Indiscriminate CD3 binding molecules

Abstract

The present disclosure relates to the field of antibodies. Specifically, the present disclosure relates to the field of producing therapeutic antibodies. More specifically, the present disclosure relates to anti-CD3 heavy chain variable regions that can pair with multiple different light chain variable regions to form functional CD3-binding domains, as well as CD3-binding domains and CD3-binding sites that include such anti-CD3 heavy chain variable regions.

Description

[Technical Field]

[0001] The present disclosure relates to the field of antibodies. Specifically, the present disclosure relates to the field of producing therapeutic antibodies. More specifically, the present disclosure relates to anti-CD3 heavy chain variable regions (also referred to herein as CD3 heavy chain variable regions) that can pair with multiple different light chain variable regions to form functional CD3-binding domains, as well as CD3-binding domains and CD3-binding sites that include such CD3 heavy chain variable regions. [Background technology]

[0002] Monoclonal antibodies that bind to human CD3 were among the first antibodies developed for therapeutic use in humans. Monoclonal CD3-binding antibodies are typically used, for example, in transplant rejection, due to their immunosuppressive qualities. Antibodies that are multispecific for CD3 on T cells and at least one surface target antigen on cancer cells can connect any type of T cell to cancer cells, regardless of T cell receptor specificity, costimulation, or peptide antigen presentation. Such multispecific T cell-engaging antibodies show great promise in the treatment of various cancers and neoplastic growths.

[0003] It is an object of the present disclosure to provide a CD3 heavy chain variable region that can pair with multiple different light chain variable regions to form a functional CD3-binding domain, i.e., to provide a promiscuous CD3 heavy chain variable region. Such CD3 heavy chain variable regions are particularly useful for generating multispecific T cell-engaging antibodies. They are likely to be successfully combined with the light chain variable region of any binding domain generated against any tumor-associated antigen (TAA). Thus, multispecific antibodies comprising such CD3-binding domains and TAA-binding domains can be efficiently produced in a single host cell as common light chain antibodies. Furthermore, promiscuous CD3 heavy chain variable regions can be easily combined with the light chain variable regions of clinically successful antibodies. Multispecific antibodies comprising such CD3-binding domains and binding domains of clinically successful antibodies have the potential to, for example, extend clinical success to other therapeutic areas, provide increased tumor specificity and therefore safety, and / or be more effective in eradicating tumor cells. Summary of the Invention

[0004] In certain embodiments, the present disclosure provides a polypeptide comprising a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO:4, or having at least 70% sequence identity thereto.

[0005] In certain embodiments, the present disclosure provides a CD3 binding domain comprising a polypeptide described herein.

[0006] In certain embodiments, the present disclosure provides a group of antigen binding proteins that bind to human CD3, wherein each antigen binding protein in the group comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises an HCDR3 having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:4.

[0007] In certain embodiments, the present disclosure provides a binding site comprising a polypeptide described herein, or a CD3 binding domain described herein.

[0008] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of a polypeptide described herein, or a CD3 binding domain described herein, or a binding site described herein, and a pharmaceutically acceptable carrier.

[0009] In certain embodiments, the present disclosure provides a polypeptide described herein, or a CD3 binding domain described herein, or a binding site described herein, or a pharmaceutical composition described herein, for use in therapy.

[0010] In certain embodiments, the present disclosure provides a polypeptide described herein, or a CD3 binding domain described herein, or a binding site described herein, or a pharmaceutical composition described herein, for use in the treatment of cancer.

[0011] In certain embodiments, the present disclosure provides methods for treating a disease, comprising administering to an individual in need thereof an effective amount of a polypeptide described herein, or a CD3 binding domain described herein, or a binding site described herein, or a pharmaceutical composition described herein.

[0012] In certain embodiments, the present disclosure provides methods for treating cancer, comprising administering to an individual in need thereof an effective amount of a polypeptide described herein, or a CD3 binding domain described herein, or a binding site described herein, or a pharmaceutical composition described herein.

[0013] In certain embodiments, the present disclosure provides a nucleic acid comprising a sequence encoding a polypeptide described herein.

[0014] In certain embodiments, the present disclosure provides a vector comprising a nucleic acid described herein.

[0015] In certain embodiments, the present disclosure provides a cell comprising a nucleic acid described herein.

[0016] In certain embodiments, the present disclosure provides cells that produce a polypeptide described herein, or a CD3 binding domain described herein, or a binding site described herein. DETAILED DESCRIPTION OF THE INVENTION

[0017] One of the objectives of the present disclosure is to provide novel CD3 heavy chain variable regions useful for the generation of therapeutic antibodies, particularly T cell-engaging antibodies. This objective is met by the provision of several polypeptides, and binding domains and binding sites comprising these polypeptides.

[0018] In certain embodiments, the polypeptide of the present disclosure is a heavy chain variable region that can pair with multiple different light chain variable regions to form a functional binding domain that binds to CD3. This property of heavy chain variable regions is referred to in the art as promiscuity. The inventors have identified a group containing particularly promiscuous anti-CD3 heavy chain variable regions and related anti-CD3 heavy chain variable regions. These CD3 heavy chain variable regions are likely to pair well with the light chain variable regions of any binding domain generated against any tumor-associated antigen (TAA). Therefore, multispecific antibodies containing such CD3-binding domains and TAA-binding domains can be efficiently produced in a single host cell as common light chain antibodies. Furthermore, the inventors have shown that the promiscuous CD3 heavy chain variable regions of the present disclosure can be easily combined with the light chain variable regions of several clinically successful antibodies. Multispecific antibodies comprising such a CD3-binding domain and the binding domain of a clinically successful antibody have the potential, for example, to extend clinical success to other therapeutic areas, provide increased tumor specificity and therefore safety, and / or be more effective in eradicating tumor cells. Thus, the CD3 heavy chain variable regions of the present disclosure are particularly useful for generating multispecific T cell-engaging antibodies.

[0019] In certain embodiments, the present disclosure provides a polypeptide comprising a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 4, or having at least 70% sequence identity thereto. In certain embodiments, the present disclosure provides a polypeptide comprising a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 4, or having at least 70% sequence identity thereto, and having the same length as SEQ ID NO: 4.

[0020] 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 binds to CD3 when combined with a suitable light chain or an antigen-binding portion thereof. For example, the antigen-binding portion of an immunoglobulin heavy chain can be a heavy chain variable region having a CH1 region, or a heavy chain variable 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.

[0021] In certain embodiments, the polypeptide binds to human CD3 when combined with a suitable light chain or antigen-binding portion thereof. The amino acid sequence of the signal peptide and extracellular domain of human CD3δ is provided herein as SEQ ID NO: 113, and the amino acid sequence of the signal peptide and extracellular domain of human CD3ε is provided herein as SEQ ID NO: 114. Binding to human CD3 can be determined by comparison to background signal and / or negative control in an ELISA assay. For example, a polypeptide, binding domain, antigen-binding protein, or binding site binds to human CD3 if it has a binding signal at least two-fold higher than the background signal and / or a binding activity at least two-fold higher than the negative control.

[0022] Antigen binding can be expressed in terms of specificity and affinity. Specificity determines which antigen or epitope thereof is specifically bound by a binding domain or site. Affinity is a measure of the strength of binding to a particular antigen or epitope.

[0023] The present inventors have identified a group of polypeptides, specifically binding domains comprising heavy chain variable regions, in which the binding domain comprises an 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 sites 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 sites comprising such polypeptides, are grouped based on having at least 70% sequence identity in the HCDR3 and the same HCDR3 length. In certain embodiments, such polypeptides, or binding domains or binding sites comprising such polypeptides, are grouped based on having at least 70% sequence identity in the HCDR3, the same HCDR3 length, and being derived from the same heavy chain variable region V gene segment.

[0024] "Percent identity (%)" herein with respect to a nucleic acid or amino acid sequence is defined as the percentage of residues in a candidate sequence that are identical to the residues in a selected sequence after aligning the sequences for optimal comparison purposes. To optimize the alignment between these two sequences, gaps can be introduced into either of the two sequences being compared. Such alignment can be performed over the entire length of the sequences being compared. Alternatively, alignment can be performed over a shorter length, for example, about 20, about 50, about 100 or more nucleic acids / bases or amino acids. Alignment can also be performed over individual CDR sequences. Sequence identity is the percentage of perfect matches between these two sequences over the reported aligned region.

[0025] Comparison of sequences and determination of the percentage of sequence identity between two sequences can be achieved using a mathematical algorithm. Those skilled in the art will recognize that several different computer programs are available for aligning two sequences and determining the identity between two sequences (Kruskal, JB (1983) An overview of sequence comparison In D. Sankoff, and JB Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley). The percentage of sequence identity between two amino acid or nucleic acid sequences can be determined using the Needleman and Wunsch algorithm for aligning two sequences (Needleman, SB and Wunsch, CD (1970) J. Mol. Biol. 48, 443-453). The Needleman-Wunsch algorithm is implemented in the computer program NEEDLE. For purposes of this disclosure, the NEEDLE program from the EMBOSS package is used to determine percent identity of amino acid and nucleic acid sequences (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) pp276-277, http: / / emboss.bioinformatics.nl / ). For protein sequences, the substitution matrix used is EBLOSUM62. For DNA sequences, DNAFULL is used. The parameters used are a gap open penalty of 10 and a gap extension penalty of 0.5.

[0026] After alignment by the program NEEDLE as described above, the percentage of sequence identity between the query sequence and the sequences of the disclosure is calculated as follows: the number of corresponding positions in the alignment represents the number of identical amino acids or identical nucleotides in both sequences divided by the total number of alignments after subtracting the total number of gaps in the alignment.

[0027] In certain embodiments, a polypeptide of the present disclosure comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 2, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 3, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 4, wherein each of the HCDRs may comprise one, two, or three amino acid variations.

[0028] In certain embodiments, a polypeptide of the disclosure comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 2, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 3, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 4, wherein each of the HCDRs may contain one, two, or at most three amino acid variations. In certain embodiments, a polypeptide of the disclosure comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 2, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 3, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 4, wherein two of the HCDRs may contain one, two, or at most three amino acid variations. In certain embodiments, the two HCDRs are HCDR1 and HCDR2. In certain embodiments, a polypeptide of the present disclosure comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 2, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 3, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 4, wherein one of the HCDRs may contain one, two, or at most three amino acid variations. In certain embodiments, the one HCDR is HCDR1 or HCDR2.

[0029] The heavy chain variable region of a polypeptide of the disclosure can include a limited number of non-conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or an unlimited number of conservative amino acid substitutions.

[0030] In certain embodiments, the polypeptides of the present disclosure also include variants thereof, in which each HCDR may contain one, two, or three amino acid variations. In certain embodiments, only one or two HCDRs may contain one, two, or three non-conservative amino acid variations. In certain embodiments, such variants do not contain amino acid variations in HCDR3. In certain embodiments, the amino acid variations are conservative amino acid substitutions.

[0031] Typically, conservative amino acid substitutions involve amino acid variations with homologous amino acid residues, which are residues that share similar characteristics or properties. Homologous amino acids are known in the art as a common method for making amino acid substitutions in antibody binding domains without significantly affecting antibody binding or function; see, for example, handbooks such as Lehninger (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), which are incorporated herein in their entirety. In determining whether an amino acid can be replaced with a conservative amino acid, an evaluation can typically be made of factors such as, but not limited to, (a) the structure of the polypeptide backbone in the area of ​​the substitution, e.g., sheet or helix structure, (b) the charge or hydrophobicity of the molecule at the target site, and / or (c) the bulkiness of the side chain. If a residue can be replaced with a residue that shares common characteristics, such as a similar side chain or similar charge or hydrophobicity, then such a residue is preferred as a replacement. For example, the following groups can be determined: (1) non-polar: Ala (A), Gly (G), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H).Alternatively, amino acids can be grouped as follows: (1) aromatic: Phe (F), Trp (W), Tyr (Y); (2) polar: Leu (L), Val (V), Ile (I), Ala (A), Met (M); (3) aliphatic: Ala (A), Val (V), Leu (L), Ile (I); (4) acidic: Asp (D), Glu (E); (5) basic: His (H), Lys (K), Arg (R); and (6) polar: Gln (Q), Asn (N), Ser (S), Thr (T), Tyr (Y). Alternatively, amino acid residues can be divided into groups based on common side chain properties: (1) hydrophobic: Met (M), Ala (A), Val (V), Leu (L), Ile (I); (2) neutral hydrophilic: Cys (C), Ser (S), Thr (T), Asn (N), Gln (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).

[0032] Substitution with another amino acid residue from the same group is preferred. Thus, conservative amino acid substitutions can involve exchanging a member of one of these classes for another member of the same class. Typically, the variation does not result in, or does not substantially result in, loss of binding specificity of the binding domain for its intended target.

[0033] Additional types of amino acid variations include variations resulting from somatic hypermutation or affinity maturation. Binding variants encompassed by the present disclosure include somatically hypermutated or affinity-matured heavy chain variable regions derived from the same VH gene segment as the heavy chain variable regions described herein by sequence, where the variants have amino acid variations in one, two, or all three HCDRs, including non-conservative and / or conservative amino acid substitutions. Conventional methods for affinity maturation of antibody binding domains are widely known in the art; see, for example, 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).

[0034] In certain embodiments, a polypeptide of the present disclosure comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:1, or having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity thereto.

[0035] In certain embodiments, the polypeptides of the present disclosure also include variants that contain one or more variations in the framework regions in addition to the variations in the HCDRs referenced above. The variations can be any type of amino acid variation described herein, such as conservative or non-conservative amino acid substitutions resulting from somatic hypermutation or affinity maturation. In certain embodiments, the polypeptides of the present disclosure do not contain variations in the CDR regions but contain one or more variations 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 present disclosure include: - comprising 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, wherein the heavy chain variable region comprises an HCDR1 amino acid sequence set forth in SEQ ID NO:2, an HCDR2 amino acid sequence set forth in SEQ ID NO:3, and an HCDR3 amino acid sequence set forth in SEQ ID NO:4.

[0036] In certain embodiments, polypeptides of the present disclosure are generated with the light chain VK1-39 / JK1. Polypeptides of the present disclosure can be paired with any suitable light chain. In certain embodiments, a 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) 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, a suitable light chain comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO:5. In certain embodiments, a suitable light chain is the light chain of urelumab, cusatuzumab, fresolimumab, inotuzumab, lemzoparimab, magrolimab, ofatumumab, olaratumab, omburtamab, gatipotuzumab (pancomab), tobetumab, trastuzumab, or nivolumab.

[0037] In certain embodiments, polypeptides of the present disclosure further comprise a CH1 region. In certain embodiments, polypeptides of the present disclosure further comprise a CH1 region, a hinge, a CH2 region, and a CH3 region. Suitable CH1 regions include, but are not limited to, the CH1 region having the amino acid sequence set forth in SEQ ID NO: 33. Suitable hinges include, but are not limited to, the hinge having the amino acid sequence set forth in SEQ ID NO: 32. Suitable CH2 and CH3 regions include, but are not limited to, the CH2 region having the amino acid sequence set forth in SEQ ID NO: 34 or 35, and the CH3 region having the amino acid sequence set forth in SEQ ID NO: 36.

[0038] The CH1, hinge, CH2, CH3, and / or CL can be modified according to methods known in the art to obtain favorable antibody characteristics, including, for example, promoting heterodimerization of different heavy chains, improving heavy chain-light chain pairing, and enhancing or reducing immune cell effector function. The CH3 region can include or lack a terminal lysine residue to improve manufacturability.

[0039] In certain embodiments, the present disclosure provides a CD3 binding domain comprising a polypeptide described herein.

[0040] In certain embodiments, the CD3 binding domain of the present disclosure further comprises a polypeptide comprising a light chain variable region, which in certain embodiments is an antibody light chain variable region.

[0041] In certain embodiments, the CD3 binding domain of the present disclosure is an antibody Fab domain.

[0042] In certain embodiments, the present disclosure provides a group of antigen binding proteins that bind to human CD3, wherein each antigen binding protein in the group comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an HCDR3 having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the present disclosure provides a group of antigen binding proteins that bind to human CD3, wherein each antigen binding protein in the group comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an 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 disclosure provides a group of antigen binding proteins that bind to human CD3, wherein each antigen binding protein in the group comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an HCDR3 having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:4, 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.

[0043] In certain embodiments, the antigen binding protein is an antibody or an antigen-binding fragment thereof.

[0044] The binding domain or antigen-binding protein of the present disclosure may comprise any suitable light chain, including, but not limited to, consensus light chains known in the art. In certain embodiments, the binding domain or antigen-binding protein of the present disclosure comprises the consensus light chain VK1-39 / JK1, or a variant thereof with a limited number of non-conservative amino acid substitutions, such as 1, 2, or 3, or an unlimited number of conservative amino acid substitutions.

[0045] In certain embodiments, a binding domain or antigen binding protein of the present disclosure 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, a binding domain or antigen binding protein of the present disclosure comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 5, or a variant having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity thereto.

[0046] In certain embodiments, a binding domain or antigen binding protein of the present disclosure 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 a binding domain or antigen binding protein of the present disclosure, including variants thereof, may comprise one, two, or three amino acid variations in each of the LCDRs. In certain embodiments, the amino acid variations are conservative amino acid substitutions.

[0047] The light chain or light chain variable region containing these LCDRs and / or light chain variable regions may 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" according to the present disclosure refers to a light chain that can pair with multiple different heavy chains, e.g., heavy chains with different antigen or epitope binding specificities. Common light chains are particularly useful, for example, in generating bispecific or multispecific antibodies, where antibody production is more efficient when all binding domains contain the same light chain. The term "common light chain" encompasses light chains that are identical or have some amino acid sequence differences, but in which the binding specificity of the full-length antibody is not affected. For example, it is possible, within the definition of a common light chain used herein, to prepare or find light chains that are not identical but are still functionally equivalent by using, for example, conservative amino acid changes, well-established variations that introduce amino acid changes in regions known or shown to not contribute, or only partially contribute, to binding specificity when paired with a heavy chain.

[0048] Apart from the common light chains containing the LCDRs and / or light chain variable regions mentioned above, other common light chains known in the art may also be used. Examples of such common light chains include, but are not limited to, VK1-39 / JK5, ​​which 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. 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: 13, each of which may contain one, two, or three amino acid variations, 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 having 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, and VK3-15 / JK1 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, and each of the LCDRs may comprise one, two, or three amino acid variations, 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 having 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, and VK3-20 / JK1 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 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: 23, and each of the LCDRs may comprise one, two, or three amino acid variations, 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: 23, or having 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: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively, and VL3-21 / JL3 comprises a light chain variable region comprising the light chain CDR1 (LCDR1), the light chain CDR2 (LCDR2), and the light chain CDR3 (LCDR3) of the light chain variable region having the amino acid sequence set forth in SEQ ID NO: 28. 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 the light chain variable region having the amino acid sequence set forth in SEQ ID NO: 28, wherein each of the LCDRs may comprise one, two, or three amino acid variations, 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: 28, or having 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: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively.

[0049] VK1-39 is an abbreviation for immunoglobulin variable kappa 1-39 gene. This gene is also known as immunoglobulin kappa variable 1-39, IGKV139, IGKV1-39, and IgVκ1-39. The external IDs for this gene are HGNC:5740, Entrez Gene:28930, and Ensembl:ENSG00000242371. The amino acid sequence of VK1-39 is given 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. Preferred VJ region sequences are designated as VK1-39 / JK1 (SEQ ID NO: 12) and VK1-39 / JK5 (SEQ ID NO: 13), alternatively designated IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01 (designations from the IMGT database world wide web at imgt.org). These designations are exemplary and encompass allelic variants of the gene segments.

[0050] VK3-15 is an abbreviation for immunoglobulin variable kappa 3-15 gene. This gene is also known as immunoglobulin kappa variable 3-15, IGKV315, IGKV3-15, or IgVκ3-15. The external identifiers for this gene are HGNC:5816, Entrez Gene:28913, and Ensembl:ENSG00000244437. The amino acid sequence of VK3-15 is given as SEQ ID NO:17. This is the sequence of the V region. A V region can be combined with one of five J regions. A preferred VJ region sequence is shown as VK3-15 / JK1 (SEQ ID NO:18), with an alternative designation of Vκ3-15*01 / IGJκ1*01 (designation by the IMGT database world wide web at imgt.org). This designation is exemplary and encompasses allelic variants of the gene segment.

[0051] VK3-20 is an abbreviation for immunoglobulin variable kappa 3-20 gene. This gene is also known as immunoglobulin kappa variable 3-20, IGKV320, IGKV3-20, or IgVκ3-20. The external identifiers for this gene are HGNC:5817, Entrez Gene:28912, and Ensembl:ENSG00000239951. The amino acid sequence of VK3-20 is shown as SEQ ID NO:22. This is the sequence of the V region. A V region can be combined with one of five J regions. A preferred VJ region sequence is shown as VK3-20 / JK1 (SEQ ID NO:23), with an alternative name being IgVκ3-20*01 / IGJκ1*01 (designation by the IMGT database world wide web at imgt.org). This designation is exemplary and encompasses allelic variants of the gene segment.

[0052] VL3-21 is an abbreviation for immunoglobulin variable lambda 3-21 gene. This gene is also known as immunoglobulin lambda variable 3-21, IGLV321, IGLV3-21, or IgVλ3-21. The external identifiers for this gene are HGNC:5905, Entrez Gene:28796, and Ensembl:ENSG00000211662.2. The amino acid sequence of VL3-21 is given 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 preferred VJ region sequence is shown as VL3-21 / JL3 (SEQ ID NO:28), with an alternative name being IgVλ3-21 / IGJλ3 (designation from the IMGT database world wide web at imgt.org). This designation is exemplary and encompasses allelic variants of the gene segment.

[0053] Furthermore, any light chain variable region of an antibody available in the art may be used, such as any other light chain variable region that can be easily obtained from an antibody display library, for example, by exhibiting antigen-binding activity when paired with a polypeptide of the present disclosure.

[0054] In certain embodiments, the binding domain or antigen-binding protein of the present disclosure comprises the light chain or light chain variable region of urelumab, cusatuzumab, fresolimumab, inotuzumab, lemzoparimab, magrolimab, ofatumumab, olaratumab, omburtamab, gatipotuzumab (pancomab), tobetumab, trastuzumab, or nivolumab. The sequences of the variable regions of these light chains are provided herein. The light chain variable region used in accordance with the present disclosure may contain one or more sequence variations that do not significantly alter the binding and functional activity of the resulting antibody, i.e., an equivalent light chain variable region.

[0055] In certain embodiments, the CD3-binding domain of the present disclosure, or an antigen-binding protein within the group of antigen-binding proteins of the present disclosure, comprises a light chain in which the light chain variable region comprises the CDR1, CDR2, and CDR3 sequences of a light chain selected from the light chains of urelumab, cusatuzumab, fresolimumab, inotuzumab, lemzoparimab, magrolimab, ofatumumab, olaratumab, omburtamab, gatipotuzumab (pancomab), tobetumab, trastuzumab, and nivolumab. CDR sequences may be determined according to any numbering system known in the art, including, but not limited to, IMGT and Kabat. CDR sequences according to IMGT are shown in italics in the sequence listings herein, and CDR sequences according to Kabat are underlined.

[0056] In certain embodiments, the CD3 binding domain of the disclosure or an antigen binding protein within the group of antigen binding proteins of the disclosure comprises a light chain, wherein 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: 5; - 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: 41; - 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: 42; - 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: 43 or SEQ ID NO: 68; - 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: 45 or SEQ ID NO: 69; - 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: 49; - 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: 50; - 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: 52; - 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: 53; - 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: 56; - 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: 57; - 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: 58 or SEQ ID NO: 70; - 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: 59 or SEQ ID NO: 71, or - 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: 64 or SEQ ID NO: 72.

[0057] In certain embodiments, the CD3 binding domain of the disclosure or an antigen binding protein within the group of antigen binding proteins of the disclosure comprises a light chain, wherein the light chain variable region comprises: a light chain CDR1 (LCDR1) of SEQ ID NO: 6, a light chain CDR2 (LCDR2) of SEQ ID NO: 7, and a light chain CDR3 (LCDR3) of SEQ ID NO: 8, a light chain CDR1 (LCDR1) of SEQ ID NO: 73, a light chain CDR2 (LCDR2) of SEQ ID NO: 74, and a light chain CDR3 (LCDR3) of SEQ ID NO: 75, a light chain CDR1 (LCDR1) of SEQ ID NO: 76, a light chain CDR2 (LCDR2) of SEQ ID NO: 77, and a light chain CDR3 (LCDR3) of SEQ ID NO: 78, a light chain CDR1 (LCDR1) of SEQ ID NO: 79, a light chain CDR2 (LCDR2) of SEQ ID NO: 80, and a light chain CDR3 (LCDR3) of SEQ ID NO: 81 or SEQ ID NO: 82, a light chain CDR1 (LCDR1) of SEQ ID NO: 83, a light chain CDR2 (LCDR2) of SEQ ID NO: 84, and a light chain CDR3 (LCDR3) of SEQ ID NO: 85, a light chain CDR1 (LCDR1) of SEQ ID NO: 86, a light chain CDR2 (LCDR2) of SEQ ID NO: 87, and a light chain CDR3 (LCDR3) of SEQ ID NO: 88, a light chain CDR1 (LCDR1) of SEQ ID NO: 89, a light chain CDR2 (LCDR2) of SEQ ID NO: 90, and a light chain CDR3 (LCDR3) of SEQ ID NO: 91; a light chain CDR1 (LCDR1) of SEQ ID NO: 92, a light chain CDR2 (LCDR2) of SEQ ID NO: 93, and a light chain CDR3 (LCDR3) of SEQ ID NO: 94, a light chain CDR1 (LCDR1) of SEQ ID NO: 95, a light chain CDR2 (LCDR2) of SEQ ID NO: 96, and a light chain CDR3 (LCDR3) of SEQ ID NO: 97, a light chain CDR1 (LCDR1) of SEQ ID NO: 98, a light chain CDR2 (LCDR2) of SEQ ID NO: 99, and a light chain CDR3 (LCDR3) of SEQ ID NO: 100, a light chain CDR1 (LCDR1) of SEQ ID NO: 101, a light chain CDR2 (LCDR2) of SEQ ID NO: 102, and a light chain CDR3 (LCDR3) of SEQ ID NO: 103, a light chain CDR1 (LCDR1) of SEQ ID NO: 104, a light chain CDR2 (LCDR2) of SEQ ID NO: 105, and a light chain CDR3 (LCDR3) of SEQ ID NO: 106, a light chain CDR1 (LCDR1) of SEQ ID NO: 107, a light chain CDR2 (LCDR2) of SEQ ID NO: 108, and a light chain CDR3 (LCDR3) of SEQ ID NO: 109, or - a light chain CDR1 (LCDR1) of SEQ ID NO: 110, a light chain CDR2 (LCDR2) of SEQ ID NO: 111, and a light chain CDR3 (LCDR3) of SEQ ID NO: 112.

[0058] In certain embodiments, the light chain variable region of the CD3 binding domain or antigen binding protein of the present disclosure, including variants thereof, may comprise one, two, or three amino acid variations in each of the LCDRs. In certain embodiments, the light chain variable region of the CD3 binding domain or antigen binding protein of the present disclosure, including variants thereof, may comprise one, two, or at most three amino acid variations in each of the LCDRs. In certain embodiments, the amino acid variations are conservative amino acid substitutions.

[0059] In certain embodiments, the light chain variable region of a CD3 binding domain or antigen binding protein of the disclosure comprises the amino acid sequence set forth in SEQ ID NO: 5, 41, 42, 43, 45, 49, 50, 52, 53, 56, 57, 58, 59, or 64, or an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity thereto.

[0060] In certain embodiments, the binding domains or antigen-binding proteins of the present disclosure also include variants that contain one or more variations in the framework regions in addition to the variations in the LCDRs referenced above. The variations are preferably conservative amino acid substitutions. In certain embodiments, the binding domains or antigen-binding proteins of the present disclosure do not contain variations in the LCDR regions but contain one or more variations 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.

[0061] In certain embodiments, the CD3 binding domain of the present disclosure, or an antigen binding protein within the group of antigen binding proteins, may further comprise a CL region. Any CL domain, particularly a human CL, may be used. An example of a suitable CL domain is provided by the amino acid sequence provided as SEQ ID NO:9.

[0062] In certain embodiments, the present disclosure provides a binding site comprising a polypeptide described herein, or a CD3 binding domain described herein.

[0063] "Binding moiety" refers to a proteinaceous molecule and includes, for example, all antibody formats available in the art, such as, for example, full length IgG antibodies, immunoconjugates, diabodies, BiTEs, Fab fragments, scFv, tandem scFv, single domain antibodies (such as VHH and VH), minibodies, scFab, scFv-zippers, nanobodies, DART molecules, TandAbs, Fab-scFv, F(ab)'2, F(ab)'2-scFv2, and intrabodies, and any other antibody format known to one of skill in the art.

[0064] In certain embodiments, the binding site of the present disclosure is a monospecific binding site, specifically a monospecific antibody. A monospecific antibody according to the present disclosure is an antibody comprising one or more binding domains with specificity for a single target, in any antibody format. In certain embodiments, the monospecific binding site of the present disclosure is a bivalent monospecific antibody. In certain embodiments, the monospecific binding site of the present disclosure may further comprise an Fc region or a portion thereof. In certain embodiments, the monospecific binding site of the present disclosure is an IgG1 antibody.

[0065] In certain embodiments, the binding site of the present disclosure is a multispecific antibody. A multispecific antibody according to the present disclosure is an antibody comprising at least two binding domains with specificity for at least two different targets or epitopes. In certain embodiments, the multispecific antibody of the present disclosure is a bispecific antibody. In certain embodiments, the multispecific antibody of the present disclosure is a bivalent bispecific antibody. In certain embodiments, the multispecific antibody of the present disclosure is a trivalent bispecific antibody. In certain embodiments, the multispecific antibody of the present disclosure is a trispecific antibody. In certain embodiments, the multispecific antibody of the present disclosure is a trivalent trispecific antibody. In certain embodiments, the multispecific antibody of the present disclosure may further comprise an Fc region or a portion thereof. In certain embodiments, the multispecific binding site of the present disclosure is an IgG1 antibody.

[0066] The "Fc region" typically comprises hinge, CH2, and CH3 regions. Suitable hinge, CH2, and CH3 regions are as described herein. The Fc region mediates antibody effector functions, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). Depending on the use of a therapeutic antibody or Fc fusion protein, it may be desirable to reduce or increase effector functions.

[0067] In certain embodiments, a binding site comprising a polypeptide or binding domain of the present disclosure has Fc effector function. In certain embodiments, a binding site comprising a polypeptide or binding domain of the present disclosure has improved Fc effector function. In certain embodiments, a binding site comprising a polypeptide or binding domain of the present disclosure exhibits antibody-dependent cell-mediated cytotoxicity (ADCC).

[0068] Binding sites such as antibodies can be engineered to improve ADCC activity (for a discussion, see Kubota T et al. Cancer Sci. 2009; 100(9):1566-72). For example, if the antibody itself has low ADCC activity, the ADCC activity of the antibody can be improved by slightly modifying the antibody's constant region (Junttila TT. et al. Cancer Res. 2010; 70(11):4481-9). Alterations can also be made to improve storage or production, or to remove C-terminal lysines (Kubota T et al. Cancer Sci. 2009; 100(9):1566-72). Another way to improve the ADCC activity of antibodies is by enzymatically interfering with the glycosylation pathway, which generates reduced fucose (von Horsten HH. et al. Glycobiology. 2010; 20(12):1607-18). Alternatively or additionally, several other strategies can be used to achieve improved ADCC, including, for example, glycoengineering (Kyowa Hakko / Biowa, GlycArt (Roche), and Eureka Therapeutics) and mutagenesis, all of which aim to improve Fc binding to the low-affinity activating FcγRIIIa and / or reduce binding to the low-affinity inhibitory FcγRIIb. In certain embodiments, the binding sites of the present disclosure exhibit improved antibody-dependent cell-mediated cytotoxicity (ADCC). In certain embodiments, the binding sites of the present disclosure are defucosylated.

[0069] The constant regions of the binding sites of the present disclosure may comprise one or more variations that modulate a property of the binding site other than its binding properties for the target antigen. For example, the constant regions of a multispecific binding site may comprise one or more variations that favor heterodimerization of two different heavy chains over homodimerization, and / or the constant regions of the binding site may comprise one or more variations that reduce or improve effector function, preferably one or more variations that reduce effector function.

[0070] In certain embodiments, the present disclosure provides nucleic acids useful for producing the polypeptides, binding domains, or binding sites of the present disclosure. In certain embodiments, such nucleic acids comprise nucleic acid sequences encoding the polypeptides described herein. In certain embodiments, the nucleic acids of the present disclosure may further comprise nucleic acid sequences encoding a CH1 region, and preferably a hinge, CH2, and CH3 region. In certain embodiments, the nucleic acids of the present disclosure may further comprise at least one nucleic acid sequence encoding a light chain variable region, and preferably a CL region. In certain embodiments, the light chain variable region may be a light chain variable region described herein.

[0071] In certain embodiments, the present disclosure provides vectors comprising the nucleic acids of the present disclosure, useful for producing the binding domains or binding sites of the present disclosure. In certain embodiments, such vectors comprise a nucleic acid sequence encoding a polypeptide described herein. In certain embodiments, a vector of the present disclosure may further comprise a nucleic acid sequence encoding a CH1 region, and preferably a hinge, CH2, and CH3 region. In certain embodiments, a vector of the present disclosure may further comprise at least one nucleic acid sequence encoding a light chain variable region, and preferably a CL region. In certain embodiments, the light chain variable region may be a light chain variable region described herein.

[0072] In certain embodiments, the present disclosure also provides cells comprising the vectors described herein. In certain embodiments, the present disclosure also provides cells comprising a nucleic acid, e.g., a vector, comprising a sequence encoding a polypeptide described herein. In certain embodiments, such a nucleic acid, e.g., a vector, may further comprise a nucleic acid sequence encoding a CH1 region, and preferably a hinge, CH2, and CH3 region. In certain embodiments, such a nucleic acid, e.g., a vector, may further comprise a nucleic acid sequence encoding a light chain variable region, and preferably a CL region. In certain embodiments, the light chain variable region may be a light chain variable region described herein.

[0073] In certain embodiments, the present disclosure also provides cells that produce the polypeptides, binding domains, or binding sites described herein. In certain embodiments, such cells may be recombinant cells that have been transformed with a nucleic acid, e.g., a vector, of the present disclosure. In certain embodiments, the cells of the present disclosure comprise a nucleic acid sequence, e.g., a vector, comprising a sequence encoding a polypeptide described herein. In certain embodiments, the nucleic acid sequence, e.g., a vector, further comprises a nucleic acid sequence encoding a CH1 region, and preferably a hinge, CH2, and CH3 region. In certain embodiments, the cells of the present disclosure further comprise at least one nucleic acid, e.g., a vector, comprising a sequence encoding a light chain variable region, specifically a light chain variable region described herein, and preferably a CL region.

[0074] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of a polypeptide described herein, or a CD3 binding domain described herein, or a binding site described herein, and a pharmaceutically acceptable carrier.

[0075] In certain embodiments, the present disclosure provides a polypeptide described herein, or a CD3 binding domain described herein, or a binding site described herein, or a pharmaceutical composition described herein, for use in therapy.

[0076] In certain embodiments, the present disclosure provides a polypeptide described herein, or a CD3 binding domain described herein, or a binding site described herein, or a pharmaceutical composition described herein, for use in the treatment of cancer.

[0077] In certain embodiments, the present disclosure provides methods for treating a disease, comprising administering to an individual in need thereof an effective amount of a polypeptide described herein, or a CD3 binding domain described herein, or a binding site described herein, or a pharmaceutical composition described herein.

[0078] In certain embodiments, the present disclosure provides methods for treating cancer, comprising administering to an individual in need thereof an effective amount of a polypeptide described herein, or a CD3 binding domain described herein, or a binding site described herein, or a pharmaceutical composition described herein.

[0079] As used herein, the terms "individual," "subject," and "patient" are used interchangeably and refer to a mammal, such as a human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, cow, horse, pig, etc., specifically a human subject with cancer.

[0080] As used herein, the terms "treat," "treating," and "treatment" refer to any type of intervention or process performed on a subject or administering an active agent or combination of active agents to a subject for the purpose of curing or ameliorating a disease or symptom in the subject, or for the purpose of producing a positive therapeutic response. As used herein, a "positive therapeutic response" refers to treatment that produces a beneficial effect, such as reversing, alleviating, ameliorating, inhibiting, or delaying a symptom, complication, condition, or biochemical manifestation associated with a disease, as well as preventing the onset, progression, development, severity, or recurrence of a symptom, complication, condition, or biochemical manifestation associated with a disease, such as amelioration of at least one symptom of a disease or disorder, e.g., cancer. A beneficial effect can take the form of an improvement over a baseline, including an improvement over measurements or observations made before initiating treatment according to the method. For example, a beneficial effect can take the form of delaying, stabilizing, halting, or reversing the progression of cancer in a subject at any clinical stage, as evidenced 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 tumor metastasis, slow or stop tumor growth, and / or prevent or slow tumor recurrence or relapse.

[0081] The term "therapeutic amount" or "effective amount" refers to an amount of a drug or combination of drugs to treat 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 tumor recurrence.

[0082] As used herein, an effective amount of an agent or composition is one that, for example, (i) can reduce the number of cancer cells, (ii) can reduce tumor size, (iii) can inhibit, slow, delay to some extent, and stop cancer cell invasion into peripheral organs, (iv) can inhibit tumor metastasis, (v) can inhibit tumor growth, (vi) can prevent or delay tumor onset and / or recurrence, and / or (vii) can alleviate to some extent one or more symptoms associated with cancer.

[0083] The effective amount may vary depending on factors such as the stage of the disease, age, sex, and weight of the individual being treated, and the ability of the agent or combination of agents to elicit a desired response in the individual, which can be readily assessed by an ordinarily skilled physician or other health care worker.

[0084] An effective amount can be administered to a subject in one or more administrations.

[0085] An effective amount can also include an amount that balances any toxic or detrimental effects of the agent or combination of agents with the beneficial effects.

[0086] The term "agent" refers to a therapeutically active substance, in this case a polypeptide, binding domain, or binding site of the present disclosure, or a pharmaceutical composition of the present disclosure.

[0087] As used herein, the word "comprises" and its conjugations are used in their open-ended sense, meaning that the items following the word are included, but items not specifically mentioned are not excluded.

[0088] The articles "a" and "an" are used herein to refer to one or to more than one of the grammatical object of the article. By way of example, "an element" means one or more elements.

[0089] The reference herein to a patent document or other matter shall not be deemed an admission that the document or matter is known or that the information it contains was part of the common general knowledge at the priority date of any of the claims.

[0090] All patents and literature references cited herein are incorporated by reference in their entirety.

[0091] It should be noted that, unless otherwise stated herein, the amino acid positions assigned to the HCDRs and frameworks of the variable regions of antibodies or antibody fragments are designated by the Kabat numbering system (see Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991)), and the numbering of the LCDRs and frameworks of the variable regions of antibodies or antibody fragments is according to Kabat or IMGT (discussed in Giudicelli et al., Nucleic Acids Res. 25:206-21 1 1997). Amino acids in the constant regions are indicated by the EU numbering system.

[0092] The accession number is provided primarily to provide a method for further target identification; the actual sequence of the bound protein may vary due to mutations in the encoding gene, such as those that occur in some cancers. The antigen-binding site of the binding domain or binding moiety of the present disclosure can bind to antigens and their various variants, such as those expressed by some antigen-positive immune cells or tumor cells. HGNC stands for HUGO Gene Nomenclature Committee. The number following the abbreviation is the accession number by which information about the gene and the protein encoded by the gene can be retrieved from the HGNC database. Entrez Gene provides the accession number or gene ID by 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 the accession number by which information about the gene or the protein encoded by the gene can be obtained from the Ensembl database. Ensembl is a collaborative project between EMBL-EBI and the Wellcome Trust Sanger Institute to develop a software system for generating and maintaining automated annotations for selected eukaryotic genomes.

[0093] When reference is made herein to a gene or protein, preferably reference is made to the human form of that gene or protein. When reference is made herein to a gene or protein, reference is made to both the native gene or protein as well as variant forms of that gene or protein that may be detected in tumors, cancers, etc., preferably human tumors, cancers, etc. [Brief explanation of the drawings]

[0094] [Figure 1]Binding of SC5Ab1 in combination with cLC or in combination with the light chains of olaratumumab, ofatumumab, urelumab, cusatuzumab, inotuzumab, fresolimumab, trastuzumab, or daratumumab in HPB-ALL cells measured as mean fluorescence intensity (MFI) by FACS at different IgG concentrations. [Figure 2] A) Digital capillary electrophoresis gel image of SC5Ab1 combined with cLC or combined with the light chains of cusatuzumab, fresolimumab, ofatumumab, or trastuzumab, B) Electropherogram of SC5Ab1 combined with the light chain of fresolimumab. [Figure 3] Binding of SC5Ab1 in combination with cLC or in combination with the light chains of cusatuzumab, fresolimumab, ofatumumab, or trastuzumab in HPB-ALL cells measured as mean fluorescence intensity (MFI) by FACS at different IgG concentrations. [Figure 4] Thermal stability of IgG containing the VL region of cLC or the VH region of antibody SC5Ab1 paired with the light chains of cusatuzumab, fresolimumab, ofatumumab, or trastuzumab. Stability was characterized by Tm (continuous line) and Tagg (dotted line). Tm was measured by the change in intrinsic protein fluorescence, expressed as the geometric centered mean (BCM) in the spectral range of 250 to 720 nm, and the aggregation temperature Tagg was measured by static light scattering (SLS) at 266 nm using the UNcle system (Unchained Labs). [Example]

[0095] In an example used to illustrate the present disclosure, but not intended to limit the present disclosure in any way, binding domains are screened in an IgG1 format comprising a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1, a CH1 having the amino acid sequence set forth in SEQ ID NO: 33, a hinge having the amino acid sequence set forth in SEQ ID NO: 32, a CH2 having the amino acid sequence set forth in SEQ ID NO: 34, and a CH3 having the amino acid sequence set forth in SEQ ID NO: 36, and a light chain comprising a light chain variable region as further specified herein, and a CL having the amino acid sequence set forth in SEQ ID NO: 9.

[0096] Control antibodies used in the examples include: a positive control anti-CD3 antibody that is a bivalent monospecific antibody comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 37 and two light chains having the amino acid sequences set forth in SEQ ID NO: 66; a negative control IgG1 antibody (RSV-G), which is a bivalent monospecific antibody comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 38 and two light chains having the amino acid sequences set forth in SEQ ID NO: 10; - a negative control IgG1 antibody (TT), which is a bivalent monospecific antibody comprising two heavy chains having the amino acid sequences set forth in SEQ ID NO: 39 and two light chains having the amino acid sequences set forth in SEQ ID NO: 10.

[0097] Example 1 - Pairing of the CD3 heavy chain with the light chains of cetuximab, trastuzumab, and nivolumab The objective of this study was to evaluate whether the heavy chain of a CD3 antibody generated with a light chain having the amino acid sequence set forth in SEQ ID NO: 10 could be combined with other light chains while preserving the specificity and affinity of the original antibody.

[0098] Binding domains, antibodies, and heavy chain variable regions with binding specificity for human CD3 were obtained by immunizing transgenic mice (MeMo® mice) containing the common IGKV1-39 light chain with human CD3 antigenic sites, including the use of TCR / CD3-containing lipoparticles (e.g., as described in WO2020 / 204708), different forms of DNA, protein, and cell-based antigen delivery. Antibodies that bind to human CD3 were grouped into distinct superclusters based on the use of the same VH V gene segment and with at least 70% sequence identity in the HCDR3 and the same HCDR3 length.

[0099] The heavy chain variable region (VH) sequence of one representative from each supercluster was combined with the light chain variable region (VL) sequences of cetuximab, trastuzumab, and nivolumab. These three commercially available antibodies bind to different targets and have diverse VLs, as shown in Table 1. The VH sequences were also combined with a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 5 to serve as a parental control. [Table 1]

[0100] ELISA Binding of CD3 antibodies to CD3-containing virus-like particles (VLPs) was assessed by ELISA.

[0101] HEK293 huCD3 (Integral Molecular; Catalog No. INT-2131B), HEK293 cyCD3 (Integral Molecular; Catalog No. INT-2132B), and HEK293 "null" (Integral Molecular; Catalog No. INT-2128B) biotinylated VLPs were coated onto ELISA plates at 5 units (U) / well overnight at 4°C. Wells were blocked with 4% nonfat milk (Marvel) in 1x PBS. CD3 or control antibodies in 1% nonfat milk / 1x PBS were added to huCD3 and cyCD3 bio-VLPs using seven 3-fold dilutions starting at 5 μg / ml. CD3 or control antibodies in 1% nonfat milk / 1x PBS were added to "null" bio-VLPs at a single concentration of 5 μg / ml. The antibodies were incubated at room temperature (RT) for 1 hour.

[0102] Control antibodies included a positive control anti-CD3 antibody used to confirm coating of the CD3-containing bio-VLPs, and a negative control IgG1 (RSV-G) antibody used to confirm specificity of binding. Control antibodies were used at 5 μg / ml.

[0103] Antibodies were detected with goat anti-huIgG(Fc)HRP-conjugated secondary antibody (Bethyl Labs; catalog no. A80-104P) in 1% nonfat milk / 1×PBS at 1:2000 and incubated for 1 h at room temperature.

[0104] Wells were washed three times with 1x PBS between steps and five times with 1x PBS before development. All washing steps were performed using buffer without Tween-20. Development involved the addition of 100 μl of TMB solution (eBioscience, catalog number 00-4201-56). The reaction was stopped with 100 μl of 0.5 M (1 N) H2SO4 (Fisher Chemical, catalog number J / 8430 / 15).

[0105] The OD was read at 450 nm using a BioTek Elx808 ELISA plate reader. ELISA titration data were analyzed with GraphPad Prism.

[0106] Antibodies that showed binding by ELISA compared to their respective parental controls were further screened by FACS.

[0107] FACS The binding and relative affinity of CD3 antibodies to huCD3 was determined by FACS.

[0108] 293FF cells transiently transfected to express (hu)CD3:TCR cultured in FreeStyle™ 293 Expression Medium (Gibco, Cat. No. 12338-018) were added to 0.5 × 10 6 HPB-ALL cells endogenously expressing huCD3 were seeded at 1 × 10 cells / well and cultured in RPMI1640 medium (Gibco, Cat. No. 21875-091) containing FBS (Gibco, Cat. No. A3160801) and penstrep (Gibco, Cat. No. 15140-122). 6 cells / well.

[0109] CD3 antibody or positive control anti-CD3 antibody was added using eight half-log (3.16-fold) serial dilutions starting at 10 μg / ml. A negative control IgG1 antibody (RSV-G) was added at a single concentration of 10 μg / ml. Antibodies in FACS buffer (0.5% FBS / EDTA 1:1000 / 1×PBS) were incubated on ice for 30 minutes.

[0110] Antibodies were detected with a goat anti-huIgG PE secondary antibody (Invitrogen, Cat. No. H10104) at 1:100 in FACS buffer (0.5% FBS / EDTA 1:1000 / 1×PBS) incubated on ice for 30 min.

[0111] FACS analysis was performed (BD Accuri™) and the mean fluorescence intensity (MFI) of each antibody was plotted as a function of the logarithm of the antibody concentration using GraphPad Prism software using a nonlinear regression with robust fit, asymmetric (five parameter) equation.

[0112] AUC values ​​were determined and used to assess the relative affinity of the antibodies to huCD3. FACS results were considered positive if the MFI value was more than two-fold higher than the negative control IgG1 antibody (RSV-G).

[0113] A summary of the ELISA and FACS results is shown in Table 2. When combined with one or more of the light chains of cetuximab, trastuzumab, and nivolumab, a select number of antibodies retain CD3 binding, but with lower affinity than when the VH is paired with their native light chain (cLC). Antibody SC5Ab1, which comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1, shows similar or higher binding to CD3 when combined with the light chain variable region of nivolumab than when combined with its native light chain. [Table 2]

[0114] Example 2 - Pairing of CD3 heavy chains with light chains of several commercially or publicly available antibodies Essentially, the study of Example 1 was repeated with the light chains of the commercially or publicly available antibodies listed in Table 3. [Table 3]

[0115] ELISA CD3 antibodies were screened to determine whether they could specifically bind to CD3. ELISA plates (Greiner Bio-One, catalog no. 655061) were coated with 2.5 μg / ml and 2 μg / ml of CD3δε-FC or tetanus toxoid (TT) (AJ Vaccines, catalog no. 2674) in PBS, respectively. The coated plates were incubated overnight at 4°C, washed twice with freshly prepared wash buffer (0.05% Tween 20 in PBS; Merck, catalog no. 8.22184.0500), and blocked with block buffer (2% BSA in PBS; Sigma, catalog no. A3294-500g) for 1 hour at room temperature. CD3 or control antibodies diluted in block buffer were added to the wells at a concentration of 5 μg / ml. The antibodies were incubated at room temperature for 1 hour. Control antibodies included a positive control anti-CD3 antibody used to confirm CD3 antigen coating and a negative control IgG1 antibody (TT) used to confirm binding specificity. Before adding the secondary antibody, wells were washed three times in wash buffer. Anti-hu IgG HRP antibody (BD, catalog no. 555788) diluted 1:2000 in blocking buffer was added to the wells for 1 hour of incubation at RT. Before color development, wells were washed three times in wash buffer. 50 μl of freshly prepared TMB substrate solution by mixing 1x Reagent A:1x Reagent B (BD OptEIA TMB Substrate Reagent Set from BD, catalog no. 555214) was added to each well for up to 10 minutes, after which the reaction was stopped with 50 μl of 1 M H2SO4 (Merck, catalog no. 1.00731). Titration data were analyzed using a BioTek Elx808 ELISA plate reader with OD450 nm readout and GraphPad Prism software.

[0116] Antibodies that showed more than 3-fold background binding to CD3 and less than 3-fold background binding to TT were selected for further FACS analysis.

[0117] FACS The binding and relative affinity of CD3 antibodies to huCD3 was determined by FACS analysis on HPB-ALL cells. 10% heat-inactivated (hi) fetal bovine serum (FBS) (Sigma, Cat. No. F7524) was added to 0.05–0.2 × 10 6 HPB-ALL cells were cultured in RPMI 1640 (Thermo Fisher Scientific, Cat. No. 21875) seeded at 100 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 antibody, positive control anti-CD3 antibody, or negative control IgG1 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 half-log serial dilutions starting at 10 μg / ml. After incubation and washing for 30 minutes, anti-hu IgG R-PE secondary antibody (Invitrogen, Cat. No. H10104) was added at 1:100, followed by a 30-minute incubation and washing step, followed by FACS analysis. All incubation and washing steps were performed in ice-cold FACS buffer. FACS measurements were performed using iQue VBR; Intellicyt, and ForeCyt software to express mean fluorescence intensity (MFI).

[0118] For data analysis, all binding curves were grouped by VH, plotted, and AUC was calculated (GraphPad Prism software). The AUC of VHs with commercially or publicly available antibody light chains was compared to the same VHs combined with their original light chains.

[0119] A summary of the ELISA results is shown in Table 4. These data indicate that antibody SC5Ab1 has the most promiscuous VH. FACS data for this VH is provided in Figure 1.

[0120] Example 3 - Further characterization of antibodies comprising the heavy chain variable region of antibody SC5Ab1 and the light chain variable regions of several commercially or publicly available antibodies The heavy chain variable region (VH) of antibody SC5Ab1 was combined with the light chain variable regions (VL) of commercially or publicly available antibodies cusatuzumab, fresolimumab, ofatumumab, and trastuzumab. The VH region of antibody SC5Ab1 was also combined with a VL region having the amino acid sequence set forth in SEQ ID NO: 5 to serve as a parental control.

[0121] To determine the molecular weight sizing and percent purity of IgG, IgG samples were analyzed by LabChip (LabChip GXII Touch HT; Perkin Elmer) under non-reducing conditions using the Protein Clear HR Reagent kit (Dye Solution, Sample Buffer, Protein Gel Matrix, Protein Ladder, Lower Marker, Wash Buffer; Perkin Elmer CLS960014) and Protein Express Assay LabChip for use with the GXII Touch HT (Perkin Elmer 760499) according to the manufacturer's instructions. Protein characterization was performed using LabChip RX reviewer software. 250 nanograms of each sample was analyzed.

[0122] The binding capacity of IgG to HPB-ALL cells was tested as described in Example 2, except here the negative control was an IgG1 antibody targeting RSV-G.

[0123] The melting temperature (Tm) and aggregation temperature (Tagg) of non-cLC IgG and the corresponding parent 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 250-720 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) at a final concentration of 100 μg / mL, or assay controls at 250 μg / mL. 8.8 μL of IgG solution or PBS was loaded into a microcuvette (Unis, product code 201-1009). The manufacturer's instructions were followed for assembly of the UniSeal (product code 201-1009 / 201-1013) and UniFrame (product code 201-1012) and loading into the UNcle system. The IgG sample was heated from 25 to 95°C at 0.3°C / min with a 1-min equilibration time before the temperature increase. UNcle Analysis v5.01 software was used for data analysis and generation of melting and aggregation curves.

[0124] The data show that the heavy chain variable region (VH) of antibody SC5Ab1 pairs well with multiple non-cognate light chain variable regions, as demonstrated by the major band (150 kD) corresponding to IgG, as shown in Figure 2A. Manual inspection of individual chromatograms revealed that additional bands did not correspond to protein peaks, as seen, for example, in the case of fresolimumab (Figure 2B).

[0125] In conclusion, antibodies comprising the VH region of antibody SC5Ab1 paired with the VL regions of most commercially or publicly available antibodies retain sufficient CD3 binding (Figure 3) and exhibit a good developability profile, with thermal stability comparable to that when paired with its cognate VL region (SEQ ID NO: 5) (see Figure 4). [Table 4-1] [Table 4-2]

[0126] array SEQ ID NO: 1 - Heavy chain variable region EVQLVESGAEVKKPGESLKISCKASGYSFTRFYIGWVRQMPGKGLEWMGLIYPGDSDTKYSPSFQGQVTISVDKSINTAYLQWNSLKASDTAMYYCARHIRYFDWFSHYYYMDVWGKGTMVTVSS SEQ ID NO:2 - Heavy chain CDR1 according to Kabat RFYIG SEQ ID NO: 3 - Heavy chain CDR2 according to Kabat LIYPGDSDTKYSPSFQG SEQ ID NO: 4 - Heavy chain CDR3 according to Kabat HIRYFDWFSHYYYMDV SEQ ID NO: 5 - Light chain variable region cLC [ka] QSISSY Light chain CDR2 according to SEQ ID NO: 7-IMGT AAS Light chain CDR3 according to SEQ ID NO: 8-IMGT QQSYSTPPT SEQ ID NO: 9-CL region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 10 - Light chain cLC DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 11 - V region VK1-39 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP SEQ ID NO: 12-VK1-39 / JK1 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK SEQ ID NO: 13 - Light chain variable region DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK Light chain CDR1 according to SEQ ID NO: 14-IMGT QSISSY Light chain CDR2 according to SEQ ID NO: 15-IMGT AAS Light chain CDR3 according to SEQ ID NO: 16-IMGT QQSYSTPPIT SEQ ID NO: 17 - V region VK3-15 EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWP SEQ ID NO: 18 - Light chain variable region EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPWTFGQGTKVEIK Light chain CDR1 according to SEQ ID NO: 19-IMGT QSVSSN Light chain CDR2 according to SEQ ID NO: 20-IMGT GAS Light chain CDR3 according to SEQ ID NO: 21-IMGT QQYNNWPWT SEQ ID NO: 22 - V region VK3-20 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP SEQ ID NO:23 - Light chain variable region EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK Light chain CDR1 according to SEQ ID NO: 24-IMGT QSVSSSY Light chain CDR2 according to SEQ ID NO: 25-IMGT GAS Light chain CDR3 according to SEQ ID NO: 26-IMGT QQYGSSPWT SEQ ID NO: 27 - V region VL3-21 SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGSSDH SEQ ID NO:28 - Light chain variable region SYVLTQPPSVSVAPGETARITCGGDNIGRKSVYWYQQKSGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDGSSDHWVFGGGTKLTVL Light chain CDR1 according to SEQ ID NO: 29-IMGT NIGRKS Light chain CDR2 according to SEQ ID NO: 30-IMGT YDS Light chain CDR3 according to SEQ ID NO: 31-IMGT QVWDGSSDHWV SEQ ID NO:32—hinge region EPKSCDKTHTCPPCP SEQ ID NO: 33 - CH1 region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV SEQ ID NO: 34 - CH2 region APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK SEQ ID NO: 35 - CH2-DM region APELGRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK SEQ ID NO: 36 - CH3 region GQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 37 - Heavy chain positive control anti-CD3 antibody EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWA YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 38 - Heavy chain negative control IgG1 antibody (RSV-G) EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVISYDGSTKYSADSLKGRFTISRDNSKNTLYLQMNSLRADDTTAVYYCAKEGWSFDSSGYRSWFD SWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 39 - Heavy chain negative control IgG1 antibody (TT) EVQLVETGAEVKKPGASVKVSCKASDYIFTKYDINWVRQAPGQGLEWMGWMSANTGNTGYAQKFQGRVTMTRDTSINTAYMELSSLTSGDTAVYFCARSSLFKTETAPYYHFAL DVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 40 - Light chain variable region cetuximab DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK SEQ ID NO: 41 - Light chain variable region Trastuzumab [ka] SEQ ID NO: 42 - Light chain variable region Nivolumab [ka] SEQ ID NO: 43 - Light chain variable region urelumab [ka] SEQ ID NO: 44 - Light chain variable region milatuzumab DIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHVPPTFGAGTRLEIK SEQ ID NO: 45 - Light chain variable region cusatuzumab [ka] SEQ ID NO: 46 - Light chain variable region Belantamab DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLEIK SEQ ID NO: 47 - Light chain variable region daratumumab EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK SEQ ID NO: 48 - Light chain variable region epratuzumab DIQLTQSPSSLSASVGDRVTMSCKSSQSVLYSANHKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYLSSWTFGGGTKLEIK SEQ ID NO: 49 - Light chain variable region fresolimumab [ka] SEQ ID NO: 50 - Light chain variable region Inotuzumab [ka] SEQ ID NO: 51 - Light chain variable region KA3 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQMHPRAPKTFGQGTKVEIK SEQ ID NO: 52 - Light chain variable region lemzoparimab [ka] SEQ ID NO: 53 - Light chain variable region magrolimab [ka] SEQ ID NO: 54 - Light chain variable region monalizumab DIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLIYNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPRTFGGGTKVEIK SEQ ID NO: 55 - Light chain variable region obinutuzumab DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIK SEQ ID NO: 56 - Light chain variable region ofatumumab [ka] SEQ ID NO: 57 - Light chain variable region olaratumab [ka] SEQ ID NO: 58 - Light chain variable region omburtamab [ka] SEQ ID NO: 59 - Light chain variable region pancomab [ka] SEQ ID NO: 60 - Light chain variable region prosalizumab DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTFLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTRLEIK SEQ ID NO: 61 - Light chain variable region rituximab QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK SEQ ID NO: 62 - Light chain variable region sibrotuzumab DIVMTQSPDSLAVSLGERATINCKSSQSLLYSRNQKNYLAWYQQKPGQPPKLLIFWASTRESGVPDRFSGSGFGTDFTLTISSLQAEDVAVYYCQQYFSYPLTFGQGTKVEIK SEQ ID NO: 63 - Light chain variable region tafasitamab DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIK SEQ ID NO: 64 - Light chain variable region tobetumab [ka] SEQ ID NO: 65 - Light chain variable region urocupulumab DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFVTYYCQQYNSYPRTFGQGTKVEIK SEQ ID NO: 66 - Light chain variable region H2C QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL SEQ ID NO: 67 - Light chain variable region hOKT3 DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQIT SEQ ID NO: 68 - Light chain variable region [ka] SEQ ID NO: 69 - Light chain variable region [ka] SEQ ID NO: 70 - Light chain variable region [ka] SEQ ID NO: 71 - Light chain variable region [ka] SEQ ID NO: 72 - Light chain variable region [ka] QDVNTA Light chain CDR2 according to SEQ ID NO: 74-IMGT SAS Light chain CDR3 according to SEQ ID NO: 75-IMGT QQHYTTPPT Light chain CDR1 according to SEQ ID NO: 76-IMGT QSVSSY Light chain CDR2 according to SEQ ID NO: 77-IMGT DAS Light chain CDR3 according to SEQ ID NO: 78-IMGT QQSSNWPRT Light chain CDR1 according to SEQ ID NO: 79-IMGT QSVSSY Light chain CDR2 according to SEQ ID NO: 80-IMGT DAS Light chain CDR3 according to SEQ ID NO: 81-IMGT QQRSNWPPALT Light chain CDR3 according to SEQ ID NO: 82-IMGT QQRSNWPPALTFC Light chain CDR1 according to SEQ ID NO: 83-IMGT SGSVTSDNF Light chain CDR2 according to SEQ ID NO: 84-IMGT NTN Light chain CDR3 according to SEQ ID NO: 85-IMGT ALFISNPSVE Light chain CDR1 according to SEQ ID NO: 86-IMGT QSLGSSY Light chain CDR2 according to SEQ ID NO: 87-IMGT GAS Light chain CDR3 according to SEQ ID NO: 88-IMGT QQYADSPIT Light chain CDR1 according to SEQ ID NO: 89-IMGT QSLANSYGNTF Light chain CDR2 according to SEQ ID NO: 90-IMGT GIS Light chain CDR3 according to SEQ ID NO: 91-IMGT LQGTHQPYT Light chain CDR1 according to SEQ ID NO: 92-IMGT QSVLYAGNNRNY Light chain CDR2 according to SEQ ID NO: 93-IMGT QAS Light chain CDR3 according to SEQ ID NO: 94-IMGT QQYYTPPLA Light chain CDR1 according to SEQ ID NO: 95-IMGT QSIVYSNGNTY Light chain CDR2 according to SEQ ID NO: 96-IMGT KVS Light chain CDR3 according to SEQ ID NO: 97-IMGT FQGSHVPYT Light chain CDR1 according to SEQ ID NO: 98-IMGT QSVSSY Light chain CDR2 according to SEQ ID NO: 99-IMGT DAS Light chain CDR3 according to SEQ ID NO: 100-IMGT QQRSNWPIT Light chain CDR1 according to SEQ ID NO: 101-IMGT QSVSSY Light chain CDR2 according to SEQ ID NO: 102-IMGT DAS Light chain CDR3 according to SEQ ID NO: 103-IMGT QQRSNWPPA Light chain CDR1 according to SEQ ID NO: 104-IMGT QSISDY Light chain CDR2 according to SEQ ID NO: 105-IMGT YAS Light chain CDR3 according to SEQ ID NO: 106-IMGT QNGHSFPLT Light chain CDR1 according to SEQ ID NO: 107-IMGT KSLLHSNGITY Light chain CDR2 according to SEQ ID NO: 108-IMGT QMS Light chain CDR3 according to SEQ ID NO: 109-IMGT AQNLELPPT Light chain CDR1 according to SEQ ID NO: 110-IMGT QSFSRY Light chain CDR2 according to SEQ ID NO: 111-IMGT AAS Light chain CDR3 according to SEQ ID NO: 112-IMGT QQTYSNPPIT SEQ ID NO: 113: human CD3d MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELD SEQ ID NO: 114 - Human CD3e MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD

Claims

1. A polypeptide comprising a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO:4 or having at least 70% sequence identity thereto.

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

3. the polypeptide comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO:2, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO:3, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO:4; 3. The polypeptide of claim 1 or 2, wherein each of the HCDRs can contain one, two, or at most three amino acid variations.

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

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

6. The polypeptide of any one of claims 1 to 5, further comprising a hinge, 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 comprising a light chain variable region.

9. A group of antigen binding proteins that bind to human CD3, wherein each antigen binding protein in said group comprises a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region comprises an HCDR3 having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:

4.

10. the light chain variable region - 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: 5; - 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: 41; - 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: 42; - 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: 43 or 68; - 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: 45 or 69; - 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: 49; - 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: 50; - 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: 52; - 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: 53; - 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: 56; - 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: 57; - 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: 58 or 70; - 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: 59 or 71, or - 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: 64 or 72; or a variant thereof, which may contain one, two, or at most three amino acid variations, wherein each of said LCDRs is a CD3 binding domain of claim 8 or a group of antigen binding proteins of claim 9.

11. 11. The group of CD3 binding domains or antigen binding proteins according to claim 10, wherein the light chain variable region comprises an amino acid sequence as set forth 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 an amino acid sequence with at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity thereto.

12. 12. The group of CD3 binding domains or antigen binding proteins according to claim 10 or 11, further comprising a CL region.

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

14. 14. The binding site of claim 13, wherein the binding site is a multispecific binding site, in particular 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 site according to claim 13 or 14, and a pharmaceutically acceptable carrier.

16. 16. 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 site according to claim 13 or 14, or a pharmaceutical composition according to claim 15, for use in therapy.

17. 16. 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 site according to claim 13 or 14, or a pharmaceutical composition according to claim 15, for use in the treatment of cancer.

18. 19. A method for treating a disease, comprising administering to an individual in need thereof 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 site according to claim 13 or 14, or a pharmaceutical composition according to claim 15.

19. 19. A method for treating cancer, comprising administering to an individual in need thereof 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 site according to claim 13 or 14, or a 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 of claim 20.

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

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

24. A cell comprising the nucleic acid of claim 20.

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

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

27. A cell producing 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 site according to claim 13 or 14.

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

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