Anti-DLL3 / CD3 bispecific antibody and use thereof

Abstract

Provided are a DLL3 / CD3 bispecific antibody and the use thereof. The antibody has a reduced CD3-binding activity, an improved affinity for the DLL3 target, an enhanced tumor killing ability, and a relatively low level of cytokine release.

Description

Anti-DLL3 / CD3 bispecific antibodies and their uses Technical Field

[0001] This application belongs to the field of biomedicine, specifically relating to a bispecific antibody targeting CD3 and DLL3 and its uses. Background Technology

[0002] Lung cancer is one of the most common malignant tumors worldwide and a leading cause of cancer death. Small cell lung cancer accounts for 13%–15% of all new lung cancer cases. It is characterized by rapid growth, high invasiveness, early regional and distant metastasis, and poor prognosis. Small cell lung cancer can be divided into two stages: limited-stage (LS-SCLC) and extensive-stage (ES-SCLC). Approximately two-thirds of patients are diagnosed at the extensive-stage. Although the cancer cells in small cell lung cancer patients are highly sensitive to chemotherapy and radiotherapy, with an overall response rate of ≥50% with chemotherapy, most patients relapse, and few survive for more than two years. The median overall survival for patients with extensive-stage SCLC is approximately 10 months, with a 2-year survival rate of less than 5% and a 5-year survival rate of only 2%. In clinical practice, combining immune checkpoint inhibitors with chemotherapy drugs for SCLC can provide long-term and durable clinical benefits, but this is limited to a small percentage of patients and the absolute benefit is not significant. SCLC urgently needs new treatment methods, better drugs, and more rationally driven clinical trials using biomarkers.

[0003] Studies have found that DLL3 (Delta-Like Ligand 3, DLL3) is highly expressed in over 80% of small cell lung cancer (SCLC) patients, and high DLL3 expression in SCLC is negatively correlated with patient survival; that is, the higher the DLL3 expression level, the lower the patient's survival. DLL3 is a single-pass transmembrane protein composed of 619 amino acids. Its complete structure includes one DSL domain, one intracellular domain, and six epidermal growth factor-like domains. This target is almost not expressed in normal tissues; however, it is specifically and abundantly expressed in small cell lung cancer, thus DLL3 shows promise as a potential therapeutic target for lung cancer. Currently, several drugs targeting DLL3 are under development, mainly including bispecific antibodies, cell therapy, and ADC drugs. The DLL3 / CD3 bispecific antibody Tarlatamab has demonstrated excellent clinical data in previously treated SCLC patients and received accelerated FDA approval in May 2024 for second- and third-line treatment of extensive-stage SCLC, showing a significant improvement in historical survival data compared to later-line treatments. Tarlatamab is currently in Phase 3 clinical trials in first-line ES-SCLC and LS-SCLC, aiming to cover more advanced SCLC patients. Although Tarlatamab has shown very durable responses in clinical trials, offering new hope for patients with extensive-stage small cell lung cancer, TRAEs (transient etiologies) resulted in 16% of patients needing to discontinue treatment and 4% needing to stop treatment. Therefore, there is an urgent need to develop safer, more effective, and higher-quality drugs. Attached Figure Description

[0004] The novel features of this application are specifically set forth in the appended claims. To better understand the features and advantages of this application, detailed descriptions will be provided below through embodiments.

[0005] Figure 1 shows the binding activity of CD3 monoclonal antibody to CD3 protein on the surface of Jurkat cells;

[0006] Figure 2 shows a schematic diagram of the structure of the bispecific antibody of this application;

[0007] Figure 3A shows the binding affinity of the bispecific antibody to human CD3 protein;

[0008] Figure 3B shows the binding activity of the bispecific antibody to the DLL3 protein on the surface of SHP77 cells;

[0009] Figure 3C shows the binding activity of the bispecific antibody to the DLL3 protein on the surface of NCI-H82 cells;

[0010] Figure 3D shows the binding activity of the bispecific antibody to the CD3 protein on the surface of Jurkat cells;

[0011] Figure 4A shows the bispecific antibody-mediated activation of the Jurkat-NFAT reporter gene system in SHP77 cells;

[0012] Figure 4B shows the bispecific antibody-mediated activation of the Jurkat-NFAT reporter gene system in NCI-H82 cells;

[0013] Figure 5A shows the killing effect of bispecific antibody-mediated PBMCs on SHP77 cells (TDCC);

[0014] Figure 5B shows the bispecific antibody-mediated killing effect of PBMCs on NCI-H82 cells (TDCC);

[0015] Figure 6A shows the release of cytokines mediated by bispecific antibody in the SHP77 cell TDCC system.

[0016] Figure 6B shows the release of cytokines mediated by bispecific antibody in the NCI-H82 cell TDCC system.

[0017] Figure 7A shows the level of T cell activation associated with the bispecific antibody TDCC (SHP77 cells);

[0018] Figure 7B shows the level of T cell activation associated with the bispecific antibody TDCC (NCI-H82 cells);

[0019] Figure 8 shows the nonspecific activation of PBMCs by the bispecific antibody;

[0020] Figure 9 shows the tumor growth curves of the SHP77 local PBMC model under DLL3-CD3 bispecific antibody administration. In Figure 9, n=7, 10C vs Vehicle: *** P<0.001 **** P<0.0001.

[0021] Figure 10 shows the tumor weight scatter plot of the SHP77 local PBMC model under the administration of DLL3-CD3 dual antibody;

[0022] Figure 11 shows the weight change curve of the SHP77 local PBMC model under the administration of DLL3-CD3 dual antibody;

[0023] Figure 12 shows the tumor growth curves of the NCI-H82 local PBMC model under DLL3-CD3 bispecific antibody administration; where n = 7, 10C (5 nmol / kg) vs Vehicle: ** P<0.01 **** P<0.0001; 10C (3nmol / kg) vs Vehicle: ##P<0.01 #### P<0.0001.

[0024] Figure 13 shows the tumor weight scatter plot of the NCI-H82 local PBMC model under the administration of DLL3-CD3 dual antibody;

[0025] Figure 14 shows the weight change curve of the NCI-H82 local PBMC model under the administration of DLL3-CD3 dual antibody.

[0026] Invention Details

[0027] definition

[0028] kon (1 / Ms): refers to the binding rate constant; kon represents the rate constant for the formation of a complex (RL) by the binding of two reactants (such as acceptor R and ligand L). In the International System of Units (SI), the unit of kon is megahertz (M). -1 ·s -1 (i.e., 1 / (M·s)). If the concentration of both substances is 1M (moles per liter), then the number of moles that increase in the concentration of the complex formed by their combination per second is kon.

[0029] kdis (1 / s): refers to the dissociation rate constant; kdis represents the rate at which a complex (such as a receptor-ligand complex, enzyme-substrate complex, or coordination compound) dissociates into its components. It reflects the stability of intermolecular binding: the larger the kdis, the faster the dissociation and the less stable the binding; the smaller the kdis, the slower the dissociation and the more stable the binding. The unit of kdis is 1 / second (s⁻¹), also known as the reciprocal of the second. This indicates what percentage of the complex dissociates per second. Assuming a complex has a kdis = 0.5 s⁻¹, this does not mean that 0.5 units of the complex dissociate in 1 second, but rather that 50% of the complex dissociates per second.

[0030] Knobs-into-Holes Mutation: Knobs-into-Holes (KiH) is a classic and mature protein engineering technique primarily used to solve the challenge of heterodimerization of heavy chains in bispecific antibodies (BsAbs). Its core principle is steric hindrance. This technique creates an interlocking structure of a "knob" and a "hole" by making specific mutations at the CH3 domain interface of two different antibody heavy chains (HC). Knob: Mutates a smaller amino acid (e.g., threonine, Thr) to a larger amino acid (e.g., tryptophan, Trp). Hole: Mutates a larger amino acid (e.g., tyrosine, Tyr) to a smaller amino acid (e.g., threonine, Thr). This "knob-holes" combination utilizes steric hindrance, forcing the two different heavy chains to bind and form a heterodimer, rather than a homodimer.

[0031] BLI stands for Biolayer Interferometry; it is a label-free detection technology based on optical interference. It monitors the interactions between biomolecules (such as proteins and nucleic acids) in real time by shining white light onto the sensor surface and detecting changes in the interference pattern of the reflected light. It is mainly used to determine intermolecular affinity, binding rate constant (ka / kd), and affinity constant (KD), and is widely applied in drug development, antibody engineering, and vaccine evaluation.

[0032] In this application, “AMG757-CD3” refers to the scFv form formed by the CD3 light and heavy chain variable region sequence in the AMG757 (see US20170037130A1, SEQ ID NO:520) bispecific antibody, the sequence of which can be found in Tables 3 and 4 of this application.

[0033] In this application, “AMG757” refers to the AMG757 bispecific antibody in US20170037130A1, the sequence of which can be found in SEQ ID NO:520 in US20170037130A1. Detailed Implementation

[0034] The novel CD3 antibody or its antigen-binding fragment provided in this application has particularly advantageous properties, such as reduced binding activity to CD3. This application also provides a DLL3 / CD3 bispecific antibody with particularly advantageous properties, such as increased DLL3 target affinity, enhanced tumor-killing ability, and lower cytokine release levels.

[0035] In a first aspect, this application provides a CD3 antibody or an antigen-binding fragment thereof, comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the light chain variable region and the heavy chain variable region comprise:

[0036] HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:11 and LCDR3 shown in SEQ ID NO:17;

[0037] The VH comprises an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:42, and the VL comprises an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:43; or the VH comprises an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:3, and the VL comprises an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:4.

[0038] In one embodiment, the antigen-binding fragment is selected from Fab fragments, Fab′ fragments, Fab′-SH fragments, F(ab′)2 fragments, Fv fragments, and scFv fragments; optionally, the antigen-binding fragment is an antigen-binding fragment derived from a monoclonal antibody and / or a humanized antibody.

[0039] Secondly, this application provides a bispecific antigen-binding molecule, comprising the aforementioned immunoglobulin domain targeting CD3 and an immunoglobulin domain targeting tumor cell surface antigens; optionally, the immunoglobulin domain targeting tumor cell surface antigens is an antigen-binding fragment of a DLL3 antibody.

[0040] In one embodiment, the bispecific antigen-binding molecule includes (a) a first immunoglobulin domain targeting CD3, (b) a second immunoglobulin domain targeting DLL3, and (c) a third immunoglobulin domain targeting DLL3; optionally, the first immunoglobulin domain is an scFv structure, and the second and third immunoglobulin domains are a Fab domain and an scFv domain, respectively.

[0041] In one specific embodiment, in the bispecific antigen-binding molecule, the antigen-binding fragment of the CD3 antibody includes a light chain variable region (VL) and a heavy chain variable region (VH), wherein the light chain variable region and the heavy chain variable region include: HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:17;

[0042] Optionally, the VH comprises an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:42, and the VL comprises an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:43; or the VH comprises an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:3, and the VL comprises an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:4.

[0043] In one specific embodiment, in the bispecific antigen-binding molecule, the antigen-binding fragment of the DLL3 antibody includes a light chain variable region (VL) and a heavy chain variable region (VH), wherein the light chain variable region and the heavy chain variable region include:

[0044] HCDR1 shown in SEQ ID NO:30, HCDR2 shown in SEQ ID NO:31, HCDR3 shown in SEQ ID NO:32, LCDR1 shown in SEQ ID NO:33, LCDR2 shown in SEQ ID NO:34 and LCDR3 shown in SEQ ID NO:35; and / or HCDR1 shown in SEQ ID NO:36, HCDR2 shown in SEQ ID NO:37, HCDR3 shown in SEQ ID NO:38, LCDR1 shown in SEQ ID NO:39, LCDR2 shown in SEQ ID NO:40 and LCDR3 shown in SEQ ID NO:41;

[0045] Optionally, the VH segment of the antigen-binding fragment of the DLL3 antibody contains an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:26, and the VL segment contains an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:27; and / or

[0046] The VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:28, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:29.

[0047] In one specific embodiment, in the bispecific antigen-binding molecule, the immunoglobulin domain targeting CD3 includes HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:17.

[0048] The antigen-binding fragment of the DLL3 antibody comprises HCDR1 shown in SEQ ID NO:30, HCDR2 shown in SEQ ID NO:31, HCDR3 shown in SEQ ID NO:32, LCDR1 shown in SEQ ID NO:33, LCDR2 shown in SEQ ID NO:34, and LCDR3 shown in SEQ ID NO:35; and / or comprises HCDR1 shown in SEQ ID NO:36, HCDR2 shown in SEQ ID NO:37, HCDR3 shown in SEQ ID NO:38, LCDR1 shown in SEQ ID NO:39, LCDR2 shown in SEQ ID NO:40, and LCDR3 shown in SEQ ID NO:41.

[0049] In one specific embodiment, in the bispecific antigen-binding molecule, the first immunoglobulin domain targeting CD3 comprises HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:17; the second immunoglobulin domain targeting DLL3 comprises HCDR1 shown in SEQ ID NO:30, HCDR2 shown in SEQ ID NO:31, HCDR3 shown in SEQ ID NO:32, LCDR1 shown in SEQ ID NO:33, LCDR2 shown in SEQ ID NO:34, and LCDR3 shown in SEQ ID NO:35; and the third immunoglobulin domain targeting DLL3 comprises HCDR1 shown in SEQ ID NO:36, HCDR2 shown in SEQ ID NO:37, HCDR3 shown in SEQ ID NO:38, LCDR1 shown in SEQ ID NO:39, LCDR2 shown in SEQ ID NO:40, and LCDR3 shown in SEQ ID NO:41.

[0050] Optionally, the first immunoglobulin domain targeting CD3 is an scFv domain, which includes HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:17; the second immunoglobulin domain targeting DLL3 is a Fab domain, which includes HCDR1 shown in SEQ ID NO:30, HCDR2 shown in SEQ ID NO:31, HCDR3 shown in SEQ ID NO:32, LCDR1 shown in SEQ ID NO:33, LCDR2 shown in SEQ ID NO:34, and LCDR3 shown in SEQ ID NO:35; and the third immunoglobulin domain targeting DLL3 is an scFv domain, which includes HCDR1 shown in SEQ ID NO:36, HCDR2 shown in SEQ ID NO:37, HCDR3 shown in SEQ ID NO:38, LCDR1 shown in SEQ ID NO:39, and SEQ ID NO:17. LCDR2 shown in NO:40 and LCDR3 shown in SEQ ID NO:41.

[0051] In one specific embodiment, in the bispecific antigen-binding molecule, the VH of the first immunoglobulin domain targeting CD3 contains an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:42, and the VL contains an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:43; or the VH of the first immunoglobulin domain targeting CD3 contains an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:3, and the VL contains an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:43; The amino acid sequence shown in NO:4 has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:26; the VH of the second immunoglobulin domain targeting DLL3 contains an amino acid sequence or its amino acid sequence has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:27, and the VL contains an amino acid sequence or its amino acid sequence has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:27; and the VH of the third immunoglobulin domain targeting DLL3 contains an amino acid sequence or its amino acid sequence has an identity with the amino acid sequence shown in SEQ ID NO:27. The amino acid sequence shown in NO:28 has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO:29; and VL contains or has an amino acid sequence that has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO:29.

[0052] Optionally, the first immunoglobulin domain targeting CD3 is an scFv domain, wherein its VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:42, and its VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:43; the second immunoglobulin domain targeting DLL3 is a Fab domain, wherein its VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:26, and its VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:43; The amino acid sequence shown in NO:27 has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:28; and the third immunoglobulin domain targeting DLL3 is an scFv domain, wherein its VH contains or its amino acid sequence has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:28, and its VL contains or its amino acid sequence has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:29.

[0053] In one specific embodiment, the bispecific antigen-binding molecule comprises the following peptide chains (1)-(3):

[0054] peptide chain (1) N'-DLL3VL-CL-C';

[0055] peptide chain (2)N'-DLL3VH-CH1-Fc-C'; and

[0056] Peptide chain (3)N'-DLL3'VL-DLL3'VH-CD3VL-CD3VH-Fc-C', N'-CD3VL-CD3VH-DLL3'VL-DLL3'VH-Fc- C', N'-DLL3'VH-DLL3'VL-CD3VH-CD3VL-Fc-C' or N'-CD3VH-CD3VL-DLL3'VH-DLL3'VL-Fc-C';

[0057] Where VH represents the heavy chain variable region, DLL3VH and DLL3'VH represent the heavy chain variable regions of the antigen-binding fragments targeting DLL3, CD3VH represents the heavy chain variable region of the antigen-binding fragments targeting CD3; DLL3VL and DLL3'VL represent the light chain variable regions of the antigen-binding fragments targeting DLL3, CD3VL represents the light chain variable region of the antigen-binding fragments targeting CD3; Fc contains CH2 and CH3; CH1, CH2, and CH3 represent domains 1, 2, and 3 of the heavy chain constant region, respectively; CL represents the light chain constant region.

[0058] Optionally, the Fc region comprises a natural Fc sequence or a non-natural Fc sequence; optionally, the Fc region is a human Fc region; further optionally, the Fc region is the Fc region of an IgG1, IgG2, IgG3, or IgG4 antibody; even further optionally, the Fc region is an IgG1 Fc region; even further optionally, the Fc region comprises one or more mutations selected from those weakening the antibody ADCC effect, those weakening the antibody CDC effect, knocks-in-holes mutations, and disulfide bond mutations. Further, the Fc region comprises one or more of L234A, L235A, P329A, P331S, S354C, T366W, Y349C, T366S, L368A, and Y407V.

[0059] In one embodiment, in the bispecific antigen-binding molecule, CH1 comprises an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:44.

[0060] The CL comprises, or has an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:45; and / or

[0061] The Fc contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the amino acid sequences shown in SEQ ID NO:46-49.

[0062] In one embodiment, the bispecific antigen-binding molecule comprises peptide chain (1)-peptide chain (3);

[0063] The peptide chain (1) comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:50;

[0064] The peptide chain (2) comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 51 or 53;

[0065] The peptide chain (3) comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 52 or 54;

[0066] Optionally, the peptide chain (1)-peptide chain (3) comprises or has an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NO: 50, 51, and 52; or

[0067] The peptide chain (1)-peptide chain (3) comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences shown in SEQ ID NO:50, 53 and 54.

[0068] This application also provides a DLL3 antibody or an antigen-binding fragment thereof, the DLL3 antibody or the antigen-binding fragment thereof comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the light chain variable region and the heavy chain variable region comprise:

[0069] HCDR1 shown in SEQ ID NO:36, HCDR2 shown in SEQ ID NO:37, HCDR3 shown in SEQ ID NO:38, LCDR1 shown in SEQ ID NO:39, LCDR2 shown in SEQ ID NO:40, and LCDR3 shown in SEQ ID NO:41.

[0070] In one embodiment, the light chain variable region (VL) and heavy chain variable region (VH) of the DLL3 antibody or its antigen-binding fragment include:

[0071] VH contains an amino acid sequence or its amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:28, and VL contains an amino acid sequence or its amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:29.

[0072] This application also provides a bispecific antigen-binding molecule comprising the DLL3 antibody or its antigen-binding fragment.

[0073] Furthermore, the bispecific antigen-binding molecule also includes an immunoglobulin domain that targets CD3.

[0074] Furthermore, the bispecific antigen-binding molecule includes (a) a first immunoglobulin domain targeting CD3, (b) a second immunoglobulin domain targeting DLL3, and (c) a third immunoglobulin domain targeting DLL3; optionally, the first immunoglobulin domain is an scFv structure, and the second and third immunoglobulin domains are a Fab domain and an scFv domain, respectively.

[0075] Furthermore, the bispecific antigen-binding molecule peptide chain comprises the following peptide chains (1)-(3):

[0076] peptide chain (1) N'-DLL3VL-CL-C';

[0077] peptide chain (2)N'-DLL3VH-CH1-Fc1-hole-C'; and

[0078] peptide chain (3)N'-DLL3'VL-DLL3'VH-CD3VL-CD3VH-Fc2-knob-C';

[0079] In one specific embodiment of this application, the first immunoglobulin domain includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region include: HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:17.

[0080] In one specific embodiment, the light chain variable region (VL) and heavy chain variable region (VH) of the first immunoglobulin domain include:

[0081] The VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:42, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:43; and / or

[0082] The VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:3, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:4.

[0083] In one specific embodiment, the second immunoglobulin domain includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region include: HCDR1 shown in SEQ ID NO:30, HCDR2 shown in SEQ ID NO:31, HCDR3 shown in SEQ ID NO:32, LCDR1 shown in SEQ ID NO:33, LCDR2 shown in SEQ ID NO:34, and LCDR3 shown in SEQ ID NO:35.

[0084] In one specific embodiment, the light chain variable region (VL) and heavy chain variable region (VH) of the second immunoglobulin domain include:

[0085] The VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:26, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:27.

[0086] In one specific embodiment, the third immunoglobulin domain includes a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region include: HCDR1 shown in SEQ ID NO:36, HCDR2 shown in SEQ ID NO:37, HCDR3 shown in SEQ ID NO:38, LCDR1 shown in SEQ ID NO:39, LCDR2 shown in SEQ ID NO:40, and LCDR3 shown in SEQ ID NO:41.

[0087] In one specific embodiment, the light chain variable region (VL) and heavy chain variable region (VH) of the third immunoglobulin domain include:

[0088] The VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:28, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:29.

[0089] In one specific embodiment, CH1 comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:44;

[0090] The CL comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:45;

[0091] The Fc1 comprises or has an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:46 or 48; and / or

[0092] The Fc2 comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:47 or 49.

[0093] In one specific embodiment, the bispecific antigen-binding molecule comprises peptide chain (1)-peptide chain (3);

[0094] The peptide chain (1) comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:50;

[0095] The peptide chain (2) comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 51 or 53;

[0096] The peptide chain (3) comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 52 or 54;

[0097] Preferably, the peptide chain (1)-peptide chain (3) comprises or has an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NO: 50, 51, and 52; or

[0098] The peptide chain (1)-peptide chain (3) comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences shown in SEQ ID NO:50, 53 and 54.

[0099] Thirdly, this application provides an isolated nucleic acid comprising a nucleic acid sequence encoding any of the aforementioned CD3 antibodies or their antigen-binding fragments, or bispecific antigen-binding molecules.

[0100] Fourthly, this application provides a vector comprising the nucleic acid.

[0101] Fifthly, this application provides an isolated host cell comprising the nucleic acid or vector.

[0102] In a sixth aspect, this application provides a composition comprising any of the aforementioned CD3 antibodies or their antigen-binding fragments, or the aforementioned bispecific antigen-binding molecules, or the aforementioned nucleic acids, or vectors, or host cells; optionally, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0103] In a seventh aspect, this application provides the use of the CD3 antibody or its antigen-binding fragment, the bispecific antigen-binding molecule, the nucleic acid, the vector, or the host cell in the manufacture of a medicament for treating or delaying the progression of an individual's cancer; optionally, the cancer is DLL3-positive cancer; further optionally, the cancer is lung cancer; and even more optionally, the cancer is small cell lung cancer.

[0104] Eighthly, this application provides a method for treating or delaying cancer progression in a subject in need, comprising administering the bispecific antigen-binding molecule to the subject; optionally, wherein the cancer is DLL3-positive cancer; further optionally, the cancer is lung cancer; and even more optionally, the cancer is small cell lung cancer.

[0105] The technical solution of this application has the following advantages:

[0106] 1. The CD3 antibody or its antigen-binding fragment has particularly advantageous properties, such as reduced binding activity to CD3.

[0107] 2. The DLL3 / CD3 bispecific antibody of this application also has one or more of the following advantages: increased affinity for the DLL3 target, enhanced Jurkat reporter gene activation activity, enhanced tumor killing ability, and reduced cytokine release levels.

[0108] 3. The DLL3-CD3 bispecific antibody developed in this application has shown in preclinical studies to have stronger affinity for DLL3 and lower affinity for CD3 antibodies than the commercially available AMG757 bispecific antibody. It has a milder effect, target-dependent T-cell activation, and reduced toxicity while maintaining long-lasting anticancer activity. It is expected to improve tumor tissue distribution, reduce side effects, and expand the administration window without losing efficacy.

[0109] Example

[0110] Example 1: Design, production and identification of anti-DLL3 antibody or its antigen-binding fragment sequence

[0111] 1.1: Design and production of anti-DLL3 antibodies or their antigen-binding fragment sequences

[0112] Murine 33B10E8 and 25G10A9 antibodies were obtained through hybridoma screening and analyzed using Discovery Studio and... Homology modeling was performed using Antibody Modeling. Through structural simulation and rational design, a human frame region most closely resembling the murine antibody frame region was obtained. The CDRs of the light and heavy chains were then transplanted into the frame sequences of the matching human light and heavy chain genes, respectively, resulting in the humanized antibodies Hab33B10E8 and Hab25G10A9. These were then analyzed using Discovery Studio and... Antibody Modeling was used to construct 3D models and analyze whether there were any sites where replacing mouse amino acids with human amino acids at framework positions would affect binding and / or CDR conformation. Reverse mutations were performed, and the resulting DLL3 antibody or its antigen-binding fragment sequence is shown in Table 1.

[0113] Table 1: Sequences of DLL3 antibodies or their antigen-binding fragments (CDRs numbered in kabat format)

[0114] 1.2: Identification of anti-DLL3 antibody or its antigen-binding fragment

[0115] The binding affinity of humanized antibodies to DLL3 on the cell surface was assessed using SHP77 or NCI-H82 cells. Cells were seeded at a density of 2E6 / mL, 100 μL / well in 96-well plates. 66.7 nM of antibody was added, and the cells were incubated at 4°C for 1 h. Goat anti-human secondary antibody (Invitrogen, A-11013) was added, and the cells were labeled at 4°C in the dark for 45 min. After cell collection and washing, fluorescence intensity was read by flow cytometry. The results are shown in Table 2. The humanized antibodies can bind to the DLL3 protein on the cell surface. Hab33B10E8 and Hab25G10A9 antibodies showed better affinity for DLL3 than the control molecule Rovalpituzumab (Baiying Biotechnology, B717301).

[0116] Table 2: Binding activity of humanized antibodies to DLL3 on cell surface

[0117] Example 2: Design, production, and identification of anti-CD3 antibodies or their antigen-binding fragment sequences

[0118] 2.1: Design and production of anti-CD3 antibodies or their antigen-binding fragment sequences

[0119] The CDR region of a CD3 antibody with CD3 receptor recognition capability was mutated to obtain the CD3 monoclonal antibody variable region. This variable region was then constructed into an scFv form and added to a eukaryotic expression vector containing hIgG1. The Fc sequence of this vector incorporated amino acid mutations at L234A / L235A / P329A / P331S (according to Kabat's "EU" number). The CD3 light and heavy chain variable region sequences of the AMG757 (patent number: US20170037130A1, SEQ ID NO:520) bispecific antibody were also constructed into an scFv form and used as a positive control. The obtained eukaryotic expression vector was transiently transformed into CHO cells and cultured for 6 days. The cell supernatant was collected by centrifugation and purified using a protein A column to obtain the target antibodies, named #6, #7, and AMG757-CD3 (i.e., CD3 in AMG757). Specific sequences are shown in Tables 3-4.

[0120] Table 3: Variable region sequences of CD3 antibody heavy and light chains (underlined regions are CDR regions, encoded using kabat encoding)

[0121] Table 4: Complete scFv sequences and Fc region sequences of #6, #7, and AMG757-CD3

[0122] 2.2: BLI assay to determine the affinity of CD3 monoclonal antibody for human and cynomolgus monkey CD3 protein

[0123] Using ProA biosensor ( ProA Biosensors (Sartorius, catalog number 18-5012) immobilized CD3 monoclonal antibodies on biosensors to a binding response threshold of 0.3 nm. After a 120-second baseline step, the sensors were immersed in recombinant human CD3 protein (ACRO Biosystem, CDE-H5223) or cynomolgus monkey CD3 protein (ACRO Biosystem, CDE-C5226) diluted with 0.02% PBST buffer. The initial protein concentration was 100 nM, with 3-fold serial dilutions and 7 gradients. The binding and dissociation times were 120 s and 300 s, respectively. The affinity data of the antigen and antibody were calculated by regression analysis using analysis software based on the binding curves. AMG757-CD3 showed the best binding affinity to recombinant human CD3 protein, #7 showed weaker binding affinity to recombinant human CD3 protein than AMG757-CD3, and #6 did not bind to recombinant human CD3 protein. The results are shown in Tables 5-6.

[0124] Table 5: Affinity of CD3 monoclonal antibody to recombinant human CD3 protein as determined by BLI

[0125] Table 6: Affinity of CD3 monoclonal antibody to recombinant monkey CD3 protein as determined by BLI

[0126] 2.3: Binding activity of CD3 monoclonal antibody to CD3 protein on the surface of Jurkat cells

[0127] The binding affinity of CD3 monoclonal antibodies to CD3 on the cell surface was assessed using Jurkat cells. Cells were seeded at a density of 2E6 / mL, 100 μL / well in 96-well plates. Four-fold serial dilutions of CD3 antibody (0-250 nM) were added, and the cells were incubated at 4°C for 1 h. Goat anti-human secondary antibody (Invitrogen, A11013) was added, and the cells were labeled at 4°C in the dark for 45 min. After cell collection and washing, the fluorescence intensity was read by flow cytometry. A stronger fluorescence signal indicated higher antibody affinity to the target. The experimental results are shown in Figure 1 and Table 7. The results show that the binding affinity of molecules #6 and #7 was lower than that of AMG757-CD3, while the binding affinity of #7 was higher than that of #6.

[0128] Table 7: Binding ability of CD3 antibody to CD3 on the surface of Jurkat cells

[0129] Example 3: Production and Identification of Anti-DLL3 / CD3 Bispecific Antibody

[0130] 3.1. Production of anti-DLL3 / CD3 bispecific antibodies

[0131] Bispecific antibodies were constructed using the DLL3 antibody sequence from Example 1 and the #7 molecule from Example 2. The structures of the bispecific antibodies are shown in Figure 2 and Table 8. The nucleic acid encoding the bispecific antibody molecule was constructed into a eukaryotic expression vector containing hIgG1. The obtained eukaryotic expression vector was transiently transfected into CHO cells and cultured at 37°C and 5% CO2 for 6 days. The cell supernatant was collected by centrifugation and purified by protein A column to obtain the target antibodies, named 10B and 10C, respectively. Their sequences are shown in Tables 9-11.

[0132] Table 8: Chain structure of DLL3 / CD3 bispecific antibodies

[0133] Table 9: Antibody variable region sequences used in anti-DLL3 / CD3 bispecific antibodies (underlined regions are CDR regions, encoded using kabat encoding).

[0134] Table 10: Constant region sequences used in anti-DLL3 / CD3 bispecific antibodies

[0135] Table 11: Complete sequences of anti-DLL3 / CD3 bispecific antibodies

[0136] 3.2: Identification of anti-DLL3 / CD3 bispecific antibodies

[0137] 3.2.1: ELISA detection of the affinity between DLL3 / CD3 bispecific antibody and human CD3 protein

[0138] The affinity of DLL3 / CD3 bispecific antibody for human CD3 protein (Acro, CDE-H5223) was determined using ELISA. Human CD3 protein was diluted to 0.5 μg / mL and coated onto the microplate. After blocking, 100 μL of DLL3 / CD3 bispecific antibody (0-10 nM) diluted 4-fold in each well was added, and the plate was incubated at 37°C for 1 h. Then, 100 μL of goat anti-human secondary antibody (Jackson, 109-036-098) was added to each well and incubated for another 1 h. TMB was added for color development, and the results were read using a microplate reader. The results were fitted using GraphPad and are shown in Figure 3A and Table 12. The affinity of DLL3 / CD3 bispecific antibody molecules 10B and 10C for human CD3 protein was lower than that of AMG757 (patent number: US20170037130A1, SEQ ID NO:520).

[0139] Table 12: Affinity of DLL3 / CD3 bispecific antibody to human CD3 protein as detected by ELISA

[0140] 3.2.2: BLI assay to detect the affinity of DLL3 / CD3 bispecific antibody for human / monkey CD3 protein

[0141] The affinity of DLL3 / CD3 bispecific antibodies for recombinant human CD3 protein (Acro, CDE-H5223) and cynomolgus monkey CD3 protein (Acro, CDE-C5226) was determined using BLI. Using a ProA biosensor, the DLL3 / CD3 bispecific antibodies were immobilized onto the biosensor to a binding response threshold of 0.3 nm. After a 120-s baseline step, the sensor was immersed in human / monkey CD3 protein diluted with 0.02% PBST buffer. The initial CD3 protein concentration was 100 nM, with 3-fold serial dilutions and 7 gradients. The binding and dissociation times of the immobilized material with the analyte were 120 s and 300 s, respectively. Affinity data for the antigen and antibody were calculated using regression analysis software based on the binding curves. The results are shown in Table 13. The affinity of DLL3 / CD3 bispecific antibody molecules 10B and 10C for human / monkey CD3 protein was lower than that of AMG757.

[0142] Table 13: BLI detection of the affinity of DLL3 / CD3 bispecific antibody molecules for human or cynomolgus monkey CD3 protein

[0143] 3.2.3: BLI assay to detect the affinity of DLL3 / CD3 bispecific antibody for human or cynomolgus monkey DLL3 protein

[0144] The affinity of the DLL3 / CD3 bispecific antibody for recombinant human DLL3 protein (Acro, DL3-H52H4) and cynomolgus monkey DLL3 protein (Acro, DL3-C52H3) was determined using BLI. The DLL3 / CD3 bispecific antibody was immobilized on the ProA biosensor to a binding response threshold of 0.3 nm. After a 120-s baseline step, the sensor was immersed in human / monkey DLL3 protein diluted with 0.02% PBST buffer. The initial concentration of human / monkey DLL3 protein was 100 nM, with 3-fold serial dilutions and 7 gradients. The binding and dissociation times of the immobilized material and analyte were 120 s and 300 s, respectively. The affinity data of the antigen and antibody were calculated by regression analysis using the binding curves. The results are shown in Table 14. The affinity of DLL3 / CD3 bispecific antibody molecules 10B and 10C for human / monkey DLL3 protein was significantly higher than that of AMG757.

[0145] Table 14: BLI assay for affinity of bispecific antibody molecules to human or cynomolgus monkey DLL3 protein

[0146] 3.2.4: Binding activity of DLL3 / CD3 bispecific antibody to DLL3 protein on the surface of SHP77 cells or NCI-H82 cells or CD3 protein on the surface of Jurkat cells.

[0147] The binding activity of DLL3 / CD3 bispecific antibodies to DLL3 protein on the cell surface was evaluated using human small cell lung cancer cell lines NCI-H82 (Nanjing Kebai, CBP61483) / SHP77 (Nanjing Kebai, CBP60151), and the binding activity of DLL3-CD3 bispecific antibodies to CD3 protein on the cell surface was evaluated using Jurkat cells.

[0148] Adjust the density of SHP77, NCI-H82, or Jurkat cells to 2E6 / mL, and add 100 μL / well to each 96-well plate. Centrifuge at 2000 rpm for 2 min, discard the supernatant, and add 100 μL of 4-fold serially diluted DLL3-CD3 bispecific antibody (0-62.5 nM) to each well. Incubate at 4℃ for 1 h. Collect cells, add 100 μL of goat anti-human secondary antibody (Invitrogen, A-11013) to each well, label at 4℃ in the dark for 45 min, collect cells, wash, and read fluorescence intensity by flow cytometry. Fit antibody dose-dependent binding curves using GraphPad. The experimental results are shown in Figures 3B, 3C, and 3D. The binding activity of bispecific antibody molecules 10B and 10C to the cell surface DLL3 protein is significantly higher than that of AMG757, while their binding activity to the cell surface CD3 protein is lower than that of AMG757.

[0149] 3.2.5: Activation activity of DLL3 / CD3 bispecific antibody against Jurkat reporter gene

[0150] The Jurkat-NFAT-Luc luciferase system, which endogenously expresses human CD3, was used. SHP77 or NCI-H82 cells, which endogenously express DLL3, were used as target cells. The activity of the DLL3 / CD3 bispecific antibody was detected using a reporter gene system. Cells in logarithmic growth phase were harvested, and the Jurkat-NFAT-Luc cell density was adjusted to 3.2E6 / mL, and the SHP77 / NCI-H82 cell density was adjusted to 4E5 / mL. 25 μL of each cell was seeded into 96-well plates, resulting in an effector-target cell ratio of 8:1. 50 μL of a 3-fold serially diluted DLL3 / CD3 bispecific antibody sample (0-10 nM) was added to each well, and the cells were incubated at 37℃ for 16 h in a 5% CO2 incubator. 100 μL of Bright-Glo was added to each well. TM (Promega, E2620), incubate at room temperature for 3-5 minutes, then read the fluorescence value using a microplate reader.

[0151] The results obtained by fitting with GraphPad are shown in Figures 4A and 4B and Table 15. Jurkat reporter gene activation can be induced only when target cells NCI-H82 or SHP77 are present. The Jurkat reporter gene activation activity of DLL3 / CD3 bispecific antibodies 10B and 10C is better than that of AMG757.

[0152] Table 15: Activation activity of bispecific antibody molecules against the Jurkat reporter gene

[0153] 3.2.6: DLL3 / CD3 bispecific antibody against DLL3 + Evaluation of the tumor cell killing ability and T cell activation.

[0154] 3.2.6.1: DLL3 / CD3 bispecific antibody against DLL3 + The killing ability of tumor cells

[0155] Peripheral blood mononuclear cells (PBMCs) were used as effector cells, and tumor cells SHP77 or NCI-H82 expressing DLL3 on their cell surface were used as target cells to evaluate the killing ability of DLL3 / CD3 bispecific antibodies. PBMCs were resuscitated, and the PBMC cell density was adjusted to 3.2E6 / mL, and the target cell density was adjusted to 4E5 / mL. 25 μL of each cell was seeded into 96-well plates, resulting in an effector-target cell ratio of 8:1. 50 μL of a 3-fold serially diluted DLL3 / CD3 bispecific antibody sample (0-30000 pM) was added to each well, and the plates were incubated at 37℃ for 24 h in a 5% CO2 incubator. 100 μL of CTG (Cell Titer Glo, Promega, G7572) was added to each well to detect the killing effect of the DLL3 / CD3 bispecific antibody on tumor cells. After incubation at room temperature for 3-5 min, the fluorescence values ​​were read using a microplate reader. The formula for calculating the kill rate based on the readings is as follows: Kill rate (%) = (Fluorescence value of blank control group - Fluorescence value of experimental group) / Fluorescence value of blank control group * 100%. The results, obtained through GraphPad fitting, are shown in Figures 5A and 5B and Table 16. Both DLL3 / CD3 bispecific antibody molecules 10B and 10C can mediate effective killing of T cells, and their killing activity is superior to AMG757.

[0156] Table 16: Killing activity of bispecific antibodies against tumor cells

[0157] 3.2.6.2: Evaluation of T cell activation (assessment of the presence of target cells and cytokine levels)

[0158] The evaluation of T cell activation by bispecific antibodies was conducted using PBMCs as effector cells and SHP77 or NCI-H82 as target cells, cultured as described in Example 3.2.6.1. After 24 h of culture, cell samples were centrifuged at 300 g for 10 min, and the supernatant was collected to detect cytokine concentrations (BioLegend cytokine assay kit information: BioLegend, 431815; 430515; 430215; 430116). Results obtained by GraphPad fitting are shown in Figures 6A and 6B and Table 17. The DLL3 / CD3 bispecific antibody induced T cell cytokine secretion, with molecules 10B and 10C showing milder effects compared to AMG757. Furthermore, Figures 6A-6B are labeled with the EC values ​​corresponding to molecules 10B, 10C, and AMG757 during TDCC (T cell-mediated tumor killing) as shown in Table 16. 50Concentrations are detailed on the horizontal axis indicated by the arrows, where ↓ represents 10C; ↓ represents 10B; and ↓ represents AMG757. It can be seen that at their respective EC50 concentrations, for SHP77 cells (Figure 6A), 10C and 10B showed significantly lower IL2 release than AMG757, while their IL6, TNFα, and IFNγ releases were similar. For NCI-H82 cells (Figure 6B), 10C showed significantly lower TNFα and IFNγ release than AMG757, and 10B showed significantly lower IFNγ release than AMG757, while their IL2 and IL6 releases were similar. It can be seen that 10B and 10C of this application exhibit stronger cell lysis activity (lower EC50). 50 (Value), but the relative cytokine release level is lower or similar to that of AMG757.

[0159] Table 17: Cytokine Release Accompanying TDCC

[0160] 3.2.6.3: Evaluation of T cell activation (presence of target cells, evaluation of T cell activation markers CD25 and CD69)

[0161] Evaluation of T cell activation by bispecific antibodies: PBMCs were used as effector cells, and SHP77 or NCI-H82 cells were used as target cells. The culture method was as described in Example 3.2.6.1. After culturing for 24 h, the cells were collected by centrifugation. 100 μl of T cell activation detection antibody (Invitrogen: CD3-eFluor™ 450, 48-0037-42; CD4-PE, 12-0049-42; CD8-APC-eFluor™ 780, 47-0088-42; CD25-APC, 17-0257-42; CD69-FITC, 11-0699-42) was added to each well. The cells were incubated at 4 °C in the dark for 30 min. The supernatant was washed and discarded, and the cells were detected by flow cytometry.

[0162] The results, obtained by fitting with GraphPad, are shown in Figure 7A (SHP77 cells) and Figure 7B (NCI-H82 cells). In the presence of target cells, the dual antibody can induce CD4+. + and CD8 + T cell activation: the bispecific antibody 10C has a higher activation capacity than AMG757, while the activation capacity of 10B is similar to that of AMG757.

[0163] 3.2.6.4: Evaluation of T cell activation (no evaluation of target cells or cytokine levels)

[0164] Under target cell-free conditions, the non-specific activation of bispecific antibodies was evaluated by detecting PBMC cytokine release levels using ELISA. 96-well cell culture plates were coated with 125 μL / well serially diluted test antibody (0-320 nM, 3-fold dilution) and incubated overnight at 4°C. After washing the cell culture plates twice with PBS, PBMC cells were added to adjust the cell density to 1E6 / mL, 250 μL / well, and cultured at 37°C and 5% CO2 for 24 h. The cell culture supernatant was collected by centrifugation at 300g for 10 min, and the cytokine levels in the supernatant were measured according to the BioLegend Cytokine Detection Kit (same as 3.2.6.2).

[0165] The results obtained by fitting with GraphPad are shown in Figure 8 and Table 18. Under the condition of lack of target cells, the release level of AMG757 cytokines at high concentrations is higher than that of 10B and 10C molecules, that is, the release of cytokines by 10B and 10C molecules is milder than that by AMG757 molecules.

[0166] Table 18: Nonspecific activation of PBMCs by bispecific antibodies

[0167] 3.2.7 In vivo antitumor efficacy of bispecific antibodies

[0168] 3.2.7.1: NCG mouse SHP77 xenograft model

[0169] Small cell lung cancer (PBMC) cells were subcutaneously inoculated into severely immunodeficient mice with NCG to construct a local PBMC model (SHP77). The efficacy of the DLL3 / CD3 dual antibody was evaluated. The tumor cell to PBMC ratio was 8:1. Approximately 10 days post-inoculation, mice were randomly assigned to two groups based on tumor growth: a treatment group and a solvent control group, with 7 mice in each group. The first administration was given 10 days post-inoculation (D0). Intraperitoneal administration was performed on D0 and D3 at a dose of 3 nmol / kg. Tumors were weighed twice weekly, and their volume was measured using the formula: minor axis * minor axis * major axis / 2. The experiment concluded approximately 4 weeks after the first administration, with tumor tissue collected and weighed.

[0170] The in vivo efficacy results in mice are shown in Figures 9 and 10. The 10C molecule has a significant inhibitory effect on tumors (P<0.0001). As shown in Figure 11, the tumor-bearing mice tolerated the administered dose well and did not experience adverse reactions such as weight loss.

[0171] 3.2.7.2: NCG mouse NCI-H82 xenograft model

[0172] Small cell lung cancer cells (PBMCs) were subcutaneously inoculated into severely immunodeficient mice with NCG to construct an NCI-H82 local PBMC model for evaluating the efficacy of the DLL3 / CD3 dual antibody. The tumor cell to PBMC mixture ratio was 2:1. Two groups (n=7 per group) were established: an administration group and a solvent control group. The day after tumor inoculation was designated D0. Intraperitoneal administration was performed on D1 and D8 at doses of 3 nmol / kg and 5 nmol / kg, respectively. Tumors were weighed twice weekly, and their volume was measured using the formula: minor axis * minor axis * major axis / 2. The experiment concluded approximately 4 weeks after the first administration, with tumor tissue collected and weighed.

[0173] The in vivo efficacy results in mice are shown in Figures 12 and 13. The 10C molecule has a significant inhibitory effect on tumors (P<0.0001). As shown in Figure 14, the tumor-bearing mice tolerated the administered dose well and did not experience adverse reactions such as weight loss.

Claims

1. A CD3 antibody or an antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region (VL) and the heavy chain variable region (VH) comprise: HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:11 and LCDR3 shown in SEQ ID NO:

17.

2. The CD3 antibody or its antigen-binding fragment according to claim 1, wherein the VH comprises an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:42, and the VL comprises an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:43; or the VH comprises an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:3, and the VL comprises an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:

4.

3. The CD3 antibody or its antigen-binding fragment according to claim 1 or 2, wherein the antigen-binding fragment is selected from Fab fragment, Fab′ fragment, Fab′-SH fragment, F(ab′)2 fragment, Fv fragment and scFv fragment; optionally, the antigen-binding fragment is an antigen-binding fragment derived from a monoclonal antibody and / or a humanized antibody.

4. A bispecific antigen-binding molecule comprising an immunoglobulin domain targeting CD3 and an immunoglobulin domain targeting tumor cell surface antigens; wherein the immunoglobulin domain targeting CD3 is selected from the antigen-binding fragment of the CD3 antibody according to any one of claims 1-3; Optionally, the immunoglobulin domain targeting tumor cell surface antigens is an antigen-binding fragment of the DLL3 antibody.

5. The bispecific antigen-binding molecule according to claim 4, wherein the bispecific antigen-binding molecule comprises (a) a first immunoglobulin domain targeting CD3, (b) a second immunoglobulin domain targeting DLL3, and (c) a third immunoglobulin domain targeting DLL3. Optionally, the first immunoglobulin domain is an scFv domain, and the second and third immunoglobulin domains are a Fab domain and an scFv domain, respectively.

6. The bispecific antigen-binding molecule according to any one of claims 4-5, wherein the antigen-binding fragment of the DLL3 antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region comprise: The HCDR1 shown in SEQ ID NO:30, the HCDR2 shown in SEQ ID NO:31, the HCDR3 shown in SEQ ID NO:32, the LCDR1 shown in SEQ ID NO:33, the LCDR2 shown in SEQ ID NO:34, and the LCDR3 shown in SEQ ID NO:35; and / or the HCDR1 shown in SEQ ID NO:36, the HCDR2 shown in SEQ ID NO:37, the HCDR3 shown in SEQ ID NO:38, the LCDR1 shown in SEQ ID NO:39, the LCDR2 shown in SEQ ID NO:40, and the LCDR3 shown in SEQ ID NO:41; Optionally, the VH segment of the antigen-binding fragment of the DLL3 antibody contains an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:26, and the VL segment contains an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:27; and / or The VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:28, and the VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:

29.

7. The bispecific antigen-binding molecule according to any one of claims 4-6, wherein the target CD3 immunoglobulin domain comprises HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:17; The antigen-binding fragment of the DLL3 antibody comprises HCDR1 shown in SEQ ID NO:30, HCDR2 shown in SEQ ID NO:31, HCDR3 shown in SEQ ID NO:32, LCDR1 shown in SEQ ID NO:33, LCDR2 shown in SEQ ID NO:34, and LCDR3 shown in SEQ ID NO:35; and / or comprises HCDR1 shown in SEQ ID NO:36, HCDR2 shown in SEQ ID NO:37, HCDR3 shown in SEQ ID NO:38, LCDR1 shown in SEQ ID NO:39, LCDR2 shown in SEQ ID NO:40, and LCDR3 shown in SEQ ID NO:

41.

8. The bispecific antigen-binding molecule according to any one of claims 5-7, wherein the first immunoglobulin domain targeting CD3 comprises HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:17; the second immunoglobulin domain targeting DLL3 comprises HCDR1 shown in SEQ ID NO:30, HCDR2 shown in SEQ ID NO:31, HCDR3 shown in SEQ ID NO:32, LCDR1 shown in SEQ ID NO:33, LCDR2 shown in SEQ ID NO:34, and LCDR3 shown in SEQ ID NO:35; and the third immunoglobulin domain targeting DLL3 comprises HCDR1 shown in SEQ ID NO:36, HCDR2 shown in SEQ ID NO:37, HCDR3 shown in SEQ ID NO:38, LCDR1 shown in SEQ ID NO:39, LCDR2 shown in SEQ ID NO:40, and LCDR3 shown in SEQ ID NO:

17. LCDR3 as shown in NO:41; Optionally, the first immunoglobulin domain targeting CD3 is an scFv domain, which includes HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:17; the second immunoglobulin domain targeting DLL3 is a Fab domain, which includes HCDR1 shown in SEQ ID NO:30, HCDR2 shown in SEQ ID NO:31, HCDR3 shown in SEQ ID NO:32, LCDR1 shown in SEQ ID NO:33, LCDR2 shown in SEQ ID NO:34, and LCDR3 shown in SEQ ID NO:35; and the third immunoglobulin domain targeting DLL3 is an scFv domain, which includes HCDR1 shown in SEQ ID NO:36, HCDR2 shown in SEQ ID NO:37, HCDR3 shown in SEQ ID NO:38, LCDR1 shown in SEQ ID NO:39, and SEQ ID NO:

17. LCDR2 shown in NO:40 and LCDR3 shown in SEQ ID NO:

41.

9. The bispecific antigen-binding molecule according to any one of claims 5-8, The VH domain targeting the first immunoglobulin domain of CD3 contains an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:42, and the VL domain contains an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:43; or the VH domain targeting the first immunoglobulin domain of CD3 contains an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:3, and the VL domain contains an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:42 ...H domain contains an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or The amino acid sequence shown in NO:4 has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:26; the VH of the second immunoglobulin domain targeting DLL3 contains an amino acid sequence or its amino acid sequence has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:27, and the VL contains an amino acid sequence or its amino acid sequence has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:27; and the VH of the third immunoglobulin domain targeting DLL3 contains an amino acid sequence or its amino acid sequence has an identity with the amino acid sequence shown in SEQ ID NO:

27. The amino acid sequence shown in NO:28 has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO:29; and VL contains or has an amino acid sequence that has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO:

29. Optionally, the first immunoglobulin domain targeting CD3 is an scFv domain, wherein its VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:42, and its VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:43; the second immunoglobulin domain targeting DLL3 is a Fab domain, wherein its VH contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:26, and its VL contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:43; The amino acid sequence shown in NO:27 has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:28; and the third immunoglobulin domain targeting DLL3 is an scFv domain, wherein its VH contains or its amino acid sequence has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:28, and its VL contains or its amino acid sequence has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:

29.

10. The bispecific antigen-binding molecule according to any one of claims 4-9, comprising the following peptide chains (1)-(3): peptide chain (1) N'-DLL3VL-CL-C'; peptide chain (2)N'-DLL3VH-CH1-Fc-C'; and Peptide chain (3)N'-DLL3'VL-DLL3'VH-CD3VL-CD3VH-Fc-C', N'-CD3VL-CD3VH-DLL3'VL-DLL3'VH-Fc- C', N'-DLL3'VH-DLL3'VL-CD3VH-CD3VL-Fc-C' or N'-CD3VH-CD3VL-DLL3'VH-DLL3'VL-Fc-C'; Where VH represents the heavy chain variable region, DLL3 VH and DLL3'VH represent the heavy chain variable regions of the antigen-binding fragments targeting DLL3, CD3 VH represents the heavy chain variable region of the antigen-binding fragments targeting CD3; DLL3 VL and DLL3'VL represent the light chain variable regions of the antigen-binding fragments targeting DLL3, CD3VL represents the light chain variable regions of the antigen-binding fragments targeting CD3; Fc contains CH2 and CH3; CH1, CH2, and CH3 represent domains 1, 2, and 3 of the heavy chain constant region, respectively; CL represents the light chain constant region. Optionally, the Fc region comprises a natural Fc sequence or a non-natural Fc sequence; optionally, the Fc region is a human Fc region; further optionally, the Fc region is the Fc region of an IgG1, IgG2, IgG3, or IgG4 antibody; even further optionally, the Fc region is an IgG1 Fc region; even further optionally, the Fc region comprises one or more mutations selected from those that weaken the antibody ADCC effect, those that weaken the antibody CDC effect, knocks-into-holes mutations, and disulfide bond mutations.

11. The bispecific antigen-binding molecule according to claim 10, wherein the CH1 comprises or has an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:44; The CL comprises, or has an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:45; and / or The Fc contains or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in any of SEQ ID NO:46-49.

12. The bispecific antigen-binding molecule according to any one of claims 10-11, wherein the bispecific antigen-binding molecule comprises peptide chain (1)-peptide chain (3); The peptide chain (1) comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:50; The peptide chain (2) comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 51 or 53; The peptide chain (3) comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 52 or 54; Optionally, the peptide chain (1)-peptide chain (3) comprises or has an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NO: 50, 51, and 52; or The peptide chain (1)-peptide chain (3) comprises or has an amino acid sequence that is at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequences shown in SEQ ID NO:50, 53 and 54.

13. An isolated nucleic acid comprising a nucleic acid sequence encoding a CD3 antibody or an antigen-binding fragment thereof as claimed in any one of claims 1-3 or a bispecific antigen-binding molecule as claimed in any one of claims 4-12.

14. A vector comprising the nucleic acid according to claim 13.

15. An isolated host cell comprising the nucleic acid of claim 13 or the vector of claim 14.

16. A composition comprising a CD3 antibody or an antigen-binding fragment thereof according to any one of claims 1-3, or a bispecific antigen-binding molecule according to any one of claims 4-12, or a nucleic acid according to claim 13, or a vector according to claim 14, or a host cell according to claim 15; optionally, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable vector.

17. Use of the CD3 antibody or antigen-binding fragment thereof of any one of claims 1-3, the bispecific antigen-binding molecule of any one of claims 4-12, the nucleic acid of claim 13, the vector of claim 14, or the host cell of claim 15 in the manufacture of a medicament for treating or delaying the progression of individual cancer; optionally, wherein the cancer is a DLL3-positive cancer; further optionally, the cancer is lung cancer; and even more optionally, the cancer is small cell lung cancer.

18. A method of treating or delaying cancer progression in a subject in need, comprising administering to the subject the bispecific antigen-binding molecule of any one of claims 4-12; optionally, wherein the cancer is DLL3-positive cancer; further optionally, wherein the cancer is lung cancer; and even more optionally, wherein the cancer is small cell lung cancer.

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