CD3-specific deimmunized antibodies

Abstract

The present invention provides affinity-optimized and deimmunized human antibodies that specifically bind to CD3. The present disclosure also provides bispecific antibodies, including human antibodies optimized for T cell activation, etc. The present invention further relates to methods for generating human antibodies and their use in treating disease.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to fully human antibodies (including structurally modified variants thereof) that bind to CD3, which have reduced binding potential to HLA proteins and thus have a reduced risk of eliciting an immune response in humans once administered. The present invention further provides bispecific antibodies utilizing the CD3-specific antibodies disclosed herein, methods of producing such antibodies, and methods of using same in the treatment of disease, such as the treatment of cancer. [Background technology]

[0002] background CD3 (cluster of differentiation 3) is a protein complex consisting of at least four invariant peptide chains non-covalently associated with the T cell receptor (TCR) on the surface of T cells, typically referred to as the CD3 antigen complex. The CD3 antigen complex plays a key role in T cell activation when antigen binds to the T cell receptor. CD3 has been extensively explored as a drug target. The therapeutic concept based on bispecific antibodies targeting CD3 relies on the simultaneous binding to cell surface antigens present on tumor cells and CD3 present on cytotoxic T cells, aiming at direct tumor cell killing by the bound cytotoxic T cells (Miller and Kontermann, Bispecific antibodies for cancer immunotherapy: Current perspectives. BioDrugs 2010, 24(2):89-98).

[0003] One of the major problems of CD3 targeted therapy is its dose-limiting toxicity due to off-target T cell activation. Such toxicity is essentially driven by the inherent ability of CD3 antibodies to stimulate T cells regardless of the presence of target cells. In fact, many side effects observed in the context of CD3-based antibody therapy are believed to be related to incorrect T cell function, such as the concomitant production of cytokines that can lead to toxic cytokine release syndrome. Therefore, to reduce such toxicity, it is well established that, on the one hand, it is possible to engage CD3 present on T cells in a monovalent manner by using antibody scaffolds that lack the ability to bind to Fc receptors on accessory cells such as monocytes, B cells, and NK cells. This approach avoids cross-linking of the CD3 complex present on T cells and its subsequent activation. A large number of antibodies that bind to CD3 have been described in the art. These include antibodies of rodent origin, such as OKT-3 (Kung P. et al., Science, 1979 Oct 19;206(4416):347-9) and SP34 (Yoshino N. et al., Exp. Anim 49:97-110, 2000; Conrad ML. et al., Cytometry 71A:925-33, 2007), as well as their humanized derivatives. Besides, a huge group of de novo generated human or humanized antibodies with specificity for CD3 have been described in the art. Due to their murine origin, SP34 or OKT-3 induce strong human anti-mouse antibody (HAMA) immunogenic reactions in non-immunosuppressed humans, thereby limiting their potential administration and also potentially causing dangerous allergic reactions. Typically, such immune responses require the uptake of a therapeutic (foreign) protein (i.e., a therapeutic antibody) by antigen-presenting cells (APCs). Once inside such cells, the protein is processed and released fragments of the protein (certain short peptide sequences form complexes with MHC class II molecules) are presented on the cell surface. If such complexes are recognized by binding of T cell receptors present on T cells, such cells can be activated to produce stimulatory cytokines.Cytokines induce the differentiation of B cells into mature antibody-producing cells. In addition, such T cell responses may also mediate other adverse effects on the patient, such as inflammation and possibly allergic reactions. However, it is understood that certain peptides found to bind to MHC class II molecules will not provoke an immune response because they are recognized as "self" in the organism to which the final protein is administered. Such peptides are found, for example, in germline human immunoglobulin variable region protein sequences.

[0004] Therefore, humanized CD3-specific antibodies with reduced immunogenicity in humans have been designed. A common aspect of the humanization process is the introduction of significant portions of the exact same amino acid sequence as present in the human antibody protein, thus involving, for example, the grafting of the CDRs of a non-human antibody into the closest germline human acceptor antibody framework. A significant drawback associated with the humanization process is the significant reduction in binding affinity of the resulting humanized antibody, as well as poor productivity and stability. Restoration of the original properties is typically achieved by a process of repeated back-mutation of human residues with amino acids at the same positions in the non-human donor antibody. However, such back-mutation may again increase the risk of induction of an immunogenic response in humans.

[0005] Thus, the use of human antibodies seems to be an ideal solution to overcome the above-mentioned shortcomings of using murine or humanized antibodies, since immune responses are generally not initiated against circulating autologous proteins such as immunoglobulins. However, human antibodies may still provoke immune responses or be immunogenic when administered to a particular individual. As an example, recombinant human antibodies selected from human phage display libraries may still provoke immune responses, since they may utilize non-germline encoded protein sequences, such as consensus framework sequences derived from human framework sequence analysis. Besides, variations introduced in the CDR regions may cause immune responses (particularly during affinity maturation of the initial selected antibodies). Such selected CDR amino acid sequences may have similarity to foreign proteins and thus provide epitopes that are generally considered unavailable to the immune system.

[0006] As outlined above, it is of paramount importance for therapeutics targeting CD3 to avoid any undesired T cell activation. An immune response against a therapeutic CD3-specific antibody can restore bivalent or multivalent CD3 binding and undesired cross-linking of CD3 complexes on T cells and thus their activation. Therefore, the present invention mainly incorporates a new approach to provide optimized fully human antibodies specific for CD3, characterized by the removal of potential T cell epitopes present in the CDR regions of the parent fully human antibody counterpart, and therefore with a reduced risk of immune response selection once administered in humans. Summary of the Invention [Means for solving the problem]

[0007] Summary of the Invention The present disclosure provides affinity-optimized human CD3-specific antibodies and deimmunized variants thereof, which have their complementarity determining regions (CDRs) modified to reduce their immunogenicity in humans or to reduce their risk of eliciting an immune response in humans once administered.

[0008] In one embodiment, the present disclosure provides: i. Below (a) heavy chain complementarity determining region (HCDR) 1 comprising the amino acid sequence of GFSFGSHYMS (SEQ ID NO: 1); (b) an HCDR2 comprising the amino acid sequence of NINQIGYSSYYVESVKG (SEQ ID NO: 2), NINQIGYSSYYGESVKG (SEQ ID NO: 3), or NINQIGYSSYYEESVKG (SEQ ID NO: 4); and (c) an HCDR3 comprising the amino acid sequence of GYSAEFAHRSGLDV (SEQ ID NO: 5), GYSDEFATRSGLDV (SEQ ID NO: 6), GYSEEFAHRSGLDV (SEQ ID NO: 7), GYSDEFAKRSGLDV (SEQ ID NO: 8), or GYSDEFAHRSGLDV (SEQ ID NO: 9); A heavy chain variable region (VH) comprising: ii. Below (a) a light chain complementarity determining region (LCDR) 1 comprising the amino acid sequence of SGSSSNIGSNYVY (SEQ ID NO: 10); (b) an LCDR2 comprising the amino acid sequence of RNNQRPS (SEQ ID NO: 11); and (c) an LCDR3 comprising the amino acid sequence of AGWSRSLHGAV (SEQ ID NO: 12) or AGWSRELHGAV (SEQ ID NO: 13); A variable light chain region (VL) comprising The present invention provides an isolated human antibody or antigen-binding fragment thereof specific for cluster of differentiation 3 (CD3), comprising:

[0009] In one embodiment, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for CD3, wherein the antibody or antigen-binding fragment thereof cross-reactively binds to cynomolgus monkey CD3.

[0010] In one embodiment, the present disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for CD3, wherein the antibody or antigen-binding fragment is a deimmunized antibody.

[0011] In one embodiment, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for CD3, wherein the antibody or antigen-binding fragment thereof has a reduced risk of eliciting an immune response in humans.

[0012] In one embodiment, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for CD3, wherein the antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, and / or a VL comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:20 or SEQ ID NO:21.

[0013] In one embodiment, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for CD3, wherein the VH and VL are selected from the following: i. a VH comprising the amino acid sequence of SEQ ID NO: 14 and a VL comprising the amino acid sequence of SEQ ID NO: 20; ii. VH comprising the amino acid sequence of SEQ ID NO: 15 and VL comprising the amino acid sequence of SEQ ID NO: 21; iii. VH comprising the amino acid sequence of SEQ ID NO: 16 and VL comprising the amino acid sequence of SEQ ID NO: 21; iv. VH comprising the amino acid sequence of SEQ ID NO: 17 and VL comprising the amino acid sequence of SEQ ID NO: 21; v. A VH comprising the amino acid sequence of SEQ ID NO: 18 and a VL comprising the amino acid sequence of SEQ ID NO: 21; and vi. VH comprising the amino acid sequence of SEQ ID NO: 19 and VL comprising the amino acid sequence of SEQ ID NO: 21; is selected from the group consisting of:

[0014] In one embodiment of the present disclosure, the isolated human antibody or antigen-binding fragment thereof specific for CD3 is a recombinant antibody or antigen-binding fragment thereof. In one embodiment of the present disclosure, the isolated human antibody or antigen-binding fragment thereof specific for CD3 is a monoclonal antibody or antigen-binding fragment thereof.

[0015] In one embodiment of the present disclosure, the isolated antigen-binding fragment specific for CD3 is a Fab, Fab', (Fab')2, Fv, or scFv. In one embodiment of the present disclosure, the isolated human antibody specific for CD3 antibody is a full-length antibody.

[0016] In one embodiment, the present disclosure provides a bispecific antibody comprising a first antigen-binding fragment of an isolated human antibody specific for CD3 of the present disclosure and a second antigen-binding fragment of the antibody that binds to a different target antigen than the first antigen-binding fragment. In one embodiment of the present disclosure, the second antigen-binding fragment binds to a cell surface antigen, particularly a tumor-associated cell surface antigen.

[0017] In one embodiment of the present disclosure, the isolated human antibody specific for CD3 of the present disclosure or a bispecific antibody comprising an antigen-binding fragment of the isolated human antibody specific for CD3 of the present disclosure comprises an Fc region comprising one or more amino acid substitutions that reduce binding to Fc receptor and / or effector function.

[0018] In one embodiment, the present disclosure provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated human antibody or antigen-binding fragment thereof specific for CD3, or a bispecific antibody comprising an antigen-binding fragment of an isolated human antibody specific for CD3 of the present disclosure.

[0019] In one embodiment, the present disclosure provides a vector composition comprising a vector or vectors comprising a nucleic acid sequence or sequences of the present disclosure. In one embodiment, the present disclosure provides a host cell comprising the vector composition of the present disclosure. In one aspect, the host cell is a mammalian cell. In one aspect, the host cell is a prokaryotic cell.

[0020] In one aspect, the disclosure provides a method for producing an antibody or antigen-binding fragment thereof according to the disclosure, comprising culturing the host cell under conditions suitable for expression of the antibody, and optionally recovering the antibody or antigen-binding fragment thereof. In a further aspect, the disclosure provides an antibody or antigen-binding fragment thereof produced by a method described herein.

[0021] In one embodiment, the present disclosure provides a pharmaceutical composition comprising an isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure, or a bispecific antibody comprising an antigen-binding fragment of an isolated human antibody specific for CD3 according to the present disclosure, and a pharma- ceutical acceptable carrier or excipient.

[0022] In one embodiment, the present disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for CD3 for use as a pharmaceutical agent, or a pharmaceutical composition according to the present disclosure.

[0023] The claimed antibodies or antigen-binding fragments thereof have utility. Additionally, the claimed methods for generating such antibodies or antigen-binding fragments thereof have utility. The claimed antibodies or antigen-binding fragments thereof are used to target T cells expressing CD3 and to stimulate T cell activation, e.g., in situations where T cell-mediated killing is beneficial or desirable. In particular, the claimed antibodies or antigen-binding fragments thereof are of therapeutic use, such as for the treatment of cancer. [Brief description of the drawings]

[0024] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A] The basic structure of the bispecific 2+1 Fab2-Fc-scFv antibody format of Example 1 is depicted. The bispecific antibody format encompasses an aglycosylated monoclonal IgG1 backbone and one additional scFv antibody, with the N-terminus of the VL of the scFv fused to the C-terminus of one IgG heavy chain via a peptide linker. To promote heterodimerization of the two different heavy chains, knob-into-hole mutations were introduced into both CH3-Fc domains. In addition to these mutations, the Fc region contained amino acid substitutions that resulted in the Fc region being unable to interact with and complement the IgG Fc receptor (Fc gamma receptor). In this example, both Fab arms of the bispecific antibody bind HER2, while the scFv is specific for CD3. [Figure 1B] The basic structure of the bispecific 1+1 Fab2-Fc antibody format of Example 2 is depicted. This bispecific antibody format reflects the typical Y-shape of conventional IgG molecules, with one Fab arm binding to the tumor target and the other Fab arm binding to CD3. The bispecific 1+1 Fab2-Fc antibody was generated in vitro by Fab arm exchange. To enable the production of bispecific antibodies by this method, source IgG1 molecules carrying single mutations in the CH3 domain were generated: one source IgG1 antibody carrying the F405L mutation (i.e., CD3-specific antibody), the other source IgG1 antibody carrying the K409R mutation (i.e., anti-HER antibody). In addition to these mutations, the source IgG1 antibody contained substitutions that rendered the Fc region unable to interact with the IgG Fc receptor (Fc gamma receptor) and complement. [Diagram 2]Basic structure of the bispecific Fab2-Fv-Fc antibody format of Example 3 and Example 4. This bispecific antibody format is constructed from an aglycosylated monoclonal human IgG1 antibody scaffold and one additional Fv fragment incorporated between the Fc region of the IgG1 scaffold and two Fab arms. In this example, both Fab arms bind to HER2, while the "extra" Fv fragment contains the variable region of an antibody specific for CD3 according to the present disclosure. Also depicted are different improved peptide linkers that were used to connect the additional Fv fragment to the Fab arms and the Fc region. [Diagram 3] Cell binding of mammalian produced bispecific antibodies specific for HER2 and CD3 according to Example 2.2 comprising variable domains of an affinity matured or crosscloned CD3 specific antibody according to the disclosure. Cell binding (signal / background) to CD3 positive Jurkat cells as determined by flow cytometry is shown as a function of bispecific antibody concentration. [Figure 4A] Figure 4A shows a cytotoxicity assay of a mammalian-produced bispecific antibody with specificity for HER2 and CD3 according to Example 2.4 comprising variable domains of an affinity matured or cross-cloned CD3-specific antibody according to the present disclosure against HER2-expressing SKBR3 cells in the presence of human T cells from one donor. The cytotoxic activity of human T cells is assessed by measuring the fluorescence of incorporated CellToxGreen. The graph shows the relative fluorescence level of HER2-expressing SKBR3 cells as a function of bispecific antibody concentration. [Figure 4B] Figure 4B: Cytotoxicity assay of mammalian-produced bispecific antibodies specific for HER2 and CD3 according to Example 2.4 comprising variable domains of affinity matured or cross-cloned CD3-specific antibodies according to the present disclosure against HER2-positive MCF-7 cells in the presence of human T cells from one donor. The cytotoxic activity of human T cells is assessed by measuring the fluorescence of internalized CellToxGreen. The graph shows the relative fluorescence level of MCF-7 cells as a function of bispecific antibody concentration. [Diagram 5]T cell activation assay of mammalian produced bispecific antibodies specific for HER2 and CD3 according to Example 2.5 comprising variable domains of affinity matured or cross-cloned CD3 specific antibodies according to the present disclosure. T cell activation is determined by evaluation of CD69 expression on CD8 positive T cells assessed by flow cytometry. The percentage of CD69+ activated CD8+ T cells from three different donors is shown as a function of bispecific antibody concentration. [Figure 6] Cytotoxicity assay of mammalian produced bispecific antibodies specific for HER2 and CD3 according to example 3.6 comprising the variable domains of one cross-clonal CD3 specific antibody (CD3-MABopt-cc) of example 2 and two of the most potent linker combinations (linker combination 2 and linker combination 5) based on the first disclosed linker combination P. Killing of HER2 expressing SKBR3 cells in the presence of human T cells from one donor is shown. Cytotoxic activity of human T cells is assessed by measuring the fluorescence of incorporated CellToxGreen. The graph shows the relative fluorescence level of HER2 expressing SKOV-3 cells as a function of bispecific antibody concentration. [Figure 7A] Figure 7A: T cell activation assay of mammalian produced bispecific antibodies specific for HER2 and CD3 according to example 3.7 comprising the variable domains of one cross-clonal CD3 specific antibody (CD3-MABopt-cc) of example 2 and six linker combinations (linker combinations 1-6) based on the first disclosed linker combination P. T cell activation is determined by evaluation of CD69 expression on CD4+ T cells assessed by flow cytometry. Graph showing the mean percentage of CD69+ activated CD4+ T cells from three different donors as a function of bispecific antibody concentration. [Figure 7B]FIG. 7B: T cell activation assay of mammalian produced bispecific antibodies specific for HER2 and CD3 according to example 3.7 comprising the variable domains of one cross-clonal CD3 specific antibody (CD3-MABopt-cc) of example 2 and six linker combinations (linker combinations 1-6) based on the first disclosed linker combination P. T cell activation is determined by evaluation of CD69 expression on CD8+ T cells assessed by flow cytometry. Graph showing the mean percentage of CD69+ activated CD8+ T cells from three different donors as a function of bispecific antibody concentration. [Figure 8]Epibase™ (Lonza, Epibase Version: v3.0) in silico screening results of the HCDR3 region of the cross-clonal antibody CD3-MABopt-cc from Example 2 for the identification of potential T cell epitopes. The human antibody germline coding sequence region was excluded from the analysis. In the analysis, the entire VH sequence is split into overlapping 10mer peptides, each shifted by one amino acid. The left panel of FIG. 8 lists the analyzed 10mer peptides starting from amino acid position 90 to position 112 on the VH of CD3-MABopt-cc across its entire HCDR3 region. The potential peptide / HLA binding of each analyzed 10mer peptide is determined against the HLA class II allotypes of the major Caucasian DRB1 alleles shown in the upper panel / column of FIG. 8. For each DRB1 allotype, its natural occurrence frequency is provided (e.g., DRB1*01:01:15%). According to the examples, peptide 93 is predicted to bind with medium (M) affinity to three allotypes of DRB1 alleles and strong (S) affinity to two allotypes of DRB1 alleles. Such peptides reflect "T cell epitopes" as defined herein. The risk score of 24.5 provided for this peptide is calculated as the sum of the natural frequency of allotypes of DRB1 alleles that bind such peptide (e.g., for peptide 93: 24+6+12+6+5=53 (rounded), exactly: 51.9). In summary, 10 T cell epitopes (peptides 92, 93, 94, 95, 97, 100, 102, 104, 110, 112) and two hotspots can be assigned to the HCDR3 region of CD3-MABopt-cc (the first hotspot spans peptides 92-97, the second hotspot spans peptides 110-112). Hotspots reflect the accumulation of nearby T cell epitopes. Such hotspots are identified based on "4 over 3", which means that at least four allotypes of the DRB1 allele must bind with moderate or strong affinity to at least two of the three consecutive analyzed 10-mer peptides. Additionally, at most one 10-mer peptide not identified as a T cell epitope can be part of a hotspot.Therefore, not every identified T cell epitope must be part of a hotspot (see, for example, peptides 100, 102, or 104 in FIG. 8). On the other hand, peptides that are not identified as T cell epitopes may still be part of a hotspot (see, for example, peptide 96 or peptide 111 in FIG. 4). Each analyzed 10-mer peptide that is part of a hotspot is defined as an H-line. The cumulative risk score of a hotspot can be calculated as the sum of the risk scores determined for each T cell epitope within the hotspot, and is defined herein as "H-score". As an example, the second hotspot in HCDR3 consists of three H-lines. Thus, the H-score is calculated by the sum of the risk scores of each T cell epitope covered by this hotspot, e.g., 27.8+60.3=88. [Figure 9] Epibase™ in silico mutation analysis: Effect of in silico single amino acid substitutions (upper panel) at each HCDR3 position (positions 92-112) of CD3-MABopt-cc on the number of T cell epitopes of the VH of CD3-MABopt-cc (left panel of FIG. 4) and the corresponding absolute risk score (right panel of FIG. 4). The numbers with bold borders correspond to the amino acid substitutions selected for gene synthesis of the respective VH variants of CD3-MABopt-cc. Amino acid substitutions that led to a reduction of 2 or more T cell epitopes while allowing to make conservative amino acid substitutions were preferably selected. Besides, care was taken not to introduce potential post-translational modification sites ("PTM motifs") in the CDR regions. According to the examples, in HCDR3, 42 single amino acid substitutions (single point variants) were selected for gene synthesis. [Figure 10]Summary of biophysical and functional properties of 27 preferred CDR single point variants of CD3-MABopt-cc according to their characterization in bispecific Fab2-Fv-Fc antibody format in Example 4. For each substitution, the reduction of T cell epitopes in VH or VL of CD3-MABopt-cc is shown, as well as the monomer content and yield of purified bispecific antibody preparations, ELISA binding and affinity to recombinant human CD3 epsilon. The last column of Figure 10 represents the variants selected for combinatorial in silico mutational analysis. The first row (wt (parent)) represents the functional properties determined in bispecific antibodies comprising the variable domains of antibody CD3-MABopt-cc. [Figure 11] Epibase™ in silico combinatorial mutation analysis. Exemplary results of 54 combinatorial amino acid substitutions in the HCDR1-3 region of CD3-MABopt-cc resulting in a reduction of three hotspots. The first row of the table shown represents the risk parameters (absolute score, hotspots, absolute H score, and absolute H line) of the VH of CD3-MABopt-cc. For each combinatorial variant, the reduction of each risk parameter is provided (as a delta value) relative to the determined risk parameter of the VH of CD3-MABopt-cc. [Figure 12] Summary of biophysical and functional properties of 33 preferred combination variants of CD3-MABopt-cc according to the characterization in bispecific Fab2-Fv-Fc antibody format of Example 4. The first row represents the functional properties determined for bispecific antibodies comprising VH and VL of CD3-MABopt-cc. For each combination variant, the reduction of each risk parameter (absolute score, hotspot, absolute H-score, and absolute H-line) is provided (as delta value) relative to the determined risk parameter of VH of CD3-MABopt-cc. Also shown are the monomer content of purified bispecific antibody preparations, ELISA binding and affinity to recombinant human CD3 epsilon antigen, as well as functional activity in receptor gene assays on SKOV-3 cells at two bispecific antibody concentrations. [Figure 13]Cytotoxicity assay according to Example 4.12 in one donor of mammalian produced bispecific antibody BissIg_21_CD3-MABdeimm_3 comprising the most preferred deimmunized combination variant VH and VL sequences of CD3-MABdeimm_3 relative to bispecific antibody BissIg_21_CD3-MABopt_cc comprising the unmodified CD3 specific variable domain of CD3-MABopt_cc. Cytotoxic activity of human T cells is assessed by measuring the fluorescence of incorporated CellToxGreen. The graph shows the relative fluorescence level of HER2 expressing SKOV-3 cells as a function of bispecific antibody concentration. [Figure 14A] FIG. 14A shows an exemplary T cell activation assay according to Example 4.13 assay of bispecific antibodies BissIg_21_CD3-MABdeimm_1, BissIg_21_CD3-MABdeimm_2, BissIg_21_CD3-MABdeimm_3, BissIg_21_CD3-MABdeimm_4, BissIg_21_CD3-MABdeimm_5, which encompass the five most preferred deimmunized combination variant VH and VL sequences relative to bispecific antibody BissIg_21_CD3-MABopt_cc, which contains the unmodified CD3-specific variable domain of CD3-MABopt_cc. As a positive control, results of murine CD3-specific OKT-3 IgG are shown. T cell activation is determined by evaluation of CD69 expression on CD4+ T cells assessed by flow cytometry. The mean percentage of CD69+ activated CD4+ T cells from one donor is shown as a function of bispecific antibody concentration. [Figure 14B]FIG. 14B shows an exemplary T cell activation assay according to Example 4.13 assay of bispecific antibodies BissIg_21_CD3-MABdeimm_1, BissIg_21_CD3-MABdeimm_2, BissIg_21_CD3-MABdeimm_3, BissIg_21_CD3-MABdeimm_4, BissIg_21_CD3-MABdeimm_5, which encompass the five most preferred deimmunized combination variant VH and VL sequences relative to the bispecific antibody BissIg_21_CD3-MABopt_cc, which contains the unmodified CD3-specific variable domain of CD3-MABopt_cc. As a positive control, results of murine CD3-specific OKT-3 IgG are shown. T cell activation is determined by evaluation of CD69 expression on CD8+ T cells assessed by flow cytometry. The mean percentage of CD69+ activated CD8+ T cells from one donor is shown as a function of bispecific antibody concentration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Detailed Description of the Disclosure definition "CD3" refers to an antigen expressed on T cells as part of the multimolecular T cell receptor (TCR), which consists of a homodimer or heterodimer formed from the association of two of four receptor chains: CD3 epsilon (CD3 epsilon), CD3 delta, CD3 zeta, and CD3 gamma.

[0026] Human CD3 epsilon (or human CD3e) has the amino acid sequence of UniProt P07766: [ka] has.

[0027] The mature extracellular domain of human CD3 epsilon without the signal sequence comprises amino acid residues 22 to 126 and consists of the following: [ka] It has the amino acid sequence:

[0028] Cynomolgus CD3 epsilon (or cynoCD3e) has the amino acid sequence of UniProt Q95LI5: [ka] has.

[0029] The mature extracellular domain of cynomolgus CD3 epsilon without the signal sequence comprises amino acid residues 22 to 198 and is as follows: [ka] It has the amino acid sequence:

[0030] The term "about" when used in connection with a particular stated numerical value means that the value can vary within 1% from the stated value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0031] The term "antigen" or "target antigen" as used herein means any molecule of interest to which one of the binding sites present in an antibody can bind. Typically, an antigen is a peptide, a protein, or any other proteinaceous molecule. Alternatively, an antigen can be any other organic or inorganic molecule, such as carbohydrates, fatty acids, lipids, dyes, fluorophores, etc.

[0032] The term "antibody" as used herein refers to an antigen-interacting protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with an antigen. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, and chimeric antibodies. Antibodies may be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Both the light and heavy chains are divided into regions of structural and functional homology.The structure and location of immunoglobulin variable domains, such as CDRs, can be defined using well-known numbering schemes, such as the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia (e.g., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services (1991), eds. Kabat et al.; Lazikani et al., (1997) J. Mol. Bio. 273:927-948); Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 US Department of Health and Human Services; Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:877-883; and Al-Lazikani et al. al., (1997) J. Mol. Biol. 273:927-948. As used herein, the term "antibody" is intended to include monospecific antibodies as well as bispecific and multispecific antibodies.

[0033] As used herein, the term "antibody fragment" or "antigen-binding fragment" of an antibody refers to one or more portions of an antibody that retain the ability to specifically interact with an antigen (e.g., by binding, steric hindrance, spatial distribution stabilization). Examples of antibody fragments or antigen-binding fragments include, but are not limited to, Fab fragments, which are monovalent fragments consisting of the VL, VH, CL, and CH1 domains; F(ab)2 fragments, which are bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments consisting of the VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; dAb fragments consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and isolated complementarity determining regions (CDRs). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be joined by a synthetic linker using recombinant techniques, resulting in a single protein chain in which the VL and VH domains pair to form a monovalent molecule (known as single-chain Fv (scFv), see, for example, Bird et al., (1988) Science 242:423-426, and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antibody fragment" or "antigen-binding fragment". These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, and bis-scFv (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23:1126-1136).Antibody fragments can be grafted into scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies). Antibody fragments or antigen-binding fragments can be incorporated into single-chain molecules containing tandem Fv segment pairs (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form antigen-binding site pairs (Zapata et al., (1995) Protein Eng. 8:1057-1062 and U.S. Pat. No. 5,641,870).

[0034] The term "Fc region" as used herein refers to two Fc region subunits capable of stably associating with each other to form a dimeric C-terminal region of an immunoglobulin. Thus, the two Fc region subunits (e.g., the first and second Fc region subunits) are complementary to each other. The Fc region of a regular IgG molecule exists as a dimer, each subunit of which contains the CH2 and CH3 IgG heavy chain constant domains.

[0035] As used herein, "Fc region subunit" refers to one of two polypeptides that form a dimeric Fc region of an immunoglobulin, i.e., a polypeptide that comprises a C-terminal constant region of an immunoglobulin heavy chain capable of stable self-association. Thus, the two Fc region subunits (e.g., the first and second Fc region subunits) that form a dimeric Fc region are complementary to each other. For example, an IgG Fc region subunit comprises an IgG CH2 and an IgG CH3 constant domain. This term includes native sequence Fc region subunits and variant Fc region subunits. Although the boundaries of an IgG heavy chain Fc region subunit may vary slightly, human IgG heavy chain Fc region subunits are usually defined as extending from Cys226 or from Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region subunit may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0036] As used herein, "human antibody" or "human antibody fragment" or "human antigen-binding fragment" includes antibodies and antibody fragments having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region is also derived from such sequences. Human origin includes, for example, antibodies containing human germline sequences, or mutated human germline sequences, or consensus framework sequences derived from human framework sequence analysis, e.g., those described in Knappik et al., (2000) J Mol Biol 296:57-86). Thereby, said human antibodies can be obtained from technology platforms that include antibodies derived from human germline genes, either generated by PCR amplification of VHA / L repertoires isolated from B cells, or are generated synthetically. Technology platforms include library-based approaches that include human immunoglobulin genes displayed on phage, ribosomes, or yeast. Each display technology is standard in the scientific community. Furthermore, immunization of transgenic mice carrying a human immunoglobulin repertoire is another approach to generate human antibodies against an antigen of interest. Antibodies or fragments thereof selected from antibody libraries based on the MorphoSys HuCAL® concept (Knappik et al., (2000) J Mol Biol 296:57-86) or the Ylanthia® concept library (Tiller et al. mAbs 5:3,1-26; May / June (2013) and U.S. Patent No. 8,728,981) are considered fully human.

[0037] The term "isolated" refers to a compound that can be, for example, an antibody or antibody fragment or antigen-binding fragment that is substantially free of other antibodies or antibody fragments having different antigen specificities. Thus, in some aspects, the antibody provided is an isolated antibody that is separated from antibodies of different specificities. An isolated antibody can be a monoclonal antibody. An isolated antibody can be a recombinant monoclonal antibody. However, an isolated antibody that specifically binds to a target epitope, isoform, or variant has cross-reactivity to other related antigens, for example, those from other species (e.g., species homologs).

[0038] The term "recombinant antibody" as used herein includes all antibodies or antigen-binding fragments prepared, expressed, engineered, or isolated by means that do not occur in nature, such as antibodies isolated from host cells transformed to express the antibody, antibodies selected and isolated from recombinant combinatorial human antibody libraries, and antibodies prepared, expressed, engineered, or isolated by any other means involving splicing all or part of the sequence of a human immunoglobulin gene to other DNA sequences, or antibodies isolated from a transgenic or transchromosomal animal (e.g., a mouse) of human immunoglobulin genes or a hybridoma prepared therefrom. Preferably, such recombinant antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when animals transgenic for human Ig sequences are used) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived and related to human germline VH and VL sequences, but are sequences that may not naturally occur within the in vivo human antibody germline repertoire. The recombinant antibody can be a monoclonal antibody.

[0039] As used herein, the term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. The monoclonal antibodies disclosed herein can be made, for example, by the hybridoma method described in Kohler et al.; Nature, 256:495 (1975), or isolated from phage libraries using the techniques described herein. Other methods for preparing clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art (see, for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel et al., eds., John Wiley and Sons, New York). Other exemplary methods for producing other monoclonal antibodies are provided in the Examples herein.

[0040] As used herein, an antibody "specifically binds to an antigen" or "specifically binds to an antigen" means "specific for" or "specifically recognizes" an antigen, even if such an antibody can distinguish such an antigen from one or more reference antigens. This is because binding specificity is a relative, not absolute, property. For example, a standard ELISA assay can be performed. Scoring can be done by standard color development (e.g. secondary antibody with horseradish peroxide and tetramethylbenzidine in combination with hydrogen peroxide). The reaction in a particular well is scored by optical density, e.g. at 450 nm. A typical background (=negative reaction) can be 0.1 OD and a typical positive reaction can be 1 OD. This means that the positive / negative difference can be more than 10-fold. Typically, the determination of binding specificity is performed using a set of about 3-5 unrelated antigens such as milk powder, BSA, transferrin, etc., rather than a single reference antigen.

[0041] As used herein, "antibodies or antigen-binding fragments thereof that bind to CD3" or "anti-CD3 antibodies or antigen-binding fragments thereof" or "antibodies or antigen-binding fragments thereof specific for CD3" includes antibodies and antibody fragments thereof or antigen-binding fragments thereof that specifically recognize one or more CD3 subunits (e.g., epsilon), as well as antibodies and antibody fragments thereof that specifically recognize dimeric complexes of two CD3 subunits (e.g., gamma / epsilon, delta / epsilon). The antibodies and antigen-binding fragments thereof of the present disclosure may bind to soluble CD3 and / or cell surface expressed CD3. Soluble CD3 includes native CD3 proteins that lack a transmembrane domain and are otherwise not associated with a cell membrane, as well as recombinant CD3 protein variants, such as monomeric and dimeric CD3 constructs.

[0042] As used herein, the term "cell surface" refers to one or more proteins expressed on the surface of a cell in vitro or in vivo such that at least a portion of the protein is exposed to the extracellular side of the cell membrane and is accessible to an antigen-binding portion or fragment of an antibody. "Cell surface expressed CD3" includes CD3 protein contained within the context of a functional T cell receptor in the cell membrane. The term "cell surface expressed CD3" includes CD3 protein expressed on the surface of a cell as part of a homodimer or heterodimer (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). The term "cell surface expressed CD3" also includes CD3 chains (e.g., CD3 epsilon) that are expressed alone, without other CD3 chain types, on the surface of a cell. "Cell surface expressed CD3" can include or consist of CD3 protein expressed on the surface of a cell that normally expresses CD3 protein. Alternatively, "cell surface expressed CD3" can include or consist of CD3 protein expressed on the surface of a cell that does not normally express human CD3 on its surface but has been artificially engineered to express CD3 on its surface.

[0043] The terms "cross-reactively bind" or "cross-reactive" are used interchangeably herein and refer to an antibody or antigen-binding fragment that has the ability to specifically bind to two or more antigens. For example, an antibody according to the present disclosure cross-reactively binds to cynomolgus CD3, such as cynomolgus CD3 epsilon.

[0044] As used herein, the term "affinity" refers to the strength of interaction between an antibody and its target at a single site. Within each site, the binding region of the antibody interacts with its target at numerous sites through weak non-covalent forces; the greater the interaction, the stronger the affinity.

[0045] As used herein, "K D The term "K d Against K a The ratio of (i.e., K d / K a ) and expressed as a molar concentration (M). For example, the K D Values ​​can be determined using methods well established in the art. For example, the K D The method for determining is SET (soluble equilibrium titration) or surface plasmon resonance using a biosensor system such as the Biacore® system. In the present disclosure, antibodies specific for CD3 epsilon polypeptides are typically detected at concentrations of 5×10 -2 Less than M, 10 -2 Less than M, 5×10 -3 Less than M, 10 -3 Less than M, 5×10 -4 Less than M, 10 -4 Less than M, 5×10 -5 Less than M, 10 -5 Less than M, 5×10 -6 Less than M, 10 -6 Less than M, 5×10 -7 Less than M, 10 -7 Less than M, 5×10- 8 Less than M, 10 -8 Less than M, 5×10-9 Less than M, 10 -9 Less than M, 5×10 -10 Less than M, 10 -10 Less than M, 5×10 -11 Less than M, 10 -11 Less than M, 5×10 -12 Less than M, 10 -12 Less than M, 5×10 -13 Less than M, 10 -13 Less than M, 5×10 -14 Less than M, 10 -14 Less than M, 5×10 -15 Less than M or 10 -15 The dissociation rate constant (K D )(k off / k on ).

[0046] The compositions of the present disclosure may be used for therapeutic or prophylactic purposes. Thus, the present disclosure includes pharmaceutical compositions containing the antibodies (or antigen-binding fragments thereof) disclosed herein and a pharma- ceutically acceptable carrier or excipient. In a related aspect, the present disclosure provides a method of treating cancer. Such a method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition containing the antibodies (or antigen-binding fragments thereof) described herein. The present disclosure provides a method of treatment comprising administering to a subject in need thereof a therapeutically effective amount of a human antibody or antigen-binding fragment thereof specific for CD3 disclosed herein. As used herein, a "therapeutically effective amount" or "effective amount" refers to the amount of a CD3-specific antibody required to elicit a desired biological response. According to the present disclosure, a therapeutically effective amount is the amount of a CD3-specific antibody or antigen-binding fragment thereof required to treat and / or prevent a disease.

[0047] The term "cell proliferative disorder" or "proliferative disorder" refers to a disorder associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. In one embodiment, the cell proliferative disorder is a tumor. The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.

[0048] The term "tumor-associated antigen" refers to an antigen that is expressed or present on the surface of a tumor or tumor stromal cells.

[0049] By "increase" is meant the ability to cause an overall increase of, for example, 20% or more, 50% or more, or 75%, 85%, 90%, 95% or more.

[0050] The term "EC50" as used herein means the concentration of an antibody or antibody fragment or bispecific antibody that induces an assay response halfway between the baseline and maximum. It represents the antibody concentration at which 50% of the maximum effect is observed.

[0051] The term "IC50" as used herein means the concentration of an antibody or antibody fragment or bispecific antibody that inhibits a response in an assay halfway between the maximum response and the baseline. It represents the antibody concentration that reduces a given response by 50%.

[0052] The terms "inhibition", "inhibit", "reduction", "reducing", "neutralization", "neutralizing" and the like refer to the reduction or cessation of any phenotypic property (e.g., binding, biological activity, or function) or the reduction or cessation of the incidence or extent or likelihood of that property. "Inhibition", "reduction", "neutralization", and the like need not necessarily be complete, so long as it is detectable using an appropriate assay. In some embodiments, "reduce", "inhibit", and the like refer to the ability to cause a reduction of 20% or more. In another embodiment, "reduce" or "inhibit" refers to the ability to cause a reduction of 50% or more. In yet another embodiment, "reduce", "inhibit", and the like refer to the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more.

[0053] "Administered" or "administration" includes, but is not limited to, delivery of an agent in an injectable form, such as by intravenous, intramuscular, intradermal, subcutaneous routes, or by mucosal routes, such as by nasal sprays or aerosols for inhalation, or by ingestible solutions, capsules, tablets, etc. Preferably, administration is by injectable form.

[0054] As used herein, "treatment", "treat", "treating" and the like refer to clinical intervention in an attempt to alter the natural history of a disease in a treated subject, and can be performed either for prophylactic purposes or during clinical pathology. Desirable treatment effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, diminution of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some aspects, the antibodies or antigen-binding fragments thereof of the present disclosure are used to delay disease onset or slow the progression of the disease.

[0055] The term "multispecific" means that an antibody is capable of specifically binding to two or more different antigens. Typically, a multispecific antibody is composed of two or more antigen-binding sites, each specific for a different antigen or epitope.

[0056] The term "bispecific" means that an antibody is capable of specifically binding to two different antigens. Typically, a bispecific antibody contains two antigen-binding sites, each specific for a different antigen or epitope.

[0057] As used herein, terms such as "first" and "second" are used to distinguish between each component type or types when more than one is present. The use of these terms is not intended to imply a specific order or orientation unless expressly stated to that effect.

[0058] As used herein, an "amino acid residue" or "amino acid" may be represented by either its full name or the standard three letter or one letter amino acid code. "Naturally occurring amino acid" means the following amino acids:

[0059] [Table 1]

[0060] "Effector function" means a biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0061] "Species" as used in the context of the present invention means any mammal, including rodents such as mice and rats, and primates such as cynomolgus monkeys (Macaca fascicularis), rhesus monkeys (Macaca mulatta), and humans (Homo sapiens). Preferably, the subject is a primate, and most preferably a human.

[0062] "Deimmunization" refers to a method of changing amino acids in a given antibody sequence that are predicted to effectively bind to HLA molecules so that they no longer bind to HLA and thus are no longer capable of stimulating a T cell response. Deimmunization therefore renders a given protein or polypeptide non-immunogenic or less immunogenic for a given species. The elimination of T cell epitopes from proteins has been previously disclosed (see WO 98 / 52976). One suitable technique for deimmunizing antibodies is described, for example, in WO 00 / 34317 or WO 2003 / 105058. In therapeutic antibodies, preferably all potential T cell epitopes are removed while retaining the functional activity of the unmodified parent antibody.

[0063] As used herein, a "deimmunized" antibody refers to an antibody that has undergone deimmunization. A "deimmunized" antibody may be less immunogenic or non-immunogenic in a given species when used in vivo compared to the unmodified parent antibody.

[0064] The term "T cell epitope" or "potential T cell epitope" or "MHC class II binding motif" as used herein refers to a specific 10-mer peptide sequence within a given protein or polypeptide sequence that either binds with reasonable efficiency to an MHC class II molecule, or binds strongly in the form of a peptide:MHC complex to a T cell receptor from a species that takes up the therapeutic protein, or exhibits the ability to stimulate T cells via presentation on MHC class II. Potential T cell epitopes can be determined by any computational or physical method that establishes MHC binding.

[0065] The term "hotspot" as used herein refers to a region within a given antibody heavy or light chain variable region where adjacent (predicted) T cell epitopes accumulate. Hotspots according to the present disclosure have been identified via the 4-over-3 algorithm detailed in FIG. 8 description and Example 4.1, which requires that at least four allotypes of DRB1 alleles bind with medium (M) or strong (S) affinity to at least two of three consecutive analyzed 10-mer peptides. Additionally, at most one 10-mer peptide not identified as a T cell epitope may be part of a hotspot.

[0066] The term "risk score" as used herein provides a numerical value as a risk measure of a T cell epitope (analyzed 10-mer peptide) causing an immune reaction or immune response in a given population, e.g., Caucasian population. The higher the value, the higher the risk. The risk score of a T cell epitope is calculated as the sum of the naturally occurring population frequencies of DRB1 alleles (HLA allotypes) to which such T cell epitope (10-mer peptide) binds. The risk score is determined by a computational method described in Example 4 herein using a commercially available in silico screening tool: Epibase™, Epibase version: v3.0 (Lonza). The basic method is described in WO 2003 / 105058.

[0067] The term "absolute risk score" or "score" as used herein provides a numerical value as a measure of risk of all analyzed polypeptides to cause an immune reaction or immune response in a given population, e.g., a Caucasian population. The "absolute risk score" is calculated as the sum of all individual risk scores determined for all T cell epitopes present in the analyzed polypeptide sequence, e.g., the entire VH or VL sequence of an antibody.

[0068] As used herein, "H-line" refers to an analyzed 10-mer peptide sequence of one of the antibody heavy or light chain variable regions that has been identified as being part of a hotspot.

[0069] As used herein, "H line number" provides a numerical value for the total number of analyzed 10-mer peptide sequences of antibody heavy or light chain variable regions that were identified as being part of a hotspot.

[0070] As used herein, "absolute H line number" provides the numerical value of the sum of the H line numbers that are a portion of all hotspots identified in a polypeptide.

[0071] As used herein, an "H-score" provides a numerical value as a measure of risk of a hotspot to cause an immune reaction or response in a given human population, e.g., a Caucasian population. The H-score is calculated as the sum of the individual risk scores determined for all T-cell epitopes that are part of the hotspot.

[0072] As used herein, an "absolute H-score" provides a numerical value as a cumulative risk measure of all hotspots identified in a given antibody heavy or light chain variable region to elicit an immune reaction or response in a given human population, e.g., a Caucasian population. The absolute H-score is calculated as the sum of the risk scores determined for all T-cell epitopes that are part of all hotspots identified in a given antibody heavy or light chain variable region.

[0073] Embodiments of the present disclosure Affinity optimized human antibodies specific for CD3 according to the present disclosure are listed in Tables 5 and 6. Deimmunized human antibodies specific for CD3 according to the present disclosure are listed in Table 7. In one aspect, the present disclosure relates to an isolated human antibody or antigen-binding fragment thereof specific for CD3 comprising six CDRs as set forth in any one of the antibodies listed in Tables 5-7. In one aspect, the present disclosure relates to an isolated human antibody or antigen-binding fragment thereof comprising a VH and a VL as set forth in any one of the antibodies listed in Tables 5-7.

[0074] In one embodiment, the present disclosure provides: i. Below (a) heavy chain complementarity determining region (HCDR) 1 comprising the amino acid sequence of GFSFGSHYMS (SEQ ID NO: 1); (b) an HCDR2 comprising the amino acid sequence of NINQIGYSSYYVESVKG (SEQ ID NO: 2), NINQIGYSSYYGESVKG (SEQ ID NO: 3), or NINQIGYSSYYEESVKG (SEQ ID NO: 4); and (c) an HCDR3 comprising the amino acid sequence of GYSAEFAHRSGLDV (SEQ ID NO: 5), GYSDEFATRSGLDV (SEQ ID NO: 6), GYSEEFAHRSGLDV (SEQ ID NO: 7), GYSDEFAKRSGLDV (SEQ ID NO: 8), or GYSDEFAHRSGLDV (SEQ ID NO: 9); A heavy chain variable region (VH) comprising: ii. Below (d) a light chain complementarity determining region (LCDR) 1 comprising the amino acid sequence of SGSSSNIGSNYVY (SEQ ID NO: 10); (e) an LCDR2 comprising the amino acid sequence of RNNQRPS (SEQ ID NO: 11); and (f) an LCDR3 comprising the amino acid sequence of AGWSRSLHGAV (SEQ ID NO: 12) or AGWSRELHGAV (SEQ ID NO: 13); A variable light chain region (VL) comprising The present invention provides an isolated human antibody or antigen-binding fragment thereof specific for cluster of differentiation 3 (CD3), comprising:

[0075] In one embodiment, the present disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for CD3, a) an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 22; b) an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 23, and c) an HCDR3 region comprising the amino acid sequence of SEQ ID NO:5; and d) an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 10; e) an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 11, and f) an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 12; VL, including

[0076] In one embodiment, the present disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for CD3, a) an HCDR1 region comprising the amino acid sequence of SEQ ID NO:1; b) an HCDR2 region comprising the amino acid sequence of SEQ ID NO:2, and c) an HCDR3 region comprising the amino acid sequence of SEQ ID NO:5; and d) an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 10; e) an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 11, and f) an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 24; VL, including

[0077] In one embodiment, the present disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for CD3, a) an HCDR1 region comprising the amino acid sequence of SEQ ID NO:1; b) an HCDR2 region comprising the amino acid sequence of SEQ ID NO:2, and c) an HCDR3 region comprising the amino acid sequence of SEQ ID NO:5; and d) an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 10; e) an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 11, and f) an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 12; VL, including

[0078] In one embodiment, the present disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for CD3, a) an HCDR1 region comprising the amino acid sequence of SEQ ID NO:1; b) an HCDR2 region comprising the amino acid sequence of SEQ ID NO:4, and c) an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 7; and d) an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 10; e) an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 11, and f) an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 13; VL, including

[0079] In one embodiment, the present disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for CD3, a) an HCDR1 region comprising the amino acid sequence of SEQ ID NO:1; b) an HCDR2 region comprising the amino acid sequence of SEQ ID NO:3, and c) an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 9; and d) an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 10; e) an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 11, and f) an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 13; VL, including

[0080] In one embodiment, the present disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for CD3, a) an HCDR1 region comprising the amino acid sequence of SEQ ID NO:1; b) an HCDR2 region comprising the amino acid sequence of SEQ ID NO:3, and c) an HCDR3 region comprising the amino acid sequence of SEQ ID NO:6; and d) an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 10; e) an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 11, and f) an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 13; VL, including

[0081] In one embodiment, the present disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for CD3, a) an HCDR1 region comprising the amino acid sequence of SEQ ID NO:1; b) an HCDR2 region comprising the amino acid sequence of SEQ ID NO:3, and c) an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 7; and d) an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 10; e) an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 11, and f) an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 13; VL, including

[0082] In one embodiment, the present disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for CD3, a) an HCDR1 region comprising the amino acid sequence of SEQ ID NO:1; b) an HCDR2 region comprising the amino acid sequence of SEQ ID NO:3, and c) an HCDR3 region comprising the amino acid sequence of SEQ ID NO:8; and d) an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 10; e) an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 11, and f) an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 13; VL, including

[0083] In one embodiment, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for cluster of differentiation 3 (CD3), comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. In one embodiment, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for cluster of differentiation 3 (CD3), comprising a light chain variable region (VL) comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21. In one embodiment, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for cluster of differentiation 3 (CD3), comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and a light chain variable region (VL) comprising an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21. In one embodiment, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for cluster of differentiation 3 (CD3), comprising a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. In one embodiment, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for cluster of differentiation 3 (CD3), comprising a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:20 or SEQ ID NO:21.In one embodiment, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for cluster of differentiation 3 (CD3), comprising a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:20 or SEQ ID NO:21.

[0084] In one embodiment, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for cluster of differentiation 3 (CD3), comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL are as follows: i. a VH comprising the amino acid sequence of SEQ ID NO: 25 and a VL comprising the amino acid sequence of SEQ ID NO: 20, and ii. VH comprising the amino acid sequence of SEQ ID NO: 14 and VL comprising the amino acid sequence of SEQ ID NO: 26; is selected from the group consisting of:

[0085] In one embodiment, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for cluster of differentiation 3 (CD3), comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL are as follows: i. a VH comprising the amino acid sequence of SEQ ID NO: 14 and a VL comprising the amino acid sequence of SEQ ID NO: 20; ii. VH comprising the amino acid sequence of SEQ ID NO: 15 and VL comprising the amino acid sequence of SEQ ID NO: 21; iii. VH comprising the amino acid sequence of SEQ ID NO: 16 and VL comprising the amino acid sequence of SEQ ID NO: 21; iv. VH comprising the amino acid sequence of SEQ ID NO: 17 and VL comprising the amino acid sequence of SEQ ID NO: 21; v. A VH comprising the amino acid sequence of SEQ ID NO: 18 and a VL comprising the amino acid sequence of SEQ ID NO: 21; and vi. VH comprising the amino acid sequence of SEQ ID NO: 19 and VL comprising the amino acid sequence of SEQ ID NO: 21; is selected from the group consisting of:

[0086] In one embodiment, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for cluster of differentiation 3 (CD3), comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 20.

[0087] In one embodiment, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for cluster of differentiation 3 (CD3), comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 21.

[0088] In one embodiment, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for cluster of differentiation 3 (CD3), comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 16 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 21.

[0089] In one embodiment, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for cluster of differentiation 3 (CD3), comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 21.

[0090] In one embodiment, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for cluster of differentiation 3 (CD3), comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 21.

[0091] In one embodiment, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for cluster of differentiation 3 (CD3), comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 19 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 21.

[0092] In one embodiment, the isolated human antibody or antigen-binding fragment thereof specific for CD3 is a monoclonal antibody or antigen-binding fragment. In another embodiment, the isolated human antibody or antigen-binding fragment thereof is a recombinant antibody or antigen-binding fragment. In one embodiment, the isolated human antibody or antigen-binding fragment thereof specific for CD3 is a synthetic antibody or antigen-binding fragment. In one embodiment of the present disclosure, the CD3-specific antibody or antigen-binding fragment thereof is a full-length IgG of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0093] specificity In one embodiment, the present disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for cluster of differentiation 3 (CD3). In one aspect, the isolated human antibody or antigen-binding fragment thereof specific for CD3 of the present disclosure is specific for human CD3. In one aspect, the isolated human antibody or and antigen-binding fragment thereof specific for CD3 of the present disclosure is specific for cynomolgus CD3. In one aspect, the isolated human antibody or and antigen-binding fragment thereof specific for CD3 of the present disclosure is specific for human and cynomolgus CD3.

[0094] In one embodiment of the present disclosure, the isolated human antibody or antigen-binding fragment thereof specific for CD3 of the present disclosure is specific for human CD3 epsilon. In one embodiment, the isolated human antibody or antigen-binding fragment thereof specific for CD3 of the present disclosure is specific for cynomolgus CD3 epsilon. In one embodiment, the isolated human antibody or antigen-binding fragment thereof specific for CD3 of the present disclosure is specific for human and cynomolgus CD3 epsilon. In one embodiment, the isolated human antibody or antigen-binding fragment thereof specific for CD3 of the present disclosure is specific for human and cynomolgus CD3 epsilon.

[0095] In one aspect, the present disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for human CD3 according to the present disclosure, where the antibody or antigen-binding fragment thereof cross-reactively binds to cynomolgus CD3. In one aspect, the present disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for human CD3 epsilon, where the antibody or antigen-binding fragment thereof cross-reactively binds to cynomolgus CD3 epsilon. In one aspect, the present disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for CD3, where the antibody or antigen-binding fragment thereof specifically binds to human CD3 epsilon. In one aspect of the present disclosure, the isolated human antibody or antigen-binding fragment thereof specific for CD3 specifically binds to human and cynomolgus CD3 epsilon.

[0096] In one aspect, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for human CD3, wherein the human CD3 is human CD3 epsilon comprising the amino acid sequence of SEQ ID NO: 45 or SEQ ID NO: 46. In one aspect, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for human CD3, wherein the isolated human antibody or antigen-binding fragment thereof specifically binds to a human CD3 epsilon polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 41, and SEQ ID NO: 43.

[0097] In one aspect, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for human CD3, wherein the cyno CD3 is cyno CD3 epsilon comprising the amino acid sequence of SEQ ID NO: 47 or SEQ ID NO: 48. In one aspect, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for human CD3, wherein the isolated human antibody or antigen-binding fragment thereof specifically binds to a cyno CD3 epsilon polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 42, and SEQ ID NO: 44.

[0098] In one aspect, the present disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for human CD3, wherein the isolated human antibody or antigen-binding fragment thereof specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 42, and SEQ ID NO: 44. In one aspect of the present disclosure, the isolated human antibody or antigen-binding fragment thereof specific for CD3 of the present disclosure specifically binds to the extracellular domain human CD3 epsilon. In one aspect, the isolated human antibody or antigen-binding fragment thereof specific for CD3 of the present disclosure specifically binds to the extracellular domain human and cynomolgus CD3 epsilon. In one aspect of the present disclosure, the extracellular domain of human CD3 epsilon comprises the amino acid sequence of SEQ ID NO: 46. In a further aspect, the extracellular domain of cynomolgus CD3 epsilon comprises the amino acid sequence of SEQ ID NO: 48. In one aspect, the disclosure relates to an isolated human antibody or antigen-binding fragment thereof specific for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44. In one aspect, the disclosure relates to an isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the disclosure that specifically binds to a polypeptide encoded by SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 41, or SEQ ID NO: 43, and a polypeptide encoded by SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 42, or SEQ ID NO: 44.

[0099] In one embodiment of the disclosure, the isolated human antibody or antigen-binding fragment thereof specific for CD3 comprises: i. Below (a) HCDR1 comprising the amino acid sequence of GFSFGSHYMS (SEQ ID NO: 1); (b) an HCDR2 comprising the amino acid sequence of NINQIGYSSYYVESVKG (SEQ ID NO: 2), NINQIGYSSYYGESVKG (SEQ ID NO: 3), or NINQIGYSSYYEESVKG (SEQ ID NO: 4); and (c) an HCDR3 comprising the amino acid sequence of GYSAEFAHRSGLDV (SEQ ID NO: 5), GYSDEFATRSGLDV (SEQ ID NO: 6), GYSEEFAHRSGLDV (SEQ ID NO: 7), GYSDEFAKRSGLDV (SEQ ID NO: 8), or GYSDEFAHRSGLDV (SEQ ID NO: 9); A heavy chain variable region (VH) comprising: ii. Below (d) LCDR1 comprising the amino acid sequence of SGSSSNIGSNYVY (SEQ ID NO: 10); (e) an LCDR2 comprising the amino acid sequence of RNNQRPS (SEQ ID NO: 11); and (f) an LCDR3 comprising the amino acid sequence of AGWSRSLHGAV (SEQ ID NO: 12) or AGWSRELHGAV (SEQ ID NO: 13); A variable light chain region (VL) comprising Includes.

[0100] In one embodiment of the disclosure, the isolated human antibody or antigen-binding fragment thereof specific for CD3 comprises: i. a VH comprising the amino acid sequence of SEQ ID NO: 14 and a VL comprising the amino acid sequence of SEQ ID NO: 20; ii. VH comprising the amino acid sequence of SEQ ID NO: 15 and VL comprising the amino acid sequence of SEQ ID NO: 21; iii. VH comprising the amino acid sequence of SEQ ID NO: 16 and VL comprising the amino acid sequence of SEQ ID NO: 21; iv. VH comprising the amino acid sequence of SEQ ID NO: 17 and VL comprising the amino acid sequence of SEQ ID NO: 21; v. A VH comprising the amino acid sequence of SEQ ID NO: 18 and a VL comprising the amino acid sequence of SEQ ID NO: 21; and vi. VH comprising the amino acid sequence of SEQ ID NO: 19 and VL comprising the amino acid sequence of SEQ ID NO: 21; The VH and VL are selected from the group consisting of:

[0101] Kinetics In one aspect, the disclosure relates to an isolated human antibody or antigen-binding fragment thereof specific for human CD3, wherein the antibody or antigen-binding fragment thereof comprises an antibody KH and VL comprising the amino acid sequences of SEQ ID NO:25 and SEQ ID NO:26, respectively. D Lower K compared to D and has monovalent affinity for human CD3 epsilon peptides comprising SEQ ID NO:41 and / or SEQ ID NO:43.

[0102] In one aspect, the disclosure relates to an isolated human antibody or antigen-binding fragment thereof specific for human CD3, wherein the antibody or antigen-binding fragment thereof has a K of 10 nM or less, e.g., 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.1 nM or less, 0.2 nM, or 0.1 nM or less. D and has monovalent affinity for human CD3 epsilon peptides comprising SEQ ID NO:41 and / or SEQ ID NO:43.

[0103] In one aspect, the disclosure relates to an isolated human antibody or antigen-binding fragment thereof specific for human CD3, wherein the antibody or antigen-binding fragment thereof has a K of about 10 nM, about 9 nM, 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, about 0.1 nM. D and has monovalent affinity for human CD3 epsilon peptide comprising SEQ ID NO:41 or SEQ ID NO:43.

[0104] In one aspect, the disclosure relates to an isolated human antibody or antigen-binding fragment thereof specific for CD3, wherein the antibody or antigen-binding fragment thereof has a K of 0.1 nM to 10 nM. D and has monovalent affinity for the human CD3 epsilon polypeptide comprising SEQ ID NO:41 or SEQ ID NO:34.

[0105] In certain embodiments, the monovalent affinity is determined in an scFv, Fv, or Fab. In one embodiment, the monovalent affinity is determined as described in Example 2.1, Example 3.4, Example 4.4, or Example 4.8 herein. In one embodiment, the monovalent affinity is determined in an antibody format described in Example 2, Example 3, or Example 4 herein.

[0106] In one aspect, the isolated human antibody or antigen-binding fragment thereof specific for cluster of differentiation 3 (CD3) comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 21.

[0107] cell binding In one aspect, the disclosure relates to an isolated human antibody or antigen-binding fragment thereof specific for human CD3, wherein the antibody or antigen-binding fragment thereof has an EC 50 It specifically binds to human Jurkat cells (ATCC #TIB-152) at concentrations

[0108] In one aspect, the disclosure relates to an isolated human antibody or antigen-binding fragment thereof specific for human CD3, wherein the antibody or antigen-binding fragment thereof has an EC of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM. 50 It specifically binds to human Jurkat cells (ATCC #TIB-152) at concentrations

[0109] In one embodiment, the EC 50 The concentration is determined by FACS assay as described in Example 2.2 or Example 3.5 herein. 50 The concentration is determined in a Fab format. 50The concentration is determined in an antibody Fv format. 50 The concentrations are determined in a bispecific antibody format according to Example 2 or Example 3 comprising an isolated human antibody or antigen-binding fragment thereof specific for human CD3 according to the present disclosure.

[0110] In one aspect, the isolated human antibody or antigen-binding fragment thereof specific for cluster of differentiation 3 (CD3) comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 21.

[0111] ELISA binding In one embodiment, the present disclosure provides an EC of 1 to 40 nM, preferably 1 to 15 nM. 50 The present invention provides an isolated human antibody or antigen-binding fragment thereof specific for CD3, which specifically binds to a recombinant human CD3 epsilon polypeptide comprising SEQ ID NO: 41 or SEQ ID NO: 43 at a concentration of 50 nM or less, and is preferably an antibody or antigen-binding fragment thereof having an EC50 concentration of less than 40 nM, preferably less than 15 nM. 50 and specifically binds to a recombinant human CD3 epsilon polypeptide having SEQ ID NO: 41 or SEQ ID NO: 43. In one embodiment, the antibody or antigen-binding fragment thereof has an EC determined with an antibody or antigen-binding fragment thereof specific for CD3 comprising a VH of SEQ ID NO: 14 and a VL of SEQ ID NO: 20 or a VH of SEQ ID NO: 25 and a VL of SEQ ID NO: 26. 50 EC at a concentration of about 1 / 0.5, about 1 / 1, about 1 / 1.5, about 1 / 2, about 1 / 2.5, or about 1 / 3 of the concentration 50 and specifically binds to a recombinant human CD3 epsilon polypeptide having SEQ ID NO:41 or SEQ ID NO:43.

[0112] In one embodiment, the EC 50 The concentration is determined as described in Example 4.7 herein. 50 The concentration is determined by ELISA assay. 50The concentration is determined by antibody Fv. 50 The concentrations are determined in a bispecific antibody format according to Example 4, which comprises an isolated human antibody or antigen-binding fragment thereof specific for human CD3 according to the present disclosure.

[0113] In one aspect, the isolated human antibody or antigen-binding fragment thereof specific for cluster of differentiation 3 (CD3) comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 21.

[0114] safety In one aspect, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for CD3, wherein the antibody or antigen-binding fragment thereof induces less upregulation of CD69 expression on CD4+ and / or CD8+ T cells compared to an isolated human antibody or antigen-binding fragment thereof specific for CD3 comprising a VH of SEQ ID NO: 14 and a VL of SEQ ID NO: 20.

[0115] In one aspect, the disclosure provides an isolated human antibody, or antigen-binding fragment thereof, that specifically binds to CD3 expressed on CD4+ and / or CD8+ T cells and induces upregulation of CD69 expression on a lower number of CD4+ and / or CD8+ T cells compared to an antibody, or antigen-binding fragment thereof, specific for CD3 comprising a VH of SEQ ID NO: 14 and a VL of SEQ ID NO: 20.

[0116] In certain embodiments, the upregulation of CD69 on CD4+ and / or CD8+ T cells is determined by the method described in Example 4.13 herein. In certain embodiments, the upregulation of CD69 is determined with a bispecific antibody according to Example 4, comprising an isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure. In one embodiment, the antibody is a bispecific antibody. In one embodiment, the bispecific antibody monovalently binds to CD3 expressed on CD4+ and / or CD8+ positive T cells. In one embodiment, the CD69 upregulation is determined with an Fv fragment. In one embodiment, the CD3 specific antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof listed in Table 8.

[0117] In one aspect, the isolated human antibody or antigen-binding fragment thereof specific for cluster of differentiation 3 (CD3) comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 21.

[0118] Deimmunization A T cell epitope is a specific peptide sequence within a polypeptide sequence that either binds reasonably efficiently to MHC class II molecules, or binds strongly in the form of a peptide:MHC complex to T cell receptors from a species that ingests the (therapeutic) protein or polypeptide, or shows the ability to stimulate T cells via presentation on MHC class II. A (potential) T cell epitope can be measured by any computational or physical method that establishes MHC binding. However, it is understood that certain peptides found to bind to MHC class II molecules will not elicit an immune response because they will be recognized as "self" in the organism to which the protein is administered. Such peptides are found, for example, in germline human immunoglobulin variable region protein sequences. The present disclosure incorporates an approach to designing improved human antibodies specific for CD3 by removal of potential T cell epitopes present in parent human antibodies specific for CD3. This involves the selection of amino acid substitutions that allow removal of the identified T cell epitopes, and the testing of a range of variant molecules with different amino acid substitutions. The principle of the present disclosure is the modification of the primary CDR sequences of a human CD3 specific antibody by the identification of potential T cell epitopes and subsequent modifications within the CDRs to eliminate such potential T cell epitopes. The primary sequence of a therapeutic antibody can be analyzed for the presence of T cell epitopes by any suitable means. The method of the present disclosure uses a computational screening method provided by Lonza (Epibase™, Epibase version: v3.0, WO 2003 / 105058).

[0119] In one aspect, the disclosure provides an isolated deimmunized human antibody or antigen-binding fragment thereof specific for CD3. In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof specific for CD3 that is an isolated deimmunized human antibody or antigen-binding fragment thereof specific for CD3. In one aspect, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for CD3 that is less immunogenic or non-immunogenic in humans when compared to an antibody or antigen-binding fragment thereof specific for CD3 comprising a VH of SEQ ID NO: 14 and a VL of SEQ ID NO: 20.

[0120] In one aspect, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for CD3, wherein the antibody or antigen-binding fragment has a reduced risk of eliciting an immune response or an immunogenic response in a human. In one aspect, the disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for CD3, wherein the antibody or antigen-binding fragment has a reduced risk of eliciting a human anti-human antibody response in a human.

[0121] In one embodiment, the antibody or antigen-binding fragment thereof has a reduced risk of eliciting an immune response or an immunogenic response in a human once administered to the human. In one embodiment, the reduced risk is based on the risk of eliciting an immune response or an immunogenic response in a human of a human antibody or antigen-binding fragment thereof comprising a VH of SEQ ID NO: 14 and a VL of SEQ ID NO: 20. In one embodiment of the present disclosure, the reduced risk in a human is determined as described in Example 4 herein. In one embodiment of the present disclosure, the reduced risk is determined with the VH and / or VL of a human antibody or antigen-binding fragment thereof specific for CD3 of the present disclosure.

[0122] In one embodiment of the disclosure, the risk or reduced risk is provided as a number determined by the number of T cell epitopes, risk score, absolute score, number of H lines, absolute number of H lines, H score, and absolute H score, and / or number of hot spots, all of which are defined herein. In one embodiment of the disclosure, the risk or reduced risk is based on a risk determined for a human antibody or antigen-binding fragment thereof comprising a VH of SEQ ID NO: 14 and a VL of SEQ ID NO: 20. In one embodiment, the number was determined by the number of T cell epitopes, risk score, absolute score, number of H lines, absolute number of H lines, H score, and absolute H score, and / or number of hot spots using the in silico T cell epitope screening tool Epibase™, Epibase version: v3.0 (Lonza) based on WO 2003 / 105058, which is incorporated herein in its entirety. In one embodiment, the allele set is major Caucasian DRB1 alleles. In one embodiment, the risk is determined at various allotypes of the Caucasian DRB1 allele. In one embodiment, the filter set is for human antibody germline sequences. In one embodiment, the risk is determined by excluding human antibody germline sequences present in the VH and / or VL of the human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure. In one embodiment, the risk is determined at the HCDR1, HCDR2, HCDR3, and LCDR3 regions of the human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure. In one embodiment, the risk excludes the risk of human antibody germline sequences in eliciting an immune response in humans. In one embodiment, the selected population is Caucasian. In one embodiment, the isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure has a reduced risk of selecting an immune response or an immunogenic response in humans of the Caucasian population.In one aspect, an isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure has a reduced risk of eliciting an immune response or an immunogenic response in humans of the Caucasian population compared to the risk of an isolated human antibody or antigen-binding fragment thereof specific for CD3 comprising a VH of SEQ ID NO: 14 and a VL of SEQ ID NO: 20.

[0123] In one embodiment, an isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure has a VH with an absolute score as determined herein within the range of about 380 to 450, preferably about 390 to 425. In one embodiment, an isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure has a VH with an absolute score of less than 440, less than 430, less than 420, less than 410, less than 400, or less than 395. In one embodiment, an isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure has a VH with a reduced absolute score compared to a VH comprising SEQ ID NO: 14. In one embodiment, the reduced absolute score is reduced by more than 200, e.g., more than 210, 215, 220, 230, 235, 240, 245, or 250.

[0124] In one embodiment, an isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure has a VH with an absolute H score in the range of about 330 to 370, preferably about 335 to 365. In one embodiment, an isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure has a VH with an absolute H score of about 335 or 365. In one embodiment, an isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure has a VH with an absolute H score of less than 370, less than 364, less than 360, less than 355, less than 350, less than 345, less than 340, or less than 335. In one embodiment, an isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure has a VH with a reduced absolute H score compared to a VH comprising SEQ ID NO: 14. In one embodiment, the reduced absolute H-score is reduced by more than 200, e.g., by more than 210, 215, 220, 230, 235, 240, 245, or 250.

[0125] In one embodiment, the isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure has a VH with an absolute H line of 14. In one embodiment, the isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure has a VH with a reduced absolute H line compared to a VH comprising SEQ ID NO: 14. In one embodiment, the reduced absolute H line is reduced by 11. In one embodiment, the isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure has a VH with a reduced number of absolute hot spots of 4. In one embodiment, the isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure has a VH with a reduced number of absolute hot spots compared to a VH comprising SEQ ID NO: 14. In one embodiment, the reduced number of absolute hot spots is reduced by 2. In one embodiment, the isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure has a VL with an absolute score and / or absolute H score of 62. In one embodiment, an isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure has a VL with a reduced absolute score and / or absolute H score compared to a VH comprising SEQ ID NO: 14. In one embodiment, an isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure has a VL with an absolute H line number of 2. In one embodiment, an isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure has a VL with an absolute hotspot number of 0.

[0126] In one embodiment, the isolated human antibody or antigen-binding fragment thereof specific for CD3 has the following structure: i. a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 21; ii. VH comprising the amino acid sequence of SEQ ID NO: 16 and VL comprising the amino acid sequence of SEQ ID NO: 21; iii. VH comprising the amino acid sequence of SEQ ID NO: 17 and VL comprising the amino acid sequence of SEQ ID NO: 21; iv. a VH comprising the amino acid sequence of SEQ ID NO: 18 and a VL comprising the amino acid sequence of SEQ ID NO: 21; and v. VH comprising the amino acid sequence of SEQ ID NO: 19 and VL comprising the amino acid sequence of SEQ ID NO: 21; The VH and VL are selected from the group consisting of:

[0127] multispecific antibodies The isolated human antibodies or antigen-binding fragments thereof specific for CD3 according to the present disclosure should preferentially be used in bi- or multispecific antibody formats to target CD3-expressing cytotoxic T cells and stimulate cytotoxic T cell activation in situations where T cell-mediated killing of a specific cell type, such as, for example, a tumor cell, is beneficial or desirable.

[0128] The isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. In one aspect, the present disclosure relates to an isolated human antibody or antigen-binding fragment thereof specific for CD3, where the antibody or antigen-binding fragment thereof is fused to a heterologous protein or polypeptide. In one aspect, the present disclosure relates to a fusion protein comprising an isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure and a heterologous protein or polypeptide. For example, the antibody or antigen-binding fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, e.g., another antibody or antigen-binding fragment thereof, to generate a bispecific or multispecific antibody having a second or, optionally, a third binding specificity. Bispecific or multispecific antibodies capable of binding to more than one antigen are of great interest for therapeutic applications. In another aspect, the present disclosure provides an isolated human antibody or antigen-binding fragment thereof specific for CD3, wherein the antibody is a monospecific, bispecific, or multispecific antibody or antigen-binding fragment thereof. A multispecific antibody may contain antibodies or antigen-binding fragments thereof specific for different epitopes on the same target antigen, or may contain antibodies or antigen-binding fragments thereof specific for two or more target antigens. In the context of a bispecific or multispecific antibody according to the present disclosure, the cell surface target antigen may be a tumor-associated antigen (TAA). Non-limiting examples of tumor-associated antigens include, for example, antigens expressed on the surface of tumor or cancerous cells.Exemplary multispecific antibody formats that can be used in the context of the present disclosure include, but are not limited to, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, nobs-into-holes, common light chain (such as a nobs-into-holes common light chain), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, Mab2 bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6,1-11), and hemibodies (see, e.g., Stuhler et al. Nat Commun. 2019;10:5387). Preferably, the isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure is used in the format described in Example 3 or Example 5 herein or in WO 2020 / 115115.

[0129] In one aspect, the present disclosure provides a bispecific or multispecific antibody comprising an antigen-binding fragment of a human antibody specific for CD3 of the present disclosure and a second antigen-binding fragment of a second antibody that binds to a different antigen than the first antigen-binding fragment. In one aspect of the present disclosure, the second antigen-binding fragment binds to a cell surface antigen. In one aspect, the cell surface target antigen is a tumor-associated antigen. In one aspect, the first antigen-binding fragment binds to CD3 present or expressed on an immune effector cell. In one aspect, the immune effector cell is a T cell. In one aspect, the T cell is a cytotoxic T cell. In one aspect, the present disclosure provides a bispecific or multispecific antibody comprising a human antibody or antigen-binding fragment thereof specific for CD3 of the present disclosure, wherein the bispecific or multispecific antibody mediates redirected T cell-mediated killing of target antigen-expressing cells. In certain aspects, the target cell killing can be determined by methods described herein, such as those described in Example 4.12.

[0130] In an embodiment of the disclosure, a bispecific or multispecific antibody of the disclosure specifically binds to CD3 expressed on a T cell and a second antigen present on a cell other than a T cell. In certain embodiments, the bispecific or multispecific antibody activates a T cell following binding to CD3 expressed on a T cell and a second antigen present on a target cell other than a T cell. In certain embodiments, the activated T cell is capable of exerting a cytotoxic and / or apoptotic effect on other cells. In one embodiment, the disclosure provides a bispecific or multispecific antibody comprising a human antibody or antigen-binding fragment thereof specific for CD3 of the disclosure, wherein the bispecific or multispecific antibody induces human T cell proliferation in the presence of a cell expressing a cell surface target antigen following binding to CD3 expressed on a T cell and a cell surface target expressing cell.

[0131] Linker Optimization In one embodiment, a bispecific antibody according to the present disclosure is composed of three Fv regions. This is achieved by using a regular immunoglobulin (e.g., IgG) antibody structure (two heavy chains associated with two light chains to form two Fv regions) incorporating an additional Fv region between the two Fab arms and the Fc portion of the regular immunoglobulin structure. In one embodiment, a bispecific antibody according to the present disclosure has the general structure depicted in FIG. 2.

[0132] In one aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: a) a first Fab comprising a first Fv region that specifically binds to a first antigen; b) a second Fv region that specifically binds to a second antigen, and c) a second Fab comprising a third Fv region that specifically binds to a third antigen, and d) an Fc domain composed of a first and a second Fc domain subunit; Including, i. the C-terminus of a first Fab heavy chain is fused to the N-terminus of the VH or VL of a second Fv region via a first peptide linker; ii. the C-terminus of the VH or VL of the second Fv region is fused to the N-terminus of the first Fc region subunit via a second peptide linker; iii. the N-terminus of the second Fc region subunit is fused to the C-terminus of a complementary variable domain of a second Fv region via a fourth peptide linker; and iv. the C-terminus of a second Fab heavy chain is fused to the N-terminus of the VH or VL of a second Fv region via a third peptide linker, whereby the first and second Fabs are fused to distinguishable variable regions of the second Fv region; Bispecific antibodies are provided.

[0133] In one embodiment, the C-terminus of the CH1 domain of the first Fab is fused to the N-terminus of the VH or VL of the second Fv region via a first peptide linker, where the first and second Fabs are fused to distinguishable variable domains of the second Fv region. In one embodiment, the C-terminus of the CH1 domain of the first Fab is fused to the N-terminus of the VH of the second Fv region via a first peptide linker, where the first and second Fabs are fused to distinguishable variable domains of the second Fv region. In one embodiment, the C-terminus of the CH1 domain of the first Fab is fused to the N-terminus of the VL of the second Fv region via a first peptide linker, where the first and second Fabs are fused to distinguishable variable domains of the second Fv region.

[0134] In one embodiment, the C-terminus of the CH1 domain of the second Fab is fused to the N-terminus of the VH or VL of the second Fv region via a third peptide linker, where the first and second Fabs are fused to distinguishable variable domains of the second Fv region. In one embodiment, the C-terminus of the CH1 domain of the second Fab is fused to the N-terminus of the VH of the second Fv region via a third peptide linker, where the first and second Fabs are fused to distinguishable variable domains of the second Fv region. In one embodiment, the C-terminus of the CH1 domain of the second Fab is fused to the N-terminus of the VL of the second Fv region via a third peptide linker, where the first and second Fabs are fused to distinguishable variable domains of the second Fv region.

[0135] In one embodiment, the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the VL of the second Fv region via a first peptide linker and the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the VH of the second Fv region via a third peptide linker. In one embodiment, the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the VH of the second Fv region via a first peptide linker and the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the VL of the second Fv region via a third peptide linker.

[0136] In one embodiment, a bispecific antibody according to the present disclosure comprises four polypeptides: a) the first polypeptide comprises a light chain of a first Fab, b) the second polypeptide comprises, from its N-terminus to its C-terminus: i. the heavy chain of a first Fab, ii. a first peptide linker; iii. the VL of a second Fv region, iv. a second peptide linker, and v. the first Fc region subunit, Including, c) the third polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a second Fab, ii. a third peptide linker; iii. the VH of a second Fv region; iv. a fourth peptide linker; v. a second Fc region subunit; and d) the fourth polypeptide comprises the light chain of the second Fab.

[0137] In one embodiment, a bispecific antibody according to the present disclosure comprises four polypeptides: a) the first polypeptide comprises a light chain of a first Fab, b) the second polypeptide comprises, from its N-terminus to its C-terminus: i. the heavy chain of a first Fab, ii. a first peptide linker; iii. the VH of a second Fv region; iv. a second peptide linker, and iii. a first Fc domain subunit; Including, c) the third polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a second Fab, ii. a third peptide linker; iii. the VL of a second Fv region, iv. a fourth peptide linker, and v. a second Fc region subunit; Including, d) the fourth polypeptide comprises the light chain of the second Fab.

[0138] In one embodiment of the present disclosure, the first, second, third, and fourth peptide linkers are selected from the following: a) GGSGGSGGS (SEQ ID NO: 30) b) GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:31), c) AQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 33), d) AHPAAPAPAHPAAPAPAHGH (SEQ ID NO: 32); e) PKAAP (SEQ ID NO: 36); f) PKAAPSVTLFPPSSEELQAN (SEQ ID NO: 34), g) ASTKGP (SEQ ID NO: 37), and h) ASTKGPSVFPLAPSSKSTSG (SEQ ID NO: 35), The amino acid sequence is selected from the group consisting of:

[0139] In one embodiment of the disclosure, the second and fourth peptide linkers are fused C-terminally to the amino acid sequence of DKTHTCPPCP (SEQ ID NO: 38). In one embodiment of the disclosure, the second and fourth peptide linkers additionally comprise the amino acid sequence of DKTHTCPPCP (SEQ ID NO: 38) at their C-terminus.

[0140] In one embodiment of the present disclosure, the second and fourth peptide linkers are: a) PKAAPDKTHTCPPCP (SEQ ID NO: 76); b) ASTKGPDKTHTCPPCP (SEQ ID NO: 77), and c) AQPAAPAPDAHEAPAPAQGSDKTHTCPPCP (SEQ ID NO: 78); d) PKAAPSVTLFPPSSEELQANDKTHTCPPCP (SEQ ID NO: 79); e) ASTKGPSVFPLAPSSKSTSGDKTHTCPPCP (SEQ ID NO: 80), The amino acid sequence is selected from the group consisting of:

[0141] In one aspect of the present disclosure, a) the first and third peptide linkers comprise the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:31), the second peptide linker comprises the amino acid sequence of PKAAP (SEQ ID NO:36), and the fourth peptide linker comprises the amino acid sequence of ASTKGP (SEQ ID NO:37); or b) the first and third peptide linkers comprise the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:31), and the second and fourth peptide linkers comprise the amino acid sequence AQPAAPAPDAHEAPAPAQGS (SEQ ID NO:33); or c) the first and third peptide linkers comprise the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 31), the second peptide linker comprises the amino acid sequence PKAAPSVTLFPPSSEELQAN (SEQ ID NO: 34), and the fourth peptide linker comprises the amino acid sequence ASTKGPSVFPLAPSSKSTSG (SEQ ID NO: 35); or d) the first and third peptide linkers comprise the amino acid sequence of AHPAAPAPAHPAAPAPAHGH (SEQ ID NO: 32), the second peptide linker comprises the amino acid sequence of PKAAPSVTLFPPSSEELQAN (SEQ ID NO: 34), and the fourth peptide linker comprises the amino acid sequence of ASTKGPSVFPLAPSSKSTSG (SEQ ID NO: 35); or e) the first and third peptide linkers comprise the amino acid sequence of AQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 33), the second peptide linker comprises the amino acid sequence of PKAAPSVTLFPPSSEELQAN (SEQ ID NO: 34), and the fourth peptide linker comprises the amino acid sequence of ASTKGPSVFPLAPSSKSTSG (SEQ ID NO: 35); or f) the first and third peptide linkers comprise the amino acid sequence GGSGGSGGS (SEQ ID NO: 30), the second peptide linker comprises the amino acid sequence PKAAPSVTLFPPSSEELQAN (SEQ ID NO: 34), and the fourth peptide linker comprises the amino acid sequence ASTKGPSVFPLAPSSKSTSG (SEQ ID NO: 35).

[0142] In one aspect of the present disclosure, a) the first and third peptide linkers comprise the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:31), the second peptide linker comprises the amino acid sequence of PKAAPDKTHTCPPCP (SEQ ID NO:76), and the fourth peptide linker comprises the amino acid sequence of ASTKGPDKTHTCPPCP (SEQ ID NO:77); or b) the first and third peptide linkers comprise the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:31), and the second and fourth peptide linkers comprise the amino acid sequence AQPAAPAPDAHEAPAPAQGSDKTHTCPPCP (SEQ ID NO:78); or c) the first and third peptide linkers comprise the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:31), the second peptide linker comprises the amino acid sequence PKAAPSVTLFPPSSEELQANDKTHTCPPCP (SEQ ID NO:79), and the fourth peptide linker comprises the amino acid sequence ASTKGPSVFPLAPSSKSTSGDKTHTCPPCP (SEQ ID NO:80); or d) the first and third peptide linkers comprise the amino acid sequence of AHPAAPAPAHPAAPAPAHGH (SEQ ID NO: 32), the second peptide linker comprises the amino acid sequence of PKAAPSVTLFPPSSEELQANDKTHTCPPCP (SEQ ID NO: 79), and the fourth peptide linker comprises the amino acid sequence of ASTKGPSVFPLAPSSKSTSGDKTHTCPPCP (SEQ ID NO: 80); or e) the first and third peptide linkers comprise the amino acid sequence of AQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 33), the second peptide linker comprises the amino acid sequence of PKAAPSVTLFPPSSEELQANDKTHTCPPCP (SEQ ID NO: 79), and the fourth peptide linker comprises the amino acid sequence of ASTKGPSVFPLAPSSKSTSGDKTHTCPPCP (SEQ ID NO: 80); or f) the first and third peptide linkers comprise the amino acid sequence GGSGGSGGS (SEQ ID NO: 30), the second peptide linker comprises the amino acid sequence PKAAPSVTLFPPSSEELQANDKTHTCPPCP (SEQ ID NO: 79), and the fourth peptide linker comprises the amino acid sequence ASTKGPSVFPLAPSSKSTSGDKTHTCPPCP (SEQ ID NO: 80).

[0143] In one embodiment of the present disclosure, the first and third peptide linkers comprise the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 31), and the second and fourth peptide linkers comprise the amino acid sequence AQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 33). In one embodiment of the present disclosure, the first and third peptide linkers comprise the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 31), and the second and fourth peptide linkers comprise the amino acid sequence AQPAAPAPDAHEAPAPAQGSDKTHTCPPCP (SEQ ID NO: 78).

[0144] In one embodiment of the present disclosure, the second Fv region specifically binds to CD3. In one embodiment, the second Fv is specific for CD3, particularly human CD3, more particularly human CD3 epsilon.

[0145] In one embodiment of the disclosure, the second Fv is i. Below (a) heavy chain complementarity determining region (HCDR) 1 comprising the amino acid sequence of GFSFGSHYMS (SEQ ID NO: 1); (b) an HCDR2 comprising the amino acid sequence of NINQIGYSSYYVESVKG (SEQ ID NO: 2), NINQIGYSSYYGESVKG (SEQ ID NO: 3), or NINQIGYSSYYEESVKG (SEQ ID NO: 4); and (c) an HCDR3 comprising the amino acid sequence of GYSAEFAHRSGLDV (SEQ ID NO: 5), GYSDEFATRSGLDV (SEQ ID NO: 6), GYSEEFAHRSGLDV (SEQ ID NO: 7), GYSDEFAKRSGLDV (SEQ ID NO: 8), or GYSDEFAHRSGLDV (SEQ ID NO: 9); A heavy chain variable region (VH) comprising: ii. Below (d) a light chain complementarity determining region (LCDR) 1 comprising the amino acid sequence of SGSSSNIGSNYVY (SEQ ID NO: 10); (e) an LCDR2 comprising the amino acid sequence of RNNQRPS (SEQ ID NO: 11); and (f) an LCDR3 comprising the amino acid sequence of AGWSRSLHGAV (SEQ ID NO: 12) or AGWSRELHGAV (SEQ ID NO: 13); A variable light chain region (VL) comprising Includes.

[0146] In one embodiment of the disclosure, the second Fv is i. a VH comprising the amino acid sequence of SEQ ID NO: 14 and a VL comprising the amino acid sequence of SEQ ID NO: 20; ii. VH comprising the amino acid sequence of SEQ ID NO: 15 and VL comprising the amino acid sequence of SEQ ID NO: 21; iii. VH comprising the amino acid sequence of SEQ ID NO: 16 and VL comprising the amino acid sequence of SEQ ID NO: 21; iv. VH comprising the amino acid sequence of SEQ ID NO: 17 and VL comprising the amino acid sequence of SEQ ID NO: 21; v. A VH comprising the amino acid sequence of SEQ ID NO: 18 and a VL comprising the amino acid sequence of SEQ ID NO: 21; and vi. VH comprising the amino acid sequence of SEQ ID NO: 19 and VL comprising the amino acid sequence of SEQ ID NO: 21; The VH and VL are selected from the group consisting of:

[0147] In one embodiment of the present disclosure, the light chain of the first Fab or the second Fab comprises the VL and CL of the first Fab or the second Fab, respectively. In one embodiment of the present disclosure, the light chain of the first Fab and the second Fab are identical. In one embodiment of the present disclosure, the heavy chain of the first Fab and the second Fab are identical. In one embodiment of the present disclosure, the first Fab and the second Fab are identical.

[0148] In one embodiment, the first and second Fc region subunits form an Fc region. In one embodiment, the Fc region is an IgG1 Fc region. In one embodiment, the IgG1 Fc region is a human IgG1 Fc region. In one embodiment, the Fc region comprises one or more amino acid modifications that facilitate association of the first and second Fc region subunits. In one embodiment, using EU index numbering, in the CH3 domain of the first Fc region subunit, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and the serine residue at position 354 is replaced with a cysteine ​​residue (S354C), and in the CH3 domain of the second Fc region subunit, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), the threonine residue at position 366 is replaced with a serine residue (T366S), the leucine residue at position 368 is replaced with an alanine residue (L368A), and the tyrosine residue at position 349 is replaced with a cysteine ​​residue (Y349C). In one embodiment of the present disclosure, the Fc region of a bispecific antibody of the present disclosure has reduced binding affinity to an Fc receptor and / or C1q and / or has reduced effector function. In one embodiment of the disclosure, the Fc region comprises one or more amino acid mutations in each Fc region subunit, said one or more amino acid mutations being selected from the group consisting of: L234A, L235E, G237A, A330S, and P331S, using the EU index numbering. In one embodiment, at least five amino acid residues in each Fc region subunit at positions corresponding to positions L234, L235, G237, A330, P331 in human IgG1, using the EU index numbering, are mutated to A, E, A, S, and S, respectively.

[0149] In one embodiment, the first antigen and the third antigen are identical. In one embodiment, the first and third antigens are tumor associated antigens. In one embodiment, the first and third antigens are tumor associated antigens expressed on tumor cells or cancerous cells. In one embodiment, the second antigen is an immune cell associated antigen. In one embodiment, the second antigen is expressed on an immune cell. In one embodiment, the second antigen is expressed on an immune effector cell. In one embodiment, the second antigen is expressed on a cytotoxic T cell. In one embodiment, the second antigen is CD3. In one embodiment, the second antigen is CD3 epsilon. In one embodiment, the second antigen is human CD3. In one embodiment, the second antigen is human CD3 epsilon. In one embodiment, a bispecific antibody according to the present disclosure provides bivalent binding to the first antigen and monovalent binding to the second antigen. In one embodiment, the bispecific antibody is a trivalent bispecific antibody.

[0150] efficacy In certain aspects, the present disclosure provides a bispecific antibody according to the present disclosure comprising a human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure, wherein the bispecific antibody mediates target cell killing of a target antigen-expressing cell. In one aspect, the target cell killing is mediated in the presence of cytotoxic T cells. In certain aspects, the bispecific antibody comprises a second antigen-binding fragment of an antibody that specifically binds to a cell surface target antigen. In certain aspects, the target antigen is a tumor-associated antigen. In one aspect, the target antigen is HER2. In one aspect, the second antigen-binding fragment of the antibody binds to HER2 (UniProtKB - P04626). In one aspect, the target cell is a tumor cell or a cancer cell. In one embodiment, the target cells are SKOV-3 (ATCC® HTB-77™), SKBR3 (ATCC® HTB-30™), or MCF-7 (ATCC® HTB-22™) cells. In one particular embodiment, target cell killing is determined by the methods described herein in Example 4.13.

[0151] treatment In one aspect, the present disclosure provides an isolated human antibody or antigen-binding fragment thereof or bispecific antibody specific for CD3 according to the present disclosure for use as a pharmaceutical agent. In one aspect, the present disclosure relates to an isolated human antibody or antigen-binding fragment thereof or bispecific antibody specific for CD3 according to the present disclosure for use in the preparation or manufacture of a pharmaceutical agent. In one aspect, the present disclosure provides an isolated human antibody or antigen-binding fragment thereof or bispecific antibody specific for CD3 according to the present disclosure for use in enhancing immune function in a subject with a cell proliferative disorder.

[0152] In one aspect, the present disclosure provides a method of treating or delaying the progression of a cell proliferative disorder in a subject in need thereof, comprising administering to the subject an effective amount of an isolated human antibody or antigen-binding fragment thereof or bispecific antibody specific for CD3 according to the present disclosure. In one aspect, the isolated human antibody or antigen-binding fragment thereof or bispecific antibody specific for CD3 according to the present disclosure is for use in treating or delaying the progression of a cell proliferative disorder in a subject in need thereof. In one aspect, the cell proliferative disorder is cancer.In one embodiment, the cancer includes, but is not limited to, the following: esophageal cancer, gastric cancer, small intestine cancer, colon cancer, colorectal cancer, breast cancer, non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), B-cell lymphoma, B-cell leukemia, multiple myeloma, renal cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), myeloma, leukemia ... AML), chronic lymphocytic leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom's macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt's lymphoma (BL), B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, unclassifiable splenic lymphoma / leukemia, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia variant, Waldenstrom's macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt's lymphoma (BL), B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, hairy cell leukemia variant, Waldenstrom's macroglobulinemia (WM), Trehm's macroglobulinemia, plasma cell myeloma, isolated plasmacytoma of bone, extraskeletal plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, pediatric follicular lymphoma, primary cutaneous follicle center lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, primary CNS DLBCL, primary cutaneous DLBCL leg type, EBV-positive DLBCL in the elderly, DLBCL with chronic inflammation, lymphomatoid granulomatosis, primary The present invention is selected from the group consisting of primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma occurring in HHV8-associated multicentric Castleman disease, primary effusion lymphoma: unclassifiable B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, and unclassifiable B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma.

[0153] In one aspect, the present disclosure provides the use of an isolated human antibody or antigen-binding fragment thereof, or bispecific antibody specific for CD3 according to the present disclosure for the manufacture of a pharmaceutical agent for treating or delaying progression of a cell proliferative disorder. In one aspect, the present disclosure provides the use of an isolated human antibody or antigen-binding fragment thereof, or bispecific antibody specific for CD3 according to the present disclosure for the manufacture of a pharmaceutical agent for enhancing immune function in a subject having a cell proliferative disorder. In one aspect, the present disclosure provides a method of treating a subject in need with an isolated human antibody or antigen-binding fragment thereof, or bispecific antibody specific for CD3 according to the present disclosure.

[0154] In one aspect, an isolated human antibody or antigen-binding fragment thereof, or bispecific antibody specific for CD3 according to the present disclosure, or a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof, or bispecific antibody, is administered subcutaneously, intravenously, intramuscularly, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.

[0155] composition In one aspect, the present disclosure provides a pharmaceutical composition comprising an isolated human antibody or antigen-binding fragment thereof or bispecific antibody specific for CD3 according to the present disclosure and a pharma- ceutically acceptable carrier or excipient. Such carriers or excipients are well known in the art, and the skilled artisan will find the optimal formulation and route of administration for treating a subject with an antibody or antigen-binding fragment thereof according to the present disclosure. In one aspect, the present disclosure relates to the use of a pharmaceutical composition comprising an isolated human antibody or antigen-binding fragment thereof or bispecific antibody specific for CD3 according to the present disclosure in the preparation of a medicament for the treatment of a disease. In one aspect, the present disclosure relates to the use of said pharmaceutical composition for the treatment of a disease. In one aspect, the present disclosure provides a method for treating a cell proliferative disease in a subject, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an isolated human antibody or antigen-binding fragment thereof or bispecific antibody specific for CD3 according to the present disclosure. In one aspect, the present disclosure provides a pharmaceutical composition comprising a combination of an isolated human antibody or antigen-binding fragment thereof or bispecific antibody specific for CD3 according to the present disclosure and a second therapeutic agent. In one aspect, the second therapeutic agent is any agent that is advantageously combined with the human antibody or antigen-binding fragment thereof or bispecific antibody according to the present disclosure. The present disclosure provides a method of treatment for stimulating T cell activation using an isolated human antibody or antigen-binding fragment thereof or bispecific antibody specific for CD3 according to the present disclosure, the method of treatment comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an isolated human antibody or antigen-binding fragment thereof or bispecific antibody specific for CD3 according to the present disclosure. The present disclosure also provides a method of treatment for redirecting T cell-mediated killing to cancerous cells or tissues using an isolated human antibody or antigen-binding fragment thereof or bispecific antibody specific for CD3 according to the present disclosure, the method of treatment comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof or bispecific antibody according to the present disclosure.

[0156] Production In another aspect, the disclosure relates to a method for producing an isolated human antibody or antigen-binding fragment thereof specific for CD3 of any of the antibodies listed in Tables 5-7.

[0157] The coding sequences for the heavy and light chains of the antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure can be recombinant DNA molecules that are introduced into an expression vector by operably linking the DNA to the necessary expression control regions (e.g., regulatory regions) required for gene expression. Those skilled in the art will appreciate that polynucleotides encoding the heavy or light chains can be cloned into different vectors or into the same vector. The vectors can be introduced into a suitable host cell, e.g., a prokaryotic cell (e.g., a bacterial cell) or a eukaryotic cell (e.g., a yeast or a mammalian cell), by methods well known in the art (see, e.g., "Current Protocols in Molecular Biology", Ausubel et al. (eds.), Greene Publishing Assoc. and John Wiley Interscience, New York, 1989 and 1992). Numerous cloning vectors are known to those skilled in the art, and the selection of an appropriate cloning vector is a matter of preference. The gene can be placed under the control of a promoter, a ribosome binding site (for bacterial expression), and optionally an operator (collectively referred to herein as "control" elements) such that the DNA sequence encoding the desired protein is transcribed into RNA in a host cell transformed with a vector containing this expression construct. The coding sequence may or may not contain a signal peptide or leader sequence. Upon expression in a host cell, an antibody or antigen-binding fragment thereof of the present disclosure is obtained. As will be appreciated by those skilled in the art, these steps can be accomplished in a variety of ways. In general, such steps typically include transforming or transfecting a suitable host cell with a nucleic acid or vector or infectious particle encoding the antibody molecule. Furthermore, such steps typically include culturing the host cell under conditions suitable for the proliferation (expansion, growth) of the host cell, the culturing step being under conditions suitable for the production (expression, synthesis) of the encoded antibody or antigen-binding fragment.Cultivation of host cells under suitable conditions for growth or expression is typically accomplished in the presence of a medium containing suitable components for cell growth or expression induction. In certain embodiments, the method for producing the antibody or antigen-binding fragment thereof of the present disclosure further comprises a step of isolating the produced antibody or antigen-binding fragment thereof from the host cells or medium. Depending on the expression system and host selected, the antibody or antigen-binding fragment thereof of the present disclosure is produced by growing a host cell transformed with the expression vector described above under conditions in which the protein of interest is expressed. The protein is then isolated from the host cells and purified. If the protein is secreted into the growth medium by the expression system, the protein can be purified directly from the medium. If the protein is not secreted, it is isolated from a cell lysate or recovered from a cell membrane fraction. The selection of appropriate growth conditions and recovery methods is within the skill of the art. The antibody or antigen-binding fragment thereof of the present disclosure can then be purified by several techniques known to those skilled in the art. It should be noted that the Fab of the present disclosure is not a naturally occurring protein. The present disclosure also provides recombinant expression vectors capable of expressing a polypeptide comprising a heavy or light chain variable region of an antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure. For example, the present disclosure includes recombinant expression vectors encoding any of the amino acid sequences listed in Tables 5-7. Also included within the scope of the present disclosure are host cells into which such vectors have been introduced, as well as methods for producing antibodies or portions thereof by culturing the host cells under conditions that permit the production of the antibodies or antigen-binding fragments thereof and recovering the antibodies and antigen-binding fragments thereof so produced.

[0158] Antigen sequence

[0159] [Table 2]

[0160] Antibody sequence

[0161] [Table 3]

[0162]

Table 4

[0163]

Table 5

[0164]

Table 6

[0165]

Table 7

[0166]

Table 8

[0167]

Table 9

[0168]

Table 10

[0169]

Table 11

[0170]

Table 12

[0171]

Table 13

[0172]

Table 14

[0173]

Table 15

[0174]

Table 16

[0175]

Table 17

[0176]

Table 18

[0177]

Table 19

[0178]

Table 20

[0179]

Table 21

[0180]

Table 22

[0181]

Table 23

[0182] [Table 24]

[0183] [Table 25]

[0184] [Table 26]

[0185] [Table 27] EXAMPLES

[0186] Working Example Example 1: Identification of affinity-optimized CD3-specific human antibodies The development of the grandparent human cynomolgus CD3 cross-reactive fully human antibody CD3-MABGP (comprising a VH of SEQ ID NO:25 and a VL of SEQ ID NO:26) is described in International Application PCT / EP2021 / 076052. GP To identify Fab fragments against human and cynomolgus CD3, the MorphoSys Ylanthia® library was used to select Fab fragments against human and cynomolgus CD3. The MorphoSys Ylanthia® library (Tiller et al. mAbs 5:3,1-26; May / June (2013) and U.S. Patent No. 8,728,981) is a commercially available phagemid library that utilizes CysDisplay® technology to display Fabs on the phage surface (Lohning et al., WO 2001 / 05950). To further increase affinity, species cross-reactivity, and biological activity, diversified Ylanthia® maturation modules previously generated using Slonomics® technology (van den Brulle et al. 2008) were used to select CD3-MABs. GPThe LCDR3 and HCDR1 / HCDR2 regions of CD3-MAB were optimized in parallel. GP For the selection of affinity-improved derivatives of , phages derived from the maturation library were subjected to three rounds of maturation panning. Panning stringency was increased by lowering the CD3 antigen concentration or the cell count of CD3-expressing cells in each panning round (Low et al. 1996). To further increase the selection stringency, in addition to antigen reduction, off-rate selection was performed using excess unbiotinylated CD3 epsilon antigen as competitor (Hawkins et al 1992). All strategies were combined with extended washing steps.

[0187] Example 1.1: SET affinity screening after affinity maturation For generation of Fab-containing crude bacterial lysates (BEL extracts) used for primary affinity screening, Fab-containing bacterial glycerol stocks were used to inoculate microtiter plates pre-filled with growth medium (2xYT containing chloramphenicol, IPTG, and low glucose). Plates were incubated at 37°C for bacterial growth and shaken overnight at 22°C for Fab expression. The next day, expression cultures were lysed by addition of BEL buffer containing borate buffer, EDTA, and lysozyme. Volumes were adjusted depending on selection plate format and application.

[0188] SET (Solution Equilibrium Titration) screening (Della Ducata et al. 2015) was performed in principle as described by Haenel et al. (Haenel et al. 2005). A fixed amount of diluted Fab-containing BEL extract was equilibrated overnight with various concentrations of CD3 epsilon antigen (hCD3e(22-118)_FLAG_chLys_avi (SEQ ID NO: 43)). The mixture was then transferred to an MSD plate pre-coated with the antigen and, after incubation and washing, a suitable MSD-Sulfo tagged detection antibody was added. The concentration of unbound Fab was subsequently quantified via ECL detection using a Sector Imager 6000 (Meso Scale Discovery | Gaithersburg | MD | USA). The clones that improved most by maturation were identified by applying the corresponding fitting model described above using XLfit (IDBS) software to process the results and estimate the affinity.

[0189] Approximately 1000 clones from the third panning round output were screened for affinity and 189 clones with a SET KD estimate of ≦40 nM against recombinant human CD3 epsilon antigen were subjected to VH or VL sequencing, as required. Sequencing resulted in the identification of 101 sequence-unique HCDR1+2 affinity matured derivatives, including CD3-MAB GP There were only 15 sequence-unique LCDR3 affinity matured derivatives.

[0190] Example 1.2: Conversion to a bispecific 2+1 Fab2-Fc-scFv antibody format and its production To determine the suitability of the sequence-unique affinity-optimized CD3-specific antibodies of Example 1.1 for use in a bispecific antibody format, a CD3-MAB of 48× sequence-unique HCDR1-3 and 3× sequence-unique LCDR3 derivatives with a SET KD estimate of ≦10 nM for human CD3 epsilon was GPThe VH and VL of the Fab2-Fc-scFv antibody were converted into a bispecific 2+1 Fab2-Fc-scFv antibody format shown in FIG. 1A. This bispecific antibody format was constructed from an aglycosylated human IgG1 backbone and contained one extra scFv fragment, with the N-terminus of the scFv VL domain fused to the C-terminus of one of the IgG heavy chains via a peptide linker. To promote heterodimerization of the two different heavy chains, knob-into-hole mutations were introduced into both CH3-Fc domains. In this example, both Fab arms of the IgG backbone bind to the tumor target HER2, while the extra scFv contains the variable domains of the CD3-specific antibody of the present disclosure. For HER2 binding, nucleotide sequences encoding the VH and VL domains from "Trastuzumab" (HERCEPTIN®) described by Baselga et al. 1998, Cancer Res 58(13):2825-2831 were used. Trastuzumab and its preparation method are described in U.S. Pat. No. 5,821,337. CD3-MAB made according to Example 1.2 opt_VL Exemplary bispecific antibody BissIg_08_#1 carrying affinity-improved VL domains of (SEQ ID NO:25 and SEQ ID NO:20) or CD3-MAB opt_VH#1 A summary of the polypeptide sequences forming an exemplary bispecific antibody BissIg_08_#2 carrying affinity-improved VH domains of (SEQ ID NO: 14 and SEQ ID NO: 26) is shown in Table 10.

[0191] All nucleic acid sequences or desired gene segments were either generated by PCR using appropriate templates or gene synthesized in-house or by an external provider as linear DNA fragments with appropriate flanking regions (e.g., suitable restriction enzyme recognition sites, linker sequences). The nucleic acid sequences or gene segments flanked by singular restriction endonuclease cleavage sites were cloned into the respective mammalian expression vectors using standard molecular biology methods. When intended for use in mammalian expression vectors, all constructs were designed with a 5'-end DNA sequence encoding a leader peptide that directs the protein for secretion in eukaryotic cells. The DNA sequences of the subcloned gene fragments were confirmed by double-stranded DNA sequencing. Eukaryotic HEK293-6E cells were transfected with mammalian expression vector DNA encoding all components of the heavy and light chains of the bispecific antibody to obtain a 2:1:1 heterodimeric bispecific antibody. Seven days after transfection, cell culture supernatants were harvested and subjected to Protein A affinity chromatography (MabSelect SURE | GE Healthcare) using a liquid handling station. Samples were left in neutral elution buffer (NaPS: 137 mM Na phosphate, 81 mM NaCl, pH 7). Samples were sterile filtered (0.2 μm pore size). Protein concentration was determined by UV spectrophotometry and antibody purity was analyzed under denaturing and reducing conditions using CE-SDS (LabChip GXII | Perkin Elmer | USA). HP-SEC was performed to analyze the bispecific antibody preparations in the native state.

[0192] In summary, 41 / 51 bispecific antibodies could be produced with acceptable quality and yield with a monomer content of >85% as determined by analytical size exclusion chromatography. Table 16 shows the results of the synthesis of the grandparent CD3 antibodies CD3-MABopt_VL and CD3-MABopt_VH#1 described above. GPFigure 1 shows a summary of the monomer content of seven produced bispecific antibody preparations containing the variable domains of either or six preferred affinity-optimized CD3 antibodies.

[0193] Affinity-optimized CD3-specific antibodies in a bispecific 2+1 Fab2-Fc-scFv antibody format; functional characterization of CD3-specific antibodies All produced bispecific antibodies of Example 1.2 have the following: · Affinity determination by surface plasmon resonance (SPR) Binding to human T cells and Jurkat cells with endogenous expression of CD3 and to J.RT-T3.5 cells without CD3 expression Functional NFAT reporter gene assay using the cancer cell line SKBR3 Functional cytotoxicity assay on SKBR3 cells The compounds were tested in in vitro assays including

[0194] Example 1.3: K via antibody capture setup D decision Kinetic characterization of the interaction between human CD3 epsilon and the 41 produced bispecific Fab2-Fc-scFv antibodies of Example 1.2 was performed in an antibody capture format, applying antigen as analyte in solution. A high-capacity capture surface was prepared by loading biotinylated MabSelect SuRe ligand (non-biotinylated ligand: GE Healthcare, 28-4018-60) onto several streptavidin sensors (fortebio, part 18-5021). Each cycle of the kinetic experiment consisted of a capture step (of one ligand on several sensors used in parallel), followed by an analyte binding step (association phase, various analyte concentrations, and assay buffer, i.e., antigen concentration 0 for blank subtraction). After binding, dissociation of bound antigen was monitored (exposing the sensors to assay buffer). At the end of each cycle, bound ligand and / or ligand-antigen complexes were removed from the sensor surface by two 20 s consecutive regeneration steps with 10 mM glycine / HCl pH 1.5 (GE Healthcare, BR 100354) while maintaining the integrity of the capture surface. The signal recorded on the sensor with the capture ligand exposed to assay buffer instead of antigen upon binding was subtracted from the sensorgrams at non-zero antigen concentrations, for example, to correct for potential dissociation of the capture ligand. Association was recorded for 300 s and dissociation was recorded for 300 s with an orbital shaking speed of 1000 rpm. DPBS (GIBCO, Ca) supplemented with 0.05% (v / v) polysorbate 20 (Merck, 8.22184.0500) and 0.1% (w / v) bovine serum albumin (Sigma, A7906) was used. 2+ None, Mg 2+None, Thermo Fisher Cat.No.14190) was used as assay buffer. The capture level of the ligand was adjusted to about 2 nM to achieve a saturation level Rmax of about 0.2 nM with the human CD3e analyte. During the kinetic experiments, seven different analyte concentrations were used for analysis (hCD3e(22-118)_F-chLys_avi (SEQ ID NO:43), applied molarities 15.6-1000 nM, 2-fold serial dilution series). Sensorgrams were evaluated with Data Analysis Software v 10 (Octet / fortebio). All sensorgrams were fitted to a 1:1 binding model to obtain k on and k off Determine the rate constant and use it to calculate K D For kinetic profiles that deviated from the expected 1:1 binding, the sensorgrams were evaluated using the best approximation to monovalent kinetics and the comment "heterogeneous binding" was annotated. These results are considered less accurate than kinetic profiles that perfectly follow the expected monovalent binding kinetics, but the K D is assumed to be a good approximation of

[0195] Overall, the monovalent affinities determined for all 41 tested affinity-optimized CD3-specific antibodies on the human CD3e antigen were in the range of 39 nM to 120 nM, whereas the grandparent antibody CD3-MAB GP showed a K of 240 nM when tested in the same bispecific antibody format. D Table 16 shows the results of CD3-MABopt_VL (BissIg_08_#1) and CD3-MABopt_VH#1 (BissIg_08_#2) GP and summarizes the results of kinetic studies determined with six preferred affinity optimized CD3 antibodies of the present disclosure.

[0196] Example 1.4: Cell binding The 41 produced bispecific Fab2-Fc-scFv antibodies of Example 1.2, including affinity-improved CD3-specific antibodies, were tested for their binding ability to CD3 positive human T cells and to the CD3 negative cell line J.RT3-T3.5.

[0197] For the isolation and purification of human T cells, human whole blood from healthy donors was collected in Li-heparin-containing S-Monovette vessels (Sarstedt). Blood was transferred to a 50 ml conical tube and mixed with an equal volume of PBS containing 2% fetal bovine serum (Sigma, #F7524) and 2 mM EDTA. The diluted blood was transferred to a SepMate-50 tube (StemCell Technologies#86450) containing 15 ml Biocoll solution (Biochrom#L6115) and centrifuged at 1200×g for 10 min. The supernatant was transferred to a 50 ml conical tube, diluted to 45 ml with PBS, and centrifuged at 300×g for 8 min. The supernatant was discarded and the cell pellet was resuspended in 1 ml PBS and the cells were counted using a Neubauer chamber. T cells were isolated and purified using the EasySep™ Human T Cell Isolation Kit (StemCell Technologies) according to the provider's instructions. CD3+ T cell purity assessment was performed by flow cytometry using anti-human CD3 PE-conjugated antibody (Biolegend #12-0037-42). Jurkat and J.RT3-T3.5 cells were resuspended and counted in Superblock (ThermoScientific, #37515) and blocked for 1 h on ice. Blocked cells were resuspended in Superblock with serially diluted bispecific antibodies (starting final concentrations: 500 nM to 0.69 nM / 0.23 nM, 1:3 serial dilutions) and incubated for 1 h on ice. Cells were washed twice in D-PBS (Gibco) containing 3% fetal bovine serum (Sigma, #F7524). Bound bispecific antibodies were detected using AlexaFluor 647-labeled goat anti-human IgG (F(ab')2 fragment specific) (Jackson Immuno Research Cat#109-606-097). Antibody staining was measured using a FACS array (Beckton Dickinson) or IntelliCyt iQue flow cytometer and analyzed with FlowJo or ForeCyt (IntelliCyt) software, respectively. ECs were calculated using four-parameter nonlinear regression analysis in Prism software (GraphPad Software Inc.).50 values ​​were calculated.

[0198] Table 16 shows the CD3-MAB opt_VL (BissIg_08_#1) and CD3-MAB opt_VH#1 (BissIg_08_#2) antibodies. GP FIG. 1 summarizes the results of cell binding studies determined with bispecific antibodies according to Example 1.2, which comprise the variable domains of the six preferred affinity-optimized CD3 antibodies according to the present disclosure. Cell binding is shown as the signal / background ratio at a selected antibody concentration of 167 nM. The affinity-improved CD3-specific antibodies are the grandparent antibody CD3-MAB GP The antibody showed significantly stronger signal intensity on human T cells when compared to that of the CD3 negative cell line J.RT3-T3.5. Furthermore, no or only very weak binding was observable to the CD3 negative cell line J.RT3-T3.5 (data not shown).

[0199] Example 1.5 Jurkat NFAT Reporter Gene Cellular Assay To evaluate the functional activity of the bispecific Fab2-Fc-scFv antibodies according to Example 1.2, Jurkat cells (ATCC #TIB-152) transiently transfected with an NFAT reporter gene construct were used as surrogate effector cells. As target cells, the HER2-positive human adenocarcinoma SKBR-3 (ATCC® HTB-30™) cell line was used. For the maintenance of the cell lines, the following growth media were used: (a) Jurkat: RPMI-1640+L-glutamine (Thermo Fisher, #21875-034) supplemented with 10% FCS (Sigma, #F7524), (b) SKBR-3: McCoy's 5a (Gibco, #26600) supplemented with 10% FCS (Sigma #F7524). SKBR-3 cells were diluted in growth medium to a density of 4E+05 cells / ml. 100 μl cell suspension corresponding to 40,000 cells was seeded into each well of a tissue culture treated 96 well plate (Corning, #3917) and incubated overnight at 37° C. and 5% CO2 in a humidified incubator. Jurkat cells were resuspended in growth medium to a concentration of 2.5E+05 cells / ml. Transfection components pGL4.30[luc2P / NFAT-RE / Hygro] reporter gene vector (Promega #9PIE848), OptiMEM-I medium (Life Technologies, #31985-047), and TransIT-LT1 transfection reagent (Mirus, #MIR2304) were incubated at RT for 15 min and then added to the Jurkat cell suspension and incubated at 37° C. and 5% CO2 in a humidified incubator for 17 h. Jurkat cells were harvested and resuspended in growth medium at a concentration of 1.2E+06 / ml. Medium was removed from coated target cells and replaced with 50 μl Jurkat cell suspension corresponding to 60,000 cells / well. Bispecific antibodies were serially diluted in Jurkat growth medium. 50 μl antibody dilutions were added to each well to give a final concentration range of 10 nM to 0.01 nM (4 step dilutions). Assay plates were incubated for 5 h at 37°C and 5% CO2 in a humidified incubator.Bright-Glo™ Reagent (Promega, #E2620) was reconstituted according to the manufacturer's instructions. The assay plate and reagents were equilibrated at room temperature. 100 μl of Bright-Glo™ Reagent was added to each well of the assay plate and mixed. Luminescence was measured using an Infinite M1000 Pro plate reader (Tecan).

[0200] Table 16 shows the CD3-MAB opt_VL (BissIg_08_#1) and CD3-MAB opt_VH#1 (BissIg_08_#2) antibodies. GP FIG. 1 summarizes the results of reporter gene assays determined with bispecific antibodies comprising variable domains of six preferred affinity-optimized CD3 antibodies according to the present disclosure. Results are shown as signal / background ratios at a bispecific antibody concentration of 1 nM. Bispecific antibodies comprising affinity-optimized CD3-specific antibodies showed little activity compared to the grandparent CD3-specific antibody CD3-MAB. GP The results showed a significant and stronger activation of the reporter gene system in Jurkat cells when compared to a bispecific antibody containing the variable domains of

[0201] Example 1.6: Cytotoxicity assay with bispecific antibodies. Forty-one bispecific Fab2-Fc-scFv antibodies, including affinity-optimized CD3-specific antibodies according to Example 1.2, were tested for their ability to mediate T-cell-dependent killing of HER2-expressing SKBR3 cells or HER2-negative MDA-MB468 cells. PBMCs were prepared as described in Example 1.4. 5,000 SKBR3 or MDA-MB468 cells were suspended in culture medium (SKBR3: McCoy's 5A medium (Gibco, #26600), 10% FCS (Sigma, #F7524); MDA-MB468: DMEM (Gibco, #10938), GlutaMax (Gibco, #35050), 10% FCS, 1x sodium pyruvate (Gibco, #11360-039)) and seeded into black 96-well assay plates (Corning, #3340) and incubated overnight at 37°C and 5% CO2. CellToxGreen dye (Promega, #G8731), serially diluted bispecific antibody constructs (final concentrations: 5 nM-100 pM), and 100,000 purified PBMCs, all diluted in assay medium containing RPMI 1640 w / o phenol red (Gibco, #32404-014), GlutaMax, and 10% fetal bovine serum, were added to the cells and incubated for 48 h at 37 °C and 5% CO2. After 72 h, cytotoxic activity was assessed by measuring the fluorescence of incorporated CellToxGreen at 485 nm excitation and 535 nm emission using a Tecan Infinite F500 device. EC 50 values ​​were calculated.

[0202] Overall, only the 24 tested bispecific antibodies containing the affinity-optimized CD3-specific variable domains of Example 1.2 mediated T cell-mediated killing of SKBR3 cells. Consistent with the findings of the RGA assay of Example 1.5, the grandparent antibody CD3-MAB GPNo killing was observed with bispecific antibodies containing the variable domains of BissIg_08_#1 and BissIg_08_#2. GP or the results of T cell-mediated redirected killing of SKBR3 cells determined with bispecific antibodies comprising the variable domains of the six preferred affinity-optimized CD3 antibodies of the present disclosure (IC 50 This is a summary of the concentrations of

[0203] Example 1.7: ELISA Binding The bispecific Fab2-Fc-scFv antibodies according to example 1.2 were tested by ELISA for their binding ability to recombinant human and cynomolgus CD3 epsilon antigens. 5 nM of recombinant human CD3e(22-49)-Fc2(K105-K330) (SEQ ID NO: 41) or 1 nM of recombinant cynomolgus CD3e(22-49)-Fc2(K105-K330) (SEQ ID NO: 42) were coated on Maxisorp plates (Nunc, #460518). The coated plates were blocked with 5% skim milk in PBS. The antibodies were serially diluted in PBS containing 0.5% skim milk and 0.5% Tween-20. Bound antibodies were detected using an alkaline phosphatase-conjugated detection antibody against human F(ab')2 fragment (Jackson Immuno Research, #109-055-097). EC was calculated using four-parameter nonlinear regression analysis in Prism software (GrapPad Software Inc.). 50 values ​​were calculated.

[0204] Table 15 shows the CD3-MAB opt_VL (BissIg_08_#1) and CD3-MAB opt_VH#1 (BissIg_08_#2) antibodies. GP and ELISA EC determined with bispecific antibodies comprising variable domains of six preferred affinity optimized CD3 antibodies according to the present disclosure. 50 The results are summarized below. Affinity-optimized CD3-specific antibodies were compared with the grandparent antibody CD3-MAB GPThe results show that the antibody exhibited 10-20 fold improved binding to human and cynomolgus CD3 epsilon antigens when compared to the antibody.

[0205] [Table 28]

[0206] Summary functional characterization of affinity-improved antibodies Grandparent antibody CD3-MAB GP Affinity maturation of the CD3-MAB with favorable properties and suitability for use in a bispecific antibody format was performed. GP Five affinity-optimized HCDR1-2 variants and one affinity-optimized LCDR3 variant were identified.

[0207] Table 16 summarizes the advantageous functional and biophysical properties of these antibodies compared to the grandparent antibodies when tested in the bispecific antibody format of Example 1.

[0208] [Table 29]

[0209] Example 2: Further optimization of affinity-improved CD3-specific antibodies - Cross-cloning of affinity-optimized variable domains and conversion to further bispecific antibody formats To further improve the functional properties of the affinity-optimized CD3-specific antibodies already identified, five crossclones were generated by combining the VHs of CD3-MABopt_VH#1 (sequence number 14), CD3-MABopt_VH#2, CD3-MABopt_VH#3, CD3-MABopt_VH#4, and CD3-MABopt_VH#5 with the VL of CD3-MABopt_VL (sequence number 20).

[0210] Subsequently, the affinity-improved antibodies CD3-MABopt_VH#1, CD3-MABopt_VH#2, CD3-MABopt_VH#3, CD3-MABopt_VH#4, CD3-MABopt_VH#5, and CD3-MABopt_VL, as well as the five crossclones generated from them, were converted into a bispecific 1+1 antibody format as shown in FIG. 1B. The bispecific antibody format of Example 2 has the typical Y-shape of a conventional IgG molecule, with one Fab arm binding to the tumor target (HER2) and the other Fab arm binding to CD3. For HER2 binding, the VH and VL domains (SEQ ID NO: 28 and SEQ ID NO: 29, respectively) of "Trastuzumab" (HERCEPTIN®) described by Baselga et al. 1998, Cancer Res 58(13):2825-2831 were used. Trastuzumab and methods for its preparation are described in US Pat. No. 5,821,337.

[0211] Bispecific 1+1 antibodies were generated in vitro by 2-MEA-induced Fab arm exchange as described in WO 2011 / 147986, WO 2011 / 131746, and WO 2013 / 060867, and by Labrijn et al. (Labrijn et al., PNAS 2013, 110:5145-50; Gramer et al., MAbs 2013, 5:962-973). To allow the production of bispecific antibodies by this method, two source IgG1 molecules carrying mutations in the CH3 domain were generated: one source IgG1 antibody carrying the F405L mutation (i.e., CD3-specific antibody), the other source IgG1 antibody carrying the K409R mutation (i.e., anti-HER2 antibody). In addition to these mutations, the source IgG1 molecule contained substitutions: L234A, L235E, G237A, A330S, and P331S ("AEASS") that result in an Fc region that is unable to interact with the IgG Fc receptor (Fc gamma receptor) and complement.

[0212] An overview of the polypeptide sequences used according to Example 2 forming two exemplary source IgG1 molecules with specificity for HER2 (IgG#1: SEQ ID NOs: 53 and 51) or CD3 (IgGopt_cc: SEQ ID NOs: 54 and 55), respectively, is given in Table 11. The CD3-specific IgG comprises the VH and VL of the cross-clone CD3-MABopt-cc (SEQ ID NOs: 14 and 20, respectively). An overview of the polypeptide sequences forming the resulting bispecific 1+1 antibody BissIg_18_opt_cc# (SEQ ID NOs: 53, 51, 54, 55) (after Fab arm exchange) is given in Table 12.

[0213] For regular production of source IgG1 molecules, eukaryotic HEK293-6E cells were transfected with mammalian expression vector DNA encoding the heavy and light chains of the source IgG molecules. Cell culture supernatants were harvested 3 or 6 days after transfection and subjected to standard Protein A affinity chromatography (MabSelect SURE | GE Healthcare). Buffer exchange into 1x Dulbecco's PBS (pH 7.2 | Invitrogen) was performed and samples were sterile filtered (0.2 μm pore size). Protein concentration was determined by UV spectrophotometry and IgG purity was analyzed under denaturing reducing and non-reducing conditions using CE-SDS (LabChip GXII | Perkin Elmer | USA). HP-SEC was performed to analyze IgG preparations in native conditions.

[0214] To generate bispecific 1+1 antibodies via Fab arm exchange, PBS buffer (phosphate buffered saline, 8.7 mM HPO4 2- , 1.8 mM HPO 4-The two source IgGs were mixed in equal masses in 100 mM Na+, 163.9 mM Na+, 140.3 mM Cl-, pH 7.4). 2-Mercaptoethylamine-HCl (2-MEA) was added to a final concentration of 75 mM and the reaction mixture was incubated at room temperature for 5 h. 2-MEA was removed by buffer exchange into PBS using a PD-10 column to allow reoxidation of the interchain disulfide bonds and formation of intact bispecific antibodies. Protein concentrations were determined by UV spectrophotometry and purity of the bispecific IgG preparations was analyzed under denaturing reducing and non-reducing conditions using CE-SDS (LabChip GXII | Perkin Elmer | USA). HP-SEC was performed to analyze the bispecific IgG preparations in native conditions.

[0215] Table 17-1 summarizes the quality control of mammalian source IgG1 molecules of affinity improved CD3 specific antibodies CD3-MABopt_VH#1, CD3-MABopt_VH#2, CD3-MABopt_VH#3, CD3-MABopt_VH#4, CD3-MABopt_VH#5, and CD3-MABopt_VL according to Example 1 before controlled Fab arm exchange (FAE). Table 17-2 summarizes the quality control of the corresponding cross-cloned CD3 specific antibodies CD3-MABopt_VH#1, CD3-MABopt_VH#2, CD3-MABopt_VH#3, CD3-MABopt_VH#4, CD3-MABopt_VH#5, and CD3-MABopt_VL according to Example 2 before controlled Fab arm exchange (FAE). opt_cc , CD3-MAB opt_cc#2 , CD3-MAB opt_cc#3 , CD3-MAB opt_cc#4 and CD3-MAB opt_cc#5 This paper summarizes the quality control of mammalian-sourced IgG1 molecules.

[0216] Overall, IgG preparations containing cross-cloned CD3-specific antibody variable domains were found to have a lower monomer content when compared to IgG containing affinity-improved single CD3-specific antibody variable domains.

[0217] [Table 30]

[0218] [Table 31]

[0219] Table 18 summarizes the quality control of bispecific IgG preparations after controlled Fab arm exchange (FAE) containing cross-clonal CD3 specific binding domains. Overall, bispecific antibody preparations showed higher monomer content when compared to the corresponding source monospecific IgG preparations.

[0220] [Table 32]

[0221] Example 2.1: K via antibody capture setup D decision Affinity determinations were performed by determining kinetic rate constants on an Octet HTX (ForteBIO, Sartorius AG) instrument. Assay buffer (DPBS (GIBCO, Ca) supplemented with 0.05% (v / v) polysorbate 20 (Merck, 8.22184.0500) and 0.1% (w / v) bovine serum albumin (Sigma, A7906)) was used. 2+ None, Mg 2+The bispecific antibody preparation of Example 2 diluted in 100 mM NaCl, Thermo Fisher Cat. No. 14190) was captured on the IgG-specific BLI sensor at a loading level of approximately 2 nm. For analysis, human CD3 epsilon antigen hCD3e(22-118)_F-chLys_avi-biotin (SEQ ID NO: 43) was diluted in assay buffer to concentrations ranging from 7.8 nM to 500 nM (stepwise 1:2 dilutions). A blank sample of assay buffer was included to reference, i.e., to correct for dissociation of the capture antibody. The association phase was recorded for 300 s, followed by a dissociation phase of 180 s. After each cycle, the biosensor was regenerated twice with 10 mM glycine HCl pH 1.7 to remove bound ligand / antibody complexes while maintaining the integrity of the capture surface. Between regeneration steps, the biosensor was washed with assay buffer for 20 s. Sensorgrams were evaluated with Data Analysis Software v 10 (Octet / fortebio). All sensorgrams were fitted to a 1:1 binding model to obtain k on and k off Determine the rate constant and use it to calculate K D For kinetic profiles that deviated from the expected 1:1 binding, the sensorgrams were evaluated using the best approximation to monovalent kinetics and the comment "heterogeneous binding" was annotated. These results are considered less accurate than kinetic profiles that perfectly follow the expected monovalent binding kinetics, but the K D is assumed to be a good approximation of

[0222] Table 19 summarizes the KD values ​​of bispecific antibodies comprising affinity optimized or cross-cloned CD3-specific binding domains according to the present disclosure. The optimized or cross-cloned CD3-specific antibodies have KD values ​​in the low single digit nanomolar range, except for the HCDR1-2 matured antibody CD3-MABopt_VH#1 (SEQ ID NO: 14 and SEQ ID NO: 26) and the LCDR3 matured antibody CD3-MABopt_VL (SEQ ID NO: 25 and SEQ ID NO: 20). DThe two antibodies recognized the recombinant human CD3 epsilon antigen with a 100% binding affinity to CD3e in the low double-digit nanomolar range. Surprisingly, the generated cross clone CD3-MABopt_cc (SEQ ID NO: 14 and SEQ ID NO: 20), constructed from the VH of CD3-MABopt_VH#1 and the VL of CD3-MABopt_VL, was found to exhibit approximately 20-30 fold improved binding affinity to CD3e when compared to its parent antibody.

[0223] [Table 33]

[0224] Example 2.2: Cell Binding The bispecific antibodies of Example 2, including affinity-improved or cross-cloned CD3-specific antibodies according to the present disclosure, were tested for their ability to bind to Jurkat (CD3+) and J.RT3-T3.5 (CD3-) cells. Target cells were mixed with serial dilutions (final concentrations: 0.1 nM to 200 nM) of bispecific antibodies in D-PBS (Gibco) containing 3% fetal bovine serum (Sigma, #F7524) and incubated on ice for 1 h. Cells were washed twice in D-PBS (Gibco) containing 3% fetal bovine serum (Sigma, #F7524) and 0.02% sodium acid. Bispecific antibodies were detected using AlexaFluor 647-labeled goat anti-human IgG (F(ab')2 fragment specific) (Jackson Immuno Research Cat#109-606-097). Antibody staining was measured using a FACS array (Beckton Dickinson) or IntelliCyt iQue flow cytometer and analyzed with FlowJo or ForeCyt (IntelliCyt) software, respectively. ECs were calculated using four-parameter nonlinear regression analysis in Prism software (GraphPad Software Inc.). 50 values ​​were calculated.

[0225] Table 20 and Figure 3 summarize the cell binding of the test bispecific antibody preparations comprising affinity matured or cross-cloned CD3-specific antibody variable domains according to the present disclosure. Overall, the CD3-specific cross-clones were found to have stronger binding to Jurkat cells when compared to their affinity-improved single counterparts. In particular, the cross-clones CD3-MABopt_cc (SEQ ID NO: 14 and SEQ ID NO: 20) exhibited the strongest binding to Jurkat cells. Binding to the CD3-negative cell line J.RT3-T3.5 was observable for the two affinity-improved CD3-specific antibodies (CD3-MABopt_VH#2 and CD3-MABopt_VH#5) and their two cross-cloned counterparts (CD3-MABopt_cc#2 and CD3- CD3-MABopt_cc#5).

[0226] [Table 34]

[0227] Example 2.3 Jurkat NFAT Reporter Gene Cellular Assay To assess the functional activity of the bispecific antibodies of Example 2, Jurkat cells (ATCC #TIB-152) transiently transfected with an NFAT reporter gene construct were used as surrogate effector cells. SKOV-3 (ATCC® HTB-77™) and MCF-7 (ATCC® HTB-22™) were used as HER2-positive target tumor cell lines. The assay was performed essentially as described in Example 1.5. For maintenance of cell lines, the following growth media were used: Jurkat: RPMI-1640 + L-glutamine (Thermo Fisher, #21875-034) supplemented with 10% FCS (Sigma, #F7524), SKOV-3: McCoy's 5a (Gibco, #26600) supplemented with 10% FCS (Sigma #F7524), MCF-7: DMEM (Gibco # 10938) supplemented with + 10% FCS (Sigma #F7524) + 1x Glutamax (Gibco #35050-061) + 1x Sodium Pyruvate (Gibco #11360-039). SKBR-3 cells were diluted to a density of 4E+05 cells / ml in growth medium. SKOV-3 and MCF-7 cells were diluted to a density of 4E+05 cells / ml in growth medium. 100 μl cell suspension corresponding to 40,000 cells was seeded into each well of a tissue culture treated 96 well plate (Corning, #3917) and incubated overnight at 37° C. and 5% CO2 in a humidified incubator. Jurkat cells were resuspended in growth medium to a concentration of 2.5E+05 cells / ml. Transfection components pGL4.30[luc2P / NFAT-RE / Hygro] reporter gene vector (Promega #9PIE848), OptiMEM-I medium (Life Technologies, #31985-047), and TransIT-LT1 transfection reagent (Mirus, #MIR2304) were incubated at RT for 15 min and then added to the Jurkat cell suspension and incubated at 37° C. and 5% CO2 in a humidified incubator for 17 h. Jurkat cells were harvested and resuspended in growth medium at a concentration of 1.2E+06 / ml.The medium was removed from the coated target cells and replaced with 50 μl Jurkat cell suspension corresponding to 60,000 cells / well. Bispecific antibodies were serially diluted in Jurkat growth medium. 50 μl antibody dilutions were added to each well to give a final concentration range of 50 nM to 0.01 nM (4 step dilutions). The assay plate was incubated for 5 h at 37°C and 5% CO2 in a humidified incubator. Bright-Glo™ Reagent (Promega, #E2620) was reconstituted according to the manufacturer's instructions. The assay plate and reagents were equilibrated at room temperature. 100 μl Bright-Glo™ Reagent was added to each well of the assay plate and mixed. Luminescence was measured using an InfiniteM1000 Pro plate reader (Tecan).

[0228] Table 21 summarizes the activation of the Jurkat cell / NFAT reporter system mediated by the bispecific antibodies of the present disclosure. Again, bispecific antibody BissIg_18_opt_cc#, which includes cross-clonal antibody CD3-MABopt_cc (SEQ ID NO: 14 and SEQ ID NO: 20), had an EC of less than 0.2 nM in the presence of both SKOV-3 and MCF-7 cells. 50 This value represented the strongest activation of the Jurkat cell / NFAT reporter system.

[0229] [Table 35]

[0230] Example 2.4: Cytotoxicity assay with bispecific antibodies. The bispecific antibodies according to Example 2 were tested for their ability to mediate T cell-dependent killing of the HER2-expressing tumor cell lines SKBR3, MCF-7, and the HER2-negative cell line MDA-MB-468. The assays were performed as described in Example 1.6. As effector cells, either human PBMC (for SKBR3) cells or purified human T cells (for MCF-7 cells) were used. Preparation and purification of human PBMC and human T cells were performed as described in Example 1.4. 5000 SKBR-3, MDA-MB-468, or MCF-7 cells were seeded in black 96-well assay plates (Corning #3340) and incubated overnight at 37° C. and 5% CO2. CellToxGreen dye (Promega, #G8731), serially diluted bispecific antibody constructs, and 100.000 purified PBMCs for SKBR-3 cells (target / effector ratio 1:20) or 50.000 purified T cells for MCF-7 cells (target / effector ratio 1:10) were all diluted in assay medium and added to the cells and incubated for 72 h at 37°C and 5% CO2. After 72 h, cytotoxic activity was assessed by measuring the fluorescence of incorporated CellToxGreen at 485 nm excitation and 535 nm emission using a Tecan Infinite F500 device. EC was calculated using a four-parameter nonlinear regression analysis in Prism software (GraphPad Software Inc.). 50 values ​​were calculated.

[0231] Table 22 and Figure 4 (one donor) summarize the T cell-mediated redirected killing of cancer cell lines mediated by the bispecific antibodies of the present disclosure. Bispecific antibodies comprising cross-clonal CD3-specific antibodies clearly demonstrated superior killing of both SKBR3 and MCF-7 cells. Among these, bispecific antibody BissIg_18_opt_cc# comprising cross-clonal antibody CD3-MABopt_cc (SEQ ID NO: 14 and SEQ ID NO: 20) exhibited the best killing activity of both cancer cell lines. Weak cell killing of HER2-negative cell line MDA-MB-468 was observable with cross-clonal CD3-MABopt_cc#4 at higher tested antibody concentrations (data not shown).

[0232] [Table 36]

[0233] Example 2.5: T cell activation in the absence of target cancer cells (high density PBMC assay) The bispecific antibody of Example 2 was tested for its ability to activate human T cells derived from human blood samples of three different donors in the absence of target cancer cells. The assay was performed under the high PBMC density pre-culture conditions proposed by Roemer et al. (Roemer et al., BLOOD, 22 DECEMBER 2011, VOLUME 118, NUMBER 26, PAGE 6772-6781). Human PBMC were prepared and purified as described above (see Example 1.4). T cells were resuspended at a density of 1E+07 cells / mL in RPMI 1640 medium (Gibco, #31870-025) supplemented with GlutaMax (Gibco, #35050-038), non-essential amino acids (Gibco, #11140-035), HEPES buffer solution (Gibco #15630-056), sodium pyruvate (Gibco, #11360-039), β-mercaptoethanol, penicillin / streptomycin (Gibco #15140-122), and human serum (Sigma, #H4522) and incubated at 37°C and 5% CO2 for 48 h. After high-density preincubation, 200,000 PBMCs in medium were mixed with equal volumes of antibodies serially diluted in medium (final concentrations: 1000 nM, 200 nM, and 40 nM) and incubated for 24 h at 37°C and 5% CO2. As a positive control, a commercially available murine IgG antibody OKT3 was used. T cell activation was assessed by evaluation of upregulation of CD69 expression on CD3-positive lymphocytes. For this, PBMCs were stained with antibodies against CD3 and CD69 conjugated to BV / PE and APC, respectively (Biolegend, #300434, #12003742, #310910). Antibody staining was measured using a NovoCyte 3000 flow cytometer (Acea Biosciences, Inc.) and analyzed using FlowJo software.

[0234] Figure 5 shows the average exemplary results of a T cell activation experiment with human CD8+ T cells obtained from three donors. As expected, a strong upregulation of CD69 expression was observable with the positive murine control IgG OKT-3. Donor-dependent activation of CD8+ T cells was observable with all tested bispecific antibodies, including affinity-optimized or cross-cloned CD3-specific antibodies. Of these, bispecific antibodies including either CD3-MABopt_VH#1 (SEQ ID NO: 14 and SEQ ID NO: 26) or its cross-cloned counterpart CD3-MABopt_cc (SEQ ID NO: 14 and SEQ ID NO: 20) exhibited the lowest level of human T cell activation in the absence of the target cancer cell line.

[0235] Example 2.6: Overview of affinity improved and cross-cloned CD3-specific antibodies. The strong affinity improved cross clone CD3-MABopt_cc (SEQ ID NO: 14 and SEQ ID NO: 20) is a cross clone of the grandparent antibody CD3-MAB GP It was identified as the most potent and safest CD3-specific antibody derived from our affinity maturation campaign.

[0236] CD3-MABopt_cc showed favorable binding to human and cynomolgus CD3 and mediated excellent cytotoxic activity against HER2 low, medium, and high expressing cancer cell lines, did not kill any HER2 negative cancer cell lines, and only low levels of T cell activation in the absence of target cells when tested in the bispecific antibody format of Example 2.

[0237] Example 3: Conversion of the optimized CD3-specific antibody CD3-MABopt-cc to an improved 2+1 Fab2-Fv-Fc antibody format Example 3.1: Preparation, production, and characterization of linker-optimized trivalent bispecific antibodies. Bispecific Fab2-Fv-Fc antibodies were generated in vitro using the bispecific antibody platform technology described in WO 2020 / 115115, which is incorporated herein in its entirety. This bispecific antibody format is constructed from an aglycosylated monoclonal human IgG1 antibody scaffold incorporating the Fc region of a regular human IgG1 molecule and one extra antibody Fv fragment inserted between the two Fab arms. The basic structure of such a bispecific antibody is provided in FIG. 2. Such a format offers the advantage of bivalent binding to target cell surface antigens such as tumor-associated antigens but monovalent binding to CD3 expressed on T cells. This format also allows for a short distance between the target cell and the cytotoxic T cell once bridged via the bispecific antibody. This narrow immunological synapse results in efficient killing of the target cell by the recruited cytotoxic T cell.

[0238] Example 3.2: Linker optimization The selection of the correct peptide linker (in terms of amino acid sequence and length) connecting the individual components of the bispecific antibody format is particularly important since such linker determines the flexibility of the extra Fv fragment and its distance from the binding region of each of the two Fab arms of the bispecific antibody. To further optimize the peptide linkers previously disclosed in WO 2020 / 115115, elongation and sequence optimization of the respective linkers was carried out.

[0239] N-terminal linker of CD3-specific Fv fragment Fusion of the C-terminus of each Fab heavy chain to the N-terminus of either the VH or VL of the incorporated CD3 specific Fv fragment was achieved using the following peptide linker: a) 9mer glycine-serine linker (GGS)3: GGSGGSGGS (SEQ ID NO: 30) b) 20mer (G4S)4 linker: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 31) c) 20mer PAPDA linker: AQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 33), or d) 20mer PAH linker: AHPAAPAPAHPAAPAPAHGH (SEQ ID NO: 32) This was achieved by using one of the following:

[0240] C-terminal linker of CD3-specific Fv fragment Fusion of the C-terminus of each of the VH and VL domains of the CD3-specific Fv fragment to the N-terminus of either of the two Fc region subunits can be achieved using the following peptide linkers: i) when the VL domain of a CD3-specific Fv fragment is fused to one of the two Fc region subunits, a) The first five amino acid residues of the CL λ constant domain: PKAAP (SEQ ID NO: 36) b) 20mer PAPDA linker: AQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 33), or c) the first 20 amino acid residues of the CLλ constant domain: PKAAPSVTLFPPSSEELQAN (SEQ ID NO: 34), or ii) when the VH domain of a CD3-specific Fv fragment is fused to one of the two Fc region subunits, a) the first 6 amino acid residues of the CH1 constant domain: ASTKGP (SEQ ID NO: 37); b) 20mer PAPDA linker: AQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 33), or c) The first 20 amino acid residues of the CH1 constant domain: ASTKGPSVFPLAPSSKSTSG (SEQ ID NO: 35) This was achieved by using one of the following:

[0241] A summary of the linker combinations that were tested is listed in Table 23. Figure 2 depicts the various peptide linkers used to connect the different components of the bispecific antibody format of Example 3.

[0242] [Table 37]

[0243] All C-terminal linkers were further extended at the C-terminus by a portion of the human IgG1 hinge sequence DKTHTCPPCP (SEQ ID NO: 38). The use of a truncated human IgG1 hinge sequence allowed for further stabilization of the heterodimeric molecule through the formation of interchain disulfide bridges between the two polypeptide chains comprising the Fab heavy chain. The Fc region was modified by introducing mutations into the CH3 domain of each Fc region subunit according to the "knob-into-holes" technique. Thereby, a polypeptide comprising one mutated CH3 domain is forced to heterodimerize with another polypeptide comprising the other complementary engineered CH3 domain. For HER2 binding, the VH and VL domains (SEQ ID NO: 28 and SEQ ID NO: 29, respectively) of "Trastuzumab" (HERCEPTIN®) described by Baselga et al. 1998, Cancer Res 58(13):2825-2831 were used. Trastuzumab and its preparation method are described in US Patent No. 5,821,337. For CD3 binding, the VH and VL of the CD3-specific crossclone CD3-MABopt-cc of Example 2 (SEQ ID NO: 14 and SEQ ID NO: 20, respectively) were used. A summary of the individual polypeptides forming the bispecific trivalent Fab2-Fv-Fc antibodies produced using the various linker combinations of Table 23 made according to Example 3 is shown in Table 13.

[0244] Example 3.3: Gene synthesis and production All nucleic acid sequences or desired gene segments were either generated by PCR using appropriate templates or gene synthesized in-house or by an external provider as linear DNA fragments with appropriate flanking regions (e.g., suitable restriction enzyme recognition sites, linker sequences). The nucleic acid sequences or gene segments flanked by singular restriction endonuclease cleavage sites were cloned into the respective expression vectors (e.g., mammalian expression vectors) or sequencing vectors using standard molecular biology methods. When intended for use in mammalian expression vectors, all constructs were designed with a 5'-end DNA sequence encoding a leader peptide directing the protein for secretion in eukaryotic cells. The DNA sequences of the subcloned gene fragments were confirmed by double-stranded DNA sequencing. For expression of the bispecific antibody, exponentially growing eukaryotic HEK293-6E cells were transfected with a mammalian vector expression system encoding all components of the bispecific antibody, resulting in a 1:1:2 ratio of two polypeptides comprising the two Fc region subunits and two polypeptides comprising the Fab light chain of trastuzumab, respectively. Cell culture supernatants were harvested 6 days after transfection and subjected to standard Protein A affinity chromatography (MabSelect SURE | GE Healthcare). Buffer exchange into 1x Dulbecco's PBS (pH 7.2 | Invitrogen) was performed and samples were sterile filtered (0.2 μm pore size). Protein concentrations were determined by UV spectrophotometry and construct purity was analyzed under denaturing reducing and non-reducing conditions using CE-SDS (LabChip GXII | Perkin Elmer | USA). HP-SEC was performed to analyze the bispecific antibody preparations in the native state. Additionally, mass spectrometry analysis confirmed the molecular mass and homogeneity of the bispecific antibodies.

[0245] Table 24 summarizes the monomer content and volumetric yields of the different preparations obtained with the produced bispecific antibodies, all of which demonstrated comparable production characteristics.

[0246] However, all bispecific antibodies using CH1 or Cλ derived peptide linkers revealed highly heterogeneous mass patterns suggesting potential O-linked glycosylation of such linkers. Only bispecific antibody BissIg_21#2 using linker combination 2, i.e., the combination of a 20mer glycine-serine linker (SEQ ID NO: 31) and a 20mer PAPDA linker (SEQ ID NO: 33), showed a homogeneous mass pattern with no potential indication of O-linked glycosylation.

[0247] [Table 38]

[0248] Example 3.4: K via antibody capture setup D decision Affinity determinations were performed by determining kinetic rate constants on an Octet HTX (ForteBIO, Sartorius AG) instrument. The various bispecific antibody preparations from Example 3 diluted in assay buffer (D-PBS, 0.05% (v / v) PS20, 0.1% (w / v) BSA) were captured on an IgG-specific BLI sensor at a loading level of approximately 2 nm. For analysis, human CD3 epsilon antigen hCD3e(22-118)_F-chLys_avi (SEQ ID NO: 43) was diluted in assay buffer to concentrations ranging from 1.56 nM to 500 nM (stepwise 1:3 dilution). Blank samples of assay buffer were included for reference, i.e. to correct for dissociation of the capture antibody. The association phase was recorded for 300 s, followed by a dissociation phase of 180 s. The sensorgrams were fitted using Octet Data Analysis Software 10.0 (ForteBio, Sartorius AG) to determine k on and k off The rate constants were determined (using a 1:1 binding model) and used to calculate the K D was calculated.

[0249] Table 25 shows the human CD3 epsilon antigen K binding profiles of the bispecific antibodies of this example using various linker combinations from Table 23. D Values ​​are summarized below. Comparable CD3 binding was observed for all linker combinations tested.

[0250] [Table 39]

[0251] Example 3.5: Cell Binding Bispecific antibodies according to Example 3 with various linker combinations in Table 23 were tested for their binding ability to CD3 positive Jurkat cells and CD3 negative J.RT3-T3.5 cells. Jurkat and J.RT3-T3.5 cells were resuspended, counted, blocked and blocked for 1 h on ice in wash buffer (DPBS+(Gibco) / 3% FBS(Sigma) / 0.02% sodium acid). Blocked cells were resuspended in serially diluted bispecific antibodies (final concentrations: 500 nM to 31 pM) in wash buffer and incubated for 1 h on ice. Cells were washed twice in wash buffer. Bound antibodies were detected using AlexaFluor 647-labeled goat anti-human IgG (F(ab')2 fragment specific) (Jackson Immuno Research Cat#109-606-097). EC was determined using a four-parameter nonlinear regression analysis in Prism software (GraphPad Software Inc.). 50 values ​​were calculated.

[0252] Table 26 shows the cell binding results (EC 50 Figure 1 summarizes the binding profiles of HER and CD3 specific bispecific antibodies with various linker combinations, when compared to the first described linker combination P, with no observed binding to CD3 negative Jurkat cells (RT3-T3.5) (data not shown).

[0253] [Table 40]

[0254] Example 3.6: Cytotoxicity assays with bispecific antibodies containing various linker combinations Bispecific antibodies according to Example 3 containing various linker combinations were tested for their ability to induce T cell-mediated killing of HER2-expressing SKOV-3 and HER-2-negative MDA-MB468 cells. Human pan-T cells from two donors were prepared and purified as described above (see Example 1.4). 5,000 HER2-expressing SKOV-3 cells were suspended in culture medium (SKOV-3: McCoy's 5A medium (ThermoFisher, #26600), 10% FCS (Sigma, #F7524), seeded in black 96-well assay plates (Corning, #3340) and incubated overnight at 37°C and 5% CO2. CellToxGreen dye (Promega, #G8731), serially diluted bispecific antibodies (0.3 nM to 1.2 pM), and 50,000 purified T cells (1:10 target / effector ratio) were all cultured in RPMI 1640. Cells were diluted in assay medium containing w / o phenol red (Gibco #32404-014), GlutaMAX, and 10% fetal bovine serum, and incubated for 72 h at 37°C and 5% CO2. Cytotoxic activity was assessed by measuring the fluorescence of incorporated CellToxGreen at 485 nm excitation and 535 nm emission using a Tecan Infinite F500 device. EC 50 values ​​were calculated.

[0255] Table 27 shows the mean IC of T cell-mediated redirected killing of SKOV-3 cells mediated by bispecific antibodies containing various linker combinations. 50Values ​​are summarized. Dose-dependent killing was observed with all bispecific antibodies. Five linker combinations resulted in improved cell killing compared to the first disclosed linker combination P (BissIg_21#P). HER2-independent killing of MDA-MB468 cells could not be observed with any of the bispecific antibodies tested (data not shown).

[0256] FIG. 6 shows exemplary SKOV-3 killing results mediated by bispecific antibodies BissIg_21#2 and BissIg_21#5 containing the most potent linker combinations 2 and 5 compared to the originally disclosed linker combination P (BissIg_21#P).

[0257] [Table 41]

[0258] Example 3.7: T cell activation in the absence of target cancer cells (high density PBMC assay) The bispecific antibodies of Example 3, containing various linker combinations, were tested for their ability to activate human T cells derived from human blood samples of three different donors in the absence of target cancer cells. The assay was performed essentially as described in Example 2.5. The bispecific antibodies were tested at final concentrations of 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, and 0.32 nM. As a positive control, the commercially available murine IgG antibody OKT3 was used. T cell activation was assessed by evaluation of the upregulation of CD69 expression on CD4+ and CD8+ T cells. For this, PBMCs were stained with antibodies against CD69, CD4, and CD8 conjugated to APC, PE, Pacific Blue, respectively (Biolegend, #310910 / #300508 / #300928). Antibody staining was measured using a NovoCyte 3000 flow cytometer (Acea Biosciences, Inc.) and analyzed using FlowJo software.

[0259] The average results of the T cell activation experiments obtained from the three donors are shown in Figure 7A for CD4+ / CD8- T cells and in Figure 7B for CD4- / CD8+ T cells. As expected, a strong upregulation of CD69 expression on CD4+ and CD8+ T cells was observable with the positive control IgG OKT-3. In the absence of HER2-expressing target cells, no activation of CD4+ T cells was observable with any of the tested bispecific antibodies, even at the highest tested concentration of 1 μM (antibody concentrations decrease from left to right). With some of the tested linker combinations, a slight donor-dependent activation of CD8+ T cells was observed, but only at the highest tested antibody concentration of 1 μM. The most significant activation was observable with the first disclosed linker combination P (BissIg_21#P), linker combination 1 (BissIg_21#1), and linker combination 6 (BissIg_21#6).

[0260] Example 3.8: Summary Linker Optimization All bispecific 2+1 Fab2-Fv-Fc antibodies of Example 3 utilizing the new linker combinations were found to have similar production characteristics and binding to CD3 when compared to the bispecific antibody containing the first described linker combination P (BissIg_21#P).

[0261] However, bispecific antibodies BissIg_21#1, BissIg_21#2, BissIg_21#3, BissIg_21#4, and BissIg_21#5, containing linker combinations 1, 2, 3, 4, and 5, respectively, demonstrated improved efficacy in T cell-mediated killing of SKOV-3 cells and less target-independent T cell activation when compared to BissIg_21#P, containing the first described linker combination P. However, all bispecific antibodies containing CH1 or Cλ-based peptide linkers demonstrated signs of O-glycosylation after mammalian production as determined by mass spectrometry analysis. Only bispecific antibody BissIg_21#2, containing linker combination 2 encompassing the extended 20-mer peptide linker (G4S)4 (SEQ ID NO: 31) and 20aa-PAPDA (SEQ ID NO: 33), did not show any signs of O-glycosylation. Therefore, linker combination 2 was identified as the most preferred linker combination to be used in the improved bispecific 2+1 Fab2-Fv-Fc antibody format of Example 3.

[0262] Example 4: Optimized CD3-specific antibody CD3-MAB opt-cc Development of deimmunized variant antibodies CD3-MAB of Example 2 having VH of sequence ID 14 and VL of SEQ ID NO: 20 opt-cc A deimmunized variant of a human CD3-specific antibody was prepared. This antibody is characterized by germline-encoded human framework regions of its VH and VL, as well as germline-encoded LCDR1 and LCDR2 regions. The following examples include CD3-MAB opt-cc Deimmunization of the non-human germline-encoded HCDR1, HCDR2, HCDR3, and LCDR3 regions of is described.

[0263] Example 4.1: CD3-MAB opt-cc Identification of the number of (potential) T cell epitopes, H lines, and hot spots in the CDR regions of CD3-MAB opt-ccThe amino acid sequences of VH (SEQ ID NO: 14) and VL (SEQ ID NO: 20) of were analyzed for the number of potential T cell epitopes, H lines and hot spots by using an in silico T cell epitope screening tool (Lonza, Epibase™, Epibase version: v3.0), the basic computing routines of which are described in WO 2003 / 105058, which is incorporated herein in its entirety. This screening tool allows the identification of potential T cell epitopes in biotherapeutic proteins such as antibodies. The structural properties of HLA receptors together with experimentally determined binding affinities are used in the tool to predict potential peptide / HLA binding, a prerequisite for T cell activation.

[0264] For analysis, the entire VH or VL sequence is split into overlapping 10-mer peptides, each shifted by one amino acid (herein referred to as "analysis 10-mer peptides"). The potential peptide / HLA binding of each 10-mer peptide was determined with HLA class II allotypes of the major Caucasian DRB1 allele. Human antibody germline coding sequence regions were excluded from the analysis.

[0265] Epibase™ Analysis Results From the results of the in silico “search” process, CD3-MAB opt-ccThe presence of six hotspots in the HCDR1, HCDR2, HCDR3 regions of CD3-MABopt-cc and one hotspot in the LCDR3 region of CD3-MABopt-cc was revealed. Hotspots reflect the accumulation of close / adjacent T cell epitopes. Such hotspots were identified based on the “4 over 3” algorithm detailed in FIG. 8 legend and FIG. 8, which requires that at least four allotypes of DRB1 alleles bind to at least two of three consecutive analyzed 10-mer peptides with medium (M) or strong (S) affinity. Besides, at most one 10-mer peptide that is not identified as a T cell epitope may be part of a hotspot. Therefore, not every identified T cell epitope must be part of a hotspot (see, for example, peptides 100, 102, or 104 in FIG. 4). On the other hand, peptides that are not identified as T cell epitopes may still be part of a hotspot (see, for example, peptides 96 or 111 in FIG. 4). Each 10-mer peptide that is part of a hotspot is defined as an H-line. The absolute risk score of a hotspot can be calculated as the sum of the risk scores determined for each T-cell epitope within the hotspot, and is provided as the "H-score."

[0266] Tables 28 and 29 show CD3-MAB opt-cc We provide a summary of the overall immunogenicity risk parameters determined in the VH and VL of the LCDR1+2 regions. As these regions are germline coding sequences, no hotspots were assigned to these regions.

[0267] [Table 42]

[0268] [Table 43]

[0269] CD3-MAB opt-ccAn exemplary detailed screening analysis of the HCDR3 region of is shown in Figure 8 (see figure legend of Figure 8 for detailed description). In summary, in the HCDR3 region, two hot spots were identified, covering seven T cell epitopes and nine H lines, with an H score of 236. In the HCDR2 region, three hot spots were identified, covering 10 T cell epitopes and 12 H lines, with an H score of 254.1, and in the HCDR1 region, one hot spot was identified, covering three T cell epitopes and four H lines, with an H score of 89.9. In the LCDR3 region, one hot spot was identified, covering six T cell epitopes and seven H lines, with an H score of 188.8.

[0270] Example 4.2: Antibody CD3-MAB with single amino acid substitutions in the CDR regions opt-cc Generation of deimmunized variant VH and VL sequences CD3-MAB opt-cc In silico Epibase™ mutational analysis was performed to remove or reduce previously identified hot spots, T cell epitopes, and / or H lines from the CDR regions of CD3-MAB. opt-cc Each amino acid position in VH and VL of was substantially substituted / randomized with each of the naturally occurring amino acid residues, except for cysteine, proline, and histidine. Preferably, amino acid substitutions that resulted in a reduction in the number of T cell epitopes by ≧2 within the identified hotspots, and that preferably allowed conservative amino acid substitutions, e.g., to select amino acids of similar charge or polarity, were selected for gene synthesis of the deimmunized variant VH and VL antibody sequences. Additionally, care was taken to avoid introducing potential post-translational modification sites ("PTM motifs") in the CDR regions.

[0271] Figure 9 depicts the effect of single amino acid substitutions at each HCDR3 position of CD3-MABopt-cc on the number of T cell epitopes (left panel of Figure 4) and the corresponding absolute risk score (right panel of Figure 4) compared to the unmodified parent sequence. Figure 10 depicts the effect of favorable single amino acid substitutions on the number of T cell epitopes in 24 actually produced and characterized bispecific antibodies (as described below) containing VH or VL single point mutant variants.

[0272] In total, CD3-MAB opt-cc 92 physical VH or VL single point variants of were generated by a PCR-based mutagenesis strategy. Briefly, linear DNA fragments were generated by PCR using suitable oligonucleotides carrying advantageous mutations and homologous overlapping sequences, and subsequently cloned into the corresponding mammalian bispecific antibody expression vectors encoding the bispecific 2+1 Fab2-Fv-Fc antibody format described in Example 3 with specificity for HER2 and CD3. The VH variants were generated using the CD3-MAB opt-cc (SEQ ID NO: 20) of the parent unmodified VL of CD3-MABopt-cc, whereas the VL variants were combined with the parent unmodified VH of CD3-MABopt-cc (SEQ ID NO: 14). The bispecific antibodies were produced as previously described in Example 3.

[0273] Overall, approximately 50% of the bispecific antibodies could be produced with an acceptable monomer content of >85% (see Table 30). Figure 10 summarizes the yields and final monomer content of bispecific antibody preparations containing the 24 preferred single point variants referenced above.

[0274] [Table 44]

[0275] Surprisingly, it was found that amino acid exchanges (even when conservative) at certain CDR positions have a strong negative effect on the productivity of bispecific antibodies, i.e., leading to an increased aggregation tendency. Table 31 shows the results of the CD3-MABs that resulted in a significant decrease in the monomer content of the corresponding produced bispecific antibodies. opt-cc The positions of substituted amino acids within the CDRs of are depicted.

[0276] [Table 45]

[0277] Example 4.3: ELISA Binding 46 produced bispecific single point variant antibodies according to Example 4.2 with >85% monomer content were characterized for ELISA binding to human CD3 epsilon antigen (hCD3e(22-118)_F-chLys_avi (SEQ ID NO: 43) coated on Maxisorp plates (Nunc, #460518). Final antibody concentrations were set at 50, 10, 2, 0.4 nM, respectively. Bound antibodies were detected using an alkaline phosphatase-conjugated detection antibody against human F(ab')2 fragment (Jackson Immuno Research, #109-055-097). EC 50 values ​​were calculated.

[0278] Figure 10 shows the ELISA EC of 24 bispecific single point variant antibodies against the human CD3 epsilon antigen. 50 This is a summary of the estimates and the parent antibody CD3-MAB opt-cc reveals similar and even better binding to CD3 when compared to EC 50 Estimates + sum of signal to background ratios across all tested antibody concentrations are provided).

[0279] Example 4.4: K via antibody capture setup D decision Affinity determination by determining kinetic rate constants was performed with bispecific single-point variant antibodies as already described in Example 3.4. Different bispecific antibody samples diluted in assay buffer (1% BPBS + 0.05% Tween 20) were captured on an IgG-specific BLI sensor at a loading level of approximately 2 nM. For analysis, human CD3 epsilon antigen hCD3e(22-118)_F-chLys_avi (SEQ ID NO: 43) was diluted in assay buffer to a concentration in the range of 200 nM to 3.1 nM. A blank sample of assay buffer was included for reference, i.e. to correct for dissociation of the capture antibody. The association phase was recorded for 300 s, followed by a dissociation phase of 180 s. The sensorgrams were fitted using Octet Data Analysis Software 10.0 (ForteBio, Sartorius AG) to determine k on and k off The rate constants were determined (using a 1:1 binding model) and used to calculate the K D was calculated.

[0280] FIG. 10 shows the K of 24 preferred bispecific single point variant antibodies against the human CD3 epsilon antigen. D These variants were compared with the parent antibody CD3-MAB opt-cc The monovalent affinity was found to be in the single-digit nanomolar range, comparable to that of

[0281] Example 4.5: Overview of deimmunized single point variants of CD3-MABopt-cc Figure 10 summarizes the biophysical and functional properties of 24 preferred bispecific single point variant antibodies (5 LCDR3 variants and 19 HCDR variants) selected based on productivity and ELISA binding. All variants, when tested in the bispecific antibody format described herein, have reduced immunogenicity risk as determined by reduced T cell epitope number, H line number, absolute score, and H score, but are more potent than the parent CD3-MAB. opt-ccIt was found that the biophysical and functional properties of

[0282] Of the 19 single-point variants (listed in Table 32), 14 were selected for the combinatorial T-cell epitope Epibase™ analysis described below (see Example 4.6), since in only 5 of the 19 single-point variants a reduction in the number of hotspots was determinable. Table 32 summarizes the impact of each of the selected 14 single-point variants on the immunogenicity risk parameters compared to those of the parent antibody. Of note, only substitutions at HCDR2 position V61 and HCDR1 position Y33 resulted in a reduction in the number of hotspots.

[0283] CD3-MAB opt-cc For the VL of the CD3-MAB, the LCDR3 variant S95E was selected as the only preferred VL variant to be used in combination with any other VH variant. This single point LCDR3 variant results in a reduction of four H lines in LCDR3 and, when tested in the bispecific antibody format of Example 4.2, was associated with a CD3-MAB opt-cc It was shown to have similar functional and biophysical properties to the parent antibody VH.

[0284] [Table 46]

[0285] Example 4.6 Generation of Deimmunized Sequences by Combinatorial Amino Acid Substitution Variants ("Combinatorial Variants") CD3-MAB opt-cc To further reduce the number of potential T cell epitopes and other immunogenicity risk parameters in the VH-CDR regions of CD3-MAB, an in silico Epibase™ mutational analysis was performed. In this case, each of the 14 previously identified favorable HCDR single point variants in Table 32 was found to be highly potent in the CD3-MAB opt-ccThe wild-type residues of VH-CDR were combined with each other at virtually every position, including the wild-type residues of VH-CDR. Thus, 251 new combinatorial VH-CDR sequence variants (including double, triple, and quadruple mutations) were screened for potential T cell epitopes.

[0286] Results of Epibase™ screening analysis of combinatorial mutation variants FIG. 11 shows that CD3-MAB caused the greatest reduction of the three hot spots on various immunogenicity risk parameters. opt-cc The table depicts exemplary results of the effect of 54 combinatorial amino acid substitutions in the VH_HCDR1-3 region of CD3-MAB. opt-cc (denoted as "wt(parent)") VH risk parameters: absolute score, number of hotspots, absolute H score, and absolute H line number are shown. Surprisingly, the combination variant that was crucial for reducing the number of hotspots, absolute H line number, and absolute H score could be assigned to the variant combination Y33W(HCDR1)+wtY56(HCDR2)+V61D / E / orG(HCDR2).

[0287] All 54 VH combination variants that resulted in a reduction of 3 hot spots, and 40 additional variant combinations that resulted in a reduction of 2 hot spots (not shown) and revealed the greatest reduction in H line number and absolute score, were selected for cloning and production in a bispecific antibody format as described in Example 3. For this, each of the 54 combination VH variants was expressed together with a VL variant containing the LCDR3 variant S95E (SEQ ID NO: 21). 25 / 94 produced bispecific combination variant antibodies were found to have a monomer content of >85%. Figure 12 summarizes the monomer content of 33 preferred bispecific combination variant antibody preparations as determined by analytical size exclusion chromatography.

[0288] Example 4.7: ELISA binding of bispecific antibodies to recombinant human CD3 epsilon. All 94 produced bispecific combinatorial variant antibodies of Example 4.6 were tested for ELISA binding to recombinant CD3 epsilon antigen as previously described in Example 4.3. In summary, 46 bispecific combinatorial variant antibodies were found to bind to the parental antibody CD3-MAB opt-cc EC<30 nM compared to 50 Figure 12 shows the ELISA EC values ​​of the 33 preferred bispecific combination variants determined against the human CD3 epsilon antigen. 50 It is a collection of values.

[0289] Example 4.8: K via antibody capture setup D decision Affinity determination by determination of kinetic rate constants for the 46 bispecific combination variants of Example 4.6 that revealed better ELISA binding to CD3 (Example 4.7) was performed as previously described in Example 4.4. Overall, the bispecific combination variant antibodies were found to have monovalent affinity for CD3 epsilon in the mid-1- to low-2-digit nanomolar range. Parental antibody CD3-MAB opt-cc Bispecific antibodies (K D Improved binding to CD3 better than 100 nM (KD = 5 nM) was not achievable. Figure 12 summarizes the KD values ​​of 33 preferred bispecific single-point variant antibodies against the human CD3 epsilon antigen. These variants were compared with the parent antibody CD3-MAB opt-cc K in the range of 5–11 nM, comparable to the affinities determined for corresponding bispecific antibodies, including D was found to be a value.

[0290] Example 4.9 Jurkat NFAT Reporter Gene Cellular Assay To evaluate the functional activity of the 46 bispecific combination variant antibodies of Example 4.8 with specificity for HER2 and CD3, a Jurkat NFAT reporter gene cell assay was performed. Jurkat cells (ATCC #TIB-152) stably transfected with an NFAT reporter gene construct were used as surrogate effector cells. The HER2 positive human SKOV-3 (ATCC® HTB-77) tumor cell line was used as tumor target cells. The assay was performed as described in Example 2.3 above. The bispecific antibodies were serially diluted (4 step dilutions) to a final assay concentration range of 50 nM to 0.5 nM.

[0291] Most of the bispecific combination variant antibodies induced luciferase activity in the presence of SKOV-3 cells. However, none of the bispecific antibodies tested induced any significant increase in EC 50 The parent antibody CD3-MAB did not show significant activity in terms of opt-cc The results showed that the maximum luciferase activity level was not exceeded by the bispecific antibody containing

[0292] Figure 12 summarizes the RGA results for the 33 preferred bispecific combination variant antibodies. Results are provided as signal / background values ​​at the two tested antibody concentrations and therefore the antibodies were sorted accordingly. The top row of the table shows the parent bispecific antibody CD3-MAB, which represents the highest signal intensity for this antibody at antibody concentrations of 50 nM and 0.5 nM. opt-cc Overall, a positive correlation can be inferred between the activity of RGA and its binding affinity to CD3.

[0293] Example 4.10 Exploratory Scale Production Thirty-three preferred bispecific combination variants of Example 4.9 were selected for larger scale production. Selection criteria included K D These included values ​​(Example 4.8), functional activity in a reporter gene assay (Example 4.9), and monomer content after smaller scale production (Example 4.6).

[0294] Eukaryotic HEK293-6E cells were transfected with mammalian expression vector DNA encoding both the heavy and light chains of the bispecific antibodies. Cell culture supernatants were harvested 6 days after transfection and subjected to standard Protein A affinity chromatography (MabSelect SURE | GE Healthcare). A buffer exchange into 1x Dulbecco's PBS (pH 7.2 | Invitrogen) was performed and samples were sterile filtered (0.2 μm pore size). Protein concentration and purity of the bispecific antibodies were determined as previously described.

[0295] Figure 12 summarizes the percentage of monomeric content in the 33 produced bispecific combinatorial variant antibody preparations. Fifteen of these preparations were found to have greater than 90% monomeric antibody content.

[0296] Example 4.11: Selection of the most suitable combination variant CD3 specific antibodies CD3-specific antibody CD3-MAB opt-cc Parallel deimmunization of the LCDR3 and HCDR1-3 regions of VH / VL resulted in the identification of five favorable deimmunized VH / VL variants. These variants emerged as the top 10 performing bispecific antibodies in each of the above-mentioned assays in Example 4 and were further characterized in T cell-mediated cytotoxicity assays (Example 4.12) and their ability to induce T cell activation in the absence of target cells (Example 4.13).

[0297] These five aforementioned deimmunized CD3-specific antibodies are referred to herein as follows: CD3-MABdeimm_1, CD3-MABdeimm_2, CD3-MABdeimm_3, CD3-MABdeimm_4, and CD3-MABdeimm_5. The VH, VL, and CDR sequences of each of the five deimmunized antibodies are provided in Table 7. The corresponding bispecific antibodies comprising the five deimmunized antibodies are referred to herein as follows, respectively: BissIg_21#CD3-MABdeimm_1, BissIg_21#CD3-MABdeimm_2, BissIg_21#CD3-MABdeimm_3, BissIg_21#CD3-MABdeimm_4, and BissIg_21#CD3-MABdeimm_5. A summary of the individual polypeptides forming the bispecific antibodies made according to Example 4 is provided in Table 14. Table 29 provides a summary of the biophysical and functional properties as well as immunogenicity risk parameters of these five deimmunized CD3-specific antibodies and the corresponding bispecific antibodies.

[0298] Example 4.12: Redirected T cell cytotoxicity mediated by bispecific antibodies containing five preferred deimmunized CD3-specific antibodies. The bispecific combination variant antibodies BissIg_21#CD3-MABdeimm_1, BissIg_21#CD3-MABdeimm_2, BissIg_21#CD3-MABdeimm_3, BissIg_21#CD3-MABdeimm_4, and BissIg_21#CD3-MABdeimm_5 were tested for their ability to induce T cell-mediated killing of tumor cells upon binding to CD3 and HER2. The method was carried out as described in Example 3.6. As target cells, 5,000 HER2-expressing SKOV-3 cells were used to give a target / effector cell ratio of 1:10.

[0299] Table 33 shows the IC of T cell-mediated killing by all five tested bispecific combination variant antibodies. 50The values ​​are summarized in Table 1. Concentration-dependent killing of HER2-highly expressing SKOV-3 cells could be confirmed for all five bispecific antibodies, which were comparable to those of the bispecific antibodies, including the parental antibody CD3-MABopt-cc. Figure 13 depicts exemplary SKOV-3 killing results with T cells obtained from one donor and the most preferred combination variant antibody CD3-MABdeimm_3 (BissIg_21#CD3-MABdeimm_3) relative to the parental antibody CD3-MABopt-cc (BissIg_21#CD3-MABoptcc).

[0300] Example 4.13: T cell activation of bispecific antibodies in the absence of target cancer cells (high density PBMC assay) To evaluate the safety of deimmunized CD3-specific antibodies, five corresponding bispecific combination variant antibodies BissIg_21#CD3-MABdeimm_1 (CD3-MABdeimm_1), BissIg_21#CD3-MABdeimm_2 (CD3-MABdeimm_2), BissIg_21#CD3-MABdeimm_3 (CD3-MABdeimm_3), BissIg_21#CD3-MABdeimm_4 (CD3-MABdeimm_4), and BissIg_21#CD3-MABdeimm_5 (CD3-MABdeimm_5) were tested for their ability to activate human T cells in the absence of target cancer cells. The assay was performed similarly to that under Example 3.7 above. Human blood samples were drawn from two different donors. T cell activation was assessed by evaluation of upregulation of CD69 expression on CD4+ or CD8+ T cells.

[0301] Table 33 qualitatively summarizes the T cell activation capabilities of the five bispecific combination variant antibodies. T cell activation was not observable with any of the bispecific combination variant antibodies, even at the highest tested antibody concentration, in the absence of HER2 expressing target cells. Donor-dependent activation of T cells was observable with the bispecific antibody BissIg_21#CD3-MABoptcc, including the parent antibody CD3-MABopt-cc, at higher tested concentrations. The positive control OKT-3 strongly induced CD69 expression in both CD4+ and CD8+ T cells within the range of 76-78% at an IgG concentration of 20 nM. Exemplary results of T cell activation experiments with one donor are shown in FIG. 14. FIG. 14A depicts the results of CD4+ / CD8- T cell activation, and FIG. 14B shows the results of CD4- / CD8+ T cell activation. These results clearly demonstrate the remarkable safety profile of the deimmunized human CD3-specific antibodies of the present disclosure.

[0302] Example 4.14: Overview of deimmunized combination variants: The present invention provides for the first time a CDR deimmunization approach in a fully human antibody with specificity for CD3. An overview of the functional and biophysical properties of the five preferred deimmunized variants CD3-MABdeimm_1, CD3-MABdeimm_2, CD3-MABdeimm_3, CD3-MABdeimm_4, and CD3-MABdeimm_5, as well as their parental human CD3-specific antibody CD3-MABopt-cc, identified in Example 2, is provided in Table 33. deimmun_3 was identified as the most preferred deimmunized CD3 specific antibody of the present invention. CDR engineering of the parent antibody CD3-MABopt-cc resulted in a total reduction of two hotspots in the HCDR2 region and one hotspot in the LCDR3 region of this antibody. Besides, a significant reduction in the number of T cell epitopes, the number of H lines, the absolute risk score, and the absolute H score could be achieved. Therefore, CD3-MAB deimmun_3 has a crucial aspect of T cell-engaging therapy in that the risk of inducing an immunogenic response in humans once administered is significantly reduced. deimmun_3This remarkable safety profile of is further strengthened by its inability to induce T cell activation in the absence of target cells. Of note, CDR engineering did not result in loss of specificity, functional activity and productivity when compared to the parent antibody CD3-MABopt-cc. This is even more remarkable as CDR engineering affects the HCDR3 region of CD3-MABopt-cc, the CDR most relevant for antigen recognition.

[0303] [Table 47]

[0304] [Table 48]

Claims

1. i. below (a) Heavy chain complementarity determination region (HCDR) 1 containing the amino acid sequence of GFSFGSHYMS (SEQ ID NO: 1), (b) HCDR2 containing the amino acid sequence of NINQIGYSSYYVESVKG (SEQ ID NO: 2), NINQIGYSSYYGESVKG (SEQ ID NO: 3), or NINQIGYSSYYEEESVKG (SEQ ID NO: 4), and (c) HCDR3 containing the amino acid sequence of GYSAEFAHRSGLDV (SEQ ID NO: 5), GYSDEFAATRSGLDV (SEQ ID NO: 6), GYSEEEFAHRSGLDV (SEQ ID NO: 7), GYSDEFAKRSGLDV (SEQ ID NO: 8), or GYSDEFAHRSGLDV (SEQ ID NO: 9), Heavy chain variable region (VH) including, ii. below (a) Light chain complementarity determination region (LCDR) 1 containing the amino acid sequence of SGSSSNIGSNYVY (SEQ ID NO: 10), (b) LCDR2 containing the amino acid sequence of RNNQRPS (SEQ ID NO: 11), (c) LCDR3 containing the amino acid sequence of AGWSRSLHGAV (SEQ ID NO: 12) or AGWSRELHGAV (SEQ ID NO: 13), Variable light chain region (VL) including An isolated human antibody or its antigen-binding fragment that is specific to differentiated cluster 3 (CD3), including the above.

2. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or its antigen-binding fragment cross-reacts with cynomolgus monkey CD3.

3. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or its antigen-binding fragment is a deimmune antibody.

4. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment has a reduced risk of immune response selection in humans.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein VH contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and / or VL contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO:

21.

6. The aforementioned VH and VL are as follows: i. VH containing the amino acid sequence of SEQ ID NO: 14 and VL containing the amino acid sequence of SEQ ID NO: 20, ii. VH containing the amino acid sequence of SEQ ID NO: 15 and VL containing the amino acid sequence of SEQ ID NO: 21, iii. VH containing the amino acid sequence of SEQ ID NO: 16 and VL containing the amino acid sequence of SEQ ID NO: 21, iv. VH containing the amino acid sequence of SEQ ID NO: 17 and VL containing the amino acid sequence of SEQ ID NO: 21, v. VH containing the amino acid sequence of SEQ ID NO: 18 and VL containing the amino acid sequence of SEQ ID NO: 21, and vi. VH containing the amino acid sequence of SEQ ID NO: 19 and VL containing the amino acid sequence of SEQ ID NO: 21, An antibody or antigen-binding fragment thereof according to claim 1, selected from the group consisting of the following.

7. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment thereof is a recombinant antibody or antigen-binding fragment thereof.

8. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody or an antigen-binding fragment thereof.

9. The antigen-binding fragment is Fab, Fab', (Fab') ２ The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is Fv or scFv.

10. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a full-length antibody.

11. A bispecific antibody comprising an antigen-binding fragment of an antibody according to claim 1, and a second antigen-binding fragment of an antibody that binds to a target antigen different from the first antigen-binding fragment.

12. The bispecific antibody according to claim 11, wherein the second antigen-binding fragment binds to a cell surface antigen, specifically a tumor-associated cell surface antigen.

13. The antibody or antigen-binding fragment thereof according to claim 1, or the bispecific antibody according to claim 11, comprising an Fc region containing one or more amino acid substitutions that reduce binding to an Fc receptor and / or effector function.

14. A nucleic acid composition comprising one or more nucleic acid sequences encoding the antibody or antigen-binding fragment thereof described in claim 1, or the bispecific antibody described in claim 11.

15. A vector composition comprising one or more vectors comprising one nucleic acid sequence or multiple nucleic acid sequences as described in claim 14.

16. A host cell comprising the vector composition according to claim 15.

17. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in claim 1, or the bispecific antibody described in claim 11, and a pharmaceutically acceptable carrier or excipient.

18. An antibody or its antigen-binding fragment according to claim 1, or a bispecific antibody according to claim 11, or a pharmaceutical composition according to claim 17, for use as a pharmaceutical agent.

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