Specific binding molecules

Abstract

The present invention relates to antibodies which bind to CD3, particularly antibodies and fragments thereof, with improved properties.

Description

[0001] SPECIFIC BINDING MOLECULES

[0002] Field of invention

[0003] The present invention relates to antibodies which bind to CD3, particularly antibodies and fragments thereof, with improved properties. The present invention also relates to compositions, uses and methods relating to the antibodies which bind to CD3.

[0004] Background to the invention

[0005] The antibody UCHT1 is a clinically relevant agonist molecule that binds to an antigen on CD3. UCHT1 was originally obtained from mouse and was subsequently humanised (Shalaby et al., J Exp Med. 1992 Jan 1; 175(1 ):217-25; Zhu and Carter, J Immunol. 1995 Aug 15; 155(4): 1903-10; US5821337). However, the fully humanised variant showed reduced affinity for target antigen on CD3. To retain the original binding affinity a minimally humanised variant was identified and deemed suitable for therapeutic use (see Example 3, US8075890).

[0006] More recently, the minimally humanised variant (termed U0 herein) has been used in the construction of bispecific T cell engaging molecules, in which an scFv fragment of the anti-CD3 antibody is fused to a soluble T cell receptor that binds to a disease relevant pMHC antigen on a target cell. Such molecules include, for example, those of the ImmTAC® platform (WO2011001152, WO2010133828). The drug KIMMTRAK® (tebentafusp-tebn) is an ImmTAC molecule comprising the minimally humanised UCHT1 scFv fragment, and is approved by the FDA for the for treatment of HLA-A*02:01 -positive patients with unresectable or metastatic uveal melanoma.

[0007] A further engineered variant of the minimally humanised UCHT1 molecule (U0) identified mutations in the variable heavy chain that conferred improved therapeutic properties on a bispecific T cell engager (increased therapeutic window and maximum T cell activation) as described in W02020157210, and termed U28 herein.

[0008] Even with the clinical success of bispecific molecules comprising UCHT1, the opportunity remains, however, to further minimise the risk of anti-drug antibodies (ADAs) arising in patients as a result of sequence elements of non-human origin within the minimally humanised UCHT1. Therefore, there is a need for further humanised variants of UCHT1, and multi-specific molecules such as T cell engagers incorporating these variants, which have reduced risk of ADAs while retaining other advantageous therapeutic properties. Such engineering requires balancing against other factors. For example, a narrow CD3 affinity optimum exists for maximal T cell activation for T cell engaging bispecific molecules (Robertson et al., Clin Exp Immunol. 2024 Feb 7;215(2):105-119), making it complicated to engineer UCHT1 while maintaining maximum activity.

[0009] Summary of Invention In a first aspect, there is provided an antibody or antigen binding fragment thereof that specifically binds to CD3 and comprises light chain CDRs selected from the following combinations:

[0010] a) VLCDR1 is SEQ ID NO: 29, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO:34, b) VLCDR1 is SEQ ID NO: 30, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 34, c) VLCDR1 is SEQ ID NO: 31, VLCDR2 is SEQ ID NO: 33, VLCDR3 is SEQ ID NO: 34, or d) VLCDR1 is SEQ ID NO: 29, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 106 and / or heavy chain CDRs selected from the following combinations:

[0011] a) VHCDR1 is SEQ ID NO: 35, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 42, b) VHCDR1 is SEQ ID NO: 35, VHCDR2 is SEQ ID NO: 41, VHCDR3 is SEQ ID NO: 43, c) VHCDR1 is SEQ ID NO: 35, VHCDR2 is SEQ ID NO: 40, VHCDR3 is SEQ ID NO: 42, d) VHCDR1 is SEQ ID NO: 36, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 42, e) VHCDR1 is SEQ ID NO: 35, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 109, f) VHCDR1 is SEQ ID NO: 107, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 42, g) VHCDR1 is SEQ ID NO: 108, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 42, or

[0012] h) VHCDR1 is SEQ ID NO: 108, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 109

[0013] In a further aspect of the invention there is provided a multi-specific binding molecule comprising:

[0014] I) an antibody or antigen binding fragment thereof that specifically binds to CD3 of the invention, and

[0015] ii) a targeting moiety.

[0016] In a further aspect of the invention, there is provided a nucleic acid encoding the antibody or antigen binding fragment thereof that specifically binds to CD3 of the invention or the multi-specific binding molecule of the invention.

[0017] In a further aspect of the invention, there is provided an expression vector comprising the nucleic acid of the invention.

[0018] In a further aspect of the invention, there is provided a host cell comprising the nucleic acid or the vector of the invention, wherein the nucleic acid is present as a single open reading frame or a distinct open reading frame encoding each chain of the antibody or multi-specific binding molecule.

[0019] In a further aspect of the invention, there is provided a pharmaceutical composition comprising an antibody or antigen binding fragment thereof that specifically binds to CD3 of the invention, a multispecific binding molecule of the invention, a nucleic acid of the invention, an expression vector of the invention, or a host cell of the invention.

[0020] In a further aspect of the invention, there is provided a method of making an antibody or antigen binding fragment thereof that specifically binds to CD3 of the invention, or a multi-specific binding molecule of the invention, comprising maintaining a host cell of the invention under optimal conditions for expression of a nucleic acid and, optionally, isolating the antibody or antigen binding fragment thereof that specifically binds to CD3 or the multi-specific binding molecule.

[0021] In a further aspect of the invention, there is provided an antibody or antigen binding fragment thereof that specifically binds to CD3 of the invention, a multi-specific binding molecule of the invention, a nucleic acid of the invention, an expression vector of the invention, a host cell of the invention, or a pharmaceutical composition of the invention for use as a medicament.

[0022] In a further aspect of the invention, there is provided a method of treatment comprising administering an antibody or antigen binding fragment thereof that specifically binds to CD3 of the invention, a multi-specific binding molecule of the invention, a nucleic acid of the invention, an expression vector of the invention, a host cell of the invention, or a pharmaceutical composition of the invention to a patient in need thereof.

[0023] Detailed description

[0024] The invention provides an antibody which binds to CD3, and antigen binding fragments thereof which bind to CD3. As used herein, the terms “antibody”, “antibody fragment” and “antigen binding fragment” refer to immunoglobulin molecules and immunologically active portions or fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding domain that specifically binds an antigen. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antigen binding domain. Said binding domain may be formed from the association of a variable heavy chain with a variable light chain. The antigen binding fragment may comprise a heavy chain and may not comprise a light chain. The antibodies and fragments can be derived from natural sources, or they may be partly or wholly synthetically produced. Examples of antibodies or fragments thereof are the immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies. Antibodies may be polyclonal or monoclonal. A monoclonal antibody may be referred to herein as “mAb”.

[0025] It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementary determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. A hybridoma or other cell producing an antibody may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.

[0026] As antibodies or fragments thereof can be modified in a number of ways, the term “antibody” and “antibody fragment” should be construed as covering any specific binding molecule or substance having an antigen binding domain with the required specificity. Thus, “antibody” and “antibody fragment” covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, humanised antibodies, including any polypeptide comprising an antigen binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Methods of cloning and expression of chimeric antibodies are known in the art. A humanised antibody may be a modified antibody having the variable regions of a non-human, e.g. murine, antibody and the constant region (or parts thereof) of a human antibody.

[0027] It has been shown that fragments of a whole antibody can perform the function of binding antigens. Included within the invention are fragments of the antibody of the invention which bind to CD3. Examples of binding fragments are (I) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (ill) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment, which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab’)2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site; (viii) bispecific single chain Fv dimers; (ix) “diabodies”, multivalent or multispecific fragments constructed by gene fusion; (x) Variable Heavy Chain of Heavy Chain-only Antibody (VHH), a single-domain antibody fragment derived from the heavy chain of an antibody, typically derived from camelids, also called nanobodies; and (xi) Variable New Antigen Receptors (VNARs), a single-domain antibody fragment derived from the immune system of cartilaginous fish.

[0028] The antibody, antigen binging fragment or antibody fragment of the invention specifically bind CD3. The antibody fragment which binds CD3, or antigen binding fragment that specifically binds CD3 may have a higher, lower or substantially the same affinity for CD3 as a full length antibody which binds CD3. Methods to measure binding to a target antigen are known in the art.

[0029] Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g. by a peptide linker) but unable to associate with each other to form an antigen binding site: antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer.

[0030] The term “antibody” also encompasses bispecific antibodies. These may be conventional bispecific antibodies, which can be manufactured in a variety of ways known in the art, e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain “Janusins” described in Traunecker et al., EMBO Journal 10:3655-3659 (1991).

[0031] Bispecific diabodies, as opposed to bispecific whole antibodies, may also be useful because they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.

[0032] An “antigen binding domain” is the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. The antigen binding fragment of the invention may comprise an antigen binding domain. An antigen binding domain may be provided by one or more antibody variable domains. An antigen binding domain may comprise (I) an antibody heavy chain variable region (VH), or (ii) an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Antibody variable domains each comprise three canonical complementarity-determining regions (CDR1, CDR2, CDR3). An antigen binding domain may comprise CDR1, CDR2 and CDR3 of the VL, and / or the CDR1, CDR2, and CDR3 of the VH. The antibody fragment which binds CD3 may comprise an antigen binding domain.

[0033] “Specific” is generally used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s), and, e.g., has less than about 30%, preferably 20%, 10%, or 1% cross-reactivity with any other molecule. The term is also applicable e.g. for an antigen binding domain that specifically binds to or is specific for a particular epitope within an antigen. The term “multi-specific” is used herein to refer to a binding molecule, an antibody or antigen binding fragment thereof capable of binding at least two targets or antigens. A multi-specific binding molecule may bind at least two, at least 3, at least 4 or more targets. Each target may be on the same cell or a different cell.

[0034] Sequences disclosed herein may be modified to enhance a specific property or characteristic. For example, to increase or decrease affinity, increase or decrease stability, increase or decrease melting temperature and / r increase or decrease charge. Suitable mutations and methods of introducing said mutations are known in the art.

[0035] Sequences

[0036] Reference is made herein to the following sequences.

[0037] Minimally humanised UCHT1 (U0) with CDR regions underlined

[0038] VL (SEQ ID NO:1) AIQMTQSPSS LSASVGDRVT ITCRASQDIR NYLNWYQQKP GKAPKLLIYY TSRLESGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQQ GNTLPWTFGQ GTKVEIK VH (CDR regions underlined) (SEQ ID NO:2)

[0039] EVQLVESGGG LVQPGGSLRL SCAASGYSFT GYTMNWVRQA PGKGLEWVAL INPYKGVSTY NQKFKDRFTI SVDKSKNTAY LQMNSLRAED TAVYYCARSG YYGDSDWYFD VWGQGTLVTV SS

[0040] Modified UCHT1 with improved therapeutic window as disclosed in W02020157210 (U28) with CDRs underlined (IMGT)

[0041] VL (SEQ ID NO:3)

[0042] AIQMTQSPSS LSASVGDRVT ITCRASQDIR NYLNWYQQKP GKAPKLLIYY TSRLESGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQQ GNTLPWTFGQ GTKVEIK VH (SEQ ID NO:4)

[0043] EVQLVESGGG LVQPGGSLRL SCAASGYSFT GYAMNWVRQA PGKGLEWVAL INPYKGVSTY NQKFKDRFTF SVDKSKNTAY LQMNSLRAED TAVYYCARSG YYGDSDWYFD VWGQGTLVTV SS

[0044] U28 with CDRs underlined (Kabat)

[0045] VL (SEQ ID NO:3)

[0046] AIQMTQSPSS LSASVGDRVT ITCRASQDIR NYLNWYQQKP GKAPKLLIYY TSRLESGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQQ GNTLPWTFGQ GTKVEIK VH (SEQ ID NO:4)

[0047] EVQLVESGGG LVQPGGSLRL SCAASGYSFT GYAMNWVRQA PGKGLEWVAL INPYKGVSTY NQKFKDRFTF SVDKSKNTAY LQMNSLRAED TAVYYCARSG YYGDSDWYFD VWGQGTLVTV SS

[0048] Human germline

[0049] IGKV1-6*01-J1 (SEQ ID NO:5)

[0050] AIQMTQSPSS LSASVGDRVT ITCRASQGIR NDLGWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCLQ DYNYPWTFGQ GTKVEIK IGHV3-23*01 D4-23*01 J2: (SEQ ID NO:6)

[0051] EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYAMSWVRQA PGKGLEWVSA ISGSGGSTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKSD YGGNSYWYFD LWGRGTLVTV SS

[0052] U28 with residues different to human VL in bold, with CDRs underlined (IMGT)

[0053] VL (SEQ ID NO:3)

[0054] AIQMTQSPSS LSASVGDRVT ITCRASQDIR NYLNWYQQKP GKAPKLLIYY TSRLESGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQQ GNTLPWTFGQ GTKVEIK VH (SEQ ID NO:4)

[0055] EVQLVESGGG LVQPGGSLRL SCAASGYSFT GYAMNWVRQA PGKGLEWVAL INPYKGVSTY NQKFKDRFTF SVDKSKNTAY LQMNSLRAED TAVYYCARSG YYGDSDWYFD VWGQGTLVTV SS

[0056] Humanised UCHT1 (U42) with CDRs underlined

[0057] VL (SEQ ID NO:7) AIQMTQSPSS LSASVGDRVT ITCRASQDIR NYLNWYQQKP GKAPKLLIYY TSRLESGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQQ GNTLPWTFGQ GTKVEIK VH (SEQ ID N0:8)

[0058] EVQLVESGGG LVQPGGSLRL SCAASGYRFT GYLMNWVRQA PGKGLEWVAL INPYKGVSTY NQKFKDRFTF SVDKSKNTAY LQMNSLRAED TAVYYCARSG YYGDSDWYFD VWGQGTLVTV SS

[0059] Humanised UCHT1 U112 (non-human residues in bold) with CDRs underlined

[0060] VL (SEQ ID NO:9)

[0061] AIQMTQSPSS LSASVGDRVT ITCRASQDIR NYLNWYQQKP GKAPKLLIYY TSRLESGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQQ GNTLPWTFGQ GTKVEIK VH (SEQ ID NO:10)

[0062] EVQLLESGGG LVQPGGSLRL SCAASGYRFT GYLMNWVRQA PGKGLEWVSA INPYKGSTYY ADSVKGRFTF SRDNSKNTAY LQMNSLRAED TAVYYCARSG YYGDSDWYFD LWGRGTLVTV SS

[0063] Humanised UCHT1 U123 (non-human residues in bold) with CDRs underlined

[0064] VL (SEQ ID NO:11)

[0065] AIQMTQSPSS LSASVGDRVT ITCRASQGIR NYLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ GNTLPWTFGQ GTKVEIK VH (SEQ ID NO:12)

[0066] EVQLLESGGG LVQPGGSLRL SCAASGYRFT GYLMNWVRQA PGKGLEWVSA INPYKGSTYY ADSVKGRFTF SRDNSKNTAY LQMNSLRAED TAVYYCARSG YYGDSDWYFD LWGRGTLVTV SS

[0067] Humanised UCHT1 U144 (non-human residues in bold) with CDRs underlined

[0068] VL (SEQ ID NO:13)

[0069] AIQMTQSPSS LSASVGDRVT ITCRASQGIR KYLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ GNTLPWTFGQ GTKVEIK VH (SEQ ID NO:14)

[0070] EVQLLESGGG LVQPGGSLRL SCAASGYRFT GYLMNWVRQA PGKGLEWVSA INPYKGSTYY ADSVKGRFTF SRDNSKNTAY LQMNSLRAED TAVYYCARSG YYGDSDWYFD LWGRGTLVTV SS

[0071] Humanised UCHT1 U152 (non-human residues in bold) with CDRs underlined

[0072] VL (SEQ ID NO:15)

[0073] AIQMTQSPSS LSASVGDRVT ITCRASQGIR NYLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ GNTLPWTFGQ GTKVEIK VH (SEQ ID NO:16)

[0074] EVQLLESGGG LVQPGGSLRL SCAASGYRFT RYLMNWVRQA PGKGLEWVSA INPYKGSTYY ADSVKGRFTF SRDNSKNTAY LQMNSLRAED TAVYYCARSG YYGDSDWYFD LWGRGTLVTV SS

[0075] Humanised UCHT1 U154 (non-human residues in bold) with CDRs underlined

[0076] VL (SEQ ID NO:17) AIQMTQSPSS LSASVGDRVT ITCRASQGIR NYLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ GNTLPWTFGQ GTKVEIK VH (SEQ ID NO:18)

[0077] EVQLLESGGG LVQPGGSLRL SCAASGYRFT GYLMNWVRQA PGKGLEWVSA INPYKGSSYY ADSVKGRFTF SRDNSKNTAY LQMNSLRAED TAVYYCARSG YYGDSDWYFD LWGRGTLVTV SS

[0078] Humanised UCHT1 U165 with CDRs underlined

[0079] VL (SEQ ID NO: 70) AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQGNTLPWTFGCGTKVEIK VH (SEQ ID NO: 71) EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKCLEWVSAINPYKGSTYYADSVKGRFTFSRD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGRGTLVTVSS

[0080] Humanised UCHT1 U166 with CDRs underlined

[0081] VL (SEQ ID NO: 72) AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTL T I S S LQPEDFAT YYCQQGNTLPWTFGQGTKVE I K VH (SEQ ID NO: 73) EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKGLEWVSAINPYKGSTYYADSVKGRFTFSRD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGRGTLVTVSS

[0082] Humanised UCHT1 U167 with CDRs underlined

[0083] VL (SEQ ID NO: 74) AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL T I S S LQPEDFAT YYCQQGNTLPWTFGQGTKVE I K VH (SEQ ID NO:75) EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKGLEWVSAINPYKGSTYYADSVKGRFTFSRD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGQGTLVTVSS

[0084] Humanised UCHT1 U170 with CDRs underlined

[0085] VL (SEQ ID NO: 76) AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL T I S S LQPEDFATYYCQQGNTLPWTFGQGTKVE I K VH (SEQ ID NO:77) EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKGLEWVSAINPYKGSTYYADSVKGRFTFSVD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGRGTLVTVSS

[0086] Humanised UCHT1 U175 with CDRs underlined

[0087] VL (SEQ ID NO: 78) AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL T I S S LQPEDFAT YYCQQGNTLPWTFGQGTKVE I K VH (SEQ ID NO:79) EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKGLEWVSAINPYKGSTYYADSVKGRFTFSRD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDDDWYFDLWGRGTLVTVSS

[0088] Humanised UCHT1 U179 with CDRs underlined

[0089] VL (SEQ ID NO: 80) AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL T I S S LQPEDFAT YYCQQGNTDPWTFGQGTKVE I K VH (SEQ ID NO:81) eVQLLESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKGLEWVSAINPYKGSTYYADSVKGRFTFSRD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGRGTLVTVSS

[0090] Humanised UCHT1 U181 with CDRs underlined

[0091] VL (SEQ ID NO: 82) AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQGNTLPWTFGCGTKVEIK VH (SEQ ID NO:83) EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKCLEWVSAINPYKGSTYYADSVKGRFTFSRD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGRGTLVTVSS

[0092] Humanised UCHT1 U182 with CDRs underlined

[0093] VL (SEQ ID NO: 84) AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQGNTLPWTFGCGTKVEIK VH (SEQ ID NO:85) EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKCLEWVSAINPYKGSTYYADSVKGRFTFSRD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGQGTLVTVSS

[0094] Humanised UCHT1 U183 with CDRs underlined

[0095] VL (SEQ ID NO: 86) AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQGNTLPWTFGCGTKVEIK VH (SEQ ID NO:87) EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKCLEWVSAINPYKGSTYYADSVKGRFTFSRD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGQGTLVTVSS

[0096] Humanised UCHT1 U184 with CDRs underlined

[0097] VL (SEQ ID NO: 88) AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQGNTLPWTFGCGTKVEIK VH (SEQ ID NO:89) EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKCLEWVSAINPYKGSTYYADSVKGRFTFSVD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGQGTLVTVSS

[0098] Humanised UCHT1 U185 with CDRs underlined

[0099] VL (SEQ ID NO: 90) AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQGNTLPWTFGCGTKVEIK VH (SEQ ID NO:91) EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKCLEWVSAINPYKGSTYYADSVKGRFTFSRD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDDDWYFDLWGQGTLVTVSS

[0100] Humanised UCHT1 U186 with CDRs underlined

[0101] VL (SEQ ID NO: 92) AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTL T I S S LQPEDFAT YYCQQGNTLPWTFGQGTKVE I K VH (SEQ ID NO:93) EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKGLEWVSAINPYKGSTYYADSVKGRFTFSRD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGQGTLVTVSS

[0102] Humanised UCHT1 U189 with CDRs underlined

[0103] VL (SEQ ID NO: 94) AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQGNTDPWTFGCGTKVEIK VH (SEQ ID NO:95) EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKCLEWVSAINPYKGSTYYADSVKGRFTFSRD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGQGTLVTVSS

[0104] Humanised UCHT1 U190 with CDRs underlined

[0105] VL (SEQ ID NO: 96) AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQGNTLPWTFGCGTKVEIK VH (SEQ ID NO:97) EVQLLESGGGLVQPGGSLRLSCAASGFRFTGYLMNWVRQAPGKCLEWVSAINPYKGSTYYADSVKGRFTFSRD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGQGTLVTVSS

[0106] Humanised UCHT1 U192 with CDRs underlined VL (SEQ ID NO: 98) AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTL T I S S LQPEDFAT YYCQQGNTLPWTFGQGTKVE I K VH (SEQ ID NO:99) EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYAMNWVRQAPGKGLEWVSAINPYKGSTYYADSVKGRFTFSRD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGQGTLVTVSS

[0107] Humanised UCHT1 U193 with CDRs underlined

[0108] VL (SEQ ID NO: 100) AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTL T I S S LQPEDFAT YYCQQGNTDPWTFGQGTKVE I K VH (SEQ ID NO:101) EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKGLEWVSAINPYKGSTYYADSVKGRFTFSRD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGQGTLVTVSS

[0109] Humanised UCHT1 U194 with CDRs underlined

[0110] VL (SEQ ID NO: 102) AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTL T I S S LQPEDFAT YYCQQGNTLPWTFGQGTKVE I K VH (SEQ ID NO: 103) EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKGLEWVSAINPYKGSTYYADSVKGRFTFSRD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDDDWYFDLWGQGTLVTVSS

[0111] Humanised UCHT1 U195 with CDRs underlined

[0112] VL (SEQ ID NO: 104) AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTL T I S S LQPEDFATYYCQQGNTDPWTFGQGTKVE I K VH (SEQ ID NO: 105) EVQLLESGGGLVQPGGSLRLSCAASGFRFTGYLMNWVRQAPGKGLEWVSAINPYKGSTYYADSVKGRFTFSRD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGQGTLVTVSS

[0113] Humanised UCHT1 U196 with CDRs underlined

[0114] VL (SEQ ID NO: 121) AIQMTQSPSSLSASVGDRVTITCRASQGIRNYLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTL T I S S LQPEDFATYYCQQGNTLPWTFGQGTKVE I K VH (SEQ ID NO: 122) EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKGLEWVSAINPYKGSTYYADSVKGRFTFSRD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGQGTLVTVSS

[0115] Humanised UCHT1 U197 with CDRs underlined

[0116] VL (SEQ ID NO: 123) AIQMTQSPSSLSASVGDRVTITCRASQGIRNYLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTL T I S S LQPEDFAT YYCQQGNTLPWTFGQGTKVE I K VH (SEQ ID NO: 124) EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYAMNWVRQAPGKGLEWVSAINPYKGSTYYADSVKGRFTFSRD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDLWGQGTLVTVSS

[0117] Humanised UCHT1 U198 with CDRs underlined

[0118] VL (SEQ ID NO: 125) AIQMTQSPSSLSASVGDRVTITCRASQGIRNYLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTL T I S S LQPEDFAT YYCQQGNTLPWTFGQGTKVE I K VH (SEQ ID NO: 126) EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYLMNWVRQAPGKGLEWVSAINPYKGSTYYADSVKGRFTFSRD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDDDWYFDLWGQGTLVTVSS

[0119] Humanised UCHT1 U199 with CDRs underlined

[0120] VL (SEQ ID NO: 127) AIQMTQSPSSLSASVGDRVTITCRASQGIRKYLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTL T I S S LQPEDFATYYCQQGNTLPWTFGQGTKVE I K VH (SEQ ID NO: 128) EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYAMNWVRQAPGKGLEWVSAINPYKGSTYYADSVKGRFTFSRD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDDDWYFDLWGQGTLVTVSS

[0121] Humanised UCHT1 U201 with CDRs underlined

[0122] VL (SEQ ID NO: 129) AIQMTQSPSSLSASVGDRVTITCRASQGIRNYLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTL T I S S LQPEDFATYYCQQGNTLPWTFGQGTKVE I K VH (SEQ ID NO: 130) EVQLLESGGGLVQPGGSLRLSCAASGYRFTGYAMNWVRQAPGKGLEWVSAINPYKGSTYYADSVKGRFTFSRD NSKNTAYLQMNSLRAEDTAVYYCARSGYYGDDDWYFDLWGQGTLVTVSS

[0123] VL-VH-linker (SEQ ID NO:19)

[0124] GGGGSGGGGSGGGGSGGGGSGGGS

[0125] VL-VH-linker (SEQ ID NO: 68)

[0126] A SGGSGGGGSG GGGSGGGGSG GGS

[0127] Exemplary two chain PIWIL TCR antiCD3 bispecific fusion molecule

[0128] TCR alpha chain (SEQ ID NO:20)

[0129] LAKTTQPISM DSYEGQEVNI TCSHNYIAAN DFITWYQQFP SQGPRFFIQG YKTNVQNEVA SLFIPADRKS STLSLPRVSL SDTAVYYCLA WGGTDLLPFG TGTRLQVFPN IQNPDPAVYQ LRDSKSSDKS VCLFTDFDSQ TNVSQSKDSD VYITDKCVLD MRSMDFKSNS AVAWSNKSDF ACANAFNNSI IPEDT

[0130] U144 scFv fused to N terminus of TCR beta chain via a G4S linker (italics, SEQ ID NO 59) (SEQ ID NO:21)

[0131] U144 scFv underlined, comprising VL and VH sequences of SEQ ID Nos 13 and 14 respectively, with a linker of SEQ ID NO: 19

[0132] AIQMTQSPSS LSASVGDRVT ITCRASQGIR KYLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ GNTLPWTFGQ GTKVEIKGGG GSGGGGSGGG GSGGGGSGGG SEVQLLESGG GLVQPGGSLR LSCAASGYRF TGYLMNWVRQ APGKGLEWVS AINPYKGSTY YADSVKGRFT FSRDNSKNTA YLQMNSLRAE DTAVYYCARS GYYGDSDWYF DLWGRGTLVT VSSGGGGSEA GVAQSPRYKI IEKRQSVAFW CNPISGHGTL YWYQQILGQG PKLLIQFHEN GVVDDSQLPK DRFSAERLKG VDSTLKIQPA KLEDSAVYLC ASSVDWVGDG ERQYFGPGTR LLVLEDLKNV FPPEVAVFEP SEAEISHTQK ATLVCLATGF YPDHVELSWW VNGKEVHSGV CTDPQPLKEQ PALNDSRYAL SSRLRVSATF WQDPRNHFRC QVQFYGLSEN DEWTQDRAKP VTQIVSAEAW GRAD

[0133] Alternatively, U183 scFv fused to N terminus of TCR beta chain via a G4S linker (italics, SEQ ID NO 59) (SEQ ID NO:116)

[0134] U183 scFv underlined, comprising VL and VH sequences of SEQ ID Nos 86 and 87 respectively, with a linker of SEQ ID NO: 19

[0135] AIQMTQSPSS LSASVGDRVT ITCRASQGIR KYLNWYQQKP GKAPKLLIYA ASSLESGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ GNTLPWTFGC GTKVEIKGGG GSGGGGSGGG GSGGGGSGGG SEVQLLESGG GLVQPGGSLR LSCAASGYRF TGYLMNWVRQ APGKGLEWVS AINPYKGSTY YADSVKGRFT FSRDNSKNTA YLQMNSLRAE DTAVYYCARS GYYGDSDWYF DLWGQGTLVT VSSGGGGSEA GVAQSPRYKI IEKRQSVAFW CNPISGHGTL YWYQQILGQG PKLLIQFHEN GVVDDSQLPK DRFSAERLKG VDSTLKIQPA KLEDSAVYLC ASSVDWVGDG ERQYFGPGTR LLVLEDLKNV FPPEVAVFEP SEAEISHTQK ATLVCLATGF YPDHVELSWW VNGKEVHSGV CTDPQPLKEQ PALNDSRYAL SSRLRVSATF WQDPRNHFRC QVQFYGLSEN DEWTQDRAKP VTQIVSAEAW GRAD

[0136] Exemplary single chain MAGEA4 TCRm antiCD3 bispecific fusion molecule - SEQ ID NO:22 Comprises U144 scFv (underlined; VL SEQ ID NO 13, VH SEQ ID NO 14, linker is SEQ ID NO 67) fused to the C terminus of a TCRm VHH domain (SEQ ID NO 67) via a linker (italics) EVQLVQSGAE VKKPGSSVKV SCKASGGTFS SYAISWVRQA PGQGLEWMGR IIPILGIANY AQKFQGRVTI TADKSTSTAY MELSSLRSED TAVYYCARVG YCSSTSCLEY YYGMDVWGQG TTVTVSSAAA GSAIQMTQSP SSLSASVGDR VTITCRASQG IRKYLNWYQQ KPGKAPKLLI YAASSLQSGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC QQGNTLPWTF GQGTKVEIKA SGGSGGGGSG GGGSGGGGSG GGSEVQLLES GGGLVQPGGS LRLSCAASGY RFTGYLMNWV RQAPGKGLEW VSAINPYKGS TYYADSVKGR FTFSRDNSKN TAYLQMNSLR AEDTAVYYCA RSGYYGDSDW YFDLWGRGTL VTVSS Exemplary single chain MAGEA4 TCRm antiCD3 bispecific fusion molecule - SEQ ID NO:117 Comprises U183 scFv (underlined; VL SEQ ID NO 86, VH SEQ ID NO 87, linker is SEQ ID NO 67) fused to the C terminus of a TCRm VHH domain (SEQ ID NO 67) via a linker (italics) EVQLVQSGAE VKKPGSSVKV SCKASGGTFS SYAISWVRQA PGQGLEWMGR IIPILGIANY AQKFQGRVTI TADKSTSTAY MELSSLRSED TAVYYCARVG YCSSTSCLEY YYGMDVWGQG TTVTVSSAAA GSAIQMTQSP SSLSASVGDR VTITCRASQG IRKYLNWYQQ KPGKAPKLLI YAASSLESGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC QQGNTLPWTF GCGTKVEIKG GGGSGGGGSG GGGSGGGGSG GGSEVQLLES GGGLVQPGGS LRLSCAASGY RFTGYLMNWV RQAPGKCLEW VSAINPYKGS TYYADSVKGR FTFSRDNSKN TAYLQMNSLR AEDTAVYYCA RSGYYGDSDW YFDLWGQGTL VTVSS

[0137] Exemplary single chain PRAME TCR-antiCD3 bispecific fusion molecule with Fc domain (SEQ ID NO:23)

[0138] Comprises U144 scFv (underlined; VL SEQ ID NO 13, VH SEQ ID NO 14, linker is SEQ ID NO 19) fused via a G4S linker (italics, SEQ ID NO 59) to a TCRβ chain, a first Fc domain, a TCRα chain and a second Fc domain.

[0139] AIQMTQSPSS LSASVGDRVT ITCRASQGIR KYLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ GNTLPWTFGQ GTKVEIKGGG GSGGGGSGGG GSGGGGSGGG SEVQLLESGG GLVQPGGSLR LSCAASGYRF TGYLMNWVRQ APGKGLEWVS AINPYKGSTY YADSVKGRFT FSRDNSKNTA YLQMNSLRAE DTAVYYCARS GYYGDSDWYF DLWGRGTLVT VSSGGGGSDG GITQSPKYLF RKEGQNVTLS CEQNLNHDAM YWYRQDPGQG LRLIYYSQIM GDEQKGDIAE GYSVSREKKE SFPLTVTSAQ KNPTAFYLCA SSWWTGGASP IRFGPGTRLT VTEDLKNVFP PEVAVFEPSE AEISHTQKAT LVCLATGFYP DHVELSWWVN GKEVHSGVCT DPQPLKEQPA LQDSRYALSS RLRVSATFWQ DPRNHFRCQV QFYGLSENDE WTQDRAKPVT QIVSAEAWGR ADGGGSGGGG EPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YGSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL SCAVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LVSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GKGGGSGGGG GDAKTTQPNS MESNEEEPVH LPCQHSTISG TDYIHWYRQL PSQGPEYVIH GLTSNVNNRM ASLAIAEDRK SSTLILHRAT LRDAAVYYCI LILGHSRLGN YIATFGKGTK LSVIPNIQNP DPAVYQLRDS KSSDKSVCLF TDFDSQTQVS QSKDSDVYIT DKCVLDMRSM DFKSNSAVAW SQKSDFACAN AFQNSIIPED TGGGSGGGGE PKSSDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY GSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRD ELTKNQVSLW CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K

[0140] Exemplary single chain PRAME TCR-antiCD3 bispecific fusion molecule with Fc domain (SEQ ID NO:118) Comprises U183 scFv (underlined; VL SEQ ID NO 86, VH SEQ ID NO 87, linker is SEQ ID NO 19) fused via a G4S linker (italics, SEQ ID NO 59) to a TCRβ chain, a first Fc domain, a TCRα chain and a second Fc domain.

[0141] AIQMTQSPSS LSASVGDRVT ITCRASQGIR KYLNWYQQKP GKAPKLLIYA ASSLESGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ GNTLPWTFGC GTKVEIKGGG GSGGGGSGGG GSGGGGSGGG SEVQLLESGG GLVQPGGSLR LSCAASGYRF TGYLMNWVRQ APGKCLEWVS AINPYKGSTY YADSVKGRFT FSRDNSKNTA YLQMNSLRAE DTAVYYCARS GYYGDSDWYF DLWGQGTLVT VSSGGGGSDG GITQSPKYLF RKEGQNVTLS CEQNLNHDAM YWYRQDPGQG LRLIYYSQIM GDEQKGDIAE GYSVSREKKE SFPLTVTSAQ KNPTAFYLCA SSWWTGGASP IRFGPGTRLT VTEDLKNVFP PEVAVFEPSE AEISHTQKAT LVCLATGFYP DHVELSWWVN GKEVHSGVCT DPQPLKEQPA LQDSRYALSS RLRVSATFWQ DPRNHFRCQV QFYGLSENDE WTQDRAKPVT QIVSAEAWGR ADGGGSGGGG EPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YGSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL SCAVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LVSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GKGGGSGGGG GDAKTTQPNS MESNEEEPVH LPCQHSTISG TDYIHWYRQL PSQGPEYVIH GLTSNVNNRM ASLAIAEDRK SSTLILHRAT LRDAAVYYCI LILGHSRLGN YIATFGKGTK LSVIPNIQNP DPAVYQLRDS KSSDKSVCLF TDFDSQTQVS QSKDSDVYIT DKCVLDMRSM DFKSNSAVAW SQKSDFACAN AFQNSIIPED TGGGSGGGGE PKSSDKTHTC PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY GSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRD ELTKNQVSLW CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K

[0142] Exemplary two chain PRAME TCR antiCD3 bispecific fusion molecule

[0143] TCR alpha chain (SEQ ID NO:24)

[0144] GDAKTTQPNS MESNEEEPVH LPCNHSTISG TDYIHWYRQL PSQGPEYVIH GLTSNVNNRM ASLAIAEDRK SSTLILHRAT LRDAAVYYCI LILGHSRLGN YIATFGKGTK LSVIPNIQNP DPAVYQLRDS KSSDKSVCLF TDFDSQTNVS QSKDSDVYIT DKCVLDMRSM DFKSNSAVAW SNKSDFACAN AFNNSIIPED T

[0145] U144 scFv (underlined) fused to N terminus of TCR beta chain via a G4S linker (italics, SEQ ID NO 59) (SEQ ID NO:25). U144 scFv comprises VL SEQ ID NO 13, VH SEQ ID NO 14, linker is SEQ ID NO 19

[0146] AIQMTQSPSS LSASVGDRVT ITCRASQGIR KYLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ GNTLPWTFGQ GTKVEIKGGG GSGGGGSGGG GSGGGGSGGG SEVQLLESGG GLVQPGGSLR LSCAASGYRF TGYLMNWVRQ APGKGLEWVS AINPYKGSTY YADSVKGRFT FSRDNSKNTA YLQMNSLRAE DTAVYYCARS GYYGDSDWYF DLWGRGTLVT VSSGGGGSDG GITQSPKYLF RKEGQNVTLS CEQNLNHDAM YWYRQDPGQG LRLIYYSQIM GDEQKGDIAE GYSVSREKKE SFPLTVTSAQ KNPTAFYLCA SSWWTGGASP IRFGPGTRLT VTEDLKNVFP PEVAVFEPSE AEISHTQKAT LVCLATGFYP DHVELSWWVN GKEVHSGVCT DPQPLKEQPA LNDSRYALSS RLRVSATFWQ DPRNHFRCQV QFYGLSENDE WTQDRAKPVT QIVSAEAWGR AD

[0147] Alternatively, U183 scFv (underlined) fused to N terminus of TCR beta chain via a G4S linker (italics, SEQ ID NO 59) (SEQ ID NO:119). U183 scFv comprises VL SEQ ID NO 86, VH SEQ ID NO 87, linker is SEQ ID NO 19

[0148] AIQMTQSPSS LSASVGDRVT ITCRASQGIR KYLNWYQQKP GKAPKLLIYA ASSLESGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ GNTLPWTFGC GTKVEIKGGG GSGGGGSGGG GSGGGGSGGG SEVQLLESGG GLVQPGGSLR LSCAASGYRF TGYLMNWVRQ APGKCLEWVS AINPYKGSTY YADSVKGRFT FSRDNSKNTA YLQMNSLRAE DTAVYYCARS GYYGDSDWYF DLWGQGTLVT VSSGGGGGDG GITQSPKYLF RKEGQNVTLS CEQNLNHDAM YWYRQDPGQG LRLIYYSQIM GDEQKGDIAE GYSVSREKKE SFPLTVTSAQ KNPTAFYLCA SSWWTGGASP IRFGPGTRLT VTEDLKNVFP PEVAVFEPSE AEISHTQKAT LVCLATGFYP DHVELSWWVN GKEVHSGVCT DPQPLKEQPA LNDSRYALSS RLRVSATFWQ DPRNHFRCQV QFYGLSENDE WTQDRAKPVT QIVSAEAWGR AD

[0149] Exemplary single chain MAGEA4 TCRm antiCD3 bispecific fusion molecule - SEQ ID NO:69 U144 scFv (underlined; comprises VL SEQ ID NO 13, VH SEQ ID NO 14, linker SEQ ID NO 19) fused to the C terminus of a TCRm VHH domain (SEQ ID NO 67) via a GS linker (italics) EVQLVQSGAE VKKPGSSVKV SCKASGGTFS SYAISWVRQA PGQGLEWMGR IIPILGIANY AQKFQGRVTI TADKSTSTAY MELSSLRSED TAVYYCARVG YCSSTSCLEY YYGMDVWGQG TTVTVS SAAA GSAIQMTQSP SSLSASVGDR VTITCRASQG IRKYLNWYQQ KPGKAPKLLI YAASSLQSGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC QQGNTLPWTF GQGTKVEIKGGGG SGGGGSG GGGSGGGGSGG GGSEVQLLES GGGLVQPGGS LRLSCAASGY RFTGYLMNWV RQAPGKGLEW VSAINPYKGS TYYADSVKGR FTFSRDNSKN TAYLQMNSLR AEDTAVYYCA RSGYYGDSDW YFDLWGRGTL VTVSS

[0150] Exemplary single chain MAGEA4 TCRm antiCD3 bispecific fusion molecule - SEQ ID NQ:120 U183 scFv (underlined; comprises VL SEQ ID NO 86, VH SEQ ID NO 87, linker SEQ ID NO 19) fused to the C terminus of a TCRm VHH domain (SEQ ID NO 67) via a GS linker (italics) EVQLVQSGAE VKKPGSSVKV SCKASGGTFS SYAISWVRQA PGQGLEWMGR IIPILGIANY AQKFQGRVTI TADKSTSTAY MELSSLRSED TAVYYCARVG YCSSTSCLEY YYGMDVWGQG TTVTVSSAAA GSAIQMTQSP SSLSASVGDR VTITCRASQG IRKYLNWYQQ KPGKAPKLLI YAASSLESGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC QQGNTLPWTF GCGTKVEIKG GGGSGGGGSG GGGSGGGGSG GGSEVQLLES GGGLVQPGGS LRLSCAASGY RFTGYLMNWV RQAPGKGLEW VSAINPYKGS TYYADSVKGR FTFSRDNSKN TAYLQMNSLR AEDTAVYYCA RSGYYGDSDW YFDLWGQGTL VTVSS

[0151] Exemplary HLA-A*02 restricted peptides

[0152] MAGEA4 (SEQ ID NO:26)

[0153] GVYDGREHTV PRAME (SEQ ID NO:27)

[0154] SLLQHLIGL PIWIL (SEQ ID NO:28)

[0155] SLSNRLYYL

[0156] CDRs

[0157] The antibodies that bind CD3 disclosed herein comprise light chain Complementarity Determining Regions (CDRs) VLCDR1, VLCDR2 and VLCDR3, and heavy chain CDRs VHCDR1, VHCDR2, VHCDR3. CDRs are defined according to the international ImMunoGeneTics information system (IMGT) (LeFranc et al., Nucleic Acids Res. 2009 Jan;37(Database issue): D1006-12).

[0158] As used herein, the terms U183, U144, U42, U112, U123, U152, U154, U165, U166, U167, U170, U175, U179, U181, U182, U184, U185, U186, U189, U190, U192, U193, U194, and U195, U196, U197, U198, U199, AND U201 refer to sequences of antibodies of the invention, as defined by the sequences above.

[0159] The antibody of the invention or antigen binding fragment thereof which binds CD3 may comprise any combination of VLCDRs disclosed herein. The antibody or antigen binding fragment thereof which binds CD3 may comprise any VLCDR1, any VLCDR2 and any VLCDR3 selected from the table below.

[0160] VLCDR1 VLCDR2 VLCDR3

[0161] QGIRKY (SEQ ID NO: 29) AAS (SEQ ID NO: 32) QQGNTLPWT(SEQ ID NO: 34) QGIRNY (SEQ ID NO: 30) YTS (SEQ ID NO: 33) QQGNTDPWT (SEQ ID NO: 106) QDIRNY (SEQ ID NO: 31)

[0162]

[0163] The antibody or antigen binding fragment thereof which binds CD3 may comprise a combination of VLCDRs selected from the following,

[0164] a) VLCDR1 is SEQ ID NO: 29, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO:34, b) VLCDR1 is SEQ ID NO: 30, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 34, c) VLCDR1 is SEQ ID NO: 31, VLCDR2 is SEQ ID NO: 33, VLCDR3 is SEQ ID NO: 34, or d) VLCDR1 is SEQ ID NO: 29, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 106, or a sequence with at least 70 % identity thereto.

[0165] Preferably, the antibody or antigen binding fragment thereof which binds CD3 comprises a combination of CDRs in which VLCDR1 is SEQ ID NO: 29, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO:34, or a sequence with at least 70 % identity thereto. The antibody or antigen binding fragment thereof which binds CD3 may comprise any combination of VHCDRs disclosed herein. The antibody or antigen binding fragment thereof which binds CD3 may comprise any VHCDR1, any VHCDR2 and any VHCDR3 selected from the table below.

[0166] VHCDR1 VHCDR2 VHCDR3

[0167] GYRFTGYL (SEQ ID INPYKGST (SEQ ID NO: ARSGYYGDSDWYFDL (SEQ ID NO: NO: 35) 39) 42)

[0168] GYRFTRYL(SEQ ID INPYKGSS (SEQ ID NO: ARSGYYGDSDWYFDV (SEQ ID NO: NO: 36) 40) 43)

[0169] GYSFTGYA (SEQ ID INPYKGVS (SEQ ID NO: ARSGYYGDDDWYFDL (SEQ ID NO: NO: 37) 41) 109)

[0170] GYSFTGYT (SEQ ID

[0171] NO: 38)

[0172] GFRFTGYL (SEQ ID

[0173] NO: 107)

[0174] GYRFTGYA (SEQ ID

[0175]

[0176] NO: 108)

[0177] The antibody or antigen binding fragment thereof which binds CD3 may comprise a combination of VHCDRs selected from the following

[0178] a) VHCDR1 is SEQ ID NO: 35, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 42, b) VHCDR1 is SEQ ID NO: 35, VHCDR2 is SEQ ID NO: 41, VHCDR3 is SEQ ID NO: 43, c) VHCDR1 is SEQ ID NO: 35, VHCDR2 is SEQ ID NO: 40, VHCDR3 is SEQ ID NO: 42, d) VHCDR1 is SEQ ID NO: 36, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 42, e) VHCDR1 is SEQ ID NO: 35, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 109, f) VHCDR1 is SEQ ID NO: 107, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 42, g) VHCDR1 is SEQ ID NO: 108, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 42, or

[0179] h) VHCDR1 is SEQ ID NO: 108, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 109, or a sequence with at least 70 % identity thereto.

[0180] Preferably, the antibody or antigen binding fragment thereof which binds CD3 comprises a combination of CDRs in which VHCDR1 SEQ ID NO: 35, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 42, or a sequence with at least 70 % identity thereto.

[0181] The antibody or antigen binding fragment thereof which binds CD3 may comprise any combination of VLCDRs and VHCDRs disclosed herein, or a sequence with at least 70 % identity thereto. The antibody or antigen binding fragment thereof which binds CD3 may comprise any combination of VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2 and VHCDR3 disclosed herein. The antibody or antigen binding fragment thereof that binds CD3 may comprise any of the following combinations of CDRs:

[0182] a) VLCDR1 is SEQ ID NO:29, VLCDR2 is SEQ ID NO:32, VLCDR3 is SEQ ID NO:34, VHCDR1 is SEQ ID NO:35, VHCDR2 is SEQ ID NO:39, VHCDR3 is SEQ ID NO:42, b) VLCDR1 is SEQ ID N0:31, VLCDR2 is SEQ ID NO:33, VLCDR3 is SEQ ID NO:34, VHCDR1 is SEQ ID NO:35, VHCDR2 is SEQ ID N0:41, VHCDR3 is SEQ ID NO:43,

[0183] c) VLCDR1 is SEQ ID N0:31, VLCDR2 is SEQ ID NO:33, VLCDR3 is SEQ ID NO: 34, VHCDR1 is SEQ ID NO:35, VHCDR2 is SEQ ID NO:39, VHCDR3 is SEQ ID NO:42,

[0184] d) VLCDR1 is SEQ ID NQ:30, VLCDR2 is SEQ ID NO:32, VLCDR3 is SEQ ID NO:34, VHCDR1 is SEQ ID NO:35, VHCDR2 is SEQ ID NO:39, VHCDR3 is SEQ ID NO:42,

[0185] e) VLCDR1 is SEQ ID NQ:30, VLCDR2 is SEQ ID NO:32, VLCDR3 is SEQ ID NO:34, VHCDR1 is SEQ ID NO:36, VHCDR2 is SEQ ID NO:39, VHCDR3 is SEQ ID NO:42,

[0186] f) VLCDR1 is SEQ ID NQ:30, VLCDR2 is SEQ ID NO:32, VLCDR3 is SEQ ID NO:34, VHCDR1 is SEQ ID NO:35, VHCDR2 is SEQ ID NQ:40, VHCDR3 is SEQ ID NO:42,

[0187] g) VLCDR1 is SEQ ID NO: 29, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 106, VHCDR1 is SEQ ID NO: 35, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 42,

[0188] h) VLCDR1 is SEQ ID NO: 29, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 106, VHCDR1 is SEQ ID NO: 107, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 42,

[0189] i) VLCDR1 is SEQ ID NO:29, VLCDR2 is SEQ ID NO:32, VLCDR3 is SEQ ID NO:34, VHCDR1 is SEQ ID NO:35, VHCDR2 is SEQ ID NO:39, VHCDR3 is SEQ ID NQ:109

[0190] j) VLCDR1 is SEQ ID NO: 29, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 34, VHCDR1 is SEQ ID NO: 107, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 42,

[0191] k) VLCDR1 is SEQ ID NO: 29, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 34, VHCDR1 is SEQ ID NO: 108, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 42,

[0192] l) VLCDR1 is SEQ ID NO: 30, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 34, VHCDR1 is SEQ ID NO: 35, VHCDR2 is SEQ ID NO: 39, and VHCDR3 is SEQ ID NO: 42, m) VLCDR1 is SEQ ID NO: 30, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 34, VHCDR1 is SEQ ID NO: 108, VHCDR2 is SEQ ID NO: 39, and VHCDR3 is SEQ ID NO: 42, n) VLCDR1 is SEQ ID NO: 30, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 34, VHCDR1 is SEQ ID NO: 35, VHCDR2 is SEQ ID NO: 39, and VHCDR3 is SEQ ID NO: 109, o) VLCDR1 is SEQ ID NO: 29, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 34, VHCDR1 is SEQ ID NO: 108, VHCDR2 is SEQ ID NO: 39, and VHCDR3 is SEQ ID NO: 109, or p) VLCDR1 is SEQ ID NO: 30, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 34, VHCDR1 is SEQ ID NO: 108, VHCDR2 is SEQ ID NO: 39, and VHCDR3 is SEQ ID NO: 109, or a sequence with at least 70 % identity thereto.

[0193] Preferably, the antibody or antigen binding fragment thereof that binds CD3 comprises a combination of CDRs in which VLCDR1 is SEQ ID NO:29, VLCDR2 is SEQ ID NO:32, VLCDR3 is SEQ ID NO:34, VHCDR1 is SEQ ID NO:35, VHCDR2 is SEQ ID NO:39, VHCDR3 is SEQ ID NO:42.

[0194] Frameworks

[0195] The CDRs may be provided in an antibody variable domain framework sequence. The framework sequence may be a mouse framework sequence or a human framework sequence or a humanised framework sequence or any other suitable framework. The framework may be a human or humanised framework sequence. Human or humanised frameworks have substantially the amino acid sequence of a human immunoglobulin. In certain cases, mouse frameworks, human frameworks and humanised frameworks may be mixed in any combination.

[0196] Preferably, the antibody or antigen binding fragment thereof which binds CD3 comprises a light chain variable domain (VL) and / or a heavy chain variable domain (VH). Preferably, the VL domain comprises an overall sequence VLFW1-VLCDR1-VLFW2-VLCDR2-VLFW3-VLCDR3-VLFW4, wherein VLFW1, VLFW2, VLFW3, and VLFW4 are VL framework (VLFW) sequences 1 to 4 respectively. The antibody or antigen binding fragment thereof which binds CD3 may comprise any combination of VLFW sequences described herein or a sequence with at least 70 % identity thereto. The antibody or antigen binding fragment thereof which binds CD3 may comprise any VLFW1, any VLFW2, VLFW3 and any VLFW4 selected from the table below.

[0197] VLFW1 VLFW2 VLFW3 VLFW4 AIQMTQSPSSLSASVGD LNWYQQKPGK SLQSGVPSRFSGSGSGTDFT FGQGTKVEIK RVTITCRAS (SEQ ID NO: APKLLIY (SEQ LTISSLQPEDFATYYC(SEQ ID (SEQ ID NO: 44) ID NO: 45) NO: 46) 48)

[0198] RLESGVPSRFSGSGSGTDYT FGCGTKVEIK LTISSLQPEDFATYYC (SEQ (SEQ ID NO: ID NO: 47) 111) SLESGVPSRFSGSGSGTDFT LTISSLQPEDFATYYC (SEQ

[0199] ID NO: 110)

[0200]

[0201] The VL domain may comprise:

[0202] a VLFW1 sequence of SEQ ID NO: 44,

[0203] a VLFW2 sequence of SEQ ID NO: 45,

[0204] a VLFW3 sequence selected from SEQ ID NO: 46, SEQ ID NO: 47 or SEQ ID NO: 110, and a VLFW4 sequence of SEQ ID NO: 48 or SEQ ID NO: 111,

[0205] or a sequence with at least 70 % identity thereto.

[0206] The VL domain may comprise a combination of any of the VLFW sequences disclosed herein. For example, the VL domain may comprise VLFW sequences in which VLFW1 is SEQ ID NO: 44; VLFW2 is SEQ ID NO: 45, VLFW3 is SEQ ID NO: 46, and VLFW4 is SEQ ID NO: 48, or sequences with at least 70 % identity thereto. Alternatively, the VL domain may comprise a combination of VLFW sequences in which VLFW1 is SEQ ID NO: 44; VLFW2 is SEQ ID NO: 45, VLFW3 is SEQ ID NO: 47, and VLFW4 is SEQ ID NO: 48, or sequences with at least 70 % identity thereto. Alternatively, the VL domain may comprise a combination of VLFW sequences in which VLFW1 is SEQ ID NO: 44; VLFW2 is SEQ ID NO: 45, VLFW3 is SEQ ID NO: 110, and VLFW4 is SEQ ID NO: 48, or sequences with at least 70 % identity thereto. Alternatively, the VL domain may comprise a combination of VLFW sequences in which VLFW1 is SEQ ID NO: 44; VLFW2 is SEQ ID NO: 45, VLFW3 is SEQ ID NO: 110, and VLFW4 is SEQ ID NO: 111, or sequences with at least 70 % identity thereto.

[0207] Preferably, the VH domain comprises an overall sequence as follows: VHFW1-VHCDR1-VHFW2-VHCDR2-VHFW3-VHCDR3-VHFW4, wherein VHFW1, VHFW2, VHFW3, and VHFW4 are framework (FW) sequences 1 to 4 respectively. The antibody or antigen binding fragment thereof which binds CD3 may comprise any combination of VHFW sequences described herein or a sequence with at least 70 % identity thereto. The antibody or antigen binding fragment thereof which binds CD3 may comprise any VHFW1, any VHFW2, VHFW3 and any VHFW4 selected from the table below.

[0208] VHFW1 VHFW2 VHFW3 VHFW4 EVQLLESGGGLVQP MNWVRQAPGKG YY ADSVKGRFTF WGRGTLVTVSS GGSLRLSCAAS (SEQ LEWVSA (SEQ ID SRDNSKNTAY (SEQ ID NO: 55) ID NO: 49) NO:51) LQMNSLRAED TAVYYC

[0209] (SEQ ID NO: 53)

[0210] EVQLVESGGGLVQP MNWVRQA TYNQKFKDRFTFSVDKSKN WGQGTLVTVSS GGSLRLSCAAS GKGLEWVAL TAYLQMNSLRAEDTAVYYC (SEQ ID NO: 56) (SEQ ID NO: 50) (SEQ ID NO: 52) (SEQ ID NO: 54)

[0211] MNWVRQAPGKC TY NQKFKDRFTI WGQGTLVTVSS LEWVSA (SEQ ID SVDKSKNTAY (SEQ ID NO: 114) NO: 112) LQMNSLRAED TAVYYC

[0212] (SEQ ID NO: 57) YYADSVKGRFTFSVDNSKN TAYLQMNSLRAEDTAVYYC

[0213] (SEQ ID NO: 113)

[0214]

[0215] The VH domain may comprise a combination of any VHFW sequences disclosed herein, or a sequence with at least 70% identity thereto. For example the VHFW1 may comprise

[0216] a VHFW1 sequence selected from SEQ ID NO: 49 or SEQ ID NO:50,

[0217] a VHFW2 sequence selected from SEQ ID NO:51, SEQ ID NO:52 or SEQ ID NO: 112. a VHFW3 sequence selected from SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:57, or SEQ ID NO: 113, and

[0218] a VHFW4 sequence selected from SEQ ID NO:55, SEQ ID NO:56 or SEQ ID NO: 114, or a sequence with at least 70 % identity thereto.

[0219] The VH domain may comprise a combination of VHFW sequences in which VHFW1 is SEQ ID NO:49, VHFW2 is SEQ ID NO:51, VHFW3 is SEQ ID NO:53 and VHFW4 is SEQ ID NO:55, or a sequence with at least 70% identity thereto. Alternatively, the VH domain may comprise a combination of VHFW sequences in which VHFW1 is SEQ ID NO:50, VHFW2 is SEQ ID NO:52, VHFW3 is SEQ ID NO:54, and VHFW4 is SEQ ID NO:56, or sequences with at least 70 % identity thereto. Alternatively, the VH domain may comprise a combination of VHFW sequences in which VHFW1 is SEQ ID NO: 49, VHFW2 is SEQ ID NO:112, VHFW3 is SEQ ID NO:53, and VHFW4 is SEQ ID NO:55, or sequences with at least 70 % identity thereto. Alternatively, the VH domain may comprise a combination of VHFW sequences in which VHFW1 is SEQ ID NO:49, VHFW2 is SEQ ID NO:51, VHFW3 is SEQ ID NO:53, and VHFW4 is SEQ ID NO:114, or sequences with at least 70 % identity thereto. Alternatively, the VH domain may comprise a combination of VHFW sequences in which VHFW1 is SEQ ID NO:49, VHFW2 is SEQ ID NO:51, VHFW3 is SEQ ID NO:113, and VHFW4 is SEQ ID NO:55, or sequences with at least 70 % identity thereto. Alternatively, the VH domain may comprise a combination of VHFW sequences in which VHFW1 is SEQ ID NO:49, VHFW2 is SEQ ID NO:112, VHFW3 is SEQ ID NO:53, and VHFW4 is SEQ ID NO:114, or sequences with at least 70 % identity thereto. Alternatively, the VH domain may comprise a combination of VHFW sequences in which VHFW1 is SEQ ID NO:49, VHFW2 is SEQ ID NO:112, VHFW3 is SEQ ID NO:113, and VHFW4 is SEQ ID NO:114, or sequences with at least 70 % identity thereto.

[0220] The antibody or antigen binding fragment thereof that binds CD3 may comprise a VL sequence selected from SEQ ID NO: 86, SEQ ID NO: 13, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO: 82, SEQ ID NO:84, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127 and SEQ ID NO:129, or a sequence with at least 70 % identity thereto.

[0221] The antibody or antigen binding fragment thereof which binds CD3 may comprise a VH sequence selected from SEQ ID NO: 87, SEQ ID NO:14, SEQ ID NO:8, SEQ ID NQ:10, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, and SEQ ID NQ:130, or a sequence with at least 70 % identity thereto.

[0222] The antibody or antigen binding fragment thereof which binds CD3 may comprise any combination of any VL and VH sequence described herein. For example the antibody or antigen binding fragment thereof which binds CD3 may comprise a VL and VH selected from the following combinations: SEQ ID NO: 86 and SEQ ID NO: 87, SEQ ID NO: 13 and SEQ ID NO:14, SEQ ID NO:7 and SEQ ID NO: 8, SEQ ID NO:9 and SEQ ID NQ:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:15 and SEQ ID NO: 16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:70 and SEQ ID NO: 71, SEQ ID NO:72 an SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO:75, SEQ ID NO:76 and SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:79, SEQ ID NO:80 and SEQ ID NO:81, SEQ ID NO:82 and SEQ ID NO:83, SEQ ID NO:84 and SEQ ID NO:85, SEQ ID NO:88 and SEQ ID NO:89, SEQ ID NO:90 and SEQ ID NO:91, SEQ ID NO:92 and SEQ ID NO:93, SEQ ID NO:94 and SEQ ID NO:95, SEQ ID NO:96 and SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:99, SEQ ID NO:100 and SEQ ID NO:101, SEQ ID NO:102 and SEQ ID NO:103, SEQ ID NO: 104 and SEQ ID NO:105, SEQ ID NO: 121 and SEQ ID NO:122, SEQ ID NO:123 and SEQ ID NO:124, SEQ ID NO:125 and SEQ ID NO:126, SEQ ID NO: 127 and SEQ ID NO: 128, and SEQ ID NO: 129 and SEQ ID NO: 130, or a sequence with at least 70 % identity thereto.

[0223] Preferably, the antibody or antigen binding fragment thereof that binds CD3 may comprise the VL and VH sequences of SEQ ID NO: 86 and SEQ ID NO: 87, or SEQ ID NO: 13 and SEQ ID NO:14 or a sequence with at least 70 % identity thereto. More preferably, the antibody or antigen binding fragment thereof that binds CD3 may comprise the VL and VH sequences of SEQ ID NO: 86 and SEQ ID NO:87 or a sequence with at least 70 % identity thereto.

[0224] The immunoglobin VL and VH domains of the antibody or antigen binding fragment thereof that binds CD3 may be connected via linker. The linker may be any amino acid sequence, preferably 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. A preferred linker comprises a sequence with the formula (GGGGS)n, optionally in addition to other amino acids. Suitable linkers are known in the art. The linker may be (GGGGS)3, (GGGGS)4 or preferably (GGGGS)5. A more preferred linker is (GGGGS)4+GGGS.

[0225] The antigen binding fragment that specifically binds CD3 may be in the form of a scFv fragment. The antibody or antigen binding fragment thereof of the invention may preferably bind the CD3 epsilon sub-unit of CD3

[0226] The antibodies of the present invention or antigen binding fragment thereof that specifically binds CD3 may comprise antibody constant regions or parts thereof. For example, the antibody or fragment thereof that specifically binds CD3 may be attached at the C-terminal end to antibody light chain constant domains including human CKor CK chains. The antibody or antigen binding fragment thereof that binds CD3 may be attached to an antibody light chain constant domain via the VL domain. Similarly, the antibody or fragment thereof that binds CD3 may be attached at their C-terminal end to all or part of an immunoglobulin heavy chain derived from any antibody isotype, e.g. IgG, IgA, IgE and IgM and any of the isotype sub-classes, particularly lgG1 and lgG4. The antibody or antigen binding fragment thereof that binds CD3 may be attached to all or part of the immunoglobulin heavy chain via the VH domain.

[0227] Multi-specific binding molecules

[0228] The antibody or antigen binding fragment thereof that specifically binds CD3 of the invention may be part of a multi-specific binding molecule. The multi-specific binding molecule may comprise an antibody or antigen binding fragment thereof that binds CD3 according to the invention and a targeting moiety. The multi-specific binding molecule may further comprise any suitable further moiety.

[0229] The targeting moiety may be a T cell receptor (TCR), an antibody, an antigen binding fragment thereof, or other specific binding molecule. The T cell receptor (TCR) may be a heterodimeric alpha / beta or gamma / delta TCR polypeptide pair. The T cell receptor (TCR) may be a single chain or multi-chain TCR polypeptide. The arrangement of the antibody or antigen binding fragment thereof that binds CD3 and targeting moiety can be in any known format (such as described in WO2025 / 133349 and Brinkman et al., Mabs. 2017 Feb-Mar; 9(2): 182-212).

[0230] The antibody or antigen binding fragment thereof which binds CD3 may be fused to the C or N terminus of the targeting moiety. For example, when the targeting moiety is a TCR, the antibody or antigen binding fragment thereof that binds CD3 may be fused to the N or C terminus of the alpha or beta chain of the T cell receptor (TCR). I antibody or antigen binding fragment thereof may be fused to the N terminus of the beta chain of the TCR. The multi-specific binding molecule may be in the form of a diabody, in which the TCR-Va is attached to antiCD3-VL and the TCR-Vb is attached to the antiCD3VH and vice versa. Alternatively, the antibody or antigen binding fragment thereof may be linked to the N terminus of the TCR beta chain variable domain.

[0231] In some cases, the antibody that specifically binds CD3 or antigen binding fragment thereof that specifically binds CD3 may be fused to the C or N terminus of the targeting moiety. In other cases, the antibody or antigen binding fragment thereof that binds CD3 is fused to the C or N terminus of the targeting moiety via a linker.

[0232] The antibody or antigen binding fragment thereof that binds CD3 may be fused to a T cell receptor (TCR) or a TCR-like antibody, optionally via a linker, which may be a polypeptide linker. Polypeptide linker sequences are usually flexible, in that they are made up of amino acids such as glycine, alanine and serine which do not have bulky side chains likely to restrict flexibility. Usable or optimum lengths of linker sequences are easily determined. Often the linker sequence will be less than about 12, such as less than 10, or from 5-10 amino acids in length The linker may be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. A preferred linker includes a sequence with the formula (GGGGS)n, optionally in addition to other amino acids.

[0233] When a TCR is present, the TCR may comprise a non-native di-sulphide bond between the constant region of the alpha chain and the constant region of the beta chain. The TCR may bind to MHC in complex with a peptide antigen. Preferably, the peptide antigen is any disease associated antigen. The peptide antigen may be any tumour associated antigen, or any infectious disease associated antigen. The peptide antigen may be a peptide derived from GP100, NYESO, MAGEA4, or PRAME as described in WO2011001152, WO2017109496, WO2017175006 and WO2018234319. A suitable TCR may have an amino acid sequence as defined in WO2011001152, WO2017109496, WO2017175006 and WO2018234319; the sequences of the TCRs disclosed therein are incorporated by reference.

[0234] The multi-specific binding molecule may comprise an antibody or antigen binding fragment thereof that specifically binds CD3 comprising any of the sequences of the invention. The targeting moiety may comprise a TCR with an amino acid sequence as defined in WO2011001152, WO2017109496, WO2017175006 and WO2018234319. For example, the antibody or antigen binding fragment thereof that binds CD3 may comprise a combination of CDRs in which VLCDR1 is SEQ ID NO:29, VLCDR2 is SEQ ID NO:32, VLCDR3 is SEQ ID NO:34, VHCDR1 is SEQ ID NO:35, VHCDR2 is SEQ ID NO:39, VHCDR3 is SEQ ID NO:42. The multi-specific binding molecule may comprise a sequence corresponding to (a) SEQ ID Nos: 20 and 21, (b) SEQ ID NO: 22, (c) SEQ ID NO: 23, (d) SEQ ID NOs: 24 and 25, (e) SEQ ID NO: 69, (f) SEQ ID Nos: 20 and 116, (g) SEQ ID NO: 117, (h) SEQ ID NO: 118, (i) SEQ ID Nos: 24 and 119, and (j) SEQ ID NO: 120, or a sequence with at least 70% identity thereto.

[0235] The antibody or antigen binding fragment thereof which binds CD3 may be fused to the C or N terminus of the targeting moiety, optionally via a linker. The multi-specific binding molecule may be constructed via N-terminal or C-terminal fusion to the antibody or fragment thereof which binds CD3, optionally via either the VL or VH domains. Further domains may be fused to the multi-specific binding molecule, optionally via linkers. Linker sequences are usually flexible, in that they are made up primarily of amino acids such as glycine, alanine and serine, which do not have bulky side chains likely to restrict flexibility. Alternatively, linkers with greater rigidity may be desirable. Usable or optimum lengths of linker sequences may be easily determined. Often the linker sequence will be less than about 12, such as less than 10, or from 2-10 amino acids in length. The linker may be any amino acid sequence, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. Examples of suitable linkers include, but are not limited to: GGGSGGGG (SEQ ID NO: 58), GGGGS (SEQ ID NO: 59), GGGSG (SEQ ID NO: 60), GGSGG(SEQ ID NO: 61), GSGGG (SEQ ID NO: 62), GSGGGP (SEQ ID NO: 63), GGEPS (SEQ ID NO: 64), GGEGGGP (SEQ ID NO: 65), GGEGGGSEGGGS (SEQ ID NO: 66) (as described in WQ2010 / 133828), and ASGGSGGGGSG GGGSGGGGSGGGS (SEQ ID NO 68). A preferred linker includes a sequence with the formula (GGGGS)n, optionally in addition to other amino acids.

[0236] The multi-specific binding molecule may be a T cell engager. As used herein, the term “T cell engager” relates to any multi-specific binding molecule capable of binding to (i) a T cell via the antibody or fragment thereof that binds CD3 of the invention, and (ii) a target cell via the targeting moiety. The multi-specific binding molecule may be a BiTE (bispecific T cell engager), or a DART (Dual-affinity re-targeting antibody). The targeting moiety may be an antibody or antigen binding fragment thereof, a T-cell receptor or binding fragment thereof, or other specific binding molecule. The targeting moiety may be a TCR-like antibody (also called a TCR mimic, or TCRm). As used herein and discussed above, the term antigen binding fragment includes functional fragments, variants and derivatives thereof.

[0237] The term “TCR” or “T cell receptor” includes functional fragments, variants and derivatives thereof. For example, the targeting moiety may be a single chain TCR (scTCR), a TCR variable domain, such as Valpha or Vbeta, or a soluble TCR. In the case of a TCR, the TCR binds to a peptide-MHC complex where the peptide is from an antigen associated with a diseased or cancerous state, and is presented on the surface of the cell in the context of MHC

[0238] The multi-specific binding molecule may further comprise a half life extending moiety or domain. The half-life extending domain may comprise a first IgG Fc region (FC1) and a second IgG Fc region (FC2), wherein the FC1 region and FC2 region dimerise to form an Fc domain. A “half-life extending domain”, as used herein, refers to a protein domain for extending the half-life of the multi-domain binding protein, relative to a multi-domain binding protein lacking the half-life extending domain. As used herein, the term “Fc region” is used to refer to a region of a single polypeptide chain comprising at least a CH2 domain and a CH3 domain sequence, whereas the term “Fc domain” refers to a dimer of two Fc regions (i.e., FC1 and FC2).

[0239] The targeting moiety may be a TCR-like antibody, or antigen binding fragment thereof. TCR-like antibodies may be called TCR mimics, or TCRm. TCR-like antibodies recognise peptides when in complex with MHC (such complexed are known as pMHC). A TCR-like antibody may specifically bind to a peptide when in complex with MHC. The multi-specific binding molecule of the invention may comprise a TCR-like antibody, and an antibody or antigen binding fragment thereof that specifically binds CD3.

[0240] “Identity” as known in the art is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. While there exist a number of methods to measure identity between two polypeptide or two polynucleotide sequences, methods commonly employed to determine identity are codified in computer programs. Preferred computer programs to determine identity between two sequences include, but are not limited to, GCG program package (Devereux, et al., Nucleic Acids Research, 12, 387 (1984), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. 215, 403 (1990)).

[0241] Preferably, the amino acid sequence of the protein has at least 70% identity, using the default parameters of the BLAST computer program, at the amino acid level, to the amino acid sequences disclosed herein. More preferably, the protein sequence may have at least 75%, 80%, 85%, 90% and still more preferably 95% (still more preferably at least 96%, 97% 98% or 99%) herein.

[0242] As will be obvious to those skilled in the art, it may be possible to truncate, or extend, the sequences provided at the C-terminus and / or N-terminus thereof, by 1, 2, 3, 4, 5 or more residues, without substantially affecting the functional characteristics of the specific binding molecule. The sequences provided at the C-terminus and / or N-terminus thereof may be truncated or extended by 1, 2, 3, 4 or 5 residues. All such variants are encompassed by the present invention.

[0243] TCRs described herein may be ap heterodimers or y5 heterodimers. Alpha-beta heterodimeric TCRs usually comprise an alpha chain TRAC constant domain sequence and / or a beta chain TRBC1 or TRBC2 constant domain sequence. The constant domains may be full-length by which it is meant that extracellular, transmembrane and cytoplasmic domains are present, or they may be in soluble format (i.e. having no transmembrane or cytoplasmic domains). One or both of the constant domains may contain mutations, substitutions or deletions relative to the native TRAC and / or TRBC1 / 2 sequences. The term TRAC and TRBC1 / 2 also encompasses natural polymorphic variants, for example N to K at position 4 of TRAC (Bragado et al International immunology. 1994 Feb;6(2):223-30).

[0244] For soluble TCRs, the constant domain sequences may be modified by truncation or substitution to delete the native disulphide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2. The alpha and / or beta chain constant domain sequence(s) may have an introduced disulphide bond between residues of the respective constant domains, as described, for example, in WO 03 / 020763. The alpha and beta constant domains may be modified by substitution of cysteine residues at position Thr 48 of TRAC and position Ser 57 of TRBC1 or TRBC2, the said cysteines forming a disulphide bond between the alpha and beta constant domains of the TCR. TRBC1 or TRBC2 may additionally include a cysteine to alanine mutation at position 75 of the constant domain and an asparagine to aspartic acid mutation at position 89 of the constant domain. One or both of the extracellular constant domains present in an ap heterodimer may be truncated at the C terminus or C termini, for example by up to 15, or up to 10, or up to 8 or fewer amino acids. One or both of the extracellular constant domains present in an ap heterodimer may be truncated at the C terminus or C termini by, for example, up to 15, or up to 10 or up to 8 amino acids. The C terminus of the alpha chain extracellular constant domain may be truncated by 8 amino acids.

[0245] The constant domains of an ap or y5 heterodimeric TCR may be full length, having both transmembrane and cytoplasmic domains. Such TCRs may contain a disulphide bond corresponding to that found in nature between the constant domains. Additionally, or alternatively, a non-native disulphide bond may be present between the extracellular constant domains. Said non-native disulphide bonds are further described in W003020763 and W006000830. The non-native disulphide bond may be between position Thr 48 of TRAC and position Ser 57 of TRBC1 or TRBC2. One or both of the constant domains may contain one or more mutations substitutions or deletions relative to the native TRAC and / or TRBC1 / 2 sequences. TCRs with full-length constant domains are preferable for use in adoptive therapy.

[0246] Single chain formats of TCRs include, but are not limited to, ap TCR polypeptides of the Va-L-Vp, Vp-L-Va, Va-Ca-L-Vp, Va-L-Vp-Cp, or Va-Ca-L-Vp-Cp types, wherein Va and Vp are TCR a and p variable regions respectively, Ca and Cp are TCR a and p constant regions respectively, and L is a linker sequence (WO 2004 / 033685; WO9918129). Where present, one or both of the constant domains may be full length, or they may be truncated and / or contain mutations as described above. The single chain TCRs may be soluble. Single chain TCRs may have an introduced disulphide bond between residues of the respective constant domains.

[0247] The multi-specific molecule of the invention may comprise a soluble TCR, and an antibody or antigen binding fragment thereof that binds CD3, wherein said soluble TCR binds to a peptide-major histocompatibility complex (pMHC) and comprises a TCR alpha chain variable domain (Va) and a TCR beta chain variable domain (Vp); wherein said antibody that binds CD3 or fragment thereof that binds CD3 may comprise an antibody light chain variable domain (VL), and an antibody heavy chain variable domain (VH); and wherein said VH is linked to the N-terminus of the Vp. The multi-specific molecule may not be greater than 60 kDa in size. The multi-specific molecule may be a single chain molecule. The single chain multi-specific molecule may have a format selected from the group consisting of: (i) VL-VH-Vp-Va; (ii) VH-Vp-Va-VL; and (iii) Va-VL-VH-Vp. The multi-specific molecule may be a dual chain molecule. The dual chain multi-specific molecule may comprise: (I) a first chain comprising VH-Vp; and (ii) a second chain comprising Va-VL. The Vp may be linked to the VL via a disulphide bond. Two or more of the Va, Vp, VH and VL may be linked via a linker and / or an IgG hinge sequence. The format may be as described in WO2025 / 133349.

[0248] Antibodies or antigen binding fragments thereof or multi-specific binding molecules may be associated (covalently or otherwise) with a PK (pharmacokinetic) modifying moiety. Examples of PK modifying moieties include, but are not limited to, PEG, PASylation, albumin, and albumin binding domains, and / or unstructured polypeptides. Further PK modifying moieties include antibody Fc fragments.

[0249] Antibodies or antigen binding fragments thereof and variants / analogues which are suitable for use in the compositions and methods described herein include minibodies, Fab fragments, F(ab’)2 fragments, dsFv and scFv fragments, diabodies, Nanobodies™ (these constructs, marketed by Ablynx (Belgium), comprise synthetic single immunoglobulin variable heavy domain derived from a camelid (e.g. camel or llama) antibody) and Domain Antibodies (Domantis (Belgium), comprising an affinity matured single immunoglobulin variable heavy domain or immunoglobulin variable light domain) or alternative protein scaffolds that exhibit antibody like binding characteristics such as Affibodies (Affibody (Sweden), comprising engineered protein A scaffold) or Anticalins (Pieris (Germany)), comprising engineered anticalins) to name but a few. Antibody also includes TCR-like antibodies (Chang et al., Expert Opin Biol Ther. 2016 Aug; 16(8):979-87 and Dahan et al., Expert Rev Mol Med. 2012 Feb 24;14:e6).

[0250] Linkage of the targeting moiety and antibody or fragment thereof which binds CD3 of the first aspect may be via covalent or non-covalent attachment. Covalent attachment may be direct, or indirect via a linker sequence. Linker sequences are usually flexible, in that they are made up primarily of amino acids such as glycine, alanine and serine, which do not have bulky side chains likely to restrict flexibility. Alternatively, linkers with greater rigidity may be desirable. Usable or optimum lengths of linker sequences may be easily determined. Often the linker sequence will be less than about 12, such as less than 10, or from 2-10 amino acids in length, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. Examples of suitable linkers that may be used in the invention include, but are not limited to: GGGSGGGG, GGGGS, GGGSG, GGSGG, GSGGG, GSGGGP, GGEPS, GGEGGGP, and GGEGGGSEGGGS (as described in WO2010 / 133828).

[0251] Sequences disclosed herein may be modified to enhance a specific property or characteristic. For example, to increase or decrease affinity, increase or decrease stability, increase or decrease melting temperature and / r increase or decrease charge. Suitable mutations and methods of introducing said mutations are known in the art.

[0252] Methods

[0253] The antibody or fragment thereof that binds CD3 and multi-specific binding molecules according to the invention may be used in a method of treatment or diagnosis of the human or animal body, such as a method of treatment of a condition in a patient (preferably human) which comprises administering to said patient an effective amount of an antibody, antibody fragment or multi-specific binding molecule of the invention. The invention also provides an antibody or fragment thereof that binds CD3 and / or multi-specific binding molecule of the present invention for use in medicine, as well as the use of an antibody or fragment thereof that binds CD3 and / or multi-specific binding molecule of the present invention in the manufacture of a medicament for the diagnosis or treatment of a disease or tumour.

[0254] As used herein, “treatment” includes any regime that can benefit a human or non-human animal, preferably mammal. The treatment may be in respect of an existing condition or may be prophylactic (preventative treatment).

[0255] “Isolated” refers to the state in which the antibody or fragment thereof or multi-specific binding molecules of the invention or nucleic acid encoding such binding molecules will preferably be, in accordance with the present invention. Molecules and nucleic acid will generally be free or substantially free of material with which they are naturally associated such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practised in vitro or in vivo. Antibodies, fragments thereof, multi-specific binding molecules and nucleic acid may be formulated with diluents or adjuvants and still for practical purposes be isolated - for example, the molecules will normally be mixed with gelatin or other carriers if used to coat microtitre plates for use in immunoassays, or will be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy. Antibodies, fragments thereof and multi-specific binding molecules may be glycosylated, either naturally or by systems of heterologous eukaryotic cells, or they may be (for example if produced by expression in a prokaryotic cell) unglycosylated.

[0256] Furthermore, the antibody or antigen binding fragment thereof or multi-specific binding molecule of the present invention may be administered alone or in combination with other treatments and / or active agents, either simultaneously or sequentially, dependent upon the condition to be treated. The method of treatment may further include administering separately, in combination, or sequentially, an additional agent. The agent may be a anti-neoplastic agent. Examples of such agents are known in the art and may include immune activating agents and / or T cell modulating agents. Thus, the present invention further provides products containing the antibody or fragment thereof or multispecific binding molecule of the present invention and an active agent as a combined preparation for simultaneous, separate or sequential use in the treatment of a disease or tumour. Active agents may include chemotherapeutic or cytotoxic agents including, 5-Fluorouracil, cisplatin, Mitomycin C, oxaliplatin and tamoxifen, which may operate synergistically with the antibody or fragment thereof or multi-specific binding molecule of the present invention. Other active agents may include suitable doses of pain relief drugs such as non-steroidal anti-inflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opitates such as morphine, or anti-emetics.

[0257] The antibody or antigen binding fragment thereof or multi-specific binding molecule of the present invention will usually be administered in the form of a composition or pharmaceutical composition, which may comprise at least one component in addition to the antibody or fragment thereof or multispecific binding molecule. The pharmaceutical composition may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, diluent, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. intravenous.

[0258] The compositions are preferably administered to an individual in a “therapeutically effective amount”, this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. The antibody or antigen binding fragment thereof or multi-specific binding molecule of the present invention may be generated wholly or partly by chemical synthesis. The antibody or antigen binding fragment thereof or multi-specific binding molecule can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available, or they may be prepared in solution, by the liquid phase method or by any combination of solid-phase, liquid phase and solution chemistry, e.g. by first completing the respective peptide portion and then, if desired and appropriate, after removal of any protecting groups being present, by introduction of the residue X by reaction of the respective carbonic or sulfonic acid or a reactive derivative thereof.

[0259] Another convenient way of producing an antibody or antigen binding fragment thereof or multispecific binding molecule according to the present invention is to express the nucleic acid encoding it, by use of nucleic acid in an expression system.

[0260] The present invention further provides an isolated nucleic acid encoding an antibody or fragment thereof of the present invention, or a multi-specific binding molecule of the invention. Nucleic acid includes DNA and RNA. In a preferred aspect, the present invention provides a nucleic acid which codes for an antibody or fragment thereof or multi-specific binding molecule of the invention as defined above. The skilled person will be able to determine substitutions, deletions and / or additions to such nucleic acids which will still provide an antibody or fragment thereof of the present invention or a multi-specific binding molecule of the invention.

[0261] The present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one nucleic acid as described above. The present invention also provides a recombinant host cell which comprises one or more constructs as above. As mentioned, a nucleic acid encoding an antibody or fragment thereof or multi-specific binding molecule of the invention forms an aspect of the present invention, as does a method of production of the antibody or fragment thereof or multi-specific binding molecule which method comprises expression from encoding nucleic acid therefor. Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression, the antibody or fragment thereof or multi-specific binding molecule may be isolated and / or purified using any suitable technique, then used as appropriate.

[0262] Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others. A common, preferred bacterial host is E. coli. The expression of antibodies and antibody fragments in prokaryotic cells such as E. coli is well established in the art. Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of a specific binding molecule.

[0263] Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. ‘phage, or phagemid, as appropriate. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are known.

[0264] Thus, a further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein. A still further aspect provides a method comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene.

[0265] The nucleic acid of the invention may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.

[0266] The present invention also provides a method which comprises using a construct as stated above in an expression system in order to express a specific binding molecule or polypeptide as above.

[0267] Antibodies or antigen binding fragments thereof or multi-specific binding molecules of the present invention can be used in methods of diagnosis and treatment of cancers or infectious diseases, preferably in human or animal subjects. Examples of cancers include but are not limited to liquid tumours such as leukaemias, lymphomas and myeloma and solid tumours including bladder, breast, cervix, colorectal, esophogeal endometrial gastric, glioblastoma, liver, melanoma, lung, ovarian, pancreatic, prostate, sarcoma, thyroid. Examples of infectious diseases include but are not limited to those caused by HIV, HBV, TB and HCV.

[0268] When used in diagnosis, antibodies, antigen binding fragments or multi-specific binding molecules of the invention may be labelled with a detectable label, for example a radiolabel such as131I or99Tc, which may be attached to molecules of the invention using conventional chemistry known in the art of antibody imaging. Labels also include enzyme labels such as horseradish peroxidase. Labels further include chemical moieties such as biotin which may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin.

[0269] Antibodies or antigen binding fragments thereof or multi-specific binding molecules of the present invention may be used in a method of decreasing anti-drug antibody formation in a patient. The antibody or antigen binding fragment thereof that binds CD3 may be preferentially included in known drugs or therapies instead of other anti-CD3 antibodies in order to reduce ADAs in a patient. The antibody or fragment thereof of the invention may be used to replace another anti-CD3 antibody in any appropriate therapy. For example, an antibody or fragment thereof that binds CD3 of the invention may be used in a known T cell engager to reduce ADA. The inventors explicitly contemplate the use of the antibody or fragment thereof of the invention instead of another anti-CD3 antibody to reduce the immunogenicity of a composition comprising an anti-CD3 antibody.

[0270] Methods to determine binding affinity (inversely proportional to the equilibrium constant KD) and binding half life (expressed as T ) are known to those skilled in the art. Preferably, binding affinity and binding half-life are determined using Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI), for example using a BIAcore instrument or Octet instrument, respectively. It will be appreciated that doubling the affinity results in halving the KD. T is calculated as In2 divided by the off-rate (koff). Therefore, doubling of T results in a halving in koff. KD and koff values. To account for variation between independent measurements, and particularly for interactions with dissociation times in excess of 20 hours, the binding affinity and or binding half-life of a given molecule may be measured several times, for example 3 or more times, using the same assay protocol, and an average of the results taken. To compare binding data between two samples (i.e. two different molecules and / or two preparations of the same molecule) it is preferable that measurements are made using the same assay conditions (e.g. temperature), such as those described in WO2018234319.

[0271] For administration to patients, the antibodies, fragments thereof, multi-specific binding molecules, nucleic acids, expression vectors or cells of the invention may be provided as part of a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. This pharmaceutical composition may be in any suitable form, (depending upon the desired method of administering it to a patient). It may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms.

[0272] The pharmaceutical composition may be adapted for administration by any appropriate route, such as parenteral (including subcutaneous, intramuscular, intrathecal or intravenous), enteral (including oral or rectal), inhalation or intranasal routes. Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions. Nucleic acids, expression vectors, host cells and methods of production as described above in relation to the first aspect are also contemplated in relation to the other aspects described herein. It will be understood that any and all features described above in respect of the first aspect are equally applicable to any further aspects of the invention. Preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis.

[0273] The prior art documents mentioned herein are incorporated by reference to the fullest extent permitted by law.

[0274] The present invention will be further illustrated in the following figures and Examples which are given for illustration purposes only and are not intended to limit the invention in any way.

[0275] Description of the drawings

[0276] Fig 1: Response curves showing IFNy release in the presence of Ag+ cell line, A375 (top) and Ag-cell line, TKYNU (bottom), with increasing concentrations of PRAME TCR-antiCD3 bispecific protein (termed ImmTAC) incorporating the indicated anti-CD3 variant.

[0277] Fig 2: Response curves showing IFNy release in the presence of Ag+ cell line, KATOIII (top) and Ag- cell line, CL11 PIWIL K / O (bottom), with increasing concentrations of PIWIL TCR-antiCD3 bispecific protein (termed ImmTAC) incorporating the indicated anti-CD3 variant.

[0278] Examples

[0279] The following examples describe antibodies, antigen binding fragments thereof, or multi-specific binding molecules of the invention. Multi-specific binding molecules of the invention may be referred to as TCR-antiCD3-bispecific proteins.

[0280] Example 1 - Methods

[0281] Engineering and production of humanised scFv fragments and bispecific anti-CD3 fusion proteins For humanisation, human germline alleles were selected based on their likely frequency in the patient population and overall similarity to the template sequence. Pseudogenes were avoided. Mutations were introduced by site directed mutagenesis and the resulting DNA sequences cloned into to suitable vector for protein expression. For construction of anti-CD3 fusion proteins, scFv fragments of humanised UCHT1 were fused via a linker to the N terminus of the beta chain of a soluble TCR or a TCR mimic antibody (TCRm) VH domain. An Fc domain was also included were indicated. Anti-CD3 scFv fragments and TCRm antiCD3 fusion proteins were expressed and purified from E. coll periplasm, briefly proteins were purified directly from the medium using a Nickel column (5ml His-excel Cytivia) followed by Cation exchange (5ml CaptoSP cytivia). Proteins were then concentrated then further purified by size exclusion chromatography (SUP 200) in PBS. TCR-antiCD3 fusion molecules were expressed and produced in E. coll inclusion bodies, refolded, and purified by anion exchange and size exclusion chromatography. TCR-antiCD3 fusion molecules comprising an Fc domain were expressed in CHO cells using the Thermo ExpiCHO™ transient expression protocol. Briefly, cultured cells were diluted to a concentration of 6 x106prior to transfection. Cells were harvested on day 14 post transfection, with temperature shift to 32°C day 1 post transfection. Feed additions were performed on day 1 and day 5 post transfection. Clarification was performed with two successive centrifugation steps, followed by a filtration step. Proteins were subsequently purified by Protein A followed by size exclusion chromatography. For use in SPR measurements, the extracellular domains of CD3 delta and CD3 epsilon were expressed in E. coll as a single chain construct with 3x[GGGGS]GGS linker and with an Avi-Tag added at the C-terminus. The protein was subsequently expressed and refolded from inclusion bodies. Refolded protein was then purified by anion exchange chromatography followed by size exclusion chromatography, and biotinylated with Bir A enzyme.

[0282] Binding affinity

[0283] Target binding was assessed by Surface Plasmon Resonance (SPR) using a Biacore instrument. Biotinylated CD3εδ was immobilized on to a streptavidin-coupled CM-5 sensor chip. Equilibrium binding constants were determined using serial dilutions. For high affinity interactions binding parameters were determined by single cycle kinetics analysis. Measurements were performed at 37°C, unless otherwise indicated, in Dulbecco’s PBS buffer, supplemented with 0.005% P20.

[0284] ELISpot

[0285] Assays were performed using a human IFNy or GrB ELISpot kit (BD Biosciences) according to the manufacturer’s instructions. Peripheral blood mononuclear cells (PBMC), isolated from fresh donor blood, were used as effector cells. The effector target ratio was less than or equal to 1:1. Data were plotted using PRISM software and EC50 values were calculated from the curves.

[0286] ADA assay

[0287] Serum samples were obtained from patients treated with a soluble TCR bispecific molecule targeting PRAME (IMCF 106C) (clinical trial #NCT04262466). Detection of specific binding ADAs from patient derived serum samples was performed using a bridging immunoassay based on the formation of soluble immuno-complexes comprising biotinylated IMCF106C, ADAs from serum samples, and Sulfo-Tag™ reporter labelled IMCF106C in solution. ADA complexes were captured on streptavidin-coated 96-well MULTI-ARRAY® (1-spot) SECTOR plates (Meso Scale Discovery) and then detected in a quasi-quantitative manner via ECL signal generation. Specific responses were confirmed by addition of unlabelled IMCF106C as a competitive inhibitor of ADA complex formation. Confirmed positive ADA samples were subsequently characterized for relative titer. The ADA response to anti-CD3 scFv fragments was assessed by replacing IMCF106C as the competitive inhibitor. A reduction in the amount of signal inhibition indicated a reduction in specificity for the ADA. Example 2 - Design and construction of improved humanised anti-CD3 variants

[0288] The U28 variant of UCHT1 (SEQ ID Nos: 3 and 4) was used as a template for humanisation. Two prior identified affinity enhancing mutations were engineered into CDR1 of VH (R at position 28 and L at position 33 with ref to SEQ ID NO:4). Using the human germline alleles IGKV1-6*01-J1 and IGHV3-23*01, D4-23*01, J2 as corresponding light and heavy chains respectively, non-human residues in both the framework regions and CDRs of the template variable domains were mutated to the corresponding human residue. Mutations were initially introduced into the VH and the VL domains separately, making two series of sequentially more humanised variants. Variants were then tested for retention of binding affinity by surface plasmon resonance (SPR). The mutated VH and VL domains giving rise to the strongest affinities were combined and additional single site mutations were introduced to further increase affinity.

[0289] Additional variants were generated with mutations selected from Q101C, G176C, K32N, Q55E, R246Q, R204V, S237D, L95D, Y159F, and L165A (with reference to SEQ ID NO: 21). The addition of a Q101C and G176C introduces a disulphide bond. In some instances, the disulphide bond may increase stability of the variant. The addition of any of Q55E, R204V, and / or R246Q mutation may reduce the overall positive charge of the variant. This may reduce the isoelectric point (pl). The addition of K32N, L95D, L165A and / or S237D mutation may reduce affinity of the variant. The addition of Y159F may increase melting temperature (Tm) of the variant.

[0290] A list of various exemplary mutations and the rationale therefor can be found in Table 1. Any of these mutations may be combined together to achieve the desired change in property. Any variant disclosed herein may be further modified by the addition of any of the mutations in Table 1. For example, in order to reduce pl and increase stability, a Q101C, Q176C and R246Q mutation could be included. Such properties can be considered when generating anti-CD3 scFvs to be incorporated, for example, as one domain into a multi-specific molecule, or one domain into a bi-specific molecule, or as a molecule to be utilised in combination with one or more other molecules. Such properties can be further considered in the context of developability, administration and / or therapeutic efficacy of such anti-CD3 scFvs, ora molecule or combination of molecules comprising such anti-CD3 scFvs..

[0291] Table 1: List of exemplary mutations

[0292] Mutation

[0293] Q101C and G176C Allows for disulphide bond formation for

[0294] increased stability

[0295] Q55E reduces positive charge (reduce pl)

[0296] R246Q reduces positive charge (reduce pl)

[0297] R204V reduces positive charge (reduce pl)

[0298] S237D reduces affinity

[0299] L95D reduces affinity

[0300] Y159F increases Tm

[0301]

[0302] L165A reduces affinity

[0303] K32N reduces affinity

[0304]

[0305] Exemplary improved humanised anti-CD3 variants having certain combinations of mutations, where the mutation combination has been introduced into the U144 molecule, include the following:

[0306] Table 2:

[0307] Mutant ID Mutation Combination

[0308] U144 Humanised UCHT1

[0309] Q101C and G176C allows for disulphide bond formation for U165

[0310] stability

[0311] U166 Q55E mutation reduces positive charge (reduce pl)

[0312] U167 R246Q mutation reduces positive charge (reduce pl)

[0313] U170 R204V mutation reduces positive charge (reduce pl)

[0314] U175 S237D mutation reduces affinity

[0315] U179 L95D mutation reduces affinity

[0316] Q101C and G176C allows for disulphide bond formation for U181

[0317] stability combined with Q55E to reduce pl

[0318] Q101C and G176C allows for disulphide bond formation for U182

[0319] stability combined with R246Q to reduce pl

[0320] Q101C and G176C allows for disulphide bond formation for U183

[0321] stability combined with Q55E and R246Q to reduce pl

[0322] Q101C and G176C allows for disulphide bond formation for U184

[0323] stability combined with Q55E, R204V, R246Q to reduce pl

[0324] Q101C and G176C allows for disulphide bond formation for U185 stability combined with Q55E and R246Q to reduce pl, and S237D to reduce affinity

[0325] U186 Q55E and R246Q to reduce pl

[0326] Q101C and G176C allows for disulphide bond formation for U189 stability combined with Q55E and R246Q to reduce pl, and L95D to reduce affinity

[0327] Q101C and G176C allows for disulphide bond formation for U190 stability combined with Q55E and R246Q to reduce pl, and Y159F to increase Tm

[0328] U192 Q55E and R246Q to reduce pl, and L165Ato reduce affinity

[0329] U193 Q55E and R246Q to reduce pl, and L95D to reduce affinity

[0330] U194 Q55E and R246Q to reduce pl, and S237D to reduce affinity

[0331] U195 Q55E and R246Q to reduce pl, L95D to reduce affinity, and Y159F to increase Tm

[0332]

[0333] Example 3 - Assessment of target binding for improved humanised anti-CD3 variants Humanised variants from Example 2 were produced as scFv fragments as described in the methods. Additionally, humanised variants were used to produce TCR and TCRm anti-CD3 fusion proteins as described. Sequences of the anti-CD3 scFv fragments are indicated in the table below, as well as tables 7 and 8 below. Sequences of the VL and VH domains in the fusion molecules tested in this example are provided in SEQ IDs 3 to 18. The TCR / TCRm end of the test molecules recognise HLA-A*02 restricted peptides from an oncology target (MAGEA4) is provided by SEQ ID NO 67. The peptide target is provided by SEQ ID NO: 26.

[0334] Binding of each molecule to CD3εδ was assessed by surface plasmon resonance at 37°C using a Biacore instrument. Binding affinity and binding half life are provided in Table 3 and Table 4.

[0335] Table 3

[0336] scFv alone MAGEA4 TCRm- antiCD3 fusion

[0337] I. D. SEQ ID of VL, VH, No. non-human KDT1 / 2(s) KDT1 / 2(s)

[0338] targeting moiety residues (nM) (nM)

[0339] U28 3, 4, 67 44 46.9 135 20 270

[0340] U112 9, 10, 67 30 - - 20.8 228

[0341] U123 11, 12, 67 24 - - 63.2 91

[0342] U144 13, 14, 67 25 25.7 105 15.2 222

[0343] U152 15, 16, 67 25 - - 27 123

[0344] U154 17, 18, 67 25 32.6 66 30.7 100

[0345]

[0346] 1Rmax fixed @ 160 RU

[0347] Table 4: scFV alone

[0348] Mutant ID SEQ ID of KD (nM) ti / 2 (min)

[0349] VH / VL

[0350] U144 13 / 14 4.8 3.5

[0351] U165 70 / 71 2.0 4.3

[0352] U166 72 / 73 4.5 2.2

[0353] U167 74 / 75 3.8 2.1

[0354] U170 76 / 77 5.9 2

[0355] U175 78 / 79 25.7 0.5

[0356] U179 80 / 81 NT NT

[0357] U181 82 / 83 NT NT

[0358] U182 84 / 85 NT NT

[0359] U183 86 / 87 2.5 4

[0360]

[0361] U184 88 / 89 4.4 3

[0362] U185 90 / 91 28.0 0.72

[0363] U186 92 / 93 5.4 2.1

[0364] U189 94 / 95 17.5 0.7

[0365] U190 96 / 97 2.1 2.8

[0366] U192 98 / 99 NT NT

[0367] U193 100 / 101 NT NT

[0368] U194 102 / 103 NT NT

[0369] U195 104 / 105 NT NT

[0370] U196 121 / 122 NT NT

[0371] U197 123 / 124 NT NT

[0372] U198 125 / 126 NT NT

[0373] U199 127 / 128 NT NT

[0374] U201 129 / 130 NT NT

[0375]

[0376] NT = not tested

[0377] These data demonstrate the production of improved variants of UCHT1 having both increased humanisation and retaining a comparable affinity for CD3 (low nanomolar range).

[0378] Example 4 - Assessment of functional activity for improved humanised anti-CD3 variants cellular testing

[0379] To confirm that the improved humanised variants also retain comparable functional characteristics to the minimally humanised template antibody, bispecific fusion molecules incorporating the variants were assessed for their ability to activate T cells using an IFNy ELISpot assay as a read out of T cell activation. Assays were performed in the presence of antigen positive and antigen negative cells to confirm the specificity of the response. In this example three different TCR-antiCD3 fusion molecules were tested. Full length sequences of the molecules fused to humanised antiCD3 variant U144 are provided in SEQ IDs 20 and 21, 22, 23, and 24 and 25. The TCR end of the test molecules recognised HLA-A*02 restricted peptides from oncology targets (PRAME (peptide SEQ ID NO 27) or PIWIL (peptide SEQ ID NO 28)).

[0380] For the PRAME and PIWIL TCR-antiCD3 fusions, ELISpot assays were performed using A375 or KATOIII cells as antigen positive cells respectively. Antigen negative cells were TKYNU and CL11 with PIWIL knockout respectively. Figures 2 and 3 shows response curves obtained for the indicated antiCD3 variants. EC50 values against antigen positive cells were calculated from the curves and provided in the table below. These data indicate that the humanised variants of the invention have comparable, and in some cases increased potency against antigen positive cells, while retaining similar specificity at concentrations below 1nM. Table 5

[0381] PRAME EC50(pM) Ag+ PIWIL EC50(pM) Ag+

[0382] anti-CD3

[0383] cells (A375) cells (KATOIII)

[0384] UO 586.6 NT

[0385] U28 193.4 73

[0386] U123 225.5 147

[0387] U144 117.7 59

[0388] U154 158.5 89

[0389]

[0390] NT - not tested

[0391] Example 5 - Assessment of risk of ADA formation by humanised antiCD3 variants

[0392] A bridging immunoassay was used to determine whether the new humanised variants demonstrated a reduction in specific ADA recognition relative to LIO. Assays were performed as described in Example 1 using patient-derived ADA positive serum samples from a phase I clinical trial of IMCF106C. AntiCD3 scFv fragments were used as competitive inhibitors of ADA complex formation.

[0393] The table below indicates the relative depletion in ADA complex formation in the presence of the indicated antiCD3 variants. A depletion of >50% indicated the presence of highly specific ADAs. Four out of eight patient serum samples showed high levels of ADAs specific for U0 (indicated in bold). For the newly humanized molecules three out of the four demonstrated a significant reduction in depletion (patient samples 1-3) indicating that the part of the scFv these patients ADAs were recognizing has been removed. Samples from patients 1-3 also had high antibody titres, indicating a potential negative impact of the ADAs on PK in vivo. The 4th sample (from patient 8) had a very low titre and therefore the ADAs were unlikely to have significant impact on PK.

[0394] Table 6

[0395] Patient ADA Titre % depletion

[0396] UO U144 U152 U154 1 8192 -77.1 -17.6 -5.9 -15.1 2 32768 -63.5 -5.4 1.1 -10.6 3 16384 -56.4 -10.7 -6.4 -4.4 4 4096 -28.4 -14 -6.5 -4.5 5 4096 -18.5 -2.3 8.4 11.8 6 16384 -0.8 -3.2 3.5 0.1 7 8192 -34.4 -11 -8.7 -7.6 8 256 -95.8 -85.7 -82.5 -67.5

[0397]

[0398] In summary, the data presented indicate that the humanized antiCD3 variants described herein offer improved therapeutic potential by reducing the risk of ADA formation without compromising functional activity.

[0399] Table 7 – Light chain CDR and Framework SEQ ID NOs of antibodies that bind CD3

[0400] FW1 CDR1 FW2 CDR2 FW3 CDR3 FW4 Full length U144 44 29 45 32 46 34 48 13 U0 44 31 45 33 47 34 48 1 U28 44 31 45 33 47 34 48 3 U42 44 31 45 33 47 34 48 7 U112 44 31 45 33 47 34 48 9 U123 44 30 45 32 46 34 48 11 U152 44 30 45 32 46 34 48 15 U154 44 30 45 32 46 34 48 17 U165 44 29 45 32 46 34 111 70 U166 44 29 45 32 110 34 48 72 U167 44 29 45 32 46 34 48 74 U170 44 29 45 32 46 34 48 76 U175 44 29 45 32 46 34 48 78 U179 44 29 45 32 46 106 48 80 U181 44 29 45 32 110 34 111 82 U182 44 29 45 32 46 34 111 84 U183 44 29 45 32 110 34 111 86 U184 44 29 45 32 110 34 111 88 U185 44 29 45 32 110 34 111 90 U186 44 29 45 32 110 34 48 92 U189 44 29 45 32 110 106 111 94 U190 44 29 45 32 110 34 111 96 U192 44 29 45 32 110 34 48 98 U193 44 29 45 32 110 106 48 100 U194 44 29 45 32 110 34 48 102 U195 44 29 45 32 110 106 48 104 U196 44 30 45 32 110 34 48 121 U197 44 30 45 32 110 34 48 123 U198 44 30 45 32 110 34 48 125 U199 44 29 45 32 110 34 48 127 U201 44 30 45 32 110 34 48 129

[0401]

[0402] Table 8 – Heavy chain CDR and Framework SEQ ID NOs of antibodies that bind CD3 FW1 CDR1 FW2 CDR2 FW3 CDR3 FW4 Full length U144 49 35 51 39 53 42 55 14 UO 50 38 52 41 57 43 56 2 U28 50 37 52 41 54 43 56 4 U42 50 35 52 41 54 43 56 8 U112 49 35 51 39 53 42 55 10 U123 49 35 51 39 53 42 55 12 U152 49 36 51 39 53 42 55 16 U154 49 35 51 40 53 42 55 18 U165 49 35 112 39 53 42 55 71 U166 49 35 51 39 53 42 55 73 U167 49 35 51 39 53 42 114 75 U170 49 35 51 39 113 42 55 77 U175 49 35 51 39 53 109 55 79 U179 49 35 51 39 53 42 55 81 U181 49 35 112 39 53 42 55 83 U182 49 35 112 39 53 42 114 85 U183 49 35 112 39 53 42 114 87 U184 49 35 112 39 113 42 114 89 U185 49 35 112 39 53 109 114 91 U186 49 35 51 39 53 42 114 93 U189 49 35 112 39 53 42 114 95 U190 49 107 112 39 53 42 114 97 U192 49 108 51 39 53 42 114 99 U193 49 35 51 39 53 42 114 101 U194 49 35 51 39 53 109 114 103 U195 49 107 51 39 53 42 114 105 U196 49 35 51 39 53 42 114 122 U197 49 108 51 39 53 42 114 124 U198 49 35 51 39 53 109 114 126 U199 49 108 51 39 53 109 114 128 U201 49 108 51 39 53 109 114 130

[0403]

Claims

Claims1. An antibody or antigen binding fragment thereof that specifically binds to CD3 and comprises light chain CDRs selected from the following combinations:a) VLCDR1 is SEQ ID NO: 29, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO:34, b) VLCDR1 is SEQ ID NO: 30, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 34, c) VLCDR1 is SEQ ID NO: 31, VLCDR2 is SEQ ID NO: 33, VLCDR3 is SEQ ID NO: 34, or d) VLCDR1 is SEQ ID NO: 29, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 106 and / or heavy chain CDRs selected from the following combinations:a) VHCDR1 is SEQ ID NO: 35, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 42, b) VHCDR1 is SEQ ID NO: 35, VHCDR2 is SEQ ID NO: 41, VHCDR3 is SEQ ID NO: 43, c) VHCDR1 is SEQ ID NO: 35, VHCDR2 is SEQ ID NO: 40, VHCDR3 is SEQ ID NO: 42, d) VHCDR1 is SEQ ID NO: 36, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 42, e) VHCDR1 is SEQ ID NO: 35, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 109, f) VHCDR1 is SEQ ID NO: 107, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 42, g) VHCDR1 is SEQ ID NO: 108, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 42, orh) VHCDR1 is SEQ ID NO: 108, VHCDR2 is SEQ ID NO: 39, VHCDR3 is SEQ ID NO: 109.

2. The antibody or antigen binding fragment of claim 1, whereina) VLCDR1 is SEQ ID NO:29, VLCDR2 is SEQ ID NO:32, VLCDR3 is SEQ ID NO:34, VHCDR1 is SEQ ID NO:35, VHCDR2 is SEQ ID NO:39, and VHCDR3 is SEQ ID NO:42,b) VLCDR1 is SEQ ID NO:31, VLCDR2 is SEQ ID NO:33, VLCDR3 is SEQ ID NO:34, VHCDR1 is SEQ ID NO:35, VHCDR2 is SEQ ID NO:41, and VHCDR3 is SEQ ID NO:43,c) VLCDR1 is SEQ ID NO:31, VLCDR2 is SEQ ID NO:33, VLCDR3 is SEQ ID NO: 34, VHCDR1 is SEQ ID NO:35, VHCDR2 is SEQ ID NO:39, and VHCDR3 is SEQ ID NO:42,d) VLCDR1 is SEQ ID NO:30, VLCDR2 is SEQ ID NO:32, VLCDR3 is SEQ ID NO:34, VHCDR1 is SEQ ID NO:35, VHCDR2 is SEQ ID NO:39, and VHCDR3 is SEQ ID NO:42,e) VLCDR1 is SEQ ID NO:30, VLCDR2 is SEQ ID NO:32, VLCDR3 is SEQ ID NO:34, VHCDR1 is SEQ ID NO:36, VHCDR2 is SEQ ID NO:39, and VHCDR3 is SEQ ID NO:42,f) VLCDR1 is SEQ ID NO:30, VLCDR2 is SEQ ID NO:32, VLCDR3 is SEQ ID NO:34, VHCDR1 is SEQ ID NO:35, VHCDR2 is SEQ ID NO:40, and VHCDR3 is SEQ ID NO:42,g) VLCDR1 is SEQ ID NO: 29, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 106, VHCDR1 is SEQ ID NO: 35, VHCDR2 is SEQ ID NO: 39, and VHCDR3 is SEQ ID NO: 42,h) VLCDR1 is SEQ ID NO: 29, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 106, VHCDR1 is SEQ ID NO: 107, VHCDR2 is SEQ ID NO: 39, and VHCDR3 is SEQ ID NO: 42,i) VLCDR1 is SEQ ID NO:29, VLCDR2 is SEQ ID NO:32, VLCDR3 is SEQ ID NO:34, VHCDR1 is SEQ ID NO:35, VHCDR2 is SEQ ID NO:39, and VHCDR3 is SEQ ID NO:109,j) VLCDR1 is SEQ ID NO: 29, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 34, VHCDR1 is SEQ ID NO: 107, VHCDR2 is SEQ ID NO: 39, and VHCDR3 is SEQ ID NO: 42,k) VLCDR1 is SEQ ID NO: 29, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 34, VHCDR1 is SEQ ID NO: 108, VHCDR2 is SEQ ID NO: 39, and VHCDR3 is SEQ ID NO: 42,l) VLCDR1 is SEQ ID NO: 30, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 34, VHCDR1 is SEQ ID NO: 35, VHCDR2 is SEQ ID NO: 39, and VHCDR3 is SEQ ID NO: 42, m) VLCDR1 is SEQ ID NO: 30, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 34, VHCDR1 is SEQ ID NO: 108, VHCDR2 is SEQ ID NO: 39, and VHCDR3 is SEQ ID NO: 42, n) VLCDR1 is SEQ ID NO: 30, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 34, VHCDR1 is SEQ ID NO: 35, VHCDR2 is SEQ ID NO: 39, and VHCDR3 is SEQ ID NO: 109, o) VLCDR1 is SEQ ID NO: 29, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 34, VHCDR1 is SEQ ID NO: 108, VHCDR2 is SEQ ID NO: 39, and VHCDR3 is SEQ ID NO: 109, or p) VLCDR1 is SEQ ID NO: 30, VLCDR2 is SEQ ID NO: 32, VLCDR3 is SEQ ID NO: 34, VHCDR1 is SEQ ID NO: 108, VHCDR2 is SEQ ID NO: 39, and VHCDR3 is SEQ ID NO: 109.

3. The antibody or antigen binding fragment thereof of claim 1 or claim 2, comprising a VL domain, wherein the VL domain comprises a sequence selected from SEQ ID NO:86, SEQ ID NO: 13, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO: 82, SEQ ID NO:

84. SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127 and SEQ ID NO:129.

4. The antibody or antigen binding fragment thereof of any preceding claim, comprising a VH domain, wherein the VH domain comprises a sequence selected from SEQ ID NO: 87, SEQ ID NO:14, SEQ ID NO:8, SEQ ID NQ:10, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, and SEQ ID NQ:130.

5. The antibody or antigen binding fragment thereof of claim 3 or claim 4, wherein the VL and VH domains are selected from a from SEQ ID NO: 86 and SEQ ID NO:87, SEQ ID NO: 13 and SEQ ID NO:14, SEQ ID NO:7 and SEQ ID NO: 8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:15 and SEQ ID NO: 16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:70 and SEQ ID NO: 71, SEQ ID NO:72 an SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO:75, SEQ ID NO:76 and SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:79, SEQ ID NO:80 and SEQ ID NO:81, SEQ ID NO:82 and SEQ ID NO:83, SEQ ID NO:84 and SEQ ID NO:85, SEQ ID NO:88 and SEQ ID NO:89, SEQ ID NO:90 and SEQ ID NO:91, SEQ ID NO:92 and SEQ ID NO:93, SEQ ID NO:94 and SEQ ID NO:95, SEQ ID NO:96 and SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:99, SEQ ID NO:100 and SEQ ID NO:101, SEQ ID NO:102 and SEQ ID NO:103, SEQ ID NO: 104 and SEQ ID NO:105, SEQ ID NO: 121 and SEQ ID NO:122, SEQ ID NO:123 and SEQ ID NO:124, SEQID NO:125 and SEQ ID NO:126, SEQ ID NO:127 and SEQ ID NO:128, and SEQ ID NO:129 and SEQ ID NO:130.

6. The antibody or antigen binding fragment thereof of claim 3, 4 or 5, comprising a VL domain of sequence SEQ ID NO: 13 and a VH domain of sequence SEQ ID NO 14.

7. The antibody or antigen binding fragment thereof of claim 3, 4 or 5, comprising a VL domain of sequence SEQ ID NO: 86 and a VH domain of sequence SEQ ID NO 87.

8. The antibody or antigen binding fragment thereof of any one of claims 3 to 7, wherein the VL domain and VH domain are connected by a linker.

9. A multi-specific binding molecule comprising:I) an antibody or antigen binding fragment thereof that specifically binds to CD3 according to any preceding claim, andii) a targeting moiety10. The multi-specific binding molecule of claim 9, which is a T cell engager.

11. The multi-specific binding molecule of claim 9 or claim 10, wherein the targeting moiety is selected from the group consisting of an antibody or antigen binding fragment thereof, a TCR or fragment thereof, or TCRm or binding fragment thereof.

12. The multi-specific binding molecule of any one of claims 9 to 11, further comprising a half life extending moiety.

13. The multi-specific binding molecule of any one of claims 9 to 12, wherein the antibody or antigen binding fragment thereof that specifically binds to CD3 according to any one of claims 1 to 8 is fused to the C or N terminus of the targeting moiety, optionally via a linker.

14. The multi-specific binding molecule of any one of claims 9 to 13, comprising a sequence selected from (a) SEQ ID NOs: 20 and 21, (b) SEQ ID NO: 22, (c) SEQ ID NO: 23, (d) SEQ ID NOs: 24 and 25, (e) SEQ ID NO: 69, (f) SEQ ID Nos: 20 and 116, (g) SEQ ID NO: 117, (h) SEQ ID NO: 118, (i) SEQ ID Nos: 24 and 119, and (j) SEQ ID NO: 120.

15. A nucleic acid encoding the antibody or antigen binding fragment thereof that specifically binds to CD3 according to any one of claims 1 to 8, or the multi-specific binding molecule according to any one of claims 9 to 14.

16. An expression vector comprising the nucleic acid of claim 15.

17. A host cell comprising the nucleic acid of claim 15 or the vector of claim 16, wherein the nucleic acid is present as a single open reading frame or a distinct open reading frame encoding each chain of the antibody or multi-specific binding molecule.

18. A pharmaceutical composition comprising an antibody or antigen binding fragment thereof that specifically binds to CD3 according to any one of claims 1 to 8, a multi-specific binding molecule according to any one of claims 9 to 14, a nucleic acid according to claim 15, an expression vector according to claim 16, or a host cell according to claim 17.

19. A method of making an antibody or antigen binding fragment thereof that specifically binds to CD3 according to any one of claims 1 to 8, or a multi-specific binding molecule according to any one of claims 9 to 14, comprising maintaining the host cell of claim 17 under optimal conditions for expression of the nucleic acid and, optionally, isolating the antibody or antigen binding fragment thereof that specifically binds to CD3 or the multi-specific binding molecule.

20. An antibody or antigen binding fragment thereof that specifically binds to CD3 according to any one of claims 1 to 8, a multi-specific binding molecule according to any one of claims 9 to 14, a nucleic acid according to claim 15, an expression vector according to claim 16, host cell according to claim 17, or pharmaceutical composition according to claim 18 for use as a medicament.

21. A method of treatment comprising administering an antibody or antigen binding fragment thereof that specifically binds to CD3 according to any one of claims 1 to 8, a multi-specific binding molecule according to any one of claims 9 to 14, a nucleic acid according to claim 15, an expression vector according to claim 16, a host cell according to claim 17, or a pharmaceutical composition of claim 18 to a patient in need thereof.

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