MODIFIED FC FRAGMENT, ANTIBODY COMPRISING SAME, AND APPLICATION THEREOF

FIELD OF THE INVENTION

The present disclosure relates to the field of antibodies. Specifically, the present disclosure relates to modified Fc fragments and antibodies containing them.

BACKGROUND OF THE INVENTION

Human natural antibodies, such as IgG1, IgG2, IgG3 and IgG4, have the ability to bind to FcγR. Human FcγR is divided into FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), wherein each type of the receptor is correspondingly expressed on the surface of different monocytes, and each type of the receptor is further divided into a, b, c and other subtypes. Through the binding of its own Fc to FcγR, natural antibodies produce the following immunological effect functions: antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-mediated cytotoxicity (CDC), and the like. In the process of antibody drug research, for some types of antibodies, it is necessary to reduce the ability to bind to FcγR in order to reduce the production of ADCC, ADCP and CDC, thereby reducing the toxicity and side effects of the antibody. In some special cases, such as anti-CD3 antibodies, especially multifunctional antibodies, can target both tumor cells and T lymphocytes expressed by CD3, and it is necessary to remove the binding to FcγR as much as possible in order to reduce the toxic and side effect caused by the release of a large number of cytokines produced by the non-specific activation of T cells.

The multifunctional antibody is an antibody or antibody-like molecule with multiple different binding specificities. The multifunctional antibody can be widely used in biomedicine, particularly in immunotherapy against tumors. Currently, a focus of immunotherapy research is how to use multifunctional antibody-mediated cytotoxicity to kill cells of interest. Multifunctional antibodies can be designed to target both tumor cells and effector cells, and simultaneously stimulate effector cells to kill tumor cells.

Multifunctional antibodies can be prepared by methods such as chemical engineering, cell engineering, and genetic engineering. The advantage of genetic engineering is that antibodies can be easily engineered to design and produce many multifunctional antibody fragments with different formats, including dimers, tanderm ScFv and single-chain dimers and the derivatives thereof (see Jin and Zhu, “The design and engineering of IgG-Like bispecific antibodies”, RE Kontermann (eds), Bispecific antibodies). These multifunctional antibodies do not have an IgG Fc domain, and thus their ability to penetrate into tumors is increased due to the small size; however, the multifunctional antibodies have a relatively short half-life in the body and lack ADCC effect, which is related to the constant region of antibodies.

Currently, there are some technologies of Fc modification that can reduce the binding ability of Fc to FcγR, for example: (1) as mentioned in “Curr Opin Biotechnol. 2011 December; 22(6):858-67. Bypassing glycosylation engineering aglycosylated fulllength IgG antibodies for human therapy”, the glycosylation of Fc can be effectively removed by mutating the asparagine at position 297 (N297) of human IgG1 Fc, thereby reducing the binding to FcγR; (2) as mentioned in the patent application “WO2009100309A2”, the amino acids at position 234 (leucine L234), position 235 (leucine L235) and position 331 (proline P331) of the Fc of human IgG1 were mutated to phenylalanine, glutamine and serine (L234F/L235E/P331S) respectively, thereby reducing the binding to FcγR; (3) as mentioned in the patent application “US20130078249A1”, the amino acids at position 234 (leucine L234), position 235 (leucine L235) and position 331 (proline P329) of the Fc of human IgG1 were mutated to alanine, alanine and glycine (L234A/L235A/P329G) respectively, thereby reducing the binding to FcγR; (4) as mentioned in “Protein Eng Des Sel. 2016 October; 29(10):457-466. Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions”, the leucine at position 234 (L234) and the leucine at position 235 (L235) of the Fc of human IgG1 were mutated to alanine and alanine (L234A/L235A) respectively, thereby reducing the ability to bind to FcγR; (5) as mentioned in the patent application “WO2011066501A1”, the amino acids at positions 234 (valine V234), 237 (glycine G237), 238 (proline P238), 268 (histidine H268), 309 (valine V309), 330 (alanine A330) and 331 (proline P331) of the Fc of human IgG2 were mutated to alanine, alanine, serine, alanine, leucine, serine and serine (V234A/G237A/P238S/H268A/V309L/A330S/P331S) respectively, thereby reducing the ability to bind to FcγR.

However, when applied to the structure of multifunctional antibodies, the above-mentioned Fc modification technology cannot completely eliminate the non-specific activation of T cells by anti-CD3 antibodies (such as L234F/L235E/P331S mutation, L234A/L235A/P329G mutation and L234A/L235A mutation in human IgG1), and modification at certain sites may lead to poor stability of the antibody (for example, the mutation of asparagine at position 297 of human IgG1 may remove glycosylation). In addition, as mentioned in “J Immunol 2003 170:3134-3138; Human IgG2 can form covalent dimers”, human IgG2 is prone to dimerization to form a tetravalent complex with a molecular weight of 3001(D, and the amino acid mutations at up to 7 positions in human IgG2 (such as V234A/G237A/P238S/H268A/V309L/A3305/P3315) may increase the risk of immunogenicity.

In order to solve the above problems, it is necessary to reduce the binding ability of Fc to FcγR to remove non-specific activation of T cells (such as by anti-CD3 antibodies), and it is also necessary to avoid the deterioration of antibody stability and the increase of immunogenicity. The present disclosure provides a new modification method of Fc, wherein the entire CH2 domain in the Fc of human IgG1 is replaced with the CH2 domain of human IgG2, and the amino acid residue of said domain is mutated, which can significantly reduce the binding ability of antibodies to FcγR, eliminate the non-specific activation of T cells by antibodies (such as anti-CD3 antibodies) more effectively, and maintain good stability of antibodies.

SUMMARY OF THE INVENTION

The present disclosure provides a method of Fc modification, wherein the entire CH2 domain of the constant region of human IgG1 is replaced with the CH2 domain of human IgG2, and preferably several amino acid residues in the replaced CH2 domain are substituted.

The residue substitution of CH2 can effectively reduce the binding ability of the antibody to FcγR. When one binding site of the antibody is CD3, the replacement of CH2 can also significantly reduce the non-specific activation of T cells by the antibody.

The present disclosure provides three kinds of multifunctional antibody and method for preparing thereof, and the specific structures of the multifunctional antibody are shown in FIGS. 1 to 3.

Specifically, the present disclosure relates to the following aspects:

Item 1. A polypeptide comprising or consisting of a modified Fc fragment, wherein the Fc fragment is derived from human IgG1, and a constant region CH2 domain of the Fc fragment is replaced with a CH2 domain of human IgG2, and wherein the CH2 domain of human IgG2 is shown as SEQ ID NO:94.

Item 2. The polypeptide of item 1, wherein the CH2 domain of the Fc fragment further comprises a mutation position selected from the group consisting of C229, D265, D270 or any combination thereof, according to Kabat numbering.

Item 3. The polypeptide of item 2, wherein the mutation is a mutation at position C229, preferably the mutation is selected from the group consisting of C229A, C229G, C229P, C229S, C229V, C229L, C229I, C229T, C229M, C229N, C229Q, C229D, C229E, C229K, C229R, C229F, C229Y, C229W or C229H, preferably C229S, C229A, C229G and C229P.

Item 4. The polypeptide of item 2, wherein the mutation is a mutation at position D265, preferably the mutation is selected from the group consisting of D265A, D265G, D265P, D265S, D265V, D265L, D265I, D265T, D265M, D265N, D265Q, D265E, D265K, D265R, D265F, D265Y, D265W or D265H, preferably D265A.

Item 5. The polypeptide of item 2, wherein the mutation is a mutation at position D270, preferably the mutation is selected from the group consisting of D270A, D270G, D270P, D270S, D270V, D270L, D270I, D270T, D270M, D270N, D270Q, D270E, D270K, D270R, D270F, D270Y, D270W or D270H, preferably D270A.

Item 6. The polypeptide of any one of items 2-5, wherein the mutation is a combination mutation of any two of positions C229, D265 and D270, or a combination mutation of positions C229, D265 and D270, preferably a combination mutation of positions D265 and D270.

Item 7. The polypeptide of any one of items 2-5, wherein the CH2 domain of the Fc fragment further comprises a mutation selected from the group consisting of G327, T339 or a combination thereof.

Item 8. The polypeptide of item 6, wherein the mutation is selected from G327A, G327V, G327L, G327I and/or T339A.

Item 9. The polypeptide of any one of items 2-8, wherein the mutation is selected from C229P/G327A, C229P/T339A, D270A/G327A, D270A/T339A, D270A/G327A/T339A or C229P/D265A/D270A.

Item 10. The polypeptide of any one of items 2-5, wherein the sequence of the CH2 domain of the Fc fragment is selected from the group consisting of SEQ ID NOs: 94 to 101 and 122 to 176.

Item 11. An antibody or antigen-binding fragment thereof comprising the polypeptide of any one of items 1-10, wherein the antibody is selected from a monospecific antibody, a multispecific antibody, more preferably a bispecific antibody; preferably, the antigen binding fragment is selected from Fab, Fab′, F(ab′)₂, Fd, Fv, dAb, Fab/c, a complementary determining region (CDR) fragment, a single-chain antibody (eg, scFv), a diabody or a domain antibody.

Item 12. The antibody of item 11, wherein the antibody specifically binds to an antigen selected from the group consisting of a tumor antigen, a viral or bacterial antigen, an endotoxin, and an immune antigen; preferably, the tumor antigen is selected from PD-L1 (preferably as shown in SEQ ID NO: 118), B7-H3, SLAMF7 (preferably as shown in SEQ ID NO: 119), CD38 (preferably as shown in SEQ ID NO: 116), EpCAM, CEA (preferably as shown in SEQ ID NO: 120), CD19 and BCMA (preferably as shown in SEQ ID NO: 117); the immune antigen is selected from CD3 (preferably as shown in SEQ ID NO: 121), CD16A, CD47 and NKG2D.

Item 13. The antibody of item 12, which is an asymmetric bispecific antibody comprising a light chain, a heavy chain and a fusion peptide 1, wherein the fusion peptide 1 comprises a scFv and an Fc fragment, the antibody has a light chain-heavy chain pair, and a heavy chain-fusion peptide 1 pair, and each pair forms an interchain disulfide bond; the light chain-heavy chain pair targets a tumor antigen, and the ScFv in the fusion peptide 1 targets an immune cell antigen.

Item 14. The antibody of item 12, which is an asymmetric trivalent bispecific antibody comprising two light chains, one heavy chain and one fusion peptide 2, and having a light chain-heavy chain pair, a light chain-fusion peptide 2 pair, and a heavy chain-fusion peptide 2 pair, wherein each pair forms an interchain disulfide bond; the fusion peptide 2 includes a heavy chain variable region VH, a first constant region of heavy chain CH1, ScFv and Fc, wherein the ScFv is located between CH1 and Fc and is linked by a linker, the light chain-heavy chain pair targets a tumor antigen, the pair of VH-CH1 in the fusion peptide 2 and light chain targets the same tumor antigen, and the ScFv targets an immune cell antigen.

Item 15. The antibody of item 12, which is an asymmetric trivalent bispecific antibody comprising a fusion heavy chain, a cross light chain, a heavy chain and a light chain, and having a light chain-heavy chain pair, a light chain-fusion heavy chain pair, a cross light chain-fusion heavy chain pair, and a fusion heavy chain-heavy chain pair, wherein each pair forms an interchain disulfide bond;the light chain includes a first light chain variable region VLm and a light chain constant region CL;the fusion heavy chain includes a first heavy chain variable region VHm, a first constant region of heavy chain CH1, a second heavy chain variable region VHs, a light chain constant region CL and Fc, wherein the VHs and CL are linked by a linker to form a peptide “VHs-linker-CL”, and wherein the “VHs-linker-CL” is located between CH1 and Fc and is linked by a linker/hinge;the cross light chain comprises a second light chain variable region VLs and CH1; VLs and CH1 are linked by a linker; the VLm-VHm pair targets a tumor antigen, and the VLs-VHs pair targets an immune cell antigen.

Item 16. The antibody of any one of items 13-15, has two different CH3, and the two CH3 are paired in a form of “knob-in-hole” or/and “salt bridge” to form a heterodimerization, preferably the sequence of CH3 domain is shown in SEQ ID NOs: 102 to 115.

Item 17. The antibody of any one of items 13-16, wherein the light chain-heavy chain pair or the VLm-VHm pair is selected from the group consisting of:

(1) SEQ ID NO: 12 and SEQ ID NO: 11 which target a tumor antigen B7-H3;

(2) SEQ ID NO: 14 and SEQ ID NO: 13 which target a tumor antigen B7-H3;

(3) SEQ ID NO: 16 and SEQ ID NO: 15 which target a tumor antigen CD38;

(4) SEQ ID NO: 18 and SEQ ID NO: 17 which target a tumor antigen CD38;

(5) SEQ ID NO: 20 and SEQ ID NO: 19 which target a tumor antigen CD38;

(6) SEQ ID NO: 22 and SEQ ID NO: 21 which target a tumor antigen EpCAM;

(7) SEQ ID NO: 24 and SEQ ID NO: 23 which target a tumor antigen EpCAM;

(8) SEQ ID NO: 26 and SEQ ID NO: 25 which target a tumor antigen BCMA;

(9) SEQ ID NO: 28 and SEQ ID NO: 27 which target a tumor antigen BCMA;

(10) SEQ ID NO: 30 and SEQ ID NO: 29 which target a tumor antigen BCMA;

(11) SEQ ID NO: 32 and SEQ ID NO: 31 which target a tumor antigen PD-L1;

(12) SEQ ID NO: 34 and SEQ ID NO: 33 which target a tumor antigen PD-L1;

(13) SEQ ID NO: 36 and SEQ ID NO: 35 which target a tumor antigen PD-L1;

(14) SEQ ID NO: 38 and SEQ ID NO: 37 which target a tumor antigen CD19;

(15) SEQ ID NO: 40 and SEQ ID NO: 39 which target a tumor antigen SLAMF7;

(16) SEQ ID NO: 42 and SEQ ID NO: 41 which target a tumor antigen CEA;

(17) SEQ ID NO: 2 and SEQ ID NO: 1 which target an immune antigen CD3;

(18) SEQ ID NO: 4 and SEQ ID NO: 3 which target an immune antigen CD3;

(19) SEQ ID NO: 6 and SEQ ID NO: 5 which target an immune antigen CD3;

(20) SEQ ID NO: 8 and SEQ ID NO: 7 which target an immune antigen CD3; and

(21) SEQ ID NO: 10 and SEQ ID NO: 9 which target an immune antigen CD3.

Item 18. The antibody of any one of items 13-17, wherein the antibody is selected from the group consisting of:

(1) PDL1-M1-G2, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 69, SEQ ID NO: 94 and SEQ ID NO: 109;the heavy chain comprises or consists of SEQ ID NO: 31, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 94 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 32 and SEQ ID NO: 75;

(2) PDL1-M1-SG2, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 69, SEQ ID NO: 95 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 31, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 95 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 32 and SEQ ID NO: 75;

(3) CD38-M1-G2, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 71, SEQ ID NO: 94 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 15, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 94 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 16 and SEQ ID NO: 75;

(4) CD38-M1-SG2, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 71, SEQ ID NO: 95 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 15, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 95 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 16 and SEQ ID NO: 75;

(5) CD38-M1-SG2-1, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 71, SEQ ID NO: 95 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 19, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 95 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 20 and SEQ ID NO: 75;

(6) CD38-M1-G2-3, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 71, SEQ ID NO: 94 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 19, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 94 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 20 and SEQ ID NO: 75;

(7) CD38-M1-SG2-2, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 71, SEQ ID NO: 95 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 17, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 95 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 18 and SEQ ID NO: 75;

(8) CD38-M1-G2-2, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 71, SEQ ID NO: 94 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 17, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 94 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 18 and SEQ ID NO: 75;

(9) CD38-M1-G2-1, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 9, SEQ ID NO: 54, SEQ ID NO: 10, SEQ ID NO: 68, SEQ ID NO: 94 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 15, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 94 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 16 and SEQ ID NO: 75;

(10) CD38-M1-SG2-3, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 9, SEQ ID NO: 54, SEQ ID NO: 10, SEQ ID NO: 68, SEQ ID NO: 95 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 15, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 95 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 16 and SEQ ID NO: 75;

(11) CD38-M1-AG2, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 9, SEQ ID NO: 54, SEQ ID NO: 10, SEQ ID NO: 68, SEQ ID NO: 96 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 15, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 96 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 16 and SEQ ID NO: 75;

(12) CD38-M1-GG2, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 9, SEQ ID NO: 54, SEQ ID NO: 10, SEQ ID NO: 68, SEQ ID NO: 97 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 15, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 97 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 16 and SEQ ID NO: 75;

(13) CD38-M1-PG2, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 9, SEQ ID NO: 54, SEQ ID NO: 10, SEQ ID NO: 68, SEQ ID NO: 98 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 15, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 98 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 16 and SEQ ID NO: 75;

(14) CD38-M1-DG2, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 9, SEQ ID NO: 54, SEQ ID NO: 10, SEQ ID NO: 68, SEQ ID NO: 99 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 15, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 99 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 16 and SEQ ID NO: 75;

(15) CD38-M1-G2D, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 9, SEQ ID NO: 54, SEQ ID NO: 10, SEQ ID NO: 68, SEQ ID NO: 100 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 15, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 100 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 16 and SEQ ID NO: 75;

(16) CD38-M1-DG2D, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 9, SEQ ID NO: 54, SEQ ID NO: 10, SEQ ID NO: 68, SEQ ID NO: 101 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 15, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 101 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 16 and SEQ ID NO: 75;

(17) CD38-M1-G2D-1, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 71, SEQ ID NO: 100 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 17, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 100 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 18 and SEQ ID NO: 75;

(18) M1IC-SG2, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 9, SEQ ID NO: 54, SEQ ID NO: 10, SEQ ID NO: 68, SEQ ID NO: 95 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 95 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(19) M1IC-G2, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 9, SEQ ID NO: 54, SEQ ID NO: 10, SEQ ID NO: 68, SEQ ID NO: 94 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 94 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(20) M1IC-AG2, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 9, SEQ ID NO: 54, SEQ ID NO: 10, SEQ ID NO: 68, SEQ ID NO: 96 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 96 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(21) M1IC-GG2, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 9, SEQ ID NO: 54, SEQ ID NO: 10, SEQ ID NO: 68, SEQ ID NO: 97 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 97 and SEQ ID NO: 108;the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(22) M1IC-PG2, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 9, SEQ ID NO: 54, SEQ ID NO: 10, SEQ ID NO: 68, SEQ ID NO: 98 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 98 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(23) M1IC-DG2, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 9, SEQ ID NO: 54, SEQ ID NO: 10, SEQ ID NO: 68, SEQ ID NO: 99 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 99 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(24) M1IC-G2D, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 9, SEQ ID NO: 54, SEQ ID NO: 10, SEQ ID NO: 68, SEQ ID NO: 100 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 100 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(25) M1IC-DG2D, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 9, SEQ ID NO: 54, SEQ ID NO: 10, SEQ ID NO: 68, SEQ ID NO: 101 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 101 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(26) M1IC-G2-1, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 94 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 94 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(27) M1IC-SG2-1, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 95 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 95 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(28) M1IC-AG2-1, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 96 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 96 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(29) M1IC-GG2-1, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 97 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 97 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(30) M1IC-PG2-1, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 98 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 98 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(31) M1IC-DG2-1, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 99 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 99 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(32) M1IC-G2D-1, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 100 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 100 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(33) M1IC-DG2D-1, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 101 and SEQ ID NO: 109; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 101 and SEQ ID NO: 108; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(34) M1IC-DG2D-1A, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 101 and SEQ ID NO: 107; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 101 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(35) M1IC-DG2D-1B, which comprises or consists of a fusion peptide 1, a heavy chain and a light chain;wherein the fusion peptide 1 comprises or consists of SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 101 and SEQ ID NO: 115; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 101 and SEQ ID NO: 114; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(36) BCMA-M2-G2, which comprises or consists of a fusion peptide 2, a heavy chain and a light chain;wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 29, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 94 and SEQ ID NO: 107; the heavy chain comprises or consists of SEQ ID NO: 29, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 94 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 30 and SEQ ID NO: 75;

(37) BCMA-M2-SG2, which comprises or consists of a fusion peptide 2, a heavy chain and a light chain;wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 29, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 95 and SEQ ID NO: 107; the heavy chain comprises or consists of SEQ ID NO: 29, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 95 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 30 and SEQ ID NO: 75;

(38) BCMA-M2-AG2, which comprises or consists of a fusion peptide 2, a heavy chain and a light chain;wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 29, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 96 and SEQ ID NO: 107; the heavy chain comprises or consists of SEQ ID NO: 29, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 96 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 30 and SEQ ID NO: 75;

(39) BCMA-M2-GG2, which comprises or consists of a fusion peptide 2, a heavy chain and a light chain;wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 29, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 97 and SEQ ID NO: 107; the heavy chain comprises or consists of SEQ ID NO: 29, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 97 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 30 and SEQ ID NO: 75;

(40) BCMA-M2-PG2, which comprises or consists of a fusion peptide 2, a heavy chain and a light chain;wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 29, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 98 and SEQ ID NO: 107; the heavy chain comprises or consists of SEQ ID NO: 29, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 98 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 30 and SEQ ID NO: 75;

(41) BCMA-M2-DG2, which comprises or consists of a fusion peptide 2, a heavy chain and a light chain;wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 29, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 99 and SEQ ID NO: 107; the heavy chain comprises or consists of SEQ ID NO: 29, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 99 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 30 and SEQ ID NO: 75;

(42) BCMA-M2-G2D, which comprises or consists of a fusion peptide 2, a heavy chain and a light chain;wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 29, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 100 and SEQ ID NO: 107; the heavy chain comprises or consists of SEQ ID NO: 29, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 100 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 30 and SEQ ID NO: 75;

(43) BCMA-M2-DG2D, which comprises or consists of a fusion peptide 2, a heavy chain and a light chain;wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 29, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 101 and SEQ ID NO: 107; the heavy chain comprises or consists of SEQ ID NO: 29, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 101 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 30 and SEQ ID NO: 75;

(44) M2IC-G2, which comprises or consists of a fusion peptide 2, a heavy chain and a light chain;wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 94 and SEQ ID NO: 107; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 94 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(45) M2IC-SG2, which comprises or consists of a fusion peptide 2, a heavy chain and a light chain;wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 95 and SEQ ID NO: 107; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 95 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(46) M2IC-AG2, which comprises or consists of a fusion peptide 2, a heavy chain and a light chain;wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 96 and SEQ ID NO: 107; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 96 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(47) M2IC-GG2, which comprises or consists of a fusion peptide 2, a heavy chain and a light chain;wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 97 and SEQ ID NO: 107; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 97 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(48) M2IC-PG2, which comprises or consists of a fusion peptide 2, a heavy chain and a light chain;wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 98 and SEQ ID NO: 107; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 98 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(49) M2IC-DG2, which comprises or consists of a fusion peptide 2, a heavy chain and a light chain;wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 99 and SEQ ID NO: 107; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 99 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(50) M2IC-G2D, which comprises or consists of a fusion peptide 2, a heavy chain and a light chain;wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 100 and SEQ ID NO: 107; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 100 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(51) M2IC-DG2D, which comprises or consists of a fusion peptide 2, a heavy chain and a light chain;wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 101 and SEQ ID NO: 107; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 101 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(52) CEA-M3-G2, which comprises or consists of a fusion heavy chain, a cross light chain, a heavy chain and a light chain;wherein the fusion heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 94 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 94 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 42 and SEQ ID NO: 75;

(53) CEA-M3-SG2, which comprises or consists of a fusion heavy chain, a cross light chain, a heavy chain and a light chain;wherein the fusion heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 95 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 95 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 42 and SEQ ID NO: 75;

(54) CEA-M3-AG2, which comprises or consists of a fusion heavy chain, a cross light chain, a heavy chain and a light chain; wherein the fusion heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 96 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 96 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 42 and SEQ ID NO: 75;

(55) CEA-M3-GG2, which comprises or consists of a fusion heavy chain, a cross light chain, a heavy chain and a light chain;wherein the fusion heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 97 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 97 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 42 and SEQ ID NO: 75;

(56) CEA-M3-PG2, which comprises or consists of a fusion heavy chain, a cross light chain, a heavy chain and a light chain;wherein the fusion heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 98 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 98 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 42 and SEQ ID NO: 75;

(57) CEA-M3-DG2, which comprises or consists of a fusion heavy chain, a cross light chain, a heavy chain and a light chain;wherein the fusion heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 99 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82;the heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 99 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 42 and SEQ ID NO: 75;

(58) CEA-M3-G2D, which comprises or consists of a fusion heavy chain, a cross light chain, a heavy chain and a light chain;wherein the fusion heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 100 and SEQ ID NO: 107;the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 100 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 42 and SEQ ID NO: 75;

(59) CEA-M3-DG2D, which comprises or consists of a fusion heavy chain, a cross light chain, a heavy chain and a light chain;wherein the fusion heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 101 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 101 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 42 and SEQ ID NO: 75;

(60) M3IC-G2, which comprises or consists of a fusion heavy chain, a cross light chain, a heavy chain and a light chain;wherein the fusion heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 94 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 94 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(61) M3IC-SG2, which comprises or consists of a fusion heavy chain, a cross light chain, a heavy chain and a light chain;wherein the fusion heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 95 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 95 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(62) M3IC-AG2, which comprises or consists of a fusion heavy chain, a cross light chain, a heavy chain and a light chain;wherein the fusion heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 96 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 96 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(63) M3IC-GG2, which comprises or consists of a fusion heavy chain, a cross light chain, a heavy chain and a light chain;wherein the fusion heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 97 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 97 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(64) M3IC-PG2, which comprises or consists of a fusion heavy chain, a cross light chain, a heavy chain and a light chain;wherein the fusion heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 98 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 98 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(65) M3IC-DG2, which comprises or consists of a fusion heavy chain, a cross light chain, a heavy chain and a light chain;wherein the fusion heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 99 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 99 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(66) M3IC-G2D, which comprises or consists of a fusion heavy chain, a cross light chain, a heavy chain and a light chain;wherein the fusion heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 100 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 100 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(67) M3IC-DG2D, which comprises or consists of a fusion heavy chain, a cross light chain, a heavy chain and a light chain;wherein the fusion heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 101 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 101 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75;

(68) CD3mAb-G2, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 94 and SEQ ID NO: 102;

(69) CD3mAb-SG2, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 95 and SEQ ID NO: 102;

(70) CD3mAb-AG2, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 96 and SEQ ID NO: 102;

(71) CD3mAb-GG2, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 97 and SEQ ID NO: 102;

(72) CD3mAb-PG2, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 98 and SEQ ID NO: 102;

(73) CD3mAb-G2-C229L, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 128 and SEQ ID NO: 102;

(74) CD3mAb-G2-C229F, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 129 and SEQ ID NO: 102;

(75) CD3mAb-G2-C229R, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 130 and SEQ ID NO: 102;

(76) CD3mAb-G2-C229V, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 131 and SEQ ID NO: 102;

(77) CD3mAb-G2-C229Q, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 132 and SEQ ID NO: 102;

(78) CD3mAb-G2-C229K, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 133 and SEQ ID NO: 102;

(79) CD3mAb-G2-C229D, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 134 and SEQ ID NO: 102;

(80) CD3mAb-G2-C229I, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 135 and SEQ ID NO: 102;

(81) CD3mAb-G2-C229Y, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 136 and SEQ ID NO: 102;

(82) CD3mAb-G2-C229N, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 137 and SEQ ID NO: 102;

(83) CD3mAb-G2-C229M, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 138 and SEQ ID NO: 102;

(84) CD3mAb-G2-C229T, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 139 and SEQ ID NO: 102;

(85) CD3mAb-G2-C229H, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 140 and SEQ ID NO: 102;

(86) CD3mAb-G2-C229E, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 141 and SEQ ID NO: 102;

(87) CD3mAb-G2-C229W, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 142 and SEQ ID NO: 102;

(88) CD3mAb-G2-C229L, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 128 and SEQ ID NO: 102;

(89) CD3mAb-DG2, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 99 and SEQ ID NO: 102;

(90) CD3mAb-G2-D265P, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 143 and SEQ ID NO: 102;

(91) CD3mAb-G2-D265K, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 144 and SEQ ID NO: 102;

(92) CD3mAb-G2-D265S, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 145 and SEQ ID NO: 102;

(93) CD3mAb-G2-D265F, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 146 and SEQ ID NO: 102;

(94) CD3mAb-G2-D265R, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 147 and SEQ ID NO: 102;

(95) CD3mAb-G2-D265L, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 148 and SEQ ID NO: 102;

(96) CD3mAb-G2-D265G, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 149 and SEQ ID NO: 102;

(97) CD3mAb-G2-D265T, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 150 and SEQ ID NO: 102;

(98) CD3mAb-G2-D265Y, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 151 and SEQ ID NO: 102;

(99) CD3mAb-G2-D265W, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 152 and SEQ ID NO: 102;

(100) CD3mAb-G2-D265H, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 153 and SEQ ID NO: 102;

(101) CD3mAb-G2-D265V, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 154 and SEQ ID NO: 102;

(102) CD3mAb-G2-D265Q, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 155 and SEQ ID NO: 102;

(103) CD3mAb-G2-D265E, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 156 and SEQ ID NO: 102;

(104) CD3mAb-G2-D265M, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 157 and SEQ ID NO: 102;

(105) CD3mAb-G2-D265N, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 158 and SEQ ID NO: 102;

(106) CD3mAb-G2-D265I, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 159 and SEQ ID NO: 102;

(107) CD3mAb-G2D, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 100 and SEQ ID NO: 102;

(108) CD3mAb-G2-D270L, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 160 and SEQ ID NO: 102;

(109) CD3mAb-G2-D270R, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 161 and SEQ ID NO: 102;

(110) CD3mAb-G2-D270P, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 162 and SEQ ID NO: 102;

(111) CD3mAb-G2-D270G, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 163 and SEQ ID NO: 102;

(112) CD3mAb-G2-D270V, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 164 and SEQ ID NO: 102;

(113) CD3mAb-G2-D270H, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 165 and SEQ ID NO: 102;

(114) CD3mAb-G2-D270Y, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 166 and SEQ ID NO: 102;

(115) CD3mAb-G2-D270I, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 167 and SEQ ID NO: 102;

(116) CD3mAb-G2-D270E, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 168 and SEQ ID NO: 102;

(117) CD3mAb-G2-D270F, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 169 and SEQ ID NO: 102;

(118) CD3mAb-G2-D270K, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 170 and SEQ ID NO: 102;

(119) CD3mAb-G2-D270W, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 171 and SEQ ID NO: 102;

(120) CD3mAb-G2-D270S, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 172 and SEQ ID NO: 102;

(121) CD3mAb-G2-D270T, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 173 and SEQ ID NO: 102;

(122) CD3mAb-G2-D270Q, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 174 and SEQ ID NO: 102;

(123) CD3mAb-G2-D270M, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 175 and SEQ ID NO: 102;

(124) CD3mAb-G2-D270N, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 176 and SEQ ID NO: 102;

(125) CD3mAb-PG2-GA, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 122 and SEQ ID NO: 102;

(126) CD3mAb-PG2-TA, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 123 and SEQ ID NO: 102;

(127) CD3mAb-G2D-GA, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 124 and SEQ ID NO: 102;

(128) CD3mAb-G2D-TA, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 125 and SEQ ID NO: 102;

(129) CD3mAb-G2D-GATA, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 126 and SEQ ID NO: 102; or (130) CD3mAb-PDG2D, which comprises or consists of a light chain and a heavy chain, wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 127 and SEQ ID NO: 102.

Item 19. A conjugate of the antibody of any one of items 11-18, wherein the conjugate comprises substance A to which the antibody is coupled or fused, and the substance A is selected from therapeutic agents, prodrugs, proteins (such as enzymes), viruses, lipids, biological response modifiers (such as immunomodulators), PEG, hormones, oligonucleotides, diagnostic agents, cytotoxic agents, which can be drugs or toxins, ultrasound enhancers, non-radioactive labels, detectable labels, such as chemiluminescent labeled compounds (such as luminol, isoluminol, thermal acridinium esters, imidazoles, acridinium salts and oxalates), or fluorescent emitting metals (such as 152Eu, or lanthanide marker).

Item 20. A polynucleotide encoding the polypeptide of any one of items 1-10.

Item 21. A cell comprising the polynucleotide of item 20.

Item 22. A composition, preferably a pharmaceutical composition, which comprises the polypeptide of any one of items 1-10, or the antibody of any one of items 11-18.

Item 23. A method for delivering a polypeptide or an antibody to a mammalian (preferably human) subject without inducing antibody-dependent cytotoxicity, comprising administering the polypeptides of any one of items 1-10 or the antibody of any one of items 11-18 to the subject.

Item 24. Use of the polypeptide of any one of items 1-10 or the antibody of any one of items 11-18 in preparation of a drug that does not induce antibody-dependent cytotoxicity after administration to a mammalian (preferably human) subject.

TECHNICAL SOLUTION

The antibody has a Fc fragment of heavy chain constant region, wherein the Fc comprises a hinge, a CH2 and a CH3, and wherein the CH2 domain comprises one or more substitutions, which can significantly reduce the binding ability of Fc fragments to Fcγ receptors (FcγR) and reduce non-specific activation of T cells by the antibody (such as anti-CD3 antibody).

In certain aspects, the Fc of the antibody comprises a CH3 domain, and the CH3 domain comprises one or more substitutions that form knobs-into-holes structure pairing between two different CH3 domains.

After substitution at both CH2 and CH3 in the Fc fragment, the binding ability of the Fc fragment to the FcγR can be significantly reduced and the non-specific activation of T cells by the antibody (such as anti-CD3 antibodies) is reduced.

In certain aspects, the antibody has a light chain-heavy chain pair or a VLm-VHm pair and those pairs are specific for tumor antigens. In one aspect, the tumor antigen is selected from PD-L1, SLAMF7, B7-H3, CEA, CD38, EpCAM, CD19, BCMA and the like. In one aspect, the light chain-heavy chain pair or VLm-VHm pair is specific for proteins that are overexpressed on tumor cells compared to the corresponding non-tumor cells.

In certain aspects, the light chain-heavy chain pair or VLm-VHm pair of the antibody is specific for viruses or bacteria.In one aspect, the light chain-heavy chain pair or VLm-VHm pair is specific for endotoxin.

In some aspects, the antibody has a fusion peptide or light and heavy chain variable region pair (VLs-VHs pair), and the fusion peptide or light and heavy chain variable region pair are specific for immune cell surface antigens, and the surface antigens are CD3, CD16A , CD47, NKG2D, etc.

In certain aspects, the immune cell is selected from a T cell, a CIK cell, a NKT cell, a B cell, a monocyte, a macrophage, a neutrophil, a dendritic cell, a macrophage, a natural killer cell, an eosinophil, a basophil and a mast cell.

In certain aspects, compared to the wild-type antibody fragment, the heavy chain, the fusion heavy chain, and/or the Fc fragment of the fusion peptide comprise one or more substitutions which form a structure pairing of Knobs-into-holes between the heavy chain and the fusion peptide, or between the heavy chain and the fusion heavy chain. This pairing can significantly improve the heterodimer pairing efficiency of the heavy chain and the fusion peptide.

In certain aspects, the heavy chain, the fusion heavy chain, and/or the Fc fragment of the fusion peptide comprise one or more substitutions which form a pairing of salt-bridge between the heavy chain and the fusion peptide, or between the heavy chain and the fusion heavy chain. This pairing can significantly improve the heterodimer pairing efficiency of the heavy chain and the fusion peptide.

In certain aspects, the CH2 domain in the fusion peptide is located between the scFv fragment and the CH3 domain. In one aspect, the fusion peptide does not comprise a CH1 domain.

In certain aspects, there is a light chain constant region between the VH and hinge region of the fused heavy chain.

In certain aspects, the fusion heavy chain and the cross light chain are paired, and the cross light chain has a VL-CH1 structure.

In one embodiment, the present disclosure also provides a composition comprising the antibody in any one of the above embodiments. In one aspect, the composition further comprises a carrier, which is a pharmaceutically acceptable carrier.

In another embodiment, the present disclosure provides a complex which comprises the antibody in any one of the above embodiments that binds to one or more antigens.

The present disclosure further provides a method for preparing the antibody.

Definitions

It is to be noted that an indefinite quantity of “a” or “an” entity refers to one or more of that entity; for example, “a multifunctional antibody” shall be understood to represent one or more multifunctional antibodies. As such, the terms “a” (or “an”), “one or more,” and “at least one” defined indefinitely can be used interchangeably herein.

As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are all included within the definition of “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the post-expression modified products of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or may be produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It may be generated by any manner, including by chemical synthesis.

As used herein, the term “recombinant” as it pertains to polypeptides or polynucleotides refers to a form of the polypeptide or polynucleotide that does not exist naturally, a non-limiting example of which can be achieved by combining polynucleotides or polypeptides that would not normally occur together.

“Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, though preferably less than 25% identity, with one of the sequences of the present disclosure.

A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined by using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, which use the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Detailed information of these programs can be found at the following Internet address: http://www.ncbi nlm nih gov/blast/Blast.cgi, last accessed on May 21, 2008. Biologically equivalent polynucleotides are those having the above specified percent homology and encoding a polypeptide having the same or similar biological activity.

The term “encode” as it is applied to polynucleotides refers to a polynucleotide which “encodes” a polypeptide and which, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof. An antisense strand is a complement of such a nucleic acid, and an encoding sequence can be deduced therefrom.

As used herein, an “antibody” or “antigen-binding polypeptide” refers to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof. Thus the term “antibody” includes any protein or peptide containing a specific molecule, wherein the specific molecule comprises at least a portion of an immunoglobulin molecule having biological activity of binding to an antigen. Examples of such include, but are not limited to a complementary determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein.

The terms “antibody fragment” or “antigen-binding fragment”, as used herein, is a portion of an antibody, such as F(ab′)₂, F(ab)₂, Fab′, Fab, Fv, Fd, dAb, Fab/c, scFv and the like. Regardless of structure, an antibody fragment that binds to the same antigen is recognized as an intact antibody. The term “antibody fragment” includes aptamers, spiegelmers, and diabodies. The term “antibody fragment” also includes any synthetic or genetically engineered protein that acts like an antibody that can binds to a specific antigen to form a complex.

A “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins. In some aspects, the regions are connected with a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of a constant region and introduction of the linker. ScFv molecules are known in the art and are described, e.g., in U.S. Pat. No. 5,892,019.

The term antibody encompasses various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgD, IgE or IgY, respectively. The immunoglobulin subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc. are well characterized and functionally specific. Modified versions of each of these classes and isotypes are readily recognized by those skilled in the art in view of the present disclosure and, accordingly, are within the scope of the present disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons, and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. The four chains are typically joined by disulfide bonds in a “Y” configuration wherein the light chains support the heavy chains starting at the mouth of the “Y” and extend through the variable region.

Antibodies, antigen-binding polypeptides, variants or derivatives thereof in the present disclosure include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primatized antibodies, or chimeric antibodies, single chain antibodies, antigen-binding fragments, e.g., Fab, Fab′ and F(ab′)2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to LIGHT antibodies as disclosed herein) Immunoglobulin or antibody molecules of the disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class may be bound to either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide bonds or non-covalent bonds when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences extend from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into structural regions and functional homology regions. The terms “constant” and “variable” are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VL) and heavy (VH) chain determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. Generally, the number of the constant region domains increases as they become more distal from the antigen-binding site or amino-terminus of the antibody. The N-terminal portion is a variable region and the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.

As indicated above, the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain, or subset of the complementary determining regions (CDRs), of an antibody combine to form a variable region that defines a three dimensional antigen-binding site. This tetravalent antibody structure forms an antigen-binding site present at the end of each arm of the Y configuration. More specifically, the antigen-binding site is defined by three CDRs on each of the VH and VL chains (i.e. CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). In some examples, e.g., for certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, the intact immunoglobulin molecule may consist of heavy chains only, without light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993).

In naturally occurring antibodies, the six “complementary determining regions” or “CDRs” present in each antigen-binding domain are short, non-contiguous amino acid sequences that are specifically positioned to form the antigen-binding domain as it is assumed three dimensional configuration of the antibody is located in an aqueous environment. The other amino acids in the antigen-binding domains are referred to as “framework” regions and show less inter-molecular variability. The framework regions largely adopt a β-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β-sheet structure. Thus, framework regions act to form a scaffold that positions the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope of the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its homologous epitope. The amino acids comprising the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by those skilled in the art, since they have been precisely defined (see “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol., 196:901-917 (1987), which are incorporated herein by reference in their full text).

In the case where there are two or more definitions of a term which is used and/or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term “complementary determining region” (“CDR”) to describe the non-contiguous antigen-binding sites found within the variable region of both heavy and light chain polypeptides. This particular region has been described in the U.S. Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al., J. MoI. Biol. 196:901-917 (1987), which are incorporated herein by reference in their full text. According to definitions of Kabat and Chothia, the CDR includes overlapping amino acid residues or substructures of amino acid residues when compared with each other. Nevertheless, application of each definition of CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth in the table below as a comparison. The exact number of residues which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR if the variable region amino acid sequence of the antibody is provided.

TABLE 1

Definition

of antibody variable region

Kabat
Chothia

CDR-H1
31-35
26-32

CDR-H2
50-65
52-58

CDR-H3
95-102
95-102

CDR-L1
24-34
26-32

CDR-L2
50-56
50-52

CDR-L3
89-97
91-96

Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. Those skilled in the art can unambiguously assign this “Kabat numbering” system to any variable domain sequence, without depending on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

In addition to table above, the CDR regions as described by the Kabat number system are: CDR-H1 begins from the amino acid approximately at position 31 (i.e., approximately 9 residues after the first cysteine residue), includes approximately 5 to 7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins from the fifteenth residue after the end of CDR-H1, includes approximately 16 to 19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins from approximately the 33rd amino acid residue after the end of CDR-H2; includes 3 to 25 amino acids; and ends at the sequence W-G-X-G, where X is any amino acid. CDR-L1 begins from the residue approximately at position 24 (i.e., following a cysteine residue); includes approximately 10 to 17 residues; and ends at the next tryptophan residue. CDR-L2 begins from approximately the sixteenth residue after the end of CDR-L1 and includes approximately 7 residues. CDR-L3 begins from approximately the thirty-third residue after the end of CDR-L2 (i.e., following a cysteine residue); includes approximately 7 to 11 residues and ends at the sequence F or W-G-X-G, where X is any amino acid.

Antibodies disclosed herein may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region may be derived from condricthoid (e.g., from sharks).

As used herein, the term “heavy chain constant region” includes amino acid sequences derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in the present disclosure may comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the present disclosure comprises a polypeptide chain with a CH3 domain. Further, an antibody for use in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As set forth above, it will be understood by those skilled in the art that the heavy chain constant region may be modified such that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.

The heavy chain constant region of an antibody disclosed herein may be derived from different immunoglobulin molecules. For example, a heavy chain constant region of a polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another embodiment, a heavy chain constant region can comprise a hinge region derived, in part, from an IgG1 molecule and, in part, from an IgG3 molecule. In another embodiment, a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule.

As used herein, the term “light chain constant region” includes amino acid sequences derived from antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or constant lambda domain.

A “light chain-heavy chain pair” refers to the collection of a light chain and heavy chain that can form a dimer through a disulfide bond between the CL domain and the CH1 domain of the light chain.

As previously indicated, the subunit structures and three dimensional configuration of the constant regions of the various classes of immunoglobulin are well known. As used herein, the term “VH domain” includes the amino terminal variable domain of an immunoglobulin heavy chain and the term “CH1 domain” includes the first (mostly amino terminal) constant region of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino terminal of the hinge region of an immunoglobulin heavy chain molecule.

As used herein the term “CH2 domain” includes a portion of the heavy chain molecule that extends, e.g., from residue at about position 244 to residue at position 360 of an antibody according to conventional numbering system (residues at position 244 to 360, according to Kabat numbering system; and residues at position 231 to 340, according to EU numbering system; see Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983). The CH2 domain is unique because it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of an intact natural IgG molecule. It is also documented that the CH3 domain extends from the CH2 domain to the C-terminal of an IgG molecule and comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

As used herein the term “disulfide bond” includes the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a sulfydryl group that can form a disulfide bond or bridge with a second sulfydryl group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to positions 239 and 242 under the Kabat numbering system (position 226 or 229, under EU numbering system).

As used herein, the term “chimeric antibody” is intended to refer to any antibody wherein the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial or modified in accordance with the present disclosure) is obtained from a second species. In certain examples, the target binding region or site will be derived from a non-human source (e.g. mouse or primate) and the constant region is derived from human

As used herein, “percent humanization” is calculated by determining the number of framework amino acid differences (i.e., non-CDR difference) between the humanized domain and the germline domain, subtracting that number from the total number of amino acids, and then dividing that by the total number of amino acids and multiplying by 100.

The term “specifically binds” or “has specificity to” generally means that an antibody binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope, and when it binds to the epitope, the binding via its antigen-binding domain is easier than binding via a random, unrelated antigen epitope. The term “specificity” is used herein to determine the relative affinity of a certain antibody binding to a certain epitope. For example, antibody “A” may be deemed to have a higher specificity for a given antigen epitope than that of antibody “B,” or antibody “A” may be said to bind to antigen epitope “C” with a higher specificity than it has for related epitope “D.”

As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the subject is to prevent or slow down (alleviate) an undesired physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilizing (i.e., not worsening) state of disease, delaying or slowing disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival without treatment. Conditions in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

Multifunctional Antibody

Embodiments of the present disclosure provide a variety of multifunctional antibodies, which comprise two different antigen-binding polypeptide units. The antibody domain that binds to the antigen is Fab, or ScFv, or non-covalent pairs between the variable region of the heavy chain (VH) and the variable region of the light chain (VL). In particular, these multifunctional antibodies all have a Fc fragment of heavy chain constant region of antibody. Wherein the Fc contains: (1) a hinge, (2) a heavy chain second constant region (CH2), and/or a heavy chain third constant region (CH3). Both the hinge and CH3 are the corresponding domains of human IgG1 type, and the CH3 undergoes “knob-into-hole” mutation, and the CH2 is the corresponding CH2 domain of human IgG2 type.

Any of the antibodies or polypeptides described above may further include additional polypeptides, thereby constituting a fusion protein or fusion peptide, e.g., an encoded polypeptide as described herein, a signal peptide of the antibody constant region used to direct secretion, or other heterologous polypeptides as described herein.

It will also be understood by those skilled in the art that the antibodies as disclosed herein may be modified such that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived. For example, a polypeptide or amino acid sequence derived from a designated protein may be similar to the original sequence, e.g., having a certain percent identity to the original sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the original sequence.

Furthermore, nucleotide or amino acid substitutions, deletions, or insertions leading to conservative substitutions or changes at “non-essential” amino acid regions may be made. For example, a polypeptide or amino acid sequence derived from a designated protein may be identical to the original sequence except for one or more independent amino acid substitutions, insertions, or deletions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more independent amino acid substitutions, insertions, or deletions. In certain embodiments, a polypeptide or amino acid sequence derived from a designated protein has 1 to 5, 1 to 10, 1 to 15, or 1 to 20 independent amino acid substitutions, insertions, or deletions relative to the original sequence.

In other embodiments, the antigen-binding polypeptides of the present disclosure may contain conservative amino acid substitutions.

A “conservative amino acid substitution” is one in which the amino acid residue is substituted with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in an immunoglobulin polypeptide is preferably substituted with other amino acid residue from the same side chain family In another embodiment, a amino acid sequence can be substituted by a structurally similar amino acid sequence that differs in order and/or composition of side chain family members.

Non-limiting examples of conservative amino acid substitutions are provided in the table below, wherein a similarity score of 0 or higher indicates conservative substitution between two amino acids.

TABLE 2

Non-limiting list of conservative amino acid substitutions

C
G
P
S
A
T
D
E
N
Q
H
K
R
V
M
I
L
F
Y
W

W
−8
−7
−6
−2
−6
−5
−7
−7
−4
−5
−3
−3
2
−6
−4
−5
−2
0
0
17

Y
0
−5
−5
−3
−3
−3
−4
−4
−2
−4
0
−4
−5
−2
−2
−1
−1
7
10

F
−4
−5
−5
−3
−4
−3
−6
−5
−4
−5
−2
−5
−4
−1
0
1
2
9

L
−6
−4
−3
−3
−2
−2
−4
−3
−3
−2
−2
−3
−3
2
4
2
6

I
−2
−3
−2
−1
−1
0
−2
−2
−2
−2
−2
−2
−2
4
2
5

M
−5
−3
−2
−2
−1
−1
−3
−2
0
−1
−2
0
0
2
6

V
−2
−1
−1
−1
0
0
−2
−2
−2
−2
−2
−2
−2
4

R
−4
−3
0
0
−2
−1
−1
−1
0
1
2
3
6

K
−5
−2
−1
0
−1
0
0
0
1
1
0
5

H
−3
−2
0
−1
−1
−1
1
1
2
3
6

Q
−5
−1
0
−1
0
−1
2
2
1
4

N
−4
0
−1
1
0
0
2
1
2

E
−5
0
−1
0
0
0
3
4

D
−5
1
−1
0
0
0
4

T
−2
0
0
1
1
3

A
−2
1
1
1
2

S
0
1
1
1

P
−3
−1
6

G
−3
5

C
12

In some embodiments, the present disclosure provides an antibody conjugate, and the antibody may bind to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.

The antibodies may be conjugated or fused to a therapeutic agent, which may include a detectable label such as a radioactive label, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, a combination thereof and other such agents known in the art.

The antibody can be detectably labeled by coupling it to a chemiluminescent compound. The presence of a chemiluminescent-labeled antigen-binding polypeptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

The antibodies can also be detectably labeled using fluorescence emitting metals such as 152Eu, or other lanthanide series labels. These metals can be attached to the antibody by using metal chelating groups such as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various moieties to an antibody are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2^ndEd.), Robinson et al., (eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. (52:119-58 (1982)).

Methods of Preparing Antibodies

Methods of preparing antibodies are well known in the art and described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptides of the present disclosure are fully human. Fully human antibodies can be prepared using techniques described in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to prepare such antibodies are described in U.S. Pat. Nos: 6,150,584; 6,458,592; 6,420,140 which are incorporated by reference in their full text.

The binding specificity of antigen-binding polypeptides of the present disclosure can be determined by in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).

Alternatively, techniques described for the production of single-chain units (U.S. Pat. No. 4,694,778; Bird, Science 242:423-442 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 55:5879-5883 (1988); and Ward et al., Nature 334:544-554 (1989)) can be used to produce single-chain units of the present disclosure. Single-chain units are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single-chain fusion peptide. Techniques for producing functional Fv fragments in E. coli may also be used (Skerra et al., Science 242: 1038-1041 (1988)).

Examples of techniques which can be used to produce single-chain Fvs (scFvs) and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., Proc. Natl. Sci. USA 90:1995-1999 (1993); and Skerra et al., Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entities.

A humanized antibody is an antibody molecule that is derived from a non-human species antibody and binds the desired antigen, and the antibody molecule has one or more complementary determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. Often, framework residues in a human framework region will be changed by substitution with corresponding residues from a CDR donor antibody, preferably to improve, antigen-binding ability. These framework substitutions are identified by methods well known in the art, e.g., by modeling interactions between the CDR and framework residues to identify framework residues important for antigen-binding and sequence comparison in order to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entities) Antibodies can be humanized by using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication No. WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., Proc. Natl. Sci. USA 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332, which is incorporated by reference in its entity).

By using routine recombinant DNA techniques, one or more CDRs of the antigen-binding polypeptides of the present disclosure, may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278:457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes a polypeptide that specifically binds to at least one antigen epitope of a desired polypeptide, e.g., LIGHT. Preferably, one or more amino acid substitutions may be performed within the framework regions, and, preferably, the amino acid substitutions improve binding ability of an antibody to an antigen. Additionally, such methods may be used to obtain amino acid substitutions or deletions of one or more variable region cysteine residues participating in forming an intrachain disulfide bond, thereby generating antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present disclosure and within the scope of the prior art.

In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA:851-855 (1984); Neuberger et al., Nature 372:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule can be used to link a human antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity. As used herein, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.

Yet another highly efficient means for generating recombinant antibodies is disclosed by Newman, Biotechnology 10: 1455-1460 (1992). Specifically, this technique results in the generation of primatized antibodies that contain monkey variable domains and human constant sequences. This reference is incorporated by reference in its full text herein. Moreover, this technique is also described in commonly assigned U.S. Pat. Nos. 5,658,570, 5,693,780 and 5,756,096 each of which is incorporated herein by reference.

Alternatively, antibody-producing cell lines may be selected and cultured using techniques well known to those skilled in the art. Such techniques are described in a variety of laboratory manuals and primary publications. In this respect, techniques suitable for use in the disclosure are described in Current Protocols in Immunology, Coligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991) which is herein incorporated by reference in its full text, including supplement reference.

Additionally, standard techniques known to those skilled in the art can be used to introduce mutations in the nucleotide sequence encoding an antibody of the present disclosure, including, but not limited to, site-directed mutagenesis and PCR-mediated mutations which result in amino acid substitutions. Preferably, the variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the reference variable heavy chain region, CDR-H1, CDR-H2, CDR-H3, variable light chain region, CDR-L1, CDR-L2, or CDR-L3. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity.

Structure Information of the Antibody

Monospecific antibodies are symmetrical antibodies including two identical light chains and two identical heavy chains. The light chain and the heavy chain are connected by a disulfide bond and target a corresponding antigen, and the heavy chain and the heavy chain are connected by a disulfide bond; the entire antibody has a “Y” structure. The light chain includes a light chain variable region (VL) and a light chain constant region (Lc); and the heavy chain includes a heavy chain variable region (VH) and a heavy chain constant region, wherein the heavy chain constant region includes a CH1 and a Fc, and the Fc includes a hinge, a CH2 and a CH3.

Multifunctional antibody structure 1 is an asymmetric bispecific antibody including a light chain, a heavy chain and a fusion peptide 1, wherein the fusion peptide 1 includes a scFv and a Fc fragment; such diabody has a light-heavy chain pair, and a heavy chain-fusion peptide 1 pair, wherein each pair forms an interchain disulfide bond; the light chain-heavy chain pair targets the tumor antigen, and the ScFv in the fusion peptide 1 targets an immune cell antigen.

FIG. 1A is a structural schematic diagram of multifunctional antibody structure 1, and FIG. 1B is a schematic diagram of the primary protein structure of each component of the antibody.

Multifunctional antibody structure 2 is an asymmetric trivalent bispecific antibody comprising two light chains, one heavy chain and one fusion peptide 2, and having a light chain-heavy chain pair, a light chain-fusion peptide 2 pair, and a heavy chain-fusion peptide 2 pair, wherein each pair forms an interchain disulfide bond; the fusion peptide 2 includes a heavy chain variable region (VH), a first constant region of heavy chain (CH1), ScFv and Fc, wherein the ScFv is located between CH1 and Fc and is connected by a linker; the light chain-heavy chain pair targets the tumor antigen, the pairing of VH-CH1 in the fusion peptide 2 and light chain targets the same tumor antigen, and the ScFv targets an immune cell antigen.

FIG. 2A is a structural schematic diagram of multifunctional antibody structure 2, and FIG. 2B is a schematic diagram of the primary protein structure of each component of the antibody.

Multifunctional antibody structure 3 is an asymmetric trivalent bispecific antibody comprising a fusion heavy chain, a cross light chain, a heavy chain and a light chain, and having a light chain-heavy chain pair, a light chain-fusion heavy chain pair, a cross light chain-fusion heavy chain pair, and a fusion heavy chain-heavy chain pair, wherein each pair forms an interchain disulfide bond; the light chain includes a first light chain variable region (VLm) and a light chain constant region (CL); the fusion heavy chain includes a first heavy chain variable region (VHm), a first constant region of heavy chain (CH1), a second heavy chain variable region (VHs), a light chain constant region (CL) and an Fc, wherein the VHs and CL are connected by a linker to form a peptide “VHs-linker-CL”, and the “VHs-linker-CL” is located between CH1 and Fc and is connected by a linker/hinge; the cross light chain contains a second light chain variable region (VLs) and a CH1, the VLs and the CH1 are connected by a linker; the VLm-VHm pair targets a tumor antigen, and the VLs-VHs pair targets an immune cell antigen.

FIG. 3A is a structural schematic diagram of multifunctional antibody structure 3, and FIG. 3B is a schematic diagram of the primary protein structure of each component of the antibody.

The above three multifunctional antibody structures all have an Fc fragment comprising a CH2 and/or a CH3, wherein the CH2 is the natural sequence of CH2 of human IgG2 or the sequence modified by amino acid point mutations, and the specific sequences of some CH2 are shown in SEQ ID NOs: 83 to 101 and 122 to 176; each multifunctional antibody has two different CH3, and the two CH3 are paired with a form of “knob-into-hole” or/and “salt bridge” to form a heterodimer, and the seqence of CH3 is shown in SEQ ID NOs: 102 to 115.

The Variable Regions of Antibody equence

TABLE 3

Variable region sequence of anti-CD3 antibody

Variable region amino acid sequence of antibody variable region of anti-CD3 (The amino

Antibody
acids underlined in bold are CDR regions)

code

SEQ

SEQ

(Sequence

ID

ID

source)
VHs
NO
VLs
NO

2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAP
1
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYAN
2

GKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLY

WVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGK

LQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLV

AALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVE

TVSS

IK

2j5a
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAP
3
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYAN
4

GKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLY

WFQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGK

LQMNSLRAEDTAVYYCARHGNFGNSYVSWAAYWGQGTLV

AALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVE

TVSS

IK

OKT3
QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQR
5
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQ
6

PGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYM

QKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLT

QLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS

ISGMEAEDAATYYCQQWSSNPFTFGSGTKLEIN

L2K
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRP
7
DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQ
8

GQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQ

QKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTI

LSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS

SSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAP
9
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPN
10

GKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY

WVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGK

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVT

AALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLT

VSS

VL

(1) Antibodies Targeting Tumor-Associated Antigens

TABLE 4

Variable region sequence of anti-B7-H3 antibody

Variable region amino acid sequence of anti-B7-H3 antibody (The amino acids underlined

Antibody
in bold are CDR regions

code

SEQ

SEQ

(Sequence

ID

ID

source)
VHm
NO
VLm
NO

8H9
QVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRP
11
DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQ
12

EQGLEWIGWIFPGDGSTQYNEKFKGKATLTTDTSSSTAYMQ

QKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINS

LSRLTSEDSAVYFCARQTTATWFAYWGQGTLVTVSS

VEPEDVGVYYCQNGHSFPLTFGAGTKLELK

BRCA69D
QVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQR
13
DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQ
14

PGQGLEWIGTIYPGDGDTRYTQKFKGKATLTADKSSSTAYM

QKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTI

QLSSLASEDSAVYYCARRGIPRLWYFDVWGAGTTVTVSS

DNLEQEDIATYFCQQGNTLPPTFGGGTKLEIK

TABLE 5

Variable region sequences of anti-CD38 antibody

Variable region amino acid sequences of anti-CD38 antibody

Antibody
(The amino acids underlined in bold are CDR regions

code

SEQ

SEQ

(Sequence

ID

ID

source)
VHm
NO
VLm
NO

Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPG
15
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQ
16

KGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQM

KPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISS

NSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS

LEPEDFAVYYCQQRSNWPPTFGQGTKVEIK

MOR
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAP
17
DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQ
18

GKGLEWVSGISGDPSNTYYADSVKGRFTISRDNSKNTLYLQ

KPGQAPVLVIYGDSKRPSGIPERFSGSNSGNTATLTISG

MNSLRAEDTAVYYCARDLPLVYTGFAYWGQGTLVTVSS

TQAEDEADYYCQTYTGGASLVFGGGTKLTVLGQ

2F5
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAP
19
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQ
20

GQGLEWMGRVIPFLGIANSAQKFQGRVTITADKSTSTAYMD

QKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTIS

LSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVTVSS

SLQPEDFATYYCQQYNSYPRTFGQGTKVEIK

TABLE 6

Variable region sequences of anti-EpCAM antibody

Variable region amino acid sequences of anti-EpCAM antibody

Antibody
(The amino acids underlined in bold are CDR regions

code

SEQ

SEQ

(Sequence

ID

ID

source)
VHm
NO
VLm
NO

3-17I
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAP
21
EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQ
22

GQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYME

QKPGQAPRLIIYGASTTASGIPARFSASGSGTDFTLTIS

LSSLRSEDTAVYYCARGLLWNYWGQGTLVTVSS

SLQSEDFAVYYCQQYNNWPPAYTFGQGTKLEIK

2-6
EVQLVESGPELKKPGETVKISCKASGYTFTDYSMHWVKQAP
23
DIQMTQSPSSLSASLGERVSLTCRASQEISVSLSWWQ
24

GKGLKWMGWINTETGEPTYADDFKGRFAFSLETSASTAYLQ

EPDGTIKRLIYATSTLDSGVPKRFSGSRSGSDYSLTISS

INNLKNEDTATYFCARTAVYWGQGTTVTVSS

LESEDFVDYYCLQYASYPWTFGGGTKLEIK

TABLE 7

Variable region sequences

of anti-BCMA antibody

Variable region amino acid

sequences of anti-BCMA

antibody (The amino acids

underlined in bold are

Antibody
CDR regions)

code

SEQ

SEQ

(Sequence

ID

ID

source)
VHm
NO
VLm
NO

B50
QVQLVQSGAE
25
DIVMTQTPLS
26

VKKPGASVKV

LSVTPGQPAS

SCKASGYSFP

ISCKSSQSLV

DYYIN
WVRQA

HSNGNTYLH
W

PGQGLEWMGW

YLQKPGQSPQ

IYFASGNSEY

LLIYKVSNRF

NQKFTG
RVTM

S
GVPDRFSGS

TRDTSINTAY

GSGTDFTLKI

MELSSLTSED

SRVEAEDVGI

TAVYFCASLY

YYCSQSSIYP

DYDWYFDV
WG

WT
FGQGTKLE

QGTMVTVSS

IK

B140153
QVQLVQSGAE
27
LPVLTQPPSA
28

VKKPGSSVKV

SGTPGQRVTI

SCKASGGTFS

SCSGRSSNIG

SYA
ISWVRQA

SNS
VNWYRQL

PGQGLEWMGR

PGAAPKLLIY

IIPILGIA
NY

SNN
QRPPGVP

AQKFQGRVTI

VRFSGSKSGT

TADKSTSTAY

SASLAISGLQ

MELSSLRSED

SEDEATYYCA

TAVYYCARGG

TWDDNLNVHY

YYSHDMWSED

V
FGTGTKVTV

WGQGTLVTVS

LG

S

B69
QLQLQESGPG
29
SYVLTQPPSV
30

LVKPSETLSL

SVAPGQTARI

TCTVSGGSIS

TCGGNNIGSK

SGSYFWG
WIR

SVH
WYQQPPG

QPPGKGLEWI

QAPVVVVYDD

GSIYYSGITY

SDRPS
GIPER

YNPSLKS
RVT

FSGNSNGNTA

ISVDTSKNQF

TLTISRVEAG

SLKLSSVTAA

DEAVYYCQVW

DTAVYYCARH

DSSSDHVV
FG

DGAVAGLFDY

GGTKLTVL

WGQGTLVTVS

S

TABLE 8

Variable region sequences

of anti-PD-L1 antibody

Variable region amino acid

sequences of anti-PD-L1

antibody (The amino acids

underlined in bold are CDR

regions)

Antibody

code

SEQ

SEQ

(Sequence

ID

ID

source)
VHm
NO
VLm
NO

S70
EVQLVESGGG
31
DIQMTQSPSS
32

LVQPGGSLRL

LSASVGDRVT

SCAASGFTFS

ITCRASQDVS

DSWIH
WVRQA

TAVA
WYQQKP

PGKGLEWVAW

GKAPKLLIYS

ISPYGGSTYY

ASFLYS
GVPS

ADSVKG
RFTI

RFSGSGSGTD

SADTSKNTAY

FTLTISSLQP

LQMNSLRAED

EDFATYYCQQ

TAVYYCARRH

YLYHPAT
FGQ

WPGGFDY
WGQ

GTKVEIK

GTLVTVSS

Avelumab
EVQLLESGGG
33
QSALTQPASV
34

LVQPGGSLRL

SGSPGQSITI

SCAASGFTFS

SCTGTSSDVG

SYIMM
WVRQA

GYNYVS
WYQQ

PGKGLEWVSS

HPGKAPKLMI

IYPSGGITFY

YDVSNRPSGV

ADTVKG
RFTI

SNRFSGSKSG

SRDNSKNTLY

NTASLTISGL

LQMNSLRAED

QAEDEADYYC

TAVYYCARIK

SSYTSSSTRV

LGTVTTVDY
W

FGTGTKVTVL

GQGTLVTVSS

12A4
QVQLVQSGAE
35
EIVLTQSPAT
36

VKKPGSSVKV

LSLSPGERAT

SCKTSGDTFS

LSCRASQSVS

TYAIS
WVRQA

SYLA
WYQQKP

PGQGLEWMGG

GQAPRLLIYD

IIPIFGKAHY

ASNRAT
GIPA

AQKFQG
RVTI

RFSGSGSGTD

TADESTSTAY

FTLTISSLEP

MELSSLRSED

EDFAVYYCQQ

TAVYFCARKF

RSNWPT
FGQG

HFVSGSPFGM

TKVEIK

DV
WGQGTTVT

VSS

TABLE 9

Variable region sequences

of anti-CD 19 antibody

Variable region amino acid

sequences of anti-CD 19

antibody (The amino acids

underlined in bold are

Antibody
CDR regions)

code

SEQ

SEQ

(Sequence

ID

ID

source)
VHm
NQ
VLm
NO

M208
EVQLVESGGG
37
DIVMTQSPAT
38

LVKPGGSLKL

LSLSPGERAT

SCAASGYTFT

LSCRSSKSLQ

SYVMH
WVRQA

NVNGNTYLY
W

PGKGLEWIGY

FQQKPGQSPQ

INPYNDGTKY

LLIYRMSNLN

NEKFQGRVTI

S
GVPDRFSGS

SSDKSISTAY

GSGTEFTLTI

MELSSLRSED

SSLEPEDFAV

TAMYYCARGT

YYCMQHLEYP

YYYCTRVFDY

IT
FGAGTKLE

WGQGTLVTVS

IK

TABLE 10

Variable region sequences

of anti-SLAMF7 antibody

Variable region amino acid

sequences of anti-SLAMF7

antibody (The amino acids

underlined in bold are

Antibody
CDR regions)

code

SEQ

SEQ

(Sequence

ID

ID

source)
VHm
NO
VLm
NO

Elotuzumab
HVQLVESGGG
39
DIQMTQSPSS
40

LVQPGGSLRL

LSASVGDRVT

SCAASGFDFS

ITCKASQDVG

RYWMS
WVRQA

IA
VAWYQQKP

PGKGLEWIGE

GKVPKLLIYW

INPDSST
INY

AS
TRHTGVPD

APSLKDKFII

RFSGSGSGTD

SRDNAKNSLY

FTLTISSLQP

LQMNSLRAED

EPVATYYCQQ

TAVYYCARPD

YSSYPYT
FGQ

GNYWYFDV
WG

GTKVEIK

QGTLVTVSS

TABLE 11

Variable region sequences

of anti-CEA antibody

Variable region

amino acid

sequences of

anti-CEA antibody

(The amino acids

Underlined in bold

Antibody
are CDR regions)

code

SEQ

SEQ

(Sequence

ID

ID

source)
VHm
NO
VLm
NO

HPRIA3
QVQLVQSGSE
41
DIQMTQSPSS
42

LKKPGASVKV

LSASVGDRVT

SCKASGYTFT

ITCKASQNVG

VFGMN
WVRQA

TNVA
WYQQKP

PGQGLEWMGW

GKAPKLLIYS

INTKTGKATY

ASYRYS
GVPS

VKKFKG
RFVF

RFSGSGSGTD

SLDTSVSTAY

FTFTISSLQP

LQISSLKADD

EDIATYYCHQ

TAVYYCARWD

YYTYPLFT
FG

FYDYVKAMDY

QGTKVEIK

WGQGTTVTVS

S

TABLE 12

Variable region sequences of

anti-luciferase antibody

Variable region

amino acid sequences

of anti-luciferase antibody

(The amino acids underlined

Antibody
in bold are CDR regions)

code

SEQ

SEQ

(Sequence

ID

ID

source)
VHm
NO
VLm
NO

4420
EVKLDETGGG
43
DVVMTQTPLS
44

LVQPGRPMKL

LPVSLGDQAS

SCVASGFTFS

ISCRSSQSLV

DYWMNWVRQS

HSNGNTYLR
W

PEKGLEWVAQ

YLQKPGQSPK

IRNKPYNYET

VLIYKVSNRF

YYSDSVKG
RF

S
GVPDRFSGS

TISRDDSKSS

GSGTDFTLKI

VYLQMNNLRV

SRVEAEDLGV

EDMGIYYCTG

YFCSQSTHVP

SYYGMDY
WGQ

WT
FGGGTKLE

GTSVTVSS

IK

Sequences of Other Domains

(1) Amino acid sequences of linker domains

TABLE 13

Amino acid sequences of linkers

Domain
Code
Amino acid sequence
SEQ ID NO

Linker
Lin1
SS
45

Lin2
AS
46

Lin3
GGGGS
47

Lin4
GGGSAAA
48

Lin5
GGGGSAS
49

Lin6
GRPGSGRPGS
50

Lin7
GGGGSGGGGS
51

Lin8
GKSSGSGSESKS
52

Lin9
GSTSGSGKSSEGKG
53

Lin10
GGGGSGGGGSGGGGS
54

Lin11
GGGGSDKTHTSPPS
55

Lin12
EPKSSDKTHTSPPS
56

Lin13
GGGGSGGGGSGGGGSAS
57

Lin14
GSTSGSGKSSEGSGSTKG
58

Lin15
GSTSGSGKPGSGEGSTKG
59

Lin16
GGGGSGGGGSGGGGSGGGGS
60

Lin15
DKTHTSPPSGGGGSGGGGS
61

Lin16
APAPAPAPAPAP
62

Lin17
AEAAAKEAAAKA
63

Lin18
GGGGSGGGGSGGGGSGGGGS
64

GGGGSGGGGSAS

Lin19
AGGGSGGGGSGGGGSGGGGS
65

GGGGSGGGGSGGGGSAS

(2) Amino acid sequences of hinge domains

TABLE 14

Amino acid sequences of hinges

Domain
Code
Amino acid sequence
SEQ ID NO

Hinge
Hin1
DKTHTCP
66

Hin2
EPKSSDKTHTCP
67

Hin3
GGGGSDKTHTCP
68

Hin4
RGRGSDKTHTCP
69

Hin5
DGDGSDKTHTCP
70

Hin6
GRGRGSDKTHTCP
71

Hin7
ASTRGRGSDKTHTCP
72

Hin8
GQPDGDASDKTHTCP
73

Hin9
DKTHT
74

(3) Amino acid sequences of CL domain of light chain constant region

TABLE 15

Amino acid sequences of CL

SEQ

ID

Domain
NO
Amino acid sequence
NO

CL
Lcl
RTVAAPSVFIFPPSD
75

EQLKSGTASVVCLLN

NFYPREAKVQWKVDN

ALQSGNSQESVTEQD

SKDSTYSLSSTLTLS

KADYEKHKVYACEVT

HQGLSSPVTKSFNRG

EC

Lc2
GQPKANPTVTLFPPS
76

SEELQANKATLVCLI

SDFYPGAVTVAWKAD

GSPVKAGVETTKPSK

QSNNKYAASSYLSLT

PEQWKSHRSYSCQVT

HEGSTVEKTVAPTEC

S

Lc3
GQPKAAPSVTLFPPS
77

SEELQANKATLVCLI

SDFYPGAVTVAWKAD

SSPVKAGVETTTPSK

QSNNKYAASSYLSLT

PEQWKSHRSYSCQVT

HEGSTVEKTVAPTEC

S

Lc4
GQPKAAPSVTLFPPS
78

SEELQANKATLVCLI

SDFYPGAVTVAWKAD

SSPAKAGVETTTPSK

QSNNKYAASSYLSLT

PEQWKSHKSYSCQVT

HEGSTVEKTVAPTEC

S

Lc5
GQPKAAPSVTLFPPS
79

SEELQANKATLVCLI

SDFYPGAVKVAWKAD

GSPVNTGVETTTPSK

QSNNKYAASSYLSLT

PEQWKSHRSYSCQVT

HEGSTVEKTVAPAEC

S

Lc6
GQPKAAPTVTLFPPS
80

SEELQANKATLVCLI

SDFYPGAVKVAWKAD

SSPAKAGVETTTPSK

QSNNKYAASSYLSLT

PEQWKSHKSYSCQVT

HEGSTVEKTVAPTEC

S

Lc7
VAAPSVFIFPPSDEQ
81

LKSGTASVVCLLNNF

YPREAKVQWKVDNAL

QSGNSQESVTEQDSK

DSTYSLSSTLTLSKA

DYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC

(4) Amino acid sequences of CH1 domain of heavy chain constant region

TABLE 16

Amino acid sequences of CH1

SEQ

ID

Domain
Code
Amino acid sequence
NO

CH1
CH1
ASTKGPSVFPLAPSSK
82

STSGGTAALGCLVKD

YFPEPVTVSWNSGAL

TSGVHTFPAVLQSSG

LYSLSSVVTVPSSSL

GTQTYICNVNHKPSN

TKVDKKVEPKSC

Specific Information of Fc Modification

Fc amino acids are numbered according to the Kabat numbering.The “Kabat numbering” refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).The specific numbering is shown in the table below:

TABLE 17

Fc amino acid numbering based on Kabat numbering system

216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241

E
P
K
S
C
D
K
T
H
T
C
P
P
C
P
A
P
E
L
L
G
G
P
S
V
F

242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267

L
F
P
P
K
P
K
D
T
L
M
I
S
R
T
P
E
V
T
C
V
V
V
D
V
S

268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293

H
E
D
P
E
V
K
F
N
W
Y
V
D
G
V
E
V
H
N
A
K
T
K
P
R
E

294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319

E
Q
Y
N
S
T
Y
R
V
V
S
V
L
T
V
L
H
Q
D
W
L
N
G
K
E
Y

320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345

K
C
K
V
S
N
K
A
L
P
A
P
I
E
K
T
I
S
K
A
K
G
Q
P
R
E

346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371

P
Q
V
Y
T
L
P
P
S
R
D
E
L
T
K
N
Q
V
S
L
T
C
L
V
K
G

372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397

F
Y
P
S
D
I
A
V
E
W
E
S
N
G
Q
P
E
N
N
Y
K
T
T
P
P
V

398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423

L
D
S
D
G
S
F
F
L
Y
S
K
L
T
V
D
K
S
R
W
Q
Q
G
N
V
F

424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448

S
C
S
V
M
H
E
A
L
H
N
H
Y
T
Q
K
S
L
S
L
S
P
G
K
—

wherein,

amino acids at positions 221 to 227 are the hinge domain,

amino acids at positions 228 to 340 are the CH2 domain of the second constant region of the heavy chain,

amino acids at positions 341 to 447 are the CH3 domain of the third constant region of the heavy chain.

In one aspect, the CH2 domain contains one or more substitutions to reduce the binding ability of Fc to FcγR. The amino acid residues that can be substituted include, but are not limited to, E233, L234, L235, G236, D265, D270, K274, Y296, N297, Y300, L309, A327, P329, P331, A339. Non-limiting examples of these substitution combinations are listed in the table below:

TABLE 18

CH2 amino acid substitution combination that reduces the binding

ability of Fc to FcγR

Combination

No.
Substitutions on CH2

CH2-1
L234A, L235A

CH2-2
L234A, L235A, P329G

CH2-3
L234F, L235E, P331S

CH2-4
L234F, L235E, P331A

CH2-5
N297A

CH2-6
N297G

CH2-7
N297Q

CH2-8
D265A, N297Q, A327Q

CH2-9
Completely deletion of CH2 domain

CH2-10
E233P, L234V, L235A, G236-*, K274Q, Y296F, Y300F,

L309V, A327G, A339T

CH2-11
C229S, E233P, L234V, L235A, G236-*, K274Q, Y296F,

Y300F, L309V, A327G, A339T

CH2-12
C229A, E233P, L234V, L235A, G236-*, K274Q, Y296F,

Y300F, L309V, A327G, A339T

CH2-13
C229G, E233P, L234V, L235A, G236-*, K274Q, Y296F,

Y300F, L309V, A327G, A339T

CH2-14
C229P, E233P, L234V, L235A, G236-*, K274Q, Y296F,

Y300F, L309V, A327G, A339T

CH2-15
E233P, L234V, L235A, G236-*, D265A, K274Q, Y296F,

Y300F, L309V, A327G, A339T

CH2-16
E233P, L234V, L235A, G236-*, D270A, K274Q, Y296F,

Y300F, L309V, A327G, A339T

CH2-17
E233P, L234V, L235A, G236-*, D265A, D270A, K274Q,

Y296F, Y300F, L309V, A327G, A339T

*Represents that the residue at that position is deleted.

The sequence and numbering of the Fc after deleting the glycine residue at position 236 in combination 10 in the above table are as follows:

TABLE 19

Fc (combination 10 of table 6) amino acid numbering after residue deletion based on kabat numbering system

216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241

E
P
K
S
C
D
K
T
H
T
C
P
P
C
P
A
P
P
V
A
—
G
P
S
V
F

242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267

L
F
P
P
K
P
K
D
T
L
M
I
S
R
T
P
E
V
T
C
V
V
V
D
V
S

268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293

H
E
D
P
E
V
Q
F
N
W
Y
V
D
G
V
E
V
H
N
A
K
T
K
P
R
E

294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319

E
Q
F
N
S
T
F
R
V
V
S
V
L
T
V
V
H
Q
D
W
L
N
G
K
E
Y

320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345

K
C
K
V
S
N
K
G
L
P
A
P
I
E
K
T
I
S
K
T
K
G
Q
P
R
E

346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371

P
Q
V
Y
T
L
P
P
S
R
D
E
L
T
K
N
Q
V
S
L
T
C
L
V
K
G

372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397

F
Y
P
S
D
I
A
V
E
W
E
S
N
G
Q
P
E
N
N
Y
K
T
T
P
P
V

398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423

L
D
S
D
G
S
F
F
L
Y
S
K
L
T
V
D
K
S
R
W
Q
Q
G
N
V
F

424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448

S
C
S
V
M
H
E
A
L
H
N
H
Y
T
Q
K
S
L
S
L
S
P
G
K
—

In the above table, the glycine at position 236 (G236) is deleted, but the amino acid numbering “236” is retained, which is represented by “−”.

The CH3 domain of the antibody can be modified to improve the efficiency of heterodimer pairing. For example, in some aspects, compared with the wild-type antibody fragments, the Fc fragment of the heavy chain of the monovalent unit and/or the Fc fragment of the fusion peptide may contain one or more substitutions, which form a knob-into-hole. The knob-into-hole configuration is known in the art. See, for example, Ridgway et al., “‘Knob-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization,” Protein Engineering 9(7):617-21 (1996).

On the one hand, T366 on a CH3 domain is substituted with a relatively large amino acid residue, such as tyrosine (Y) or tryptophan (W). Then, Y407 on the other CH3 domain can be substituted with a relatively small amino acid residue, such as threonine (T), alanine (A) or valine (V). Some non-limiting examples of these substitution combinations are shown in table 20 below.

TABLE 20

Fc amino acid substitution combinations forms

a knob-in-hole structure between different fc

to improve the efficiency of heterodimer pairing

Combination
Substitutions on
Substitutions on

No.
one CH3
another CH3

CH3-1
T366W
Y407A

CH3-2
T366W
Y407V

CH3-3
T366Y
Y407A

CH3-4
T366Y
Y407V

CH3-5
T366W
T366S, L368A, Y407V

In one aspect, one of the CH3 domains contains one or more substitutions, which are substituted with positively charged amino acid residues under physiological conditions, while another CH3 domain contains one or more substitutions, which are substituted with one or more negatively charged amino acid residues under physiological conditions. In one aspect, the positively charged amino acid residues may be arginine (R), histidine (H) or lysine (K). On the other hand, the negatively charged amino acid residues may be aspartic acid (D) or glutamic acid (E). The amino acid residues that can be substituted include, but are not limited to, D356, L368, K392, D399, and K409. Non-limiting examples of these substitution combinations are listed in Table 21 below.

TABLE 21

CH3 amino acid substitution combinations forms an ionic bond

between different Fc to improve the

efficiency of heterodimer pairing

Combination
Substitutions on
Substitutions on

No.
one CH3
another CH3

CH3-6
D356K D399K
K392D K409D

CH3-7
L368R D399K
K392D K409D

CH3-8
L368K D399K
K392D K409D

CH3-9
L368R D399K
K409D

CH3-10
L368K D399K
K409D

CH3-11
L368R
K409D

CH3-12
L368K
K409D

On one hand, 5354 on one CH3 domain is substituted with cysteine, and Y349 on another CH3 domain is also substituted with cysteine. The two residues at the substituted position form a disulfide bond.The following table shows an example of this substitution combination.

TABLE 22

CH3 amino acid substitution combinations

forms a disulfide bond between different Fc to improve

the efficiency of heterodimer pairing

Combination
Substitutions on
Substitutions on

No.
one CH3
another CH3

CH3-13
S354C
Y349C

In certain aspects, the antibody may comprise a CH2 that reduces the binding to FcγR or a CH3 that improves the heterodimer pairing, or both.

On one hand, H435 and Y436 on one CH3 domain are substituted with arginine and phenylalanine, respectively. Such substitution results in a significant reduction of the binding ability of Fc to protein A, thereby leading to different protein A-binding activities between heterodimers and homodimers, and thus it is easy to separate the two components in the process of affinity chromatography. An example of this substitution combination is shown in the following table.

TABLE 23

One amino acid substitution on

CH3 leads to a decrease in binding ability to

protein a

Combination

No.
Substitution on CH3

CH3-14
H435R, Y436F

In the above Table 18 to Table 23, amino acid substitution combination in different domains can be constructed according to the “hinge-CH2-CH3” to form an intact Fc fragment, which satisfies the following requirements: (1) reducing the binding ability to FcγR, (2) facilitating the formation of heterodimers, (3) changing the binding ability to protein A.

Examples of Some Specific Fc Sequences

TABLE 24

CH2 amino acid sequence of Fc with reduced

FcγR binding ability

SEQ

Combination
Amino acid sequence
ID

No.
of CH2
NO

WT
PCPAPELLGGPSVFLFPPKP
83

KDTLMISRTPEVTCVVVDVS

HEDPEVKFNWYVDGVEVHNA

KTKPREEQYNSTYRVVSVLT

VLHQDWLNGKEYKCKVSNKA

LPAPIEKTISKAK

AAG
PCPAPEAAGGPSVFLFPPKP
84

KDTLMISRTPEVTCVVVDVS

HEDPEVKFNWYVDGVEVHNA

KTKPREEQYNSTYRVVSVLT

VLHQDWLNGKEYKCKVSNKA

LGAPIEKTISKAK

FES
PCPAPEFEGGPSVFLFPPKP
85

KDTLMISRTPEVTCVVVDVS

HEDPEVKFNWYVDGVEVHNA

KTKPREEQYNSTYRVVSVLT

VLHQDWLNGKEYKCKVSNKA

LPASIEKTISKAK

FEG
PCPAPEFEGGPSVFLFPPKP
86

KDTLMISRTPEVTCVVVDVS

HEDPEVKFNWYVDGVEVHNA

KTKPREEQYNSTYRVVSVLT

VLHQDWLNGKEYKCKVSNKA

LPAGIEKTISKAK

N297A
PCPAPELLGGPSVFLFPPKP
87

KDTLMISRTPEVTCVVVDVS

HEDPEVKFNWYVDGVEVHNA

KTKPREEQYASTYRVVSVLT

VLHQDWLNGKEYKCKVSNKA

LPAPIEKTISKAK

N297G
PCPAPELLGGPSVFLFPPKP
88

KDTLMISRTPEVTCVVVDVS

HEDPEVKFNVVYVDGVEVHN

AKTKPREEQYGSTYRVVSVL

TVLHQDWLNGKEYKCKVSNK

ALPAPIEKTISKAK

N297Q
PCPAPELLGGPSVFLFPPKP
89

KDTLMISRTPEVTCVVVDVS

HEDPEVKFNWYVDGVEVHNA

KTKPREEQYQSTYRVVSVLT

VLHQDWLNGKEYKCKVSNKA

LPAPIEKTISKAK

LALA
PCPAPEAAGGPSVFLFPPKP
90

KDTLMISRTPEVTCVVVDVS

HEDPEVKFNWYVDGVEVHNA

KTKPREEQYNSTYRVVSVLT

VLHQDWLNGKEYKCKVSNKA

LPAPIEKTISKAK

LALANQ
PCPAPEAAGGPSVFLFPPKP
91

KDTLMISRTPEVTCVVVDVS

HEDPEVKFNWYVDGVEVHNA

KTKPREEQYQSTYRVVSVLT

VLHQDWLNGKEYKCKVSNKA

LPAPIEKTISKAK

AAQ
PCPAPELLGGPSVFLFPPKP
92

KDTLMISRTPEVTCVVVAVS

HEDPEVKFNWYVDGVEVHNA

KTKPREEQYASTYRVVSVLT

VLHQDWLNGKEYKCKVSNKQ

LPAPIEKTISKAK

AQQ
PCPAPELLGGPSVFLFPPKP
93

KDTLMISRTPEVTCVVVAVS

HEDPEVKFNWYVDGVEVHNA

KTKPREEQYQSTYRVVSVLT

VLHQDWLNGKEYKCKVSNKQ

LPAPIEKTISKAK

G2CH2
PCPAPPVAGPSVFLFPPKPK
94

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

SG2CH2
PSPAPPVAGPSVFLFPPKPK
95

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

AG2CH2
PAPAPPVAGPSVFLFPPKPK
96

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

GG2CH2
PGPAPPVAGPSVFLFPPKPK
97

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

PG2CH2
PPPAPPVAGPSVFLFPPKPK
98

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

DG2CH2
PCPAPPVAGPSVFLFPPKPK
99

DTLMISRTPEVTCVVVAVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

G2DCH2
PCPAPPVAGPSVFLFPPKPK
100

DTLMISRTPEVTCVVVDVSH

EAPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

DG2DCH2
PCPAPPVAGPSVFLFPPKPK
101

DTLMISRTPEVTCVVVAVSH

EAPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

PG2-GA
PPPAPPVAGPSVFLFPPKPK
122

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKAL

PAPIEKTISKTK

PG2-TA
PPPAPPVAGPSVFLFPPKPK
123

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKAK

G2D-GA
PCPAPPVAGPSVFLFPPKPK
124

DTLMISRTPEVTCVVVDVSH

EAPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKAL

PAPIEKTISKTK

G2D-TA
PCPAPPVAGPSVFLFPPKPK
125

DTLMISRTPEVTCVVVDVSH

EAPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKAK

G2D-GATA
PCPAPPVAGPSVFLFPPKPK
126

DTLMISRTPEVTCVVVDVSH

EAPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKAL

PAPIEKTISKAK

PDG2D
PPPAPPVAGPSVFLFPPKPK
127

DTLMISRTPEVTCVVVAVSH

EAPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

C229LG2CH2
PLPAPPVAGPSVFLFPPKPK
128

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

C229FG2CH2
PFPAPPVAGPSVFLFPPKPK
129

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

C229RG2CH2
PRPAPPVAGPSVFLFPPKPK
130

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

C229VG2CH2
PVPAPPVAGPSVFLFPPKPK
131

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

C229QG2CH2
PQPAPPVAGPSVFLFPPKPK
132

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

C229KG2CH2
PKPAPPVAGPSVFLFPPKPK
133

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

C229DG2CH2
PDPAPPVAGPSVFLFPPKPK
134

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

C229IG2CH2
PIPAPPVAGPSVFLFPPKPK
135

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

C229YG2CH2
PYPAPPVAGPSVFLFPPKPK
136

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

C229NG2CH2
PNPAPPVAGPSVFLFPPKPK
137

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

C229MG2CH2
PMPAPPVAGPSVFLFPPKPK
138

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

C229TG2CH2
PTPAPPVAGPSVFLFPPKPK
139

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

C229HG2CH2
PHPAPPVAGPSVFLFPPKPK
140

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

C229EG2CH2
PEPAPPVAGPSVFLFPPKPK
141

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

C229WG2CH2
PWPAPPVAGPSVFLFPPKPK
142

DTLMISRTPEVTCVVVDVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D265PG2CH2
PCPAPPVAGPSVFLFPPKPK
143

DTLMISRTPEVTCVVVPVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D265KG2CH2
PCPAPPVAGPSVFLFPPKPK
144

DTLMISRTPEVTCVVVKVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D265SG2CH2
PCPAPPVAGPSVFLFPPKPK
145

DTLMISRTPEVTCVVVSVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D265FG2CH2
PCPAPPVAGPSVFLFPPKPK
146

DTLMISRTPEVTCVVVFVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D265RG2CH2
PCPAPPVAGPSVFLFPPKPK
147

DTLMISRTPEVTCVVVRVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D265LG2CH2
PCPAPPVAGPSVFLFPPKPK
148

DTLMISRTPEVTCVVVLVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D265GG2CH2
PCPAPPVAGPSVFLFPPKPK
149

DTLMISRTPEVTCVVVGVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D265TG2CH2
PCPAPPVAGPSVFLFPPKPK
150

DTLMISRTPEVTCVVVTVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D265YG2CH2
PCPAPPVAGPSVFLFPPKPK
151

DTLMISRTPEVTCVVVYVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D265WG2CH2
PCPAPPVAGPSVFLFPPKPK
152

DTLMISRTPEVTCVVVWVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D265HG2CH2
PCPAPPVAGPSVFLFPPKPK
153

DTLMISRTPEVTCVVVHVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D265VG2CH2
PCPAPPVAGPSVFLFPPKPK
154

DTLMISRTPEVTCVVVVVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D265QG2CH2
PCPAPPVAGPSVFLFPPKPK
155

DTLMISRTPEVTCVVVQVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D265EG2CH2
PCPAPPVAGPSVFLFPPKPK
156

DTLMISRTPEVTCVVVEVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D265MG2CH2
PCPAPPVAGPSVFLFPPKPK
157

DTLMISRTPEVTCVVVMVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D265NG2CH2
PCPAPPVAGPSVFLFPPKPK
158

DTLMISRTPEVTCVVVNVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D265IG2CH2
PCPAPPVAGPSVFLFPPKPK
159

DTLMISRTPEVTCVVVIVSH

EDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D270LG2CH2
PCPAPPVAGPSVFLFPPKPK
160

DTLMISRTPEVTCVVVDVSH

ELPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D270RG2CH2
PCPAPPVAGPSVFLFPPKPK
161

DTLMISRTPEVTCVVVDVSH

ERPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D270PG2CH2
PCPAPPVAGPSVFLFPPKPK
162

DTLMISRTPEVTCVVVDVSH

EPPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D270GG2CH2
PCPAPPVAGPSVFLFPPKPK
163

DTLMISRTPEVTCVVVDVSH

EGPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D270VG2CH2
PCPAPPVAGPSVFLFPPKPK
164

DTLMISRTPEVTCVVVDVSH

EVPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D270HG2CH2
PCPAPPVAGPSVFLFPPKPK
165

DTLMISRTPEVTCVVVDVSH

EHPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D270YG2CH2
PCPAPPVAGPSVFLFPPKPK
166

DTLMISRTPEVTCVVVDVSH

EYPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D270IG2CH2
PCPAPPVAGPSVFLFPPKPK
167

DTLMISRTPEVTCVVVDVSH

EIPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D270EG2CH2
PCPAPPVAGPSVFLFPPKPK
168

DTLMISRTPEVTCVVVDVSH

EEPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D270FG2CH2
PCPAPPVAGPSVFLFPPKPK
169

DTLMISRTPEVTCVVVDVSH

EFPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D270KG2CH2
PCPAPPVAGPSVFLFPPKPK
170

DTLMISRTPEVTCVVVDVSH

EKPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D270WG2CH2
PCPAPPVAGPSVFLFPPKPK
171

DTLMISRTPEVTCVVVDVSH

EWPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D270SG2CH2
PCPAPPVAGPSVFLFPPKPK
172

DTLMISRTPEVTCVVVDVSH

ESPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D270TG2CH2
PCPAPPVAGPSVFLFPPKPK
173

DTLMISRTPEVTCVVVDVSH

ETPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D270QG2CH2
PCPAPPVAGPSVFLFPPKPK
174

DTLMISRTPEVTCVVVDVSH

EQPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D270MG2CH2
PCPAPPVAGPSVFLFPPKPK
175

DTLMISRTPEVTCVVVDVSH

EMPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

D270NG2CH2
PCPAPPVAGPSVFLFPPKPK
176

DTLMISRTPEVTCVVVDVSH

ENPEVQFNWYVDGVEVHNAK

TKPREEQFNSTFRVVSVLTV

VHQDWLNGKEYKCKVSNKGL

PAPIEKTISKTK

TABLE 25

CH3 amino acid sequence of Fc

that forms a heterodimer

Combi-
Amino acid
SEQ
Amino acid
SEQ

nation
Sequence
ID
Sequence
ID

No.
of CH3-A
NO
of CH3-B
NO

WT
GQPREPQVYT
102
GQPREPQVYT
103

LPPSRDELTK

LPPSRDELTK

NQVSLTCLVK

NQVSLTCLVK

GFYPSDIAVE

GFYPSDIAVE

WESNGQPENN

WESNGQPENN

YKTTPPVLDS

YKTTPPVLDS

DGSFFLYSKL

DGSFFLYSKL

TVDKSRWQQG

TVDKSRWQQG

NVFSCSVMHE

NVFSCSVMHE

ALHNHYTQKS

ALHNHYTQKS

LSLSPGK

LSLSPGK

W:SAV
GQPREPQVYT
104
GQPREPQVYT
105

LPPSRDELTK

LPPSRDELTK

NQVSLWCLVK

NQVSLSCAVK

GFYPSDIAVE

GFYPSDIAVE

WESNGQPENN

WESNGQPENN

YKTTPPVLD

YKTTPPVLDS

SDGSF

DGSFFLVSKL

FLYSKLTVDK

TVDKSRWQQG

SRWQQGNVFS

NVFSCSVMHE

CSVMHEALHN

ALHNHYTQKS

HYTQKSLSLS

LSLSPGK

PGK

CSAVRF:
GQPREPQVCT
106
GQPREPQVYT
107

CW
LPPSRDELTK

LPPCRDELTK

NQVSLSCAVK

NQVSLWCLVK

GFYPSDIAVE

GFYPSDIAVE

WESNGQPENN

WESNGQPENN

YKTTPPVLDS

YKTTPPVLDS

DGSFFLVSKL

DGSFFLYSKL

TVDKSRWQQG

TVDKSRWQQG

NVFSCSVMHE

NVFSCSVMHE

ALHNRFTQKS

ALHNHYTQKS

LSLSPGK

LSLSPGK

CW:CSAV
GQPREPQVYT
108
GQPREPQVCT
109

LPPCRDELTK

LPPSRDELTK

NQVSLWCLVK

NQVSLSCAVK

GFYPSDIAV

GFYPSDIAVE

EWESNGQP

WESNGQPENN

ENNYKTTPPV

YKTTPPVLDS

LDSDGSFFLY

DGSFFLVSKL

SKLTVDKSRW

TVDKSRWQQG

QQGNVFSCSV

NVFSCSVMHE

MHEALHNHYT

ALHNHYTQKS

QKSLSLSPGK

LSLSPGK

WDD:RKA
GQPREPQVYT
110
GQPREPQVYT
111

LPPSRDELTK

LPPSRDELTK

NQVSLWCLVK

NQVSLTCRVK

GFYPSDIAVE

GFYPSDIAVE

WESNGQPENN

WESNGQPENN

YDTTPPVLDS

YKTTPPVLKS

DGSFFLYSDL

DGSFFLASKL

TVDKSRWQQG

TVDKSRWQQG

NVFSCSVMHE

NVFSCSVMHE

ALHNHYTQKS

ALHNHYTQKS

LSLSPGK

LSLSPGK

DD:KK
GQPREPQVYT
112
GQPREPQVYT
113

LPPSRDELTK

LPPSRKELTK

NQVSLTCLVK

NQVSLTCLVK

GFYPSDIAVE

GFYPSDIAVE

WESNGQPENN

WESNGQPENN

YDTTPPVLDS

YKTTPPVLKS

DGSFFLYSDL

DGSFFLYSKL

TVDKSRWQQG

TVDKSRWQQG

NVFSCSVMHE

NVFSCSVMHE

ALHNHYTQKS

ALHNHYTQKS

LSLSPGK

LSLSPGK

CSAV:
GQPREPQVCT
114
GQPREPQVYT
115

CWRF
LPPSRDELTK

LPPCRDELTK

NQVSLSCAVK

NQVSLWCLVK

GFYPSDIAVE

GFYPSDIAVE

WESNGQPENN

WESNGQPENN

YKITPPVLDS

YKTTPPVLDS

DGSFFLVSKL

DGSFFLYSKL

TVDKSRWQQG

TVDKSRWQQG

NVFSCSVMHE

NVFSCSVMHE

ALHNHYTQKS

ALHNRFTQKS

LSLSPGK

LSLSPGK

Specific Sequences of Antigens

TABLE 26

Amino acid sequence

of tumor antigen

Name of tumor

SEQ

Antigen

ID

(Source)
Amino acid sequence
NO

Human CD38
VPRWRQQWSGPGTTKRFPET
116

(Source:
VLARCVKYTEIHPEMRHVDC

UniProtKB-
QSVWDAFKGAFISKHPCNIT

P28907)
EEDYQPLMKLGTQTVPCNKI

LLWSRIKDLAHQFTQVQRDM

FTLEDTLLGYLADDLTWCGE

FNTSKINYQSCPDWRKDCSN

NPVSVFWKTVSRRFAEAACD

VVHVMLNGSRSKIFDKNSTF

GSVEVHNLQPEKVQTLEAWV

IHGGREDSRDLCQDPTIKEL

ESIISKRNIOFSCKNIYRPD

KFLOCVKNPEDSSCTSEI

Human BCMA
MLQMAGQCSQNEYFDSLLHA
117

(Source:
CIPCQLRCSSNTPPLTCQRY

UniProtKB-
CNASVTNSVKGTNA

Q02223)

Human PD-L1
FTVTVPKDLYVVEYGSNMTI
118

(Source:
ECKFPVEKQLDLAALIVYWE

UniProtKB-
MEDKNIIQFVHGEEDLKVQH

Q9NZQ7)
SSYRQRARLLKDQLSLGNAA

LQITDVKLQDAGVYRCMISY

GGADYKRITVKVNAPYNKIN

QRILVVDPVTSEHELTCQAE

GYPKAEVIWTSSDHQVLSGK

TTTTNSKREEKLFNVTSTLR

INTTTNEIFYCTFRRLDPEE

NHTAELVIPELPLAHPPNER

Human SLAMF7
SGPVKELVGSVGGAVTFPLK
119

(Source:
SKVKQVDSIVWTFNTTPLVT

UniProtKB-
IQPEGGTIIVTQNRNRERVD

Q9NQ25)
FPDGGYSLKLSKLKKNDSGI

YYVGIYSSSLQQPSTQEYVL

HVYEHLSKPKVTMGLQSNKN

GTCVTNLTCCMEHGEEDVIY

TWKALGQAANESHNGSILPI

SWRWGESDMTFICVARNPVS

RNFSSPILARKLCEGAADDP

DSSM

human CEA
KLTIESTPFNVAEGKEVLLL
120

(Source:
VHNLPQHLFGYSWYKGERVD

UniProtKB-
GNRQIIGYVIGTQQATPGPA

P06731)
YSGREIIYPNASLLIQNIIQ

NDTGFYTLHVIKSDLVNEEA

TGQFRVYPELPKPSISSNNS

KPVEDKDAVAFTCEPETQDA

TYLWWVNNQSLPVSPRLQLS

NGNRTLTLFNVTRNDTASYK

CETQNPVSARRSDSVILNVL

YGPDAPTISPLNTSYRSGEN

LNLSCHAASNPPAQYSWFVN

GTFQQSTQELFIPNITVNNS

GSYTCQAHNSDTGLNRTTVT

TITVYAEPPKPFITSNNSNP

VEDEDAVALTCEPEIQNTTY

LWWVNNQSLPVSPRLQLSND

NRTLTLLSVTRNDVGPYECG

IQNKLSVDHSDPVILNVLYG

PDDPTISPSYTYYRPGVNLS

LSCHAASNPPAQYSWLIDGN

IQQHTQELFISNITEKNSGL

YTCQANNSASGHSRTTVKTI

TVSAELPKPSISSNNSKPVE

DKDAVAFTCEPEAQNTTYLW

WVNGQSLPVSPRLQLSNGNR

TLTLFNVTRNDARAYVCGIQ

NSVSANRSDPVTLDVLYGPD

TPIISPPDSSYLSGANLNLS

CHSASNPSPQYSWRINGIPQ

QHTQVLFIAKITPNNNGTYA

CFVSXLATGRNNSIVKSITV

SASGTSPGLSA

TABLE 27

Amino acid sequence of

immune cell antigen

Name of immune

SEQ

cell antigen
Amino acid
ID

(Source))
sequence
NO

Human CD3ϵ
DGNEEMGGITQTPYKVSI
121

(Source:
SGTTVILTCPQYPGSEIL

UniProtKB-
WQHNDKNIGGDEDDKNIG

P07766)
SDEDHLSLKEFSELEQSG

YYVCYPRGSKPEDANFYL

YLRARVCENCMEMD

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a structural schematic diagram of multifunctional antibody 1, wherein FIG. 1A is a structural schematic diagram of multifunctional antibody 1; FIG. 1B is a schematic diagram of the primary protein structure of each component of the multifunctional antibody 1.

FIG. 2 is a structural schematic diagram of multifunctional antibody 2, wherein FIG. 2A is a structural schematic diagram of multifunctional antibody 2; FIG. 2B is a schematic diagram of the primary protein structure of each component of the multifunctional antibody 2.

FIG. 3 is a structural schematic diagram of multifunctional antibody 3, wherein FIG. 3A is a structural schematic diagram of multifunctional antibody 3; FIG. 3B is a schematic diagram of the primary protein structure of each component of the multifunctional antibody 3.

FIG. 4 shows the in vitro killing ability of different multifunctional antibodies with the multifunctional antibody structure 1 on non-small cell lung cancer cell H358.

FIG. 5 shows the in vitro killing ability of different isotype control antibodies with the multifunctional antibody structure 1 on non-small cell lung cancer cell H358.

FIG. 6 shows the in vitro killing ability of CD38xCD3 antibody with the multifunctional antibody structure 1 on multiple myeloma cells MC/CAR.

FIG. 7 shows the in vitro non-specific killing ability of the isotype control antibody with the multifunctional antibody structure 1 on multiple myeloma cells MC/CAR.

FIG. 8 shows the activation of T cells in PBMC by the isotype control antibody with the multifunctional antibody structure 1 and the ratio of CD3+CD69+ T cells.

FIG. 9 shows the activation of T cells in PBMC by the isotype control antibody with the multifunctional antibody structure 1 and the ratio of CD3+CD25+ T cells.

FIG. 10 shows the binding ability of the isotype control antibody with the multifunctional antibody structure 1 to FcγR1 and the detection of activated fluorescent signal of Jurkat-luciferase cells.

FIG. 11 shows the binding ability of the isotype control antibody with the multifunctional antibody structure 1 to FcγR2 and the detection of activated fluorescent signal of Jurkat-luciferase cells.

FIG. 12 shows the binding ability of the isotype control antibody with the multifunctional antibody structure 1 to FcγR3A and the detection of activated fluorescent signal of Jurkat-luciferase cells.

FIG. 13 shows the in vitro killing ability of different multifunctional antibodies with the multifunctional antibody structure 2 on myeloma cells U266B1.

FIG. 14 shows the in vitro killing ability of different isotype control antibodies with the multifunctional antibody structure 2 on myeloma cells U266B1.

FIG. 15 shows the activation of T cells in PBMC by the isotype control antibody with the multifunctional antibody structure 2 and the ratio of CD3+CD69+ T cells.

FIG. 16 shows the activation of T cells in PBMC by the isotype control antibody with the multifunctional antibody structure 2 and the ratio of CD3+CD25+ T cells.

FIG. 17 shows the in vitro killing ability of CEAxCD3 antibody with the multifunctional antibody structure 3 on gastric cancer cell MKN-45.

FIG. 18 shows the in vitro killing ability of isotype control antibodies with the multifunctional antibody structure 3 on gastric cancer cell MKN-45.

FIG. 19 shows the activation of T cells in PBMC by the isotype control antibody with the multifunctional antibody structure 3 and the ratio of CD3+CD69+ T cells.

FIG. 20 shows the activation of T cells in PBMC by the isotype control antibody with the multifunctional antibody structure 3 and the ratio of CD3+CD25+ T cells.

FIG. 21 shows the accelerated thermal stability detection of different antibodies with the multifunctional antibody structure 1 treated at 40° C. for 14 days.

FIG. 22 shows the acid resistance detection of different antibodies with multifunctional antibody structure 1.

FIG. 23 shows the accelerated thermal stability detection of different antibodies with the multifunctional antibody structure 2 treated at 40° C. for 14 days.

FIG. 24 shows the acid resistance detection of different antibodies with multifunctional antibody structure 2.

FIG. 25 shows the accelerated thermal stability detection of different antibodies with the multifunctional antibody structure 3 treated at 40° C. for 14 days.

FIG. 26 shows the acid resistance detection of different antibodies with multifunctional antibody structure 3.

FIG. 27 is a structural schematic diagram of monoclonal antibodies.

FIG. 28 shows the binding of different Fc-modified 4420 mAbs to macrophages by flow cytometry detection and analysis.

FIG. 29 shows the binding of different Fc-modified 4420 mAbs to B cells by flow cytometry detection and analysis.

FIG. 30 shows the binding of different Fc-modified 4420 mAbs to NK cells by flow cytometry detection and analysis.

FIG. 31 shows the degree of activation of T cells in PBMC by CD3 monoclonal antibody with the residue at position 229 of Fc substituted by different amino acid residues via flow cytometry after the CH2 domain of the Fc of IGG1 CD3 monoclonal antibody is substituted with CH2 of IGG2, wherein (A) shows the ratio of CD69+in T cells, (B) shows the ratio of CD25+ in T cells.

FIG. 32 shows the degree of activation of T cells in PBMC by CD3 monoclonal antibody with the residue at position 265 of Fc substituted by different amino acid residues via flow cytometry after the CH2 domain of the Fc of IGG1 CD3 monoclonal antibody is substituted with CH2 of IGG2, wherein (A) shows the ratio of CD69+ in T cells, (B) shows the ratio of CD25+ in T cells.

FIG. 33 shows the degree of activation of T cells in PBMC by CD3 monoclonal antibody with the residue at position 270 of Fc substituted by different amino acid residues via flow cytometry after the CH2 domain of the Fc of IGG1 CD3 monoclonal antibody is substituted with CH2 of IGG2, wherein (A) shows the ratio of CD69+in T cells, (B) shows the ratio of CD25+ in T cells.

FIG. 34 shows the degree of activation of T cells in PBMC by CD3 monoclonal antibody with multiple site mutations of Fc via flow cytometry after the CH2 domain of the Fc of IGG1 CD3 monoclonal antibody is substituted with CH2 of IGG2, wherein (A) shows the ratio of CD69+in T cells, (B) shows the ratio of CD25+ in T cells.

DETAILED DESCRIPTION OF THE INVENTION

The specific embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings. The present disclosure can be implemented in many other ways which are different from those described herein, and those skilled in the art can make similar improvements without departing from the spirit of the present disclosure. Therefore, the protection scope of the present disclosure is defined by the claims, shall not be limited by the Examples disclosed below.

EXAMPLE 1
Preparation Of An Antibody
A. Construction Of Antibody Expression Plasmid

According to the sequences in Tables 28 to 30, the coding sequence DNA was synthesized by Wuhan Genecreate and cloned into the vector pcDNA3.1 (purchased from Invitrogen). Then the vector was transformed into Trans10 competent cells (purchased from Beijing TransGen Biotech). After sequencing, the expression plasmid was obtained.

The construction of the specific expression plasmids involved is as follows:

- 1) The multifunctional antibody structure 1 in FIG. 1 involves construction of three plasmids. The three plasmids are a light chain expression plasmid (pL), a heavy chain expression plasmid (pH), and a fusion peptide 1 expression plasmid (pF1) respectively.
- 2) The multifunctional antibody structure 2 in FIG. 2 involves construction of three plasmids.The three plasmids are a light chain expression plasmid (pL), a heavy chain expression plasmid (pH), and a fusion peptide 2 expression plasmid (pF2) respectively.
- 3) The multifunctional antibody structure 3 in FIG. 3 involves construction of four plasmids.The four plasmids are a light chain expression plasmid (pL), a heavy chain expression plasmid (pH), a cross light chain expression plasmid (pcL) and a fusion heavy chain expression plasmid (pFH) respectively.

B. Method Of Expressing Multifunctional Antibody

Two transient transfection expression systems, CHO-S (purchased from Gibco) or 293E (purchased from ATCC), were used for transfection.

The co-transfected plasmid DNA was as follows:

- 1) To express the multifunctional antibody 1 shown in FIG. 1, the plasmids pL, pH and pF1 were required to be co-transfected into CHO-S or 293E cells for expression;
- 2) To express the multifunctional antibody 2 shown in FIG. 2, the plasmids pL, pH and pF2 were required to be co-transfected into CHO-S or 293E cells for expression;
- 3) To express the multifunctional antibody 3 shown in FIG. 3, the plasmids pL, pH, pcL and pFH were required to be co-transfected into CHO-S or 293E cells for expression.

In general, if two plasmids were co-transfected for expression, the mole ratio of the two plasmids may be 1:1, or any other ratio; if three plasmids were co-transfected for expression, the mole ratio of the three plasmids may be 1:1:1, or any other ratio; if four plasmids were used, the mole ratio of the four plasmids may be 1:1:1:1, or any other ratio.

C. Method Of Purifying Multifunctional Antibody:

Antibody purification method mainly includes affinity chromatography, ion exchange chromatography, hydrophobic chromatography and molecular sieves, which are routine operations in the art. For details, please refer to the Molecular Cloning Experiment Guide. The first step of the purification method in the Example was protein A affinity chromatography, and then ion exchange chromatography was used to remove aggregates, so that the final protein purity reached to more than 95%.

The codes of some specifically expressed antibodies and the amino acid sequences of corresponding variable regions of antibody are shown in the following table:

TABLE 28

Code and amino acid sequence of some antibodies with multifunctional antibody structure 1

SEQ

Antibody
Poly-

ID

Code
Peptide
Domain
Code
Amino acid sequence (CDR is underlined in hold)
NO

PDL1-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
9

M1-NQ
Peptide 1

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLTGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED
10

EAEYYCVLWYSNRWVFGGGTKLTVL

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQD
89

WLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-b
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKLTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

CSAV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
S70
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQM
31

Chain

NSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQD
89

WLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-a
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

CSAV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
S70
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
32

Chain

QQYLYHPAT
FGQGTKVEIK

CL
CH1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

PDL1-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRPPSKNTLYLQ
1

M1-NQ-1
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin4
RGRGSDKTHTCP
69

CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQD
89

WLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-b
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

CSAV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
S70
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQM
31

Chain

NSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
N297Q
PCPAPELLCGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQD
89

WLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-a
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

CSAV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
S70
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
32

Chain

QQYLYHPAT
FGQGTKVEIK
75

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

PDL1-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGPTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

M1-G2
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin4
RGRGSDKTHTCP
69

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
94

LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

CSAV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
S70
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQM
31

Chain

NSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
94

LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
S70
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
32

Chain

QQYLYHPAT
FGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSITITSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

PDL1-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

M1-
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

SG2

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin4
RGRGSDKTHTCP
69

CH2
SG2CH
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
95

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

CSAV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
S70
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQM
31

Chain

NSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
SG2CH2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
95

LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
S70
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
32

Chain

QQYLYHPAT
FGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

PDL1-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

M1-FES
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin4
RGRGSDKTHTCP
69

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
85

WLNGKEYKCKVSNKALPASIEKTISKAK

CH3-b
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

CSAV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
S70
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQM
31

Chain

NSLRAEDTAVYYCARRHWPGQFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
85

WLNGKEYKCKVSNKALPASIEKTISKAK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
S70
DIQMTQSPSSUSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
32

Chain

QQYLYHPAT
FGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

Pdl1-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGPTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

M1-Lala
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin4
RGRGSDKTHTCP
69

CH2
LALA
PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
90

DWLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-b
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKITPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

CSAV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
S70
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAW1SPYGGSTYYADSVKGRFTISADTSKNTAYLQM
31

Chain

NSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
LALA
PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
90

DWLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
S70
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
32

Chain

QQYLYHPAT
FGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

PDL1-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

M1-WT
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQKPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDK
2

AEYYCALWYSNLWVFGGGIKVEIK

Hinge1
Hin4
RGRGSDKTHTCP
69

CH2
WT
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
83

WLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-b
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKlTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

CSAV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
S70
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQM
31

Chain

NSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
WT
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
83

WLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
S70
DIQMTQSPSSLSASVGDRVTlTCRASQDVSTAVAWYQQKPGKAPKLLlYSASFLYSGVPSRFSGSGSGTDFrLTISSLQPEDFATYYC
32

Chain

QQYLYHPAT
FGQGTKVElK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYBKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

CD38-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

M1-
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS
54

FES

Linker1
Lin10
GGGGSGGGGSGGGGS

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AFYYCALWYSNLWVFGGGTKVFIK

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
85

WLNGKEYKCKVSNKALPASIEKTISKAK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPEXNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMN
15

Chain

SLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
85

WLNGKEYKCKVSNKALPASIEKTISKAK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKITPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQ
16

Chain
CL
Lc1

QRSNWPPT
FGQGTKVEIK
75

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

CD38-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

M1-G2
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS
54

Linker1
Lin10
GGGGSGGGGSGGGGS

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin6
GRGRGSDKTHTCP
71

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
94

LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMN
15

Chain

SLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
94

LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQ
16

Chain

QRSNWPPT
FGQGTKVEIK
75

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

CD38-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLKWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

M1-
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

SG2

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin6
GRGRGSDKTHTCP
71

CH2
SG2CH
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
95

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMN
15

Chain

SLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS
82

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
SG2CH
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
95

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSYSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQ
16

Chain

QRSNWPPT
FGQGTKVEIK
75

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

CD38-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

M1-
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

SG2-1

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTWTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin6
GRGRGSDKTHTCP
71

CH2
SG2CH
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
95

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
2F5
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGRVIPFLGIANSAQKFQGRVHTADKSTSTAYMDL
19

Chain

SSLRSEDTAVYYCARDDIAALGPFDYWGQCTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
SG2CH
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPKVQFNWYVDGVEVHNAKTKPREKQFNSTFRVVSVLTVVHQDW
95

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKITPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
2F5
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY
20

Chain

CQQYNSYPRTFGQGTKVEIK
75

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

CD38-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

M1-G2-
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS
54

3

Linker1
Lin10
GGGGSGGGGSGGGGS

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin6
GRGRGSDKTHTCP
71

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
94

LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
2F5
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGRVIPFLGIANSAQKFQGRVnTADKSTSTAYMDL
19

Chain

SSLRSEDTAVYYCARDDIAALGPFDYWGQCTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
94

LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
2F5
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY
20

Chain

CQQYNSYPRTFGQGTKVEIK
75

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSUSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

CD38-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

M1-
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

SG2-2

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin6
GRGRGSDKTHTCP
71

CH2
SG2CH2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
95

LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
MOR
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVSGISGDPSNTYYADSVKGRFTISRDNSKNTLYLQM
17

Chain

NSLRAEDTAVYYCARDLPLVYTGFAYWGQGTLVTVSSASTKGPS

CH1
CH1
VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
SG2CH2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
95

LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
MOR
DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSKRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYC
18

Chain

QTYTGGASLV
FGGGTKLTVLGQ

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

CD38-
Fusion
VHs
2a5
QVQLVFSGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLFWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

M1-G2-
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

2

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin6
GRGRGSDKTHTCP
71

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTERVVSVLTVVHQDW
94

LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

CSAV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
MOR
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVSGISGDPSNTYYADSVKGRFTISRDNSKNTLYLQM
17

Chain

NSLRAEDTAVYYCARDLPLVYTQFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW

LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
94

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
MOR
DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSKRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYC
18

Chain

QTYTGGASLV
FGGGTKLTVLGQ

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

CD38
Fusion
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGQTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

M1-WT
Peptide 1

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin6
GRGRGSDKTHTCP
71

CH2
WT
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
83

WLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMN
15

Chain

SLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
WT
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
83

WLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQ
16

Chain

QRSNWPPT
FGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

CD38-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
1

M1-
Peptide 1

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS

FES-1

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGQTKFLAPGTPARFSGSLLGGKAALTLSGVQPED
10

EAEYYCVLWYSNRWVFGGGTKLTVL

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
85

WLNGKEYKCKVSNKALPASIEKTISKAK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMN
15

Chain

SLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
85

WLNGKEYKCKVSNKALPASIEKTISKAK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQ
16

Chain

QRSNWPPT
FGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

CD38
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

M1-WT
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGQTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin6
GRGRGSDKTHTCP
71

CH2
WT
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
83

WLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMN
15

Chain

SLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
WT
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
83

WLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQ
16

Chain

QRSNWPPT
FGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

CD38-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
Q

M1-
Peptide 1

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS

FES-1

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGQTKFLAPGTPARFSGSLLGGKAALTLSGVQPED
10

EAEYYCVLWYSNRWVFGGGTKLTVL

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
85

WLNGKEYKCKVSNKALPASIEKTISKAK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMN
15

Chain

SLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
85

WLNGKEYKCKVSNKALPASIEKTISKAK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQ
16

Chain

QRSNWPPT
FGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

CD38-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASCFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
9

M1-
Peptide 1

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
54

G2-1

Linker1
Lin10
GGGGSGGGGSGGGGS

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED
10

EAEYYCVLWYSNRWVFGGGTKLTVL

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
94

LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMN
15

Chain

SLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
94

LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDPTLTISSLEPEDFAVYYCQ
16

Chain

QRSNWPPT
FGQGTKVEIK
75

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

CD38-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
9

M1-
Peptide 1

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
54

SG2-3

Linker1
Lin10
GGGGSGGGGSGGGGS

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED
10

EAEYYCVLWYSNRWVFGGGTKLTVL

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
SG2CH
PSPAPPVAGPSVFLFPPKPKDILMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
95

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKLTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMN
15

Chain

SLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
SG2CH
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
95

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSCiTDFTLTISSLEPEDFAVYYCQ
16

Chain

QRSNWPPT
FGQGTKVEIK
75

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

CD38-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
9

M1-
Peptide 1

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
54

AG2

Linker1
Lin10
GGGGSGGGGSGGGGS

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED
10

EAEYYCVLWYSNRWVFGGGTKLTVL
68

Hinge1
Hin3
GGGGSDKTHTCP

CH2
AG2CH
PAPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
96

2
WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMN
15

Chain

SLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
AG2CH
PAPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
96

2
WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQ
16

Chain

QRSNWPPT
FGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
9

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

CD38-
Fusion
VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED
10

M1-
Peptide 1

EAEYYCVLWYSNRWVFGGGTKLTVL

GG2

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
GG2CH2
PGPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
97

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMN
15

Chain

SLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
GG2CH
PGPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
97

2
WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQ
16

Chain

QRSNWPPT
FGQGTKVEIK
75

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

CD38-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
9

M1-
Peptide 1

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
54

PG2

Linker1
Lin10
GGGGSGGGGSGGGGS

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED
10

EAEYYCVLWYSNRWVFGGGTKLTVL
68

Hinge1
Hin3
GGGGSDKTHTCP

CH2
PG2CH
PPPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
98

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMN
15

Chain

SLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
PG2CH
PPPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
98

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLnSSLEPEDFAVYYCQ
16

Chain
CL
Lc1

QRSNWPPT
FGQGTKVEIK

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

CD38-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
9

M1-
Peptide 1

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS

DG2

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED
10

EAEYYCVLWYSNRWVFGGGTKLTVL

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
DG2CH
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
99

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMN
15

Chain

SLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
DG2CH
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
99

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQ
16

Chain

QRSNWPPT
FGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

CD38-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
9

M1-
Peptide 1

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
54

G2D

Linker1
Lin10
GGGGSGGGGSGGGGS

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED
10

EAEYYCVLWYSNRWVFGGGTKLTVL

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
G2DCH
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
100

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMN
15

Chain

SLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
G2DCH
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
100

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQ
16

Chain

QRSNWPPT
FGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLILSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

CD38-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
9

M1-
Peptide 1

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS

DG2D

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED

EAEYYCVLWYSNRWVFGGGTKLTVL
10

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
DG2DC
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
101

H2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCrLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMN
15

Chain

SLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
DG2DC
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
101

H2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDPTLTISSLEPHDFAVYYCQ
16

Chain

QRSNWPPT
FGQGTKVEIK
75

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

CD38-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAVINWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
9

M1-
Peptide 1

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
54

FES-3

Linker1
Lin10
GGGGSGGGGSGGGGS

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED
10

EAEYYCVLWYSNRWVFGGGTKLTVL

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
85

WLNGKEYKCKVSNKALPASIEKTISKAK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
MOR
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVSGISGDPSNTYYADSVKGRFTISRDNSKNTLYLQM
17

Chain

NSLRAEDTAVYYCARDLPLVYTGFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
85

WLNGKEYKCKVSNKALPASIEKTISKAK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
M0R
DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSKRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYC
18

Chain

QTYTGGASLV
FGGGTKLTVLGQ
77

CL
Lc3
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVEITTPSKQSNNKYAASSYLSLTPEQWKSHRSYS

CQVTHEGSTVEKTVAPTECS

CD38-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

M1-
Peptide 1
Linker1
Lin10
MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS
54

G2D-1

GGGGSGGGGSGGGGS

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin6
GRGRGSDKTHTCP
71

CH2
G2DCH
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
100

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCRLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
MOR
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVSGISGDPSNTYYADSVKGRFTISRDNSKNTLYLQM
17

Chain

NSLRAEDTAVYYCARDLPLVYTQFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
G2DCH
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
100

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
MOR
DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSKRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYC
18

Chain

QTYTGGASLV
FGGGTKLTVLGQ

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
9

SG2
Peptide 1

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED
10

EAEYYCVLWYSNRWVFGGGTKLTVL

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
SG2CH
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
95

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAITSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
SG2CH
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPKVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
95

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVHAE
44

Chain

DLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
9

G2
Peptide 1
Linker1
Lin10
LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
54

GGGGSGGGGSGGGGS

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED
10

EAEYYCVLWYSNRWVFGGGTKLTVL

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
94

LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
94

LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKITPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain

DLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNPYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
9

FES
Peptide 1

LQMNNLKTEDTAVYYCVRHGNFQNSYISYWAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED
10

EAEYYCVLWYSNRWVFGGGTKLTVL

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
85

WLNGKEYKCKVSNKALPASIEKTISKAK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
85

WLNGKEYKCKVSNKALPASIEKTISKAK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQtPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain
CL
Lc1
DLGVYFCSQSTHVPWTFGGGTKLEIK
75

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
9

NA
Peptide 1

LQMNNLKTEDTAVYYCVRHQNpQN§YISYWAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED
10

EAEYYCVLWYSNRWVFGGGTKLTVL

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
N297A
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQD
87

WLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
N297A
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQD
87

WLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKITPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain

DLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

NQ
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYYSWFAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQD
89

WLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCRGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC
66

Hinge2
Hin1
DKTHTCP
89

CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQD

WLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKITPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain

DLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
9

AAG
Peptide 1

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
54

Linker1
Lin10
GGGGSGGGGSGGGGS

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED
10

EAEYYCVLWYSNRWVFGGGTKLTVL

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
AAG
PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
84

DWLNGKEYKCKVSNKALGAPIEKTISKAK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
AAG
PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
84

DWLNGKEYKCKVSNKALGAPIEKTISKAK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQLTLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFRLKISRVEAE
44

Chain

DLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSYKDRFTISRDDSKNTAY
9

AG2
Peptide 1

LQMNNLKTEDTAVYYCVRHgNFgNSYISYWAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTTSGVQPED
10

EAEYYCVLWYSNRWVFGGGTKLTVL

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
AG2CH
PAPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
96

2
WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain
CH1
CH1
QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS
82

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
AG2CH
PAPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
96

2
VVLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKITPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain
CL
Lc1
DLGVYFCSQSTHVPWTFGGGTKLEIK
75

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
9

GG2
Peptide 1

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
54

Linker1
Lin10
GGGGSGGGGSGGGGS

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALrLSGVQPED
10

EAEYYCVLWYSNRWVFGGGTKLTVL

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
GG2CH
PGPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
97

2
WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
GG2CH
PGPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
97

2
WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain

DLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
9

PG2
Peptide 1

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED
10

VLs
I2C
EAEYYCVLWYSNRWVFGGGTKLTVL

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
PG2CH
PPPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
98

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTTSRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
PG2CH
PPPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
98

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain
CL
Lc1
DLGVYFCSQSTHVPWTFGGGTKLEIK
75

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNVATYYADSVKDRFTISRDDSKNTAY
9

DG2
Peptide 1

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED
10

EAEYYCVLWYSNRWVFGGGTKLTVL

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
DG2CH
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
99

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
DG2CH
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
99

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain

DLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
9

G2D
Peptide 1

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED
10

EAEYYCVLWYSNRWVFGGGTKLTVL

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
G2DCH
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
100

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
G2DCH
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
100

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain
CL
Lc1
DLGVYFCSQSTHVPWTFGGGTKLEIK
75

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

MlIC-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
9

DG2D
Peptide 1

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
54

Linker1
Lin10
GGGGSGGGGSGGGGS

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED
10

EAEYYCVLWYSNRWVFGGGTKLTVL
68

Hinge1
Hin3
GGGGSDKTHTCP

CH2
DG2DC
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
101

H2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
DG2DC
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
101

H2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain

DLGVYFCSQSTHVPWTFGGGTKLEIK
75

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
I2C
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY
9

WT
Peptide 1

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS
54

Linker1
Lin10
GGGGSGGGGSGGGGS

VLs
I2C
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED
10

EAEYYCVLWYSNRWVFGGGTKLTVL
68

Hinge1
Hin3
GGGGSDKTHTCP

CH2
WT
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
83

WLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQIYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
WT
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
83

WLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain

DLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

MlIC-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

FES-1
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
FES
PCPAPEFECGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
85

WLNGKEYKCKVSNKALPASIEKTISKAK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKITPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFITSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
85

WLNGKEYKCKVSNKALPASIEKTISKAK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain

DLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

AAG-1
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
AAG
PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
84

DWLNGKEYKCKVSNKALGAPIEKTISKAK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
AAG
PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
84

DWLNGKEYKCKVSNKALGAPIEKTISKAK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain

DLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

G2-1
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
94

LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain
CH1
CH1
QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS
82

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
94

LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain

DLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

SG2-1
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
SG2CH
PSPAPPVAGPSVPLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
95

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGPTFSDYYVMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRPTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGVIDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
SG2CH
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
95

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain

DLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

AG2-1
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVVFGGGTKVEIK

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
AG2CH
PAPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
96

2
WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
AG2CH
PAPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
96

2
WLNGKEYKCKVSNKGLPAPIEKTISKTK

GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain
CL
Lc1
DLGVYFCSQSTHVPWTFGGGTKLEIK
75

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

GG2-1
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
GG2CH
PGPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
97

2
WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
Cw:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
GG2CH
PGPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
97

2
WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain

DLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

PG2-1
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS
54

Linker1
Lin10
GGGGSGGGGSGGGGS

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
PG2CH
PPPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
98

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTESDYVVMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRETISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
PG2CH
PPPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
98

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain

DLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

DG2-1
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
DG2CH
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
99

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
DG2CH
PCPAPPVAGPSVFLFPPKPKDILMISRIPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKIKPREEQFNSIFRVVSVLTVVHQDW
99

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKITPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

light
VLm
4420
DVVMIQIPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain

DLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

G2D-1
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS
54

Linker1
Lin10
GGGGSGGGGSGGGGS

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
G2DCH
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
100

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
G2DCH
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
100

2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain

DLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

DG2D-
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

1

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
DG2DC
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
101

H2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
DG2DC
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
101

H2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRVVYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain

DLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

DG2D-
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

1A

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
DG2DC
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
101

H2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVR
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
107

F:CW
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGPTFSDYWMNWVRQSPEKGLHWVAQIRNKPYNYETYYSDSVKGRPTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
DG2DC
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
101

H2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVR
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
106

F:CW
SVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain

DLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lcl
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

DG2D-
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

1B

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
DG2DC
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
101

H2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAV:C
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
115

WRF
VMHEALHNRFTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
DG2DC
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
101

H2
LNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAV:C
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
114

WRF
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain

DLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

M1IC-
Fusion
VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQ
1

WT-1
Peptide 1

MNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker1
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDE
2

AEYYCALWYSNLWVFGGGTKVEIK

Hinge1
Hin3
GGGGSDKTHTCP
68

CH2
WT
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
83

WLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-b
CW:CS
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSC
109

AV
SVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYL
43

Chain

QMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
82

KPSNTKVDKKVEPKSC

Hinge2
Hin1
DKTHTCP
66

CH2
WT
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
83

WLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-a
CW:CS
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
108

AV
SVMHEALHNHYTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
44

Chain

DLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
75

ACEVTHQGLSSPVTKSFNRGEC

TABLE 29

Code and amino acid sequence of some antibodies with multifunctional antibody structure 2

custom-character

Antibody Code
Polypeptide
Domain
Code
Amino acid sequence (CDR is underlined in bold)

custom-character

BCMA-
Fusion
VHm
B69
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSV
29

M2-WT
Peptide

TAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSS

2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
WT
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
83

DWLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
B69
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSV
29

Chain

TAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
WT
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
83

DWLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
B69
SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSDRPSGIPERFSGNSNGNTATLTISRVEAGDEAVY
30

Chain

YCQVWDSSSDHVVFGGGTKLTVL

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

BCMA-
Fusion
VHm
B69
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSV
29

M2-
Peptide

TAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSS

FES
2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
85

DWLNGKEYKCKVSNKALPASIEKTISKAK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
B69
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSV
29

Chain

TAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
85

DWLNGKEYKCKVSNKALPASIEKTISKAK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
B69
SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSDRPSGIPERFSGNSNGNTATLTISRVEAGDEAVY
30

Chain

YCQVWDSSSDHVVFGGGTKLTVL

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

BCMA-
Fusion
VHm
B69
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSV
29

M2-G2
Peptide

TAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSS

2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
94

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
B69
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSV
29

Chain

TAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
94

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
B69
SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSDRPSGIPERFSGNSNGNTATLTISRVEAGDEAVY
30

Chain

YCQVWDSSSDHVVFGGGTKLTVL

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

BCMA-
Fusion
VHm
B69
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSV
29

M2-
Peptide

TAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSS

SG2
2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
SG2CH2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
95

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
B69
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSV
29

Chain

TAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
SG2CH2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
95

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
B69
SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSDRPSGIPERFSGNSNGNTATLTISRVEAGDEAVY
30

Chain

YCQVWDSSSDHVVFGGGTKLTVL

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

BCMA-
Fusion
VHm
B69
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSV
29

M2-
Peptide

TAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSS

AG2
2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
AG2CH2
PAPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
96

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
B69
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSV
29

Chain

TAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
AG2CH2
PAPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
96

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
B69
SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSDRPSGIPERFSGNSNGNTATLTISRVEAGDEAVY
30

Chain

YCQVWDSSSDHVVFGGGTKLTVL

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

BCMA-
Fusion
VHm
B69
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSV
29

M2-
Peptide

TAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSS

GG2
2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
GG2CH2
PGPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
97

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
B69
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSV
29

Chain

TAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
GG2CH2
PGPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
97

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
B69
SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSDRPSGIPERFSGNSNGNTATLTISRVEAGDEAVY
30

Chain

YCQVWDSSSDHVVFGGGTKLTVL

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

BCMA-
Fusion
VHm
B69
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSV
29

M2-
Peptide

TAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSS

PG2
2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
PG2CH2
PPPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
98

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
B69
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSV
29

Chain

TAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
PG2CH2
PPPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
98

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
B69
SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSDRPSGIPERFSGNSNGNTATLTISRVEAGDEAVY
30

Chain

YCQVWDSSSDHVVFGGGTKLTVL

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

BCMA-
Fusion
VHm
B69
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSV
29

M2-
Peptide

TAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSS

DG2
2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
DG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
99

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
B69
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSV
29

Chain

TAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
DG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
99

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
B69
SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSDRPSGIPERFSGNSNGNTATLTISRVEAGDEAVY
30

Chain

YCQVWDSSSDHVVFGGGTKLTVL

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

BCMA-
Fusion
VHm
B69
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSV
29

M2-
Peptide

TAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSS

G2D
2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
G2DCH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
100

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
B69
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSV
29

Chain

TAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
G2DCH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
100

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
B69
SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSDRPSGIPERFSGNSNGNTATLTISRVEAGDEAVY
30

Chain

YCQVWDSSSDHVVFGGGTKLTVL

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

BCMA-
Fusion
VHm
B69
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSV
29

M2-
Peptide

TAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSS

DG2D
2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
DG2DCH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
101

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
B69
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSV
29

Chain

TAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
DG2DCH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
101

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
B69
SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSDRPSGIPERFSGNSNGNTATLTISRVEAGDEAVY
30

Chain

YCQVWDSSSDHVVFGGGTKLTVL

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

M2IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

WT
Peptide

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
WT
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
83

DWLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

Chain

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
WT
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
83

DWLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEA
44

Chain

EDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

M2IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

AAG
Peptide

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
AAG
PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH
84

QDWLNGKEYKCKVSNKALGAPIEKTISKAK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

Chain

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
AAG
PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH
84

QDWLNGKEYKCKVSNKALGAPIEKTISKAK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEA
44

Chain

EDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

M2IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

NA
Peptide

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
N297A
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQ
87

DWLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

Chain

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
N297A
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQ
87

DWLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEA
44

Chain

EDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

M2IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

FES
Peptide

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
85

DWLNGKEYKCKVSNKALPASIEKTISKAK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

Chain

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
85

DWLNGKEYKCKVSNKALPASIEKTISKAK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEA
44

Chain

EDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

M2IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

G2
Peptide

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
94

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

Chain

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
94

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEA
44

Chain

EDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

M2IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

SG2
Peptide

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
SG2CH2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
95

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

Chain

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
SG2CH2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
95

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEA
44

Chain

EDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

M2IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

AG2
Peptide

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
AG2CH2
PAPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
96

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

Chain

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
AG2CH2
PAPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
96

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEA
44

Chain

EDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

M2IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

GG2
Peptide

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
GG2CH2
PGPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
97

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

Chain

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
GG2CH2
PGPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
97

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEA
44

Chain

EDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

M2IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

PG2
Peptide

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
PG2CH2
PPPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
98

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

Chain

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
PG2CH2
PPPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
98

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEA
44

Chain

EDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

M2IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

DG2
Peptide

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
DG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
99

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

Chain

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
DG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
99

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEA
44

Chain

EDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

M2IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

G2D
Peptide

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
G2DCH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
100

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

Chain

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
G2DCH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
100

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEA
44

Chain

EDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

M2ic-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

Dg2d
Peptide

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

2
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge1
Hin9
DKTHT
74

Linker2
Lin4
GGGSAAA
48

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYL
1

QMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker3
Lin10
GGGGSGGGGSGGGGS
54

VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPED
2

EAEYYCALWYSNLWVFGGGTKVEIK

Hinge2
Hin3
GGGGSDKTHTCP
68

CH2
DG2DCH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
101

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
107

CW
CSVMHEALHNHYTQKSLSLSPGK

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVY
43

Chain

LQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
DG2DCH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD
101

WLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFS
106

CW
CSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEA
44

Chain

EDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

TABLE 30

Code and amino acid sequence of some antibodies with multifunctional antibody structure 3

custom-character

Antibody Code
Polypeptide
Domain
Code
Amino acid sequence (CDR is underlined in bold)

custom-character

CEA-
Fusion
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

M3-
Heavy

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

WT
Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
WT
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
83

HQDWLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

Chain

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
WT
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
83

HQDWLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
hPR1A3
DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIA
42

Chain

TYYCHQYYTYPLFTFGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

CEA-
Fusion
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

M3-
Heavy

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

AAG
Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
AAG
PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
84

LHQDWLNGKEYKCKVSNKALGAPIEKTISKAK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

Chain

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
AAG
PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
84

LHQDWLNGKEYKCKVSNKALGAPIEKTISKAK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
hPR1A3
DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIA
42

Chain

TYYCHQYYTYPLFTFGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

CEA-
Fusion
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

M3-
Heavy

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

NA
Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
N297A
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL
87

HQDWLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

Chain

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
N297A
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL
87

HQDWLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
hPR1A3
DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIA
42

Chain

TYYCHQYYTYPLFTFGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

CEA-
Fusion
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

M3-
Heavy

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

FES
Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
85

HQDWLNGKEYKCKVSNKALPASIEKTISKAK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

Chain

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
85

HQDWLNGKEYKCKVSNKALPASIEKTISKAK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
hPR1A3
DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIA
42

Chain

TYYCHQYYTYPLFTFGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

CEA-
Fusion
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

M3-
Heavy

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

G2
Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTERVVSVLTVVH
94

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

Chain

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTERVVSVLTVVH
94

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
hPR1A3
DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIA
42

Chain

TYYCHQYYTYPLFTFGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

CEA-
Fusion
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

M3-
Heavy

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

SG2
Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
SG2CH2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTERVVSVLTVVH
95

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

Chain

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
SG2CH2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTERVVSVLTVVH
95

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
hPR1A3
DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIA
42

Chain

TYYCHQYYTYPLFTFGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

CEA-
Fusion
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

M3-
Heavy

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

AG2
Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
AG2CH2
PAPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH
96

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

Chain

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
AG2CH2
PAPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH
96

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
hPR1A3
DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIA
42

Chain

TYYCHQYYTYPLFTFGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

CEA-
Fusion
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

M3-
Heavy

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

GG2
Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
GG2CH2
PGPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTERVVSVLTVVH
97

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

Chain

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
GG2CH2
PGPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTERVVSVLTVVH
97

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
hPR1A3
DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIA
42

Chain

TYYCHQYYTYPLFTFGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

CEA-
Fusion
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

M3-
Heavy

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

PG2
Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
PG2CH2
PPPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTERVVSVLTVVH
98

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

Chain

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
PG2CH2
PPPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTERVVSVLTVVH
98

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
hPR1A3
DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIA
42

Chain

TYYCHQYYTYPLFTFGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

CEA-
Fusion
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

M3-
Heavy

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

DG2
Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
DG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTERVVSVLTVVH
99

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

Chain

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
DG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH
99

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
hPR1A3
DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIA
42

Chain

TYYCHQYYTYPLFTFGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

CEA-
Fusion
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

M3-
Heavy

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

G2D
Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
G2DCH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH
100

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

Chain

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
G2DCH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH
100

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
hPR1A3
DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIA
42

Chain

TYYCHQYYTYPLFTFGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

CEA-
Fusion
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

M3-
Heavy

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

DG2D
Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
DG2DCH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTERVVSVLTVVH
101

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRFVFSLDTSVSTA
41

Chain

YLQISSLKADDTAVYYCARWDFYDYVEAMDYWGQGTTVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
DG2DCH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTERVVSVLTVVH
101

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
hPR1A3
DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIA
42

Chain

TYYCHQYYTYPLFTFGQGTKVEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

M3IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

WT
Heavy

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
WT
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
83

HQDWLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

Chain

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
WT
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
83

HQDWLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSGISLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISR
44

Chain

VEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

M3IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

AAG
Heavy

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
AAG
PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
84

LHQDWLNGKEYKCKVSNKALGAPIEKTISKAK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

Chain

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
AAG
PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
84

LHQDWLNGKEYKCKVSNKALGAPIEKTISKAK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISR
44

Chain

VEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

M3IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

NA
Heavy

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
N297A
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL
87

HQDWLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

Chain

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
N297A
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL
87

HQDWLNGKEYKCKVSNKALPAPIEKTISKAK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISR
44

Chain

VEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

M3IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

FES
Heavy

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
85

HQDWLNGKEYKCKVSNKALPASIEKTISKAK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

Chain

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
85

HQDWLNGKEYKCKVSNKALPASIEKTISKAK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISR
44

Chain

VEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

M3IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

G2
Heavy

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTERVVSVLTVVH
94

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

Chain

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTERVVSVLTVVH
94

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSGISLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISR
44

Chain

VEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

M3IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

SG2
Heavy

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
SG2CH2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH
95

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

Chain

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
SG2CH2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH
95

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISR
44

Chain

VEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

M3IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

AG2
Heavy

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
AG2CH2
PAPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH
96

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

Chain

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
AG2CH2
PAPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH
96

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSGISLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISR
44

Chain

VEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

M3IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

GG2
Heavy

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
GG2CH2
PGPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH
97

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

Chain

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
GG2CH2
PGPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH
97

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISR
44

Chain

VEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

M3IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

PG2
Heavy

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
PG2CH2
PPPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH
98

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

Chain

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
PG2CH2
PPPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH
98

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISR
44

Chain

VEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

M3IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

DG2
Heavy

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
DG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH
99

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

Chain

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
DG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH
99

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISR
44

Chain

VEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

M3IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

G2D
Heavy

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
G2DCH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTERVVSVLTVVH
100

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

Chain

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
G2DCH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTERVVSVLTVVH
100

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISR
44

Chain

VEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

M3IC-
Fusion
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

DG2D
Heavy

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

Chain
CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Linker4
Lin7
GGGGSGGGGS
51

VHs
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT
1

LYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

Linker5
Lin2
AS
46

CL
Lc7
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
81

YACEVTHQGLSSPVTKSFNRGEC

Hinge4
Hin1
DKTHTCP
66

CH2
DG2DCH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTERVVSVLTVVH
101

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-b
CSAVRF:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
107

CW
FSCSVMHEALHNHYTQKSLSLSPGK

Crosslight
VLs
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQ
2

Chain

PEDEAEYYCALWYSNLWVFGGGTKVEIK

Linker6
Lin1
SS
45

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSS
43

Chain

VYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
82

VNHKPSNTKVDKKVEPKSC

Hinge3
Hin1
DKTHTCP
66

CH2
DG2DCH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH
101

QDWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3-a
CSAVRF:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV
106

CW
FSCSVMHEALHNRFTQKSLSLSPGK

Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISR
44

Chain

VEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
75

KVYACEVTHQGLSSPVTKSFNRGEC

EXAMPLE 2
Detection of Antibody Biological Activity

1. Cell Affinity

1) Cell preparation: T cells isolated from CD3-positive human whole blood were used for a CD3 end affinity detection of multifunctional antibody molecule; positive tumor cells of corresponding antigen were used for detecting affinity of tumor antigen; for example, CD38-positive MM. 1S cells (purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) or RPMI 8226 cells (purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were used for CD38 antigen detection, and PD-L1 positive H358 cells (purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were used for PD-L1 antigen detection, and so on. The cells were resuspended with 1% FBS-PBS, adjusted to a density of 4×10⁶/ml, and 50 μl of cells were taken from each well, and then plated at 2×10⁵per well. Cell plating was operated on ice

2) Addition of the antibody: according to the experimental design, the antibody was diluted gradually, and dilution of antibody was operated on ice. For example, the initial concentration of antibody for dilution was 3000 nM, and the antibody was diluted by 3 times with 11 concentration gradients. The diluted antibody was added to a cell well at 50 μl per well, mixed well, and shaked and incubated at 4° C. 1100 rpm/min for 2 h; after washing, the cells were resuspend in 1% FBS-PBS, and a diluted secondary antibody PE anti-human IgG FC (Biolegend, 409304) was added with a final concentration of 8 ug/ml and a volume of 50 μl/well. At the same time, a well only added with cells and secondary antibody was set as a control, the components were mixed well, and incubated under shaking at 1100 rpm/min at 4° C. for 1 h;

3) Washing and fixation: after the washed cells were resuspended in 1% FBS-PBS, 2% paraformaldehyde was added into each well to fix the cells at room temperature;

4) Flow cytometry detection: the cells were resuspended with 1% FBS-PBS, and detected on a flow cytometer;

5) Data analysis: the data was analyzed by a flow analysis software FlowJo 7.6 thereby obtaining an average fluorescence intensity of the specific antibody concentration, plotted by a Graphpad Prism 5 with the antibody concentration (nM) as an abscissa and the average fluorescence intensity as an ordinate; the EC50 value was calculated by the method of One site—Specific binding, and the EC50 value indicated the cell affinity of the antibody to the corresponding target antigen.

TABLE 31

Cell affinity and transient transfection expression level of some

antibodies with the multifunctional antibody structure 1

Affinity to
Affinity to
Transient

Human
Monkey
Transfection

CD3
CD3
Expression

Affinity to
Positive T
Positive T
Level in

Tumor Cell
Cell
Cell
293E

Antibody Code
(Nm)
(Nm)
(Nm)
(Mg/L)

PDL1-M1-NQ
1.48
3.61
6.89
67.9

PDL1-M1-NQ-1
0.95
145
168
33.4

PDL1-M1-G2
1.29
191
239
87.7

PDL1-M1-SG2
1.06
177
194
76.8

PDL1-M1-FES
1.01
200
207
41.4

PDL1-M1-LALA
1.38
153
196
62.6

PDL1-M1-WT
1.41
135
161
88.3

CD38-M1-FES
1.53
192
291
67.4

CD38-M1-G2
2.69
91.9
142
60.2

CD38-M1-SG2
1.76
107
159
62.8

CD38-M1-WT
2.33
129
150
28.4

CD38-M1-SG2-1
11.9
88.1
94.13
89.9

CD38-M1-SG2-2
106
437
461
67.1

CD38-M1-G2-2
104
478
491
70.5

CD38-M1-G2-3
10.2
91.7
103
96.5

CD38-M1-FES-1
3.20
8.50
9.18
29.6

CD38-M1-G2-1
2.10
9.21
12.61
78.2

CD38-M1-SG2-3
1.54
6.48
7.59
55.2

CD38-M1-AG2
1.79
6.19
10.19
63.0

CD38-M1-GG2
1.87
7.31
10.42
69.8

CD38-M1-PG2
2.33
8.16
11.47
59.9

CD38-M1-DG2
2.40
8.22
11.04
50.4

CD38-M1-G2D
2.16
8.67
10.08
69.2

CD38-M1-DG2D
1.62
6.48
7.05
53.6

CD38-M1-FES-3
11.1
20
34.05
39.2

CD38-M1-G2D-1
100
405
420
77.2

M1IC-FES
No binding
7.61
11.39
62.5

M1IC-NA
No binding
7.68
9.63
52.3

M1IC-NQ
No binding
194
378
50.9

M1IC-AAG
No binding
5.94
8.79
55.2

M1IC-G2
No binding
6.65
10.39
58.7

M1IC-SG2
No binding
9.38
13.24
63.3

M1IC-AG2
No binding
11.0
14.2
50.0

M1IC-GG2
No binding
9.93
13.0
64.5

M1IC-PG2
No binding
11.27
17.8
52.6

M1IC-DG2
No binding
7.52
14.5
59.9

M1IC-G2D
No binding
5.77
10.6
64.9

M1IC-DG2D
No binding
9.37
16.0
58.9

MlIC-WT
No binding
7.15
19.8
55.5

M1IC-FES-1
No binding
170
295
54.2

M1IC-AAG-1
No binding
152
205
47.4

M1IC-SG2-1
No binding
122
222
87.5

M1IC-G2-1
No binding
127
165
44.6

M1IC-AG2-1
No binding
163
197
69.9

M1IC-GG2-1
No binding
168
166
55.6

M1IC-PG2-1
No binding
116
146
52.6

M1IC-DG2-1
No binding
120
130
71.3

M1IC-G2D-1
No binding
101
143
69.8

M1IC-DG2D-1
No binding
128
181
70.3

M1IC-WT-1
No binding
153
174
78.8

Note: PDL1-M1 series of molecules were antibodies targeting both PD-L1 and CD3 with multifunctional antibody structure 1; CD38-M1 series of molecules were antibodies targeting both CD38 and CD3 with multifunctional antibody structure 1; M1IC series of molecules were all isotype control antibodies with multifunctional antibody structure 1 and targeting CD3 and luciferase (without tumor targeting ability).

TABLE 32

Cell affinity and transient transfection expression level of some antibodies

with the multifunctional antibody structure 2

Transient

Affinity to
Affinity to
Affinity to
Transfection

Human
Human CD3
Monkey CD3
Expression

Tumor Cell
Positive T Cell
Positive T Cell
Level in293E

Antibody Code
(Nm)
(Nm)
(Nm)
(Mg/L)

BCMA-M2-WT
20.3
588
676
62.5

BCMA-M2-FES
15.3
532
630
37.2

BCMA-M2-G2
19.1
515
591
53.8

BCMA-M2-SG2
17.0
400
461
48.4

BCMA-M2-AG2
23.8
613
668
50.0

BCMA-M2-GG2
23.6
448
636
44.6

BCMA-M2-PG2
24.0
599
716
48.3

BCMA-M2-DG2
17.4
550
501
53.0

BCMA-M2-G2D
19.2
351
510
59.7

BCMA-M2-DG2D
18.8
534
641
41.7

M2IC-WT
No binding
499
540
81.0

M2IC-AAG
No binding
515
528
59.8

M2IC-NA
No binding
547
561
35.9

M2IC-FES
No binding
411
506
40.2

M2IC-G2
No binding
408
488
75.5

M2IC-SG2
No binding
481
525
70.7

M2IC-AG2
No binding
535
607
51.6

M2IC-GG2
No binding
556
538
43.3

M2IC-PG2
No binding
467
656
70.8

M2IC-DG2
No binding
357
570
44.7

M2IC-G2D
No binding
367
538
72.1

M2IC-DG2D
No binding
452
519
67.6

Note: BCMA-M2 series were antibodies targeting both BCMA and CD3 with multifunctional antibody structure 2; M2IC series were all isotype control antibodies with multifunctional antibody structure 2 and targeting CD3 and luciferase (without tumor targeting ability).

TABLE 33

Cell affinity and transient transfection expression level of some antibodies

with the multifunctional antibody structure 3

Affinity to
Affinity to
Affinity to
Transient

Human
Human CD3
Monkey CD3
Transfection

Tumor Cell
Positive T Cell
Positive T Cell
Expression

Antibody Code
(Nm)
(Nm)
(Nm)
Level in 293E

CEA-M3-SG2
30.48
631
669
76.3

CEA-M3-G2
36.30
638
755
70.9

CEA-M3-WT
35.82
624
757
91.1

CEA-M3-AAG
30.12
646
662
67.1

CEA-M3-NA
32.13
688
798
58.1

CEA-M3-FES
42.90
639
732
63.2

CEA-M3-AG2
38.07
602
757
56.0

CEA-M3-GG2
39.15
695
762
59.5

CEA-M3-PG2
41.94
654
838
79.7

CEA-M3-DG2
31.89
609
634
58.6

CEA-M3-G2D
38.35
670
699
73.0

CEA-M3-DG2D
34.18
611
775
49.8

M3IC-SG2
No binding
677
681
67.4

M3IC-G2
No binding
701
771
70.4

M3IC-WT
No binding
637
709
67.0

M3IC-AAG
No binding
606
659
42.3

M3IC-NA
No binding
739
935
43.2

M3IC-FES
No binding
662
671
51.9

M3IC-AG2
No binding
674
747
52.4

M3IC-GG2
No binding
609
866
54.7

M3IC-PG2
No binding
696
712
72.1

M3IC-DG2
No binding
710
704
52.4

M3IC-G2D
No binding
607
848
69.9

M3IC-DG2D
No binding
638
747
63.9

Note: CEA-M3 series were antibodies targeting both CEA and CD3 with multifunctional antibody structure 3; M3IC series were all isotype control antibodies with multifunctional antibody structure 3 and targeting CD3 and luciferase (without tumor targeting ability).

Based on the above data, it can be seen that for the same antibody structure and antibody variable region sequence, there is no significant difference in the binding activity of the antibody to the target with different modifications to the Fc, and there is no significant difference in the expression amount of each antibody.

2. In Vitro Killing

1) Sufficient amount of tumor cells were taken to prepare a single cell suspension;

2) CFSE staining of tumor cells: a certain amount of cell suspension was taken for centrifuge (300×g, 5 min) to remove the supernatant; 2 ml of CFSE solution prepared with PBS was added with a final concentration of 5 μM; the cells were incubated in a 5% CO₂, 37° C. incubator for 15 minutes; the cells were taken out, washed with PBS, centrifuged at 300×g for 5 min to remove the supernatant, and then washed repeatly for three times, resuspended in a complete medium, and then the suspension was taken to count the cells;

3) Tumor cell plating: the cells were resuspended with a complete medium to a density of 2×10⁵/ml, and then added into a 96-well plate at 2×10⁴cells/well (i.e., 100 μl/well);

4) Addition of effector cell PBMC (peripheral blood mononuclear cells, isolated from human whole blood): the effector cells were resuspended in a complete medium which was used by the tumor cells, and a corresponding number of effector cells was added according to the E:T in the experimental design with a volume of 50 μl/well;

5) Addition of diluted antibody: according to the experimental design, the highest concentration of antibody was 10 μg/ml, since the antibody was supplemented with a volume of 50 μl which accounted for ¼ of the total volume of 200 μl, the antibody was required to be prepared at 4 times of the final concentration before supplement, so the antibody should be diluted to 40 μg/ml, and then subjected to 10-fold dilution from 40 μg/ml with 9 gradients, and the antibody was added with a volume of 50 μl/well;

6) the 96-well plate was observed under a microscope to ensure that the cells were evenly dispersed in the culture wells, and then the cells were cultured in a 5% CO2, 37° C. incubator for detection;

7) treatment of adherent cells after reaching the detection time: the cell supernatant was aspirated and washed with 30 μl/well PBS, and the washing liquid was aspirated and added into the previously aspirated supernatant; 30 μl/well of trypsin was added into the cell well, and the cells were digested in a 5% CO2, 37° C. incubator for 3 to 5 min; the supernatant collected previously was added, the cells in each well were pipetted to a single cell suspension; the suspension cells were treated by pipetting and mixing for several times;

8) PI (final concentration of 10 μg/ml) was added at 10 μl/well for each sample 10 to 15 min before flow cytometry;

9) the samples were detected by flow cytometer; the result of flow cytometer was analyzed by a FlowJo software, the data was output in a Microsoft Excel, and analyzed by GraphPad; the ratio of CFSE and PI double-positive cells to CFSE-positive cells was the target cell-killing rate which was calculated as follows:

target cell-killing rate (%)=the number of PI and CFSE double-positive cells/the number of CFES positive cells×100%

10) Calculation of the killing ability of antibodies against tumor cells: the target cell killing rate at each antibody concentration was calculated according to the calculation formula of target cell killing rate, and plotted with the antibody concentration as an abscissa and the target cell killing rate as an ordinate, the data were analyzed by Graphpad Prism 5, and the EC50 value was calculated by log(agonist) vs. response—Variable slope, indicating the cell killing ability of the antibody.

3. T cell activation (antibody+PBMC co-culture system)

- 1) PBMC were separated from the whole blood of healthy volunteers by density gradient centrifugation, and then added into a tumor cell plate according to the E:T in the experimental design;
- 2) the antibody was subjected to a series of 3-fold concentration gradient dilution according to the experimental design, and each concentration of antibody was added;
- 3) the 96-well plate was incubated in a 37° C., 5% CO₂incubator until the detection time; the suspended PBMC cells were collected, added with corresponding CD3, CD69, CD25 detection antibodies (all purchased from BD), incubated for 1 hour, washed to remove excess antibodies, and then resuspended, subjected to flow cytometric detection to obtain the ratio of CD3 and CD69 double-positive cells or the ratio of CD3 and CD25 double-positive cells, that is, the activation ratio of T cells in PBMC induced by the antibody, which was calculated as follows:

ratio of CD3 and CD69 double-positive cells (%)=the number of CD3 and CD69 double-positive cells/the total number of CD3 positive-cells×100%

ratio of CD3 and CD25 double-positive cells (%)=the number of CD3 and CD25 double-positive cells/the total number of CD3 positive-cells×100%

Nonlinear fitting (log(agonist) vs. response—Variable slope) was performed by a “GraphPad Prism 5” software with diabody concentration as an abscissa and CD3&CD69 (CD3&CD25)% value as an ordinate, to calculate the T cell activation curve and EC50 value.

4. T cell activation (antibody+tumor cell+PBMC co-culture system)

- 1) Tumor cells in good culture condition were collected and prepared as a single cell suspension, and then plated into a 96-well plate at 2*10⁴/well;
- 2) PBMC were separated from the whole blood of healthy volunteers by density gradient centrifugation, and then added into a tumor cell plate according to the E:T in the experiment design;
- 3) the antibody was subjected to a series of concentration gradient dilution according to the experimental design, and each concentration of antibody was added;
- 4) the 96-well plate was incubated in a 37° C., 5% CO₂incubator until the detection time, the suspended PBMC cells were collected, added with corresponding CD3, CD69, CD25 detection antibodies, incubated for 1 hour, washed to remove excess antibodies, and then resuspended, subjected to flow cytometric detection to obtain the ratio of CD3 and CD69 double-positive cells or the ratio of CD3 and CD25 double-positive cells, that is, the activation ratio of T cells in PBMC induced by the antibody, which was calculated as follows:

ratio of CD3 and CD69 double-positive cells (%)=the number of CD3 and CD69 double-positive cells/the total number of CD3 positive-cells×100%

ratio of CD3 and CD25 double-positive cells (%)=the number of CD3 and CD25 double-positive cells/the total number of CD3 positive-cells×100%

5. Jurkat-luciferase cell activation

- 1) Different FcγRs (FcγR1, FcγR2, FcγR3A (all purchased from ACRO Biosystems)) were coated onto a microplate reader with a protein concentration of 1 ug/ml and a volume of 100 μl/well, and incubated overnight at 4° C.; the coating solution was discarded and the plate was washed with PBS;
- 2) 40 ul of antibodies with different concentrations (1˜10000 ng/ml) and a certain amount of Jurkat-luciferase cells (2×10⁶/ml, 40 ul/well) were added into each well of the microplate reader, and incubated for 6 h at 37° C.;
- 3) fluorescence was displayed according to the instruction of the kit Bio-Glo Luciferase Assay System (Cat. No. G7940, Promega), and the fluorescence signal value was detected by a fluorescence microplate reader (LUX 3020, Thermo).

Results

1. The killing ability of the PD-L1×CD3 antibody with multifunctional antibody structure 1 to non-small cell lung cancer cells H358 (PD-Ll positive expression, Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) was shown in FIG. 4 (wherein the ratio of the number of effector cells, human PBMC, to the number of target cells, H358, was 10:1, the treatment time was 48 h, and PD-L1 mAb was purchased from Roche). hIgG was a IgG antibody isolated from human serum as a negative control, and E+T was a negative control without adding any antibodies, which were also applied below.

TABLE 34

EC50 value of the PD-L1 × CD3 antibody with multifunctional antibody

structure 1 in killing tumor cells H358

Type
Antibody Code
Maximum Killing (%)
EC50 (pM)

PD-L1 × CD3
PDL1-M1-NQ
69.48
8.779

Antibody With
PDL1-M1-NQ-1
57.08
14.67

Multifunctional
PDL1-M1-G2
69.09
7.264

Antibody
PDL1-M1-SG2
70.77
10.52

Structure 1
PDL1-M1-FES
71.97
9.646

PDL1-M1-LALA
73.18
10.36

PDL1-M1-WT
73.82
11.84

PD-L1 mAb
7.06
Not calculated

None
hIgG
7.14
Not calculated

None
E + T
7.84
None

As can be seen from FIG. 4 and Table 34, if the antibody sequence with multifunctional antibody structure 1 is the same, different modification of Fc has no significant difference in the killing ability to tumor cell H358, which indicates that the modification of Fc does not affect the cytotoxicity of the antibody with double-targeting function, that is, does not affect the efficacy of the antibody.

2. The killing ability of the isotype control antibody (4420×CD3) with multifunctional antibody structure 1 to non-small cell lung cancer cells H358 was shown in FIG. 5 (wherein the ratio of the number of effector cells, human PBMC, to the number of target cells, H358, was 10:1, and the treatment time was 48 h).

TABLE 35

EC50 Value Of The Isotype Control Antibody

With Multifunctional

Antibody Structure 1 In Killing Tumor Cells H358

Maximum
EC50

Type
Antibody Code
Killing (%)
(pM)

Isotype Control
M1IC-FES-1
No Significant
None

Antibody

Killing

4420 ×
M1IC-SG2-1
No Significant
None

CD3 With

Killing

Multifunctional
M1IC-G2-1
No Significant
None

Antibody

Killing

Structure 1
M1IC-NQ
16.17
Not

calculated

M1IC-WT
52.07
1153

M1IC-AG2
No Significant
None

Killing

M1IC-GG2
No Significant
None

Killing

M1IC-PG2
No Significant
None

Killing

M1IC-DG2
No Significant
None

Killing

M1IC-G2D
No Significant
None

Killing

M1IC-DG2D
No Significant
None

Killing

PD-L1
PD-L1 mAb
No Significant
None

Monoclonal

Killing

Antibody

None
hIgG
No Significant
None

Killing

None
E + T
No Significant
None

Killing

As can be seen from FIG. 5 and Table 35, the isotype control antibodies subjected to different Fc modifications have different killing abilities on tumor cells H358. The isotype control antibody itself is only CD3-targeted, not tumor antigen-targeted, therefore the killing of the isotype control antibody is caused by non-specific activation of CD3-positive T cells, wherein, M1IC-WT has significant killing ability, indicating that the Fc of antibody (with corresponding CH2 of WT, SEQ ID NO: 83) can lead to significant non-specific activation of T cells; M1IC-NQ has a significantly higher maximum killing, indicating that it can lead to specific activation of T cells when the CH2 corresponding to Fc is N297Q (SEQ ID NO: 89); other antibodies do not activate T cells, indicating that the modification of Fc significantly reduces the non-specific activation of T cells.

3. The killing ability of CD38×CD3 antibody with multifunctional antibody structure 1 to multiple myeloma cells MC/CAR (CD38 positive, purchased from ATCC) (wherein the ratio of the number of effector cells human PBMC to the number of target cells MC/CAR was 5:1; the treatment time was 48 h; the CD38 mAb was CD38 monoclonal antibody control, and the sequence of CD38 mAb was: VL was SEQ ID NO: 16, CL was SEQ ID NO: 75, and VH was SEQ ID NO : 15, CH1 was SEQ ID NO: 82, hinge was SEQ ID NO: 66, CH2 was SEQ ID NO: 83, CH3 was SEQ ID NO: 102)was shown in FIG. 6.

TABLE 36

EC50 Value Of CD38 × CD3 Antibody With Multifunctional Antibody

Structure 1 In Killing Tumor Cells MC/CAR

Type
Antibody Code
Maximum Killing (%)
EC50 (pM)

CD38 × CD3
CD38-M1-FES
64.02
1.092

Antibody With
CD38-M1-G2
64.04
0.9789

Multifunctional
CD38-M1-SG2
65.34
1.213

Antibody Structure
CD38-M1-WT
61.45
1.002

1
CD38-M1-SG2-1
63.4
1.116

CD38-M1-SG2-2
55.32
30.27

CD38-M1-G2-2
63.57
47.65

CD38-M1-G2-3
64.38
1.006

CD38-M1-FES-1
62.69
1.01

CD38-M1-G2-1
76.99
1.654

CD38-M1-SG2-3
66.9
1.466

CD38-M1-FES-2
61.47
1.825

CD38-M1-NA
61.49
1.452

CD38-M1-FES-3
58.14
0.7104

CD38-M1-AG2
65.29
1.403

CD38-M1-GG2
66.34
1.664

CD38-M1-PG2
65.67
1.419

CD38-M1-DG2
67.22
1.369

CD38-M1-G2D
67.3
1.365

CD38-M1-DG2D
67.32
1.918

CD38 Monoclonal
CD38 mAb
10.66
~6.023

Antibody

None
hIgG
0.75
None

None
E + T
0.87
None

As can be seen from FIG. 6 and Table 36, for a multifunctional antibody with the same variable region sequence, different modification of Fc has no significant difference in the killing ability to MC/CAR tumor cells, which indicates that the modification of Fc does not affect the cytotoxicity of the antibody with double-targeting function, that is, does not affect the efficacy of the antibody.

4. The killing ability of the isotype control antibody (4420×CD3) with multifunctional antibody structure 1 to multiple myeloma cells MC/CAR (CD38 positive) (wherein the ratio of the number of effector cells, human PBMC, to the number of target cells, MC/CAR, was 5:1, and the treatment time was 48 h) was shown in FIG. 7.

TABLE 37

EC50 value of the isotype control antibody with multifunctional antibody

structure 1 in non-specific killing of tumor cells MC/CAR

Type
Antibody Code
Maximum Killing (%)
EC50 (pM)

M1IC-G2
24.4
Not calculated

M1IC-SG2
19.5
Not calculated

M1IC-AG2
No Significant
Not calculated

Killing

M1IC-GG2
No Significant
Not calculated

Killing

M1IC-PG2
No Significant
Not calculated

Killing

M1IC-DG2
No Significant
Not calculated

Killing

M1IC-G2D
No Significant
Not calculated

Killing

M1IC-DG2D
No Significant
Not calculated

Killing

M1IC-WT
36.1
Not calculated

M1IC-SG2-1
15.73
Not calculated

M1IC-G2-1
No Significant
Not calculated

Killing

M1IC-AG2-1
No Significant
Not calculated

Killing

M1IC-GG2-1
No Significant
Not calculated

Killing

M1IC-PG2-1
No Significant
Not calculated

Killing

M1IC-DG2-1
No Significant
Not calculated

Killing

M1IC-G2D-1
No Significant
Not calculated

Killing

M1IC-DG2D-1
No Significant
Not calculated

Killing

M1IC-WT-1
37.5
Not calculated

CD38 Monoclonal
CD38 mAb
10.66
Not calculated

Antibody

None
hIgG
No Significant
Not calculated

Killing

None
E + T
No Significant
none

Killing

As can be seen from FIG. 7 and Table 37, the isotype control antibodies subjected to different Fc modifications have different killing abilities to tumor cells MC/CAR. The isotype control antibody itself is only CD3-targeted, not tumor antigen-targeted, therefore the killing of the isotype control antibody is caused by non-specific activation of CD3-positive T cells, wherein, M1IC-WT has a significant killing ability, indicating that the Fc of antibody (with corresponding CH2 of WT) can lead to significant non-specific activation of T cells. In contrast, the isotype control antibody containing the modified Fc of the present disclosure only leads to weak non-specific activation of T cells or can completely avoid non-specific activation of T cells.

5. Activation experiment of T cells in peripheral blood mononuclear cells by isotype control antibody (4420 ×CD3) with multifunctional antibody structure 1. The results were shown in FIG. 8 and FIG. 9.

TABLE 38

EC50 value of the isotype control antibody with multifunctional antibody

structure 1 in activation of Tcells in PBMC

CD3+ CD69+ T Cells
CD3+ CD25+ T Cells

Antibody
Maximum
EC50
Maximum
Maximum

Code
Activation (%)
(pM)
Activation (%)
Activation (%)

Type
M1IC-G2
83.4
3030
No significant
Not calculated

activation

M1IC-SG2
44.00
4132
No significant
Not calculated

activation

M1IC-AG2
No significant
Not calculated
No significant
Not calculated

activation

activation

M1IC-GG2
No significant
8459
No significant
Not calculated

activation

activation

M1IC-PG2
No significant
Not calculated
No significant
Not calculated

activation

activation

M1IC-DG2
No significant
Not calculated
No significant
Not calculated

activation

activation

M1IC-G2D
No significant
Not calculated
No significant
Not calculated

activation

activation

M1IC-DG2D
No significant
Not calculated
No significant
Not calculated

activation

activation

M1IC-WT
82.33
1.252
50.97
0.2317

M1IC-G2-1
38.5
~7960
No significant
Not calculated

activation

M1IC-SG2-1
32.33
2799
No significant
Not calculated

activation

M1IC-AG2-1
No significant
Not calculated
No significant
Not calculated

activation

activation

M1IC-GG2-1
No significant
Not calculated
No significant
Not calculated

activation

activation

M1IC-PG2-1
No significant
Not calculated
No significant
Not calculated

activation

activation

M1IC-DG2-1
No significant
Not calculated
No significant
Not calculated

activation

activation

M1IC-G2D-1
No significant
Not calculated
No significant
Not calculated

activation

activation

M1IC-DG2D-1
No significant
Not calculated
No significant
Not calculated

activation

activation

M1IC-WT-1
78.53
~0.8307
52.59
0.04556

None
h-IgG
No significant
Not calculated
No significant
Not calculated

activation

activation

As can be seen from FIG. 8, FIG. 9 and Table 38, among the above isotype control antibodies in PBMCs, M1IC-WT had the strongest non-specific activation of T cells, M1IC-SG2, M1IC-G2, M1IC-SG2-1 and M1IC-G2-1 had weaker non-specific activation of T cells, and M1IC-AG2/GG2/DG2/G2D/DG2D had no activation of T cells. It showed that when the antibody with multifunctional antibody structure 1 were CD3-targeted and CH2 was SG2CH2 (SEQ ID NO: 95) or G2CH2 (SEQ ID NO: 94), the antibody had a weakened non-specific activation of T cells; when the isotype control antibodies with multifunctional antibody structure 1 were AG2CH2 (SEQ ID NO: 96), GG2CH2 (SEQ ID NO: 97), PG2CH2 (SEQ ID NO: 98), DG2CH2 (SEQ ID NO: 99), G2DCH2 (SEQ ID NO: 100) and DG2DCH2 (SEQ ID NO: 101), it was more effective to avoid non-specific activation of T cells.

6. Activation experiment of Jurkat-luciferase cells by isotype control antibody (4420×CD3) with multifunctional antibody structure 1. The results were shown in FIG. 10, FIG. 11 and FIG. 12.

If the anti-CD3 antibody bound to the immobilized Fc receptor and then bound to Jurkat-luciferase cells with CD3 surface antigen, the cells can be activated and fluorescent signals can be detected. Stronger fluorescence signals indicated higher activation of cells, and further indicated that the binding of the antibody to Fc receptor was stronger. As can be seen from FIGS. 10 to 12, among the above isotype control antibodies, M1IC-WT-1 has the strongest activation to Jurkat-luciferase cells; when the immobilized antigen was FcγR2, M1IC-FES-1 had a relatively significant activation to Jurkat-luciferase cells; the activation of Jurkat-luficerase cells by M1IC-AG2-1, M1IC-GG2-1, M1IC-PG2-1, M1IC-DG2-1, M1IC-G2D-1, M1IC-DG2D-1, M1IC-DG2D-1A and M1IC-DG2D-1B was very weak and there was no significant difference between the activation by the above antibodies. It was shown that when the CH2 of isotype control antibody with multifunctional antibody structure 1 were AG2CH2 (SEQ ID NO: 96), GG2CH2 (SEQ ID NO: 97), PG2CH2 (SEQ ID NO: 98), DG2CH2 (SEQ ID NO: 99) , G2DCH2 (SEQ ID NO: 100) and DG2D (SEQ ID NO: 101), it was more effective to avoid non-specific activation of T cells than FES (SEQ ID NO: 85).

7. The killing ability of the BCMAxCD3 antibody with multifunctional antibody structure 2 to myeloma cell U266B1 (BCMA positive expression, China Center for Type Culture Collection) (wherein the ratio of the number of effector cells, human PBMC, to the number of target cells, U266B1, was 5:1, and the treatment time was 48 h) were shown in FIG. 13 and FIG. 14.

TABLE 39

Killing effect and EC50 value of BCMA × CD3 antibody with multifunctional

antibody structure 2 and isotype control antibody in killing tumor cells U266B1

Antibody
Maximum Killing
EC50

Type
Code
(%)
(pM)

BCMA × CD3
BCMA-M2-SG2
66.94
1.83

Antibody With
BCMA-M2-G2
66.99
3.092

Multifunctional
BCMA-M2-FES
68.29
2.004

Antibody
BCMA-M2-WT
69.21
1.678

Structure 2
BCMA-M2-
71.26
1.724

AG2

BCMA-M2-
71.51
1.552

GG2

BCMA-M2-PG2
71.92
1.768

BCMA-M2-
73.79
1.612

DG2

BCMA-M2-
73.08
1.587

G2D

BCMA-M2-
71.82
1.672

DG2D

None
E + T
None
None

As can be seen from FIG. 13 and Table 39, for the BCMA×CD3 antibody with multifunctional antibody structure 2, different modification of Fc can significantly kill the tumor cell U266B1, and there was no significant difference in killing effect, which indicated that the modification of Fc did not affect the cytotoxicity of the antibody with double-targeting function, that is, did not affect the efficacy of the antibody.

TABLE 40

Killing effect and EC50 value of isotype control antibody (4420 × CD3) with

multifunctional antibody structure 2 in killing tumor cells U266B1

Antibody
Maximum Killing

Type
Code
(%)
EC50 (pM)

M2IC-WT
50.15
1240

M2IC-G2
27.93
~1604

M2IC-SG2
No
None

Significant

Killing

M2IC-AG2
No
None

Significant

Killing

M2IC-GG2
No
None

Significant

Killing

M2IC-PG2
No
None

Significant

Killing

M2IC-DG2
No
None

Significant

Killing

M2IC-G2D
No
None

Significant

Killing

M2IC-DG2D
No
None

Significant

Killing

None
E + T
None
None

As can be seen from FIG. 14 and Table 40, for the antibodies with multifunctional antibody structure 2 in which the sequence of variable region was the same and the Fc was subjected to different modifications, the isotype control antibody M2IC-WT can significantly kill U266B1, indicating that the Fc can lead to non-specific activation of T cells; when the CH2 of Fc was G2CH2 (SEQ ID NO: 94), a weaker killing effect was observed, indicating that it may lead to a weaker non-specific activation of T cells; when the CH2 of Fc was SG2CH2 (SEQ ID NO: 95), AG2CH2 (SEQ ID NO: 96), GG2CH2 (SEQ ID NO: 97), PG2CH2 (SEQ ID NO: 98), DG2CH2 (SEQ ID NO: 99), G2DCH2 (SEQ ID NO: 100) and DG2DCH2 (SEQ ID NO: 101), there was no cell killing, indicating that these five Fcs did not lead to non-specific activation of T cells.

8. Activation experiment of T cells in peripheral blood mononuclear cells (PBMC) by isotype control antibody with multifunctional antibody structure 2 (treatment time was 48 h). The results were shown in FIG. 15 and FIG. 16.

TABLE 41

Activation ratio and EC50 value of the isotype control antibody with

multifunctional antibody structure 2 in activation of T cells in PBMC

CD3+ CD69+ T cells
CD3+ CD25+ T cells

Maximum
EC50
Maximum
EC50

Type
Antibody Code
Activation (%)
(pM)
Activation (%)
(pM)

4420 × CD3
M2IC-WT
32.61
13211
25.44
8008

isotype control
M2IC-G2
13.15
Not
21.65
Not

antibody with

calculated

calculated

multifunctional
M2IC-SG2
12.49
Not
No
Not

antibody

calculated
significant
calculated

structure 2

activation

M2IC-AG2
No
Not
No
Not

significant
calculated
significant
calculated

activation

activation

M2IC-GG2
No
Not
No
Not

significant
calculated
significant
calculated

activation

activation

M2IC-PG2
No
Not
No
Not

significant
calculated
significant
calculated

activation

activation

M2IC-DG2
No
Not
No
Not

significant
calculated
significant
calculated

activation

activation

M2IC-G2D
No
Not
No
Not

significant
calculated
significant
calculated

activation

activation

M2IC-DG2D
No
Not
No
Not

significant
calculated
significant
calculated

activation

activation

None
hIgG
3.75
Not
3.114
Not

calculated

calculated

As can be seen from FIG. 15, FIG. 16 and Table 41, in the absence of relevant tumor cells, the analysis of the maximum activation showed that M2IC-WT had a significant non-specific activation to T cells, and M2IC-G2 and M2IC-SG2 had a weakened non-specific activation to T cells, and the remaining antibodies did not have a significant activation to T cells. When the CH2 of Fc was AG2CH2 (SEQ ID NO: 96), GG2CH2 (SEQ ID NO: 97), PG2CH2 (SEQ ID NO: 98), DG2CH2 (SEQ ID NO: 99), G2DCH2 (SEQ ID NO: 100) and DG2DCH2 (SEQ ID NO: 101), there was no cell killing, indicating that these five Fc did not lead to non-specific activation of T cells.

9. The killing ability of the CEA×CD3 antibody with multifunctional antibody structure 3 to gastric cancer cell MKN-45 (CEA positive expression, Basic Medical Cell Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences) (wherein the ratio of the number of effector cells, human PBMC, to the number of target cells, MKN-45, was 5:1, and the treatment time was 48 h) were shown in FIG. 17 and FIG. 18.

TABLE 42

Killing effect and EC50 value of CEA × CD3 antibody with multifunctional

antibody structure 3 in killing tumor cells MKN-45

Antibody
Maximum Killing
EC50

Type
Code
(%)
(pM)

CEA × CD3
CEA-M3-WT
46.33
259.0

Antibody With
CEA-M3-AAG
46.55
249.0

Multifunctional
CEA-M3-NA
50.28
260.0

Antibody Structure
CEA-M3-FES
48.95
368.8

3
CEA-M3-G2
49.6
409.3

CEA-M3-SG2
47.76
255.8

CEA-M3-AG2
48.84
256.0

CEA-M3-GG2
45.62
253.0

CEA-M3-PG2
47.9
263.9

CEA-M3-DG2
47.53
358.5

CEA-M3-G2D
46.87
250.4

CEA-M3-
45.39
287.8

DG2D

None
E + T
4.68
None

As can be seen from FIG. 17 and Table 42, when the sequence of CEAxCD3 antibody with multifunctional antibody structure 3 was identical, different modification of Fc had no significant difference in the killing ability to tumor cell MKN-45, which indicated that the modification of Fc did not affect the cytotoxicity of the antibody with double-targeting function, that is, did not affect the efficacy of the antibody.

TABLE 43

Killing effect and EC50 value of isotype control antibody (4420 × CD3) with

multifunctional antibody structure 3 in killing tumor cells MKN-45

Maximum Killing
EC50

Type
Antibody Code
(%)
(pM)

Isotype Control
M3IC-WT
29.09
Not calculated

Antibody With
M3IC-G2
17.60
Not calculated

Multifunctional
M3IC-SG2
12.43
Not calculated

Antibody Structure 3
M3IC-AG2
No Significant
Not calculated

Killing

M3IC-GG2
No Significant
Not calculated

Killing

M3IC-PG2
No Significant
Not calculated

Killing

M3IC-DG2
No Significant
Not calculated

Killing

M3IC-G2D
No Significant
Not calculated

Killing

M3IC-DG2D
No Significant
Not calculated

Killing

None
E + T
No Significant
None

Killing

As can be seen from FIG. 18 and Table 43, the maximum killing showed that the antibody M3IC-WT had a significant killing; M3IC-G2 and M3IC-SG2 had a weaker killing, while the other antibodies had no significant killing, indicating that the modification of CH2 of Fc of the multifunctional antibody structure 3 to G2CH2 (SEQ ID NO: 94) and SG2CH2 (SEQ ID NO: 95) had the effect of weakening the non-specific activation of T cells compared with the control M3IC-WT, while the following five Fc, AG2CH2 (SEQ ID NO: 96), GG2CH2 (SEQ ID NO: 97), PG2CH2 (SEQ ID NO: 98), DG2CH2 (SEQ ID NO: 99), G2DCH2 (SEQ ID NO: 100) and DG2DCH2 (SEQ ID NO: 101), did not lead to non-specific activation of T cells .

10. Activation experiment of T cells in peripheral blood mononuclear cells(PBMC) by isotype control antibody (4420×CD3) with multifunctional antibody structure 3 (treatment time was 48 h). The results were shown in FIG. 19 and FIG. 20.

TABLE 44

Activation ratio and EC50 value of the isotype control antibody with

multifunctional antibody structure 3 in activation of T cells in PBMC

CD3+ CD69+ T cells
CD3+ CD25+ T cells

Maximum

Maximum

Activation

Activation
EC50

Type
Antibody Code
(%)
EC50 (pM)
(%)
(pM)

4420 × CD3
M3IC-WT
41.2
Not calculated
26.75
Not

Isotype Control

calculated

Antibody With
M3IC-G2
26.97
Not calculated
14.21
Not

Multifunctional

calculated

Antibody
M3IC-SG2
14.90
Not calculated
No significant
Not

Structure 3

activation
calculated

M3IC-AG2
12.59
Not calculated
No significant
Not

activation
calculated

M3IC-GG2
9.026
Not calculated
No significant
Not

activation
calculated

M3IC-PG2
9.869
Not calculated
No significant
Not

activation
calculated

M3IC-DG2
10.39
Not calculated
No significant
Not

activation
calculated

M3IC-G2D
6.614
Not calculated
No significant
Not

activation
calculated

M3IC-
10.26
Not calculated
No significant
Not

DG2D

activation
calculated

None
hIgG
3.75
Not calculated
3.114
Not

calculated

As can be seen from FIG. 19, FIG. 20 and Table 44, among the isotype control antibodies with multifunctional antibody structure 3, M3IC-WT had the strongest significant activation to T cells, and M3IC-G2 had a weaker activation to T cells. When CH2 was G2CH2 (SEQ ID NO: 94), the antibody showed a weakened non-specific activation to T cell as compared with M2IC-WT, and the remaining antibodies showed a even weaker activation to T cells as compared with M2IC-WT, wherein, the CH2 was most preferably G2DCH2 (SEQ ID NO: 100).

EXAMPLE 3
Detection of Antibody Stability

Experimental Steps:

A. The specific steps of accelerated thermal stability test at 40° C. were:

1) the sample was substituted into a buffer, and the constituents of the buffer was 20 mM citric acid, pH 5.5, and the sample concentration was adjusted to 1 mg/mL;

2) each sample was divided into 500 μL per tube (6 tubes in total) and placed in a 40° C. water bath after being sealed. On day 0, day 3, day 5, day 7, day 10, and day 14, samples were taken for HPLC-SEC. The water bath time was 14 days in total.

B. Acid resistance test, also known as low-pH stability, was a test to detect whether the antibody molecule can maintain its original state after being treated in an acidic environment for a period of time and then being neutralized to physiological conditions. The specific steps were:

When antibody molecules were subjected to protein A affinity chromatography, the eluted antibody solution was not neutralized in the acid elution step (citrate buffer at pH 3.5 was used). After being kept in the buffer for a period of time, samples were taken at 30 min and 60 mM, and 1/10 volume of 1M Tris-HCl (pH 8.0) was added for neutralization, and the sample was tested by HPLC-SEC.

Results

1. The test results of 40° C. accelerated thermal stability test of the multifunctional antibody structure 1 were shown in FIG. 21.

As can be seen from FIG. 21, the purity of the different antibodies with multifunctional antibody structure 1 remained above 90% after being treated at 40° C. for 14 days, without a large amount of aggregation or degradation. Among them, the purity of the antibody with N297Q as the CH2 of Fc was significantly reduced as compared to the purity of other antibodies, and there was no significant difference among other CH2 modified antibodies, which indicated that the multifunctional antibody structure 1 had a good thermal stability when CH2 was G2CH2 (SEQ ID NO: 94), SG2CH2 (SEQ ID NO: 95), AG2CH2 (SEQ ID NO: 96), GG2CH2 (SEQ ID NO: 97), PG2CH2 (SEQ ID NO: 98), DG2CH2 (SEQ ID NO: 99), G2DCH2 (SEQ ID NO: 100) and DG2DCH2 (SEQ ID NO: 101).

2. The results of acid resistance test of the multifunctional antibody structure 1 were shown in FIG. 22.

As can be seen from FIG. 22, for the different Fc-modified antibodies with multifunctional antibody structure 1, the antibody purity did not change significantly after being treated under low pH conditions for 60 minutes, and there was no significant difference between different antibodies, indicating the above-mentioned Fc-modified multifunctional antibody structure 1 had a good acid resistance.

As can be seen from FIG. 21 and FIG. 22, the antibody with multifunctional antibody structure 1 (wherein the CH2 of the antibody was G2CH2 (SEQ ID NO: 94), SG2CH2 (SEQ ID NO: 95), AG2CH2 (SEQ ID NO: 96), GG2CH2 (SEQ ID NO: 97), PG2CH2 (SEQ ID NO: 98), DG2CH2 (SEQ ID NO: 99), G2DCH2 (SEQ ID NO: 100) and DG2DCH2 (SEQ ID NO: 101)) had a good thermal stability and acid resistance.

3. The results of 40° C. accelerated thermal stability test of the multifunctional antibody structure 2 were shown in FIG. 23 .

As can be seen from FIG. 23, if the CH2 of the multifunctional antibody structure 2 was N297A (SEQ ID NO: 87), the purity of antibody after being treated at 40° C. for 14 days was decreased significantly; the purity of other antibodies did not change significantly, and there was no significant difference between antibodies, which proved that these antibodies with multifunctional antibody structure 2 other than M2IC-NA had a good accelerated thermal stability.

4. The results of acid resistance test of the multifunctional antibody structure 2 were shown in FIG. 24.

As can be seen from FIG. 24, for the different Fc-modified antibodies with multifunctional antibody structure 1, the antibody purity did not change significantly after being treated under low pH conditions for 60 minutes, and there was no significant difference between different antibodies, indicating that the above-mentioned Fc-modified multifunctional antibody structure 2 had a good acid resistance.

As can be seen from FIG. 23 and FIG. 24, the antibody with multifunctional antibody structure 2 (wherein the CH2 of the antibody was G2CH2 (SEQ ID NO: 94), SG2CH2 (SEQ ID NO: 95), AG2CH2 (SEQ ID NO: 96), GG2CH2 (SEQ ID NO: 97), PG2CH2 (SEQ ID NO: 98), DG2CH2 (SEQ ID NO: 99), G2DCH2 (SEQ ID NO: 100) and DG2DCH2 (SEQ ID NO: 101)) had a good thermal stability and acid resistance.

5. The results of 40° C. accelerated thermal stability test of the multifunctional antibody structure 3 were shown in FIG. 25.

As can be seen from FIG. 25, the purity of different antibodies with multifunctional antibody structure 3 did not change significantly after being treated at 40° C. for 14 days, and there was no significant difference between the antibodies, which proved that the above-mentioned antibodies with multifunctional antibody structure 3 had a good accelerated thermal stability.

6. The results of acid resistance test of the multifunctional antibody structure 3 were shown in FIG. 26.

As can be seen from FIG. 26, for the different Fc-modified antibodies with multifunctional antibody structure 3, the antibody purity did not change significantly after being treated under low pH conditions for 60 minutes, and there was no significant difference between different antibodies, indicating that the above-mentioned Fc-modified multifunctional antibody structure 3 had a good acid resistance.

As can be seen from FIG. 25 and FIG. 26, the antibody with multifunctional antibody structure 3 (wherein the CH2 of the antibody was G2CH2 (SEQ ID NO: 94), SG2CH2 (SEQ ID NO: 95), AG2CH2 (SEQ ID NO: 96), GG2CH2 (SEQ ID NO: 97), PG2CH2 (SEQ ID NO: 98), DG2CH2 (SEQ ID NO: 99), G2DCH2 (SEQ ID NO: 100) and DG2DCH2 (SEQ ID NO: 101)) had a good thermal stability and acid resistance.

EXAMPLE 4
Binding Experiment Of Fe-modified Monoclonal Antibodies And FcγR-Expressing Cells

1. Preparation of Monoclonal Antibodies

(1) In the present disclosure, the prepared monoclonal antibody had the same or similar structure as the natural antibody such as human IgG1, IgG2, IgG3 or IgG4 subtype, and the structure was in a form of symmetrical “Y”, with a bivalent Fc domain binding to the same target antigen and the corresponding subtype.

FIG. 27 showed a schematic diagram of the specific structure.

(2) The construction of expression plasmid, the transfection expression and the purification method of the monoclonal antibody were consistent with the methods of multifunctional antibody involved in Example 1 of the present disclosure, and the used sequences were shown in Table 45.

TABLE 45

Codes and the amino acid sequences of variable region of some monoclonal antibodies

Antibody

code
Polypeptide
Domain
Code
Amino acid sequence (CDR is underlined in bold)

custom-character

4420
Light
VL
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVE
44

mAb-
Chain

AEDLGVYFCSQSTHVPWTFGGGTKLEIK

WT

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

Heavy
VH
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSV
43

Chain

YLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
WT
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH
83

QDWLNGKEYKCKVSNKALPAPIEKTISKAK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVES
102

CSVMHEALHNHYTQKSLSLSPGK

4420
Light
VL
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVE
44

mAb-
Chain

AEDLGVYFCSQSTHVPWTFGGGTKLEIK

G2

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

Heavy
VH
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSV
43

Chain

YLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQ
94

DWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
102

CSVMHEALHNHYTQKSLSLSPGK

4420
Light
VL
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRESGSGSGTDFTLKISRVE
44

mAb-
Chain

AEDLGVYFCSQSTHVPWTFGGGTKLEIK

SG2

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

Heavy
VH
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSV
43

Chain

YLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
SG2CH2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQ
95

DWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
102

CSVMHEALHNHYTQKSLSLSPGK

4420
Light
VL
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVE
44

mAb-
Chain

AEDLGVYFCSQSTHVPWTFGGGTKLEIK

AG2

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

Heavy
VH
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSV
43

Chain

YLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
AG2CH2
PAPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQ
96

DWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
102

CSVMHEALHNHYTQKSLSLSPGK

4420
Light
VL
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVE
44

mAb-
Chain

AEDLGVYFCSQSTHVPWTFGGGTKLEIK

GG2

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

Heavy
VH
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSV
43

Chain

YLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
GG2CH2
PGPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQ
97

DWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
102

CSVMHEALHNHYTQKSLSLSPGK

4420
Light
VL
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVE
44

mAb-
Chain

AEDLGVYFCSQSTHVPWTFGGGTKLEIK

PG2

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

Heavy
VH
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSV
43

Chain

YLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
PG2CH2
PPPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQ
98

DWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
102

CSVMHEALHNHYTQKSLSLSPGK

4420
Light
VL
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVE
44

mAb-
Chain

AEDLGVYFCSQSTHVPWTFGGGTKLEIK

DG2

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

Heavy
VH
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSV
43

Chain

YLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
DG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQ
99

DWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
102

CSVMHEALHNHYTQKSLSLSPGK

4420
Light
VL
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVE
44

mAb-
Chain

AEDLGVYFCSQSTHVPWTFGGGTKLEIK

G2D

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

Heavy
VH
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSV
43

Chain

YLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
G2DCH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQ
100

DWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
102

CSVMHEALHNHYTQKSLSLSPGK

4420
Light
VL
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVE
44

mAb-
Chain

AEDLGVYFCSQSTHVPWTFGGGTKLEIK

DG2D

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
75

YACEVTHQGLSSPVTKSFNRGEC

Heavy
VH
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSV
43

Chain

YLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
82

HKPSNTKVDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
DG2DCH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQ
101

DWLNGKEYKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVFS
102

CSVMHEALHNHYTQKSLSLSPGK

2. Binding Experiment of Monoclonal Antibody To FcγR-Expressing Cells

(1) There are three types of FcγR that bind to human IgG antibody Fc: FcγRI, FcγRII and FcγRIII. Among them, FcγRI (also known as CD64) and FcγRIIA are expressed on macrophages and neutrophils; FcγRIIB is expressed on B cells, and FcγRIIIA (also known as CD16A) is expressed on natural killer (NK) cells. The surface marker of dendritic cells is CD83, the surface marker of macrophages is CD14, the surface marker of B cells is CD20, and the surface marker of NK cells is CD56.

Nine monoclonal antibodies in Table 45 were prepared and then were labeled with biotin. The negative control was 4420 antibody with Fab structure (wherein, VL was SEQ ID NO: 44, CL was SEQ ID NO: 75, VH was SEQ ID NO: 43, CH1 was SEQ ID NO: 82) and without Fc, and was also biotin-labeled.

PBMCs were isolated from the blood of healthy donors and divided into three groups:

a) the binding ability of 4420 mAb Fc to macrophages was detected in group 1; PBMC and PE-labeled anti-CD14 antibody (purchased from Thermofisher) and the aforementioned 9 biotin-labeled 4420 mAbs (three concentrations of 500 μg/ml, 50 μg/ml and 5 μg/ml for each antibody) were incubated for 2 h (at room temperature), washed for 3 times, then incubated for 30 minutes (at room temperature) after being added with FITC-labeled avidin, washed for 5 times and then subjected to flow cytometry; PE fluorescent cell population was circled, and then the average fluorescence intensity of FITC was analyzed;

b) the binding ability of 4420 mAb Fc to B cells was detected in group 2, the steps were the same as in a), except that PE-labeled anti-CD20 antibody (purchased from Thermofisher) was used instead of PE-labeled anti-CD14 antibody;

c) the binding ability of 4420 mAb Fc to NK cells was detected in group 3, the steps were the same as in a), except that anti-CD56 antibody (purchased from Thermofisher) was used instead of PE-labeled anti-CD14 antibody.

The results of flow cytometry were shown in FIGS. 28 to 30. As can be seen from FIG. 28, 4420mAb-WT significantly bound to to macrophages; and if CH2 was G2CH2 (SEQ ID NO:94), SG2CH2 (SEQ ID NO:95), AG2CH2 (SEQ ID NO:96), GG2CH2 (SEQ ID NO:97), PG2CH2 (SEQ ID NO:98), DG2CH2 (SEQ ID NO:99), G2DCH2 (SEQ ID NO:100) and DG2DCH2 (SEQ ID NO: 101), the binding to FcγRI or FcγRIIA was significantly reduced or lost.

As can be seen from FIG. 29, 4420mAb-WT significantly bound to to B cells; compared with 4420mAb-WT, if the CH2 of the Fc of antibody was SG2CH2 (SEQ ID NO: 95), G2CH2 (SEQ ID NO: 94), AG2CH2 (SEQ ID NO: 96), GG2CH2 (SEQ ID NO: 97), PG2CH2 (SEQ ID NO: 98), DG2CH2 (SEQ ID NO: 99), G2DCH2 (SEQ ID NO: 100) and DG2DCH2 (SEQ ID NO: 101), the binding to FcγRIIB was significantly reduced or lost.

As can be seen from FIG. 30, only 4420mAb-WT had a significant binding to NK, indicating that if the CH2 of the Fc was SG2CH2 (SEQ ID NO: 95), G2CH2 (SEQ ID NO: 94), AG2CH2 (SEQ ID NO: 96), GG2CH2 (SEQ ID NO: 97), PG2CH2 (SEQ ID NO: 98), DG2CH2 (SEQ ID NO: 99), G2DCH2 (SEQ ID NO: 100) and DG2DCH2 (SEQ ID NO: 101), the binding to FcγRIIIA was significantly reduced or lost.

Based on FIGS. 28 to 30, it can be seen that if the CH2 of Fc was modified to G2CH2 (SEQ ID NO: 94), SG2CH2 (SEQ ID NO: 95), AG2CH2 (SEQ ID NO: 96), GG2CH2 (SEQ ID NO: 97), PG2CH2 (SEQ ID NO: 98), DG2CH2 (SEQ ID NO: 99), G2DCH2 (SEQ ID NO: 100) and DG2DCH2 (SEQ ID NO: 101), the binding function of antibody Fc to its receptor FcγRI, FcγRIIA, FcγRIIB and FcγRIIIA was greatly reduced or lost. It is known that the activation of T cells by CD3 antibody is largely caused by the binding of antibody Fc to FcγR on the surface of other cells. In the present disclosure, the Fc was innovatively modified, wherein the CH2 domain in the Fc of human IgG1 was substituted with a CH2 domain of human IgG2, preferably subjected to substitution of several amino acid residues; and antibodies or fusion proteins with significantly reduced or even lost binding ability to FcγR were obtained; and these Fc modification methods did not affect the stability and biological activity of the antibody itself.

EXAMPLE 5
Activation Experiment of T Cells by Anti-CD3 Monoclonal Antibody

1. Preparation of Monoclonal Antibodies

See Part 1 “Preparation of monoclonal antibodies” in Example 4, and the used sequences were shown in Table 46.

TABLE 46

Codes and amino acid sequences of variable region of some monoclonal antibodies

Antibody

SEQ ID

Code
Poplypeptide
Domain
Code
Amino acid sequence (CDR is underlined in bold)
NO

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

WT

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
WT
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
83

EYKCKVSNKALPAPIEKTISKAK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

FES

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
85

EYKCKVSNKALPASIEKTISKAK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

AAG

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
AAG
PCPAPEAAGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
84

KEYKCKVSNKALGAPIEKTISKAK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
G2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
94

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

SG2

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
SG2CH2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
95

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

AG2

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
AG2CH2
PAPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
96

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

GG2

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
GG2CH2
PGPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
97

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

PG2

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
PG2CH2
PPPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
98

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

C229L

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
C229LG2CH2
PLPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
128

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

C229F

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
C229FG2CH2
PFPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
129

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

C229R

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
C229RG2CH2
PRPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
130

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

C229V

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
C229VG2CH2
PVPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
131

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

C229Q

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
C229QG2CH2
PQPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
132

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

C229K

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNEGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
C229KG2CH2
PKPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
133

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

C229D

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
C229DG2CH2
PDPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
134

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

C229I

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
C229IG2CH2
PIPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
135

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

C229Y

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
C229YG2CH2
PYPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
136

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

C229N

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
C229NG2CH2
PNPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
137

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

C229M

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
C229MG2CH2
PMPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
138

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

C229T

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
C229TG2CH2
PTPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
139

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

C229H

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
C229HG2CH2
PHPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
140

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

C229E

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
C229EG2CH2
PEPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
141

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

C229W

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
C229WG2CH2
PWPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK
142

EYKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

DG2

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
DG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
99

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D265P

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D265PG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVPVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
143

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D265K

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D265KG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVKVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
144

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D265S

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D265SG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
145

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D265F

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D265FG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVFVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
146

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D265R

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D265RG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVRVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
147

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARESGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIEPPSDEQLKSGTASVVCLLNNEYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D265L

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D265LG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVLVSHEDPEVQFNWYVDGVEVHNAKTKPREEQENSTERVVSVLTVVHQDWLNGKE
148

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARESGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIEPPSDEQLKSGTASVVCLLNNEYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D265G

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D265GG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVGVSHEDPEVQFNWYVDGVEVHNAKTKPREEQENSTERVVSVLTVVHQDWLNGKE
149

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARESGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIEPPSDEQLKSGTASVVCLLNNEYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D265T

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

Hinge
Hin1
DKTHTCP
66

CH2
D265TG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVTVSHEDPEVQFNWYVDGVEVHNAKTKPREEQENSTERVVSVLTVVHQDWLNGKE
150

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D265Y

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D265YG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVYVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
151

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D265W

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D265WG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVWVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
152

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D265H

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D265HG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVHVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
153

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D265V

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D265VG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVVVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
154

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D265Q

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D265QG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVQVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
155

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D265E

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D265EG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVEVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
156

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D265M

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D265MG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVMVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
157

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D265N

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D265NG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVNVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
158

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D265I

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D265IG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVIVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
159

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2D

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
G2DCH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
100

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D270L

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D270LG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHELPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
160

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D270R

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D270RG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHERPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
161

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D270P

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D270PG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEPPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
162

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D270G

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D270GG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
163

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D270V

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D270VG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEVPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
164

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D270H

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D270HG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEHPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
165

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D270Y

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D270YG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEYPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
166

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D270I

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D270IG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEIPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
167

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D270E

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D270EG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEEPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
168

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D270F

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D270FG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEEPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
169

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D270K

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D270KG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEKPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
170

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D270W

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D270WG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEWPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
171

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D270S

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D270SG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHESPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
172

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D270T

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D270TG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHETPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
173

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D270Q

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D270QG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEQPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
174

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D270M

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS
1

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D270MG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEMPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
175

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

D270N

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
D270NG2CH2
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHENPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
176

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

PG2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

GA

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
PG2-
PPPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH
122

GA
QDWLNGKEYKCKVSNKALPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

PG2-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

TA

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
PG2-
PPPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH
123

TA
QDWLNGKEYKCKVSNKGLPAPIEKTISKAK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2D-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

GA

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
G2D-
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
124

GA
YKCKVSNKALPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2D-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

TA

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
G2D-TA
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
125

YKCKVSNKGLPAPIEKTISKAK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

G2D-

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

GATA

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
G2D-
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
126

GATAYKCKVSNKALPAPIEKTISKAK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

CD3
Light
VL
2a5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
2

mAb-
Chain

ALWYSNLWV
FGGGTKVEIK

PDG2D

CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
75

QGLSSPVTKSFNRGEC

Heavy
VH
2a5
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLR
1

Chain

AEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS

CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
82

VDKKVEPKSC

Hinge
Hin1
DKTHTCP
66

CH2
PDG2D
PPPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVAVSHEAPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE
127

YKCKVSNKGLPAPIEKTISKTK

CH3
WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
102

LHNHYTQKSLSLSPGK

2. Activation of T Cells in PBMC by CD3 Monoclonal Antibody

The method in the Example is the same as that of “3. T cell activation (antibody+PBMC co-culture system)” in “Example 2: Detection of antibody biological activity”. The results were shown in Tables 47 to 48 and FIGS. 31 to 34.

TABLE 47

Activation of T cells in PBMC by anti-CD3 monoclonal

antibodies with single point mutated Fc

Maximum Activation
Maximum Activation

Ratio (%) Of
Ratio (%) Of

Antibody Code
CD3+ CD69+ T Cells
CD3+ CD25+ T Cells

CD3mAb-WT
72.28
50.78

CD3mAb-G2
23.17
11.14

CD3mAb-SG2
25.39
12.41

CD3mAb-AG2
21.46
12.03

CD3mAb-GG2
19.22
9.845

CD3mAb-PG2
20.77
9.591

CD3mAb-G2-C229L
52.2
14.803

CD3mAb-G2-C229F
14.68
5.25

CD3mAb-G2-C229R
34.41
14.78

CD3mAb-G2-C229V
46.1
22.94

CD3mAb-G2-C229Q
35.77
16.04

CD3mAb-G2-C229K
31.73
11.73

CD3mAb-G2-C229D
45.73
24.27

CD3mAb-G2-C229I
48.74
26.35

CD3mAb-G2-C229Y
48.21
25.53

CD3mAb-G2-C229N
45.81
19.53

CD3mAb-G2-C229M
37.2
16.05

CD3mAb-G2-C229T
30.77
11.31

CD3mAb-G2-C229H
35.49
15.38

CD3mAb-G2-C229E
38.9
16.88

CD3mAb-G2-C229W
42.06
22.29

CD3mAb-DG2
24.84
9.6

CD3mAb-G2-D265P
36.91
11.15

CD3mAb-G2-D265K
34.12
9.819

CD3mAb-G2-D265S
35.15
13.19

CD3mAb-G2-D265F
46.8
20.15

CD3mAb-G2-D265R
41.17
13.471

CD3mAb-G2-D265L
35.58
10.36

CD3mAb-G2-D265G
39
12.22

CD3mAb-G2-D265T
34.99
9.867

CD3mAb-G2-D265Y
37.11
12.8

CD3mAb-G2-D265W
30.84
8.829

CD3mAb-G2-D265H
30.4
8.31

CD3mAb-G2-D265V
32.87
11.7

CD3mAb-G2-D265Q
34.17
10.54

CD3mAb-G2-D265E
25.59
5.316

CD3mAb-G2-D265M
32.27
10.24

CD3mAb-G2-D265N
27.28
9.442

CD3mAb-G2-D265I
27.46
11.51

CD3mAb-G2D
22.78
11.41

CD3mAb-G2-D270L
30.97
10.6

CD3mAb-G2-D270R
33.92
13.39

CD3mAb-G2-D270P
39.38
18.98

CD3mAb-G2-D270G
32.78
11.41

CD3mAb-G2-D270V
20.44
5.258

CD3mAb-G2-D270H
24.41
7.125

CD3mAb-G2-D270Y
27.48
6.706

CD3mAb-G2-D270I
26.51
5.853

CD3mAb-G2-D270E
57.36
41.08

CD3mAb-G2-D270F
27.18
5.516

CD3mAb-G2-D270K
26.95
6.55

CD3mAb-G2-D270W
25.45
6.546

CD3mAb-G2-D270S
26.01
6.332

CD3mAb-G2-D270T
22.55
6.742

CD3mAb-G2-D270Q
29.21
9.175

CD3mAb-G2-D270M
22.28
6.278

CD3mAb-G2-D270N
22.4
5.406

PBMC
none
none

As can be seen from Table 47 and FIGS. 31 to 33, for a IgG1 type CD3 monoclonal antibody in which the CH2 domain of Fc was substituted with a CH2 of IgG2 and then the cysteine(C) at position 229, the aspartic acid (D) at position 265 or the the aspartic acid (D) at position 270 was substituted with other essential amino acids, T cell activation can be significantly reduced as compared with the antibody with CH1 domain of Fc.

TABLE 48

Activation of T cells in PBMC by anti-CD3 monoclonal

antibodies with multiple point mutated Fc

CD3+ CD69+
CD3+ CD25+

T Cells
T Cells

Maximum
Maximum

Antibody Code
Activation (%)
Activation (%)

CD3mAb-WT
70.45
37.29

CD3mAb-FES
41.75
12.62

CD3mAb-AAG
33.05
11.75

CD3mAb-G2
29.17
11.14

CD3mAb-SG2
31.39
12.41

CD3mAb-AG2
31.9
12.03

CD3mAb-GG2
30.61
9.845

CD3mAb-PG2
31.56
9.591

CD3mAb-DG2
30.84
9.6

CD3mAb-G2D
28.78
11.41

CD3mAb-PG2-GA
37.36
12.1

CD3mAb-PG2-TA
28.75
9.479

CD3mAb-G2D-GA
27.57
8.237

CD3mAb-G2D-TA
27.75
9.435

CD3mAb-G2D-GATA
31.6
8.595

CD3mAb-PDG2D
31.89
11.85

PBMC
none
none

As can be seen from Table 48 and FIG. 34, after the CH2 domain of Fc of the IgG1-type CD3 monoclonal antibody was substituted with the CH2 of IgG2, some amino acids were substituted as follows: (1) no substitution; (2) single-site substitution, C229S, C229A, C229G, C229P, D265A, D270A; (3) two-sites substitution, C229P/G327A, C229P/T339A, D270A/G327A, D270A/T339A; (4) three-sites substitution, D270A/G327A/T339A, C229P/D265A/D270A, etc.; it can be seen that activation of T cells was significantly weakened by the above-mentioned four types of substituted monoclonal antibodies as compared with wild-type IgG1, and the weakening was even more significant than that of FES.

MODIFIED FC FRAGMENT, ANTIBODY COMPRISING SAME, AND APPLICATION THEREOF

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