Hybrid CD3 binding molecules

Abstract

The invention relates to hybrid CD3 binding molecules. The present disclosure relates to the field of antibodies. It relates in particular to the field of the preparation of therapeutic antibodies. More particularly, it relates to anti-CD3 heavy chain variable regions capable of pairing with a plurality of different light chain variable regions to form a functional CD3 binding domain, as well as CD3 binding domains and CD3 binding moieties comprising such anti-CD3 heavy chain variable regions.

Description

[0001] This application is a divisional application of the Chinese patent application No. 202380089930.6 entitled “Hybrid CD3 binding molecules”, the original application being PCT International Application PCT / NL2023 / 050691 filed on December 28, 2023, which entered the Chinese national phase on June 30, 2025. TECHNICAL FIELD

[0002] The present disclosure relates to the field of antibodies. It particularly relates to the field of therapeutic antibodies. It more particularly relates to anti-CD3 heavy chain variable regions (herein also referred to as CD3 heavy chain variable regions) that can pair with multiple different light chain variable regions to form functional CD3 binding domains, to CD3 binding domains, and to CD3 binding moieties comprising such anti-CD3 heavy chain variable regions. BACKGROUND

[0003] Monoclonal antibodies that bind to human CD3 were among the first antibodies developed for therapeutic use in humans. Monoclonal CD3 binding antibodies are often used for their immunosuppressive properties, for example for transplant rejection. Antibodies that are multi-specific for CD3 on T cells and at least one surface target antigen on cancer cells are able to link any type of T cell to a cancer cell, independent of T cell receptor specificity, costimulation or peptide antigen presentation. Such multi-specific T-cell engaging antibodies show considerable promise for use in treating various cancers and tumor growth.

[0004] It is an object of the present invention to provide CD3 heavy chain variable regions that can pair with multiple different light chain variable regions to form functional CD3 binding domains, i.e. to provide hybrid CD3 heavy chain variable regions. Such CD3 heavy chain variable regions are particularly useful for the generation of multi-specific T cell engaging antibodies. They have a high likelihood of being successfully combined with the light chain variable region of any binding domain generated against any tumor associated antigen (TAA). Thus, multi-specific antibodies comprising such CD3 binding domains and TAA binding domains can be efficiently produced in a common light chain antibody format in a single host cell. Furthermore, hybrid CD3 heavy chain variable regions can easily be combined with the light chain variable region of a clinically successful antibody. Multi-specific antibodies comprising such CD3 binding domains and the binding domain of a clinically successful antibody, for example, have the potential to extend this clinical success experience to other therapeutic areas, providing increased tumor specificity and thus safety, and / or more effective eradication of tumor cells. SUMMARY

[0005] In certain embodiments, the present invention provides a polypeptide comprising a heavy chain CDR3 (HCDR3) having an amino acid sequence as set forth in SEQ ID NO: 4; or having at least 70% sequence identity thereto.

[0006] In certain embodiments, the present application provides a CD3 binding domain comprising a polypeptide as described herein.

[0007] In certain embodiments, the present application provides a set of antigen binding proteins that bind human CD3, wherein each antigen binding protein within the set comprises a heavy chain variable region comprising a HCDR3 having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4.

[0008] In certain embodiments, the present application provides a binding moiety comprising a polypeptide as described herein, or a CD3 binding domain as described herein.

[0009] In certain embodiments, the present application provides a pharmaceutical composition comprising an effective amount of a polypeptide as described herein, or a CD3 binding domain as described herein, or a binding moiety as described herein, and a pharmaceutically acceptable carrier.

[0010] In certain embodiments, the present application provides a polypeptide as described herein, or a CD3 binding domain as described herein, or a binding moiety as described herein, or a pharmaceutical composition as described herein, for use in therapy.

[0011] In certain embodiments, the present application provides a polypeptide as described herein, or a CD3 binding domain as described herein, or a binding moiety as described herein, or a pharmaceutical composition as described herein, for use in cancer therapy.

[0012] In certain embodiments, the present application provides a method of treating a disease, comprising administering to a subject in need thereof an effective amount of a polypeptide as described herein, or a CD3 binding domain as described herein, or a binding moiety as described herein, or a pharmaceutical composition as described herein.

[0013] In certain embodiments, the present application provides a method of treating cancer, comprising administering to a subject in need thereof an effective amount of a polypeptide as described herein, or a CD3 binding domain as described herein, or a binding moiety as described herein, or a pharmaceutical composition as described herein.

[0014] In certain embodiments, the present application provides a nucleic acid comprising a sequence encoding a polypeptide as described herein.

[0015] In certain embodiments, the present application provides a vector comprising a nucleic acid as described herein.

[0016] In certain embodiments, the present application provides a cell comprising a nucleic acid as described herein.

[0017] In certain embodiments, the present application provides a cell producing a polypeptide as described herein, or a CD3 binding domain as described herein, or a binding moiety as described herein. DETAILED DESCRIPTION

[0018] It is an object of the present application to provide novel CD3 heavy chain variable regions which can be used for the production of therapeutic antibodies, in particular for the production of T cell engaging antibodies. This object is achieved by providing several polypeptides as well as binding domains and binding moieties comprising these polypeptides.

[0019] In certain embodiments, the polypeptides of the present application are heavy chain variable regions which can be paired with a plurality of different light chain variable regions and form a functional binding domain binding CD3. The nature of such heavy chain variable regions is referred to in the art as promiscuity. The present inventors have identified an anti-CD3 heavy chain variable region, as well as a group of related anti-CD3 heavy chain variable regions, which are particularly promiscuous. These CD3 heavy chain variable regions have a high likelihood of successfully combining with the light chain variable region of any binding domain produced against any tumor associated antigen (TAA). A multi-specific antibody comprising such a CD3 binding domain and a TAA binding domain can be efficiently produced in a single host cell in the form of a common light chain antibody. Furthermore, the present inventors have shown that the promiscuous CD3 heavy chain variable regions of the present application can easily be combined with the light chain variable regions of several clinically successful antibodies. A multi-specific antibody comprising such a CD3 binding domain and a binding domain of a clinically successful antibody, for example, has the potential to extend the clinical success experience to other therapeutic areas, to provide increased tumor specificity and thus safety, and / or to more effectively eradicate tumor cells. Thus, the CD3 heavy chain variable regions of the present disclosure are particularly useful for the production of multi-specific T cell engaging antibodies.

[0020] In certain embodiments, the present application provides a polypeptide comprising a heavy chain CDR3 (HCDR3) having an amino acid sequence as set forth in SEQ ID NO: 4; or at least 70% sequence identity thereto. In certain embodiments, the present application provides a polypeptide comprising a heavy chain CDR3 (HCDR3) having an amino acid sequence as set forth in SEQ ID NO: 4; or at least 70% sequence identity thereto and having the same length as SEQ ID NO: 4.

[0021] In certain embodiments, the polypeptide is an immunoglobulin heavy chain or an antigen binding portion thereof. In certain embodiments, the polypeptide is an immunoglobulin heavy chain or an antigen binding portion thereof that, when combined with an appropriate light chain or antigen binding portion thereof, binds to CD3. For example, the antigen binding portion of an immunoglobulin heavy chain can be a heavy chain variable region, or a heavy chain variable region with a CHl region. The antigen binding portion of a light chain can be, for example, a light chain variable region. In certain embodiments, the light chain is an immunoglobulin light chain.

[0022] In certain embodiments, the polypeptide binds to human CD3 when combined with an appropriate light chain or antigen binding portion thereof. The amino acid sequence of the signal peptide and extracellular domain of human CD3 delta is provided herein as SEQ ID NO: 113, and the amino acid sequence of the signal peptide and extracellular domain of human CD3 epsilon is provided herein as SEQ ID NO: 114. Binding to human CD3 can be determined in an ELISA assay by comparison to background signal and / or negative control groups. For example, the polypeptide, binding domain, antigen binding protein, or binding portion binds to human CD3 when it has a binding signal that is at least two-fold higher than the background signal and / or a binding activity that is at least two-fold higher than the negative control group.

[0023] Antigen binding can be expressed in terms of specificity and affinity. Specificity determines to which antigen or epitope thereof a binding domain or binding portion specifically binds. Affinity is a measure of the strength of binding to a particular antigen or epitope.

[0024] The present inventors have identified a group of binding domains comprising a polypeptide, in particular a heavy chain variable region, comprising a HCDR3 having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, such polypeptides, or binding domains or binding portions comprising such polypeptides, are grouped based on at least 70% sequence identity in the HCDR3. In certain embodiments, such polypeptides, or binding domains or binding portions comprising such polypeptides, are grouped based on at least 70% sequence identity in the HCDR3 and have the same length as the HCDR3. In certain embodiments, such polypeptides, or binding domains or binding portions comprising such polypeptides, are grouped based on at least 70% sequence identity in the HCDR3, have the same length as the HCDR3, and are derived from the same heavy chain variable region V gene segment.

[0025] Reference to the "percent (%) identity" of a nucleic acid or amino acid sequence as used herein defines the percentage of identical residues between the candidate sequence and the selected sequence after alignment of the sequences for optimal comparison. To optimize alignment of two sequences, white spaces can be introduced in either of the two sequences being compared. Such alignment can be over the entire length of the sequences being compared. Alternatively, alignment can be over a shorter length, such as over about 20, about 50, about 100 or more nucleic acids / bases or amino acids. Alignment can also be over individual CDR sequences. Sequence identity is the percentage of identical matches between two sequences over the reported alignment interval.

[0026] Comparison of sequences and determination of the percent sequence identity between two sequences can be accomplished using mathematical algorithms. The skilled person will be aware that there are several different computer programs available for aligning two sequences and determining the identity between two sequences ((Kruskal, J. B. (1983) An overview of sequence comparison In D. Sankoff and J. B. Kruskal, (Eds.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44, Addison Wesley). Sequence identity between two amino acid sequences or nucleic acid sequences can be determined using the Needleman and Wunsch algorithm for the alignment of two sequences (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purposes of the present application, the percent identity of amino acid and nucleic acid sequences was determined using the NEEDLE program in the EMBOSS suite (version 2.8.0, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden J. and Bleasby, A. Trends in Genetics 16, (6) pp. 276-277, http: / / emboss.bioinformatics.nl / ). For protein sequences, EBLOSUM62 was used for the substitution matrix. For DNA sequences, DNAFULL was used. The parameters used were a gap open penalty of 10 and a gap extension penalty of 0.5.

[0027] After alignment by the program NEEDLE as described above, the percentage of sequence identity between the query sequence and the sequence of the present application is calculated as follows: the number of positions at which the two sequences exhibit identical amino acids or identical nucleotides in the alignment divided by the total length of the alignment minus the total number of gaps in the alignment.

[0028] In certain embodiments, the polypeptide of the present application comprises a heavy chain CDR1 (HCDR1) having an amino acid sequence as set forth in SEQ ID NO: 2, a heavy chain CDR2 (HCDR2) having an amino acid sequence as set forth in SEQ ID NO: 3, and a heavy chain CDR3 (HCDR3) having an amino acid sequence as set forth in SEQ ID NO: 4, wherein each of the HCDRs can contain one, two, or up to three amino acid mutations.

[0029] In certain embodiments, the polypeptide of the present application comprises a heavy chain CDR1 (HCDR1) having an amino acid sequence as set forth in SEQ ID NO: 2, a heavy chain CDR2 (HCDR2) having an amino acid sequence as set forth in SEQ ID NO: 3, and a heavy chain CDR3 (HCDR3) having an amino acid sequence as set forth in SEQ ID NO: 4, wherein each of the HCDRs can contain one, two, or up to three amino acid mutations. In certain embodiments, the polypeptide of the present application comprises a heavy chain CDR1 (HCDR1) having an amino acid sequence as set forth in SEQ ID NO: 2, a heavy chain CDR2 (HCDR2) having an amino acid sequence as set forth in SEQ ID NO: 3, and a heavy chain CDR3 (HCDR3) having an amino acid sequence as set forth in SEQ ID NO: 4, wherein two of the HCDRs can contain one, two, or up to three amino acid mutations. In certain embodiments, the two of the HCDRs are HCDR1 and HCDR2. In certain embodiments, the polypeptide of the present application comprises a heavy chain CDR1 (HCDR1) having an amino acid sequence as set forth in SEQ ID NO: 2, a heavy chain CDR2 (HCDR2) having an amino acid sequence as set forth in SEQ ID NO: 3, and a heavy chain CDR3 (HCDR3) having an amino acid sequence as set forth in SEQ ID NO: 4, wherein one of the HCDRs can contain one, two, or up to three amino acid mutations. In certain embodiments, the one of the HCDRs is HCDR1 or HCDR2.

[0030] The heavy chain variable region of the polypeptide of the present application can comprise a limited number, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, non-conservative amino acid substitutions, or an unlimited number of conservative amino acid substitutions.

[0031] In certain embodiments, the polypeptides of the application also include variants thereof, wherein each of the HCDRs can comprise one, two, or three amino acid mutations. In certain embodiments, only one or two of the HCDRs can comprise one, two, or three non-conservative amino acid mutations. In certain embodiments, such variants do not comprise amino acid mutations in HCDR3. In certain embodiments, the amino acid mutations are conservative amino acid substitutions.

[0032] Generally, conservative amino acid substitutions involve mutations between amino acids and their cognate amino acid residues that have similar characteristics or properties. Cognate amino acids are known in the art and are routine methods of making amino acid substitutions in antibody binding domains without significantly affecting the binding or function of the antibody, for example, see Lehninger et al. manual (Nelson, David L., and Michael M. Cox. 2017. Lehninger Principles of Biochemistry. 7th ed. New York, NY: W.H. Freeman) or Stryer (Berg, J., Tymoczko, J., Stryer, L., and Stryer, L., 2007. Biochemistry. New York: W.H. Freeman), the entire contents of which are incorporated herein. In determining whether an amino acid can be substituted for a conservative amino acid substitution, the following factors can generally be evaluated, for example, but not limited to: (a) the polypeptide backbone structure, for example, folded or helical conformation, in the region of the substitution, (b) the charge or hydrophobicity of the molecule at the site of the target, and / or (c) the volume of the side chain. If the residue can be substituted for a residue having common characteristics, for example, similar side chains or similar charge or hydrophobicity, then the residue is a preferred substitution. For example, the following groups can be determined: (1) nonpolar: Ala (A), Gly (G), Val (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H). In addition, amino acids can be grouped as follows: (1) aromatic: Phe (F), Trp (W), Tyr (Y); (2) nonpolar: Leu (L), Val (V), He (I), Ala (A), Met (M); (3) aliphatic: Ala (A), Val (V), Leu (L), He (I); (4) acidic: Asp (D), Glu (E); (5) basic: His (H), Lys (K), Arg (R); and (6) polar: Gin (Q), Asn (N), Ser (S), Thr (T), Tyr (Y).Alternatively, amino acid residues can be grouped based on common side-chain properties: (1) hydrophobic: Met (M), Ala (A), Val (V), Leu (L), He (I); (2) neutral hydrophilic: Cys (C), Ser (S), Thr (T), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: His (H), Lys (K), Arg (R); (5) residues that influence chain orientation: Gly (G), Pro (P); and (6) aromatic: Trp (W), Tyr (Y), Phe (F).

[0033] It is preferred that substitution is with another amino acid residue in the same group. Thus, conservative amino acid substitution can involve the exchange of members of these classes to another member of the same class. Generally, the mutation will not or substantially not result in loss of binding specificity of the binding domain to its intended target.

[0034] Additional types of amino acid mutations include mutations resulting from somatic hypermutation or affinity maturation. Binding variants encompassed by the present invention include heavy chain variable regions that are derived from the same VH gene segment as the heavy chain variable regions described by the sequences herein, which variants have amino acid mutations, including non-conservative and / or conservative amino acid substitutions in one, two, or all three HCDRs. Conventional methods for affinity maturing antibody binding domains are well known in the art, e.g., see Tabasinezhad M, et al. (Trends in therapeutic antibody affinity maturation: From in-vitro towards next-generation sequencing approaches. Immunol Lett. 2019 Aug;212:106-113).

[0035] In certain embodiments, the polypeptides of the present invention comprise a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 1, or at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity thereto.

[0036] In certain embodiments, the polypeptides of the application also comprise variants that comprise one or more mutations in the framework regions in addition to the mutations in the HCDRs noted above. The mutations can be any type of amino acid mutation described herein, such as conservative amino acid substitutions or non-conservative amino acid substitutions resulting from, for example, somatic hypermutation or affinity maturation. In certain embodiments, the polypeptides of the application do not comprise mutations in the CDR regions, but comprise one or more mutations in the framework regions. Such variants have at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to the sequences disclosed herein. Thus, in certain embodiments, the polypeptides of the application comprise:

[0037] - a heavy chain variable region having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, the heavy chain variable region comprising HCDR1 of the amino acid sequence set forth in SEQ ID NO: 2; HCDR2 of the amino acid sequence set forth in SEQ ID NO: 3; and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 4.

[0038] In certain embodiments, the polypeptides of the application are produced with the light chain VK1-39 / JK1. The polypeptides of the application can be paired with any suitable light chain. In certain embodiments, the suitable light chain is the light chain VK1-39 / JK1. This light chain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3), which have the amino acid sequences set forth in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. In certain embodiments, the suitable light chain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the suitable light chain is the light chain of urelumab, custatuzumab, fresolimumab, inotuzumab, lemzoparlimab, magrolimab, ofatumumab, olaratumab, omburtamab, gatipotuzumab (pankomab), tovetumab, trastuzumab, or nivolumab.

[0039] In certain embodiments, the polypeptides of the application further comprise a CH1 region. In certain embodiments, the polypeptides of the application further comprise a CH1 region, a hinge region, a CH2 region, and a CH3 region. Suitable CH1 regions include, but are not limited to, a CH1 region having an amino acid sequence as set forth in SEQ ID NO: 33. Suitable hinges include, but are not limited to, a hinge having an amino acid sequence as set forth in SEQ ID NO: 32. Suitable CH2 and CH3 regions include, but are not limited to, a CH2 region having an amino acid sequence as set forth in SEQ ID NO: 34 or 35, and a CH3 region having an amino acid sequence as set forth in SEQ ID NO: 36.

[0040] CH1, hinge, CH2, CH3, and / or CL can be modified according to methods known in the art in order to obtain advantageous antibody properties, including, for example, to promote heterodimerization of different heavy chains, to enhance heavy chain-light chain pairing, and to enhance or reduce immune cell effector functions. The CH3 region can comprise a terminal lysine residue, or lack a terminal lysine residue, to improve manufacturability.

[0041] In certain embodiments, the application provides a CD3 binding domain comprising a polypeptide described herein.

[0042] In certain embodiments, the CD3 binding domains of the application further comprise a polypeptide comprising a light chain variable region. In certain embodiments, the light chain variable region is an antibody light chain variable region.

[0043] In certain embodiments, the CD3 binding domains of the application are antibody Fab domains.

[0044] In certain embodiments, the present application provides a set of antigen-binding proteins that bind human CD3, wherein each antigen-binding protein within the set comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a HCDR3 having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the present application provides a set of antigen-binding proteins that bind human CD3, wherein each antigen-binding protein within the set comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a HCDR3 having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4, and wherein the heavy chain variable region of each antigen-binding protein has the same HCDR3 length. In certain embodiments, the present application provides a set of antigen-binding proteins that bind human CD3, wherein each antigen-binding protein within the set comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a HCDR3 having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4, and wherein the heavy chain variable region of each antigen-binding protein has the same HCDR3 length, and wherein the heavy chain variable region of each antigen-binding protein is derived from the same heavy chain variable region V gene segment.

[0045] In certain embodiments, the antigen-binding protein is an antibody or antigen-binding fragment thereof.

[0046] In certain embodiments, the binding domain or antigen-binding protein of the present application comprises any suitable light chain, including but not limited to a common light chain known in the art. In certain embodiments, the binding domain or antigen-binding protein of the present application comprises the common light chain VK1-39 / JK1, or a variant thereof having a limited number of, e.g., 1, 2, or 3, non-conservative amino acid substitutions, or an unlimited number of conservative amino acid substitutions.

[0047] In certain embodiments, the binding domain or antigen-binding protein of the present application comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 5, or a variant thereof. In certain embodiments, the binding domain or antigen-binding protein of the present application comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 5, or a variant thereof having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity thereto.

[0048] In certain embodiments, the binding domain or antigen binding protein of the application comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. In certain embodiments, the light chain variable region of the binding domain or antigen binding protein of the application further comprises variants thereof, wherein each of the LCDRs can comprise one, two, or three amino acid mutations. In certain embodiments, the amino acid mutations are conservative amino acid substitutions.

[0049] The light chain, or light chain variable region comprising these LCDRs, and / or the light chain variable region can be, for example, a light chain referred to in the art as VK1-39 / JK1. This is a common light chain. The term "common light chain" in the context of the present application refers to a light chain that is able to pair with multiple different heavy chains, for example, heavy chains having different antigen or epitope binding specificities. Common light chains are particularly useful for the production of, for example, bispecific or multispecific antibodies, where antibody production is more efficient when all binding domains comprise the same light chain. The term "common light chain" encompasses light chains that are identical or have some amino acid sequence differences that do not affect the binding specificity of the full-length antibody. For example, within the scope of the common light chain definition used herein, non-identical but functionally identical light chains can be made or found, for example, by using well-established mutations that introduce conservative amino acid changes, amino acid changes in regions known or shown to not contribute, or only partially contribute, to binding specificity when paired with a heavy chain, and similar changes.

[0050] In addition to the common light chains comprising the LCDRs and / or light chain variable regions described above, other common light chains known in the art can be used. Examples of such common light chains include, but are not limited to: VK1-39 / JK5, which comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the light chain variable region comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 13, wherein each of the LCDRs can contain one, two, or three amino acid mutations, e.g., substitutions. In certain embodiments, the light chain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 13, or at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity thereto. In certain embodiments, the light chain comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; VK3-15 / JK1, which comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the light chain comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 18, wherein each of the LCDRs can contain one, two, or three amino acid mutations, e.g., substitutions. In certain embodiments, the light chain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 18, or at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity thereto. In certain embodiments, the light chain comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) having the amino acid sequences set forth in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively; VK3-20 / JK1, which comprises a light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) of a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 23.In certain embodiments, the heavy chain comprises a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19, wherein each of the HCDRs can comprise one, two, or three amino acid mutations, e.g., substitutions. In certain embodiments, the heavy chain comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 19, or at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity thereto. In certain embodiments, the heavy chain comprises a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; VH3-21 / JH3 comprising a heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23, wherein each of the HCDRs can comprise one, two, or three amino acid mutations, e.g., substitutions. In certain embodiments, the heavy chain comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 23, or at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity thereto. In certain embodiments, the heavy chain comprises a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) having the amino acid sequences set forth in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively.

[0051] VK1-39 is an abbreviation for the immunoglobulin variable Kappa 1-39 gene. The gene is also known as immunoglobulin kappa variable 1-39; IGKV139; IGKV1-39; IgV kappa 1-39. External IDs for the gene are HGNC: 5740; Entrez Gene: 28930; Ensembl: ENSG00000242371. The amino acid sequence of VK1-39 is shown as SEQ ID NO: 11. This is the sequence of the V region. The V region can be combined with one of five J regions. Suitable VJ-region sequences are shown as VK1-39 / JK1 (SEQ ID NO: 12) and VK1-39 / JK5 (SEQ ID NO: 13); alternative names are IgV kappa 1-39*01 / IGJ kappa 1*01 or IgV kappa 1-39*01 / IGJ kappa 5*01 (nomenclature according to IMGT database worldwide web imgt.org). These names are illustrative and encompass allelic variants of the gene segment.

[0052] K3-15 is an abbreviation for the immunoglobulin variable Kappa 3-15 gene. The gene is also known as immunoglobulin kappa variable 3-15; IGKV315; IGKV3-15; IgV kappa 3-15. External IDs for the gene are HGNC: 5816; Entrez Gene: 28913; Ensembl: ENSG00000244437. The amino acid sequence of VK3-15 is shown as SEQ ID NO: 17. This is the sequence of the V region. The V region can be combined with one of five J regions. A suitable VJ-region sequence is shown as VK3-15 / JK1 (SEQ ID NO: 18); an alternative name is V kappa 3-15*01 / IGJ kappa 1*01 (nomenclature according to IMGT database worldwide web imgt.org). These names are illustrative and encompass allelic variants of the gene segment.

[0053] VK3-20 is an abbreviation for the immunoglobulin variable kappa 3-20 gene. The gene is also known as immunoglobulin kappa variable 3-20; IGKV320; IGKV3-20; IgV kappa 3-20. The gene has the external IDs HGNC: 5817; Entrez Gene: 28912; Ensembl: ENSG00000239951. The amino acid sequence of VK3-20 is shown as SEQ ID NO: 22. This is the sequence of the V region. The V region can be combined with one of five J regions. A suitable VJ-region sequence is shown as VK3-20 / JK1 (SEQ ID NO: 23); an alternative name is IgV kappa 3-20*01 / IGJ kappa 1*01 (nomenclature according to IMGT database worldwide web imgt.org). This name is exemplary and encompasses allelic variants of this gene segment.

[0054] VL3-21 is an abbreviation for the immunoglobulin variable lambda 3-21 gene. The gene is also known as immunoglobulin variable lambda 3-21; IGLV321; IGLV3-21; IgV lambda 3-21. The gene has the external IDs HGNC: 5905; Entrez Gene: 28796; Ensembl: ENSG00000211662.2. The amino acid sequence of VL3-21 is shown as SEQ ID NO: 27. This is the sequence of the V region. The V region can be combined with one of five J regions. A suitable VJ-region sequence is shown as VL3-21 / JL3 (SEQ ID NO: 28); an alternative name is IgV lambda 3-21 / IGJ lambda 3 (nomenclature according to IMGT database worldwide web imgt.org). This name is exemplary and encompasses allelic variants of this gene segment.

[0055] Furthermore, any light chain variable region of an antibody available in the art can be used, as well as any other light chain variable region that is readily available, e.g. from antibody display libraries, when paired with a polypeptide of the application, shows antigen binding activity.

[0056] In certain embodiments, the binding domain or antigen binding protein of the application comprises the light chain or light chain variable region of urelumab, custatuzumab, fresolimumab, inotuzumab, lemzoparlimab, magrolimab, ofatumumab, olaratumab, omburtamab, gatipotuzumab (pankomab), tovetumab, trastuzumab, or nivolumab. The variable region sequences of these light chains are provided herein. The light chain variable region used in the application can comprise one or more sequence mutations that do not significantly alter the binding and functional activity of the resulting antibody, i.e., an equivalent light chain variable region.

[0057] In certain embodiments, the CD3 binding domain of the application or the antigen binding protein within the group of antigen binding proteins of the application comprises a light chain whose light chain variable region comprises the CDR1, CDR2, and CDR3 sequences of a light chain selected from the group consisting of urelumab, custatuzumab, fresolimumab, inotuzumab, lemzoparlimab, magrolimab, ofatumumab, olaratumab, omburtamab, gatipotuzumab (pankomab), tovetumab, trastuzumab, or nivolumab. The CDR sequences can be determined according to any numbering system known in the art, including but not limited to: IMGT and Kabat. The CDR sequences according to IMGT numbering are indicated in italics in the sequence listing herein; the CDR sequences according to Kabat numbering are indicated in bold. underlined

[0058] In certain embodiments, the CD3 binding domain of the application or the antigen binding protein within the group of antigen binding proteins of the application comprises a light chain whose light chain variable region comprises:

[0059] - the light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) of a light chain variable region having the amino acid sequence of SEQ ID NO: 5; ​

[0060] - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 40;

[0061] - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 42;

[0062] - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 43 or 68;

[0063] - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 45 or 69;

[0064] - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 49;

[0065] - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 50;

[0066] - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 52;

[0067] - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 53;

[0068] - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 56;

[0069] - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having an amino acid sequence of SEQ ID NO: 53 or 54;

[0070] - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having an amino acid sequence of SEQ ID NO: 58 or 70;

[0071] - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having an amino acid sequence of SEQ ID NO: 59 or 71 ; or

[0072] - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having an amino acid sequence of SEQ ID NO: 64 or 72.

[0073] In certain embodiments, the CD3 binding domain of the application or the antigen binding protein within the set of antigen binding proteins of the application comprises a heavy chain, which heavy chain variable region comprises:

[0074] - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having an amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively;

[0075] - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having an amino acid sequence of SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 75, respectively;

[0076] - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having an amino acid sequence of SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78, respectively;

[0077] - a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) of a heavy chain variable region having an amino acid sequence of SEQ ID NO: 79, SEQ ID NO: 80, and SEQ ID NO: 81 or SEQ ID NO: 82, respectively;

[0078] - a heavy chain CDR1 (HD R1) as set forth in SEQ ID NO: 1, a heavy chain CDR2 (HD R2) as set forth in SEQ ID NO: 2, and a heavy chain CDR3 (HD R3) as set forth in SEQ ID NO: 3;

[0079] - a heavy chain CDR1 (HD R1) as set forth in SEQ ID NO: 4, a heavy chain CDR2 (HD R2) as set forth in SEQ ID NO: 5, and a heavy chain CDR3 (HD R3) as set forth in SEQ ID NO: 6;

[0080] - a heavy chain CDR1 (HD R1) as set forth in SEQ ID NO: 7, a heavy chain CDR2 (HD R2) as set forth in SEQ ID NO: 8, and a heavy chain CDR3 (HD R3) as set forth in SEQ ID NO: 9;

[0081] - a heavy chain CDR1 (HD R1) as set forth in SEQ ID NO: 10, a heavy chain CDR2 (HD R2) as set forth in SEQ ID NO: 11, and a heavy chain CDR3 (HD R3) as set forth in SEQ ID NO: 12;

[0082] - a heavy chain CDR1 (HD R1) as set forth in SEQ ID NO: 13, a heavy chain CDR2 (HD R2) as set forth in SEQ ID NO: 14, and a heavy chain CDR3 (HD R3) as set forth in SEQ ID NO: 15;

[0083] - a heavy chain CDR1 (HD R1) as set forth in SEQ ID NO: 16, a heavy chain CDR2 (HD R2) as set forth in SEQ ID NO: 17, and a heavy chain CDR3 (HD R3) as set forth in SEQ ID NO: 18;

[0084] - a heavy chain CDR1 (HD R1) as set forth in SEQ ID NO: 19, a heavy chain CDR2 (HD R2) as set forth in SEQ ID NO: 20, and a heavy chain CDR3 (HD R3) as set forth in SEQ ID NO: 21;

[0085] - a heavy chain CDR1 (HD R1) as set forth in SEQ ID NO: 22, a heavy chain CDR2 (HD R2) as set forth in SEQ ID NO: 23, and a heavy chain CDR3 (HD R3) as set forth in SEQ ID NO: 24;

[0086] - a heavy chain CDR1 (HCDR1) as set forth in SEQ ID NO: 102, a heavy chain CDR2 (HCDR2) as set forth in SEQ ID NO: 103, and a heavy chain CDR3 (HCDR3) as set forth in SEQ ID NO: 104; or

[0087] - a heavy chain CDR1 (HCDR1) as set forth in SEQ ID NO: 105, a heavy chain CDR2 (HCDR2) as set forth in SEQ ID NO: 106, and a heavy chain CDR3 (HCDR3) as set forth in SEQ ID NO: 109.

[0088] In certain embodiments, the light chain variable region of the CD3 binding domain or antigen binding protein of the application further comprises variants thereof, wherein each of the LCDRs can comprise one, two, or three amino acid mutations. In certain embodiments, the light chain variable region of the CD3 binding domain or antigen binding protein of the application further comprises variants thereof, wherein each of the LCDRs can contain one, two, or up to three amino acid mutations. In certain embodiments, the amino acid mutations are conservative amino acid substitutions.

[0089] In certain embodiments, the light chain variable region of the CD3 binding domain or antigen binding protein of the application comprises an amino acid sequence as set forth in SEQ ID NO: 5; 41; 42; 43; 45; 49; 50; 52; 53; 56; 57; 58; 59; or 64, or at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity thereto.

[0090] In certain embodiments, the binding domain or antigen binding protein of the application further comprises variants that comprise one or more mutations in the framework regions in addition to the mutations in the LCDRs mentioned above. The mutations are preferably conservative amino acid substitutions. In certain embodiments, the binding domain or antigen binding protein of the application does not comprise mutations in the LCDR regions, but comprises one or more mutations in the framework regions. Such variants have at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity to the sequences disclosed herein.

[0091] In certain embodiments, the antigen binding proteins within the CD3 binding domain or antigen binding protein set of the application can further comprise a CL region. Any CL domain can be used, particularly a human CL. An example of a suitable CL domain is provided by the amino acid sequence set forth in SEQ ID NO: 9.

[0092] In certain embodiments, the application provides a binding moiety comprising a polypeptide described herein or a CD3 binding domain described herein.

[0093] A "binding moiety" refers to a proteinaceous molecule and includes, for example, all antibody formats available in the art, such as full-length IgG antibodies, immunoconjugates, diabodies, BiTEs, Fab fragments, scFv, tandem scFv, single domain antibodies (such as VHH and VH), minibodies, scFab, scFv-zipper, nanobodies, DART molecules, TandAbs, Fab-scFv, F(ab)'2, F(ab)'2-scFv2, and intrabodies, as well as any other antibody format known to one of ordinary skill in the art.

[0094] In certain embodiments, the binding moiety of the present application is a monospecific binding moiety, in particular a monospecific antibody. The monospecific antibody of the present application is an antibody of any antibody format comprising one or more binding domains specific for a single target. In certain embodiments, the monospecific binding moiety of the present application is a bivalent monospecific antibody. In certain embodiments, the monospecific binding moiety of the present application can further comprise an Fc region or a portion thereof. In certain embodiments, the monospecific binding moiety of the present application is an IgGl antibody.

[0095] In certain embodiments, the binding moiety of the present application is a multispecific antibody. The multispecific antibody of the present application is an antibody comprising at least two binding domains specific for at least two different targets or epitopes. In certain embodiments, the multispecific antibody of the present application is a bispecific antibody. In certain embodiments, the multispecific antibody of the present application is a bivalent bispecific antibody. In certain embodiments, the multispecific antibody of the present application is a trivalent bispecific antibody. In certain embodiments, the multispecific antibody of the present application is a trispecific antibody. In certain embodiments, the multispecific antibody of the present application is a trivalent trispecific antibody. In certain embodiments, the multispecific binding moiety of the present application can further comprise an Fc region or a portion thereof. In certain embodiments, the multispecific binding moiety of the present application is an IgGl antibody.

[0096] An "Fc region" generally comprises a hinge region, a CH2 region, and a CH3 region. Suitable hinge regions, CH2 regions, and CH3 regions are as described herein. The Fc region mediates effector functions of antibodies, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). Depending on the application of the therapeutic antibody or Fc fusion protein, it can be desirable to reduce or increase the effector functions.

[0097] In certain embodiments, the binding moiety comprising a polypeptide or binding domain of the application has Fc effector function. In certain embodiments, the binding moiety comprising a polypeptide or binding domain of the application has enhanced Fc effector function. In certain embodiments, the binding moiety comprising a polypeptide or binding domain of the application exhibits antibody-dependent cell-mediated cytotoxicity (ADCC).

[0098] Binding moieties (e.g., antibodies) can be engineered to enhance ADCC activity (for a review, see Kubota T et al., Cancer Sci. 2009; 100(9): 1566-72). For example, when an antibody has low ADCC activity by itself, the ADCC activity of the antibody can be enhanced by slightly modifying the constant region of the antibody (Junttila TT. et al., Cancer Res. 2010; 70(11): 4481-9). Changes are also sometimes made to enhance storage or production, or to remove a C-terminal lysine (Kubota T et al., Cancer Sci. 2009; 100(9): 1566-72). Another method of enhancing ADCC activity of an antibody is by enzymatic interference with the glycosylation pathway, resulting in reduced fucose (von Horsten HH. et al., Glycobiology. 2010; 20(12): 1607-18). Alternatively or additionally, various other strategies can be used to achieve ADCC enhancement, including glycoengineering (Kyowa Hakko / Biowa, GlycArt (Roche), and Eureka Therapeutics) and mutagenesis, all of which seek to enhance binding of the Fc to low affinity activating FcyRIIIa, and / or reduce binding to low affinity inhibitory FcyRIIb. In certain embodiments, the binding moiety of the application exhibits enhanced antibody-dependent cell-mediated cytotoxicity (ADCC). In certain embodiments, the binding moiety of the application is afucosylated.

[0099] The constant region of a binding moiety of the application can comprise one or more mutations that modulate a property of the binding moiety other than binding properties to the target antigen. For example, the constant region of the multispecific binding moiety can comprise one or more mutations that favor heterodimerization of two different heavy chains over homodimerization, and / or the constant region of the binding moiety can comprise one or more mutations that reduce or enhance effector function, preferably one or more mutations that reduce effector function.

[0100] In certain embodiments, the present application provides a nucleic acid that can be used to produce a polypeptide, binding domain, or binding moiety of the present application. In certain embodiments, the nucleic acid comprises a nucleic acid sequence that encodes a polypeptide described herein. In certain embodiments, the nucleic acid of the present application can further comprise a nucleic acid sequence that encodes a CHI region, and preferably a hinge region, a CH2 and a CH3 region. In certain embodiments, the nucleic acid of the present application can further comprise at least one nucleic acid sequence that encodes a light chain variable region, and the light chain variable region is preferably a CL region. In certain embodiments, the light chain variable region can be a light chain variable region described herein.

[0101] In certain embodiments, the present application provides a vector that comprises a nucleic acid of the present application that can be used to produce a binding domain or binding moiety of the present application. In certain embodiments, the vector comprises a nucleic acid sequence that encodes a polypeptide described herein. In certain embodiments, the vector of the present application can further comprise a nucleic acid sequence that encodes a CHI region, and preferably a hinge region, a CH2 and a CH3 region. In certain embodiments, the vector of the present application can further comprise at least one nucleic acid sequence that encodes a light chain variable region, and the light chain variable region is preferably a CL region. In certain embodiments, the light chain variable region can be a light chain variable region described herein.

[0102] In certain embodiments, the present application also provides a cell that comprises a vector as described herein. In certain embodiments, the present application also provides a cell that comprises a nucleic acid (e.g., a vector) that comprises a sequence that encodes a polypeptide described herein. In certain embodiments, such a nucleic acid (e.g., a vector) can further comprise a nucleic acid sequence that encodes a CHI region, and preferably a hinge, a CH2 and a CH3 region. In certain embodiments, such a nucleic acid (e.g., a vector) can further comprise a nucleic acid sequence that encodes a light chain variable region, and the light chain variable region is preferably a CL region. In certain embodiments, the light chain variable region can be a light chain variable region described herein.

[0103] In certain embodiments, the present application also provides a cell that produces a polypeptide, binding domain, or binding moiety described herein. In certain embodiments, such a cell can be a recombinant cell that has been transformed with a nucleic acid (e.g., a vector) of the present application. In certain embodiments, a cell of the present application comprises a nucleic acid sequence, e.g., a vector, that comprises a sequence that encodes a polypeptide described herein. In certain embodiments, the nucleic acid sequence, e.g., a vector, further comprises a nucleic acid sequence that encodes a CHI region, and preferably a hinge, a CH2 and a CH3 region. In certain embodiments, a cell of the present application further comprises at least one nucleic acid, e.g., a vector, that comprises a sequence that encodes a light chain variable region, and in particular a light chain variable region described herein (and preferably a CL region).

[0104] In certain embodiments, the present application provides a pharmaceutical composition comprising an effective amount of a polypeptide described herein, or a CD3 binding domain described herein, or a binding moiety described herein, and a pharmaceutically acceptable carrier.

[0105] In certain embodiments, the present application provides a polypeptide as described herein, or a CD3 binding domain as described herein, or a binding moiety as described herein, or a pharmaceutical composition as described herein, for use in therapy.

[0106] In certain embodiments, the present application provides a polypeptide as described herein, or a CD3 binding domain as described herein, or a binding moiety as described herein, or a pharmaceutical composition as described herein, for use in treating cancer.

[0107] In certain embodiments, the present application provides a method of treating a disease, comprising administering to an individual in need thereof an effective amount of a polypeptide as described herein, or a CD3 binding domain as described herein, or a binding moiety as described herein, or a pharmaceutical composition as described herein.

[0108] In certain embodiments, the present application provides a method of treating cancer, comprising administering to an individual in need thereof an effective amount of a polypeptide as described herein, or a CD3 binding domain as described herein, or a binding moiety as described herein, or a pharmaceutical composition as described herein.

[0109] As used herein, the terms“individual,”“subject,” and“patient” are used interchangeably and refer to a mammal, for example, a human, a mouse, a rat, a hamster, a guinea pig, a rabbit, a cat, a dog, a monkey, a cow, a horse, a pig, and the like, particularly a human individual having cancer.

[0110] The term“treat,”“treating,” and“treatment” as used herein refer to any type of intervention or process carried out on or administered to an individual with an active agent or combination of active agents with the objective of curing or improving a disease or its symptoms or producing a positive therapeutic response. As used herein, a“positive therapeutic response” refers to treatment that produces a beneficial effect, e.g., reversal, lessening, amelioration, inhibition or slowing of symptoms, complications, conditions or biochemical indicia associated with a disease, as well as prevention of the onset, progression, development, severity or recurrence of symptoms, complications, conditions or biochemical indicia associated with a disease, such as, for example, improvement of at least one symptom of a disease or condition, e.g., cancer. The beneficial effect can take the form of an improvement over baseline, including an improvement over a measurement or observation made prior to the initiation of treatment according to the method. For example, the beneficial effect can take the form of slowing, stabilizing, halting or reversing the progression of cancer in an individual at any clinical stage, evidenced, for example, by a reduction or elimination of clinical or diagnostic symptoms of the disease or markers of cancer. Effective treatment may, for example, reduce tumor size, reduce the presence of circulating tumor cells, reduce or prevent metastasis of a tumor, slow or arrest tumor growth, and / or prevent or delay tumor reoccurrence or recurrence.

[0111] The term "therapeutic amount" or "effective amount" refers to the amount of an agent or combination of agents that treats a disease, such as cancer. In some embodiments, a therapeutic amount is an amount sufficient to delay tumor progression. In some embodiments, a therapeutic amount is an amount sufficient to prevent or delay recurrence of a tumor.

[0112] As used herein, an effective amount of an agent or composition is, for example, an amount that: (i) reduces the number of cancer cells; (ii) reduces tumor size; (iii) inhibits, delays, slows, and can stop cancer cell infiltration into peripheral organs; (iv) inhibits tumor metastasis; (v) inhibits tumor growth; (vi) prevents or delays occurrence and / or recurrence of a tumor; and / or (vii) relieves to some extent one or more symptoms associated with the cancer.

[0113] An effective amount can vary depending on, for example, the disease state, age, sex, and weight of the individual to be treated, and the ability of the agent or combination of agents to elicit a desired response in the individual, which can readily be determined by one of ordinary skill in the art of medicine or other health care worker.

[0114] An effective amount can be administered to an individual in one or more administrations.

[0115] An effective amount can also include an amount that balances any toxic or deleterious effects of the agent or composition of agents with the beneficial effects.

[0116] The term "agent" refers to a therapeutically active substance, in the context of the present application a polypeptide, binding domain or binding moiety of the present application, or a pharmaceutical composition of the present application.

[0117] As used herein, "comprising" and its variants are used in their non-limiting sense to mean that the subsequent recited item is included, but that other items not specifically recited are not excluded.

[0118] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to one or at least one) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.

[0119] The citation of any patent or other matter in this specification is not intended as an admission that the patent or other matter is known or constitutes prior art in any jurisdiction.

[0120] All patents and references cited in this specification are hereby incorporated by reference in their entirety.

[0121] It is noted that in the present specification, unless otherwise indicated, amino acid positions of HCDRs and frameworks in the variable region of an antibody or antibody fragment are designated according to the numbering of Kabat (see Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991)) and the numbering of LCDRs and frameworks in the variable region of an antibody or antibody fragment is according to Kabat or IMGT (discussed in Giudicelli et al., Nucleic Acids Res. 25: 206-21 1 1997). Amino acids in the constant region are denoted according to the EU numbering system.

[0122] The provided accession numbers are provided mainly to provide further means of identifying the targets, the actual sequence of the bound protein can vary, for example due to mutations in the encoding gene, for example mutations that occur in some cancers or similar conditions. The antigen binding site of the binding domain or binding moiety of the present application can bind to the antigen and to a variety of variants thereof, for example expressed by some antigen positive immune cells or tumor cells. HGNC stands for HUGO Gene Nomenclature Committee. The number after the abbreviation is the accession number, through which information about the gene and the protein encoded by the gene can be retrieved from the HGNC database. Entrez Gene provides accession numbers or gene IDs, through which information about the gene or the protein encoded by the gene can be retrieved from the NCBI (National Center for Biotechnology Information) database. Ensembl provides accession numbers, through which information about the gene or the protein encoded by the gene can be retrieved from the Ensembl database. Ensembl is a joint project between EMBL-EBI and the Wellcome Trust Sanger Institute to develop a software system that can automatically annotate selected eukaryotic genomes.

[0123] When a gene or protein is mentioned herein, it is preferably meant the human form of the gene or protein. When a gene or protein is mentioned herein, it is meant both the natural gene or protein and a variant form of the gene or protein that can be detected in a tumor, cancer or similar condition, preferably in a human tumor, cancer and similar condition. BRIEF DESCRIPTION OF DRAWINGS

[0125] Figure 1- Binding of SC5Ab1 in combination with the cLC or light chain of olaratumab, ofatumumab, urelumab, cusatuzumab, inotuzumab, fresolimumab, trastuzumab or daratumumab on HPB-ALL cells, measured by FACS as mean fluorescence intensity (MFI) at different IgG concentrations.

[0126] Figure 2 - A) Digital capillary electrophoresis gel image of SC5Ab1 in combination with the cLC or light chain of cusatuzumab, fresolimumab, ofatumumab or trastuzumab; B) Electropherogram of SC5Ab1 in combination with the light chain of fresolimumab.

[0127] Figure 3 - Binding of SC5Ab1 in combination with the cLC or light chain of cusatuzumab, fresolimumab, ofatumumab or trastuzumab on HPB-ALL cells, measured by FACS as mean fluorescence intensity (MFI) at different IgG concentrations.

[0128] Figure 4 - Thermal stability of IgG comprising the VH region of antibody SC5Ab1 paired with the VL region of the cLC of cusatuzumab, fresolimumab, ofatumumab or trastuzumab or light chain. Stability is identified by Tm (solid line) and Tagg (dashed line). Tm is measured by changes in endogenous protein fluorescence, expressed as barycentric mean (BCM) of the spectral range between 250 and 720 nm, and Tagg is measured by static light scattering (SLS) at 266 nm using the UNcle system (Unchained Labs).

[0129] Example

[0130] In the various examples used to illustrate the application, but not intended to limit the application in any way, the binding domains were screened in the form of IgGl comprising a heavy chain comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1, CH1 having the amino acid sequence of SEQ ID NO: 33, a hinge having the amino acid sequence of SEQ ID NO: 32, CH2 having the amino acid sequence of SEQ ID NO: 34, and CH3 having the amino acid sequence of SEQ ID NO: 36, and a light chain comprising a light chain variable region as specified further herein, and CL having the amino acid sequence of SEQ ID NO: 9.

[0131] The control antibodies used in the various examples include:

[0132] - a positive control anti-CD3 antibody which is a bivalent monospecific antibody comprising two heavy chains having the amino acid sequence of SEQ ID NO: 37 and two light chains having the amino acid sequence of SEQ ID NO: 66;

[0133] - a negative control IgGl antibody (RSV-G) which is a bivalent monospecific antibody comprising two heavy chains having the amino acid sequence of SEQ ID NO: 38 and two light chains having the amino acid sequence of SEQ ID NO: 10;

[0134] - a negative control IgGl antibody (TT) which is a bivalent monospecific antibody comprising two heavy chains having the amino acid sequence of SEQ ID NO: 39 and two light chains having the amino acid sequence of SEQ ID NO: 10.

[0135] Example 1 - CD3 heavy chain pairing with light chains of cetuximab, trastuzumab and nivolumab

[0136] The aim of this study was to evaluate whether the heavy chain of a CD3 antibody produced with a light chain having the amino acid of SEQ ID NO: 10 could be combined with other light chains, while retaining the specificity and affinity of the original antibody.

[0137] Binding domains, antibodies and heavy chain variable regions with binding specificity for human CD3 were obtained by immunizing genetically modified mice (MeMo® mice) comprising a common IGKV1-39 light chain with human CD3 antigen portions, including the use of TCR / CD3 containing lipid particles (e.g. as described in WO 2020 / 204708), different forms of DNA, protein and cell-based antigen delivery. Antibodies binding human CD3 were grouped into different superclusters based on the use of the same VH V gene segment and having at least 70% sequence identity in the HCDR3 and the same HCDR3 length.

[0138] The representative heavy chain variable region (VH) sequence of each supercluster was combined with the light chain variable region (VL) sequence of cetuximab, trastuzumab and nivolumab. These three commercially available antibodies bind different targets and have different VLs, as shown in Table 1. The VH sequence was also combined with a light chain variable region having the amino acid sequence shown as SEQ ID NO: 5 as a parental control group.

[0139] Table 1 - Selected commercially available antibodies combined with CD3 VH. Abbreviations: mo - mouse, hu - human.

[0140] light chain target VL VL sequence cetuximab EGFR IGKV5-48*01 _mo SEQ ID NO: 40 trastuzumab HER2 IGKV1 -39*01 _hu SEQ ID NO: 41 nivolumab PD-1 IGKV3-11 *01 _hu SEQ ID NO: 42 parental - IGKV1 -39*01 _hu SEQ ID NO: 5

[0141] ELISA

[0142] Binding of CD3 antibodies to virus-like particles (VLPs) containing CD3 was assessed in an ELISA.

[0143] HEK293 huCD3 (Integral Molecular; Cat. No. INT-2131B), HEK293 cyCD3 (Integral Molecular; Cat. No. INT-2132B), and HEK293“empty” (Integral Molecular; Cat. No. INT-2128B) biotinylated VLPs were coated on ELISA plates at 5 units (U) per well at 4°C overnight. The wells were blocked with 4% skimmed milk (Marvel) in lx PBS solution. CD3 antibodies or control antibodies in 1% skimmed milk / lx PBS were added to huCD3 and cyCD3 bio-VLPs using a seven-step, three-fold dilution starting at 5 pg / ml. CD3 antibodies or control antibodies in 1% skimmed milk / lx PBS were added to“empty” bio-VLPs at a single concentration of 5 pg / ml. Antibodies were incubated for 1 hour at room temperature (RT).

[0144] Control antibodies include: a positive control anti-CD3 antibody to confirm coating of bio-VLPs with CD3, and a negative control IgGl (RSV-G) antibody to confirm binding specificity. Control antibodies were used at a concentration of 5 pg / ml.

[0145] Antibodies were detected with a goat anti-huIgG (Fc) HRP-conjugated secondary antibody (Bethyl Labs; Cat No. A80-104P) at 1% Skim milk / 1x PBS solution at 1:2000 and left to incubate for 1 hour at room temperature.

[0146] Wells were washed 3 times with IX PBS between steps and 5 times with IX PBS before color development. All wash steps were performed using buffers without Tween-20. Color development was performed by adding 100 mΐ TMB solution (eBioscience, Cat No. 00-4201-56). The reaction was stopped with 100 mΐ of 0.5M (1N) H2SO4 (Fisher Chemical, Cat No. J / 8430 / 15).

[0147] Readings were taken at O.D. 450 nm using a BioTek Elx808 ELISA plate reader. ELISA titration data was analyzed in GraphPad Prism.

[0148] Antibodies that showed binding in ELISA were further screened in FACS compared to their respective parental control groups.

[0149] FACS

[0150] Binding and relative affinity of CD3 antibodies to huCD3 was determined by FACS.

[0151] 293FF cells were transiently transfected to express (hu)CD3:TCR and cultured in FreeStyle™ 293 Expression Medium (Gibco, Cat No. 12338-018) at 0.5x10 6 HPB-ALL cells that endogenously express huCD3 were cultured in RPMI 1640 Medium (Gibco, Cat No. 21875-091) with FBS (Gibco, Cat No. A3160801) and penstrep (Gibco, Cat No. 15140-122) at 1x10 6 HPB-ALL cells that endogenously express huCD3 were cultured in RPMI 1640 Medium (Gibco, Cat No. 21875-091) with FBS (Gibco, Cat No. A3160801) and penstrep (Gibco, Cat No. 15140-122) at 1x10

[0152] CD3 antibody or positive control anti-CD3 antibody serial dilutions were added starting at 10 pg / ml with eight steps, half-log (3.16-fold) dilutions. Negative control IgG1 antibody (RSV-G) was added at a single concentration of 10 pg / ml. Antibodies were incubated in FACS buffer (0.5% FBS / EDTA 1 : 1000 / 1 x PBS) for 30 minutes on ice.

[0153] Antibodies were detected with goat anti-huIgG PE secondary antibody (Invitrogen, Cat. No. H10104) at 1 : 100 and incubated in FACS buffer (0.5% FBS / EDTA 1 : 1000 / 1 x PBS) for 30 minutes on ice.

[0154] FACS analysis was performed (BD Accuri™) and the mean fluorescence intensity (MFI) of each antibody was plotted as a function of the log of the antibody concentration using a non-linear regression, asymmetric (five-parameter) equation with robust fitting using GraphPad prism software.

[0155] AUC values were determined and used to assess the relative affinity of the antibodies for huCD3. FACS results were considered positive when the MFI values were more than two-fold higher than the negative control IgG1 antibody (RSV-G).

[0156] A summary of the ELISA and FACS results is shown in Table 2. A select number of antibodies retained CD3 binding when combined with one or more light chains of Cetuximab, Trastuzumab, and Nivolumab, although the affinity was lower than when the VH was paired with its original light chain (cLC). Antibody SC5Abl, which comprises a heavy chain variable region having the amino acid sequence shown as SEQ ID NO: 1, showed similar or higher binding to CD3 when combined with the light chain variable region of Nivolumab than when combined with its original light chain.

[0157] Table 2. Results of ELISA and FACS. N.D. not detected.

[0158]

[0159]

[0160] Example 2 - Pairing of CD3 heavy chain with light chains of several commercially available or publicly available antibodies

[0161] Essentially, the studies of Example 1 were repeated using the light chains of commercially available or publicly available antibodies listed in Table 3.

[0162] Table 3. Commercially available or publicly available antibodies used in this study, their light chain variable region (or sequence variants thereof).

[0163]

[0164]

[0165]

[0166] ELISA

[0167] CD3 antibodies were screened to determine if the antibodies were able to specifically bind to CD3. ELISA plates (Greiner Bio-One, Cat. No. 655061) were coated with CD3δε-FC or tetanus toxoid (TT) (AJ Vaccines, Cat. No. 2674) at 2.5 pg / ml and 2 pg / ml in PBS, respectively. Coated plates were left overnight at 4°C, washed twice with freshly prepared wash buffer (0.05% Tween 20; Merck, Cat. No. 8.22184.0500, prepared in PBS) and blocked with blocking buffer (2% BSA; Sigma, Cat. No. A3294-500g, prepared in PBS) for 1 hour at room temperature. CD3 antibodies or control antibodies were added to each well at a concentration of 5 pg / ml diluted in blocking buffer. Antibodies were left for 1 hour at room temperature. Control antibodies included a positive control anti-CD3 antibody to confirm CD3 antigen coating and a negative control IgG1 antibody (TT) to confirm binding specificity. Each well was washed three times with wash buffer before the addition of secondary antibody. An anti-human IgG HRP antibody (BD, Cat. No. 555788) diluted 1 :2000 in blocking buffer was added to each well and left for 1 hour at room temperature. Each well was washed 3 times with wash buffer before development. 50ul of TMB substrate solution, freshly prepared by mixing 1x Reagent A: 1x Reagent B (BD OptEIA TMB Substrate Reagent Set from BD, Cat. No. 555214) was added to each well for up to 10 minutes before the reaction was stopped with 50ul 1M H2SO4 (Merck, Cat. No. 1.00731). A BioTek Elx808 ELISA plate reader was used to read the O.D. 450 readings and titration data was analysed using GraphPad prism software.

[0168] Antibodies showing CD3 binding higher than three times background and TT binding lower than three times background were selected for further FACS analysis.

[0169] FACS

[0170] Binding and relative affinity of CD3 antibodies to huCD3 was determined by FACS analysis of HPB-ALL cells. HPB-ALL cells were cultured in RPMI1640 (Thermo Fisher, Cat. No. 21875) containing 10% heat inactivated (h.i.) fetal bovine serum (FBS) (Sigma, Cat. No. F7524) at 0.05-0.2 x 106 cells / well. To block Fc function, cells were incubated with FACS blocking buffer (0.5% BSA, Sigma-Aldrich, Cat. No. A3294, and 2 mM EDTA, Invitrogen, Cat. No. 15575-020 + 3% rabbit serum, Sigma-Aldrich, Cat. No. R9133). CD3 antibodies, positive control anti-CD3 antibody or negative control IgGl antibody (TT) were prepared in FACS buffer (0.5% BSA, Sigma-Aldrich, Cat. No. A3294 and 2 mM EDTA, Invitrogen, Cat. No. 15575-020) and added to the cells in eight steps, half-log serial dilutions starting at a concentration of 10 pg / ml. Cells were incubated for 30 min and washed, then anti-hu IgG R-PE secondary antibody (Invitrogen, Cat. No. H10104) was added at 1 : 100, incubated for 30 min and washed before FACS analysis. All incubation and washing steps were done in ice-cold FACS buffer. FACS measurements were performed using an iQue VBR; Intellicyt and ForeCyt software to express mean fluorescence intensity (MFI).

[0171] For data analysis, all binding curves were plotted according to VH grouping and AUC was calculated (GraphPad prism software). The AUC of a VH with a commercially available or publicly available antibody light chain was compared to the AUC of the same VH in combination with its original light chain.

[0172] A summary of the ELISA results is shown in Table 4. These data show that antibody SC5Ab1 has the most promiscuous VH. FACS data for this VH are provided in Figure 1 .

[0173] Example 3 - Further identification of antibodies comprising the heavy chain variable region of antibody SC5Ab1 and the light chain variable region of several commercially available or publicly available antibodies

[0174] The heavy chain variable region (VH) of antibody SC5Abl was combined with the light chain variable region (VL) of commercially available or publicly available antibodies cusatuzumab, fresolimumab, ofatumumab or trastuzumab. The VH region of antibody SC5Abl was also combined with a VL region having the amino acid sequence as set forth in SEQ ID NO: 5 as a parental control group.

[0175] To determine the molecular weight size and purity percentage of the IgG, the Labchip (LabChip GXII Touch HT; Perkin Elmer) analysis was used, under non-reducing conditions using Protein Clear HR Reagent Kit (dye solution, sample buffer, protein gel matrix, protein marker, low molecular weight marker, wash buffer; Perkin Elmer CLS960014) and Protein Express Assay LabChip, using GXII Touch HT (Elmer 760499) to analyze the IgG samples according to the manufacturer's instructions; Labchip RX reviewer software was used for protein identification. 250 ng of each sample was used for analysis.

[0176] The binding ability of the IgG was tested on HPB-ALL cells as described in Example 2, except that the negative control here was an IgG1 antibody targeting RSV-G.

[0177] The melting temperature (Tm) and aggregation temperature (Tagg) of non-cLC IgG and their corresponding parental cLC IgG were measured using the Uncle system (Unchained Labs, product code 200-1037). Tm was measured by the change in intrinsic fluorescence of amino acids upon heating in the spectral range of 250 to 720 nm, while Tagg was measured by static light scattering (SLS) at 266 nm. IgG sample solutions were prepared in PBS pH 7.4 (1x, Gibco, catalog number 10010-031) to a final concentration of 100 µg / mL, or 250 µg / mL for the control group. 8.8 μL of IgG solution or PBS was dispensed into a microvolume cuvette (Unis, product code 201-1009). Assembly of the uni, seal (product code 201-1009 / 201-1013), and uni frame (product code 201-1012), and loading onto the UNcle system, was performed according to the manufacturer's instructions. IgG samples were heated from 25 °C to 95 °C at a rate of 0.3 °C / min, with an equilibration time of 1 minute before heating. Data analysis was performed using UNcle Analysis v5.01 software, generating dissociation and aggregation curves.

[0178] like Figure 2A As shown, the data indicates that the heavy chain variable region (VH) of antibody SC5Ab1 pairs well with multiple, non-homologous light chain variable regions, as shown by the main band corresponding to IgG (150 kD). Manual examination of each graph revealed that additional bands (e.g., observed in the non-hematoxylin and eosinophil (fresolimumab) cases) did not correspond to protein spikes. Figure 2B ).

[0179] In summary, antibodies containing the VH region of antibody SC5Ab1 that pair with the VL region of most commercially available or publicly available antibodies can maintain sufficient CD3 binding. Figure 3 It exhibits good exploitability and comparable thermal stability when paired with its homologous VL region (SEQ ID NO: 5) (see [link to relevant documentation]). Figure 4 ).

[0180]

[0181]

[0182] sequence

[0183] SEQ ID NO: 1 - Heavy chain variable region

[0184] EVQLVESGAEVKKPGESLKISCKASGYSFTRFYIGWVRQMPGKGLEWMGLIYPGDSDTKYSPSFQGQVTISVDKSINTAYLQWNSLKASDTAMYYCARHIRYFDWFSHYYYMDVWGKGTMVTVSS

[0185] SEQ ID NO: 2 - Heavy chain CDR1 according to Kabat numbering

[0186] RFYIG

[0187] SEQ ID NO: 3 - Heavy chain CDR2 according to Kabat numbering

[0188] LIYPGDSDTKYSPSFQG

[0189] SEQ ID NO: 4 - Heavy chain CDR3 according to Kabat numbering

[0190] HIRYFDWFSHYYYMDV

[0191] SEQ ID NO: 5 - Light chain variable region cLC

[0192] DIQMTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYSTPPT FGQGTKVEIK

[0193] SEQ ID NO: 6 - Light chain CDR1 according to IMGT numbering

[0194] QSISSY

[0195] SEQ ID NO: 7 - Light chain CDR2 according to IMGT numbering

[0196] AAS

[0197] SEQ ID NO: 8 - Light chain CDR3 according to IMGT numbering

[0198] QQSYSTPPT

[0199] SEQ ID NO: 9 - CL region

[0200] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0201] SEQ ID NO: 10 - Light chain cLC

[0202] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0203] SEQ ID NO: 11 - V region VK1-39

[0204] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP

[0205] SEQ ID NO: 12 - VK1-39 / JK1

[0206] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK

[0207] SEQ ID NO: 13 - Light chain variable region

[0208] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

[0209] SEQ ID NO: 14 - Light chain CDR1 according to IMGT numbering

[0210] QSISSY

[0211] SEQ ID NO: 15 - Light chain CDR2 according to IMGT numbering

[0212] AAS

[0213] SEQ ID NO: 16 - Light chain CDR3 according to IMGT numbering

[0214] QQSYSTPPIT

[0215] SEQ ID NO: 17 - V region VK3-15

[0216] EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWP

[0217] SEQ ID NO: 18 - Light chain variable region

[0218] EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPWTFGQGTKVEIK

[0219] SEQ ID NO: 19 - Light chain CDR1 according to IMGT numbering

[0220] QSVSSN

[0221] SEQ ID NO: 20 - Light chain CDR2 according to IMGT numbering

[0222] GAS

[0223] SEQ ID NO: 21 - Light chain CDR3 according to IMGT numbering

[0224] QQYNNWPWT

[0225] SEQ ID NO: 22 - V region VK3-20

[0226] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP

[0227] SEQ ID NO: 23 - Light chain variable region

[0228] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK

[0229] SEQ ID NO: 24 - Light chain CDR1 according to IMGT numbering

[0230] QSVSSSY

[0231] SEQ ID NO: 25 - Light chain CDR2 according to IMGT numbering

[0232] GAS

[0233] SEQ ID NO: 26 - Light chain CDR3 according to IMGT numbering

[0234] QQYGSSPWT

[0235] SEQ ID NO: 27 - V region VL3-21

[0236] SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGSSDH

[0237] SEQ ID NO: 28 - Light chain variable region

[0238] SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGSSDHWVFGGGTKLTVL

[0239] SEQ ID NO: 29 - Light chain CDR1 according to IMGT numbering

[0240] NIGRKS

[0241] SEQ ID NO: 30 - Light chain CDR2 according to IMGT numbering

[0242] YDS

[0243] SEQ ID NO: 31 - Light chain CDR3 according to IMGT numbering

[0244] QVWDGSSDHWV

[0245] SEQ ID NO: 32 - Hinge region

[0246] EPKSCDKTHTCPPCP

[0247] SEQ ID NO: 33 - CHI region

[0248] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV

[0249] SEQ ID NO: 34 - CH2 region

[0250] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0251] SEQ ID NO: 35 - CH2-DM region

[0252] APELGRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

[0253] SEQ ID NO: 36 - CH3 region

[0254] GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0255] SEQ ID NO: 37 - Heavy chain positive control anti-CD3 antibody

[0256] EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0257] SEQ ID NO: 38 - Heavy chain negative control IgGl antibody (RSV-G)

[0258] EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVISYDGSTKYSADSLKGRFTISRDNSKNTLYLQMNSLRADDTAVYYCAKEGWSFDSSGYRSWFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0259] SEQ ID NO: 39 - Heavy chain negative control IgGl antibody (TT)

[0260] EVQLVETGAEVKKPGASVKVSCKASDYIFTKYDINWVRQAPGQGLEWMGWMSANTGNTGYAQKFQGRVTMTRDTSINTAYMELSSLTSGDTAVYFCARSSLFKTETAPYYHFALDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0261] SEQ ID NO: 40 - Light chain variable region cetuximab

[0262] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK

[0263] SEQ ID NO: 41 - Light chain variable region trastuzumab

[0264] DIQMTQSPSSLSASVGDRVTITC RASQDVNTAVA WYQQKPGKAPKLLIY SASFLYS GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC QQHYTTPPT FGQGTKVEIK

[0265] SEQ ID NO: 42 - Light chain variable region nivolumab

[0266] EIVLTQSPATLSLSPGERATLSC RASQSVSSYLA WYQQKPGQAPRLLIY DASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQSSNWPRT FGQGTKVEIK

[0267] SEQ ID NO: 43 - Light chain variable region of ulocuplumab

[0268] EIVLTQSPATLSLSPGERATLSC RASQSVSSYLA WYQQKPGQAPRLLIY DASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPPALT FGGGTKVEIK

[0269] SEQ ID NO: 44 - Light chain variable region of miravatuzumab

[0270] DIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHVPPTFGAGTRLEIK

[0271] SEQ ID NO: 45 - Light chain variable region of golimumab

[0272] QAVVTQEPSLTVSPGGTVTLTC GLKSGSVTSDNFPT WYQQTPGQAPRLLIY NTNTRHS GVPDRFSGSILGNKAALTITGAQADDEAEYFC ALFISNPSVE FGGGTQLEIK

[0273] SEQ ID NO: 46 - Light chain variable region of belantumab

[0274] DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIK

[0275] SEQ ID NO: 47 - Light chain variable region of daratumumab

[0276] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK

[0277] SEQ ID NO: 48 - Light chain variable region Ipilimumab

[0278] DIQLTQSPSSLSASVGDRVTMSCKSSQSVLYSANHKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYLSSWTFGGGTKLEIK

[0279] SEQ ID NO: 49 - Light chain variable region Fosnetupinab

[0280] ETVLTQSPGTLSLSPGERATLSC RASQSLGSSYLA WYQQKPGQAPRLLIY GASSRAP GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYADSPIT FGQGTRLEIK

[0281] SEQ ID NO: 50 - Light chain variable region Inotuzumab

[0282] DVQVTQSPSSLSASVGDRVTITC RSSQSLANSYGNTFLS WYLHKPGKAPQLLIY GISNRFS GVPDRFSGSGSGTDFTLTISSLQPEDFATYYC LQGTHQPYT FGQGTKVEIK

[0283] SEQ ID NO: 51 - Light chain variable region KA3

[0284] DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQMHPRAPKTFGQGTKVEIK

[0285] SEQ ID NO: 52 - Light chain variable region Ligelizumab

[0286] DIVMTQSPDSLAVSLGERATINC KSSQSVLYAGNNRNYLAWYQQKPGQPPKLLIN QASTRAS GVPDRFSGSGSGTEFTLIISSLQAEDVAIYYC QQYYTPPLA FGGGTKLEIK

[0287] SEQ ID NO: 53 - Light chain variable region Morolimumab

[0288] DIVMTQSPLSLPVTPGEPASISC RSSQSIVYSNGNTYLG WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC FQGSHVPYT FGQGTKLEIK

[0289] SEQ ID NO: 54 - Light chain variable region Monalizumab

[0290] DIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLIYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPRTFGGGTKVEIK

[0291] SEQ ID NO: 55 - Light chain variable region Obinutuzumab

[0292] DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIK

[0293] SEQ ID NO: 56 - Light chain variable region Ofatumumab

[0294] EIVLTQSPATLSLSPGERATLSC RASQSVSSYLA WYQQKPGQAPRLLIY DASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPIT FGQGTRLEIK

[0295] SEQ ID NO: 57 - Light chain variable region Orlamumab

[0296] EIVLTQSPATLSLSPGERATLSC RASQSVSSYLA WYQQKPGQAPRLLIY DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPPA FGQGTKVEIK

[0297] SEQ ID NO: 58 - Light chain variable region obinutuzumab

[0298] EIVMTQSPATLSVSPGERVSLSC RASQSISDYLH WYQQKSHESPRLLIK YASQSIS GIPARFSGSGSGSEFTLTINSVEPEDVGVYYC QNGHSFPLT FGQGTKLEIK

[0299] SEQ ID NO: 59 - Light chain variable region galiximab

[0300] DIVMTQAAFSNPVTLGTSASISC RSSKSLLHSNGITYFF WYLQKPGLSPQLLIY QMSNLAS GVPDRFSSSGSGTDFTLRISRVEAEDVGVYYC AQNLELPPT FGGGTKLEIK

[0301] SEQ ID NO: 60 - Light chain variable region lorvotuzumab mertansine

[0302] DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTFLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTRLEIK

[0303] SEQ ID NO: 61 - Light chain variable region lintuzumab

[0304] QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK

[0305] SEQ ID NO: 62 - Light chain variable region sevirumab

[0306] DIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQKNYLAWYQQKPGQPPKLLIFWASTRESGVPDRFSGSGFGTDFTLTISSLQAEDVAVYYCQQYFSYPLTFGQGTKVEIK

[0307] SEQ ID NO: 63 - Light chain variable region Talizumab

[0308] DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIK

[0309] SEQ ID NO: 64 - Light chain variable region Tocilizumab

[0310] DIQMTQSPSSLSASVGDRVSITC RPSQSFSRYIN WYQQKPGKAPKLLIH AASSLVG GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQTYSNPPIT FGQGTRLEIK

[0311] SEQ ID NO: 65 - Light chain variable region Ulocuplumab

[0312] DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFVTYYCQQYNSYPRTFGQGTKVEIK

[0313] SEQ ID NO: 66 - Light chain variable region H2C

[0314] QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL

[0315] SEQ ID NO: 67 - Light chain variable region (hOKT3)

[0316] DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQIT

[0317] SEQ ID NO: 68 - Light chain variable region

[0318] EIVLTQSPATLSLSPGERATLSC RASQSVSSYLA WYQQKPGQAPRLLIY DASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPPALT FCGGTKVEIK

[0319] SEQ ID NO: 69 - Light chain variable region

[0320] QAVVTQEPSLTVSPGGTVTLTC GLKSGSVTSDNFPT WYQQTPGQAPRLLIY NTNTRHS GVPDRFSGSILGNKAALTITGAQADDEAEYFC ALFISNPSVE FGGGTQLTVL

[0321] SEQ ID NO: 70 - Light chain variable region

[0322] DIVMTQSPATLSVTPGDRVSLSC RASQSISDYLH WYQQKSHESPRLLIK YASQSIS GIPSRFSGSGSGSDFTLSINSVEPEDVGVYYC QNGHSFPLT FGAGTKLELK

[0323] SEQ ID NO: 71 - Light chain variable region

[0324] DIVMTQSPLSNPVTPGEPASISC RSSKSLLHSNGITYFF WYLQKPGQSPQLLIY QMSNLAS GVPDRFSGSGSGTDFTLRISRVEAEDVGVYYC AQNLELPPT FGQGTKVEIK

[0325] SEQ ID NO: 72 - Light chain variable region

[0326] DIQMTQSPSSLSASVGDRVSITC RPSQSFSRYIN WYQQKPGKAPKLLIHAASSLVG GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQTYSNPPIT FGQGTRLEMK

[0327] SEQ ID NO: 73 - Light chain CDR1 according to IMGT numbering

[0328] QDVNTA

[0329] SEQ ID NO: 74 - Light chain CDR2 according to IMGT numbering

[0330] SAS

[0331] SEQ ID NO: 75 - Light chain CDR3 according to IMGT numbering

[0332] QQHYTTPPT

[0333] SEQ ID NO: 76 - Light chain CDR1 according to IMGT numbering

[0334] QSVSSY

[0335] SEQ ID NO: 77 - Light chain CDR2 according to IMGT numbering

[0336] DAS

[0337] SEQ ID NO: 78 - Light chain CDR3 according to IMGT numbering

[0338] QQSSNWPRT

[0339] SEQ ID NO: 79 - Light chain CDR1 according to IMGT numbering

[0340] QSVSSY

[0341] SEQ ID NO: 80 - Light chain CDR2 according to IMGT numbering

[0342] DAS

[0343] SEQ ID NO: 81 - Light chain CDR3 according to IMGT numbering

[0344] QQRSNWPPALT

[0345] SEQ ID NO: 82 - Light chain CDR3 according to IMGT numbering

[0346] QQRSNWPPALTFC

[0347] SEQ ID NO: 83 - Light chain CDR1 according to IMGT numbering

[0348] SGSVTSDNF

[0349] SEQ ID NO: 84 - Light chain CDR2 according to IMGT numbering

[0350] NTN

[0351] SEQ ID NO: 85 - Light chain CDR3 according to IMGT numbering

[0352] ALFISNPSVE

[0353] SEQ ID NO: 86 - Light chain CDR1 according to IMGT numbering

[0354] QSLGSSY

[0355] SEQ ID NO: 87 - Light chain CDR2 according to IMGT numbering

[0356] GAS

[0357] SEQ ID NO: 88 - Light chain CDR3 according to IMGT numbering

[0358] QQYADSPIT

[0359] SEQ ID NO: 89 - Light chain CDR1 according to IMGT numbering

[0360] QSLANSYGNTF

[0361] SEQ ID NO: 90 - Light chain CDR2 according to IMGT numbering

[0362] GIS

[0363] SEQ ID NO: 91 - Light chain CDR3 according to IMGT numbering

[0364] LQGTHQPYT

[0365] SEQ ID NO: 92 - Light chain CDR1 according to IMGT numbering

[0366] QSVLYAGNNRNY

[0367] SEQ ID NO: 93 - Light chain CDR2 according to IMGT numbering

[0368] QAS

[0369] SEQ ID NO: 94 - Light chain CDR3 according to IMGT numbering

[0370] QQYYTPPLA

[0371] SEQ ID NO: 95 - Light chain CDR1 according to IMGT numbering

[0372] QSIVYSNGNTY

[0373] SEQ ID NO: 96 - Light chain CDR2 according to IMGT numbering

[0374] KVS

[0375] SEQ ID NO: 97 - Light chain CDR3 according to IMGT numbering

[0376] FQGSHVPYT

[0377] SEQ ID NO: 98 - Light chain CDR1 according to IMGT numbering

[0378] QSVSSY

[0379] SEQ ID NO: 99 - Light chain CDR2 according to IMGT numbering

[0380] DAS

[0381] SEQ ID NO: 100 - Light chain CDR3 according to IMGT numbering

[0382] QQRSNWPIT

[0383] SEQ ID NO: 101 - Light chain CDR1 according to IMGT numbering

[0384] QSVSSY

[0385] SEQ ID NO: 102 - Light chain CDR2 according to IMGT numbering

[0386] DAS

[0387] SEQ ID NO: 103 - Light chain CDR3 according to IMGT numbering

[0388] QQRSNWPPA

[0389] SEQ ID NO: 104 - Light chain CDR1 according to IMGT numbering

[0390] QSISDY

[0391] SEQ ID NO: 105 - Light chain CDR2 according to IMGT numbering

[0392] YAS

[0393] SEQ ID NO: 106 - Light chain CDR3 according to IMGT numbering

[0394] QNGHSFPLT

[0395] SEQ ID NO: 107 - Light chain CDR1 according to IMGT numbering

[0396] KSLLHSNGITY

[0397] SEQ ID NO: 108 - Light chain CDR2 according to IMGT numbering

[0398] QMS

[0399] SEQ ID NO: 109 - Light chain CDR3 according to IMGT numbering

[0400] AQNLELPPT

[0401] SEQ ID NO: 110 - Light chain CDR1 according to IMGT numbering

[0402] QSFSRY

[0403] SEQ ID NO: 111 - Light chain CDR2 according to IMGT numbering

[0404] AAS

[0405] SEQ ID NO: 112 - Light chain CDR3 according to IMGT numbering

[0406] QQTYSNPPIT

[0407] SEQ ID NO: 113 - Human CD3d

[0408] MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELD

[0409] SEQ ID NO: 114 - Human CD3e

[0410] MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD

Claims

1. A polypeptide comprising a heavy chain CDR3 (HCDR3) having an amino acid sequence as shown in SEQ ID NO: 4; or having at least 70% sequence identity with it.

2. The polypeptide of claim 1, wherein the polypeptide comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence shown in SEQ ID NO: 2, a heavy chain CDR2 (HCDR2) having the amino acid sequence shown in SEQ ID NO: 3, and a heavy chain CDR3 (HCDR3) having the amino acid sequence shown in SEQ ID NO: 4; or having at least 70% sequence identity with said HCDR3.

3. The polypeptide according to claim 1 or 2, wherein the polypeptide comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence shown in SEQ ID NO: 2, a heavy chain CDR2 (HCDR2) having the amino acid sequence shown in SEQ ID NO: 3, and a heavy chain CDR3 (HCDR3) having the amino acid sequence shown in SEQ ID NO:

4. Each HCDR may contain one, two, or up to three amino acid mutations.

4. The polypeptide according to any one of claims 1 to 3, wherein the polypeptide comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 1, or having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with it.

5. The polypeptide according to any one of claims 1 to 4, further comprising a CH1 region.

6. The polypeptide according to any one of claims 1 to 5, further comprising a hinge region, a CH2 region and a CH3 region.

7. A CD3 binding domain comprising a polypeptide according to any one of claims 1 to 6.

8. The CD3 binding domain of claim 7, wherein the CD3 binding domain further comprises a polypeptide containing a light chain variable region.

9. An antigen-binding protein group that binds to human CD3, wherein each antigen-binding protein in the group comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR3 having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:

4.

10. The CD3 binding domain of claim 8 or the antigen-binding proteome of claim 9, wherein the light chain variable region comprises: - Light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) having the light chain variable region as shown in SEQ ID NO: 5; - Light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 41; - Light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 42; - Light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 43 or 68; - Light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 45 or 69; - Light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 49; - Light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 50; - Light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 52; - Light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 53; - Light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 56; - Light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) having the light chain variable region as shown in SEQ ID NO: 57; - Light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 58 or 70; - Light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) having the light chain variable region as shown in SEQ ID NO: 59 or 71; or - Light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 64 or 72; Or its variants, wherein each LCDR may contain one, two or up to three amino acid mutations.

11. The CD3 binding domain or antigen-binding protein group according to claim 10, wherein the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 5; 41; 42; 43 or 68; 45 or 69; 49; 50; 52; 53; 56; 57; 58 or 70; 59 or 71; or 64 or 72, or has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with it.

12. The CD3 binding domain or antigen-binding proteome according to claim 10 or 11, further comprising a CL region.

13. A binding portion comprising a polypeptide according to any one of claims 1 to 6, or a CD3 binding domain according to claim 7 or 8.

14. The binding portion according to claim 13, wherein the binding portion is a multispecific binding portion, particularly a bispecific or trispecific antibody.

15. A pharmaceutical composition comprising an effective amount of a polypeptide according to any one of claims 1 to 6, or a CD3 binding domain according to claim 7 or 8, or a binding moiety according to claim 13 or 14, and a pharmaceutically acceptable carrier.

16. The polypeptide according to any one of claims 1 to 6, or the CD3 binding domain according to claim 7 or 8, or the binding portion according to claim 13 or 14, or the pharmaceutical composition according to claim 15, for therapeutic purposes.

17. The polypeptide according to any one of claims 1 to 6, or the CD3 binding domain according to claim 7 or 8, or the binding portion according to claim 13 or 14, or the pharmaceutical composition according to claim 15, for cancer treatment.

18. A method of treating a disease, comprising administering to an individual in need an effective amount of the polypeptide according to any one of claims 1 to 6, or the CD3 binding domain according to claim 7 or 8, or the binding portion according to claim 13 or 14, or the pharmaceutical composition according to claim 15.

19. A method of treating cancer, comprising administering to an individual in need an effective amount of the polypeptide according to any one of claims 1 to 6, or the CD3 binding domain according to claim 7 or 8, or the binding portion according to claim 13 or 14, or the pharmaceutical composition according to claim 15.

20. A nucleic acid comprising a sequence encoding a polypeptide according to any one of claims 1 to 6.

21. A vector comprising the nucleic acid according to claim 20.

22. The vector according to claim 21, wherein the vector further comprises at least one nucleic acid sequence encoding a light chain variable region, preferably a CL region.

23. The carrier of claim 22, wherein the light chain variable region is a light chain variable region comprising a light chain CDR as defined in claim 10, or a light chain variable region as defined in claim 11.

24. A cell comprising the nucleic acid according to claim 20.

25. The cell of claim 24, wherein the cell further comprises at least one nucleic acid, the nucleic acid comprising a sequence encoding a light chain variable region, preferably a CL region.

26. The cell of claim 25, wherein the light chain variable region is a light chain variable region comprising a light chain CDR as defined in claim 10, or a light chain variable region as defined in claim 11.

27. A cell that produces a polypeptide according to any one of claims 1 to 6, or a CD3 binding domain according to claim 7 or 8, or a binding moiety according to claim 13 or 14.

28. The cell of claim 27, wherein the cell is a recombinant cell comprising the vector of any one of claims 21 to 23.

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