Optimized CD3 antigen-binding domain

Abstract

This disclosure relates to an antibody or fragment thereof comprising an antigen-binding domain capable of binding to a CDS protein or fragment thereof. This disclosure also relates to such antibodies that bind to CDS having an affinity optimized for inducing T cell activation but without being associated with excessive cytokine release and decreased tolerance. This disclosure also relates to methods for producing these antibodies and their therapeutic use.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims the interests of U.S. Provisional Patent Application No. 63 / 495,127, filed on April 10, 2023, which is incorporated herein by reference in its entirety.

[0002] (Reference to electronically submitted sequence listings) The contents of the electronically submitted sequence listing (name: IOTS-102-WO Sequence Listing.xml, size: 109,086 bytes, date of creation: March 29, 2024), filed with this application, are incorporated herein by reference in their entirety.

[0003] (Field of invention) This disclosure relates to antibodies or fragments thereof that include an antigen-binding domain capable of binding to the CD3 protein or a fragment thereof. This disclosure also relates to methods for producing these antibodies and their therapeutic use. [Background technology]

[0004] T cells recognize antigen peptides via a complex of heterodimeric T cell receptor (TCR) α and β chains, in combination with four CD3 subunits represented as ε, γ, δ, and ζ (Kindt et al., 2007). After TCR-mediated antigen recognition, CD3 is essential for transmitting TCR-induced signaling via the immunoreceptor tyrosine activation motif (ITAM) (Letourmeur et al., 1992). Further antibodies, particularly multispecific antibodies, that can bind to CD3 and transmit TCR-induced signaling in a safe and effective manner are still needed. [Overview of the project]

[0005] Therefore, there is a need in the art for CD3-binding antibodies that have optimized affinity to induce T cell activation but without excessive cytokine release and reduced tolerance. This disclosure includes a broad selection and affinity maturation program for isolating a panel of CD3 antibodies capable of binding to CD3 and inducing T cell activation. Such molecules are expected to achieve therapeutic benefits by ensuring effective T cell engagement, but without the safety concerns associated with antibodies that bind to CD3 with high affinity or cytokine release profiles, thereby making these molecules suitable for the treatment of cancer as immunotherapeutic agents.

[0006] Accordingly, in one embodiment, an antibody comprising an antigen-binding domain capable of binding to a CD3 protein or a fragment thereof is provided herein, wherein the CD3 antigen-binding domain comprises a heavy chain variable (VH) region as described in any one of the following: VH area containing the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 90, HCDR2 having the amino acid sequence of SEQ ID NO: 91, and HCDR3 having the amino acid sequence of SEQ ID NO: 92, VH area containing the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 82, HCDR2 having the amino acid sequence of SEQ ID NO: 83, and HCDR3 having the amino acid sequence of SEQ ID NO: 84, VH area containing the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 78, HCDR2 having the amino acid sequence of SEQ ID NO: 79, and HCDR3 having the amino acid sequence of SEQ ID NO: 80, VH area containing the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 66, HCDR2 having the amino acid sequence of SEQ ID NO: 67, and HCDR3 having the amino acid sequence of SEQ ID NO: 68, VH region comprising the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 70, HCDR2 having the amino acid sequence of SEQ ID NO: 71, and HCDR3 having the amino acid sequence of SEQ ID NO: 72, VH region comprising the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 74, HCDR2 having the amino acid sequence of SEQ ID NO: 75, and HCDR3 having the amino acid sequence of SEQ ID NO: 76, VH region comprising the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 86, HCDR2 having the amino acid sequence of SEQ ID NO: 87, and HCDR3 having the amino acid sequence of SEQ ID NO: 88, VH region comprising the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 94, HCDR2 having the amino acid sequence of SEQ ID NO: 95, and HCDR3 having the amino acid sequence of SEQ ID NO: 96, and VH region comprising the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 98, HCDR2 having the amino acid sequence of SEQ ID NO: 99, and HCDR3 having the amino acid sequence of SEQ ID NO: 100, And the CD3 antigen-binding domain comprises a heavy chain variable (VH) region as described in any one of the following: VL region comprising the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 46, HCDR2 having the amino acid sequence of SEQ ID NO: 47, and HCDR3 having the amino acid sequence of SEQ ID NO: 48, VL region comprising the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 58, HCDR2 having the amino acid sequence of SEQ ID NO: 59, and HCDR3 having the amino acid sequence of SEQ ID NO: 60, and VL area containing the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 62, HCDR2 having the amino acid sequence of SEQ ID NO: 63, and HCDR3 having the amino acid sequence of SEQ ID NO: 64.

[0007] In some cases, the CD3 antigen-binding domain includes a VH region and a VL region containing one of the following CDR sets: i. HCDR1 having the amino acid sequence of SEQ ID NO: 90, ii. HDCR2 having the amino acid sequence of SEQ ID NO: 91, iii. HCDR3 having the amino acid sequence of SEQ ID NO: 92, iv. LCDR1 having the amino acid sequence of SEQ ID NO: 46, v. LCDR2 having the amino acid sequence of SEQ ID NO: 47, vi. LCDR3 having the amino acid sequence of SEQ ID NO: 48, or i. HCDR1 having the amino acid sequence of SEQ ID NO: 82, ii. HDCR2 having the amino acid sequence of SEQ ID NO: 83, iii. HCDR3 having the amino acid sequence of SEQ ID NO: 84, iv. LCDR1 having the amino acid sequence of SEQ ID NO: 46, v. LCDR2 having the amino acid sequence of SEQ ID NO: 47, vi. LCDR3 having the amino acid sequence of SEQ ID NO: 48, or i. HCDR1 having the amino acid sequence of SEQ ID NO: 78, ii. HDCR2 having the amino acid sequence of SEQ ID NO: 79, iii. HCDR3 having the amino acid sequence of SEQ ID NO: 80, iv. LCDR1 having the amino acid sequence of SEQ ID NO: 62, v. LCDR2 having the amino acid sequence of SEQ ID NO: 63, vi. LCDR3 having the amino acid sequence of SEQ ID NO: 64.

[0008] In some cases, the VH domain of the CD3 antigen-binding domain contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 77, SEQ ID NO: 89, or SEQ ID NO: 81. In some cases, the VL domain of the CD3 antigen-binding domain contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 45, or SEQ ID NO: 61.

[0009] In some cases, the CD3 antigen-binding domain includes: i. A VH region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to sequence number 77, and a VL region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to sequence number 61, ii A VH region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to sequence number 89, and a VL region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to sequence number 45, iii. A VH region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to sequence number 81, and a VL region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to sequence number 45.

[0010] As described above, the CD3 antigen-binding domain finds particular utility when used in the context of multispecific antibodies that contain an antigen-binding domain capable of binding to another target. For example, the CD3 antigen-binding domain described herein may form part of a multispecific antibody capable of binding to CD3 and another target, such as a tumor-associated antigen (TAA). Examples of suitable TAAs are described herein.

[0011] Therefore, in some cases, the antibody further contains a target antigen-binding domain. In some cases, the target antigen-binding domain can bind to tumor-associated antigens (TAAs). In some cases, the antibody is in a "2+1" format, where the antibody contains a single CD3 antigen-binding domain that binds monovalently to CD3 and two target antigen-binding domains that bind divalently to a target (e.g., a TAA). In some cases, the antibody is in a trivalent, triplicate format containing three antigen-binding domains.

[0012] With regard to generating multispecific antibodies containing multiple antigen-binding arms formed from different light and heavy chains, indiscriminate pairing of heavy and light chains can present a challenge. As described herein, amino acid residues at the interface between lambda light and heavy chains into which charge pairs can be introduced have been identified, and it has been demonstrated that the introduction of these lambda charge pairs can favorably improve chain pairing beyond what has been achieved in previous antibody formats. As further described herein, these charge variants can be used to efficiently produce multispecific antibodies in different formats, including a "2+1" bispecific format and a trivalent triple specific format.

[0013] Therefore, the antigen-binding domain in the antibody (e.g., the CD3 antigen-binding arm) contains a lambda charge pair between the constant light chain lambda region (CLλ) and the heavy chain constant region 1 (CH1) of the antigen-binding arm, and the antigen-binding domain is as follows: (i) Position 117 of CLλ and position 141 of CH1, (ii) Position 117 of CLλ and position 185 of CH1, (iii) Position 119 of CLλ and position 128 of CH1, (iv) Position 134 of CLλ and position 128 of CH1, (v) Position 134 of CLλ and position 145 of CH1, (vi) Position 134 of CLλ and position 183 of CH1, (vii) Position 136 of CLλ and position 185 of CH1, (viii) Position 178 of CLλ and position 173 of CH1, (ix) Including one or more lambda charge pairs located at the 117th position of CLλ and the 187th position of CH1, The lambda charge pair comprises a positively charged amino acid residue optionally selected from arginine, lysine, or histidine located at one position of the lambda charge pair, and a negatively charged amino acid residue optionally selected from aspartic acid, glutamic acid, serine, or threonine located at the other position of the lambda charge pair, and The numbering follows the EU index.

[0014] In some cases, the antigen-binding arm of an antibody (e.g., the CD3 antigen-binding arm) contains a lambda charge pair located at position 117 of CLλ and position 141 of CH1, the lambda charge pair comprising a positively charged amino acid residue selected from arginine, lysine, or histidine located at one position of the lambda charge pair, and an uncharged amino acid residue selected from aspartic acid, glutamic acid, serine, or threonine located at the other position of the lambda charge pair.

[0015] As further described herein, charge pairing can be combined with other approaches to promote light chain pairing, for example, to further increase the correct assembly of the desired trispecific antibody.

[0016] In some cases, the antibody comprises a CD3 antigen-binding domain and a CD8 antigen-binding domain. For example, the antibody may comprise a CD3 antigen-binding domain, a TAA antigen-binding domain, and a CD8 antigen-binding domain. The CD8 antigen-binding domain may be a VHH molecule (e.g., fused to the 2+1 bispecificity format described herein). The CD8 antigen-binding domain may also be an antigen-binding ("Fab") arm (e.g., one of the antigen-binding arms in the trispecificity antibody format described herein).

[0017] Pharmaceutical compositions comprising antibodies described herein and further defined herein are also provided. Antibody molecules and pharmaceutical compositions, all as defined herein, for use in methods of treating the human or animal body, for example, in methods of treating cancer, are also provided. Methods of treating cancer, comprising administering antibodies or pharmaceutical compositions as defined herein, are also provided.

[0018] Nucleic acids, vectors, and host cells as defined herein are also provided. Methods for producing antibodies as defined herein are also provided. This disclosure encompasses combinations of the embodiments and features described herein, unless such combinations are obviously impossible or expressly avoided. [Brief explanation of the drawing]

[0019] Next, examples and experiments demonstrating the principles of this disclosure will be explained with reference to the attached drawings. [Figure 1A] (A) shows the parental AZ Vκ+AZ VH IgG1-TM antibody, which binds to HPB-ALL cells expressing human CD3 (hCD3+). [Figure 1B] (B) shows the parental AZ Vκ+AZ VH IgG1-TM antibody. (B) shows the parental anti-CD3 AZ Vκ+AZ VH IgG1-TM antibody that binds to HSC-F cells expressing cynomolgus monkey CD3 (cyCD3+). [Figure 1C](C) shows the parental AZ Vκ+AZ VH IgG1-TM antibody. (C) shows the parental anti-CD3 AZ Vκ+AZ VH IgG1-TM antibody that binds to Jurkat hCD3+ cells. [Figure 1D] (D) shows the parental AZ Vκ+AZ VH IgG1-TM antibody. (D) shows the parental anti-CD3 AZ Vκ+AZ VH IgG1-TM antibody that binds to T cell receptor (TCR) knockout (KO) Jurkat cells. [Figure 2A] Flow cytometry measures the binding of mutants to CD3+ cells resulting from the mitigation of potential sequence defects in CDR L1-2 and CDR H1-2. (A) shows the binding of the parental anti-CD3 antibody and the mutant to HPB-ALL cells expressing human CD3 (hCD3+). [Figure 2B] (B) shows the binding of mutants resulting from the mitigation of potential sequence defects in CDR L1-2 and CDR H1-2 to CD3+ cells, as measured by flow cytometry. (B) shows the binding of the parental anti-CD3 antibody and the AZ mutant to HSC-F cells expressing cynomolgus monkey CD3 (cyCD3+). [Figure 2C] Flow cytometry measures the binding of mutants to CD3+ cells resulting from the mitigation of potential sequence defects in CDR L1-2 and CDR H1-2. (C) shows the binding of the parental anti-CD3 antibody and the mutant to Jurkat hCD3+ cells. [Figure 2D] (D) shows the binding of mutants resulting from the mitigation of potential sequence defects in CDR L1-2 and CDR H1-2 to CD3+ cells, as measured by flow cytometry. (D) shows the binding of the parental anti-CD3 antibody and AZ mutant to T cell receptor (TCR) knockout (KO) Jurkat cells. [Figure 3A] The results of profiling anti-CD3 mutants regarding CD3+ cell binding are shown. (A) Binding to HPB-ALL (hCD3+) cells and (B) Binding to HSC-F (cyCD3+) cells were measured by flow cytometry. [Figure 3B]The results of profiling anti-CD3 mutants regarding CD3+ cell binding are shown. (A) Binding to HPB-ALL (hCD3+) cells and (B) Binding to HSC-F (cyCD3+) cells were measured by flow cytometry. [Figure 4] This report presents the results of profiling anti-CD3 variants for EGFR+ cytotoxicity. The cytotoxicity profiles of EGFR-CD3 variant antibodies were evaluated by flow cytometry after 24-hour exposure of T cells to EGFR+NCI H358. Data representing the parental anti-CD3 variant are indicated by filled black circles. Variants based on data 17, 26, and 29 representing AZ Vκ are shown in dark gray, medium gray, and light gray, respectively. [Figure 5] This report presents the results of profiling anti-CD3 variants for CD4+ T cell activation. The CD4+ T cell activation profiles of EGFR-CD3 variant antibodies were evaluated by flow cytometry after 24-hour exposure of T cells to EGFR+NCI H358. Data representing the parental anti-CD3 variant are indicated by filled black circles. Variants based on data 17, 26, and 29 representing AZ Vκ are shown in dark gray, medium gray, and light gray, respectively. [Figure 6] This report presents the results of profiling anti-CD3 variants for CD8+ T cell activation. The CD8+ T cell activation profiles of EGFR-CD3 variant antibodies were evaluated by flow cytometry after 24-hour exposure of T cells to EGFR+NCI H358. Data representing the parental anti-CD3 variant are indicated by filled black circles. Variants based on data 17, 26, and 29 representing AZ Vκ are shown in dark gray, medium gray, and light gray, respectively. [Figure 7]The results of profiling anti-CD3 variants for IL-6 release are shown. The IL-6 cytokine release profiles of EGFR-CD3 variant antibodies were evaluated using a ProcartaPlex multiplex immunoassay after 24 hours of exposure of T cells to EGFR+NCI H358. Data representing the parental anti-CD3 variant are indicated by filled black circles. Variants based on data 17, 26, and 29 representing AZ Vκ are shown in dark gray, medium gray, and light gray, respectively. [Figure 8] The results of profiling anti-CD3 variants for TNF-α release are shown. The TNF-α cytokine release profiles of EGFR-CD3 variant antibodies were evaluated using a ProcartaPlex multiplex immunoassay after 24 hours of exposure of T cells to EGFR+NCI H358. Data representing the parental anti-CD3 variant are indicated by filled black circles. Variants based on data 17, 26, and 29 representing AZ Vκ are shown in dark gray, medium gray, and light gray, respectively. [Figure 9] The results of profiling anti-CD3 variants for IFN-γ release are shown. IFN-γ cytokine release profiles of EGFR-CD3 variant antibodies were evaluated using a ProcartaPlex multiplex immunoassay after 24 hours of exposure of T cells to EGFR+NCI H358. Data representing the parental anti-CD3 variant are indicated by filled black circles. Variants based on data 17, 26, and 29 representing AZ Vκ are shown in dark gray, medium gray, and light gray, respectively. [Figure 10] The results of profiling anti-CD3 variants for IL-2 release are shown. The IL-2 cytokine release profiles of EGFR-CD3 variant antibodies were evaluated using a ProcartaPlex multiplex immunoassay after 24 hours of exposure of T cells to EGFR+NCI H358. Data representing the parental anti-CD3 variant are indicated by filled black circles. Variants based on data 17, 26, and 29 representing AZ Vκ are shown in dark gray, medium gray, and light gray, respectively. [Figure 11] The results of profiling anti-CD3 variants for IL-10 release are shown. The IL-10 cytokine release profiles of EGFR-CD3 variant antibodies were evaluated using a ProcartaPlex multiplex immunoassay after 24 hours of exposure of T cells to EGFR+NCI H358. Data representing the parental anti-CD3 variant are indicated by filled black circles. Variants based on data 17, 26, and 29 representing AZ Vκ are shown in dark gray, medium gray, and light gray, respectively. [Figure 12] The results of profiling anti-CD3 variants for FasL release are shown. FasL cytokine release profiles of EGFR-CD3 variant antibodies were evaluated using a ProcartaPlex multiplex immunoassay after 24 hours of exposure of T cells to EGFR+NCI H358. Data representing the parental anti-CD3 variant are indicated by filled black circles. Variants based on data 17, 26, and 29 representing AZ Vκ are shown in dark gray, medium gray, and light gray, respectively. [Figure 13] The results of profiling anti-CD3 mutants for granzyme A release are shown. The granzyme A cytokine release profiles of EGFR-CD3 mutant antibodies were evaluated using a ProcartaPlex multiplex immunoassay after 24 hours of exposure of T cells to EGFR+NCI H358. Data representing the parental anti-CD3 mutant are shown as filled black circles. Mutants based on data 17, 26, and 29 representing AZ Vκ are shown in dark gray, medium gray, and light gray, respectively. [Figure 14]The results of profiling anti-CD3 mutants for granzyme B release are shown. The granzyme B cytokine release profiles of EGFR-CD3 mutant antibodies were evaluated using a ProcartaPlex multiplex immunoassay after T cells were exposed to EGFR+NCI H358 for 24 hours. Data representing the parental anti-CD3 mutant are shown as filled black circles. Mutants based on data 17, 26, and 29 representing AZ Vκ are shown in dark gray, medium gray, and light gray, respectively. [Figure 15] The results of profiling anti-CD3 variants for IL-17A release are shown. The IL-17A cytokine release profiles of EGFR-CD3 variant antibodies were evaluated using a ProcartaPlex multiplex immunoassay after 24 hours of exposure of T cells to EGFR+NCI H358. Data representing the parental anti-CD3 variant are indicated by filled black circles. Variants based on data 17, 26, and 29 representing AZ Vκ are shown in dark gray, medium gray, and light gray, respectively. [Figure 16] The results of profiling anti-CD3 variants for perforin release are shown. Perforin cytokine release profiles of EGFR-CD3 variant antibodies were evaluated using a ProcartaPlex multiplex immunoassay after 24 hours of exposure of T cells to EGFR+NCI H358. Data representing the parental anti-CD3 variant are indicated by filled black circles. Variants based on data 17, 26, and 29 representing AZ Vκ are shown in dark gray, medium gray, and light gray, respectively. [Figure 17A] This study demonstrates EGFR+-dependent cytotoxicity and T cell activation for selected anti-CD3 variants. The cytotoxicity (A), CD4+ T cell activation (B), and CD8+ T cell activation (C) profiles of selected EGFR-CD3 variant antibodies were evaluated by flow cytometry after 24 hours of T cell exposure to EGFR+MDA-MB-468. The results show an EGFR-dependent response. [Figure 17B]This study demonstrates EGFR+-dependent cytotoxicity and T cell activation for selected anti-CD3 variants. The cytotoxicity (A), CD4+ T cell activation (B), and CD8+ T cell activation (C) profiles of selected EGFR-CD3 variant antibodies were evaluated by flow cytometry after 24 hours of T cell exposure to EGFR+MDA-MB-468. The results show an EGFR-dependent response. [Figure 17C] This study demonstrates EGFR+-dependent cytotoxicity and T cell activation for selected anti-CD3 variants. The cytotoxicity (A), CD4+ T cell activation (B), and CD8+ T cell activation (C) profiles of selected EGFR-CD3 variant antibodies were evaluated by flow cytometry after 24 hours of T cell exposure to EGFR+MDA-MB-468. The results show an EGFR-dependent response. [Figure 18A] This study demonstrates that anti-CD3 mutants exhibit reduced T cell activation compared to parental anti-CD3 in the absence of EGFR+ cells. CD4+(A) and CD8+(B) T cell activation profiles were evaluated by flow cytometry after 48 hours of exposure to immobilized EGFR-CD3 mutant antibodies. Data representing parental anti-CD3 mutants are shown as filled bars. Mutants based on data 17, 26, and 29 representing AZ Vκ are shown as dark gray, medium gray, and light gray bars, respectively. [Figure 18B] This study demonstrates that anti-CD3 mutants exhibit reduced T cell activation compared to parental anti-CD3 in the absence of EGFR+ cells. CD4+(A) and CD8+(B) T cell activation profiles were evaluated by flow cytometry after 48 hours of exposure to immobilized EGFR-CD3 mutant antibodies. Data representing parental anti-CD3 mutants are shown as filled bars. Mutants based on data 17, 26, and 29 representing AZ Vκ are shown as dark gray, medium gray, and light gray bars, respectively. [Figure 19-1]The dynamics of the anti-CD3 mutant are shown. Reaction rates to the soluble form of CD3 were obtained using an Octet384 instrument. The dissociation constant KD was calculated as the koff / kon ratio from a nonlinear fit of the data. [Figure 19-2] The dynamics of the anti-CD3 mutant are shown. Reaction rates to the soluble form of CD3 were obtained using an Octet384 instrument. The dissociation constant KD was calculated as the koff / kon ratio from a nonlinear fit of the data. [Figure 19-3] The dynamics of the anti-CD3 mutant are shown. Reaction rates to the soluble form of CD3 were obtained using an Octet384 instrument. The dissociation constant KD was calculated as the koff / kon ratio from a nonlinear fit of the data. [Figure 20] The diagram includes schematic representations of DuetMab antibodies containing charge pairs. The “hole” HC on the left is disulfide-bonded to kappa LC via native cysteine ​​and contains a kappa charge pair (e.g., S183K / V133E) indicated by a minus ("-") sign on the kappa LC and a plus ("+") sign on the “hole” HC. The “knob” HC on the right is disulfide-bonded to lambda LC via modified cysteine ​​and contains a lambda charge pair indicated by a plus ("+") sign on the lambda LC and a minus ("-") sign on the “knob” HC. [Figure 21]This diagram includes a schematic representation of the DuetMab "2+1" antibody containing charge pairs. The "knob" HC on the right is disulfide-bonded to the lambda LC via a modified cysteine ​​and contains a lambda charge pair indicated by a plus ("+") sign on the lambda LC and a minus ("-") sign on the "knob" HC. The CH1 and VH regions of this knob HC and the lambda LC form a "CD3 antigen binding arm". The "hole" HC on the left is disulfide-bonded to the kappa LC via a native cysteine ​​and contains a kappa charge pair (e.g., S183K / V133E) indicated by a minus ("-") sign on the kappa LC and a plus ("+") sign on the "hole" HC. The third antigen-binding arm is fused by a peptide linker from its CH1 N-terminus to the C-terminus of the "knob" HC. The CH1 of the third antigen-binding arm is disulfide-bonded to kappa LC via native cysteine ​​and contains a kappa charge pair. As indicated by the different shading, the first antigen-binding arm binds to the first epitope (CD3), and the second and third binding arms bind to a second different epitope. [Figure 22]This diagram contains a schematic representation of the "TriMab" triple-specific antibody containing charge pairs. The "knob" HC on the right is disulfide-bonded to the lambda LC via a modified cysteine ​​and contains a lambda charge pair indicated by a plus ("+") sign on the lambda LC and a minus ("-") sign on the "knob" HC. The CH1 and VH regions of this knob HC and lambda LC form the "first antigen binding arm." The "hole" HC on the left is disulfide-bonded to the kappa LC via a native cysteine ​​and contains a kappa charge pair indicated by a minus ("-") sign on the kappa LC and a plus ("+") sign on the "hole" HC. The CH1 and VH regions of this "hole" HC and kappa LC form the "second antigen binding arm." The third antigen binding arm is fused from the N-terminus of its CH1 to the C-terminus of the "knob" HC by a peptide linker. The CH1 of the third antigen-binding arm is disulfide-bonded to kappa LC via a modified cysteine ​​and contains a kappa charge pair having the opposite charge to that of the second antigen-binding arm, this kappa charge pair consisting of the negatively charged amino acid residue ("-") of CH1 and the positively charged amino acid residue ("+") in kappa LC. As indicated by the different shading, the first antigen-binding arm (dark shading) binds to the first epitope, the second antigen-binding arm (light shading) binds to the second epitope, and the third binding arm (shaded shading) binds to the third epitope. [Modes for carrying out the invention]

[0020] Next, aspects and examples of this disclosure will be described with reference to the accompanying drawings. Further aspects and examples will be apparent to those skilled in the art. All documents referenced herein are incorporated herein by reference.

[0021] antibody This disclosure relates to antibodies. The antibodies relating to this disclosure may be provided in an isolated form, meaning that they are free from contaminants such as antibodies that can bind to other polypeptides and / or serum components.

[0022] The terms “antibody” or “antibody molecule” refer to immunoglobulins, whether naturally occurring or partially or entirely synthetically produced. Antibodies can be human or humanized. In some embodiments, antibodies are monoclonal antibodies. Examples of antibodies include immunoglobulin isotypes such as immunoglobulin G (IgG), and their isotype subclasses such as IgG1, IgG2, IgG3, and IgG4, as well as fragments thereof.

[0023] As used herein, the term “antibody” includes multispecific antibodies and antibody fragments. A multispecific antibody is an antibody containing at least two antigen-binding domains, each capable of binding to a different target. An “antibody fragment” refers to a portion of a full-length antibody, insofar as it indicates binding to a relevant target molecule(s). Typically, an antibody fragment contains an antigen-binding region or its variable region.

[0024] Antibodies are typically composed of two different types of polypeptide chains: one called the heavy chain and the other the light chain. Naturally occurring monospecific antibodies consist of two identical heavy chains and two identical light chains. The two heavy chains are linked to each other by disulfide bonds, and each heavy chain is typically linked to a light chain by disulfide bonds. The disulfide bonds linking the light and heavy chains are sometimes called "intra-chain" disulfide bonds to distinguish them from "inter-chain" disulfide bonds present within individual heavy and light chain polypeptides.

[0025] The formation of disulfide bonds between cysteine ​​residues occurs during the folding of many proteins entering the secretory pathway. When a polypeptide chain breaks down, adjacent cysteines can form a covalent bond during a process catalyzed by members of the protein disulfide isomerase family. As used herein, the terms “disulfide link” or “disulfide linked” refer to a single covalent bond formed from the coupling of thiol groups, particularly cysteine ​​residues. In some embodiments, the covalent bond between two cysteines is located between the two sulfur atoms of each residue. However, depending on the environment, not all protein species can always have a disulfide present, for example, in the event of disulfide reduction. Therefore, the terms “disulfide link” or “disulfide linked” (whether natural or modified) also refer, in some embodiments, to the presence of two cysteine ​​residues that can form a disulfide bond, regardless of whether they are actually linked at individual points in time.

[0026] All light chains in natural antibodies are either "lambda λ" or "kappa κ" light chains, which differ in their amino acid sequences. A light chain consists of a single constant light chain region (CL) and a single light chain variable region (VL). An example of a constant light chain lambda region (CLλ) amino acid sequence is provided as SEQ ID NO: 105, and an example of a constant light chain kappa region (CLκ) amino acid sequence is provided as SEQ ID NO: 106. The light chains used in the antibodies described herein may be chimeric light chains, for example, containing CLλ and VLκ.

[0027] The IgG heavy chain consists of a heavy variable (VH) region and three heavy chain constant regions (CH1, CH2, and CH3), with a further "hinge region" between CH1 and CH2. An example of the IgG1 CH1 region amino acid sequence is provided as SEQ ID NO: 101. An example of the IgG1 CH2 amino acid sequence is provided as SEQ ID NO: 102. An example of the IgG1 CH3 amino acid sequence is provided as SEQ ID NO: 103. An example of the heavy chain amino acid sequence including CH1, the hinge, CH2, and CH3 is provided as SEQ ID NO: 104.

[0028] Unless otherwise specified, amino acid residue positions in constant domains, including amino acid sequences, substitutions, deletions, and insertions, described herein are numbered according to EU numbering (Edelman, 2007).

[0029] The light chain associates with VH and CH1 to form an "antigen binding arm" and, as herein referred to, a "target binding arm," and the variable domains in the antigen binding arm interact to form an "antigen binding domain."

[0030] The "antigen-binding domain" represents a portion of a molecule that binds to all or part of a target antigen, and generally contains six complementarity-determining regions (CDRs), three of which are in the VH region: HCDR1, HCDR2, and HCDR3, and three in the VL region: LCDR1, LCDR2, and LCDR3. Together, the six CDRs define a paratope of the antigen-binding domain, which is a portion of the antigen-binding domain that binds to the target antigen.

[0031] The VH and VL regions contain a framework region (FR) on either side of each CDR, which provides a scaffold for the CDR. From the N-terminus to the C-terminus, the VH region has the following structure: N-terminus-[HFR1]-[HCDR1]-[HFR2]-[HCDR2]-[HFR3]-[HCDR3]-[HFR4]-C-terminus, and the VL region has the following structure: N-terminus-[LFR1]-[LCDR1]-[LFR2]-[LCDR2]-[LFR3]-[LCDR3]-[LFR4]-C-terminus. Typically, the antigen-binding domain contains both the VH and VL regions.

[0032] The “antigen-binding arm” described herein also includes an antigen-binding domain (e.g., a VH region and a VL region), as well as CH1 and a constant light chain (i.e., at least one constant domain and one variable domain of the heavy chain and light chain, respectively), the constant light chain being disulfide-bonded to CH1. A monoclonal monospecific IgG antibody molecule comprises two antigen-binding arms, each capable of binding to the same target (i.e., divalent to a single target).

[0033] Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, and Fv fragments. Diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies are antibodies formed from these antibody fragments.

[0034] Traditionally, these fragments were induced by proteolytic digestion of complete antibodies using techniques well known in the art. However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and scFv antibody fragments are all expressed in Escherichia coli (E. coli) and can be secreted from E. coli, thus enabling the easy production of large quantities of these fragments. In one embodiment, antibody fragments can be isolated from antibody phage libraries discussed elsewhere in this specification. Fab'-SH fragments can also be recovered directly from E. coli and chemically conjugated to form F(ab')2 fragments. F(ab')2 fragments can also be isolated directly from recombinant host cell cultures. Other techniques for the production of antibody fragments are well known in the art. In certain embodiments, the antibodies provided herein contain a single-chain Fv fragment (scFv) or other antigen-binding domains.

[0035] In some cases, the antibody fragments described herein include antibodies containing domain antibodies, for example, small functional binding units of an antibody corresponding to the loadable regions of the VH and VL chains of a human antibody.

[0036] CD3 antigen-binding domain This disclosure provides antibodies comprising an antigen-binding domain capable of binding to the CD3 protein or a fragment thereof. Such an antigen-binding domain is also referred to herein as a “CD3 antigen-binding domain.” In some cases, antibodies comprising an antigen-binding domain bind monovalently to CD3 (for example, an antibody contains only a single CD3 antigen-binding domain).

[0037] CD3 (differentiation antigen group 3) is a protein complex composed of four subunits: the CD3γ chain, the CD3δ chain, and two CD3ε chains. CD3 associates with the T cell receptor and the ζ chain to generate an activation signal in T lymphocytes.

[0038] The CD3 antigen-binding domain includes a CDR of an antibody capable of binding to CD3. In some cases, the CD3 antigen-binding domain is the VH region and / or VL region of an antibody that binds to CD3, as exemplified herein, or includes a VH region and / or VL region having at least 70% identity thereto.

[0039] In some cases, the CD3 antigen-binding domain includes the VH region described in any one of (1) to (9) below.

[0040] An antibody comprising an antigen-binding domain capable of binding to the CD3 protein or a fragment thereof, wherein the CD3 antigen-binding domain comprises a heavy chain variable (VH) region as described in any one of the following: (1) VH area containing the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 90, HCDR2 having the amino acid sequence of SEQ ID NO: 91, and HCDR3 having the amino acid sequence of SEQ ID NO: 92, (2) VH area containing the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 82, HCDR2 having the amino acid sequence of SEQ ID NO: 83, and HCDR3 having the amino acid sequence of SEQ ID NO: 84, (3) VH area containing the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 78, HCDR2 having the amino acid sequence of SEQ ID NO: 79, and HCDR3 having the amino acid sequence of SEQ ID NO: 80, (4) VH area containing the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 66, HCDR2 having the amino acid sequence of SEQ ID NO: 67, and HCDR3 having the amino acid sequence of SEQ ID NO: 68, (5) VH area containing the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 70, HCDR2 having the amino acid sequence of SEQ ID NO: 71, and HCDR3 having the amino acid sequence of SEQ ID NO: 72, (6) VH area containing the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 74, HCDR2 having the amino acid sequence of SEQ ID NO: 75, and HCDR3 having the amino acid sequence of SEQ ID NO: 76, (7) VH area containing the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 86, HCDR2 having the amino acid sequence of SEQ ID NO: 87, and HCDR3 having the amino acid sequence of SEQ ID NO: 88, (8) VH area containing the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 94, HCDR2 having the amino acid sequence of SEQ ID NO: 95, and HCDR3 having the amino acid sequence of SEQ ID NO: 96, and (9) VH area containing the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 98, HCDR2 having the amino acid sequence of SEQ ID NO: 99, and HCDR3 having the amino acid sequence of SEQ ID NO: 100.

[0041] In some cases, the CD3 antigen-binding domain includes the VL region described in any one of the following (1) to (3): (10) VL area containing the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 46, HCDR2 having the amino acid sequence of SEQ ID NO: 47, and HCDR3 having the amino acid sequence of SEQ ID NO: 48, (11) VL area containing the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 58, HCDR2 having the amino acid sequence of SEQ ID NO: 59, and HCDR3 having the amino acid sequence of SEQ ID NO: 60, and (12) VL area containing the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 62, HCDR2 having the amino acid sequence of SEQ ID NO: 63, and HCDR3 having the amino acid sequence of SEQ ID NO: 64.

[0042] In some cases, the CD3 antigen-binding domain includes the VH region described in any one of (1) to (9) above and the VL region described in any one of (10) to (12). For example, the CD3 antigen-binding domain is (1) The VH region containing the CDR described in (10), and the VL region containing the CDR described in (10), (2) The VH region containing the CDR described in (2), and the VL region containing the CDR described in (10), (3) The VH region containing the CDR described in (3), and the VL region containing the CDR described in (10), (4) The VH region containing the CDR described in (4), and the VL region containing the CDR described in (10), (5) The VH region containing the CDR described in (5), and the VL region containing the CDR described in (10), (6) The VH region containing the CDR described in (6), and the VL region containing the CDR described in (10), (7) The VH region containing the CDR described in (7), and the VL region containing the CDR described in (10), (8) The VH region containing the CDR described in (8), and the VL region containing the CDR described in (10), (9) The VH region containing the CDR described in (9), and the VL region containing the CDR described in (10), (1) The VH region containing the CDR described in (11), and the VL region containing the CDR described in (11), (2) The VH region containing the CDR described in (2), and the VL region containing the CDR described in (11), (3) The VH region containing the CDR described in (3), and the VL region containing the CDR described in (11), (4) The VH region containing the CDR described in (4), and the VL region containing the CDR described in (11), (5) The VH region containing the CDR described in (5), and the VL region containing the CDR described in (11), (6) The VH region containing the CDR described in (6), and the VL region containing the CDR described in (11), (7) The VH region containing the CDR described in (7), and the VL region containing the CDR described in (11), (8) The VH region containing the CDR described in (8), and the VL region containing the CDR described in (11), (9) The VH region containing the CDR described in (9), and the VL region containing the CDR described in (11), (1) The VH region containing the CDR described in (12), and the VL region containing the CDR described in (12), (2) The VH region containing the CDR described in (2), and the VL region containing the CDR described in (12), (3) The VH region containing the CDR described in (3), and the VL region containing the CDR described in (12), (4) The VH region containing the CDR described in (4), and the VL region containing the CDR described in (12), (5) The VH region containing the CDR described in (5), and the VL region containing the CDR described in (12), (6) The VH region containing the CDR described in (6), and the VL region containing the CDR described in (12), (7) The VH region containing the CDR described in (7), and the VL region containing the CDR described in (12), (8) The VH region containing the CDR described above, and the VL region containing the CDR described above, or (9) may include a VH region containing the CDR described in (9), and a VL region containing the CDR described in (12).

[0043] In some cases, the CD3 antigen-binding domain does not include the VH region containing the CDR described in (1) and the VL region containing the CDR described in (12).

[0044] In some cases, the CD3 antigen-binding domain includes: (13) The VH region including the CDR described in (3), and the VL region including the CDR described in (12), (14) The VH region containing the CDR described in (1), and the VL region containing the CDR described in (10), or (15) A VH region containing the CDR described in (2), and a VL region containing the CDR described in (10).

[0045] In some cases, the CD3 antigen-binding domain includes the VH region described in any one of (16) to (24) below. (16)(1) includes the CDR described above, and includes an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO: 89, or at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, and a VH region, A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 81, or at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, (18)(3) includes the CDR described above, and includes an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO: 77, or at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, and a VH region, A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 65, or at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 69, or at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 73, or at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, including the CDR described in (21)(6), A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 85, or at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, including the CDR described in (22)(7), A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 93, or at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, including the CDR described in (23)(8), A VH region comprising the CDR described in (24)(9), and containing an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO: 97, or at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0046] In some cases, the CD3 antigen-binding domain includes the VL region described in any one of (25) to (27) below: A VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 45, or at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, including the CDR described in (25)(10), A VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 57, or at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, A VL region comprising the CDR described in (27)(12), and containing an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO: 61, or at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0047] In some cases, the CD3 antigen-binding domain includes the VH region described in any one of (16) to (24) above and the VL region described in any one of (25) to (27). For example, the CD3 antigen-binding domain is The VH region described in (16) and the VL region described in (25), The VH region described in (17) and the VL region described in (25), The VH region described in (18) and the VL region described in (25), The VH region described in (19) and the VL region described in (25), The VH region described in (20) and the VL region described in (25), The VH region described in (21) and the VL region described in (25), The VH region described in (22) and the VL region described in (25), The VH region described in (23) and the VL region described in (25), The VH region described in (24) and the VL region described in (25), The VH region described in (16) and the VL region described in (26), The VH region described in (17) and the VL region described in (26), The VH region described in (18) and the VL region described in (26), The VH region described in (19) and the VL region described in (26), The VH region described in (20) and the VL region described in (26), The VH region described in (21) and the VL region described in (26), The VH region described in (22) and the VL region described in (26), The VH region described in (23) and the VL region described in (26), The VH region described in (24) and the VL region described in (26), The VH region described in (16) and the VL region described in (27), The VH region described in (17) and the VL region described in (27), The VH region described in (18) and the VL region described in (27), The VH region described in (19) and the VL region described in (27), The VH region described in (20) and the VL region described in (27), The VH region described in (21) and the VL region described in (27), The VH region described in (22) and the VL region described in (27), The VH region described in (23) and the VL region described in (27), or The VH region described in (24) and the VL region described in (27) may also be included.

[0048] In some cases, the CD3 antigen-binding domain does not include the VH region described in (16) and the VL region described in (27).

[0049] In some cases, the CD3 antigen-binding domain includes: (28) the VH region described in (18), and the VL region described in (27), (29) the VH region described in (16), and the VL region described in (25), or (30) the VH region described in (17), and the VL region described in (25).

[0050] The antibodies described herein may exhibit reduced toxicity due to lower binding affinities, while maintaining T cell activation and thus ensuring T cell involvement. The antibodies described herein may be characterized by a CD3 antigen-binding domain having a specific affinity for CD3. The binding affinity of an antibody molecule for a cognate antigen such as human CD3 (e.g., recombinant human CD3εδ such as CDD-H82W6 available from Acro Biosystems) can be determined by surface plasmon resonance (SPR) using, for example, Octet analysis or Biacore. The binding affinity can be determined using an antibody, for example, as part of a multispecific antibody molecule comprising a CD3 antigen-binding domain and another antigen-binding domain. Alternatively, the binding affinity can be determined using an antibody that is monospecific for CD3.

[0051] Binding affinity is typically K 8 , 7 , d , 8 , 7 , , d ,

[0052] , (equilibrium dissociation constant between the antigen-binding domain and its antigen) and is measured. As is well understood, the lower the K d value, the higher the binding affinity of the antigen-binding domain. For example, an antigen-binding domain that binds to a target with a K d of 10 nM is considered to bind to the same target with a higher affinity than an antigen-binding domain that binds to the same target with a K d of 100 nM.

[0052] In some cases, the CD3 antigen-binding domain binds to human CD3 with an affinity having a K 8 of 1×10 7 M or more, 9×10 7 M or more, 8×10 d M or more. In some cases, the CD3 antigen-binding domain is 1×10 8M~1×10 6 , 9×10 7 M~1×10 6 , or 8×10 7 M~1×10 6 K d It binds to human CD3 with affinity. In some cases, the CD3 antigen-binding domain is 1 × 10⁻¹⁶ 8 M~2×10 6 , 9×10 7 M~2×10 6 , or 8×10 7 M~2×10 6 K d It binds to human CD3 with affinity.Optionally, K d This is measured using surface plasmon resonance, for example, by using Octet analysis as described in the examples.

[0053] The CD3 antigen-binding domain may bind to human CD3 with an affinity similar to that of the antigen-binding domain described in (28), (29), or (30) above. For example, the CD3 antigen-binding domain may have a K factor that is less than 5 times, less than 4 times, less than 3 times, less than 2 times, less than 1 time, or less than 0.5 times different from the antigen-binding domain described in (28), (29), or (30) above. d It may bind to human CD3 with an affinity that has the following properties.

[0054] The CD3 antigen-binding domain may be classified as one that can specifically bind to CD3. The term "specific" may refer to a situation where the antigen-binding domain does not show any significant binding to its specific binding partner(s), in this case, molecules other than CD3. Such molecules are called "non-target molecules." The term "specific" can also be applied when an antibody molecule is specific to a particular epitope held by many antigens (for example, an epitope on CD3), in which case the antibody molecule can bind to various antigens that hold the epitope.

[0055] In some cases, the CD3 antigen-binding domain is considered to exhibit no significant binding to non-target molecules if the degree of binding to non-target molecules is less than approximately 10% of the binding to the target, as measured, for example, by ELISA, SPR, Bio-Layer Interferometry (BLI), Microscale Thermophoresis (MST), or radioimmunoassay (RIA). Alternatively, binding specificity may be reflected in terms of binding affinity, where the CD3 antigen-binding domain described herein binds to CD3 with an affinity at least 0.1 orders of magnitude greater than its affinity to another non-target molecule. In some cases, the CD3 antigen-binding domain described herein binds to CD3 with an affinity at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0 orders of magnitude greater than its affinity to another non-target molecule.

[0056] The CD3 antigen-binding domain may be further classified according to its ability to induce T cell activation. Methods for measuring T cell activation are well known in the art. For example, T cell activation may be evaluated by measuring the upregulation of activation markers such as CD69 and / or CD25 on CD4+ T cells and / or CD8+ T cells using flow cytometry. CD69 is a membrane-bound type II C-lectin receptor and is an early activation marker for T cell activation due to its rapid appearance on the surface of the plasma membrane after T cell activation (Cibrian (Horton) et al., 2017). CD25 is a component of the IL-2 receptor, which is important in T cell proliferation, and is an important late activation marker in T cell proliferation and activation; upregulation of CD25 on the cell surface indicates late T cell activation. Alternative methods for measuring T cell proliferation are envisioned, such as measuring T cell proliferation or measuring the production of effector cytokines (e.g., IFN-γ and TNF-α) (Zappasodi (Horton) et al., 2020).

[0057] Therefore, T cell activation can be measured by using flow cytometry to calculate the percentage of CD4+ T cells and / or CD8+ T cells that are positive for CD69 and / or CD25, as performed in the examples. For example, antigen-binding domains that induce upregulation of CD69 and CD25 in 20% of CD4+ T cells result in decreased T cell activity compared to antigen-binding domains that induce upregulation of CD69 and CD25 in 80% of CD4+ T cells, as measured by flow cytometry.

[0058] As described herein, multispecific antibodies that bind to a target and CD3 are intended to induce T cell activation only upon the association of both target (non-T cell) cells and T cells. Off-target T cell activity may occur when a multispecific antibody induces T cell activation without binding to the target cell. Off-target T cell activation can be measured in T cell activation assays in the absence of cells expressing the target antigen, using a multispecific antibody molecule that includes a CD3 antigen-binding domain and another antigen-binding domain that binds to a different target antigen (e.g., a tumor-associated antigen (TAA) antigen-binding domain).

[0059] In some cases, the CD3 antigen-binding domains described herein exhibit reduced off-target T cell activity compared to a control antigen-binding domain having a VH domain sequence indicated as SEQ ID NO: 5 and a VL domain sequence indicated as SEQ ID NO: 1. In some cases, the CD3 antigen-binding domains described herein exhibit at least 2-fold, at least 3-fold, and at least 4-fold lower off-target T cell activity compared to a control antigen-binding domain having a VH domain sequence indicated as SEQ ID NO: 5 and a VL domain sequence indicated as SEQ ID NO: 1. Off-target T cell activation can be determined using antibodies in a T cell activation assay in the absence of binding to target cells. In some cases, off-target T cell activation is determined by calculating the percentage of CD4+ T cells and / or CD8+ T cells that are positive for CD69 and / or CD25 using flow cytometry in the absence of target cells expressing the target antigen to which the antibody can bind.

[0060] In some cases, the CD3 antigen-binding domains described herein exhibit T cell activity measured by cytokine release by CD4+ and / or CD8+ T cells upon CD3 binding, and T cell activity is reduced compared to the parental anti-CD3 control antibody. The cytokines may be selected from the group consisting of IL-6, TNF-α, INF-γ, IL-10, FasL, granzyme A, granzyme B, IL-17A, and perforin. Methods for measuring cytokine release are well known in the art and include, for example, measuring the concentration of cytokines in the supernatant of T cell cultures using immunoassays such as ELISA. Keeping cytokine release, particularly pro-inflammatory cytokines, under control after T cell activation is beneficial because it reduces the risk of cytokine release syndrome (CRS).

[0061] Overactivation of immune cells such as T cells can lead to CRS, a severe condition characterized by excessive secretion of pro-inflammatory cytokines. In some cases, the antibodies described herein exhibit reduced cytotoxicity compared to parental anti-CD3 control antibodies. Assays for measuring cytotoxicity may include measuring cell death by flow cytometry using propidium iodide (Crowley et al., 2016) or using dyes that covalently bind to free amines on the cell surface and inside the cell (such as CellTrace®).

[0062] multispecific antibodies As described above, the CD3 antigen-binding domain is useful when used in a multispecific antibody format. For example, a multispecific antibody may contain the CD3 antigen-binding domain described herein and a target antigen-binding domain that can bind to targets other than CD3. The target antigen-binding domain may be able to bind to tumor-associated antigens (TAAs). Such a multispecific antibody can be used to simultaneously bind to TAAs expressed on cancer cells and CD3 on T cells, thereby forcing a transient interaction between target cells and T cells, leading to crosslinking, T cell activation, and subsequent antigen-dependent T cell death of the target cells.

[0063] In some cases, the antibody comprises a CD3 antigen-binding domain and a target antigen-binding domain. In some cases, the antibody further comprises an antigen-binding domain (also referred to herein as a TAA antigen-binding domain) capable of binding to TAA. The antibody may be able to bind monovalently to CD3 and monovalently to TAA (for example, the antibody comprises one CD3 antigen-binding domain and one TAA antigen-binding domain).

[0064] Examples of TAA include AFP, a n b3 (vitronectin receptor), a nb6, B-cell maturation agent (BCMA), CA125 (MUC16), CD4, CD20, CD22, CD33, CD52, CD56, CD66e, CD80, CD140b, CD227 (MUC1), EGFR (HER1), EpCAM, GD3 ganglioside, HER2, prostate-specific membrane antigen antigen, PSMA), prostate specific antigen (PSA), CD5, CD19, CD21, CD25, CD37, CD30, CD33, CD45, HLA-DR, anti-idiotype, carcinoembryonic antigen (CEA), such as carcinoembryonic antigen-related cell adhesion molecule 5. Examples include 5, CEACAM5), TAG-72, folate-binding proteins, A33, G250, ferritin, glycolipids such as gangliosides, carbohydrates such as CA-125, IL-2 receptor, fibroblast activation protein (FAP), IGF1 R, B7H3, B7H4, PD-L1, CD200, EphA2, c-Met, and mesothelin or its variants. In some embodiments, TAA includes EGFR, HER2, STEAP2, GPC3, and c-Met.

[0065] In some cases, antibodies include a CD3 antigen-binding domain and a CD8 antigen-binding domain. For example, an antibody may include a CD3 antigen-binding domain, a TAA antigen-binding domain, and a CD8 antigen-binding domain.

[0066] CD8 (differentiation antigen group 8) is a dimer consisting of a pair of CD8 chains. The most common form of CD8 is composed of a CD8-α chain and a CD8-β chain. CD8 acts as a co-receptor on MCHI-restricted T cells, binding to a nearly invariant region of MHCI at a site different from where the T cell receptor binds. +It acts to enhance the antigen sensitivity of T cells. While we do not wish to be bound by theory, including an antigen-binding domain in a multispecific antibody that can bind to CD8 is a possible approach. + This is thought to enable preferential activation of T cells, which may offer superior therapeutic efficacy.

[0067] In some cases, multispecific antibodies are in a "2+1" format. In the "2+1 format," the antibody contains an antigen-binding domain that binds monovalently to a first epitope and two further antigen-binding domains that bind divalently to a second epitope, where the second epitope is distinct from the first. The 2+1 format of multispecific antibodies is well-suited for CD3 binding because the goal is to bind monovalently to the CD3 protein so that the T cell receptor is crosslinked and activated only upon binding to the target cell. Therefore, in some cases, the antibody comprises a CD3 antigen-binding domain as described herein and two target antigen-binding domains that can bind to the same target (e.g., TAA).

[0068] In the "2+1" format, one of the two target antigen-binding domains is fused to one of the other antigen-binding domains present in the antibody, typically via a peptide linker. Suitable peptide linkers are well known in the art and may consist of 5-100 amino acids, 5-50 amino acids, 5-25 amino acids, or 5-15 amino acids. The peptide linker is mainly formed from glycine and serine amino acid residues and may include the amino acid sequence GGGGS (SEQ ID NO: 113) or SGGGGS (SEQ ID NO: 114). In one embodiment, the peptide linker includes or consists of (GGGGS)2 (SEQ ID NO: 115). In some cases, one of the target antigen-binding domains is fused to the CD3 antigen-binding domain (e.g., via a peptide linker).

[0069] Furthermore, multispecific antibodies comprising a CD8 antigen-binding region in addition to the antigen-binding arm of a "2+1" bispecific antibody are described herein. Such antibodies may also be trispecific tetravalent antibodies: one CD3 antigen-binding domain conjugates monovalently to CD3, two target antigen-binding domains conjugate bivalently to a target (e.g., TAA), and a CD8 antigen-binding domain (e.g., VHH) conjugates monovalently to CD8.

[0070] In some cases, the CD8 antigen-binding domain is a single-domain antibody, such as a heavy-chain variable (VH) domain lacking CH1 and a light chain. Heavy-chain variable domains derived from naturally occurring light-chain-lacking heavy-chain antibodies are referred to herein as VHH to distinguish them from the VH of conventional quadruple-chain immunoglobulins. These VHH molecules may originate from antibodies produced in camelid species such as camels, alpacas, dromedaries, llamas, and guanacos. Non-camelid species can also naturally produce light-chain-lacking heavy-chain antibodies, and such VHHs are also included.

[0071] Similar to other non-human antibody fragments, the amino acid sequence of camelid VHH can be modified by recombination to obtain a sequence that more closely mimics the human sequence, i.e., "humanized," thereby reducing the antigenicity of camelid VHH to humans. Furthermore, key elements derived from camelid VHH can be transferred to the human VH domain to obtain a camelidized human VH domain.

[0072] The CD8 antigen-binding domain (e.g., VHH) may be fused to any of the other antigen-binding domains present in the antibody, typically via a peptide linker. Suitable peptide linkers are well known in the art and may consist of 5-100 amino acids, 5-50 amino acids, 5-25 amino acids, or 5-15 amino acids. The peptide linker is mainly formed from glycine and serine amino acid residues and may contain the amino acid sequence GGGGS (SEQ ID NO: 113) or SGGGGS (SEQ ID NO: 114). In one embodiment, the peptide linker contains or consists of (GGGGS)2 (SEQ ID NO: 115). In some cases, the CD8 antigen-binding domain is fused to one of the target antigen-binding domains.

[0073] In some embodiments, the CD8 antigen-binding domain (e.g., VHH) includes the following complementarity-determining region (CDR): i. HCDR1 having the amino acid sequence of SEQ ID NO: 109, SEQ ID NO: 116, or SEQ ID NO: 117, ii. HCDR2 having the amino acid sequence of SEQ ID NO: 110, iii. HCDR3 having the amino acid sequence of SEQ ID NO: 111.

[0074] In some embodiments, the first antigen-binding arm includes a VH region containing an amino acid sequence having at least 70% sequence identity, or at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 112, SEQ ID NO: 118, SEQ ID NO: 119, or SEQ ID NO: 120.

[0075] The VHH proteins of SEQ ID NOs. 118–120 are derivatives of SEQ ID NOs. 112 modified to remove a deamidation site, including one within the CDR1 sequence (see the modified CDR1 sequences of SEQ ID NOs. 116 and 117, which are derivatives of SEQ ID NOs. 109). Therefore, these modified VHH proteins may exhibit improved stability compared to the original VHH of SEQ ID NOs. 112.

[0076] Furthermore, the multispecific antibodies described herein are trispecific trivalent antibodies. In this format, the antibody contains three antigen-binding domains, each monovalently bound to a different epitope. Thus, in some cases, the antibody comprises a CD3 antigen-binding domain, a target (e.g., TAA) antigen-binding domain, and a third antigen-binding domain, as described herein. The third antigen-binding domain may be a CD8 antigen-binding domain, or a target (e.g., TAA) antigen-binding domain that binds to a different target (e.g., TAA) than the target antigen-binding domain. In this trispecific format, each antigen-binding domain comprises a light chain, VH, and CH1 (i.e., at least one constant domain and one variable domain of each light chain), the light chain being disulfide-bonded to CH1 (i.e., each antigen-binding domain is an antigen-binding arm).

[0077] In some embodiments, one of the antigen-binding arms of an antigen1 / antigen2 / CD3 TriMab can bind to an epitope on antigen1 or antigen2 that does not induce cytotoxicity. In some embodiments, the antigen1-binding arm of an antigen1 / antigen2 / CD3 TriMab induces cytotoxicity, while the antigen2-binding arm acts as an anchoring arm that does not have the ability to induce cytotoxicity in cells expressing only antigen2. In some embodiments, the antigen2-binding arm of an antigen1 / antigen2 / CD3 TriMab induces cytotoxicity, while the antigen1-binding arm acts as an anchoring arm that does not have the ability to induce cytotoxicity in cells expressing only antigen1. The inability of the anchoring arm to induce cytotoxicity may be due, for example, to binding to a distal membrane epitope that interferes with the ability to form an active immunological synapse.

[0078] The newly identified lambda charge pair, used in conjunction with the kappa charge pair, can be used to produce a triply specific antibody containing three different antigen-binding arms. In particular, the following was observed: - Efficient pairing of the first antigen-binding arm can be achieved by using the lambda charge pair between CH1 and CLλ of the first antigen-binding arm (e.g., the CD3 antigen-binding arm described herein). - Efficient pairing of the second antigen-binding arm can be achieved by using the kappa charge pair between CH1 and CLκ of the second antigen-binding arm (e.g., the target antigen-binding arm described herein), and y - Efficient pairing of the third antigen-binding arm (e.g., a CD8-targeted antigen-binding arm, or a target antigen-binding domain that binds to a different target than the second antigen-binding arm (e.g., TAA)) is achieved by using the kappa charge pair between CH1 and CLκ of the third antigen-binding arm, where the charged amino acid residue is in the opposite configuration to that of the second antigen-binding arm.

[0079] Having a kappa charge pair in the opposite configuration to the kappa charge pair of the second antigen-binding arm means that if the kappa charge pair of the second antigen-binding arm contains a positively charged amino acid residue on CH1 and a negatively charged amino acid residue on CLκ, then the kappa charge pair of the third antigen-binding arm contains a negatively charged amino acid residue on CH1 and a positively charged amino acid residue on CLκ.

[0080] Steady-state region As described herein, the antigen-binding domain (e.g., the VH and VL regions as defined above) may further include constant domains derived from the heavy and light chains. The antigen-binding domain further including CH1 and a constant light chain, wherein the constant light chain is disulfide-bonded to CH1, is also referred to herein as the "antigen-binding arm" or "Fab region."

[0081] In some cases, the antibodies described herein include one or more immunoglobulin heavy chain constant (CH) regions. In some cases, the CH is or is derived from a heavy chain constant sequence of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM.

[0082] The IgG heavy chain consists of a heavy variable (VH) region and three heavy chain constant regions (CH1, CH2, and CH3), with a further "hinge region" between CH1 and CH2. An example of the IgG1 CH1 region amino acid sequence is provided as SEQ ID NO: 101. An example of the IgG1 CH2 amino acid sequence is provided as SEQ ID NO: 102. An example of the IgG1 CH3 amino acid sequence is provided as SEQ ID NO: 103. An example of the heavy chain amino acid sequence including CH1, the hinge, CH2, and CH3 is provided as SEQ ID NO: 104.

[0083] Each antigen-binding domain may further contain additional heavy chain regions, namely one or more of CH1, hinge, CH2, and CH3. In some cases, the antigen-binding domain contains the complete heavy chain (i.e., VH, CH1, hinge, CH2, and CH3).

[0084] In some cases, the antigen-binding domain includes a CH1 region having an amino acid sequence with at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 101, or at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0085] In some cases, the antigen-binding domain includes a CH2 region having an amino acid sequence with at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 102, or at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0086] In some cases, the antigen-binding domain includes a CH3 region having an amino acid sequence with at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 103, or at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0087] In some cases, the antigen-binding domain includes a heavy chain constant region having an amino acid sequence with at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 104, or at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0088] In some cases, the antibodies described herein include an immunoglobulin light chain constant (CL) region or a fragment thereof. In some cases, the CL region includes a lambda constant (CLλ) region having, for example, at least 70% sequence identity to SEQ ID NO: 105, or at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some cases, the CL region includes a kappa stationary (CLκ) region having, for example, at least 70% sequence identity with respect to sequence number 106, or at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0089] In some cases, the antibody comprises a complete light chain containing, or comprising, the VL region and the CL region as described herein. For example, the antibody may also comprise a heavy chain containing the VL region of the CD3 antigen-binding domain and the CL region (e.g., the CLλ region or CLκ region) as described herein.

[0090] The CH, CL, heavy chain and / or light chain of the antibodies described herein may include one or more modifications for, for example, to inhibit or reduce Fc effector function, to promote the formation of heterodimeric antibody molecules, to increase the effectiveness of congeneral heavy- and light chain pairing, and / or to assist in conjugate formation, as described in more detail below.

[0091] Charge pair In some cases, the antigen-binding arm ("Fab region") of an antibody described herein includes charge pairs located in positions intended to facilitate the association of the light and heavy chains, including a charge pair located in the light chain region (e.g., the constant light chain region) and the other charge pair located in the heavy chain region (e.g., the constant heavy chain region 1 (CH1)). A "lambda charge pair" means a charge pair in which a positively charged or uncharged amino acid residue is located in the lambda light chain. A "kappa charge pair" means a charge pair in which a positively charged or uncharged amino acid residue in the light chain is located in the kappa light chain.

[0092] While we do not wish to be constrained by theory, it is thought that the oppositely charged amino acid residues in the charge pair increase the attractive force of the heavy chain to the light chain in the antigen-binding arm, thereby promoting the formation of an antigen-binding arm with the correct heavy and light chains.

[0093] In some embodiments, at least one of the amino acid residues of the charge pair is modified into an antigen-binding arm (i.e., at least one amino acid residue of the pair is not a wild-type amino acid residue). In some embodiments, both amino acid residues of the charge pair are modified into antigen-binding arms (i.e., neither amino acid residue of the pair is a wild-type amino acid residue).

[0094] Charged amino acid residues are typically found in nature. Examples of naturally occurring positively charged amino acid residues according to this disclosure include arginine, lysine, and histidine. Examples of naturally occurring negatively charged amino acid residues according to this disclosure include glutamic acid, serine, threonine, and aspartic acid. Although serine and threonine are often described as "uncharged" in the art, they have isoelectric points less than 6 and are therefore partially negatively charged at neutral pH. For the purposes of charged pairs as described herein, serine and threonine are examples of negatively charged amino acid residues (along with glutamic acid and aspartic acid).

[0095] Therefore, a charge pair may include a positively charged amino acid residue selected from arginine, lysine, or histidine located at one position of the charge pair, and a negatively charged amino acid residue selected from aspartic acid, glutamic acid, serine, or threonine located at the other position of the charge pair. For example, a charge pair may include any one of the following amino acid residue pairs. Arginine and aspartic acid, Arginine and glutamic acid, Arginine and serine, Arginine and threonine, Lysine and aspartic acid, Lysine and glutamic acid, Lysine and serine, Lysine and threonine, Histidine and aspartic acid, Histidine and glutamic acid, Histidine and serine, and Histidine and threonine.

[0096] In some cases, the positively charged amino acid residue in the charge pair is located on the light chain, and the uncharged amino acid residue in the charge pair is located on the heavy chain. In other cases, the uncharged amino acid residue is located on the light chain, and the positively charged amino acid residue in the charge pair is located on the heavy chain. By introducing charge pairs at certain positions, the correct pairing of light and heavy chains in the antigen-binding arm can be improved.

[0097] In some cases, the antigen-binding arm (e.g., the CD3 antigen-binding arm) includes a lambda charge pair. As demonstrated herein, amino acid residues at the interface between the lambda LC and HC into which charge pairs can be introduced have been identified, and it has been demonstrated that the introduction of these lambda charge pairs can favorably improve chain pairing beyond what was achieved in previous antibody formats lacking lambda charge pairs. In some cases, the lambda charge pair includes a positively charged or uncharged amino acid residue at positions 117, 119, 134, 136, or 178 of the constant light chain lambda region (CLλ). In some embodiments, the lambda charge pair includes a positively charged or uncharged amino acid residue at positions 141, 185, 128, 145, 183, 185, 173, or 187 of CH1, with numbering following EU numbering.

[0098] In some cases, the lambda charge pair is as follows: (i) Position 117 of CLλ and position 141 of CH1, (ii) Position 117 of CLλ and position 185 of CH1, (iii) Position 119 of CLλ and position 128 of CH1, (iv) Position 134 of CLλ and position 128 of CH1, (v) Position 134 of CLλ and position 145 of CH1, (vi) Position 134 of CLλ and position 183 of CH1, (vii) Position 136 of CLλ and position 185 of CH1, (viii) Position 178 of CLλ and position 173 of CH1, (ix) Located at one or more of the pair of positions 117 of CLλ and 187 of CH1.

[0099] In some cases, the lambda charge pair is located at position 117 of CLλ and position 141 of CH1. For example, the lambda charge pair may be selected from the following list. a. Arginine at position 117 of CLλ, and aspartic acid at position 141 of CH1, b. Arginine at position 117 of CLλ, and glutamic acid at position 141 of CH1, c. Arginine at position 117 of CLλ, and serine at position 141 of CH1, d. Arginine at position 117 of CLλ, and threonine at position 141 of CH1, e. Lysine at position 117 of CLλ, and aspartic acid at position 141 of CH1, f. Lysine at position 117 of CLλ, and glutamic acid at position 141 of CH1, g. Lysine at position 117 of CLλ, and serine at position 141 of CH1, and h. Lysine at position 117 of CLλ, and threonine at position 141 of CH1.

[0100] In some cases, the lambda charge pair is selected from any one of a. to f. in the list above. In some embodiments, the lambda charge pair is selected from any one of a. to e. in the list above. In some embodiments, the lambda charge pair is selected from any one of a, b, and e in the list above. In some embodiments, the lambda charge pair is a.

[0101] In some cases, the lambda charge pair is located at position 117 of CLλ and position 185 of CH1. For example, the lambda charge pair may be selected from the following list. a. Arginine at position 117 of CLλ, and aspartic acid at position 185 of CH1, b. Arginine at position 117 of CLλ, and glutamic acid at position 185 of CH1, c. Arginine at position 117 of CLλ, and serine at position 185 of CH1, d. Arginine at position 117 of CLλ, and threonine at position 185 of CH1, e. Lysine at position 117 of CLλ, and aspartic acid at position 185 of CH1, f. Lysine at position 117 of CLλ, and glutamic acid at position 185 of CH1, g. Lysine at position 117 of CLλ, and serine at position 185 of CH1, and h. Lysine at position 117 of CLλ, and threonine at position 185 of CH1.

[0102] In some cases, the lambda charge pair is located at position 119 of CLλ and position 128 of CH1. For example, the lambda charge pair may be selected from the following list. a. Arginine at position 119 of CLλ, and aspartic acid at position 128 of CH1, b. Arginine at position 119 of CLλ, and glutamic acid at position 128 of CH1, c. Arginine at position 119 of CLλ, and serine at position 128 of CH1, d. Arginine at position 119 of CLλ, and threonine at position 128 of CH1, e. Lysine at position 119 of CLλ, and aspartic acid at position 128 of CH1, f. Lysine at position 119 of CLλ, and glutamic acid at position 128 of CH1, g. Lysine at position 119 of CLλ, and serine at position 128 of CH1, and h. Lysine at position 119 of CLλ, and threonine at position 128 of CH1.

[0103] In some embodiments, the lambda charge pair is located at position 134 of CLλ and position 128 of CH1. For example, the lambda charge pair may be selected from the following list. a. Arginine at position 134 of CLλ, and aspartic acid at position 128 of CH1, b. Arginine at position 134 of CLλ, and glutamic acid at position 128 of CH1, c. Arginine at position 134 of CLλ, and serine at position 128 of CH1, d. Arginine at position 134 of CLλ, and threonine at position 128 of CH1, e. Lysine at position 134 of CLλ, and aspartic acid at position 128 of CH1, f. Lysine at position 134 of CLλ, and glutamic acid at position 128 of CH1, g. Lysine at position 134 of CLλ, and serine at position 128 of CH1, h. Lysine at position 134 of CLλ, and threonine at position 128 of CH1.

[0104] In some cases, the lambda charge pair is located at position 134 of CLλ and position 145 of CH1. For example, the lambda charge pair may be selected from the following list. a. Arginine at position 134 of CLλ, and aspartic acid at position 145 of CH1, b. Arginine at position 134 of CLλ, and glutamic acid at position 145 of CH1, c. Arginine at position 134 of CLλ, and serine at position 145 of CH1, d. Arginine at position 134 of CLλ, and threonine at position 145 of CH1, e. Lysine at position 134 of CLλ, and aspartic acid at position 145 of CH1, f. Lysine at position 134 of CLλ, and glutamic acid at position 145 of CH1, g. Lysine at position 134 of CLλ, and serine at position 145 of CH1, h. Lysine at position 134 of CLλ, and threonine at position 145 of CH1.

[0105] In some cases, the lambda charge pair is located at position 134 of CLλ and position 183 of CH1. For example, the lambda charge pair may be selected from the following list. a. Arginine at position 134 of CLλ, and aspartic acid at position 183 of CH1, b. Arginine at position 134 of CLλ, and glutamic acid at position 183 of CH1, c. Arginine at position 134 of CLλ, and serine at position 183 of CH1, d. Arginine at position 134 of CLλ, and threonine at position 183 of CH1, e. Lysine at position 134 of CLλ, and aspartic acid at position 183 of CH1, f. Lysine at position 134 of CLλ, and glutamic acid at position 183 of CH1, g. Lysine at position 134 of CLλ, and serine at position 183 of CH1, and h. Lysine at position 134 of CLλ, and threonine at position 183 of CH1.

[0106] In some cases, the lambda charge pair is aspartic acid or serine at the lysine position 134 of CLλ and at the 183 position of CH1.

[0107] In some cases, the lambda charge pair is located at position 136 of CLλ and position 185 of CH1. For example, the lambda charge pair may be selected from the following list. a. Arginine at position 136 of CLλ, and aspartic acid at position 185 of CH1, b. Arginine at position 136 of CLλ, and glutamic acid at position 185 of CH1, c. Arginine at position 136 of CLλ, and serine at position 185 of CH1, d. Arginine at position 136 of CLλ, and threonine at position 185 of CH1, e. Lysine at position 136 of CLλ, and aspartic acid at position 185 of CH1, f. Lysine at position 136 of CLλ, and glutamic acid at position 185 of CH1, g. Lysine at position 136 of CLλ, and serine at position 185 of CH1, h. Lysine at position 136 of CLλ, and threonine at position 185 of CH1.

[0108] In some cases, the lambda charge pair is located at position 178 of CLλ and position 173 of CH1. For example, the lambda charge pair can be selected from the following list. a. Arginine at position 178 of CLλ, and aspartic acid at position 173 of CH1, b. Arginine at position 178 of CLλ, and glutamic acid at position 173 of CH1, c. Arginine at position 178 of CLλ, and serine at position 173 of CH1, d. Arginine at position 178 of CLλ, and threonine at position 173 of CH1, e. Lysine at position 178 of CLλ, and aspartic acid at position 173 of CH1, f. Lysine at position 178 of CLλ, and glutamic acid at position 173 of CH1, g. Lysine at position 178 of CLλ, and serine at position 173 of CH1, h. Lysine at position 178 of CLλ, and threonine at position 173 of CH1.

[0109] In some cases, the antigen-binding arm containing lambda charge pairs contains two or more lambda charge pairs. For example, the antigen-binding arm may contain 2, 3, 4, 5, 6, 7, 8, or 9 lambda charge pairs at positions (i) to (ix) above.

[0110] In some cases, the antigen-binding arm in an antibody contains a kappa charge pair. Introducing the kappa charge pair at certain positions can improve the correct pairing of the light and heavy chains in the antigen-binding arm. As described above, the kappa charge pair refers to a positively charged amino acid residue and an uncharged amino acid residue, one of which is located on the kappa light chain (e.g., CLκ) and the other on the heavy chain of the antigen-binding arm (e.g., CH1), in a position intended to facilitate the association of the light chain of the antigen-binding arm with CH1.

[0111] In some cases, the antigen-binding arm contains a kappa charge pair located at position 133 of CLκ and position 183 of CH1. In some cases, the loaded amino acid residue in the kappa charge pair is at position 133 of CLκ, and the positively charged amino acid residue in the kappa charge pair is at position 183 of CH1. In other cases, the positively charged amino acid residue in the kappa charge pair is at position 133 of CLκ, and the loaded amino acid residue in the kappa charge pair is at position 183 of CH1. In some cases, the loaded amino acid residue (e.g., position 133 of CLκ) is glutamic acid, and the positively charged amino acid residue (e.g., position 183 of CH1) is lysine. As mentioned elsewhere, this numbering follows EU numbering.

[0112] In some cases, one of the antigen-binding arms in an antibody contains a lambda charge pair, and another (different) antigen-binding arm in the antibody contains a kappa charge pair. As illustrated herein, the CD3 antigen-binding arm may contain a lambda charge pair, and the target antigen-binding arm (e.g., the TAA antigen-binding arm) may contain a kappa charge pair. Alternatively, the target antigen-binding arm (e.g., the TAA antigen-binding arm) may contain a lambda charge pair, and the CD3 antigen-binding arm may contain a kappa charge pair.

[0113] In this specification, a singular reference (e.g., "a" or "the") to a charge pair or domain also includes multiple charge pairs or multiple domains, unless the context clearly indicates otherwise.

[0114] The charge pairs described herein may be combined with other strategies to promote heterodimerization in order to further increase the correct pairing of heavy-chain and light-chain polypeptides.

[0115] Non-limiting examples of strategies for promoting heterodimerization are described in more detail below and include disulfide modifications at the CH1 / CL interface, introduction of additional charge pairs (e.g., kappa charge pairs), and Fc region modifications such as knob-into-hole, as well as the use of strategies that enable fractionation and purification.

[0116] Fc region modification In some cases, the antibodies described herein include one or more modifications in one or more of the CH1, CH2, and CH3 domains that promote the formation of heterodimeric antibody molecules by promoting the formation of Fc regions present in the antibody. This may include the Knobs into Holes (KiH) strategy based on single amino acid substitutions in the CH3 domain that promote heavy chain heterodimerization, as described by Ridgway, 1996. The knob mutant heavy chain CH3 has a small amino acid substituted with a larger amino acid, thereby creating a bump (knob) on the surface of the CH3 domain, while the hole mutant has a large amino acid substituted with a smaller amino acid, thereby creating a cavity (hole) on the surface of the CH3 domain. Further modifications may also be introduced to stabilize the association between heavy chains.

[0117] Examples of CH3 modifications to enhance heterodimerization include the "hole" mutation Y407V / T366S / L368A on one Fc region and the "knob" mutation T366W on the other Fc region. These may further include stabilizing the cystine mutation Y349C (e.g., on the Fc region with the "hole" mutation) and stabilizing the S354C mutation on the other Fc region (e.g., on the Fc region with the "knob" mutation).

[0118] Therefore, in one case, the substitution to generate the knob is the substitution of tryptophan at position 366, and the substitution to generate the hole is one or more of the following: i) Substitution with valine at position 407, ii) Substitution with serine at position 366, and iii) Substitution with alanine at position 368.

[0119] For example, one Fc region may include modifications to enable fractional elution by protein A chromatography, as described by Tustian (Barton), 2016. In short, one of the Fc regions may include modifications to enable fractional elution by protein A chromatography. * This modification includes removing the bond to the heterodimer FcFc (called * This enables the selective purification of bispecific products. Fc * Suitable modifications for generating the region include substitution of H435 with arginine and substitution of Y436 with phenylalanine.

[0120] Other Fc modifications that may be used in addition to those used to enhance heterodimerization include those that reduce or inhibit the binding of antibody molecules to one or more Fcγ receptors and / or complement, such as FcγRI, FcγRIIa, FcγRIIb, and FcγRIII. Such mutations reduce or inhibit Fc effector function. Mutations that reduce or inhibit the binding of antibody molecules to one or more Fcγ receptors and / or complement are well known, such as the L234F / L235E / P331S “triple mutation” or “TM” (according to the European Union numbering rules) described by Organosesyan et al., Acta Crystallogr D Biol Crystallogr 64(6):700-704, 2008.

[0121] In some embodiments, the CH2 domain of one or both of the constant domains of the immunoglobulin heavy chain contains the following substitution: E233P / L234V / L235A / G236del / S267K. This combination of mutations may be referred to herein as an "Fc effector null mutation."

[0122] Other suitable Fc region amino acid substitutions or modifications are well known in the art and include, for example, a triple substitution (M252Y / S254T / T256E, referred to as the "YTE" or "YTE" mutation) numbered according to the EU index, such as in Kabat, of methionine (M) to tyrosine (Y) substitution at position 252, serine (S) to threonine (T) substitution at position 254, and threonine (T) to glutamic acid (E) substitution at position 256 (M252Y / S254T / T256E) (see, e.g., U.S. Patent No. 7,658,921, U.S. Patent Application Publication No. 2014 / 0302058, and Yu et al., Antimicrob. Agents Chemother., 61(1):e01020-16 (2017) (each of these is incorporated herein by reference in whole)). This combination of mutations may extend the half-life of the antibody.

[0123] Triple mutations, Fc effector null mutations, and YTE mutations, if present, may be located in one or both of the heavy chain constant domains. Typically, if present, they are located in both of the heavy chain constant domains.

[0124] In some embodiments, the Fc region includes YTE mutations and triple mutations. In other embodiments, the Fc region includes YTE mutations and Fc effector null mutations.

[0125] Modified disulfide In some cases, antibodies contain engineered disulfides. "Engineered disulfide" means that the natural interchain disulfide bond at the CH1-CL interface of the antigen-binding arm (e.g., 220 of CH1 and 212 of LC), and optionally one or more additional antigen-binding arms, are replaced by engineered (non-natural) interchain disulfides. Engineered disulfides are typically formed by modifying cysteine ​​at CL of the light chain and the corresponding CH1 of the heavy chain, replacing the cysteine ​​that normally forms the interchain disulfide. Disclosures relating to the introduction of engineered disulfides into antibodies for the purpose of promoting heterodimerization can be found, for example, in U.S. Patent No. 9,527,927, which is incorporated herein by reference in its entirety.

[0126] In some cases, a disulfide bond is formed between the light chain and at least one CH1 of the antigen-binding arm between a pair of modified cysteines of the light chain and the CH1 of its antigen-binding arm.

[0127] In some cases, (i) A disulfide bond is formed between the light chain of the CD3 antigen-binding arm and CH1 between the light chain of the CD3 antigen-binding arm and the pair of cysteines modified to CH1, and a disulfide bond is formed between the light chain of the target antigen-binding arm and CH1 between the pair of native cysteines. A schematic diagram of this embodiment is shown in Figure 20, or (ii) A disulfide bond is formed between the light chain of the target antigen-binding arm and CH1, between the light chain of the target antigen-binding arm and the pair of cysteines modified to CH1, and a disulfide bond is formed between the light chain of the CD3 antigen-binding arm and CH1, between the pair of native cysteines.

[0128] In some cases, if the antibody is in the “2+1” format as described herein, (i) The disulfide bond between the light chain of the CD3 antigen-binding arm and CH1 is formed between the light chain of the CD3 antigen-binding arm and a pair of cysteine ​​modified to CH1, and the disulfide bond between the light chains of the two target antigen-binding arms and CH1 is formed between a pair of native cysteine. A schematic diagram of this embodiment is shown in Figure 21. (ii) Disulfide bonds between the light chains of the two target antigen-binding arms and CH1 are formed between the pair of cysteine ​​modified to the light chains of the two target antigen-binding arms and CH1, and disulfide bonds between the light chain of the CD3 antigen-binding arm and CH1 are formed between the pair of native cysteine. (iii) All three antigen-binding arms contain modified disulfides, but the cysteine ​​modified in the CD3 antigen-binding arm is located in a different position (e.g., in the light chain) than the cysteine ​​modified in the target antigen-binding arm.

[0129] In some cases, if the antibody is in the triple-specific format described herein, the disulfide bond between the light chain of the first antigen-binding arm (e.g., the CD3 antigen-binding arm) and CH1 is formed between the light chain of the first antigen-binding arm and a pair of cysteines modified to CH1; the disulfide bond between the light chain of the second antigen-binding arm (e.g., the target antigen-binding arm) and CH1 is formed between a pair of native cysteines of the second antigen-binding arm; and the disulfide bond between the light chain of the third antigen-binding arm (e.g., the CD8 target antigen-binding arm, or a target antigen-binding domain that binds to a different target than the second antigen-binding arm) and CH1 is formed between the light chain of the first antigen-binding arm and a pair of cysteines modified to CH1. A schematic diagram of this example is shown in Figure 22.

[0130] If both the first and third antigen-binding arms contain modified disulfides, it is desirable that the modified disulfide introduced into the third antigen-binding arm be different from the modified disulfide introduced into the first antigen-binding arm, as this is thought to further improve correct pairing. Therefore, optionally, the cysteine ​​pair inserted into the third antigen-binding arm is located at a different amino acid residue position than the cysteine ​​pair inserted into the first antigen-binding arm.

[0131] As described above, the light chain may contain CLλ or CLκ. In some cases, the pair of modified cysteines of CLλ and CH1 are located at position 122 of CLλ and position 126 of CH1, the same CLλ contains a non-cysteine ​​residue at position 212, and the same CH1 contains a non-cysteine ​​residue at position 220. In some cases, the non-cysteine ​​residue is valine.

[0132] An exemplary amino acid sequence of CLλ containing the modified cysteine ​​is provided as SEQ ID NO: 107, and an exemplary amino acid sequence of CH1 containing the corresponding modified cysteine ​​for forming the modified disulfide is provided as SEQ ID NO: 108.

[0133] In some cases, a pair of modified cysteines in the constant light chain kappa region (CLκ) and CH1 are located at position 121 of CLκ and position 126 of CH1, the same CLκ containing a non-cysteine ​​residue at position 214, and the same CH1 containing a non-cysteine ​​residue at position 220. In some cases, the non-cysteine ​​residue is valine.

[0134] In some cases, the antibody is in the “DuetMab” format, as described, for example, in U.S. Patent No. 9,527,927. DuetMab includes a modified disulfide to enhance correct heavy / light chain pairing and an Fc region modification to enhance correct heterodimerization of the heavy chain.

[0135] In some embodiments, the antibody is The above-mentioned CD3 antigen-binding arm including the first Fc region, and The above-mentioned target antigen-binding arm includes a second Fc region, The first Fc region and the second Fc region include modifications to promote heterodimerization of the first Fc region and the second Fc region.

[0136] In some embodiments, the antibody is The above-mentioned CD3 antigen-binding arm including the first Fc region, and The above-mentioned target antigen-binding arm includes a second Fc region, The first and second Fc regions include modifications to promote heterodimerization of the first and second Fc regions, and the antibody contains the modified disulfide.

[0137] In some embodiments, the antibody is The CD3 antigen-binding arm includes the lambda charge pair and the first Fc region, and The above-mentioned target antigen-binding arm includes a second Fc region, The first Fc region and the second Fc region include modifications to promote heterodimerization of the first Fc region and the second Fc region.

[0138] In some embodiments, the antibody is The CD3 antigen-binding arm includes the above-mentioned lambda charge pair and the first Fc region, and The above-mentioned target antigen-binding arm includes a second Fc region, The first and second Fc regions include modifications to promote heterodimerization of the first and second Fc regions, and the antibody contains the modified disulfide.

[0139] In some embodiments, the antibody is The CD3 antigen-binding arm includes the above-mentioned lambda charge pair and the first Fc region, and It includes the above-mentioned kappa charge pair and a target antigen-binding arm comprising a second Fc region, The first Fc region and the second Fc region include modifications to promote heterodimerization of the first Fc region and the second Fc region.

[0140] In some embodiments, multispecific antibodies are The CD3 antigen-binding arm includes the above-mentioned lambda charge pair and the first Fc region, and It includes the above-mentioned kappa charge pair and a target antigen-binding arm comprising a second Fc region, The first and second Fc regions include modifications to promote heterodimerization of the first and second Fc regions, and the multispecific antibody contains the modified disulfide.

[0141] Non-limiting examples of antibodies, including lambda charge pairs, kappa charge pairs, modified disulfides, and modifications to promote heterodimerization of the first and second Fc regions, are provided in the examples.

[0142] Sequence identity and mutation In some cases, the CLλ described herein comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 105 or SEQ ID NO: 107. In some cases, the CLλ comprises an amino acid sequence of SEQ ID NO: 105 or SEQ ID NO: 107 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications.

[0143] In some cases, the CLκ described herein comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 106. In some cases, the CLκ comprises an amino acid sequence of SEQ ID NO: 106 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications.

[0144] In some cases, CH1 contains an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 101 or SEQ ID NO: 108. In some cases, CH1 contains an amino acid sequence of SEQ ID NO: 101 or SEQ ID NO: 108 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications.

[0145] In some cases, CH2 contains an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 102. In some cases, CH2 contains an amino acid sequence of SEQ ID NO: 102 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications.

[0146] In some cases, CH3 contains an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 103. In some cases, CH3 contains an amino acid sequence of SEQ ID NO: 103 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications.

[0147] One, two, three, four, five, six, seven, eight, nine, or ten amino acid modifications in the constant regions (CLλ, CLκ, CH1, CH2, and CH3) may be added to the above modifications for introducing the charge pair, modified disulfide, and / or Fc region modifications. For example, compared to the wild-type CLλ described in SEQ ID NO: 105, the CLλ used in the antibody may contain a lambda charge pair mutation, a modified disulfide (e.g., S122C and C212V), and one, two, three, four, five, six, seven, eight, nine, or ten further amino acid modifications. As another example, compared to the wild-type CH1 provided in SEQ ID NO: 101, the CH1 used in the antibody may contain a lambda charge mutation, a modified disulfide (e.g., F126C, C220V), and one, two, three, four, five, six, seven, eight, nine, or ten further amino acid modifications.

[0148]

[0148] In some cases, the CD3 antigen-binding domain comprises a VH region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence defined in (16)-(24) above. In some cases, the CD3 antigen-binding domain comprises a VH region having the amino acid sequence defined in (16)-(24) above with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications.

[0149]

[0148] In some cases, the CD3 antigen-binding domain comprises a VL region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence defined in (25)-(27) above. In some cases, the CD3 antigen-binding domain comprises a VL region having the amino acid sequence defined in (25)-(27) above with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications.

[0150]

[0148] The CLλ region described herein comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 105 or SEQ ID NO: 107. In some cases, CLλ comprises the amino acid sequence of SEQ ID NO: 105 or SEQ ID NO: 107 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications.

[0151]

[0148] In some cases, the CD3 antigen-binding domain comprises a VH region described in any one of (16)-(24) below.

[0152]

[0148] The amino acid modification may be an insertion, substitution, or deletion. In some embodiments, the amino acid modification is a substitution of an amino acid residue with any other naturally occurring or non-naturally occurring amino acid residue.

[0153]

[0148] Naturally occurring residues may be classified into classes based on common side-chain characteristics. 1) Nonpolar aliphatic compounds: Glycine (G), methionine (M), alanine (A), valine (V), leucine (L), isoleucine (I), 2) Polarity: Cysteine ​​(C), asparagine (N), glutamine (Q), proline (P), 3) Polarity, partial load: serine (S), threonine (T), 4) Acidic (loaded electricity): Aspartic acid (D), Glutamic acid (E), 5) Basic (positively charged): Histidine (H), Lysine (K), Arginine I, 6) Aromatic compounds: tryptophan (W), tyrosine (Y), phenylalanine (F).

[0154] As described above, serine (S) and threonine (T) have isoelectric points less than 6 and are partially negatively charged at neutral pH; therefore, they are classified as “polar, partially negatively charged” in this specification.

[0155] Amino acid substitutions may be conservative amino acid substitutions. Conservative amino acid substitutions may include the exchange of one member of these classes with another member of the same class. For example, a conservative amino acid substitution may be a substitution using the acidic amino acid glutamic acid (E) instead of the acidic amino acid aspartic acid (D).

[0156] Nucleic acids, vectors, and host cells One or more nucleic acids encoding the antibodies described herein are also provided herein. In some cases, the nucleic acids are purified or isolated, for example, from other nucleic acids or naturally occurring biological materials. Those skilled in the art will not have difficulty preparing such nucleic acid molecules using methods well known in the art.

[0157] In some cases, one or more nucleic acids encode the light chain and / or CH1 as described herein. The one or more nucleic acids encoding CH1 may further encode other heavy chain domains, e.g., hinge, CH2 and CH3, or they may encode the complete heavy chain.

[0158] This disclosure also provides one or more vectors comprising nucleic acids encoding antibodies or fragments thereof as described herein. Suitable vectors can be selected or constructed containing appropriate regulatory sequences, including a promoter sequence, a terminator fragment, a polyadenylation sequence, an enhancer sequence, a marker gene, and optionally other sequences. Preferably, the vector contains appropriate regulatory sequences for driving nucleic acid expression in host cells. The vector may optionally be a plasmid, a virus, such as a phage, or a phagemid.

[0159] Antibodies may be produced from light chain vectors and heavy chain vectors. The light chain vector may contain nucleic acids encoding one of the light chains in the antibody and nucleic acids encoding the other light chain, which may be present on the vector as separate cassettes (e.g., each operably connected to a different promoter). Similarly, the heavy chain vector may be used to encode both the CH1 (and Fc region, if present) of one antigen-binding arm and the CH1 (and Fc region, if present) of the other antigen-binding arm, which may be present on the vector as separate cassettes. Alternatively, separate vectors may be used to encode the light chains, CH1, and Fc region (if present), respectively.

[0160] The nucleic acid molecules or vectors described herein may be introduced into host cells. Techniques for introducing nucleic acids or vectors into host cells are well established in the art, and any suitable technique may be used. Various host cells suitable for the production of recombinant antibody molecules are well known in the art and include bacterial, yeast, insect, or mammalian host cells. In some embodiments, the host cell is a mammalian cell such as a CHO, NS0, or HEK cell, e.g., a HEK293 cell. In some embodiments, the host cell is a CHO cell.

[0161] Methods for producing antibodies Methods for producing the antibodies described herein are also provided herein. Techniques for purifying recombinant antibody molecules are well known in the art and include, for example, high-performance liquid chromatography, high-performance protein liquid chromatography, ion-exchange chromatography, and affinity chromatography, for example, using or conjugating protein A or protein L to an affinity tag. In some cases, purification is carried out using affinity chromatography (e.g., protein A affinity chromatography). In some cases, purification further includes light chain affinity chromatography (in addition to, for example, protein A chromatography).

[0162] The method may also include formulating an antibody molecule into a pharmaceutical composition with optionally pharmaceutically acceptable excipients or other substances listed below.

[0163] treatment The antibodies described herein may be useful for therapeutic purposes, such as the treatment of cancer.

[0164] The antibodies described herein may be used in methods for treating the human or animal body. Relevant aspects of this disclosure are: (i) Antibodies described herein for use as pharmaceuticals, (ii) Antibodies described herein for use in methods of treating diseases or disorders, (iii) Antibodies described herein in the manufacture of pharmaceuticals for use in the treatment of disease or disorder, and (iv) A method for treating a disease or disorder in an individual, comprising administering to the individual a therapeutically effective amount of an antibody described herein.

[0165] The individual may be a patient, preferably a human patient.

[0166] The treatment may be any treatment or therapy that achieves any desired therapeutic effect, such as inhibiting or delaying the progression of the condition, and may include a reduction in the rate of progression, cessation of the rate of progression, improvement of the condition, cure or remission of the condition (either partially or completely), prevention, improvement, delay, reduction or cessation of one or more symptoms and / or signs of the condition, or extension of the survival of the individual or patient beyond what would be expected in the absence of treatment.

[0167] Preventive measures (i.e., treatments as prophylaxis) are also included. For example, individuals that are susceptible to or at risk of developing or recurring a disease such as cancer may be treated as described herein. Such treatments may prevent or delay the development or recurrence of the disease in the individual.

[0168] Antibodies may be administered alone, but antibodies or fragments thereof are usually administered in the form of a pharmaceutical composition which may contain at least one component in addition to the antibody. Therefore, another aspect of this disclosure provides a pharmaceutical composition comprising an antibody as described herein. Methods for formulating antibodies into a pharmaceutical composition are also provided.

[0169] In addition to the antibody, the pharmaceutical composition may contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of a subject (e.g., a human) without excessive toxicity, irritation, allergic response, or other problems or complications and that exhibit a reasonable benefit / risk ratio.

[0170] Administration may be a "therapeutically effective amount," which is sufficient to show benefit to an individual. The actual amount administered, as well as the rate and duration of administration, will depend on the nature and severity of what is being treated, the particular individual being treated, the individual's clinical condition, the cause of the disorder, the site to which the composition is being delivered, the type of antibody molecule, the method of administration, and the scheduling of administration.

[0171] Features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, which are expressed in terms of a particular form, or of means for performing the disclosed function, or of a method or process for obtaining the disclosed result, may, if necessary, be utilized separately or in any combination of such features in its various forms for carrying out the disclosure.

[0172] Although the disclosure has been described in conjunction with the foregoing exemplary cases, many equivalent modifications and variations will be apparent to those skilled in the art when the disclosure is provided. Accordingly, the foregoing exemplary cases of the disclosure are considered to be illustrative and not limiting. Various changes to the disclosure may be made without departing from the spirit and scope of the disclosure.

[0173] To avoid any ambiguity, any theoretical explanations provided herein are provided for the purpose of improving the reader's understanding. Any section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein.

[0174] Throughout this Spec., including the claims, unless the context requires otherwise, the words “comprise” and “include,” as well as variations such as “comprises,” “comprising,” and “including,” will be understood to mean the inclusion of the integer or step or group of integers or steps described, but not the exclusion of any other integer or step or group of integers or steps.

[0175] Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple objects unless the context clearly indicates otherwise. Ranges may be expressed herein as “about” one particular value and / or “about” another particular value. Where such ranges are expressed, the alternative aspects include from one particular value and / or the other particular value. Similarly, where a value is expressed as an approximation by the use of the antecedent “about,” it will be understood that a particular value forms an alternative aspect. The term “about” with respect to numbers is optional and means, for example, ±10%.

[0176] [Table 1-1]

[0177] [Table 1-2]

[0178] [Table 1-3]

[0179] [Table 1-4]

[0180] [Table 1-5]

[0181] [Table 1-6] [Examples]

[0182] Example 1 - Materials and Method Cells and culture medium CD3-expressing cell lines, including HPB-ALL, HSC-F, Jurkat, and Jurkat TCR KO, as well as EGFR-expressing cell lines, including NCI H358 and MDA-MB468, were obtained from the American Type Culture Collection. All cell lines except MDA-MB-468 were maintained in RPMI 1640 medium supplied with 10% heat-inactivated FBS and cultured in a 37°C incubator supplied with 80% humidity and 5% CO2. MDA-MB-468 cells were maintained in Leibovitz's L-15 medium supplied with 10% heat-inactivated FBS and cultured in a 37°C incubator supplied with 80% humidity.

[0183] Antibody expression construct For affinity and cross-reactivity evaluation, preliminary anti-CD3 mutants were generated in the bivalent monospecific IgG1™ (L234F / L235E / P331S) format. DNA encoding Vκ and VH of promising anti-CD3 mutants was obtained from Integrated DNA Technologies (IDT, Coralville, IA) and assembled into properly digested pOE-IgG1™ (κLC) using NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs, catalog number E2621).

[0184] To produce monovalent bispecific antibodies, the inventors used the DuetMab platform (Mazor et al., 2015). This included an EGFR-CD3 DuetMab expression construct encoding the variable domain of anti-EGFR GA201 (Gerdes et al., 2013) and a variable domain derived from the generated CD3 sequence. Since both anti-EGFR and anti-CD3 antibodies were derived from a κ-containing parent antibody, pDuetLight vectors were constructed encoding anti-EGFR GA201 VL with a κ constant domain and wild-type cysteine, and anti-CD3 VL with a λ constant domain containing S121C. Furthermore, pDuetHeavy vectors encoding anti-EGFR GA201 VH and anti-CD3 VH variants were constructed. These pDuetHeavy vectors encoded IgG1™ (L234F / L235E / P331S) to eliminate Fc-mediated effector function. Only sequence-validated expression constructs were used in transient transfection for antibody production.

[0185] Antibody expression and purification Preliminary anti-CD3 mutants were produced from transient transfection of HEK293F cells using 293 Fectin™ (Life Technologies) in serum-free FreeStyle293™ medium, according to the manufacturer's protocol. The HEK293F cell culture supernatant containing the antibody was collected 6 days after transfection, filtered through a 0.22 μm sterile filter, and the antibody concentration was measured by a Protein A biosensor on an Octet384 instrument (ForteBio) according to the manufacturer's protocol. The antibody was purified using MabSelect SuRe resin (GE Healthcare), eluted with Pierce IgG elution buffer, pH 2.0, and buffer-replaced with phosphate-buffered saline (PBS), pH 7.2 (Life Technologies). Antibody purity was assessed by HP-SEC, generally yielding monomers with >95% purity.

[0186] DuetMabs were prepared by transiently co-transfecting CHO-K1 cells with pDuetHeavy and pDuetLight-based expression vectors using lipofectamine and proprietary culture medium. The CHO-K1 cell culture supernatant was collected 10 or 12 days post-transfection, filtered, and the antibody content in the supernatant was quantified. DuetMabs were first purified by MabSelect SuRe affinity chromatography, and the buffer was replaced with PBS, pH 7.2. DuetMabs were then evaluated for light chain mispairing by Bioanalyzer A analysis (Agilent), and further purified by LambdaFabSelect (Cytiva) affinity chromatography according to the manufacturer's protocol if necessary. After replacing the buffer with PBS, pH 7.2, DuetMabs were loaded onto a Superdex 200 16 / 600 column, and the monomer-containing fraction was collected. Next, DuetMabs were evaluated for purification and light chain mispairing using HP-SEC and Bioanalyzer A, respectively. All DuetMabs exhibited >98% monomers, >95% correct light chain pairing, and <0.5 mg / Eu endotoxin.

[0187] HP-SEC Antibodies were analyzed using HP-SEC to determine the levels of purity, aggregates, monomers, and fragments. The sample (100 μg in PBS) was injected into an Agilent 1200 series high-performance liquid chromatography (HPLC) instrument and separated using a TSKgel G3000SW xl size exclusion column (Tosoh Bioscience #08541). The mobile phase was 100 mM sodium phosphate (pH 6.8), and the sample flow rate was 1 mL / min. Proteins were detected using absorbance at 280 nm.

[0188] Flow cytometry measurement Specific cell surface antigen binding was determined using flow cytometry. Human CD3 and cynomolgus monkey CD3 binding were evaluated using human leukemia T cell lines HPB-ALL, Jurkat, Jurkat TCR KO, and cynomolgus monkey T cell line HSC-F, respectively. In short, 1 × 10⁻⁶ 5 Cells were suspended in FACS buffer (1×PBS, pH 7.2 supplemented with 2% heat-inactivated FBS, 0.1% sodium azide, and 2 mM EDTA) and placed in each well of a round-bottom 96-well plate. Antibodies were diluted to various concentrations using FACS buffer and added to the cells. Antibodies and cells were incubated at 4°C for 30 minutes and then washed twice with FACS buffer. Antibody cell surface antigen binding was detected using an Alexa Fluor 647-conjugated goat F(ab')2 fragment specific to human IgG Fcγ fragments. Data were collected using a BD FACSYMyOphony A3 Cell Analyzer (BD Biosciences). Data were analyzed with FlowJo v10.6.1 and plotted using GraphPad Prism v9.0.0.

[0189] Analysis of EGF receptor density Quantitative analysis of receptor density on tumor cell lines was performed by flow cytometry using a BD FACSYmyphony® A3 Cell Analyzer (BD Biosciences). Briefly, anti-EGFR human IgG1 was first labeled with Alexa Fluor 647 using a protein labeling kit (Invitrogen) according to the manufacturer's instructions. Antibody concentration and the fluorescent dye to protein (F:P) ratio were determined by an ND-1000 spectrophotometer (NanoDrop). Exfoliated tumor cells were washed and resuspended in ice-cold FACS buffer (1×PBS pH 7.2, 2% thermally inactivated FBS, 2 mM EDTA, and 0.1% sodium azide). Then, 2×10⁶ cells were incubated with saturation concentration (≧20 μg / mL) of Alexa Fluor 647 conjugated antibody at 4°C for 1 hour. After washing with FACS buffer, cells were fixed in ice-cold 1.8% paraformaldehyde (PFA), and bound antibodies were detected using a BD FACSymphony A3 Cell Analyzer with BD FACSDiva® software. The results were analyzed using FlowJo analysis software (Tree Star). To quantify EGFR receptor density on cells, Quantum Alexa Fluor 647 MESF (Molecules of Equivalent Soluble Fluorochrome) beads (Bangs Laboratories) were analyzed using a flow cytometer with similar settings to establish a standard curve. The calculated MESF was divided by the antibody F:P ratio using the QuickCal program (Bangs Laboratories) to obtain corrected antibody binding capacity (ABC).

[0190] Cytotoxicity and T cell activation T cell-mediated cytotoxicity and T cell activation were evaluated by flow cytometry. Cell-specific growth media supplemented with 50 μM β-mercaptoethanol (RPMI 1640 + 10% FBS for NCI H358, or L-15 + 10% FBS for MDA-MB-468) were used as assay media. Total 1 × 10⁶ 4 Individual EGFR+ target cells (NCI H358 or MDA-MB-468) were stained with CellTrace Violet (ThermoFisher Scientific) and seeded in each well of a tissue-culture-treated round-bottom 96-well plate. Effector cells (PBMCs derived from healthy donors) were added in an E:T ratio of 10:1, and various concentrations of antibodies were added. After incubation at 37°C for 1 day in a humidified incubator (5% CO2 for NCI H358, or 0% CO2 for MDA-MB-468), all cells from the cytotoxicity assay were harvested, stained, and analyzed by flow cytometry using Symphony A3 (BD). EGFR+ target cells were identified by CellTrace Violet staining, and T cells were identified by CD4+ or CD8+ staining. T cell activation was determined by CD69+ and CD25+ staining. Data were analyzed using FlowJo v10.6.1. The cytotoxicity of cells was normalized to the minimum cytotoxicity value without antibody treatment and plotted using GraphPad Prism v9.0.0.

[0191] Cytokine release assay Cytokines released into the supernatant from cytotoxic assays were evaluated according to the manufacturer's protocol using ProcartaPlex Human, NHP, and Canine Mix&Match Panels Luminex Kit (ThermoFisher Scientific). Plates were read using the Bio-Plex 3D Suspension Array System (Bio-Rad) with Luminex xPONENT software. Data were analyzed and plotted using GraphPad Prism v9.0.0.

[0192] Evaluation of T cell activation in the absence of EGFR+ cells To evaluate the level of T cell activation induced by anti-CD3 mutants in the absence of EGFR+ cells, the inventors used a plate-based PBMC incubation assay. Flat-bottom 96-well plates were coated overnight at 4°C with various concentrations of T cell engager (50, 5.0, 0.5 nM) in 50 μL. The plates were then washed twice with PBS, pH 7.2, and 150,000 PBMCs in 200 μL of AIM V medium were added. The PBMCs were incubated at 37°C for 48 hours. The PBMCs were then transferred to 96-well round-bottom plates, centrifuged, and the cell pellet was washed twice in PBS. CD69 and CD25 expression were stained by flow cytometry using anti-CD2, anti-CD3, anti-CD4, anti-CD8, anti-CD25, and anti-CD69 antibody panels.

[0193] Kinetic measurements The binding kinetics of DuetMabs based on anti-CD3 mutants were determined using Octet analysis. Biotinylated recombinant human CD3εδ (Acro Biosystems, CDD-H82W6) was immobilized on a streptavidin sensor at a concentration of 1 μg / mL, followed by a 2-fold dilution of anti-CD3 mutant DuetMabs. For the parental anti-CD3 DuetMabs, concentrations from 300 to 4.7 nM were evaluated. For the anti-CD3 mutant DuetMabs, concentrations of either 5 μM to 78.1 nM or 8 μM to 125 nM were used. The kinetics were analyzed using a 1:1 antibody:antigen binding model.

[0194] AC-SINS Antibody self-assembly was evaluated using affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) as described in other literature (Dippel et al., 2023). Briefly, 5 μL of nanoparticles were mixed with 45 μL of purified antibody at 50 ug / mL in PBS, pH 7.2 or HSA buffer [20 mM histidine, 120 mM sucrose, 80 mM arginine, pH 6] in a 384-well plate. Nanoparticles were also mixed with buffer only (no antibody) as a control. Absorbance was measured at 490–700 nm using a SPECTROstar Nano UV / vis plate reader. The wavelength of the peak absorbance was calculated using MARS data analysis software and used to determine the wavelength shift compared to the nanoparticle-only control.

[0195] Differential scanning fluorescence quantification The thermal fusion temperature of DuetMabs was evaluated using differential scanning fluorescence (DSF). The measurement was performed using a previously established method with minor modifications (Shan et al., 2018). Samples were prepared by combining 20 μL of 1 mg / mL protein sample in PBS (pH 7.2) with 5 μL of 40-fold diluted SYPRO Orange dye (Invitrogen S-6651) in PBS (pH 7.2) in a 96-well PCR plate. The plates were sealed and measured using a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). The samples were subjected to an initial equilibrium step at 25°C for 2 minutes, followed by a temperature gradient to 99°C in increments of 0.05°C / second. Fluorescence emission was monitored using a FAM filter set. The Tm value for each sample was calculated using the Boltzmann method with Protein Thermal Shift software (Applied Biosystems).

[0196] Accelerated stability and thermal stress testing For accelerated stability testing, samples were diluted to 1 mg / mL in PBS, pH 7.2, and incubated at either 4°C or 45°C for 2 weeks. The samples were then analyzed by HP-SEC. The percentages of monomers, aggregates, and fragments for each sample were calculated based on curve integration using HPLC ChemStation software (Agilent). Changes in monomer, aggregate, and fragment content were calculated from the difference between samples incubated at 45°C versus 4°C.

[0197] Example 2 - Construction of the parent antibody The inventors generated AZ Vκ+AZ VH IgG1-TM and evaluated its binding to CD3+ cells, including HPB ALL (high human CD3+), Jurkat (human CD3+), Jurkat (TCR KO, human CD3-), and HSC-F (high cynomolgus monkey CD3+), by flow cytometry. The results are summarized in Figure 1. Figure 1 shows that AZ Vκ+AZ VH IgG1-TM maintained the cross-reactivity of the parent antibody to human and cynomolgus monkey CD3 (Figures 1A and B), and demonstrated by the lack of binding to TCR KO Jurkat cells, thus maintaining CD3 cross-reactivity. + This demonstrates selective binding to cells (see Figures 1C and D).

[0198] Example 3 - Reducing the burden on the CD-R Potential sequence tendencies pose risks to large-scale manufacturing. For example, NN and NG deamidation sites were found within CDR L1, tryptophan oxidation sites within CDR H1, and DK isomerization and NS deamidation sites within CDR H2. Therefore, early CDR engineering efforts focused on mitigating these tendencies.

[0199] To reduce the NN and NG deamidation sites within CDR L1, the NNG sequence is

[0200] [Table 2] A mutated construct was generated, but the underlined amino acid is different from the parent / anti-CD3. Furthermore, constructs in which phenylalanine in CDR L2 was mutated to alanine or tyrosine were also generated, but here alanine is human IGKV2D-28 * 01 Present in the germline, tyrosine is a hydrophobic amino acid with a structure similar to phenylalanine.

[0201] Similarly, to mitigate the tryptophan oxidation site within CDR H1, constructs were generated in which tryptophan was mutated to tyrosine. Furthermore, the threonine-lysine at the FW1-CDR H1 junction and the terminal histidine of CDR H1 were modified with human IGHV3-15. * We generated constructs with mutations in serine-asparagine and serine, respectively, present in the germline. To mitigate potential problems within CDR H2, we used DK.

[0202] [Table 3] NS

[0203] [Table 4] It was mutated.

[0204] Next, combinations of Vκ and VH mutants were generated, and their binding to HPB ALL (high human CD3+), Jurkat (human CD3+), Jurkat (TCR KO, human CD3-), and HSC-F (high cynomolgus monkey CD3+) was evaluated by flow cytometry. The results are summarized in Figure 2, which shows that the mutants exhibit affinity spectra to human and cynomolgus monkey CD3+ cells, and importantly, the mutants maintain binding to cynomolgus monkey CD3+ cells.

[0205] By prioritizing leads that maintained binding to CD3+ cells, we further identified mutants that specifically modified CDR L3 and CDR H3 to confer desired cytotoxicity, T cell activation, and cytokine release profiles.

[0206] Example 4 - Alanine scanning of CDR L3 and CDR H3 To identify residues within CDR L3 and CDR H3 directly and indirectly involved in the anti-CD3 paratope, alanine scanning mutagenesis was performed in these regions, and the binding of mutants to CD3+ cells was evaluated by flow cytometry. The results were interpreted as follows: (1) Mutants that abolished binding to CD3+ cells contained mutations that disrupted the anti-CD3 paratope; (2) Mutants that showed reduced binding contained mutations that simply confused the paratope; and (3) Mutants that showed unaffected binding contained mutations that were not involved in the paratope.

[0207] The results show that Vκ variants containing G91A or F96A (Kabat numbering) abolished CD3+ cell binding, variants containing G89A, Q90A, and T92A showed slightly reduced binding, and variants containing Q93A, Y94A, P95A, or T97A had no effect on binding. Similarly, VH variants containing Y97A and A98L abolished CD3+ cell binding, while variants containing R93A, G94A, V95A, Y96A, P100aA, F100bA, or Y102A showed slightly reduced binding, and variants containing L99A, S100A, or D101A showed an unaffected binding profile.

[0208] Example 5 - Functional characterization of anti-CD3 mutants in related bispecificity formats Binding to CD3+ cells is merely a prerequisite for T cell engager function; the objective is to optimize the functional activity of the CD3-binding arm within the context of the relevant bispecific T cell engager. Therefore, monovalent bispecific antibodies were constructed based on well-established DuetMab platforms (Mazor et al., 2015; Wang et al., 2020; Dovedi et al., 2021) to determine the functional effects of anti-CD3 variants on affinity, cytotoxicity, T cell activation, and cytokine release. These DuetMabs consisted of anti-EGFR and anti-CD3 variant-binding arms.

[0209] It is important to note that functional characterization of anti-CD3 variants was necessary to identify variants with appropriate affinity, cytotoxicity, T cell activation, and cytokine release. Ultimately, three light chain variants (AZ Vκ17, AZ Vκ26, and AZ Vκ29) and nine heavy chain variants (AZ VH R75, AZ VH R79, AZ VH R82, AZ VH SN75, AZ VH SN79, AZ VH SN82, AZ VH E75, AZ VH E79, and AZ VH E82) were selected as anti-CD3 variant compositions for further investigation. Twenty-seven anti-CD3 variants derived from these Vκ and VH combinations were extensively characterized.

[0210] The EGFR-CD3 mutant DuetMabs was characterized for its CD3+ cell binding affinity and functionally profiled for cytotoxicity, T cell activation, and cytokine release upon exposure to EGFR+ cells. To enable direct comparison, all functional assays were completed in a single batch of frozen peripheral blood mononuclear cells (PBMCs) to avoid inherent donor heterogeneity in newly isolated PBMCs. The selected batch of PBMCs reflects the median reactivity of the PBMCs investigated. Furthermore, we evaluated the functional activity of all mutants using low-EGFR+ NCI H358 cells (3.1 × 10⁴ ± receptor / cell) (Mazor et al., 2017), and selected mutants using high-EGFR+ MDA-MB-468 (1.1 × 10⁶ ± 2.8 × 10⁴ receptor / cell).

[0211] The results are shown in Figures 3 to 17. Cultured DuetMabs containing anti-CD3 mutants exhibit variable affinity for CD3+ cells, cytotoxicity of EGFR+ cells, T cell activation, and cytokine release profiles. Furthermore, the functional response induced by EGFR-CD3 mutant DuetMabs was EGFR-dependent. For example, cultured DuetMabs that showed little activity upon exposure to low-EGFR+ NCI H358 showed enhanced activity against high-EGFR+ MDA-MB-468. Therefore, the optimal anti-CD3 mutant composition for T cell engagers can be selected based on the desired TAA density or TAA-CD3 format (1+1, 2+1, 1+1+1, etc.).

[0212] Example 6 - Evaluation of T cell activation in the absence of target cells We evaluated the performance of anti-CD3 mutant-based T cell engagers in the presence of EGFR+ target cells and T cells. However, since premature T cell activation can lead to undesirable toxicity and cytokine release syndrome (CRS) in vivo, it was also important to understand the behavior of anti-CD3 mutant-containing T cell engagers in the absence of EGFR+ cells.

[0213] To evaluate the level of T cell activation induced by our anti-CD3 mutant-containing T cell engagers in the absence of EGFR+ cells, we used a plate-based PBMC incubation assay. In this assay, PBMCs were incubated in 96-well plates pre-coated with various concentrations of candidate T cell engagers (50, 5.0, 0.5 nM). After 48 hours of exposure, the level of T cell activation of CD4+ and CD8+ T cells was characterized by flow cytometry using CD25 and CD69 markers. The results are summarized in Figure 18, showing that all anti-CD3 mutant-containing T cell engagers exhibited reduced T cell activation compared to anti-CD3-based controls.

[0214] Example 7: Dynamic Measurement The binding dynamics of anti-CD3 mutant antibodies were determined using Octet analysis. The results are shown in Figure 19 and Table 1. The mutants exhibited a 10- to 100-fold decrease in human CD3 affinity.

[0215] [Table 5-1]

[0216] [Table 5-2]

[0217] Table 1: Dynamic measurements of the soluble form of CD3 were obtained using an Octet384 instrument. The dissociation constant KD was obtained from a nonlinear fit of the data.off / k on It was calculated as a ratio.

[0218] Example 8 - Development Poorly performing antibodies correlate with poor clinical success, making it increasingly common to identify and mitigate risks before lead selection. For this purpose, anti-CD3 variants were further characterized for expression titer, nonspecific binding, reversible self-association, thermal stability, and aggregation and fragmentation tendencies after thermal stress. The results of these evaluations are summarized in Table 2.

[0219] [Table 6]

[0220] Table 2: Summary of the developmental profile of EGFR-CD3 variant antibodies. Prior to biochemical, biophysical, and biological characterization, antibodies were purified by protein A affinity chromatography followed by light chain affinity chromatography, and then subjected to preparative SEC for aggregate removal.

[0221] Purified anti-CD3 mutant-containing cultured DuetMabs exhibiting over 95% precisely paired light chains and over 98% monomers were fully characterized for nonspecific binding, reversible self-association, and thermal stability. To evaluate nonspecific binding, anti-CD3 mutant-based cultured DuetMabs were evaluated using flow cytometry-based baculovirus particle (BVP) ELISA and human embryonic kidney 293 (HEK293) cell binding assays. CD3 is not expressed in either the insect cells used to generate baculovirus particles or in HEK293; instead, these assays evaluate the "stickiness" of the antibody being tested. Our evaluation of the parental antibody showed a moderate risk of nonspecific binding to baculovirus particles but no nonspecific binding to HEK293 cells. In an assay evaluating the nonspecific binding of anti-CD3 variants, only the AZ Vκ29+AZ VH R75 variant showed a risk of nonspecific binding, and the pattern (nonspecific binding to baculovirus particles, but not to HEK293) was similar to that of the parent antibody, albeit at a lower level. Next, the thermal stability of the anti-CD3 variants was evaluated by differential scanning fluorescence quantification. Both the parent antibody and all anti-CD3 variants had a melting onset temperature of approximately 49-50°C (T 開始 ) has a melting temperature of approximately 61°C (T m ) presented with the following symptoms.

[0222] In addition to these developmental feasibility assessments, the anti-CD3 variants were evaluated using accelerated stability testing. Here, the antibodies were incubated at 45°C for 14 days, and the samples were evaluated for fragmentation and aggregation before and after heat stress.

[0223] Example 9 - Multispecific antibody containing charge pairs To improve correct chain pairing beyond what alternative disulfides can achieve in the DuetMab setting (see PCT International Publication WO2013 / 096291 incorporated herein by reference), charge pairs were designed using amino acids involved in the lambda light chain (LC)-heavy chain (HC) interface. The following positions were evaluated as lambda light chain amino acids involved in interface formation with the CH1 domain: T117, F119, S122, E124, E125, K130, T132, V134, L136, S138, D139, E161, T163, S166, Q168, A174, S176, Y178, S180, and in connection therewith, the following heavy chain CH1 domain The amino acids involved in interface formation with the lambda light chain CL domain are: S124, F126, L128, A129, S131, S132, K133, S134, A141, G143, L145, K147, D148, H168, F170, P171, V173, Q175, S176, S181, S183, V185, T187, V211, and K213.

[0224] These amino acids were investigated individually or in pairs, in combination, or alone, using alternative interchain disulfides or leaving the disulfides in their natural state. The introduction of positively or partially positively charged amino acids means substituting the amino acid present at that position with lysine and arginine, and possibly asparagine, glutamine, or histidine. The introduction of negatively or partially negatively charged amino acids means substituting the amino acid present at that position with aspartic acid, glutamic acid, serine, threonine, and possibly asparagine or glutamine. Adding histidine residues to some of these positions makes it possible to introduce pH-dependent CH1-CL interactions.

[0225] Nine pair combinations at the lambda LC-HC interface that meet the above criteria are provided in Table 3 as non-exhaustive examples, and the improved pairings described herein were tested.

[0226] Table 3. All mutations presented here are specific to lambda light chain-containing molecules and are expected to function as lambda charge pairs. Furthermore, the opposite charge pair [i.e., V134(D,E,S,T)-L128(R,K,H)] is also expected to provide a preferred pairing. Net uncharged side chains containing amino acids such as asparagine and glutamine have been found to be involved in the formation of hydrogen bonds with both positively and negatively charged amino acids, as well as with each other, and can therefore be used for substitutions with either positive or negative partial charges.

[0227] [Table 7]

[0228] To construct DuetMab antibodies with charge pair mutations at the heavy-light chain interface, the pDuet-heavy-chain and pDuet-light-chain plasmids described in PCT International Publication No. WO2013 / 096291 and Mazor et al., 2015, were used as the backbone vectors. Briefly, the pDuet-Heavy vector contained two human gamma monoheavy-chain (HC) cassettes to support HC heterodimerization. The former heavy chain possessed a "hole" set mutation (T366S / L368A / Y407V) and a stabilizing mutation (Y349C) in the CH3 domain, while the latter possessed a complementary "knob" mutation (T366W) and a stabilizing mutation (S354C) in CH3. However, the order of the cassettes could be easily reversed. The pDuet-Light vector contains two human light chain (LC) cassettes, the former containing a kappa constant domain (Cκ) and the latter containing a lambda constant domain (Cλ). The pDuet-Heavy and pDuet-Light vectors also contain mutations to remove the native interchain disulfide bond in CH1 / Cλ and provide an alternative disulfide bond indicated herein as "V12 DS" or "V12". Mutation F126C / C220V was introduced into the CH1 domain of the "knob" heavy chain, and mutation S122C / C212V was introduced into the lambda constant domain. The amino acid sequences of the constant domains in the exemplary DuetMab antigen backbone (before introduction of charge mutations) are provided below.

[0229] The “knob-and-hole” sets of mutations and stabilizing / alternative disulfide bonds used herein are provided merely as examples. Those skilled in the art may use any other combination of “knob-and-hole” techniques and / or mutations for stabilizing / alternative disulfide bonds known in the art to support HC heterodimerization.

[0230] To construct a pDuet-Heavy vector with a charge mutation, the "Hole" heavy chain was cloned into the pDuet-Heavy vector using restriction cloning techniques with BssHII / HindIII by a synthetic DNA fragment of the VH-CH1-CH2-CH3 domain containing the above mutation for the "Hole" heavy chain. Optionally, the "Hole" heavy chain contained the charge mutation S183K in the CH1 domain. The "Knob" heavy chain was cloned into the vector using restriction cloning techniques with BsrGI / EcoRI by a synthetic DNA fragment of the VH-CH1-CH2-CH3 domain containing the above mutation for the "Knob" heavy chain. The "knob" heavy chains were optionally selected to contain one of the following charge mutations in the CH1 domain: L128D, L128E, L128S, L128T, A141D, A141E, A141S, A141T, L145D, L145E, L145S, L145T, S183D, V185D, V185E, V185S, V185T, V173D, V173E, V173S, and V173T.

[0231] To construct pDuet-Light with charge mutations, the κ light chain was cloned into the pDuet-Light vector using a synthetic DNA fragment of the VL-Cκ domain with BssHII / NheI restriction cloning technology. Optionally, the stationary kappa (Cκ) domain contained the charge mutation V133E. The lambda light chain was cloned into the pDuet-Light vector using a synthetic DNA fragment of the VL-Cλ domain containing the above-mentioned S122C / C212V mutation for the lambda light chain with BsrGI / EcoRI restriction cloning technology. Optionally, the stationary lambda (Cλ) domain contained one of the charge mutations V117R, V117K, F119R, F119K, V134R, V134K, L136R, L136K, Y178R, and Y178K. The light chain variable domain (VL) can be either a variable kappa domain (Vκ) or a variable lambda domain (Vλ).

[0232] All constructs were transiently expressed in CHO cells in suspension using PEI-MAX (Polysciences, Inc., Warrington, PA) as the transfection reagent and grown in in-house prepared CHO medium. Vectors containing the following charge pair combinations were used for antibody expression in these studies. A schematic diagram of the constructed DuetMabs containing the charge pairs is shown in Figure 20. Table 4 below provides details of the various lambda charge pair DuetMabs produced that target CD3 and EGFR.

[0233] [Table 8]

[0234] The culture medium was collected 7–13 days after transfection and filtered through a 0.22 μm sterile filter. The antibody concentration in the culture supernatant was measured using an Octet 384 instrument with a protein A sensor (Sartorius, Gottingen, Germany) according to the manufacturer's protocol. The antibodies were purified by either protein A magnetic bead affinity purification (Genscript, Piscataway, NJ) or standard protein A affinity chromatography (Cytiva, Marlborough, MA), followed by light chain affinity chromatography if necessary, according to the manufacturer's protocol, and then buffer exchange in PBS (pH 7.2). The purity and oligomeric state of the purified molecules were determined by microfluidic electrophoresis and analytical size exclusion chromatography (see method below). Protein aggregates were removed by preparative SEC. The concentration of the purified antibodies was determined by reading the absorbance at 280 nm using the theoretically determined extinction coefficient.

[0235] Analytical SEC-HPLC (Agilent 1260 Infinity HPLC system) was performed using a TSK-gel G3000SWxL column (Tosoh Biosciences, King of Prussia, PA) to determine the oligomeric state of the purified molecules. Preparative SEC-HPLC was performed using a Superdex 200 column (Cytiva) to remove protein aggregates.

[0236] To evaluate the ratio of kappa light chains to lambda light chains in the antibody, microfluidic electrophoresis was performed using a Bioanalyzer according to the manufacturer's protocol (Agilent, Santa Clara, CA), and the percentage of the correct light chain ratio was calculated based on this.

[0237] Table 5 summarizes the expression and biochemical profiles of selected charge-pair mutants in EGFR / CD3 DuetMabs. Charge-pair mutants #33, #34, #35, #36, and #41 showed improved correct LC ratios compared to controls #1 and #2.

[0238] [Table 9]

[0239] Example 10 - Production of a 2+1 bispecific antibody containing a lambda charge pair The p2+1-heavy chain vector was constructed on the pDuet-heavy chain skeleton described in Example 9. To construct the p2+1-heavy chain vector with charge mutations, the "hole" heavy chain was cloned into the vector by BssHII / HindIII as previously described. The "knob" heavy chain was cloned into the vector using restriction cloning techniques with BsrGI / EcoRI with synthetic DNA fragments of the VH-CH1-VH-CH1-CH2-CH3 domain, where the preceding VH-CH1 segment corresponds to the sequence found on the "hole" heavy chain, and the subsequent VH-CH1 segment contained charge mutations A141D and V12 DS in VH and CH1 for different targets. The pDuet-Light vector is common to both the Duet2(2+1) bispecific construct and the DuetMab construct.

[0240] All Duet2(2+1) bispecific constructs were transiently expressed and purified for the DuetMab molecule as described above.

[0241] A schematic diagram of the constructed Duet2(2+1) including charge pairs is shown in Figure 21.

[0242] Table 6 summarizes the expression and biochemical profiles of EGFR / CD3 and NIP228 / CD3 Duet2(2+1) bispecific molecules possessing selected charge pair sets, produced in 500 mL cell cultures. NIP228 is a nonspecific control-binding domain. For further analysis, DuetMabs were further purified by light chain affinity chromatography to remove mispaired byproducts, and aggregates were removed by preparative SEC.

[0243] [Table 10]

[0244] Table 7 summarizes the thermal stability and accelerated stability profiles of EGFR / CD3 and NIP228 / CD3 Duet2(2+1) bispecific molecules as determined by differential scanning fluorescence (DSF). The Duet2(2+1) bispecific molecules did not show any concerns regarding aggregation or fragmentation after thermal stress.

[0245] [Table 11]

[0246] Table 8 shows the subunit mass spectral data for EGFR / CD3 and NIP228 / CD3 Duet2(2+1) bispecific molecules. Molecular integrity and LC / HC association identity of each variant were confirmed by alignment of theoretical and measured masses.

[0247] [Table 12]

[0248] Table 9 shows the thermal stability studies of EGFR / CD3 and NIP228 / CD3 Duet2(2+1) bispecific molecules using differential scanning calorimetry (DSC) analysis. Table 14 lists the deconvoluted TM and approximate T start values ​​for the Duet2(2+1) bispecific molecules.

[0249] [Table 13]

[0250] The EGFR / CD3 Duet2(2+1) bispecific molecule was tested in cytotoxic assays involving T cells, EGFR-expressing target cells, and activated CD8 and CD4 T cells, demonstrating the functional nature of Duet2(2+1).

[0251] Example 11 - Production of a triplicate antibody containing lambda and kappa charge pairs The pTriMab-heavy chain vector was constructed on the pDuet-heavy chain skeleton described in Example 9 above. To construct a pTriMab-heavy chain vector with a charge mutation, the "hole" heavy chain was cloned into the vector by BssHII / HindIII as previously described, and the VH-CH1 segment in the "hole" heavy chain was defined as "Fab1" having the VH and CH1 charge mutation S183K for the first target. The "knob" heavy chain was cloned into the vector using restriction cloning techniques with BsrGI / EcoRI with synthetic DNA fragments of the VH-CH1-VH-CH1-CH2-CH3 domain. The preceding VH-CH1 segment of the "knob" heavy chain was defined as "Fab2" having the VH and charge mutation S183E for the second target, and an optional V12 DS for CH1. The subsequent VH-CH1 segment of the "knob" heavy chain was defined as "Fab3," which has charge mutations A141D and V12 DS in VH and CH1 for a third target.

[0252] The vectors pTriMab-light chain 1 and pTriMab-light chain 2 were constructed on the pDuet-light chain skeleton described in Example 1 above. To construct pTriMab-light chain 1 with a charge mutation, the κ light chain of Fab1 was cloned into the vector by BssHII / NheI as previously described, and the Cκ domain contained the charge mutation V133E. The second (lambda)LC cassette in pTriMab-light chain 1 was removed.

[0253] For the construction of pTriMab-light chain 2 with charge mutations, the kappa light chain of Fab2 was cloned into a vector by BssHII / NheI as previously described, and the Cκ domain contained the charge mutation V133K and, optionally, V12 DS (S121C / C214V for Cκ). The λ light chain of Fab3 was cloned into a vector by BsrGI / EcoRI as previously described, and the Cλ domain contained the charge mutation T117R and V12 DS.

[0254] All TriMab constructs were transiently expressed and purified as described above for the DuetMab molecule. A schematic diagram of the constructed TriMab, including the charge pair, is shown in Figure 22.

[0255] Table 15 summarizes the expression and biochemical profiles of EGFR / Her2 / CD3 TriMab possessing a selected set of charge pairs, produced in 500 mL cell cultures. For further analysis, TriMab was further purified by protein A affinity chromatography, and aggregates were removed by preparative CHT column (an incompressible mixed-mode chromatography medium using cation exchange and calcium-affinity interactions).

[0256] Table 15: EGFR / Her2 / CD3 TriMab trispecific antibodies with charge mutations. The kappa-to-lambda ratio was calculated by band density from capillary gel electrophoresis under reducing conditions using Agilent Protein 80 Chip. Monomer content was calculated by size exclusion chromatogram for analysis of complete molecules. CHT ceramic hydroxyapatite was purified using the mixed-mode method.

[0257] [Table 14]

[0258] Table 16 summarizes the thermal stability and accelerated stability profiles of EGFR / Her2 / CD3 TriMab molecules as determined by differential scanning fluorescence (DSF). EGFR / Her2 / CD3 TriMab molecules showed no signs of aggregation or fragmentation after thermal stress.

[0259] Table 16: Overview of the development potential of EGFR / Her2 / CD3 TriMab with charge mutations. The kappa-to-lambda ratio was calculated by band density from capillary gel electrophoresis under reducing conditions using Agilent Protein 80 Chip. Monomer content was calculated by size exclusion chromatogram for analysis of the complete molecule.

[0260] [Table 15]

[0261] Table 17 reports the subunit mass spectral data for EGFR / Her2 / CD3 TriMab. Molecular integrity and LC / HC association identity of each peak were confirmed by alignment of theoretical and measured masses.

[0262] [Table 16]

[0263] Table 18 reports on the thermal stability study of EGFR / Her2 / CD3 TriMab using differential scanning calorimetry (DSC) analysis and lists the deconvoluted TM and approximated Tonset values ​​of EGFR / Her2 / CD3 TriMab.

[0264] [Table 17]

[0265] The EGFR / Her2 / CD3 TriMab molecule was tested in cytotoxic assays involving T cells and cells positive for either EGFR alone ("single-positive" cells) or both EGFR and HER2 ("double-positive" cells). EGFR / Her2 / CD3 TriMab demonstrated the functional nature of this TriMab format by inducing cell death in both single-positive and double-positive cells, as well as activation of CD8 and CD4 T cells.

Claims

1. An antibody comprising an antigen-binding domain capable of binding to a CD3 protein or a fragment thereof, wherein the CD3 antigen-binding domain is a heavy chain variable (VH) region as described in any one of the following: The VH area containing the following CD-R: HCDR1 having the amino acid sequence of SEQ ID NO: 90, HCDR2 having the amino acid sequence of SEQ ID NO: 91, and HCDR3 having the amino acid sequence of SEQ ID NO: 92, The VH area containing the following CD-R: HCDR1 having the amino acid sequence of SEQ ID NO: 82, HCDR2 having the amino acid sequence of SEQ ID NO: 83, and HCDR3 having the amino acid sequence of SEQ ID NO: 84, The VH area containing the following CD-R: HCDR1 having the amino acid sequence of SEQ ID NO: 78, HCDR2 having the amino acid sequence of SEQ ID NO: 79, and HCDR3 having the amino acid sequence of SEQ ID NO: 80, The VH area containing the following CD-R: HCDR1 having the amino acid sequence of SEQ ID NO: 66, HCDR2 having the amino acid sequence of SEQ ID NO: 67, and HCDR3 having the amino acid sequence of SEQ ID NO: 68, The VH area containing the following CD-R: HCDR1 having the amino acid sequence of SEQ ID NO: 70, HCDR2 having the amino acid sequence of SEQ ID NO: 71, and HCDR3 having the amino acid sequence of SEQ ID NO: 72, The VH area containing the following CD-R: HCDR1 having the amino acid sequence of SEQ ID NO: 74, HCDR2 having the amino acid sequence of SEQ ID NO: 75, and HCDR3 having the amino acid sequence of SEQ ID NO: 76, The VH area containing the following CD-R: HCDR1 having the amino acid sequence of SEQ ID NO: 86, HCDR2 having the amino acid sequence of SEQ ID NO: 87, and HCDR3 having the amino acid sequence of SEQ ID NO: 88, The VH area containing the following CD-R: HCDR1 having the amino acid sequence of SEQ ID NO: 94, HCDR2 having the amino acid sequence of SEQ ID NO: 95, and HCDR3 having the amino acid sequence of SEQ ID NO: 96, and The VH area containing the following CD-R: HCDR1 having the amino acid sequence of SEQ ID NO: 98, HCDR2 having the amino acid sequence of SEQ ID NO: 99, and HCDR3 having the amino acid sequence of SEQ ID NO: 100, Furthermore, the CD3 antigen-binding domain is a heavy chain variable (VH) region as described in any one of the following: The VL area containing the following CDR: HCDR1 having the amino acid sequence of SEQ ID NO: 46, HCDR2 having the amino acid sequence of SEQ ID NO: 47, and HCDR3 having the amino acid sequence of SEQ ID NO: 48, The VL area containing the following CDR: HCDR1 having the amino acid sequence of SEQ ID NO: 58, HCDR2 having the amino acid sequence of SEQ ID NO: 59, and HCDR3 having the amino acid sequence of SEQ ID NO: 60, and The VL area containing the following CDR: HCDR1 having the amino acid sequence of SEQ ID NO: 62, HCDR2 having the amino acid sequence of SEQ ID NO: 63, and An antibody containing HCDR3 having the amino acid sequence of SEQ ID NO:

64.

2. The CD3 antigen-binding domain comprises a VH region and a VL region containing one of the following sets of CDRs, or vii. HCDR1 having the amino acid sequence of SEQ ID NO: 90, viiii. HDCR2 having the amino acid sequence of SEQ ID NO: 91, ix. HCDR3 having the amino acid sequence of SEQ ID NO: 92, x. LCDR1 having the amino acid sequence of SEQ ID NO: 46, xi. LCDR2 having the amino acid sequence of SEQ ID NO: 47, xi. LCDR3 having the amino acid sequence of SEQ ID NO: 48, or vii. HCDR1 having the amino acid sequence of SEQ ID NO: 82, viiii. HDCR2 having the amino acid sequence of SEQ ID NO: 83, ix. HCDR3 having the amino acid sequence of SEQ ID NO: 84, x. LCDR1 having the amino acid sequence of SEQ ID NO: 46, xi. LCDR2 having the amino acid sequence of SEQ ID NO: 47, xi. LCDR3 having the amino acid sequence of SEQ ID NO: 48, or vii. HCDR1 having the amino acid sequence of SEQ ID NO: 78, viiii. HDCR2 having the amino acid sequence of SEQ ID NO: 79, ix. HCDR3 having the amino acid sequence of SEQ ID NO: 80, x. LCDR1 having the amino acid sequence of SEQ ID NO: 62, xi. LCDR2 having the amino acid sequence of SEQ ID NO: 63, xi. The antibody according to claim 1, comprising LCDR3 having the amino acid sequence of SEQ ID NO:

64.

3. The antibody according to claim 1 or 2, wherein the CD3 antigen-binding domain includes a VH region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 77, SEQ ID NO: 89, or SEQ ID NO:

81.

4. The antibody according to any one of claims 1 to 3, wherein the CD3 antigen-binding domain includes a VL region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 45 or SEQ ID NO:

61.

5. The CD3 antigen-binding domain, A VH region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to sequence number 77, and a VL region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to sequence number 61, A VH region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 89, and a VL region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 45, or The antibody according to any one of claims 1 to 4, comprising a VH region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 81, and a VL region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO:

45.

6. The CD3 antigen-binding domain is the K of the control antigen-binding domain that binds to human CD3. ｄ In comparison, i. 10 to 200 times higher, ii. 15 to 200 times higher, iii. 20 to 200 times higher, iv. K is 25 to 200 times higher ｄ It binds to human CD3 with affinity, The antibody according to any one of claims 1 to 5, wherein the control antigen domain binding comprises the VH domain sequence of SEQ ID NO: 5 and the VL domain sequence of SEQ ID NO:

1.

7. The antibody according to any one of claims 1 to 6, wherein the CD3 antigen-binding domain exhibits reduced off-target T cell activation compared to a control antigen-binding domain, the control antigen domain binding comprises the VH domain sequence of SEQ ID NO: 5 and the VL domain sequence of SEQ ID NO: 1, and optionally, off-target T cell activation is determined in a T cell activation assay in the absence of association with target cells.

8. The antibody according to any one of claims 1 to 7, further comprising a target antigen-binding domain.

9. The antibody according to claim 8, wherein the target antigen-binding domain can bind to tumor-associated antigens (TAAs).

10. The aforementioned TAA is AFP, a ｎ b3 (vitronectin receptor), a ｎ b ６ , B cell maturation factor (BCMA), CA125 (MUC16), CD4, CD20, CD22, CD33, CD52, CD56, CD66e, CD80, CD140b, CD227 (MUC1), EGFR (HER1), EpCAM, GD3 ganglioside, HER2, prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), CD5, CD19, CD21, CD25, CD37, CD30, CD33, CD45, HLA-DR, anti-idiotype, carcinoembryonic antigen (CEA), e.g., carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), TAG-72, folate-binding protein, A33, G250, ferritin, glycolipids such as gangliosides, carbohydrates such as CA-125, IL-2 receptor, fibroblast-activating protein (FAP), IGF1 The antibody according to claim 9, selected from the list consisting of R, B7H3, B7H4, PD-L1, CD200, EphA2, c-Met, and mesothelin.

11. The antibody according to claim 9, wherein the TAA is EGFR, HER2, STEAP2, GPC3, and c-Met.

12. The antibody according to any one of claims 8 to 11, wherein one of the antigen-binding domains comprises CH1 and a lambda constant (CLλ) region, and optionally the CD3 antigen-binding domain comprises CH1 and the CLλ region.

13. The antibody according to claim 12, wherein the CLλ comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% relative to SEQ ID NO:

105.

14. The antigen-binding domain, which includes the CLλ region, includes a lambda charge pair, and optionally, the lambda charge pair is a pair at the following positions in the antigen-binding domain: (i) Position 117 of CLλ and position 141 of CH1, (ii) Position 117 of CLλ and position 185 of CH1, (iii) Position 119 of CLλ and position 128 of CH1, (iv) Position 134 of CLλ and position 128 of CH1, (v) Position 134 of CLλ and position 145 of CH1, (vi) Position 134 of CLλ and position 183 of CH1, (vii) Position 136 of CLλ and position 185 of CH1, (viiii) Position 178 of CLλ and position 173 of CH1, (ix) Located at one or more of the pairs of positions 117 of CLλ and 187 of CH1, The lambda charge pair comprises a positively charged amino acid residue optionally selected from arginine, lysine, or histidine located at one position of the lambda charge pair, and a negatively charged amino acid residue optionally selected from aspartic acid, glutamic acid, serine, or threonine located at the other position of the lambda charge pair, and The antibody according to claim 12 or 13, wherein the numbering follows the EU index.

15. The lambda charge pair is located at position 117 of CLλ and position 141 of CH1, and optionally the lambda charge pair is from the following list, a. Arginine at position 117 of CLλ, and aspartic acid at position 141 of CH1, b. Arginine at position 117 of CLλ, and glutamic acid at position 141 of CH1, c. Arginine at position 117 of CLλ, and serine at position 141 of CH1, d. Arginine at position 117 of CLλ, and threonine at position 141 of CH1, e. The antibody according to claim 14, selected from lysine at position 117 of CLλ and aspartic acid at position 141 of CH1.

16. The antibody according to any one of claims 12 to 15, wherein the other antigen-binding domain comprises CH1 and a kappa constant CLκ region, and optionally the target antigen-binding domain comprises CH1 and the CLκ region.

17. The antibody according to claim 16, wherein the CLκ is present in an amount of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% relative to SEQ ID NO:

106.

18. The antibody according to claim 16 or 17, wherein the antigen-binding domain including the CLκ region includes a kappa charge pair, and optionally the kappa charge pair is located at position 133 of the CLκ and position 183 of the CH1.

19. (i) The disulfide bond between the light chain of the CD3 antigen-binding arm and the CH1 is formed between the light chain of the CD3 antigen-binding arm and a pair of cysteine ​​modified to CH1, and the disulfide bond between the light chain of the target antigen-binding arm and the CH1 is formed between a pair of natural cysteine, or (ii) The antibody according to any one of claims 8 to 18, wherein the disulfide bond between the light chain of the target antigen-binding arm and the CH1 is formed between the light chain of the target antigen-binding arm and a pair of cysteine ​​modified to CH1, and the disulfide bond between the light chain of the CD3 antigen-binding arm and the CH1 is formed between a pair of native cysteine.

20. The antibody according to claim 21, wherein the light chain of the CD3 antigen-binding arm and the pair of cysteines modified in CH1 are located at position 122 of the light chain of the CD3 antigen-binding arm and position 126 of CH1, the light chain of the CD3 antigen-binding arm contains a non-cysteine ​​residue at position 212, and CH1 of the CD3 antigen-binding arm contains a non-cysteine ​​residue at position 220, and optionally the non-cysteine ​​residue is valine.

21. The antibody according to any one of claims 8 to 20, wherein the CD3 antigen-binding region further comprises a first Fc region, and the target antigen-binding arm further comprises a second Fc region.

22. The antibody according to claim 21, comprising modifications in the first Fc region and the second Fc region to promote heterodimerization of the first Fc region and the second Fc region.

23. The antibody according to claim 22, wherein the modification is located at the CH3 in the Fc region.

24. The antibody according to claim 23, wherein the modification of the CH3 in one of the first Fc region and the second Fc region is a substitution of an amino acid residue with a larger side chain, thereby generating a bump (knob) on the surface of the CH3 domain, and the modification of the CH3 in the other Fc region is a substitution of an amino acid residue with a smaller side chain, thereby generating a cavity (hole) on the surface of the CH3 domain, and optionally, the CH3 domain containing the bump (knob) is part of the first heavy chain polypeptide, and the CH3 domain containing the cavity (hole) is part of the second heavy chain.

25. The substitution for generating the knob is the substitution of tryptophan at position 366, and the substitution for generating the hole is the following: i) Substitution with valine at position 407, ii) Substitution with serine at position 366, and iii) The antibody according to claim 24, wherein one or more substitutions for alanine at position 368.

26. The antibody according to claim 24 or 25, wherein the CH3 domain containing the knob contains cysteine ​​at position 354, and the CH3 domain containing the cavity contains cysteine ​​at position 349.

27. At least one of the Fc regions is subjected to the following amino acid substitutions: (a) L234F / L235E / P331S, (b) E233P / L234V / L235A / G236del / S267K, and / or (c) The antibody according to any one of claims 21 to 26, comprising M252Y / S254T / T256E.

28. The antibody according to any one of claims 8 to 27, comprising two antigen-binding domains capable of binding to the same target.

29. It further comprises a CD8 antigen-binding domain, and optionally the CD8 antigen-binding domain is VHH, and (1) The following complementarity determination regions (CDRs): HCDR1 having the amino acid sequence of SEQ ID NO: 109, SEQ ID NO: 116, or SEQ ID NO: 117, HCDR2 having the amino acid sequence of SEQ ID NO: 110, and HCDR3 having the amino acid sequence of SEQ ID NO: 111, or (2) The antibody according to claim 28, comprising either a VH region containing an amino acid sequence having at least 70% sequence identity with respect to the amino acid sequence of SEQ ID NO: 112, SEQ ID NO: 118, SEQ ID NO: 119, or SEQ ID NO: 120, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

30. The antibody according to any one of claims 8 to 27, further comprising a third antigen-binding domain, wherein optionally the third antigen-binding domain binds to CD8 or to a target different from the target antigen-binding domain.

31. One or more nucleic acids encoding an antibody according to any one of claims 1 to 30.

32. A vector comprising the nucleic acid described in claim 31.

33. An isolated host cell comprising the nucleic acid described in claim 31 or the vector described in claim 32.

34. A method for producing an antibody according to any one of claims 1 to 30, comprising culturing a recombinant host cell according to claim 33 under conditions for the production of an antibody molecule.

35. The method according to claim 34, further comprising isolating and / or purifying the antibody molecule.

36. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 30 and a pharmaceutically acceptable carrier.

37. A method for treating a disease in a patient requiring treatment for the disease, comprising administering to the patient an effective amount of an antibody according to any one of claims 1 to 30, or a pharmaceutical composition according to claim 36.

38. The method according to claim 37, wherein the disease is cancer.

39. An antibody for use as a pharmaceutical, according to any one of claims 1 to 30, or a pharmaceutical composition according to claim 36, wherein the antibody is optionally a multispecific antibody.

40. An antibody for use in the treatment of cancer, according to any one of claims 1 to 30, or a pharmaceutical composition according to claim 36, wherein the antibody is optionally a multispecific antibody.

41. Use of an antibody according to any one of claims 1 to 30, or a pharmaceutical composition according to claim 36, for the manufacture of a pharmaceutical for the treatment of the aforementioned cancer, wherein the antibody is optionally a multispecific antibody.

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