Method for screening cell strains aiming at asymmetric multispecific antibody

Abstract

The invention relates to a cell strain screening method aiming at an asymmetric multispecific antibody. According to the method, a product expressed by a multispecific antibody CHO cell with a Titin chain and an Obscurin chain is detected in a cell strain screening process, so that multispecific antibody molecules which are correctly paired can be conveniently and quickly identified in the early stage of cell strain screening.

Description

[0001] This application claims the priority of Chinese Patent Application No. CN202311850404.5, filed on December 29, 2023. Technical Field

[0002] This disclosure belongs to the field of biotechnology. More specifically, this disclosure relates to a method for screening cell lines for asymmetric multispecific antibodies, and particularly to a method for screening cell lines of asymmetric multispecific antibodies with Titin chains and Obscurin chains by using homogeneous time-resolved fluorescence technology. Background Art

[0003] The statements herein only provide background information related to this disclosure and do not necessarily constitute prior art.

[0004] Based on conventional asymmetric multispecific antibodies, heterodimers formed by Titin chains and Obscurin chains in cells are used to replace CH1 and CL in a certain antibody to form an asymmetric multispecific antibody. This antibody platform can effectively prevent mismatching between the heavy chain and the light chain of the asymmetric multispecific antibody (WO2021139758A1, WO2022237882A1). However, during the screening process of cell lines, there are still various by-products such as heavy chain mismatching, light chain dropping, and half antibodies. Some of these by-products are difficult to remove during downstream purification, which not only increases the production cost but also may affect the speed of drug advancement to the clinic. These by-products with abnormal structures cannot be screened by an efficient method in the early stage of cell line screening, which will increase the difficulty of screening cell lines of asymmetric multispecific antibodies, resulting in problems such as an increase in the screening throughput and an extension of the screening cycle of asymmetric bispecific antibodies.

[0005] Homogeneous time-resolved fluorescence technology (HTRF) is a general time-resolved fluorescence resonance energy transfer technology, which combines two technologies: time-resolved fluorescence (TRF) and fluorescence resonance energy transfer (FRET). Compared with traditional ELISA or SDS-PAGE, this method is convenient to operate and easy to achieve high-throughput detection. In addition, due to the integration of time-resolved fluorescence technology, the influence of the background of culture supernatant or buffer on the detection accuracy during the detection process can be reduced. This method has currently been applied to the screening of traditional monoclonal antibodies.

[0006] Although the homogeneous time-resolved fluorescence technique can be used for the screening of monoclonal antibody cell lines, in the screening of asymmetric multispecific antibodies, this method cannot effectively distinguish impurities in the antibodies, such as by-products like mismatches, half-antibodies, and light chain loss, etc. Therefore, it cannot meet the current requirements for the screening of multispecific antibody cells.

[0007] This disclosure aims to establish a homogeneous time-resolved fluorescence technique detection method based on a multispecific antibody platform with Titin chain and Obscurin chain, to detect the products expressed by CHO cells of multispecific antibodies with Titin chain and Obscurin chain during the cell line screening process, so as to facilitate the rapid identification of correctly paired multispecific antibody molecules at the early stage of cell line screening. Summary of the Invention

[0008] This disclosure provides a method for screening cell lines for asymmetric multispecific antibodies, and the method includes the following steps:

[0009] Step 1. Obtain an anti-Titin&Obscurin antibody that binds to the asymmetric multispecific antibody;

[0010] Step 2. Detection method development: Label the anti-Titin&Obscurin antibody and the anti-human light chain λ or κ antibody with a donor and a receptor respectively, and pair them one by one for detection. The pairing schemes are as follows:

[0011] (1) Anti-Titin&Obscurin-Eu or Anti-Titin&Obscurin-Tb, and Anti-κ-d2 or Anti-κ-XL665, or

[0012] (2) Anti-κ-Eu or Anti-κ-Tb, and Anti-Titin&Obscurin-d2 or Anti-Titin&Obscurin-XL665, or

[0013] (3) Anti-Titin&Obscurin-Eu or Anti-Titin&Obscurin-Tb, and Anti-λ-d2 or Anti-λ-XL665, or

[0014] (4) Anti-λ-Eu or Anti-λ-Tb, and Anti-Titin&Obscurin-d2 or Anti-Titin&Obscurin-XL665;

[0015] Wherein the donor is Eu 3+ cryptate or Tb 2+ cryptate (which can be abbreviated as Eu or Tb), and the receptor is d2 or XL665;

[0016] Step 3. Detect the energy transfer signal using a microplate reader. The higher the energy transfer signal of a sample, the higher the content of correctly paired antibodies in the sample, and the corresponding cell line is a high-quality cell line.

[0017] In some embodiments, in the cell line screening method as described above, the anti-human light chain λ or κ antibody is a commercially available anti-human light chain λ or κ antibody.

[0018] The present disclosure provides a method for screening cell lines for asymmetric multispecific antibodies, the method comprising the following steps:

[0019] Step 1. Obtain an anti-Titin&Obscurin antibody that binds to an asymmetric multispecific antibody.

[0020] Step 2. Detection method development: Label the anti-Titin&Obscurin antibody and a commercially available anti-human light chain λ or κ antibody with a donor and a receptor respectively, and pair them one by one for detection. The pairing scheme is as follows:

[0021] (1) Anti-Titin&Obscurin-Eu or Tb and Anti-κ-d2 or XL665,

[0022] (2) Anti-κ-Eu or Tb and Anti-Titin&Obscurin-d2 or XL665,

[0023] (3) Anti-Titin&Obscurin-Eu or Tb and Anti-λ-d2 or XL665, or

[0024] (4) Anti-λ-Eu or Tb and Anti-Titin&Obscurin-d2 or XL665;

[0025] wherein the donor is Eu 3+ cryptate or Tb 2+ cryptate (which can be abbreviated as Eu or Tb), and the receptor is d2 or XL665;

[0026] Step 3. Detect the energy transfer signal using a microplate reader. The higher the energy transfer signal of a sample, the higher the content of correctly paired antibodies in the sample, and the corresponding cell line is a high-quality cell line.

[0027] In some embodiments, in the cell line screening method as described above, Step 1 includes immunizing mice with Titin and / or Obscurin proteins in the asymmetric multispecific antibody, and obtaining an anti-Titin&Obscurin antibody that binds to the asymmetric multispecific antibody by means of hybridoma.

[0028] In some embodiments, in the cell line screening method as described in any one of the preceding items, wherein the Titin and / or Obscurin protein is a Titin and Obscurin-Fc fusion protein.

[0029] In some embodiments, in the cell line screening method as described in any one of the preceding items, wherein the Titin protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 168 to SEQ ID NO: 186, and the Obscurin protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 187 to SEQ ID NO: 227.

[0030] In some embodiments, in the cell line screening method as described in any one of the preceding items, wherein step 1 comprises immunizing a mouse with the Titin and Obscurin-Fc fusion protein in an asymmetric multispecific antibody, and obtaining an anti-Titin&Obscurin antibody that binds to the asymmetric multispecific antibody by means of a hybridoma.

[0031] In some embodiments, in the cell line screening method as described in any one of the preceding items, wherein the Titin and Obscurin-Fc fusion proteins have the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 3, respectively.

[0032] In some embodiments, in the cell line screening method as described in any one of the preceding items, a step of adding cell supernatant or a test substance to a microplate is further included between step 1 and step 2.

[0033] In some embodiments, in the cell line screening method as described in any one of the preceding items, wherein the sample is cell supernatant or a test substance.

[0034] In some embodiments, in the cell line screening method as described in any one of the preceding items, wherein the detection method in step 2 is time-resolved fluorescence resonance energy transfer technology.

[0035] In some embodiments, in the cell line screening method as described in any one of the preceding items, wherein the detection method in step 2 is homogeneous time-resolved fluorescence technology.

[0036] In some embodiments, in the cell line screening method as described in any one of the preceding items, wherein the pairing scheme in step 2 comprises:

[0037] (1) Anti-Titin&Obscurin-Eu or Anti-Titin&Obscurin-Tb, and Anti-κ-d2, or

[0038] (2) Anti-κ-Eu or Anti-κ-Tb and Anti-Titin&Obscurin-d2, or

[0039] (3) Anti-Titin&Obscurin-Eu or Anti-Titin&Obscurin-Tb and Anti-λ-d2, or

[0040] (4) Anti-λ-Eu or Tb and Anti-Titin&Obscurin-d2.

[0041] In some embodiments, the cell line screening method as described in any of the preceding items, wherein the pairing scheme in step 2 comprises:

[0042] (1) Anti-Titin&Obscurin-Eu and Anti-κ-d2, or

[0043] (2) Anti-Titin&Obscurin-Tb and Anti-κ-d2, or

[0044] (3) Anti-κ-Eu and Anti-Titin&Obscurin-d2, or

[0045] (4) Anti-κ-Tb and Anti-Titin&Obscurin-d2, or

[0046] (5) Anti-Titin&Obscurin-Eu and Anti-λ-d2, or

[0047] (6) Anti-Titin&Obscurin-Tb and Anti-λ-d2, or

[0048] (7) Anti-λ-Eu and Anti-Titin&Obscurin-d2, or

[0049] (8) Anti-λ-Tb and Anti-Titin&Obscurin-d2.

[0050] In some embodiments, the cell line screening method as described in any of the preceding items, wherein the pairing scheme in step 2 comprises:

[0051] (1) Anti-Titin&Obscurin-Eu and Anti-κ-d2,

[0052] (2) Anti-Titin&Obscurin-Tb and Anti-κ-d2,

[0053] (3) Anti-κ-Eu and Anti-Titin&Obscurin-d2, or

[0054] (4) Anti-κ-Tb and Anti-Titin&Obscurin-d2.

[0055] In some embodiments, in the cell line screening method as described in any of the preceding items, wherein the pairing scheme in step 2 comprises: Anti-Titin&Obscurin-Eu and Anti-κ-d2.

[0056] In some embodiments, in the cell line screening method as described in any of the preceding items, wherein the Eu 3+ cryptate or Tb 2+ The pairing concentration ratio of the cryptate-labeled antibody molecule and the d2 or XL665-labeled antibody molecule is 0.5:1, 0.5:2, 0.5:4, 0.5:6, 0.5:8, 1:1, 1:2, 1:4, 1:6, 1:8 or 2:2. In some embodiments, in the cell line screening method as described in any of the preceding items, wherein the Eu 3+ cryptate or Tb 2+ The pairing concentration ratio of the cryptate-labeled antibody molecule and the d2 or XL665-labeled antibody molecule is 1:1.

[0057] In some embodiments, in the cell line screening method as described in any of the preceding items, wherein the Eu 3+ cryptate or Tb 2+ The pairing concentration ratio of the cryptate-labeled anti-Titin&Obscurin antibody molecule and the d2 or XL665-labeled anti-κ or anti-λ antibody molecule is 0.5:1, 0.5:2, 0.5:4, 0.5:6, 0.5:8, 1:1, 1:2, 1:4, 1:6, 1:8 or 2:2. In some embodiments, in the cell line screening method as described in any of the preceding items, wherein the Eu 3+ cryptate or Tb 2+ The pairing concentration ratio of the cryptate-labeled anti-Titin&Obscurin antibody molecule and the d2 or XL665-labeled anti-κ or anti-λ antibody molecule is 1:1. In some embodiments, in the cell line screening method as described in any of the preceding items, wherein the Eu 3+ The pairing concentration ratio of the cryptate-labeled anti-Titin&Obscurin antibody and the d2-labeled anti-κ antibody is 1:1.

[0058] In some embodiments, in the cell line screening method as described in any of the preceding items, wherein the Eu3+ cryptate or Tb 2+ The paired concentration ratios of cryptate-labeled anti-kappa or anti-lambda antibodies and d2 or XL665-labeled anti-Titin&Obscurin antibodies are 0.5:1, 0.5:2, 0.5:4, 0.5:6, 0.5:8, 1:1, 1:2, 1:4, 1:6, 1:8 or 2:2. In some embodiments, in the cell line screening method as described in any of the preceding items, the paired concentration ratio is 1:1.

[0059] In some embodiments, in the cell line screening method as described in any of the preceding items, step 2 further comprises the step of mixing and incubating the anti-Titin&Obscurin antibody and the commercial anti-human kappa or lambda light chain antibody with the cell supernatant or the analyte.

[0060] In some embodiments, in the cell line screening method as described in any of the preceding items, the incubation time is 2 hours or more. In some embodiments, the incubation time is 2 to 12 hours. In some embodiments, in the cell line screening method as described in any of the preceding items, the incubation is at room temperature for 2 hours or overnight at 4°C. In some embodiments, in the cell line screening method as described in any of the preceding items, the incubation is at 4°C for 12 hours.

[0061] In some embodiments, in the cell line screening method as described in any of the preceding items, step 2 further comprises the operation of using the target multispecific antibody as a standard.

[0062] In some embodiments, in the cell line screening method as described in any of the preceding items, step 3 further comprises the operation of plotting an energy transfer signal-antibody concentration standard curve according to the detection results.

[0063] In some embodiments, in the cell line screening method as described in any of the preceding items, the energy transfer signal is determined by the fluorescence ratio, and the fluorescence ratio is calculated according to Em665 / Em620 / 10 4 Calculate.

[0064] In some embodiments, in the cell line screening method as described in any of the preceding items, in step 3, the fluorescence intensities of the antibodies are detected at 620 nm and 665 nm respectively; then the fluorescence ratio is calculated according to the formula Em665 / Em620 / 10 4 Calculate the fluorescence ratio.

[0065] In some embodiments, in the cell line screening method as described in any of the preceding items, in step 3, first detect Eu at 620 nm 3+ cryptate or Tb 2+The fluorescence intensity of the anti-Titin&Obscurin antibody labeled with cryptate; then detect the fluorescence intensity of the anti-λ or anti-κ antibody labeled with d2 or XL665 at 665 nm; then calculate the fluorescence ratio according to the formula Em665 / Em620 / 10 4 Calculate the fluorescence ratio.

[0066] In some embodiments, in the cell line screening method as described in any of the preceding items, wherein in step 3, first detect the Eu 3+ cryptate or Tb 2+ The fluorescence intensity of the anti-Titin&Obscurin antibody labeled with cryptate; then detect the fluorescence intensity of the anti-λ or anti-κ antibody labeled with d2 at 665 nm; finally calculate the fluorescence ratio according to the formula Em665 / Em620 / 10 4 Calculate the fluorescence ratio.

[0067] In some embodiments, in the cell line screening method as described in any of the preceding items, wherein in step 3, first detect the Eu 3+ cryptate or Tb 2+ The fluorescence intensity of the anti-Titin&Obscurin antibody labeled with cryptate; then detect the fluorescence intensity of the anti-κ antibody labeled with d2 at 665 nm; finally calculate the fluorescence ratio according to the formula Em665 / Em620 / 10 4 Calculate the fluorescence ratio.

[0068] In some embodiments, in the cell line screening method as described in any of the preceding items, wherein in step 3, first detect the Eu 3+ cryptate or Tb 2+ The fluorescence intensity of the anti-λ or κ antibody labeled with cryptate; then detect the fluorescence intensity of the anti-Titin&Obscurin antibody labeled with d2 or XL665 at 665 nm; then calculate the fluorescence ratio according to the formula Em665 / Em620 / 10 4 Calculate the fluorescence ratio.

[0069] In some embodiments, in the cell line screening method as described in any of the preceding items, wherein in step 3, first detect the Eu 3+ cryptate or Tb 2+ The fluorescence intensity of the anti-λ or κ antibody labeled with cryptate; then detect the fluorescence intensity of the anti-Titin&Obscurin antibody labeled with d2 at 665 nm; finally calculate the fluorescence ratio according to the formula Em665 / Em620 / 10 4 Calculate the fluorescence ratio.

[0070] In some embodiments, in the cell line screening method as described in the previous item, in step 3, Eu 3+ cryptate or Tb 2+ the fluorescence intensity of the cryptate-labeled anti-κ antibody is detected first at 620 nm; then the fluorescence intensity of the d2-labeled anti-Titin&Obscurin antibody is detected at 665 nm; then the fluorescence ratio is calculated according to the formula Em665 / Em620 / 10 4 Calculate the fluorescence ratio.

[0071] In some embodiments, in the cell line screening method as described in the previous item, in step 2, the cell culture supernatant to be detected is diluted to 5 μg / mL and added to a 384-well detection plate, 10 μL / well; the multi-specific antibody standard is diluted to 15 μg / mL and then serially diluted 2-fold, with a total of 6 points, and added to a 384-well detection plate, 10 μL / well; the Eu 3+ cryptate-labeled anti-Titin&Obscurin antibody and the d2-labeled anti-κ antibody, each 5 μL / well, are added to the above samples respectively; after mixing, incubate at room temperature for 2 hours or overnight at 4°C.

[0072] In some embodiments, in the cell line screening method as described in the previous item, the asymmetric multi-specific antibody to be detected is an asymmetric multi-specific antibody having a Titin chain and an Obscurin chain.

[0073] In some embodiments, in the cell line screening method as described in the previous item, the multi-specific antibody has at least one substituted Fab, which contains a Titin chain and an Obscurin chain capable of forming a dimer. In some embodiments, in the cell line screening method as described in the previous item, the substituted Fab is such that the original CH1 and CL of the Fab are replaced by the Obscurin chain and the Titin chain respectively, or the original CH1 and CL of the Fab are replaced by the Titin chain and the Obscurin chain respectively.

[0074] In some embodiments, in the cell line screening method as described in the previous item, the asymmetric multi-specific antibody further comprises a κ or λ light chain. In some embodiments, the asymmetric multi-specific antibody further comprises a κ light chain.

[0075] In some embodiments, the cell line screening method as described in the previous item, wherein the Titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 168 to SEQ ID NO: 186, and the Obscurin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 187 to SEQ ID NO: 227.

[0076] In some embodiments, the cell line screening method as described in the previous item, wherein the asymmetric multispecific antibody to be detected is an asymmetric bispecific antibody.

[0077] In some embodiments, the cell line screening method as described in the previous item, wherein the anti-Titin&Obscurin antibody binds to the Titin protein and / or the Obscurin protein. In some embodiments, the Titin protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 168 to SEQ ID NO: 186, and the Obscurin protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 187 to SEQ ID NO: 227. In some specific embodiments, the anti-Titin&Obscurin antibody binds to the Titin protein as shown in SEQ ID NO: 4, and / or the Obscurin protein as shown in SEQ ID NO: 3.

[0078] In some embodiments, the cell line screening method as described in the previous item, wherein the anti-Titin&Obscurin antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0079] a) The heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences in SEQ ID NO: 24, and the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences in SEQ ID NO: 23; or

[0080] b) The heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences in SEQ ID NO: 12, and the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences in SEQ ID NO: 11; or

[0081] c) The heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences in SEQ ID NO: 16, and the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences in SEQ ID NO: 15; or

[0082] d) The heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences in the sequence of SEQ ID NO: 20, and the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences in the sequence of SEQ ID NO: 19; or

[0083] e) The heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences in the sequence of SEQ ID NO: 22, and the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences in the sequence of SEQ ID NO: 21; or

[0084] f) The heavy chain variable region comprises 1, 2, or 3 amino acid sequences of HCDR in the sequence of SEQ ID NO: 26, and the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences in the sequence of SEQ ID NO: 25; or

[0085] g) The heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences in the sequence of SEQ ID NO: 30, and the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences in the sequence of SEQ ID NO: 29; or

[0086] h) The heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences in the sequence of SEQ ID NO: 32, and the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences in the sequence of SEQ ID NO: 31; or

[0087] i) The heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences in the sequence of SEQ ID NO: 6, and the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences in the sequence of SEQ ID NO: 5; or

[0088] j) The heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences in the sequence of SEQ ID NO: 8, and the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences in the sequence of SEQ ID NO: 7; or

[0089] k) The heavy chain variable region comprises 1, 2, or 3 amino acid sequences of HCDR in the sequence of SEQ ID NO: 10, and the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences in the sequence of SEQ ID NO: 9; or

[0090] l) The heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences in the SEQ ID NO: 14 sequence, and the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences in the SEQ ID NO: 13 sequence; or

[0091] m) The heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences in the SEQ ID NO: 18 sequence, and the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences in the SEQ ID NO: 17 sequence; or

[0092] n) The heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences in the SEQ ID NO: 28 sequence, and the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences in the SEQ ID NO: 27 sequence.

[0093] In some embodiments, in the method for screening cell lines as described in any of the preceding items, the anti-Titin&Obscurin antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the HCDR3 amino acid sequence in the SEQ ID NO: 24, 12, 16, 20, 22, 26, 30, 32, 6, 8, 10, 14, 18 or 28 sequence.

[0094] In some embodiments, in the method for screening cell lines as described in any of the preceding items, the anti-Titin&Obscurin antibody comprises a light chain variable region, wherein the light chain variable region comprises the LCDR3 amino acid sequence in the SEQ ID NO: 23, 11, 15, 19, 21, 25, 29, 31, 5, 7, 9, 13, 17 or 27 sequence.

[0095] In some embodiments, in the method for screening cell lines as described in any of the preceding items, the anti-Titin&Obscurin antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0096] a) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 24, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 23; or

[0097] b) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 12, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 11; or

[0098] c) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 16, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 15; or

[0099] d) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 20, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 19; or

[0100] e) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 22, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 21; or

[0101] f) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 26, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 25; or

[0102] g) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 30, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 29; or

[0103] h) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 32, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 31; or

[0104] i) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 6, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 5; or

[0105] j) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 8, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 7; or

[0106] k) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 10, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 9; or

[0107] l) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 14, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 13; or

[0108] m) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 18, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 17; or

[0109] n) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 28, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 27.

[0110] In some embodiments, the method for screening cell lines as described in any of the previous items, wherein the anti-Titin&Obscurin antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0111] a) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 24, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 23; or

[0112] b) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 12, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 11; or

[0113] c) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 16, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 15; or

[0114] d) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 20, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 19; or

[0115] e) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 22, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 21; or

[0116] f) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 26, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 25; or

[0117] g) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 30, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 29; or

[0118] h) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 32, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 31.

[0119] In some embodiments, in the method for screening cell lines as described in any one of the preceding items, the anti-Titin&Obscurin antibody comprises, wherein:

[0120] a) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 113, HCDR2 comprises the amino acid sequence of SEQ ID NO: 114, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 115, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 116, LCDR2 comprises the amino acid sequence of SEQ ID NO: 117, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 118; or

[0121] b) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 77, HCDR2 comprises the amino acid sequence of SEQ ID NO: 78, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 79, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 80, LCDR2 comprises the amino acid sequence of SEQ ID NO: 81, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 82; or

[0122] c) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 89, HCDR2 comprises the amino acid sequence of SEQ ID NO: 90, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 91, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 92, LCDR2 comprises the amino acid sequence of SEQ ID NO: 93, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 94; or

[0123] d) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 101, HCDR2 comprises the amino acid sequence of SEQ ID NO: 102, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 103, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 104, LCDR2 comprises the amino acid sequence of SEQ ID NO: 105, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 106; or

[0124] e) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 107, HCDR2 comprises the amino acid sequence of SEQ ID NO: 108, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 109, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 110, LCDR2 comprises the amino acid sequence of SEQ ID NO: 111, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 112; or

[0125] f) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59, HCDR2 comprises the amino acid sequence of SEQ ID NO: 119, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 120, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 121, LCDR2 comprises the amino acid sequence of SEQ ID NO: 122, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 123; or

[0126] g) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 130, HCDR2 comprises the amino acid sequence of SEQ ID NO: 131, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 132, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 133, LCDR2 comprises the amino acid sequence of SEQ ID NO: 134, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 135; or

[0127] h) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 71, HCDR2 comprises the amino acid sequence of SEQ ID NO: 136, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 137, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 138, LCDR2 comprises the amino acid sequence of SEQ ID NO: 139, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 76; or

[0128] i) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59, HCDR2 comprises the amino acid sequence of SEQ ID NO: 60, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 61, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62, LCDR2 comprises the amino acid sequence of SEQ ID NO: 63, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 64; or

[0129] j) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 65, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 66, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 67, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 68, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 69, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 70; or

[0130] k) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 71, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 72, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 73, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 74, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 75, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 76; or

[0131] l) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 83, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 84, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 85, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 86, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 87, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 88; or

[0132] m) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 95, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 96, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 97, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 98, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 99, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 100; or

[0133] n) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 124, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 125, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 126, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 127, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 128, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 129.

[0134] In some embodiments, the cell line screening method as described in any of the preceding items, wherein the anti-Titin&Obscurin antibody comprises:

[0135] a) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 113, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 114, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 115, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 116, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 117, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 118; or

[0136] b) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 77, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 78, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 79, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 80, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 81, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 82; or

[0137] c) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 89, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 90, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 91, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 92, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 93, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 94; or

[0138] d) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 101, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 102, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 103, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 104, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 105, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 106; or

[0139] e) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 107, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 108, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 109, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 110, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 111, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 112; or

[0140] f) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 119, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 120, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 121, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 122, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 123; or

[0141] g) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 130, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 131, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 132, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 133, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 134, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 135; or

[0142] h) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 71, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 136, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 137, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 138, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 139, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 76.

[0143] In some embodiments, the cell line screening method as described in any one of the preceding items, wherein the anti-Titin&Obscurin antibody comprises:

[0144] The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 113, HCDR2 comprises the amino acid sequence of SEQ ID NO: 114, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 115, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 116, LCDR2 comprises the amino acid sequence of SEQ ID NO: 117, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 118.

[0145] In some embodiments, the cell line screening method as described in any of the preceding items, wherein the anti-Titin&Obscurin antibody comprises:

[0146] The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 77, HCDR2 comprises the amino acid sequence of SEQ ID NO: 78, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 79, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 80, LCDR2 comprises the amino acid sequence of SEQ ID NO: 81, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 82.

[0147] In some embodiments, the cell line screening method as described in any of the preceding items, wherein the anti-Titin&Obscurin antibody comprises:

[0148] a) The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 113, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 114, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 115, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 116, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 117, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 118; or

[0149] b) The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 77, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 78, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 79, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 80, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 81, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 82; or

[0150] c) The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 89, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 90, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 91, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 92, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 93, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 94; or

[0151] d) The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 101, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 102, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 103, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 104, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 105, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 106; or

[0152] e) The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 107, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 108, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 109, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 110, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 111, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 112; or

[0153] f) The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 59, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 119, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 120, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 121, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 122, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 123; or

[0154] g) The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 130, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 131, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 132, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 133, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 134, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 135; or

[0155] h) The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 71, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 136, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 137, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 138, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 139, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 76.

[0156] In some embodiments, the cell line screening method as described in any one of the preceding items, wherein the anti-Titin&Obscurin antibody comprises:

[0157] The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 113, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 114, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 115, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 116, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 117, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 118.

[0158] In some embodiments, the cell line screening method as described in any one of the preceding items, wherein the anti-Titin&Obscurin antibody comprises:

[0159] The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 77, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 78, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 79, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 80, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 81, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 82.

[0160] In some embodiments, the cell line screening method as described in any one of the preceding items, wherein the anti-Titin&Obscurin antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0161] a) The heavy chain variable region comprises SEQ ID NO: 24, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 23, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0162] b) The heavy chain variable region comprises SEQ ID NO: 12, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 11, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0163] c) The heavy chain variable region comprises SEQ ID NO: 16, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 15, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0164] d) The heavy chain variable region comprises SEQ ID NO: 20, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 19, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0165] e) The heavy chain variable region comprises SEQ ID NO: 22, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 21, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0166] f) The heavy chain variable region comprises SEQ ID NO: 26, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 25, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0167] g) The heavy chain variable region comprises SEQ ID NO: 30, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 29, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0168] h) The heavy chain variable region comprises SEQ ID NO: 32, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 31, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0169] i) The heavy chain variable region comprises SEQ ID NO: 6, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 5, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0170] j) The heavy chain variable region comprises SEQ ID NO: 8, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 7, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0171] k) The heavy chain variable region comprises SEQ ID NO: 10, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 9, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0172] l) The heavy chain variable region comprises SEQ ID NO: 14, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 13, or an amino acid sequence having at least 80% (such as at least 85%, at least 91%, at least 92%, at least 90%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0173] m) The heavy chain variable region comprises SEQ ID NO: 18, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 17, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0174] n) The heavy chain variable region comprises SEQ ID NO: 28, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 27, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith.

[0175] In some embodiments, the cell line screening method as described in any of the preceding items, wherein the anti-Titin&Obscurin antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0176] a) The heavy chain variable region comprises SEQ ID NO: 24, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 23, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0177] b) The heavy chain variable region comprises SEQ ID NO: 12, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 11, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0178] c) The heavy chain variable region comprises SEQ ID NO: 16, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 15, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0179] d) The heavy chain variable region comprises SEQ ID NO: 20, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 19, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0180] e) The heavy chain variable region comprises SEQ ID NO: 22, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 21, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0181] f) The heavy chain variable region comprises SEQ ID NO: 26, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 25, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0182] g) The heavy chain variable region comprises SEQ ID NO: 30, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 29, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0183] h) The heavy chain variable region comprises SEQ ID NO: 32, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 31, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith.

[0184] In some embodiments, the method for screening cell lines as described in any of the preceding items, wherein the anti-Titin&Obscurin antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0185] a) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 24, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 23; or

[0186] b) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 11; or

[0187] c) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 16, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15; or

[0188] d) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 20, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 19; or

[0189] e) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 22, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 21; or

[0190] f) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 25; or

[0191] g) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 30, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 29; or

[0192] h) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 32, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 31; or

[0193] i) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 6, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 5; or

[0194] j) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 7; or

[0195] k) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9; or

[0196] l) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 14, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13; or

[0197] m) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 18, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 17; or

[0198] n) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 28, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 27.

[0199] In some embodiments, the method for screening cell lines as described in any one of the preceding items, wherein the anti-Titin&Obscurin antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0200] a) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 24, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 23; or

[0201] b) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 11; or

[0202] c) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 16, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15; or

[0203] d) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 20, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 19; or

[0204] e) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 22, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 21; or

[0205] f) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 25; or

[0206] g) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 30, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 29; or

[0207] h) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 32, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 31.

[0208] In some embodiments, in the cell line screening method as described in any one of the preceding items, the anti-Titin&Obscurin antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 24, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 23.

[0209] In some embodiments, in the cell line screening method as described in any one of the preceding items, the anti-Titin&Obscurin antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 11.

[0210] In some embodiments, the cell line screening method as described in the previous item, wherein the anti-Titin&Obscurin antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region consists of SEQ ID NO: 24, and the amino acid sequence of the light chain variable region consists of SEQ ID NO: 23.

[0211] In some embodiments, the cell line screening method as described in the previous item, wherein the anti-Titin&Obscurin antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region consists of SEQ ID NO: 12, and the amino acid sequence of the light chain variable region consists of SEQ ID NO: 11.

[0212] In some embodiments, the cell line screening method as described in the previous item, wherein the anti-Titin&Obscurin antibody comprises a heavy chain and a light chain, wherein:

[0213] a) the heavy chain comprises SEQ ID NO: 158, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 159, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith;

[0214] b) the heavy chain comprises SEQ ID NO: 146, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 147, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0215] c) The heavy chain comprises SEQ ID NO: 150, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 151, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0216] d) The heavy chain comprises SEQ ID NO: 154, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 155, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0217] e) The heavy chain comprises SEQ ID NO: 156, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 157, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0218] f) The heavy chain comprises SEQ ID NO: 160, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 161, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0219] g) The heavy chain comprises SEQ ID NO: 164, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 165, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0220] h) The heavy chain comprises SEQ ID NO: 166, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 167, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0221] i) The heavy chain comprises SEQ ID NO: 140, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 141, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0222] j) The heavy chain comprises SEQ ID NO: 142, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 143, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0223] k) The heavy chain comprises SEQ ID NO: 144, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 145, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0224] l) The heavy chain comprises SEQ ID NO: 148, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 149, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0225] m) The heavy chain comprises SEQ ID NO: 152, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 153, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0226] n) The heavy chain comprises SEQ ID NO: 162, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 163, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith.

[0227] In some embodiments, the cell line screening method as described in any one of the preceding items, wherein the anti-Titin&Obscurin antibody comprises a heavy chain and a light chain, wherein:

[0228] a) The heavy chain comprises SEQ ID NO: 158, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 159, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith;

[0229] b) The heavy chain comprises SEQ ID NO: 146, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 147, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0230] c) The heavy chain comprises SEQ ID NO: 150, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 151, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0231] d) The heavy chain comprises SEQ ID NO: 154, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 155, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0232] e) The heavy chain comprises SEQ ID NO: 156, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 157, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0233] f) The heavy chain comprises SEQ ID NO: 160, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 161, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0234] g) The heavy chain comprises SEQ ID NO: 164, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 165, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0235] h) The heavy chain comprises SEQ ID NO: 166, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 167, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith.

[0236] In some embodiments, the method for screening cell lines as described in any of the preceding items, wherein the anti-Titin&Obscurin antibody comprises a heavy chain and a light chain, wherein:

[0237] a) The heavy chain comprises the amino acid sequence of SEQ ID NO: 158, and the light chain comprises the amino acid sequence of SEQ ID NO: 159; or

[0238] b) The heavy chain comprises the amino acid sequence of SEQ ID NO: 146, and the light chain comprises the amino acid sequence of SEQ ID NO: 147; or

[0239] c) The heavy chain comprises the amino acid sequence of SEQ ID NO: 150, and the light chain comprises the amino acid sequence of SEQ ID NO: 151; or

[0240] d) The heavy chain comprises the amino acid sequence of SEQ ID NO: 154, and the light chain comprises the amino acid sequence of SEQ ID NO: 155; or

[0241] e) The heavy chain comprises the amino acid sequence of SEQ ID NO: 156, and the light chain comprises the amino acid sequence of SEQ ID NO: 157; or

[0242] f) The heavy chain comprises the amino acid sequence of SEQ ID NO: 160, and the light chain comprises the amino acid sequence of SEQ ID NO: 161; or

[0243] g) The heavy chain comprises the amino acid sequence of SEQ ID NO: 164, and the light chain comprises the amino acid sequence of SEQ ID NO: 165; or

[0244] h) The heavy chain comprises the amino acid sequence of SEQ ID NO: 166, and the light chain comprises the amino acid sequence of SEQ ID NO: 167; or

[0245] i) The heavy chain comprises the amino acid sequence of SEQ ID NO: 140, and the light chain comprises the amino acid sequence of SEQ ID NO: 141; or

[0246] j) The heavy chain comprises the amino acid sequence of SEQ ID NO: 142, and the light chain comprises the amino acid sequence of SEQ ID NO: 143; or

[0247] k) The heavy chain comprises the amino acid sequence of SEQ ID NO: 144, and the light chain comprises the amino acid sequence of SEQ ID NO: 145; or

[0248] l) The heavy chain comprises the amino acid sequence of SEQ ID NO: 148, and the light chain comprises the amino acid sequence of SEQ ID NO: 149; or

[0249] m) The heavy chain comprises the amino acid sequence of SEQ ID NO: 152, and the light chain comprises the amino acid sequence of SEQ ID NO: 153; or

[0250] n) The heavy chain comprises the amino acid sequence of SEQ ID NO: 162, and the light chain comprises the amino acid sequence of SEQ ID NO: 163.

[0251] In some embodiments, the method for screening cell lines as described in any of the preceding items, wherein the anti-Titin&Obscurin antibody comprises a heavy chain and a light chain, wherein:

[0252] a) The heavy chain comprises the amino acid sequence of SEQ ID NO: 158, and the light chain comprises the amino acid sequence of SEQ ID NO: 159; or

[0253] b) The heavy chain comprises the amino acid sequence of SEQ ID NO: 146, and the light chain comprises the amino acid sequence of SEQ ID NO: 147; or

[0254] c) The heavy chain comprises the amino acid sequence of SEQ ID NO: 150, and the light chain comprises the amino acid sequence of SEQ ID NO: 151; or

[0255] d) The heavy chain comprises the amino acid sequence of SEQ ID NO: 154, and the light chain comprises the amino acid sequence of SEQ ID NO: 155; or

[0256] e) The heavy chain comprises the amino acid sequence of SEQ ID NO: 156, and the light chain comprises the amino acid sequence of SEQ ID NO: 157; or

[0257] f) The heavy chain comprises the amino acid sequence of SEQ ID NO: 160, and the light chain comprises the amino acid sequence of SEQ ID NO: 161; or

[0258] g) The heavy chain comprises the amino acid sequence of SEQ ID NO: 164, and the light chain comprises the amino acid sequence of SEQ ID NO: 165; or

[0259] h) The heavy chain comprises the amino acid sequence of SEQ ID NO: 166, and the light chain comprises the amino acid sequence of SEQ ID NO: 167.

[0260] In some embodiments, the method for screening cell lines as described in any of the preceding items, wherein the anti-Titin&Obscurin antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 158, and the light chain comprises the amino acid sequence of SEQ ID NO: 159.

[0261] In some embodiments, the method for screening cell lines as described in any of the preceding items, wherein the anti-Titin&Obscurin antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 146, and the light chain comprises the amino acid sequence of SEQ ID NO: 147.

[0262] In some embodiments, the cell line screening method as described in the previous item, wherein the anti-Titin&Obscurin antibody is CED2208, CED2184, CED2190, CED2202, CED2205, CED2211, CED2217, CED2220, CED2175, CED2178, CED2181, CED2187, CED2193 or CED2214 antibody. In some embodiments, the cell line screening method as described in the previous item, wherein the anti-Titin&Obscurin antibody is CED2208, CED2184, CED2190, CED2202, CED2205, CED2211, CED2217 or CED2220 antibody. In some embodiments, the cell line screening method as described in the previous item, wherein the anti-Titin&Obscurin antibody is CED2208 or CED2184 antibody.

[0263] In the cell line screening method of the present disclosure, when the analyte (the presence of multispecific or bispecific antibodies in the cell line) is directly or indirectly bound by the fluorophore donor or acceptor at the same time, there are two excitation lights at 620 nm and 665 nm, otherwise there is only one excitation light at 620 nm.

[0264] On the other hand, the present disclosure provides an anti-Titin&Obscurin antibody, which comprises a heavy chain variable region and a light chain variable region, wherein:

[0265] a) The heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences in SEQ ID NO: 24, and the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences in SEQ ID NO: 23; or

[0266] b) The heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences in SEQ ID NO: 12, and the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences in SEQ ID NO: 11; or

[0267] c) The heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences in SEQ ID NO: 16, and the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences in SEQ ID NO: 15; or

[0268] d) The heavy chain variable region contains 1, 2, or 3 HCDR amino acid sequences in the SEQ ID NO: 20 sequence, and the light chain variable region contains 1, 2, or 3 LCDR amino acid sequences in the SEQ ID NO: 19 sequence; or

[0269] e) The heavy chain variable region contains 1, 2, or 3 HCDR amino acid sequences in the SEQ ID NO: 22 sequence, and the light chain variable region contains 1, 2, or 3 LCDR amino acid sequences in the SEQ ID NO: 21 sequence; or

[0270] f) The heavy chain variable region contains 1, 2, or 3 amino acid sequences of HCDR in the SEQ ID NO: 26 sequence, and the light chain variable region contains 1, 2, or 3 LCDR amino acid sequences in the SEQ ID NO: 25 sequence; or

[0271] g) The heavy chain variable region contains 1, 2, or 3 HCDR amino acid sequences in the SEQ ID NO: 30 sequence, and the light chain variable region contains 1, 2, or 3 LCDR amino acid sequences in the SEQ ID NO: 29 sequence; or

[0272] h) The heavy chain variable region contains 1, 2, or 3 HCDR amino acid sequences in the SEQ ID NO: 32 sequence, and the light chain variable region contains 1, 2, or 3 LCDR amino acid sequences in the SEQ ID NO: 31 sequence; or

[0273] i) The heavy chain variable region contains 1, 2, or 3 HCDR amino acid sequences in the SEQ ID NO: 6 sequence, and the light chain variable region contains 1, 2, or 3 LCDR amino acid sequences in the SEQ ID NO: 5 sequence; or

[0274] j) The heavy chain variable region contains 1, 2, or 3 HCDR amino acid sequences in the SEQ ID NO: 8 sequence, and the light chain variable region contains 1, 2, or 3 LCDR amino acid sequences in the SEQ ID NO: 7 sequence; or

[0275] k) The heavy chain variable region contains 1, 2, or 3 amino acid sequences of HCDR in the SEQ ID NO: 10 sequence, and the light chain variable region contains 1, 2, or 3 LCDR amino acid sequences in the SEQ ID NO: 9 sequence; or

[0276] l) The heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences in the SEQ ID NO: 14 sequence, and the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences in the SEQ ID NO: 13 sequence; or

[0277] m) The heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences in the SEQ ID NO: 18 sequence, and the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences in the SEQ ID NO: 17 sequence; or

[0278] n) The heavy chain variable region comprises 1, 2, or 3 HCDR amino acid sequences in the SEQ ID NO: 28 sequence, and the light chain variable region comprises 1, 2, or 3 LCDR amino acid sequences in the SEQ ID NO: 27 sequence.

[0279] In some embodiments, the anti-Titin&Obscurin antibody as described above comprises a heavy chain variable region, wherein the heavy chain variable region comprises the HCDR3 amino acid sequence in the SEQ ID NO: 24, 12, 16, 20, 22, 26, 30, 32, 6, 8, 10, 14, 18 or 28 sequence.

[0280] In some embodiments, the anti-Titin&Obscurin antibody as described above comprises a light chain variable region, wherein the light chain variable region comprises the LCDR3 amino acid sequence in the SEQ ID NO: 23, 11, 15, 19, 21, 25, 29, 31, 5, 7, 9, 13, 17 or 27 sequence.

[0281] In some embodiments, the anti-Titin&Obscurin antibody as described in any of the preceding items comprises a heavy chain variable region and a light chain variable region, wherein:

[0282] a) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 24, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 23; or

[0283] b) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 12, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 11; or

[0284] c) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 16, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 15; or

[0285] d) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 20, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 19; or

[0286] e) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 22, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 21; or

[0287] f) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 26, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 25; or

[0288] g) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 30, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 29; or

[0289] h) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 32, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 31; or

[0290] i) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 6, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 5; or

[0291] j) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 8, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 7; or

[0292] k) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 10, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 9; or

[0293] l) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 14, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 13; or

[0294] m) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 18, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 17; or

[0295] n) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 28, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 27.

[0296] In some embodiments, the anti-Titin&Obscurin antibody as described in any of the previous items, wherein:

[0297] a) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 24, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 23; or

[0298] b) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 12, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 11; or

[0299] c) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 16, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 15; or

[0300] d) The HCDR1, HCDR2 and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 20, and the LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 19; or

[0301] e) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 22, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 21; or

[0302] f) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 26, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 25; or

[0303] g) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 30, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 29; or

[0304] h) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively contain the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 32, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively contain the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 31.

[0305] In some embodiments, the anti-Titin&Obscurin antibody as described in any preceding item, wherein the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are defined according to the same numbering rules selected from Kabat, IMGT, Chothia, AbM, and Contact. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are defined according to the Kabat numbering rules. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are defined according to the IMGT numbering rules. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are defined according to the Chothia numbering rules. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are defined according to the AbM numbering rules. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are defined according to the Contact numbering rules.

[0306] In some embodiments, the anti-Titin&Obscurin antibody as described in any preceding item, wherein:

[0307] a) the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 113, HCDR2 comprises the amino acid sequence of SEQ ID NO: 114, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 115, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 116, LCDR2 comprises the amino acid sequence of SEQ ID NO: 117, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 118; or

[0308] b) the HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 77, HCDR2 comprises the amino acid sequence of SEQ ID NO: 78, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 79, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 80, LCDR2 comprises the amino acid sequence of SEQ ID NO: 81, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 82; or

[0309] c) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 89, HCDR2 comprises the amino acid sequence of SEQ ID NO: 90, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 91, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 92, LCDR2 comprises the amino acid sequence of SEQ ID NO: 93, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 94; or

[0310] d) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 101, HCDR2 comprises the amino acid sequence of SEQ ID NO: 102, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 103, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 104, LCDR2 comprises the amino acid sequence of SEQ ID NO: 105, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 106; or

[0311] e) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 107, HCDR2 comprises the amino acid sequence of SEQ ID NO: 108, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 109, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 110, LCDR2 comprises the amino acid sequence of SEQ ID NO: 111, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 112; or

[0312] f) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59, HCDR2 comprises the amino acid sequence of SEQ ID NO: 119, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 120, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 121, LCDR2 comprises the amino acid sequence of SEQ ID NO: 122, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 123; or

[0313] g) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 130, HCDR2 comprises the amino acid sequence of SEQ ID NO: 131, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 132, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 133, LCDR2 comprises the amino acid sequence of SEQ ID NO: 134, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 135; or

[0314] h) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 71, HCDR2 comprises the amino acid sequence of SEQ ID NO: 136, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 137, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 138, LCDR2 comprises the amino acid sequence of SEQ ID NO: 139, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 76; or

[0315] i) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59, HCDR2 comprises the amino acid sequence of SEQ ID NO: 60, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 61, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 62, LCDR2 comprises the amino acid sequence of SEQ ID NO: 63, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 64; or

[0316] j) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 65, HCDR2 comprises the amino acid sequence of SEQ ID NO: 66, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 67, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 68, LCDR2 comprises the amino acid sequence of SEQ ID NO: 69, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 70; or

[0317] k) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 71, HCDR2 comprises the amino acid sequence of SEQ ID NO: 72, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 73, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 74, LCDR2 comprises the amino acid sequence of SEQ ID NO: 75, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 76; or

[0318] (l) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 83, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 84, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 85, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 86, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 87, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 88; or

[0319] (m) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 95, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 96, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 97, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 98, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 99, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 100; or

[0320] n) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 124, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 125, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 126, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 127, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 128, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 129.

[0321] In some embodiments, the anti-Titin&Obscurin antibody as described in any one of the preceding items, wherein:

[0322] a) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 113, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 114, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 115, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 116, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 117, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 118; or

[0323] b) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 77, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 78, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 79, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 80, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 81, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 82; or

[0324] c) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 89, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 90, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 91, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 92, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 93, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 94; or

[0325] d) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 101, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 102, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 103, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 104, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 105, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 106; or

[0326] e) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 107, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 108, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 109, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 110, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 111, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 112; or

[0327] f) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59, HCDR2 comprises the amino acid sequence of SEQ ID NO: 119, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 120, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 121, LCDR2 comprises the amino acid sequence of SEQ ID NO: 122, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 123; or

[0328] g) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 130, HCDR2 comprises the amino acid sequence of SEQ ID NO: 131, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 132, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 133, LCDR2 comprises the amino acid sequence of SEQ ID NO: 134, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 135; or

[0329] h) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 71, HCDR2 comprises the amino acid sequence of SEQ ID NO: 136, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 137, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 138, LCDR2 comprises the amino acid sequence of SEQ ID NO: 139, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 76.

[0330] In some embodiments, the method for screening cell lines as described in any one of the preceding items, wherein the anti-Titin&Obscurin antibody comprises:

[0331] The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 113, HCDR2 comprises the amino acid sequence of SEQ ID NO: 114, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 115, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 116, LCDR2 comprises the amino acid sequence of SEQ ID NO: 117, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 118.

[0332] In some embodiments, the anti-Titin&Obscurin antibody as described in any one of the preceding items, wherein:

[0333] The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 77, HCDR2 comprises the amino acid sequence of SEQ ID NO: 78, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 79, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 80, LCDR2 comprises the amino acid sequence of SEQ ID NO: 81, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 82.

[0334] In some embodiments, the anti-Titin&Obscurin antibody as described in any one of the preceding items, wherein:

[0335] a) The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 113, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 114, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 115, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 116, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 117, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 118; or

[0336] b) The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 77, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 78, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 79, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 80, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 81, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 82; or

[0337] c) The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 89, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 90, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 91, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 92, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 93, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 94; or

[0338] d) The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 101, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 102, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 103, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 104, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 105, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 106; or

[0339] e) The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 107, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 108, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 109, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 110, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 111, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 112; or

[0340] f) The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 59, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 119, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 120, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 121, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 122, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 123; or

[0341] g) The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 130, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 131, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 132, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 133, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 134, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 135; or

[0342] h) The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 71, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 136, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 137, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 138, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 139, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 76.

[0343] In some embodiments, the anti-Titin&Obscurin antibody as described in any one of the preceding items, wherein:

[0344] The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 113, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 114, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 115, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 116, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 117, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 118.

[0345] In some embodiments, the anti-Titin&Obscurin antibody as described in any one of the preceding items, wherein:

[0346] The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 77, HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 78, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 79, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 80, LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 81, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 82.

[0347] In some embodiments, for the anti-Titin&Obscurin antibody as described in any one of the preceding items, HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and LCDR1, LCDR2 and LCDR3 of the light chain variable region are defined according to the Kabat numbering rule.

[0348] In some embodiments, the anti-Titin&Obscurin antibody as described in any of the preceding items is a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody. In some embodiments, the anti-Titin&Obscurin antibody as described in any of the preceding items comprises a heavy chain variable region and a light chain variable region, wherein:

[0349] a) the heavy chain variable region comprises SEQ ID NO: 24, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 23, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0350] b) the heavy chain variable region comprises SEQ ID NO: 12, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 11, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0351] c) the heavy chain variable region comprises SEQ ID NO: 16, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 15, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0352] d) The heavy chain variable region comprises SEQ ID NO: 20, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 19, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0353] e) The heavy chain variable region comprises SEQ ID NO: 22, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 21, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0354] f) The heavy chain variable region comprises SEQ ID NO: 26, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 25, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0355] g) The heavy chain variable region comprises SEQ ID NO: 30, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 29, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0356] h) The heavy chain variable region comprises SEQ ID NO: 32, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 31, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0357] i) The heavy chain variable region comprises SEQ ID NO: 6, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 5, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0358] j) The heavy chain variable region comprises SEQ ID NO: 8, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 7, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0359] k) The heavy chain variable region comprises SEQ ID NO: 10, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 9, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0360] l) The heavy chain variable region comprises SEQ ID NO: 14, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 13, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0361] m) The heavy chain variable region comprises SEQ ID NO: 18, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 17, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0362] n) The heavy chain variable region comprises SEQ ID NO: 28, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 27, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith.

[0363] In some embodiments, the anti-Titin&Obscurin antibody as described in any of the preceding items, wherein:

[0364] a) the heavy chain variable region comprises SEQ ID NO: 24, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 23, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0365] b) the heavy chain variable region comprises SEQ ID NO: 12, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 11, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0366] c) the heavy chain variable region comprises SEQ ID NO: 16, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 15, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0367] d) The heavy chain variable region comprises SEQ ID NO: 20, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto, and the light chain variable region comprises SEQ ID NO: 19, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto; or

[0368] e) The heavy chain variable region comprises SEQ ID NO: 22, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto, and the light chain variable region comprises SEQ ID NO: 21, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto; or

[0369] f) The heavy chain variable region comprises SEQ ID NO: 26, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto, and the light chain variable region comprises SEQ ID NO: 25, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto; or

[0370] g) The heavy chain variable region comprises SEQ ID NO: 30, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto, and the light chain variable region comprises SEQ ID NO: 29, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto; or

[0371] h) The heavy chain variable region comprises SEQ ID NO: 32, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain variable region comprises SEQ ID NO: 31, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith.

[0372] In some embodiments, the anti-Titin&Obscurin antibody as described in any one of the preceding items, wherein:

[0373] a) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 24, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 23; or

[0374] b) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 11; or

[0375] c) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 16, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15; or

[0376] d) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 20, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 19; or

[0377] e) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 22, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 21; or

[0378] f) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 25; or

[0379] g) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 30, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 29; or

[0380] h) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 32, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 31; or

[0381] i) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 6, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 5; or

[0382] j) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 7; or

[0383] k) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9; or

[0384] l) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 14, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13; or

[0385] m) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 18, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 17; or

[0386] n) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 28, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 27.

[0387] In some embodiments, the anti-Titin&Obscurin antibody as described in any of the preceding items, wherein:

[0388] a) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 24, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 23; or

[0389] b) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 11; or

[0390] c) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 16, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15; or

[0391] d) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 20, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 19; or

[0392] e) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 22, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 21; or

[0393] f) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 26, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 25; or

[0394] g) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 30, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 29; or

[0395] h) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 32, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 31.

[0396] In some embodiments, the anti-Titin&Obscurin antibody as described in any of the preceding items, wherein:

[0397] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 24, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 23.

[0398] In some embodiments, the anti-Titin&Obscurin antibody as described in any of the preceding items, wherein:

[0399] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 11.

[0400] In some embodiments, the anti-Titin&Obscurin antibody as described in any of the preceding items, wherein:

[0401] The amino acid sequence of the heavy chain variable region consists of SEQ ID NO: 24, and the amino acid sequence of the light chain variable region consists of SEQ ID NO: 23.

[0402] In some embodiments, the anti-Titin&Obscurin antibody as described in any of the preceding items, wherein:

[0403] The amino acid sequence of the heavy chain variable region consists of SEQ ID NO: 12, and the amino acid sequence of the light chain variable region consists of SEQ ID NO: 11.

[0404] In some embodiments, the anti-Titin&Obscurin antibody as described in any of the preceding items, wherein the anti-Titin&Obscurin antibody is an antigen-binding fragment. In some embodiments, the antigen-binding fragment is Fab, Fab′, F(ab')2, Fd, Fv, scFv, dsFv or dAb.

[0405] In some embodiments, the anti-Titin&Obscurin antibody as described in any of the preceding items, wherein the anti-Titin&Obscurin antibody comprises a heavy chain constant region and a light chain constant region. In some embodiments, the anti-Titin&Obscurin antibody as described in any of the preceding items, wherein the heavy chain constant region is the heavy chain constant region of IgG1, IgG2, IgG3, IgG4 or a variant thereof. In some embodiments, the anti-Titin&Obscurin antibody as described in any of the preceding items, wherein the heavy chain constant region is the murine IgG2a heavy chain constant region. In some embodiments, the anti-Titin&Obscurin antibody as described in any of the preceding items, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the anti-Titin&Obscurin antibody as described in any of the preceding items, wherein the light chain constant region is the light chain constant region of lambda, kappa or a variant thereof. In some embodiments, the anti-Titin&Obscurin antibody as described in any of the preceding items, wherein the light chain constant region is the murine kappa light chain constant region. In some embodiments, the anti-Titin&Obscurin antibody as described in any of the preceding items, wherein the light chain constant region comprises the amino acid sequence of SEQ ID NO: 34.

[0406] In some embodiments, the anti-Titin&Obscurin antibody as described in any of the preceding items, wherein:

[0407] a) the heavy chain comprises SEQ ID NO: 158, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto, and the light chain comprises SEQ ID NO: 159, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto;

[0408] b) The heavy chain comprises SEQ ID NO: 146, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 147, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0409] c) The heavy chain comprises SEQ ID NO: 150, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 151, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0410] d) The heavy chain comprises SEQ ID NO: 154, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 155, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0411] e) The heavy chain comprises SEQ ID NO: 156, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 157, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0412] f) The heavy chain comprises SEQ ID NO: 160, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 161, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0413] g) The heavy chain comprises SEQ ID NO: 164, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 165, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0414] h) The heavy chain comprises SEQ ID NO: 166, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 167, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0415] i) The heavy chain comprises SEQ ID NO: 140, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 141, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0416] j) The heavy chain comprises SEQ ID NO: 142, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto, and the light chain comprises SEQ ID NO: 143, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto; or

[0417] k) The heavy chain comprises SEQ ID NO: 144, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto, and the light chain comprises SEQ ID NO: 145, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto; or

[0418] l) The heavy chain comprises SEQ ID NO: 148, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto, and the light chain comprises SEQ ID NO: 149, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto; or

[0419] m) The heavy chain comprises SEQ ID NO: 152, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto, and the light chain comprises SEQ ID NO: 153, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto; or

[0420] n) The heavy chain comprises SEQ ID NO: 162, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto, and the light chain comprises SEQ ID NO: 163, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto.

[0421] In some embodiments, the anti-Titin&Obscurin antibody as described in any one of the preceding items, wherein:

[0422] a) The heavy chain comprises SEQ ID NO: 158, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto, and the light chain comprises SEQ ID NO: 159, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto;

[0423] b) The heavy chain comprises SEQ ID NO: 146, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 147, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0424] c) The heavy chain comprises SEQ ID NO: 150, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 151, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0425] d) The heavy chain comprises SEQ ID NO: 154, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 155, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0426] e) The heavy chain comprises SEQ ID NO: 156, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises SEQ ID NO: 157, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0427] f) The heavy chain comprises the amino acid sequence of SEQ ID NO: 160, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises the amino acid sequence of SEQ ID NO: 161, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0428] g) The heavy chain comprises the amino acid sequence of SEQ ID NO: 164, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises the amino acid sequence of SEQ ID NO: 165, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith; or

[0429] h) The heavy chain comprises the amino acid sequence of SEQ ID NO: 166, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith, and the light chain comprises the amino acid sequence of SEQ ID NO: 167, or an amino acid sequence having at least 80% (such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity therewith.

[0430] In some embodiments, the anti-Titin&Obscurin antibody as described in any of the preceding items, wherein:

[0431] a) The heavy chain comprises the amino acid sequence of SEQ ID NO: 158, and the light chain comprises the amino acid sequence of SEQ ID NO: 159; or

[0432] b) The heavy chain comprises the amino acid sequence of SEQ ID NO: 146, and the light chain comprises the amino acid sequence of SEQ ID NO: 147; or

[0433] c) The heavy chain comprises the amino acid sequence of SEQ ID NO: 150, and the light chain comprises the amino acid sequence of SEQ ID NO: 151; or

[0434] d) The heavy chain comprises the amino acid sequence of SEQ ID NO: 154, and the light chain comprises the amino acid sequence of SEQ ID NO: 155; or

[0435] e) The heavy chain comprises the amino acid sequence of SEQ ID NO: 156, and the light chain comprises the amino acid sequence of SEQ ID NO: 157; or

[0436] f) The heavy chain comprises the amino acid sequence of SEQ ID NO: 160, and the light chain comprises the amino acid sequence of SEQ ID NO: 161; or

[0437] g) The heavy chain comprises the amino acid sequence of SEQ ID NO: 164, and the light chain comprises the amino acid sequence of SEQ ID NO: 165; or

[0438] h) The heavy chain comprises the amino acid sequence of SEQ ID NO: 166, and the light chain comprises the amino acid sequence of SEQ ID NO: 167; or

[0439] i) The heavy chain comprises the amino acid sequence of SEQ ID NO: 140, and the light chain comprises the amino acid sequence of SEQ ID NO: 141; or

[0440] j) The heavy chain comprises the amino acid sequence of SEQ ID NO: 142, and the light chain comprises the amino acid sequence of SEQ ID NO: 143; or

[0441] k) The heavy chain comprises the amino acid sequence of SEQ ID NO: 144, and the light chain comprises the amino acid sequence of SEQ ID NO: 145; or

[0442] l) The heavy chain comprises the amino acid sequence of SEQ ID NO: 148, and the light chain comprises the amino acid sequence of SEQ ID NO: 149; or

[0443] m) The heavy chain comprises the amino acid sequence of SEQ ID NO: 152, and the light chain comprises the amino acid sequence of SEQ ID NO: 153; or

[0444] n) The heavy chain comprises the amino acid sequence of SEQ ID NO: 162, and the light chain comprises the amino acid sequence of SEQ ID NO: 163.

[0445] In some embodiments, the anti-Titin&Obscurin antibody as described in the previous item, wherein:

[0446] a) the heavy chain comprises the amino acid sequence of SEQ ID NO: 158, and the light chain comprises the amino acid sequence of SEQ ID NO: 159; or

[0447] b) the heavy chain comprises the amino acid sequence of SEQ ID NO: 146, and the light chain comprises the amino acid sequence of SEQ ID NO: 147; or

[0448] c) the heavy chain comprises the amino acid sequence of SEQ ID NO: 150, and the light chain comprises the amino acid sequence of SEQ ID NO: 151; or

[0449] d) the heavy chain comprises the amino acid sequence of SEQ ID NO: 154, and the light chain comprises the amino acid sequence of SEQ ID NO: 155; or

[0450] e) the heavy chain comprises the amino acid sequence of SEQ ID NO: 156, and the light chain comprises the amino acid sequence of SEQ ID NO: 157; or

[0451] f) the heavy chain comprises the amino acid sequence of SEQ ID NO: 160, and the light chain comprises the amino acid sequence of SEQ ID NO: 161; or

[0452] g) the heavy chain comprises the amino acid sequence of SEQ ID NO: 164, and the light chain comprises the amino acid sequence of SEQ ID NO: 165; or

[0453] h) the heavy chain comprises the amino acid sequence of SEQ ID NO: 166, and the light chain comprises the amino acid sequence of SEQ ID NO: 167.

[0454] In some embodiments, the anti-Titin&Obscurin antibody as described in the previous item, wherein:

[0455] the heavy chain comprises the amino acid sequence of SEQ ID NO: 158, and the light chain comprises the amino acid sequence of SEQ ID NO: 159.

[0456] In some embodiments, the anti-Titin&Obscurin antibody as described in the previous item, wherein:

[0457] the heavy chain comprises the amino acid sequence of SEQ ID NO: 146, and the light chain comprises the amino acid sequence of SEQ ID NO: 147.

[0458] In some embodiments, the anti-Titin&Obscurin antibodies as described in any of the preceding items are CED2208, CED2184, CED2190, CED2202, CED2205, CED2211, CED2217, CED2220, CED2175, CED2178, CED2181, CED2187, CED2193 or CED2214 antibodies. In some embodiments, the anti-Titin&Obscurin antibodies as described in any of the preceding items are CED2208, CED2184, CED2190, CED2202, CED2205, CED2211, CED2217 or CED2220 antibodies. In some embodiments, the anti-Titin&Obscurin antibodies as described in any of the preceding items are CED2208 or CED2184 antibodies.

[0459] In another aspect, the present disclosure provides a method for screening cell lines using the anti-Titin&Obscurin antibodies as described in any of the preceding items.

[0460] In some embodiments, the method comprises the following steps:

[0461] Step 1. Obtain the anti-Titin&Obscurin antibody that binds to the asymmetric multispecific antibody as described in any of the preceding items;

[0462] Step 2. Detection method development: Label the anti-Titin&Obscurin antibody and the commercial anti-human light chain λ or κ antibody with a donor and a receptor respectively, and pair them one by one for detection. The pairing schemes are as follows:

[0463] (1) Anti-Titin&Obscurin-Eu or Anti-Titin&Obscurin-Tb, and Anti-κ-d2 or Anti-κ-XL665, or

[0464] (2) Anti-κ-Eu or Anti-κ-Tb, and Anti-Titin&Obscurin-d2 or Anti-Titin&Obscurin-XL665, or

[0465] (3) Anti-Titin&Obscurin-Eu or Anti-Titin&Obscurin-Tb, and Anti-λ-d2 or Anti-λ-XL665, or

[0466] (4) Anti-λ-Eu or Anti-λ-Tb, and Anti-Titin&Obscurin-d2 or Anti-Titin&Obscurin-XL665;

[0467] wherein the donor is Eu 3+ cryptate or Tb 2+ cryptate, and the acceptor is d2 or XL665;

[0468] Step 3. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the energy transfer signal. The higher the energy transfer signal of a sample, the higher the content of correctly paired antibodies in the sample, and the corresponding cell line is a high-quality cell line. Description of the Drawings

[0469] Figure 1A Shows the ELISA experimental results of the binding of anti-Titin&Obscurin antibody to the EGFR-cMET bispecific antibody.

[0470] Figure 1B Shows the ELISA experimental results of the binding of anti-Titin&Obscurin antibody to the BCMA-CD3 bispecific antibody.

[0471] Figure 2A Shows the experimental results of the mutual influence of the binding forces of anti-Titin&Obscurin antibody and anti-Kappa antibody when binding to the EGFR-cMET bispecific antibody simultaneously.

[0472] Figure 2B Shows the experimental results of the mutual influence of the binding forces of anti-Titin&Obscurin antibody and anti-Kappa antibody when binding to the BCMA-CD3 bispecific antibody simultaneously.

[0473] Figure 3A Shows the experimental results of the fluorescence ratio corresponding to the EGFR-cMET bispecific antibody cell line detected by the method disclosed herein.

[0474] Figure 3B Shows the experimental results of the fluorescence ratio corresponding to the BCMA-CD3 bispecific antibody cell line detected by the method disclosed herein.

[0475] Figure 4A Shows the standard curve graph of the fluorescence ratio obtained by the detection method disclosed herein and the concentration of the PSMA-CD28 bispecific antibody standard.

[0476] Figure 4B Shows the relationship graph between the fluorescence ratio of the PSMA-CD28 bispecific antibody cell line detected by the method disclosed herein and the mass spectrometry detection results.

[0477] Figure 5AThe standard curve graph of the fluorescence ratio obtained by using the detection method disclosed herein and the concentration of the PSMA-CD3 bispecific antibody standard product.

[0478] Figure 5B The relationship graph between the fluorescence ratio detected by using the method disclosed herein for the PSMA-CD3 bispecific antibody cell line and the mass spectrometry detection result.

[0479] Figure 6A The standard curve graph of the fluorescence ratio obtained by using the detection method disclosed herein and the concentration of the GPC3-γδT bispecific antibody standard product.

[0480] Figure 6B The relationship graph between the fluorescence ratio detected by using the method disclosed herein for the GPC3-γδT bispecific antibody cell line and the mass spectrometry detection result.

[0481] Figure 7A The standard curve graph of the fluorescence ratio obtained by using the detection method disclosed herein and the concentration of the MUC1-EGFR bispecific antibody standard product.

[0482] Figure 7B The relationship graph between the fluorescence ratio of the MUC1-EGFR bispecific antibody cell line obtained by using the detection method disclosed herein and the mass spectrometry detection result.

[0483] Figure 8A The relationship graph between the fluorescence ratio of the GPC3-γδT bispecific antibody cell line obtained by using the detection method disclosed herein and the SEC purity.

[0484] Figure 8B The relationship graph between the fluorescence ratio of the MUC1-EGFR bispecific antibody cell line obtained by using the detection method disclosed herein and the SEC purity. DETAILED DESCRIPTION OF THE INVENTION

[0486] TERMS

[0487] To facilitate a better understanding of the present disclosure, certain technical and scientific terms are described below. Unless otherwise clearly defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0488] The singular forms "a", "an", and "the" as used in the specification and claims include plural referents unless the context clearly dictates otherwise.

[0489] Unless the context clearly requires otherwise, in the patent specification and claims, the words "comprising", "having", "including", etc. shall be construed in the sense of "including but not limited to", rather than in an exclusive or exhaustive sense.

[0490] The term "and / or" means including both the meanings of "and" and "or". For example, the phrase "A, B and / or C" is intended to cover each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0491] The term "TR-FRET" refers to time-resolved fluorescence resonance energy transfer, which combines two techniques: fluorescence resonance energy transfer (FRET) and time-resolved (TR). The TR-FRET technique utilizes the energy transfer between two fluorophores, which are respectively called the energy donor (Donor, lanthanide elements such as europium or terbium, etc.) and the energy acceptor (Acceptor, fluorophores such as FITC or 5-FAM, etc.). The emission spectrum of the former overlaps with the excitation spectrum of the latter. The donor is excited by an external energy source (such as a laser). If its acceptor is within a sufficiently close distance, energy can be resonantly transferred to the acceptor. The acceptor is excited and emits emission light of a specific wavelength. The donor and the acceptor are respectively conjugated to two biomolecules that can specifically bind. The binding of the biomolecules can bring the donor and the acceptor closer, resulting in energy transfer. The TR technique utilizes the unique properties of lanthanide elements in rare earth elements, and their fluorescence lasts longer than ordinary fluorescence. The half-life of ordinary fluorescence is in the nanosecond range, while the half-life of lanthanide elements is in the millisecond range, with a difference of six orders of magnitude. Therefore, during detection, there is a time delay of about 100 microseconds for TR, so that the ordinary background fluorescence signal in the reaction system is almost zero. Thus, the background of TR is very low, reflecting the actual situation of the sample.

[0492] The term "HTRF" is the abbreviation of homogeneous time-resolved fluorescence assay, which is a further improvement of the TR-FRET technique (refer to patents WO1987000927A1, WO1989005813A1, EP0180492B1 and US5527684A). The commonly used energy donors in HTRF are cryptates of europium (Eu) and terbium (Tb). In this cryptate, Eu and Tb are permanently embedded in a cage with a very stable structure, which was invented by Professor J.M. Lehn and won the Nobel Prize in 1987. There are also two commonly used energy acceptors in HTRF, namely XL665 and d2, which have the same optical properties but different molecular weights. The former has a molecular weight of 10KD, that is, allophycocyanin (APC); the latter has a molecular weight of 1KD and is conjugated with Eu 3+Highly compatible with cryptate compounds. HTRF measures the emitted light at two different wavelengths, namely 620 nm (donor) and 665 nm (acceptor).

[0493] When two fluorophores approach each other due to biomolecular interactions, part of the energy captured by the cryptate compound at excitation is released, and the emission wavelength is 620 nm; another part of the energy is transferred to the acceptor, and the emission wavelength is 665 nm. Therefore, when biomolecular interactions occur, there are two excitation lights at 620 nm and 665 nm; when there is no interaction, there is only one excitation light at 620 nm.

[0494] The three-letter and single-letter codes of amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0495] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that have been modified later, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon to which hydrogen, carboxyl, amino, and R groups are attached), such as homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics are chemical compounds that have a structure different from the general chemical structure of amino acids, but act in a manner similar to naturally occurring amino acids.

[0496] The term "asymmetric" means that the antibodies of the present invention cannot be divided into two symmetric parts. Specifically, certain natural antibodies (e.g., IgG) contain two identical heavy chains and two identical light chains, and the part composed of one heavy chain and one light chain is symmetric with the part composed of another heavy chain and another light chain.

[0497] The term "multispecific antibody" refers to an antibody that has binding specificity for at least two different epitopes on the same antigen or two different antigens. Multispecific antibodies can be made into full-length antibodies or antibody fragments (e.g., F(ab’)2 bispecific antibodies) or combinations thereof (e.g., full-length antibodies plus additional scFv or Fab fragments). Engineered antibodies with two, three, or more (e.g., four) functional antigen-binding sites have also been reported (see, e.g., US2002 / 0004587A1).

[0498] The term "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, antibody fragments, and antigen-binding fragments (or antigen-binding portions), provided that they exhibit the desired antigen-binding activity.

[0499] The term "bispecific antibody" refers to an antibody (including an antibody or its antigen-binding fragment, such as a single-chain antibody) that is capable of specifically binding to two different antigens or at least two different antigenic epitopes of the same antigen. The prior art has disclosed bispecific antibodies with various structures, which can be classified into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies according to the integrity of the IgG molecule, into bivalent, trivalent, tetravalent or more-valent bispecific antibodies according to the number of antigen-binding regions, and into symmetric-structure bispecific antibodies and asymmetric-structure bispecific antibodies according to whether the structure is symmetric. Among them, bispecific antibodies based on antibody fragments, such as Fab fragments lacking the Fc fragment, form bispecific antibodies by binding two or more Fab fragments in one molecule, which have lower immunogenicity, small molecular weight, and high tumor tissue permeability. Typical antibody structures of this type are such as F(ab)2, scFv-Fab, (scFv)2-Fab; IgG-like bispecific antibodies (e.g., having an Fc fragment), such antibodies have a relatively large molecular weight. The Fc fragment helps in the purification of the antibody and improves its solubility and stability. The Fc portion may also bind to the receptor FcRn, increasing the serum half-life of the antibody. Typical bispecific antibody structure models are such as KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, 1Fab-IgG TDB, FynomAb, two-in-one / DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb, etc. (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585-608 (2019); Chen S1 et al., J Immunol Res. 2019 Feb 11; 2019:4516041).

[0500] The term "natural antibody" refers to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetrameric proteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N- to the C-terminus, each heavy chain has a variable region (VH), also known as the variable heavy domain, the heavy chain variable region, followed by the heavy chain constant region, and the natural IgG heavy chain constant region typically contains three constant domains (CH1, CH2, and CH3). Similarly, from the N- to the C-terminus, each light chain has a variable region (VL), also known as the variable light domain, or the light chain variable domain, followed by a constant light domain (the light chain constant region, CL).

[0501] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to that of a natural antibody or having a heavy chain with an Fc region as defined herein. The light chain of a natural intact antibody includes the variable region VL and the constant region CL, with VL at the amino terminus of the light chain, and the light chain constant region includes the κ chain and the λ chain; the heavy chain includes the variable region VH and the constant regions (CH1, CH2, and CH3), with VH at the amino terminus of the heavy chain and the constant regions at the carboxyl terminus, where CH3 is closest to the carboxyl terminus of the polypeptide, and the heavy chain can belong to any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0502] The term antibody "variable region" or "variable domain" refers to the domain in the antibody heavy or light chain that is involved in antibody binding to antigen. In this article, the variable region of the antibody heavy chain (VH) and the variable region of the light chain (VL) each contain four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). Among them, the term "complementarity-determining region" or "CDR" refers to the region within the variable domain that mainly contributes to antigen binding; "framework" or "FR" refers to the variable domain residues other than the CDR residues. VH contains 3 CDR regions: HCDR1, HCDR2, and HCDR3; VL contains 3 CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus (also known as the N-terminus) to the carboxyl terminus (also known as the C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0503] The amino acid sequence boundaries of the CDR can be determined by various well-known methods, such as: the "Kabat" numbering rule (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, Bethesda, MD), the "Chothia" numbering rule, the "ABM" numbering rule, the "contact" numbering rule (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains[J]. 2001) and the ImMunoGenTics (IMGT) numbering rule (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9:2278), etc.; the correspondence between various numbering systems is well-known to those skilled in the art. Exemplarily, as shown in Table 1 below.

[0504] Table 1. Relationships between CDR numbering systems

[0505] CDR IMGT Kabat AbM Chothia Contact HCDR1 27-38 31-35 26-35 26-32 30-35 HCDR2 56-65 50-65 50-58 52-56 47-58 HCDR3 105-117 95-102 95-102 95-102 93-101 LCDR1 27-38 24-34 24-34 24-34 30-36 LCDR2 56-65 50-56 50-56 50-56 46-55 LCDR3 105-117 89-97 89-97 89-97 89-96

[0506] Unless otherwise specified, the "Kabat" numbering rule is applicable to the variable regions and CDRs in the embodiments of this disclosure. Although in specific embodiments, the Kabat numbering rule is used to define amino acid residues, the corresponding technical solutions of other numbering systems will be regarded as equivalent technical solutions.

[0507] The term "antibody fragment" refers to a molecule different from a complete antibody that contains a portion of the complete antibody and binds to the antigen to which the complete antibody binds. Examples of antibody fragments include, but are not limited to, Fv, dsFv, Fab, Fab′, Fab′-SH, Fd, F(ab′)2, single-domain antibodies (sdAb, such as VH, VL, VHH or V HH )), single-chain Fab (scFab), diabodies, linear antibodies, single-chain antibodies (such as scFv, sc(Fv)2); and multispecific antibodies formed by antibody fragments.

[0508] The term "antigen-binding fragment" encompasses full-length antibodies, Fab, modified Fab, Fab', Fab'-SH, modified Fab', F(ab')2, Fv, dsFv, Fab-Fv, Fab-dsFv, Fd, single-domain antibodies (sdAb, such as VH or VL or VHH), single-chain Fab (scFab), single-chain antibodies (such as scFv, sc(Fv)2), diabodies, linear antibodies, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the foregoing (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for generating and preparing these antigen-binding fragments are well known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0509] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including native Fc regions and engineered Fc regions. In some embodiments, the Fc region comprises two subunits that are the same or different. In some embodiments, the Fc region of a human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. Suitable Fc regions for the antibodies described herein include the Fc regions of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some embodiments, the boundaries of the Fc region can also vary, such as deletion of the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) or deletion of the C-terminal glycine and lysine of the Fc region (residues 446 and 447 according to the EU numbering system). Unless otherwise specified, the numbering convention for the Fc region is the EU numbering system, also known as the EU index.

[0510] The term "Titin chain" refers to a peptide segment of Titin protein that is 78-118 amino acids in length and contains the Titin Ig-like 152 domain, or a functional variant thereof, which is capable of binding to the Obscurin Ig-like 1 or Obscurin-like Ig-like 1 domain to form a dimerization complex.

[0511] The term "Obscurin chain" refers to a peptide segment of 87-117 amino acids on the Obscurin protein that contains the Obscurin Ig-like 1 domain or its functional variant, or a peptide segment of 78-118 amino acids on the Obscurin-like 1 protein that contains the Obscurin-like Ig-like 1 domain or its functional variant.

[0512] The Obscurin chain can bind to the Titin chain to form a dimerization complex. The Titin chain and the Obscurin chain of the present disclosure can be used to replace CH1 and CL in Fab, respectively, to form a replaced Fab (Fab-S), and this replacement does not affect the binding of the antibody to the antigen (WO2021139758A1, WO2022237882A1).

[0513] In the present disclosure, the term "anti-Titin&Obscurin antibody" is used in the broadest sense and can be an anti-Titin antibody, an anti-Obscurin antibody, or an anti-Titin and anti-Obscurin antibody, as long as it exhibits the ability to bind to a multispecific antibody having a HOT-Ig structure. In some embodiments, the HOT-Ig structure is derived from WO2021139758A1, WO2022237882A1 (incorporated herein by reference in its entirety).

[0514] In the present disclosure, the terms kappa and κ can be used interchangeably; the terms lambda and λ can be used interchangeably.

[0515] In the present disclosure, the term "Anti-Titin&Obscurin-Eu" refers to a cryptate-labeled anti-Titin&Obscurin antibody with donor Eu. 3+ The term "Anti-Titin&Obscurin-Tb" refers to a cryptate-labeled anti-Titin&Obscurin antibody with donor Tb. 2+ The term "Anti-Titin&Obscurin-d2" refers to a receptor d2-labeled anti-Titin&Obscurin antibody.

[0516] The term "Anti-κ-d2" refers to a receptor d2-labeled anti-human light chain κ chain antibody. The term "Anti-κ-Eu" refers to a cryptate-labeled anti-human light chain κ chain antibody with donor Eu. 3+ The term "Anti-κ-Tb" refers to a cryptate-labeled anti-human light chain κ chain antibody with donor Tb. 2+ The term "Anti-κ-Tb" refers to a cryptate-labeled anti-human light chain κ chain antibody.

[0517] The term "Anti-λ-d2" refers to an anti-human light chain λ antibody labeled with acceptor d2. The term "Anti-λ-Eu" refers to a donor Eu 3+ cryptate-labeled anti-human light chain λ antibody. The term "Anti-λ-Tb" refers to a donor Tb 2+ cryptate-labeled anti-human light chain λ antibody.

[0518] The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, the binding "affinity" refers to the intrinsic binding affinity, which reflects the 1:1 interaction between the members of a binding pair (e.g., antibody-antigen). The affinity of molecule X for its ligand Y can generally be represented by the dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.

[0519] The term "antigen" refers to a molecule or molecular moiety that can be selectively bound by a binding agent such as an antigen-binding protein (including, for example, an antibody), and that can additionally be used in an animal to generate an antibody capable of binding to the antigen. An antigen can have one or more epitopes that can interact with different antigen-binding proteins (e.g., antibodies).

[0520] The term "epitope" refers to a region (area or region) on an antigen to which an antibody or its antigen-binding fragment can specifically bind. An epitope can be formed by a continuous string of amino acids (linear epitope) or contain non-contiguous amino acids (conformational epitope), e.g., becoming spatially proximal due to the folding of the antigen (i.e., the tertiary folding of a proteinaceous antigen). The difference between conformational and linear epitopes is that in the presence of a denaturing solvent, the binding of an antibody to a conformational epitope is lost. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be carried out using routine methods in the art, such as, but not limited to, alanine scanning, peptide blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking.

[0521] The terms "capable of specifically binding", "specifically binds", or "binds" mean that an antibody is capable of binding to a particular antigen or an epitope of that antigen with a higher affinity compared to other antigens or epitopes. Generally, an antibody binds with approximately 1×10 -7 M or less (e.g., approximately 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11bind to an antigen or an epitope within an antigen with a dissociation equilibrium constant (KD) of M or less. In some embodiments, the KD of the antibody-antigen binding is 10% or less (e.g., 1%) of the KD of the antibody binding to a non-specific antigen (e.g., BSA, casein). Known methods can be used to measure KD, e.g., by measured by surface plasmon resonance assays. However, an antibody that specifically binds to an antigen or an epitope within an antigen may have cross-reactivity to other related antigens, e.g., to corresponding antigens from other species (homologs) such as human or monkey, e.g., cynomolgus macaque (Macaca fascicularis) (cynomolgus, cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp), or common marmoset (Callithrix jacchus) (marmoset).

[0522] The term “does not bind” means that an antibody is unable to bind to an antigen or its epitope in the manner of specific binding described above. For example, when an antibody binds to an antigen or its epitope with a dissociation equilibrium constant (KD) of about 1×10 -6 M or greater.

[0523] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, having similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). "Isolated" nucleic acid molecules are those separated from the components of their natural environment. Isolated nucleic acid molecules include nucleic acid molecules contained in cells that ordinarily contain the nucleic acid molecule, but wherein the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. Isolated nucleic acids encoding a polypeptide or fusion protein refer to one or more nucleic acid molecules encoding the polypeptide or fusion protein, including such one or more nucleic acid molecules in a single vector or separate vectors, and such one or more nucleic acid molecules present at one or more locations in a host cell. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly recited. Specifically, as detailed below, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues.

[0524] The terms "polypeptide" and "protein" are used interchangeably herein and refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses its conservatively modified variants.

[0525] The term "sequence identity" refers to the degree (percentage) to which the amino acids / nucleic acids of two sequences are the same when the two sequences are optimally aligned, introducing gaps as necessary to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. To determine the percentage of sequence identity, the alignment may be carried out by techniques known in the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters appropriate for measuring the alignment, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared.

[0526] The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, such as an adeno-associated virus vector (AAV or AAV2), into which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of the host cell after being introduced into the host cell, and thus replicate along with the host genome. The term "expression vector" or "expression construct" refers to a vector that can transform a host cell and contains a nucleic acid sequence that directs and / or controls (along with the host cell) the expression of one or more heterologous coding regions to which it is operably linked. Expression constructs can include, but are not limited to, sequences that affect or control transcription, translation, and RNA splicing of the coding region to which they are operably linked in the presence of introns.

[0527] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells", which include the primary transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be completely identical to the parental cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity selected or screened for in the original transformed cell are included herein. Host cells include prokaryotic and eukaryotic host cells, where eukaryotic host cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse, and hamster cells, including, but not limited to, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, simian kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, the genus Pichia, Saccharomyces cerevisiae, the genus Saccharomyces, Hansenula polymorpha, the genus Kluyveromyces, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, the genus Fusarium, Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa, the genus Aspergillus, and Yarrowia lipolytica.

[0528] As used in this disclosure, the terms "cell", "cell line", and "cell culture" are used interchangeably and all such names include progeny. Thus, the terms "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It should also be understood that due to deliberate or inadvertent mutations, not all progeny have exactly the same DNA content. Mutant progeny that have the same function or biological activity as the original transformed cell are included.

[0529] "Optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and this description includes instances where the event or circumstance occurs or does not occur.

[0530] The cell line screening method of the present disclosure

[0531] The present disclosure provides a method for screening cell lines for asymmetric multispecific antibodies, which has many advantageous characteristics, such as being able to rapidly identify correctly paired multispecific antibody molecules (such as bispecific antibodies) at an early stage of cell line screening.

[0532] Exemplary cell line screening method

[0533] Exemplarily, a method for screening cell lines for asymmetric multispecific antibodies provided by the present disclosure, the method comprising the following steps:

[0534] Step 1. Obtain anti-Titin&Obscurin antibodies that bind to asymmetric multispecific antibodies;

[0535] Step 2. Detection method development: Label the anti-Titin&Obscurin antibody and the commercial anti-human light chain λ or κ antibody with a donor and a receptor respectively, and pair them one by one for detection. The pairing scheme is as follows:

[0536] (1) Anti-Titin&Obscurin-Eu or Tb and Anti-κ-d2; or

[0537] (2) Anti-κ-Eu or Tb and Anti-Titin&Obscurin-d2;

[0538] wherein the donor is Eu 3+ cryptate or Tb 2+ cryptate (abbreviated as Eu or Tb), the receptor is d2 or XL665; Step 3. Use a microplate reader to detect the energy transfer signal. The higher the energy transfer signal in the sample, the higher the content of correctly paired antibodies, and the corresponding cell line is a high-quality cell line.

[0539] Exemplarily, in the cell line screening method of the present disclosure, step 1 includes immunizing mice with Titin and Obscurin-Fc fusion proteins in an asymmetric multispecific antibody platform, and obtaining anti-Titin&Obscurin antibodies that bind to the asymmetric multispecific antibody platform by means of hybridomas.

[0540] In some embodiments, the cell line screening method as described above, wherein the Titin and Obscurin-Fc fusion proteins have the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 3, respectively.

[0541] Exemplarily, in the cell line screening method of the present disclosure, the detection method in step 2 is homogeneous time-resolved fluorescence technology.

[0542] Exemplarily, in the cell line screening method of the present disclosure, the pairing scheme in step 2 includes:

[0543] (1) Anti-Titin&Obscurin-Eu and Anti-κ-d2, or

[0544] (2) Anti-Titin&Obscurin-Tb and Anti-κ-d2, or

[0545] (3) Anti-κ-Eu and Anti-Titin&Obscurin-d2, or

[0546] (4) Anti-κ-Tb and Anti-Titin&Obscurin-d2.

[0547] Exemplarily, in the cell line screening method of the present disclosure, wherein the Eu 3+ cryptate or Tb 2+ cryptate donor-labeled molecule and the d2 or XL665 acceptor-labeled molecule have a pairing concentration ratio of 0.5:1, 0.5:2, 0.5:4, 0.5:6, 0.5:8, 1:1, 1:2, 1:4, 1:6, 1:8 or 2:2. In some embodiments, in the cell line screening method as described above, wherein the Eu 3+ cryptate or Tb 2+ cryptate donor-labeled molecule and the d2 or XL665 acceptor-labeled molecule have a pairing concentration ratio of 1:1. In some embodiments, in the cell line screening method as described above, wherein the pairing concentration ratio of the Eu 3+ cryptate-labeled anti-Titin&Obscurin antibody and the d2-labeled anti-κ antibody is 1:1.

[0548] Exemplarily, in the cell line screening method of the present disclosure, step 2 further includes a step of mixing and incubating the anti-Titin&Obscurin antibody and a commercial anti-human light chain λ or κ antibody with the cell supernatant or the analyte.

[0549] Exemplarily, in the cell line screening method of the present disclosure, the incubation time is 2 hours or more; preferably, the incubation time is 2 to 12 hours.

[0550] Exemplarily, in the cell line screening method of the present disclosure, the incubation is incubation at room temperature for 2 hours or overnight incubation at 4°C.

[0551] Exemplarily, in the cell line screening method of the present disclosure, the incubation is incubation at 4°C for 12 hours.

[0552] Exemplarily, in the cell line screening method of the present disclosure, step 2 further includes a step using the target multispecific antibody as a standard.

[0553] Exemplarily, in the cell line screening method of the present disclosure, the energy transfer signal is determined by a fluorescence ratio, and the fluorescence ratio is calculated according to Em665 / Em620 / 10 4 Calculate.

[0554] In some embodiments, in the cell line screening method as described above, step 3 further includes an operation of plotting a standard curve of energy transfer signal - antibody concentration according to the detection results.

[0555] Exemplarily, in the cell line screening method of the present disclosure, in step 3, the emission light of the antibody is detected at 620 nm and 665 nm respectively; then the fluorescence ratio is calculated according to the formula Em665 / Em620 / 10 4 Calculate the fluorescence ratio.

[0556] Exemplarily, in the cell line screening method of the present disclosure, in step 3, first, the emission light of the Eu 3+ cryptate or Tb 2+ cryptate-labeled anti-Titin&Obscurin antibody is detected at 620 nm; then the emission light of the d2-labeled anti-κ antibody is detected at 665 nm; then the fluorescence ratio is calculated according to the formula Em665 / Em620 / 10 4 Calculate the fluorescence ratio.

[0557] Exemplarily, in the cell line screening method of the present disclosure, in step 3, first, the emission light of the Eu 3+ cryptate-labeled anti-Titin&Obscurin antibody is detected at 620 nm; then the emission light of the d2-labeled anti-κ antibody is detected at 665 nm; then the fluorescence ratio is calculated according to the formula Em665 / Em620 / 10 4 Calculate the fluorescence ratio.

[0558] Exemplarily, in the cell line screening method of the present disclosure, in step 3, first, the emission light of the Tb 2+Emission light of the cryptate-labeled anti-Titin&Obscurin antibody; then detect the emission light of the d2-labeled anti-κ antibody at 665 nm; then calculate the fluorescence ratio according to the formula Em665 / Em620 / 10 4 Calculate the fluorescence ratio.

[0559] Exemplarily, in the cell line screening method of the present disclosure, in step 3, first detect Eu 3+ cryptate or Tb 2+ Emission light of the cryptate-labeled anti-κ antibody; then detect the emission light of the d2-labeled anti-Titin&Obscurin antibody at 665 nm; then calculate the fluorescence ratio according to the formula Em665 / Em620 / 10 4 Calculate the fluorescence ratio.

[0560] Exemplarily, in the cell line screening method of the present disclosure, in step 3, first detect Eu 3+ Emission light of the cryptate-labeled anti-κ antibody; then detect the emission light of the d2-labeled anti-Titin&Obscurin antibody at 665 nm; finally calculate the fluorescence ratio according to the formula Em665 / Em620 / 10 4 Calculate the fluorescence ratio.

[0561] Exemplarily, in the cell line screening method of the present disclosure, in step 3, first detect Tb 2+ Emission light of the cryptate-labeled anti-κ antibody; then detect the emission light of the d2-labeled anti-Titin&Obscurin antibody at 665 nm; then calculate the fluorescence ratio according to the formula Em665 / Em620 / 10 4 Calculate the fluorescence ratio.

[0562] Exemplarily, in the cell line screening method of the present disclosure, in step 2, dilute the cell culture supernatant to be detected to 5 μg / mL, add it to a 384-well detection plate, 10 μL / well; dilute the multispecific antibody standard to 15 μg / mL, then perform serial two-fold dilutions for a total of 6 points, add it to a 384-well detection plate, 10 μL / well; add the Eu 3+ cryptate-labeled anti-Titin&Obscurin antibody and d2-labeled anti-κ antibody diluted to 1×, 5 μL / well each, into the above samples respectively; after mixing, incubate at room temperature for 2 hours or overnight at 4°C.

[0563] Exemplarily, in the cell line screening method of the present disclosure, the asymmetric multispecific antibody to be detected is an asymmetric multispecific antibody having a Titin chain and an Obscurin chain.

[0564] Exemplarily, the cell line screening method of the present disclosure, wherein the multispecific antibody has at least one replaced Fab, which comprises a Titin chain and an Obscurin chain capable of forming a dimer. In some embodiments, the cell line screening method as described above, wherein the replaced Fab has its original CH1 and CL of the Fab replaced by the Obscurin chain and the Titin chain, respectively, or the original CH1 and CL of the Fab are replaced by the Titin chain and the Obscurin chain, respectively.

[0565] In some embodiments, the cell line screening method as described above, wherein the Titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 168 to 186, and the Obscurin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 187 to 227.

[0566] Exemplarily, the cell line screening method of the present disclosure, wherein the asymmetric multispecific antibody to be detected is an asymmetric bispecific antibody.

[0567] Exemplarily, the cell line screening method of the present disclosure, wherein the anti-Titin&Obscurin antibody comprises a heavy chain variable region and a light chain variable region:

[0568] a) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 24, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 23; or

[0569] b) The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region respectively comprise the amino acid sequences of HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 12, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region respectively comprise the amino acid sequences of LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 11.

[0570] Exemplarily, the cell line screening method of the present disclosure, wherein the anti-Titin&Obscurin antibody comprises:

[0571] a) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 113, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 114, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 115, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 116, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 117, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 118; or

[0572] b) The HCDR1 of the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 77, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 78, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 79, and the LCDR1 of the light chain variable region comprises the amino acid sequence of SEQ ID NO: 80, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 81, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 82.

[0573] Exemplarily, in the cell line screening method of the present disclosure, the anti-Titin&Obscurin antibody comprises:

[0574] a) The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 113, the HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 114, and the HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 115, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 116, the LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 117, and the LCDR3 has an amino acid sequence consisting of SEQ ID NO: 118; or

[0575] b) The HCDR1 of the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 77, the HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 78, and the HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 79, and the LCDR1 of the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 80, the LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 81, and the LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 82.

[0576] Exemplarily, in the cell line screening method of the present disclosure, the anti-Titin&Obscurin antibody comprises a heavy chain variable region and a light chain variable region:

[0577] a) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 24, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 23; or

[0578] b) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 11.

[0579] Exemplarily, in the method for screening cell lines disclosed herein, the anti-Titin&Obscurin antibody comprises a heavy chain variable region and a light chain variable region:

[0580] a) The amino acid sequence of the heavy chain variable region consists of SEQ ID NO: 24, and the amino acid sequence of the light chain variable region consists of SEQ ID NO: 23; or

[0581] b) The amino acid sequence of the heavy chain variable region consists of SEQ ID NO: 12, and the amino acid sequence of the light chain variable region consists of SEQ ID NO: 11.

[0582] Exemplarily, in the method for screening cell lines disclosed herein, the anti-Titin&Obscurin antibody comprises a heavy chain and a light chain:

[0583] a) The heavy chain comprises the amino acid sequence of SEQ ID NO: 158, and the light chain comprises the amino acid sequence of SEQ ID NO: 159; or

[0584] b) The heavy chain comprises the amino acid sequence of SEQ ID NO: 146, and the light chain comprises the amino acid sequence of SEQ ID NO: 147.

[0585] Exemplarily, in the method for screening cell lines disclosed herein, the anti-Titin&Obscurin antibody is the CED2208 or CED2184 antibody.

[0586] Fab Modification

[0587] In one aspect, in the asymmetric multispecific antibody to be detected disclosed herein, the Fab is a substituted Fab, and the substituted Fab comprises a heavy chain variable region, a light chain variable region, a Titin chain, and an Obscurin chain. In the substituted Fab, the original CH1 and CL of the Fab are replaced by the Titin chain and the Obscurin chain. Exemplarily, the sequences of the Titin chain and the Obscurin chain are shown in Table 2-1 and Table 2-2.

[0588] Table 2-1. Amino Acid Sequence of Titin Chain

[0589]

[0590]

[0591] Table 2-2. Amino acid sequence of the Obscurin chain

[0592]

[0593]

[0594]

[0595] Kit

[0596] In another aspect of the present disclosure, a kit is provided, which comprises materials that can be used to detect a multi-specific antibody cell line having a Titin chain and an Obscurin chain structure. The kit comprises a container and a label or a package insert on or associated with the container. Optionally, or in addition, the kit may further comprise a second (or third) container, which contains a pharmaceutically acceptable buffer. From a commercial and user perspective, it may further include other required materials, including but not limited to other buffers, diluents, filters, etc. Detailed implementation manners

[0597] Examples

[0598] The present disclosure is further described below in conjunction with examples, but these examples do not limit the scope of the present disclosure. For the experimental methods without specific conditions noted in the examples of the present disclosure, they are generally carried out under conventional conditions, such as the antibody technology experimental manual of Cold Spring Harbor, the molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer; for the reagent materials without specific sources noted, they are obtained by purchasing from the market.

[0599] Example 1. Preparation of Titin and Obscurin detection proteins

[0600] Using the Titin protein and Obscurin protein (T.16 and O.28) in patent WO2022237882A1 as templates, the amino acid sequences of the detection fusion proteins used in the present disclosure were designed: The Titin protein or Obscurin protein was fused with a His tag and constructed into a vector respectively. It was transiently or stably transfected and expressed in CHO cells, and the protein was purified to obtain the detection protein used in the present disclosure.

[0601] The sequence of the Titin protein with a His tag (abbreviation: T-His) was used as a detection reagent:

[0602] >T-His

[0603]

[0604] Note: The underlined part is the 6×His tag, and the rest is the Titin protein.

[0605] The sequence of Obscurin protein with His tag (abbreviation: O-His), as a detection reagent:

[0606] >O-His

[0607]

[0608] Note: The underlined part is the 6×His tag, and the rest is the Obscurin protein.

[0609] The human Fc bispecific antibodies containing Titin chain and Obscurin chain used in this disclosure are BCMA-CD3 bispecific antibody, EGFR-cMET bispecific antibody, PSMA-CD28 bispecific antibody, PSMA-CD3 bispecific antibody, MUC1-EGFR bispecific antibody and GPC3-γδT bispecific antibody.

[0610] The BCMA-CD3 bispecific antibody is from WO2022156739A1 (Obscurin chain on the heavy chain, Titin chain on the light chain):

[0611] >BCMA-CD3 heavy chain 1

[0612] EIQLVQSGSELKKPGASVKVSCKASGYTFTEYPIHWVRQAPGQVFEWMGMIYTDTGEPTYAEEFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCLRGPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0613] SEQ ID NO: 35

[0614] >BCMA-CD3 heavy chain 2

[0615]

[0616] >BCMA-CD3 light chain 1

[0617]

[0618] >BCMA-CD3 light chain 2

[0619]

[0620] Note: Single underlines indicate Titin chains; double underlines indicate Obscurin chains; italics indicate Kappa light chains.

[0621] The EGFR-cMET bispecific antibody is from WO2023284829A1 (Titin chain on the heavy chain, Obscurin chain on the light chain):

[0622] >EGFR-cMET heavy chain 1

[0623] QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGTLVTVSSGGGGSGGGGQVQLVESGGGVVQPGRSLRLSCAASGFSLSDYGVHWVRQAPGKGLEWLAVIWSGGSTNYADAFVSRLTISKDNSKNTVYLQMNSLRAEDTAVYYCARNHDNPYIYAMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0624] SEQ ID NO: 39

[0625] >EGFR-cMET heavy chain 2

[0626]

[0627] >EGFR-cMET light chain 1

[0628]

[0629] >EGFR-cMET light chain 2

[0630]

[0631] Note: Single underlines indicate Titin chains; double underlines indicate Obscurin chains; italics indicate Kappa light chains.

[0632] The PSMA-CD28 bispecific antibody is from WO2023198015A1 (Titin chain on the heavy chain, Obscurin chain on the light chain):

[0633] >PSMA-CD28 heavy chain 1

[0634]

[0635] >PSMA-CD28 heavy chain 2

[0636] EVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYMNWVRQAPGQRLEWMGEINPSTGDTTYNQKFKAKVTLTVDKSASTAYMELSSLRSEDTAVYYCASPYYDLTPFLYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0637] SEQ ID NO: 44

[0638] >PSMA-CD28 light chain 1

[0639]

[0640] >PSMA-CD28 light chain 2

[0641]

[0642]

[0643] Note: Single underlines indicate Titin chains; double underlines indicate Obscurin chains; italics indicate Kappa light chains.

[0644] The PSMA-CD3 bispecific antibody is from WO2023147784A1 (Obscurin chain on the heavy chain, Titin chain on the light chain):

[0645] >PSMA-CD3 heavy chain 1

[0646] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMAWVRQAPGKGLEWVASINYDGSSTYYLDSLKSRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAGSDGYWAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0647] SEQ ID NO: 47

[0648] >PSMA-CD3 heavy chain 2

[0649]

[0650] >PSMA-CD3 light chain 1

[0651]

[0652] >PSMA-CD3 light chain 2

[0653]

[0654]

[0655] Note: Single underline is the Titin chain; double underline is the Obscurin chain; italic is the Kappa light chain.

[0656] The MUC1-EGFR bispecific antibody is from CN202311772280.3 (Titin chain on the heavy chain, Obscurin chain on the light chain):

[0657] >MUC1-EGFR heavy chain 1

[0658] EVQLVQSGAEVKKPGASVKVSCKASGYTFTTYGVPWVRQAPGQGLEWMGDIYPRSGNTYYNEKFKGRVTMTADKSTDTVYMELSSLRSEDTAVYYCAREDYDNYPYALDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0659] SEQ ID NO: 51

[0660] >MUC1-EGFR heavy chain 2

[0661]

[0662] >MUC1-EGFR light chain 1

[0663]

[0664]

[0665] >MUC1-EGFR light chain 2

[0666]

[0667] Note: Single underlines indicate Titin chains; double underlines indicate Obscurin chains; italics indicate Kappa light chains.

[0668] The GPC3-γδT bispecific antibody is from CN202311518247.8 (Obscurin chain on the heavy chain, Titin chain on the light chain):

[0669] >GPC3-γδT heavy chain 1

[0670]

[0671] >GPC3-γδT heavy chain 2

[0672] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0673] SEQ ID NO: 56

[0674] >GPC3-γδT light chain 1

[0675]

[0676] >GPC3-γδT light chain 2

[0677]

[0678] Note: Single underlines indicate the Titin chain; double underlines indicate the Obscurin chain; italics indicate the Kappa light chain.

[0679] Example 2. Preparation of antigens for Titin and Obscurin immunization: CED2026 + CED2027

[0680] Using the Titin chain and Obscurin chain in WO2022237882A1 as templates, design the amino acid sequences of the immunogenic antigens used in this disclosure; fuse the Obscurin chain with murine Fc and the Titin chain without a tag into vectors respectively, and co-express to form the Titin / Obscurin fusion protein CED2026+2027; transiently or stably express in CHO cells, and purify the protein to obtain the immunogenic antigen protein of this disclosure.

[0681] >Obscurin-Fc(CED2027)

[0682]

[0683] Note: The underlined part is the murine IgG2a Fc part, and the non-underlined part is the Obscurin chain.

[0684] The light chain sequence of the Titin protein as a heterodimer:

[0685] >Titin(CED2026)

[0686] GIPPKIECLPIDISIDEGKVLTVASAFTGEPTPEVTWSTGGRKIHSQEQGRFHIENTDDSTTLTIKDVQKQDGGLYTLTLRNEFGSDSATVNIHIRSI

[0687] SEQ ID NO: 4

[0688] Example 3. Purification of Titin / Obscurin-related recombinant proteins

[0689] 1. Purification steps of the recombinant protein with a His tag:

[0690] The cell expression supernatant sample was centrifuged at high speed to remove impurities, and the buffer was replaced with PBS. Imidazole was added to a final concentration of 5 mM. The nickel column was equilibrated with PBS solution containing 5 mM imidazole and rinsed with 2 - 5 column volumes. The replaced cell supernatant sample was passed through a Ni Sepharose excel column (GE, 17 - 3712 - 02). The column was rinsed with PBS solution containing 5 mM imidazole until the A280 reading dropped to the baseline. Then, the chromatography column was rinsed with PBS + 10 mM imidazole to remove non - specifically bound miscellaneous proteins, and the effluent was collected. Subsequently, the target protein was eluted with PBS solution containing 300 mM imidazole, and the elution peak was collected. The collected eluate was concentrated and further purified by gel filtration chromatography on Superdex200 (GE, 28 - 9893 - 35), with PBS as the mobile phase. The polymer peak was removed, and the elution peak was collected. The obtained protein was identified by electrophoresis, peptide mapping, and LC - MS as correct and then aliquoted for use. O - His (SEQ ID NO: 2) or T - His (SEQ ID NO: 1) with a His - tag was obtained and used as a detection reagent for the antibodies disclosed herein.

[0691] 2. Purification steps of Titin / Obscurin - Fc fusion protein (CED2026 + 2027):

[0692] After co - expression of tag - free Titin and Obscurin - Fc, the cell expression supernatant sample was centrifuged at high speed to remove impurities, and the supernatant was subjected to MabSelect PrismA affinity chromatography. The MabSelect PrismA chromatography column was regenerated with 0.5 M NaOH and equilibrated with 20 mM PB, 1 M NaCl, pH 7.4. After binding the supernatant, it was washed with 20 mM PB, 1 M NaCl, pH 7.4 until the A280 reading dropped to the baseline. Miscellaneous proteins were washed with 50 mM Tris - HCl, 0.5 M Arg, pH 8.5 buffer and then with 20 mM PB, pH 7.4. The target protein was eluted at pH 3.6, and the pH was adjusted to 5.0 with 1 M Tris. The obtained protein was identified by electrophoresis, peptide mapping, and LC - MS as correct, and the endotoxin met the standard. This method was used to obtain high - purity Titin and Obscurin - Fc fusion protein (CED2026 + 2027), and this method can also be used to purify other antibody proteins disclosed herein.

[0693] Example 4. Screening of anti - Titin&Obscurin antibodies

[0694] 1. Immunization of mice:

[0695] Anti-human Titin & Obscurin antibodies were produced by immunizing mice. The experimental mice were Balb / c white mice, female, 6 - 8 weeks old (Beijing Vital River Laboratory Animal Technology Co., Ltd., animal production license number: SCXK(Beijing)2012 - 0001). Breeding environment: SPF level. After the mice were purchased, they were raised in the laboratory environment for 1 week, with a 12 / 12 hour light / dark cycle adjustment, temperature 20 - 25°C; humidity 40 - 60%. The mice that had adapted to the environment were immunized according to the following protocol. The immunizing antigen was Titin and Obscurin-Fc heterodimer CED2026 + 2027 (SEQ ID NO: 3 and SEQ ID NO: 4).

[0696] Immunization protocol: Use Gold Adjuvant (Sigma Cat No.T2684) Thermo Alum (Thermo Cat No.77161) adjuvants for cross-immunization. The antigen and adjuvant The preparation ratio of Gold Adjuvant was 1:1, and the preparation ratio of the antigen and Thermo Alum was 3:1, 50 μg / animal / time (primary immunization), 50 μg / animal / time (booster immunization). After the antigen was emulsified, inoculation was carried out on days 0, 14, 28, and 42. On day 0, 50 μg / animal of the emulsified antigen was injected intraperitoneally (i.p.). On day 14, 25 μg / animal of the emulsified antigen was injected subcutaneously (s.c.) at multiple points. On days 28 and 42, depending on the lumps on the back and abdominal swelling, the antigen was injected into the back or intraperitoneally. Blood was taken on days 21 and 49, and the antibody titer in the mouse serum was determined by ELISA method. After the 4th immunization, 1 - 3 mice with high antibody titer and a titer tending to plateau in the serum (titer 1250K) were selected for splenocyte fusion. Three days before splenocyte fusion, booster immunization was carried out, and 50 μg / animal of the antigen solution prepared with normal saline was injected intraperitoneally (i.p.).

[0697] 2. Splenocyte fusion:

[0698] The optimized electrofusion method was used to fuse splenic lymphocytes with myeloma cells Sp2 / 0 cells( CRL-8287 TM ) to obtain hybridoma cells.

[0699] According to the splenocyte count results, the fused hybridoma cells were adjusted to 3×10 5 -4×10 5The cells with a density of / mL were resuspended in complete medium (IMDM medium containing 20% FBS, 1×HAT, and 1×OPI), and 150 μL / well was inoculated into a 96-well plate. After incubation at 37 °C and 5% CO2 for 4 - 5 days, the supernatant was removed, and 200 μL / well of HT complete medium (IMDM medium containing 20% FBS, 1×HT, and 1×OPI) was added. After culturing at 37 °C and 5% CO2 for 2 days, ELISA detection was performed.

[0700] 3. Hybridoma preparation:

[0701] According to the growth density of hybridoma cells, the culture supernatant of hybridoma was detected by the binding ELISA method. The cells in the wells with positive binding were amplified, cryopreserved, stored, and subcloned one to two times until single cell clones were obtained.

[0702] 4. Obtaining the variable region sequences of anti-Titin&Obscurin antibodies by ELISA screening:

[0703] The culture supernatant after hybridoma fusion was taken, and ELISA detection was performed using human Fc bispecific antibodies containing Titin chains and Obscurin chains with different spatial structures. The hybridoma cells in the positive wells were selected for expansion culture. The human Fc bispecific antibody protein containing Titin chains and Obscurin chains was diluted to a concentration of 1 μg / mL with PBS buffer at pH 7.4, and 50 μL / well was added to a 96-well ELISA plate (Corning, Cat No.CLS3590 - 100EA) and incubated overnight in a refrigerator at 4 °C. After discarding the liquid, the plate was washed 6 times with PBST (PBS at pH 7.4 containing 0.1% tween - 20); 300 μL / well of 1% BSA (SIGMA, Cat No.B2064 - 100G) blocking solution diluted with PBST was added and incubated in an incubator at 37 °C for 3 hours for blocking. After the blocking was completed, the blocking solution was discarded, and the plate was washed 6 times with PBST buffer. The hybridoma culture supernatant diluted with 1% BSA - PBST was added and incubated in an incubator at 37 °C for 2 hours. After the incubation was completed, the plate was washed 6 times with PBST, 50 μL / well of anti - mouse IgG (H + L) - HRP secondary antibody (Jackson Immunorsearch, Cat No.115 - 035 - 003) diluted with 1% BSA - PBST was added, and incubated at 37 °C for 1 hour. After washing the plate 6 times with PBST, 50 μL / well of TMB chromogenic substrate (SeraCare, Cat No.5120 - 0077) was added and incubated at room temperature for 5 - 10 minutes. Then, 50 μL / well of 1M H2SO4 was added to terminate the reaction, and the absorbance was read at a wavelength of 450 nm using a SpectraMax microplate reader (Molecular Devices).

[0704] According to the ELISA-detected positive clones, the corresponding mouse hybridoma cells were selected, and total RNA was extracted using RNAiso Plus (TaKaRa CatNo.9109). Reverse transcription was performed using the III First-Strand Synthesis SuperMix kit (Invitrogen Cat No.18080400) to obtain cDNA. Primers for library construction were designed and synthesized according to the IMGT database (Nanjing GenScript Biotechnology Co., Ltd.). The variable region fragments of the antibody heavy and light chains were obtained through two rounds of PCR reactions. Taq DNA Polymerase (Takara CatNo.R001B) was used for all PCR reactions.

[0705] >6G11-G1-L light chain variable region sequence

[0706]

[0707]

[0708] >6G11-G1-H heavy chain variable region sequence

[0709]

[0710] >7D8-F10-L light chain variable region sequence

[0711]

[0712] >7D8-F10-H heavy chain variable region sequence

[0713]

[0714] >4B11-A11-L light chain variable region sequence

[0715]

[0716] >4B11-A11-H heavy chain variable region sequence

[0717]

[0718] >2E6-G3-L4 light chain variable region sequence

[0719]

[0720] >2E6-G3-H2 heavy chain variable region sequence

[0721]

[0722] >7A3-C5-L Variable Region Sequence of Light Chain

[0723]

[0724]

[0725] >7A3-C5-H1 Variable Region Sequence of Heavy Chain

[0726]

[0727] >9G10-E11-L Variable Region Sequence of Light Chain

[0728]

[0729] >9G10-E11-H1 Variable Region Sequence of Heavy Chain

[0730]

[0731] >8B12-F7-L7 Variable Region Sequence of Light Chain

[0732]

[0733] >8B12-F7-H1 Variable Region Sequence of Heavy Chain

[0734]

[0735] >10C10-G5-L Variable Region Sequence of Light Chain

[0736]

[0737] >10C10-G5-H Variable Region Sequence of Heavy Chain

[0738]

[0739] >10G1-B8-L Variable Region Sequence of Light Chain

[0740]

[0741] >10G1-B8-H Variable Region Sequence of Heavy Chain

[0742]

[0743] >5B11-D1-L Variable Region Sequence of Light Chain

[0744]

[0745] >5B11-D1-H Variable Region Sequence of Heavy Chain

[0746]

[0747] >4C2-H7-L variable region sequence of light chain

[0748]

[0749] >4C2-H7-H variable region sequence of heavy chain

[0750]

[0751] >10A8-C2-L variable region sequence of light chain

[0752]

[0753] >10A8-C2-H variable region sequence of heavy chain

[0754]

[0755] >1H3-D9-L variable region sequence of light chain

[0756]

[0757] >1H3-D9-H variable region sequence of heavy chain

[0758]

[0759] >8F5-E10-L variable region sequence of light chain

[0760]

[0761] >8F5-E10-H variable region sequence of heavy chain

[0762]

[0763] Note: Single underline: CDR region; where CDR is numbered according to Kabat.

[0764] 5. Preparation of Anti-Titin & Obscurin Antibodies

[0765] Using the screened variable region sequences of the heavy and light chains of the anti-Titin&Obscurin antibodies (SEQ ID NO: 5 to SEQ ID NO: 32) as the antibody variable region sequences, the murine IgG2a heavy chain constant region (SEQ ID NO: 33) as the antibody heavy chain constant region sequence, and the murine κ light chain constant region (SEQ ID NO: 34) as the antibody light chain constant region sequence, the detection antibodies used in this disclosure were designed; the heavy and light chains were respectively constructed onto vectors, and the final expression vector was formed through enzymatic digestion and ligation, and the anti-Titin&Obscurin antibody was expressed; transient or stable transfection and expression were carried out in CHO cells, and the protein was purified to obtain the final protein for detection in this disclosure.

[0766] >Murine IgG2a heavy chain constant region

[0767] AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0768] SEQ ID NO: 33

[0769] >Murine κ (kappa) light chain constant region

[0770] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0771] SEQ ID NO: 34

[0772] Table 3. CDR sequences of anti-Titin&Obscurin antibodies

[0773]

[0774]

[0775]

[0776] Heavy and light chain sequences of the selected anti-Titin&Obscurin antibodies:

[0777] >CED2175 heavy chain

[0778]

[0779] >CED2175 light chain

[0780]

[0781] >CED2178 heavy chain

[0782]

[0783]

[0784] >CED2178 light chain

[0785] >CED2181 heavy chain

[0786]

[0787] >CED2181 light chain

[0788]

[0789] >CED2184 heavy chain

[0790]

[0791]

[0792] >CED2184 light chain

[0793]

[0794] >CED2187 heavy chain

[0795]

[0796] >CED2187 light chain

[0797]

[0798] >CED2190 heavy chain

[0799]

[0800]

[0801] > CED2190 light chain

[0802]

[0803] > CED2193 heavy chain

[0804]

[0805] > CED2193 light chain

[0806]

[0807] > CED2202 heavy chain

[0808]

[0809]

[0810] > CED2202 light chain

[0811]

[0812] > CED2205 heavy chain

[0813]

[0814] > CED2205 light chain

[0815]

[0816] > CED2208 heavy chain

[0817]

[0818]

[0819] > CED2208 light chain

[0820]

[0821] > CED2211 heavy chain

[0822]

[0823] > CED2211 light chain

[0824]

[0825] > CED2214 heavy chain

[0826]

[0827]

[0828] >CED2214 light chain

[0829]

[0830] >CED2217 heavy chain

[0831]

[0832] >CED2217 light chain

[0833]

[0834] >CED2220 heavy chain

[0835]

[0836]

[0837] >CED2220 light chain

[0838]

[0839] Note: The double underline is the signal peptide sequence, and the italic is the antibody constant region.

[0840] Example 5. ELISA experiment on the specific binding of anti-Titin&Obscurin antibodies to Titin and Obscurin proteins with different spatial structures

[0841] Using PBS buffer at pH 7.4, dilute the EGFR-cMET bispecific antibody or BCMA-CD3 bispecific antibody as the detection substrate to a concentration of 1 μg / mL, and add it to a 96-well enzyme-linked immunosorbent assay (ELISA) plate (Corning, Cat No. CLS3590-100EA) at a volume of 50 μL / well. Incubate it in a refrigerator at 4°C for 16 - 18 hours. After discarding the liquid, add 300 μL / well of 1% BSA (SIGMA, Cat No. B2064-100G) blocking solution diluted with PBST, and incubate it in an incubator at 37°C for 2 hours for blocking. After the blocking is completed, discard the blocking solution, and wash the plate 6 times with PBST buffer (PBS at pH 7.4 containing 0.1% tween-20). Then add the above-screened anti-Titin&Obscurin antibodies: CED2184, CED2202, CED2205, CED2208, CED2211, CED2217, CED2220, CED2190 diluted with PBST gradient to the plate, and place it in an incubator at 37°C for 1 hour. After the incubation is completed, wash the plate 6 times with PBST, add 50 μL / well of anti-mouse IgG (H+L)-HRP secondary antibody (Jackson Immunorsearch, Cat No. 115-035-003) diluted with 1% BSA-PBST, and incubate it at 37°C for 1 hour. After washing the plate 6 times with PBST, add 50 μL / well of TMB chromogenic substrate (SeraCare, Cat No. 5120-0077), incubate it at room temperature for 5 - 10 minutes, add 50 μL / well of 1M H2SO4 to terminate the reaction, and read the absorbance value at a wavelength of 450 nm using a SpectraMax microplate reader (Molecular Devices). The experimental results are as Figure 1A and Figure 1B shown.

[0842] According to the binding experimental results of different anti-Titin&Obscurin antibodies with substrates having different Titin&Obscurin spatial structures ( Figure 1A and Figure 1B ), it can be seen that the candidate antibodies have good binding effects on substrates with different Titin&Obscurin spatial structures (Titin on the heavy chain, Obscurin on the light chain or Obscurin on the heavy chain, Titin on the light chain).

[0843] Example 6. Experiment on the influence of the binding force of anti-Titin&Obscurin antibody and anti-kappa antibody on the binding of detection antibody

[0844] Use PBS buffer at pH 7.4 to dilute the EGFR-cMET bispecific antibody or BCMA-CD3 bispecific antibody as the detection substrate to a concentration of 1 μg / mL, and add it to a 96-well ELISA plate (Corning, Cat No. CLS3590-100EA) at a volume of 50 μL / well. Incubate in a 4°C refrigerator for 16 - 18 hours. After discarding the liquid, add 300 μL / well of 1% BSA (SIGMA, Cat No. B2064-100G) blocking solution diluted with PBST, and incubate in a 37°C incubator for 2 hours for blocking. After the blocking is completed, discard the blocking solution, and wash the plate 6 times with PBST buffer (pH 7.4 PBS containing 0.1% tween-20). Then, add the anti-Titin&Obscurin antibody and anti-kappa antibody (commercial antibody, from Abcam, catalog number: ab288283) diluted with PBST gradient, and place it in a 37°C incubator for 1 hour. After the incubation is completed, wash the plate 6 times with PBST, add 50 μL / well of anti-mouse IgG (H+L)-HRP secondary antibody (Jackson Immunorsearch, Cat No. 115-035-003) diluted with 1% BSA-PBST, and incubate at 37°C for 1 hour. After washing the plate 6 times with PBST, add 50 μL / well of TMB chromogenic substrate (SeraCare, Cat No. 5120-0077), incubate at room temperature for 5 - 10 minutes, add 50 μL / well of 1M H2SO4 to terminate the reaction, and read the absorbance at a wavelength of 450 nm using a SpectraMax ELISA reader (Molecular Devices). The experimental results are as Figure 2A and Figure 2B shown.

[0845] The experimental results show that when the anti-Titin&Obscurin antibody and the anti-kappa antibody act on the antibody to be detected simultaneously, they do not affect each other's binding ability to the corresponding binding sites of the antibody to be detected, that is, there is no steric hindrance effect when the two antibodies act on the antibody to be detected simultaneously.

[0846] Example 7. Detection of Antibody Pairs Labeled with Donor or Receptor

[0847] This disclosure involves a total of 3 different types of antibodies, anti-human light chain κ chain antibody, anti-human light chain λ antibody, and anti-Titin&Obscurin antibody. Among them, the anti-human light chain κ chain antibody and the anti-human light chain λ antibody are respectively labeled with receptor d2 (Anti-κ-d2, Anti-λ-d2), or donor Eu 3+ cryptate (Anti-κ-Eu, Anti-λ-Eu), or donor Tb 2+cryptate(Anti-κ-Tb, Anti-λ-Tb); The anti-Titin and anti-Obscurin antibodies are respectively labeled with the donor Eu 3+ cryptate(Anti-Titin&Obscurin-Eu), or the donor Tb 2+ cryptate(Anti-Titin&Obscurin-Tb), or the acceptor d2(Anti-Titin&Obscurin-d2). Screen the optimal donor-acceptor pairing method. The pairing scheme is as shown in Table 4 below:

[0848] Table 4. Donor-Acceptor Pairing Scheme

[0849]

[0850] Scheme: Pairing detection with different labeling methods of Anti-Titin&Obscurin / Anti-κ / λ

[0851] (1) Add the standards (EGFR-cMET bispecific antibody or BCMA-CD3 bispecific antibody) with different Titin and Obscurin spatial structures to be detected into a 384-well microplate, 10 μL / well; the initial concentration of the standard is 15 μg / mL, and it is diluted 5-fold at a 2-fold dilution; the protein concentration of the cell culture supernatant to be detected is diluted to 5 μg / mL;

[0852] (2) Mix the two detection reagents of 1×Anti-Titin&Obscurin-Eu or Tb and 1×Anti-κ-d2, 1×Anti-κ-Eu or Tb and 1×Anti-Titin&Obscurin-d2, 1×Anti-Titin&Obscurin-Eu or Tb and 1×Anti-λ-d2, 1×Anti-λ-Eu or Tb and 1×Anti-Titin&Obscurin-d2 in a 1:1 ratio;

[0853] (3) Add the mixed two reagents into the microplate containing the sample to be detected, 10 μL / well, and centrifuge and mix at 1500 rpm for 5 minutes;

[0854] (4) Let the mixed sample stand at room temperature for 2 hours or overnight;

[0855] (5) Set the fluorescence wavelength: Emission 665 nm / 620 nm, read the fluorescence value, HTRF fluorescence ratio = Em665 / Em620 / 10 4 .

[0856] The test results are as Figure 3A and Figure 3B shown.

[0857] In this disclosure, when an asymmetric multispecific antibody containing Titin chain and Obscurin chain structures is bound by antibodies labeled with a donor and a receptor simultaneously, the donor is excited to generate emission light at 620 nm, and transfers its emission light energy to the receptor, causing the receptor to emit emission light at 665 nm. When the two sides of the multispecific antibody are correctly paired, the donor will transfer energy to the receptor, thus obtaining an emission light signal at 665 nm; while when the multispecific antibody is mismatched, a half-antibody, or has a light chain dropped (kappa or lambda light chain, Obscurin chain, or Titin chain dropped), there is only emission light at 620 nm. Therefore, the higher the concentration of correctly paired antibodies in the asymmetric multispecific antibody sample, the higher the ratio of Em665 / Em620. When the sample is diluted to the same concentration, high-quality cell lines are selected according to the fluorescence ratio. The higher the ratio, the higher the concentration of correctly paired asymmetric multispecific antibodies in the sample. According to the detection results ( Figure 3A and Figure 3B ), in this disclosure, anti-Titin&Obscurin antibodies labeled with a receptor or a donor can both be paired with anti-kappa antibodies labeled with a donor or a receptor to detect asymmetric multispecific antibodies. Among them, the pairing effect of CED2208-Eu and anti-κ-d2 is the best.

[0858] Example 8. Purity Detection of PSMA-CD28 Bispecific Antibody

[0859] The PSMA-CD28 bispecific antibody is an asymmetric bispecific antibody containing a kappa light chain and Titin&Obscurin structures. The method established in this disclosure is used to detect the correct pairing efficiency of the antibody in the cell culture supernatant expressing the bispecific antibody.

[0860] (1) Dilute the cell culture supernatant to be detected to 5 μg / mL and add it to a 384-well detection plate, 10 μL / well; dilute the PSMA-CD28 bispecific antibody standard to 15 μg / mL, and then perform serial two-fold dilutions for a total of 6 points and add them to a 384-well detection plate, 10 μL / well; add the CED2208-Eu and Anti-κ-d2 antibodies diluted to 1×, 5 μL / well respectively to the above samples; centrifuge and mix at 1500 rpm for 5 minutes; incubate the mixed samples at room temperature for 2 hours or overnight at 4°C.

[0861] (2) Set the fluorescence wavelengths: Emission 665 nm / 620 nm, read the fluorescence values, and calculate the fluorescence ratio according to Em665 / Em620 / 10 4 to calculate the fluorescence ratio.

[0862] (3) According to Em665 / Em620 / 10 4Plot a standard curve using the values and the concentrations of the standards. The standard curve is as shown in Figure 4A as follows.

[0863] From the HTRF fluorescence ratios, it can be seen that the quality of different cell culture supernatants detected by the method disclosed herein is basically consistent with the trend of the corresponding sample mass spectrometry detection results (Table 5 and Figure 4B ), indicating the feasibility of the method disclosed herein.

[0864] Table 5. Fluorescence ratios of correctly composed components of PSMA-CD28 samples detected by the HTRF method and the proportion of correctly composed components detected by mass spectrometry

[0865]

[0866] Example 9. Purity detection of PSMA-CD3 bispecific antibody

[0867] The PSMA-CD3 bispecific antibody is an asymmetric bispecific antibody containing a κ light chain and an Obscurin & Titin structure. Use the method established in this disclosure to detect the correct pairing efficiency of the antibody in the cell culture supernatant expressing the bispecific antibody.

[0868] (1) Dilute the cell culture supernatant to be detected to 5 μg / mL and add it to a 384-well detection plate, 10 μL / well; dilute the PSMA-CD3 bispecific antibody standard to 15 μg / mL, and then perform a two-fold serial dilution for a total of 6 points, and add it to a 384-well detection plate, 10 μL / well; add 5 μL / well of the CED2208-Eu and Anti-κ-d2 antibodies diluted to 1× to the above samples respectively; centrifuge and mix at 1500 rpm for 5 minutes; incubate the mixed samples at room temperature for 2 hours or overnight at 4°C.

[0869] (2) Set the fluorescence wavelength: Emission 665 nm / 620 nm, read the fluorescence value, and calculate the fluorescence ratio according to Em665 / Em620 / 10 4 .

[0870] (3) Plot a standard curve according to the Em665 / Em620 / 10 4 value and the concentration of the standard. The standard curve is as shown in Figure 5A as follows.

[0871] From the HTRF fluorescence ratios, it can be seen that the quality of different cell culture supernatants detected by the method disclosed herein is basically consistent with the detection results of the corresponding sample mass spectrometry (Table 6 and Figure 5B ), indicating the feasibility of the method disclosed herein.

[0872] Table 6. Fluorescence ratios of correctly composed components of PSMA-CD3 samples detected by the HTRF method and the proportion of correctly composed components detected by mass spectrometry

[0873]

[0874]

[0875] Example 10. Purity Detection of GPC3-γδT Bispecific Antibody

[0876] The GPC3-γδT bispecific antibody is an asymmetric bispecific antibody containing a κ light chain and an Obscurin&Titin structure. The method established in this disclosure is used to detect the correct pairing efficiency of the antibody in the cell culture supernatant expressing the bispecific antibody.

[0877] (1) Dilute the cell culture supernatant to be detected to 5 μg / mL and add it to a 384-well detection plate, 10 μL per well; dilute the GPC3-γδT bispecific antibody standard to 10 μg / mL, then perform a two-fold serial dilution for a total of 6 points and add it to a 384-well detection plate, 10 μL per well; add 5 μL per well of the CED2208-Eu and Anti-κ-d2 antibodies diluted to 1× to the above samples respectively; centrifuge and mix at 1500 rpm for 5 minutes; incubate the mixed samples at room temperature for 2 hours or overnight at 4°C.

[0878] (2) Set the fluorescence wavelength: Emission 665 nm / 620 nm, read the fluorescence value, and calculate the fluorescence ratio according to Em665 / Em620 / 10 4 Calculate the fluorescence ratio.

[0879] (3) Draw a standard curve based on the Em665 / Em620 / 10 4 value and the concentration of the standard. The standard curve is as shown in Figure 6A shown.

[0880] From the HTRF fluorescence ratio, it can be seen that the quality of different cell culture supernatants detected by the method of this disclosure is basically consistent with the detection results of the corresponding sample mass spectrometry (Table 7 and Figure 6B ), indicating the feasibility of the method of this disclosure.

[0881] Table 7. Fluorescence Ratio of Correct Components Detected by HTRF Method for GPC3-γδT Samples and Proportion of Correct Components Detected by Mass Spectrometry

[0882]

[0883]

[0884]

[0885]

[0886] Example 11. Purity Detection of MUC1-EGFR Bispecific Antibody

[0887] MUC1-EGFR is an asymmetric bispecific antibody containing κ light chain and Titin&Obscurin structure. The method established in this disclosure is used to detect the correct pairing efficiency of the antibody in the cell culture supernatant expressing the bispecific antibody.

[0888] (1) Dilute the cell culture supernatant to be detected to 5 μg / mL and add it to a 384-well detection plate, 10 μL / well; dilute the MUC1-EGFR standard to 10 μg / mL, then perform a two-fold serial dilution for a total of 5 points and add it to a 384-well detection plate, 10 μL / well; add 5 μL / well of CED2208-Eu and Anti-κ-d2 antibodies diluted to 1× to the above samples respectively; centrifuge and mix at 1500 rpm for 5 minutes; incubate the mixed samples at room temperature for 2 hours or overnight at 4°C.

[0889] (2) Set the fluorescence wavelength: Emission 665 nm / 620 nm, read the fluorescence value, and calculate the fluorescence ratio according to Em665 / Em620 / 10 4 Calculate the fluorescence ratio.

[0890] (3) Draw a standard curve based on the Em665 / Em620 / 10 4 value and the concentration of the standard product. The standard curve is as Figure 7A shown.

[0891] From the HTRF fluorescence ratio, it can be seen that the quality of different cell culture supernatants detected by the method of this disclosure is basically consistent with the detection results of the corresponding sample mass spectrometry (Table 8 and Figure 7B ), indicating the feasibility of the method of this disclosure.

[0892] Table 8. Fluorescence Ratio of Correct Components Detected by HTRF Method for MUC1-EGFR Samples and Proportion of Correct Components Detected by Mass Spectrometry

[0893]

[0894]

[0895]

[0896] Example 12. Verification of the Correlation between HTRF Fluorescence Ratio and SEC

[0897] According to the comparison between the HTRF fluorescence ratio and the correct components detected by mass spectrometry, the high and low trends of the two are basically the same, verifying the applicability of the HTRF method in screening the quality of asymmetric multispecific antibodies containing Titin chain and Obscurin chain structures; however, it was also found during the detection that for some samples with relatively high mass spectrometry results, the corresponding HTRF fluorescence ratio was relatively low. It is speculated that this may be related to the aggregate components in the asymmetric antibody. The HTRF method can distinguish aggregates during detection, while mass spectrometry cannot distinguish aggregate structures. The consistency analysis was performed on the HTRF method detection results and SEC purity of some samples of the GPC3-γδT bispecific antibody and the MUC1-EGFR bispecific antibody. The results are shown in Table 9 and Table 10, Figure 8A and Figure 8B as shown below.

[0898] Table 9. Fluorescence ratio of the correct components detected by the HTRF method for the GPC3-γδT bispecific antibody samples and the proportion of the correct components detected by SEC

[0899]

[0900]

[0901]

[0902] Table 10. Fluorescence ratio of the correct components detected by the HTRF method for the MUC1-EGFR samples and the proportion of the correct components detected by SEC

[0903] MP Number HTRF Fluorescence Ratio SEC Main Peak (%) MUC1-EGFR-ADC-MP1 9.158 89.5 MUC1-EGFR-ADC-MP2 9.708 87.4 MUC1-EGFR-ADC-MP3 8.947 87.1 MUC1-EGFR-ADC-MP4 9.491 86 MUC1-EGFR-ADC-MP5 4.942 85.4 MUC1-EGFR-ADC-MP6 10.739 81.2 MUC1-EGFR-ADC-MP7 10.107 79.4 MUC1-EGFR-ADC-MP8 8.776 77.1 MUC1-EGFR-ADC-MP9 4.359 73.9 MUC1-EGFR-ADC-MP10 9.668 72.9 MUC1-EGFR-ADC-MP13 2.727 51.4

[0904] The results show that the HTRF fluorescence ratio is positively correlated with the SEC purity of the antibody to be detected, verifying the phenomenon that the consistency between some HTRF fluorescence ratios and mass spectrometry detection results is poor. It shows that the HTRF detection method disclosed in this disclosure is more accurate than mass spectrometry detection.

[0905] Although the above invention has been described in detail with the aid of drawings and examples for the sake of clear understanding, the description and examples should not be construed as limiting the scope of this disclosure. The disclosures of all patents and scientific literatures cited herein are clearly incorporated by reference in their entirety.

Claims

1. A method for screening cell lines for asymmetric multispecific antibodies, the method comprising the following steps: Step 1. Obtaining anti-Titin & Obscurin antibodies combined with asymmetric multispecific antibodies; Step 2. Development of detection method: The anti-Titin & Obscurin antibody and the anti-human light chain λ or κ antibody are respectively labeled with the donor and the acceptor, and paired one by one for detection. The pairing scheme is as follows: (1) Anti-Titin & Obscurin-Eu or Anti-Titin & Obscurin-Tb, and Anti-κ-d2 or Anti-κ-XL665, or (2) Anti-κ-Eu or Anti-κ-Tb, and Anti-Titin & Obscurin-d2 or Anti-Titin & Obscurin-XL665, or (3) Anti-Titin & Obscurin-Eu or Anti-Titin & Obscurin-Tb, and Anti-λ-d2 or Anti-λ-XL665, or (4) Anti-λ-Eu or Anti-λ-Tb, and Anti-Titin & Obscurin-d2 or Anti-Titin & Obscurin-XL665; The donor is Eu 3+ cryptate or Tb 2+ Cryptate, the receptor is d2 or XL665; Step 3. Use an ELISA reader to detect the energy transfer signal. The higher the energy transfer signal, the higher the content of correctly paired asymmetric multispecific antibodies in the sample, and the corresponding cell line is a high-quality cell line.

2. The method according to claim 1, wherein the step 1 comprises the operation of immunizing mice with titin and / or obscurin proteins, and obtaining anti-titin & obscurin antibodies combined with asymmetric multispecific antibodies by hybridoma; preferably, the titin protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 168 to SEQ ID NO: 186, and the obscurin protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 187 to SEQ ID NO:

227.

3. The method according to claim 1 or 2, wherein the sample is cell supernatant or an analyte.

4. The method according to any one of claims 1 to 3, wherein the pairing scheme of step 2 comprises: (1) Anti-Titin & Obscurin-Eu or Anti-Titin & Obscurin-Tb, and Anti-κ-d2, or (2) Anti-κ-Eu or Anti-κ-Tb, and Anti-Titin & Obscurin-d2, or (3) Anti-Titin & Obscurin-Eu or Anti-Titin & Obscurin-Tb, and Anti-λ-d2, or (4) Anti-λ-Eu or Anti-λ-Tb and Anti-Titin & Obscurin-d2; Preferably, The pairing scheme of step 2 includes: (1) Anti-Titin & Obscurin-Eu and Anti-κ-d2, or (2) Anti-Titin & Obscurin-Tb and Anti-κ-d2, or (3) Anti-κ-Eu and Anti-Titin & Obscurin-d2, or (4) Anti-κ-Tb and Anti-Titin&Obscurin-d2; More preferably, the pairing scheme of step 2 comprises: Anti-Titin & Obscurin-Eu and Anti-κ-d2. The method according to any one of claims 1 to 4, wherein the anti-human light chain λ or κ antibody is a commercial anti-human light chain λ or κ antibody.

6. The method according to claim 5, wherein step 2 further comprises the operation of mixing and incubating the anti-Titin & Obscurin antibody and the commercial anti-human light chain λ or κ antibody with the cell supernatant or the test substance.

7. The method according to claim 6, wherein the incubation time is 2 hours or more; preferably, the incubation time is 2 hours to 12 hours.

8. The method according to claim 7, wherein the incubation is incubation at room temperature for 2 hours or incubation at 4°C overnight; preferably, the incubation is incubation at room temperature for 2 hours or incubation at 4°C overnight for 12 hours.

9. The method according to claim 1, wherein the step 2 further comprises an operation using the target multispecific antibody as a standard.

10. The method according to claim 1, wherein the detection method in step 2 is time-resolved fluorescence resonance energy transfer technology; preferably homogeneous time-resolved fluorescence technology.

11. The method according to claim 1, wherein the step 3 further comprises the operation of drawing an energy transfer signal-antibody concentration standard curve according to the detection results.

12. The method according to claim 1, wherein the energy transfer signal is determined by a fluorescence ratio, wherein the fluorescence ratio is determined according to Em665 / Em620 / 10 4 calculate.

13. The method according to any one of claims 1 to 12, wherein the asymmetric multispecific antibody is an asymmetric multispecific antibody having a titin chain and an obscurin chain; preferably, wherein the multispecific antibody has at least one replaced Fab, which comprises a titin chain and an obscurin chain capable of forming a dimer; more preferably, wherein the replaced Fab is a Fab in which the original CH1 and CL are replaced by the obscurin chain and the titin chain, respectively, or the original CH1 and CL are replaced by the titin chain and the obscurin chain, respectively.

14. The method of claim 13, wherein the asymmetric multispecific antibody further comprises a kappa or lambda light chain; preferably, the asymmetric multispecific antibody further comprises a kappa light chain.

15. The method of claim 13 or 14, wherein the asymmetric multispecific antibody is a bispecific antibody.

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