromatography can be performed in a fixed bed or a fluidised bed.

The term “gradient mode chromatography” refers to a chromatography method wherein the proportion of the “elution” buffer (buffer B) is increased from 0% to 100% in a graduai or stepwise manner.

The terms “capture-elution mode chromatography” or “capture-elution purification mode” or “capture-elution purification” refers to a chromatography method wherein the proportion of the “elution” buffer (buffer B) is not increased from 0% to 100% in a graduai or stepwise manner but rather directly applied at a 100% after capture and optionally a wash step with running buffer (buffer A).

Development of hetero-dimeric immunoglobulins targeting CD3

The présent invention provides an epitope binding région that binds the CD3 protein complex comprising the heavy and light chain CDRs as described supra and further comprising a heavy chain variable framework région that is the product of or derived from human gene IGHV3-23*04 (SEQ ID NO: 22). The heavy chain variable framework région comprises at least one amino acid modification from the corresponding framework région of the heavy chain variable région of the corresponding murine antibody OKT3 comprising the amino acid sequence of SEQ ID NO: 18. Preferably the amino acid modification is an amino acid substitution. Typically, no more than seven, preferably no more than six, preferably no more than five, preferably no more than four, more preferably no more than three, even more preferably no more than two, most preferably no more than one amino acid modifications are performed within a framework région. In some embodiments the présent disclosure provides an epitope binding région that binds to the CD3 protein complex, wherein the amino acid modification of the framework régions of the heavy chain variable région comprise an amino acid substitution at amino acid position selected from the group consisting of: 34, 48, 49, 58, 69, 71 and 73 and wherein the amino acid position of each group member is indicated according to the Kabat numbering. Preferably, amino acid substitutions of the framework régions ofthe heavy chain variable région are selected from the group consisting of: I34M, V48I, A49G, R58N, R58Y, I69L, A71T and T73K. Preferred amino acid substitution of the framework régions of the heavy chain variable région are at amino acid positions selected from the group consisting of 34, 49 and 71. More preferred amino acid substitutions of the framework régions of the heavy chain variable région are selected from the group consisting of I34M, A49G and A71T.

In a further aspect, the epitope binding région of the first polypeptide that binds the CD3 protein complex comprises a light chain variable framework région that is the product of or derived from a human gene selected from the group consisting of: IGKV1-39*01 (SEQ ID NO: 23) and IGKV3-20*01 (SEQ ID NO: 24). The light chain variable framework région comprises at least one amino acid modification from the corresponding framework région of the light chain variable région of the corresponding murine antibody OKT3 comprising the amino acid sequence of SEQ ID NO: 19. Preferably the amino acid modification is an amino acid substitution. Typically, no more than eight, preferably no more than seven, preferably no more than six, preferably no more than five, preferably no more than four, more preferably no more than three, even more preferably no more than two, most preferably no more than one amino acid modifications are perfonned within a framework région. In some embodiments the présent disclosure provides an epitope binding région that binds to the CD3 protein complex, wherein the amino acid modification of the framework régions of the light chain variable région sequence comprises an amino acid substitution at amino acid position selected from the group consisting of: 4, 33, 34, 46, 47, 66, 71 and 96. Preferably, amino acid substitutions of the framework régions of the light chain variable région are selected from the group consisting of: M4L, V33M, A34N, L46R, L47W, R66G, F71Y and P96F. Preferred amino acid substitution of the framework régions of the light chain variable région are at amino acid positions selected from the group consisting of 4, 46 and 47. More preferred amino acid substitutions of the framework régions of the light chain variable région are selected from the group consisting of M4L, L46R, L47W and F71 Y. In some embodiments the epitope binding région of the first polypeptide that binds to the CD3 protein complex may comprise amino acid modifications of the framework régions of the heavy chain variable région sequence as set out above and amino acid modifications of the framework régions of the light chain variable région sequence as set out above.

The présent disclosure also provides an antibody or fragment thereof that binds to the CD3 protein complex that comprises a heavy chain sequence selected from the group consisting of SEQ ID NOs: 27 to 38, 64-68 and 359, preferably selected consisting of SEQ ID NO: 359. The présent disclosure also provides an antibody or fragment thereof that binds to the CD3 protein complex that comprises a light chain sequence selected from the group consisting of SEQ ID NOs: 39 to 47, 69 to 90 and 360 preferably consisting of SEQ ID NO: 360.

Given that each of these heavy and light chain variable région sequences can bind to the CD3 protein complex, the heavy and light chain variable région sequences can be “mixed and matched” to create anti-CD3 binding molécules of the invention. CD3 binding of such “mixed and matched” antibodies can be tested using the binding assays described e.g. in the Examples.

Engineering of the immunoglobulin constant région to promote hetero-dimer formation over homo-dimer formation

Methods to produce hetero-dimeric immunoglobulins are known in the art and one of the simplest methods relies on expressing the two distinct immunoglobulin chains in a single cell (WO95/33844, Lindhofer H & Thierfelder S). Without engineering, this straightforward method is limited by the formation of homo-dimeric species over the hetero-dimer of interest (Kufer P et al., (2004) Trends Biotechnol., 22(5): 238-244). When using complementary technologies that will enhance heavy chain hetero-dimerization (Merchant AM et al., (1998) Nat. Biotechnol., 16(7): 677-681), greater hetero-dimer production can be achieved but still results in the production of a significant amount of undesirable homo-dimers (Jackman J et al., (2010) J Biol Chem., 285(27):20850-9, Klein C et al., (2012) MAbs, 4(6):653-63). The présent invention therefore utilises the BEAT® technology described method (PCT publication No: WO2012/131555), which is based on a unique concept of bio-mimicry that exhibit superior hetero-dimerisation over prior art methods. The BEAT technology is based on an interface exchange between naturally occurring homo or hetero-dimeric immunoglobulin domain pairs to create new hetero-dimers which can be used as building blocks for Fc-based bispecific antibodies.

In one aspect, the présent invention provides a hetero-dimeric immunoglobulin or fragment thereof comprising first and second polypeptides comprising an engineered immunoglobulin constant région with a modified CH3 domain having a protein-protein interface, wherein the protein-protein interface of the first polypeptide comprises an amino acid substitution at a position selected from the group consisting of: 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88 and 90 (IMGT® numbering), and wherein the protein-protein interface of the second polypeptide comprises an amino acid substitution at position 84.4 and at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88 and 90 (IMGT® numbering).

In a further embodiment, the présent invention provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the first and second polypeptides comprise an engineered immunoglobulin constant région with a modified CH3 domain having a protein-protein interface, wherein the protein-protein interface of the first polypeptide comprises an amino acid substitution at position 88 and at a position selected from the group consisting of: 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86 and 90 (IMGT® numbering), and wherein the protein-protein interface of the second polypeptide comprises an amino acid substitution at position 85.1 and/or 86 and at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 84.4, 88 and 90 (IMGT® numbering), wherein the amino acid residue substituted at position 88 in the first engineered immunoglobulin constant région is interacting with the amino acid residue substituted at position 85.1 and/or 86 in the second engineered immunoglobulin constant région, wherein the amino acid position of each group member is indicated according to the IMGT® numbering.

Preferably the amino acid residue which is substituted in the protein-protein interface of the first engineered immunoglobulin constant région at position 88 is 88W and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to IMGT® numbering. More preferably, the amino acid residue which is substituted in the protein-protein interface of the first engineered immunoglobulin constant région at position 88 is 88W and wherein the further amino acid residue substituted in the protein-protein interface of the first engineered immunoglobulin constant région is selected from the group consisting of: 3A, 20V, 20T, 20A, 20N, 20Q, 20E, 20S, 20K, 20W, 22A, 22G, 22T, 22L, 221, 22V, 26R,

26Q, 26T, 26K, 26V, 26S, 26N, 26E, 79Y, 85.1T, 85.1M, 85.1A, 85.1S, 85.1R, 85.1H, 85.1K,

85.1F, 85.IC, 85.1N, 85.1W, 86S, 861, 86T, 86H, 86Q, 86V, 86W, 86Y, 86F and 90N, wherein the amino acid position is indicated according to the IMGT® numbering.

Preferably the amino acid residue which is substituted at position 85 and 86 in the proteinprotein interface of the second engineered immunoglobulin constant région is selected from the group consisting of: 85.1 A, 85.1 S, 85.IC and 86S and conservative amino acid substitutions thereof (IMGT® numbering). More preferably the amino acid residue which is substituted in the protein-protein interface of the second engineered immunoglobulin constant région is selected from the group consisting of: 85.1 A, 85.1 S, 85.IC and 86S and wherein the further amino acid residue substituted in the protein-protein interface of the second engineered immunoglobulin constant région is selected from the group consisting of: 3E, 5A, 7F, 20T, 22V, 26T, 81D, 84L, 84.2E, 88R and 90R and conservative amino acid substitutions thereof (IMGT® numbering).

In a preferred embodiment the amino acid residue which is substituted in the protein-protein interface of the first engineered immunoglobulin constant région at position 88 is 88W and wherein the further amino acid residue substituted in the protein-protein interface of the first engineered immunoglobulin constant région is: 3A, 20K, 22V, 26T, 79Y, 85.1 S, 86V and 90N and, wherein the amino acid residues which are substituted in the protein-protein interface of the second engineered immunoglobulin constant région at positions 85.1 and 86 are 85.IA, 85.IS or 85.IA and 86S and wherein the further amino acid residue substituted in the protein-protein interface of the second engineered immunoglobulin constant région is: 3E, 5A, 7F, 20T, 22V, 26T, 81D, 84L, 84.2E, 84.4Q, 88R and 90R (IMGT® numbering).

In an alternative embodiment, the présent invention provides a hetero-dimeric immunoglobulin or fragment thereof, wherein the first and second polypeptides comprise an engineered immunoglobulin constant région with a modified CH3 domain having a proteinprotein interface, wherein the protein-protein interface of the first polypeptide comprises an amino acid substitution at position 20, and at a position selected from the group consisting of: 3, 5, 7, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88 and 90 and, wherein the protein-protein interface of the second polypeptide comprises an amino acid substitution at position 26 and at a position selected from the group consisting of: 3, 22, 27, 79, 81, 84, 85.1, 86, and 88, wherein the amino acid residue substituted at position 20 in the first engineered immunoglobulin constant région is interacting with the amino acid residue substituted at position 26 in the second engineered immunoglobulin constant région, wherein the amino acid position of each group member is indicated according to the IMGT® numbering.

Preferably the amino acid residues which are substituted in the protein-protein interface of the first engineered immunoglobulin chain comprise the amino acid residues at positions 20 and 22, and optionally a further amino acid residue at a position selected from the group consisting of: 3, 5, 7, 26, 27, 79, 81, 84, 84.2, 84.4, 85.1, 86, 88 and 90 and, wherein the amino acid residues which are substituted in the protein-protein interface of the second engineered immunoglobulin chain comprise the amino acid residues at positions 26 and at a further position selected from the group consisting of: 3, 5, 7, 20, 22, 27, 79, 81, 84, 84.2, 84.4, 85.1, 86, 88 and 90, wherein the amino acid position of each group member is indicated according to the IMGT® numbering. Preferably the amino acid residues which are substituted in the protein-protein interface of the first engineered immunoglobulin chain comprise the amino acid residues at positions 20 and 22, and optionally a further amino acid residue at a position selected from the group consisting of: 3, 5, 7, 26, 27, 79, 81, 84, 84.2, 84.4, 85.1, 86, 88 and 90 and, wherein the amino acid residues which are substituted in the protein-protein interface of the second engineered immunoglobulin chain comprise the amino acid residues at positions 26 and 86 and optionally at a further position selected from the group consisting of 3, 5, 7, 20, 22, 27, 79, 81, 84, 84.2, 84.4, 85.1, 88 and 90, wherein the amino acid position of each group member is indicated according to the IMGT® numbering.

More preferably the amino acid residue which is substituted at position 20 in the proteinprotein interface of the first engineered immunoglobulin constant région is selected from the group consisting of 20V, 20T, 20A, 20N, 20Q, 20K, 20S, 20W and 20E and wherein the further amino acid residue substituted in the protein-protein interface of the first engineered immunoglobulin constant région is selected from the group consisting of 3A, 22A, 22G, 22L, 221, 22V, 22T, 26K, 26R, 26Q, 26T, 26V, 26S, 26N, 26E, 79Y, 85.1W, 85.1F, 85.1T, 85.IM, 85.IA, 85.1S, 85.1R, 85.1H, 85.1K, 85.1C, 85.1N, 86W, 86Y, 86S, 861, 86H, 86Q, 86V, 86T, 86F, 88Q, 88L, 88V, 88R, 88E, 88T, 881, 88Y, 88K, 88W and 90N, and wherein the amino acid residue which is substituted at position 26 in the protein-protein interface of the second engineered immunoglobulin constant région is selected from the group consisting of 26T and

26E and conservative amino acid substitutions thereof, wherein the amino acid position is indicated according to the IMGT® numbering.

In a most preferred embodiment the amino acid residue which is substituted in the proteinprotein interface of the first engineered immunoglobulin constant région at position 20 is 20K and wherein the further amino acid residue substituted in the protein-protein interface of the first engineered immunoglobulin constant région is 3 A, 22V, 26T, 79Y, 85.1 S, 86V, 88W and 90N and, wherein the amino acid residues which are substituted in the protein-protein interface of the second engineered immunoglobulin constant région at position 26 is 26T and wherein the further amino acid residue substituted in the protein-protein interface of the second engineered immunoglobulin constant région is 3E, 5 A, 7F, 20T, 22V, 81D, 84L, 84.2E, 84.4Q, 85.1C/S/A, 86S, 88R and 90R (IMGT® numbering).

Development ofhetero-dimeric immunoglobulins targeting CD3 and a disease associated antigen

As a first step (Example 1), the substitutions that reduce or abrogate binding to Protein A were assayed in homo-dimeric immunoglobulins based on FAB or scFv fragments. It was found that the presence of a variable heavy chain domain of the VH3 subclass within the heavy chain which has substitutions for reduced or no binding to Protein A, hampers any differential affinity methods based on Protein A. Solutions to these major impediments were found in the forms of framework substitutions that reduce or abrogate Protein A binding to the VH3 subclass for the differential affinity methods based on Protein A.

In a second step (Example 2.1), a humanised antibody targeting the human CD3 (epsilon subunit) was generated by grafting the CDRs of a murine anti-CD3 antibody onto IGVH3-23 and IGVK1 or IGVK3 human germline frameworks. The best humanised variants had the Protein A binding site présent in their VH domain abrogated using a G65S or N82aS substitution (Kabat numbering). These variants were formatted as FAB or scFv fragments.

In a third step, antigen binding sites of antibodies targeting disease associated antigens were generated. CDRs of murine antibodies could be grafted onto the human germline frameworks

IGVH3-23 and IGVK1 (Examples 2.3, 2.4 and 2.6-2.10). Altematively CDRs of antibodies isolated from phage display libraries could be based on the VH3 variable domain subclass or grafted onto the human germline frameworks IGVH3-23 and IGVK1 (Examples 2.5 and 2.6).

The Protein A binding site in the VH domain of the epitope binding région was abrogated using the G65S orN82aS substitutions (Kabat numbering).

In a fourth step, hetero-dimeric antibodies were produced based on the BEAT® technology (as described in WO2012/131555) in which the anti-CD3 antibody from Example 2.1 and the epitope binding région of the disease associated antigen as described in Examples 2.2-2.10 were used in an scFv-FAB format or vice versa (Example 3.1). Since a différence in the number of Protein A binding sites between homo- and hetero-dimeric species can be used to isolate the hetero-dimeric species by Protein A chromatography, the bispecifîc antibodies of the présent invention were engineered to resuit in one of the two homo-dimeric species having no Protein A binding site and therefore no binding to Protein A resin. Furthermore, in order to improve the safety profile of the BEAT antibodies, the Fc receptor binding was reduced or eliminated by engineering the two substitutions L234A and L235A (EU numbering) into the lower hinge région of the Fc région.

Examples

Materials and Methods

Construction of expression vectors for transient mammalian cell expression cDNAs encoding the different polypeptide chains in part or in full were first gene synthetized by GENEART AG (Regensburg, Germany) and modified using standard molecular biology techniques. PCR products were digested with appropriate DNA restriction enzymes, purified and ligated in a modified pcDNA3.1 plasmid (Invitrogen AG, Zug, Switzerland) carrying a CMV promoter and a bovine hormone poly-adenylation (poly(A)) previously digested with the same DNA restriction enzymes . Ail polypeptide chains were independently ligated in this expression vector where sécrétion was driven by the murine VJ2C leader peptide.

Expression of recombinant proteins

Antibodies, ScFv-Fc fusion proteins, BEAT antibodies and antigens were expressed as described below unless otherwise indicated. For transient expression, equal quantifies of each engineered chains vectors were co-transfected into suspension-adapted HEK293-EBNA cells (ATCC-LGL standards, Teddington, UK; Cat. No: CRL-10852) using Polyethyleneimine (PEI; Sigma, Buchs, Switzerland). Typically, 100ml of cells in suspension at a density of 0.81.2 million cells per ml is transfected with a DNA-PEI mixture. When recombinant expression vectors encoding each engineered chain genes are introduced into the host cells, the immunoglobulin construct is produced by further culturing the cells for a period of 4 to 5 days to allow for sécrétion into the culture medium (EX-CELL 293, HEK293-serum-free medium (Sigma), supplemented with 0.1% pluronic acid, 4mM glutamine and 0.25pg/ml geneticin). Cell-free culture supematants containing the secreted immunoglobulins were prepared by centrifugation followed by stérile filtration and used for further analysis.

Differential Protein A affïnity chromatography (Example 1)

Purification ofFc 133 fragment and homo-dimeric scFv-Fc immunoglobulins Capture-elution mode chromatography

Supematants were conditioned with 0.1 volume (V) of IM Tris-HCl pH 8.0 prior purification. Protein G Sepharose™ 4 Fast Flow (GE Healthcare Europe GmbH, Glattbrugg, Switzerland; catalogue number 17-0618-01) was added to conditioned supematants. Mixtures were incubated ovemight at 4°C. After incubation, bound proteins were washed with lOCVs of

PBS pH 7.4, eluted with 4 column volumes (CVs) of 0.1M Glycine pH 3.0 and neutralised with 0.1V of IM Tris-H Cl pH8.0. Supematant, flow through and elution fractions were analysed under non-reduced conditions by SDS-PAGE (NuPAGE Bis-Tris 4-12% acrylamide,

Invitrogen AG, Basel, Switzerland).

Gradient mode chromatography

Post production, cell-culture supematants containing the Fc 133 fragment were first purified in capture-elution mode chromatography using Protein G Sepharose™ 4 Fast Flow (above). Eluted material from capture-elution mode chromatography were subsequently loaded onto a lml HiTrap™ Mab Select SuRe™ Protein A column (Protein A binding site mutants). The column was pre-equilibrated in 0.2M phosphate citrate buffer pH 8.0 and operated on an ÀKTApurifier™ chromatography system (both column and instrument from GE Healthcare Europe GmbH; column catalogue No: 11-0034-93) at a flow rate of lml/min. Elution was performed with a pH linear gradient combining various amounts of two buffers (running buffer (A): 0.2M phosphate citrate buffer pH 8.0 and elution buffer (B): 0.04M phosphate citrate buffer pH 3.0. The linear gradient went from 0% B to 100% B in five column volumes (CVs). Eluted fractions were neutralised with 0.1 V of IM Tris-HCl pH 8.0. Supematant, flow through and elution fractions were analysed under non-reduced conditions by SDS-PAGE (NuPAGE Bis-Tris 4-12% acrylamide, Invitrogen AG, Basel, Switzerland).

Purification ofhomo-dimeric FAB-Fc immunoglobulins andFAB fragments.

Post production, cell culture supematants were conditioned with 0.1 V of IM Tris-HCl pH 8.0. Protein L resin (Genescript, Piscataway, USA) was added to the conditioned supematant and incubated ovemight at 4°C. After incubation, bound proteins were washed with ten CVs of PBS pH7.4, eluted with 4CVs of 0.1M Glycine pH 3.0, and finally neutralised with 0.1 V of IM Tris-HCl pH 8.0. To assess Protein A binding, Protein L purified FAB were injected on a lml HiTrap MabSelect™ column (GE Healthcare Europe GmbH, Glattbrugg, Switzerland) at pH8.0 (Citric acid/Na2HPO4 buffer). Elution was performed with a pH linear gradient combining various amounts of two buffers (running buffer (A): 0.2 M phosphate citrate buffer pH8.0 and elution buffer (B): 0.04 M phosphate citrate buffer pH3.0). The linear gradient went from 0% B to 100% B in 5CVs. Eluted fractions were neutralised with 0.1V of IM TrisHCl pH8.0. Supematant, flow through and elution fractions were analysed under non-reduced conditions by SDS-PAGE (NuPAGE Bis-Tris 4-12% acrylamide, fnvitrogen AG, Basel,

Switzerland). · .

Purification and testing ofVH3 based homo-dimeric FAB-Fc and scFv-Fc immunoglobulins abrogated for Protein A binding in their Fc and VH3 domains.

Purification scheme included a capture-elution mode chromatography followed by a gradient mode chromatography according to the procedure described above.

Differential Protein A affinity chromatography (Examples 1 & 3)

Post production, cell-free supematants were loaded onto a 1ml HiTrap™ MabSelect SuRe™ Protein A column pre-equilibrated in 0.2M phosphate citrate buffer pH 6.0 and operated on an ÀKTApurifier™ chromatography system (both from GE Healthcare Europe GmbH; column Cat. No: 11-0034-93) at a flow rate of lml/min. Running buffer was 0.2 M phosphate citrate buffer pH 6. Elution of the hetero-dimer of interest was performed using 20 mM sodium citrate buffer pH 4 whilst homo-dimeric species were eluted with 0.1 M glycine, pH3.0. Elution was followed by OD reading at 280 nm; fraction containing the hetero-dimer of interest were pooled and neutralized with 0.1 volume of IM Tris pH 8.0 (Sigma). Supematant, flow through and elution fractions were analysed under non-reduced conditions by SDS-PAGE (NuPAGE Bis-Tris 4-12% acrylamide, Invitrogen AG, Basel, Switzerland).

Differential Scanning Calorimetry (DSC)

The thermal stabilities of antibodies were compared using calorimetric measurements. Calorimetric measurements were carried out on a VP-DSC differential scanning microcalorimeter (MicroCal-GE Healthcare Europe GmbH, Glattbrugg, Switzerland). The cell volume was 0.128 ml, the heating rate was l°C/min and the excess pressure was kept at 64 p.s.i. Ail protein fragments were used at a concentration of 1-0.5 mg/ml in PBS (pH 7.4). The molar heat capacity of each protein was estimated by comparison with duplicate samples containing identical buffer from which the protein had been omitted. The partial molar heat capacities and melting curves were analysed using standard procedures. Thermograms were baseline corrected and concentration normalized before being further analysed using a NonTwo State model in the software Origin v7.0.

The expected melting profiles for the human IgG subclasses are known (Garber E & Demarest SJ (2007) Biochem Biophys Res Commun, 355(3): 751-7) and ail profiles hâve been shown to contain three unfolding transitions corresponding to the independent unfolding of the CH2, CH3 and F AB domains. Of the four human IgG subclasses, IGHG1 has the most stable CH3 domain (~85°C); while other subclasses CH3 domains are less stable, although none are known to melt below 70°C. Similarly, ail subclasses are known to hâve a melting température of ~70°C for the CH2 domain.

Purity assessment by capillary gel electrophoresis (Example 3.2)

Non-reduced sample préparation pg of desalted protein sample was buffered in SDS sample buffer (Beckman Coulter International S.A., Nyon, Switzerland; IgG Purity Kit, Cat. No: Al0663) containing 5 mM Iodoacetamide (Sigma). A 10-kDa internai standard was added to the samples. The samplemixtures were heated at 70°C for 10 min.

Capillary gel electrophoresis

Following sample préparation, samples were run on a ProteomeLab PA 800 (Beckman Coulter International S.A., Nyon, Switzerland) fitted with a photodiode array detector (DAD) set at 220 nm. Bare-fused silica capillaries of 50 pm ID x 30.2 cm (20.2 cm effective length to detector) were used as séparation medium. Sample injection and séparation were performed at constant voltages of 5 and 15 kV, respectively, with reverse polarity in SDS-molecular weight gel buffer. The data were recorded at a rate of 2 Hz and current was stable during séparation. Capillary and samples were thermo-stated at 25°C.

Affinity measurements by SPR (Example 1)

SPR testing of FAB fragments abrogatedfor Protein A binding cDNA encoding the human HER2 extracellular région fused to an IGHG1 Fc fragment was cloned into an expression vector similar to the heavy and light expression vectors described above and transiently transfected in HEK293E cells using the PEI method (see PCT Publication No: WO2012131555). Supematants were conditioned with 0.1V of 1 M Tris-HCl pH8.0 and the antigen purified by Protein A capture-elution chromatography as described in Example 1. For SPR experiments, a monoclonal mouse anti-human IgG (Fc) antibody sensor chip was used, this allowed for the capture the Fc fused recombinant HER2 antigen in the correct orientation (Human Antibody Capture Kit, catalogue number BR-1008-39, GE Healthcare Europe GmbH). Measurements were recorded on a BIAcore™ 2000 instrument (GE Healthcare Europe GmbH, Glattbrugg, Switzerland). Different dilutions of anti-HER2 FAB (50, 25, 12.5, 6.25, 3.13,1.57, 0.78, 0.39nM) wereinjected over the sensor chip for 4min at 30pl/min. For each measurement, after seven minutes of dissociation, a 3M MgCl₂solution was injected for 1min at 30 μΐ/min for régénération. Data (sensorgram: fc2-fcl) were fitted with a 1:1 Langmuir. To account for the experimental variations in captured HER2-Fc at the beginning of each measurement, the Rmax value was set to local in ail fits. Measurements were performed in duplicate, and included zero-concentration samples for referencing. Both Chi2 and residual values were used to evaluate the quality of a fit between the experimental data and individual binding models

Affinity measurements by SPR (Examples 2 & 3)

SPR analysis was used to measure the association and dissociation rate constants for the binding kinetics of the different antibodies (murine and humanized antibodies). The binding kinetics of antibodies were measured on a BIAcore 2000 instrument (BIAcore-GE Healthcare Europe GmbH, Glattbrugg, Switzerland) at room température and analysed with the BiaEvaluation software (version 4.1, BIAcore-GE Healthcare Europe GmbH).

Measurements were performed on CM5 sensor chips (GE Healthcare Europe GmbH, Cat. No: BR-1000-14) individually coupled with the ligand of interest using a commercial amine coupling kit (GE Healthcare Europe GmbH, Cat. No: BR-1000-50). Protein G ligand was from Pierce (Thermo Fisher Scientific-Perbio Science S.A., Lausanne, Switzerland, Cat. No: 21193).

Data (sensorgram: fc2-fcl) were fitted with a 1:1 Langmuir model with or without mass transfer as indicated. In capture experiments, to account for the experimental variations in at the beginning of each measurement, the Rmax value was set to local in ail fits. Dissociation times were of at least 350 seconds. Measurements were performed in triplicate and included zero-concentration samples for referencing. Both Chi2 and residual values were used to evaluate the quality of a fit between the experimental data and individual binding models.

Affinity measurements on HPB-ALL cells by FACS

HPB-ALL cells (DSMZ, Braunschweig, Germany, Cat. No: ACC483) were used as CD3 positive cell line for FACS staining. HPB-ALL were maintained in RPMI 1640 supplemented with 10% FCS and 100 U/ml Penicillin and 100ug/ml streptomycin. 100 μΐ dilution sériés of the chimeric OKT3 antibody and humanized variants were incubated with 4x10⁵ HPB-all cells in PBS supplemented with 1% BSA and 0.1% Sodium Azide (referred as FACS buffer) for 45 min on ice. An irrelevant human IgGl was used as isotype control and the chimeric OKT3 antibody as positive control. After washing, cells were incubated with a 1/200 dilution of anti-Human Fc-PE (EBioscience, Vienna, Austria) for 45 min on ice. Cells were then washed again and resuspended in 200ul FACS buffer. The relative mean fluorescence of each sample was measured on FACSCalibur (BD Biosciences, Allschwil, Switzerland ) Results are summarized in FIG. 9 as the relative staining of HBP-ALL compared to the chimeric OKT3 antibody.

Cell-lines for in vitro assays

Human HER2 positive cell lines Human cells expressing HER2 antigen hâve been described in PCT Publication No: W02010108127. HER2 positive human cell lines as used herein were as follows: BT474 (ATCC-LGL standards; Cat. No: HTB-20)

Culture conditions: RPMI medium supplemented with 10% heat-inactivated FBS, 1% penicillin-streptomycin (Invitrogen AG, Cat. No: 10378-016), 1% sodium pyruvate solution (PAA Laboratories, Pasching, Austria; Cat. No: SI 1-003), 1% MEM Non-Essential Amino Acids (PAA Laboratories, Cat. No: Mll-00dsmz3) and 1% GlutaMAX-1 (Invitrogen AG, Cat. No: 35050-038) in 150 cm² tissue culture flask (TPP, Trasadingen, Switzerland; Cat. No: 90150). Cells were passaged twice per week.

JIMT-1 (DSMZ, Braunschweig, Germany, Cat. No: ACC589)

Culture conditions: Dulbeco’s modified essential medium (DMEM (IX)) + GlutaMAX-1 (Invitrogen AG, Cat. No: 31966-012), supplemented with 10% heat-inactivated FBS, 1% penicillin-streptomycin (Invitrogen AG, Cat. No: 10378-016), 1% sodium pyruvate solution (PAA Laboratories, Cat. No: SI 1-003), 1% MEM Non-Essential Amino Acids (PAA Laboratories, Cat. No: Ml 1-003) and 1% GlutaMAX-1 (Invitrogen AG, Cat. No: 35050-038). Cells were passaged 2-3 times per week.

MDA-MB-231 (ATCC-LGL standards; Cat. No: HTB-26).

Culture conditions: same culture conditions as JIMT-1.

HT-1080 (ATCC-LGL standards; Cat. No: CCL-121).

Culture conditions: HT1080 cells are cultured in EMEM medium supplemented with 10% heat-inactivated FBS, 1% penicillin-streptomycin (Invitrogen AG, Cat. No: 10378-016), and 1% glutamine (Invitrogen AG, Cat. No: 25030-024). The cells are cultured at split three times a week (1 in 6 dilution).

NCI-N87 (ATCC-LGL standards; Cat. No: CRL-5822).

Culture conditions: NCI-N87 cells are cultured in RPMI1640 medium with 10% heatinactivated FBS, 1% penicillin-streptomycin (Invitrogen AG, Cat. No: 10378-016), 1% sodium pyruvate solution (PAA Laboratories, Pasching, Austria; Cat. No: SI 1-003), 1% MEM Non-Essential Amino Acids (PAA Laboratories, Cat. No: Ml l-00dsmz3), and 1% glutamine (Invitrogen AG, Cat. No: 25030-024). The cells are split twice a week (1 in 3 dilution).

Human CD38 positive cell lines

Human cells expressing CD38 antigen hâve been described in PCT Publication Nos: W02005103083, W02008047242, WO2011154453 and WO2012092612. CD38 positive human cell lines as used herein were as follows:

Stable recombinant CHO[CD38] cells

A gene coding for human CD38 was ordered at Source Biosciences (Berlin, Germany, Cat.No.: IRAU37D11, 4309086). Human CD38 was amplified using primers adding a kozak sequence, a start codon followed by a signal peptide (murine V leader) to the 5’ end and a Nhel restriction site to the 3’ end. The amplicon was eut using Nhel and HindlII and cloned into the expression cassette of pTl, a pcDNA3.1 (Invitrogen AG) derived vector developed in-house. The expression cassette of pTl links the expression of the gene of interest with expression of GFP and PAC (the gene for puromycin résistance) using two IRES (internai ribosome entry sites) on a polycistronic mRNA. A midiprep of the plasmid was prepared and the cloned CD38 open reading frame was confirmed by DNA sequencing. Suspension CHO-S cells (Invitrogen AG) were transfected using polyethyleneimine (JetPEI®, Polyplustransfection, Illkirch, France) in 50 ml bioreactor format (TubeSpin 50 bioreactors, TPP,

Trasadingen, Switzerland). For this purpose, exponential growing cells were seeded in OptiMEM medium (Invitrogen AG, Cat. No.: 31985-047). A JetPEI®:DNA complex was added to the cells. After 5 h incubation of the cells with the JetPEI®:DNA complex at 37°C under shaking (200 RPM) for endocytosis, one volume of culture medium PowerCHO2 (Lonza, distributor RUWAG Lifescience, Bettlach, Switzerland, Cat. No:BE12-771Q) supplemented with 4 mM Gin was added to the cell suspension. The cells were then incubated on a shaken platform at 37°C, 5% CO2 and 80% humidity. One day after transfection the cells were seeded in 96 well plates at different concentrations in sélective medium containing puromycin (Sigma, Cat. No: P8833-25mg). After approximately 14 days of sélection under static conditions, 46 high GFP expressing cell pools were expanded as suspension cultures using TubeSpin 50 bioreactors. Once successfully adapted to suspension, the cells were analysed for CD38 by FACS. Stable CHO[CD38] clones with a homogenous CD38 staining profile were selected and used herein.

Other CD38 positive cell lines included: NCI-H929 (ATCC-LGL standards; Cat. No: CRL-9068).

Namalwa (ATCC-LGL standards; Cat. No: CRL-1432) U266 (ATCC-LGL standards; Cat. No: TIB-196) RPMI 8226 (ATCC-LGL standards; Cat. No: CCL-155) Culture conditions: RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 1% penicillin-streptomycin (Invitrogen AG) and 1% GlutaMAX-1 (Invitrogen AG) Raji (ATCC-LGL standards; Cat. No: CCL-86) Daudi (ATCC-LGL standards; Cat. No: CCL-213)

Human 0X40 positive cell lines

Human cells expressing 0X40 antigen hâve been described in PCT Publication No: W02013008171.

Peripheral blood mononuclear cells (PBMCs) and HBP-ALL are examples of human 0X40 positive cell lines.

Stable recombinant CHO[OX40] cells were used herein. A recombinant CHO cell line carrying a synthetic cDNA coding for human 0X40 was engineered using a similar protocol to that of the stable recombinant CHO[CD38] cell line described above.

Human CD20 positive cell lines

Human cells expressing CD20 antigen hâve been described in PCT Publications No: WO2010095031. An example of CD20+ cancer cells is the Daudi cancer cell-line (ATCCLGL standards; Cat. No: CCL-213), these B lymphoblast cancer cells are cultured in RPMI 1640 medium (Sigma) supplemented with 20% FBS and 1%P/S; 1% L-Glut; 1% Na-Pyr and 1% NEAA. The cells are cultured at 37°C with 5% CO2 supplémentation.

Human EGFR positive cell lines

Human cells expressing EGFR antigen hâve been described in PCT Publication No: W02010108127. An example of EGFR+ cancer cells is the HT-29 cancer cell-line (ATCCLGL standards; Cat. No: HTB-38), these colorectal cancer cells are cultured are cultured in McCoy's 5A medium (Sigma) supplemented with 10% FBS and 1%P/S; 1% L-Glut; 1% NaPyr and 1% NEAA. The cells are cultured at 37°C with 5% CO2 supplémentation.

Human CD 19 positive cell lines

Human cells expressing CD19 antigen hâve been described in PCT Publication No:

WO2010/095031. Namalwa (ATCC-LGL standards; Cat. No: CRL-1432) and Raji (ATCCLGL standards; Cat. No: CCL-86) are examples of human CD20 positive cell lines.

Human membrane IgE positive cell lines

PCT Publication No: WO2010/033736 on page 71 describes a method to class switch human PBMCs into IgE producing B cells by adding interleukin-4 (IL-4) and anti-CD40 antibody.

Recombinant target antigens

Human CD3 gamma-epsilon-Fc fusion protein

A cDNA encoding the human CD3 gamma extracellular région (UniProt accession No: P09693 residues 23-103 (SEQ ID NO: 184); UniProt Consortium (2013) Nucleic Acids Res., 41 (Database issue): D43-7; http://www.uniprot.org/) fused to the human CD3 epsilon extracellular région (UniProt accession No: P07766, residues 22-118 (SEQ ID NO: 185)) by a 26-residue peptide linker (sequence: GSADDAKKDAAKKDDAKKDDAKKDGS; SEQ ID NO: 186) was first synthetized by GENE ART AG (Regensburg, Germany). This synthetic gene was fused to a human IgGl Fc portion using standard overlap PCR techniques and a human IgGl Fc cDNA template also obtain from Geneart AG. The resulting cDNA was cloned in the modified pcDNA3.1 plasmid mentioned above.

For transient expression of the CD3 gamma-epsilon-Fc protein (SEQ ID NO: 187), the recombinant vector was transfected into suspension-adapted HEK-EBNA cells (ATCC-CRL10852) using Polyethyleneimine (PEI) as described above. The CD3 gamma-epsilon-Fc construct was then purified from cell-free supematant using recombinant Streamline rProtein A media (GE Healthcare Europe GmbH, Glattbrugg, Switzerland) and used for further analysis.

Human and cynomolgus monkey CD3 epsilon l-26_Fc fusion proteins

A cDNA encoding the human CD3 epsilon peptide 1-26 (UniProt accession No: P07766, amino acids 23-48, SEQ ID NO: 188) and a cDNA encoding the cynomolgus CD3 epsilon peptide 1-26 (UniProt accession No: Q95LI5, amino acids 22-47, SEQ ID NO: 189) were PCR amplified from synthetic cDNAs obtained from GENEART A.G. for the human and cynomolgus monkey CD3 epsilon extracellular régions, respectively. The amplified products were subsquently fused to a human IgGl Fc portion using standard overlap PCR techniques. The human IgGl Fc cDNA template was obtained from Geneart AG. The resulting cDNA were cloned in the modified pcDNA3.1 plasmid mentioned above.

For transient expression of human and cynomolgus CD3 epsilon constructs (SEQ ID NO: 190 and 191, respectively), the recombinant vectors were transfected into suspension-adapted HEK-EBNA cells (ATCC-CRL-10852) using Polyethyleneimine (PEI) as described above. The CD3 epsilon fusion constructs were then purified from cell-free supematant using recombinant Streamline rProtein A media (GE Healthcare Europe GmbH, Glattbrugg, Switzerland) and used for further analysis. These two fusion proteins are referred herein as the human and cynomolgus monkey CD3 epsilon l-26_Fc fusion proteins.

Human HER2 extracellular région

Préparations of HER2 soluble extracellular région hâve been described in PCT Publication No: WO2012131555. Human HER2 soluble extracellular région fused to a poly-histidine tag (referred herein as HER2-his) or fused to a human IgGl Fc région (referred herein as HER2Fc) were prepared.

Human and cynomolgus monkey CD38 extracellular régions

A cDNA for human CD38 was obtained from Source Biosciences (Erwin-Negelein-Haus, Germany, Cat. No.: IRAU37D11, 4309086), its extracellular région (UniProt accession No: P28907 residues 43-300) was PCR amplified and cloned into an in-house expression vector derived from pcDNA3.1 (Invitrogen AG). This expression vector encompassed akozak sequence and a start codon followed by the murine VJ2C leader peptide to the 5’ end and a 6His-tag to the 3 ’ end of its multiple cloning site. The soluble extracellular région of human CD38 fused to a 6-His-tag (SEQ ID NO: 192) was expressed and purified as follows: one volume of RPMI1640 medium (PAA Laboratories, Cat. No: El 5-039) containing HEK cells, 0.1% pluronic acid (Invitrogen AG), expression vector and polyethylenimine (JetPEI®, Polyplus-transfection, Illkirch, France) was incubated in a shaker flask at 37°C, 5% CO₂ and 80% humidity. One volume of ExCell293 medium supplemented with 6 mM glutamine was added to the mixture after 4 hours and incubation continued further for a total of 5 days. Post production, cell-ffee supematant was prepared by centrifugation and filtrated using 0.2 pm filters, pH was adjusted at 7.4 (4°C) using Tris 1 M pH 8.7. Ni-Sepharose Excell beads (GE Healthcare, Cat. No: 17-3712-03) were added to the solution and incubated ovemight at 4°C under agitation. The solution was loaded on an Econo-Column (Bio-Rad Laboratories AG, Reinach, Switzerland, Cat. No: 737-4252) for gravity-flow purification. The beads were washed in PBS (2x), 20 mM imidazole and the protein was eluted in PBS, 500 mM Imidazole. Eluted fractions were pooled and buffer exchanged for PBS with two dialysis steps at 4°C. The purified human CD38 extracellular région was filtrated using 0.22 pm syringe filters.

Using the methods as described above the soluble extracellular région of cynomolgus monkey CD38 antigen fused to a 6-His-tag (SEQ ID NO: 193) was cloned, expressed and purified.

Human 0X40 extracellular région

A method to préparé the soluble extracellular région of human 0X40 has been described in PCT Publication No: W02013008171.

Human EGFR extracellular région

An example of EGFR soluble extracellular région antigen préparation has been described in PCT Publication No: WO2012131555.

In vitro T cell redirection killing assay

Préparation of peripheral blood mononuclear cells

To produce peripheral blood mononuclear cells (PBMCs), blood filters containing human leukocytes were collected from the Blood Collection Centre in La Chaux-de-Fonds, Switzerland (Centre de Transfusion Sanguine et Laboratoire de Sérologie, rue Sophie-Mairet 29, CH-2300). Cells were removed from the filters by back-flushing with 60 ml of PBS containing 10 U/ml of liquemin (Drossapharm AG, Lucem, Switzerland). PBMCs were then purified with 50 mL Blood-Sep-Filter Tubes (Brunschwig, Basel, Switzerland) following manufacturer’s instructions. Tubes were centrifuged for 20 min at 800g at room température (without brake) and the cells were collected from the interface. Cells were washed 3x with Roswell Park Memorial Institute (RPMI, PAA Laboratoires, Pasching, Austria) medium without FBS or phosphate buffered Saline (PBS). PBMCs were resuspended at 10e6 cells/mL in RDL medium (RPMI supplemented with 10% heat inactivated Fêtai bovine sérum (FBS) and penicillin/streptomycin) and were cultured ovemight at 37°C in a 5% CO2 incubator prior to the assay.

T cell préparations

T cell purification was performed directly after the PBMC isolation using pan-T cell isolation kit II (Myltenyi Biotec GmbH, Bergisch Gladbach, Germany, Cat. No: 130-091-156) following manufacturer’s instructions. After purification, T cells were resuspended at 10e6 cells/mL in RDL medium and cultured ovemight at 37°C in a 5% CO2 incubator prior assay.

Assay readouts

Two different readouts which gave highly comparable results were used to quantify the redirected killing.

A flow cytometry method, referred herein as RDL-FACS method, based on fluorescencecytometry as described in Schlereth B et al. ((2005) Cancer Res, 65: 2882-2889), Moore PA et al. ((2011) Blood, 117(17): 4542-51) and Friedrich M et al. ((2012) Mol Cancer Ther, 11 : 2664-2673). Target cells were harvested, counted, washed once and resuspended at 5x10e6 cells/mL in PBS+1 μΜ Carboxyfluorescein succinimidyl ester (CFSE, Sigma). Cells were incubated 15 min at 37°C with gentle agitation every 5 min. CFSE loaded cells were washed 3x with RDL medium and resuspended at 2x10e5 cells/mL in RDL medium. PBMCs were harvested, counted and resuspended at 2x10e6 cells/mL in RDL medium. Antibodies serial dilutions (3x solutions) were prepared in RDL medium. Target cells (50 μΐ/well), T cells (50 μΐ/well) and 3x antibody solutions (50 μΐ/well) were distributed in flat-bottom 96-well plate (TPP, Trasadingen, Switzerland). The effector: target ratio was 10:1. The plates were incubated for 48h in a 5% CO₂ incubator at 37°C. After incubation the plates were centrifuged for 3 min at 300g, the supematants were discarded by flicking the plates. The plates were washed once with 200 μΐ of PBS, centrifuged again and the PBS was discarded. A pre-warmed solution of accutase (Invitrogen AG) was added and the plates were incubated 10 min at 37°C. The detached adhèrent cells were resuspended by pipetting up and down after addition of 100 pL of RDL medium. The solution was transferred into a U-bottom 96-well plate (TPP). The U-bottom plates were centrifuged for 3 min at 300 g, the supematants were discarded and the cells were resuspended in 200μ1 of cold FACS buffer (PBS + 2% FBS + 10% Versene) supplemented with 7-AAD (Becton Dickinson AG, Allschwil, Switzerland) at a 1/40 dilution. The plates were immediately acquired on a Guava easyCyte™ Flow Cytometer (Millipore AG, Zug, Switzerland). For each well, the absolute number of living target cells was determined by gating on CFSE positive 7ADD négative population using Flowjo® software (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The percentage of spécifie cytotoxicity for each sample was determined using the condition in which only target cells were incubated as baseline. The EC50 values were determined using nonlinear variable slope régression method with Prism software (GraphPad software, La Jolla, CA, USA). The percentage of spécifie re-directed lysis (RDL) was calculated by subtracting the percentage of spécifie cytotoxicity of the condition without antibody to the conditions where a test antibody was added.

A cell viability method, referred herein as RDL-MTS method based on a colorimétrie method to assess cell viability as described in in Bühler P et al. ((2008) Cancer Immunol Immunother, 57: 43-52, Labrijn AF et al. ((2013) Proc Natl Acad Sci USA, 110(13): 5145-50) and PCT Publication No: WO2012143524. Target cells were harvested, counted, washed once and resuspended at 2x10e5 cells/ml in RDL medium. PBMCs were harvested, counted and resuspended at 2x10e6 cells/mL in RDL medium. Antibodies serial dilutions (3x solutions) were prepared in RDL medium. Target cells (50 μΐ/well), T cells (50 μΐ/well) and 3x antibody solutions (50 μΐ/well) were distributed in flat-bottom 96-well plate (TPP). The effector: target ratio was 10:1. The plates were incubated for 48 h in a 5% CO₂ incubator at 37°C. After incubation the supematants were discarded and the plates were washed 3 fîmes with 200 pL of PBS to remove the PBMCs and 100 μΐ of RDL medium was then added to each well. The readout was done using CellTiter 96® kit (Promega AG, Dübendorf, Switzerland) according to manufacturer’s instructions. Briefly, 10-20 μΐ of MTS reagent was added to each well and the plates were incubated 2-6h in a 5% CO₂ incubator at 37°C. The 490 nm absorbance was then read on a BioTek synergy plate reader (BioTek AG, Luzem, Switzerland). The percentage of spécifie killing was calculated using this formula: Spécifie killing = 100 x [(SDSp)/(SD-MD)]. SD is the absorbance measured in spontaneous death condition where target cells were incubated alone. Sp is the absorbance measured in each test condition (target cells + PBMCs + antibody). MD is the absorbance measured in the maximum death condition in which target cells were lysed by 3 ffeeze and thaw cycles. The percentage of spécifie redirected lysis (RDL) was calculated by subtracting the percentage spécifie cytotoxicity of the condition without antibody to the conditions where a test antibody was added. The EC50 values were determined using nonlinear variable slope régression method with Prism software (GraphPad software).

Xenograft model

JIMT-1 xenografts

Cells lines and reagents

Breast carcinoma JIMT-1 cell line was obtained from DSMZ (Cat. No: ACC589). Cells were maintained in DMEM (IX) with GlutaMAX™-l (Invitrogen AG, Cat. No: 31966-021) supplemented with 10% heat-inactivated fêtai bovine sérum (FBS) (AMIMED, London, UK, Cat. No: Z10834P), 1% penicillin-streptomycin (Invitrogen AG, Cat. No: 10378-016), 1% sodium pyruvate solution (PAA Laboratories, Cat. No: SI 1-003), 1% MEM Non-Essential Amino Acids (PAA Laboratories, Cat. No: Ml 1-003) and 1% GlutaMAX™-l (Invitrogen AG, Cat. No: 35050-038). Cells were split twice a week with StemPro Accutase (Invitrogen AG, Cat. No: Al 1105-01).

Peripheral blood mononuclear cells (PMBC) were collected from blood filters containing human leukocytes from the Blood Collection Centre in La Chaux-de-Fonds, Switzerland (Centre de Transfusion Sanguine et Laboratoire de Sérologie, rue Sophie-Mairet 29, CH2300). Cells were removed from the filters by back flushing with 60 ml of PBS containing 10 U/mL of liquemin (Drossapharm AG, Lucem, Switzerland). PBMCs were then isolated with 50 ml Blood-Sep-Filter Tubes (Brunschwig, Basel, Switzerland) following manufacturer’s instructions: tubes were centrifuged 20 min at 800 g at RT (without brake) and the cells were collected from the interface. Cells were washed 3 times with Roswell Park Memorial Institute medium without FBS (RPMI, Invitrogen AG, Cat. No: 21875-091). PBMCs were resuspended at 10e6 cells/ml in RPMI medium supplemented with 10% FBS (AMIMED), 1% penicillin-streptomycin (Invitrogen AG) and were cultured ovemight at 37°C under 5% CO2. Target cells were harvested, counted, washed once and resuspended at 5x10e6 cells/ml in PBS.

Mice and experimental conditions

In vivo experiments were performed in 5-week-old immunodeficient NOD.CB17/AlhnRjPrkdcscid/Rj (NOD/SCID) female mice characterized by T cell, B cell and natural killer cell deficiency (Janvier Labs, St Berthevin, France). The mice were maintained under stérile and standardized environmental conditions in standard rodent micro-isolator cages (20 +/- 1°C room température, 50 ± 10% relative humidity, 12 hours light dark rhythm). Mice received irradiated food, bedding and 0.22 pm-filtered drinking water. Ail experiments were done according to the Swiss Animal Protection Law with permission from the responsible cantonal authorities (Neuchâtel Canton, Switzerland). In compliance with the Animal Protection Law, mice had to be euthanized when tumor volumes exceeded 2000 mm³. Statistical analysis of the mean tumor volume of the corresponding treatment groups versus the vehicle control group was done by ANOVA one way and Bonferroni parametric tests.

Ail mice were depilated before engraftment with VEET cream (Reckitt Benckiser AG, Wallisellen, Switzerland) on the right flank. Photographe and weight measurements of mice were performed on the day of engraftment and later once a week. For each animal, 5x10e6 human PBMC were mixed with 5x10e6 JIMT-1 breast carcinoma cells in a final volume of 0.2 ml PBS. Four different PBMC donors were included. The PBMC/JIMT-1 mixture was subcutaneously injected into the right flank of each NOD/SCID mouse. A control group with 5x10e6 JIMT-1 breast carcinoma cells in a final volume of 0.2 ml PBS without any human PBMC was included. For each group of ten JIMT-1/PBMC engrafted animais (one group per PBMC donor), five animais were intravenously treated with HER2/CD3-1 bispecific antibody at 0.05mg/kg 3 hours after engraftment using a volume of 100 μΐ. Treatment was repeated 3 times per week, every two days, during two weeks. Tumors were measured twice a week with a caliper in two perpendicular dimensions and tumor volumes were calculated according to the following formula: tumor volume = [(width2 x length) / 2],

Example 1: Détermination of mutations that reduce or abrogate binding to Protein A in VH3 subclass

Methods to abrogate Protein A binding in immunoglobulin constant région are known (Lindhofer H. et al., (1995) J hnmunol, 155(1): 219-225; US6,551,592; Jendeberg L. et al., (1997) J hnmunol Methods, 201(1): 25-34; PCT Publication No: W02010151792).To assess the use of Protein A abrogating methods in full-length homo-dimeric immunoglobulins, an anti-HER2 homo-dimeric immunoglobulin based on a mixed IGHG1-IGHG3 Fc format and the corresponding Fc 133 control fragment were prepared. The anti-HER2 homo-dimeric immunoglobulin was formatted similarly to a naturally occurring antibody and consisted of a F AB fragment with anti-HER2 specificity fused to a Fc 133 fragment (a Fc sequence originating from the naturally occurring human IGHG3 isotype wherein the hinge sequence was substituted for the entire hinge sequence from the naturally occurring human IGHG1 isotype, abbreviated Fc 133 with SEQ ID NO:1 - wherein the numerals in the name correspond to the immunoglobulin gamma isotype subclass of each domain in the order of: hinge/CH2/CH3; the corresponding full-length anti-HER2 immunoglobulin being referred herein as anti-HER2 FAB-Fc 133; heavy chain with SEQ ID NO: 2 and light chain with SEQ ID NO: 3). Post transfection, the anti-HER2 FAB-Fc 133 homo-dimer and Fc 133 fragment were assayed for Protein A binding by gradient chromatography according to the protocol described in the Materials and Methods Section. As shown in FIG. 3 and FIG. 4A, the Fc 133 fragment did not bind the commercial MabSelect SuRe™ Protein A resin (GE Healthcare Europe GmbH) while the anti-HER2 FAB-Fc 133 homo-dimer was able to bind.

To assess the contribution of the FAB constant domains, the anti-HER2 homo-dimer described above was reformatted as an anti-HER2 scFv-Fc molécule wherein the scFv unit consisted of the parent immunoglobulin variable domains fused by a 15 amino-acid linker (abbreviated herein as anti-HER2 scFv-Fc 133; heavy chain with SEQ ID NO: 4). The resulting anti-HER2 scFv-Fc 133 homo-dimer was therefore identical to the parent anti-HER2 FAB-Fc 133 homo-dimeric immunoglobulin but lacked the CH1 and CK constant domains. As shown in FIG. 4B, the scFv-Fc 133 homo-dimer exhibited Protein A binding as observed with the parent anti-HER2 homo-dimeric immunoglobulin. This finding led to the conclusion that the variable domains of the anti-HER2 FAB fragment were responsible for hampering the efficacy of the methods abrogating Protein A binding in the Fc portion of homo-dimeric immunoglobulins. More importantly, it was concluded that Protein A binding within variable domains of homo-dimeric immunoglobulins will prevent the préparation of hetero-dimeric immunoglobulins based on Protein A differential purification techniques.

Ail five domains of Protein A are known to bind the variable heavy chain domains from the VH3 variable domain subclass (Jansson B et al., (1998) FEMS Immunol. Med. Microbiol., 20(1): 69-78), a feature which is known to hamper the préparation of VH3 based FAB fragments following papain digestion of whole antibody molécules, since Protein A binds both VH3 based FAB and Fc fragments. The heavy chain variable domain found in the homodimeric anti-HER2 immunoglobulin or its scFv-Fc version belongs to the VH3-23 subclass, and explains why these homo-dimeric molécules bound Protein A in spite of having no Protein A binding site within their engineered Fc régions.

VH3 based immunoglobulins or fragments thereof are of major importance to the biotechnology industry. VH3 based molécules hâve been extensively developed since their ability to bind Protein A facilitâtes their functional pre-screening, and as such many synthetic or donor based phage display libraries or transgenic animal technologies used for antibody discovery are based on the VH3 domain subclass. In addition VH3 based molécules are often selected for their good expression and stability over other known heavy chain variable domain subclasses. A recombinant version of Protein A which does not bind VH3 based FAB fragments has been developed and commercialized by GE Healthcare under the trade name MabSelect SuRe™.

Since the MabSelect SuRe™ resin was used herein for the Protein A binding assessment of the two homo-dimeric anti-HER2 immunoglobulins discussed above, it was concluded that the MabSelect SuRe™ resin was unsuitable for the préparation of hetero-dimeric immunoglobulins having at least one VH3 variable domain when using Protein A differential purification techniques - since homo-dimeric species having no Protein A binding in their Fc régions will still bind Protein A through their VH3 domains.

To investigate substitutions that would abrogate or reduce Protein A binding frontVH3 based homo-dimeric immunoglobulins or fragments thereof, VH3 based FAB variants will need to be assayed for Protein A binding. Although the MabSelect SuRe™ resin type is known to lack VH3 domain subclass binding, another commercial Protein A resin known as MabSelect™ does bind the VH3 domain subclass (also from GE healthcare) and was selected to analyse VH3 based FAB variants for Protein A binding.

The use of the MabSelect™ resin was validated by preparing a recombinant anti-HER2 FAB fragment derived from the parent anti-HER2 homo-dimeric immunoglobulin described earlier that is known to be of the VH3-23 variable domain subclass (abbreviated herein as anti-HER2 FAB; heavy chain with SEQ ID NO: 5 and light chain with SEQ ID NO: 3), and assaying the fragment onto the MabSelect™ and MabSelect SuRe™ columns (having a light chain based on the VK subclass I, the FAB fragment was first purified in capture-elution mode using protein L chromatography before Protein A gradient chromatography was performed on MabSelect™ or MabSelect SuRe™ columns, protocol for both columns followed the protocol described the Materials and Methods section). As shown in FIG. 4C, the VH3 based antiHER2 FAB only bound to the MabSelect™ column, confirming that the MabSelect SuRe™ resin lacks binding to the VH3 based FAB fragments; at least as far as monomeric VH3 based FAB fragments are concemed, and further contrasted with the results observed earlier for the VH3 based homo-dimeric immunoglobulins with engineered Fc régions having no binding to Protein A. Conversely, the anti-HER2 FAB showed strong binding to the MabSelect™ column which offered the possibility to assay for VH3 based FAB variants that would hâve no or reduced Protein A binding.

To abrogate Protein A binding in VH3 based FAB fragments, critical Protein A binding residues in VH3 domains were identified from the crystal structure of a human FAB fragment in complex with the D domain of Protein A (PDB code: IDEE; www.pdb.org; Graille M et al., (2000) Proc Natl Acad Sci USA, 97(10): 5399-5404). This analysis was used as a starting point for rational design wherein the nature of the substitutions undertaken was based on sequence comparison between Protein A binding and non-Protein A binding VH subclasses from human origin. FIG. 5 shows an alignment of one représentative framework for each human heavy chain variable domain subclass. Amino acid positions 15,17, 19, 57, 59, 64, 65, 66, 68, 70, 81, 82a, and 82b (Kabat numbering) were identified as part of the protein-protein interaction interface between the D domain of Protein A and the VH3 based FAB fragment in the IDEE structure. Amongst human VH subclasses, VH3 is the only subclass to bind Protein A, and residues at équivalent amino acid sequence positions in other subclasses were selected to be the source of the substitutions with the view to abrogate or reduce Protein A binding while having potentially reduce immunogenicity - since these substitutions involved the replacement of one residue with another naturally occurring residue at the same équivalent amino acid position found in a non-Protein A binding human VH subclass.

Mutations were introduced in the sequence of the aforementioned anti-HER2 FAB fragment by standard PCR based mutagenesis techniques, the following substitutions were made: G65S (heavy chain with SEQ ID NO:6 and light chain with SEQ JD NO: 3), R66Q (heavy chain with SEQ ID NO: 7 and light chain with SEQ ID NO: 3), T68V (heavy chain with SEQ ID NO: 8 and light chain with SEQ ID NO: 3), Q81E (heavy chain with SEQ ID NO: 9 and light chain with SEQ ID NO: 3), N82aS (heavy chain with SEQ ID NO: 10 and light chain with SEQ ID NO: 3), and the combination R19G/T57A/Y59A (heavy chain with SEQ ID NO: 11 and light chain with SEQ ID NO: 3).

In addition, the T57A substitution (heavy chain with SEQ ID NO: 12 and light chain with SEQ ID NO: 3), and T57E substitution (heavy chain with SEQ ID NO: 13 and light chain with SEQ JD NO: 3) were made. T57A was previously shown to abrogate Protein A binding in WO2010075548, and T57E was designed to introduce a charged residue that may disrupt the VH3-Protein A interaction. Having a light chain based on the VK subfamily I, FAB mutants were first purified in capture-elution mode using Protein L chromatography, and further assayed for Protein A binding using the MabSelect™ column operated under gradient mode as described in the Materials and Methods section. FIG. 6 shows that only T57A, T57E, G65S, Q81E, N82aS and the combination R19G/T57A/Y59A abrogated or reduced antiHER2 FAB binding to the MAbSelect™ resin. Substitutions G65S, Q81E and N82aS are preferred when abrogating Protein A binding in VH3 based FAB fragments since these mutations substitute for the sequence équivalent residue found in non-Protein A binding VH subclasses thereby potentially reducing immunogenicity.

Antibody affïnity and specificity is essentially confined to the CDR régions, however, framework substitutions may impact on antibody properties as shown in the case of several humanized antibodies. To assess if the above substitutions may impact the specificity and/or the affmity of VH3 derived antibodies, two of the preferred FAB mutants were assayed for HER2 antigen binding by Surface Plasmon Résonance (SPR). SPR measurements with recombinant HER2 antigen were performed as described in the Materials and Methods section. Both preferred mutants showed identical binding to the HER2 antigen when compared to the original FAB molécule (FIG. 7) demonstrating that the substitutions had not impact in terms of specificity or affmity. It is therefore expected that these substitutions could be broadly used to engineer out Protein A binding in VH3 derived antibody molécules without significant loss of antigen binding.

Two of these preferred substitutions were introduced in the anti-HER2 homo-dimeric immunoglobulin and anti-HER2 scFv-Fc molécule described earlier, and variants were assayed for binding onto the MabSelect SuRe™ resin. The following variants were prepared: anti-HER2 scFv(G65S)-Fc 133 (heavy chain with SEQ PD NO: 14), anti-HER2 scFv(N82aS)Fc 133 (heavy chain with SEQ ID NO: 15), anti-HER2 FAB(G65S)-Fc 133 (heavy chain with SEQ ID NO: 16 and light chain with SEQ ID NO: 3), and anti-HER2 FAB(N82aS)-Fc 133 (heavy chain with SEQ ID NO: 17 and light chain with SEQ ID NO: 3).

FIG. 8 shows the profiles from the MabSelect SuRe™ chromatography for ail four mutants. Ail variants now displayed reduced to almost no binding to the MabSelect SuRe™ column indicating a successful removal of Protein A binding with the previously identified substitutions. More importantly, it was concluded that when combined with Protein A differential purification techniques, substitutions which abrogate or reduce VH3 based FAB affmity for Protein A will allow the préparation of hetero-dimeric immunoglobulins wherein at least one VH3 variable domain is présent.

Example 2: Antigen binding sites that target the human CD3 antigen, tumour associated antigens and inflammatory cell surface antigens

Antigen binding sites against the human CD3 antigen

The human CD3 epsilon subunit was selected to drive T cell redirect killing via bispecific engagement.

Humanized variants of the mouse OKT3 antibody

The anti-human CD3 epsilon antigen binding site used herein was derived from the mouse OKT3 antibody (Muromonab-CD3, trade name Orthoclone OKT3, marketed by JanssenCilag and subsequently discontinued; murine variable heavy chain and light chain domains with SEQ ID NO: 18 and 19, respectively). OKT3 murine variable domains were humanized and formatted as scFv and FAB fragments.

Humanization followed the method described by Jung S & Plückthun A (1997, Protein Eng, 10(8): 959-66) to produce a highly stable humanized variant that would be suitable for both FAB and scFv formatting. The method makes use of the highly stable pair of VH/VL domains found in the Herceptin® antibody (rhuMAbHER2, huMAB4D5-8, trastuzumab or trade name Herceptin®; US Patent publication No.5,821,337; variable heavy chain and light chain domains with SEQ ID NO: 20 and 21, respectively) and follows the workflow of a humanization process onto fixed frameworks (Almagro JC & Fransson J (2008), Front Biosci, 13: 1619-33). Since the Herceptin® antibody is originally derived from the highly stable human families of germline ffamework VH3 and VK1, germline frameworks from these two families can be equally used as a source of fixed frameworks. Altematively, the human VK3 germline light chain ffamework family can be used instead of VK1 as it also has good stability properties (Ewert S et al., (2003) J Mol Biol, 325: 531-553). In addition to mouse antibodies, human antibodies can be engineered using this fixed ffamework method to improve stability. Preferred is the use of the human germline ffamework IGHV3-23*04, IGKVl-39*01 and IGKV3-20*01 having SEQ ID NO: 22, 23 and 24, respectively (referenced according to IMGT® (the international ImMunoGeneTics information system (Leffanc MP et al. (1999) Nucleic Acids Res, 27(1): 209-12; Ruiz M et al. (2000) Nucleic Acids Res, 28(1): 219-21; Leffanc MP (2001) Nucleic Acids Res, 29(1): 207-9; Leffanc MP (2003) Nucleic Acids Res, 31(1): 307-10; Leffanc MP et al., (2005) Dev Comp Immunol,

29(3): 185-203; Kaas Q et al., (2007) Briefings in Functional Genomics & Proteomics, 6(4):

253-64; http://www.imgt.org).

To this aim a first humanized antibody was constructed wherein the CDRs in the variable domains of the Herceptin® antibody were respectively replaced with the CDRs from the mouse OKT3 antibody and benchmarked against a chimera of the mouse OKT3 antibody (variable heavy chain and light chain with SEQ ID NO: 25 and 26, and referred herein as the chimeric OKT3 antibody).

The prototype antibody (variable heavy chain and light chain with SEQ ID NO: 27 and 39, and abbreviated VH/VL) had increased production levels in transient expression tests and increased FAB stability as measured by differential scanning calorimetry but had no binding to HPB-ALL cells (assessed by médian fluorescence intensity in FACS experiments, see Materials and Methods section), a human CD3 epsilon positive T cell tumour line (FIG. 9A).

Based on a 3D model of the first prototype pair of variable domains, a subset of back mutations (from CDR grafted Herceptin® prototype to mouse OKT3 sequence) were selected and tested: I34M, V48I, A49G, R58N, R58Y, I69L, A71T and T73K in the variable heavy chain domain and M4L, V33M, A34N, L46R, L47W, R66G, F71Y and P96F in the variable light chain (Kabat numbering). Note that the R58N substitution corresponds to a CDR grafted Herceptin® prototype-to-mouse OKT3 mutation while the R58Y substitution corresponds to a CDR grafted Herceptin® prototype-to-human IGHV3-23*04 germline substitution. The engineering strategy with regard to the combination of substitutions was based on the complementarity of the different substitutions in terms of their putative influence on CDR régions and/or variable domain packing and/or immunogenicity.

In a first approach, ail candidates were formatted as human IgGl antibodies. Best variants were selected according to expression levels, FAB fragment thermo-stability and ability to bind HPB-ALL cells by FACS. Best humanized variants had the Protein A binding site présent within their VH domain abrogated using the G65S or N82aS substitution. This engineering step was needed to further produce safe T cell retargeting BEAT antibodies free of anti-CD3 homo-dimers.

Back mutations in the VH of: I34M, A49G and A71T along with back mutations in the VL of: M4L, L46R, L47W and F71Y restored affinity. Best combinations of variable domains were VH8/VL4, VH8/VL8, VH11/VL4 and VH11/VL8 as these retained most of parental cell binding (FIG. 9A-C). In addition, combinations VH8/VL8 (variable domains with SEQ JD NO: 48 and 51, respectively) and VH11/VL8 (variable domains with SEQ ID NO: 49 and 51, respectively) had enhanced FAB stability and (+2.8°C and +1.6°C, respectively, FIG. 9D).

Finally, best humanized variants were also reformatted as scFv-Fc fusions and transiently expressed. Variants were ranked in terms of their relative affinity, stability, expression levels in transient transfection in this format (FIG. 9E). Best combinations of variable domains in a scFv-Fc fusion format were similar to the combinations identified in an antibody format: VH8-VL4 (scFv fragment with SEQ ID NO: 58) and VH8-VL8 (scFv fragment with SEQ ID NO: 59). Both scFv fragments had good thermal stability with the scFv-Fc fusion format (FIG. 9F).

Humanized variants of the mouse SP34 antibody

The mouse antibody SP34 was first described in 1985 (Pessano S et al., (1985) EMBO J, 4(2):337-44). It was produced by a hybridoma obtained from mice immunised with denatured protein extracts from HPB-ALL cells, the antibody has human specificity and cross-reactivity to cynomolgus monkey. SP34 epitopes on human and cynomolgus monkey CD3 epsilon subunit are known.

Following the methods and work flow described in this example supra, humanized VH and VL domains for the murine SP34 antibody having a VH domain with SEQ ID NO: 60 and a VL domain with SEQ ID NO: 61 were engineered via CDR grafting onto the VH3-23 and VK3 germline frameworks, respectively. The resulting VH3 based variable domains can be further abrogated for Protein A binding using the G65S or N82aS substitutions (Kabat numbering) depending on their usage in a BEAT antibody format.

To this aim a first humanized antibody was constructed wherein the CDRs in the variable domains of a human antibody having a germline VH3 heavy chain domain and a germline VK3 light chain domain were respectively replaced with the CDRs from the mouse SP34 antibody. The resulting humanized antibody was used a starting point for further affinity improvement and benchmarked against a chimera of the SP34 antibody (heavy chain and light chain with SEQ ID NO: 62 and 63, respectively, and referred herein as the chimeric SP34 antibody).

The prototype antibody (variable heavy chain and light chain with SEQ ID NO: 64 and 69, and abbreviated VH1/VL1) had a low binding to human CD3 epsilon l-26_Fc fusion protein (assessed by SPR, see Materials and Methods section and FIG. 10A).

Based on a 3D model of the first prototype pair of variable domains, a subset of substitutions that corresponded to either back mutations between the CDR grafted human germline VH3/VK3 prototype and mouse SP34 sequence (human-to-mouse or mouse-to-human substitutions) or rationally designed amino acid changes was selected. The following changes were made and tested in various combinations: WlOOeF, and WlOOeY in the variable heavy chain domain and A2I, S25A, T27A, G27aA, V27cA, T28A, T29A, S30A, N31A, Y32A, E38Q, F44P, G46L, T51 A, N52A, K53A, R54A, P56A, L66G, D69T, F87Y, Q89A, W91F, Y92A, S93A, N94A, and Q100G in the variable light chain (Kabat numbering; see FIG. 10A). The engineering strategy with regard to the combination of substitutions was based on the complementarity of the different substitutions in terms of their putative influence on CDR régions and/or variable domain packing and/or immunogenicity and/or impact on transient expression in mammalian cells.

In a first approach, ail candidates were formatted as human IgGl antibodies and later further tested in a scFv-Fc fusion protein format (FIG. 10B) with some variants having the Protein A binding site présent within their VH domain abrogated using the G65S. Best humanized candidates were selected according to expression levels and ability to bind the human and cynomolgus monkey CD3 epsilon l-26_Fc fusion proteins by SPR.

Preferred combinations of heavy chain and light chain variable domains with regard to antigen binding and recombinant expression were as follows: VH1 (SEQ ID NO: 101) or VH2 (SEQ ID NO: 102) or VH3 (SEQ ID NO: 103) or VH5 (SEQ ID NO: 104) paired with light chains domains VL21 (SEQ ID NO: 105) and VL32 (SEQ ID NO: 106).

HER2

Bispecific antibodies that would redirect T cells to kill HER2 positive cancer cells are useful to treat different forms of human breast cancer. Anti-HER2 antibodies hâve been described (Blumenthal GM et al., (2013) Clin Cancer Res, 19(18): 4911-6) with some being currently used in the clinic or currently under clinical investigations in humans (Tsang RY & Finn RS (2012) Br J Cancer, 106(1): 6-13).

The anti-HER2 antigen binding site as used herein was derived from the recombinant humanized anti-HER2 antibody Herceptin® (see section 1.1) formatted as a FAB fragment (FAB heavy chain fragment with SEQ ID NO: 5 and light chain SEQ ID NO: 3) or a scFv fragment (SEQ ID NO: 107). In some formats, the Protein A binding présent in the VH domain of the humanized anti-HER2 antibody 4D5 (VH3 domain subclass) was abrogated using the G65S substitution (FAB fragment with heavy chain having SEQ ID NO: 108 and light chain SEQ ID NO: 3 or scFv fragment with SEQ ID NO: 109) or the N82aS substitution (FAB fragment with heavy chain having SEQ ID NO: 110 and light chain SEQ ID NO: 3 or scFv fragment with SEQ ID NO: 111).

CD38

CD38 is a type II transmembrane glycoprotein which is normally found on hemopoietic cells and in solid tissues. CD38 is also expressed in a variety of malignant hematological diseases. Bispecific antibodies that would redirect T cells to kill CD38 positive cancer cells will be useful to treat a variety of malignant hematological diseases, including multiple myeloma, Bcell chronic lymphocytic leukaemia, B-cell acute lymphocytic leukaemia, Waldenstrôm’s macroglobulinemia, primary systemic amyloidosis, mantle-cell lymphoma, prolymphocytic/myelocytic leukaemia, acute myeloid leukaemia, chronic myeloid leukaemia, follicular lymphoma, NK-cell leukaemia and plasma-cell leukaemia. Several anti-CD38 antibodies hâve been described as research reagents or therapeutic candidates (PCT Publication No: W02006099875). Amongst the best characterized anti-human CD38 antibodies are OKT-10 and HB-7 mouse hybridomas (Hoshino S et al., (1997) J Immunol, 158(2): 741-7).

In a first approach, anti-human CD38 antigen binding sites can be derived from mouse hybridomas OKT10 (variable heavy chain and light chain with SEQ ID NO: 112 and 113, respectively) or HB-7 (variable heavy chain and light chain with SEQ ID NO: 114 and 115, respectively) and humanized versions thereof which can be further formatted as a FAB or scFv fragments. Following the methods and work flow described in Example 2.1, humanized VH and VL domains for the HB-7 hybridoma are can engineered via CDR grafting onto the VH3-23 and VK1 germline frameworks, respectively.

In a second approach, following the so-called best-fit humanization method described by Almagro JC & Fransson J (Front Biosci, (2008) 13: 1619-33), best-fit humanized VH and VL domains for the HB-7 hybridoma were engineered via CDR grafting onto the human IGHV459*03 and IGKVl-NLl*01 germline frameworks, respectively (referenced according to IMGT®sz.ipra). Humanized VH and VL variants with different degree of back mutations were investigated in silico and one preferred sélection of humanized VH and VL was transiently expressed as a human IgGl format and referred herein as humanized HB-7 best-fit VH (SEQ ID NO: 116) and VL (SEQ ID NO: 117) domains. The following mouse back mutations were introduced: (VH) S35H, I37V, I48L, V67L, V71K, T73N, F78V, Y91F and (VL): M4L, L48I, Y49S, T69K (Kabat numbering).

The humanized HB-7 best-fit antibody (heavy chain with SEQ ID NO: 118 and light chain with SEQ ID NO: 119) stained CHO[CD38] recombinant cells by FACS (data not shown). The humanized HB-7 best-fit antibody had a binding affinity for the CD38 extracellular région similar to that of the chimeric HB-7 antibody (heavy chain with SEQ ID NO: 120 and light chain with SEQ ID NO: 121) when assayed by SPR (KDs of 3.6 and 2.5 nM, respectively; FIG. 11A (chimeric) and FIG. 11B (humanized)). Surprisingly, the humanized HB-7 best-fit antibody displayed a signifîcant enhancement (+14.6°C) in FAB fragment stability compared to the chimeric HB-7 antibody as judged from calorimetry profiles (76.4°C (chimeric) vs 91.0°C (humanized), FIG. 11F).

In a third approach, mice immunized with the human CD38 extracellular domain and human CD38+ cells were used to generate novel hybridoma candidates against human CD38. Methods to generate hybridomas are known and the methods used herein were similar to methods disclosed in PCT Publication No: W02013008171. The 9G7 mouse antibody candidate had a high affinity for both human and cynomolgus monkey CD38 (variable heavy chain and light chain with SEQ ID NO: 122 and 123, respectively). This mouse antibody was first humanized according the methods described in this example supra. Using the best-fit approach, the germline VH framework IGHV2-5*09 and VK framework IGKVl-33*01 (referenced according to IMGT®j«pra) were selected as a starting point for the humanization process. Post CDR grafting, the first antibody prototype (fonnatted as a human IgGl isoptype, heavy chain SEQ ID NO: 124 and light chain with SEQ ID NO: 125) exhibited a strong binding to human CD38 only three fold lower than the mouse parental antibody as judged by SPR (chimeric 9G7 antibody with heavy chain SEQ ID NO: 126 and light chain with SEQ ID NO: 127; KD of 0.3 nM and 1 nM for the chimeric 9G7 antibody (data not shown) and first humanized prototype (data not shown), respectively). Affinity improved by two fold upon introduction of the F36Y back mutation in the variable light chain of the antibody (Kabat numbering) (the resulting antibody is referred herein as the humanized 9G7 best-fit antibody with heavy chain SEQ ID NO: 124 and light chain with SEQ ID NO: 128; KD of 0.5 nM for human CD38, FIG. 1 IC). The humanized 9G7 best-fit antibody also exhibited ahigh affinity for the cynomolgus monkey CD38 antigen (KD of 3.2 nM, data not shown), and an enhanced F AB thermo-stability (F AB Tm from DSC scans) over the chimeric 9G7 antibody (94°C vs. 82.2°C for the humanized 9G7 best-fit antibody and the chimeric 9G7 antibody, respectively; see FIG. 1 IG). The humanized 9G7 best-fit antibody has heavy chain variable domain with SEQ ID NO: 129 and light chain variable domain with SEQ ID NO: 130.

In addition, the 9G7 mouse antibody was humanized following the best-framework approach via CDR grafting onto the VH3-23 and VK1 germline frameworks. Humanized VH and VL variants with different degree of back mutations were investigated in silico and one preferred sélection of humanized VH and VL combination was transiently expressed as a human IgGl antibody (the resulting antibody is referred herein as the humanized 9G7 best-framework antibody with heavy chain SEQ ID NO: 131 and light chain with SEQ ID NO: 132). The following mouse back mutations were introduced: (VH) A24F, V37I, V48L, S49A, F67L, R71K, N73T, L78V, and K94R, and (VL) F36Y (Kabat numbering). This antibody exhibited a strong binding to human CD38 and cynomolgus monkey CD38 with affinity constants similar to that of the humanized 9G7 best-fit antibody (KD of 0.4 and 1 nM for human and cynomolgus monkey CD38, respectively; FIG. 11D). FAB thermo-stability (FAB Tm from DSC scans) was also very similar to that of the 9G7 best-fit F36Y humanized variant (89.2°C, see FIG. 11H). FIG. 11J summarizes the different humanized 9G7 antibodies described above. The humanized 9G7 best-framework antibody has heavy chain variable domain with SEQ ID NO: 133 and light chain variable domain with SEQ DD NO: 134.

In a fourth approach, an antibody phage library was screened to generate additional scFv fragments against human CD38. The library had a diversity based on the naturally occurring human V genes. This donor derived antibody phage display library used cDNAs amplified from blood lymphocytes originating from 48 human donors of which 70% had an autoimmune disease (vasculitis, systemic lupus erythematosus, spondiloarthropathy, rheumatoid arthritis and scleroderma). Library construction followed the protocol described by Schofield et al. (2007, Genome Biol., 8(11): R254) with a total diversity of 2.53 x 1 Oel 0 clones. ScFv fragments recognizing human and/or cynomolgus monkey CD38 were isolated from this donor derived phage display library as follows. ScFv fragments were isolated in a sériés of repeated sélection cycles on recombinantly derived human and/or cynomolgus monkey CD38 antigens (see Materials and Methods section). Methods to screen antibody phage display libraries are known (Viti F et al., (2000) Methods Enzymol, 326: 480-505). Briefly, following incubation with the library, the immobilised antigen which had been previously coated on a plastic immunotube (ovemight in PBS at a concentration of 20 pg/ml) or captured on streptavidin beads (when using a biotin labelled form of the antigen, antigen captured at a concentration of 50 nM throughout the sélection process), bound phages were recovered whist unbound phages were washed away. Bound phages were rescued as described by Marks et al (Marks JD et al., (1991) J Mol Biol, 222(3): 581-97) and the sélection process repeated three times. Over one thousand clones from the second and third round of panning were expressed and analysed by ELIS A against the human and cynomolgus monkey CD38 antigens. Positive clones were subjected to DNA sequencing and some of the unique clones were further analysed for their ability to bind cell lines expressing human CD38. Following a first round of panning on a biotin labelled version of the human CD38 antigen immobilized on streptavidin beads and a second round of panning on a biotin labelled version of the cynomolgus monkey CD38 antigen immobilized on streptavidin beads, one preferred scFv fragment (clone No 767) having a variable heavy chain sequence with SEQ ID NO: 135 and a variable light chain with SEQ ID NO: 136 was selected for its ability to bind both human and cynomolgus monkey CD38. When formatted as a human IgGl antibody, clone 767 had a KD of about 300 nM for human CD38 (FIG. 1 IE) and about 1.2 μΜ for cynomolgus monkey CD38 (data not shown) (clone 767 IgGl antibody is referred herein as human 767 antibody with heavy chain SEQ ID NO: 137 and light chain with SEQ ID NO: 138). FAB thermo17758 stability (FAB Tm from DSC scans) was 70.2°C (FIG. 111). Clone 767 VH domain belongs to the VH3 domain subclass.

0X40

A bispecific antibody targeting CD3 and 0X40 will allow targeting and déplétion of activated T lymphocytes. In this combination, the activated T lymphocytes, which express both CD3 and 0X40 molécules, will engage into a mutual killing process resulting in rapid cell disappearance. Co-engagement of human CD3 and 0X40 by a bispecific antibody may achieve an effective élimination of pathogenic T cells in a short time frame. 0X40 is a member of the TNFR-superfamily of receptors and was first identified in 1987 as a 50 kDa glycoprotein expressed on activated CD4+ T cells from the rat (Paterson DJ et al., (1987) Mol. Immunol. 24: 1281-90). Unlike CD28, 0X40 is not constitutively expressed on naïve T cells but is induced after engagement of the T-Cell Receptor (TCR). 0X40 is a secondary costimulatory molécule, expressed after 24 to 72 hours following activation; its ligand, OX40L, is also not expressed on resting antigen presenting cells, but is expressed following their activation.

The mouse anti-human 0X40 antibody disclosed in PCT Publication No: W02013008171 (heavy chain and light chain domains with SEQ ID NO: 139 and 140, respectively) can be used as a source of anti-human 0X40 antigen binding site. A humanized version of this antibody based on the best-fit humanization method is also disclosed in PCT Publication No: WO2013008171 (heavy chain and light chain domains with SEQ ID NO: 141 and 142, respectively and with both antibodies being amendable for reformatting into a BEAT format.

Following the methods and work flow described in Example 2.1, humanized VH and VL domains for the anti-human 0X40 hybridoma are engineered via CDR grafting onto the VH323 and VK1 germline fiameworks, respectively. The resulting VH3 based variable domains are further abrogated for Protein A binding using the G65S or N82aS substitutions (Kabat numbering) depending on their usage in a BEAT antibody format. Only two humanized VH and VL domains were investigated differing by their different degree of back mutations. Back mutations were identified from sequence alignments between the parent antibody variable domains and a CDR grafted VH3 and VK1 similar to the first prototype antibody and the approach described in Example 2.1. These CDR grafted variable domains hâve no back mutations and are referred to herein as mingrafts. These sequences were then further modified to include ail the back mutations identified from the previous alignaient and resulted in modified variable domain sequences referred to herein as maxgrafts. The resulting sequences are summarized below:

Humanized and stabilized anti-OX40 VH having no back mutations; abbreviated humanized anti-OX40/mingraft VH (SEQ ID NO: 278).

Humanized and stabilized anti-OX40 VH having ail possible back mutations; abbreviated humanized anti-OX40/maxgraft VH (SEQ ID NO: 279).

Humanized and stabilized anti-OX40 VL having no back mutations; abbreviated humanized anti-OX40/mingraft VL (SEQ ID NO: 280).

Humanized and stabilized anti-OX40 VL having ail possible back mutations; abbreviated humanized anti-OX40/maxgraft VL (SEQ ID NO: 281).

CD20

Bispecific antibodies that would redirect T cells to kill CD20 expressing B cells can be useful to treat different forms of human lymphomas cancers. Several anti-human CD20 antibodies hâve been described as research reagents or therapeutic candidates. Amongst the best characterized anti-human CD20 antibodies are the chimeric rituximab antibody and humanized variants thereof (chimeric rituximab antibody, trade name Rituxan®, PCT Publication No: WO1994011026; mouse VH domain of SEQ ID NO: 143 and VL domain of SEQ ID NO: 144).

Following the methods and work flow described in Example 2.1, humanized VH and VL domains for the rituximab chimeric antibody are engineered via CDR grafting onto the VH323 and VK1 germline ffameworks, respectively. The resulting VH3 based variable domains are further abrogated for Protein A binding using the G65S or N82aS substitutions (Kabat numbering) depending on their usage in a BEAT antibody format. Two humanized VH and VL domains are investigated differing by their different degree of back mutations. Back mutations were identified from sequence alignments between the parent antibody variable domains and a CDR grafted VH3 and VK1 similar to the first prototype antibody and the approach described in Example 2.1. These CDR grafted variable domains hâve no back mutations and are referred to herein as mingrafts. These sequences were then further modified to include ail the back mutations identified from the previous alignaient and resulted in modified variable domain sequences referred to herein as maxgrafts. The resulting sequences are summarized below:

Humanized and stabilized Rituximab VH having no back mutations; abbreviated humanized Rituximab/mingraft VH (SEQ ID NO: 282).

Humanized and stabilized Rituximab VH having ail possible back mutations; abbreviated humanized Rituximab/maxgraft VH (SEQ ID NO: 283).

Humanized and stabilized Rituximab VL having no back mutations; abbreviated humanized Rituximab/mingraft VL (SEQ ID NO:284).

Humanized and stabilized Rituximab VL having ail possible back mutations; abbreviated humanized Rituximab/maxgraft VL (SEQ ID NO: 285).

EGFR

Bispecifîc antibodies that would redirect T cells to kill EGFR positive cancer cells can be useful to treat different forms of human cancers, preferably human pancreatic cancers and human colon cancers. Several anti-human EGFR antibodies hâve been described as research reagents or therapeutic candidates. Amongst the best characterized anti-human EGFR antibodies are the chimeric cetuximab antibody and humanized variants thereof. (chimeric cetuximab antibody, trade name Erbitux®, C225, IMC-C225; PCT Publication No: WO199640210; mouse VH domain with SEQ ID NO: 145 and mouse VL domain with SEQ ID NO: 146).

Following the methods and work flow described in Example 2.1, humanized VH and VL domains for the Erbitux® chimeric antibody are engineered via CDR grafting onto the VH323 and VK1 germline frameworks, respectively. The resulting VH3 based variable domains are further abrogated for Protein A binding using the G65S or N82aS substitutions (Kabat numbering) depending on their usage in a BEAT antibody format. Two humanized VH and VL domains are investigated differing by their different degree of back mutations. Back mutations were identified from sequence alignments between the parent antibody variable domains and a CDR grafted VH3 and VK1 similar to the first prototype antibody and the approach described in Example 2.1. These CDR grafted variable domains hâve no back mutations and are referred to herein as mingrafts. These sequences were then further modified to include ail the back mutations identified from the previous alignment and resulted in modified variable domain sequences referred to herein as maxgrafts. The resulting sequences are summarized below:

Humanized and stabilized Erbitux VH having no back mutations; abbreviated humanized Erbitux/mingraft VH (SEQ ID NO: 286).

Humanized and stabilized Erbitux VH having ail possible back mutations; abbreviated humanized Erbitux/maxgrafit VH (SEQ ID NO: 287).

Humanized and stabilized Erbitux VL having no back mutations; abbreviated humanized Erbitux/mingraft VL (SEQ ID NO: 288).

Humanized and stabilized Erbitux VL having ail possible back mutations; abbreviated humanized Erbitux/maxgraft VL (SEQ ID NO: 289).

Another well characterized anti-human EGFR antibody is the human panitumumab antibody and humanized variants thereof (human panitumumab antibody, trade name Vectibix®, PCT Publication No: WO2012138997; mouse VH domain with SEQ ID NO: 290 and mouse VL domain with SEQ ID NO: 291).

Following the methods and work flow described in Example 2.1, humanized VH and VL domains for the Vectibix® chimeric antibody are engineered via CDR grafting onto the VH323 and VK1 germline frameworks, respectively. The resulting VH3 based variable domains are further abrogated for Protein A binding using the G65S or N82aS substitutions (Kabat numbering) depending on their usage in a BEAT antibody format. Two humanized VH and VL domains are investigated differing by their different degree of back mutations. Back mutations were identified from sequence alignments between the parent antibody variable domains and a CDR grafted VH3 and VK1 similar to the first prototype antibody and the approach described in Example 2.1. These CDR grafted variable domains hâve no back mutations and are referred to herein as mingrafts. These sequences were then further modified to include ail the back mutations identified from the previous alignment and resulted in modified variable domain sequences referred to herein as maxgrafts. The resulting sequences are summarized below:

Humanized and stabilized Vectibix VH having no back mutations; abbreviated humanized Vectibix /mingrafit VH (SEQ ID NO: 292).

Humanized and stabilized Vectibix VH having ail possible back mutations; abbreviated humanized Vectibix /maxgraft VH (SEQ ID NO: 293).

Humanized and stabilized Vectibix VL having no back mutations; abbreviated humanized

Vectibix /mingraft VL (SEQ ID NO: 294).

Humanized and stabilized Vectibix VL having ail possible back mutations; abbreviated humanized Vectibix /maxgraft VL (SEQ ID NO: 295).

CD19

Bispecific antibodies that would redirect T cells to kill CD 19 expressing B cells will be useful to treat different forms of human blood and myeloid cancers. The human CD 19 molécule is a structurally distinct cell surface receptor expressed on the surface of human B cells, including, but not limited to, pre-B cells, B cells in early development (i.e., immature B cells), mature B cells through terminal différentiation into plasma cells and malignant B cells. CD 19 is expressed by most pre-B acute lymphoblastic leukemias (ALL), non-Hodgkin's lymphomas, B cell chronic lymphocytic leukemias (CLL), pro-lymphocytic leukemias, hairy cell leukemias, common acute lymphocytic leukemias and some Null-acute lymphoblastic leukemias (Nadler LM et al. (1983) J hnmunol, 131: 244-250; Anderson KC et al., (1984) Blood, 63: 1424-1433; Loken MR et al. (1987) Blood, 70: 1316-1324; Uckun FM et al. (1988) Blood, 71: 13-29; Scheuermann RH & Racila E (1995) Leuk Lymphoma, 18: 385397). The expression of CD19 on plasma cells further suggests it may be expressed on differentiated B cell tumors such as multiple myeloma, plasmacytomas, Waldenstrom's tumors (Grossbard ML et al. (1998) Br J Haematol, 102: 509-15; Treon SP et al. (2003) Semin Oncol, 30: 248-52).

Humanized anti-human CD19 antibodies described in PCT Publication No: W02010/095031 utilise the VH3-23 and VK1 variable domain frameworks and can be used to produce bispecific antibodies as described in Example 2.1. The humanized anti-human CD 19 antibody having a VH domain with SEQ ID NO: 296 and a VL domain with SEQ ID NO: 297 is used and further abrogated for Protein A binding using the G65S or N82aS substitutions (Kabat numbering) depending on its use in a BEAT antibody format.

IgE

Bispecific antibodies that would redirect T cells to kill membrane bound IgE positive B cells can be useful to treat different inflammatory disease such as asthma or fibrosis. Several antihuman IgE antibodies hâve been described as research reagents or therapeutic candidates. Amongst the best characterized anti-human IgE antibodies are the Omalizumab antibody (trade name Xolair®, USPTO publication No: US6,761,889, US6,329,509 and US20080003218A1; Presta LG et al., (1993) J lmmunol, 151: 2623-2632; humanized VH domain with SEQ ID NO: 298 and VL domain with SEQ ID NO: 299) and variants thereof.

Following the methods and work flow described in Example 2.1, humanized VH and VL domains for the Omalizumab antibody are engineered via CDR grafting onto the VH3-23 and VK1 germline ffameworks, respectively. The resulting VH3 based variable domains are further abrogated for Protein A binding using the G65S or N82aS substitutions (Kabat numbering) depending on their usage in a BEAT antibody format. Two stabilized VH and VL domains are investigated differing by their different degree of back mutations. Back mutations were identified from sequence alignments between the parent antibody variable domains and a CDR grafted VH3 and VK1 similar to the first prototype antibody and the approach described in Example 2.1. These CDR grafted variable domains hâve no back mutations and are referred to herein as mingrafts. These sequences were then further modified to include ail the back mutations identified from the previous alignment and resulted in modified variable domain sequences referred to herein as maxgrafts. The resulting sequences are summarized below:

Stabilized Omalizumab VH having no back mutations; abbreviated stabilized Omalizumab/mingraft VH (SEQ ID NO: 300).

Stabilized Omalizumab VH having ail possible back mutations; abbreviated stabilized Omalizumab/maxgraft VH (SEQ ID NO: 301).

Stabilized Omalizumab VL having no back mutations; abbreviated stabilized Omalizumab/mingraft VL (SEQ ID NO: 302).

Stabilized Omalizumab VL having ail possible back mutations; abbreviated stabilized Omalizumab/maxgraft VL (SEQ ID NO: 303).

Another example of anti-human IgE antibody is the mouse antibody Bswl7 (Vogel M et al., (2004) J Mol Biol, 341(2): 477-89; mouse VH domain with SEQ ID NO: 304 and mouse VL domain with SEQ ID NO: 305).

Following the methods and work flow described in Example 2.1, humanized VH and VL domains for the humanized Bswl7 antibody are engineered via CDR grafting onto the VH323 and VK1 germline frameworks, respectively. The resulting VH3 based variable domains are further abrogated for Protein A binding using the G65S or N82aS substitutions (Kabat numbering) depending on their usage in a BEAT antibody format. Two stabilized VH and VL domains are investigated differing by their different degree of back mutations. Back mutations were identified from sequence alignements between the parent antibody variable domains and a CDR grafted VH3 and VK1 similar to the first prototype antibody and the approach described in Example 2.1. These CDR grafted variable domains hâve no back mutations and are referred to herein as mingrafts. These sequences were then further modified to include ail the back mutations identified from the previous alignment and resulted in modified variable domain sequences referred to herein as maxgrafts. The resulting sequences are summarized below:

Stabilized Bswl7 VH having no back mutations; abbreviated stabilized Bswl7/mingraft VH (SEQ ID NO: 306).

Stabilized Bswl7 VH having ail possible back mutations; abbreviated stabilized Bswl7/maxgraft VH (SEQ ID NO: 307).

Stabilized Bswl7 VL having no back mutations; abbreviated stabilized Bswl7/mingraft VL (SEQ ID NO: 308).

Stabilized Bswl7 VL having ail possible back mutations; abbreviated stabilized Bswl7/maxgraft VL (SEQ ID NO: 309).

Example 3: Production of T cell retargeting hetero-dimeric immunoglobulins

3.1 BEAT® technology and built-in purification system

BEAT antibodies are heavy chain hetero-dimers based on a unique concept of bio-mimicry that exhibit superior hetero-dimerization over the “knob-into-hole” technology (PCT publication No: WO2012131555). The BEAT platform is based on an exchange of interface amino acids at 3D équivalent positions between naturally occurring homo or hetero-dimeric immunoglobulin domain pairs to create new hetero-dimers that can be used as building blocks for Fc-based bispecific antibodies. The technology allows for the design of Fc-based bispecific antibodies using any type of antigen binding scaffold. A scFv-FAB format is used herein to design Fc-based bispecific antibodies without the need to develop a common light chain for both antigen binding sites.

Since BEAT antibodies are heavy chain hetero-dimers, it is needed to distinguish between the two different heavy chains. These are referred herein as BTA and BTB chains. BTA and BTB chains as used herein encompass an antigen binding site, a human IgGl hinge région, a CH2 domain originating from human IgGl or IgG3 isotype and a modified CH3 domain originating from human IgGl or IgG3 isotype. Some of the BTA and BTB CH3 domains were identical or modified variants of the domains described in PCT Publication No: WO2012131555. BTA and BTB CH3 domains were selected from the groups consisting of: (BTA) SEQ ID NO: 147,148, 149, 153, 154, and 155, and (BTB) SEQ ID NO: 150,151, 152, 156, 157, and 158. Preferred BTA-BTB CH3 domain pairings are selected from the group consisting of: SEQ ID NO: 147 with SEQ ED NO: 150, SEQ ID NO: 148 with SEQ ID NO: 150, SEQ ID NO: 149 with SEQ ID NO: 151, SEQ ID NO: 147 with SEQ ID NO: 152, and SEQ ID NO: 148 with SEQ ID NO: 152. Most preferred BTA-BTB CH3 domain pairings are selected from the group consisting of: SEQ ID NO: 147 with SEQ ID NO: 156, SEQ LD NO: 148 with SEQ ID NO: 156, SEQ ID NO: 154 with SEQ ID NO: 150, and SEQ ID NO: 154 with SEQ JD NO: 152.

As described above, BEAT heavy chain hetero-dimers with an asymmetrical binding to Protein A can be created using parental domains from immunoglobulin isotypes having no binding to Protein A (PCT publication No: WO2012131555). A différence in the number of

Protein A binding sites between homo- and hetero-dimeric species is particularly useful to résolve these molecular species by Protein A chromatography. To avoid a residual binding that will interfère with species séparation by Protein A chromatography, it is necessary to abrogate any secondary Protein A binding sites which are naturally found within the VH3 subclass of human heavy chain variable domains. When antigen binding sites originate from the VH3 family, abrogation of their Protein A binding site can be achieved through the G65S or N82aS substitutions (Kabat numbering).

When preparing a bispecific antibody encompassed by the présent invention, using one antigen binding site of VH3 origin and one antigen binding site from a non VH3 origin, the antigen binding site of VH3 origin needs to be located on the heavy chain that does bind Protein A in its Fc région. Altematively, the antigen binding site of VH3 origin can be substituted with the N82aS substitution or G65S substitution or équivalent substitutions thereof to abrogate Protein A binding. When preparing a bispecific antibody from the présent invention using a pair of antigen binding sites of VH3 origin, the only possibility is to abrogate Protein A binding in at least one of the VH3 based antigen binding sites through the amino acid substitutions described above. Preferably, bispecific antibodies from the présent invention are engineered to create one of the two homo-dimer without Protein A binding site. More preferably, bispecific antibodies from the présent invention are engineered to create one homo-dimer without Protein A binding site, and the other homo-dimer having a substantial différence in its number of Protein A binding sites (at least one Protein A binding site, preferably two Protein A binding sites) over the hetero-dimer of interest.

Mechanisms of toxicity triggered by monospecific anti- human CD3 epsilon antibodies hâve been under extensive investigation; direct mechanisms hâve been linked to affinity, epitope and valency of the antibodies but indirect mechanisms of toxicity hâve also been described. These indirect mechanisms of toxicity are mediated by the Fc région of the anti- human CD3 epsilon antibodies which interact with Fc receptor expressing immune cells and lead to transient T cell activation and cytokine release. With a goal to improve safety, BEAT antibodies targeting human CD3 epsilon were abrogated for Fc-receptor binding in their lower hinge région. Fc receptor binding was abrogated or reduced using the L234A and L235A substitutions (EU numbering; Strohl WR et al., (2009) Curr Opin Biotechnol, 20(6): 685-91); which are often referred as the LALA substitutions.

Examples of BEAT antibodies encompassing at least one VH3 domain abrogated for

Protein A binding

Examples of HER2/CD3 targeting BEAT antibodies

Anti-HER2 and anti-CD3 epsilon arms can be formatted either as a scFv-Fc type of heavy chains consisting of a scFv fragment fused to a BEAT chain or as a heavy chain consisting of a FAB fragment fused to a BEAT chain similar to that of a naturally occurring antibody. The F AB based heavy chain requires its association with its cognate light chain to assemble into a functional antigen binding site.

L234A and L235A substitutions were introduced in CH2 régions and residual Protein A binding was abrogated within using the G65S or N82aS substitutions (Kabat numbering) when appropriate. Examples of BEAT antibodies targeting both human HER2 antigen and human CD3 epsilon were formatted as follows:

A first BEAT HER2/CD3 antibody was engineered using a combination of antigen binding sites described in Example 2.1 and 2.2 for the anti-human CD3 epsilon and the anti-human HER2 arms, respectively. The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 159) encompassing a variable heavy chain région with the N82aS substitution (Kabat numbering), a CH1 γΐ région, a γΐ hinge région, a γ3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 47). This heavy chain encompassed part of a human IgG3 Fc région and therefore had no binding to Protein A but since the heavy chain used herein had its heavy chain variable domain originating from the VH3 domain subclass, the VH domain was mutated to include the N82aS substitution thereby removing any additional Protein A binding sites within the heavy chain. The anti-human HER2 arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 160) encompassing a scFv fragment, a CH1 yl région, ayl hinge région, a γΐ CH2 région with L234A and L235A substitutions (EU numbering), and a γΐ based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT HER2/CD3-1 antibody (FIG. 12A format A).

A second BEAT HER2/CD3 antibody was engineered using a combination of antigen binding sites described in Example 2.1 and 2.2 for the anti-human CD3 epsilon and the anti-human HER2 arms, respectively. The anti-human HER2 arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 161) encompassing a variable heavy chain région, a CH1 γΐ région, a γΐ hinge région, a γΐ CH2 région with L234A and L235A substitutions (EU numbering), and a γΐ based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 3). The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 162) encompassing a scFv fragment, a CH1 γΐ région, a γΐ hinge région, a γ3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ3 based BEAT CH3 domain. This heavy chain encompassed part of a human IgG3 Fc région and therefore had no binding to Protein A but since the heavy chain used herein had its heavy chain variable domain originating from the VH3 domain subclass, the VH domain was mutated to include the N82aS substitution thereby removing any additional Protein A binding sites within the heavy chain. This bispecific antibody is referred herein as BEAT HER2/CD3-2 antibody (FIG. 12A format B).

A third BEAT HER2/CD3 antibody was engineered using a combination of antigen binding sites described in Example 2.1 and 2.2 for the anti-human CD3 epsilon and the anti-human HER2 arms, respectively. The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 163) encompassing a variable heavy chain domain with the G65S substitution (Kabat numbering), a CH1 γΐ région, ayl hinge région, ay3 CH2 région with L234A and L235A substitutions (EU numbering), and ay3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 47). This heavy chain encompassed part of a human IgG3 Fc région and therefore had no binding to Protein A but since the heavy chain used herein had its heavy chain variable domain originating from the VH3 domain subclass, the VH domain was mutated to include the G65S substitution thereby removing any additional Protein A binding sites within the heavy chain. The anti-human HER2 arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 164) encompassing a scFv fragment, a CH1 yl région, ayl hinge région, aγΐ CH2 région with L234A and L235A substitutions (EU numbering), and a γΐ based BEAT CH3 domain. The scFv portion of the bispecific antibody was further stabilised using an engineered disulfide bond between the heavy and light chain domains at Kabat position heavy chain 44 (G44C) and light chain 100 (Q100C) as described in PCT publication No WO

1994029350. This bispecific antibody is referred herein as BEAT HER2/CD3-3 antibody (FIG. 12B format C).

A fourth BEAT HER2/CD3 antibody was engineered using a combination of antigen binding sites described in Example 2.1 and 2.2 for the anti-human CD3 epsilon and the anti-human HER2 arms, respectively. The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 165) encompassing a variable heavy chain domain, a CH1 yl région, a γΐ hinge région, a y 1 CH2 région with L234A and L235A substitutions (EU numbering), and a y 1 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 166). This heavy chain and light assembly encompassed a humanized version of the anti-human CD3 epsilon antibody (SP34) as described in PCT Publication No: W02008119565. The anti-human HER2 arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 167) encompassing a scFv fragment, a CH1 γ 1 région, a γ 1 hinge région, a γ 3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ 3 based BEAT CH3 domain. This heavy chain encompassed part of a human IgG3 Fc région and therefore had no binding to Protein A but since the heavy chain used herein had its heavy chain variable domain originating from the VH3 domain subclass, the VH domain was mutated to include the N82aS substitution thereby removing any additional Protein A binding sites within the heavy chain. This bispecific antibody is referred herein as BEAT HER2/CD3(SP34) antibody (FIG. 12B format D).

A fifth BEAT HER2/CD3 antibody was engineered using a combination of antigen binding sites described in Example 2.1 and 2.2 for the anti-human CD3 epsilon and the anti-human HER2 arms, respectively. The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 168) encompassing a variable heavy chain domain, a CH1 yl région, a γΐ hinge région, a γ 1 CH2 région with L234A and L235A substitutions (EU numbering), and a y 1 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 89). This arm of the bispecific antibody encompassed the variable domains ofthe humanized SP34 VH1/VL21 antibody described in Ex ample 2.1. The anti-human HER2 arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 167) encompassing a scFv fragment, a CH1 y 1 région, a y 1 hinge région, a y 3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ 3 based BEAT CH3 domain. This arm is équivalent to the BEAT HER2/CD3(SP34) antiHER2 arm described above (see FIG. 12B format D). The heavy chain encompassed part of a human IgG3 Fc région and therefore had no binding to Protein A but since the heavy chain used herein had its heavy chain variable domain originating from the VH3 domain subclass, the VH domain was mutated to include the N82aS substitution thereby removing any additional Protein A binding sites within the heavy chain. This bispecific antibody is referred herein as BEAT HER2/CD3(SP34-Kappal) antibody (FIG. 12C format E).

BEAT HER2/CD3-1, BEAT HER2/CD3-2, BEAT HER2/CD3-3, BEAT HER2/CD3(SP34), and BEAT HER2/CD3(SP34-Kappal) antibodies were expressed transiently, purified and tested in vitro for their affinity towards the HER2 and CD3 epsilon antigens, their stability and their ability to redirect T cell killing. Transient expression yields were in the range of 515 mg/1 of culture supematant for ail BEAT antibodies. Importantly, ail bispecific antibodies exhibited very low level of homo-dimeric contaminants in their préparation after a single Protein A chromatography step.

Since ail these BEAT antibodies were designed with both arm encompassing a VH3 domain, only abrogation of Protein A binding in at least one VH3 domain allowed to readily purify the hetero-dimer of interest using the one of the preferred differential purification method (see FIG. 2E). An example of differential Protein A purification trace for the BEAT HER2/CD3-1 antibody is shown in FIG. 13, and FIG. 14 shows the capillary electrophoresis profile of the purified hetero-dimer. Only a marginal content of homo-dimeric contaminants can be identified from this profile. Homo-dimers of the heavy chain formatted to carry a FAB portion are not found since these do not bind Protein A. Homo-dimers of the heavy chain formatted to carry the scFv fragments are found in a marginal proportion (2.5%), resulting in a heterodimer content of 97% after a single Protein A chromatography step. BEAT HER2/CD3-2, BEAT HER2/CD3-3, BEAT HER2/CD3(SP34), and BEAT HER2/CD3(SP34-Kappal) antibodies purified to similar levels of homogeneity and purity after a single Protein A chromatography step. The BEAT HER2/CD3-3 antibodies showed a proportion of disulfide bonded hetero-dimer aggregates after Protein A chromatography (27%) that were removed by cation exchange chromatography.

To further demonstrate that abrogation of Protein A binding within VH3 based heavy chain hetero-dimers greatly impacts on post Protein A chromatography purity, a BEAT HER2/CD3 antibody was engineered without the aforementioned N82aS substitution. FIG. 15A and 15B show the SDS-PAGE analysis of eluted Protein A chromatography fractions for the BEAT HER2/CD3-1 and its non N82aS substituted version, respectively. At pH 4, the eluted fraction for the non N82aS substituted version exhibits an additional band corresponding to homo-dimers of the heavy chain formatted to carry a FAB arm (FIG. 15B) while the N82aS substituted BEAT HER2/CD3 version does not (FIG. 15A), since the heavy chain formatted to carry a FAB arm has no binding to Protein A in its Fc région (Fc région based on human IgG3 isotype), it can only be deduced that the VH3 based variable domains found in this homo-dimeric species are responsible for Protein A binding. This resuit clearly demonstrates the utility of abrogating Protein A binding within VH3 based heavy chain hetero-dimers.

Both BEAT HER2/CD3-1 and BEAT HER2/CD3-2 antibodies had similar KD values for the human HER2 and human CD3 epsilon antigens. KD values were in the range of 0.50 - 2 nM for the human HER2 antigen and 1-2 μΜ for the human CD3 epsilon antigen (measured by SPR using the human CD3gamma-epsilon-Fc construct (see Materials and Methods section; FIG. 16A and 16B). DSC profiles for the two bispecific antibodies were similar, in both case the scFv portions either engaging human HER2 or human CD3 epsilon had retained their good thermo-stability profiles with Tm in the range of 68°C. FAB portions in both antibodies had Tm in the range of 82-83°C (FIG. 16C).

Another example of BEAT antibodies targeting both human HER2 antigen and human CD3 epsilon using the humanized Herceptin VH and VL sequences is formatted as foliows: a BEAT HER2/CD3 is engineered using a combination of antigen binding sites described in Example 2.1 and 2.2 for the anti-human CD3 epsilon and the anti-human HER2 antigen binding sites, respectively.

The anti-human HER2 arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 310) encompassing a variable heavy chain région, a CH1 γΐ région, a γΐ hinge région, a γ3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 3). This heavy chain encompasses part of a human IgG3 Fc région and therefore has no binding to Protein A but since the heavy chain used herein has its heavy chain variable domain originating from a VH3 ffamework, the VH domain is mutated to include the G65S substitution thereby removing any additional Protein A binding sites within the heavy chain.

The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 γΐ région, ayl hinge région, a yl CH2 région with L234A and L235A substitutions (EU numbering), and a γΐ based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT HER2/CD3(SP34-Kappa2) antibody.

In vitro T cell killing assays

The mechanism of action of BEAT HER2/CD3 antibodies is based on targeting cytotoxic T cell killing towards targeted cells by bridging the CD3 antigen on the cell surface of cytotoxic T cells and the HER2 antigen expressed on targeted cells.

The potency of BEAT HER2/CD3-1 and BEAT HER2/CD3-2 antibodies to redirect T cell killing was measured using a flow cytometry based method (referred herein as RDL-FACS method) or a colorimétrie based method (referred herein as RDL-MTS method).

The high expressing HER2 cell line JIMT-1, a Herceptin® (trastuzumab) résistant breast carcinoma cell line, the high expressing HER2 cell line BT-474, a Herceptin® (trastuzumab) sensitive breast carcinoma cell line and the low HER2 expressing breast adenocarcinoma cell line MDA-MB-231 were individually cultured during 48 h in the presence of human PBMCs and serial dilutions of BEAT HER2/CD3-1 or -2 antibodies or control antibodies.

In these assays, human PBMCs from blood donations were used a source of cytotoxic T lymphocytes. An effector to target cells ratio of 10:1 was used in ail assays. Négative controls were provided in the form of samples without antibody treatment (target cells and human PBMCs only). The cytotoxicity was determined using the RDL-FACS or RDL-MTS methods after the incubation period (see Materials and Methods section). The results showed that control antibodies did not trigger spécifie T cell-mediated cytotoxicity. In contrast BEAT HER2/CD3-1 and -2 antibodies induced a very potent, dose dépendent, tumor target cell death. Maximum killing was close to 100%. Both readout methods methods gave close results. Donor-to-donor variability accounted for about a tenfold different in EC50 between the methods. Measured EC50S correlated to the level of HER2 antigen expression by the target cell lines.

BT-474 cells express the most HER2 antigen and EC50S for both BEAT HER2/CD3-1 and -2 antibodies were in the sub-picomolar to picomolar range (0.6 and 2 pM, respectively, FIG. 17A). JIMT-1 cells hâve masked HER2 antigen on their cell surface (Nagy P et al. (2005), Cancer Res, 65(2): 473-482) and consequently exhibit low Herceptin® binding in spite of having high HER2 expression. Surprisingly, both BEAT HER2/CD3-1 and -2 antibodies had EC50S in the picomolar range against JIMT-1 cells as measured by the RDL-MTS method (21 and 16 pM, respectively; FIG. 17B). When measured with the RDL-FACS method, the BEAT HER2/CD3-1 antibody had an EC50 of 1.4 pM. Low HER2 expressing breast adenocarcinoma cell line MDA-MB-231 was less sensitive than the previous two cell lines with both antibodies exhibiting sub-nanomolar EC50S (both values close to 0.2 nM; FIG. 17C). When measured with the RDL-FACS method, the BEAT HER2/CD3-1 antibody had an EC50 of 0.08 nM. Taken together, these results show that BEAT HER2/CD3-1 and -2 antibodies were highly potent at redirecting T cell killing against various HER2 expressing breast cancer cell lines.

The BEAT HER2/CD3(SP34) antibody encompassed a humanized version of the anti-human CD3 epsilon antibody (SP34) described in PCT Publication No: W02008119565. The ability of this BEAT antibody format to redirect T cell killing towards HER2+ cells was investigated in vitro. Two different HER2+ cell lines were used in killing assays, a high HER2 expressing cell line (NCI-N87) and a low HER2 expressing cell line (HT-1080) (See Materials and Methods section). FIG. 17D-E show T cell redirected killing of NCI-N87 and HT-1080 cells by the BEAT HER2/CD3(SP34) antibody, respectively. The assays used an effector cells to target cells ratio of 10 to 1, and the RDL-MTS readout method after a 48h incubation period (see Materials and Methods section). The results show that the BEAT HER2/CD3(SP34) antibody was highly potent at redirecting T cell killing against HER2+ cell lines with EC50S of 0.35 and 29 pM when targetingNCI-N87 and HT-1080 cells, respectively.

The BEAT HER2/CD3(SP34-Kappal) antibody encompassed the humanized version of the anti-human CD3 epsilon antibody (SP34-Kappal) VH1/VL21 described in Example 2.1. The ability of this BEAT antibody format to redirect T cell killing towards HER2+ cells was investigated in vitro. Two different HER2+ cell lines were used in killing assays, a high HER2 expressing cell line (NCI-N87) and a low HER2 expressing cell line (HT-1080) (See Materials and Methods section). FIG. 17F-G show T cell redirected killing of NCI-N87 and

HT-1080 cells by the BEAT HER2/CD3(SP34-ICappal) antibody, respectively. The assays used an effector cells to target cells ratio of 10 to 1, and the RDL-MTS readout method after a 48h incubation period (see Materials and Methods section). The results show that the BEAT HER2/CD3(SP34-Kappal) antibody was highly potent at redirecting T cell killing against HER2+ cell lines with EC50S of 0.46 and 338 pM when targeting NCI-N87 and HT-1080 cells, respectively.

In vivo efficacy studies

JIMT-1 xenografts

The in vivo efficacy of the BEAT HER2/CD3-1 antibody was investigated using a JIMT1/PBMC xenograft model. Human PBMCs from blood donations were used a source of cytotoxic T lymphocytes. Herceptin® résistant breast carcinoma JIMT-1 cells were mixed at a 1:1 ratio with non-stimulated human PBMCs (four different donors) and subsequently injected subcutaneously in immunodeficiency (NOD/SCID) mice. Following engraftment, animais were treated with the BEAT HER2/CD3-1 antibody intravenously three times per week during two weeks. Antibody treatment started 3 hours after engraftment and continued on day 2, 4, 7, 9 and 11 thereafter.

To assess tumour growth without PBMCs, one cohort out of five was inoculated subcutaneously with 5x10e6 JIMT-1 cells in the absence of human PBMCs, whereas the remaining cohorts were subcutaneously injected with mixtures of 5x10e6 JIMT-1 cells mixed with 5x10e6 non-stimulated human PBMCs from healthy donors.

Human PBMCs, in the absence of antibody did not show a négative effect on tumour growth (FIG. 18A). Treatment with the BEAT HER2/CD3-1 antibody, in the presence of human effector cells induced a complété suppression oftumour growth in most ofthe animais (18/20 tumours, FIG. 18B-C). Eighteen days after the last day of treatment, only 11% of tumours (2/18) started to grow again. These data show very clearly the potent antitumor efficacy of the BEAT HER2/CD3-1 antibody.

Examples of CD38/CD3 targeting BEAT antibodies

Anti-CD38 and anti-CD3 epsilon arms can be formatted either as a scFv-Fc type of heavy chains consisting of a scFv fragment fused to a BEAT chain or as a heavy chain consisting of a FAB fragment fused to a BEAT chain similar to that of a naturally occurring antibody. The FAB based heavy chain requires its association with its cognate light chain to assemble into a functional antigen binding site.

L234A and L235A substitutions were introduced in CH2 régions and residual Protein A binding was abrogated within using the G65S or N82aS substitutions (Kabat numbering) when appropriate. Examples of BEAT antibodies targeting both human CD38 antigen and human CD3 epsilon were formatted as follows:

A first example of BEAT antibodies targeting both human CD38 antigen and human CD3 epsilon using the humanized HB7 bestfit VH and VL sequences was formatted as follows: A BEAT CD38/CD3 antibody was engineered using a combination of antigen binding sites described in Example 2.1 and 2.3 for the anti-human CD3 epsilon and the anti-human CD38 arms, respectively. The anti-human CD38 arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 169) encompassing a variable heavy chain région, a CH1 γΐ région, ayl hinge région, ayl CH2 région with L234A and L235A substitutions (EU numbering), and a γΐ based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 119). The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 162) encompassing a scFv fragment, a CH1 γΐ région, a γΐ hinge région, a γ3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ3 based BEAT CH3 domain. This heavy chain encompassed part of a human IgG3 Fc région and therefore had no binding to Protein A but since the heavy chain used herein had its heavy chain variable domain originating from a VH3 framework, the VH domain was mutated to include the N82aS substitution thereby removing any additional Protein A binding sites within the heavy chain. This arm is équivalent to the BEAT HER2/CD3-2 anti-CD3 epsilon arm described above (see FIG. 12A format B). The bispecific antibody is referred herein as BEAT CD38-HB7bestfit/CD3 antibody (FIG. 19 format A).

The BEAT CD38-HB7bestfit/CD3 antibody was expressed transiently, purified and tested in vitro for its affinity towards the CD38 and CD3 epsilon antigens, its stability and its ability to

100 redirect T cell killing. The KD value was 3.2 nM for the human CD38 antigen (measured by

SPR; FIG. 20A). DSC profiles for the bispecific antibody showed good thermo-stability profiles with a Tm of approximately 68°C for the scFv portion. The FAB portion had a Tm of approximately 91°C (FIG. 20B).

CD38 expressing cell lines (see Materials and Methods section) were used to assess redirected T cell killing in assays similar to that of described in Example 3.2.1. FIG. 21 shows T cell redirected killing of RPMI 8226 myeloma cells using the BEAT CD38-HB7bestfit/CD3 antibody. Note that the assay used purified T cells as effector cells with an effector cells to target cells ratio of 10 toi. When measured with the RDL-FACS method, the BEAT CD38HB7bestfit/CD3 antibody had an EC50 of 2.2 pM (mean of 2 donors, 48h incubation).

A second example of BEAT antibodies targeting both human CD38 antigen and human CD3 epsilon using the human clone 767 VH and VL sequences was formatted as follows: a BEAT CD38/CD3 antibody was engineered using a combination of antigen binding sites described in Example 2.1 and 2.3 for the anti-human CD3 epsilon and the anti-human CD38 arms, respectively. The anti-human CD38 arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 170) encompassing a variable heavy chain région, a CH1 yl région, a yl hinge région, a γΐ CH2 région with L234A and L235A substitutions (EU numbering), and a yl based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 138). The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 171) encompassing a scFv fragment, a CH1 yl région, a yl hinge région, a y3 CH2 région with L234A and L235A substitutions (EU numbering), and a y3 based BEAT CH3 domain. This heavy chain encompassed part of a human IgG3 Fc région and therefore had no binding to Protein A but since the heavy chain used herein had its heavy chain variable domain originating from a VH3 framework, the VH domain was mutated to include the G65S substitution thereby removing any additional Protein A binding sites within the heavy chain. This bispecific antibody is referred herein as BEAT CD38-767/CD3 antibody (FIG. 19 format B).

The BEAT CD38-767/CD3 antibody was expressed transiently, purified and tested in vitro for its affinity towards the CD38 and CD3 epsilon antigens, its stability and its ability to redirect T cell killing. CD38 expressing cell lines (see Materials and Methods section) were

101 used to assess redirected T cell killing in assays similar to that of described in Example 3.2.1.

FIG. 22 shows T cell redirected killing of Daudi cells using the BEAT CD38-767/CD3 antibody. Note that the assay used human PBMCs as effector cells with an effector cells to target cells ratio of 10:1. When measured with the RDL-FACS method, the BEAT CD38767/CD3 antibody had an EC50 of 244 pM (mean of 3 donors, 24h incubation).

Another example of BEAT antibodies targeting both human CD38 antigen and human CD3 epsilon using the humanized 9G7 best-ffamework VH and VL sequences is formatted as follows: a BEAT CD38/CD3 is engineered using a combination of antigen binding sites described in Example 2.1 and 2.3 for the anti-human CD3 epsilon and the anti-human CD38 antigen binding sites, respectively.

The anti-human CD38 arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 312) encompassing a variable heavy chain région, a CH1 γΐ région, ayl hinge région, a γ3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 132). This heavy chain encompasses part of a human IgG3 Fc région and therefore has no binding to Protein A but since the heavy chain used herein has its heavy chain variable domain originating from a VH3 framework, the VH domain is mutated to include the G65S substitution thereby removing any additional Protein A binding sites within the heavy chain. The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 yl région, ayl hinge région, a γΐ CH2 région with L234A and L235A substitutions (EU numbering), and a yl based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT CD389G7bestframework/CD3 (SP3 4-Kappa2) antibody.

Another example of BEAT antibodies targeting both human CD38 antigen and human CD3 epsilon using the human clone 767 VH and VL sequences is formatted as follows: a BEAT CD38/CD3 is engineered using a combination of antigen binding sites described in Example 2.1 and 2.3 for the anti-human CD3 epsilon and the anti-human CD38 antigen binding sites, respectively.

The anti-human CD38 arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 313) encompassing a variable heavy chain région, a CH1 γΐ région, a γΐ

102 hinge région, a y3 CH2 région with L234A and L235A substitutions (EU numbering), and a y3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 138): This heavy chain encompasses part of a human IgG3 Fc région and therefore has no binding to Protein A but since the heavy chain used herein has its heavy chain variable domain originating from a VH3 framework, the VH domain is mutated to include the G65S substitution thereby removing any additional Protein A binding sites within the heavy chain. The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 γΐ région, a γΐ hinge région, ayl CH2 région with L234A and L235A substitutions (EU numbering), and ayl based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT CD3 8-767 /CD3(SP34-Kappa2) antibody.

Examples of 0X40 /CD3 targeting BEAT antibodies

Anti-OX40 and anti-CD3 epsilon arms can be formatted either as a scFv-Fc type of heavy chains consisting of a scFv fragment fused to a BEAT chain or as a heavy chain consisting of a FAB fragment fused to a BEAT chain similar to that of a naturally occurring antibody. The FAB based heavy chain requires its association with its cognate light chain to assemble into a functional antigen binding site.

L234A and L235A substitutions were introduced in CH2 régions and residual Protein A binding was abrogated within using the G65S or N82aS substitutions (Kabat numbering) when appropriate. Examples of BEAT antibodies targeting both human 0X40 antigen and human CD3 epsilon were formatted as foliows:

An example of BEAT OX40/CD3 antibody was engineered using a combination of antigen binding sites described in Example 2.1 and 2.4 for the anti-human CD3 epsilon and the antihuman 0X40 anus, respectively. The anti-human 0X40 arm of the hetero-dimeric immunoglobulin used the variable domains of the humanized anti-human 0X40 antibody disclosed in PCT Publication No: W02013008171 (variable heavy chain and light chain domains with SEQ ID NO: 141 and 142, respectively) and consisted of a BEAT heavy chain (SEQ ID NO: 172) encompassing a variable heavy chain région, a CH1 γΐ région, a yl hinge région, ayl CH2 région with L234A and L235A substitutions (EU numbering), and ayl based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 173). The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consisted of a BEAT

103 heavy chain (SEQ ID NO: 162) encompassing a scFv fragment, a CH1 γΐ région, ayl hinge région, a γ3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ3 based BEAT CH3 domain. This heavy chain encompassed part of a human IgG3 Fc région and therefore had no binding to Protein A but since the heavy chain used herein had its heavy chain variable domain originating from a VH3 framework, the VH domain was mutated to include the N82aS substitution thereby removing any additional Protein A binding sites within the heavy chain. This arm is équivalent to the BEAT HER2/CD3-2 anti-CD3 epsilon arm described above (see FIG. 12A format B). The bispecific antibody is referred herein as BEAT OX40/CD3 antibody (FIG. 23).

The ability of the BEAT OX40/CD3 antibody to redirect T cell killing towards 0X40+ cells was investigated in vitro. The stable recombinant CHO[OX40] cell line was used in killing assays. FIG. 24 show T cell redirected killing of stable recombinant CHO[OX40] cells by the BEAT OX40/CD3 antibody. The assays used human PBMCs as effector cells with an effector cells to target cells ratio of 20 to 1, and the RDL-MTS readout method after a 48h incubation period (see Materials and Methods section). The results show that the BEAT OX40/CD3 antibody was highly potent at redirecting T cell killing against the stable recombinant CHO[OX40] cells with an EC50 of 0.5 nM (mean of 3 donors).

Another example of BEAT antibodies targeting both human 0X40 antigen and human CD3 epsilon using the humanized anti-OX40/maxgraft VH and VL sequences is formatted as foliows: a BEAT OX40/CD3 is engineered using a combination of antigen binding sites described in Example 2.1 and 2.4 for the anti-human CD3 epsilon and the anti-human 0X40 antigen binding sites, respectively.

The anti-human 0X40 arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 314) encompassing a variable heavy chain région, a CH1 γΐ région, a γΐ hinge région, a γ3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 315). This heavy chain encompasses part of a human IgG3 Fc région and therefore has no binding to Protein A but since the heavy chain used herein has its heavy chain variable domain originating from a VH3 framework, the VH domain is mutated to include the G65S substitution thereby removing any additional Protein A binding sites within the heavy chain.

104

The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 γΐ région, a γΐ hinge région, a yl CH2 région with L234A and L235A substitutions (EU numbering), and a γΐ based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT OX40maxgraft/CD3(SP34-Kappa2) antibody.

Another example of BEAT antibodies targeting both human 0X40 antigen and human CD3 epsilon using the humanized anti-OX40/mingraft VH and VL sequences is formatted as foliows: a BEAT OX40/CD3 is engineered using a combination of antigen binding sites described in Example 2.1 and 2.4 for the anti-human CD3 epsilon and the anti-human 0X40 antigen binding sites, respectively.

The anti-human 0X40 arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 316) encompassing a variable heavy chain région, a CH1 γΐ région, a γΐ hinge région, a γ3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 317). This heavy chain encompasses part of a human IgG3 Fc région and therefore has no binding to Protein A but since the heavy chain used herein has its heavy chain variable domain originating from a VH3 framework, the VH domain is mutated to include the G65S substitution thereby removing any additional Protein A binding sites within the heavy chain. The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 γΐ région, a γΐ hinge région, ayl CH2 région with L234A and L235A substitutions (EU numbering), and ayl based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT OX40mingraft/CD3 (SP34-Kappa2) antibody.

Examples of CD20 /CD3 targeting BEAT antibodies

An example of BEAT antibodies targeting both human CD20 antigen and human CD3 epsilon using the mouse rituximab antibody VH and VL sequences was formatted as follows: A BEAT CD20/CD3 was engineered using a combination of antigen binding sites described in Example 2.1 and 2.5 for the anti-human CD3 epsilon and the anti-human CD20 arms, respectively.

105

An example of BEAT antibodies targeting both human CD20 antigen and human CD3 epsilon using the humanized rituximab/maxgraft VH and VL sequences is formatted as follows: a

BEAT CD20/CD3 is engineered using a combination of antigen binding sites described in

Example 2.1 and 2.5 for the anti-human CD3 epsilon and the anti-human CD20 antigen binding sites, respectively.

The anti-human CD20 arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 318) encompassing a variable heavy chain région, a CH1 γΐ région, a γΐ hinge région, a γ3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 319). This heavy chain encompasses part of a human IgG3 Fc région and therefore has no binding to Protein A but since the heavy chain used herein has its heavy chain variable domain originating from a VH3 Framework, the VH domain is mutated to include the G65S substitution thereby removing any additional Protein A binding sites within the heavy chain. The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 γΐ région, ayl hinge région, a γΐ CH2 région with L234A and L235A substitutions (EU numbering), and a γΐ based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT CD20maxgraft /CD3(SP34-Kappa2) antibody.

Another example of BEAT antibodies targeting both human CD20 antigen and human CD3 epsilon using the humanized rituximab/mingraft VH and VL sequences is formatted as follows: a BEAT CD20/CD3 is engineered using a combination of antigen binding sites described in Example 2.1 and 2.5 for the anti-human CD3 epsilon and the anti-human CD20 antigen binding sites, respectively.

The anti-human CD20 arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 320) encompassing a variable heavy chain région, a CH1 γΐ région, a γΐ hinge région, a γ3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 321). This heavy chain encompasses part of a human IgG3 Fc région and therefore has no binding to Protein A but since the heavy chain used herein has its heavy chain variable domain

106 originating from a VH3 framework, the VH domain is mutated to include the G65S substitution thereby removing any additional Protein A binding sites within the heavy chain. The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 γΐ région, a γΐ hinge région, a γΐ CH2 région with L234A and L235A substitutions (EU numbering), and a yl based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT CD20mingraft/CD3(SP34-Kappa2) antibody.

Examples of EGFR/CD3 targeting BEAT antibodies

Anti-EGFR and anti-CD3 epsilon arms can be formatted either as a scFv-Fc type of heavy chains consisting of a scFv fragment fused to a BEAT chain or as a heavy chain consisting of a FAB fragment fused to a BEAT chain similar to that of a naturally occurring antibody. The FAB based heavy chain requires its association with its cognate light chain to assemble into a functional antigen binding site.

L234A and L235A substitutions were introduced in CH2 régions and residual Protein A binding was abrogated within using the G65S or N82aS substitutions (Kabat numbering) when appropriate. Examples of BEAT antibodies targeting both human EGFR antigen and human CD3 epsilon were formatted as follows:

An example of BEAT antibodies targeting both human EGFR and human CD3 epsilon antigens is formatted as follows: a BEAT EGFR/CD3 is engineered using a combination of antigen binding sites described in Example 2.1 and 2.6 for the anti-human CD3 epsilon and the anti-human EGFR arms, respectively. The anti-human EGFR arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 174) based on the mouse Erbitux antibody variable domains (mouse variable heavy and light chain domains with SEQ ID NO: 145 and 146, respectively) that encompassed a variable heavy chain région, a CH1 γΐ région, ayl hinge région, aγΐ CH2 région with L234A and L235A substitutions (EU numbering), and a γΐ based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 175). The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 171) encompassing a scFv fragment, a CH1 yl région, ayl hinge région, ay3 CH2 région with L234A and L235A substitutions (EU numbering), and a y3 based BEAT CH3 domain. This heavy chain encompassed part of a

107 human IgG3 Fc région and therefore had no binding to Protein A but since the heavy chain used herein had its heavy chain variable domain originating from a VH3 framework, the VH domain was mutated to include the G65S substitution thereby removing any additional Protein A binding sites within the heavy chain. This arm is équivalent to the BEAT CD38767/CD3 anti-CD3 epsilon arm described above (see FIG. 19 format B). The bispecific antibody is referred herein as BEAT EGFR/CD3 antibody (FIG. 25).

The BEAT EGFR /CD3 antibody was transiently expressed, purified and tested in vitro for its ability to redirect T cell killing against human EGFR+ cell lines. The HT-29 cell line was used in killing assays. FIG. 26 show T cell redirected killing of HT-29 cells by the BEAT EGFR /CD3 antibody. The assays used human PBMCs as effector cells with an effector cells to target cells ratio of 10 to 1, and the RDL-MTS readout method after a 48h incubation period (see Materials and Methods section). The results show that the BEAT EGFR/CD3 antibody was highly potent at redirecting T cell killing against HT-29 cells with an EC50 of 70.6 pM (mean of 4 donors).

Another example of BEAT antibodies targeting both human EGFR antigen and human CD3 epsilon using the humanized Erbitux/maxgraft VH and VL sequences is formatted as follows: a BEAT EGFR/CD3 is engineered using a combination of antigen binding sites described in Example 2.1 and 2.6 for the anti-human CD3 epsilon and the anti-human EGFR antigen binding sites, respectively.

The anti-human EGFR arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 322) encompassing a variable heavy chain région, a CH1 γΐ région, a γΐ hinge région, a γ3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 323). This heavy chain encompasses part of a human IgG3 Fc région and therefore has no binding to Protein A but since the heavy chain used herein has its heavy chain variable domain originating from a VH3 framework, the VH domain is mutated to include the G65S substitution thereby removing any additional Protein A binding sites within the heavy chain. The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 γΐ région, a γΐ hinge région, a γΐ CH2 région with L234A and L235A substitutions (EU numbering), and a γΐ

108 based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT EGFRcetuxmaxgraft/CD3 (SP3 4-Kappa2) antibody.

Another example of BEAT antibodies targeting both human EGFR antigen and human CD3 epsilon using the humanized Erbitux/mingraft VH and VL sequences is formatted as follows: a BEAT EGFR/CD3 is engineered using a combination of antigen binding sites described in Example 2.1 and 2.6 for the anti-human CD3 epsilon and the anti-human EGFR antigen binding sites, respectively.

The anti-human EGFR arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 324) encompassing a variable heavy chain région, a CH1 γΐ région, a γΐ hinge région, a γ3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 325). This heavy chain encompasses part of a human IgG3 Fc région and therefore has no binding to Protein A but since the heavy chain used herein has its heavy chain variable domain originating fforn a VH3 framework, the VH domain is mutated to include the G65S substitution thereby removing any additional Protein A binding sites within the heavy chain. The anti-human CD3 epsilon part of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 γΐ région, a γΐ hinge région, a yl CH2 région with L234A and L235A substitutions (EU numbering), and a γΐ based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT EGFRcetuxmingraft/CD3(SP34-Kappa2) antibody.

Another example of BEAT antibodies targeting both human EGFR antigen and human CD3 epsilon using the humanized Vectibix/maxgraft VH and VL sequences is formatted as follows: a BEAT EGFR/CD3 is engineered using a combination of antigen binding sites described in Example 2.1 and 2.6 for the anti-human CD3 epsilon and the anti-human EGFR antigen binding sites, respectively.

The anti-human EGFR arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 326) encompassing a variable heavy chain région, a CH1 γΐ région, a γΐ hinge région, a γ3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 327). This heavy chain encompasses part of a human IgG3 Fc région and therefore has no binding to

109

Protein A but since the heavy chain used herein has its heavy chain variable domain originating from a VH3 framework, the VH domain is mutated to include the G65S substitution thereby removing any additional Protein A binding sites within the heavy chain.

The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 γΐ région, a γΐ hinge région, a γΐ CH2 région with L234A and L235A substitutions (EU numbering), and a γΐ based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT EGFRpanimaxgraft/CD3(SP34-Kappa2) antibody.

Another example of BEAT antibodies targeting both human EGFR antigen and human CD3 epsilon using the humanized Vectibix /mingraft VH and VL sequences is formatted as follows: a BEAT EGFR/CD3 is engineered using a combination of antigen binding sites described in Example 2.1 and 2.6 for the anti-human CD3 epsilon and the anti-human EGFR antigen binding sites, respectively.

The anti-human EGFR arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 328) encompassing a variable heavy chain région, a CH1 γΐ région, a γΐ hinge région, a γ3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 329). This heavy chain encompasses part of a human IgG3 Fc région and therefore has no binding to Protein A but since the heavy chain used herein has its heavy chain variable domain originating from a VH3 framework, the VH domain is mutated to include the G65S substitution thereby removing any additional Protein A binding sites within the heavy chain. The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 yl région, ayl hinge région, a γΐ CH2 région with L234A and L235A substitutions (EU numbering), and a γΐ based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT EGFRpanimingraft/CD3(SP34-Kappa2) antibody.

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Examples of CD19/CD3 BEAT antibodies

Anti-CD 19 and anti-CD3 heavy chains can be fonnatted either as a scFv-Fc type of heavy chains consisting of a scFv fragment fused to a first BEAT chain or as a heavy chain consisting of a FAB fragment fused to a first BEAT chain similar to that of a naturally occurring antibody. The FAB based heavy chain requires its association with its cognate light chain to assemble into a functional antigen binding site. L234A and L235A substitutions were introduced in CH2 régions and residual Protein A binding was abrogated within using the G65S or N82aS substitutions (Kabat numbering) when appropriate. An example of BEAT antibodies targeting both human CD 19 antigen and human CD3 epsilon using anti-CD 19 VH and VL sequences described in WO2010095031 is formatted as follows:

An example of BEAT CD19/CD3 is engineered using a combination of antigen binding sites described in Example 2.1 and 2.7 for the anti-human CD3 epsilon and the anti-human CD19 antigen binding sites, respectively.

The anti-human CD 19 arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 330) encompassing a variable heavy chain région, a CH1 γΐ région, a γΐ hinge région, a γ3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 331). This heavy chain encompasses part of a human IgG3 Fc région and therefore has no binding to Protein A but since the heavy chain used herein has its heavy chain variable domain originating from a VH3 framework, the VH domain is mutated to include the G65S substitution thereby removing any additional Protein A binding sites within the heavy chain. The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 γΐ région, ayl hinge région, a γΐ CH2 région with L234A and L235A substitutions (EU numbering), and a γΐ based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT CD19/CD3(SP34-Kappa2) antibody.

CD19 expressing cell lines described in PCT Publication No: W02010/095031 are used to assess redirected T cell killing in assays similar to that of described in Example 3.2.1.

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Examples ofIgE/CD3 BEAT antibodies '

Anti-IgE and anti-CD3 heavy chains can be fonnatted either as a scFv-Fc type of heavy chains consisting of a scFv fragment fused to a first BEAT chain or as a heavy chain consisting of a FAB fragment fused to a first BEAT chain similar to that of a naturally occurring antibody. The FAB based heavy chain requires its association with its cognate light chain to assemble into a functional antigen binding site. L234A and L235A substitutions were introduced in CH2 régions and residual Protein A binding was abrogated within using the G65S or N82aS substitutions (Kabat numbering) when appropriate.

BEAT IgE/CD3 antibodies are engineered using a combination of antigen binding sites described in Example 2.1 and 2.8 for the anti-human CD3 epsilon and the anti-human IgE antigen binding sites, respectively.

Cell lines expressing IgE on their cell surface are described in PCT Publication No: W02010/033736 and can used to assess redirected T cell killing in assays similar to that of described in Example 3.2.1.

An example of BEAT antibodies targeting both human IgE antigen and human CD3 epsilon using the stabilized omalizumab/maxgraft VH and VL sequences is formatted as follows: The anti-human IgE arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 332) encompassing a variable heavy chain région, a CH1 γΐ région, ayl hinge région, a γ3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 333). This heavy chain encompasses part of a human IgG3 Fc région and therefore has no binding to Protein A but since the heavy chain used herein has its heavy chain variable domain originating from a VH3 framework, the VH domain is mutated to include the G65S substitution thereby removing any additional Protein A binding sites within the heavy chain. The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 yl région, a yl hinge région, a γΐ CH2 région with L234A and L235A substitutions (EU numbering), and a yl based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT IgEomalimaxgraft/CD3(SP34-Kappa2) antibody.

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Another example of BEAT antibodies targeting both human IgE antigen and human CD3 epsilon using the stabilized omalizumab/mingraft VH and VL sequences is formatted as follows:

The anti-human IgE arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 334) encompassing a variable heavy chain région, a CH1 γΐ région, a γΐ hinge région, a γ3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 335). This heavy chain encompasses part of a human IgG3 Fc région and therefore has no binding to Protein A but since the heavy chain used herein has its heavy chain variable domain originating from a VH3 framework, the VH domain is mutated to include the G65S substitution thereby removing any additional Protein A binding sites within the heavy chain. The anti-human CD3 epsilon ann of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 γΐ région, a γΐ hinge région, a γΐ CH2 région with L234A and L235A substitutions (EU numbering), and a γΐ based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT IgEomalimingraft/CD3 (SP3 4-Kappa2)antibody.

Another example of BEAT antibodies targeting both human IgE antigen and human CD3 epsilon using the stabilized Bswl7/maxgraft VH and VL sequences is formatted as follows: The anti-human IgE arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 336) encompassing a variable heavy chain région, a CH1 γΐ région, a γΐ hinge région, a γ3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 337). This heavy chain encompasses part of a human IgG3 Fc région and therefore has no binding to Protein A but since the heavy chain used herein has its heavy chain variable domain originating from a VH3 framework, the VH domain is mutated to include the G65S substitution thereby removing any additional Protein A binding sites within the heavy chain. The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 γΐ région, ayl hinge région, a γΐ CH2 région with L234A and L235A substitutions (EU numbering), and a γΐ

113 based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT IgEbswl7maxgraft/CD3(SP34-Kappa2) antibody.

Another example of BEAT antibodies targeting both human IgE antigen and human CD3 epsilon using the stabilized Bswl7/mingraft VH and VL sequences is formatted as follows: The anti-human IgE arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 338) encompassing a variable heavy chain région, a CH1 γΐ région, a yl hinge région, a y3 CH2 région with L234A and L235A substitutions (EU numbering), and a y3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 339). This heavy chain encompasses part of a human IgG3 Fc région and therefore has no binding to Protein A but since the heavy chain used herein has its heavy chain variable domain originating from a VH3 framework, the VH domain is mutated to include the G65S substitution thereby removing any additional Protein A binding sites within the heavy chain. The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 γΐ région, ayl hinge région, ayl CH2 région with L234A and L235A substitutions (EU numbering), and ayl based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT IgE bswl7mingraft/CD3(SP34-Kappa2) antibody.

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Examples of BEAT antibodies encompassing only one VH3 domain

Examples of CD38/CD3 targeting BEAT antibodies

An example of BEAT antibodies targeting both human CD38 antigen and human CD3 epsilon using the humanized HB7/bestfit VH and VL sequences was formatted as follows: a BEAT CD38/CD3 was engineered using a combination of antigen binding sites described in Example 2.1 and 2.3 for the anti-human CD3 epsilon and the anti-human CD38 arms, respectively. The anti-human CD38 arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 176) encompassing a variable heavy chain domain, a CH1 γΐ région, a γΐ hinge région, a γ3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 119). This heavy chain had no binding to Protein A as it encompassed part of a human IgG3 Fc région and had its heavy chain variable domain originating from a non-VH3 domain subclass. The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 177) encompassing a scFv fragment, a CH1 γΐ région, a γΐ hinge région, a γΐ CH2 région with L234A and L235A substitutions (EU numbering), and a γΐ based BEAT CH3 domain. This heavy chain and light assembly encompassed a humanized version of the anti-human CD3 epsilon antibody (SP34) as described in PCT Publication No: W02008119565. This BEAT antibody format is referred herein as BEAT CD38-HB7bestfit/CD3(SP34) antibody (FIG. 27 format A).

The ability of the BEAT CD38-HB7bestfit/CD3(SP34) antibody to redirect T cell killing towards CD38+ cells was investigated in -vitro. The CD38+ B lymphoblast cell line Daudi was used in killing assays. FIG. 28 show T cell redirected killing of Daudi cells by the BEAT CD38-HB7bestfit/CD3(SP34) antibody. The assays used human PBMCs as effector cells with an effector cells to target cells ratio of 10 to 1, and the RDL-FACS readout method after a 24h incubation period (see Materials and Methods section). The results show that the BEAT CD38-HB7bestfit/CD3(SP34) antibody was highly potent at redirecting T cell killing against the Daudi CD38+ cell line with an EC50 of 1.8 pM (mean of 3 donors).

A second example of BEAT antibodies targeting both human CD38 antigen and human CD3 epsilon using the humanized 9G7 best-fit VH and VL sequences (SEQ ID NO: 129 and 130,

115 respecitively) was formatted as follows: a BEAT CD38/CD3 was engineered using a combination of antigen binding sites described in Example 2.1 and 2.3 for the anti-human CD3 epsilon and the anti-human CD38 arms, respectively. The anti-human CD38 arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 178) encompassing a variable heavy chain domain, a CH1 yl région, ayl hinge région, ay3 CH2 région with L234A and L235A substitutions (EU numbering), and a y3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 128). This heavy chain had no binding to Protein A as it encompassed part of a human IgG3 Fc région and had its heavy chain variable domain originating from a non-VH3 domain subclass. The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 179) encompassing a scFv fragment, a CH1 yl région, ayl hingerégion, ayl CH2 région with L234A and L235A substitutions (EU numbering), and a yl based BEAT CH3 domain. This arm of the bispecific antibody encompassed the variable domains of the humanized SP34 VH5/VL32 antibody described in Example 2.1. This BEAT antibody format is referred herein as BEAT CD38-9G7best-fit/CD3(SP34-Kappa2) antibody (FIG. 27 format B). CD38-9G7best-fit/CD3(SP34-Kappa2) antibody had a KD value of 18 nM for the human CD3 l-26_Fc fusion protein (FIG. 29).

The ability of the BEAT CD38-9G7best-fit/CD3(SP34-Kappa2) antibody to redirect T cell killing towards CD38+ cells was investigated in vitro. The CD38+ B lymphoblast cell line Daudi was used in killing assays. FIG. 30 show T cell redirected killing of Daudi cells by the BEAT CD38-9G7best-fit/CD3(SP34-Kappa2) antibody. The assays used human PBMCs as effector cells with an effector cells to target cells ratio of 10 to 1, and the RDL-FACS readout method after a 24h incubation period (see Materials and Methods section). The results show that the BEAT CD38-9G7best-fit/CD3(SP34-Kappa2) antibody was highly potent at redirecting T cell killing against the Daudi CD38+ cell line with an EC₅₀ of 2 pM (mean of 3 donors).

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Examples of OX40/CD3 targeting BEAT antibodies

An example of BEAT antibodies targeting both human 0X40 antigen and human CD3 epsilon using the humanized anti-OX40 antibody VH and VL sequences (PCT Publication No: W02013008171) is formatted as follows:

A BEAT OX40/CD3 is engineered using a combination of antigen binding sites described in Example 2.1 and 2.4 for the anti-human CD3 epsilon and the anti-human 0X40 antigen binding sites, respectively.

The anti-human 0X40 arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 340) encompassing a variable heavy chain région, a CH1 γΐ région, a yl hinge région, a y3 CH2 région with L234A and L235A substitutions (EU numbering), and a y3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 173). This heavy chain has no binding to Protein A as it encompasses part of a human IgG3 Fc région and has its heavy chain variable domain originating from a non-VH3 domain subclass.

The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 yl région, ayl hinge région, a γΐ CH2 région with L234A and L235A substitutions (EU numbering), and a yl based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT OX40/CD3(SP34-Kappa2) antibody.

Human 0X40 expressing cell lines described above are used to assess redirected T cell killing in assays similar to that of described in Example 3.2.4.

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Examples of CD20/CD3 targeting BEAT antibodies

The anti-human CD20 arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 180) based on the mouse rituximab antibody variable domains (mouse variable heavy and light chain domains with SEQ ID NO: 143 and 144, respectively) that encompassed a variable heavy chain région, a CH1 yl région, a yl hinge région, a y3 CH2 région with L234A and L235A substitutions (EU numbering), and ay3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 181). This heavy chain had no binding to Protein A as it encompassed part of a human IgG3 Fc région and had its heavy chain variable domain originating from a non-VH3 domain subclass.

The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consisted of a BEAT heavy chain (SEQ ID NO: 177) encompassing a scFv fragment, a CH1 yl région, a γΐ hinge région, a γΐ CH2 région with L234A and L235A substitutions (EU numbering), and a yl based BEAT CH3 domain. This arm is équivalent to the BEAT CD38-HB7bestfit/CD3 antiCD3 epsilon arm described above (see FIG. 27 format A). This scFv fragment encompassed a humanized version of the anti-human CD3 epsilon SP34 antibody as described in PCT Publication No: WO2008119565 (VH and VL domains with SEQ ID NO: 182 and 183, respectively). This BEAT antibody format is referred herein as BEAT CD20/CD3(SP34) antibody (FIG. 31).

The BEAT CD20/CD3(SP34) antibody was transiently expressed, purified and tested in vitro for its ability to redirect T cell killing against human CD20+ cell lines. The CD38+ B lymphoblast cell line Daudi was used in killing assays. FIG. 32 show T cell redirected killing of Daudi cells by the BEAT CD20/CD3(SP34) antibody. The assays used human PBMCs as effector cells with an effector cells to target cells ratio of 10 to 1, and the RDL-FACS readout method after a 24h incubation period (see Materials and Methods section). The results show that the BEAT CD20/CD3(SP34) antibody was highly potent at redirecting T cell killing against Daudi cells with an EC50 of 25 pM (mean of 3 donors).

118

Another example of BEAT antibodies targeting both human CD20 antigen and human CD3 epsilon using the chimeric rituximab antibody VH and VL sequences is formatted as follows: a BEAT EGFR/CD3 is engineered using a combination of antigen binding sites described in Example 2.1 and 2.5 for the anti-human CD3 epsilon and the anti-human CD20 antigen binding sites, respectively.

The anti-human CD20 arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 341) encompassing a variable heavy chain région, a CH1 yl région, a γΐ hinge région, a y3 CH2 région with L234A and L235A substitutions (EU numbering), and a y3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 181). This heavy chain has no binding to Protein A as it encompasses part of a human IgG3 Fc région and has its heavy chain variable domain originating from a non-VH3 domain subclass.

The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 yl région, ayl hinge région, ayl CH2 région with L234A and L235A substitutions (EU numbering), and ayl based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT CD20/CD3(SP34-Kappa2) antibody.

Examples of EGFR/CD3 targeting BEAT antibodies

An example of BEAT antibodies targeting both human EGFR antigen and human CD3 epsilon using the mouse Erbitux antibody VH and VL sequences is formatted as follows: a BEAT EGFR/CD3 is engineered using a combination of antigen binding sites described in Example 2.1 and 2.6 for the anti-human CD3 epsilon and the anti-human EGFR antigen binding sites, respectively.

The anti-human EGFR arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 342) encompassing a variable heavy chain région, a CH1 yl région, a yl hinge région, a y3 CH2 région with L234A and L235A substitutions (EU numbering), and a y3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 175). This heavy chain has no binding to Protein A as it encompasses part of a human IgG3 Fc région and has its heavy chain variable domain originating from a non-VH3 domain subclass.

119

The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 γΐ région, a yl hinge région, a yl CH2 région with L234A and L235A substitutions (EU numbering), and a yl based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT EGFRcetux/CD3(SP34-Kappa2) antibody.

Another example of BEAT antibodies targeting both human EGFR antigen and human CD3 epsilon using the human Vectibix antibody VH and VL sequences is formatted as follows: a BEAT EGFR/CD3 is engineered using a combination of antigen binding sites described in Example 2.1 and 2.6 for the anti-human CD3 epsilon and the anti-human EGFR antigen binding sites, respectively.

The anti-human EGFR arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 343) encompassing a variable heavy chain région, a CH1 yl région, a yl hinge région, a y3 CH2 région with L234A and L235A substitutions (EU numbering), and a y3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 344). This heavy chain has no binding to Protein A as it encompasses part of a human IgG3 Fc région and has its heavy chain variable domain originating from a non-VH3 domain subclass.

The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 yl région, a yl hinge région, a yl CH2 région with L234A and L235A substitutions (EU numbering), and a yl based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT EGFRpani/CD3(SP34-Kappa2) antibody.

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Examples of IgE/CD3 targeting BEAT antibodies

An example of BEAT antibodies targeting both human IgE antigen and human CD3 epsilon using the humanized omalizumab antibody VH and VL sequences is formatted as follows: a BEAT IgE/CD3 is engineered using a combination of antigen binding sites described in Example 2.1 and 2.8 for the anti-human CD3 epsilon and the anti-human IgE antigen binding sites, respectively.

The anti-human IgE arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 345) encompassing a variable heavy chain région, a CH1 γΐ région, a γΐ hinge région, a γ3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 346). This heavy chain has no binding to Protein A as it encompasses part of a human IgG3 Fc région and has its heavy chain variable domain originating from a non-VH3 domain subclass.

The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 γΐ région, a yl hinge région, a γΐ CH2 région with L234A and L235A substitutions (EU numbering), and a γΐ based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT IgEomali/CD3 (SP3 4-Kappa2) antibody.

Another example of BEAT antibodies targeting both human IgE antigen and human CD3 epsilon using the mouse Bswl7 antibody VH and VL sequences is formatted as follows: a BEAT IgE/CD3 is engineered using a combination of antigen binding sites described in Example 2.1 and 2.8 for the anti-human CD3 epsilon and the anti-human IgE antigen binding sites, respectively.

The anti-human IgE arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 347) encompassing a variable heavy chain région, a CH1 γΐ région, a yl hinge région, a γ3 CH2 région with L234A and L235A substitutions (EU numbering), and a γ3 based BEAT CH3 domain assembled with its cognate light chain (SEQ ID NO: 348). This heavy chain has no binding to Protein A as it encompasses part of a human IgG3 Fc région and has its heavy chain variable domain originating from a non-VH3 domain subclass.

The anti-human CD3 epsilon arm of the hetero-dimeric immunoglobulin consists of a BEAT heavy chain (SEQ ID NO: 311) encompassing a scFv fragment, a CH1 yl région, a γΐ hinge

121 région, a γΐ CH2 région with L234A and L235A substitutions (EU numbering), and a γΐ based BEAT CH3 domain. This bispecific antibody is referred herein as BEAT IgEbswl7 /CD3(SP34-Kappa2) antibody.

Membrane IgE expressing cell lines described are used to assess redirected T cell killing in assays similar to that of described above.

Sequence listing

SEQ ID NO: 1 - Fc 133	DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCW VDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPE NNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHE ALHNRFTQKSLSLSPGK
SEQ ID NO: 2 - anti- HER2 FAB-Fc 133 heavy chain	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQM NSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCWVDVSHEDPEVQFKWYVDGVEVHNAKT KPREEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSR WQQGNIFSCSVMHEALHNRFTQKSLSLSPGK
SEQ ID NO: 3 - anti- HER2 light chain	DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGK APKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYY CQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS WCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 4 - anti- HER2 scFv-Fc 133	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQM NSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGG GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVN TAVAWYQQKPGKAPKLLIYSASFLYSTVPSRFSGSRSGTDFTL TISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRGGGGTDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVS HEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRWSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYN TTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHN RFTQKSLSLSPGK
SEQ ID NO: 5 - anti-	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG

122

HER2 FAB heavy chain	KGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQM NSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSC
SEQ ID NO: 6 - antiHER! FAB G65S heavy chain	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYTRYADSVKSRFTISADTSKNTAYLQMN SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSC
SEQ ID NO: 7 - antiHER! FAB R66Q heavy chain	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYTRYADSVKGQFTISADTSKNTAYLQM NSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSC
SEQ ID NO: 8 - antiHER! FAB T6 8 V heavy chain	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYTRYADSVKGRFVISADTSKNTAYLQM NSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSC
SEQ ID NO: 9 - antiHER! FAB Q81E heavy chain	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLEM NSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSC
SEQ ID NO: 10 - anti- HER! FAB N82aS heavy chain	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMS SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSC
SEQIDNO: 11 - anti- HER2 FAB R19G/T57A/Y59A heavy chain	EVQLVESGGGLVQPGGSLGLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYARAADSVKGRFTISADTSKNTAYLQM NSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSC
SEQ ID NO: 12 - antiHER2 FAB T57A heavy chain	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYARYADSVKGRFTISADTSKNTAYLQM NSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSC
SEQ ID NO: 13 - antiHER2 FAB T57E heavy	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYERYADSVKGRFTISADTSKNTAYLQM

123

chain	NSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSC
SEQ ID NO: 14 - anti- HER2 scFv(G65S)-Fc 133 heavy chain	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYTRYADSVKSRFTISADTSKNTAYLQMN SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGG SGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNT AVAWYQQKPGKAPKLLIYSASFLYSTVPSRFSGSRSGTDFTLT ISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRGGGGTDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSH EDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRWSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNT TPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNR FTQKSLSLSPGK
SEQIDNO: 15-antiHER2 scFv(N82aS)-Fc 133 heavy chain	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMS SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGG SGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNT AVAWYQQKPGKAPKLLIYSASFLYSTVPSRFSGSRSGTDFTLT ISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRGGGGTDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRWSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNT TPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNR FTQKSLSLSPGK
SEQ ID NO: 16 - anti- HER2 FAB(G65S)-Fc 133 heavy chain	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYTRYADSVKSRFTISADTSKNTAYLQMN SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTK GPSVTPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTKP REEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQ QGNIFSCSVMHEALHNRFTQKSLSLSPGK
SEQ LD NO: 17 - antiHER2 FAB(N82aS)-Fc 133 heavy chain	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMS SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTKP REEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQ

124

	QGNIFSCSVMHEALHNRFTQKSLSLSPGK
SEQ ID NO: 18 - OKT3 heavy chain variable domain	QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQR PGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQ LSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS
SEQ ID NO: 19 - OKT3 light chain variable domain	QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTS PKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAAT YYCQQWSSNPFTFGSGTKLEIN
SEQ ID NO: 20 Herceptin heavy chain variable domain	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQM NSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
SEQ ID NO: 21 Herceptin light chain variable domain	DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGK APKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYY CQQHYTTPPTFGQGTKVEIK
SEQ ID NO: 22 Human germlime heavy chain variable domain IGHV3-23*04	EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISG—SGGSTYYADSVKGRFTISRDNSKNTLYL QMNSLRAEDTAVYYCAK
SEQ ID NO: 23 Human germline light chain variable domain IGKVl-39*01	DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKA PKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQSYSTP
SEQ ID NO: 24 Human germline light chain variable domain IGKV3-20*01	EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQ APRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVY YCQQYGSSP
SEQ ID NO: 25 - Chimeric OKT3 heavy chain IgGl	QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQR PGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQ LSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 26 - Chimeric OKT3 human kappa light chain	QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTS PKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAAT YYCQQWSSNPFTFGSGTKLEINRTVAAPSVFIFPPSDEQLKSGT ASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
SEQ ID NO: 27 - OKT3 humanized heavy chain with VH domain	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTIHWVRQAPG KGLEWVAYINPSRGYTRYADSVKGRFTISADTSKNTAYLQM NSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP

125

	REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 28 - OKT3 humanized heavy chain with VH1 domain	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTIHWVRQAPG KGLEWVGYINPSRGYTRYADSVKGRFTISADTSKNTAYLQM NSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 29 - OKT3 humanized heavy chain with VH2 domain	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP GKGLEWVGYINPSRGYTRYADSVKGRFTISADTSKNTAYLQ MNSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 30 - OKT3 humanized heavy chain with VH3 domain	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTIHWVRQAPG KGLEWVGYINPSRGYTRYADSVKGRFTISTDTSKNTAYLQMN SLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 31 - OKT3 humanized heavy chain with VH4 domain	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP GKGLEWVGYINPSRGYTRYADSVKGRFTISTDTSKNTAYLQM NSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 32 - OKT3	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP

126

humanized heavy chain with VH5 domain	GKGLEWVGYINPSRGYTRYADSVKGRFTLSTDKSKNTAYLQ MNSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 33 - OKT3 humanized heavy chain with VH6 domain	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP GKGLEWIGYINPSRGYTRYADSVKGRFTLSTDKSKNTAYLQM NSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 34 - OKT3 humanized heavy chain with VH7 domain	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP GKGLEWVGYINPSRGYTNYADSVKGRFTLSTDKSKNTAYLQ MNSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 35 - OKT3 humanized heavy chain with VH8 domain	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP GKGLEWIGYINPSRGYTYYADSVKGRFTLSTDKSKNTAYLQ MNSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 36 - OKT3 humanized heavy chain with VH9 domain	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP GKGLEWIGYINPSRGYTYYADSVKSRFTLSTDKSKNTAYLQM NSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT

127

	LMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 37 - OKT3 humanized heavy chain with VH 10 domain	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP GKGLEWIGYINPSRGYTYYADSVKSRATLSTDKSKNTAYLQ MNSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 38 - OKT3 humanized heavy chain with VH 11 domain	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP GKGLEWIGYINPSRGYTYYADSVKGRFTLSTDKSKNTAYLQ MSSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ LD NO: 39 - OKT3 humanized light chain with VL domain	DIQMTQSPSSLSASVGDRVTITCRASSSVSYVAWYQQKPGKA PKLLIYDTSKLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYY CQQWSSNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C
SEQ ID NO: 40 - OKT3 humanized light chain withVLl domain	DIQMTQSPSSLSASVGDRVTITCRASSSVSYVAWYQQKPGKA PKLLIYDTSKLYSGVPSRFSGSRSGTDYTLTISSLQPEDFATYY CQQWSSNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C
SEQ ID NO: 41 - OKT3 humanized light chain with VL2 domain	DIQLTQSPSSLSASVGDRVTITCRASSSVSYVAWYQQKPGKAP KLLIYDTSKLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYC QQWSSNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS WCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 42 - OKT3 humanized light chain with VL3 domain	DIQLTQSPSSLSASVGDRVTITCRASSSVSYVAWYQQKPGKAP KLLIYDTSKLYSGVPSRFSGSRSGTDYTLTISSLQPEDFATYYC QQWSSNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS WCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

128

SEQ ID NO: 43 - OKT3 humanized light chain with VL4 domain	DIQLTQSPSSLSASVGDRVTITCRASSSVSYVAWYQQKPGKAP KRWIYDTSKLYSGVPSRFSGSRSGTDYTLTISSLQPEDFATYY CQQWSSNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C
SEQ ID NO: 44 - OKT3 humanized light chain with VL5 domain	DIQLTQSPSSLSASVGDRVTITCRASSSVSYMNWYQQKPGKA PKLLIYDTSKLYSGVPSRFSGSRSGTDYTLTISSLQPEDFATYY CQQWSSNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C
SEQ ID NO: 45 - OKT3 humanized light chain with VL6 domain	DIQLTQSPSSLSASVGDRVTITCRASSSVSYVAWYQQKPGKAP KRWIYDTSKLYSGVPSRFSGSRSGTDYTLTISSLQPEDFATYY CQQWSSNPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C
SEQ ID NO: 46 - OKT3 humanized light chain with VL7 domain	DIQLTQSPSSLSASVGDRVTITCRASSSVSYMNWYQQKPGKA PKLLIYDTSKLYSGVPSRFSGSRSGTDYTLTISSLQPEDFATYY CQQWSSNPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C
SEQ ID NO: 47 - OKT3 humanized light chain with VL8 domain	DIQLTQSPSSLSASVGDRVTITCRASSSVSYVAWYQQKPGKAP KRWIYDTSKLYSGVPSRFSGSGSGTDYTLTISSLQPEDFATYY CQQWSSNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C
SEQ ID NO: 48 - OKT3 humanized VH8 domain	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP GKGLEWIGYINPSRGYTYYADSVKGRFTLSTDKSKNTAYLQ MNSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSS
SEQ ID NO: 49 - OKT3 humanized VH 11 domain	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP GKGLEWIGYINPSRGYTYYADSVKGRFTLSTDKSKNTAYLQ MSSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSS
SEQ ID NO: 50 - OKT3 humanized VL4 domain	DIQLTQSPSSLSASVGDRVTITCRASSSVSYVAWYQQKPGKAP KRWIYDTSKLYSGVPSRFSGSRSGTDYTLTISSLQPEDFATYY CQQWSSNPPTFGQGTKVEIK
SEQ ID NO: 51 - OKT3 humanized VL8 domain	DIQLTQSPSSLSASVGDRVTITCRASSSVSYVAWYQQKPGKAP KRWIYDTSKLYSGVPSRFSGSGSGTDYTLTISSLQPEDFATYY CQQWSSNPPTFGQGTKVEIK
SEQ ID NO: 52 - scFv fragment mouse OKT3 - human IgGl Fc fusion	QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQR PGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQ LSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGG SGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSY MNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSL TISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINGGGGTDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVS

129

	HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK
SEQ JD NO: 53 - scFv fragment humanized OKT3 VH5-VL3 human IgGl Fc fusion	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP GKGLEWVGYINPSRGYTRYADSVKGRFTLSTDKSKNTAYLQ MNSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSGGG GSGGGGSGGGGSGGGASDIQLTQSPSSLSASVGDRVTITCRAS SSVSYVAWYQQKPGKAPKLLIYDTSKLYSGVPSRFSGSRSGT DYTLTISSLQPEDFATYYCQQWSSNPPTFGQGTKVEIKGGGGT DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCW VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRW SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK
SEQ ID NO: 54 - scFv fragment humanized OKT3 VH6-VL4 human IgGl Fc fusion	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP GKGLEWIGYINPSRGYTRYADSVKGRFTLSTDKSKNTAYLQM NSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGASDIQLTQSPSSLSASVGDRVTITCRASS SVSYVAWYQQKPGKAPKRWIYDTSKLYSGVPSRFSGSRSGTD YTLTISSLQPEDFATYYCQQWSSNPPTFGQGTKVEIKGGGGTD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVW DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK
SEQ ID NO: 55 - scFv fragment humanized OKT3 VH6-VL5 human IgGl Fc fusion	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP GKGLEWIGYINPSRGYTRYADSVKGRFTLSTDKSKNTAYLQM NSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGASDIQLTQSPSSLSASVGDRVTITCRASS SVSYMNWYQQKPGKAPKLLIYDTSKLYSGVPSRFSGSRSGTD YTLTISSLQPEDFATYYCQQWSSNPPTFGQGTKVEIKGGGGTD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVW DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK
SEQ ID NO: 56 - scFv fragment humanized OKT3 VH8-VL4 human IgGl Fc fusion	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP GKGLEWIGYINPSRGYTYYADSVKGRFTLSTDKSKNTAYLQ MNSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSGGG GSGGGGSGGGGSGGGASDIQLTQSPSSLSASVGDRVTITCRAS SSVSYVAWYQQKPGKAPKRWIYDTSKLYSGVPSRFSGSRSGT DYTLTISSLQPEDFATYYCQQWSSNPPTFGQGTKVEIKggggtdkt htcppcpapellggpsvflfppkpkdtlmisrtpevtcvwdvshedpevkfiiwyvdgvev hnaktkpreeqynstyrwsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprep

130

	qvytlppsrdeltknqvsltclvkgfypsdiavewesngqpeimykttppvldsdgsfflysklt vdksrwqqgnvfscsvmhealhnhytqkslslspgk
SEQ ID NO: 57 - scFv fragment humanized OKT3 VH8-VL8 human IgGl Fc fusion	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP GKGLEWIGYINPSRGYTYYADSVKGRFTLSTDKSKNTAYLQ MNSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSGGG GSGGGGSGGGGSGGGASDIQLTQSPSSLSASVGDRVTITCRAS SSVSYVAWYQQKPGKAPKRWIYDTSKLYSGVPSRFSGSGSGT DYTLTISSLQPEDFATYYCQQWSSNPPTFGQGTKVEIKggggtdkt htcppcpapellggpsvflfppkpkdtlmisrtpevtcvwdvshedpevkfnwyvdgvev hnaktkpreeqynstyrwsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprep qvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfïlysklt vdksrwqqgnvfscsvmhealhnhytqkslslspgk
SEQ JD NO: 58 - scFv fragment humanized OKT3 VH8-VL4	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP GKGLEWIGYINPSRGYTYYADSVKGRFTLSTDKSKNTAYLQ MNSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSGGG GSGGGGSGGGGSGGGASDIQLTQSPSSLSASVGDRVTITCRAS SSVSYVAWYQQKPGKAPKRWIYDTSKLYSGVPSRFSGSRSGT DYTLTISSLQPEDFATYYCQQWSSNPPTFGQGTKVEIK
SEQ ID NO: 59 - scFv fragment humanized OKT3 VH8-VL8	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP GKGLEWIGYINPSRGYTYYADSVKGRFTLSTDKSKNTAYLQ MNSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSGGG GSGGGGSGGGGSGGGASDIQLTQSPSSLSASVGDRVTITCRAS SSVSYVAWYQQKPGKAPKRWIYDTSKLYSGVPSRFSGSGSGT DYTLTISSLQPEDFATYYCQQWSSNPPTFGQGTKVEIK
SEQ ID NO: 60 - Mouse anti-human CD3 epsilon SP34 VH domain	EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYL QMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVT VSA
SEQ ID NO: 61 - Mouse anti-human CD3 epsilon SP34 VL domain	QAWTQESALTTSPGETVTLTCRSS-TGAVTTSNYANWVQEK PDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTED EAIYFCALWYSNLWVFGGGTKLTVL
SEQ ID NO: 62 Chimeric SP34 heavy chain IgGl	EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYL QMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVT VSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 63 Chimeric SP34 light chain (mouse V lambda - human lambda constant domain)	QAWTQESA-LTTSPGETVTLTCRSSTGAVTTSNYANWVQEK PDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTED EAIYFCALWYSNLWVFGGGTKLTVLRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 64 - SP34	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP

131

humanized heavy chain with VH1 domain	GKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTTVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 65 - SP34 humanized heavy chain with VH2 domain	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNTLYL QMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTTVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 66 - SP34 humanized heavy chain with VH3 domain	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNTLYL QMNSLRAEDTAVYYCVRHGNFGNSYVSYFAYWGQGTTVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 67 - SP34 humanized heavy chain with VH4 domain	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNTLYL QMNSLRAEDTAVYYCVRHGNFGNSYVSFFAYWGQGTTVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 68 - SP34 humanized heavy chain with VH5 domain	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSYFAYWGQGTTVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

132

	FPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 69 - SP34 humanized light chain with VL1 domain	EAWTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQEK PGQAFRGLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSED FAVYFCQLWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 70 - SP34 humanized light chain with VL2 domain	EAWTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQEK PGQAFRGLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSED FAVYFCALWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 71 - SP34 humanized light chain with VL3 domain	EAWTQS-ATLSVSPGERATLSCRSSTGAVTTSNYANWVQEK PGQAFRGLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSED FAVYFCQLWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 72 - SP34 humanized light chain with VL4 domain	EAVVTQS-ATLSVSPGERATLSCRSSTGAVTTSNYANWVQEK PGQAFRGLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSED FAVYFCALWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 73 - SP34 humanized light chain with VL5 domain	EIWTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQEKP GQAFRGLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSEDF AVYFCALWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 74 - SP34 humanized light chain with VL6 domain	EAWTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQEK PGQAPRGLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSED FAVYFCALWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 75 - SP34 humanized light chain with VL7 domain	EIWTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQEKP GQAPRGLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSEDF AVYFCALWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 76 - SP34 humanized light chain	EAWTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQEK PGQAFRGLIGGANKRAPGVPARFSGSGSGDEATLTISSLQSED

133

with VL8 domain	FAVYFCALWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 77 - SP34 humanized light chain with VL9 domain	EIWTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQEKP GQAFRGLIGGANKRAPGVPARFSGSGSGDEATLTISSLQSEDF AVYFCALWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 78 - SP34 humanized light chain with VL10 domain	EAWTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQEK PGQAFRGLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSED FAVYYCALWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC
SEQ ID NO: 79 - SP34 humanized light chain withVLll domain	EAWTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQEK PGQAFRGLIGGANKRAPGVPARFSGSGSGTEATLTISSLQSED FAVYFCALWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 80 - SP34 humanized light chain with VL12 domain	EAWTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQEK PGQAFRGLIGGANKRAPGVPARFSGSLSGTEATLTISSLQSEDF AVYFCALWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 81 - SP34 humanized light chain with VL 13 domain	EAWTQSPATLSVSPGERATLSCRASTGAVTTSNYANWVQEK PGQAFRGLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSED FAVYFCALWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 82 - SP34 humanized light chain with VL14 domain	EAWTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQEK PGQAFRLLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSEDF AVYFCALWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 83 - SP34 humanized light chain with VL15 domain	EAWTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQQK PGQAFRGLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSED FAVYFCALWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 84 - SP34 humanized light chain with VL16 domain	EIWTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQEKP GQAFRGLIGGANKRAPGVPARFSGSLSGTEATLTISSLQSEDF AVYFCALWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQL

134

	KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 85 - SP34 humanized light chain with VL17 domain	EIWTQSPATLSVSPGERATLSCRASTGAVTTSNYANWVQEK PGQAFRGLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSED FAVYFCALWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 86 - SP34 humanized light chain with VL18 domain	EIWTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQEKP GQAFRGLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSEDF AVYFCALWYSNLWVFGGGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 87 - SP34 humanized light chain with VL19 domain	EIWTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQEKP GQAFRGLIGGANKRAPGVPARFSGSLSGTEATLTISSLQSEDF AVYYCALWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 88 - SP34 humanized light chain with VL20 domain	EIWTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQQK PGQAFRGLIGGANKRAPGVPARFSGSLSGTEATLTISSLQSEDF AVYYCALWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 89 - SP34 humanized light chain withVL21 domain	EIWTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQEKP GQAFRGLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSEDF AVYYCALWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 90 - SP34 humanized light chain with VL22 domain	EIWTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQQK PGQAFRGLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSED FAVYYCALWYSNLWVFGQGTKLEIKRTVAAPSVFIFPPSDEQ LKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC
SEQ ID NO: 91 - scFv fragment humanized SP34 VH2-VL21 human IgGl Fc fusion	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNTLYL QMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTTVTV SSGGGGSGGGGSGGGGSEIVVTQSPATLSVSPGERATLSCRSS TGAVTTSNYANWVQEKPGQAFRGLIGGANKRAPGVPARFSG SLSGDEATLTISSLQSEDFAVYYCALWYSNLWVFGQGTKLEI KGGGGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW

135

	ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 92 - scFv fragment humanized SP34 VH3-VL23 human IgGl Fc fusion	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNTLYL QMNSLRAEDTAVYYCVRHGNFGNSYVSYFAYWGQGTTVTV SSGGGGSGGGGSGGGGSEIWTQSPATLSVSPGERATLSCRSS TGAVTTSNYANWVQEKPGQAFRGLIGGANKRAPGVPARFSG SLSGDEATLTISSLQSEDFAVYYCALFYSNLWVFGQGTKLEIK GGGGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 93 - scFv fragment humanized SP34 VH4-VL23 human IgGl Fc fusion	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNTLYL QMNSLRAEDTAVYYCVRHGNFGNSYVSFFAYWGQGTTVTV SSGGGGSGGGGSGGGGSEIVVTQSPATLSVSPGERATLSCRSS TGAVTTSNYANWVQEKPGQAFRGLIGGANKRAPGVPARFSG SLSGDEATLTISSLQSEDFAVYYCALFYSNLWVFGQGTKLEIK GGGGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 94 - scFv fragment humanized SP34 VH5-VL23 human IgGl Fc fusion	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSYFAYWGQGTTVT VSSGGGGSGGGGSGGGGSEIWTQSPATLSVSPGERATLSCRS STGAVTTSNYANWVQEKPGQAFRGLIGGANKRAPGVPARFS GSLSGDEATLTISSLQSEDFAVYYCALFYSNLWVFGQGTKLEI KGGGGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 95 - scFv fragment humanized SP34 VH1-VL27 human IgGl Fc fusion	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTTVT VSSGGGGSGGGGSGGGGSEIWTQSPATLSVSPGERATLSCRS STGAVTTSAAANWVQEKPGQAFRGLIGGANKRAPGVPARFS GSLSGDEATLTISSLQSEDFAVYYCALWYSNLWVFGQGTKLE IKGGGGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF

136

	SCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 96 - scFv fragment humanized SP34VH1-VL28 human IgGl Fc fusion	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTTVT VSSGGGGSGGGGSGGGGSEIWTQSPATLSVSPGERATLSCRS STGAVTTSNYANWVQEKPGQAFRGLIGGAAARAPGVPARFS GSLSGDEATLTISSLQSEDFAVYYCALWYSNLWVFGQGTKLE IKGGGGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 97 - scFv fragment humanized SP34 VH1-VL29 human IgGl Fc fusion	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTTVT VSSGGGGSGGGGSGGGGSEIWTQSPATLSVSPGERATLSCRS STGAVTTSNYANWVQEKPGQAFRGLIGGANKAAAGVPARFS GSLSGDEATLTISSLQSEDFAVYYCALWYSNLWVFGQGTKLE IKGGGGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 98 - scFv fragment humanized SP34 VH1-VL30 human IgGl Fc fusion	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTTVT VSSGGGGSGGGGSGGGGSEIWTQSPATLSVSPGERATLSCRS STGAVTTSNYANWVQEKPGQAFRGLIGGANKRAPGVPARFS GSLSGDEATLTISSLQSEDFAVYYCALWAANLWVFGQGTKLE IKGGGGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 99 - scFv fragment humanized SP34VH1-VL31 human IgGl Fc fusion	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTTVT VSSGGGGSGGGGSGGGGSEIWTQSPATLSVSPGERATLSCRS STGAVTTSNYANWVQEKPGQAFRGLIGGANKRAPGVPARFS GSLSGDEATLTISSLQSEDFAVYYCALWYSALWVFGQGTKLE IKGGGGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK

137

SEQ ID NO: 100 - scFv fragment humanized SP34 VH5-VL32 human IgGl Fc fusion	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSYFAYWGQGTTVT VSSGGGGSGGGGSGGGGSEIWTQSPATLSVSPGERATLSCRS STGAVTAANYANWVQEKPGQAFRGLIGGANKRAPGVPARFS GSLSGDEATLTISSLQSEDFAVYYCALFYSNLWVFGQGTKLEI KGGGGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK
SEQIDNO: 101 -SP34 humanized VH 1 domain	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTTVT VSS
SEQIDNO: 102-SP34 humanized VH2 domain	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNTLYL QMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTTVTV SS
SEQIDNO: 103 -SP34 humanized VH3 domain	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNTLYL QMNSLRAEDTAVYYCVRHGNFGNSYVSYFAYWGQGTTVTV SS
SEQIDNO: 104-SP34 humanized VH5 domain	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSYFAYWGQGTTVT VSS
SEQIDNO: 105 - SP34 humanized VL21 domain	EIWTQSPATLSVSPGERATLSCRSSTGAVTTSNYANWVQEKP GQAFRGLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSEDF AVYYCALWYSNLWVFGQGTKLEIK
SEQIDNO: 106-SP34 humanized VL32 domain	EIWTQSPATLSVSPGERATLSCRSSTGAVTAANYANWVQEK PGQAFRGLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSED FAVYYCALFYSNLWVFGQGTKLEIK
SEQIDNO: 107Humanized anti-HER2 antibody 4D5 - scFv fragment	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQM NSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGG GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVN TAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTL TISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK
SEQIDNO: 108 - Humanized anti-HER2 antibody 4D5 - FAB fragment heavy chain (VH-VHl)with VH:G65S substitution	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYTRYADSVKSRFTISADTSKNTAYLQMN SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
SEQIDNO: 109- Humanized anti-HER2 antibody 4D5 - scFv	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYTRYADSVKSRFTISADTSKNTAYLQMN SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGG

138

fragment with VH:G65S substitution	SGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNT AVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLT ISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK
SEQIDNO: 110Humanized anti-HER2 antibody 4D5 - FAB fragment heavy chain (VH-VHl)with VH:N82aS substitution	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMS SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
SEQIDNO: 111 Humanized anti-HER2 antibody 4D5 - scFv fragment with VH:N82aS substitution	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMS SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGG SGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNT AVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLT ISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK
SEQIDNO: 112 - OKTIO mouse VH domain	QVELVESGGSLKLSCAASGFDFSRSWMNWVRQAPGKGLEWI GEINPDSSTINYTTSLKDKFIISRDNAKNTLYLQMTKVRSEDTA LYYCARYGNWFPYWGQGTLVTVSS
SEQIDNO: 113 OKTIO mouse VL domain	DILMTQSQKIMPTSVGDRVSVTCKASQNVDTNVAWYQQKPG QSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTITNVQSEDLA EYFCQQYDSYPLTFGAGTKLDLKR
SEQIDNO: 114-HB-7 mouse VH domain	KVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGK GLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNS LQADDTAIYFCAKTLITTGYAMDYWGQGTTVTVSS
SEQIDNO: 115-HB-7 mouse VL domain	DIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNA PRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYY CQQYWSTPTFGGGTKLEIK
SEQIDNO: 116 - Humanized HB-7 bestfit VH domain	QVQLQESGPGLVKPSETLSLTCTVSGFSLISYGVHWVRQPPGK GLEWLGVIWRGGSTDYNAAFMSRLTISKDNSKNQVSLKLSSV TAADTAVYFCAKTLITTGYAMDYWGQGTLVTVSS
SEQIDNO: 117 - Humanized HB-7 bestfit VL domain	DIQLTQSPSSLSASVGDRVTITCRASEDIYNRLAWYQQKPGKA PKLLISGATSLETGVPSRFSGSGSGKDYTLTISSLQPEDFATYY CQQYWSTPTFGQGTKLEIK
SEQIDNO: 118 - Humanized HB-7 bestfit heavy chain	QVQLQESGPGLVKPSETLSLTCTVSGFSLISYGVHWVRQPPGK GLEWLGVIWRGGSTDYNAAFMSRLTISKDNSKNQVSLKLSSV TAADTAVYFCAKTLITTGYAMDYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQIDNO: 119 - Humanized HB-7 bestfit light chain	DIQLTQSPSSLSASVGDRVTITCRASEDIYNRLAWYQQKPGKA PKLLISGATSLETGVPSRFSGSGSGKDYTLTISSLQPEDFATYY CQQYWSTPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS WCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

139

SEQ ID NO: 120 - Chimeric HB-7 heavy chain IgGl	KVQLQESGPSLVQPSQRLSITCTVSGFSLISYGVHWVRQSPGK GLEWLGVIWRGGSTDYNAAFMSRLSITKDNSKSQVFFKMNS LQADDTAIYFCAKTLITTGYAMDYWGQGTTVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQIDNO: 121 - Chimeric HB-7 human kappa light chain	DIELTQSPSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGNA PRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYY CQQYWSTPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 122 - 9G7 mouse VH domain	QVTLKESGPGILQPSQTLSLTCSFSGLSLSTSGKGVGWIRQPSG KGLEWLAHIWWDDDKRYNPALKSRLTISKDTSSNQVFLKIAS VDTADTATYYCARIELGRSYVMDYWGQGTTVTVSS-
SEQ ID NO: 123 - 9G7 mouse VL domain	DIVMTQSHKFMSTSVGDRVSISCKASQDVITSVAWFQQKPGQ SPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVY YCQQHYTIPLTFGAGTKLELK
SEQ ID NO: 124 - Humanized 9G7 best-fit heavy chain	QVTLKESGPTLVKPTQTLTLTCTFSGLSLSTSGKGVGWIRQPP GKALEWLAHIWWDDDKRYNPALKSRLTITKDTSKNQWLT MTNMDPVDTATYYCARIELGRSYVMDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK-
SEQ ID NO: 125 Humanized 9G7 best-fit first prototype light chain	DIQMTQSPSSLSASVGDRVTITCQASQDVITSVAWFQQKPGK APKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYY CQQHYTIPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS WCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 126 - Chimeric 9G7 heavy chain IgGl	QVTLKESGPGILQPSQTLSLTCSFSGLSLSTSGKGVGWIRQPSG KGLEWLAHIWWDDDKRYNPALKSRLTISKDTSSNQVFLKIAS VDTADTATYYCARIELGRSYVMDYWGQGTTVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK

140

SEQ ID NO: 127 Chimeric 9G7 human kappa light chain	DIVMTQSHKFMSTSVGDRVSISCKASQDVITSVAWFQQKPGQ SPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVY YCQQHYTIPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGT ASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
SEQ ID NO: 128 Humanized 9G7 best-fit light chain (prototype light chain with F36Y substitution)	DIQMTQSPSSLSASVGDRVTITCQASQDVITSVAWYQQKPGK APKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYY CQQHYTIPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS WCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 129 Humanized 9G7 best-fit VH domain	QVTLKESGPTLVKPTQTLTLTCTFSGLSLSTSGKGVGWIRQPP GKALEWLAHIWWDDDKRYNPALKSRLTITKDTSKNQWLT MTNMDPVDTATYYCARIELGRSYVMDYWGQGTLVTVSS
SEQIDNO: 130 - Humanized 9G7 best-fit VL domain	DIQMTQSPSSLSASVGDRVTITCQASQDVITSVAWFQQKPGK APKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYY CQQHYTIPLTFGQGTKLEIK
SEQIDNO: 131 Humanized 9G7 bestframework heavy chain	EVQLVESGGGLVQPGGSLRLSCAFSGLSLSTSGKGVGWIRQA PGKGLEWLAHIW-WDDDKRYNPALKSRLTISKDTSKNTVYL QMNSLRAEDTAVYYCARIELGRSYVMDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 132 - Humanized 9G7 bestftamework light chain	DIQMTQSPSSLSASVGDRVTITCRASQDVITSVAWFQQKPGKA PKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQHYTIPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS WCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQIDNO: 133 Humanized 9G7 bestframework VH domain	EVQLVESGGGLVQPGGSLRLSCAFSGLSLSTSGKGVGWIRQA PGKGLEWLAHIW-WDDDKRYNPALKSRLTISKDTSKNTVYL QMNSLRAEDTAVYYCARIELGRSYVMDYWGQGTLVTVSS
SEQIDNO: 134Humanized 9G7 bestframework VL domain	DIQMTQSPSSLSASVGDRVTITCRASQDVITSVAWFQQKPGKA PKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQHYTIPLTFGQGTKVEIK
SEQIDNO: 135 - Human clone 767 VH domain	QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAP GKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQ MNSLRAEDTAVYYCAREGRTGYFDYWGQGTLVTVSS
SEQIDNO: 136 - Human clone 767 VL domain	QSVLTQPPSASGTPGQRVTISCSGSTSNIGTNYVYWYQQLPGT APKLLIYRNDQRPSGVPDRFSGSKSGTSASLAISGLRSEDEAD YYCAAWDDSRSGVYAFGTGTKVTVL
SEQIDNO: 137Human 767 heavy chain	QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAP GKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQ MNSLRAEDTAVYYCAREGRTGYFDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT

141

	SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQIDNO: 138Human 767 light chain	QSVLTQPPSASGTPGQRVTISCSGSTSNIGTNYVYWYQQLPGT APKLLIYRNDQRPSGVPDRFSGSKSGTSASLAISGLRSEDEAD YYCAAWDDSRSGVYAFGTGTKVTVLRTVAAPSVFIFPPSDEQ LKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC
SEQIDNO: 139 Mouse anti-human 0X40 antibody VH domain from W02013/008171	QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSG KGLEWLAHIWWDDDKYYNTALKSGLTISKDTSKNQVFLKIA SVDTTDTATYYCARIDWDGFAYWGQGTLVTVSS
SEQ ID NO: 140 Mouse anti-human 0X40 antibody VL domain from W02013/008171	QIVLTQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSP KPWIYATSNLASGVPARFSGSGSGTSYSLTINRVEAEDAATYY CQQWSSNPWTFGGGTKLEIK
SEQ ID NO: 141 Humanized anti-human 0X40 antibody VH domain from W02013/008171	QVTLKESGPALVKPTQTLTLTCSFSGFSLSTSGMGVGWIRQPP GKALEWIAHIWWDDDKYYNTALKTRLTISKDTSKNQWLTM TNMDPVDTATYYCARIDWDGFAYWGQGTLVTVSS
SEQ ID NO: 142 Humanized anti-human 0X40 antibody VL domain from W02013/008171	EIVLTQSPATLSLSPGERATLSCRASSSVSYMHWYQQKPGQAP RPWIYATSNRATGIPARFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPWTFGQGTKVEIK
SEQIDNO: 143 rituximab mouse VH domain	QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQT PGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYM QLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA
SEQ ID NO: 144 rituximab mouse VL domain	QIVLSQSPAILSASPGEKVTMETTCRASSSVSYIHWFQQKPGSS PKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATY YCQQWTSNPPTFGGGTKLEIK
SEQ ID NO: 145 cetuximab mouse VH domain	QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPG KGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNS LQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA
SEQ ID NO: 146 cetuximab mouse VL domain	DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSP RLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQ QNNNWPTTFGAGTKLELK
SEQ ID NO: 147 - BTA CH3 NO: 1 Original BTA 11	GQPREPQVYTLPPSRDELTKNQVKLVCLVTGFYPSDIAVEWE SNGQPENNYYTTPPVLDSDGSFSLVSWLNVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK~~~~~~--------~~~~~~~~

142


SEQ ID NO: 148 - BTA CH3 NO: 2 BTA FTO 11	GQPREPAVYTLPPSRDELTKNQVKLVCLVTGFYPSDIAVEWE SNGQPENNYYTTPPVLDSDGSFSLVSWLNVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK---——-----
SEQ ID NO: 149 - BTA CH3 NO: 3 BTA FTO 33 411D	GQPREPAVYTLPPSREEMTKNQVKLVCLVTGFYPSDIAVEWE SSGQPENNYYTTPPMLDSDGSFSLVSWLDVDKSRWQQGNIFS CSVMHEALHNRFTQKSLSLSPGK—---———
SEQIDNO: 150-BTB CH3 NO: 1 Original BTB	GQPREPEVATFPPSRDELTKNQVTLVCLVTGFYPSDIAVEWES NGQPENNYKTDPPLLESDGSFALSSRLRVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK~~~~~~~~~~~~~~~~~~~~~---
SEQID NO: 151 - BTB CH3 NO: 2 BTB 401R 11	GQPREPEVATFPPSRDELTKNQVTLVCLVTGFYPSDIAVEWES NGQPENNYKTDPPLLESRGSFALSSRLRVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK~~~——
SEQIDNO: 152-BTB CH3 No: 3 BTB 401Q 11	GQPREPEVATFPPSRDELTKNQVTLVCLVTGFYPSDIAVEWES NGQPENNYKTDPPLLESQGSFALSSRLRVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK~~~~~~~~~~~~~~~~~~~~~~~~
SEQID NO: 153 - BTA CH3 NO: 4 BTA 11 FTO N411T	GQPREPAVYTLPPSRDELTKNQVKLVCLVTGFYPSDIAVEWE SNGQPENNYYTTPPVLDSDGSFSLVSWLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK
SEQIDNO: 154-BTA CH3 NO: 5 BTA 33 FTO	GQPREPAVYTLPPSREEMTKNQVKLVCLVTGFYPSDIAVEWE SSGQPENNYYTTPPMLDSDGSFSLVSWLNVDKSRWQQGNIFS CSVMHEALHNRFTQKSLSLSPGK
SEQIDNO: 155-BTA CH3 NO: 6 BTA 33 FTO N411T	GQPREPAVYTLPPSREEMTKNQVKLVCLVTGFYPSDIAVEWE SSGQPENNYYTTPPMLDSDGSFSLVSWLTVDKSRWQQGNIFS CSVMHEALHNRFTQKSLSLSPGK
SEQIDNO: 156-BTB CH3 No: 4 BTB 33 D401Q	GQPREPEVATFPPSREEMTKNQVTLVCLVTGFYPSDIAVEWES SGQPENNYNTDPPLLESQGSFALSSRLRVDKSRWQQGNIFSCS VMHEALHNRFTQKSLSLSPGK
SEQIDNO: 157-BTB CH3 No: 5 BTB 11 D401Q R411T	GQPREPEVATFPPSRDELTKNQVTLVCLVTGFYPSDIAVEWES NGQPENNYKTDPPLLESQGSFALSSRLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK
SEQIDNO: 158-BTB CH3 No: 6 BTB	GQPREPEVATFPPSREEMTKNQVTLVCLVTGFYPSDIAVEWES SGQPENNYNTDPPLLESQGSFALSSRLTVDKSRWQQGNIFSCS

143

33 D401Q R411T	VMHEALHNRFTQKSLSLSPGK
SEQIDNO: 159BEAT HER2/CD3-1 antibody FAB heavy chain (CD3 epsilon arm - humanized 0KT3 with N82aS substitution)	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP GKGLEWIGYINPSRGYTYYADSVKGRFTLSTDKSKNTAYLQ MSSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCWVDVSHEDPEVQFKWYVDGVEVHNAKT KPREEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGFY PSDIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDKS RWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK
SEQIDNO: 160- BEAT HER2/CD3-1 antibody scFv heavy chain (HER2 arm)	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQM NSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGG GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVN TAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTL TISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRGGGGTDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPEVA TFPPSRDELTKNQVTLVCLVTGFYPSDIAVEWESNGQPENNY KTDPPLLESDGSFALSSRLRVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK
SEQIDNO: 161 BEAT HER2/CD3-2 antibody FAB heavy chain (HER2 arm)	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQM NSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAST NGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPAVYTLPPSRDELTKNQVKLVCLVTGFY PSDIAVEWESNGQPENNYYTTPPVLDSDGSFSLVSWLNVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQIDNO: 162BEAT HER2/CD3-2 antibody scFv heavy chain (CD3 epsilon arm - humanized OKT3 with N82aS substitution)	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP GKGLEWIGYINPSRGYTYYADSVKGRFTLSTDKSKNTAYLQ MSSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSGGG GSGGGGSGGGGSGGGASDIQLTQSPSSLSASVGDRVTITCRAS SSVSYVAWYQQKPGKAPKRWIYDTSKLYSGVPSRFSGSGSGT DYTLTISSLQPEDFATYYCQQWSSNPPTFGQGTKVEIKGGGGT DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCW VDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRWS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREP EVATFPPSREEMTKNQVTLVCLVTGFYPSDIAVEWESSGQPE NNYNTDPPLLESDGSFALSSRLRVDKSRWQQGNIFSCSVMHE ALHNRFTQKSLSLSPGK
SEQIDNO: 163 BEAT HER2/CD3-3	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP GKGLEWIGYINPSRGYTYYADSVKSRFTLSTDKSKNTAYLQM

144

antibody FAB heavy chain(CD3 epsilon arm humanized OKT3 with G65S substitution)	NSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTK PREEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGFYPS DIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDKSR WQQGNIFSCSVMHEALHNRFTQKSLSLSPGK
SEQIDNO: 164BEAT HER2/CD3-3 antibody scFv heavy chain (HER2 arm with additional disulfide bond)	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KCLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQM NSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGG GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVN TAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTL TISSLQPEDFATYYCQQHYTTPPTFGCGTKVEIKGGGGTDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPEVAT FPPSRDELTKNQVTLVCLVTGFYPSDIAVEWESNGQPENNYK TDPPLLESQGSFALSSRLRVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK
SEQIDNO: 165 - BEAT HER2/CD3(SP34) antibody FAB heavy chain(CD3 epsilon arm humanized SP34 VH from W02008/119565)	EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPAVYTLPPSRDELTKNQVKLVC LVTGFYPSDIAVEWESNGQPENNYYTTPPVLDSDGSFSLVSW LNVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQIDNO: 166- BEAT HER2/CD3(SP34) antibody FAB light chain(CD3 epsilon arm humanized SP34 VL from W02008/119565)	QTWTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKP GQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNRWVFGGGTKLTVLGRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC
SEQIDNO: 167 - BEAT HER2/CD3(SP34) antibody scFv heavy chain (HER2 arm with N82aS substitution)	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMS SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGG SGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNT AVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLT ISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGTDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSH EDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRWSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPEVATF PPSREEMTKNQVTLVCLVTGFYPSDIAVEWESSGQPENNYNT

145

	DPPLLESQGSFALSSRLRVDKSRWQQGNIFSCSVMHEALHNR FTQKSLSLSPGK
SEQIDNO: 168 - BEAT HER2/CD3(SP34Kappal) antibody FAB heavy chain(CD3 epsilon arm - humanized SP34VH1)	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTTVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPAVYTLPPSRDELTKNQVKLVC LVTGFYPSDIAVEWESNGQPENNYYTTPPVLDSDGSFSLVSW LNVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQIDNO: 169 - BEAT CD38HB7bestfit/CD3 antibody FAB heavy chain (CD38 arm humanized HB-7 bestfit)	QVQLQESGPGLVKPSETLSLTCTVSGFSLISYGVHWVRQPPGK GLEWLGVIWRGGSTDYNAAFMSRLTISKDNSKNQVSLKLSSV TAADTAVYFCAKTLITTGYAMDYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPAVYTLPPSRDELTKNQVKLVCLVTGFYPSD IAVEWESNGQPENNYYTTPPVLDSDGSFSLVSWLNVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQIDNO: 170BEAT CD38-767/CD3 antibody FAB heavy chain (CD38 arm human clone 767)	QVQLQESGPGLVKPSETLSLTCTVSGFSLISYGVHWVRQPPGK GLEWLGVIWRGGSTDYNAAFMSRLTISKDNSKNQVSLKLSSV TAADTAVYFCAKTLITTGYAMDYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTKPR EEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGFYPS DIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDKSR WQQGNIFSCSVMHEALHNRFTQKSLSLSPGK
SEQ ID NO: 171 BEAT CD38-767/CD3 antibody scFv heavy chain(CD3 epsilon arm humanized OKT3 with G65S substitution)	EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVT VSSGGGGSGGGGSGGGGSQTWTQEPSLTVSPGGTVTLTCGS STGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSG SLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLT VLGGGGGTDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISR TPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPEVATFPPSRDELTKNQVTLVCLVTGFYPSDIAVE WESNGQPENNYKTDPPLLESQGSFALSSRLRVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK
SEQIDNO: 172- BEAT OX40/CD3	QVTLKESGPALVKPTQTLTLTCSFSGFSLSTSGMGVGWIRQPP GKALEWIAHIWWDDDKYYNTALKTRLTISKDTSKNQWLTM

146

antibody FAB heavy chain (0X40 arm with humanized anti-human 0X40 VH domain from W02013/008171)	TNMDPVDTATYYCARIDWDGFAYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPAVYTLPPSRDELTKNQVKLVCLVTGFYPSD IAVEWESNGQPENNYYTTPPVLDSDGSFSLVSWLNVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQIDNO: 173 - BEAT OX40/CD3 antibody FAB light chain (0X40 arm with humanized anti-human 0X40 VL domain from W02013/008171)	EIVLTQSPATLSLSPGERATLSCRASSSVSYMHWYQQKPGQAP RPWIYATSNRATGIPARFSGSGSGTDYTLTISSLEPEDFAVYYC QQWSSNPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS WCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQIDNO: 174BEAT EGFR/CD3 antibody FAB heavy chain (EGFR arm with mouse Erbitux VH domain)	QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPG KGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNS LQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPAVYTLPPSRDELTKNQVKLVCLVTGFYPSD IAVEWESNGQPENNYYTTPPVLDSDGSFSLVSWLNVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQIDNO: 175 BEAT EGFR/CD3 antibody FAB light chain (EGFR arm with mouse Erbitux VL domain)	DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSP RLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQ QNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQIDNO: 176BEATCD38HB7bestfit/CD3(SP34)a ntibody FAB heavy chain (CD38 arm humanized HB-7 bestfit)	QVQLQESGPGLVKPSETLSLTCTVSGFSLISYGVHWVRQPPGK GLEWLGVIWRGGSTDYNAAFMSRLTISKDNSKNQVSLKLSSV TAADTAVYFCAKTLITTGYAMDYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTKPR EEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGFYPS DIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDKSR WQQGNIFSCSVMHEALHNRFTQKSLSLSPGK
SEQIDNO: 177BESTCD38HB7bestfit/CD3(SP34) antibody scFv heavy chain (CD3 arm -	EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVT VSSGGGGSGGGGSGGGGSQTWTQEPSLTVSPGGTVTLTCGS STGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSG

147

humanized SP34 VH/VL domains from W02008/119565)	SLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLT VLGGGGGTDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISR TPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPEVATFPPSRDELTKNQVTLVCLVTGFYPSDIAVE WESNGQPENNYKTDPPLLESQGSFALSSRLRVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK
SEQIDNO: 178BEAT CD389G7bestfit/CD3(SP34)a ntibody FAB heavy chain (CD38 arm humanized 9G7 best-fit VH)	QVTLKESGPTLVKPTQTLTLTCTFSGLSLSTSGKGVGWIRQPP GKALEWLAHIWWDDDKRYNPALKSRLTITKDTSKNQWLT MTNMDPVDTATYYCARIELGRSYVMDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAK TKPREEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGF YPSDIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDK SRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK
SEQ ID NO: 179 - BEAT CD389G7bestfit/CD3(SP34Kappa2) antibody scFv heavy chain (CD3 arm humanized SP34 VH5/VL32)	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTTVT VSSGGGGSGGGGSGGGGSEIWTQSPATLSVSPGERATLSCRS STGAVTTSNYANWVQEKPGQAFRGLIGGANKRAPGVPARFS GSLSGDXATLTISSLQSEDFAVYYCALWYSNLWVFGQGTKLE IKGGGGTDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPEVATFPPSRDELTKNQVTLVCLVTGFYPSDLAVE WESNGQPENNYKTDPPLLESQGSFALSSRLRVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK
SEQIDNO: 180 - BEAT CD20/CD3 antibody FAB heavy chain	QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQT PGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYM QLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAK TKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGF YPSDIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDK SRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK
SEQIDNO: 181 BEAT CD20/CD3 antibody FAB light chain	QIVLSQSPAILSASPGEKVTMETTCRASSSVSYIHWFQQKPGSS PKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATY YCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGT ASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
SEQIDNO: 182Humanized SP34 VH	QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQT PGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYM

148

domain from W02008/119565	QLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA
SEQIDNO: 183 Humanized SP34 VL domain from W02008/119565	QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQT PGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYM QLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA
SEQIDNO: 184 Human CD3 gamma extracellular région	QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIG FLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRM CQNCIELNAATIS
SEQIDNO: 185 Human CD3 epsilon extracellular région	DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKN IGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDA NFYLYLRARVCENCMEMD
SEQIDNO: 186-26residue peptide linker	GSADDAKKDAAKKDDAKKDDAKKDGS
SEQIDNO: 187 Human CD3 gammaepsilon-Fc fusion protein	QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIG FLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRM GSADDAKKDAAKKDDAKKDDAKKDGSQDGNEEMGGITQTP YKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSD EDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVGG GGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR WSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG
SEQIDNO: 188Human CD3 epsilon 126 amino acid sequence	QDGNEEMGGITQTPYKVSISGTTVIL
SEQIDNO: 189 - Cynomolgus monkey CD3 epsilon 1-26 amino acid sequence	QDGNEEMGSITQTPYQVSISGTTVIL
SEQIDNO: 190Human CD3 epsilon 126 Fc fusion	QDGNEEMGGITQTPYKVSISGTTVILGGGGTDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK
SEQIDNO: 191 Cynomolgus monkey CD3 epsilon 1-26 Fc fusion	QDGNEEMGSITQTPYQVSISGTTVILGGGGTDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK
SEQIDNO: 192- Human CD38 extracellular région	VPRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQ SVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLW SRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSK

149

fused to a polyhistine tag - amino acid sequence	INYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDWHVML NGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDS RDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSS CTSEIHHHHHH
SEQIDNO: 193 Cynomolgus monkey CD38 extracellular région fused to a polyhistine tag - amino acid sequence	VPRWRQQWSGSGTTSRFPETVLARCVKYTEVHPEMRHVDCQ SVWDAFKGAFISKYPCNITEEDYQPLVKLGTQTVPCNKTLLW SRIKDLAHQFTQVQRDMFTLEDMLLGYLADDLTWCGEFNTF EINYQSCPDWRKDCSNNPVSVFWKTVSRRFAETACGWHVM LNGSRSKIFDKNSTFGSVEVHNLQPEKVQALEAWVIHGGRED SRDLCQDPTIKELESIISKRNIRFFCKNIYRPDKFLQCVKNPEDS SCLSGIHHHHHH
SEQID NO: 194- Mouse anti- human CD3 epsilon OKT3 CDR H1	GYTFTRYT
SEQIDNO: 195 - Mouse anti- human CD3 epsilon OKT3 CDR H2	INPSRGYT
SEQIDNO: 196 - Mouse anti- human CD3 epsilon OKT3 CDR H3	ARYYDDHYCLDY
SEQID NO: 197 - Mouse anti- human CD3 epsilon OKT3 CDR Ll	SSVSY
SEQIDNO: 198 - Mouse anti- human CD3 epsilon OKT3 CDR L2	DTS
SEQID NO: 199 - Mouse anti- human CD3 epsilon OKT3 CDR L3	QQWSSNPPT
SEQ ID NO: 200 Mouse anti- human CD3 epsilon SP34 CDRH1	GFTFNTYA
SEQ ID NO: 201 - Mouse anti- human CD3 epsilon SP34 CDRH2	IRSKYNNYAT
SEQ ID NO: 202 Mouse anti- human CD3 epsilon SP34 CDRH3	VRHGNFGNSYVSWFAY
SEQ ED NO: 203 Mouse anti- human CD3 epsilon SP34 CDR Ll	TGAVTTSNY
SEQ ID NO: 204 Mouse anti- human CD3 epsilon SP34 CDR L2	GTN
SEQ ED NO: 205 Mouse anti- human CD3 epsilon SP34 CDR L3	ALWYSNLWV
SEQ ID NO: 206 - Herceptin	GFNIKDTY

150

(trastuzumab) CDR H1

SEQ ID NO: 207 Herceptin (trastuzumab)

IYPTNGYT

CDRH2

SEQ ID NO: 208 Herceptin (trastuzumab)

SRWGGDGFYAMDY

CDRH3

SEQ ID NO: 209 Herceptin (trastuzumab)

QDVNTA

CDR L1

SEQIDNO: 210Herceptin (trastuzumab)

SAS

CDRL2

SEQIDNO: 211 Herceptin (trastuzumab)

QQHYTTPPT

CDRL3

SEQ ID NO: 212 Mouse anti- human

GFSLISYG

CD38 HB-7 CDR H1

SEQ ID NO: 213 Mouse anti- human

IWRGGST

CD38 HB-7 CDR H2

SEQ ID NO: 214 Mouse anti- human

AKTLITTGYAMDY

CD38HB-7 CDR H3

SEQIDNO: 215Mouse anti- human

EDIYNR

CD38 HB-7 CDR L1

SEQIDNO: 216Mouse anti- human

GAT

CD38 HB-7 CDR L2

SEQIDNO: 217Mouse anti- human

QQYWSTPT

CD38 HB-7 CDR L3

SEQIDNO: 218Mouse anti- human

GFDFSRSW

CD38 OKTIOCDRHl

SEQIDNO: 219Mouse anti- human

INPDSSTI

CD38 OKTIO CDRH2

SEQ ID NO: 220 Mouse anti- human

ARYGNWFPY

CD38 ΘΚΤ10 CDRH3

SEQ ID NO: 221 Mouse anti- human

QNVDTN

CD38 OKTIO CDR L1

SEQ ID NO: 222 Mouse anti- human

SAS

151

CD38 OKTIO CDR L2

SEQ ID NO: 223 Mouse anti- human

QQYDSYPLTFGAGTK

CD38 OKTIO CDRL3

SEQ ID NO: 224 Mouse anti- human

GLSLSTSGKG

CD38 9G7CDRH1

SEQ ID NO: 225 Mouse anti- human

IWWDDDK

CD38 9G7 CDR H2

SEQ ID NO: 226 Mouse anti- human

ARIELGRSYVMDY

CD38 9G7CDRH3

SEQ ID NO: 227 Mouse anti- human

QDVITS

CD38 9G7 CDR L1

SEQ ID NO: 228 Mouse anti- human

SAS

CD38 9G7 CDR L2

SEQ ID NO: 229 Mouse anti- human

QQHYTIPLT

CD38 9G7 CDR L3

SEQ ID NO: 230 Human anti- human

GFTFSSYW

CD38 767 CDRH1

SEQIDNO: 231 Human anti- human

IKQDGSEK

CD38767 CDR H2

SEQ ID NO: 232 Human anti- human

AREGRTGYFDY

CD38767 CDR H3

SEQ ID NO: 233 Human anti- human

TSNIGTNY

CD3 8767 CDR L1

SEQ ID NO: 234 Human anti- human

RND

CD38767 CDR L2

SEQIDNO: 235 Human anti- human

AAWDDSRSGVYA

CD38767 CDR L3

SEQ ID NO: 236 Mouse anti-human

GFSLSTSGMG

0X40 CDR HI from

W02013/008171

SEQ ID NO: 237 Mouse anti-human

IWWDDDK

0X40 CDR H2 from

W02013/008171

	152
SEQ ID NO: 238 Mouse anti-human	ARIDWDGFAY

0X40 CDR H3 from

W02013/008171

SEQ ID NO: 239 Mouse anti-human

SSVSY

0X40 CDR L1 from

W02013/008171

SEQ ID NO: 240 Mouse anti-human

ATS

0X40 CDR L2 from

W02013/008171

SEQ ID NO: 241 Mouse anti-human

QQWSSNPWT

0X40 CDR L3 from

W02013/008171

SEQ ID NO: 242 Rituxan (rituximab)

GYTFTSYN

CDRH1

SEQ ID NO: 243 Rituxan (rituximab)

IYPGNGDT

CDRH2

SEQ ID NO: 244 Rituxan (rituximab)

ARSTYYGGDWYFNV

CDRH3

SEQ ID NO: 245 Rituxan (rituximab)

ASSSVSY

CDR L1

SEQ ID NO: 246 Rituxan (rituximab)

ATS

CDRL2

SEQ LD NO: 247 Rituxan (rituximab)

QQWTSNPPT

CDRL3

SEQ ID NO: 248 Erbitux (cetuximab)

GFSLTNYG

CDRH1

SEQ ID NO: 249 Erbitux (cetuximab)

IWSGGNT

CDRH2

SEQ ID NO: 250 Erbitux (cetuximab)

ARALTYYDYEFAY

CDRH3

SEQ ID NO: 251 Erbitux (cetuximab)

QSIGTN

CDR L1

SEQ ED NO: 252 Erbitux (cetuximab)

YAS

CDRL2

	153
SEQ ID NO: 253 Erbitux (cetuximab)	QQNNNWPTT

CDRL3

SEQ ID NO: 254 Vectibix (panitumumab)

GGSVSSGDYY

CDRH1

SEQ ID NO: 255 Vectibix (panitumumab)

IYYSGNT

CDRH2

SEQ ID NO: 256 Vectibix (panitumumab)

VRDRVTGAFDI

CDRH3

SEQ ID NO: 257 Vectibix (panitumumab)

QDISNY

CDR L1

SEQ ID NO: 258 Vectibix (panitumumab)

DAS

CDRL2

SEQ ID NO: 259 Vectibix (panitumumab) CDRL3	QHFDHLPLA
SEQ ID NO: 260 Mouse anti-human	GVSLPDYG

CD19CDRH1 from

W02010/095031

SEQ ID NO: 261 Mouse anti-human

IWGSETT

CD 19 CDR H2 from

W02010/095031

SEQ ID NO: 262 Mouse anti-human

AKHYYYGGSYAMDY

CD 19 CDR H3 from

WO2010/095031

SEQ ID NO: 263 Mouse anti-human

QDISKY

CD19 CDR L1 from

WO2010/095031

SEQ ID NO: 264 Mouse anti-human

HTS

CD 19 CDR L2 from

WO2010/095031

SEQ ID NO: 265 Mouse anti-human

QQGATLPYT

CD19 CDR L3 from

WO2010/095031

SEQ ID NO: 266 - Bswl7 CDR H1	GFTFSSYA
SEQ ID NO: 267 - Bswl7CDRH2	ISSGNII

154

SEQ ID NO: 268 - Bswl7CDR H3	TRGRSTYGGFDH
SEQ ID NO: 269 - Bswl7 CDR L1	SSVTF
SEQ ID NO: 270 - Bswl7CDRL2	DTS
SEQIDNO: 271 - Bswl7 CDR L3	QHWSGNPLT
SEQ ID NO: 272 Omalizumab CDR H1	GYSITSGYS
SEQ ID NO: 273 - Omalizumab CDR H2	ITYDGST
SEQ ID NO: 274 Omalizumab CDR H3	ARGSHYFGHWHFAV
SEQ ID NO: 275 Omalizumab CDR L1	QSVDYDGDSY
SEQ ID NO: 276 - Omalizumab CDR L2	AAS
SEQ ID NO: 277 - Omalizumab CDR L3	QQSHEDPYT
SEQ ID NO: 278 Humanized antiOX40/mingraft VH domain	EVQLVESGGGLVQPGGSLRLSCAASGFSLSTSGMGMSWVRQ APGKGLEWVSAIWWDDDKYYADSVKGRFTISRDNSKNTLYL QMNSLRAEDTAVYYCARIDWDGFAYWGQGTLVTVSS
SEQ ID NO: 279 Humanized antiOX40/maxgraft VH domain	EVQLVESGGGLVQPGGSLRLSCAFSGFSLSTSGMGVGWIRQA PGKGLEWLAHIWWDDDKYYNTALKSGLTISKDTSKNTVYLQ MNSLRAEDTAVYYCARIDWDGFAYWGQGTLVTVSS
SEQ ID NO: 280 Humanized antiOX40/mingrafit VL domain	DIQMTQSPSSLSASVGDRVTITCRASSSVSYLNWYQQKPGKA PKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQWSSNPWTFGQGTKVEIK
SEQIDNO: 281 Humanized antiOX40/maxgraft VL domain	DIQLTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKA PKPWIYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYY CQQWSSNPWTFGQGTKVEIK
SEQ ID NO: 282 Humanized Rituximab/mingraft VH domain	EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMSWVRQAP GKGLEWVSAIYPGNGDTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSS
SEQ ID NO: 283 - Humanized Rituximab/maxgraft VN domain	EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMSWVRQAP GKGLEWIGAIYPGNGDTYYADSVKGRATLSADKSKNTAYLQ MNSLRAEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSS
SEQ ID NO: 284 Humanized Rituximab/mingraft VL domain	DIQMTQSPSSLSASVGDRVTITCRASASSSVSYLNWYQQKPG KAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQWTSNPPTFGQGTKVEIK

155

SEQ ID NO: 285 Humanized Rituximab/maxgraft VL domain	DIQLTQSPSSLSASVGDRVTITCRLSASSSVSYLNWFQQKPGK APKPWIYATSSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATY YCQQWTSNPPTFGQGTKVEIK
SEQ ID NO: 286 Humanized Erbitux/mingraft VH domain	EVQLVESGGGLVQPGGSLRLSCAASGFSLTNYGMSWVRQAP GKGLEWVSAIWSGGNTYYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCARALTYYDYEFAYWGQGTLVTVSS
SEQ ID NO: 287 Humanized Erbitux/maxgraft VH domain	EVQLVESGGGLVQPGGSLRLSCAASGFSLTNYGVHWVRQAP GKGLEWLGAIWSGGNTDYNTPFTGRLTISKDNSKNTLYLQM NSLRAEDTAVYYCARALTYYDYEFAYWGQGTLVTVSS
SEQ ID NO: 288 Humanized Erbitux/mingraft VL domain	DIQMTQSPSSLSASVGDRVTITCRASQSIGTNLNWYQQKPGK APKLLIYYASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQNNNWPTTFGQGTKVEIK
SEQ JD NO: 289 Humanized Erbitux/maxgraft VL domain	DIQLTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKA PKLLIKYASESISGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQNNNWPTTFGQGTKVEIK
SEQ ID NO: 290 Vectibix VH domain	QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQS PGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSS VTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS
SEQ ID NO: 291 Vectibix VL domain	DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGK APKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYF CQHFDHLPLAFGGGTKVEIK
SEQ ID NO: 292 Humanized Vectibix/mingraft VH domain	EVQLVESGGGLVQPGGSLRLSCAASGGSVSSGDYYMSWVRQ APGKGLEWVSAIYYSGNTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCVRDRVTGAFDIWGQGTLVTVSS
SEQ ID NO: 293 Humanized Vectibix/maxgraft VH domain	EVQLVESGGGLVQPGGSLRLSCAVSGGSVSSGDYYMSWVRQ APGKGLEWIGAIYYSGNTYYADSVKGRLTISIDTSKNTFYLQ MNSLRAEDTAVYYCVRDRVTGAFDIWGQGTLVTVSS
SEQ LD NO: 294 Humanized Vectibix/mingraft VL domain	DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGK APKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQHFDHLPLAFGQGTKVEIK
SEQ ID NO: 295 Humanized Vectibix/maxgraft VL domain	DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGK APKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYF CQHFDHLPLAFGGGTKVEIK
SEQ ID NO: 296 - antihuman CD 19 VH domain from WO2010/095031	QVQLVQSGGGWQPGRSLRLSCAASGVSLPDYGVSWVRQAP GKGLEWVAVIWGSETTYYNSALKSRFTISRDNSKNTLYLQM NSLRAEDTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS
SEQ ID NO: 297 - antihuman CD 19 VL domain from	DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGK AIKLLIYHTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQGATLPYTFGPGTKVDIK

156

WO2010/095031
SEQ ID NO: 298 - Omalizumab VH domain	EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAP GKGLEWVASITYDGSTNYADSVKGRFTISRDDSKNTFYLQMN SLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSS
SEQ ID NO: 299 Omalizumab VL domain	DIQLTQSPSSLSASVGDRVTITCRASQSVDYDGDSYMNWYQQ KPGKAPKLLIYAASYLESGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQSHEDPYTFGQGTKVEIK
SEQ ID NO: 300 Stabilized Omalizumab/maxgraft VH domain	EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAP GKGLEWVASITYDGSTNYADSVKGRFTISRDDSKNTFYLQMN SLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSS
SEQ ID NO: 301 Stabilized Omalizumab/mingraft VH domain	EVQLVESGGGLVQPGGSLRLSCAASGYSITSGYSMSWVRQAP GKGLEWVSAITYDGSTYYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSS
SEQ ID NO: 302 Stabilized Omalizumab/maxgraft VL domain	DIQLTQSPSSLSASVGDRVTITCRASQSVDYDGDSYMNWYQQ KPGKAPKLLIYAASYLESGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQSHEDPYTFGQGTKVEIK
SEQ ID NO: 303 Stabilized Omalizumab/mingraft VL domain	DIQMTQSPSSLSASVGDRVTITCRASQSVDYDGDSYLNWYQQ KPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQSHEDPYTFGQGTKVEIK
SEQ JD NO: 304- Bswl7 mouse VH domain	EVQLLESGGGFVKPGGSLKLSCWSGFTFSSYAMSWVRQTPE KRLEWVASISSGNIIYYPDNVKGRFTISRDNVRNILYLQMSSL RSEDTAMYYCTRGRSTYGGFDHWGQGTTLTVSS
SEQ ID NO: 305 - Bswl7 mouse VL domain	ELVMTQSPAIMSASPGEKVTMTCSASSSVTFIHWYRQKSGTSP KGWIYDTSKLASGVPARFSGSGSGTSYSLTISTMEAEDAATY YCQHWSGNPLTFGTGTKLELK
SEQ ID NO: 306 Humanized Bswl7/mingraft VH domain	EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISSGNIIYYADSVKGRFTISRDNSKNTLYLQMNS LRAEDTAVYYCTRGRSTYGGFDHWGQGTLVTVSS
SEQ ID NO: 307 Humanized Bswl7/maxgraft VH domain	EVQLVESGGGLVKPGGSLRLSCAVSGFTFSSYAMSWVRQAP GKGLEWVASISSGNIIYYPDNVKGRFTISRDNAKNSLYLQMNS LRAEDTAVYYCTRGRSTYGGFDHWGQGTTVTVSS
SEQ ID NO: 308 Humanized Bswl7/mingraft VL domain	DIQMTQSPSSLSASVGDRVTITCRASSSVTFLNWYQQKPGKAP KLLIYDTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QHWSGNPLTFGQGTKVEIK
SEQ ID NO: 309 Humanized Bswl7/maxgraft VL domain	DLQMTQSPSSLSASVGDRVTITCSASSSVTFLNWYQQKPGKA PWLLIYDTSSLQSGVPSRFSGSGSGTDYTLTISSMQPEDFATYY CQHWSGNPLTFGQGTKVEIK
SEQIDNO: 310- BEAT HER2/CD3(SP34- Kappa2) antibody FAB	EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPG KGLEWVARIYPTNGYTRYADSVKSRFTISADTSKNTAYLQMN SLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT

157

heavy chain (anti-HER2 FAB arm with G65S substitution BT33 LALA)	SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTK PREEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGFYPS DIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDKSR WQQGNIFSCSVMHEALHNRFTQKSLSLSPG
SEQIDNO: 311 BEAT antibody scFv heavy chain SP34Kappa2(anti-CD3 epsilon arm - humanized SP34VH5/VL32 BT11 LALA)	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSYFAYWGQGTTVT VSSGGGGSGGGGSGGGGSEIWTQSPATLSVSPGERATLSCRS STGAVTAANYANWVQEKPGQAFRGLIGGANKRAPGVPARFS GSLSGDEATLTISSLQSEDFAVYYCALFYSNLWVFGQGTKLEI KGGGGTDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPEVATFPPSRDELTKNQVTLVCLVTGFYPSDIAVEWE SNGQPENNYKTDPPLLESQGSFALSSRLRVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPG
SEQIDNO: 312BEAT CD389G7bestframwork/CD3 ( SP34-Kappa2) antibody FAB heavy chain (antiCD38 FAB arm with G65S substitution BT33 LALA)	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTTVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCWVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCL VTGFYPSDIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWL NVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG
SEQIDNO: 313BEAT CD38767/CD3(SP34-Kappa2) antibody FAB heavy chain (anti-CD38 FAB arm with G65S substitution BT33 LALA)	EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSN KALP AP IEKTISKTKGQPREP AVYTLPPSREEMTKNQVKLVCL VTGFYPSDLAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWL NVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG
SEQIDNO: 314- BEAT OX40maxgraft/CD3(SP 34-Kappa2) antibody FAB heavy chain (anti0X40 maxgraft FAB arm with G65S substitution BT33	EVQLVESGGGLVQPGGSLRLSCAFSGFSLSTSGMGVGWIRQA PGKGLEWLAHIWWDDDKYYNTALKSGLTISKDTSKNTVYLQ MNSLRAEDTAVYYCARIDWDGFAYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTKPR EEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE

158

LALA)	KTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGFYPS DIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDKSR WQQGNIFSCSVMHEALHNRFTQKSLSLSPG
SEQIDNO: 315- BEAT OX40maxgraft/CD3(SP 34-Kappa2) antibody FAB light chain (anti0X40 maxgraft FAB arm LC)	DIQLTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKA PKPWIYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYY CQQWSSNPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C
SEQIDNO: 316- BEAT OX40mingraft/CD3(SP 34-Kappa2) antibody FAB heavy chain (anti0X40 mingraft FAB arm with G65S substitution BT33 LALA)	EVQLVESGGGLVQPGGSLRLSCAASGFSLSTSGMGMSWVRQ APGKGLEWVSAIWWDDDKYYADSVKSRFTISRDNSKNTLYL QMNSLRAEDTAVYYCARIDWDGFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTK PREEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGFYPS DIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDKSR WQQGNIFSCSVMHEALHNRFTQKSLSLSPG
SEQIDNO: 317- BEAT OX40mingraft/CD3(SP 34-Kappa2) antibody FAB light chain (anti0X40 mingraft FAB arm LC)	DIQMTQSPSSLSASVGDRVTITCRASSSVSYLNWYQQKPGKA PKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQWSSNPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS WCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQIDNO: 318 - BEAT CD20maxgraft/CD3 (SP 34-Kappa2) antibody FAB heavy chain (antiCD20 maxgraft FAB arm with G65S substitution BT33 LALA)	EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMSWVRQAP GKGLEWIGAIYPGNGDTYYADSVKSRATLSADKSKNTAYLQ MNSLRAEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAK TKPREEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGF YPSDIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDK SRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG
SEQIDNO: 319- BEAT CD20maxgraft/CD3(SP 34-Kappa2) antibody FAB light chain (antiCD20 maxgraft FAB arm LC)	DIQLTQSPSSLSASVGDRVTITCRLSASSSVSYLNWFQQKPGK APKPWIYATSSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATY YCQQWTSNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
SEQ ID NO: 320 - BEAT CD20mingraft/CD3(SP3	EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMSWVRQAP GKGLEWVSAIYPGNGDTYYADSVKSRFTISRDNSKNTLYLQM NSLRAEDTAVYYCARSTYYGGDWYFNVWGQGTLVTVSSAS

159

4-Kappa2) antibody FAB heavy chain (antiCD20 mingraft FAB ami with G65S substitution BT33 LALA)	TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAK TKPREEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGF YPSDIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDK SRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG
SEQ ID NO: 321 - BEAT CD20mingraft/CD3(SP3 4-Kappa2) antibody FAB light chain (anti- CD20 mingraft FAB arm LC)	DIQMTQSPSSLSASVGDRVTITCRASASSSVSYLNWYQQKPG KAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQWTSNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC
SEQ ID NO: 322 BEAT EGFRcetuxmaxgraft/CD3 (SP34Kappa2) antibody FAB heavy chain (anti-EGFR cetuximab maxgraft FAB arm with G65S substitution BT33 LALA)	EVQLVESGGGLVQPGGSLRLSCAASGFSLTNYGVHWVRQAP GKGLEWLGAIWSGGNTDYNTPFTSRLTISKDNSKNTLYLQMN SLRAEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTKPR EEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGFYPS DIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDKSR WQQGNIFSCSVMHEALHNRFTQKSLSLSPG
SEQ ID NO: 323 BEAT EGFRcetuxmaxgraft/CD3(SP34Kappa2) antibody FAB light chain (anti-EGFR cetuximab maxgraft FAB arm)	DIQLTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKA PKLLIKYASESISGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQNNNWPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS WCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 324 BEAT EGFRcetuxmingraft/CD3(SP34Kappa2) antibody FAB heavy chain (anti-EGFR cetuximab mingraft FAB arm with G65S substitution BT33 LALA)	EVQLVESGGGLVQPGGSLRLSCAASGFSLTNYGMSWVRQAP GKGLEWVSAIWSGGNTYYADSVKSRFTISRDNSKNTLYLQM NSLRAEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTK PREEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGFYPS DIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDKSR WQQGNIFSCSVMHEALHNRFTQKSLSLSPG
SEQ ID NO: 325 BEAT EGFRcetuxmingraft/CD3 (SP3 4Kappa2) antibody FAB light chain (anti-EGFR	DIQMTQSPSSLSASVGDRVTITCRASQSIGTNLNWYQQKPGK APKLLIYYASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQNNNWPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG

160

cetuximab mingraft FAB arm)	EC
SEQ ID NO: 326 BEAT EGFRpanimaxgraft/CD3(SP34Kappa2) antibody FAB heavy chain (anti-EGFR panitumumab maxgraft FAB arm with G65S substitution BT33 LALA)	EVQLVESGGGLVQPGGSLRLSCAVSGGSVSSGDYYMSWVRQ APGKGLEWIGAIYYSGNTYYADSVKSRLTISIDTSKNTFYLQM NSLRAEDTAVYYCVRDRVTGAFDIWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTKPR EEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGFYPS DIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDKSR WQQGNIFSCSVMHEALHNRFTQKSLSLSPG
SEQ ID NO: 327 BEAT EGFRpanimaxgraft/CD3 (SP3 4Kappa2) antibody FAB light chain (anti-EGFR panitumumab maxgraft FAB arm)	DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGK APKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYF CQHFDHLPLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C
SEQ ID NO: 328 BEAT EGFRpanimingraft/CD3(SP34Kappa2) antibody FAB heavy chain (anti-EGFR panitumumab mingraft FAB arm with G65S substitution BT33 LALA)	EVQLVESGGGLVQPGGSLRLSCAASGGSVSSGDYYMSWVRQ APGKGLEWVSAIYYSGNTYYADSVKSRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCVRDRVTGAFDIWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTKPR EEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGFYPS DIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDKSR WQQGNIFSCSVMHEALHNRFTQKSLSLSPG
SEQ ID NO: 329 BEAT EGFRpanimingraft/CD3(SP34Kappa2) antibody FAB light chain (anti-EGFR panitumumab mingraft FAB arm)	DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGK APKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQHFDHLPLAFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
SEQ ID NO: 330 BEAT CD19/CD3(SP34- Kappa2) antibody FAB heavy chain (anti-CD 19 FAB arm with G65S substitution BT33 LALA)	QVQLVQSGGGWQPGRSLRLSCAASGVSLPDYGVSWVRQAP GKGLEWVAVIWGSETTYYNSALKSRFTISRDNSKNTLYLQM NSLRAEDTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAK TKPREEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGF YPSDIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDK SRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG

161

SEQ ID NO: 331 BEAT CD19/CD3(SP34- Kappa2) antibody FAB light chain (anti-CD 19 FAB arm)	DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGK AIKLLIYHTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQGATLPYTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTA SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C
SEQ ID NO: 332 BEAT IgEomalimaxgraft/CD3(SP34Kappa2) antibody FAB heavy chain (anti-IgE omalizumab maxgraft FAB arm with G65S substitution BT33 LALA)	EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAP GKGLEWVASITYDGSTNYADSVKSRFTISRDDSKNTFYLQMN SLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKT KPREEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGFY PSDIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDKS RWQQGNIFSCSVMHEALHNRFTQKSLSLSPG
SEQ ID NO: 333 BEAT IgEomalimaxgraft/CD3(SP34Kappa2) antibody FAB light chain (anti-IgE omalizumab maxgraft FAB arm)	DIQLTQSPSSLSASVGDRVTITCRASQSVDYDGDSYMNWYQQ KPGKAPKLLIYAASYLESGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQSHEDPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 334 BEAT IgEomalimingraft/CD3(SP34Kappa2) antibody FAB heavy chain (anti-IgE omalizumab mingraft FAB arm with G65S substitution BT33 LALA)	EVQLVESGGGLVQPGGSLRLSCAASGYSITSGYSMSWVRQAP GKGLEWVSAITYDGSTYYADSVKSRFTISRDNSKNTLYLQMN SLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCWVDVSHEDPEVQFKWYVDGVEVHNAKT KPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGFY PSDIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDKS RWQQGNIFSCSVMHEALHNRFTQKSLSLSPG
SEQ ID NO: 335 BEAT IgEomalimingraft/CD3 (SP 3 4Kappa2) antibody FAB light chain (anti-IgE omalizumab mingraft FAB arm)	DIQMTQSPSSLSASVGDRVTITCRASQSVDYDGDSYLNWYQQ KPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQSHEDPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 336 BEAT IgEbswl7maxgraft/CD3 (SP34Kappa2) antibody FAB heavy chain (anti-IgE omalizumab maxgraft FAB arm with G65S	EVQLVESGGGLVKPGGSLRLSCAVSGFTFSSYAMSWVRQAP GKGLEWVASISSGNHYYPDNVKSRFTISRDNAKNSLYLQMNS LRAEDTAVYYCTRGRSTYGGFDHWGQGTTVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTKPR

162

substitution BT33 LALA)	EEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGFYPS DIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDKSR WQQGNIFSCSVMHEALHNRFTQKSLSLSPG
SEQ ID NO: 337 BEAT IgEbswl7maxgraft/CD3(SP34Kappa2) antibody FAB light chain (anti-IgE omalizumab maxgraft	DLQMTQSPSSLSASVGDRVTITCSASSSVTFLNWYQQKPGKA PWLLIYDTSSLQSGVPSRFSGSGSGTDYTLTISSMQPEDFATYY CQHWSGNPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C

FAB arm)

SEQ ID NO: 338 BEAT IgEbswl7mingraft/CD3(SP34Kappa2) antibody FAB heavy chain (anti-IgE omalizumab mingraft FAB arm with G65S substitution BT33 LALA)	EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP GKGLEWVSAISSGNIIYYADSVKSRFTISRDNSKNTLYLQMNS LRAEDTAVYYCTRGRSTYGGFDHWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTKPR EEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGFYPS DIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDKSR WQQGNIFSCSVMHEALHNRFTQKSLSLSPG
SEQ ID NO: 339 BEAT IgEbswl7mingraft/CD3 (SP 3 4Kappa2) antibody FAB light chain (anti-IgE omalizumab mingraft FAB arm)	DIQMTQSPSSLSASVGDRVTITCRASSSVTFLNWYQQKPGKAP KLLIYDTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QHWSGNPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS WCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ JD NO: 340 - BEAT OX40/CD3(SP34- Kappa2) antibody FAB heavy chain (anti-OX40 1D4 FAB arm BT33 LALA)	QVTLKESGPALVKPTQTLTLTCSFSGFSLSTSGMGVGWIRQPP GKALEWIAHIWWDDDKYYNTALKTRLTISKDTSKNQWLTM TNMDPVDTATYYCARIDWDGFAYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTKPR EEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGFYPS DIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDKSR WQQGNIFSCSVMHEALHNRFTQKSLSLSPG
SEQ ID NO: 341 - BEAT CD20/CD3(SP34- Kappa2) antibody FAB heavy chain (anti-CD20 rituximab FAB arm BT33 LALA)	QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQT PGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYM QLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAK TKPREEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGF

163

	YPSDIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDK SRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG
SEQ ID NO: 342 - BEAT EGFRcetux/CD3(SP34Kappa2) antibody FAB heavy chain (anti-EGFR cetuximab FAB arm BT33 LALA)	QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPG KGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNS LQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTKPR EEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGFYPS DIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDKSR WQQGNIFSCSVMHEALHNRFTQKSLSLSPG
SEQ ID NO: 343 - BEAT EGFRpani/CD3(SP34Kappa2) antibody FAB heavy chain (anti-EGFR panitumumab FAB arm BT33 LALA)	QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQS PGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSS VTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTK. VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTKPREE QYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGFYPSDIA VEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDKSRWQQ GNIFSCSVMHEALHNRFTQKSLSLSPG
SEQ ID NO: 344 - BEAT EGFRpani/CD3(SP34- Kappa2) antibody FAB light chain (anti-EGFR panitumumab FAB arm)	DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGK APKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYF CQHFDHLPLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C
SEQ ID NO: 345 - BEAT IgEomali/CD3(SP34- Kappa2) antibody FAB heavy chain (anti-IgE omalizumab FAB arm BT33 LALA)	EVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAP GKGLEWVASITYDGSTNYADSVKGRFTISRDDSKNTFYLQMN SLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCWVDVSHEDPEVQFKWYVDGVEVHNAKT KPREEQYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGFY PSDIAVEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDKS RWQQGNIFSCSVMHEALHNRFTQKSLSLSPG
SEQ ID NO: 346 - BEAT IgEomali/CD3(SP34- Kappa2) antibody FAB light chain (anti-IgE omalizumab FAB arm)	DIQLTQSPSSLSASVGDRVTITCRASQSVDYDGDSYMNWYQQ KPGKAPKLLIYAASYLESGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQSHEDPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC
SEQ ID NO: 347 - BEAT	EVQLLESGGGFVKPGGSLKLSCWSGFTFSSYAMSWVRQTPE KRLEWVASISSGNIIYYPDNVKGRFTISRDNVRNILYLQMSSL

164

IgEbswl7/CD3(SP34Kappa2) antibody FAB heavy chain (anti-IgE Bswl7 FAB arm BT33 LALA)	RSEDTAMYYCTRGRSTYGGFDHWGQGTTLTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVQFKWYVDGVEVHNAKTKPREE QYNSTFRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKTKGQPREPAVYTLPPSREEMTKNQVKLVCLVTGFYPSDIA VEWESSGQPENNYYTTPPMLDSDGSFSLVSWLNVDKSRWQQ GNIFSCSVMHEALHNRFTQKSLSLSPG
SEQ ID NO: 348 - BEAT IgEbswl7/CD3(SP34Kappa2) antibody FAB light chain (anti-IgE Bswl7 FAB arm)	ELVMTQSPAIMSASPGEKVTMTCSASSSVTFIHWYRQKSGTSP KGWIYDTSKLASGVPARFSGSGSGTSYSLTISTMEAEDAATY YCQHWSGNPLTFGTGTKLELKRTVAAPSVFIFPPSDEQLKSGT ASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
SEQ ID NO: 349 - scFv fragment humanized SP34 VH1-VL24 human IgGl Fc fusion	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTTVT VSSGGGGSGGGGSGGGGSEIWTQSPATLSVSPGERATLSCRS SAAAVTTSNYANWVQEKPGQAFRGLIGGANKRAPGVPARFS GSLSGDEATLTISSLQSEDFAVYYCALWYSNLWVFGQGTKLE IKGGGGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK
SEQIDNO: 350-scFv fragment humanized SP34 VH1-VL25human IgGl Fc fusion	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTTVT VSSGGGGSGGGGSGGGGSEIWTQSPATLSVSPGERATLSCRS STGAAATSNYANWVQEKPGQAFRGLIGGANKRAPGVPARFS GSLSGDEATLTISSLQSEDFAVYYCALWYSNLWVFGQGTKLE IKGGGGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK
SEQID NO: 351 - scFv fragment humanized SP34 VH1-VL26human IgGl Fc fusion	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTTVT VSSGGGGSGGGGSGGGGSEIWTQSPATLSVSPGERATLSCRS STGAVTAANYANWVQEKPGQAFRGLIGGANKRAPGVPARFS GSLSGDEATLTISSLQSEDFAVYYCALWYSNLWVFGQGTKLE IKGGGGTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW

165

	ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 352 Humanized anti- human CD3 epsilon SP34 VH5 CDRH1	GFTFNTYA
SEQIDNO: 353 Humanized anti- human CD3 epsilon SP34 VH5 CDRH2	IRSKYNNYAT
SEQ ID NO: 354 - Humanized anti- human CD3 epsilon SP34 VH5 CDRH3	VRHGNFGNSYVSYFAY
SEQ ID NO: 355 Humanized anti- human CD3 epsilon SP34 VL32 CDR Ll	TGAVTAANY
SEQ ID NO: 356 Humanized anti- human CD3 epsilon SP34 VL32 CDRL2	GAN
SEQ ID NO: 357 Humanized anti- human CD3 epsilon SP34 VL32 CDRL3	ALFYSNLWV
SEQ ID NO: 358 - OKT3 humanized VH9 domain	EVQLVESGGGLVQPGGSLRLSCAASGYTFTRYTMHWVRQAP GKGLEWIGYINPSRGYTYYADSVKSRFTLSTDKSKNTAYLQM NSLRAEDTAVYYCARYYDDHYCLDYWGQGTLVTVSS
SEQ ID NO: 359 SP34 humanized IgGl heavy chain with VH5	EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCVRHGNFGNSYVSYFAYWGQGTTVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQIDNO: 360SP34 humanized Light chain with VL32	EIWTQSPATLSVSPGERATLSCRSSTGAVTAANYANWVQEK PGQAFRGLIGGANKRAPGVPARFSGSLSGDEATLTISSLQSED FAVYYCALFYSNLWVFGQGTKLEIKKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC

166

Claims

1. A hetero-dimeric immunoglobulin or fragment thereof, comprising:

(a) a first polypeptide that binds to Protein A comprising an epitope binding région that binds a first epitope and an immunoglobulin constant région; and (b) a second polypeptide that does not bind to Protein A comprising an epitope binding région, that binds a second epitope and an immunoglobulin constant région;

wherein the first and second polypeptides comprise an engineered immunoglobulin constant région with a modified CH3 domain having a protein-protein interface, wherein the proteinprotein interface of the first polypeptide comprises an amino acid substitution at a position selected from the group consisting of: 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 85.1, 86, 88 and 90 (IMGT® numbering), and wherein the protein-protein interface of the second polypeptide comprises an amino acid substitution at a position selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2, 84.4, 85.1, 86, 88 and 90 (IMGT® numbering);

wherein the epitope binding région of the first polypeptide binds the CD3 protein complex and the epitope binding région of the second polypeptide binds a disease associated antigen or wherein the epitope binding région of the first polypeptide binds a disease associated antigen and the epitope binding région of the second polypeptide binds the CD3 protein complex; and wherein the epitope binding région that binds the CD3 protein complex comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 194, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 195 and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 196, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 197, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 198 and a light chain CDR3 comprising the amino acid sequences of: SEQIDNO: 199; or wherein the epitope binding région that binds the CD3 protein complex comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 200, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 201 and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 202, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 203, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 204 and a light chain CDR3 comprising the amino acid sequences of: SEQ ID NO: 205; or

168 wherein the epitope binding région that binds the CD3 protein complex comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 352, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 353 and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 354, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 355, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 356 and a light chain CDR3 comprising the amino acid sequences ofSEQIDNO: 357.

2. A hetero-dimeric immunoglobulin or fragment thereof of claim 1, wherein the epitope binding région that binds a disease associated antigen comprises heavy chain CDR1, CDR2 and CDR3 amino acid sequences and light chain CDR1, CDR2 and CDR3 amino acid sequences, respectively, selected from the group consisting of:

i) SEQ ED NOs: 206-211;

ii) SEQ ID NOs: 212-217;

iii) SEQIDNOs: 218-223;

iv) SEQIDNOs: 224-229;

v) SEQ ED NOs: 230-235;

vi) SEQID NOs: 236-241;

vii) SEQ ID NOs: 242 - 247;

viii) SEQIDNOs: 248-253;

ix) SEQ ED NOs: 254 - 259;

x) SEQ ID NOs: 260 - 265;

xi) SEQ ID NOs: 266 - 271 ; and xii) SEQ ID NOs: 272 - 277;

3. The hetero-dimeric immunoglobulin or fragment thereof of claim 1 or 2, wherein the constant région of said second polypeptide comprises an IgG3 CH3 région.

4. The hetero-dimeric immunoglobulin or fragment thereof of claim 1, 2 or 3, wherein said protein-protein interface of the second polypeptide comprises an amino acid substitution at a position 84.4 and at least one additional amino acid substitution selected from the group consisting of 3, 5, 7, 20, 22, 26, 27, 79, 81, 84, 84.2,, 85.1, 86, 88 and 90 (IMGT® numbering).

169

5. The hetero-dimeric immunoglobulin or fragment thereof of any one of claims 1 to 4, wherein the epitope binding région that binds the CD3 protein complex comprises a heavy chain variable région comprising the amino acid sequence of SEQ ID NO: 27, and a light chain variable région comprising the amino acid sequence of SEQ ID NO: 39; or wherein the epitope binding région that binds the CD3 protein complex comprises a heavy chain variable région comprising the amino acid sequence of SEQ ID NO: 64, and a light chain variable région comprising the amino acid sequence of SEQ ID NO: 69; or wherein the epitope binding région that binds the CD3 protein complex comprises a heavy chain variable région comprising the amino acid sequence of SEQ ID NO: 104, and a light chain variable région comprising the amino acid sequence of SEQ ID NO: 106.

6. The hetero-dimeric immunoglobulin or fragment thereof of any one of claims 1 to 5, wherein said epitope binding région of said second polypeptide comprises a VH3 région and wherein said VH3 région of the second polypeptide comprises a modification that reduces or abrogates the binding of said second polypeptide to Protein A.

7. The hetero-dimeric immunoglobulin or fragment thereof of claim 6, wherein the modified VH3 région comprises an amino acid substitution selected from the group consisting of: 57, 65, 81, 82a and combination 19/57/59 (Kabat numbering).

8. The hetero-dimeric immunoglobulin or fragment thereof of claim 6 or 7, wherein the modified VH3 région comprises an amino acid substitution selected from the group consisting of: 57A, 57E, 65S, 81E, 82aS and combination 19G/57A/59A (Kabat numbering).

9. The hetero-dimeric immunoglobulin or fragment thereof of any one of claims 1 to 8, wherein the heavy chain variable framework région comprises an amino acid substitution selected from the group consisting of: I34M, V48I, A49G, R58N/Y, I69L, A71T and T73K (Kabat numbering) and the light chain variable framework région comprises an amino acid substitution selected from the group consisting of: M4L, V33M, A34N, L46R, L47W, R66G, F71Y and P96F (Kabat numbering); or wherein the heavy chain variable framework région comprises the amino acid substitutions I34M, A49G and A71T (Kabat numbering) and the

170 light chain variable framework région comprises the amino acid substitutions M4L, L46R,

L47W and F71Y (Kabat numbering).

10. The hetero-dimeric immunoglobulin or fragment thereof of any one of claims 1 to 9, wherein the heavy chain variable région comprises an amino acid substitution selected from the group consisting of: WlOOeF and WlOOeY (Kabat numbering) and the light chain variable région comprises an amino acid substitution selected from the group consisting of: A2I, S25A, T27A, G27aA, V27cA, T28A, T29A, S30A, N31A, Y32A, E38Q, F44P, G46L, T51A N52A, K53A, R54A, P56A, L66G, D69T, F87Y, Q89A, W91F, Y92A, S93A, N94A, and Q100G (Kabat numbering); or wherein the heavy chain variable région comprises the amino acid substitutions WlOOeY (Kabat numbering) and the light chain variable région comprises the amino acid substitutions A2I, T29A, S30A, T51A, F87Y, Q89A,and W91F (Kabat numbering) or light chain variable région comprises the amino acid substitutions A2I, E38Q F87Y, and Q89A.

11. The hetero-dimeric immunoglobulin or fragment thereof of any one of the preceding claims, wherein the epitope binding région of the first polypeptide is a FAB and the epitope binding région of the second polypeptide is a scFv or wherein the epitope binding région of the first polypeptide is a scFv and the epitope binding région of the second polypeptide is a FAB.

12. A hetero-dimeric immunoglobulin or fragment thereof that binds to:

171

SEQ ID NO: 47 and binds CD3 epsilon, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 164 and binds HER2;

v) the CD3 protein complex and HER2, wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 168 and is assembled with a light chain of amino acid sequence of SEQ ID NO: 89 and binds CD3 epsilon, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 167 and binds HER2;

xi) the CD3 protein complex and EGFR wherein the first polypeptide has an amino acid sequence of SEQ ID NO: 174 and is assembled with a cognate light chain of amino acid

172 sequence of SEQ ID NO: 175 and binds EGFR, and wherein the second polypeptide has an amino acid sequence of SEQ ID NO: 171 and binds CD3 epsilon;

13. An in vitro method for the production of a hetero-dimeric immunoglobulin or fragment thereof of any one of the preceding claims comprising the following steps:

ii) transfecting or co-transfecting the DNA vector(s) from (ia) or (ib) in a mammalian host cell line;

iv) contacting the cell culture supematant on a Protein A affinity chromatography resin; and

v) eluting and collecting the hetero-dimeric immunoglobulin of interest.

14. A method according to claim 13, wherein the hetero-dimeric immunoglobulin or fragment thereof found in the purified material from step (v) is at least 95% pure as determined by capillary electrophoresis.

173

Abstract

The présent invention describes novel hetero-dimeric immunoglobulins or fragments thereof which bind to CD3 and a disease associated antigen. These hetero-dimeric immunoglobulins

5 hâve been engineered to promote hetero-dimer formation during expression and can be purified to a high degree using a Protein A differential purification technique.