WO2021071319A1

WO2021071319A1 - Multispecific fusion protein and use thereof

Info

Publication number: WO2021071319A1
Application number: PCT/KR2020/013805
Authority: WO
Inventors: 박범찬; 송재호; 김나영; 장세일; 백정현; 목진걸; 송영자; 김혜난; 이재민; 장창호; 김영철; 이은진; 백기선; 이현미; 양소영; 유재님; 윤재봉; 박영우
Original assignee: 주식회사 와이바이오로직스
Priority date: 2019-10-10
Filing date: 2020-10-08
Publication date: 2021-04-15
Also published as: KR20210043475A; TW202128758A; MX2022004218A

Abstract

The present invention provides a multispecific fusion protein that does not contain an Fc region and can specifically bind to two or more antigens. In one embodiment, a multispecific fusion protein comprising the following structural formulas (I) and (II) is provided: N'-A-L1-X-C' (I); and N'-B-L1-Y-C' (II). The fusion protein may bind to cancer cells and immune cells, and may enhance the activity of immune cells. Therefore, the fusion protein can be used as an anticancer agent.

Description

Multispecific fusion proteins and uses thereof

The present invention relates to a multispecific fusion protein that binds to cells, a method for preparing a multispecific fusion protein, a composition comprising a multispecific fusion protein, and a use thereof.

Antibodies and developed materials based on antibodies are used in the treatment of various diseases and disorders today. Currently, at least 70 antibodies have been approved in the United States and the European Union, with a large number of new molecules in the preclinical and clinical trial stages. However, there is an ongoing search for new, better and safer therapeutics within the research and clinical community.

Usually, antibodies specifically bind to one epitope or one antigen. On the other hand, multispecific antibodies have the ability to bind two or more antigens or two or more epitopes (Korean Patent Publication 10-2016-0035065). However, the development of multispecific antibodies is difficult due to many technical limitations, and there are few multispecific antibodies approved as therapeutic agents. Therefore, there is still a need for better multispecific antibodies and methods for efficiently producing stable multispecific antibodies having multiple functions.

Accordingly, the present inventors studied to develop a new structure of a multispecific antibody that enhances immune activity, as a result of the heterodimer form in which the Fc region of the antibody is substituted with the antibody variable region consisting of the heavy and light chain variable regions of the antibody. The multispecific fusion protein of was developed. In addition, the present invention was completed by confirming that the fusion protein is effective as an anticancer agent.

To achieve the above object, one aspect of the present invention provides a multispecific fusion protein comprising the following structural formulas (I) and (II):

N'-A-L1-X-C' (I); And N'-B-L1-Y-C' (II)

In this case, in the structural formulas (I) and (II), the N'is the N-terminus of the fusion protein, the C'is the C-terminus of the fusion protein, and A and B are each specific for a first antigen. And L1 is a peptide linker containing at least one Cys, X is an antibody variable heavy chain (VH) region or an antibody variable light chain (VL) region, and Y is an antibody variable light chain (VL) region or an antibody variable heavy chain ( VH) region, wherein X and Y form an antibody variable region that specifically binds to a second antigen.

Another aspect of the present invention provides a pharmaceutical composition for treating or preventing cancer comprising the fusion protein as an active ingredient.

The multispecific fusion protein according to the present invention can specifically bind to two or more antigens. In addition, a multispecific fusion protein constructed to include an antibody variable region that specifically binds to immune cells and an antibody variable region that specifically binds to a cancer antigen can be used as an anticancer agent.

1 is a diagram showing the "Knob-in-Hole" structure of the variable domains of anti-CD3 UCHT1, anti-PD-L1 dervalumab, and anti-CTLA-4 ipilimumab. Here, the CDR loops are denoted as "VH CDR3", "VL CDR1", "VL CDR2", and "VL CDR3".

2 is an enlarged view showing the interaction between the variable domain fragments of UCHT1. Specifically, the structure related to the specific VH-VL interaction is shown on the right, and the variable formed by electrostatic interaction (top), hydrophobicity and knob-into-hole interaction (bottom). Domain fragments are shown.

Figure 3a is a schematic diagram of the structure of the ALiCE.

3b, 3c, 3d, 3e, and 3f are schematic structures of ACE-05, ACE-31, BiTE-05, YBL-007 and UCHT1 representing variable domains for PD-L1 and CD3 binding. .

Figure 3g is a schematic diagram of the structures of ACE-11 and ACE-19.

Figure 3h is a schematic diagram of the structure of ACE-28.

4 is an anti-PD-L1 antibody (YBL-007) having an activity similar to that of avelumab in PD-L1/PD-1 signal blocking ability.

Figure 5a shows whether the expression of ACE-HC-VH, ACE-HC-VL and ACE-LC was confirmed by SDS-PAGE and Western blotting.

Figure 5b is a view confirming ACE-18 by SDS-PAGE under reducing and non-reducing conditions.

5C and 5D are diagrams confirming ACE-11 and ACE-19 by SDS-PAGE under reducing and non-reducing conditions.

5E is a view confirming ACE-11 and ACE-19 by capillary electrophoresis (CE) under reducing and non-reducing conditions.

5F shows the results of analysis of ACE-11 and ACE-19 by SEC HPLC.

5G is a result of analyzing ACE-19 by SEC-HPLC.

5h shows the ACE-19 fraction confirmed by Native-PAGE.

5i is a view confirming the ACE-19 fraction obtained in FIG. 5g by CE under reducing and non-reducing conditions.

6 is a schematic diagram of a multispecific fusion protein capable of specifically binding to PD-L1 and CD3, ACE-LC, ACE-HC-VH and ACE-HC-VL through capillary electrophoresis analysis and size-exclusion chromatography. I confirmed it.

Figure 7a is through capillary electrophoresis analysis, confirming the ACE-LC, ACE-HC-VH and ACE-HC-VL of ACE-05. As a result of the analysis, it was confirmed that ACE-LC, ACE-HC-VH, and ACE-HC-VL were present in a 2:1:1 ratio.

7B is a result of analyzing ACE-10 and ACE-18 by SEC-HPLC.

7c is a result of confirming ACE-11 and ACE-19 with CE under reducing and non-reducing conditions.

7D is a result of confirming ACE-11 with CE in reducing and non-reducing conditions.

7E is a result of confirming ACE-19 with CE in reducing and non-reducing conditions.

7F is a result of analyzing ACE-19 by SEC-HPLC.

7G is a result of analysis by SEC-HPL after dividing ACE-19 into complex and ACE-19 using SEC-FPLC and purifying.

Figure 7h is a view confirming the presence and absence of SDS, ACE-19 and complex according to the heating conditions in Native-PAGE.

Figure 8 is the result of mass spectrometry (MS) using size-exclusion chromatography and LC-ESI/TOF. As a result, it was confirmed that ACE-05 is a homogeneous heterotetramer.

9 is a schematic diagram of ACE-00, capillary electrophoresis analysis, and size-exclusion chromatography to confirm ACE-LC, ACE-HC-VH and ACE-HC-VL.

10 is an analysis of ACE-05 using cation exchange chromatography (CEX) under different conditions.

Figure 11a confirms the thermal stability of ACE-05.

Figure 11b confirms the thermal stability of ACE-11 and ACE-19.

12 shows the stability of ACE-05 through CEX-HPLC analysis.

13 is a schematic diagram of the immunological synaptic bridge of ACE-05.

Figure 14a confirms the binding strength of ACE-05 and ACE-31.

Figure 14b confirms the binding force of ACE-19.

Figure 14c confirms the binding strength of ACE-11 and ACE-19.

Figure 15a confirms the binding strength of ACE-05 and ACE-31.

Figure 15b confirms the binding ability of ACE-19 to EGFR.

Figure 15c confirms the binding ability of ACE-19 to CD3.

Figure 16a confirms the simultaneous binding of ACE-05 to PD-L1 and CD3.

16B and 16C confirm the simultaneous binding of ACE-19 to EGFR and CD3.

Figure 17 shows the binding power of ACE-05 and ACE-31 to Jrukat T cells.

18A shows the binding power of ACE-05 and ACE-31 to Karpas-299 cells, Jrukat T cells, and Raji cells.

Figure 18b shows the binding power of ACE-18 to Raji cells.

Figure 18c shows the binding ability of ACE-18 to CD20 ⁺ Raji cells or CD20R ^- aji cells.

18D shows the amount of CD20 expression in cells by using the cell avidity of the anti-CD20 antibody in each cell.

Fig. 18e shows the ability of ACE-18 to kill Karpass-299 cells.

Figure 18f confirms the killing ability of ACE-18 on Raji cells.

Fig. 18g is a confirmation of the killing ability of ACE-18 on Toledo cells and Jeko-1 cells.

18h is a view confirming the expression level of EGFR present in the tumor through flow cytometry and the killing ability of ACE-19 on SW48 cells, HCT116 cells, and HT29 cells.

19 shows HEK cells into which the PD-L1 expression vector has been introduced.

20 shows NFAT-luciferase reporter activity was measured to evaluate the activation of Jurkat T cells of ACE-05 and ACE-31.

FIG. 21 shows the NFAT-luciferase reporter activity was measured, and the activities of ACE-05 and ACE-31 were compared with other substances.

Figure 22 is a comparison of the cytolytic ability of ACE-05, ACE-31 and BiTE-05 against PD-L1 ⁺ tumor cells (HCC827).

23 is a comparison of the cell lysis ability of ACE-05, ACE-31 and BiTE-05 against PD-L1 ⁺ tumor cells (MDA-MB-231).

24 is a flow cytometer confirming the expression of PD-L1 in HCC827 and MDA-MB-231.

Figure 25a is a measure of the anti-cancer activity of ACE-05.

Figure 25b is a measure of the killing ability against tumor cells after simultaneous administration of ACE-11 and PBMC.

Figure 26a confirms whether ACE-05 has the ability to specifically activate T cells.

Figure 26b confirms whether or not ACE-18 has the ability to specifically activate T cells.

Figure 27 confirms whether ACE-05, ACE-31 and BiTE-05 have the ability to activate T cells.

Fig. 28 shows the effect of ACE-05, ACE-31 and BiTE-05 on the release of Granzyme B in ^{CD8 + T cells.}

29 shows whether ACE-05 has the ability to specifically activate T cells.

FIG. 30 confirms whether ACE-05, ACE-31, and BiTE-05 have the ability to specifically activate T cells.

31 shows the effect of ACE-05, ACE-31 and BiTE-05 on T cell aggregation and activity.

Fig. 32 shows the effects of ACE-05, ACE-31 and BiTE-05 on T cell stimulation.

33 shows cytokines secreted from PBMCs after treatment with ACE-05, ACE-31 and BiTE-05.

Figure 34 shows the binding affinity of ACE-05 and its variants, ACE-47 (K55Q variant of ACE-05), ACE-49 (D104N variant of ACE-05), and ACE-56 (K55Q, D104N variant of ACE-05). Is measured.

Figure 35 confirms the non-target (Off Target) Jurkat T cell activation of ACE-05 and its variants.

Fig. 36 shows the ability of ACE-05 and its variants to kill PD-L1 HCC827 cancer cells.

Figure 37 shows that ACE-05, ACE-31, and BiTE-05 confirm the ability to secrete non-target cytokines in ^{CD4 +} and CD8 ^{+ T cells.}

38 is an analysis of BiTE-05 by SEC-HPLC.

39 shows the degree of activation of non-target T cells of the fusion protein according to the present example when cancer cells are not present.

40 shows whether the fusion protein according to the present embodiment non-specifically kills cancer cells.

41 shows the cancer cell killing effects of ACE-05, YBL-007 and BiTE-05 in mice.

Figure 42 shows the change in body weight of mice after administration of ACE-05, YBL-007 and BiTE-05.

43 is an analysis of the pharmacokinetics of ACE-05 and BiTE-05.

44 shows the anticancer effect according to the concentration of ACE-05.

45 shows cytokine secretion by ACE-05 in mice that do not have cancer.

46 shows the change in body weight by ACE-05 in mice that do not have cancer.

47 shows the anticancer effect of ACE-05.

48 shows the change in body weight of mice after administration of ACE-05, YBL-007 and UCHT1.

49 shows changes in tumor size after administration of ACE-05, YBL-007 and UCHT1.

^{50 shows CD45 +} lymphocytes present in tumors obtained after administration of ACE-05, YBL-007 and UCHT1.

^{FIG. 51 shows the confirmation of CD45 +} lymphocyte count and CD3 ⁺ T cell ratio after administration of ACE-05, YBL-007 and UCHT1.

52 shows the percentage of ^{CD4 +} and CD8 ⁺ T cells after administration of ACE-05, YBL-007 and UCHT1.

53 shows the percentage of ^{CD4 +} and CD8 ⁺ T cells present in the tumor through flow cytometry after administration of ACE-05, YBL-007 and UCHT1.

54 shows the amount of expression of EGFR and PD-L1 present in the tumor through flow cytometry.

55 shows the effect of ACE-19 on SW48, HT29 and HCT116 cells.

56 and 57 show ACE-05-HC-VH and ACE-05 that can be presented on MHC class I molecules to confirm the immunogenicity of ACE-05-HC-VH and ACE-05-HC-VL. This is an analysis of the peptide in -HC-VL.

58 and 59 are views confirming ACE-02 and ACE-03 by SDS-PAGE under reducing and non-reducing conditions.

FIG. 60 is a diagram showing the results of confirming ACE-05 and ACE-16 by SDS-PAGE (left) and CE (right) under reducing and non-reducing conditions.

61 to 63 are views confirming ACE-06, ACE-10, and ACE-11 by SDS-PAGE under reducing and non-reducing conditions.

Figure 64 is a view confirmed by Western blot (top and middle panels) and SDS-PAGE (bottom panels) of ACE-15 under reducing and non-reducing conditions. Here, Lane 1 is the total supernatant of the ACE-05 culture, Lane 2 is the total supernatant of the ACE-15 culture, and Lane 3 is loaded with the purified protein of ACE-05. In addition, the upper panel shows an anti-CH1 antibody, and the middle panel shows a Western blot image using an anti-Kappa antibody.

65 and 66 are diagrams confirming ACD-20 and ACE21 by SDS-PAGE and Western blot under reducing and non-reducing conditions.

67 is a view confirming ACE-23 by SDS-PAGE under reducing and non-reducing conditions.

FIG. 68 is a diagram showing the results of analyzing ACE-25 by SDS-PAGE and CE under reducing and non-reducing conditions and by SEC-HPLC.

69 is a diagram showing ACE-VH-LC, ACE-VL-LC and ACE-VH-VL-LC of ACE-26 confirmed by SDS-PAGE and Western blot under reducing and non-reducing conditions.

FIG. 70 is a diagram showing the results of analyzing ACE-26 by SDS-PAGE and CE under reducing and non-reducing conditions.

71 is a diagram showing the results of analysis of ACE-28 by SDS-PAGE, Western blot and CE under reducing and non-reducing conditions.

72 is a view showing the results of SDS-PAGE and Western blot analysis of ACE-28 containing a CH3 region under reduced and non-reducing conditions.

73 is a diagram showing the results of CE analysis of ACE-28 containing a CH3 region under reduced and non-reducing conditions.

74 and 75 are diagrams showing the results of analysis of ACE-30 and ACE-32 by SDS-PAGE, Western blot, and CE under reducing and non-reducing conditions.

76 is a diagram showing the results of analysis of ACE-33 by SDS-PAGE and CE under reducing and non-reducing conditions.

용어의 정의Definition of Terms

As used herein, the term "multispecific fusion protein" refers to a substance that binds to at least one target or antigen. Specifically, the fusion protein may specifically bind or selectively bind to an antigen, for example, when the dissociation constant (KD) is ≤ 10 ^{-7 M.} In one embodiment, the fusion protein may specifically bind to an antigen with high affinity when the ^{KD is ≤ 10 -8} M or the KD is ≤ 10 ^{-9 M.} The multispecific fusion protein may include an antibody and a molecule derived from the antibody.

In one embodiment, the multispecific fusion protein or antigen binding domain thereof may be a "humanized" form of a non-human antibody that is a chimeric antibody comprising a human immunoglobulin comprising a native CDR. In addition, a multispecific fusion protein or antigen binding domain may comprise a portion of a “fully human antibody” or “human antibody”. Also, in one embodiment, the multispecific fusion protein or antigen binding domain may be a “monoclonal antibody” or a portion thereof.

As used herein, the term "antibody" refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction. In addition, antibodies are also referred to as immunoglobulins. The antibody may mean any one selected from IgG, IgE, IgM, IgD and IgA, and may be IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2, which are subclasses of IgG. Further, the antibody may be an agonistic antibody or an antagonistic antibody.

As used herein, the term “Fab” or “Fab region” refers to an antibody region that binds to an antigen. Conventional IgG generally contains two Fab regions. Each Fab region typically consists of one variable region and one constant region of each heavy and light chain. Specifically, in the Fab region, the variable and constant regions of the heavy chain are VH and CH1 regions, and in the Fab region, the variable and constant regions of the light chain are VL and CL regions. The VH, CH1, VL and CL of the Fab region can be arranged in a variety of ways to confer antigen binding capacity according to the present disclosure.

As used herein, the term “heavy chain” refers to a polypeptide chain of about 50-70 kDa. Here, the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and the carboxy terminal portion includes a constant region. The constant region may be one of five types: alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ). Here, α, δ, and γ include about 450 amino acids, and μ and ε include about 550 amino acids.

As used herein, the term “light chain” refers to a polypeptide chain of about 25 kDa. Wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids and the carboxy-terminal portion includes a constant region. There are two types of light chain constant domains: kappa (κ) or lambda (λ). In addition, the constant region of the light chain is referred to as "CL". The heavy chain C domain (CH domain) is numbered from amino-terminus to carboxy-terminus (eg, CH1, CH2, CH3, etc.). Any of the CL and CH1 regions of these antibody classes can be used in the present disclosure. In certain embodiments, the CL and CH1 regions provided herein are of the IgG type (eg, IgG1). A representative CL region of the Fab region provided herein has the following amino acid sequence: TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 59). The representative CH1 region of the Fab region provided herein has the following amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 60).

As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain including a native Fc region, a recombinant Fc region and a variant Fc region. In one embodiment, the variant Fc region may have at least one amino acid substitution, for example about 1 to about 10 amino acid substitutions, or about 1 to about 5 amino acids substitutions compared to the native sequence Fc region. . Also. The variant Fc region may have at least about 80% homology, at least about 90% homology, at least about 95% homology with the native sequence Fc region.

As used herein, the term "antigen" is a structure capable of selectively binding to an antibody. The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring compound or a synthetic compound. Specifically, the antigen is a polypeptide, and may be a protein present on or within a cell.

As used herein, the term “vector” refers to a material for carrying or expressing a nucleic acid sequence comprising a nucleic acid sequence encoding a multispecific fusion protein (eg, an antibody) described herein. Specifically, vectors include expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes.

As used herein, the term “polynucleotide” is also referred to as “nucleic acid” and refers to a polymer of nucleotides of any length. Specifically, the polynucleotide may be DNA or RNA.

다중 특이적 융합 단백질Multispecific fusion proteins

One aspect of the present invention provides a multispecific fusion protein in which the Fc region of an antibody is substituted with a variable region of an antibody.

The present invention provides a novel multispecific fusion protein having multiple binding domains that bind to cells. These fusion proteins may be referred to herein as "antibody like cell engagers (ALICE)". The ALiCE molecule provided in the present invention has two antigen binding domains. The first antigen binding domain has two Fab regions and the second antigen binding domain has an Fv region. Examples of ALiCE molecules are shown in FIGS. 3A, 3B, 3C, 3G, and 3H.

The Fv and Fab regions can be linked through the hinge region of the heavy chain. Generally, in such molecules, the first antigen binding domain comprises a Fab region, and the second antigen binding domain is usually at a position where the CH2 and CH3 domains will be located on the native antibody structure. Attached (directly or indirectly). For example, in one embodiment, the C-terminus of the first heavy chain comprises a VH domain other than the CH2 domain, and the C-terminus of the second heavy chain comprises a VL domain other than the CH2 domain.

The multispecific fusion proteins disclosed herein provide many advantages over conventional antibodies and conventional multispecific antibodies (eg, bispecific antibodies). Due to the multiple antigen binding domains and overall configuration design, the multispecific fusion proteins provided in the present invention can be used as cell linkers providing multiple cells. For example, a first antigen binding domain can bind an antigen expressed on a first cell, and a second antigen binding domain can bind an antigen expressed on a second cell. Therefore, when the multispecific fusion protein is used, the two cells can be effectively linked.

The multispecific fusion proteins provided herein may be useful for binding and activating immune cells. For example, in one embodiment, the divalent Fab portion of the ALiCE molecule retains the function of a conventional antibody, while the second Fc-deficient monovalent antigen-binding region (i.e., the Fv region) is an effector cell of the immune system, For example, T cells can be recognized, bound, redirected and/or activated.

In another embodiment, the Fv portion of the ALiCE molecule retains the function of a conventional antibody, and the Fab region is capable of recognizing, binding, redirecting and/or activating effector cells, such as T cells, of the immune system. For example, ACE-05 and ACE-31 are both ALiCE molecules composed of anti-PD-L1 and anti-CD3 domains, but ACE-05 binds to PD-L1 through the Fab region and to CD3 through the Fv region. Combine. On the other hand, ACE-31 binds to CD3 through the Fab region and to PD-L1 through the Fv region.

In one embodiment, the absence of a properly functioning Fc region or the absence of a complete CH2 and/or CH3 region eliminates or reduces Fc-mediated effector cytotoxicity. In one embodiment, the natural interaction between the VH and VL chains of the Fv moiety can promote heterodimerization of ALiCE molecules without conferring undesirable immunogenicity through artificial manipulation.

One embodiment of the multispecific fusion protein comprises two antigen binding domains, wherein the first antigen binding domain comprises two antibody Fab regions and the second antigen binding domain comprises an antibody Fv region. I can. At this time, each of the two Fab regions includes two portions. The first portion comprises an antibody variable heavy chain (VH) region and an antibody CH1 region. In addition, the second portion comprises an antibody variable light chain (VL) region and an antibody light chain constant region (CL). Each of the two Fab regions is capable of binding to an antigen. The Fv region of the second antigen binding domain comprises a VH region and a VL region.

In addition, an embodiment of the multispecific fusion protein may include the following four polypeptides:

(a) a first polypeptide and a second polypeptide each comprising an antibody light chain,

(b) a third polypeptide comprising a first VH region, a first CH1 region, and a second VH region, and

(c) a fourth polypeptide comprising a third VH region, a second CH1 region and a VL region.

At this time, the first VH region and the first CH1 region of the first polypeptide and the third polypeptide form a first antigen-binding Fab region, and the third VH region and the second CH1 region of the second and fourth polypeptides are second. The antigen binding Fab region is formed, and the second VH region of the third polypeptide and the VL region of the fourth polypeptide form the antigen binding Fv region.

It was confirmed that the C-terminal Fv also plays an important role in the heterodimerization of two different heavy chain-like chains (third and fourth polypeptides). Because the interaction between the VH and VL regions is much stronger than that of the VL-VL interaction, VH-VL binding occurs. Thus, it can be heterodimerized to produce the multispecific fusion protein of the present application. The efficiency of heterodimerization was very high, and most of the multispecific fusion proteins expressed and purified in mammalian cells are in heterodimerized form. It was confirmed that the heterodimerization efficiency was more than 99%.

In addition, this structure provides an optimal synaptic distance between the target cell and the effector cell. The distance between the two N-terminal Fab regions and the C-terminal Fv region was estimated to be 40 Å. Moreover, the multispecific fusion proteins provided herein have more folding complexity (molecular size) than other known bispecific antibodies such as BiTE, DART and other ScFv based bispecific antibody formats, and thus improved thermodynamic stability. Is expected to have. In particular, the VH region and the VL region of the Fv region are on separate polypeptides.

다중 특이적 융합 단백질의 특징Characteristics of multispecific fusion proteins

In one embodiment, it is estimated that the distance between the two N-terminal Fab regions and the C-terminal Fv regions in the multispecific fusion proteins provided herein is in the range of about 40 Å to about 70 Å. In one embodiment, the distance between the two N-terminal Fab regions and the C-terminal Fv regions in the multispecific fusion proteins provided herein was estimated to be about 42 Å. In some other embodiments, the distance between the N-terminal two Fab regions and the C-terminal Fv regions in the multispecific fusion proteins provided herein was estimated to be about 60 Å.

As an embodiment of the multispecific fusion protein, the binding affinity of the first antigen-binding domain for the first antigen may be higher than that of the second antigen-binding domain for the second antigen. For example, the binding kinetics of ACE-05 to human PD-L1 was similar to that of the parental anti-PD-L1 antibody (ie, YBL-007 from Y-Biologics Inc.). In contrast, the binding affinity of ACE-05 for CD3 was much lower than that of the parental anti-CD3 antibody (UCHT1, BioLegend, USA).

In one embodiment, the multispecific fusion proteins provided herein are about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, 50 It is possible to bind one or more targets, antigens or epitopes with _{a dissociation constant (K D} ) of pM or less, 10 pM or less, or 1 pM or less. In one embodiment, a multispecific fusion protein provided herein is capable of binding a target, antigen or epitope with _{a K D of about 20 nM or less.} In one embodiment, the multispecific fusion protein is capable of binding to a target, antigen or epitope with _{a K D of about 10 nM or less.} In one embodiment, the multispecific fusion protein is capable of binding to a target, antigen or epitope with _{a K D of about 1 nM or less.} In one embodiment, the multispecific fusion protein is capable of binding to a target, antigen or epitope with _{a K D of about 0.5 nM or less.} In one embodiment, a multispecific fusion protein provided herein is capable of binding a target, antigen or epitope with _{a K D of about 0.1 nM or less.} In one embodiment, a multispecific fusion protein provided herein is capable of binding a target, antigen or epitope with _{a K D of about 50 pM or less.} In one embodiment, a multispecific fusion protein provided herein is capable of binding a target, antigen or epitope with _{a K D of about 25 pM or less.} In one embodiment, a multispecific fusion protein provided herein is capable of binding a target, antigen or epitope with _{a K D of about 10 pM or less.} In one embodiment, a multispecific fusion protein provided herein is capable of binding a target, antigen or epitope with _{a K D of about 1 pM or less.}

In one embodiment, the K _D of the multispecific fusion protein for the first antigen is about 2 times, 3 times, 4 times, 5 times, 6 times, 7 times that _{of the multispecific fusion protein K D for the second antigen.} , May be 8 times, 9 times, 10 times, 15 times, 20 times, 50 times or more. In some embodiments, the K _D of the multispecific fusion protein for the first antigen may be about 10, 10 ² , 10 ³ or 10 ⁴ _{times that of the multispecific fusion protein K D for the second antigen.}

The multispecific fusion proteins herein may be in chemically modified form. In one embodiment, the fusion protein is chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting groups/blockers, proteolytic cleavage and/or binding of cellular ligands or other proteins. Can be. Many of these chemical modifications can be performed by known techniques.

C 말단에 Fv가 존재하는 다중 특이적 융합 단백질Multispecific fusion protein with Fv at the C-terminus

One embodiment of the fusion protein may include the following structural formulas (I) and (II):

N'-A-L1-X-C' (I); And

N'-B-L1-Y-C' (II)

At this time, in the above structural formulas (I) and (II),

Wherein N'is the N-terminus of the fusion protein,

C'is the C-terminus of the fusion protein,

Each of A and B is specific for a first antigen,

The L1 is a peptide linker containing at least one Cys,

X is an antibody variable heavy chain (VH) region or an antibody variable light chain (VL) region,

Y is an antibody variable light chain (VL) region or an antibody variable heavy chain (VH) region,

The X and Y form an antibody variable region (Fv) that specifically binds to the second antigen.

In this case, the A and B are each antibody Fab region, (i) a first portion including an antibody variable heavy chain (VH) region and an antibody CH1 region; And (ii) an antibody variable light chain (VL) region and an antibody light chain constant region (CL).

In one embodiment, A and B may bind to the same antigen by two Fab regions. In addition, A and B may bind to the same epitope of the same antigen. In addition, A and B bind to different epitopes of the same antigen. In another embodiment, A and B bind different antigens.

In another embodiment, the first antigen binding domain and the second antigen binding domain bind different antigens, wherein the first antigen binding domain binds a first antigen and a second antigen binding domain is capable of binding a second antigen. . 3B and 3C show examples of such ALiCE molecules. Here, in the case of ACE-05, the first antigen-binding domain (two Fab regions) binds to the cancer antigen (PD-L1), and the second antigen-binding domain binds to immune cells such as T cells through antigens such as CD3. . In addition, in the case of ACE-31, the first antigen binding domain (two Fab regions) binds to immune cells such as T cells through antigens such as CD3. In addition, the second antigen binding domain binds to the cancer antigen (PD-L1). These ALiCE molecules can bind immune cells (eg, T cells) to cancer cells, and thus can be used as a therapeutic agent for cancer treatment.

구조식 (I)Structural Formula (I)

In addition, the structural formula (I) may include the following structural formulas (I') and (I''):

N'-A'-L1-X-C' (I'); And

N'-A''-C' (I'')

At this time, in the above structural formulas (I') and (I''),

A'is a heavy chain region of an antibody and includes a variable region and a CH1 region, or is a light chain region of an antibody;

A'' is a light chain region of an antibody, or as a heavy chain region of an antibody, includes a variable region and a CH1 region,

At this time, A'and A'' bind to form a variable region of the antibody, wherein the variable region specifically binds to the first antigen. In this case, the light chain region may include a light chain variable region and a light chain constant region. In addition, N', L1, X and C'are as defined above.

구조식 (II)Structural Formula (II)

In addition, the structural formula (II) may include the following structural formulas (II') and (II''):

N'-B'-L1-Y-C' (II'); And

N'-B''-C' (II'')

At this time, in the above structural formulas (II') and (II''),

B'is a heavy chain region of an antibody and includes a variable region and a CH1 region, or is a light chain region of an antibody;

B'' is the light chain region of the antibody, or as the heavy chain region of the antibody comprises a variable region and a CH1 region;

At this time, B'and B'' bind to form a variable region of the antibody, wherein the variable region specifically binds to the first antigen. In this case, the light chain region may include a light chain variable region and a light chain constant region. In addition, N', L1, Y and C'are as defined above.

N 말단에 Fv가 존재하는 다중 특이적 융합 단백질Multispecific fusion protein with Fv at the N-terminus

Another embodiment of the fusion protein may be one comprising the following structural formulas (III) and (IV):

N'-X-L2-A-C' (III)

N'-Y-L2-B-C' (IV)

In this case, in the structural formulas (III) and (IV),

Wherein N'is the N-terminus of the fusion protein,

C'is the C-terminus of the fusion protein,

Each of A and B specifically binds to a first antigen,

The L2 is a peptide linker containing at least one Cys,

In addition, the structural formula (III) may include the following structural formulas (III') and (III"):

N'-X-L1-A'-C' (III'); And

N'-A''-C' (III'')

At this time, in the above structural formulas (III') and (III''),

A'' is the light chain region of the antibody, or as the heavy chain region of the antibody comprises a variable region and a CH1 region;

At this time, A'and A'' combine to form a variable region of the antibody, wherein the variable region is specific for the first antigen.

In addition, the structural formula (IV) may include the following structural formulas (IV') and (IV''):

N'-Y-L1-B'-C' (IV'); And

N'-B''-C' (IV'')

At this time, in the structural formulas (IV') and (IV''),

At this time, B'and B'' bind to form a variable region of the antibody, wherein the variable region is specific for the first antigen.

링커Linker

In this case, the L1 and L2 may include a hinge region derived from an immunoglobulin. In addition, the L1 and L2 may include 1, 2 or 3 Cys.

Specifically, each of L1 and L2 may have the following structural formula (V):

(L1')n-hinge-(L1'')m (V)

At this time, the L1' and L1'' are each a linker consisting of 1 to 15 amino acids,

n and m are integers of 0 or 1,

The hinge is an immunoglobulin-derived hinge region.

In one embodiment, the antibody hinge region is an IgG hinge region. The IgG hinge regions provided herein can be selected from, for example, antibody hinge regions of various IgG subtypes. The table below lists exemplary IgG subtypes with core hinge sequences that can be included in the flexible peptide regions provided herein.

IgG subtypeIgG subtype	Core hinge sequenceCore hinge sequence	서열번호Sequence number
IgG1IgG1
EPKSCDKTHTCPPCPEPKSCDKTHTCPPCP	5555
IgG2 IgG2		ERKCCVECPPCPERKCCVECPPCP	5656
IgG3IgG3	ELKTPLDTTHTCPRCP(EPKSCDTPPPCPRCP)₃ ELKTPLDTTHTCPRCP(EPKSCDTPPPCPRCP) ₃	5757
IgG4IgG4	ESKYGPPCPSCPESKYGPPCPSCP	5858

Hinge Region SequenceHinge Region Sequence	서열번호Sequence number
DKTHTCPPCPPCPAPELLGGPDKTHTCPPCPPCPAPELLGGP	385385
DKTHTCPPCPAPELLGGPDKTHTCPPCPAPELLGGP	386386
DKTHTCPPCPPCPAPELLGDKTHTCPPCPPCPAPELLG	387387

The hinge can be modified to introduce additional disulfide bonds. The hinge region may contain more than two disulfide bonds. The hinge region may contain an even number of disulfide bonds such as, for example, 2, 4, 6, 8 or 10 disulfide bonds, 4, 6, 8, 10 disulfide bonds, and 4 disulfide bonds. In one embodiment, the hinge region may comprise an odd number of disulfide bonds such as 1, 3, 5, 7 or 9 disulfide bonds, 3, 5, 7 or 9 disulfide bonds, 3 disulfide bonds. . 3G shows a representative example of a multispecific fusion protein, wherein the hinge region was prepared such that two and three disulfide bonds were formed between the heavy chains of the multispecific fusion protein.

The hinge region comprises an amino acid sequence (PPC) _n , where n is an integer. In some embodiments, n can be an even number, such as 2, 4, 6, 8 or 10, for example 4, 6, 8 or 10, for example 4. In other embodiments, n can be an odd number, for example 1, 3, 5, 7, or 9, for example 3, 5, 7, or 9, for example 3. In addition, an inter-hinge disulfide bond is formed between a cysteine residue in one hinge region and a cysteine residue in another hinge region.

Each of L1' and L1'' may be a peptide consisting of 1 to 20 amino acids. Specifically, the L1′ and L1″ may be amino acids such as (G4S)p (p is an integer of 1 to 10). Specifically, L1' and L1'' may be (G4S) ₁ , (G4S) ₂ , (G4S) ₃ , or (G4S) ₄ .

The length of L1′ and/or L1″ may vary. In some embodiments, LI' and/or LI'' is 5 amino acids long. In another embodiment, L1' and/or L1'' is 9 amino acids long. In another embodiment, L1' and/or L1'' is 10 amino acids long. The table below shows representative examples of L1' and/or L1''.

Linker SequenceLinker Sequence	서열번호Sequence number
GGGGSGGGGS	112112
GGGGSGGGGSGGGGSGGGGS	113113
GGSGGGGSGGGSGGGGSG	114114

제1 항원First antigen

The first antigen may be a cancer antigen (Tumor-Specific Antigens), or a protein present on the surface of an immune cell, or may be a cytokine. The first antigen is PD-L1, PD-1, EGFR, TNFR, BCMA, CD22, CD25, CD30, CD33, CD37, CD38, CD52, CD56, CD123, cMET, DLL3, GD2, Nectin-4, RANKL, SLAMF7, TROP2, Claudin 18.2, TNFR, TNF, CD3, HER2, CD20, CD19, CTLA-4, VEGFR, VEGF, NCAM1, ICAM-1, ICAM-2, CEACAM6, Carcinoembryonic antigen (CEA), CA-125, Alphafetoprotein (AFP ), MUC-1, Epithelial tumor antigen (ETA), Melanoma-associated antigen (MAGE), Immature laminin receptor, TAG-72, HPV E6/E7, BING-4, Calcium-activated chloride channel 2, Cyclin-B1, 9D7 , Ep-CAM, EphA3, Mesothelin, SAP-1, Survivin or virus-derived antigens.

Specifically, the first antigen may be a cancer antigen. At this time, the cancer antigens are PD-L1, EGFR, BCMA, CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD38, CD52, CD56, CD123, HER2, cMET, DLL3, GD2, Nectin-4, RANKL, It may be any one selected from the group consisting of SLAMF7, TROP2, Claudin 18.2, MUC-1, Mesothelin, EpCAM and CEA.

At this time, in one embodiment, the first antigen may be a cell surface molecule that regulates T cell function. In another embodiment, the first antigen may be an immune checkpoint inhibitor. In addition, the first antigen may be a cancer antigen.

In addition, the first antigen may be a protein expressed on the surface of immune cells such as lymphocytes and monocytes. Specifically, it may be a protein expressed in cells such as T cells, B cells, dendritic cells, granulocytes, megakaryocytes, monocytes, and NK cells. In addition, the first antigen may be a protein expressed on CD8 + T cells or CD4 + T cells.

In the above structural formula, A and B can recognize the same antigen. In addition, A and B can recognize different antigens.

In one embodiment, A and/or B may specifically bind to any one antigen selected from the group consisting of PD-L1, HER2, CD19, CD20, EGFR, CD3, TNF, and CTLA-4. Specifically, A and/or B may include any one variable region selected from:

PD-L1:PD-L1:

1) VH region comprising the amino acid sequence of SEQ ID NO: 5 (VH-CDR1), SEQ ID NO: 6 (VH-CDR2) and SEQ ID NO: 7 (VH-CDR3), and SEQ ID NO: 9 (VL-CDR1), SEQ ID NO: 10 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 11 (VL-CDR3);

2) a VH region comprising the amino acid sequence of SEQ ID NO: 159 (VH-CDR1), SEQ ID NO: 160 (VH-CDR2) and SEQ ID NO: 161 (VH-CDR3), and SEQ ID NO: 171 (VL-CDR1), SEQ ID NO: 172 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 173 (VL-CDR3);

3) VH region comprising the amino acid sequence of SEQ ID NO: 159 (VH-CDR1), SEQ ID NO: 160 (VH-CDR2) and SEQ ID NO: 161 (VH-CDR3), and SEQ ID NO: 314 (VL-CDR1), SEQ ID NO: 315 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 316 (VL-CDR3);

4) VH region comprising the amino acid sequence of SEQ ID NO: 330 (VH-CDR1), SEQ ID NO: 331 (VH-CDR2) and SEQ ID NO: 332 (VH-CDR3), and SEQ ID NO: 334 (VL-CDR1), SEQ ID NO: 335 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 336 (VL-CDR3);

HER2:HER2:

5) VH region comprising the amino acid sequence of SEQ ID NO: 118 (VH-CDR1), SEQ ID NO: 119 (VH-CDR2) and SEQ ID NO: 120 (VH-CDR3), and SEQ ID NO: 121 (VL-CDR1), SEQ ID NO: 122 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 123 (VL-CDR3);

6) VH region comprising the amino acid sequence of SEQ ID NO: 276 (VH-CDR1), SEQ ID NO: 277 (VH-CDR2) and SEQ ID NO: 278 (VH-CDR3), and SEQ ID NO: 288 (VL-CDR1), SEQ ID NO: 289 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 290 (VL-CDR3);

CD19:CD19:

7) VH region comprising the amino acid sequence of SEQ ID NO: 140 (VH-CDR1), SEQ ID NO: 141 (VH-CDR2) and SEQ ID NO: 142 (VH-CDR3), and SEQ ID NO: 152 (VL-CDR1), SEQ ID NO: 153 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 154 (VL-CDR3);

8) VH region comprising the amino acid sequence of SEQ ID NO: 62 (VH-CDR1), SEQ ID NO: 63 (VH-CDR2) and SEQ ID NO: 64 (VH-CDR3), and SEQ ID NO: 66 (VL-CDR1), SEQ ID NO: 67 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 68 (VL-CDR3);

CD20:CD20:

9) VH region comprising the amino acid sequence of SEQ ID NO: 223 (VH-CDR1), SEQ ID NO: 224 (VH-CDR2) and SEQ ID NO: 225 (VH-CDR3), and SEQ ID NO: 227 (VL-CDR1), SEQ ID NO: 228 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 229 (VL-CDR3);

10) VH region comprising the amino acid sequence of SEQ ID NO: 436 (VH-CDR1), SEQ ID NO: 437 (VH-CDR2) and SEQ ID NO: 438 (VH-CDR3), and SEQ ID NO: 441 (VL-CDR1), SEQ ID NO: 442 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 443 (VL-CDR3);

EGFR:EGFR:

11) VH region comprising the amino acid sequence of SEQ ID NO: 234 (VH-CDR1), SEQ ID NO: 235 (VH-CDR2) and SEQ ID NO: 236 (VH-CDR3), and SEQ ID NO: 238 (VL-CDR1), SEQ ID NO: 239 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 240 (VL-CDR3);

12) VH region comprising the amino acid sequence of SEQ ID NO: 245 (VH-CDR1), SEQ ID NO: 246 (VH-CDR2) and SEQ ID NO: 247 (VH-CDR3), and SEQ ID NO: 249 (VL-CDR1), SEQ ID NO: 250 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 251 (VL-CDR3);

13) VH region comprising the amino acid sequence of SEQ ID NO: 256 (VH-CDR1), SEQ ID NO: 257 (VH-CDR2) and SEQ ID NO: 258 (VH-CDR3), and SEQ ID NO: 260 (VL-CDR1), SEQ ID NO: 261 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 262 (VL-CDR3);

14) VH region comprising the amino acid sequence of SEQ ID NO: 234 (VH-CDR1), SEQ ID NO: 235 (VH-CDR2) and SEQ ID NO: 236 (VH-CDR3), and SEQ ID NO: 238 (VL-CDR1), SEQ ID NO: 239 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 240 (VL-CDR3);

CD3:CD3:

15) VH region comprising the amino acid sequence of SEQ ID NO: 326 (VH-CDR1), SEQ ID NO: 327 (VH-CDR2) and SEQ ID NO: 328 (VH-CDR3), and SEQ ID NO: 338 (VL-CDR1), SEQ ID NO: 339 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 340 (VL-CDR3);

16) VH region comprising the amino acid sequence of SEQ ID NO: 330 (VH-CDR1), SEQ ID NO: 331 (VH-CDR2) and SEQ ID NO: 332 (VH-CDR3), and SEQ ID NO: 334 (VL-CDR1), SEQ ID NO: 335 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 336 (VL-CDR3);

17) VH region comprising the amino acid sequence of SEQ ID NO: 163 (VH-CDR1), SEQ ID NO: 271 (VH-CDR2) and SEQ ID NO: 165 (VH-CDR3), and SEQ ID NO: 167 (VL-CDR1), SEQ ID NO: 168 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 169 (VL-CDR3);

18) VH region comprising the amino acid sequence of SEQ ID NO: 396 (VH-CDR1), SEQ ID NO: 397 (VH-CDR2) and SEQ ID NO: 398 (VH-CDR3), and SEQ ID NO: 402 (VL-CDR1), SEQ ID NO: 403 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 404 (VL-CDR3);

19) VH region comprising the amino acid sequence of SEQ ID NO: 144 (VH-CDR1), SEQ ID NO: 145 (VH-CDR2) and SEQ ID NO: 146 (VH-CDR3), and SEQ ID NO: 148 (VL-CDR1), SEQ ID NO: 149 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 410 (VL-CDR3);

20) VH region comprising the amino acid sequence of SEQ ID NO: 144 (VH-CDR1), SEQ ID NO: 145 (VH-CDR2) and SEQ ID NO: 146 (VH-CDR3), and SEQ ID NO: 148 (VL-CDR1), SEQ ID NO: 149 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 150 (VL-CDR3);

21) VH region comprising the amino acid sequence of SEQ ID NO: 70 (VH-CDR1), SEQ ID NO: 71 (VH-CDR2) and SEQ ID NO: 72 (VH-CDR3), and SEQ ID NO: 74 (VL-CDR1), SEQ ID NO: 75 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 76 (VL-CDR3);

22) VH region comprising the amino acid sequence of SEQ ID NO: 78 (VH-CDR1), SEQ ID NO: 79 (VH-CDR2) and SEQ ID NO: 80 (VH-CDR3), and SEQ ID NO: 82 (VL-CDR1), SEQ ID NO: 83 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 84 (VL-CDR3);

23) VH region comprising the amino acid sequence of SEQ ID NO: 295 (VH-CDR1), SEQ ID NO: 296 (VH-CDR2) and SEQ ID NO: 297 (VH-CDR3), and SEQ ID NO: 299 (VL-CDR1), SEQ ID NO: 300 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 301 (VL-CDR3);

24) VH region comprising the amino acid sequence of SEQ ID NO: 78 (VH-CDR1), SEQ ID NO: 79 (VH-CDR2) and SEQ ID NO: 80 (VH-CDR3), and SEQ ID NO: 82 (VL-CDR1), SEQ ID NO: 83 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 87 (VL-CDR3);

25) VH region comprising the amino acid sequence of SEQ ID NO: 163 (VH-CDR1), SEQ ID NO: 271 (VH-CDR2) and SEQ ID NO: 165 (VH-CDR3), and SEQ ID NO: 167 (VL-CDR1), SEQ ID NO: 168 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 169 (VL-CDR3);

26) VH region comprising the amino acid sequence of SEQ ID NO: 177 (VH-CDR1), SEQ ID NO: 178 (VH-CDR2) and SEQ ID NO: 179 (VH-CDR3), and SEQ ID NO: 181 (VL-CDR1), SEQ ID NO: 182 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 183 (VL-CDR3);

27) VH region comprising the amino acid sequence of SEQ ID NO: 187 (VH-CDR1), SEQ ID NO: 188 (VH-CDR2) and SEQ ID NO: 189 (VH-CDR3), and SEQ ID NO: 191 (VL-CDR1), SEQ ID NO: 192 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 193 (VL-CDR3);

28) VH region comprising the amino acid sequence of SEQ ID NO: 177 (VH-CDR1), SEQ ID NO: 178 (VH-CDR2) and SEQ ID NO: 179 (VH-CDR3), and SEQ ID NO: 181 (VL-CDR1), SEQ ID NO: 182 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 207 (VL-CDR3);

29) VH region comprising the amino acid sequence of SEQ ID NO: 177 (VH-CDR1), SEQ ID NO: 178 (VH-CDR2) and SEQ ID NO: 211 (VH-CDR3), and SEQ ID NO: 181 (VL-CDR1), SEQ ID NO: 182 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 207 (VL-CDR3);

30) VH region comprising the amino acid sequence of SEQ ID NO: 306 (VH-CDR1), SEQ ID NO: 307 (VH-CDR2) and SEQ ID NO: 308 (VH-CDR3), and SEQ ID NO: 310 (VL-CDR1), SEQ ID NO: 311 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 312 (VL-CDR3);

31) VH region comprising the amino acid sequence of SEQ ID NO: 349 (VH-CDR1), SEQ ID NO: 350 (VH-CDR2) and SEQ ID NO: 351 (VH-CDR3), and SEQ ID NO: 353 (VL-CDR1), SEQ ID NO: 354 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 355 (VL-CDR3);

32) VH region comprising the amino acid sequence of SEQ ID NO: 359 (VH-CDR1), SEQ ID NO: 360 (VH-CDR2) and SEQ ID NO: 361 (VH-CDR3), and SEQ ID NO: 363 (VL-CDR1), SEQ ID NO: 364 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 365 (VL-CDR3);

33) VH region comprising the amino acid sequence of SEQ ID NO: 197 (VH-CDR1), SEQ ID NO: 198 (VH-CDR2) and SEQ ID NO: 199 (VH-CDR3), and SEQ ID NO: 201 (VL-CDR1), SEQ ID NO: 202 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 203 (VL-CDR3);

TNF: TNF:

34) VH region comprising the amino acid sequence of SEQ ID NO: 124 (VH-CDR1), SEQ ID NO: 125 (VH-CDR2) and SEQ ID NO: 126 (VH-CDR3), and SEQ ID NO: 127 (VL-CDR1), SEQ ID NO: 128 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 129 (VL-CDR3);

35) VH region comprising the amino acid sequence of SEQ ID NO: 280 (VH-CDR1), SEQ ID NO: 281 (VH-CDR2) and SEQ ID NO: 282 (VH-CDR3), and SEQ ID NO: 284 (VL-CDR1), SEQ ID NO: 285 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 286 (VL-CDR3);

36) VH region comprising the amino acid sequence of SEQ ID NO: 276 (VH-CDR1), SEQ ID NO: 277 (VH-CDR2) and SEQ ID NO: 278 (VH-CDR3), and SEQ ID NO: 288 (VL-CDR1), SEQ ID NO: 289 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 290 (VL-CDR3); And

CTLA-4:CTLA-4:

37) VH region comprising the amino acid sequence of SEQ ID NO: 369 (VH-CDR1), SEQ ID NO: 370 (VH-CDR2) and SEQ ID NO: 371 (VH-CDR3), and SEQ ID NO: 373 (VL-CDR1), SEQ ID NO: 374 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 375 (VL-CDR3).

In addition, A or B may include any one variable region selected from the following group:

PD-L1:PD-L1:

1) the heavy chain variable region of SEQ ID NO: 4 (VH) and the light chain variable region of SEQ ID NO: 8 (VL);

2) the heavy chain variable region of SEQ ID NO: 158 (VH) and the light chain variable region of SEQ ID NO: 170 (VL);

3) the heavy chain variable region of SEQ ID NO: 158 (VH) and the light chain variable region of SEQ ID NO: 313 (VL);

4) the heavy chain variable region of SEQ ID NO: 329 (VH) and the light chain variable region of SEQ ID NO: 333 (VL);

5) the heavy chain variable region of SEQ ID NO: 329 (VH) and the light chain variable region of SEQ ID NO: 376 (VL);

HER2:HER2:

6) the heavy chain variable region of SEQ ID NO: 51 (VH) and the light chain variable region of SEQ ID NO: 52 (VL);

7) the heavy chain variable region of SEQ ID NO: 275 (VH) and the light chain variable region of SEQ ID NO: 287 (VL);

CD19:CD19:

8) the heavy chain variable region of SEQ ID NO: 139 (VH) and the light chain variable region of SEQ ID NO: 151 (VL);

9) the heavy chain variable region of SEQ ID NO: 61 (VH) and the light chain variable region of SEQ ID NO: 65 (VL);

CD20: CD20:

10) the heavy chain variable region of SEQ ID NO: 222 (VH) and the light chain variable region of SEQ ID NO: 226 (VL);

11) the heavy chain variable region of SEQ ID NO: 435 (VH) and the light chain variable region of SEQ ID NO: 440 (VL);

EGFR: EGFR:

12) the heavy chain variable region of SEQ ID NO: 233 (VH) and the light chain variable region of SEQ ID NO: 237 (VL);

13) the heavy chain variable region of SEQ ID NO: 244 (VH) and the light chain variable region of SEQ ID NO: 248 (VL);

14) the heavy chain variable region of SEQ ID NO: 255 (VH) and the light chain variable region of SEQ ID NO: 259 (VL);

CD3:CD3:

15) the heavy chain variable region of SEQ ID NO: 325 (VH) and the light chain variable region of SEQ ID NO: 337 (VL);

16) the heavy chain variable region of SEQ ID NO: 329 (VH) and the light chain variable region of SEQ ID NO: 376 (VL);

17) the heavy chain variable region of SEQ ID NO: 162 (VH) and the light chain variable region of SEQ ID NO: 166 (VL);

18) the heavy chain variable region of SEQ ID NO: 395 (VH) and the light chain variable region of SEQ ID NO: 401 (VL);

19) the heavy chain variable region of SEQ ID NO: 406 (VH) and the light chain variable region of SEQ ID NO: 409 (VL);

20) the heavy chain variable region of SEQ ID NO: 143 (VH) and the light chain variable region of SEQ ID NO: 147 (VL);

21) the heavy chain variable region of SEQ ID NO: 69 (VH) and the light chain variable region of SEQ ID NO: 73 (VL);

22) the heavy chain variable region of SEQ ID NO: 77 (VH) and the light chain variable region of SEQ ID NO: 81 (VL);

23) the heavy chain variable region of SEQ ID NO: 294 (VH) and the light chain variable region of SEQ ID NO: 298 (VL);

24) the heavy chain variable region of SEQ ID NO: 85 (VH) and the light chain variable region of SEQ ID NO: 86 (VL);

25) the heavy chain variable region of SEQ ID NO: 176 (VH) and the light chain variable region of SEQ ID NO: 180 (VL);

26) the heavy chain variable region of SEQ ID NO: 186 (VH) and the light chain variable region of SEQ ID NO: 190 (VL);

27) the heavy chain variable region of SEQ ID NO: 176 (VH) and the light chain variable region of SEQ ID NO: 206 (VL);

28) the heavy chain variable region of SEQ ID NO: 210 (VH) and the light chain variable region of SEQ ID NO: 212 (VL);

29) the heavy chain variable region of SEQ ID NO: 215 (VH) and the light chain variable region of SEQ ID NO: 216 (VL);

30) the heavy chain variable region of SEQ ID NO: 305 (VH) and the light chain variable region of SEQ ID NO: 309 (VL);

31) the heavy chain variable region of SEQ ID NO: 348 (VH) and the light chain variable region of SEQ ID NO: 352 (VL);

32) the heavy chain variable region of SEQ ID NO: 358 (VH) and the light chain variable region of SEQ ID NO: 362 (VL);

33) the heavy chain variable region of SEQ ID NO: 196 (VH) and the light chain variable region of SEQ ID NO: 200 (VL);

TNF:TNF:

34) the heavy chain variable region of SEQ ID NO: 53 (VH) and the light chain variable region of SEQ ID NO: 54 (VL);

35) the heavy chain variable region of SEQ ID NO: 279 (VH) and the light chain variable region of SEQ ID NO: 283 (VL);

36) the heavy chain variable region of SEQ ID NO: 275 (VH) and the light chain variable region of SEQ ID NO: 287 (VL); And

CTLA-4:CTLA-4:

37) The heavy chain variable region of SEQ ID NO: 368 (VH) and the light chain variable region of SEQ ID NO: 372 (VL).

제2 항원Second antigen

The second antigen may be cancer antigens (Tumor-Specific Antigens), proteins present on the surface of immune cells, or cytokines. Second antigens are PD-L1, PD-1, EGFR, BCMA, CD22, CD25, CD30, CD33, CD37, CD38, CD52, CD56, CD123, cMET, DLL3, GD2, Nectin-4, RANKL, SLAMF7, TROP2, Claudin 18.2, TNFR, TNF, CD3, HER2, CD20, CD19, CTLA-4, VEGFR, VEGF, NCAM1, ICAM-1, ICAM-2, CEACAM6, Carcinoembryonic antigen (CEA), CA-125, Alphafetoprotein (AFP), MUC-1, Epithelial tumor antigen (ETA), Melanoma-associated antigen (MAGE), Immature laminin receptor, TAG-72, HPV E6/E7, BING-4, Calcium-activated chloride channel 2, Cyclin-B1, 9D7, Ep -CAM, EphA3, Mesothelin, SAP-1, Survivin or virus derived antigen. Specifically, the second antigen may be a protein present on the surface of an immune cell.

Fv 형성 영역Fv formation area

It is characterized in that the above-described X and Y combine to form the Fv of the antibody. In this case, the Fv may specifically bind to a predetermined antigen. In this case, X and Y may be a light chain variable region or a heavy chain variable region specific for an antigen. In addition, X and Y may include light chain and heavy chain CDRs.

The X and Y combine to form Fv, and the Fv may specifically bind to the above-described second antigen. In one embodiment, the Fv formed by binding of X and Y specifically binds to any one antigen selected from the group consisting of PD-L1, HER2, CD19, CD20, EGFR, CD3, TNF, and CTLA-4. can do. Specifically, X and Y may each include a variable heavy chain (VH) region or a variable light chain (VL) region of any one variable region selected from the following group:

PD-L1:PD-L1:

HER2:HER2:

CD19:CD19:

CD20:CD20:

EGFR:EGFR:

CD3:CD3:

TNF: TNF:

CTLA-4:CTLA-4:

Fv 형성 영역Fv formation area

In addition, X or Y may be a variable heavy (VH) region or a variable light (VL) region of any one variable region selected from the following group:

PD-L1:PD-L1:

HER2:HER2:

CD19:CD19:

CD20: CD20:

EGFR: EGFR:

CD3:CD3:

TNF:TNF:

CTLA-4:CTLA-4:

CH3이 추가된 Fv 영역 Fv area to which CH3 is added

The X and/or Y may further include CH3. At this time, CH3 may be an immunoglobulin-derived CH3 region. At this time, CH3 may be bonded to the N-terminus or C-terminus of X and/or Y. Specifically, CH3 may be included in both the C-terminus of X and Y. In addition, CH3 may be included only at the C-terminus of X or may be included only at the C-terminus of Y.

The presence of the CH3 region can provide the ability of the multispecific fusion protein to bind to the Fc receptor. In the CH3 region connected to the Fv region, a kiH (knobs-into-holes) structure may be introduced. 1A shows the "Knob-in-Hole" structure of the variable domains of anti-CD3 UCHT1, anti-PD-L1 dervalumab, and anti-CTLA-4 ipilimumab.

A 및 B 및 X/Y의 조합 예시Examples of combinations of A and B and X/Y

The A and B may be designed to specifically bind to any one antigen selected from the above-described first antigen. X/Y may be designed to specifically bind to any one antigen selected from the above-described second antigen.

In one embodiment, the A and B specifically bind to any one first antigen selected from the group consisting of PD-L1, EGFR, CD20, HER2, TNF, CD19, CD3 and CTLA4, and X and Y are Fv formed by binding specifically binds to any one second antigen selected from the group consisting of PD-L1, EGFR, CD20, HER2, TNF, CD19, CD3 and CTLA4, but the A and B and X and Y The formed Fv can be characterized as not binding to the same antigen.

In another embodiment, the multispecific fusion protein may specifically bind to HER2 as a first antigen and specifically bind to TNF as a second antigen.

In one embodiment, the multispecific fusion protein may specifically bind to PD-L1 as a first antigen and specifically bind to CD3 as a second antigen.

In another embodiment, the multispecific fusion protein may specifically bind to EGFR as a first antigen and specifically bind to CD3 as a second antigen.

In another embodiment, the multispecific fusion protein may specifically bind to CD20 as a first antigen and specifically bind to CD3 as a second antigen.

In one embodiment, the multispecific fusion protein may specifically bind to CD3 as a first antigen and specifically to PD-L1 as a second antigen.

In another embodiment, the multispecific fusion protein may specifically bind to HER2 as a first antigen and specifically bind to CD3 as a second antigen.

In another embodiment, the multispecific fusion protein may specifically bind to CD19 as a first antigen and specifically bind to CD3 as a second antigen.

In another embodiment, the multispecific fusion protein may specifically bind to PD-L1 as a first antigen and specifically bind to CTLA-4 as a second antigen.

In another embodiment, the multispecific fusion protein may specifically bind to PD-L1 with a first antigen and specifically bind to PD-1 with a second antigen.

In another embodiment, the multispecific fusion protein may specifically bind to PD-1 with a first antigen and specifically bind to PD-L1 with a second antigen.

In another embodiment, the multispecific fusion protein may specifically bind to CD20 as a first antigen and to CTLA-4 as a second antigen.

다중 특이성 융합 단백질에 관한 실험 결과Experimental results on multispecific fusion proteins

VH-VL 이종이량체 형성에 기반한 ALiCE 분자 생성Generation of ALiCE molecules based on VH-VL heterodimer formation

Despite the important role of the CH3 domain in antibody assembly, the present inventors found that the autonomous assembly of the VH and VL domains and the formation of a stable Fv complex not only promote the specific heterodimerization of the two HCs, but also to bind the second antigen. It is assumed that it can also be used to introduce additional sites. To test this, ACE-HC-VH and ACE-HC-VL were generated by replacing the Fc domains of the two HCs of the parent IgG with the VH and VL domains of the IgG specific for the second antigen (Fig. 3a). .

As a test system, the present inventors used anti-CD3 antibody UCHT1 and anti-PD-L1 antibody YBL-007, and the YBL-007 has PD-L1/PD-1 signal blocking ability of avelumab. It is an anti-PD-L1 antibody produced by the present inventors similar to (Fig. 4). The LC of parent YBL-007 was used for ACE-LC, and the hinge regions of VH-CH1 and YBL-007 HC were fused to VH or VL of UCHT1 to generate two HCs of ALiCE. Expression vectors of various combinations encoding ACE-HC-VH, ACE-HC-VL and ACE-LC were transfected into FreeStyle 293-F cells, and the expression of these molecules in culture medium was SDS-PAGE and Western blotting. It was analyzed as (Fig. 5).

As expected, each chain could not be expressed due to folding and secretion problems (FIG. 5, lanes 1 to 3). In addition, the ACE-HC-VH chain having a knob structure in the CDR 3 loop of the UCHT1 VH domain cannot assemble into a homodimer due to inappropriate knob-knob interaction (Fig. 5, lane 4). , Homodimer formation between the same ACE-HC-VL chains was hardly detected (Fig. 5, lane 5).

In addition, the suitably assembled complex was highly expressed and secreted only when ACE-HC-VL, ACE-HC-VH and ACE-LC were present together (FIG. 5, lane 6). The resulting ALiCE (anti-PD-L1 Fab × anti-CD3 Fv; hereinafter referred to as ACE-05) was transiently expressed in FreeStyle 293-F cells and purified by CH1 affinity chromatography, and the yield was about 20-30 mg. /L. Capillary electrophoresis analysis using the Bioanalyzer assay kit (P230 and P80 kit) under reducing and non-reducing conditions showed that the ACE-LC, ACE-HC-VH and ACE-HC-VL chains were in a 2:1:1 ratio in ACE-05. It was confirmed that it exists (Figs. 6 and 7).

Analysis results through size-exclusion chromatography and mass spectrometry (MS) using LC-ESI/TOF indicated that ACE-05 was a homogeneous heterotetramer (FIGS. 6 and 8). In particular, the mass of the observed main peak (123,997 Da) was very close to the theoretical mass of the heterotetrameric structure of ACE-05 (123,942 Da). In comparison, the mass of ACE-05-HC-VH homodimer + two ACE-05-LC, which is a homotetramer structure, was 125,597 Da, and ACE-05-HC-VL homodimer + two The mass of ACE-05-LC was 122,287 Da (Fig. 8). From these results, it was confirmed that no homotetrameric complex detectable in purified ACE-05 was present.

In addition, the present inventors described ACE-31 (anti-CD3 Fab x anti-PD-L1 Fv using UCHT1 and YBL-007) and ACE-00 (anti-HER2 mAb [Herceptin] and anti-TNF-α mAb [Humira] Anti-HER2 Fab x anti-TNF-α Fv) was generated to investigate whether the ALiCE platform could be applied to other antibody pairs in general. Similar to ACE-05, ACE-31 and ACE-00 were assembled into the corresponding heterotetrameric complex, ACE-HC-VL, ACE-HC-VH and two ACE-LCs, and secreted in a homogeneous form ( 6 and 9).

During protein expression, a small amount of aggregation (or oligomer misfolding) occurred (FIG. 10). However, these aggregates were easily removed by cation exchange chromatography (CEX), and further aggregation did not occur after CEX purification. Stability testing of ACE-05 using thermal stability analysis and exposure to various pH conditions (pH6-8) or long-term incubation at room temperature (7 days) showed that ACE-05 was of a stable and homogeneous structure similar to IgG ( 11 and 12).

T 세포 결합자로서 ALiCE의 결합 카이네틱스 및 동시 결합능Binding kinetics and simultaneous binding ability of ALiCE as a T cell combiner

The distance between the outer and inner binding domains of ALiCE was found to be about 60 Å, which is the likely distance for the formation of an immunological synaptic bridge between the tumor and effector cells (Arnett, KL et.al. , Proc Natl Acad. Sci USA , 101:16268-16273, 2004) (Figures 3 and 13). These findings suggest that unlike monovalent BiTE, two Fab arms of ALiCE can preferentially bind to tumor antigens with high avidity, followed by stem Fv engineered to a second antigen on tumor-penetrating T cells. Means can be combined.

Thus, ALiCE, acting as a tumor-specific T cell linker, can potentially improve anti-tumor efficacy while reducing off-target T-cell cytotoxicity. To test this, the binding kinetics of ACE-05 (anti-PD-L1 Fab × anti-CD3 Fv) and ACE-31 (anti-CD3 Fab × anti-PD-L1 Fv) to PD-L1 and CD3 were evaluated. Analysis by surface plasmon resonance (SPR) using the Biacore 8K system (GE Healthcare), YBL-007 (anti-PD-L1 parent antibody), UCHT1 (anti-CD3 parent antibody) against the same antigen ) And BiTE-05 (anti-PD-L1 × anti-CD3) were compared with the binding kinetics (FIGS. 14 and 15 ).

The present inventors found that the binding affinity (K _D ) of ACE-05 for PD-L1 (6.78 × 10 ^-10 M) was similar to that of YBL-007 (6.46 × 10 ^-10 M), but BiTE-05 It was confirmed to be higher than the binding affinity of (1.39 × 10 ^{-9 M).} This is probably due to the two PD-L1 binding sites in ACE-05 and YBL-007. Similarly, the binding affinity for CD3 of both ACE-31 (2.39 × 10 ^-10 M) and UCHT1 (2.65 × 10 ^{-10 M), including bivalent anti-CD3 Fab cancer, is BiTE-05 (1.01 ×} It was higher than the binding affinity of ^{10 -9 M).}

In contrast, the binding affinity of the monovalent stem Fv of ACE-05 for CD3 (2.15 × 10 ^-8 M) and ACE-31 (2.72 × 10 ^{-8 M) for PD-L1 is the parent antibody for CD3, respectively.} The binding affinity of UCHT1 (2.65 × 10 ^-10 M) and that of the parent antibody YBL-007 for PD-L1 (6.46 × 10 ^-10 M) were 40 to 80 fold lower. In addition, the binding affinity of the stem Fv of ACE-05 to CD3 and of ACE-31 to PD-L1 is the binding affinity of BiTE-05 to CD3 (1.01 × 10 ^-9 M) and BiTE to PD-L1. It was much lower than the binding affinity of -05 (1.39 × 10 ^-9 M), which may be due to steric hindrance between the Fab cancer and the second antigen-binding region of stem Fv.

However, using biolayer light interferometry (BLI) and flow cytometry (GE Healthcare) of the Octet QKe system (Pall Forte Bio), the present inventors demonstrated simultaneous binding of ACE-05 and ACE-31 to both PD-L1 and CD3. Was confirmed (FIGS. 16 and 17 ), which indicates that the binding affinity for each of the two antigens is dependent on the valency of the ALiCE paratope (an antibody molecule site attached to the antigen).

Next, the present inventors investigated the effect of the valence of ALiCE paratope on tumor and T-cell binding. The apparent binding affinity of ACE-05 or ACE-31 for PD-L1 of Karpas-299 tumor cells and CD3 of Jurkat T cells was consistent with the in vitro binding affinity for PD-L1 and CD3.

Specifically, ACE-05 compared to the binding affinity of the corresponding ACE-31 ( _{K D} =20.58 nM ^{for PD-L1 +} Karpas-299 cells _{and K D} ^{=856 pM for CD3 +} Jurkat T cells), PD It showed stronger binding to -L1 (PD-L1 ⁺ _{K D} =31.45 pM for Karpas-299 cells) and weaker binding to CD3 ( _{K D} =52.44 nM ^{for CD3 +} Jurkat T cells). On the other hand, both ACE-05 and ACE-31 ^{did not bind to PD-L1-} Raji cells (FIG. 18A).

ACE-05의 종양세포 사멸 효과 향상 및 비-특이적 T-세포의 독성 감소Enhancement of tumor cell killing effect of ACE-05 and reduction of non-specific T-cell toxicity

To compare the on-target and off-target T-cell activation by ACE-05, ACE-31 and BiTE-05, the present inventors described wild-type (WT) PD-L1 ^- HEK cells or maintain a fully operating the PD-L1 ⁺ HEK cells NFAT- luciferase reporter gene PD-1 expressing ^- Jurkat T cells were incubated with the ball (19). Then, after treatment with the T-cell binding agents ACE-05, ACE-31 or BiTE-05, NFAT-luciferase reporter activity was measured to evaluate Jurkat T cell activation.

Interestingly, ACE-05 showed the highest on-target NFAT activation when co-cultured with ^{PD-L1 +} HEK cells and PD-1 ^{-Jurkat T cells.} On the other hand, BiTE-05 and ACE-31 showed higher off-target T-cell activation than ACE-05 when co-cultured with ^{WT PD-L1-} HEK cells and PD-1 ^{-Jurkat T cells.} , It is mediated by direct binding to CD3 on Jurkat T cells in the absence of PD-L1 targeting (FIG. 20 ).

Although the binding affinity is different, as described above, since ACE-05, ACE-31, BiTE-05 and YBL-007 bound to PD-L1, the present inventors stably expressed PD-1 and NFAT-luciferase reporters. The ability to inhibit PD-L1/PD-1 activity was evaluated using Jurkat T cells and CHO-K1 cells expressing human PD-L1 and engineered cell surface proteins. At this time, the engineered cell surface protein was designed to activate cognate T-cell receptor (TCR) in an antigen-independent manner (Cheng, ZJJ et al., Cancer Res ., 75 (2015)).

In addition, T-cell activation was higher after treatment with ACE-05 or BiTE-05 during cell culture than after treatment with YBL-007, which can only interfere with PD-L1/PD-1 interaction (FIG. 21). These results are attributed to the dual-target specificity of ACE-05 and BiTE-05, which can simultaneously inhibit PD-L1/PD-1 interaction and PD-L1/CD3-mediated T-cell redirection. see.

_{In addition, the T cell activation efficiency (EC 50} =0.21 nM) of ACE-05 containing the bivalent anti-PD-L1 Fab cancer was determined by the T cell activation efficiency of BiTE-05 containing the monovalent anti-PD-L1 scFv ( EC ₅₀ =1.09 nM) 5 times higher. However, ACE-31 is much less effective than ACE-05, BiTE-05 and YBL-007 in re-activating T cells, and the affinity of ACE-31 for PD-L1 is low compared to the other three molecules. Reflects (Fig. 21).

Next, the present inventors used human effector cells (peripheral blood mononuclear cells (PBMC) or isolated T cells) to ^{ACE- for PD-L1 +} tumor cells (HCC827 or MDA-MB-231). 05, the cytolytic ability of ACE-31 and BiTE-05 was compared. ^{Consistent with NFAT reporter analysis of PD-L1 +} Jurkat T cells co-cultured with engineered CHO-K1 cells, incubation of effector cells (PBMC or CD3 ⁺ T cells) and ACE-05 was performed using PD-L1 ⁺ HCC827 (EC ₅₀ =1.97 pM) and MDA-MB-231 (EC ₅₀ =8.37 pM) cells showed the strongest target killing activity (FIGS. 22 to 26 ).

Since CD8 ⁺ cytotoxic T cells are often regarded as major effector cells in solid tumors, we tested whether ^{CD8 + T cells isolated from PBMCs were cytotoxic upon ACE-05 treatment.} Consistently, ^{incubation of CD8 +} T cells and PD-L1 ⁺ MDA-MB-231 and ACE-05 showed the most potent cytolytic activity, and direct proteolysis of target tumor cells in cooperation with perforin. ) And caspase-mediated apoptosis inducing granzyme B (Granzyme B) showed the highest level of secretion (FIGS. 27 and 28).

In addition, the present inventors investigated whether ACE-05 can stimulate the activation and expansion of human effector cells in the presence ^{of PD-L1 + tumor cells.} To this end, the present inventors ^{cultured human CD3 +} T cells with PD-L1 ⁺ MDA-MB-231 cells and 1 nM ACE-05 or IgG for 24 hours, and then monitored the surface expression of the activation markers CD69 and CD25. ^{Initial activation marker CD69 was upregulated in both CD4 +} and CD8 ⁺ T cells in the presence of ACE-05 and PD-L1 ⁺ tumor cells, but not by IgG (FIG. 29 ).

In addition, CD25, a late activation marker, ^{was highly upregulated in CD3 +} T cells by ACE-05 in the presence ^{of PD-L1 +} tumor cells (FIG. 30 ). Activation of T cells and subsequent differentiation into effector cells are also associated with T-cell clustering and aggregation (Zhou, J. et al. , PLoS One , 13:e0191634, 2018). Therefore, the present inventors cultured CytoLight-stained human CD3 ⁺ T cells with PD-L1 ⁺ MDA-MB-231 cells and 1 nM ACE-05, ACE-31, BiTE-05 or IgG for 90 hours and then cluster The area was measured.

As a result, it was confirmed that ACE-05 ^{stimulates clustering of CD3 +} T cells with respect ^{to PD-L1 +} tumor cells, and can induce T-cell activation more effectively than ACE-31 or BiTE-05 (FIG. 31 ). Moreover, ACE-05 ^{strongly induced CD3 +} T cell proliferation/expansion (Fig. 32).

On the other hand, the on-target tumor-killing ability of ACE-05 was higher than that of BiTE-05 for the two cancer cell lines (HCC827 and MDA-MB-231), but the present inventors released It was unexpectedly confirmed that interleukin (IL)-2 and interferon (IFN)-γ were significantly higher in PBMC treated with BiTE-05 than in the group treated with ACE-05 (FIG. 33). These results show that cytokines released from activated effector cells are responsible for T-cell cytotoxicity, but ^{tumor-killing activity in the presence of PD-L1 +} tumor cells is bispecific T-cell binding to PD-L1 in tumor cells. It depends on the affinity of the child, suggesting that it results in the redirection of T cells.

In order to further confirm this assumption, the inventors of the present invention have different binding affinity for CD3 (ACE-05> ACE-49> ACE-47> ACE-56), but ACE with the same binding affinity for PD-L1. The -05 variant was prepared (Figure 34). ACE-05, which has the highest affinity for CD3, showed the highest off-target T-cell activation in the NFAT reporter assay, and non-target T-cell activation was found in CD3 for the ACE-05 variant. The binding affinity for this decreased continuously with decreasing.

^{Surprisingly, the target tumor killing ability of CD3 +} T cells in the presence of PD-L1 ⁺ tumor cells was similar for the ACE-05 and ACE-05 variants (FIGS. 35 and 36 ). Previous results on the uncoupling of T cytolytic activity and toxic cytokine release by anti-HER2/CD3 bispecific antibodies (Li, J. et al. , Sci Transl Med , 11 (2019)) Consistent with, cytokine release can be disrupted with the T cell cytolytic activity of ACE-05, which can be mediated primarily by secreted granzyme B and perforin. These observations collectively suggest that, as long as ALiCE binds to CD3 with low affinity, the ability of ALiCE to bind to tumor PD-L1 for T-cell binding and immune-checkout inhibition is more important for anti-tumor effects. .

To evaluate non-target T cell activation by ACE-05, ACE-31 and BiTE-05, ACE-05, ACE-31 or BiTE- on ^{CD4 +} or CD8 ^{+ T cells in the absence of PD-L1+ tumor cells.} After treatment with 05, the secretion of cytokines IL-2 and IFN-γ was measured. Interestingly, despite the fact that the affinity of BiTE-05 for CD3 is lower than that of ACE-31, only BiTE-05 strongly induced the secretion of IL-2 and IFN-γ from ^{CD4 + T cells (Figure 37} ; IL-6 and TNF-α were not detected).

It has been reported that clustering and/or multimerization of CD3 induced by CD3 antibody binding is more important for T-cell activation than for CD3 binding affinity of CD3 antibodies (Minguet, S. et.al. , Immunity). , 26:43-54, 2007). Unlike ACE-05 and ACE-31, which are homogeneous heterotetrameric complexes, gel filtration analysis showed that the BiTE-05 formulation contained various misfolded multimeric forms (FIG. 38). Indeed, this multimeric component of BiTE-05 can directly activate CD4+ and CD8+ T cells without tumor cells (Figure 39), and nonspecific non-target T-cytotoxicity, or cytokine-release syndrome. , CRS).

In addition, ^{PD-L1 +} tumor-specific T-cell cytotoxicity of ACE-05 was confirmed by tumor-killing analysis using ^{CD3 +} T cells and PD-L1 ^{-Raji tumor cells isolated from PBMCs (FIG. 40).} Taken together, these findings suggest that the higher affinity of ACE-05 for tumor cells increased on-target T-cell activation, and the homogeneous heterotetrameric ACE-05 was reduced compared to BiTE-05. It was shown to show T-cell toxicity.

인간화 마우스 모델에서 ALiCE의 생체 내 항-종양 효능In vivo anti-tumor efficacy of ALiCE in a humanized mouse model

The in vivo anti-tumor efficacy of ALiCE was investigated in PBMC-reconstituted NCG mice transplanted with ^{PD-L1 + HCC827 tumor cells.} Briefly, PBMCs isolated from two healthy donors were implanted into female NCG mice 3 days before subcutaneous inoculation of PD-L1 ^{+ HCC827 tumor cells in the right posterior flank.} When the tumor is palpable (day 4), mice are given 3 doses of ACE-05 or BiTE-05 (0.5 mg/kg body weight), or 3 doses of YBL-007 or IgG (5.0 mg/kg body weight). It was injected intravenously.

In 9 of 10 mice treated with ACE-05, the pre-established tumors completely regressed on day 12 (FIG. 41 ). In addition, the present inventors monitored body weight changes as signs of side effects. Mice treated with BiTE-05 showed a significant decrease in body weight (~20%), whereas mice treated with ACE-05 or YBL-007 did not show significant weight loss (FIG. 42 ).

The present inventors speculated that the weight loss in the mice treated with BiTE-05 was due to the rapid increase in cytokine release after the first administration of BiTE-05. Moreover, pharmacokinetic analysis showed that ACE-05 had a much longer half-life than BiTE-05 in rats and cynomolgus monkeys (FIG. 43 ). ^{Dose de-escalating analysis revealed that PD-L1 +} HCC827 tumors completely regressed even at very low doses of ACE-05 (0.05 mg/kg body weight) (FIG. 44).

The present inventors further investigated the in vivo T cytotoxicity and anti-tumor efficacy of ACE-05 and BiTE-05 in hCD3ε transgenic (TG) mice, where the T cells were genetically engineered to express both hCD3ε and mCD3ε. Became. Both IL-2 and IFN-γ are pleiotropic cytokines, important effector molecules for anti-tumor immunity through various mechanisms.

However, immune-related side effects of IL-2 and immune-avoidance function of IFN-γ have also been reported (Berraondo, P. et. al. , Br J Cancer , 120:6-15, 2019). In addition, pro-inflammatory cytokines IL-6 and TNF-α, which are released from activated antigen presenting cells (APCs) such as macrophages, dendritic cells or B cells, are central mediators of cytokine-releasing syndrome (CRS) toxicity. It has been proposed (Shimabukuro-Vornhagen, A. et. al. , J Immunother Cancer , 6, 56 (2018)). Therefore, to evaluate non-target T-cell toxicity, we injected a single dose of ACE-05, BiTE-05 or IgG into hCD3ε TG mice and administered serum cytokines, IL-2, IFN-γ every 6 hours. , IL-6 and TNF-α were measured.

Similar to the cytokine release observed in co-culture of PBMC and HCC827 cells, non-tumor-bearing hCD3ε mice received higher levels of cytokines than ACE-05 when administered BiTE-05, particularly The release of IL-2, IFN-γ and IL-6 was induced, and the highest cytokine release level was shown 6 hours after administration (FIG. 45). In addition, the non-tumor bearing hCD3ε mice treated with BiTE-05 showed severe weight loss, whereas the mice treated with ACE-05 or IgG showed much less weight loss (Figure 46), and the low of ACE-05. Non-target cytotoxicity was demonstrated.

Consistent with the anti-tumor efficacy of ACE-05 in PBMC-reconstituted NCG mice transplanted with PD-L1 ⁺ ^{HCC827 tumor cells, hCD3ε TG mice transplanted with hPD-L1 +} CT26 tumors treated with ACE-05 It was confirmed that the tumor size was effectively reduced so as not to cause a significant change in body weight, and complete regression was caused in 1 out of 6 mice by the 15th day (FIGS. 47 and 48). Thereafter, the present inventors collected tumors from each mouse at the end of the study, and analyzed tumor-infiltrating lymphocytes (TIL).

Unlike the tumor samples collected from the YBL-007 treatment group and the UCHT1 treatment group, in the case of the ACE-05 treatment group, only two tumor samples could be analyzed due to the small size and weight of the remaining tumor (FIG. 49 ). ^{Interestingly, it was confirmed that the number of viable CD45 +} lymphocytes and CD3 ⁺ T cells in the tumor microenvironment increased when ACE-05 was treated compared to UCHT1 (FIGS. 50 and 51). In addition, ACE-05 and YBL-007 is a CD4 ⁺ T cells are not induced the proliferation and expansion of CD8 ⁺ T cells (Fig. 52 and Fig. 53), which possibly PD-1 and PD-L1 be due to inhibition of the interaction (Beyrend, G. et. al. , J Immunother Cancer , 7 , 217 (2019)).

The present inventors also investigated the T-cell class I MHC immunogenicity of the ACE-05-HC-VH and ACE-05-HC-VL chains using an in silico immunogenicity prediction tool. The immunogenicity of ACE-05-LC was not analyzed because it was identical to that of the native antibody LC. First, the present inventors investigated peptides treated with ACE-05-HC-VH and ACE-05-HC-VL that can be presented on MHC class I molecules using a percentile ranking (based on cut-off value of 0.3). (Fig. 54 and Fig. 55, left).

Thereafter, the collected peptide sequence was analyzed using MHC-I binding prediction to determine the immunogenicity score. Immunogenic peptides (score> 0) possible within the ACE-05-HC-VH and ACE-05-HC-VL chains are listed and shown in FIGS. 56 and 57 (right). Potential immunogenic peptides of the ACE-05-HC-VH and ACE-05-HC-VL chains were mainly found in the CDRs, FRs and CH1s within the variable domains.

The artificial junction connecting the hinge of ACE-05 and the second VH or VL has not been found to be an immunogenic epitope. Taken together, these results show that ACE-05 has improved anti-tumor efficacy with less promiscuous cytotoxicity through tumor-specific on-target T-cell activation and prolonged pharmacokinetic profile, indicating that PD on tumor cells On the high-affinity binding of the bivalent anti-PD-L1 Fab arm of ACE-05 to -L1 and the low-affinity binding of the monovalent anti-CD3 stem Fv to CD3 on T cells. It proved to be due.

다중 특이성 융합 단백질을 코딩하는 폴리뉴클레오티드Polynucleotides encoding multispecific fusion proteins

Another aspect of the present invention is to provide a polynucleotide encoding the structural formula (I), (I'), (I''), (II), (II') or (II'').

The polynucleotide may be in the form of RNA or DNA. It may be double-stranded or single-stranded, and if single-stranded, it may be a coding strand or a non-coding (antisense) strand.

Additionally, the polynucleotide may include a sequence encoding a marker or tag sequence. One embodiment of the tag sequence is a hexa-histidine tag.

When preparing by chemically synthesizing a nucleotide sequence, a synthetic method well known in the art, for example, a method described in Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988, can be used. , Tryester, phosphite, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods, and oligonucleotide synthesis methods on a solid support.

According to one embodiment, the polypeptide is at least about 70% with a polynucleotide encoding the formula (I), (I'), (I''), (II), (II') or (II''), At least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, At least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity. have.

In addition, the polynucleotide may additionally include a nucleic acid encoding a signal sequence or a leader sequence. The term "signal sequence" as used herein refers to a signal peptide that directs the secretion of a protein of interest. The signal peptide is cleaved after translation in the host cell. Specifically, the signal sequence is an amino acid sequence that initiates the movement of a protein through the ER (endoplasmic reticulum) membrane.

The signal sequence is well known in the art, and usually includes 16 to 30 amino acid residues, but may include more or less amino acid residues. A typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region contains 4 to 12 hydrophobic residues that fix the signal sequence through the membrane lipid bilayer during migration of the immature polypeptide.

After initiation, the signal sequence is cleaved within the lumen of the ER by cellular enzymes commonly known as signal peptidases. In this case, the signal sequence may be a tissue plasma activation (tPa), a signal sequence of Herpes simplex virus glycoprotein D (HSV gDs), or a secretion signal sequence of growth hormone. Preferably, a secretion signal sequence used in higher eukaryotic cells including mammals and the like can be used. In addition, it can be used by substituting codons with high expression frequency in host cells.

융합 단백질을 코딩하는 폴리뉴클레오티드가 적재된 벡터Vector loaded with polynucleotides encoding fusion proteins

Another aspect of the present invention is a vector comprising a polynucleotide encoding the above structural formula (I), (I'), (I''), (II), (II') or (II''). to provide.

The vector can be introduced into a host cell and recombined and inserted into the host cell genome. Or the vector is understood as a nucleic acid means comprising a polynucleotide sequence capable of spontaneously replicating as an episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

Specifically, the vector may be plasmid DNA, phage DNA, etc., commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), E. coli-derived plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis S-derived plasmids (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal virus vectors (retrovirus (retrovirus) ), adenovirus, vaccinia virus, etc.), insect virus vectors (baculovirus, etc.). Since the vector has different expression levels and modifications of proteins depending on the host cell, it is preferable to select and use the most suitable host cell for the purpose.

As used herein, the term "gene expression" or "expression" of a protein of interest is understood to mean transcription of a DNA sequence, translation of an mRNA transcript, and secretion of a fusion protein product or fragment thereof. Useful expression vectors may be RcCMV (Invitrogen, Carlsbad) or a variant thereof. The expression vector includes a human CMV (cytomegalovirus) promoter for promoting the continuous transcription of a target gene in mammalian cells, and a bovine growth hormone polyadenylation signal sequence for increasing the stable state level of RNA after transcription. can do.

융합 단백질을 발현하는 형질전환된 세포Transformed cells expressing the fusion protein

Another aspect of the present invention provides a transformed cell into which the vector has been introduced.

Host cells of the transformed cells may include, but are not limited to, prokaryotic cells, eukaryotic cells, mammals, plants, insects, fungi, or cells of cellular origin. As an example of the prokaryotic cell, E. coli may be used. In addition, yeast may be used as an example of eukaryotic cells. In addition, CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK 293 cells, HEK293T cells, etc. can be used as the mammalian cells. , It is not limited thereto, and all cells that can be used as mammalian host cells known to those skilled in the art may be used.

In addition, when introducing an expression vector into a host cell, the Hanahan method, electroporation, calcium phosphate precipitation method, which improved efficiency by using a reducing material called dimethyl sulfoxide (DMSO) in _{CaCl 2} precipitation method and CaCl _{2 precipitation method.} , Protoplasm fusion method, agitation method using silicon carbide fibers, Agrobacteria mediated transformation method, transformation method using PEG, dextran sulfate, lipofectamine and drying/inhibition mediated transformation method, and the like may be used.

As described above, the glycosylation-related gene of the host cell for optimizing the properties of the fusion protein as a therapeutic agent or for other purposes is manipulated through a method known to those skilled in the art, and the sugar chain pattern of the fusion protein (e.g., Sialic acid, fucosylation, saccharification) can be adjusted.

융합 단백질을 포함하는 약학 조성물Pharmaceutical composition comprising a fusion protein

Another aspect of the present invention is to provide a pharmaceutical composition for the treatment or prevention of cancer comprising the above-described multi-specific fusion protein as an active ingredient.

Specifically, the cancer is gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myelogenous leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and It may be any one selected from the group consisting of lymphoma.

In this case, the cancer may be overexpression of any one protein selected from the group consisting of PD-L1, EGFR, and HER2.

The preferred dosage of the pharmaceutical composition varies depending on the condition and weight of the patient, the degree of disease, the form of the drug, the route and duration of administration, but may be appropriately selected by those skilled in the art. In the pharmaceutical composition for the treatment or prevention of cancer or infectious diseases of the present invention, the active ingredient is an arbitrary amount (effective amount) according to the use, formulation, purpose of combination, etc., as long as it exhibits anti-cancer activity or can exhibit a therapeutic effect on infectious diseases. A typical effective amount will be determined within the range of 0.001% to 20.0% by weight based on the total weight of the composition. Here, "effective amount" refers to an amount of an active ingredient capable of inducing an anticancer effect or an infectious disease treatment effect. Such effective amounts can be determined empirically within the range of ordinary skill in the art.

As used herein, the term “treatment” may be used to include both therapeutic treatment and prophylactic treatment. In this case, prevention may be used in the sense of alleviating or reducing a pathological condition or disease of an individual. In one embodiment, the term “treatment” encompasses any form of dosage or application to treat a disease in mammals, including humans. In addition, the term includes inhibiting or slowing the progression of a disease or disease; Restore or repair impaired or impaired function to partially or completely alleviate the disease; Or stimulating an inefficient process; Includes the meaning of alleviating the severity of the disease.

As used herein, the term “efficacy” refers to one or more parameters such as survival over a period of time, such as 1, 5, or 10 years, or disease-free survival. Can be determined. In addition, the parameter may include suppressing the size of at least one tumor in the individual.

Pharmacokinetic parameters such as bioavailability and underlying parameters such as clearance rate can also affect efficacy. Thus, “enhanced efficacy” (eg, improved efficacy) can be attributed to improved pharmacokinetic parameters and improved efficacy, comparing clearance rates and tumor growth in test animals or human subjects, or survival, recurrence rates or disease. It can be measured by comparing parameters such as survival in the absence.

Herein, "therapeutically effective amount" or "pharmaceutically effective amount" is an amount of a compound or composition effective to prevent or treat the subject disease, which is sufficient to treat the disease at a reasonable benefit/risk ratio applicable to medical treatment, and It means the amount that does not cause side effects. The level of the effective amount is the health condition of the patient, the type of disease, the severity, the activity of the drug, the sensitivity to the drug, the method of administration, the time of administration, the route of administration and the rate of excretion, the duration of treatment, factors including the drugs used in combination or concurrently, and Other factors well known in the medical field can be determined. In one embodiment, a therapeutically effective amount refers to an amount of a drug effective to treat cancer.

In this case, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any carrier as long as it is a non-toxic material suitable for delivery to a patient. Distilled water, alcohols, fats, waxes and inert solids may be included as carriers. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.

Specifically, the pharmaceutical composition may be prepared in a parenteral formulation according to an administration route by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, "pharmaceutically acceptable" means that the application (prescription) does not have toxicity beyond adaptable without inhibiting the activity of the active ingredient.

When the pharmaceutical composition is prepared in a parenteral formulation, it may be formulated in the form of an injection, a transdermal administration, a nasal inhalation, and a suppository according to a method known in the art together with a suitable carrier. When formulated as an injection, sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof may be used as suitable carriers, preferably Ringer's solution, PBS (phosphate buffered saline) containing triethanol amine, or sterilization for injection. Water, isotonic solutions such as 5% dextrose, etc. can be used. Regarding the formulation of a pharmaceutical composition, it is known in the art, and specifically, Remington's Pharmaceutical Sciences (19th ed., 1995), etc. may be referred to. This document is considered part of this specification.

The preferred dosage of the pharmaceutical composition may be in the range of 0.01 µg to 10 g per 1 kg of body weight per day, or in the range of 0.01 mg to 1 g, depending on the patient's condition, weight, sex, age, patient severity, and route of administration. Administration can be made once a day or divided into several times. Such dosage should not be construed as limiting the scope of the invention in any aspect.

Subjects to which the pharmaceutical composition can be applied (prescribed) are mammals and humans, particularly preferably humans. In addition to the active ingredient, the pharmaceutical composition of the present application may further include any compound or natural extract, which has already been verified for safety and has a therapeutic effect on anti-cancer activity or infectious disease, in order to increase and reinforce anti-cancer activity.

병용 투여용 약학 조성물Pharmaceutical composition for combined administration

Another aspect of the present invention provides a pharmaceutical composition further comprising the above-described multispecific fusion protein and an anticancer agent.

The fusion protein includes a pharmaceutically acceptable carrier. At this time, the pharmaceutical composition may be formulated to be suitable for the route of administration to the subject.

다중 특이적 융합 단백질의 용도Use of multispecific fusion proteins

Another aspect of the present invention provides the use of the multispecific fusion protein for treating or preventing cancer.

The cancer is gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myelogenous leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer and lymphoma. Can be selected from the group.

Another aspect of the present invention provides the use of the multispecific fusion protein for use as a cell engager.

암 예방 및 치료 방법Cancer prevention and treatment methods

Another aspect of the present invention provides a method for treating or preventing cancer comprising administering the multispecific fusion protein to an individual.

The subject may be a subject suffering from cancer or an infectious disease. In addition, the individual may be a mammal, preferably a human. The multispecific fusion protein is as described above.

The multispecific fusion protein may be administered to a subject in various methods and amounts depending on the condition of the patient and the presence or absence of side effects, and the optimal administration route, dosage, and frequency of administration may be selected within an appropriate range by a person skilled in the art. In addition, the fusion protein may be administered in combination with other drugs or physiologically active substances known to have a therapeutic effect on the disease to be treated, or may be formulated in the form of a combination formulation with other drugs.

융합 단백질 생산 방법Fusion protein production method

Another aspect of the present invention provides a method for producing a multispecific fusion protein comprising culturing the transformed cells described above. Specifically, the production method comprises the steps of: i) culturing the transformed cells to obtain a culture; And ii) recovering the fusion protein from the culture.

The method of culturing the transformed cells may be performed using a method well known in the art. Specifically, the culture may be continuously cultured in a batch process or in a fed batch or repeated fed batch process.

Any method known in the art for purification of immunoglobulins to recover the fusion protein, for example chromatography (ion exchange, affinity, especially Protein A, sizing column chromatography and kappa-select affinity chromatography Thereafter, in the case of specific antigens, purification by affinity), centrifugation, differential solubility or other standard techniques for protein purification can be used. In certain embodiments, kappa-select (eg, kappa select developed by GE Healthcare Life Science) is used for purification of a Fab (kappa) fragment or a multispecific fusion protein containing a Fab fragment. Additionally, the multispecific fusion proteins provided herein can be fused to heterologous polypeptide sequences described herein or other sequences known in the art to facilitate purification.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

I. 다중 특이적 융합 단백질의 제조I. Preparation of multiple specific fusion proteins

실시예 1. 다중 특이적 융합 단백질 ALiCE의 제조Example 1. Preparation of multispecific fusion protein ALiCE

ALiCE variants ACE-05 (anti-PD-L1 Fab × anti-CD3 Fv), ACE-31 (anti-CD3 Fab × anti-PD-L1 Fv), ACE-18 (anti-CD20 Fab × CD3 Fv) and HC pair of ACE-00 (anti-HER2 Fab × anti-TNF-α Fv), ACE-HC-VH and ACE-HC-VL are parent antibodies YBL-007 (anti-PD-L1), UCHT1 (Anti-CD3), Rituximab (anti-CD20), Herceptin (anti-HER2) and Humira (anti-TNF-α) were used as templates, and VHA-CH1-hinge, VHB and VLB were encoded The sequence was amplified by PCR.

Two PCR fragment pairs VHA-VHB and CH1- hinge or VHA-VLB and CH1- hinge according to the manufacturer's protocol Electra ^TM cloning system ^{(Electra TM cloning system; ATUM)} (Engler, C. et.al., PloS one , 3 , e3647 (2008)) was used to sub-cloning into the mammalian expression vector p293F prepared in-house. Then, it was transformed into DH5α competent cells (# CP010, Enzynomics). PCR primers for constructing ALiCE HC were designed to contain a short linker (G4S) between the VHB or VLB at the hinge and 5'end, and the Sap ^{I restriction site required for the Electra TM} cloning system at the 3'end.

ACE-LC, the LC used to construct ALiCE, was the same as the change in light of the parent antibody. The sequence encoding the LC containing the leader peptide at the N-terminus was PCR-amplified and subcloned into the p293 expression vector containing the Nhe I/ Xho I restriction site. The resulting constructs were transformed with DH5α competent cells (# CP010, Enzynomics) and confirmed through sequencing.

In addition, other ALiCE variants ACE-02, ACE-03, ACE-06, ACE-10, ACE-11, ACE-16, ACE-20, ACE-21, ACE-23, ACE-25, ACE-26, ACE-28, ACE-30, ACE-32 and ACE-33 were also cloned in the same manner as described above based on the sequence described in Example 21.

실시예 2. ALiCE의 발현 및 정제Example 2. Expression and purification of ALiCE

To produce ALiCE variants ACE-00, ACE-05, ACE-18, ACE-31 and BiTE-05 containing a 6×-His tag at the C-terminus, ACE-HC- in a 1:1:2 ratio Plasmids encoding VH, ACE-HC-VL and ACE-LC, or BiTE-05 were transferred to FreeStyle 293-F cells (#R79007, ThermoFisher) in a 1:4 (w/w) DNA to PEI ratio of polyethyleneimine ( PEI; #23996-1, Polysciences) was used to form a polyplex (Xie, Q. et.al. , Cytotechnology , 65:263-271, 2013). For transient transfection, 1 mg of plasmid DNA was added to 2×10 ⁶ After transfection with Freestyle 203-F cells, they were cultured while shaking (120 rpm) using FreeStyle 293 expression medium (#12338018, Gibco) under conditions of _{37° C. and 5% CO 2.}

ALiCE molecule and BiTE-05 are purified by centrifuging the culture at 4°C for 30 minutes at 4,800 rpm, and then filtering the supernatant using a 0.22 μm TOP-filter to remove debris. I did. The supernatant containing ALiCE molecules was loaded on a CaptureSelect CH1-XL pre-packed column (#494346201, ThermoFisher), and the supernatant containing BiTE-05 was Ni-NTA agar. It was loaded on OS resin (Ni-NTA agarose resin, # R90101, ThermoFisher).

The ALiCE molecule was eluted from the column using 0.1 M glycine (pH 3.0), and BiTE-05 was eluted using 3 M imidazole/20 mM sodium phosphate (pH 6.0). The eluted ALiCE molecules and BiTE-05 were dialyzed with phosphate buffered saline (PBS; pH 7.4) using Slide-A-Lyzer Dialysis Cassette Kits (#66372, ThermoFisher). For SPR binding kinetic assay (Surface plasmon resonance binding kinetic assay), a 6×-His-tag was removed from BiTE-05 using a Thrombin Cleavage Capture kit (#69022, Merck).

In addition, other ALiCE variants ACE-02, ACE-03, ACE-06, ACE-10, ACE-11, ACE-16, ACE-20, ACE-21, ACE-23, ACE-25, ACE-26, ACE-28, ACE-30, ACE-32 and ACE-33 were also obtained in the same manner as above.

실시예 3. ALiCE 구조 분석Example 3. ALiCE structure analysis

실험예 1. 항체 구조의 PyMOL 분석Experimental Example 1. PyMOL analysis of antibody structure

The structures of anti-CD3 antibody UCHT (PBD ID: 1XIW), anti-PD-L1 antibody dervalumab, and anti-CTLA-1 antibody ipilimumab (PBD ID: 5TRU) are described in Protein Data Bank (PBD, www.rcsb. org) and visualized using PyMOL software (Schrodinger, LLC The PyMOL Molecular Graphics System, Version 1.8. (2015)).

실험예 3.2. 이종 이량체 ALiCE의 구조 분석Experimental Example 3.2. Structural analysis of heterodimer ALiCE

The ALiCE molecule was SDS-PAGE; Fully automated capillary electrophoresis (CE) using an Agilent 2100 Bioanalyzer (Agilent Technology); Analytical Size-Exclusion Chromatography (SEC) using a Superdex 200A column (GE Healthcare Life Science); And analytical cation exchange chromatography (CEX) using a MabPac SCX-10 column (ThermoFisher). Heterodimer formation by two different ALiCE HCs was evaluated by analyzing ALiCE molecules by SDS-PAGE and CE under reducing and non-reducing conditions.

For CE analysis, the protein-analytical solution mixture was loaded onto a microfluidic protein chip and separated by molecular weight using the Bioanalyzer Protein 230 assay kit (Agilent) under reducing and non-reducing conditions according to the manufacturer's protocol. I did. In addition, the stoichiometric ratio of various chains in ACE-05 was determined under reducing conditions using a Bioanalyzer Protein 80 assay kit (Agilent).

The mean and relative standard deviation (% CV) of each chain's contribution (% total) to ACE-05 determined in five independent experiments were analyzed using 2100 Expert Software (Agilent Technology), and GraphPad Prism 8 software was used. Plotted using. The quality and morphology of ALiCE molecules were analyzed by SEC (buffer, PBS pH 7.4) using Superdex 200A column (GE Healthcare Life Science) and CEX (buffer, 50 mM sodium acetate pH 5.0) using MabPac SCX-10 column (ThermoFisher). I did. Data were plotted and analyzed using GraphPad Prism 8 software.

As a result, various VH and VL including anti-CD3 antibody UCHT1 (PDB ID: 1XIW), anti-PD-L1 antibody dervalumab (PDB ID: 5X8M) and anti-CTLA4 antibody ipilimumab (PDB ID: 5TRU) The crystal structure of the complex is clear that the key determinant of VH and VL interaction is the CDR3 region of VH, which forms a "knob-into-Hole" structure and binds to the CDR1, CDR2 and CDR3 regions of VL. Proved to be (Fig. 1). In addition, it was confirmed that the hydrophobic interaction surrounded by electrostatic interactions in the VH-VL interface contributes to the autonomous assembly of the VH and VL domains and the stabilization of the Fv complex (FIG. 2).

실험예 4. ALiCE의 질량 분석Experimental Example 4. ALiCE mass spectrometry

The molecular weight of purified ACE-05 was determined by liquid chromatography-electrospray ionization (LC-ESI/TOF) through time-of-flight (TOF) analysis using a ZORBAX 300SB-C8 (2.1×50 nm; Agilent) column. Confirmed. The formic acid concentration was constant over the entire run at 0.2%, while the mobile phase consisted of a gradient of water and acetonitrile over 35 minutes, starting with 5% acetonitrile (initial conditions) to 100% acetonitrile.

The flow rate was 0.1 ml/min (min). Mass spectrometry detection was performed using a micro A-TOF III mass spectrometer (Bruker Daltonics, Germany) using electrospray ionization (ESI) as a negative mode. The following MS parameters were used: capillary voltage, 4500 V; Nebulizer pressure, 0.8 psi; Dry gas flow, 5.5/min; Dry gas temperature, 190°C.

실험예 5. ALiCE의 안정성 분석Experimental Example 5. Stability Analysis of ALiCE

The thermal stability of ACE-05, BiTE-05, YBL-007 and UCHT1 was determined by Thermofluor assay (Lavinder, JJ et.al. , J Am Chem Soc , 131:3794-3795, 2009) using SYPRO orange dye (dye). Analyzed using. Specifically, 3 μM solution of each purified antibody was mixed with 10 μl of SYPRO orange dye (#S6650, ThermoFisher) diluted 1:25, and 50 μl of each mixture was incubated at 25° C. for 30 minutes. Samples were denatured by heating at a rate of 1°C/min (min) from room temperature to 99°C using a C100 Thermal Cycler, and the amount of denatured protein stained with CYPRO dye was recorded every minute (i.e., each 1°C temperature change). Melting temperature (TM) was calculated using the CFX 96 ORM system (BioRad). Protein stability was determined by exposing CEX-purified ACE-05 to various pH conditions (

pH

6, 20 mM sodium phosphate; pH 7.4, PBS;

pH

8, 20 mM Tris-HCl) or long-term incubation at room temperature (7 days). After the analysis was evaluated by CEX-HPLC using a MabPac SCX-10 analysis column (ThermoFisher). Data were plotted using GraphPad Prism 8 software.

실시예 6. ALiCE의 결합력 측정Example 6. Measurement of binding force of ALiCE

실시예 6.1. ALiCE 분자의 결합 카이네틱스 분석Example 6.1. Binding kinetics analysis of ALiCE molecules

Binding kinetics of ALiCE molecules to various antigens was measured by surface plasmon resonance using a Biacore 8K system equipped with a certified-grade CM5 series S sensor chip (# BR100399, GE Healthcare). HEPES-buffered saline (0.01 M HEPES, 0.15 M NaCl) containing 3 mM ethylenediaminetetraacetic acid (EDTA) and 0.05% (v/v) P20 surfactant (HBS-EP+) was added to the reaction and running buffer. (#BR100669, GE Healthcare).

Antigens PD-L1-his (0.1 μg/ml; self-synthesizing in-house) and CD3εδ-flag-his (0.2 μg/ml; #CT038-H2508H, Sino Biological) were assigned to the CM5 sensor chip according to the manufacturer's instructions. It was fixed on the surface of (#BR100399, GE Healthcare). Thereafter, bispecific T-cell linkers (ACE-05, BiTE-05, and ACE-31) and parent mAb (YBL-007 and UCHT1) diluted in HBS-EP + buffer were added. Sensors immobilized with antigens at 12 different concentrations (0, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256, and 512 nM) for 300 seconds at a flow rate of 30 μl/min (min) It was applied on a chip (antigen-immobilized sensor chip). The analyte bound to the sensor chip was dissociated by washing with HBS-EP + running buffer for 300 seconds.

Both binding (M ^-1 s ^-1 , K _a ) and dissociation (S ^-1 , K _d ) were measured at 300 second intervals. The equilibrium dissociation constant (M, K _D ) was calculated as the ratio of off-rate to on-rate (k _d /k _{a ).} Global fitting of Biacore Insight Evaluation Software using a 1:1 binding model for monovalent ligand-analyte interactions and a 1:2 binding model for bivalent ligand-analyte interactions. The kinetic parameters were determined by function.

실시예 6.2. 동시 결합 분석Example 6.2. Simultaneous binding analysis

For simultaneous double binding analysis of ACE-05 and ACE-31 to CD3 and PD-L1, biolayer light interferometry (BLI) was performed with the Octet QKe system (Pall Forte Bio) (Abdiche, Y. et.al. , Anal Biochem , 377:209-217, 2008). The first ligand, PD-L1-Fc (2 µg/ml; synthesized in-house) and CD3εδ-flag-his (3 µg/ml; #CT038-H2508H, Sino Biological) have a binding of 0.5 to 1.0 Each hydrated AHC (#18-5064, Pall Forte Bio) or Ni-NTA (#18-5013, Pall Forte Bio) biosensor was loaded until nM was reached.

Thereafter, the biosensor was washed with kinetics buffer (0.1% bovine serum albumin [BSA] and 0.02% Tween-20-containing PBS) for 2 minutes (ACE-05) or 1 minute (ACE-31) to obtain unbound protein. Was removed and immersed in 30 nM ACE-05 or 15 nM ACE-31 to analyze association, and was washed with PBS for 7 minutes (ACE-05) or 2 minutes (ACE-31) to measure dissociation. . Subsequently, a second ligand, that is, 200 nM CD3εδ-flag-his (ACE-05) or 120 nM PD-L1-Fc (ACE-31), was added to the biosensor loaded with the first ligand and ACE-05 or ACE-31. The binding was measured by immersion in the containing solution, followed by washing with PBS for 3 minutes (ACE-05) or 2 minutes (ACE-31) to measure dissociation. Sensorgram data was plotted using GraphPad Prism 8 software.

II. ALiCE의 효능 확인 : In vitro assayII. Confirmation of the efficacy of ALiCE: In vitro assay

실시예 7. 유세포 분석Example 7. Flow cytometry

In order to evaluate the level of cell surface PD-L1, we used both PD-L1 ⁺ cancer cells (HCC827, MDA-MB-231, and Karpas-299) and PD-L1 ^- Raji cells at 0.5×10 ⁶ cells/ It was 100 μl and incubated with an anti-PD-L1 antibody (#55817, BD Bioscience) conjugated with phycoerythrin (PE)-Cy7 diluted 1:50 (v/v). FITC (fluorescein isothiocyanate) conjugated anti-CD3 antibody (#130-113-138, Miltenyi Biotech) was used ^{to evaluate CD3 levels on Jurkat T cells (0.5×10 6} cells/100 μl).

Cells were washed twice with 1 ml of FACS buffer (PBS containing 1% fetal bovine serum [FBS]), and cell surface expression of PD-L1 or CD3 was analyzed by flow cytometry (BD FACSCanto II). The double binding capacity of ACE-05 and ACE-31 to PD-L1 and CD3 on the cell surface was determined by CD3 ⁺ Jurkat T cells and 20 nM ACE-05, ACE-31 or ACE-18 (CD20 Fab × CD3 Fv, Ctrl-ACE). ) Was investigated by incubating for 1 hour. After washing twice with 1 ml of FACS buffer, cells were incubated with PD-L1-Fc (75 μg/100 μl) and washed twice with 1 ml of FACS buffer.

Thereafter, PD-L1-Fc was detected using an anti-human Fc antibody conjugated with Alexa 647 (#109-605-098, Jackson ImmunoResearch). The apparent binding affinity of ALiCE (ACE-05 and ACE-31) to PD-L1 ⁺ Karpas-299 and PD-L1 ^- Raji cancer cells and CD3 ⁺ ^{Jurkat T cells was determined by the above cells (0.5×10 6} cells/100). Μl) in ACE-05 (0.000932, 0.003729, 0.014915, 0.059662, 0.59459, 3.81, and 15.27 nM for Raji cells; 1.56, 15.625, 156.25, and 1562 nM for Raji cells; 1.1, 3.3 for Jurkat cells) , 9.9, 29.6, 88.9, 266.7, 800, and 2400 nM) or ACE-31 (0.594, 3.81, 15.27, 61.03, 244.37, 977.5, and 3910 nM for Karpas-299 cells; 1.56, 15.625, for Raji cells; 156.25, and 1562 nM; for Jurkat cells 0.011, 0.033, 0.101, 0.304, 0.914, 2.743, 8.320, 24.691, and 74.074 nM) were measured by treatment at the indicated concentrations.

ACE-05 or ACE-31 bound to the cell surface was detected with a flow cytometer (CytoPLEX-LX) after incubation with an antibody conjugated with Alexa 647 to a human Fab fragment (#109-606-097, Jackson Immunoresearch). . Flow cytometric data was analyzed using FlowJo 10 software (FlowJo, LLC), and geometric mean plotted using GraphPad Prism 8 software.

실시예 8. ALiCE(ACE-05 및 ACE-31)에 의한 Jurkat T 세포 활성화 분석Example 8. Analysis of Jurkat T cell activation by ALiCE (ACE-05 and ACE-31)

To evaluate whether ACE-05 and ACE-31 redirect T-cell activation to PD-L1 on the target cell surface, PD-L1 ^- WT HEK cells or genetically engineered PD-L1 ⁺ HEK Cells (7×10 ⁴ cells/well) were seeded on a white-bottom plate coated with poly-L-lysine (#P4707, Sigma). After pre-culturing the cells for 24 hours, PD-1 expressing firefly luciferase gene ^- Jurkat T cells (2×10 ⁵ cells/well) and ACE- under the control of NFAT-responsive elements 05, serial dilutions of ACE-31 or BiTE-05 were added, and the cells were incubated for 6 hours under conditions of _{37°C and 5% CO 2.} Luciferase accumulation induced by T cell activation was measured by performing a Bio-Glo Luciferase assay (#G7940, Promega) according to the manufacturer's protocol. The resulting data expressed in relative light units (RLU) was plotted and analyzed using GraphPad Prism 8 software.

실시예 9. 표적 T-세포 활성화 분석Example 9. Target T-cell activation assay

To evaluate on-target specific T cell activation, PD-L1 ⁺ MDA-BM-231 cancer cells (1×10 ⁵ cells/well) and human PBMC (#SER- PBMC-200-F, Zenbio) isolated CD3 ⁺ T cells (1×10 ⁶ cells/well) were co-cultured, but 1 nM ACE-05, BiTE-05, ACE-31 or IgG was added. CD3 ⁺ T cells were stained with Trace Far Red (#C34564, ThermoFisher) according to the manufacturer's instructions. After incubation for 24 or 48 hours, T cells were harvested, and APC conjugated anti-CD4 antibody (#130-113-210, MiltenyBiotec), FITC conjugated anti-CD8 antibody (#130-110- 677, MiltenyBiotec), PE-Vio 770 conjugated anti-CD69 antibody (#130-122-5-4, MiltenyBiotec) and PE conjugated anti-CD25 antibody (#341009, BD Bioscience). T cell subsets and activated T cells were identified by flow cytometry (BD FACSCanto II) using FlowJo 10 software (FlowJo, LLC).

실시예 10. PD-1/PD-L1 차단에 대한 생물학적 검정Example 10. Bioassay for PD-1/PD-L1 blockade

The PD-1/PD-L1 blockade bioassay was performed according to the manufacturer's protocol (#J1250, Promega). ^{Briefly, one vial of PD-L1/aAPC +} CHO-K1 cells included in the bioassay kit was suspended in recovery medium from a frozen stock (90% Ham's F-12 containing 10% FBS) and , This was seeded on a white bottom plate and incubated overnight at 37°C. Assay buffer (1% FBS) with ACE-05, BiTE-05, ACE-31, YBL-007 or IgG at concentrations of 0, 0.006, 0.032, 0.16, 0.8, 4 or 20 nM in each well. RPM 1640) containing Jurkat T cells stably expressing human PD-1 and NFAT-luciferase reporters were added. After 6 hours, NFAT-mediated luciferase activity was measured using a Bio-Glo Luciferase assay system (#G7940, Promega). RLU (relative light units) data were plotted and analyzed using GraphPad Prism 8 software.

실시예 11. 종양 살상 분석(Tumor-killing assay)Example 11. Tumor killing assay (Tumor-killing assay)

HCC827 (ATCC CRL2868), MDA-MB-231 (ATCC HTB-26), Karpas-299 (# 06072604, Sigma) and Raji (ATCC CCL86) cancer cells were added with 10% FBS under conditions of _{37°C and 5% CO 2} It was cultured using the RPMI-1640 medium. ^{PBMC and CD8 +} T cells of all healthy donors used in the present invention were purchased from AllCells (#PB004F and #PB009-3F), Zenbio (#SER-PBMC-200-F), and Lonza (#3W-270). . CD3 ⁺ T cells and CD8 ⁺ T cells were human using Pan T-cell isolation kit (#130-096-535, Miltenyi Biotec) and CD8 ⁺ T-cell isolation kit (#130-096-495, Miltenyi Biotec). It was isolated from PBMC preparation.

The cytotoxicity of PBMCs or T-cells against cancer cells expressing PD-L1 was evaluated by measuring lactate dehydrogenase (LDH) released from killed cancer cells. The on-target tumor cell-killing ability of the bispecific T cell linker is 10:1 (CD3 ⁺ T cells) or 5:1 (CD8 ⁺ T cells) of E:T (effector: target ( target)) PD-L1 ⁺ MDA-MB-231 cancer cells (1×10 ⁴ cells/well) and T cells with the indicated protein (ACE-05, ACE-31, BiTE-05, or IgG) It was investigated by culturing.

After 48-72 hours incubation, LDH released from the killed tumor cells was measured using a CytoTox96 non-radioactive cytotoxicity assay kit (#G1780, Promega) according to the manufacturer's instructions. The percentage of tumor cells killed was calculated using the following equation:

[수학식 1][Equation 1]

% Cytotoxicity (% killed tumor cells) = (experimental-target spontaneous-effector spontaneous) / (target maximum-target spontaneous) x 100%.

In addition, PBMC were also used as effector cells (E:T ratio, 25:1) to investigate ^{PD-L1 + HCC827 tumor cell-killing ability.} To evaluate non-target T-cell cytotoxicity, PD-L1 ^- HEK293 or Raji cancer cells ^{were co-cultured with CD3 +} T cells and 1 nM ACE-05, BiTE-05, ACE-31, IgG or ACE-18. . After 48-72 hours incubation, ^{LDH released from PD-L1-} cells was measured and calculated as described above.

실시예 12. T-세포 클러스터링Example 12. T-cell clustering

T-cell activation and differentiation was evaluated by accurately quantifying ^{T cells and PD-L1 +} tumor cell clustering using the IncuCyte live-cell analysis system (Sartorius, USA). CD3 ⁺ T cells were isolated from human PBMCs and labeled with CytoLight reagent (#4706, Sartorius) according to the manufacturer's protocol. PD-L1 ⁺ MDA-MB-231 cells (4×10 ³ cells/well) and CD3 ⁺ T cells with 1 nM ACE-05, ACE-31, BiTE-05 or IgG at an E:T ratio of 10:1. Co-cultured together. Live-cell images were obtained every 6 hours during the 90-hour culture period, and the average area (mm 2) of red fluorescent clusters representing T-cell activation was measured using IncuCyte software. Data obtained in quadruplicate were plotted using GraphPad Prism 8 software.

실시예 13. T-세포 확장 분석Example 13. T-cell expansion assay

Primary human T-cell expansion induced with ACE-05 in the presence of PD-L1 ^{+ tumor cells was investigated using a T-cell proliferation assay.} ^{CD3 +} T cells isolated from PBMC were stained with Cell Trace Far Red (#C34564, ThermoFisher) according to the manufacturer's instructions. PD-L1 ⁺ MDA-MB-231 cancer cells were seeded in a 24-well plate at a concentration of ^{1×10 5 cells/well.} The next day, the pre-cultured MDA-MB-231 cells were washed once with Dulbecco's PBS, which had been pre-warmed, and then replaced with an assay medium (RPMI-1640 containing 1% FBS). ^{A mixture containing CD3 +} T cells (1 × 10 ⁶ cells/well) stained with Cell Trace and 1 nM ACE-05, IgG or ACE-18 (CD20 × CD3 as negative control) was prepared in MDA- It was added to MB-231 cells. After culturing for 96 hours, T cells were harvested and analyzed by flow cytometry using the BD FACSCanto II system. The expansion of T cells was visualized based on flow cytometric data obtained using FlowJo 10 software (FlowJo, LLC).

실시예 14. 종양 세포의 부재시 다량체 T-세포 결합자에 의한 비-표적 T-세포 활성화 분석Example 14. Non-target T-cell activation assay by multimeric T-cell combiner in the absence of tumor cells

^{In the absence of target tumor cells, CD3 +} T cells isolated from human PBMC (#4W-270C, Lonza) (1× 10 ⁶ cells/well) were cultured in a medium containing 5% FBS, but 1 nM ACE-05, BiTE-05 or multimer ACE-05 was added directly. Clustered ACE-05 was prepared by mixing 5 μl CH1 beads (#1943462250, ThermoFisher) with 1 nM ACE-05. After incubation for 48 hours, T cells were harvested, and FITC conjugated with CD4 antibody (#130-114-531, MiltenyBiotec) and CD69-PE-Vio 770 antibody (#130-122-5-4, MiltenyBiotec). Labeled. T cell subsets and activated T cells were identified by flow cytometry (BD FACSCanto II) using FlowJo 10 software (FlowJo, LLC).

실시예 15. 면역세포가 분비하는 사이토카인 분석Example 15. Analysis of cytokines secreted by immune cells

Levels of cytokines, IL-2, IFN-γ, IL-6 and TNF-α, released by activated immune cells (PBMC and CD4 + T cells) in the presence or absence of PD-L1 + tumor cells Investigated. Specifically, as ^{a target condition in which PD-L1 +} HCC827 cancer cells are present, with 1 nM ACE-05, BiTE-05, ACE-31, or IgG, human PBMCs with HCC827 cancer cells 25:1 E:T Co-cultured for 72 hours at a rate. Samples were collected at 0, 6, 12, 18, 24, 48 and 72 hours after drug administration. Thereafter, the concentration of cytokines was analyzed using an ELISA kit for IL-2 (#431004, BioLegend) and IFN-γ (#431004, BioLegend).

As a non-target condition, 1 nM of ACE-05, BiTE-05, ACE-31 or IgG was directly added to ^{CD4 +} T cells isolated from human PBMC using a ^{CD4 + T cell separation kit (#130-096} -533, Miltenyi Biotec). After incubation for 72 hours, the released cytokines were analyzed by ELISA as described above.

For Granzyme B analysis, in the presence of various concentrations of ACE-05, BiTE-05, ACE-31 or IgG (0, 6.4, 32, 160, 800, 4000 pM), PBMC-derived CD8 ⁺ T cells and MDA-MB -231 cells were co-cultured at an E:T ratio of 5:1. After 48 hours, Granzyme B accumulated in the assay medium was analyzed using an ELISA kit (#DGZB00, R & D Systems). The optical density (OD) of each supernatant was measured using a microplate reader, and the concentration of cytokines was analyzed using GraphPad Prism 8 software.

III. ALiCE의 효능 확인 : In vivo assayIII. Checking the efficacy of ALiCE: In vivo assay

실시예 16. 래트 및 원숭이에서 약동학 분석Example 16. Pharmacokinetic analysis in rats and monkeys

Male Sprague-Dawley rats aged 6-7 weeks were injected with ACE-05, BiTE-05 or hIgG at a dose of 10 mg/kg via tail vein (n = 3 / group). Blood samples (150 μl each) 10 and 30 minutes after drug administration; 1, 2, 4, 8 and 24 hours; It was collected from each rat on the 3rd, 5th, and 9th days. Then, it was centrifuged for 2 minutes at 10,000-13,000 rpm. Each plasma sample was divided into 70 μl and stored at -80°C.

In addition, 20-24 month-old male cynomolgus monkeys were administered ACE-05, BiTE-05 or hIgG intravenously at a dose of 5 mg/kg (n = 3 / group). Blood samples (150 μl each) 10 and 30 minutes after drug administration; 1, 2, 4, 8 and 24 hours; Collected from each monkey on

days

2, 3, 4, 8, 12 and 15. Pharmacokinetic analysis was performed using the Gyrolab xPlore automated immunoassay system.

To isolate ACE-05 or human IgG, biotinylated anti-human IgG CH1 nanobodies were immobilized on the surface of streptavidin-coated Gyrolab Bioaffy CD200 (#P0004180, Gyros Protein Technologies), and serum samples were loaded. Similarly, biotinylated PD-L1-Fc was used to capture BiTE-05. Captured ACE-05 and human IgG were detected using an Alexa 647-conjugated anti-kappa antibody (#316514, Novus). In addition, BiTE-05 was detected using an Alexa 647-conjugated anti-His antibody (#362611, Novus).

Sample concentrations were calculated using Gyrolab software, and t _1/2 values were calculated using Phoenix WinNonlin software.

실시예 17. 인간화 마우스 모델에서 ALiCE의 항암 효과 확인Example 17. Confirmation of anticancer effect of ALiCE in humanized mouse model

The anti-tumor efficacy of ALiCE against PD-L1 + HCC827 tumors was evaluated in a PBMC-reconstituted humanized NCG (NOD/scid IL-2Rγ null) mouse model. Specifically, 7-8 week old female NCG mice (Crown Bioscience) were randomly divided into 4 groups of 10 mice each. ^{PBMCs of 5×10 6} cells/100 μl obtained from two healthy donors were implanted into the veins of each mouse. ^{After 3 days, 5 Х10 6} PD-L1 ⁺ HCC827 tumor cells were inoculated subcutaneously on the right flank of the mouse. When the tumor volume ^{reached ~ 50 mm 3} (after 4 days), mice were injected with ACE-05 or BiTE-05 every other day (Q2d, total 3 doses), or YBL-007 or IgG every 3 days. (Q3d, total 3 doses). Tumor diameter and body weight were measured every 2 days and analyzed using GraphPad Prism 8 software.

실시예 18. In vivo에서 사이토카인 분석Example 18. In vivo cytokine analysis

6-7 week old female non-tumor-bearing hCD3ε TG mice were divided into 4 groups of 6 mice each. Thereafter, 0.5 mg/kg of ACE-05 and BiTE-05 were administered to tail venous blood to each group, and 5 mg/kg of IgG was administered. Blood samples for cytokine analysis were collected from each animal at 0, 6, 12, 24 and 48 hours and stored at -80°C. Various cytokines present in each collected plasma sample were analyzed according to the protocols of BD Cytometric Bead Array (CBA) Mouse Inflammation Kit (# 552364, BD Bioscience) and Mouse Th1 / Th2 Cytokine Kit (# 551287, BD Bioscience). Cytokine levels were analyzed using GraphPad Prism 8 software.

실시예 19. 종양 침투 림프구 분석Example 19. Tumor Penetrating Lymphocyte Analysis

Tumor-infiltrating lymphocytes (TIL) were analyzed in hCD3ε TG mice bearing CT26 tumors expressing hPD-L1. ^{Specifically, 5×10 5} CT26-hPD-L1 cells were inoculated subcutaneously on the right flank of the mouse. When the subcutaneous tumor volume reaches ~90 mm ³ , start the intraperitoneal (ip) administration of the test formulation (ACE-05, 1 mg/kg; YBL-007, 3 mg/kg; UCHT1, 2 mg/kg) and , It was administered twice a week for 2 weeks (BIW, a total of 4 administrations). Tumors were obtained from mice one week after the last injection. Live lymphocytes and T cells in tumor tissue were analyzed by flow cytometry using lymphocyte markers (mCD45, mCD4, mCD8 and hCD3). The results were analyzed using GraphPad Prism 8 software.

실시예 20. In silico 면역원성 예측Example 20. In silico immunogenicity prediction

MHC-I Binding Predictions ( http://tools.iedb.org/mhci/ ) and Class , an in silico web-based prediction program, for Class I immunogenicity of ACE-05-HC-VH and ACE-05-HC-VL chains I Immunogenicity ( http://tools.iedb.org/immunogenicity/ ) was used to analyze. For MHC-I binding, epitope candidates of ACE-05-HC-VH and ACE-05-HC-VL were predicted using IEDB (Immune Epitope Database). As a peptide that can be presented to the MHC class-I molecule, 8-10 mer peptides were selected with the 0.3 percentile cutoff. Immunogenicity scores were obtained using the MHC-I binding prediction program. Immunogenic peptides present in ACE-05-HC-VH and ACE-05-HC-VL and their position were identified.

IV. 다중 특이적 융합 단백질의 예시IV. Examples of multispecific fusion proteins

실시예 21. 표적에 따른 융합 단백질의 실시예Example 21. Examples of fusion proteins according to targets

This example shows exemplary multispecific fusion proteins as provided herein, in particular multispecific fusion proteins ACE-00, ACE-02, ACE-03, ACE-04, ACE-05, ACE-09, ACE- 10, ACE-11, ACE-12, ACE-18, ACE-19, ACE-31 and many other multispecific fusion proteins have been disclosed. In addition, ACE-00r, ACE-02r, ACE-03r, ACE-04r, ACE-05r, ACE-09r, ACE-10r, ACE- 11r, ACE-12r, ACE-18r, ACE-19r, ACE-31r, and many other multispecific fusion proteins are illustrated. Components targeting the first and second antigens in each exemplary multispecific fusion protein are summarized in Table 4 below.

다중 특이적 융합 단백질Multispecific fusion proteins	타겟팅 구성요소(Targeting component)Targeting component
다중 특이적 융합 단백질Multispecific fusion proteins	제1 항원 결합 도메인First antigen binding domain	제2 항원 결합 도메인Second antigen binding domain
ACE-00ACE-00	Trastuzumab(Anti-Her2 Ab)Trastuzumab (Anti-Her2 Ab)	AdalimumabAdalimumab
ACE-01ACE-01	Anti-CD19 AbAnti-CD19 Ab	Anti-CD3 mouse OKT3Anti-CD3 mouse OKT3
ACE-02ACE-02	Anti-CD19 AbAnti-CD19 Ab	Anti-CD3 humanized 12F6Anti-CD3 humanized 12F6
ACE-03ACE-03	Anti-CD19 AbAnti-CD19 Ab	Anti-CD3 humanized OKT3Anti-CD3 humanized OKT3
ACE-04ACE-04	Anti-PD-L1 AbAnti-PD-L1 Ab	Anti-CD3 chimeric OKT3 FabAnti-CD3 chimeric OKT3 Fab
ACE-05ACE-05	Anti-PD-L1 AbAnti-PD-L1 Ab	Anti-CD3 AbAnti-CD3 Ab
ACE-06ACE-06	Anti-PD-L1 AbAnti-PD-L1 Ab	ForalumabForalumab
ACE-07ACE-07	Anti-PD-L1 AbAnti-PD-L1 Ab	Anti-CD3 UCHT1Anti-CD3 UCHT1
ACE-08ACE-08	Anti-PD-L1 AbAnti-PD-L1 Ab	Anti-CD3 2C11Anti-CD3 2C11
ACE-09ACE-09	Anti-PD-L1 AbAnti-PD-L1 Ab	Anti-CD3 UCHT1Anti-CD3 UCHT1
ACE-10ACE-10	Anti-CD20Anti-CD20	Anti-CD3 UCHT1Anti-CD3 UCHT1
ACE-11ACE-11	Cetuximab(Anti-EGFR Ab)Cetuximab (Anti-EGFR Ab)	Anti-CD3 UCHT1Anti-CD3 UCHT1
ACE-12ACE-12	Anti-PD-L1 AbAnti-PD-L1 Ab	Anti-CD3 UCHT1Anti-CD3 UCHT1
ACE-13ACE-13	Anti-PD-L1 AbAnti-PD-L1 Ab	Anti-PD-1 AbAnti-PD-1 Ab
ACE-14ACE-14	Anti-PD-L1 AbAnti-PD-L1 Ab	Anti-CD3 UCHT1Anti-CD3 UCHT1
ACE-15ACE-15	Anti-PD-L1 AbAnti-PD-L1 Ab	Anti-CD3 UCHT1Anti-CD3 UCHT1
ACE-16ACE-16	Anti-PD-L1 AbAnti-PD-L1 Ab	Anti-CD3 UCHT1Anti-CD3 UCHT1
ACE-17ACE-17	Anti-PD-L1 AbAnti-PD-L1 Ab	Anti-CD3 UCHT1Anti-CD3 UCHT1
ACE-18ACE-18	Anti-CD20 AbAnti-CD20 Ab	Anti-CD3 UCHT1Anti-CD3 UCHT1
ACE-19ACE-19	Anti-EGFR AbAnti-EGFR Ab	Anti-CD3 UCHT1Anti-CD3 UCHT1
ACE-20ACE-20	Matuzumab(Anti-EGFR Ab)Matuzumab (Anti-EGFR Ab)	Anti-CD3 UCHT1Anti-CD3 UCHT1
ACE-21ACE-21	Zalutumumab(Anti-EGFR Ab)Zalutumumab (Anti-EGFR Ab)	Anti-CD3 UCHT1Anti-CD3 UCHT1
ACE-22ACE-22	Rituximab(Anti-CD20 Ab)Rituximab (Anti-CD20 Ab)	Anti-CD3 UCHT1Anti-CD3 UCHT1
ACE-23ACE-23	Anti-PD-L1 AbAnti-PD-L1 Ab	Foralumab(Anti-CD3 Ab)Foralumab (Anti-CD3 Ab)
ACE-24ACE-24	Trastuzumab(Anti-Her2 Ab)Trastuzumab (Anti-Her2 Ab)	Adalimumab(Anti-TNF Ab)Adalimumab (Anti-TNF Ab)
ACE-25ACE-25	Trastuzumab(Anti-Her2 Ab)Trastuzumab (Anti-Her2 Ab)	Adalimumab(Anti-TNF Ab)Adalimumab (Anti-TNF Ab)
ACE-26ACE-26	Cetuximab(Anti-EGFR Ab)Cetuximab (Anti-EGFR Ab)	Anti-CD3 SP34Anti-CD3 SP34
ACE-27ACE-27	Anti-PD-L1 AbAnti-PD-L1 Ab	Anti-CD3 SP34Anti-CD3 SP34
ACE-28ACE-28	Cetuximab(Anti-EGFR Ab)Cetuximab (Anti-EGFR Ab)	Anti-CD3 UCHT1Anti-CD3 UCHT1
ACE-29ACE-29	Cetuximab(Anti-EGFR Ab)Cetuximab (Anti-EGFR Ab)	Anti-CD3 SP34Anti-CD3 SP34
ACE-30ACE-30	Anti-CD3 UCHT1Anti-CD3 UCHT1	Anti-PD-L1 AbAnti-PD-L1 Ab
ACE-31ACE-31	Anti-CD3 UCHT1Anti-CD3 UCHT1	Anti-PD-L1 AbAnti-PD-L1 Ab
ACE-32ACE-32	Cetuximab(Anti-EGFR Ab)Cetuximab (Anti-EGFR Ab)	Anti-CD3 SP34Anti-CD3 SP34
ACE-33ACE-33	Anti-CD3 UCHTAnti-CD3 UCHT	Anti-PD-L1 AbAnti-PD-L1 Ab
ACE-34ACE-34	Anti-PD-L1 AbAnti-PD-L1 Ab	Mouse Anti-CD3 Mouse Anti-CD3
ACE-35ACE-35	Anti-PD-L1 AbAnti-PD-L1 Ab	Mouse Anti-CD3 Mouse Anti-CD3
ACE-36ACE-36	Anti-PD-L1 AbAnti-PD-L1 Ab	Ipilimumab(Anti-CTLA4 Ab)Ipilimumab (Anti-CTLA4 Ab)
ACE-37ACE-37	Rituximab(Anti-CD20 Ab)Rituximab (Anti-CD20 Ab)	Ipilimumab(Anti-CTLA4 Ab)Ipilimumab (Anti-CTLA4 Ab)
ACE-38ACE-38	Cetuximab(Anti-EGFR Ab)Cetuximab (Anti-EGFR Ab)	Anti-CD3 UCHT1Anti-CD3 UCHT1
ACE-39ACE-39	Cetuximab(Anti-EGFR Ab)Cetuximab (Anti-EGFR Ab)	Anti-CD3 UCHT1Anti-CD3 UCHT1
ACE-40ACE-40	Cetuximab(Anti-EGFR Ab)Cetuximab (Anti-EGFR Ab)	Anti-CD3 UCHT1Anti-CD3 UCHT1
ACE-00rACE-00r	Adalimumab(Anti-TNF Ab)Adalimumab (Anti-TNF Ab)	Trastuzumab(Anti-Her2 Ab)Trastuzumab (Anti-Her2 Ab)
ACE-01rACE-01r	Anti-CD3 mouse OKT3Anti-CD3 mouse OKT3	Anti-CD19 AbAnti-CD19 Ab
ACE-02rACE-02r	Anti-CD3 humanized 12F6Anti-CD3 humanized 12F6	Anti-CD19 AbAnti-CD19 Ab
ACE-03rACE-03r	Anti-CD3 humanized OKT3Anti-CD3 humanized OKT3	Anti-CD19 AbAnti-CD19 Ab
ACE-04rACE-04r	Anti-CD3 chimetic OKT3 FabAnti-CD3 chimetic OKT3 Fab	Anti-PD-L1 AbAnti-PD-L1 Ab
ACE-05rACE-05r	Anti-CD3 UCHT1Anti-CD3 UCHT1	Anti-PD-L1 AbAnti-PD-L1 Ab
ACE-06rACE-06r	Foralumab(Anti-CD3 Ab)Foralumab (Anti-CD3 Ab)	Anti-PD-L1 AbAnti-PD-L1 Ab
ACE-07rACE-07r	Anti-CD3 UCHT1Anti-CD3 UCHT1	Anti-PD-L1 AbAnti-PD-L1 Ab
ACE-08rACE-08r	Anti-CD3 2C11Anti-CD3 2C11	Anti-PD-L1 AbAnti-PD-L1 Ab
ACE-09rACE-09r	Anti-CD3 UCHT1Anti-CD3 UCHT1	Anti-PD-L1 AbAnti-PD-L1 Ab
ACE-10rACE-10r	Anti-CD3 UCHT1Anti-CD3 UCHT1	Anti-CD20 AbAnti-CD20 Ab
ACE-11rACE-11r	Anti-CD3 UCHT1Anti-CD3 UCHT1	Cetuximab(Anti-EGFR Ab)Cetuximab (Anti-EGFR Ab)
ACE-12rACE-12r	Anti-CD3 UCHT1Anti-CD3 UCHT1	Anti-PD-L1 AbAnti-PD-L1 Ab
ACE-13rACE-13r	Anti-PD-1 AbAnti-PD-1 Ab	Anti-PD-L1 AbAnti-PD-L1 Ab
ACE-14rACE-14r	Anti-CD3 UCHT1Anti-CD3 UCHT1	Anti-PD-L1 AbAnti-PD-L1 Ab
ACE-15rACE-15r	Anti-CD3 UCHT1Anti-CD3 UCHT1	Anti-PD-L1 AbAnti-PD-L1 Ab
ACE-16rACE-16r	Anti-CD3 UCHT1Anti-CD3 UCHT1	Anti-PD-L1 AbAnti-PD-L1 Ab
ACE-17rACE-17r	Anti-CD3 UCHT1Anti-CD3 UCHT1	Anti-PD-L1 AbAnti-PD-L1 Ab
ACE-18rACE-18r	Anti-CD3 UCHT1Anti-CD3 UCHT1	Anti-CD20 AbAnti-CD20 Ab
ACE-19rACE-19r	Anti-CD3 UCHT1Anti-CD3 UCHT1	Anti-EGFR AbAnti-EGFR Ab
ACE-20rACE-20r	Anti-CD3 UCHT1Anti-CD3 UCHT1	Matuzumab(Anti-EGFR Ab)Matuzumab (Anti-EGFR Ab)
ACE-21rACE-21r	Anti-CD3 UCHT1Anti-CD3 UCHT1	Zalutumumab(Anti-EGFR Ab)Zalutumumab (Anti-EGFR Ab)
ACE-22rACE-22r	Anti-CD3 UCHT1Anti-CD3 UCHT1	Rituximab(Anti-CD20 Ab)Rituximab (Anti-CD20 Ab)
ACE-23rACE-23r	Foralumab(Anti-CD3 Ab)Foralumab (Anti-CD3 Ab)	Anti-PD-L1 AbAnti-PD-L1 Ab
ACE-24rACE-24r	Adalimumab(Anti-TNF Ab)Adalimumab (Anti-TNF Ab)	Trastuzumab(Anti-Her2 Ab)Trastuzumab (Anti-Her2 Ab)
ACE-25rACE-25r	Adalimumab(Anti-TNF Ab)Adalimumab (Anti-TNF Ab)	Trastuzumab(Anti-Her2 Ab)Trastuzumab (Anti-Her2 Ab)
ACE-26rACE-26r	Anti-CD3 SP34Anti-CD3 SP34	Cetuximab(Anti-EGFR Ab)Cetuximab (Anti-EGFR Ab)
ACE-27rACE-27r	Anti-CD3 SP34Anti-CD3 SP34	Anti-PD-L1 AbAnti-PD-L1 Ab
ACE-28rACE-28r	Anti-CD3 UCHT1Anti-CD3 UCHT1	CetuximabCetuximab
ACE-29rACE-29r	Anti-CD3 SP34Anti-CD3 SP34	CetuximabCetuximab
ACE-30rACE-30r	Anti-PD-L1 AbAnti-PD-L1 Ab	Anti-CD3 UCHTAnti-CD3 UCHT
ACE-31rACE-31r	Anti-PD-L1 AbAnti-PD-L1 Ab	Anti-CD3 UCHTAnti-CD3 UCHT
ACE-32rACE-32r	Anti-CD3 SP34Anti-CD3 SP34	CetuximabCetuximab
ACE-33rACE-33r	Anti-PD-L1 AbAnti-PD-L1 Ab	Anti-CD3 UCHTAnti-CD3 UCHT
ACE-34rACE-34r	Mouse Anti-CD3 Mouse Anti-CD3	Anti-PD-L1 AbAnti-PD-L1 Ab
ACE-35rACE-35r	Mouse Anti-CD3 Mouse Anti-CD3	Anti-PD-L1 AbAnti-PD-L1 Ab
ACE-36rACE-36r	IpilimumabIpilimumab	Anti-PD-L1 AbAnti-PD-L1 Ab
ACE-37rACE-37r	IpilimumabIpilimumab	RituximabRituximab
ACE-38rACE-38r	Anti-CD3 UCHT1Anti-CD3 UCHT1	CetuximabCetuximab
ACE-39rACE-39r	Anti-CD3 UCHT1Anti-CD3 UCHT1	CetuximabCetuximab
ACE-40rACE-40r	Anti-CD3 UCHT1Anti-CD3 UCHT1	CetuximabCetuximab

Formats used in amino acid sequences: BOLD : VH or VL;

BOLD and UNDERLINED : CDR;

ITALICIZED : antibody hinge region;

lower case: flexible linker;

[BRACKET]: CH1;

[ BRACKET and UNDERLINED ]: CL.

Double underlined: CH3

실시예 21.1. ACE-00의 제조Example 21.1. Preparation of ACE-00

ACE-00 contains two different heavy chain-like chains (ACE-00-VH and ACE-00-VL) and two identical light chains (ACE-00-LC). The parent antibody used to construct the anti-Her2 domain of ACE-00 is trastuzumab, and the parent antibody used to construct the anti-TNF alpha domain of ACE-00 is adalimumab. The amino acid sequence of these three types of polypeptide is as follows:

ACE-00-VH amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAAS GFNIKDTY IHWVRQAPGKGLEWVAR IYPTNGYT RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDY WGQGTLVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPAPELLGGP EVQLVESGGGLVQPGRSLRLSCAAS GFTFDDYA MHWVRQAPGKGLEWVSA ITWNSGHI DYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC AKVSYLSTASSLDY WGQGTLVTVSS (SEQ ID NO: 115)

ACE-00-VL amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAAS GFNIKDTY IHWVRQAPGKGLEWVAR IYPTNGYT RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDY WGQGTLVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPAPELLGGP DIQMTQSPSSLSASVGDRVTITCRAS QGIRNY LAWYQQKPGKAPKLLIY AAS TLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QRYNRAPYT FGQGTKVEIKR (SEQ ID NO: 116)

ACE-00-LC amino acid sequence (anti-CD19 antibody light chain) :

DIQMTQSPSSLSASVGDRVTITCRAS QDVNTA VAWYQQKPGKAPKLLIY SAS FLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYC QQHYTTPPT FGQGTKVEIK[ RSVAAPSVFIFPPSDEQLKSGTASVVCLLNNVDYPREAKSQGNSVSTYPREAKVQWKSDNSVDYPREAKVQWKDS

The VH and VL amino acid sequences for the bivalent Fab region targeting Her2 and the monovalent Fv region targeting TNF alpha are listed in Table 5:

Fab region (Anti-Her2)Fab region (Anti-Her2)	VH: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (서열번호: 51)VH: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 51)	VL: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR (서열번호: 52)VL: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR (SEQ ID NO: 52)
	CDR H1: GFNIKDTY (서열번호: 118)CDR H1: GFNIKDTY (SEQ ID NO: 118)	CDR L1: QDVNTA (서열번호: 121)CDR L1: QDVNTA (SEQ ID NO: 121)
	CDR H2: IYPTNGYT (서열번호: 119)CDR H2: IYPTNGYT (SEQ ID NO: 119)	CDR L2: SAS (서열번호: 122)CDR L2: SAS (SEQ ID NO: 122)
	CDR H3: SRWGGDGFYAMDY (서열번호: 120)CDR H3: SRWGGDGFYAMDY (SEQ ID NO: 120)	CDR L3: QQHYTTPPT (서열번호: 123)CDR L3: QQHYTTPPT (SEQ ID NO: 123)
Fv region (Anti-TNF alpha)Fv region (Anti-TNF alpha)	VH: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS (서열번호: 53)VH: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS (SEQ ID NO: 53)	VL: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKR (서열번호: 54)VL: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKR (SEQ ID NO: 54)
	CDR H1: GFTFDDYA (서열번호: 124)CDR H1: GFTFDDYA (SEQ ID NO: 124)	CDR L1: QGIRNY (서열번호: 127)CDR L1: QGIRNY (SEQ ID NO: 127)
	CDR H2: ITWNSGHI (서열번호: 125)CDR H2: ITWNSGHI (SEQ ID NO: 125)	CDR L2: AAS (서열번호: 128)CDR L2: AAS (SEQ ID NO: 128)
	CDR H3: AKVSYLSTASSLDY (서열번호: 126)CDR H3: AKVSYLSTASSLDY (SEQ ID NO: 126)	CDR L3: QRYNRAPYT (서열번호: 129)CDR L3: QRYNRAPYT (SEQ ID NO: 129)

실시예 21.2. ACE-01의 제조 Example 21.2. Preparation of ACE-01

ACE-01 contains two different heavy chain-like chains (ACE-01-VH and ACE-01-VL) and two identical light chains (ACE-01-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-01-VH amino acid sequence (SEQ ID NO: 136)

QVQLQQSGAELVRPGSSVKISCKASGYAFS SYWMN WVKQRPGQGLEWIGQ IWPGDGDT NYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFC ARRETTTVGRYYYAMDY WGQGTTVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPAPELLGGP QVQLQQSGAELARPGASVKMSCKAS GYTFTRYT MHWVKQRPGQGLEWIGY INPSRGYT NYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYC ARYYDDHYCLDY WGQGTTVTVSS

ACE-01- VL amino acid sequence (SEQ ID NO: 137)

QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPAPELLGGP QIVLTQSPAIMSASPGEKVTMTCSAS SSVSY MNWYQQKSGTSPKRWIY DTS KLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYC QQWSSNPFTF GSGTKLEINR

ACE-01-LC amino acid sequence (SEQ ID NO: 138)

DIQLTQSPASLAVSLGQRATISCKAS QSVDYDGDSY LNWYQQIPGQPPKLLIY DAS NLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHC QQSTEDPWT FGGGTKLEIK [ RTVAAPSVFIFPPSDEQLKSGTASVVCSQVLTQLKSGTASVVCSKNFQLKSGTASVVCSKNFREADS

The VH and VL amino acid sequences and CDR sequences therein for the first antigen-binding domain (divalent Fab region targeting CD19) and the second antigen-binding domain (monovalent Fv region targeting CD3) are shown in Table 6 below. Are listed:

Fab region (Anti-CD19)Fab region (Anti-CD19)	VH: QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (서열번호: 139)VH: QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (SEQ ID NO: 139)	VL: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK (서열번호: 151)VL: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK (SEQ ID NO: 151)
	CDR H1: SYWMN (서열번호: 140)CDR H1: SYWMN (SEQ ID NO: 140)	CDR L1: QSVDYDGDSY (서열번호: 152)CDR L1: QSVDYDGDSY (SEQ ID NO: 152)
	CDR H2: IWPGDGDT (서열번호: 141)CDR H2: IWPGDGDT (SEQ ID NO: 141)	CDR L2: DAS (서열번호: 153)CDR L2: DAS (SEQ ID NO: 153)
	CDR H3: ARRETTTVGRYYYAMDY (서열번호: 142)CDR H3: ARRETTTVGRYYYAMDY (SEQ ID NO: 142)	CDR L3: QQSTEDPWT (서열번호: 154)CDR L3: QQSTEDPWT (SEQ ID NO: 154)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTVTVSS (서열번호: 143)VH: QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTVTVSS (SEQ ID NO: 143)	VL: QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (서열번호: 147)VL: QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (SEQ ID NO: 147)
	CDR H1: GYTFTRYT (서열번호: 144)CDR H1: GYTFTRYT (SEQ ID NO: 144)	CDR L1: SSVSY (서열번호: 148)CDR L1: SSVSY (SEQ ID NO: 148)
	CDR H2: INPSRGYT (서열번호: 145)CDR H2: INPSRGYT (SEQ ID NO: 145)	CDR L2: DTS (서열번호: 149)CDR L2: DTS (SEQ ID NO: 149)
	CDR H3: ARYYDDHYCLDY (서열번호: 146)CDR H3: ARYYDDHYCLDY (SEQ ID NO: 146)	CDR L3: QQWSSNPFTF (서열번호: 150)CDR L3: QQWSSNPFTF (SEQ ID NO: 150)

실시예 21.3. ACE-02의 제조 Example 21.3. Preparation of ACE-02

ACE-02 contains two different heavy chain-like chains (ACE-02-VH and ACE-02-VL) and two identical light chains (ACE-02-LC). The amino acid sequence of these three types of polypeptide is as follows:

ACE-02-VH amino acid sequence :

QVQLQQSGAELVRPGSSVKISCKASGYAFS SYWM NWVKQRPGQGLEWIG QIWPGDGDTNYNGKFKG KATLTADESSSTAYMQLSSLASEDSAVYFCAR RETTTVGRYYYAMDY WGQGTTVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPAPELLGGP QVQLVQSGGGVVQPGRSLRLSCKAS GYTFTSYT MHWVRQAPGKGLEWIGY INPSSGYT KYNQKFKDRFTISADKSKSTAFLQMDSLRPEDTGVYFC ARWQDYDVYFDY WGQGTPVTVSS (SEQ ID NO: 88)

ACE-02-VL amino acid sequence :

QVQLQQSGAELVRPGSSVKISCKASGYAFS SYWMN WVKQRPGQGLEWIG QIWPGDGDTNYNGKFKG KATLTADESSSTAYMQLSSLASEDSAVYFCAR RETTTVGRYYYAMDY WGQGTTVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPAPELLGGP DIQMTQSPSSLSASVGDRVTMTCRA SSSVSY MHWYQQTPGKAPKPWIY ATS NLASGVPSRFSGSGSGTDYTLTISSLQPEDIATYYC QQWSSNPPT FGQGTKLQITR (SEQ ID NO: 89)

ACE-02-LC amino acid sequence (anti-CD19 antibody light chain) :

DIQLTQSPASLAVSLGQRATISCKAS QSVDYDGDSY LNWYQQIPGQPPKLLIY DAS NLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHC QQSTEDPWT FGGGTKLEIK [RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC] (SEQ ID NO: 90)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD19 and the second antigen binding domain monovalent Fv region of humanized 12F6 are listed in Table 7 below:

Fab region (Anti-CD19)Fab region (Anti-CD19)	VH: QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (서열번호: 61)VH: QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (SEQ ID NO: 61)	VL: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK (서열번호: 65)VL: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK (SEQ ID NO: 65)
	CDR H1: SYWMN (서열번호: 62)CDR H1: SYWMN (SEQ ID NO: 62)	CDR L1: QSVDYDGDSY (서열번호: 66)CDR L1: QSVDYDGDSY (SEQ ID NO: 66)
	CDR H2: QIWPGDGDTNYNGKFKG (서열번호: 63)CDR H2: QIWPGDGDTNYNGKFKG (SEQ ID NO: 63)	CDR L2: DAS (서열번호: 67)CDR L2: DAS (SEQ ID NO: 67)
	CDR H3: RETTTVGRYYYAMDY (서열번호: 64)CDR H3: RETTTVGRYYYAMDY (SEQ ID NO: 64)	CDR L3: QQSTEDPWT (서열번호: 68)CDR L3: QQSTEDPWT (SEQ ID NO: 68)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: QVQLVQSGGGVVQPGRSLRLSCKASGYTFTSYTMHWVRQAPGKGLEWIGYINPSSGYTKYNQKFKDRFTISADKSKSTAFLQMDSLRPEDTGVYFCARWQDYDVYFDYWGQGTPVTVSS (서열번호: 69)VH: QVQLVQSGGGVVQPGRSLRLSCKASGYTFTSYTMHWVRQAPGKGLEWIGYINPSSGYTKYNQKFKDRFTISADKSKSTAFLQMDSLRPEDTGVYFCARWQDYDVYFDYWGQGTPVTVSS (SEQ ID NO: 69)	VL: DIQMTQSPSSLSASVGDRVTMTCRASSSVSYMHWYQQTPGKAPKPWIYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDIATYYCQQWSSNPPTFGQGTKLQITR (서열번호: 73)VL: DIQMTQSPSSLSASVGDRVTMTCRASSSVSYMHWYQQTPGKAPKPWIYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDIATYYCQQWSSNPPTFGQGTKLQITR (SEQ ID NO: 73)
	CDR H1: GYTFTSYT (서열번호: 70)CDR H1: GYTFTSYT (SEQ ID NO: 70)	CDR L1: SSSVSY (서열번호: 74)CDR L1: SSSVSY (SEQ ID NO: 74)
	CDR H2: INPSSGYT (서열번호: 71)CDR H2: INPSSGYT (SEQ ID NO: 71)	CDR L2: ATS (서열번호: 75)CDR L2: ATS (SEQ ID NO: 75)
	CDR H3: ARWQDYDVYFDY (서열번호: 72)CDR H3: ARWQDYDVYFDY (SEQ ID NO: 72)	CDR L3: QQWSSNPPT (서열번호: 76)CDR L3: QQWSSNPPT (SEQ ID NO: 76)

DNA sequences encoding ACE-02-VH, ACE-02-VL and ACE-02-LC have nucleic acid sequences of SEQ ID NOs: 100 to 102, respectively.

실시예 21.4. ACE-03의 제조 Example 21.4. Preparation of ACE-03

ACE-03 consists of anti-CD19 and humanized anti-CD3 OKT3 domains. ACE-03 contains two different heavy chain-like chains (ACE-03-VH and ACE-03-VL) and two identical light chains (ACE-03-LC). The amino acid sequence of these three types of polypeptide is as follows:

ACE-03-VH amino acid sequence:

QVQLQQSGAELVRPGSSVKISCKASGYAFS SYWMN WVKQRPGQGLEWIG QIWPGDGDTNYNGKFKG KATLTADESSSTAYMQLSSLASEDSAVYFCAR RETTTVGRYYYAMDY WGQGTTVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPAPELLGGP VQLVQSGGGVVQPGRSLRLSCKAS GYTFTRYT MHWVRQAPGKGLEWIGY INPSRGYT NYNQKVKDRFTISTDKSKSTAFLQMDSLRPEDTAVYYC ARYYDDHYCLDY WGQGTPVTVSS (SEQ ID NO: 91)

ACE-03-VL amino acid sequence :

QVQLQQSGAELVRPGSSVKISCKASGYAFS SYWMN WVKQRPGQGLEWIG QIWPGDGDTNYNGKFKG KATLTADESSSTAYMQLSSLASEDSAVYFCAR RETTTVGRYYYAMDY WGQGTTVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPAPELLGGP DIQMTQSPSSLSASVGDRVTITCSAS SSVSY MNWYQQTPGKAPKRWIY DTS KLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYC QQWSSNPFT FGQGTKLQITR (SEQ ID NO: 92)

ACE-03-LC amino acid sequence (anti-CD19 antibody light chain) :

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD19 and the second antigen binding domain monovalent Fv region of humanized OKT3 are listed in Table 8 below:

Fab region (Anti-CD19)Fab region (Anti-CD19)	VH: QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (서열번호: 61)VH: QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (SEQ ID NO: 61)	VL: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK (서열번호: 65)VL: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK (SEQ ID NO: 65)
	CDR H1: SYWMN (서열번호: 62)CDR H1: SYWMN (SEQ ID NO: 62)	CDR L1: QSVDYDGDSY (서열번호: 66)CDR L1: QSVDYDGDSY (SEQ ID NO: 66)
	CDR H2: QIWPGDGDTNYNGKFKG (서열번호: 63)CDR H2: QIWPGDGDTNYNGKFKG (SEQ ID NO: 63)	CDR L2: DAS (서열번호: 67)CDR L2: DAS (SEQ ID NO: 67)
	CDR H3: RETTTVGRYYYAMDY (서열번호: 64)CDR H3: RETTTVGRYYYAMDY (SEQ ID NO: 64)	CDR L3: QQSTEDPWT (서열번호: 68)CDR L3: QQSTEDPWT (SEQ ID NO: 68)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: VQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISTDKSKSTAFLQMDSLRPEDTAVYYCARYYDDHYCLDYWGQGTPVTVSS (서열번호: 77)VH: VQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISTDKSKSTAFLQMDSLRPEDTAVYYCARYYDDHYCLDYWGQGTPVTVSS (SEQ ID NO: 77)	VL: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR (서열번호: 81)VL: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR (SEQ ID NO: 81)
	CDR H1: GYTFTRYT (서열번호: 78)CDR H1: GYTFTRYT (SEQ ID NO: 78)	CDR L1: SSVSY (서열번호: 82)CDR L1: SSVSY (SEQ ID NO: 82)
	CDR H2: INPSRGYT (서열번호: 79)CDR H2: INPSRGYT (SEQ ID NO: 79)	CDR L2: DTS (서열번호: 83)CDR L2: DTS (SEQ ID NO: 83)
	CDR H3: ARYYDDHYCLDY (서열번호: 80)CDR H3: ARYYDDHYCLDY (SEQ ID NO: 80)	CDR L3: QQWSSNPFT (서열번호: 84)CDR L3: QQWSSNPFT (SEQ ID NO: 84)

DNA sequences encoding ACE-03-VH, ACE-03-VL and ACE-03-LC have nucleic acid sequences of SEQ ID NOs: 103, 104 and 102, respectively.

실시예 21.5. ACE-04의 제조 Example 21.5. Preparation of ACE-04

ACE-04 has two different heavy chains such as ACE-04-VH (VL-CL-VH-CH1) and ACE-04-VL (VH-CH1-VL-CL) and two identical light chains (ACE-04-LC ). The amino acid sequences of these three types of polypeptides are as follows:

ACE-04-VH amino acid sequence :

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGI ANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs QVQLQQSGAELARPGASVKMSCKAS GYTFTRYT MHWVKQRPGQGLEWIGY INPSRGYT NYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYC ARYYDDHYCLDY WGQGTTVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] ( SEQ ID NO: 93)

ACE-04-VL amino acid sequence :

QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGR IIPILGI ANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs QIVLTQSPAIMSASPGEKVTMTCSAS SSVSY MNWYQQKSGTSPKRWIY DTS KLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYC QQWSSNPF TFGSGTKLEIN [RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC] ( SEQ ID NO: 94)

ACE-04-LC amino acid sequence (anti-PD-L1 antibody light chain) :

QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVL [RSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC] (SEQ ID NO: 95)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region of the chimeric OKT3 Fab region are listed in Table 9 below. have:

Fab region (Anti-PD-L1)Fab region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 4)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 4)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (서열번호: 8)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 8)
	CDR H1: GGTFSSYA (서열번호: 5)CDR H1: GGTFSSYA (SEQ ID NO: 5)	CDR L1: SSNIGAGYD (서열번호: 9)CDR L1: SSNIGAGYD (SEQ ID NO: 9)
	CDR H2: IIPILGIA (서열번호: 6)CDR H2: IIPILGIA (SEQ ID NO: 6)	CDR L2: GDI (서열번호: 10)CDR L2: GDI (SEQ ID NO: 10)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 7)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 7)	CDR L3: QSYDSSLSGGV (서열번호: 11)CDR L3: QSYDSSLSGGV (SEQ ID NO: 11)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTVTVSA (서열번호: 85)VH: QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTVTVSA (SEQ ID NO: 85)	VL: QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (서열번호: 86)VL: QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (SEQ ID NO: 86)
	CDR H1: GYTFTRYT (서열번호: 78)CDR H1: GYTFTRYT (SEQ ID NO: 78)	CDR L1: SSVSY (서열번호: 82)CDR L1: SSVSY (SEQ ID NO: 82)
	CDR H2: INPSRGYT (서열번호: 79)CDR H2: INPSRGYT (SEQ ID NO: 79)	CDR L2: DTS (서열번호: 83)CDR L2: DTS (SEQ ID NO: 83)
	CDR H3: ARYYDDHYCLDY (서열번호: 80)CDR H3: ARYYDDHYCLDY (SEQ ID NO: 80)	CDR L3: QQWSSNPF (서열번호: 87)CDR L3: QQWSSNPF (SEQ ID NO: 87)

DNA sequences encoding ACE-04-VH, ACE-04-VL and ACE-04-LC have nucleic acid sequences of SEQ ID NOs: 105, 106 and 107, respectively.

실시예 21.6. ACE-05의 제조 Example 21.6. Preparation of ACE-05

ACE-05 contains two different heavy chain-like chains (ACE-05-VH and ACE-05-VL) and two identical light chains (ACE-05-LC). ACE-05 contains a G4S linker (amino acid sequence of GGGS, SEQ ID NO: 112) in the flexible peptide region. The amino acid sequences of these three types of polypeptides are as follows:

ACE-05-VH amino acid sequence (SEQ ID NO: 213)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS

ACE-05-VL amino acid sequence (SEQ ID NO: 214)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGTVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR

ACE-05-LC amino acid sequence (SEQ ID NO: 157)

QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVLR [ SVAAPSVFIFPPSDEQLKSGTASVVCSKSVLTQLKSGTASVVCSKNFQLKSGTASVVCSKNFREADS

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 10 below. have:

Fab region (Anti-PD-L1)Fab region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (서열번호: 170)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 170)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171)CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 215)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 215)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 216)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 216)
	CDR H1: GYSFTGYT (서열번호: 177)CDR H1: GYSFTGYT (SEQ ID NO: 177)	CDR L1: QDIRNY (서열번호: 181)CDR L1: QDIRNY (SEQ ID NO: 181)
	CDR H2: INPYKGVS (서열번호: 178)CDR H2: INPYKGVS (SEQ ID NO: 178)	CDR L2: YTS (서열번호: 182)CDR L2: YTS (SEQ ID NO: 182)
	CDR H3: ARSGYYGDSDWYFD (서열번호: 211)CDR H3: ARSGYYGDSDWYFD (SEQ ID NO: 211)	CDR L3: QQGNTLPWT (서열번호: 207)CDR L3: QQGNTLPWT (SEQ ID NO: 207)

Triple transfection was performed to transfect host cells with DNA of ACE-05-VH, ACE-05-VL and ACE-05-LC (0.5: 0.5: 1 w / w ratio). DNA sequences encoding ACE-05-VH, ACE-05-VL and ACE-05-LC have nucleic acid sequences of SEQ ID NOs: 20, 21 and 22, respectively.

실시예 21.7. ACE-06의 제조 Example 21.7. Preparation of ACE-06

ACE-06 contains two different heavy chain-like chains (ACE-06-VH and ACE-06-VL) and two identical light chains (ACE-06-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-06-VH amino acid sequence (SEQ ID NO: 155)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs QVQLVESGGGVVQPGRSLRLSCAAS GFKFSGYG MHWVRQAPGKGLEWVAVI WYDGSKK YYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARQMGYWHFDL WGRGTLVTVSS

ACE-06-VL amino acid sequence (SEQ ID NO: 156)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs EIVLTQSPATLSLSPGERATLSCRAS QSVSSY LAWYQQKPGQAPRLLIY DAS NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPPLT FGGGTKVEIKR

ACE-06-LC (amino acid sequence (SEQ ID NO: 157)

QLVLTQPPSVSGAPGQRVTISCTGSQLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYDSSNIGAGYD VHWYQQLPGAAPKLLIYVHWYQQLPGAAPKLLIY GDIGDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCNRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGVQSYDSSLSGGV FGGGTKLTVLR[FGGGTKLTVLR[ SVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC]

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 11 below. have:

Fab region (Anti-PD-L1)Fab region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (서열번호: 170)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 170)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171)CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173) CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSS (서열번호: 162)VH: QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSS (SEQ ID NO: 162)	VL: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIKR (서열번호: 166)VL: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIKR (SEQ ID NO: 166)
	CDR H1: GFKFSGYG (서열번호: 163)CDR H1: GFKFSGYG (SEQ ID NO: 163)	CDR L1: QSVSSY (서열번호: 167)CDR L1: QSVSSY (SEQ ID NO: 167)
	CDR H2: IWYDGSKK (서열번호: 271)CDR H2: IWYDGSKK (SEQ ID NO: 271)	CDR L2: DAS (서열번호: 168)CDR L2: DAS (SEQ ID NO: 168)
	CDR H3: ARQMGYWHFDL (서열번호: 165)CDR H3: ARQMGYWHFDL (SEQ ID NO: 165)	CDR L3: QQRSNWPPLT (서열번호: 169)CDR L3: QQRSNWPPLT (SEQ ID NO: 169)

실시예 21.8. ACE-07의 제조 Example 21.8. Preparation of ACE-07

ACE-07 contains two different heavy chain-like chains (ACE-07-VH and ACE-07-VL) and two identical light chains (ACE-07-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-07-VH amino acid sequence (SEQ ID NO: 174)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKCLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS

ACE-07-VL amino acid sequence (SEQ ID NO: 175)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGTVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPW TFAGCTKLEIKR

ACE-07-LC amino acid sequence (SEQ ID NO: 157)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 12 below. have:

Fab region (Anti-PD-L1)Fab region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (서열번호: 170)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 170)
	CDR H1: GGTFSSYA (서열번호: 159) CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171) CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKCLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 176)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKCLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 176)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGCTKLEIKR (서열번호: 180)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGCTKLEIKR (SEQ ID NO: 180)
	CDR H1: GYSFTGYT (서열번호: 177)CDR H1: GYSFTGYT (SEQ ID NO: 177)	CDR L1: QDIRNY (서열번호: 181)CDR L1: QDIRNY (SEQ ID NO: 181)
	CDR H2: INPYKGVS (서열번호: 178)CDR H2: INPYKGVS (SEQ ID NO: 178)	CDR L2: YTS (서열번호: 182)CDR L2: YTS (SEQ ID NO: 182)
	CDR H3: ARSGYYGDSDWYFDV (서열번호: 179)CDR H3: ARSGYYGDSDWYFDV (SEQ ID NO: 179)	CDR L3: QQGNTLPW (서열번호: 183)CDR L3: QQGNTLPW (SEQ ID NO: 183)

실시예 21.9. ACE-08의 제조 Example 21.9. Preparation of ACE-08

ACE-08 contains two different heavy chain-like chains (ACE-08-VH and ACE-08-VL) and two identical light chains (ACE-08-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-08-VH amino acid sequence (SEQ ID NO: 184)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs EVQLVESGGGLVQPGKSLKLSCEAS GFTFSGYG MHWVRQAPGRCLESVAY ITSSSINI KYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYC ARFDWDKNY WGQGTMVTVSS

ACE-08-VL amino acid sequence (SEQ ID NO: 185)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs QMTQSPSSLPASLGDRVTINCQAS QDISNY LNWYQQKPGKAPKLLIY YTN KLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYC QQYYNYPWT FGPCTKLEIKR

ACE-08-LC amino acid sequence (SEQ ID NO: 157)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 13 below. have:

Fab region (Anti-PD-L1)Fab region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (서열번호: 170)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 170)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171) CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRCLESVAYITSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSS (서열번호: 186)VH: EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRCLESVAYITSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSS (SEQ ID NO: 186)	VL: QMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPCTKLEIKR (서열번호: 190)VL: QMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPCTKLEIKR (SEQ ID NO: 190)
	CDR H1: GFTFSGYG (서열번호: 187)CDR H1: GFTFSGYG (SEQ ID NO: 187)	CDR L1: QDISNY (서열번호: 191) CDR L1: QDISNY (SEQ ID NO: 191)
	CDR H2: ITSSSINI (서열번호: 188)CDR H2: ITSSSINI (SEQ ID NO: 188)	CDR L2: YTN (서열번호: 192)CDR L2: YTN (SEQ ID NO: 192)
	CDR H3: ARFDWDKNY (서열번호: 189)CDR H3: ARFDWDKNY (SEQ ID NO: 189)	CDR L3: QQYYNYPWT (서열번호: 193)CDR L3: QQYYNYPWT (SEQ ID NO: 193)

실시예 21.10. ACE-09의 제조Example 21.10. Preparation of ACE-09

ACE-09 contains two different heavy chain-like chains (ACE-09-VH and ACE-09-VL) and two identical light chains (ACE-09-LC). Compared to ACE-05, ACE-09 does not contain a G4S linker (amino acid sequence of GGGS) in the flexible peptide region. The amino acid sequences of these three types of polypeptides are as follows:

ACE-09-VH amino acid sequence (without the G4S linker) :

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYTMN WVKQSHGKNLEWMG LINPYKGVST YNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCAR SGYYGDSDWYFDV WGQGTTLTVFS (SEQ ID NO: 96)

ACE-09-VL amino acid sequence (without the G4S linker) :

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP DIQMTQTTSSLSASLGDRVTISC RASQDIRNYLN WYQQKPDGTVKLLIY YTSRLHS GVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR (SEQ ID NO: 97)

ACE-09-LC amino acid sequence (anti-PD-L1 antibody light chain): QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVL [RSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC] ( SEQ ID NO: 95)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 14 below. have:

Fab region (Anti-PD-L1)Fab region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 4)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 4)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (서열번호: 8)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 8)
	CDR H1: GGTFSSYA (서열번호: 5)CDR H1: GGTFSSYA (SEQ ID NO: 5)	CDR L1: SSNIGAGYD (서열번호: 9)CDR L1: SSNIGAGYD (SEQ ID NO: 9)
	CDR H2: IIPILGIA (서열번호: 6)CDR H2: IIPILGIA (SEQ ID NO: 6)	CDR L2: GDI (서열번호: 10)CDR L2: GDI (SEQ ID NO: 10)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 7)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 7)	CDR L3: QSYDSSLSGGV (서열번호: 11)CDR L3: QSYDSSLSGGV (SEQ ID NO: 11)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 12)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 12)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 16)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 16)
	CDR H1: GYSFTGYTMN (서열번호: 13)CDR H1: GYSFTGYTMN (SEQ ID NO: 13)	CDR L1: RASQDIRNYLN (서열번호: 17)CDR L1: RASQDIRNYLN (SEQ ID NO: 17)
	CDR H2: LINPYKGVST (서열번호: 14)CDR H2: LINPYKGVST (SEQ ID NO: 14)	CDR L2: YTSRLHS (서열번호: 18)CDR L2: YTSRLHS (SEQ ID NO: 18)
	CDR H3: SGYYGDSDWYFDV (서열번호: 15)CDR H3: SGYYGDSDWYFDV (SEQ ID NO: 15)	CDR L3: QQGNTLPWT (서열번호: 19)CDR L3: QQGNTLPWT (SEQ ID NO: 19)

The DNA sequences encoding ACE-09-VH, ACE-09-VL and ACE-09-LC have the nucleic acid sequences of SEQ ID NOs: 108, 109 and 107, respectively.

실시예 21.11. ACE-10의 제조Example 21.11. Preparation of ACE-10

ACE-10 contains two different heavy chain-like chains (ACE-10-VH and ACE-10-VL) and two identical light chains (ACE-10-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-10-VH amino acid sequence (SEQ ID NO: 23)

QVQLQQPGAELVKPGASVKMSCKAS GYTFTSYN MHWVKQTPGRGLEWIGA IYPGNGDT SYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYC ARSTYYGGDWYFNV WGAGTTVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYTMN WVKQSHGKNLEWMG LINPYKGVST YNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS

ACE-10-VL amino acid sequence (SEQ ID NO: 24)

QVQLQQPGAELVKPGASVKMSCKAS GYTFTSYN MHWVKQTPGRGLEWIGA IYPGNGDT SYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYC ARSTYYGGDWYFNV WGAGTTVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs DIQMTQTTSSLSASLGDRVTISC RASQDIRNYLN WYQQKPDGTVKLLIY YTSRLHS GVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR

ACE-10-LC amino acid sequence (SEQ ID NO: 25)

QIVLSQSPAILSASPGEKVTMTCRAS SSVSY IHWFQQKPGSSPKPWIY ATS NLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYC QQWTSNPPT FGGGTKLEIK [ RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPYPREAKVQWKVDTLSKVQWKLSKDSKSKLSVDS

The VH and VL amino acid sequences and CDR sequences therein for the bivalent Fab region targeting CD20 and the monovalent Fv region targeting CD3 are listed in Table 15 below:

Fab region (Anti-CD20)Fab region (Anti-CD20)	VH: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (서열번호: 26)VH: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 26)	VL: QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKR (서열번호: 30)VL: QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKR (SEQ ID NO: 30)
	CDR H1: GYTFTSYN (서열번호: 27)CDR H1: GYTFTSYN (SEQ ID NO: 27)	CDR L1: SSVSY (서열번호: 31)CDR L1: SSVSY (SEQ ID NO: 31)
	CDR H2: IYPGNGDT (서열번호: 28)CDR H2: IYPGNGDT (SEQ ID NO: 28)	CDR L2: ATS (서열번호: 32)CDR L2: ATS (SEQ ID NO: 32)
	CDR H3: ARSTYYGGDWYFNV (서열번호: 29)CDR H3: ARSTYYGGDWYFNV (SEQ ID NO: 29)	CDR L3: QQWTSNPPT (서열번호: 33)CDR L3: QQWTSNPPT (SEQ ID NO: 33)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 12)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 12)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 16)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 16)
	CDR H1: GYSFTGYTMN (서열번호: 13)CDR H1: GYSFTGYTMN (SEQ ID NO: 13)	CDR L1: RASQDIRNYLN (서열번호: 17)CDR L1: RASQDIRNYLN (SEQ ID NO: 17)
	CDR H2: LINPYKGVST (서열번호: 14)CDR H2: LINPYKGVST (SEQ ID NO: 14)	CDR L2: YTSRLHS (서열번호: 18)CDR L2: YTSRLHS (SEQ ID NO: 18)
	CDR H3: SGYYGDSDWYFDV (서열번호: 15)CDR H3: SGYYGDSDWYFDV (SEQ ID NO: 15)	CDR L3: QQGNTLPWT (서열번호: 19)CDR L3: QQGNTLPWT (SEQ ID NO: 19)

DNA sequences encoding ACE-10-VH, ACE-10-VL and ACE-10-LC have nucleic acid sequences of SEQ ID NOs: 34, 35 and 36, respectively.

실시예 21.12. ACE-11의 제조Example 21.12. Preparation of ACE-11

ACE-11 has the same overall structure as ACE-05 and ACE-10, and has two different heavy chain-like chains (ACE-11-VH and ACE-11-VL) and two identical light chains (ACE-11-LC). Includes. The amino acid sequences of these three types of polypeptides are as follows:

ACE-11-VH amino acid sequence (SEQ ID NO: 37)

QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYTMN WVKQSHGKNLEWMG LINPYKGVST YNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS

ACE-11-VL amino acid sequence (SEQ ID NO: 38)

QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs DIQMTQTTSSLSASLGDRVTISC RASQDIRNYLN WYQQKPDGTVKLLIY YTSRLHS GVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR

ACE-11-LC amino acid sequence (SEQ ID NO: 39)

DILLTQSPVILSVSPGERVSFSCRASDILLTQSPVILSVSPGERVSFSCRAS QSIGTNQSIGTN IHWYQQRTNGSPRLLIKIHWYQQRTNGSPRLLIK YASYAS ESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQNNNWPTTQQNNNWPTT FGAGTKLELK[FGAGTKLELK[ RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC]

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting EGFR and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 16 below:

Fab region (Anti-EGFR)Fab region (Anti-EGFR)	VH: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (서열번호: 40)VH: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 40)	VL: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKR (서열번호: 44)VL: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKR (SEQ ID NO: 44)
	CDR H1: GFSLTNYG (서열번호: 41)CDR H1: GFSLTNYG (SEQ ID NO: 41)	CDR L1: QSIGTN (서열번호: 45)CDR L1: QSIGTN (SEQ ID NO: 45)
	CDR H2: IWSGGNT (서열번호: 42) CDR H2: IWSGGNT (SEQ ID NO: 42)	CDR L2: YAS (서열번호: 46)CDR L2: YAS (SEQ ID NO: 46)
	CDR H3: ARALTYYDYEFAY (서열번호: 43) CDR H3: ARALTYYDYEFAY (SEQ ID NO: 43)	CDR L3: QQNNNWPTT (서열번호: 47)CDR L3: QQNNNWPTT (SEQ ID NO: 47)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 12)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 12)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 16)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 16)
	CDR H1: GYSFTGYTMN (서열번호: 13)CDR H1: GYSFTGYTMN (SEQ ID NO: 13)	CDR L1: RASQDIRNYLN (서열번호: 17)CDR L1: RASQDIRNYLN (SEQ ID NO: 17)
	CDR H2: LINPYKGVST (서열번호: 14)CDR H2: LINPYKGVST (SEQ ID NO: 14)	CDR L2: YTSRLHS (서열번호: 18)CDR L2: YTSRLHS (SEQ ID NO: 18)
	CDR H3: SGYYGDSDWYFDV (서열번호: 15)CDR H3: SGYYGDSDWYFDV (SEQ ID NO: 15)	CDR L3: QQGNTLPWT (서열번호: 19)CDR L3: QQGNTLPWT (SEQ ID NO: 19)

DNA sequences encoding ACE-11-VH, ACE-11-VL and ACE-11-LC have nucleic acid sequences of SEQ ID NOs: 48, 49 and 50, respectively.

실시예 21.13. ACE-12의 제조Example 21.13. Preparation of ACE-12

ACE-12 comprises a G4S linker with the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 113) and GGSGGGGSG (SEQ ID NO: 114), while ACE-05 comprises a G4S linker with the GGGGS amino acid sequence of the flexible peptide region. The amino acid sequences of these three types of polypeptides are as follows:

ACE-12-VH amino acid sequence (with 10 residues GGGGSGGGGS) :

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggsggggs EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYTMN WVKQSHGKNLEWMG LINPYKGVST YNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCAR SGYYGDSDWYFDV WGQGTTLTVFS (SEQ ID NO: 98)

ACE-12-VL amino acid sequence (with 9 residues GGSGGGGSG) :

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggsggggsg DIQMTQTTSSLSASLGDRVTISC RASQDIRNYLN WYQQKPDGTVKLLIY YTSRLHS GVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR (SEQ ID NO: 99)

ACE-12-LC amino acid sequence (anti-PD-L1 antibody light chain) :

QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVL [RSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC] (SEQ ID NO: 95)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region of UCHT1 are listed in Table 17 below:

DNA sequences encoding ACE-12-VH, ACE-12-VL and ACE-12-LC have nucleic acid sequences of SEQ ID NOs: 110, 111 and 107, respectively.

실시예 21.14. ACE-13의 제조Example 21.14. Preparation of ACE-13

ACE-13 contains two different heavy chain-like chains (ACE-13-VH and ACE-13-VL) and two identical light chains (ACE-13-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-13-VH amino acid sequence (SEQ ID NO: 194)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs QVQLVESGGGVVQPGRSLRLSCAAS GFTFLRYA MHWVRQAPGKGLEWVAV ISYDGRYK YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC TTTTFDS WGQGTLVTVSS

ACE-13-VL amino acid sequence (SEQ ID NO: 195)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs DIVMTQTPLSLPVTPGEAASISCRSS QSLLDSEDGNTY LDWYLQKPGQSPQLLIY TLS HRASGVPDRFSGSGSGTDFTLEISRVEAEDVGVYYC MQRRDFPFT FGQGTKVDIKR

ACE-13-LC amino acid sequence (SEQ ID NO: 157)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen-binding domain bivalent Fab region targeting PD-L1 and the second antigen-binding domain monovalent Fv region targeting PD-1 are shown in Table 18 below. It is listed:

Fab region (Anti-PD-L1)Fab region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (서열번호: 170)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 170)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171)CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fv region (Anti-PD-1)Fv region (Anti-PD-1)	VH: QVQLVESGGGVVQPGRSLRLSCAASGFTFLRYAMHWVRQAPGKGLEWVAVISYDGRYKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTTTTFDSWGQGTLVTVSS (서열번호: 196)VH: QVQLVESGGGVVQPGRSLRLSCAASGFTFLRYAMHWVRQAPGKGLEWVAVISYDGRYKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTTTTFDSWGQGTLVTVSS (SEQ ID NO: 196)	VL: DIVMTQTPLSLPVTPGEAASISCRSSQSLLDSEDGNTYLDWYLQKPGQSPQLLIYTLSHRASGVPDRFSGSGSGTDFTLEISRVEAEDVGVYYCMQRRDFPFTFGQGTKVDIKR (서열번호: 200)VL: DIVMTQTPLSLPVTPGEAASISCRSSQSLLDSEDGNTYLDWYLQKPGQSPQLLIYTLSHRASGVPDRFSGSGSGTDFTLEISRVEAEDVGVYYCMQRRDFPFTFGQGTKVDIKR (SEQ ID NO: 200)
	CDR H1: GFTFLRYA (서열번호: 197)CDR H1: GFTFLRYA (SEQ ID NO: 197)	CDR L1: QSLLDSEDGNTY (서열번호: 201)CDR L1: QSLLDSEDGNTY (SEQ ID NO: 201)
	CDR H2: ISYDGRYK (서열번호: 198)CDR H2: ISYDGRYK (SEQ ID NO: 198)	CDR L2: TLS (서열번호: 202)CDR L2: TLS (SEQ ID NO: 202)
	CDR H3: TTTTFDS (서열번호: 199)CDR H3: TTTTFDS (SEQ ID NO: 199)	CDR L3: MQRRDFPFT (서열번호: 203)CDR L3: MQRRDFPFT (SEQ ID NO: 203)

실시예 21.15. ACE-14의 제조Example 21.15. Preparation of ACE-14

ACE-14 contains two different heavy chain-like chains (ACE-14-VH and ACE-14-VL) and two identical light chains (ACE-14-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-14-VH amino acid sequence (SEQ ID NO: 204)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggsggggs EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKCLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS

ACE-14-VL amino acid sequence (SEQ ID NO: 205)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggsggggs DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGTVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FACGTKLEIKR

ACE-14-LC amino acid sequence (SEQ ID NO: 157)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 19 below. Has :

Fab region (Anti-PD-L1)Fab region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (서열번호: 170)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 170)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171)CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKCLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 176)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKCLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 176)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFACGTKLEIKR (서열번호: 206)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFACGTKLEIKR (SEQ ID NO: 206)
	CDR H1: GYSFTGYT (서열번호: 177)CDR H1: GYSFTGYT (SEQ ID NO: 177)	CDR L1: QDIRNY (서열번호: 181)CDR L1: QDIRNY (SEQ ID NO: 181)
	CDR H2: INPYKGVS (서열번호: 178)CDR H2: INPYKGVS (SEQ ID NO: 178)	CDR L2: YTS (서열번호: 182)CDR L2: YTS (SEQ ID NO: 182)
	CDR H3: ARSGYYGDSDWYFDV (서열번호: 179)CDR H3: ARSGYYGDSDWYFDV (SEQ ID NO: 179)	CDR L3: QQGNTLPWT (서열번호: 207)CDR L3: QQGNTLPWT (SEQ ID NO: 207)

실시예 21.16. ACE-15의 제조Example 21.16. Preparation of ACE-15

ACE-15 contains two different heavy chain-like chains (ACE-15-VH and ACE-15-VL) and two identical light chains (ACE-15-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-15-VH amino acid sequence (SEQ ID NO: 208)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGCGTTLTVFS

ACE-15-VL amino acid sequence (SEQ ID NO: 209)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGCVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR

ACE-15-LC amino acid sequence (SEQ ID NO: 157)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen-binding domain bivalent Fab region targeting PD-L1 and the second antigen-binding domain monovalent Fv region targeting CD3 are listed in Table 20 below. Has been:

Fab region (Anti-PD-L1)Fab region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (서열번호: 170)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 170)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171)CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGCGTTLTVFS (서열번호: 210)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGCGTTLTVFS (SEQ ID NO: 210)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGCVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 212)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGCVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 212)
	CDR H1: GYSFTGYT (서열번호: 177)CDR H1: GYSFTGYT (SEQ ID NO: 177)	CDR L1: QDIRNY (서열번호: 181)CDR L1: QDIRNY (SEQ ID NO: 181)
	CDR H2: INPYKGVS (서열번호: 178)CDR H2: INPYKGVS (SEQ ID NO: 178)	CDR L2: YTS (서열번호: 182)CDR L2: YTS (SEQ ID NO: 182)
	CDR H3: ARSGYYGDSDWYFD (서열번호: 211)CDR H3: ARSGYYGDSDWYFD (SEQ ID NO: 211)	CDR L3: QQGNTLPWT (서열번호: 207)CDR L3: QQGNTLPWT (SEQ ID NO: 207)

실시예 21.17. ACE-16의 제조Example 21.17. Preparation of ACE-16

ACE-16 contains two different heavy chain-like chains (ACE-16-VH and ACE-16-VL) and two identical light chains (ACE-16-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-16-VH amino acid sequence (SEQ ID NO: 1)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYTMN WVKQSHGKNLEWMG LINPYKGVST YNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS

ACE-16-VL amino acid sequence (SEQ ID NO: 2)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs DIQMTQTTSSLSASLGDRVTISC RASQDIRNYLN WYQQKPDGTVKLLIY YTSRLHS GVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR

ACE-16-LC amino acid sequence (SEQ ID NO: 3)

QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVL [ RTVAAPSVFIFPPSDEQLKSGTASVVCSKNFLTQLKSGTASVVCSKNFQLKSGTASVVCSKNFREADS

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 21 below. Has been:

Fab region (Anti-PD-L1)Fab region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 4)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 4)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (서열번호: 8)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 8)
	CDR H1: GGTFSSYA (서열번호: 5)CDR H1: GGTFSSYA (SEQ ID NO: 5)	CDR L1: SSNIGAGYD (서열번호: 9)CDR L1: SSNIGAGYD (SEQ ID NO: 9)
	CDR H2: IIPILGIA (서열번호: 6)CDR H2: IIPILGIA (SEQ ID NO: 6)	CDR L2: GDI (서열번호: 10)CDR L2: GDI (SEQ ID NO: 10)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 7) CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 7)	CDR L3: QSYDSSLSGGV (서열번호: 11)CDR L3: QSYDSSLSGGV (SEQ ID NO: 11)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 12)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 12)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 16)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 16)
	CDR H1: GYSFTGYTMN (서열번호: 13)CDR H1: GYSFTGYTMN (SEQ ID NO: 13)	CDR L1: RASQDIRNYLN (서열번호: 17)CDR L1: RASQDIRNYLN (SEQ ID NO: 17)
	CDR H2: LINPYKGVST (서열번호: 14)CDR H2: LINPYKGVST (SEQ ID NO: 14)	CDR L2: YTSRLHS (서열번호: 18)CDR L2: YTSRLHS (SEQ ID NO: 18)
	CDR H3: SGYYGDSDWYFDV (서열번호: 15)CDR H3: SGYYGDSDWYFDV (SEQ ID NO: 15)	CDR L3: QQGNTLPWT (서열번호: 19)CDR L3: QQGNTLPWT (SEQ ID NO: 19)

실시예 21.18. ACE-17의 제조Example 21.18. Preparation of ACE-17

ACE-17 contains two different heavy chain-like chains (ACE-17-VH and ACE-17-VL) and two identical light chains (ACE-17-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-17-VH amino acid sequence (SEQ ID NO: 217)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS

ACE-17-VL amino acid sequence (SEQ ID NO: 218)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGTVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR

ACE-17-LC amino acid sequence (SEQ ID NO: 157)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 22 below. have:

Fab region (Anti-PD-L1)Fab region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (서열번호: 170) VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 170)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171)CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 215)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 215)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 216)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 216)
	CDR H1: GYSFTGYT (서열번호: 177)CDR H1: GYSFTGYT (SEQ ID NO: 177)	CDR L1: QDIRNY (서열번호: 181)CDR L1: QDIRNY (SEQ ID NO: 181)
	CDR H2: INPYKGVS (서열번호: 178)CDR H2: INPYKGVS (SEQ ID NO: 178)	CDR L2: YTS (서열번호: 182)CDR L2: YTS (SEQ ID NO: 182)
	CDR H3: ARSGYYGDSDWYFD (서열번호: 211)CDR H3: ARSGYYGDSDWYFD (SEQ ID NO: 211)	CDR L3: QQGNTLPWT (서열번호: 207)CDR L3: QQGNTLPWT (SEQ ID NO: 207)

실시예 21.19. ACE-18의 제조Example 21.19. Preparation of ACE-18

ACE-18 contains two different heavy chain-like chains (ACE-18-VH and ACE-18-VL) and two identical light chains (ACE-18-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-18-VH amino acid sequence (SEQ ID NO: 219)

QVQLQQPGAELVKPGASVKMSCKAS GYTFTSYN MHWVKQTPGRGLEWIGA IYPGNGDT SYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYC ARSTYYGGDWYFNV WGAGTTVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPPCPAPELLGGP GGGGS EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS

ACE-18-VL amino acid sequence (SEQ ID NO: 220)

QVQLQQPGAELVKPGASVKMSCKAS GYTFTSYN MHWVKQTPGRGLEWIGA IYPGNGDT SYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYC ARSTYYGGDWYFNV WGAGTTVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPPCPAPELLGGP GGGGS DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGTVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR

ACE-18-LC amino acid sequence (SEQ ID NO: 221)

QIVLSQSPAILSASPGEKVTMTCRAS SSVSY IHWFQQKPGSSPKPWIY ATS NLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYC QQWTSNPPT FGGGTKLEIK [ RSVAAPSVFIFPPSDEQLKSGTKLEIK [RSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPYPREAKVQWKVDTLSKVQWKVDTLSKVQWKVT

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD20 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 23 below:

Fab region (Anti-CD20)Fab region (Anti-CD20)	VH: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (서열번호: 222)VH: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 222)	VL: QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (서열번호: 226)VL: QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 226)
	CDR H1: GYTFTSYN (서열번호: 223)CDR H1: GYTFTSYN (SEQ ID NO: 223)	CDR L1: SSVSY (서열번호: 227)CDR L1: SSVSY (SEQ ID NO: 227)
	CDR H2: IYPGNGDT (서열번호: 224)CDR H2: IYPGNGDT (SEQ ID NO: 224)	CDR L2: ATS (서열번호: 228)CDR L2: ATS (SEQ ID NO: 228)
	CDR H3: ARSTYYGGDWYFNV (서열번호: 225)CDR H3: ARSTYYGGDWYFNV (SEQ ID NO: 225)	CDR L3: QQWTSNPPT (서열번호: 229)CDR L3: QQWTSNPPT (SEQ ID NO: 229)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 215)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 215)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 216)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 216)
	CDR H1: GYSFTGYT (서열번호: 177)CDR H1: GYSFTGYT (SEQ ID NO: 177)	CDR L1: QDIRNY (서열번호: 181)CDR L1: QDIRNY (SEQ ID NO: 181)
	CDR H2: INPYKGVS (서열번호: 178)CDR H2: INPYKGVS (SEQ ID NO: 178)	CDR L2: YTS (서열번호: 182)CDR L2: YTS (SEQ ID NO: 182)
	CDR H3: ARSGYYGDSDWYFD (서열번호: 211)CDR H3: ARSGYYGDSDWYFD (SEQ ID NO: 211)	CDR L3: QQGNTLPWT (서열번호: 207)CDR L3: QQGNTLPWT (SEQ ID NO: 207)

실시예 21.20. ACE-19의 제조Example 21.20. Preparation of ACE-19

ACE-19 contains two different heavy chain-like chains (ACE-19-VH and ACE-19-VL) and two identical light chains (ACE-19-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-19-VH amino acid sequence (SEQ ID NO: 230)

QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPPCPAPELLGGP GGGGS EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS

ACE-19-VL amino acid sequence (SEQ ID NO: 231)

QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPPCPAPELLGGP GGGGS DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGTVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR

ACE-19-LC amino acid sequence (SEQ ID NO: 232)

DILLTQSPVILSVSPGERVSFSCRAS QSIGTN IHWYQQRTNGSPRLLIK YAS ESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQNNNWPTT FGAGTKLELK [ RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWHKVDNALKAQSGNSHKVDNALKLSQS

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting EGFR and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 24 below:

Fab region (Anti-EGFR)Fab region (Anti-EGFR)	VH: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (서열번호: 233)VH: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 233)	VL: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (서열번호: 237)VL: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 237)
	CDR H1: GFSLTNYG (서열번호: 234)CDR H1: GFSLTNYG (SEQ ID NO: 234)	CDR L1: QSIGTN (서열번호: 238)CDR L1: QSIGTN (SEQ ID NO: 238)
	CDR H2: IWSGGNT (서열번호: 235)CDR H2: IWSGGNT (SEQ ID NO: 235)	CDR L2: YAS (서열번호: 239)CDR L2: YAS (SEQ ID NO: 239)
	CDR H3: ARALTYYDYEFAY (서열번호: 236)CDR H3: ARALTYYDYEFAY (SEQ ID NO: 236)	CDR L3: QQNNNWPTT (서열번호: 240)CDR L3: QQNNNWPTT (SEQ ID NO: 240)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 215)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 215)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 216)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 216)
	CDR H1: GYSFTGYT (서열번호: 177)CDR H1: GYSFTGYT (SEQ ID NO: 177)	CDR L1: QDIRNY (서열번호: 181)CDR L1: QDIRNY (SEQ ID NO: 181)
	CDR H2: INPYKGVS (서열번호: 178)CDR H2: INPYKGVS (SEQ ID NO: 178)	CDR L2: YTS (서열번호: 182)CDR L2: YTS (SEQ ID NO: 182)
	CDR H3: ARSGYYGDSDWYFD (서열번호: 211)CDR H3: ARSGYYGDSDWYFD (SEQ ID NO: 211)	CDR L3: QQGNTLPWT (서열번호: 207)CDR L3: QQGNTLPWT (SEQ ID NO: 207)

실시예 21.21. ACE-20의 제조Example 21.21. Preparation of ACE-20

ACE-20 contains two different heavy chain-like chains (ACE-20-VH and ACE-20-VL) and two identical light chains (ACE-20-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-20-VH amino acid sequence (SEQ ID NO: 241)

QVQLVQSGAEVKKPGASVKVSCKAS GYTFTSHW MHWVRQAPGQGLEWIGE FNPSNGRT NYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYC ASRDYDYDCRYFDY WGQGTLVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS *

ACE-20-VL amino acid sequence (SEQ ID NO: 242)

QVQLVQSGAEVKKPGASVKVSCKAS GYTFTSHW MHWVRQAPGQGLEWIGE FNPSNGRT NYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYC ASRDYDYDCRYFDY WGQGTLVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGTVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR

ACE-20-LC amino acid sequence (SEQ ID NO: 243)

DIQMTQSPSSLSASVGDRVTITCSAS SSVTY MYWYQQKPGKAPKLLIY DTS NLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYC QQWSSHIFT FGQGTKVEIKR [ TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPYPREAKVQSKLSVDSKVQWKVDTLSKSKLSVD

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting EGFR and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 25 below:

Fab region (Anti-EGFR)Fab region (Anti-EGFR)	VH: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLEWIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDCRYFDYWGQGTLVTVSS (서열번호: 244)VH: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLEWIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDCRYFDYWGQGTLVTVSS (SEQ ID NO: 244)	VL: DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKAPKLLIYDTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIKR (서열번호: 248)VL: DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKAPKLLIYDTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIKR (SEQ ID NO: 248)
	CDR H1: GYTFTSHW (서열번호: 245)CDR H1: GYTFTSHW (SEQ ID NO: 245)	CDR L1: SSVTY (서열번호: 249)CDR L1: SSVTY (SEQ ID NO: 249)
	CDR H2: FNPSNGRT (서열번호: 246)CDR H2: FNPSNGRT (SEQ ID NO: 246)	CDR L2: DTS (서열번호: 250)CDR L2: DTS (SEQ ID NO: 250)
	CDR H3: ASRDYDYDCRYFDY (서열번호: 247)CDR H3: ASRDYDYDCRYFDY (SEQ ID NO: 247)	CDR L3: QQWSSHIFT (서열번호: 251)CDR L3: QQWSSHIFT (SEQ ID NO: 251)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 215)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 215)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 216)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 216)
	CDR H1: GYSFTGYT (서열번호: 177)CDR H1: GYSFTGYT (SEQ ID NO: 177)	CDR L1: QDIRNY (서열번호: 181)CDR L1: QDIRNY (SEQ ID NO: 181)
	CDR H2: INPYKGVS (서열번호: 178)CDR H2: INPYKGVS (SEQ ID NO: 178)	CDR L2: YTS (서열번호: 182)CDR L2: YTS (SEQ ID NO: 182)
	CDR H3: ARSGYYGDSDWYFD (서열번호: 211)CDR H3: ARSGYYGDSDWYFD (SEQ ID NO: 211)	CDR L3: QQGNTLPWT (서열번호: 207)CDR L3: QQGNTLPWT (SEQ ID NO: 207)

실시예 21.22. ACE-21의 제조Example 21.22. Preparation of ACE-21

ACE-21 contains two different heavy chain-like chains (ACE-21-VH and ACE-21-VL) and two identical light chains (ACE-21-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-21-VH amino acid sequence (SEQ ID NO: 252)

QVQLVESGGGVVQPGRSLRLSCAAS GFTFSTYG MHWVRQAPGKGLEWVAV IWDDGSYK YYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARDGITMVRGVMKDYFDY WGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPPCPAPELLGGPggggs EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS

ACE-21-VL amino acid sequence (SEQ ID NO: 253)

QVQLVESGGGVVQPGRSLRLSCAAS GFTFSTYG MHWVRQAPGKGLEWVAV IWDDGSYK YYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARDGITMVRGVMKDYFDY WGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPPCPAPELLGGPggggs DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGTVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR

ACE-21-LC amino acid sequence (SEQ ID NO: 254)

AIQLTQSPSSLSASVGDRVTITCRASAIQLTQSPSSLSASVGDRVTITCRAS QDISSAQDISSA LVWYQQKPGKAPKLLIYLVWYQQKPGKAPKLLIY DASDAS SLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYC QQFNSYPLTQQFNSYPLT FGGGTKVEIKRFGGGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting EGFR and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 26 below:

Fab region (Anti-EGFR)Fab region (Anti-EGFR)	VH: QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGTLVTVSS (서열번호: 255)VH: QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGTLVTVSS (SEQ ID NO: 255)	VL: AIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPKLLIYDASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKR (서열번호: 259)VL: AIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPKLLIYDASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKR (SEQ ID NO: 259)
	CDR H1: GFTFSTYG (서열번호: 256)CDR H1: GFTFSTYG (SEQ ID NO: 256)	CDR L1: QDISSA (서열번호: 260)CDR L1: QDISSA (SEQ ID NO: 260)
	CDR H2: IWDDGSYK (서열번호: 257)CDR H2: IWDDGSYK (SEQ ID NO: 257)	CDR L2: DAS (서열번호: 261)CDR L2: DAS (SEQ ID NO: 261)
	CDR H3: ARDGITMVRGVMKDYFDY (서열번호: 258)CDR H3: ARDGITMVRGVMKDYFDY (SEQ ID NO: 258)	CDR L3: QQFNSYPLT (서열번호: 262)CDR L3: QQFNSYPLT (SEQ ID NO: 262)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 215)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 215)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 216)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 216)
	CDR H1: GYSFTGYT (서열번호: 177)CDR H1: GYSFTGYT (SEQ ID NO: 177)	CDR L1: QDIRNY (서열번호: 181)CDR L1: QDIRNY (SEQ ID NO: 181)
	CDR H2: INPYKGVS (서열번호: 178)CDR H2: INPYKGVS (SEQ ID NO: 178)	CDR L2: YTS (서열번호: 182)CDR L2: YTS (SEQ ID NO: 182)
	CDR H3: ARSGYYGDSDWYFD (서열번호: 211)CDR H3: ARSGYYGDSDWYFD (SEQ ID NO: 211)	CDR L3: QQGNTLPWT (서열번호: 207)CDR L3: QQGNTLPWT (SEQ ID NO: 207)

실시예 21.23. ACE-22의 제조Example 21.23. Preparation of ACE-22

ACE-22 contains two different heavy chain-like chains (ACE-22-VH and ACE-22-VL) and two identical light chains (ACE-22-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-22-VH amino acid sequence (SEQ ID NO: 263)

QVQLQQPGAELVKPGASVKMSCKAS GYTFTSYN MHWVKQTPGRGLEWIGA IYPGNGDT SYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYC ARSTYYGGDWYFNV WGAGTTVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPPCPAPELLGGP EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS

ACE-22-VL amino acid sequence (SEQ ID NO: 264)

QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPPCPAPELLGGP DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGTVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR

ACE-22-LC amino acid sequence (SEQ ID NO: 221)

QIVLSQSPAILSASPGEKVTMTCRAS SSVSY IHWFQQKPGSSPKPWIY ATS NLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYC QQWTSNPPT FGGGTKLEIK [ RSVAAPSVFIFPPSDEQLKSGTKLEIK [RSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPYPREAKVQWKVDTLSKVQWKVDTLSKVQWKVDL

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD20 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 27 below:

실시예 21.24. ACE-23의 제조Example 21.24. Preparation of ACE-23

ACE-23 contains two different heavy chain-like chains (ACE-23-VH and ACE-23-VL) and two identical light chains (ACE-23-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-23-VH amino acid sequence (SEQ ID NO: 269)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QVQLVESGGGVVQPGRSLRLSCAAS GFKFSGYG MHWVRQAPGKGLEWVAV IWYDGSKK YYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARQMGYWHFDL WGRGTLVTVSS

ACE-23-VL amino acid sequence (SEQ ID NO: 270)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYAI SWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYY CAKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs EIVLTQSPATLSLSPGERATLSCRAS QSVSSY LAWYQQKPGQAPRLLIY DAS NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPPLT FGGGTKVEIKR

ACE-23-LC amino acid sequence (SEQ ID NO: 157)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 28 below. have:

Fab region (Anti-PD-L1)Fab region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (서열번호: 170)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 170)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171)CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSS (서열번호: 162)VH: QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSS (SEQ ID NO: 162)	VL: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIKR (서열번호: 166)VL: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIKR (SEQ ID NO: 166)
	CDR H1: GFKFSGYG (서열번호: 163)CDR H1: GFKFSGYG (SEQ ID NO: 163)	CDR L1: QSVSSY (서열번호: 167)CDR L1: QSVSSY (SEQ ID NO: 167)
	CDR H2: IWYDGSKK (서열번호: 271)CDR H2: IWYDGSKK (SEQ ID NO: 271)	CDR L2: DAS (서열번호: 168)CDR L2: DAS (SEQ ID NO: 168)
	CDR H3: ARQMGYWHFDL (서열번호: 165)CDR H3: ARQMGYWHFDL (SEQ ID NO: 165)	CDR L3: QQRSNWPPLT (서열번호: 169)CDR L3: QQRSNWPPLT (SEQ ID NO: 169)

실시예 21.25. ACE-24의 제조Example 21.25. Preparation of ACE-24

ACE-24 contains two different heavy chain-like chains (ACE-24-VH and ACE-24-VL) and two identical light chains (ACE-24-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-24-VH amino acid sequence (SEQ ID NO: 272)

EVQLVESGGGLVQPGGSLRLSCAAS GFNIKDTY IHWVRQAPGKGLEWVAR IYPTNGYT RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDY WGQGTLVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs EVQLVESGGGLVQPGRSLRLSCAAS GFTFDDYA MHWVRQAPGKGLEWVSA ITWNSGHI DYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC AKVSYLSTASSLDY WGQGTLVTVSS

ACE-24-VL amino acid sequence (SEQ ID NO: 273)

EVQLVESGGGLVQPGGSLRLSCAAS GFNIKDTY IHWVRQAPGKGLEWVAR IYPTNGYT RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDY WGQGTLVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPAPELLGGP DIQMTQSPSSLSASVGDRVTITCRAS QGIRNY LAWYQQKPGKAPKLLIY AAS TLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QRYNRAPYT FGQGTKVEIKR

ACE-24-LC amino acid sequence (SEQ ID NO: 274)

DIQMTQSPSSLSASVGDRVTITCRAS QDVNTA VAWYQQKPGKAPKLLIY SAS FLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYC QQHYTTPPT FGQGTKVEIK [ RSVAAPSVFIFPPSDEQLKSGTASVVCLLNNVDYPREAKVQWDTLSVDYPREAKVQWKDS

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting HER2 and the second antigen binding domain monovalent Fv region targeting TNF are listed in Table 29 below:

Fab region (Anti-HER2)Fab region (Anti-HER2)	VH: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (서열번호: 275)VH: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 275)	VL: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (서열번호: 287) VL: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 287)
	CDR H1: GFNIKDTY (서열번호: 276)CDR H1: GFNIKDTY (SEQ ID NO: 276)	CDR L1: QDVNTA (서열번호: 288) CDR L1: QDVNTA (SEQ ID NO: 288)
	CDR H2: IYPTNGYT (서열번호: 277)CDR H2: IYPTNGYT (SEQ ID NO: 277)	CDR L2: SAS (서열번호: 289)CDR L2: SAS (SEQ ID NO: 289)
	CDR H3: SRWGGDGFYAMDY (서열번호: 278)CDR H3: SRWGGDGFYAMDY (SEQ ID NO: 278)	CDR L3: QQHYTTPPT (서열번호: 290)CDR L3: QQHYTTPPT (SEQ ID NO: 290)
Fv region (Anti-TNF)Fv region (Anti-TNF)	VH: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS (서열번호: 279)VH: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS (SEQ ID NO: 279)	VL: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKR (서열번호: 283) VL: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKR (SEQ ID NO: 283)
	CDR H1: GFTFDDYA (서열번호: 280)CDR H1: GFTFDDYA (SEQ ID NO: 280)	CDR L1: QGIRNY (서열번호: 284) CDR L1: QGIRNY (SEQ ID NO: 284)
	CDR H2: ITWNSGHI (서열번호: 281)CDR H2: ITWNSGHI (SEQ ID NO: 281)	CDR L2: AAS (서열번호: 285)CDR L2: AAS (SEQ ID NO: 285)
	CDR H3: AKVSYLSTASSLDY (서열번호: 282)CDR H3: AKVSYLSTASSLDY (SEQ ID NO: 282)	CDR L3: QRYNRAPYT (서열번호: 286)CDR L3: QRYNRAPYT (SEQ ID NO: 286)

실시예 21.26. ACE-25의 제조Example 21.26. Preparation of ACE-25

ACE-25 contains two different heavy chain-like chains (ACE-25-VH and ACE-25-VL) and two identical light chains (ACE-25-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-25-VH amino acid sequence (SEQ ID NO: 291)

EVQLVESGGGLVQPGGSLRLSCAAS GFNIKDTY IHWVRQAPGKGLEWVAR IYPTNGYT RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDY WGQGTLVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGPggggsggggs EVQLVESGGGLVQPGRSLRLSCAAS GFTFDDYA MHWVRQAPGKGLEWVSA ITWNSGHI DYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC AKVSYLSTASSLDY WGQGTLVTVSS

ACE-25-VL amino acid sequence (SEQ ID NO: 273)

ACE-25-LC amino acid sequence (SEQ ID NO: 274)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting HER2 and the second antigen binding domain monovalent Fv region targeting TNF are listed in Table 30 below:

Fab region (Anti-HER2)Fab region (Anti-HER2)	VH: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (서열번호: 275)VH: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 275)	VL: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (서열번호: 287)VL: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 287)
	CDR H1: GFNIKDTY (서열번호: 276)CDR H1: GFNIKDTY (SEQ ID NO: 276)	CDR L1: QDVNTA (서열번호: 288)CDR L1: QDVNTA (SEQ ID NO: 288)
	CDR H2: IYPTNGYT (서열번호: 277)CDR H2: IYPTNGYT (SEQ ID NO: 277)	CDR L2: SAS (서열번호: 289)CDR L2: SAS (SEQ ID NO: 289)
	CDR H3: SRWGGDGFYAMDY (서열번호: 278)CDR H3: SRWGGDGFYAMDY (SEQ ID NO: 278)	CDR L3: QQHYTTPPT (서열번호: 290)CDR L3: QQHYTTPPT (SEQ ID NO: 290)
Fv region (Anti-TNF)Fv region (Anti-TNF)	VH: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS (서열번호: 279)VH: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS (SEQ ID NO: 279)	VL: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKR (서열번호: 283)VL: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKR (SEQ ID NO: 283)
	CDR H1: GFTFDDYA (서열번호: 280)CDR H1: GFTFDDYA (SEQ ID NO: 280)	CDR L1: QGIRNY (서열번호: 284)CDR L1: QGIRNY (SEQ ID NO: 284)
	CDR H2: ITWNSGHI (서열번호: 281)CDR H2: ITWNSGHI (SEQ ID NO: 281)	CDR L2: AAS (서열번호: 285)CDR L2: AAS (SEQ ID NO: 285)
	CDR H3: AKVSYLSTASSLDY (서열번호: 282)CDR H3: AKVSYLSTASSLDY (SEQ ID NO: 282)	CDR L3: QRYNRAPYT (서열번호: 286)CDR L3: QRYNRAPYT (SEQ ID NO: 286)

실시예 21.27. ACE-26의 제조Example 21.27. Preparation of ACE-26

ACE-26 contains two different heavy chain-like chains (ACE-26-VH and ACE-26-VL) and two identical light chains (ACE-26-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-26-VH amino acid sequence (SEQ ID NO: 292)

QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs EVKLLESGGGLVQPKGSLKLSCAAS GFTFNTYA MNWVRQAPGKGLEWVAR IRSKYNNYAT YYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYC VRHGNFGNSYVSWFAY WGQGTLVTVSA

ACE-26-VL amino acid sequence (SEQ ID NO: 293)

QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QAVVTQESALTTSPGETVTLTCRSS TGAVTTSNY ANWVQEKPDHLFTGLIG GTN KRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC ALWYSNLWV FGGGTKLTVL

ACE-26-LC amino acid sequence (SEQ ID NO: 232)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting EGFR and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 31 below:

Fab region (Anti-EGFR)Fab region (Anti-EGFR)	VH: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (서열번호: 233)VH: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 233)	VL: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (서열번호: 237)VL: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 237)
	CDR H1: GFSLTNYG (서열번호: 234)CDR H1: GFSLTNYG (SEQ ID NO: 234)	CDR L1: QSIGTN (서열번호: 238)CDR L1: QSIGTN (SEQ ID NO: 238)
	CDR H2: IWSGGNT (서열번호: 235)CDR H2: IWSGGNT (SEQ ID NO: 235)	CDR L2: YAS (서열번호: 239)CDR L2: YAS (SEQ ID NO: 239)
	CDR H3: ARALTYYDYEFAY (서열번호: 236)CDR H3: ARALTYYDYEFAY (SEQ ID NO: 236)	CDR L3: QQNNNWPTT (서열번호: 240)CDR L3: QQNNNWPTT (SEQ ID NO: 240)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: EVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSA (서열번호: 294)VH: EVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSA (SEQ ID NO: 294)	VL: QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL (서열번호: 298)VL: QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL (SEQ ID NO: 298)
	CDR H1: GFTFNTYA (서열번호: 295)CDR H1: GFTFNTYA (SEQ ID NO: 295)	CDR L1: TGAVTTSNY (서열번호: 299)CDR L1: TGAVTTSNY (SEQ ID NO: 299)
	CDR H2: IRSKYNNYAT (서열번호: 296)CDR H2: IRSKYNNYAT (SEQ ID NO: 296)	CDR L2: GTN (서열번호: 300)CDR L2: GTN (SEQ ID NO: 300)
	CDR H3: VRHGNFGNSYVSWFAY (서열번호: 297)CDR H3: VRHGNFGNSYVSWFAY (SEQ ID NO: 297)	CDR L3: ALWYSNLWV (서열번호: 301)CDR L3: ALWYSNLWV (SEQ ID NO: 301)

실시예 21.28. ACE-27의 제조Example 21.28. Preparation of ACE-27

ACE-27 contains two different heavy chain-like chains (ACE-27-VH and ACE-27-VL) and two identical light chains (ACE-27-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-27-VH amino acid sequence (SEQ ID NO: 302)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGPggggs EVKLLESGGGLVQPKGSLKLSCAAS GFTFNTYA MNWVRQAPGKGLEWVAR IRSKYNNYAT YYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYC VRHGNFGNSYVSWFAY WGQGTLVTVSA

ACE-27-VL amino acid sequence (SEQ ID NO: 303)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QAVVTQESALTTSPGETVTLTCRSS TGAVTTSNY ANWVQEKPDHLFTGLIG GTN KRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC ALWYSNLWV FGGGTKLTVL

ACE-27-LC amino acid sequence (SEQ ID NO: 304)

QAVVTQESALTTSPGETVTLTCRSS TGAVTTSNY ANWVQEKPDHLFTGLIG GTN KRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC ALWYSNLWV FGGGTKLTVL[ RSVAAPSVFIFPPSDEQLKSGTKLTVL[ RSVAAPSVFIFPPSDEQLKSGTGNASVVCLLNNFYPREAKSKVQSKVLTQDSKVQSKVDSKLSKVD

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 32 below. have:

Fab region (Anti-PD-L1)Fab region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL (서열번호: 313)VL: QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL (SEQ ID NO: 313)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CD TGAVTTSNY (서열번호: 314) R L1: CD TGAVTTSNY (SEQ ID NO: 314) R L1:
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GTN (서열번호: 315)CDR L2: GTN (SEQ ID NO: 315)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: ALWYSNLWV (서열번호: 316)CDR L3: ALWYSNLWV (SEQ ID NO: 316)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: EVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSA (서열번호: 305)VH: EVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSA (SEQ ID NO: 305)	VL: QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL (서열번호: 309)VL: QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL (SEQ ID NO: 309)
	CDR H1: GFTFNTYA (서열번호: 306)CDR H1: GFTFNTYA (SEQ ID NO: 306)	CDR L1: TGAVTTSNY (서열번호: 310)CDR L1: TGAVTTSNY (SEQ ID NO: 310)
	CDR H2: IRSKYNNYAT (서열번호: 307)CDR H2: IRSKYNNYAT (SEQ ID NO: 307)	CDR L2: GTN (서열번호: 311)CDR L2: GTN (SEQ ID NO: 311)
	CDR H3: VRHGNFGNSYVSWFAY (서열번호: 308)CDR H3: VRHGNFGNSYVSWFAY (SEQ ID NO: 308)	CDR L3: ALWYSNLWV (서열번호: 312)CDR L3: ALWYSNLWV (SEQ ID NO: 312)

실시예 21.29. ACE-28의 제조Example 21.29. Preparation of ACE-28

ACE-28 contains two different heavy chain-like chains (ACE-28-VH and ACE-28-VL) and two identical light chains (ACE-28-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-28-VH amino acid sequence (SEQ ID NO: 317)

QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

ACE-28-VL amino acid sequence (SEQ ID NO: 318)

QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR ALTYYDYEFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGTVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

ACE-28-LC amino acid sequence (SEQ ID NO: 232)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting EGFR and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 33 below:

실시예 21.30. ACE-29의 제조Example 21.30. Preparation of ACE-29

ACE-29 contains two different heavy chain-like chains (ACE-29-VH and ACE-29-VL) and two identical light chains (ACE-29-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-29-VH amino acid sequence (SEQ ID NO: 319)

QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPCPAPELLGGP] ggggs EVKLLESGGGLVQPKGSLKLSCAAS GFTFNTYA MNWVRQAPGKGLEWVAR IRSKYNNYAT YYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYC VRHGNFGNSYVSWFAY WGQGTLVTVSA GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

ACE-29-VL amino acid sequence (SEQ ID NO: 320)

QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPCPAPELLGGP] ggggs QAVVTQESALTTSPGETVTLTCRSS TGAVTTSNY ANWVQEKPDHLFTGLIG GTN KRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC ALWYSNLWV FGGGTKLTVL GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

ACE-29-LC amino acid sequence (SEQ ID NO: 232)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting EGFR and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 34 below:

실시예 21.31. ACE-30의 제조Example 21.31. Preparation of ACE-30

ACE-30 contains two different heavy chain-like chains (ACE-30-VH and ACE-30-VL) and two identical light chains (ACE-30-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-30-VH amino acid sequence (SEQ ID NO: 322)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS

ACE-30-VL amino acid sequence (SEQ ID NO: 323)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVLR

ACE-30-LC amino acid sequence (SEQ ID NO: 324)

DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGTVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR [ TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPYPREAKVQKLSVDSKVQWKLSDSKDSKLSVT

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting PD-L1 are listed in Table 35 below. have:

Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 325)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 325)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 337)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 337)
	CDR H1: GYSFTGYT (서열번호: 326)CDR H1: GYSFTGYT (SEQ ID NO: 326)	CDR L1: QDIRNY (서열번호: 338)CDR L1: QDIRNY (SEQ ID NO: 338)
	CDR H2: INPYKGVS (서열번호: 327)CDR H2: INPYKGVS (SEQ ID NO: 327)	CDR L2: YTS (서열번호: 339)CDR L2: YTS (SEQ ID NO: 339)
	CDR H3: ARSGYYGDSDWYFDV (서열번호: 328)CDR H3: ARSGYYGDSDWYFDV (SEQ ID NO: 328)	CDR L3: QQGNTLPWT (서열번호: 340)CDR L3: QQGNTLPWT (SEQ ID NO: 340)
Fv region (Anti-PD-L1)Fv region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 329)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 329)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (서열번호: 333)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 333)
	CDR H1: GGTFSSYA (서열번호: 330)CDR H1: GGTFSSYA (SEQ ID NO: 330)	CDR L1: SSNIGAGYD (서열번호: 334)CDR L1: SSNIGAGYD (SEQ ID NO: 334)
	CDR H2: IIPILGIA (서열번호: 331)CDR H2: IIPILGIA (SEQ ID NO: 331)	CDR L2: GDI (서열번호: 335)CDR L2: GDI (SEQ ID NO: 335)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 332)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 332)	CDR L3: QSYDSSLSGGV (서열번호: 336)CDR L3: QSYDSSLSGGV (SEQ ID NO: 336)

실시예 21.32. ACE-31의 제조Example 21.32. Preparation of ACE-31

ACE-31 contains two different heavy chain-like chains (ACE-31-VH and ACE-31-VL) and two identical light chains (ACE-31-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-31-VH amino acid sequence (SEQ ID NO: 344)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggsggggs QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS

ACE-31-VL amino acid sequence (SEQ ID NO: 345)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVLR

ACE-31-LC amino acid sequence (SEQ ID NO: 324)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting PD-L1 are listed in Table 36 below. have:

실시예 21.33. ACE-32의 제조Example 21.33. Preparation of ACE-32

ACE-32 contains two different heavy chain-like chains (ACE-32-VH and ACE-32-VL) and two identical light chains (ACE-32-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-32-VH amino acid sequence (SEQ ID NO: 342)

QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggsggggs EVKLLESGGGLVQPKGSLKLSCAAS GFTFNTYA MNWVRQAPGKGLEWVAR IRSKYNNYAT YYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYC VRHGNFGNSYVSWFAY WGQGTLVTVSA

ACE-32-VL amino acid sequence) (SEQ ID NO: 293)

ACE-32-LC amino acid sequence (SEQ ID NO: 232)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting EGFR and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 37 below:

Fab region (EGFR)Fab region (EGFR)	VH: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (서열번호: 233)VH: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 233)	VL: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (서열번호: 237)VL: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 237)
	CDR H1: GFSLTNYG (서열번호: 234)CDR H1: GFSLTNYG (SEQ ID NO: 234)	CDR L1: QSIGTN (서열번호: 238)CDR L1: QSIGTN (SEQ ID NO: 238)
	CDR H2: IWSGGNT (서열번호: 235)CDR H2: IWSGGNT (SEQ ID NO: 235)	CDR L2: YAS (서열번호: 239)CDR L2: YAS (SEQ ID NO: 239)
	CDR H3: ARALTYYDYEFAY (서열번호: 236)CDR H3: ARALTYYDYEFAY (SEQ ID NO: 236)	CDR L3: QQNNNWPTT (서열번호: 240)CDR L3: QQNNNWPTT (SEQ ID NO: 240)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: EVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSA (서열번호: 294)VH: EVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSA (SEQ ID NO: 294)	VL: QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL (서열번호: 298)VL: QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL (SEQ ID NO: 298)
	CDR H1: GFTFNTYA (서열번호: 295)CDR H1: GFTFNTYA (SEQ ID NO: 295)	CDR L1: TGAVTTSNY (서열번호: 299)CDR L1: TGAVTTSNY (SEQ ID NO: 299)
	CDR H2: IRSKYNNYAT (서열번호: 296)CDR H2: IRSKYNNYAT (SEQ ID NO: 296)	CDR L2: GTN (서열번호: 300)CDR L2: GTN (SEQ ID NO: 300)
	CDR H3: VRHGNFGNSYVSWFAY (서열번호: 297)CDR H3: VRHGNFGNSYVSWFAY (SEQ ID NO: 297)	CDR L3: ALWYSNLWV (서열번호: 301)CDR L3: ALWYSNLWV (SEQ ID NO: 301)

실시예 21.34. ACE-33의 제조Example 21.34. Preparation of ACE-33

ACE-33 contains two different heavy chain-like chains (ACE-33-VH and ACE-33-VL) and two identical light chains (ACE-33-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-33-VH amino acid sequence (SEQ ID NO: 341)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggsggggs QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS

ACE-33-VL amino acid sequence (SEQ ID NO: 323)

ACE-33-LC amino acid sequence (SEQ ID NO: 324)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting PD-L1 are listed in Table 38 below. have:

Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 325)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 325)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 337)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 337)
	CDR H1: GYSFTGYT (서열번호: 326)CDR H1: GYSFTGYT (SEQ ID NO: 326)	CDR L1: QDIRNY (서열번호: 338)CDR L1: QDIRNY (SEQ ID NO: 338)
	CDR H2: INPYKGVS (서열번호: 327)CDR H2: INPYKGVS (SEQ ID NO: 327)	CDR L2: YTS (서열번호: 339)CDR L2: YTS (SEQ ID NO: 339)
	CDR H3: ARSGYYGDSDWYFDV (서열번호: 328)CDR H3: ARSGYYGDSDWYFDV (SEQ ID NO: 328)	CDR L3: QQGNTLPWT (서열번호: 340)CDR L3: QQGNTLPWT (SEQ ID NO: 340)
Fv region (Anti-PD-L1)Fv region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 329)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 329)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR(서열번호: 333)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 333)
	CDR H1: GGTFSSYA (서열번호: 330)CDR H1: GGTFSSYA (SEQ ID NO: 330)	CDR L1: SSNIGAGYD (서열번호: 334CDR L1: SSNIGAGYD (SEQ ID NO: 334
	CDR H2: IIPILGIA (서열번호: 331)CDR H2: IIPILGIA (SEQ ID NO: 331)	CDR L2: GDI (서열번호: 335)CDR L2: GDI (SEQ ID NO: 335)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 332)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 332)	CDR L3: QSYDSSLSGGV (서열번호: 336)CDR L3: QSYDSSLSGGV (SEQ ID NO: 336)

실시예 21.35. ACE-34의 제조Example 21.35. Preparation of ACE-34

ACE-34 contains two different heavy chain-like chains (ACE-34-VH and ACE-34-VL) and two identical light chains (ACE-34-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-34-VH amino acid sequence (SEQ ID NO: 346)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs EVQLVESGGGLVQPGKSLKLSCEASGFTFS GYGMH WVRQAPGRGLESVA YITSSSINIKYADAVKG RFTVSRDNAKNLLFLQMNILKSEDTAMYYCAR FDWDKN YWGQGTMVTVSS

ACE-34-VL amino acid sequence (SEQ ID NO: 347)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs DIQMTQSPSSLPASLGDRVTINC QASQDISNYLN WYQQKPGKAPKLLIY YTNKLAD GVPSRFSGSGSGRDSSFTISSLESEDIGSYYC QQYYNYPWT FGPGTKLEIK

ACE-34-LC amino acid sequence (SEQ ID NO: 157)

QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVLR [SVAAPSVFIFPPSDEQLKSGTASVVCSKSVLTQLKSGTASVVCSKNFQLKSGTASVVCSKNFREADS

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 39 below. have:

Fab region (Anti-PD-L1)Fab region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR(서열번호: 170) VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 170)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171)CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSS (서열번호: 348)VH: EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSS (SEQ ID NO: 348)	VL: DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIK (서열번호: 352)VL: DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIK (SEQ ID NO: 352)
	CDR H1: GYGMH (서열번호: 349)CDR H1: GYGMH (SEQ ID NO: 349)	CDR L1: QASQDISNYLN (서열번호: 353)CDR L1: QASQDISNYLN (SEQ ID NO: 353)
	CDR H2: YITSSSINIKYADAVKG (서열번호: 350)CDR H2: YITSSSINIKYADAVKG (SEQ ID NO: 350)	CDR L2: YTNKLAD(서열번호: 354)CDR L2: YTNKLAD (SEQ ID NO: 354)
	CDR H3: FDWDKN (서열번호: 351)CDR H3: FDWDKN (SEQ ID NO: 351)	CDR L3: QQYYNYPWT (서열번호: 355)CDR L3: QQYYNYPWT (SEQ ID NO: 355)

실시예 21.36. ACE-35의 제조Example 21.36. Preparation of ACE-35

ACE-35 contains two different heavy chain-like chains (ACE-35-VH and ACE-35-VL) and two identical light chains (ACE-35-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-35-VH amino acid sequence (SEQ ID NO: 356)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QVKLQQSGSELGKPGASVKLSCKTSGYIFT DHYIS WVKQKPGESLQWIG NVYGGNGGTSYNQKFQG KATLTVDKISSTAYMELSSLTSEDSAIYYCAR RPVATGHAMDY WGQGIQVTVSS

ACE-35-VL amino acid sequence (SEQ ID NO: 357)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs DIVLTQTPATLSLIPGERVTMTC KTSQNIGTILH WYHQKPKEAPRALIK YASQSIP GIPSRFSGSGSETDFTLSINNLEPDDIGIYYC QQSRSWPVT FGPGTKLEIK

ACE-35-LC amino acid sequence (SEQ ID NO: 157)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 40 below. have:

Fab region (Anti-PD-L1)Fab region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR(서열번호: 170)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 170)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171)CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: QVKLQQSGSELGKPGASVKLSCKTSGYIFTDHYISWVKQKPGESLQWIGNVYGGNGGTSYNQKFQGKATLTVDKISSTAYMELSSLTSEDSAIYYCARRPVATGHAMDYWGQGIQVTVSS (서열번호: 358)VH: QVKLQQSGSELGKPGASVKLSCKTSGYIFTDHYISWVKQKPGESLQWIGNVYGGNGGTSYNQKFQGKATLTVDKISSTAYMELSSLTSEDSAIYYCARRPVATGHAMDYWGQGIQVTVSS (SEQ ID NO: 358)	VL: DIVLTQTPATLSLIPGERVTMTCKTSQNIGTILHWYHQKPKEAPRALIKYASQSIPGIPSRFSGSGSETDFTLSINNLEPDDIGIYYCQQSRSWPVTFGPGTKLEIK (서열번호: 362)VL: DIVLTQTPATLSLIPGERVTMTCKTSQNIGTILHWYHQKPKEAPRALIKYASQSIPGIPSRFSGSGSETDFTLSINNLEPDDIGIYYCQQSRSWPVTFGPGTKLEIK (SEQ ID NO: 362)
	CDR H1: DHYIS (서열번호: 359)CDR H1: DHYIS (SEQ ID NO: 359)	CDR L1: KTSQNIGTILH (서열번호: 363)CDR L1: KTSQNIGTILH (SEQ ID NO: 363)
	CDR H2: NVYGGNGGTSYNQKFQG (서열번호: 360)CDR H2: NVYGGNGGTSYNQKFQG (SEQ ID NO: 360)	CDR L2: YASQSIP (서열번호: 364)CDR L2: YASQSIP (SEQ ID NO: 364)
	CDR H3: RPVATGHAMDY (서열번호: 361)CDR H3: RPVATGHAMDY (SEQ ID NO: 361)	CDR L3: QQSRSWPVT (서열번호: 365)CDR L3: QQSRSWPVT (SEQ ID NO: 365)

실시예 21.37. ACE-36의 제조Example 21.37. Preparation of ACE-36

ACE-36 contains two different heavy chain-like chains (ACE-36-VH and ACE-36-VL) and two identical light chains (ACE-36-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-36-VH amino acid sequence (SEQ ID NO: 366)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYT MHWVRQAPGKGLEWVTF ISYDGNNK YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYC ARTGWLGPFDY WGQGTLVTVSS

ACE-36-VL amino acid sequence (SEQ ID NO: 367)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLG EIVLTQSPGTLSLSPGERATLSCRAS QSVGSSY LAWYQQKPGQAPRLLIY GAF SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPWT FGQGTKVEIK

ACE-36-LC amino acid sequence (SEQ ID NO: 157)

QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVL [ RSVAAPSVFIFPPSDEQLKSGTASVVCSKSVLTQLKSGTASVVCSKNFQLKSGTASVVCSKNFREADS

The VH and VL amino acid sequences and CDR sequences therein for the first antigen-binding domain bivalent Fab region targeting PD-L1 and the second antigen-binding domain monovalent Fv region targeting CTLA-4 are shown in Table 41 below. It is listed:

Fab region (Anti-PD-L1)Fab region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVL (서열번호: 376)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVL (SEQ ID NO: 376)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171)CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fv region (Anti-CTLA4)Fv region (Anti-CTLA4)	VH: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS (서열번호: 368)VH: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS (SEQ ID NO: 368)	VL: EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (서열번호: 372)VL: EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 372)
	CDR H1: GFTFSSYT (서열번호: 369)CDR H1: GFTFSSYT (SEQ ID NO: 369)	CDR L1: QSVGSSY (서열번호: 373)CDR L1: QSVGSSY (SEQ ID NO: 373)
	CDR H2: ISYDGNNK (서열번호: 370)CDR H2: ISYDGNNK (SEQ ID NO: 370)	CDR L2: GAF (서열번호: 374)CDR L2: GAF (SEQ ID NO: 374)
	CDR H3: ARTGWLGPFDY (서열번호: 371)CDR H3: ARTGWLGPFDY (SEQ ID NO: 371)	CDR L3: QQYGSSPWT (서열번호: 375)CDR L3: QQYGSSPWT (SEQ ID NO: 375)

실시예 21.38. ACE-37의 제조Example 21.38. Preparation of ACE-37

ACE-37 contains two different heavy chain-like chains (ACE-37-VH and ACE-37-VL) and two identical light chains (ACE-37-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-37-VH amino acid sequence (SEQ ID NO: 377)

QVQLQQPGAELVKPGASVKMSCKAS GYTFTSYN MHWVKQTPGRGLEWIGA IYPGNGDT SYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYC ARSTYYGGDWYFNV WGAGTTVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYT MHWVRQAPGKGLEWVTF ISYDGNNK YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYC ARTGWLGPFDY WGQGTLVTVSS

ACE-37-VL amino acid sequence (SEQ ID NO: 378)

QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPPCPAPELLG EIVLTQSPGTLSLSPGERATLSCRAS QSVGSSY LAWYQQKPGQAPRLLIY GAF SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPWT FGQGTKVEIK

ACE-37-LC amino acid sequence (SEQ ID NO: 221)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD20 and the second antigen binding domain monovalent Fv region targeting CTLA-4 are listed in Table 42 below. have:

Fab region (Anti-CD20)Fab region (Anti-CD20)	VH: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (서열번호: 222)VH: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 222)	VL: QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (서열번호: 226)VL: QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 226)
	CDR H1: GYTFTSYN (서열번호: 223)CDR H1: GYTFTSYN (SEQ ID NO: 223)	CDR L1: SSVSY (서열번호: 227)CDR L1: SSVSY (SEQ ID NO: 227)
	CDR H2: IYPGNGDT (서열번호: 224)CDR H2: IYPGNGDT (SEQ ID NO: 224)	CDR L2: ATS (서열번호: 228)CDR L2: ATS (SEQ ID NO: 228)
	CDR H3: ARSTYYGGDWYFNV (서열번호: 225)CDR H3: ARSTYYGGDWYFNV (SEQ ID NO: 225)	CDR L3: QQWTSNPPT (서열번호: 229)CDR L3: QQWTSNPPT (SEQ ID NO: 229)
Fv region (Anti-CTLA-4)Fv region (Anti-CTLA-4)	VH: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS (서열번호: 368)VH: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS (SEQ ID NO: 368)	VL: EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (서열번호: 372)VL: EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 372)
	CDR H1: GFTFSSYT (서열번호: 369)CDR H1: GFTFSSYT (SEQ ID NO: 369)	CDR L1: QSVGSSY (서열번호: 373)CDR L1: QSVGSSY (SEQ ID NO: 373)
	CDR H2: ISYDGNNK (서열번호: 370)CDR H2: ISYDGNNK (SEQ ID NO: 370)	CDR L2: GAF (서열번호: 374)CDR L2: GAF (SEQ ID NO: 374)
	CDR H3: ARTGWLGPFDY (서열번호: 371)CDR H3: ARTGWLGPFDY (SEQ ID NO: 371)	CDR L3: QQYGSSPWT (서열번호: 375)CDR L3: QQYGSSPWT (SEQ ID NO: 375)

실시예 21.39. ACE-38의 제조Example 21.39. Preparation of ACE-38

ACE-38 contains two different heavy chain-like chains (ACE-38-VH and ACE-38-VL) and two identical light chains (ACE-38-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-38-VH amino acid sequence (SEQ ID NO: 381)

QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS GQPREPQVYTSPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

ACE-38-VL amino acid sequence (SEQ ID NO: 382)

QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGTVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR

ACE-38-LC amino acid sequence (SEQ ID NO: 232)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting EGFR and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 43 below:

실시예 21.40. ACE-39의 제조Example 21.40. Preparation of ACE-39

ACE-39 contains two different heavy chain-like chains (ACE-39-VH and ACE-39-VL) and two identical light chains (ACE-39-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-39-VH amino acid sequence (SEQ ID NO: 383)

QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS

ACE-39-VL amino acid sequence (SEQ ID NO: 384)

QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGTVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR GQPREPQVYTSPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

ACE-39-LC amino acid sequence (SEQ ID NO: 232)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting EGFR and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 44 below:

Fab region (EGFR)Fab region (EGFR)	VH: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (서열번호: 233)VH: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 233)	VL: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (서열번호: 237)VL: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 237)
	CDR H1: GFSLTNYG (서열번호: 234)CDR H1: GFSLTNYG (SEQ ID NO: 234)	CDR L1: QSIGTN (서열번호: 238)CDR L1: QSIGTN (SEQ ID NO: 238)
	CDR H2: IWSGGNT (서열번호: 235)CDR H2: IWSGGNT (SEQ ID NO: 235)	CDR L2: YAS (서열번호: 239)CDR L2: YAS (SEQ ID NO: 239)
	CDR H3: ARALTYYDYEFAY (서열번호: 236)CDR H3: ARALTYYDYEFAY (SEQ ID NO: 236)	CDR L3: QQNNNWPTT (서열번호: 240)CDR L3: QQNNNWPTT (SEQ ID NO: 240)
Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 215)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 215)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 216)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 216)
	CDR H1: GYSFTGYT (서열번호: 177)CDR H1: GYSFTGYT (SEQ ID NO: 177)	CDR L1: QDIRNY (서열번호: 181)CDR L1: QDIRNY (SEQ ID NO: 181)
	CDR H2: INPYKGVS (서열번호: 178)CDR H2: INPYKGVS (SEQ ID NO: 178)	CDR L2: YTS (서열번호: 182)CDR L2: YTS (SEQ ID NO: 182)
	CDR H3: ARSGYYGDSDWYFD (서열번호: 211)CDR H3: ARSGYYGDSDWYFD (SEQ ID NO: 211)	CDR L3: QQGNTLPWT (서열번호: 207)CDR L3: QQGNTLPWT (SEQ ID NO: 207)

실시예 21.41. ACE-40의 제조Example 21.41. Preparation of ACE-40

ACE-40 contains two different heavy chain-like chains (ACE-40-VH and ACE-40-VL) and two identical light chains (ACE-40-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-40-VH amino acid sequence (SEQ ID NO: 381)

ACE-40-VL amino acid sequence (SEQ ID NO: 384)

ACE-40-LC amino acid sequence (SEQ ID NO: 232)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting EGFR and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 45 below:

실시예 21.42. ACE-00r의 제조Example 21.42. Preparation of ACE-00r

ACE-00r contains two different heavy chain-like chains (ACE-00r-VH and ACE-00r-VL) and two identical light chains (ACE-00r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-00r-VH amino acid sequence :

EVQLVESGGGLVQPGRSLRLSCAAS GFTFDDYA MHWVRQAPGKGLEWVSA ITWNSGHI DYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC AKVSYLSTASSLDY WGQGTLVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPAPELLGGP EVQLVESGGGLVQPGGSLRLSCAAS GFNIKDTY IHWVRQAPGKGLEWVAR IYPTNGYT RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDY WGQGTLVTVSS (SEQ ID NO: 388)

ACE-00r-VL amino acid sequence :

EVQLVESGGGLVQPGRSLRLSCAAS GFTFDDYA MHWVRQAPGKGLEWVSA ITWNSGHI DYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC AKVSYLSTASSLDY WGQGTLVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPAPELLGGP DIQMTQSPSSLSASVGDRVTITCRAS QDVNTA VAWYQQKPGKAPKLLIY SAS FLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYC QQHYTTPPT FGQGTKVEIK (SEQ ID NO: 389)

ACE-00r-LC amino acid sequence :

DIQMTQSPSSLSASVGDRVTITCRAS QGIRNY LAWYQQKPGKAPKLLIY AAS TLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QRYNRAPYT FGQGTKVEIKR [RSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC] (SEQ ID NO: 390)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting TNF and the second antigen binding domain monovalent Fv region targeting HER2 are listed in Table 46 below:

Fab region (Anti-TNF alpha)Fab region (Anti-TNF alpha)	VH: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS (서열번호: 279)VH: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS (SEQ ID NO: 279)	VL: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKR (서열번호: 283)VL: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKR (SEQ ID NO: 283)
	CDR H1: GFTFDDYA (서열번호: 280)CDR H1: GFTFDDYA (SEQ ID NO: 280)	CDR L1: QGIRNY (서열번호: 284)CDR L1: QGIRNY (SEQ ID NO: 284)
	CDR H2: ITWNSGHI (서열번호: 281)CDR H2: ITWNSGHI (SEQ ID NO: 281)	CDR L2: AAS (서열번호: 285)CDR L2: AAS (SEQ ID NO: 285)
	CDR H3: AKVSYLSTASSLDY (서열번호: 282)CDR H3: AKVSYLSTASSLDY (SEQ ID NO: 282)	CDR L3: QRYNRAPYT (서열번호: 286)CDR L3: QRYNRAPYT (SEQ ID NO: 286)
Fv region (Anti-Her2)Fv region (Anti-Her2)	VH: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID 275)VH: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID 275)	VL: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR (서열번호: 389)VL: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR (SEQ ID NO: 389)
	CDR H1:GFNIKDTY (서열번호: 276)CDR H1:GFNIKDTY (SEQ ID NO: 276)	CDR L1: QDVNTA (서열번호: 288)CDR L1: QDVNTA (SEQ ID NO: 288)
	CDR H2: IYPTNGYT (서열번호: 277)CDR H2: IYPTNGYT (SEQ ID NO: 277)	CDR L2: SAS (서열번호: 289)CDR L2: SAS (SEQ ID NO: 289)
	CDR H3: SRWGGDGFYAMDY (서열번호: 278)CDR H3: SRWGGDGFYAMDY (SEQ ID NO: 278)	CDR L3: QQHYTTPPT (서열번호: 290)CDR L3: QQHYTTPPT (SEQ ID NO: 290)

실시예 21.43. ACE-01r의 제조Example 21.43. Preparation of ACE-01r

ACE-01r contains two different heavy chain-like chains (ACE-01r-VH and ACE-01r-VL) and two identical light chains (ACE-01r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-01r-VH amino acid sequence (SEQ ID NO: 391)

QVQLQQSGAELARPGASVKMSCKAS GYTFTRYT MHWVKQRPGQGLEWIGY INPSRGYT NYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYC ARYYDDHYCLDY WGQGTTVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPAPELLGGP QVQLQQSGAELVRPGSSVKISCKASGYAFS SYWMN WVKQRPGQGLEWIGQ IWPGDGDT NYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFC ARRETTTVGRYYYAMDY WGQGTTVTVSS

ACE-01r -VL amino acid sequence (SEQ ID NO: 392)

QVQLQQSGAELARPGASVKMSCKAS GYTFTRYT MHWVKQRPGQGLEWIGY INPSRGYT NYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYC ARYYDDHYCLDY WGQGTTVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPAPELLGGP DIQLTQSPASLAVSLGQRATISCKAS QSVDYDGDSY LNWYQQIPGQPPKLLIY DAS NLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHC QQSTEDPWT FGGGTKLEIK

ACE-01r-LC amino acid sequence (SEQ ID NO: 393)

QIVLTQSPAIMSASPGEKVTMTCSAS SSVSY MNWYQQKSGTSPKRWIY DTS KLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYC QQWSSNPFTF GSGTKLEINR [ RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVFISPPSDEQLKSGTASVVCLLNNFYPREAKVFIDSCHKVDLSQDSKSKVDLSQDS

The VH and VL amino acid sequences and CDR sequences therein for the first antigen-binding domain (divalent Fab region targeting CD3) and the second antigen-binding domain (monovalent Fv region targeting CD19) are shown in Table 47 below. Are listed:

Fv region (Anti-CD19)Fv region (Anti-CD19)	VH: QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (서열번호: 139)VH: QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (SEQ ID NO: 139)	VL: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK (서열번호: 151)VL: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK (SEQ ID NO: 151)
	CDR H1: SYWMN (서열번호: 140)CDR H1: SYWMN (SEQ ID NO: 140)	CDR L1: QSVDYDGDSY (서열번호: 152)CDR L1: QSVDYDGDSY (SEQ ID NO: 152)
	CDR H2: IWPGDGDT (서열번호: 141)CDR H2: IWPGDGDT (SEQ ID NO: 141)	CDR L2: DAS (서열번호: 153)CDR L2: DAS (SEQ ID NO: 153)
	CDR H3: ARRETTTVGRYYYAMDY (서열번호: 142)CDR H3: ARRETTTVGRYYYAMDY (SEQ ID NO: 142)	CDR L3: QQSTEDPWT (서열번호: 154)CDR L3: QQSTEDPWT (SEQ ID NO: 154)
Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTVTVSS (서열번호: 143)VH: QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTVTVSS (SEQ ID NO: 143)	VL: QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (서열번호: 147)VL: QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (SEQ ID NO: 147)
	CDR H1: GYTFTRYT (서열번호: 144)CDR H1: GYTFTRYT (SEQ ID NO: 144)	CDR L1: SSVSY (서열번호: 148)CDR L1: SSVSY (SEQ ID NO: 148)
	CDR H2: INPSRGYT (서열번호: 145)CDR H2: INPSRGYT (SEQ ID NO: 145)	CDR L2: DTS (서열번호: 149)CDR L2: DTS (SEQ ID NO: 149)
	CDR H3: ARYYDDHYCLDY (서열번호: 146)CDR H3: ARYYDDHYCLDY (SEQ ID NO: 146)	CDR L3: QQWSSNPFTF (서열번호: 150)CDR L3: QQWSSNPFTF (SEQ ID NO: 150)

실시예 21.44. ACE-02r의 제조Example 21.44. Preparation of ACE-02r

ACE-02r contains two different heavy chain-like chains (ACE-02r-VH and ACE-02r-VL) and two identical light chains (ACE-02r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-02r-VH amino acid sequence :

QVQLVQSGGGVVQPGRSLRLSCKAS GYTFTSYT MHWVRQAPGKGLEWIGY INPSSGYT KYNQKFKDRFTISADKSKSTAFLQMDSLRPEDTGVYFC ARWQDYDVYFDY WGQGTPVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPAPELLGGP QVQLQQSGAELVRPGSSVKISCKASGYAFS SYWMN WVKQRPGQGLEWIGQ IWPGDGDT NYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFC ARRETTTVGRYYYAMDY WGQGTTVTVSS (SEQ ID NO: 394)

ACE-02r-VL amino acid sequence :

QVQLVQSGGGVVQPGRSLRLSCKAS GYTFTSYT MHWVRQAPGKGLEWIGY INPSSGYT KYNQKFKDRFTISADKSKSTAFLQMDSLRPEDTGVYFC ARWQDYDVYFDY WGQGTPVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPAPELLGGP DIQLTQSPASLAVSLGQRATISCKAS QSVDYDGDSY LNWYQQIPGQPPKLLIY DAS NLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHC QQSTEDPWT FGGGTKLEIK (SEQ ID NO: 399)

ACE-02r-LC amino acid sequence :

DIQMTQSPSSLSASVGDRVTMTCRA SSSVSY MHWYQQTPGKAPKPWIY ATS NLASGVPSRFSGSGSGTDYTLTISSLQPEDIATYYC QQWSSNPPT FGQGTKLQITR [ RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNVDYPREAKSQGNSVSTYPREAKVQWKTHKLSKDSQWKDS

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fvr egion targeting CD19 are listed in Table 48 below:

Fv region (Anti-CD19)Fv region (Anti-CD19)	VH: QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (서열번호: 139)VH: QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (SEQ ID NO: 139)	VL: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK (서열번호: 151)VL: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK (SEQ ID NO: 151)
	CDR H1: SYWMN (서열번호: 140)CDR H1: SYWMN (SEQ ID NO: 140)	CDR L1: QSVDYDGDSY (서열번호: 152)CDR L1: QSVDYDGDSY (SEQ ID NO: 152)
	CDR H2: QIWPGDGDTNYNGKFKG (서열번호: 141)CDR H2: QIWPGDGDTNYNGKFKG (SEQ ID NO: 141)	CDR L2: DAS (서열번호: 153)CDR L2: DAS (SEQ ID NO: 153)
	CDR H3: RETTTVGRYYYAMDY (서열번호: 142)CDR H3: RETTTVGRYYYAMDY (SEQ ID NO: 142)	CDR L3: QQSTEDPWT (서열번호: 154)CDR L3: QQSTEDPWT (SEQ ID NO: 154)
Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: QVQLVQSGGGVVQPGRSLRLSCKASGYTFTSYTMHWVRQAPGKGLEWIGYINPSSGYTKYNQKFKDRFTISADKSKSTAFLQMDSLRPEDTGVYFCARWQDYDVYFDYWGQGTPVTVSS (서열번호: 395)VH: QVQLVQSGGGVVQPGRSLRLSCKASGYTFTSYTMHWVRQAPGKGLEWIGYINPSSGYTKYNQKFKDRFTISADKSKSTAFLQMDSLRPEDTGVYFCARWQDYDVYFDYWGQGTPVTVSS (SEQ ID NO: 395)	VL: DIQMTQSPSSLSASVGDRVTMTCRASSSVSYMHWYQQTPGKAPKPWIYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDIATYYCQQWSSNPPTFGQGTKLQITR (서열번호: 401)VL: DIQMTQSPSSLSASVGDRVTMTCRASSSVSYMHWYQQTPGKAPKPWIYATSNLASGVPSRFSGSGSGTDYTLTISSLQPEDIATYYCQQWSSNPPTFGQGTKLQITR (SEQ ID NO: 401)
	CDR H1: GYTFTSYT (서열번호: 396)CDR H1: GYTFTSYT (SEQ ID NO: 396)	CDR L1: SSSVSY (서열번호: 402)CDR L1: SSSVSY (SEQ ID NO: 402)
	CDR H2: INPSSGYT (서열번호: 397)CDR H2: INPSSGYT (SEQ ID NO: 397)	CDR L2: ATS (서열번호: 403)CDR L2: ATS (SEQ ID NO: 403)
	CDR H3: ARWQDYDVYFDY (서열번호: 398)CDR H3: ARWQDYDVYFDY (SEQ ID NO: 398)	CDR L3: QQWSSNPPT (서열번호: 404)CDR L3: QQWSSNPPT (SEQ ID NO: 404)

실시예 21.45. ACE-03r의 제조Example 21.45. Preparation of ACE-03r

ACE-03r contains two different heavy chain-like chains (ACE-03r-VH and ACE-03r-VL) and two identical light chains (ACE-03r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-03r-VH amino acid sequence:

VQLVQSGGGVVQPGRSLRLSCKAS GYTFTRYT MHWVRQAPGKGLEWIGY INPSRGYT NYNQKVKDRFTISTDKSKSTAFLQMDSLRPEDTAVYYC ARYYDDHYCLDY WGQGTPVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPAPELLGGP QVQLQQSGAELVRPGSSVKISCKASGYAFS SYWMN WVKQRPGQGLEWIGQ IWPGDGDT NYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFC ARRETTTVGRYYYAMDY WGQGTTVTVSS (SEQ ID NO: 405)

ACE-03r-VL amino acid sequence :

VQLVQSGGGVVQPGRSLRLSCKAS GYTFTRYT MHWVRQAPGKGLEWIGY INPSRGYT NYNQKVKDRFTISTDKSKSTAFLQMDSLRPEDTAVYYC ARYYDDHYCLDY WGQGTPVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPAPELLGGP DIQLTQSPASLAVSLGQRATISCKAS QSVDYDGDSY LNWYQQIPGQPPKLLIY DAS NLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHC QQSTEDPWT FGGGTKLEIK (SEQ ID NO: 407)

ACE-03r-LC amino acid sequence :

DIQMTQSPSSLSASVGDRVTITCSAS SSVSY MNWYQQTPGKAPKRWIY DTS KLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYC QQWSSNPFT FGQGTKLQITR [ RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPYPREASKVQWKLSVTYPREAKVQWKLSVGDSKVQWKLSVGDSKVQWK

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting CD19 are listed in Table 49 below:

Fv region (Anti-CD19)Fv region (Anti-CD19)	VH: QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (서열번호: 139)VH: QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS (SEQ ID NO: 139)	VL: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK (서열번호: 151)VL: DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK (SEQ ID NO: 151)
	CDR H1: SYWMN (서열번호: 140)CDR H1: SYWMN (SEQ ID NO: 140)	CDR L1: QSVDYDGDSY (서열번호: 152)CDR L1: QSVDYDGDSY (SEQ ID NO: 152)
	CDR H2: IWPGDGDT (서열번호: 141)CDR H2: IWPGDGDT (SEQ ID NO: 141)	CDR L2: DAS (서열번호: 153)CDR L2: DAS (SEQ ID NO: 153)
	CDR H3: ARETTTVGRYYYAMDY (서열번호: 142)CDR H3: ARETTTVGRYYYAMDY (SEQ ID NO: 142)	CDR L3: QQSTEDPWT (서열번호: 154)CDR L3: QQSTEDPWT (SEQ ID NO: 154)
Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: VQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISTDKSKSTAFLQMDSLRPEDTAVYYCARYYDDHYCLDYWGQGTPVTVSS (서열번호: 406)VH: VQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISTDKSKSTAFLQMDSLRPEDTAVYYCARYYDDHYCLDYWGQGTPVTVSS (SEQ ID NO: 406)	VL: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR (서열번호: 409)VL: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR (SEQ ID NO: 409)
	CDR H1: GYTFTRYT (서열번호: 144)CDR H1: GYTFTRYT (SEQ ID NO: 144)	CDR L1: SSVSY (서열번호: 148)CDR L1: SSVSY (SEQ ID NO: 148)
	CDR H2: INPSRGYT (서열번호: 145)CDR H2: INPSRGYT (SEQ ID NO: 145)	CDR L2: DTS (서열번호: 149)CDR L2: DTS (SEQ ID NO: 149)
	CDR H3: ARYYDDHYCLDY (서열번호: 146)CDR H3: ARYYDDHYCLDY (SEQ ID NO: 146)	CDR L3: QQWSSNPFT (서열번호: 410)CDR L3: QQWSSNPFT (SEQ ID NO: 410)

실시예 21.46. ACE-04r의 제조Example 21.46. Preparation of ACE-04r

ACE-04r has two different heavy chains such as ACE-04r-VH (VL-CL-VH-CH1) and ACE-04r-VL (VH-CH1-VL-CL) chains and two identical light chains (ACE-04r- LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-04r-VH amino acid sequence :

QVQLQQSGAELARPGASVKMSCKAS GYTFTRYT MHWVKQRPGQGLEWIGY INPSRGYT NYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYC ARYYDDHYCLDY WGQGTTVTVSA [ ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] ( SEQ ID NO: 411)

ACE-04r-VL amino acid sequence :

QVQLQQSGAELARPGASVKMSCKAS GYTFTRYT MHWVKQRPGQGLEWIGY INPSRGYT NYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYC ARYYDDHYCLDY WGQGTTVTVSA [ ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVL [RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC] ( SEQ ID NO: 413)

ACE-04r-LC amino acid sequence :

QIVLTQSPAIMSASPGEKVTMTCSAS SSVSY MNWYQQKSGTSPKRWIY DTS KLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYC QQWSSNPF TFGSGTKLEIN [ RSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVFIPSDEQLKSGTASVVCLLNNFYPREAKVFIPSDEQLKSGTASVVCLLNNFYPREADSKVQWKVDLSQDS

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting PD-L1 are listed in Table 50 below. have:

Fv region (Anti-PD-L1)Fv region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 329)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 329)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVL (서열번호: 376)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVL (SEQ ID NO: 376)
	CDR H1: GGTFSSYA (서열번호: 330)CDR H1: GGTFSSYA (SEQ ID NO: 330)	CDR L1: SSNIGAGYD (서열번호: 334)CDR L1: SSNIGAGYD (SEQ ID NO: 334)
	CDR H2: IIPILGIA (서열번호: 331)CDR H2: IIPILGIA (SEQ ID NO: 331)	CDR L2: GDI (서열번호: 335)CDR L2: GDI (SEQ ID NO: 335)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 332)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 332)	CDR L3: QSYDSSLSGGV (서열번호: 336)CDR L3: QSYDSSLSGGV (SEQ ID NO: 336)
Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTVTVSA (서열번호: 412)VH: QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTVTVSA (SEQ ID NO: 412)	VL: QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEIN (서열번호: 415)VL: QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEIN (SEQ ID NO: 415)
	CDR H1: GYTFTRYT (서열번호: 144)CDR H1: GYTFTRYT (SEQ ID NO: 144)	CDR L1: SSVSY (서열번호: 148)CDR L1: SSVSY (SEQ ID NO: 148)
	CDR H2: INPSRGYT (서열번호: 145)CDR H2: INPSRGYT (SEQ ID NO: 145)	CDR L2: DTS (서열번호: 149)CDR L2: DTS (SEQ ID NO: 149)
	CDR H3: ARYYDDHYCLDY (서열번호: 146)CDR H3: ARYYDDHYCLDY (SEQ ID NO: 146)	CDR L3: QQWSSNPF (서열번호: 416)CDR L3: QQWSSNPF (SEQ ID NO: 416)

실시예 21.47. ACE-05r의 제조Example 21.47. Preparation of ACE-05r

ACE-05r contains two different heavy chain-like chains (ACE-05r-VH and ACE-05r-VL) and two identical light chains (ACE-05r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-05r-VH amino acid sequence (SEQ ID NO: 417)

ACE-05r-VL amino acid sequence (SEQ ID NO: 418)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVL

ACE-05r-LC amino acid sequence (SEQ ID NO: 419)

DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGTVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR [ RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPYPREAKVQWKLSVTYPREAKVQWKLSVDSKVQWKLSVT

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting PD-L1 are listed in Table 51 below. have:

Fv region (Anti-PD-L1)Fv region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 329)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 329)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVL (서열번호: 376)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVL (SEQ ID NO: 376)
	CDR H1: GGTFSSYA (서열번호: 330)CDR H1: GGTFSSYA (SEQ ID NO: 330)	CDR L1: SSNIGAGYD (서열번호: 334)CDR L1: SSNIGAGYD (SEQ ID NO: 334)
	CDR H2: IIPILGIA (서열번호: 331)CDR H2: IIPILGIA (SEQ ID NO: 331)	CDR L2: GDI (서열번호: 335)CDR L2: GDI (SEQ ID NO: 335)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 332)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 332)	CDR L3: QSYDSSLSGGV (서열번호: 336)CDR L3: QSYDSSLSGGV (SEQ ID NO: 336)
Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 325)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 325)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 337)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 337)
	CDR H1: GYSFTGYT (서열번호: 326)CDR H1: GYSFTGYT (SEQ ID NO: 326)	CDR L1: QDIRNY (서열번호: 338)CDR L1: QDIRNY (SEQ ID NO: 338)
	CDR H2: INPYKGVS (서열번호: 327)CDR H2: INPYKGVS (SEQ ID NO: 327)	CDR L2: YTS (서열번호: 339)CDR L2: YTS (SEQ ID NO: 339)
	CDR H3: ARSGYYGDSDWYFDV (서열번호: 328)CDR H3: ARSGYYGDSDWYFDV (SEQ ID NO: 328)	CDR L3: QQGNTLPWT (서열번호: 340)CDR L3: QQGNTLPWT (SEQ ID NO: 340)

실시예 21.48. ACE-06r의 제조Example 21.48. Preparation of ACE-06r

ACE-06r contains two different heavy chain-like chains (ACE-06r-VH and ACE-06r-VL) and two identical light chains (ACE-06r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-06r-VH amino acid sequence (SEQ ID NO: 420)

QVQLVESGGGVVQPGRSLRLSCAAS GFKFSGYG MHWVRQAPGKGLEWVAVI WYDGSKK YYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARQMGYWHFDL WGRGTLVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS

ACE-06r-VL amino acid sequence (SEQ ID NO: 421)

QVQLVESGGGVVQPGRSLRLSCAAS GFKFSGYG MHWVRQAPGKGLEWVAVI WYDGSKK YYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARQMGYWHFDL WGRGTLVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVLR

ACE-06r-LC amino acid sequence (SEQ ID NO: 422)

EIVLTQSPATLSLSPGERATLSCRAS QSVSSY LAWYQQKPGQAPRLLIY DAS NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPPLT FGGGTKVEIKR [ SVAAPSVFIFPPSDEQLKSGTKVEIKR [SVAAPSVFIFPPSDEQLKSGTKVEIKR [SVAAPSVFIFPPSDEQLKSGTKVEIKR [SVAAPSVFIFPPSDEQLKSGTGNASVVCLLNNFYPREADSKVQSKVLTQYPREAKVQSKVDLSKDS

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting PD-L1 are listed in Table 52 below. have:

Fv region (Anti-PD-L1)Fv region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (서열번호: 170)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 170)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171)CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSS (서열번호: 162)VH: QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSS (SEQ ID NO: 162)	VL: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIKR (서열번호: 166)VL: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIKR (SEQ ID NO: 166)
	CDR H1: GFKFSGYG (서열번호: 163)CDR H1: GFKFSGYG (SEQ ID NO: 163)	CDR L1: QSVSSY (서열번호: 167)CDR L1: QSVSSY (SEQ ID NO: 167)
	CDR H2: IWYDGSKK (서열번호: 271)CDR H2: IWYDGSKK (SEQ ID NO: 271)	CDR L2: DAS (서열번호: 168)CDR L2: DAS (SEQ ID NO: 168)
	CDR H3: ARQMGYWHFDL (서열번호: 165)CDR H3: ARQMGYWHFDL (SEQ ID NO: 165)	CDR L3: QQRSNWPPLT (서열번호: 169)CDR L3: QQRSNWPPLT (SEQ ID NO: 169)

실시예 21.49. ACE-07r의 제조Example 21.49. Preparation of ACE-07r

ACE-07r contains two different heavy chain-like chains (ACE-07r-VH and ACE-07r-VL) and two identical light chains (ACE-07r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-07r-VH amino acid sequence (SEQ ID NO: 423)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKCLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGP ggggs QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS

ACE-07r-VL amino acid sequence (SEQ ID NO: 424)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKCLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGP ggggs QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVLR

ACE-07r-LC amino acid sequence (SEQ ID NO: 425)

DIQMTQTTSSLSASLGDRVTISCRASDIQMTQTTSSLSASLGDRVTISCRAS QDIRNYQDIRNY LNWYQQKPDGTVKLLIYLNWYQQKPDGTVKLLIY YTSYTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWQQGNTLPW TFAGCTKLEIKRTFAGCTKLEIKR SVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 53 below. have:

Fv region (Anti-PD-L1)Fv region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (서열번호: 170)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 170)
	CDR H1: GGTFSSYA (서열번호: 159) CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171) CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKCLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 176)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKCLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 176)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGCTKLEIKR (서열번호: 180)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGCTKLEIKR (SEQ ID NO: 180)
	CDR H1: GYSFTGYT (서열번호: 177)CDR H1: GYSFTGYT (SEQ ID NO: 177)	CDR L1: QDIRNY (서열번호: 181)CDR L1: QDIRNY (SEQ ID NO: 181)
	CDR H2: INPYKGVS (서열번호: 178)CDR H2: INPYKGVS (SEQ ID NO: 178)	CDR L2: YTS (서열번호: 182)CDR L2: YTS (SEQ ID NO: 182)
	CDR H3: ARSGYYGDSDWYFDV (서열번호: 179)CDR H3: ARSGYYGDSDWYFDV (SEQ ID NO: 179)	CDR L3: QQGNTLPW (서열번호: 183)CDR L3: QQGNTLPW (SEQ ID NO: 183)

실시예 21.50. ACE-08r의 제조Example 21.50. Preparation of ACE-08r

ACE-08r contains two different heavy chain-like chains (ACE-08r-VH and ACE-08r-VL) and two identical light chains (ACE-08r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-08r-HC-VH amino acid sequence (SEQ ID NO: 426)

EVQLVESGGGLVQPGKSLKLSCEAS GFTFSGYG MHWVRQAPGRCLESVAY ITSSSINI KYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYC ARFDWDKNY WGQGTMVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGP ggggs QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS

ACE-08r-HC-VL amino acid sequence (SEQ ID NO: 427)

EVQLVESGGGLVQPGKSLKLSCEAS GFTFSGYG MHWVRQAPGRCLESVAY ITSSSINI KYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYC ARFDWDKNY WGQGTMVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGP ggggs QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVLR

ACE-08r-LC amino acid sequence (SEQ ID NO: 428)

QMTQSPSSLPASLGDRVTINCQASQMTQSPSSLPASLGDRVTINCQAS QDISNYQDISNY LNWYQQKPGKAPKLLIYLNWYQQKPGKAPKLLIY YTNYTN KLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYCKLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYC QQYYNYPWTQQYYNYPWT FGPCTKLEIKRFGPCTKLEIKR SVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting PD-L1 are listed in Table 54 below. have:

Fv region (Anti-PD-L1)Fv region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (서열번호: 170)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 170)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171)CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRCLESVAYITSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSS (서열번호: 186)VH: EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRCLESVAYITSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSS (SEQ ID NO: 186)	VL: QMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPCTKLEIKR (서열번호: 190)VL: QMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPCTKLEIKR (SEQ ID NO: 190)
	CDR H1: GFTFSGYG (서열번호: 187)CDR H1: GFTFSGYG (SEQ ID NO: 187)	CDR L1: QDISNY (서열번호: 191) CDR L1: QDISNY (SEQ ID NO: 191)
	CDR H2: ITSSSINI (서열번호: 188)CDR H2: ITSSSINI (SEQ ID NO: 188)	CDR L2: YTN (서열번호: 192)CDR L2: YTN (SEQ ID NO: 192)
	CDR H3: ARFDWDKNY (서열번호: 189)CDR H3: ARFDWDKNY (SEQ ID NO: 189)	CDR L3: QQYYNYPWT (서열번호: 193)CDR L3: QQYYNYPWT (SEQ ID NO: 193)

실시예 21.51. ACE-09r의 제조Example 21.51. Preparation of ACE-09r

ACE-09r-VH amino acid sequence (SEQ ID NO: 429)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS

ACE-09r-VL amino acid sequence (SEQ ID NO: 430)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVL

ACE-09r-LC amino acid sequence (SEQ ID NO: 431)

DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGTVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR [ RSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPYPREAKVQWKLSVTYPREAKVQWKLSVDSKVQWKLSVT

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting PD-L1 are listed in Table 55 below. have:

Fv region (Anti-PD-L1)Fv region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 329)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 329)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVL(서열번호: 376)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVL (SEQ ID NO: 376)
	CDR H1: GGTFSSYA (서열번호: 330)CDR H1: GGTFSSYA (SEQ ID NO: 330)	CDR L1: SSNIGAGYD (서열번호: 171)CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 331)CDR H2: IIPILGIA (SEQ ID NO: 331)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 332)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 332)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 325)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 325)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 337)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 337)
	CDR H1: GYSFTGYT (서열번호: 326)CDR H1: GYSFTGYT (SEQ ID NO: 326)	CDR L1: QDIRNY (서열번호: 338)CDR L1: QDIRNY (SEQ ID NO: 338)
	CDR H2: INPYKGVS (서열번호: 327)CDR H2: INPYKGVS (SEQ ID NO: 327)	CDR L2: YTS (서열번호: 339)CDR L2: YTS (SEQ ID NO: 339)
	CDR H3: ARSGYYGDSDWYFDV (서열번호: 328)CDR H3: ARSGYYGDSDWYFDV (SEQ ID NO: 328)	CDR L3: QQGNTLPWT (서열번호: 340)CDR L3: QQGNTLPWT (SEQ ID NO: 340)

실시예 21.52. ACE-10r의 제조Example 21.52. Preparation of ACE-10r

ACE-10r contains two different heavy chain-like chains (ACE-10r-VH and ACE-10r-VL) and two identical light chains (ACE-10r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-10r-VH amino acid sequence :

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs QVQLQQPGAELVKPGASVKMSCKAS GYTFTSYN MHWVKQTPGRGLEWIGA IYPGNGDT SYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYC ARSTYYGGDWYFNV WGAGTTVTVSA (SEQ ID NO: 432)

ACE-10r-VL amino acid sequence :

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs QIVLSQSPAILSASPGEKVTMTCRAS SSVSY IHWFQQKPGSSPKPWIY ATS NLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYC QQWTSNPPT FGGGTKLEIK (SEQ ID NO: 433)

ACE-10r-LC amino acid sequence :

DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGTVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR [ RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPYPREAKVQWKLSTSPDSKVQWKLSVGDSKVQWKLTHV

The VH and VL amino acid sequences for the bivalent Fab region targeting CD3 and the monovalent Fv region targeting CD20 and the CDR sequences therein are listed in Table 56 below:

Fv region (Anti-CD20)Fv region (Anti-CD20)	VH QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (서열번호: 222)VH QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 222)	VL QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (서열번호: 226)VL QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 226)
	CDR H1: GYTFTSYN (서열번호: 223)CDR H1: GYTFTSYN (SEQ ID NO: 223)	CDR L1: SSVSY (서열번호: 227)CDR L1: SSVSY (SEQ ID NO: 227)
	CDR H2: IYPGNGDT (서열번호: 224)CDR H2: IYPGNGDT (SEQ ID NO: 224)	CDR L2: ATS (서열번호: 228)CDR L2: ATS (SEQ ID NO: 228)
	CDR H3: ARSTYYGGDWYFNV (서열번호: 225)CDR H3: ARSTYYGGDWYFNV (SEQ ID NO: 225)	CDR L3: QQWTSNPPT (서열번호: 229)CDR L3: QQWTSNPPT (SEQ ID NO: 229)
Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 325)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 325)	VL DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 337)VL DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 337)
	CDR H1: GYSFTGYT (서열번호: 326)CDR H1: GYSFTGYT (SEQ ID NO: 326)	CDR L1: QDIRNY (서열번호: 338)CDR L1: QDIRNY (SEQ ID NO: 338)
	CDR H2: INPYKGVS (서열번호: 327)CDR H2: INPYKGVS (SEQ ID NO: 327)	CDR L2: YTS (서열번호: 339)CDR L2: YTS (SEQ ID NO: 339)
	CDR H1 ARSGYYGDSDWYFDV (서열번호: 328)CDR H1 ARSGYYGDSDWYFDV (SEQ ID NO: 328)	CDR L3: QQGNTLPWT (서열번호: 340)CDR L3: QQGNTLPWT (SEQ ID NO: 340)

실시예 21.53. ACE-11r의 제조Example 21.53. Preparation of ACE-11r

ACE-11r contains two different heavy chain-like chains (ACE-11r-VH and ACE-11r-VL) and two identical light chains (ACE-11r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-11r-VH amino acid sequence (SEQ ID NO: 434)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA

ACE-11r-VL amino acid sequence (SEQ ID NO: 439)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs DILLTQSPVILSVSPGERVSFSCRAS QSIGTN IHWYQQRTNGSPRLLIK YAS ESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQNNNWPTT FGAGTKLELK

ACE-11r-LC amino acid sequence (SEQ ID NO: 419)

DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGTVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR [ RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPYPREAKVQWKLSVTYPREAKVQWKLSVTDREAKVQWKLSVT

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting EGFR are listed in Table 57 below:

Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 325)VH: EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS (SEQ ID NO: 325)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 337)VL: DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGTVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR (SEQ ID NO: 337)
	CDR H1: GYSFTGYT(서열번호: 326)CDR H1: GYSFTGYT (SEQ ID NO: 326)	CDR L1: QDIRNY (서열번호: 338)CDR L1: QDIRNY (SEQ ID NO: 338)
	CDR H2: INPYKGVS (서열번호: 327)CDR H2: INPYKGVS (SEQ ID NO: 327)	CDR L2: YTS (서열번호: 339)CDR L2: YTS (SEQ ID NO: 339)
	CDR H3: ARSGYYGDSDWYFDV (서열번호: 328)CDR H3: ARSGYYGDSDWYFDV (SEQ ID NO: 328)	CDR L3: QQGNTLPWT (서열번호: 340)CDR L3: QQGNTLPWT (SEQ ID NO: 340)
Fv region (Anti-EGFR)Fv region (Anti-EGFR)	VH: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (서열번호: 435)VH: QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA (SEQ ID NO: 435)	VL: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (서열번호: 440)VL: DILLTQSPVILSVSPGERVSFSCRAS QSIGTN IHWYQQRTNGSPRLLIK YAS ESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQNNNWPTT FGAGTKLELK (SEQ ID NO: 440)
	CDR H1: GFSLTNYG (서열번호: 436)CDR H1: GFSLTNYG (SEQ ID NO: 436)	CDR L1: QSIGTN (서열번호: 441)CDR L1: QSIGTN (SEQ ID NO: 441)
	CDR H2: IWSGGNT (서열번호: 437)CDR H2: IWSGGNT (SEQ ID NO: 437)	CDR L2: YAS (서열번호: 442)CDR L2: YAS (SEQ ID NO: 442)
	CDR H3: ARALTYYDYEFAY (서열번호: 438)CDR H3: ARALTYYDYEFAY (SEQ ID NO: 438)	CDR L3: QQNNNWPTT (서열번호: 443)CDR L3: QQNNNWPTT (SEQ ID NO: 443)

실시예 21.54. ACE-12r의 제조Example 21.54. Preparation of ACE-12r

ACE-12r contains two different heavy chain-like chains (ACE-12r-VH and ACE-12r-VL) and two identical light chains (ACE-12r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-12r-VH amino acid sequence (SEQ ID NO: 444)

ACE-12r-VL amino acid sequence (SEQ ID NO: 445)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggsggggsg QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVL

ACE-12r-LC amino acid sequence (SEQ ID NO: 431)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting PD-L1 are listed in Table 58 below. have:

실시예 21.55. ACE-13r의 제조Example 21.55. Preparation of ACE-13r

ACE-13r contains two different heavy chain-like chains (ACE-13r-VH and ACE-13r-VL) and two identical light chains (ACE-13r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-13r-VH amino acid sequence (SEQ ID NO: 446)

QVQLVESGGGVVQPGRSLRLSCAAS GFTFLRYA MHWVRQAPGKGLEWVAV ISYDGRYK YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC TTTTFDS WGQGTLVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS

ACE-13r-VLamino acid sequence (SEQ ID NO: 447)

QVQLVESGGGVVQPGRSLRLSCAAS GFTFLRYA MHWVRQAPGKGLEWVAV ISYDGRYK YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC TTTTFDS WGQGTLVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVLR

ACE-13r-LC amino acid sequence (SEQ ID NO: 448)

DIVMTQTPLSLPVTPGEAASISCRSS QSLLDSEDGNTY LDWYLQKPGQSPQLLIY TLS HRASGVPDRFSGSGSGTDFTLEISRVEAEDVGVYYC MQRRDFPFT FGQGTKVDIKR [ SVAAPSVFIFPPSDEQLKSGTASVVCLLVNNFYPQDSGGNQSVKDYPQKREADDS

The VH and VL amino acid sequences and CDR sequences therein for the first antigen-binding domain bivalent Fab region targeting PD-1 and the second antigen-binding domain monovalent Fv region targeting PD-L1 are shown in Table 59 below. It is listed:

Fv region (Anti-PD-L1)Fv region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (서열번호: 170)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 170)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171)CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fab region (Anti-PD-1)Fab region (Anti-PD-1)	VH: QVQLVESGGGVVQPGRSLRLSCAASGFTFLRYAMHWVRQAPGKGLEWVAVISYDGRYKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTTTTFDSWGQGTLVTVSS (서열번호: 196)VH: QVQLVESGGGVVQPGRSLRLSCAASGFTFLRYAMHWVRQAPGKGLEWVAVISYDGRYKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTTTTFDSWGQGTLVTVSS (SEQ ID NO: 196)	VL: DIVMTQTPLSLPVTPGEAASISCRSSQSLLDSEDGNTYLDWYLQKPGQSPQLLIYTLSHRASGVPDRFSGSGSGTDFTLEISRVEAEDVGVYYCMQRRDFPFTFGQGTKVDIKR (서열번호: 200)VL: DIVMTQTPLSLPVTPGEAASISCRSSQSLLDSEDGNTYLDWYLQKPGQSPQLLIYTLSHRASGVPDRFSGSGSGTDFTLEISRVEAEDVGVYYCMQRRDFPFTFGQGTKVDIKR (SEQ ID NO: 200)
	CDR H1: GFTFLRYA (서열번호: 197)CDR H1: GFTFLRYA (SEQ ID NO: 197)	CDR L1: QSLLDSEDGNTY (서열번호: 201)CDR L1: QSLLDSEDGNTY (SEQ ID NO: 201)
	CDR H2: ISYDGRYK (서열번호: 198)CDR H2: ISYDGRYK (SEQ ID NO: 198)	CDR L2: TLS (서열번호: 202)CDR L2: TLS (SEQ ID NO: 202)
	CDR H3: TTTTFDS (서열번호: 199)CDR H3: TTTTFDS (SEQ ID NO: 199)	CDR L3: MQRRDFPFT (서열번호: 203)CDR L3: MQRRDFPFT (SEQ ID NO: 203)

실시예 21.56. ACE-14r의 제조Example 21.56. Preparation of ACE-14r

ACE-14r contains two different heavy chain-like chains (ACE-14r-VH and ACE-14r-VL) and two identical light chains (ACE-14r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-14r-VH amino acid sequence (SEQ ID NO: 449)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKCLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggsggggs QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS

ACE-14r-VL amino acid sequence (SEQ ID NO: 450)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKCLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggsggggs QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVLR

ACE-14r-LC amino acid sequence (SEQ ID NO: 451)

DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGTVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FACGTKLEIKR [ SVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPYPREAKVQWKLSVTYPREAKVQWKLSVTDSKVQWKLSVT

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting PD-L1 are listed in Table 60 below. have:

Fv region (Anti-PD-L1)Fv region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (서열번호: 170)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 170)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171)CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKCLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 176)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKCLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 176)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFACGTKLEIKR (서열번호: 206)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFACGTKLEIKR (SEQ ID NO: 206)
	CDR H1: GYSFTGYT (서열번호: 177)CDR H1: GYSFTGYT (SEQ ID NO: 177)	CDR L1: QDIRNY (서열번호: 181)CDR L1: QDIRNY (SEQ ID NO: 181)
	CDR H2: INPYKGVS (서열번호: 178)CDR H2: INPYKGVS (SEQ ID NO: 178)	CDR L2: YTS (서열번호: 182)CDR L2: YTS (SEQ ID NO: 182)
	CDR H3: ARSGYYGDSDWYFDV (서열번호: 179)CDR H3: ARSGYYGDSDWYFDV (SEQ ID NO: 179)	CDR L3: QQGNTLPWT (서열번호: 207)CDR L3: QQGNTLPWT (SEQ ID NO: 207)

실시예 21.57. ACE-15r의 제조Example 21.57. Preparation of ACE-15r

ACE-15r contains two different heavy chain-like chains (ACE-15r-VH and ACE-15r-VL) and two identical light chains (ACE-15r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-15r-VH amino acid sequence (SEQ ID NO: 452)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGCGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS

ACE-15r-VL amino acid sequence (SEQ ID NO: 453)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGCGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVLR

ACE-15r-LC amino acid sequence (SEQ ID NO: 454)

DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGCVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR [ SVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPYPREAKVQWKLSVTYPREAKVQWKLSVDSKVQWKLSVT

The VH and VL amino acid sequences and CDR sequences therein for the first antigen-binding domain bivalent Fab region targeting CD3 and the second antigen-binding domain monovalent Fv region targeting PD-L1 are listed in Table 61 below. Has been:

Fv region (Anti-PD-L1)Fv region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (서열번호: 170)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 170)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171)CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGCGTTLTVFS (서열번호: 210)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGCGTTLTVFS (SEQ ID NO: 210)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGCVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 212)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGCVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 212)
	CDR H1: GYSFTGYT (서열번호: 177)CDR H1: GYSFTGYT (SEQ ID NO: 177)	CDR L1: QDIRNY (서열번호: 181)CDR L1: QDIRNY (SEQ ID NO: 181)
	CDR H2: INPYKGVS (서열번호: 178)CDR H2: INPYKGVS (SEQ ID NO: 178)	CDR L2: YTS (서열번호: 182)CDR L2: YTS (SEQ ID NO: 182)
	CDR H3: ARSGYYGDSDWYFD (서열번호: 211)CDR H3: ARSGYYGDSDWYFD (SEQ ID NO: 211)	CDR L3: QQGNTLPWT (서열번호: 207)CDR L3: QQGNTLPWT (SEQ ID NO: 207)

실시예 21.58. ACE-16r의 제조Example 21.58. Preparation of ACE-16r

ACE-16r contains two different heavy chain-like chains (ACE-16r-VH and ACE-16r-VL) and two identical light chains (ACE-16r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-16r-VH amino acid sequence (SEQ ID NO: 455)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS

ACE-16r-VL amino acid sequence (SEQ ID NO: 456)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVLR

ACE-16r-LC amino acid sequence (SEQ ID NO: 457)

DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGTVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR [ SVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPYPREAKVQWKLSVTYPREAKVQWKLSVTEREAKVQWKLSVT

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting PD-L1 are listed in Table 62 below. have.

Fv region (Anti-PD-L1)Fv region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (서열번호: 170)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 170)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171)CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 215)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 215)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 216)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 216)
	CDR H1: GYSFTGYT (서열번호: 177)CDR H1: GYSFTGYT (SEQ ID NO: 177)	CDR L1: QDIRNY (서열번호: 181)CDR L1: QDIRNY (SEQ ID NO: 181)
	CDR H2: INPYKGVS (서열번호: 178)CDR H2: INPYKGVS (SEQ ID NO: 178)	CDR L2: YTS (서열번호: 182)CDR L2: YTS (SEQ ID NO: 182)
	CDR H3: ARSGYYGDSDWYFD (서열번호: 211)CDR H3: ARSGYYGDSDWYFD (SEQ ID NO: 211)	CDR L3: QQGNTLPWT (서열번호: 207)CDR L3: QQGNTLPWT (SEQ ID NO: 207)

실시예 21.59. ACE-17r의 제조Example 21.59. Preparation of ACE-17r

ACE-17r contains two different heavy chain-like chains (ACE-17r-VH and ACE-17r-VL) and two identical light chains (ACE-17r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-17r-VH amino acid sequence (SEQ ID NO: 458)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS

ACE-17r-VL amino acid sequence (SEQ ID NO: 459)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVLR

ACE-17r-LC amino acid sequence (SEQ ID NO: 457)

DIQMTQTTSSLSASLGDRVTISCRASDIQMTQTTSSLSASLGDRVTISCRAS QDIRNYQDIRNY LNWYQQKPDGTVKLLIYLNWYQQKPDGTVKLLIY YTSYTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWTQQGNTLPWT FAGGTKLEIKR[FAGGTKLEIKR[ SVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC]

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting PD-L1 are listed in Table 63 below. have.

실시예 21.60. ACE-18r의 제조Example 21.60. Preparation of ACE-18r

ACE-18r contains two different heavy chain-like chains (ACE-18r-VH and ACE-18r-VL) and two identical light chains (ACE-18r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-18r-VH amino acid sequence (SEQ ID NO: 460)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPPCPAPELLGGP GGGGS QVQLQQPGAELVKPGASVKMSCKAS GYTFTSYN MHWVKQTPGRGLEWIGA IYPGNGDT SYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYC ARSTYYGGDWYFNV WGAGTTVTVSA

ACE-18r-VL amino acid sequence (SEQ ID NO: 461)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPPCPAPELLGGP GGGGS QIVLSQSPAILSASPGEKVTMTCRAS SSVSY IHWFQQKPGSSPKPWIY ATS NLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYC QQWTSNPPT FGGGTKLEIK

ACE-18r-LC amino acid sequence (SEQ ID NO: 431)

DIQMTQTTSSLSASLGDRVTISCRASDIQMTQTTSSLSASLGDRVTISCRAS QDIRNYQDIRNY LNWYQQKPDGTVKLLIYLNWYQQKPDGTVKLLIY YTSYTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWTQQGNTLPWT FAGGTKLEIKRFAGGTKLEIKR RSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting CD20 are listed in Table 64 below.

Fv region (Anti-CD20)Fv region (Anti-CD20)	VH: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (서열번호: 222)VH: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 222)	VL: QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (서열번호: 226)VL: QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 226)
	CDR H1: GYTFTSYN (서열번호: 223)CDR H1: GYTFTSYN (SEQ ID NO: 223)	CDR L1: SSVSY (서열번호: 227)CDR L1: SSVSY (SEQ ID NO: 227)
	CDR H2: IYPGNGDT (서열번호: 224)CDR H2: IYPGNGDT (SEQ ID NO: 224)	CDR L2: ATS (서열번호: 228)CDR L2: ATS (SEQ ID NO: 228)
	CDR H3: ARSTYYGGDWYFNV (서열번호: 225)CDR H3: ARSTYYGGDWYFNV (SEQ ID NO: 225)	CDR L3: QQWTSNPPT (서열번호: 229)CDR L3: QQWTSNPPT (SEQ ID NO: 229)
Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 215)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 215)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 216)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 216)
	CDR H1: GYSFTGYT (서열번호: 177)CDR H1: GYSFTGYT (SEQ ID NO: 177)	CDR L1: QDIRNY (서열번호: 181)CDR L1: QDIRNY (SEQ ID NO: 181)
	CDR H2: INPYKGVS (서열번호: 178)CDR H2: INPYKGVS (SEQ ID NO: 178)	CDR L2: YTS (서열번호: 182)CDR L2: YTS (SEQ ID NO: 182)
	CDR H3: ARSGYYGDSDWYFD (서열번호: 211)CDR H3: ARSGYYGDSDWYFD (SEQ ID NO: 211)	CDR L3: QQGNTLPWT (서열번호: 207)CDR L3: QQGNTLPWT (SEQ ID NO: 207)

실시예 21.61. ACE-19r의 제조Example 21.61. Preparation of ACE-19r

ACE-19r contains two different heavy chain-like chains (ACE-19r-VH and ACE-19r-VL) and two identical light chains (ACE-19r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-19r-VH amino acid sequence (SEQ ID NO: 462)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPPCPAPELLGGP GGGGS QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA

ACE-19r-VL amino acid sequence (SEQ ID NO: 463)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPPCPAPELLGGP GGGGS DILLTQSPVILSVSPGERVSFSCRAS QSIGTN IHWYQQRTNGSPRLLIK YAS ESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQNNNWPTT FGAGTKLELK

ACE-19r-LC amino acid sequence (SEQ ID NO: 419)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting EGFR are listed in Table 65 below.

Fv region (Anti-EGFR)Fv region (Anti-EGFR)	VH: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (서열번호: 233)VH: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 233)	VL: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (서열번호: 237)VL: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 237)
	CDR H1: GFSLTNYG (서열번호: 234)CDR H1: GFSLTNYG (SEQ ID NO: 234)	CDR L1: QSIGTN (서열번호: 238)CDR L1: QSIGTN (SEQ ID NO: 238)
	CDR H2: IWSGGNT (서열번호: 235)CDR H2: IWSGGNT (SEQ ID NO: 235)	CDR L2: YAS (서열번호: 239)CDR L2: YAS (SEQ ID NO: 239)
	CDR H3: ARALTYYDYEFAY (서열번호: 236)CDR H3: ARALTYYDYEFAY (SEQ ID NO: 236)	CDR L3: QQNNNWPTT (서열번호: 240)CDR L3: QQNNNWPTT (SEQ ID NO: 240)
Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 215)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 215)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 216)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 216)
	CDR H1: GYSFTGYT (서열번호: 177)CDR H1: GYSFTGYT (SEQ ID NO: 177)	CDR L1: QDIRNY (서열번호: 181)CDR L1: QDIRNY (SEQ ID NO: 181)
	CDR H2: INPYKGVS (서열번호: 178)CDR H2: INPYKGVS (SEQ ID NO: 178)	CDR L2: YTS (서열번호: 182)CDR L2: YTS (SEQ ID NO: 182)
	CDR H3: ARSGYYGDSDWYFD (서열번호: 211)CDR H3: ARSGYYGDSDWYFD (SEQ ID NO: 211)	CDR L3: QQGNTLPWT (서열번호: 207)CDR L3: QQGNTLPWT (SEQ ID NO: 207)

실시예 21.62. ACE-20r의 제조Example 21.62. Preparation of ACE-20r

ACE-20r contains two different heavy chain-like chains (ACE-20r-VH and ACE-20r-VL) and two identical light chains (ACE-20r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-20r-VH amino acid sequence (SEQ ID NO: 464)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QVQLVQSGAEVKKPGASVKVSCKAS GYTFTSHW MHWVRQAPGQGLEWIGE FNPSNGRT NYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYC ASRDYDYDCRYFDY WGQGTLVTVSS

ACE-20r-VL amino acid sequence (SEQ ID NO: 465)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs DIQMTQSPSSLSASVGDRVTITCSAS SSVTY MYWYQQKPGKAPKLLIY DTS NLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYC QQWSSHIFT FGQGTKVEIKR

ACE-20r-LC amino acid sequence (SEQ ID NO: 324)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting EGFR are listed in Table 66 below.

Fv region (Anti-EGFR)Fv region (Anti-EGFR)	VH: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLEWIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDCRYFDYWGQGTLVTVSS (서열번호: 244)VH: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLEWIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDCRYFDYWGQGTLVTVSS (SEQ ID NO: 244)	VL: DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKAPKLLIYDTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIKR (서열번호: 248)VL: DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKAPKLLIYDTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIKR (SEQ ID NO: 248)
	CDR H1: GYTFTSHW (서열번호: 245)CDR H1: GYTFTSHW (SEQ ID NO: 245)	CDR L1: SSVTY (서열번호: 249)CDR L1: SSVTY (SEQ ID NO: 249)
	CDR H2: FNPSNGRT (서열번호: 246)CDR H2: FNPSNGRT (SEQ ID NO: 246)	CDR L2: DTS (서열번호: 250)CDR L2: DTS (SEQ ID NO: 250)
	CDR H3: ASRDYDYDCRYFDY (서열번호: 247)CDR H3: ASRDYDYDCRYFDY (SEQ ID NO: 247)	CDR L3: QQWSSHIFT (서열번호: 251)CDR L3: QQWSSHIFT (SEQ ID NO: 251)
Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 215)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 215)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 216)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 216)
	CDR H1: GYSFTGYT (서열번호: 177)CDR H1: GYSFTGYT (SEQ ID NO: 177)	CDR L1: QDIRNY (서열번호: 181)CDR L1: QDIRNY (SEQ ID NO: 181)
	CDR H2: INPYKGVS (서열번호: 178)CDR H2: INPYKGVS (SEQ ID NO: 178)	CDR L2: YTS (서열번호: 182)CDR L2: YTS (SEQ ID NO: 182)
	CDR H3: ARSGYYGDSDWYFD (서열번호: 211)CDR H3: ARSGYYGDSDWYFD (SEQ ID NO: 211)	CDR L3: QQGNTLPWT (서열번호: 207)CDR L3: QQGNTLPWT (SEQ ID NO: 207)

실시예 21.63. ACE-21r의 제조Example 21.63. Preparation of ACE-21r

ACE-21r contains two different heavy chain-like chains (ACE-21r-VH and ACE-21r-VL) and two identical light chains (ACE-21r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-21r-VH amino acid sequence (SEQ ID NO: 466)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QVQLVESGGGVVQPGRSLRLSCAAS GFTFSTYG MHWVRQAPGKGLEWVAV IWDDGSYK YYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARDGITMVRGVMKDYFDY WGQGTLVTVSS

ACE-21r-VL amino acid sequence (SEQ ID NO: 467)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs AIQLTQSPSSLSASVGDRVTITCRAS QDISSA LVWYQQKPGKAPKLLIY DAS SLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYC QQFNSYPLT FGGGTKVEIKR

ACE-21r-LC amino acid sequence (SEQ ID NO: 324)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting EGFR are listed in Table 67 below.

Fv region (Anti-EGFR)Fv region (Anti-EGFR)	VH: QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGTLVTVSS (서열번호: 255)VH: QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGTLVTVSS (SEQ ID NO: 255)	VL: AIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPKLLIYDASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKR (서열번호: 259)VL: AIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPKLLIYDASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKR (SEQ ID NO: 259)
	CDR H1: GFTFSTYG (서열번호: 256)CDR H1: GFTFSTYG (SEQ ID NO: 256)	CDR L1: QDISSA (서열번호: 260)CDR L1: QDISSA (SEQ ID NO: 260)
	CDR H2: IWDDGSYK (서열번호: 257)CDR H2: IWDDGSYK (SEQ ID NO: 257)	CDR L2: DAS (서열번호: 261)CDR L2: DAS (SEQ ID NO: 261)
	CDR H3: ARDGITMVRGVMKDYFDY (서열번호: 258)CDR H3: ARDGITMVRGVMKDYFDY (SEQ ID NO: 258)	CDR L3: QQFNSYPLT (서열번호: 262)CDR L3: QQFNSYPLT (SEQ ID NO: 262)
Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 215)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 215)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 216)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 216)
	CDR H1: GYSFTGYT (서열번호: 177)CDR H1: GYSFTGYT (SEQ ID NO: 177)	CDR L1: QDIRNY (서열번호: 181)CDR L1: QDIRNY (SEQ ID NO: 181)
	CDR H2: INPYKGVS (서열번호: 178)CDR H2: INPYKGVS (SEQ ID NO: 178)	CDR L2: YTS (서열번호: 182)CDR L2: YTS (SEQ ID NO: 182)
	CDR H3: ARSGYYGDSDWYFD (서열번호: 211)CDR H3: ARSGYYGDSDWYFD (SEQ ID NO: 211)	CDR L3: QQGNTLPWT (서열번호: 207)CDR L3: QQGNTLPWT (SEQ ID NO: 207)

실시예 21.64. ACE-22r의 제조Example 21.64. Preparation of ACE-22r

ACE-22r contains two different heavy chain-like chains (ACE-22r-VH and ACE-22r-VL) and two identical light chains (ACE-22r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-22r-VH amino acid sequence (SEQ ID NO: 468)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPPCPAPELLGGP QVQLQQPGAELVKPGASVKMSCKAS GYTFTSYN MHWVKQTPGRGLEWIGA IYPGNGDT SYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYC ARSTYYGGDWYFNV WGAGTTVTVSA

ACE-22r-VL amino acid sequence (SEQ ID NO: 469)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS [ ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPPCPAPELLGGP QIVLSQSPAILSASPGEKVTMTCRAS SSVSY IHWFQQKPGSSPKPWIY ATS NLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYC QQWTSNPPT FGGGTKLEIK

ACE-22r-LC amino acid sequence (SEQ ID NO: 431)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting CD20 are listed in Table 68 below.

실시예 21.65. ACE-23r의 제조Example 21.65. Preparation of ACE-23r

ACE-23r contains two different heavy chain-like chains (ACE-23r-VH and ACE-23-VL) and two identical light chains (ACE-23r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-23r-VH amino acid sequence (SEQ ID NO: 470)

QVQLVESGGGVVQPGRSLRLSCAAS GFKFSGYG MHWVRQAPGKGLEWVAV IWYDGSKK YYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARQMGYWHFDL WGRGTLVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS

ACE-23r-VL amino acid sequence (SEQ ID NO: 471)

QVQLVESGGGVVQPGRSLRLSCAAS GFKFSGYG MHWVRQAPGKGLEWVAV IWYDGSKK YYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARQMGYWHFDL WGRGTLVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVLR

ACE-23r-LC amino acid sequence (SEQ ID NO: 422)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting PD-L1 are listed in Table 69 below. have.

실시예 21.66. ACE-24r의 제조Example 21.66. Preparation of ACE-24r

ACE-24r contains two different heavy chain-like chains (ACE-24r-VH and ACE-24r-VL) and two identical light chains (ACE-24r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-24r-VH amino acid sequence (SEQ ID NO: 472)

EVQLVESGGGLVQPGRSLRLSCAAS GFTFDDYA MHWVRQAPGKGLEWVSA ITWNSGHI DYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC AKVSYLSTASSLDY WGQGTLVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs EVQLVESGGGLVQPGGSLRLSCAAS GFNIKDTY IHWVRQAPGKGLEWVAR IYPTNGYT RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDY WGQGTLVTVSS

ACE-24r-VL amino acid sequence (SEQ ID NO: 473)

EVQLVESGGGLVQPGRSLRLSCAAS GFTFDDYA MHWVRQAPGKGLEWVSA ITWNSGHI DYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC AKVSYLSTASSLDY WGQGTLVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPAPELLGGP DIQMTQSPSSLSASVGDRVTITCRAS QDVNTA VAWYQQKPGKAPKLLIY SAS FLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYC QQHYTTPPT FGQGTKVEIK

ACE-24r-LC amino acid sequence (SEQ ID NO: 390)

DIQMTQSPSSLSASVGDRVTITCRAS QGIRNY LAWYQQKPGKAPKLLIY AAS TLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QRYNRAPYT FGQGTKVEIKR [ RSVAAPSVFIFPPSDEQLKSGTASVVCLLVNFYPKDSGNQSVKDYPQKREADSQWDS

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting TNF and the second antigen binding domain monovalent Fv region targeting HER2 are listed in Table 70 below.

Fv region (Anti-HER2)Fv region (Anti-HER2)	VH: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (서열번호: 275)VH: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 275)	VL: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (서열번호: 287)VL: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 287)
	CDR H1: GFNIKDTY (서열번호: 276)CDR H1: GFNIKDTY (SEQ ID NO: 276)	CDR L1: QDVNTA (서열번호: 288)CDR L1: QDVNTA (SEQ ID NO: 288)
	CDR H2: IYPTNGYT (서열번호: 277)CDR H2: IYPTNGYT (SEQ ID NO: 277)	CDR L2: SAS (서열번호: 289)CDR L2: SAS (SEQ ID NO: 289)
	CDR H3: SRWGGDGFYAMDY (서열번호: 278)CDR H3: SRWGGDGFYAMDY (SEQ ID NO: 278)	CDR L3: QQHYTTPPT (서열번호: 290)CDR L3: QQHYTTPPT (SEQ ID NO: 290)
Fab region (Anti-TNF)Fab region (Anti-TNF)	VH: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS (서열번호: 279)VH: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS (SEQ ID NO: 279)	VL: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKR (서열번호: 283)VL: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKR (SEQ ID NO: 283)
	CDR H1: GFTFDDYA (서열번호: 280)CDR H1: GFTFDDYA (SEQ ID NO: 280)	CDR L1: QGIRNY (서열번호: 284) CDR L1: QGIRNY (SEQ ID NO: 284)
	CDR H2: ITWNSGHI (서열번호: 281)CDR H2: ITWNSGHI (SEQ ID NO: 281)	CDR L2: AAS (서열번호: 285)CDR L2: AAS (SEQ ID NO: 285)
	CDR H3: AKVSYLSTASSLDY (서열번호: 282)CDR H3: AKVSYLSTASSLDY (SEQ ID NO: 282)	CDR L3: QRYNRAPYT (서열번호: 286)CDR L3: QRYNRAPYT (SEQ ID NO: 286)

실시예 21.67. ACE-25r의 제조Example 21.67. Preparation of ACE-25r

ACE-25r contains two different heavy chain-like chains (ACE-25r-VH and ACE-25r-VL) and two identical light chains (ACE-25r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-25r-HC-VH amino acid sequence (SEQ ID NO: 474)

EVQLVESGGGLVQPGRSLRLSCAAS GFTFDDYA MHWVRQAPGKGLEWVSA ITWNSGHI DYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC AKVSYLSTASSLDY WGQGTLVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGPggggsggggs EVQLVESGGGLVQPGGSLRLSCAAS GFNIKDTY IHWVRQAPGKGLEWVAR IYPTNGYT RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDY WGQGTLVTVSS

ACE-25r-HC-VL amino acid sequence (SEQ ID NO: 475)

ACE-25r-LC amino acid sequence (SEQ ID NO: 390)

DIQMTQSPSSLSASVGDRVTITCRAS QGIRNY LAWYQQKPGKAPKLLIY AAS TLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QRYNRAPYT FGQGTKVEIKR [ RSVAAPSVFIFPPSDEQLKSGTASVVCLLVNNFYPKDSGNQSSPYKDSKREADSV

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting TNF and the second antigen binding domain monovalent Fv region targeting HER2 are listed in Table 71 below.

Fv region (Anti-HER2)Fv region (Anti-HER2)	VH: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (서열번호: 275)VH: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 275)	VL: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (서열번호: 287)VL: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 287)
	CDR H1: GFNIKDTY (서열번호: 276)CDR H1: GFNIKDTY (SEQ ID NO: 276)	CDR L1: QDVNTA (서열번호: 288)CDR L1: QDVNTA (SEQ ID NO: 288)
	CDR H2: IYPTNGYT (서열번호: 277)CDR H2: IYPTNGYT (SEQ ID NO: 277)	CDR L2: SAS (서열번호: 289)CDR L2: SAS (SEQ ID NO: 289)
	CDR H3: SRWGGDGFYAMDY (서열번호: 278)CDR H3: SRWGGDGFYAMDY (SEQ ID NO: 278)	CDR L3: QQHYTTPPT (서열번호: 290)CDR L3: QQHYTTPPT (SEQ ID NO: 290)
Fab region (Anti-TNF)Fab region (Anti-TNF)	VH: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS (서열번호: 279)VH: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS (SEQ ID NO: 279)	VL: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKR (서열번호: 283)VL: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKR (SEQ ID NO: 283)
	CDR H1: GFTFDDYA (서열번호: 280)CDR H1: GFTFDDYA (SEQ ID NO: 280)	CDR L1: QGIRNY (서열번호: 284)CDR L1: QGIRNY (SEQ ID NO: 284)
	CDR H2: ITWNSGHI (서열번호: 281)CDR H2: ITWNSGHI (SEQ ID NO: 281)	CDR L2: AAS (서열번호: 285)CDR L2: AAS (SEQ ID NO: 285)
	CDR H3: AKVSYLSTASSLDY (서열번호: 282)CDR H3: AKVSYLSTASSLDY (SEQ ID NO: 282)	CDR L3: QRYNRAPYT (서열번호: 286)CDR L3: QRYNRAPYT (SEQ ID NO: 286)

실시예 21.68. ACE-26r의 제조Example 21.68. Preparation of ACE-26r

ACE-26r contains two different heavy chain-like chains (ACE-26r-VH and ACE-26r-VL) and two identical light chains (ACE-26r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-26r-VH amino acid sequence (SEQ ID NO: 476)

EVKLLESGGGLVQPKGSLKLSCAAS GFTFNTYA MNWVRQAPGKGLEWVAR IRSKYNNYAT YYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYC VRHGNFGNSYVSWFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA *

ACE-26r-VL amino acid sequence (SEQ ID NO: 477)

EVKLLESGGGLVQPKGSLKLSCAAS GFTFNTYA MNWVRQAPGKGLEWVAR IRSKYNNYAT YYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYC VRHGNFGNSYVSWFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs DILLTQSPVILSVSPGERVSFSCRAS QSIGTN IHWYQQRTNGSPRLLIK YAS ESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQNNNWPTT FGAGTKLELK *

ACE-26r-LC amino acid sequence (SEQ ID NO: 478)

QAVVTQESALTTSPGETVTLTCRSS TGAVTTSNY ANWVQEKPDHLFTGLIG GTN KRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC ALWYSNLWV FGGGTKLTVL [ RTVAAPSVFIFPPSDEQLKSGTKLTVLNNFYPREADSKVQSKVLTQDSKVQWKVDSLGE

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting EGFR are listed in Table 72 below.

Fv region (Anti-EGFR)Fv region (Anti-EGFR)	VH: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (서열번호: 233)VH: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 233)	VL: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (서열번호: 237)VL: DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 237)
	CDR H1: GFSLTNYG (서열번호: 234)CDR H1: GFSLTNYG (SEQ ID NO: 234)	CDR L1: QSIGTN (서열번호: 238)CDR L1: QSIGTN (SEQ ID NO: 238)
	CDR H2: IWSGGNT (서열번호: 235)CDR H2: IWSGGNT (SEQ ID NO: 235)	CDR L2: YAS (서열번호: 239)CDR L2: YAS (SEQ ID NO: 239)
	CDR H3: ARALTYYDYEFAY (서열번호: 236)CDR H3: ARALTYYDYEFAY (SEQ ID NO: 236)	CDR L3: QQNNNWPTT (서열번호: 240)CDR L3: QQNNNWPTT (SEQ ID NO: 240)
Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: EVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSA (서열번호: 294)VH: EVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSA (SEQ ID NO: 294)	VL: QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL (서열번호: 298)VL: QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL (SEQ ID NO: 298)
	CDR H1: GFTFNTYA (서열번호: 295)CDR H1: GFTFNTYA (SEQ ID NO: 295)	CDR L1: TGAVTTSNY (서열번호: 299)CDR L1: TGAVTTSNY (SEQ ID NO: 299)
	CDR H2: IRSKYNNYAT (서열번호: 296)CDR H2: IRSKYNNYAT (SEQ ID NO: 296)	CDR L2: GTN (서열번호: 300)CDR L2: GTN (SEQ ID NO: 300)
	CDR H3: VRHGNFGNSYVSWFAY (서열번호: 297)CDR H3: VRHGNFGNSYVSWFAY (SEQ ID NO: 297)	CDR L3: ALWYSNLWV (서열번호: 301)CDR L3: ALWYSNLWV (SEQ ID NO: 301)

실시예 21.69. ACE-27r의 제조Example 21.69. Preparation of ACE-27r

ACE-27r contains two different heavy chain-like chains (ACE-27r-VH and ACE-27r-VL) and two identical light chains (ACE-27r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-27r-VH amino acid sequence (SEQ ID NO: 479)

EVKLLESGGGLVQPKGSLKLSCAAS GFTFNTYA MNWVRQAPGKGLEWVAR IRSKYNNYAT YYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYC VRHGNFGNSYVSWFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGPggggs QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS

ACE-27r-VL amino acid sequence (SEQ ID NO: 480)

EVKLLESGGGLVQPKGSLKLSCAAS GFTFNTYA MNWVRQAPGKGLEWVAR IRSKYNNYAT YYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYC VRHGNFGNSYVSWFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QAVVTQESALTTSPGETVTLTCRSS TGAVTTSNY ANWVQEKPDHLFTGLIG GTN KRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC ALWYSNLWV FGGGTKLTVL

ACE-27r-LC amino acid sequence (SEQ ID NO: 304)

QAVVTQESALTTSPGETVTLTCRSS TGAVTTSNY ANWVQEKPDHLFTGLIG GTN KRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC ALWYSNLWV FGGGTKLTVL [ RSVAAPSVFIFPPSDEQLKSGTKLTVLNNFYPREADSKVQSKVLTQDSKVQSKVDSKDSK

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting PD-L1 are listed in Table 73 below:

Fv region (Anti-PD-L1)Fv region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL (서열번호: 313) VL: QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL (SEQ ID NO: 313)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CD TGAVTTSNY (서열번호: 314) R L1: CD TGAVTTSNY (SEQ ID NO: 314) R L1:
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GTN (서열번호: 315)CDR L2: GTN (SEQ ID NO: 315)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: ALWYSNLWV (서열번호: 316)CDR L3: ALWYSNLWV (SEQ ID NO: 316)
Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: EVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSA (서열번호: 305)VH: EVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSA (SEQ ID NO: 305)	VL: QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL (서열번호: 309)VL: QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL (SEQ ID NO: 309)
	CDR H1: GFTFNTYA (서열번호: 306)CDR H1: GFTFNTYA (SEQ ID NO: 306)	CDR L1: TGAVTTSNY (서열번호: 310)CDR L1: TGAVTTSNY (SEQ ID NO: 310)
	CDR H2: IRSKYNNYAT (서열번호: 307)CDR H2: IRSKYNNYAT (SEQ ID NO: 307)	CDR L2: GTN (서열번호: 311)CDR L2: GTN (SEQ ID NO: 311)
	CDR H3: VRHGNFGNSYVSWFAY (서열번호: 308)CDR H3: VRHGNFGNSYVSWFAY (SEQ ID NO: 308)	CDR L3: ALWYSNLWV (서열번호: 312)CDR L3: ALWYSNLWV (SEQ ID NO: 312)

실시예 21.70. ACE-28r의 제조Example 21.70. Preparation of ACE-28r

ACE-28r contains two different heavy chain-like chains (ACE-28r-VH and ACE-28r-VL) and two identical light chains (ACE-28r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-28r-VH amino acid sequence (SEQ ID NO: 481)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

ACE-28r-VL amino acid sequence (SEQ ID NO: 482)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs DILLTQSPVILSVSPGERVSFSCRAS QSIGTN IHWYQQRTNGSPRLLIK YAS ESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQNNNWPTT FGAGTKLELK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

ACE-28r-LC amino acid sequence (SEQ ID NO: 419)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting EGFR are listed in Table 74 below.

실시예 21.71. ACE-29r의 제조Example 21.71. Preparation of ACE-29r

ACE-29r contains two different heavy chain-like chains (ACE-29r-VH and ACE-29r-VL) and two identical light chains (ACE-29r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-29r-VH amino acid sequence (SEQ ID NO: 483)

EVKLLESGGGLVQPKGSLKLSCAAS GFTFNTYA MNWVRQAPGKGLEWVAR IRSKYNNYAT YYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYC VRHGNFGNSYVSWFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPCPAPELLGGP] ggggs QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

ACE-29r-VL amino acid sequence (SEQ ID NO: 484)

EVKLLESGGGLVQPKGSLKLSCAAS GFTFNTYA MNWVRQAPGKGLEWVAR IRSKYNNYAT YYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYC VRHGNFGNSYVSWFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPCPAPELLGGP] ggggs DILLTQSPVILSVSPGERVSFSCRAS QSIGTN IHWYQQRTNGSPRLLIK YAS ESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQNNNWPTT FGAGTKLELK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

ACE-29r-LC amino acid sequence (SEQ ID NO: 478)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting EGFR are listed in Table 75 below.

실시예 21.72. ACE-30r의 제조Example 21.72. Preparation of ACE-30r

ACE-30r contains two different heavy chain-like chains (ACE-30r-VH and ACE-30r-VL) and two identical light chains (ACE-30r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-30r-VH amino acid sequence (SEQ ID NO: 485)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS

ACE-30r-VL amino acid sequence (SEQ ID NO: 486)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggs DIQMTQTTSSLSASLGDRVTISCRAS QDIRNY LNWYQQKPDGTVKLLIY YTS RLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPWT FAGGTKLEIKR

ACE-30r-LC amino acid sequence (SEQ ID NO: 487)

QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVLR [ TVAAPSVFIFPPSDEQLKSGTASVVCSHKVTELSKSGNSVCLLKNFSKLTQDSVCLLKVDKV

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 76 below. have.

Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 325)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 325)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 337)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 337)
	CDR H1: GYSFTGYT (서열번호: 326)CDR H1: GYSFTGYT (SEQ ID NO: 326)	CDR L1: QDIRNY (서열번호: 338)CDR L1: QDIRNY (SEQ ID NO: 338)
	CDR H2: INPYKGVS (서열번호: 327)CDR H2: INPYKGVS (SEQ ID NO: 327)	CDR L2: YTS (서열번호: 339)CDR L2: YTS (SEQ ID NO: 339)
	CDR H3: ARSGYYGDSDWYFDV (서열번호: 328)CDR H3: ARSGYYGDSDWYFDV (SEQ ID NO: 328)	CDR L3: QQGNTLPWT (서열번호: 340)CDR L3: QQGNTLPWT (SEQ ID NO: 340)
Fab region (Anti-PD-L1)Fab region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 329)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 329)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (서열번호: 333)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 333)
	CDR H1: GGTFSSYA (서열번호: 330)CDR H1: GGTFSSYA (SEQ ID NO: 330)	CDR L1: SSNIGAGYD (서열번호: 334)CDR L1: SSNIGAGYD (SEQ ID NO: 334)
	CDR H2: IIPILGIA (서열번호: 331)CDR H2: IIPILGIA (SEQ ID NO: 331)	CDR L2: GDI (서열번호: 335)CDR L2: GDI (SEQ ID NO: 335)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 332)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 332)	CDR L3: QSYDSSLSGGV (서열번호: 336)CDR L3: QSYDSSLSGGV (SEQ ID NO: 336)

실시예 21.73. ACE-31r의 제조Example 21.73. Preparation of ACE-31r

ACE-31r contains two different heavy chain-like chains (ACE-31r-VH and ACE-31r-VL) and two identical light chains (ACE-31r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-31r-VH amino acid sequence (SEQ ID NO: 488)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPAPELLGGP ggggsggggs EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS

ACE-31r-VL amino acid sequence (SEQ ID NO: 489)

ACE-31r-LC amino acid sequence (SEQ ID NO: 487)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting PD-L1 are listed in Table 77 below. have.

실시예 21.74. ACE-32r의 제조Example 21.74. Preparation of ACE-32r

ACE-32r contains two different heavy chain-like chains (ACE-32r-VH and ACE-32r-VL) and two identical light chains (ACE-32r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-32r-VH amino acid sequence (SEQ ID NO: 490)

EVKLLESGGGLVQPKGSLKLSCAAS GFTFNTYA MNWVRQAPGKGLEWVAR IRSKYNNYAT YYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYC VRHGNFGNSYVSWFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggsggggs QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA *

ACE-32r-VL amino acid sequence (SEQ ID NO: 491)

EVKLLESGGGLVQPKGSLKLSCAAS GFTFNTYA MNWVRQAPGKGLEWVAR IRSKYNNYAT YYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYC VRHGNFGNSYVSWFAY WGQGTLVTVSA [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs DILLTQSPVILSVSPGERVSFSCRAS QSIGTN IHWYQQRTNGSPRLLIK YAS ESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQNNNWPTT FGAGTKLELK *

ACE-32r-LC amino acid sequence (SEQ ID NO: 478)

QAVVTQESALTTSPGETVTLTCRSS TGAVTTSNY ANWVQEKPDHLFTGLIG GTN KRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFC ALWYSNLWV FGGGTKLTVL [ RTVAAPSVFIFPPSDEQLKSGTKLTVLNNFYPREADSKSCHKVLTQDSKVQWKVDSLGE

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting EGFR are listed in Table 78 below.

실시예 21.75. ACE-33r의 제조Example 21.75. Preparation of ACE-33r

ACE-33r contains two different heavy chain-like chains (ACE-33r-VH and ACE-33r-VL) and two identical light chains (ACE-33r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-33r-VH amino acid sequence (SEQ ID NO: 492)

QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggsggggs EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFDV WGQGTTLTVFS

ACE-33r-VL amino acid sequence (SEQ ID NO: 493)

ACE-33r-LC amino acid sequence (SEQ ID NO: 487)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting PD-L1 and the second antigen binding domain monovalent Fv region targeting CD3 are listed in Table 79 below. have.

Fv region (Anti-CD3)Fv region (Anti-CD3)	VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (서열번호: 325)VH: EVQLQQSGPELVKPGPSMKISCKASGYSFTGYTMNWVKQSHGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYGDSDWYFDVWGQGTTLTVFS (SEQ ID NO: 325)	VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (서열번호: 337)VL: DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFAGGTKLEIKR (SEQ ID NO: 337)
	CDR H1: GYSFTGYT (서열번호: 326)CDR H1: GYSFTGYT (SEQ ID NO: 326)	CDR L1: QDIRNY (서열번호: 338)CDR L1: QDIRNY (SEQ ID NO: 338)
	CDR H2: INPYKGVS (서열번호: 327)CDR H2: INPYKGVS (SEQ ID NO: 327)	CDR L2: YTS (서열번호: 339)CDR L2: YTS (SEQ ID NO: 339)
	CDR H3: ARSGYYGDSDWYFDV (서열번호: 328)CDR H3: ARSGYYGDSDWYFDV (SEQ ID NO: 328)	CDR L3: QQGNTLPWT (서열번호: 340)CDR L3: QQGNTLPWT (SEQ ID NO: 340)
Fab region (Anti-PD-L1)Fab region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 329)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 329)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR(서열번호: 333)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 333)
	CDR H1: GGTFSSYA (서열번호: 330)CDR H1: GGTFSSYA (SEQ ID NO: 330)	CDR L1: SSNIGAGYD (서열번호: 334CDR L1: SSNIGAGYD (SEQ ID NO: 334
	CDR H2: IIPILGIA (서열번호: 331)CDR H2: IIPILGIA (SEQ ID NO: 331)	CDR L2: GDI (서열번호: 335)CDR L2: GDI (SEQ ID NO: 335)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 332)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 332)	CDR L3: QSYDSSLSGGV (서열번호: 336)CDR L3: QSYDSSLSGGV (SEQ ID NO: 336)

실시예 21.76. ACE-34r의 제조Example 21.76. Preparation of ACE-34r

ACE-34r contains two different heavy chain-like chains (ACE-34r-VH and ACE-34r-VL) and two identical light chains (ACE-34r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-34r-VH amino acid sequence (SEQ ID NO: 494)

EVQLVESGGGLVQPGKSLKLSCEASGFTFS GYGMH WVRQAPGRGLESVA YITSSSINIKYADAVKG RFTVSRDNAKNLLFLQMNILKSEDTAMYYCAR FDWDKN YWGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS

ACE-34r-VL amino acid sequence (SEQ ID NO: 495)

EVQLVESGGGLVQPGKSLKLSCEASGFTFS GYGMH WVRQAPGRGLESVA YITSSSINIKYADAVKG RFTVSRDNAKNLLFLQMNILKSEDTAMYYCAR FDWDKN YWGQGTMVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVLR

ACE-34r-LC amino acid sequence (SEQ ID NO: 496)

DIQMTQSPSSLPASLGDRVTINC QASQDISNYLN WYQQKPGKAPKLLIY YTNKLAD GVPSRFSGSGSGRDSSFTISSLESEDIGSYYC QQYYNYPWT FGPGTKLEIK [SVAAPSVFIFPPSDEQLKSGTASVVCLLNNVDYPREAKSQGNSVSTYPREAKVQWQLSKDS

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting PD-L1 are listed in Table 80 below. have.

Fv region (Anti-PD-L1)Fv region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR(서열번호: 170) VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 170)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171)CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSS (서열번호: 348)VH: EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSS (SEQ ID NO: 348)	VL: DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIK (서열번호: 352)VL: DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIK (SEQ ID NO: 352)
	CDR H1: GYGMH (서열번호: 349)CDR H1: GYGMH (SEQ ID NO: 349)	CDR L1: QASQDISNYLN (서열번호: 353)CDR L1: QASQDISNYLN (SEQ ID NO: 353)
	CDR H2: YITSSSINIKYADAVKG (서열번호: 350)CDR H2: YITSSSINIKYADAVKG (SEQ ID NO: 350)	CDR L2: YTNKLAD(서열번호: 354)CDR L2: YTNKLAD (SEQ ID NO: 354)
	CDR H3: FDWDKN (서열번호: 351)CDR H3: FDWDKN (SEQ ID NO: 351)	CDR L3: QQYYNYPWT (서열번호: 355)CDR L3: QQYYNYPWT (SEQ ID NO: 355)

실시예 21.77. ACE-35r의 제조Example 21.77. Preparation of ACE-35r

ACE-35r contains two different heavy chain-like chains (ACE-35r-VH and ACE-35r-VL) and two identical light chains (ACE-35r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-35r-VH amino acid sequence (SEQ ID NO: 497)

QVKLQQSGSELGKPGASVKLSCKTSGYIFT DHYIS WVKQKPGESLQWIG NVYGGNGGTSYNQKFQG KATLTVDKISSTAYMELSSLTSEDSAIYYCAR RPVATGHAMDY WGQGIQVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS

ACE-35r-VL amino acid sequence (SEQ ID NO: 498)

QVKLQQSGSELGKPGASVKLSCKTSGYIFT DHYIS WVKQKPGESLQWIG NVYGGNGGTSYNQKFQG KATLTVDKISSTAYMELSSLTSEDSAIYYCAR RPVATGHAMDY WGQGIQVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVLR

ACE-35r-LC amino acid sequence) (SEQ ID NO: 499)

DIVLTQTPATLSLIPGERVTMTC KTSQNIGTILH WYHQKPKEAPRALIK YASQSIP GIPSRFSGSGSETDFTLSINNLEPDDIGIYYC QQSRSWPVT FGPGTKLEIK [SVAAPSVFIFPPSDEQLKSGTKLEIK [SVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREADSGLSQSKVLTQYPREAKVQSKVDLSKADS

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting PD-L1 are listed in Table 81 below. have.

Fv region (Anti-PD-L1)Fv region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR(서열번호: 170)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVLR (SEQ ID NO: 170)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171)CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fab region (Anti-CD3)Fab region (Anti-CD3)	VH: QVKLQQSGSELGKPGASVKLSCKTSGYIFTDHYISWVKQKPGESLQWIGNVYGGNGGTSYNQKFQGKATLTVDKISSTAYMELSSLTSEDSAIYYCARRPVATGHAMDYWGQGIQVTVSS (서열번호: 358)VH: QVKLQQSGSELGKPGASVKLSCKTSGYIFTDHYISWVKQKPGESLQWIGNVYGGNGGTSYNQKFQGKATLTVDKISSTAYMELSSLTSEDSAIYYCARRPVATGHAMDYWGQGIQVTVSS (SEQ ID NO: 358)	VL: DIVLTQTPATLSLIPGERVTMTCKTSQNIGTILHWYHQKPKEAPRALIKYASQSIPGIPSRFSGSGSETDFTLSINNLEPDDIGIYYCQQSRSWPVTFGPGTKLEIK (서열번호: 362)VL: DIVLTQTPATLSLIPGERVTMTCKTSQNIGTILHWYHQKPKEAPRALIKYASQSIPGIPSRFSGSGSETDFTLSINNLEPDDIGIYYCQQSRSWPVTFGPGTKLEIK (SEQ ID NO: 362)
	CDR H1: DHYIS (서열번호: 359)CDR H1: DHYIS (SEQ ID NO: 359)	CDR L1: KTSQNIGTILH (서열번호: 363)CDR L1: KTSQNIGTILH (SEQ ID NO: 363)
	CDR H2: NVYGGNGGTSYNQKFQG (서열번호: 360)CDR H2: NVYGGNGGTSYNQKFQG (SEQ ID NO: 360)	CDR L2: YASQSIP (서열번호: 364)CDR L2: YASQSIP (SEQ ID NO: 364)
	CDR H3: RPVATGHAMDY (서열번호: 361)CDR H3: RPVATGHAMDY (SEQ ID NO: 361)	CDR L3: QQSRSWPVT (서열번호: 365)CDR L3: QQSRSWPVT (SEQ ID NO: 365)

실시예 21.78. ACE-36r의 제조Example 21.78. Preparation of ACE-36r

ACE-36r contains two different heavy chain-like chains (ACE-36r-VH and ACE-36r-VL) and two identical light chains (ACE-36r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-36r-VH amino acid sequence (SEQ ID NO: 500)

QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYT MHWVRQAPGKGLEWVTF ISYDGNNK YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYC ARTGWLGPFDY WGQGTLVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGR IIPILGIA NYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC AKPRDGYNLVAFDI WGQGTMVTVSS

ACE-36r-VL amino acid sequence (SEQ ID NO: 501)

QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYT MHWVRQAPGKGLEWVTF ISYDGNNK YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYC ARTGWLGPFDY WGQGTLVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLG QLVLTQPPSVSGAPGQRVTISCTGS SSNIGAGYD VHWYQQLPGAAPKLLIY GDI NRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYC QSYDSSLSGGV FGGGTKLTVL

ACE-36r-LC amino acid sequence (SEQ ID NO: 502)

EIVLTQSPGTLSLSPGERATLSCRAS QSVGSSY LAWYQQKPGQAPRLLIY GAF SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPWT FGQGTKVEIK [ RSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPYPREASKVQWKVSTLSKVQWKLSKDSPWT

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CTLA-4 and the second antigen binding domain monovalent Fv region targeting PD-L1 are shown in Table 82 below. Are listed.

Fv region (Anti-PD-L1)Fv region (Anti-PD-L1)	VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (서열번호: 158)VH: QMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKPRDGYNLVAFDIWGQGTMVTVSS (SEQ ID NO: 158)	VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVL (서열번호: 376)VL: QLVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGDINRPSGVPDRFSGSKSGISASLAITGLQAEDEADYYCQSYDSSLSGGVFGGGTKLTVL (SEQ ID NO: 376)
	CDR H1: GGTFSSYA (서열번호: 159)CDR H1: GGTFSSYA (SEQ ID NO: 159)	CDR L1: SSNIGAGYD (서열번호: 171)CDR L1: SSNIGAGYD (SEQ ID NO: 171)
	CDR H2: IIPILGIA (서열번호: 160)CDR H2: IIPILGIA (SEQ ID NO: 160)	CDR L2: GDI (서열번호: 172)CDR L2: GDI (SEQ ID NO: 172)
	CDR H3: AKPRDGYNLVAFDI (서열번호: 161)CDR H3: AKPRDGYNLVAFDI (SEQ ID NO: 161)	CDR L3: QSYDSSLSGGV (서열번호: 173)CDR L3: QSYDSSLSGGV (SEQ ID NO: 173)
Fab region (Anti-CTLA4)Fab region (Anti-CTLA4)	VH: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS (서열번호: 368)VH: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS (SEQ ID NO: 368)	VL: EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (서열번호: 372)VL: EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 372)
	CDR H1: GFTFSSYT (서열번호: 369)CDR H1: GFTFSSYT (SEQ ID NO: 369)	CDR L1: QSVGSSY (서열번호: 373)CDR L1: QSVGSSY (SEQ ID NO: 373)
	CDR H2: ISYDGNNK (서열번호: 370)CDR H2: ISYDGNNK (SEQ ID NO: 370)	CDR L2: GAF (서열번호: 374)CDR L2: GAF (SEQ ID NO: 374)
	CDR H3: ARTGWLGPFDY (서열번호: 371)CDR H3: ARTGWLGPFDY (SEQ ID NO: 371)	CDR L3: QQYGSSPWT (서열번호: 375)CDR L3: QQYGSSPWT (SEQ ID NO: 375)

실시예 21.79. ACE-37r의 제조Example 21.79. Preparation of ACE-37r

ACE-37r contains two different heavy chain-like chains (ACE-37r-VH and ACE-37r-VL) and two identical light chains (ACE-37r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-37r-VH amino acid sequence (SEQ ID NO: 503)

QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYT MHWVRQAPGKGLEWVTF ISYDGNNK YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYC ARTGWLGPFDY WGQGTLVTVSS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QVQLQQPGAELVKPGASVKMSCKAS GYTFTSYN MHWVKQTPGRGLEWIGA IYPGNGDT SYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYC ARSTYYGGDWYFNV WGAGTTVTVSA

ACE-37r-VL amino acid sequence (SEQ ID NO: 504)

QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYT MHWVRQAPGKGLEWVTF ISYDGNNK YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYC ARTGWLGPFDY WGQGTLVTVSS [ ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC] DKTHTCPPCPPCPAPELLG QIVLSQSPAILSASPGEKVTMTCRAS SSVSY IHWFQQKPGSSPKPWIY ATS NLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYC QQWTSNPPT FGGGTKLEIK

ACE-37r-LC amino acid sequence (SEQ ID NO: 502)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CTLA-4 and the second antigen binding domain monovalent Fv region targeting CD20 are listed in Table 83 below. have.

Fv region (Anti-CD20)Fv region (Anti-CD20)	VH: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (서열번호: 222)VH: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 222)	VL: QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (서열번호: 226)VL: QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 226)
	CDR H1: GYTFTSYN (서열번호: 223)CDR H1: GYTFTSYN (SEQ ID NO: 223)	CDR L1: SSVSY (서열번호: 227)CDR L1: SSVSY (SEQ ID NO: 227)
	CDR H2: IYPGNGDT (서열번호: 224)CDR H2: IYPGNGDT (SEQ ID NO: 224)	CDR L2: ATS (서열번호: 228)CDR L2: ATS (SEQ ID NO: 228)
	CDR H3: ARSTYYGGDWYFNV (서열번호: 225)CDR H3: ARSTYYGGDWYFNV (SEQ ID NO: 225)	CDR L3: QQWTSNPPT (서열번호: 229)CDR L3: QQWTSNPPT (SEQ ID NO: 229)
Fab region (Anti-CTLA-4)Fab region (Anti-CTLA-4)	VH: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS (서열번호: 368)VH: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS (SEQ ID NO: 368)	VL: EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (서열번호: 372)VL: EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 372)
	CDR H1: GFTFSSYT (서열번호: 369)CDR H1: GFTFSSYT (SEQ ID NO: 369)	CDR L1: QSVGSSY (서열번호: 373)CDR L1: QSVGSSY (SEQ ID NO: 373)
	CDR H2: ISYDGNNK (서열번호: 370)CDR H2: ISYDGNNK (SEQ ID NO: 370)	CDR L2: GAF (서열번호: 374)CDR L2: GAF (SEQ ID NO: 374)
	CDR H3: ARTGWLGPFDY (서열번호: 371)CDR H3: ARTGWLGPFDY (SEQ ID NO: 371)	CDR L3: QQYGSSPWT (서열번호: 375)CDR L3: QQYGSSPWT (SEQ ID NO: 375)

실시예 21.80. ACE-38r의 제조Example 21.80. Preparation of ACE-38r

ACE-38 contains two different heavy chain-like chains (ACE-38r-VH and ACE-38r-VL) and two identical light chains (ACE-38r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-38r-VH amino acid sequence (SEQ ID NO: 505)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA GQPREPQVYTSPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

ACE-38r-VL amino acid sequence (SEQ ID NO: 506)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs DILLTQSPVILSVSPGERVSFSCRAS QSIGTN IHWYQQRTNGSPRLLIK YAS ESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQNNNWPTT FGAGTKLELK

ACE-38r-LC amino acid sequence (SEQ ID NO: 419)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting EGFR are listed in Table 84 below.

실시예 21.81. ACE-39r의 제조Example 21.81. Preparation of ACE-39r

ACE-39r contains two different heavy chain-like chains (ACE-39r-VH and ACE-39r-VL) and two identical light chains (ACE-39r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-39r-VH amino acid sequence (SEQ ID NO: 507)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA

ACE-39r-VL amino acid sequence (SEQ ID NO: 508)

EVQLQQSGPELVKPGPSMKISCKAS GYSFTGYT MNWVKQSHGKNLEWMGL INPYKGVS TYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARSGYYGDSDWYFD VWGQGTTLTVFS [ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC] DKTHTCPPCPPCPAPELLGGP ggggs DILLTQSPVILSVSPGERVSFSCRAS QSIGTN IHWYQQRTNGSPRLLIK YAS ESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQNNNWPTT FGAGTKLELK GQPREPQVYTSPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

ACE-39r-LC amino acid sequence (SEQ ID NO: 419)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting EGFR are listed in Table 85 below.

실시예 21.82. ACE-40r의 제조Example 21.82. Preparation of ACE-40r

ACE-40r contains two different heavy chain-like chains (ACE-40r-VH and ACE-40r-VL) and two identical light chains (ACE-40r-LC). The amino acid sequences of these three types of polypeptides are as follows:

ACE-40r-VH amino acid sequence (SEQ ID NO: 509)

ACE-40r-VL amino acid sequence (SEQ ID NO: 510)

ACE-40r-LC amino acid sequence (SEQ ID NO: 419)

The VH and VL amino acid sequences and CDR sequences therein for the first antigen binding domain bivalent Fab region targeting CD3 and the second antigen binding domain monovalent Fv region targeting EGFR are listed in Table 86 below.

From the foregoing, while specific embodiments have been described herein for purposes of illustration, it will be understood that various modifications may be made without departing from the spirit and scope of what is provided herein. All references mentioned above are incorporated herein by reference in their entirety.

Claims

A multispecific fusion protein in which the Fc region of the antibody is substituted with the variable region of the antibody.
The method of claim 1,

The fusion protein is a multispecific fusion protein comprising the following structural formulas (I) and (II):

N'-A-L1-X-C' (I); And

N'-B-L1-Y-C' (II)

At this time, in the above structural formulas (I) and (II),

Wherein N'is the N-terminus of the fusion protein,

C'is the C-terminus of the fusion protein,

Each of A and B specifically binds to a first antigen,

The L1 is a peptide linker containing at least one Cys,

X is an antibody variable heavy chain (VH) region or an antibody variable light chain (VL) region,

Y is an antibody variable light chain (VL) region or an antibody variable heavy chain (VH) region,

The X and Y form an antibody variable region that specifically binds to the second antigen.
The method of claim 2,

Each of A and B is an antibody Fab region,

(i) a first portion comprising an antibody variable heavy chain (VH) region and an antibody CH1 region; And

(ii) A multispecific fusion protein consisting of a second portion comprising an antibody variable light chain (VL) region and an antibody light chain constant region (CL).
The method of claim 3,

The structural formula (I) is a multi-specific fusion protein comprising the following structural formulas (I') and (I'').

N'-A'-L1-X-C' (I'); And

N'-A''-C' (I'')

At this time, in the above structural formulas (I') and (I''),

A'is a heavy chain region of an antibody and includes a variable region and a CH1 region, or is a light chain region of an antibody;

A'' is the light chain region of the antibody, or as the heavy chain region of the antibody comprises a variable region and a CH1 region;

At this time, A'and A'' bind to form a variable region of the antibody, wherein the variable region specifically binds to the first antigen,

N', L1, X and C'are the same as defined in claim 2.
The method of claim 3,

The structural formula (II) is a multi-specific fusion protein comprising the following structural formulas (II') and (II").

N'-B'-L1-Y-C' (II'); And

N'-B''-C' (II'')

At this time, in the above structural formulas (II') and (II''),

B'is a heavy chain region of an antibody and includes a variable region and a CH1 region, or is a light chain region of an antibody;

B'' is the light chain region of the antibody, or as the heavy chain region of the antibody comprises a variable region and a CH1 region;

At this time, B'and B'' bind to form a variable region of the antibody, wherein the variable region specifically binds to the first antigen,

N', L1, Y and C'are the same as defined in claim 2.
The method of claim 1,

The fusion protein is a multispecific fusion protein comprising the following structural formulas (III) and (IV):

N'-X-L2-A-C' (III); And

N'-Y-L2-B-C' (IV)

In this case, in the structural formulas (III) and (IV),

Wherein N'is the N-terminus of the fusion protein,

C'is the C-terminus of the fusion protein,

Each of A and B specifically binds to a first antigen,

The L2 is a peptide linker containing at least one Cys,

X is an antibody variable heavy chain (VH) region or an antibody variable light chain (VL) region,

Y is an antibody variable light chain (VL) region or an antibody variable heavy chain (VH) region,

The X and Y form an antibody variable region that specifically binds to the second antigen.
The method of claim 2,

The L1 is a multispecific fusion protein containing an immunoglobulin-derived hinge region.
The method of claim 2,

The L1 is a multispecific fusion protein containing 2 or 3 Cys.
The method of claim 7,

The L1 is a multispecific fusion protein having the following structural formula (V):

(L1')n-hinge-(L1'')m (V)

At this time, the L1' and L1'' are each a linker consisting of 1 to 15 amino acids,

n and m are integers of 0 or 1,

The hinge is an immunoglobulin-derived hinge region.
The method of claim 2,

The first and second antigens are PD-L1, EGFR, BCMA, CD22, CD25, CD30, CD33, CD37, CD38, CD52, CD56, CD123, cMET, DLL3, GD2, Nectin-4, RANKL, SLAMF7, respectively. TROP2, Claudin 18.2, TNFR, TNF, CD3, HER2, CD20, CD19, CTLA-4, VEGFR, VEGF, NCAM1, ICAM-1, ICAM-2, CEACAM6, Carcinoembryonic antigen (CEA), CA-125, Alphafetoprotein (AFP ), MUC-1, Epithelial tumor antigen (ETA), Melanoma-associated antigen (MAGE), Immature laminin receptor, TAG-72, HPV E6/E7, BING-4, Calcium-activated chloride channel 2, Cyclin-B1, 9D7 , Ep-CAM, EphA3, Mesothelin, SAP-1, Survivin, and any one selected from the group consisting of virus-derived antigens, a multispecific fusion protein.
The method of claim 2,

The first antigen is a cancer antigen, a multispecific fusion protein.
The method of claim 2,

The second antigen is a protein present on the surface of immune cells, a multispecific fusion protein.
The method of claim 2,

In the above structural formula, A and B recognize the same antigen, or recognize different antigens, multi-specific fusion protein.
The method of claim 13,

A or B is a multi-specific fusion protein comprising any one variable region selected from the following group:

PD-L1:

1) VH region comprising the amino acid sequence of SEQ ID NO: 5 (VH-CDR1), SEQ ID NO: 6 (VH-CDR2) and SEQ ID NO: 7 (VH-CDR3), and SEQ ID NO: 9 (VL-CDR1), SEQ ID NO: 10 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 11 (VL-CDR3);

2) a VH region comprising the amino acid sequence of SEQ ID NO: 159 (VH-CDR1), SEQ ID NO: 160 (VH-CDR2) and SEQ ID NO: 161 (VH-CDR3), and SEQ ID NO: 171 (VL-CDR1), SEQ ID NO: 172 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 173 (VL-CDR3);

3) VH region comprising the amino acid sequence of SEQ ID NO: 159 (VH-CDR1), SEQ ID NO: 160 (VH-CDR2) and SEQ ID NO: 161 (VH-CDR3), and SEQ ID NO: 314 (VL-CDR1), SEQ ID NO: 315 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 316 (VL-CDR3);

4) VH region comprising the amino acid sequence of SEQ ID NO: 330 (VH-CDR1), SEQ ID NO: 331 (VH-CDR2) and SEQ ID NO: 332 (VH-CDR3), and SEQ ID NO: 334 (VL-CDR1), SEQ ID NO: 335 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 336 (VL-CDR3);

HER2:

5) VH region comprising the amino acid sequence of SEQ ID NO: 118 (VH-CDR1), SEQ ID NO: 119 (VH-CDR2) and SEQ ID NO: 120 (VH-CDR3), and SEQ ID NO: 121 (VL-CDR1), SEQ ID NO: 122 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 123 (VL-CDR3);

6) VH region comprising the amino acid sequence of SEQ ID NO: 276 (VH-CDR1), SEQ ID NO: 277 (VH-CDR2) and SEQ ID NO: 278 (VH-CDR3), and SEQ ID NO: 288 (VL-CDR1), SEQ ID NO: 289 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 290 (VL-CDR3);

CD19:

7) VH region comprising the amino acid sequence of SEQ ID NO: 140 (VH-CDR1), SEQ ID NO: 141 (VH-CDR2) and SEQ ID NO: 142 (VH-CDR3), and SEQ ID NO: 152 (VL-CDR1), SEQ ID NO: 153 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 154 (VL-CDR3);

8) VH region comprising the amino acid sequence of SEQ ID NO: 62 (VH-CDR1), SEQ ID NO: 63 (VH-CDR2) and SEQ ID NO: 64 (VH-CDR3), and SEQ ID NO: 66 (VL-CDR1), SEQ ID NO: 67 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 68 (VL-CDR3);

CD20:

9) VH region comprising the amino acid sequence of SEQ ID NO: 223 (VH-CDR1), SEQ ID NO: 224 (VH-CDR2) and SEQ ID NO: 225 (VH-CDR3), and SEQ ID NO: 227 (VL-CDR1), SEQ ID NO: 228 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 229 (VL-CDR3);

10) VH region comprising the amino acid sequence of SEQ ID NO: 436 (VH-CDR1), SEQ ID NO: 437 (VH-CDR2) and SEQ ID NO: 438 (VH-CDR3), and SEQ ID NO: 441 (VL-CDR1), SEQ ID NO: 442 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 443 (VL-CDR3);

EGFR:

11) VH region comprising the amino acid sequence of SEQ ID NO: 234 (VH-CDR1), SEQ ID NO: 235 (VH-CDR2) and SEQ ID NO: 236 (VH-CDR3), and SEQ ID NO: 238 (VL-CDR1), SEQ ID NO: 239 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 240 (VL-CDR3);

12) VH region comprising the amino acid sequence of SEQ ID NO: 245 (VH-CDR1), SEQ ID NO: 246 (VH-CDR2) and SEQ ID NO: 247 (VH-CDR3), and SEQ ID NO: 249 (VL-CDR1), SEQ ID NO: 250 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 251 (VL-CDR3);

13) VH region comprising the amino acid sequence of SEQ ID NO: 256 (VH-CDR1), SEQ ID NO: 257 (VH-CDR2) and SEQ ID NO: 258 (VH-CDR3), and SEQ ID NO: 260 (VL-CDR1), SEQ ID NO: 261 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 262 (VL-CDR3);

14) VH region comprising the amino acid sequence of SEQ ID NO: 234 (VH-CDR1), SEQ ID NO: 235 (VH-CDR2) and SEQ ID NO: 236 (VH-CDR3), and SEQ ID NO: 238 (VL-CDR1), SEQ ID NO: 239 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 240 (VL-CDR3);

CD3:

15) VH region comprising the amino acid sequence of SEQ ID NO: 326 (VH-CDR1), SEQ ID NO: 327 (VH-CDR2) and SEQ ID NO: 328 (VH-CDR3), and SEQ ID NO: 338 (VL-CDR1), SEQ ID NO: 339 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 340 (VL-CDR3);

16) VH region comprising the amino acid sequence of SEQ ID NO: 330 (VH-CDR1), SEQ ID NO: 331 (VH-CDR2) and SEQ ID NO: 332 (VH-CDR3), and SEQ ID NO: 334 (VL-CDR1), SEQ ID NO: 335 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 336 (VL-CDR3);

17) VH region comprising the amino acid sequence of SEQ ID NO: 163 (VH-CDR1), SEQ ID NO: 271 (VH-CDR2) and SEQ ID NO: 165 (VH-CDR3), and SEQ ID NO: 167 (VL-CDR1), SEQ ID NO: 168 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 169 (VL-CDR3);

18) VH region comprising the amino acid sequence of SEQ ID NO: 396 (VH-CDR1), SEQ ID NO: 397 (VH-CDR2) and SEQ ID NO: 398 (VH-CDR3), and SEQ ID NO: 402 (VL-CDR1), SEQ ID NO: 403 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 404 (VL-CDR3);

19) VH region comprising the amino acid sequence of SEQ ID NO: 144 (VH-CDR1), SEQ ID NO: 145 (VH-CDR2) and SEQ ID NO: 146 (VH-CDR3), and SEQ ID NO: 148 (VL-CDR1), SEQ ID NO: 149 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 410 (VL-CDR3);

20) VH region comprising the amino acid sequence of SEQ ID NO: 144 (VH-CDR1), SEQ ID NO: 145 (VH-CDR2) and SEQ ID NO: 146 (VH-CDR3), and SEQ ID NO: 148 (VL-CDR1), SEQ ID NO: 149 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 150 (VL-CDR3);

21) VH region comprising the amino acid sequence of SEQ ID NO: 70 (VH-CDR1), SEQ ID NO: 71 (VH-CDR2) and SEQ ID NO: 72 (VH-CDR3), and SEQ ID NO: 74 (VL-CDR1), SEQ ID NO: 75 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 76 (VL-CDR3);

22) VH region comprising the amino acid sequence of SEQ ID NO: 78 (VH-CDR1), SEQ ID NO: 79 (VH-CDR2) and SEQ ID NO: 80 (VH-CDR3), and SEQ ID NO: 82 (VL-CDR1), SEQ ID NO: 83 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 84 (VL-CDR3);

23) VH region comprising the amino acid sequence of SEQ ID NO: 295 (VH-CDR1), SEQ ID NO: 296 (VH-CDR2) and SEQ ID NO: 297 (VH-CDR3), and SEQ ID NO: 299 (VL-CDR1), SEQ ID NO: 300 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 301 (VL-CDR3);

24) VH region comprising the amino acid sequence of SEQ ID NO: 78 (VH-CDR1), SEQ ID NO: 79 (VH-CDR2) and SEQ ID NO: 80 (VH-CDR3), and SEQ ID NO: 82 (VL-CDR1), SEQ ID NO: 83 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 87 (VL-CDR3);

25) VH region comprising the amino acid sequence of SEQ ID NO: 163 (VH-CDR1), SEQ ID NO: 271 (VH-CDR2) and SEQ ID NO: 165 (VH-CDR3), and SEQ ID NO: 167 (VL-CDR1), SEQ ID NO: 168 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 169 (VL-CDR3);

26) VH region comprising the amino acid sequence of SEQ ID NO: 177 (VH-CDR1), SEQ ID NO: 178 (VH-CDR2) and SEQ ID NO: 179 (VH-CDR3), and SEQ ID NO: 181 (VL-CDR1), SEQ ID NO: 182 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 183 (VL-CDR3);

27) VH region comprising the amino acid sequence of SEQ ID NO: 187 (VH-CDR1), SEQ ID NO: 188 (VH-CDR2) and SEQ ID NO: 189 (VH-CDR3), and SEQ ID NO: 191 (VL-CDR1), SEQ ID NO: 192 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 193 (VL-CDR3);

28) VH region comprising the amino acid sequence of SEQ ID NO: 177 (VH-CDR1), SEQ ID NO: 178 (VH-CDR2) and SEQ ID NO: 179 (VH-CDR3), and SEQ ID NO: 181 (VL-CDR1), SEQ ID NO: 182 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 207 (VL-CDR3);

29) VH region comprising the amino acid sequence of SEQ ID NO: 177 (VH-CDR1), SEQ ID NO: 178 (VH-CDR2) and SEQ ID NO: 211 (VH-CDR3), and SEQ ID NO: 181 (VL-CDR1), SEQ ID NO: 182 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 207 (VL-CDR3);

30) VH region comprising the amino acid sequence of SEQ ID NO: 306 (VH-CDR1), SEQ ID NO: 307 (VH-CDR2) and SEQ ID NO: 308 (VH-CDR3), and SEQ ID NO: 310 (VL-CDR1), SEQ ID NO: 311 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 312 (VL-CDR3);

31) VH region comprising the amino acid sequence of SEQ ID NO: 349 (VH-CDR1), SEQ ID NO: 350 (VH-CDR2) and SEQ ID NO: 351 (VH-CDR3), and SEQ ID NO: 353 (VL-CDR1), SEQ ID NO: 354 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 355 (VL-CDR3);

32) VH region comprising the amino acid sequence of SEQ ID NO: 359 (VH-CDR1), SEQ ID NO: 360 (VH-CDR2) and SEQ ID NO: 361 (VH-CDR3), and SEQ ID NO: 363 (VL-CDR1), SEQ ID NO: 364 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 365 (VL-CDR3);

33) VH region comprising the amino acid sequence of SEQ ID NO: 197 (VH-CDR1), SEQ ID NO: 198 (VH-CDR2) and SEQ ID NO: 199 (VH-CDR3), and SEQ ID NO: 201 (VL-CDR1), SEQ ID NO: 202 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 203 (VL-CDR3);

TNF:

34) VH region comprising the amino acid sequence of SEQ ID NO: 124 (VH-CDR1), SEQ ID NO: 125 (VH-CDR2) and SEQ ID NO: 126 (VH-CDR3), and SEQ ID NO: 127 (VL-CDR1), SEQ ID NO: 128 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 129 (VL-CDR3);

35) VH region comprising the amino acid sequence of SEQ ID NO: 280 (VH-CDR1), SEQ ID NO: 281 (VH-CDR2) and SEQ ID NO: 282 (VH-CDR3), and SEQ ID NO: 284 (VL-CDR1), SEQ ID NO: 285 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 286 (VL-CDR3);

36) VH region comprising the amino acid sequence of SEQ ID NO: 276 (VH-CDR1), SEQ ID NO: 277 (VH-CDR2) and SEQ ID NO: 278 (VH-CDR3), and SEQ ID NO: 288 (VL-CDR1), SEQ ID NO: 289 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 290 (VL-CDR3); And

CTLA-4:

37) VH region comprising the amino acid sequence of SEQ ID NO: 369 (VH-CDR1), SEQ ID NO: 370 (VH-CDR2) and SEQ ID NO: 371 (VH-CDR3), and SEQ ID NO: 373 (VL-CDR1), SEQ ID NO: 374 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 375 (VL-CDR3).
The method of claim 14,

A or B is a multi-specific fusion protein comprising any one variable region selected from the following group:

PD-L1:

1) the heavy chain variable region of SEQ ID NO: 4 (VH) and the light chain variable region of SEQ ID NO: 8 (VL);

2) the heavy chain variable region of SEQ ID NO: 158 (VH) and the light chain variable region of SEQ ID NO: 170 (VL);

3) the heavy chain variable region of SEQ ID NO: 158 (VH) and the light chain variable region of SEQ ID NO: 313 (VL);

4) the heavy chain variable region of SEQ ID NO: 329 (VH) and the light chain variable region of SEQ ID NO: 333 (VL);

5) the heavy chain variable region of SEQ ID NO: 329 (VH) and the light chain variable region of SEQ ID NO: 376 (VL);

HER2:

6) the heavy chain variable region of SEQ ID NO: 51 (VH) and the light chain variable region of SEQ ID NO: 52 (VL);

7) the heavy chain variable region of SEQ ID NO: 275 (VH) and the light chain variable region of SEQ ID NO: 287 (VL);

CD19:

8) the heavy chain variable region of SEQ ID NO: 139 (VH) and the light chain variable region of SEQ ID NO: 151 (VL);

9) the heavy chain variable region of SEQ ID NO: 61 (VH) and the light chain variable region of SEQ ID NO: 65 (VL);

CD20:

10) the heavy chain variable region of SEQ ID NO: 222 (VH) and the light chain variable region of SEQ ID NO: 226 (VL);

11) the heavy chain variable region of SEQ ID NO: 435 (VH) and the light chain variable region of SEQ ID NO: 440 (VL);

EGFR:

12) the heavy chain variable region of SEQ ID NO: 233 (VH) and the light chain variable region of SEQ ID NO: 237 (VL);

13) the heavy chain variable region of SEQ ID NO: 244 (VH) and the light chain variable region of SEQ ID NO: 248 (VL);

14) the heavy chain variable region of SEQ ID NO: 255 (VH) and the light chain variable region of SEQ ID NO: 259 (VL);

CD3:

15) the heavy chain variable region of SEQ ID NO: 325 (VH) and the light chain variable region of SEQ ID NO: 337 (VL);

16) the heavy chain variable region of SEQ ID NO: 329 (VH) and the light chain variable region of SEQ ID NO: 376 (VL);

17) the heavy chain variable region of SEQ ID NO: 162 (VH) and the light chain variable region of SEQ ID NO: 166 (VL);

18) the heavy chain variable region of SEQ ID NO: 395 (VH) and the light chain variable region of SEQ ID NO: 401 (VL);

19) the heavy chain variable region of SEQ ID NO: 406 (VH) and the light chain variable region of SEQ ID NO: 409 (VL);

20) the heavy chain variable region of SEQ ID NO: 143 (VH) and the light chain variable region of SEQ ID NO: 147 (VL);

21) the heavy chain variable region of SEQ ID NO: 69 (VH) and the light chain variable region of SEQ ID NO: 73 (VL);

22) the heavy chain variable region of SEQ ID NO: 77 (VH) and the light chain variable region of SEQ ID NO: 81 (VL);

23) the heavy chain variable region of SEQ ID NO: 294 (VH) and the light chain variable region of SEQ ID NO: 298 (VL);

24) the heavy chain variable region of SEQ ID NO: 85 (VH) and the light chain variable region of SEQ ID NO: 86 (VL);

25) the heavy chain variable region of SEQ ID NO: 176 (VH) and the light chain variable region of SEQ ID NO: 180 (VL);

26) the heavy chain variable region of SEQ ID NO: 186 (VH) and the light chain variable region of SEQ ID NO: 190 (VL);

27) the heavy chain variable region of SEQ ID NO: 176 (VH) and the light chain variable region of SEQ ID NO: 206 (VL);

28) the heavy chain variable region of SEQ ID NO: 210 (VH) and the light chain variable region of SEQ ID NO: 212 (VL);

29) the heavy chain variable region of SEQ ID NO: 215 (VH) and the light chain variable region of SEQ ID NO: 216 (VL);

30) the heavy chain variable region of SEQ ID NO: 305 (VH) and the light chain variable region of SEQ ID NO: 309 (VL);

31) the heavy chain variable region of SEQ ID NO: 348 (VH) and the light chain variable region of SEQ ID NO: 352 (VL);

32) the heavy chain variable region of SEQ ID NO: 358 (VH) and the light chain variable region of SEQ ID NO: 362 (VL);

33) the heavy chain variable region of SEQ ID NO: 196 (VH) and the light chain variable region of SEQ ID NO: 200 (VL);

TNF:

34) the heavy chain variable region of SEQ ID NO: 53 (VH) and the light chain variable region of SEQ ID NO: 54 (VL);

35) the heavy chain variable region of SEQ ID NO: 279 (VH) and the light chain variable region of SEQ ID NO: 283 (VL);

36) the heavy chain variable region of SEQ ID NO: 275 (VH) and the light chain variable region of SEQ ID NO: 287 (VL); And

CTLA-4:

37) The heavy chain variable region of SEQ ID NO: 368 (VH) and the light chain variable region of SEQ ID NO: 372 (VL).
The method of claim 2,

The X or Y is a multi-specific fusion protein comprising a variable heavy chain region or a variable light chain region in any one of the variable regions selected from the following group:

PD-L1:

1) VH region comprising the amino acid sequence of SEQ ID NO: 5 (VH-CDR1), SEQ ID NO: 6 (VH-CDR2) and SEQ ID NO: 7 (VH-CDR3), and SEQ ID NO: 9 (VL-CDR1), SEQ ID NO: 10 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 11 (VL-CDR3);

2) a VH region comprising the amino acid sequence of SEQ ID NO: 159 (VH-CDR1), SEQ ID NO: 160 (VH-CDR2) and SEQ ID NO: 161 (VH-CDR3), and SEQ ID NO: 171 (VL-CDR1), SEQ ID NO: 172 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 173 (VL-CDR3);

3) VH region comprising the amino acid sequence of SEQ ID NO: 159 (VH-CDR1), SEQ ID NO: 160 (VH-CDR2) and SEQ ID NO: 161 (VH-CDR3), and SEQ ID NO: 314 (VL-CDR1), SEQ ID NO: 315 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 316 (VL-CDR3);

4) VH region comprising the amino acid sequence of SEQ ID NO: 330 (VH-CDR1), SEQ ID NO: 331 (VH-CDR2) and SEQ ID NO: 332 (VH-CDR3), and SEQ ID NO: 334 (VL-CDR1), SEQ ID NO: 335 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 336 (VL-CDR3);

HER2:

5) VH region comprising the amino acid sequence of SEQ ID NO: 118 (VH-CDR1), SEQ ID NO: 119 (VH-CDR2) and SEQ ID NO: 120 (VH-CDR3), and SEQ ID NO: 121 (VL-CDR1), SEQ ID NO: 122 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 123 (VL-CDR3);

6) VH region comprising the amino acid sequence of SEQ ID NO: 276 (VH-CDR1), SEQ ID NO: 277 (VH-CDR2) and SEQ ID NO: 278 (VH-CDR3), and SEQ ID NO: 288 (VL-CDR1), SEQ ID NO: 289 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 290 (VL-CDR3);

CD19:

7) VH region comprising the amino acid sequence of SEQ ID NO: 140 (VH-CDR1), SEQ ID NO: 141 (VH-CDR2) and SEQ ID NO: 142 (VH-CDR3), and SEQ ID NO: 152 (VL-CDR1), SEQ ID NO: 153 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 154 (VL-CDR3);

8) VH region comprising the amino acid sequence of SEQ ID NO: 62 (VH-CDR1), SEQ ID NO: 63 (VH-CDR2) and SEQ ID NO: 64 (VH-CDR3), and SEQ ID NO: 66 (VL-CDR1), SEQ ID NO: 67 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 68 (VL-CDR3);

CD20:

9) VH region comprising the amino acid sequence of SEQ ID NO: 223 (VH-CDR1), SEQ ID NO: 224 (VH-CDR2) and SEQ ID NO: 225 (VH-CDR3), and SEQ ID NO: 227 (VL-CDR1), SEQ ID NO: 228 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 229 (VL-CDR3);

10) VH region comprising the amino acid sequence of SEQ ID NO: 436 (VH-CDR1), SEQ ID NO: 437 (VH-CDR2) and SEQ ID NO: 438 (VH-CDR3), and SEQ ID NO: 441 (VL-CDR1), SEQ ID NO: 442 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 443 (VL-CDR3);

EGFR:

11) VH region comprising the amino acid sequence of SEQ ID NO: 234 (VH-CDR1), SEQ ID NO: 235 (VH-CDR2) and SEQ ID NO: 236 (VH-CDR3), and SEQ ID NO: 238 (VL-CDR1), SEQ ID NO: 239 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 240 (VL-CDR3);

12) VH region comprising the amino acid sequence of SEQ ID NO: 245 (VH-CDR1), SEQ ID NO: 246 (VH-CDR2) and SEQ ID NO: 247 (VH-CDR3), and SEQ ID NO: 249 (VL-CDR1), SEQ ID NO: 250 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 251 (VL-CDR3);

13) VH region comprising the amino acid sequence of SEQ ID NO: 256 (VH-CDR1), SEQ ID NO: 257 (VH-CDR2) and SEQ ID NO: 258 (VH-CDR3), and SEQ ID NO: 260 (VL-CDR1), SEQ ID NO: 261 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 262 (VL-CDR3);

14) VH region comprising the amino acid sequence of SEQ ID NO: 234 (VH-CDR1), SEQ ID NO: 235 (VH-CDR2) and SEQ ID NO: 236 (VH-CDR3), and SEQ ID NO: 238 (VL-CDR1), SEQ ID NO: 239 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 240 (VL-CDR3);

CD3:

15) VH region comprising the amino acid sequence of SEQ ID NO: 326 (VH-CDR1), SEQ ID NO: 327 (VH-CDR2) and SEQ ID NO: 328 (VH-CDR3), and SEQ ID NO: 338 (VL-CDR1), SEQ ID NO: 339 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 340 (VL-CDR3);

16) VH region comprising the amino acid sequence of SEQ ID NO: 330 (VH-CDR1), SEQ ID NO: 331 (VH-CDR2) and SEQ ID NO: 332 (VH-CDR3), and SEQ ID NO: 334 (VL-CDR1), SEQ ID NO: 335 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 336 (VL-CDR3);

17) VH region comprising the amino acid sequence of SEQ ID NO: 163 (VH-CDR1), SEQ ID NO: 271 (VH-CDR2) and SEQ ID NO: 165 (VH-CDR3), and SEQ ID NO: 167 (VL-CDR1), SEQ ID NO: 168 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 169 (VL-CDR3);

18) VH region comprising the amino acid sequence of SEQ ID NO: 396 (VH-CDR1), SEQ ID NO: 397 (VH-CDR2) and SEQ ID NO: 398 (VH-CDR3), and SEQ ID NO: 402 (VL-CDR1), SEQ ID NO: 403 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 404 (VL-CDR3);

19) VH region comprising the amino acid sequence of SEQ ID NO: 144 (VH-CDR1), SEQ ID NO: 145 (VH-CDR2) and SEQ ID NO: 146 (VH-CDR3), and SEQ ID NO: 148 (VL-CDR1), SEQ ID NO: 149 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 410 (VL-CDR3);

20) VH region comprising the amino acid sequence of SEQ ID NO: 144 (VH-CDR1), SEQ ID NO: 145 (VH-CDR2) and SEQ ID NO: 146 (VH-CDR3), and SEQ ID NO: 148 (VL-CDR1), SEQ ID NO: 149 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 150 (VL-CDR3);

21) VH region comprising the amino acid sequence of SEQ ID NO: 70 (VH-CDR1), SEQ ID NO: 71 (VH-CDR2) and SEQ ID NO: 72 (VH-CDR3), and SEQ ID NO: 74 (VL-CDR1), SEQ ID NO: 75 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 76 (VL-CDR3);

22) VH region comprising the amino acid sequence of SEQ ID NO: 78 (VH-CDR1), SEQ ID NO: 79 (VH-CDR2) and SEQ ID NO: 80 (VH-CDR3), and SEQ ID NO: 82 (VL-CDR1), SEQ ID NO: 83 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 84 (VL-CDR3);

23) VH region comprising the amino acid sequence of SEQ ID NO: 295 (VH-CDR1), SEQ ID NO: 296 (VH-CDR2) and SEQ ID NO: 297 (VH-CDR3), and SEQ ID NO: 299 (VL-CDR1), SEQ ID NO: 300 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 301 (VL-CDR3);

24) VH region comprising the amino acid sequence of SEQ ID NO: 78 (VH-CDR1), SEQ ID NO: 79 (VH-CDR2) and SEQ ID NO: 80 (VH-CDR3), and SEQ ID NO: 82 (VL-CDR1), SEQ ID NO: 83 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 87 (VL-CDR3);

25) VH region comprising the amino acid sequence of SEQ ID NO: 163 (VH-CDR1), SEQ ID NO: 271 (VH-CDR2) and SEQ ID NO: 165 (VH-CDR3), and SEQ ID NO: 167 (VL-CDR1), SEQ ID NO: 168 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 169 (VL-CDR3);

26) VH region comprising the amino acid sequence of SEQ ID NO: 177 (VH-CDR1), SEQ ID NO: 178 (VH-CDR2) and SEQ ID NO: 179 (VH-CDR3), and SEQ ID NO: 181 (VL-CDR1), SEQ ID NO: 182 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 183 (VL-CDR3);

27) VH region comprising the amino acid sequence of SEQ ID NO: 187 (VH-CDR1), SEQ ID NO: 188 (VH-CDR2) and SEQ ID NO: 189 (VH-CDR3), and SEQ ID NO: 191 (VL-CDR1), SEQ ID NO: 192 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 193 (VL-CDR3);

28) VH region comprising the amino acid sequence of SEQ ID NO: 177 (VH-CDR1), SEQ ID NO: 178 (VH-CDR2) and SEQ ID NO: 179 (VH-CDR3), and SEQ ID NO: 181 (VL-CDR1), SEQ ID NO: 182 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 207 (VL-CDR3);

29) VH region comprising the amino acid sequence of SEQ ID NO: 177 (VH-CDR1), SEQ ID NO: 178 (VH-CDR2) and SEQ ID NO: 211 (VH-CDR3), and SEQ ID NO: 181 (VL-CDR1), SEQ ID NO: 182 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 207 (VL-CDR3);

30) VH region comprising the amino acid sequence of SEQ ID NO: 306 (VH-CDR1), SEQ ID NO: 307 (VH-CDR2) and SEQ ID NO: 308 (VH-CDR3), and SEQ ID NO: 310 (VL-CDR1), SEQ ID NO: 311 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 312 (VL-CDR3);

31) VH region comprising the amino acid sequence of SEQ ID NO: 349 (VH-CDR1), SEQ ID NO: 350 (VH-CDR2) and SEQ ID NO: 351 (VH-CDR3), and SEQ ID NO: 353 (VL-CDR1), SEQ ID NO: 354 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 355 (VL-CDR3);

32) VH region comprising the amino acid sequence of SEQ ID NO: 359 (VH-CDR1), SEQ ID NO: 360 (VH-CDR2) and SEQ ID NO: 361 (VH-CDR3), and SEQ ID NO: 363 (VL-CDR1), SEQ ID NO: 364 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 365 (VL-CDR3);

33) VH region comprising the amino acid sequence of SEQ ID NO: 197 (VH-CDR1), SEQ ID NO: 198 (VH-CDR2) and SEQ ID NO: 199 (VH-CDR3), and SEQ ID NO: 201 (VL-CDR1), SEQ ID NO: 202 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 203 (VL-CDR3);

TNF:

34) VH region comprising the amino acid sequence of SEQ ID NO: 124 (VH-CDR1), SEQ ID NO: 125 (VH-CDR2) and SEQ ID NO: 126 (VH-CDR3), and SEQ ID NO: 127 (VL-CDR1), SEQ ID NO: 128 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 129 (VL-CDR3);

35) VH region comprising the amino acid sequence of SEQ ID NO: 280 (VH-CDR1), SEQ ID NO: 281 (VH-CDR2) and SEQ ID NO: 282 (VH-CDR3), and SEQ ID NO: 284 (VL-CDR1), SEQ ID NO: 285 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 286 (VL-CDR3);

36) VH region comprising the amino acid sequence of SEQ ID NO: 276 (VH-CDR1), SEQ ID NO: 277 (VH-CDR2) and SEQ ID NO: 278 (VH-CDR3), and SEQ ID NO: 288 (VL-CDR1), SEQ ID NO: 289 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 290 (VL-CDR3); And

CTLA-4:

37) VH region comprising the amino acid sequence of SEQ ID NO: 369 (VH-CDR1), SEQ ID NO: 370 (VH-CDR2) and SEQ ID NO: 371 (VH-CDR3), and SEQ ID NO: 373 (VL-CDR1), SEQ ID NO: 374 (VL-CDR2) and a VL region comprising the amino acid sequence of SEQ ID NO: 375 (VL-CDR3).
The method of claim 2,

The X or Y is a variable heavy chain region or a variable light chain region in any one of the variable regions selected from the following group, a multi-specific fusion protein:

PD-L1:

1) the heavy chain variable region of SEQ ID NO: 4 (VH) and the light chain variable region of SEQ ID NO: 8 (VL);

2) the heavy chain variable region of SEQ ID NO: 158 (VH) and the light chain variable region of SEQ ID NO: 170 (VL);

3) the heavy chain variable region of SEQ ID NO: 158 (VH) and the light chain variable region of SEQ ID NO: 313 (VL);

4) the heavy chain variable region of SEQ ID NO: 329 (VH) and the light chain variable region of SEQ ID NO: 333 (VL);

5) the heavy chain variable region of SEQ ID NO: 329 (VH) and the light chain variable region of SEQ ID NO: 376 (VL);

HER2:

6) the heavy chain variable region of SEQ ID NO: 51 (VH) and the light chain variable region of SEQ ID NO: 52 (VL);

7) the heavy chain variable region of SEQ ID NO: 275 (VH) and the light chain variable region of SEQ ID NO: 287 (VL);

CD19:

8) the heavy chain variable region of SEQ ID NO: 139 (VH) and the light chain variable region of SEQ ID NO: 151 (VL);

9) the heavy chain variable region of SEQ ID NO: 61 (VH) and the light chain variable region of SEQ ID NO: 65 (VL);

CD20:

10) the heavy chain variable region of SEQ ID NO: 222 (VH) and the light chain variable region of SEQ ID NO: 226 (VL);

11) the heavy chain variable region of SEQ ID NO: 435 (VH) and the light chain variable region of SEQ ID NO: 440 (VL);

EGFR:

12) the heavy chain variable region of SEQ ID NO: 233 (VH) and the light chain variable region of SEQ ID NO: 237 (VL);

13) the heavy chain variable region of SEQ ID NO: 244 (VH) and the light chain variable region of SEQ ID NO: 248 (VL);

14) the heavy chain variable region of SEQ ID NO: 255 (VH) and the light chain variable region of SEQ ID NO: 259 (VL);

CD3:

15) the heavy chain variable region of SEQ ID NO: 325 (VH) and the light chain variable region of SEQ ID NO: 337 (VL);

16) the heavy chain variable region of SEQ ID NO: 329 (VH) and the light chain variable region of SEQ ID NO: 376 (VL);

17) the heavy chain variable region of SEQ ID NO: 162 (VH) and the light chain variable region of SEQ ID NO: 166 (VL);

18) the heavy chain variable region of SEQ ID NO: 395 (VH) and the light chain variable region of SEQ ID NO: 401 (VL);

19) the heavy chain variable region of SEQ ID NO: 406 (VH) and the light chain variable region of SEQ ID NO: 409 (VL);

20) the heavy chain variable region of SEQ ID NO: 143 (VH) and the light chain variable region of SEQ ID NO: 147 (VL);

21) the heavy chain variable region of SEQ ID NO: 69 (VH) and the light chain variable region of SEQ ID NO: 73 (VL);

22) the heavy chain variable region of SEQ ID NO: 77 (VH) and the light chain variable region of SEQ ID NO: 81 (VL);

23) the heavy chain variable region of SEQ ID NO: 294 (VH) and the light chain variable region of SEQ ID NO: 298 (VL);

24) the heavy chain variable region of SEQ ID NO: 85 (VH) and the light chain variable region of SEQ ID NO: 86 (VL);

25) the heavy chain variable region of SEQ ID NO: 176 (VH) and the light chain variable region of SEQ ID NO: 180 (VL);

26) the heavy chain variable region of SEQ ID NO: 186 (VH) and the light chain variable region of SEQ ID NO: 190 (VL);

27) the heavy chain variable region of SEQ ID NO: 176 (VH) and the light chain variable region of SEQ ID NO: 206 (VL);

28) the heavy chain variable region of SEQ ID NO: 210 (VH) and the light chain variable region of SEQ ID NO: 212 (VL);

29) the heavy chain variable region of SEQ ID NO: 215 (VH) and the light chain variable region of SEQ ID NO: 216 (VL);

30) the heavy chain variable region of SEQ ID NO: 305 (VH) and the light chain variable region of SEQ ID NO: 309 (VL);

31) the heavy chain variable region of SEQ ID NO: 348 (VH) and the light chain variable region of SEQ ID NO: 352 (VL);

32) the heavy chain variable region of SEQ ID NO: 358 (VH) and the light chain variable region of SEQ ID NO: 362 (VL);

33) the heavy chain variable region of SEQ ID NO: 196 (VH) and the light chain variable region of SEQ ID NO: 200 (VL);

TNF:

34) the heavy chain variable region of SEQ ID NO: 53 (VH) and the light chain variable region of SEQ ID NO: 54 (VL);

35) the heavy chain variable region of SEQ ID NO: 279 (VH) and the light chain variable region of SEQ ID NO: 283 (VL);

36) the heavy chain variable region of SEQ ID NO: 275 (VH) and the light chain variable region of SEQ ID NO: 287 (VL); And

CTLA-4:

37) The heavy chain variable region of SEQ ID NO: 368 (VH) and the light chain variable region of SEQ ID NO: 372 (VL).
The method of claim 2,

At least one of the X and Y will further comprise CH3, a multispecific fusion protein.
The method of claim 2,

The A and B specifically bind to a first antigen selected from the group consisting of PD-L1, EGFR, CD20, HER2, TNF, CD19, CD3 and CTLA4,

Fv formed by binding X and Y specifically binds to a second antigen selected from the group consisting of PD-L1, EGFR, CD20, HER2, TNF, CD19, CD3 and CTLA4,

The Fv formed of A and B and X and Y is characterized in that it does not bind to the same antigen,

Multispecific fusion protein.
A polynucleotide encoding the above structural formula (I), (I'), (I''), (II), (II') or (II'').
A vector comprising the polynucleotide of claim 20.
A transformed cell into which the vector of claim 21 has been introduced.
A pharmaceutical composition for the treatment or prevention of cancer comprising the multispecific fusion protein of any one of claims 1 to 19 as an active ingredient.
The method of claim 23,

The cancer is that any one protein selected from the group consisting of PD-L1, EGFR, and HER2 is overexpressed, a pharmaceutical composition for cancer treatment or prevention.
The method of claim 23,

The cancer is gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myelogenous leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer and lymphoma. Any one selected from the group, a pharmaceutical composition for the treatment or prevention of cancer.
The method of claim 23,

In addition, a pharmaceutical composition for the treatment or prevention of cancer that further comprises an anticancer agent.
Use of the multispecific fusion protein of any one of claims 1 to 19 for the treatment of cancer.
Use of the multispecific fusion protein of any one of claims 1 to 19 for use as a cell engager.
A method of treating cancer comprising administering the multispecific fusion protein of any one of claims 1 to 19 to an individual.