CN115850449A

CN115850449A - Antibodies and conjugates

Info

Publication number: CN115850449A
Application number: CN202211197665.7A
Authority: CN
Inventors: 须涛; 齐浩; 王超; 杨艳玲; 杜海菁; 孙艳
Original assignee: Suzhou Zhihe Biomedical Technology Co ltd
Current assignee: Suzhou Zhihe Biomedical Technology Co ltd
Priority date: 2022-09-29
Filing date: 2022-09-29
Publication date: 2023-03-28
Also published as: WO2024067670A1

Abstract

The present application relates to an isolated antigen binding protein comprising a variable antigen binding domain (VHH) of a heavy chain antibody, wherein the VHH comprises one or more pairs of engineered cysteine residues, wherein each pair of the cysteine residues independently comprises a cysteine residue at a first position and a cysteine residue at a second position, the cysteine residue at the first position being capable of pairing to form a disulfide bond with the cysteine residue at the second position.

Description

Antibodies and conjugates

Technical Field

The application relates to the field of biomedicine, in particular to an antibody and a conjugate.

Background

In recent years, antibody drugs have been used in many fields, and many diseases have been diagnosed and treated by antibody drugs. Because antibody drugs have good targeting properties, many therapeutic approaches have been developed for different types of antibodies. One type of antibody-drug conjugate/radionuclide is. And increasing the ratio of drug to antibody (DAR) can be effective in improving therapeutic efficacy.

Compared with the traditional antibody, the nano antibody has many advantages, such as smaller molecular weight which is only 12-15 kDa, strong penetrating power and good stability. Compared with human VH, the CDR3 region of the nano antibody is longer, can form a convex ring structure, can penetrate into the interior of an antigen to better combine with the antigen, and therefore has better affinity.

At present, the coupling mode of the nano antibody mostly adopts the fixed-point coupling of the lysine residue or the terminal cys of the random coupling to the antibody. Random conjugation, while increasing the DAR value, also adds a lot of uncertainty, such as relatively random conjugation sites, also creates a heterogeneous antibody-drug conjugate mixture, and is relatively complex to control, and may also affect the biological activity and stability of the sample. The advantages of end-directed conjugation over random conjugation are that the conjugation site is relatively fixed, but the relative DAR value is low and is only 1, and because cysteine is added at the end of the antibody, a polymer is easily formed, which has great challenges for the production process of naked antibody.

Disclosure of Invention

In order to overcome the technical problems in the background art, the application provides a surface cysteine fixed-point coupling technology based on a nano antibody. Compared with the two coupling modes mentioned above, the surface fixed-point coupling technology has many advantages, such as high DAR value and controllable DAR value, and can be designed into DAR2, DAR4 or DAR6 and the like according to requirements, the process is relatively simple, and higher drug loading capacity can be achieved under the condition of the same protein amount. The antibody-drug complex based on the nano-antibody can be applied to a plurality of fields, and the invention can simultaneously solve the problems of high DAR value of the traditional ADC drug and low DAR value of the nano-antibody-drug complex, thereby advancing the treatment based on the nano-antibody drug.

Briefly, the present application discloses methods for preparing antibody-drug (including but not limited to toxins, nuclides, affinity payloads, etc.) immunoconjugates that design high DAR values based on nanobodies, and discloses the diagnostic and therapeutic utility of cysteine engineered antibody drugs.

In one aspect, the present application provides an antigen binding protein comprising a variable antigen binding domain (VHH) of a heavy chain antibody, wherein the VHH comprises one or more pairs of engineered cysteine residues, wherein each pair of the engineered cysteine residues independently comprises a cysteine residue at a first position and a cysteine residue at a second position, the cysteine residue at the first position and the cysteine residue at the second position being capable of pairing to form a disulfide bond; wherein at least one of the cysteine residue at the first position and the cysteine residue at the second position is mutated.

In certain embodiments, wherein the cysteine residue at the first position and the cysteine residue at the second position are both mutated.

In certain embodiments, wherein the surface disulfide bond is capable of forming a sulfhydryl group upon reduction by a reducing agent, wherein the sulfhydryl group allows the antigen binding protein to be conjugated to a payload at the first location and/or the second location.

In certain embodiments, wherein the engineered cysteine residue does not substantially reduce the affinity of the antigen binding protein.

In certain embodiments, wherein neither the first position nor the second position is adjacent to a cysteine residue of a disulfide bond carried by the VHH.

In certain embodiments, wherein the engineered cysteine residue does not pair with a cysteine residue carried by the antigen binding protein.

In certain embodiments, wherein the first position and the second position are both located in the Framework Region (FR) of the VHH.

In certain embodiments, wherein neither the first location nor the second location is located in a loop region of the VHH.

In certain embodiments, wherein neither the first location nor the second location is located in a Complementarity Determining Region (CDR) of the VHH.

In certain embodiments, wherein the cysteine residue at the first position and the cysteine residue at the second position have a C.beta. -C.beta.of no more than about

The spatial distance of amino acid residues can be calculated predictively by Swiss-PdbViewer software.

In certain embodiments, wherein the cysteine residue at the first position and the cysteine residue at the second position are both located on the surface of the antigen binding protein.

In certain embodiments, wherein the relative solvent accessibility (ASA%) of the amino acid residue at the first position to the amino acid residue at the second position is not less than about 20%; solvent accessibility of amino acid residues was predicted from http:// cib.cf.ocha.ac.jp/bitool/ASA/website or calculated by the Swiss-PdbViewer software.

In certain embodiments, wherein the first location and the second location are each independently selected from the following: 3. 7, 9, 11, 12, 13, 17, 23, 39, 41, 66, 68, 83, 84, 87, 8, 16, 18, 25, 42, 45, 81, 82a, 82b, 85, 108, 110, 111, and 113; wherein the first location and the second location are different; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, wherein the first position is selected from: 3. 7, 9, 11, 12, 13, 17, 23, 39, 41, 66, 68, 83, 84, and 87; the second position is selected from: 8. 16, 18, 25, 42, 45, 81, 82a, 82b, 85, 108, 110, 111 and 113; the first and second positions are different; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, wherein the combination of the first and second locations is selected from at least one of the following combinations: a) 66-82b; b) 3-25; c) 7-8; d) 84-113; e) 83-85; f) 68-81; g) 17-82a; h) 41-42; i) 9-108; j) 11-110; k) 87-111; l) 13 to 16; m) 12 to 18; n) 39-45; and o) 14 to 113; wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, wherein the combination of amino acid residues at the first position and amino acid residues at the second position is selected from at least one of the following combinations: a) 66 (R/E) -82b (S/N/R/T/D/Y); b) 3 (Q) -25 (S); c) 7 (S) -8 (G); d) 84 (A/P/M) -113 (S); e) 83 (K/R/Q/S/T/E) -85 (E/D); f) 68 (T/V/A) -81 (Q/E/N); g) 17 (S) -82a (N/L/D); h) 41 (P/A/R) -42 (G); i) 9 (G) -108 (L/Q/H); j) 11 (S/L) -110 (T); k) 87 (T/A/L) -111 (V/D); l) 13 (Q/R) -16 (G); m) 12 (V/G) -18 (L); n) 39 (Q) -45 (R/C/L/H); and o) 14 (P) -113 (S); wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, wherein the VHH comprises 1, 2, 3 or 4 cysteine residues.

In certain embodiments, wherein the VHH comprises 2 pairs of cysteine residues, wherein the combination of the first and second positions in each pair of cysteine residues is selected from any two of the following combinations: a) 66-82b; b) 3-25; c) 7-8; d) 84-113; e) 83-85; f) 68-81; g) 17-82a; h) 41-42; i) 9-108; j) 11-110; k) 87-111; l) 13 to 16; m) 12 to 18; n) 39-45; and o) 14-113; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, wherein said VHH comprises 2 pairs of cysteine residues, wherein the combination of said first and second positions in each pair of cysteine residues is selected from any two of the following combinations: a) 66 (R/E) -82b (S/N/R/T/D/Y); b) 3 (Q) -25 (S); c) 7 (S) -8 (G); d) 84 (A/P/M) -113 (S); e) 83 (K/R/Q/S/T/E) -85 (E/D); f) 68 (T/V/A) -81 (Q/E/N); g) 17 (S) -82a (N/L/D); h) 41 (P/A/R) -42 (G); i) 9 (G) -108 (L/Q/H); j) 11 (S/L) -110 (T); k) 87 (T/A/L) -111 (V/D); l) 13 (Q/R) -16 (G); m) 12 (V/G) -18 (L); n) 39 (Q) -45 (R/C/L/H); and o) 14 (P) -113 (S); wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, wherein the VHH comprises 2 pairs of cysteine residues, wherein the combination of the first and second positions in each pair of cysteine residues is selected from the group consisting of: a) 3-25 and 84-113; b) 7-8 and 3-25; c) 7-8 and 84-113; and d) 11-110 and 84-113; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, wherein the VHH comprises 2 pairs of cysteine residues, wherein the combination of the first and second positions in each pair of cysteine residues is selected from the group consisting of: a) 3 (Q) -25 (S) and 84 (A/P/M) -113 (S); b) 7 (S) -8 (G) and 3 (Q) -25 (S); c) 7 (S) -8 (G) and 84 (A/P/M) -113 (S); and d) 11 (S/L) -110 (T) and 84 (A/P/M) -113 (S); wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, wherein the antigen binding protein comprises an antibody or antigen binding fragment thereof.

In certain embodiments, wherein the antibody or antigen binding fragment comprises a monoclonal antibody.

In certain embodiments, wherein the antibody or antigen-binding fragment thereof comprises a single domain antibody or a heavy chain antibody.

In certain embodiments, wherein the antibody or antigen-binding fragment comprises a chimeric antibody, a humanized antibody, and/or a fully human antibody.

In certain embodiments, wherein the antigen binding protein is a humanized VHH antibody or a fully human VHH antibody.

In certain embodiments, wherein the antigen binding protein specifically binds to a tumor antigen or a non-tumor antigen.

<xnotran> , CD19, BCMA, TSHR, CD171, CS-1, CLL-1, GD3, tn Ag, FLT3, CD38, CD123, CD44v6, B7H3, B7H4, KIT, IL-13Ra2, IL-11Ra, PSCA, PSMA, PRSS21, EGFR2, lewisY, CD24, PDGFR-beta, SSEA-4, MUC1, EGFR, NCAM, CAIX, LMP2, ephA2, GM1, sLe, GM3, TGS5, HMWMAA, FOLR1, FOLR2, TEM1/CD248, TEM7R, CLDN6, CLDN18.2, GPRC5D, CXORF61, CD97, CD179a, ALK, , PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TAARP, WT1, ETV6-AML, SPA17, XAGE1, tie 2, MAD-CT-1, MAD-CT-2, FOSL1, hTERT, ML-IAP, ERG, NA17, PAX3, AR, B1, MYCN, rhoC, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD20, CD30, HER2, ROR1, TAAG72, CD22, CD33, GD2, gp100Tn, FAP, , EPCAM, CEA, IGF-1R, ephB2, , 17, CD32b, EGFRvIII, GPNMB, GPR64, HER3, LRP6, LYPD8, NKG2D, SLC34A2, SLC39A6, SLITRK6, GUCY2C, 5T4 / TACSTD2. </xnotran>

In certain embodiments, wherein the antigen binding protein comprises an anti-PD-L1 VHH antibody, an anti-HER 2 VHH antibody, or an anti-CD 8 α VHH antibody.

In certain embodiments, wherein the anti-PD-L1 VHH antibody comprises: CDR1 of the amino acid sequence shown in SEQ ID NO. 46, CDR2 of the amino acid sequence shown in SEQ ID NO. 47 and CDR3 of the amino acid sequence shown in SEQ ID NO. 48.

In another aspect, the present application provides an antigen binding protein that specifically binds to PD-L1, comprising a variable antigen binding domain (VHH) of a heavy chain antibody, the VHH comprising a cysteine mutation at a position selected from any one or more of the following: a) 66-82b; b) 3-25; c) 7-8; d) 84-113; e) 83-85; f) 68-81; g) 17-82a; h) 41-42; i) 9-108; j) 11 to 110; k) 87-111; l) 13 to 16; m) 12 to 18; n) 39-45; and o) 14 to 113; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, it comprises a variable antigen binding domain (VHH) of a heavy chain antibody, said VHH comprising a cysteine mutation compared to the sequence set forth in SEQ ID NO. 49, said cysteine mutation position selected from any one or more of the following: a) 66 (R/E) -82b (S/N/R/T/D/Y); b) 3 (Q) -25 (S); c) 7 (S) -8 (G); d) 84 (A/P/M) -113 (S); e) 83 (K/R/Q/S/T/E) -85 (E/D); f) 68 (T/V/A) -81 (Q/E/N); g) 17 (S) -82a (N/L/D); h) 41 (P/A/R) -42 (G); i) 9 (G) -108 (L/Q/H); j) 11 (S/L) -110 (T); k) 87 (T/A/L) -111 (V/D); l) 13 (Q/R) -16 (G); m) 12 (V/G) -18 (L); n) 39 (Q) -45 (R/C/L/H); and o) 14 (P) -113 (S); wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, the antigen binding protein comprises the amino acid sequence of any one of SEQ ID NOs 50-63.

In another aspect, the present application provides an antigen binding protein that specifically binds PD-L1 comprising CDR1, CDR2 and CDR3 of a variable antigen binding domain (VHH) of a heavy chain antibody, wherein the amino acid sequence of the VHH is as set forth in any one of SEQ ID NOS: 50-63.

In another aspect, the present application provides an antigen binding protein that competes for binding to PD-L1 with an antigen binding protein described herein.

In certain embodiments, wherein the anti-CD 8 α VHH antibody comprises: CDR1 of the amino acid sequence shown in SEQ ID NO. 1, CDR2 of the amino acid sequence shown in SEQ ID NO. 2 and CDR3 of the amino acid sequence shown in SEQ ID NO. 3.

In another aspect, the present application provides an antigen binding protein that specifically binds CD8 α, comprising a variable antigen binding domain (VHH) of a heavy chain antibody, said VHH comprising a cysteine mutation compared to the sequence set forth in SEQ ID No. 4, at a position selected from any one or more of the following: a) 66-82b; b) 3-25; c) 7-8; d) 84-113; e) 83-85; f) 68-81; g) 17-82a; h) 41-42; i) 9-108; j) 11 to 110; k) 87-111; l) 13 to 16; m) 12 to 18; n) 39-45; and o) 14 to 113; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, it comprises a variable antigen binding domain (VHH) of a heavy chain antibody, said VHH comprising a cysteine mutation compared to the sequence set forth in SEQ ID NO:4 at a position selected from any one or more of the following: a) 66 (R/E) -82b (S/N/R/T/D/Y); b) 3 (Q) -25 (S); c) 7 (S) -8 (G); d) 84 (A/P/M) -113 (S); e) 83 (K/R/Q/S/T/E) -85 (E/D); f) 68 (T/V/A) -81 (Q/E/N); g) 17 (S) -82a (N/L/D); h) 41 (P/A/R) -42 (G); i) 9 (G) -108 (L/Q/H); j) 11 (S/L) -110 (T); k) 87 (T/A/L) -111 (V/D); l) 13 (Q/R) -16 (G); m) 12 (V/G) -18 (L); n) 39 (Q) -45 (R/C/L/H); and o) 14 (P) -113 (S); wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, the antigen binding protein comprises the amino acid sequence of any one of SEQ ID NOs 5-18, 20.

In another aspect, the present application provides an antigen binding protein that specifically binds CD8 a comprising CDR1, CDR2 and CDR3 of a variable antigen binding domain (VHH) of a heavy chain antibody, wherein the amino acid sequence of said VHH is set forth in any one of SEQ ID NOs 5-18, 20.

In another aspect, the present application provides an antigen binding protein that competes for binding to CD8 a with an antigen binding protein described herein.

In certain embodiments, wherein the anti-HER 2 VHH antibody comprises:

i) CDR1 of the amino acid sequence shown as SEQ ID NO. 21, CDR2 of the amino acid sequence shown as SEQ ID NO. 22 and CDR3 of the amino acid sequence shown as SEQ ID NO. 23; or

ii) CDR1 of the amino acid sequence shown in SEQ ID NO. 64, CDR2 of the amino acid sequence shown in SEQ ID NO. 65 and CDR3 of the amino acid sequence shown in SEQ ID NO. 66.

In another aspect, the present application provides an antigen binding protein that specifically binds HER2 comprising a variable antigen binding domain (VHH) of a heavy chain antibody, said VHH comprising a cysteine mutation compared to the sequence shown in SEQ ID No. 24, at a position selected from any one or more of the following: a) 66-82b; b) 3-25; c) 7-8; d) 84-113; e) 83-85; f) 68-81; g) 17-82a; h) 41-42; i) 9-108; j) 11 to 110; k) 87-111; l) 13 to 16; m) 12 to 18; n) 39-45; and o) 14 to 113; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, the VHH comprises a cysteine mutation compared to the sequence set forth in SEQ ID No. 24 at a position selected from any one or more of: a) 66 (R/E) -82b (S/N/R/T/D/Y); b) 3 (Q) -25 (S); c) 7 (S) -8 (G); d) 84 (A/P/M) -113 (S); e) 83 (K/R/Q/S/T/E) -85 (E/D); f) 68 (T/V/A) -81 (Q/E/N); g) 17 (S) -82a (N/L/D); h) 41 (P/A/R) -42 (G); i) 9 (G) -108 (L/Q/H); j) 11 (S/L) -110 (T); k) 87 (T/A/L) -111 (V); l) 13 (Q/R) -16 (G); m) 12 (V/G) -18 (L); n) 39 (Q) -45 (R/C/L/H); and o) 14 (P) -113 (S); wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, the antigen binding protein comprises the amino acid sequence of any one of SEQ ID NOs 25-45.

In another aspect, the present application provides an antigen binding protein that specifically binds HER2 comprising a variable antigen binding domain (VHH) of a heavy chain antibody, said VHH comprising a cysteine mutation compared to the sequence shown in SEQ ID NO:67, the cysteine mutation position being selected from any one or more of the following: a) 66-82b; b) 3-25; c) 7-8; d) 84-113; e) 83-85; f) 68-81; g) 17-82a; h) 41-42; i) 9-108; j) 11-110; k) 87-111; l) 13 to 16; m) 12 to 18; n) 39-45; and o) 14 to 113; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, the VHH comprises a cysteine mutation compared to the sequence set forth in SEQ ID NO 67 at a position selected from any one or more of: a) 66 (R/E) -82b (S/N/R/T/D/Y); b) 3 (Q) -25 (S); c) 7 (S) -8 (G); d) 84 (A/P/M) -113 (S); e) 83 (K/R/Q/S/T/E) -85 (E/D); f) 68 (T/V/A) -81 (Q/E/N); g) 17 (S) -82a (N/L/D); h) 41 (P/A/R) -42 (G); i) 9 (G) -108 (L/Q/H); j) 11 (S/L) -110 (T); k) 87 (T/A/L) -111 (V); l) 13 (Q/R) -16 (G); m) 12 (V/G) -18 (L); n) 39 (Q) -45 (R/C/L/H); and o) 14 (P) -113 (S); wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, the antigen binding protein comprises the amino acid sequence of any one of SEQ ID NOs 68-83.

In certain embodiments, the VHH is further affinity optimized compared to the amino acid sequence of any one of SEQ ID NOs 68-83.

In certain embodiments, it comprises the amino acid sequence set forth in any one of SEQ ID NOs 84-94.

In another aspect, the present application provides an antigen binding protein that specifically binds HER2 comprising CDR1, CDR2 and CDR3 of a variable antigen binding domain (VHH) of a heavy chain antibody, wherein the amino acid sequence of said VHH is set forth in any one of SEQ ID NOs 25-45, 68-95.

In another aspect, the present application provides an antigen binding protein that competes for binding to HER2 with an antigen binding protein described herein.

In another aspect, the present application provides a fusion polypeptide comprising an antigen binding protein as described herein,

in another aspect, the present application provides an immunoconjugate comprising an antigen binding protein as described herein, a payload, and a linker linking the payload to the antigen binding protein.

In certain embodiments, the immunoconjugate has the structure shown in formula I:

VHH-(L-D) _n (I)

Wherein L is a linker; d is a payload; n is any number from 1 to 8.

In certain embodiments, wherein the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs 5-18, 20, 25-45, 50-63, 68-95.

In certain embodiments, wherein the VHH is linked to the linker through a thiol group of a cysteine at a first position and/or a thiol group of a cysteine at a second position.

In certain embodiments, the surface disulfide bond formed by pairing a cysteine residue at a first position with a cysteine residue at a second position in the VHH is reduced to form a free cysteine, the free cysteine comprising a thiol group, and the linker is attached to the thiol group.

In certain embodiments, wherein the linker is cleavable or non-cleavable.

In certain embodiments, wherein the linker is selected from the group consisting of: 6-Maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (alaphe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4- (2-pyridylthio) pentanoate (SPP), N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), (N-succinimidyl 4-iodo-acetyl) aminobenzoate (SIAB),), SPDB, hydrazone, maleimidocaproyl and 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB); or a branched linker comprising a peptide chain and derived from o-hydroxy p-aminobenzyl alcohol, wherein the peptide chain is linked to a benzene ring through a p-amino group, the payload is linked to a benzene ring through a benzyl alcohol moiety, and the antigen binding protein is linked to a benzene ring through an o-hydroxy group.

In certain embodiments, wherein the linker comprises a chelating agent.

In certain embodiments, wherein the chelator is selected from DTPA, EDTA, NOTA, DOTA, TRAP, TETA, NETA, CB-TE2A, cyclen, cyclam, bispidine, TACN, ATSM, sarAR, amBaSar, MAG3, MAG2, HYNIC, DADT, EC, NS3, H2dedpa, HBED, DFO, PEPA or HEHA and derivatives thereof.

In certain embodiments, wherein the payload comprises a detectable label.

In certain embodiments, wherein the detectable label is selected from the group consisting of: radionuclides, fluorescers, chemiluminescent agents, bioluminescent agents, paramagnetic ions and enzymes, and combinations thereof.

In certain embodiments, wherein the detectable label comprises a radionuclide.

In certain embodiments, wherein the radionuclide comprises ¹¹⁰ In、 ¹¹¹ In、 ¹⁷⁷ Lu、 ¹⁸ F、 ⁵² Fe、 ⁶² Cu、 ⁶⁷ Cu、 ⁶⁷ Ga、 ⁶⁸ Ga、 ⁶⁸ Ge、 ⁸⁶ Y、 ⁹⁰ Y、 ⁸⁹ Zr、 ^94m Tc、 ¹²⁰ I、 ¹²³ I、 ¹²⁴ I、 ¹²⁵ I、 ¹³¹ I、 ^154-158 Gd、 ³² P、 ¹¹ C、 ¹³ N、 ¹⁵ O、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ⁵¹ Mn、 ^52m Mn、 ⁷² As、 ⁷⁵ Br、 ⁷⁶ Br、 ^82m Rb、 ⁸³ Sr or other gamma-, beta-, or positron emitters.

In certain embodiments, wherein the radionuclide is linked to the antigen binding protein by a chelator.

In certain embodiments, wherein the payload comprises a targeting moiety.

In certain embodiments, wherein the targeting moiety is selected from the group consisting of: proteins, nucleic acids, lipids, carbohydrates, and combinations thereof.

In certain embodiments, wherein the payload comprises a drug.

In certain embodiments, wherein the drug is selected from: an anti-cancer therapeutic agent, an anti-inflammatory therapeutic agent, an anti-infection therapeutic agent, an anesthetic therapeutic agent, a cytotoxic therapeutic agent, a radionuclide, an immunomodulator, a cell signal peptide, a growth factor, an enzyme, an oligonucleotide, a photoactive therapeutic agent, and any combination thereof.

In certain embodiments, wherein the anti-cancer therapeutic is selected from: cytostatics, cytotoxic nucleosides, tubulin binding agents, hormones and hormone antagonists, anti-angiogenic agents, enzyme inhibitors, gene modulators, proteasome inhibitors, pteridine, diyne, podophyllotoxin, auristatin, geldanamycin, calicheamicin, gramicin D, maytansine, neocarzinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, emetine, tinosporine, colchicine, dihydroxyanthracenedione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansine derivatives, anthracycline derivatives, bisphosphonate derivatives, leptomycin derivatives, desmosine derivatives, auristatin derivatives, duocarmycin derivatives, and any combination thereof.

In certain embodiments, the cytostatic agent is selected from: anthraquinone, DNA synthesis inhibitors, DNA-intercalators, DNA-RNA transcription regulators, ansamycinquinone, quinone derivatives, busulfan, ifosfamide, mechlorethamine, triazone, diazinon, carbazolquinone, indoloquinone E09, diazaspinyl-benzoquinone methyl DZQ, triethylenephosphoramide, nitrosourea compounds, and any combination thereof.

In certain embodiments, wherein the cytotoxic nucleoside is selected from the group consisting of: vidarabine, cytarabine, 5-fluorouracil, fludarabine, fluorouracil nucleoside, ftoracure, 6-mercaptopurine, and combinations of any of these.

In certain embodiments, wherein the tubulin-binding agent is selected from the group consisting of: paclitaxel, nocodazole, rhizoxin, dolastatin, colchicine, combretastatin, vinca alkaloids, and any combination thereof.

In certain embodiments, wherein the hormone and hormone antagonist are selected from the group consisting of: corticosteroids, progestins, estrogens, antiestrogens, androgens, aromatase inhibitors, 17- (allylamino) -17-demethoxygeldanamycin, 4-amino-I, 8-naphthalimide, apigenin, brefeldin A, cimetidine, dichloromethylene diphosphonic acid, leuprolide, luteinizing hormone releasing hormone, pifithrin-a, rapamycin, sex hormone binding globulin, thapsigargin, and any combination thereof.

In certain embodiments, wherein the anti-angiogenic agent is selected from the group consisting of: angiostatin K1-3, DL- α -difluoromethyl-ornithine, endostatin, fumagillin, genistein, minocycline, staurosporine, (+) -thalidomide, and any combination thereof.

In certain embodiments, wherein the enzyme inhibitor is selected from the group consisting of: s (+) -camptothecin, curcumin, (-) -deguelin, 5, 6-dichlorobenzene-imidazole I-beta-D-ribofuranoside, etoposide, formestane, fosstricin, hispidin, 2-imino-1-imidazolidineacetic acid, mevinolin, trichostatin A, tyrphostin AG 34, tyrphostin AG 879, and any combination thereof.

In certain embodiments, wherein the gene modulator is selected from the group consisting of: 5-aza-2' -deoxycytidine, 5-azacytidine, cholecalciferol, 4-hydroxyttamoxifen, melatonin, mifepristone, raloxifene, trans retinal, retinoic acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol, tamoxifen, troglitazone, and any combination thereof.

In certain embodiments, wherein n is any integer from 1 to 8.

In certain embodiments, wherein n is 2, 4, 6, or 8.

In another aspect, the present application provides a pharmaceutical composition comprising an antigen binding protein as described herein or a conjugate as described herein, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

In another aspect, the present application provides an isolated nucleic acid molecule comprising a polynucleotide encoding an antigen binding protein as described herein or a fusion protein as described herein.

In another aspect, the present application provides a vector comprising a nucleic acid molecule as described herein.

In another aspect, the present application provides a cell comprising a nucleic acid molecule as described herein and/or a vector as described herein.

In another aspect, the present application provides a method of making an antigen binding protein described herein, the method comprising:

i) Site-directed mutagenesis of a nucleic acid sequence of a parent antibody, wherein the mutagenesis comprises substitution of one or more pairs of amino acid residues with cysteine residues; wherein each pair of said cysteine residues comprises a cysteine residue at a first position and a cysteine residue at a second position, said cysteine residue at the first position being capable of pairing with said cysteine residue at the second position to form a disulfide bond; and

ii) expressing the antigen binding protein.

In certain embodiments, wherein the method further comprises recovering the antigen binding protein.

In certain embodiments, wherein the method further comprises purifying the antigen binding protein.

In certain embodiments, wherein the mutagenesis further comprises affinity optimization of the antibody after substitution with a cysteine residue.

In another aspect, the present application provides a method of making an antigen binding protein described herein, the method comprising: comprising culturing the cells described herein under conditions that allow expression of the antigen binding protein.

In another aspect, the present application provides a method of making a conjugate described herein, the method comprising: the antigen binding proteins described herein are conjugated to a payload.

In certain embodiments, the method comprises: reacting at least one pair of cysteines of the antigen binding protein with the linker-payload intermediate to form an antigen binding protein-payload conjugate:

VHH-(L-D) _n (I)

wherein L is a linker; d is a payload; n is any number from 1 to 8.

In certain embodiments, where n is any integer from 1 to 8.

In certain embodiments, wherein n is 2, 4, 6, or 8.

In certain embodiments, wherein the method comprises: i) Reducing and opening disulfide bonds formed by paired cysteines in the antigen binding protein to form sulfhydryl free cysteine; ii) linking the sulfhydryl group of a free cysteine with a linker to form an antigen binding protein-payload conjugate.

In certain embodiments, wherein the method comprises: i) Reducing open disulfide bonds formed by paired cysteine residues in the antigen binding protein to form free cysteines; ii) linking the sulfhydryl group of the free cysteine to a linker to form an antigen binding protein-linker intermediate; iii) Conjugating the antigen binding protein-linker intermediate to a payload to form an antigen binding protein-payload conjugate.

In another aspect, the present application provides a method of inhibiting cell proliferation comprising contacting a cell with an effective amount of an antigen binding protein described herein, a conjugate described herein, or a pharmaceutical composition described herein.

In certain embodiments, the contacting is performed in vivo or in vitro.

In another aspect, the present application provides a method of inhibiting cell proliferation comprising exposing cells in a cell culture medium to an antigen binding protein described herein, a conjugate described herein, or a pharmaceutical composition described herein.

In certain embodiments, the cell comprises a mammalian cell.

In certain embodiments, the cell comprises a tumor cell.

In another aspect, the present application provides a method of detecting, preventing and/or treating a tumor or other disease comprising administering to a subject in need thereof an antigen binding protein described herein, a conjugate described herein or a pharmaceutical composition described herein.

In certain embodiments, wherein the tumor comprises a solid tumor and a non-solid tumor.

In certain embodiments, wherein the tumor is selected from any one or more of the group consisting of: lymphoma, multiple myeloma, breast cancer, ovarian cancer, kidney cancer, endometrial cancer, melanoma, pancreatic cancer, lung cancer, gastric cancer, liver cancer, mesothelioma, esophageal cancer, head and neck cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, thymus cancer, and colorectal cancer.

In certain embodiments, the method further comprises administering to the subject an additional therapy or drug.

In certain embodiments, wherein the additional therapy is selected from the group consisting of: chemotherapy, radiation therapy, miRNA, and oligonucleotides.

In another aspect, the present application provides the use of an antigen binding protein as described herein, an immunoconjugate as described herein and/or a pharmaceutical composition as described herein for the preparation of a medicament for the treatment of a tumor or other disease.

In another aspect, the present application provides the use of an antigen binding protein as described herein, an immunoconjugate as described herein and/or a pharmaceutical composition as described herein in combination with other therapies or drugs in the manufacture of a medicament for the treatment of tumors or other diseases.

In another aspect, the present application provides a method of detecting a specific target for non-disease diagnostic purposes comprising the use of an antigen binding protein as described herein, an immunoconjugate as described herein and/or a pharmaceutical composition as described herein.

In another aspect, the present application provides a kit comprising an antigen binding protein as described herein, an immunoconjugate as described herein and/or a pharmaceutical composition as described herein, and optionally instructions for use.

In another aspect, the present application provides a kit comprising an antigen binding protein as described herein, an immunoconjugate as described herein and/or a pharmaceutical composition as described herein, and optionally an administration device.

In another aspect, the present application provides a method for screening a nanobody site-directed conjugation site, the method comprising: screening for amino acid residue pairing in VHHs at about C.beta. -C.beta.

Combinations within the range.

In certain embodiments, the method further comprises excluding amino acid residues having ASA% less than about 20% relative solvent accessibility.

In certain embodiments, the spatial distance and/or solvent accessibility of amino acid residues therein is calculated as predicted according to Swiss-PdbViewer software.

In certain embodiments, the method further comprises excluding amino acid residues located in the CDR regions, or loop regions, or near the internal disulfide bond of the VHH.

In certain embodiments, the method further comprises homology modeling the VHH prior to screening for amino acid residue pairing.

In some embodiments, the homology modeling is calculated using modeler 9 software.

In certain embodiments, the method comprises:

1) Performing homologous modeling on the VHH;

2) Screening for amino acid residues in VHH at about C.beta. -C.beta.

Combinations within the range;

3) Excluding amino acid residues having ASA% less than about 20% relative solvent accessibility;

4) Amino acid residues located in the CDR regions of the VHH, or in the loop regions, or near the internal disulfide bonds of the VHH are excluded.

Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.

Drawings

The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The drawings are briefly described as follows:

figure 1 shows multiple sequence alignment data for 90 multiple single domain antibody sequences in the PDB database.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.

Definition of terms

In the present application, the term "PD-L1" or "PD-L1" generally refers to programmed cell death 1 ligand 1, also referred to as B7 homolog 1, B7-H1, cluster of differentiation 274, (3) 274 or CD274, which upon binding to PD-1 down-regulates T cell activation and cytokine secretion. "PD-L1" includes any native PD-L1 of any vertebrate origin, including mammals, such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed PD-L1, as well as any form of PD-L1 that results from cellular processing. PD-L1 may exist as a transmembrane protein or as a soluble protein. "PD-L1" includes intact PD-L1 and fragments thereof, and also includes functional variants, isoforms, species homologs, derivatives, analogs of PD-L1, and analogs having at least one common epitope with PD-L1. The basic structure of PD-L1 includes 4 domains: an extracellular Ig-like V-type domain and an Ig-like C2-type domain, a transmembrane domain, and a cytoplasmic domain. Exemplary human PD-L1 amino acid sequences can be found under NCBI accession No. NP _001254653 or UniProt accession No. Q9NZQ 7.

In the present application, the term "HER2" generally refers to a type I transmembrane protein belonging to the epidermal growth factor receptor family, also known as c-erbB2, erbB2 or Neu. The term "HER2" also encompasses homologs, variants, and isoforms of HER2, including splice isoforms. HER2 is associated with tumor transformation in human breast cancer cells, as overexpression of HER2 protein has been detected in patients with breast, gastric, pancreatic, ovarian, peritoneal or colon cancer. The terms "HER2 positive" and "expressing HER2" are used interchangeably in this application. A "HER2 positive" tumor comprises tumor cells having higher than normal levels of HER 2. Examples of HER2 positive tumors include HER2 positive breast cancer and HER2 positive gastric cancer. Optionally, the HER2 positive is a HER2 overexpressing cancer, and in certain embodiments, the HER-2 positive cancer has an Immunohistochemistry (IHC) score of 2+ or 3+ and/or an In Situ Hybridization (ISH) amplification rate ≧ 2.0.

The term "CD8" (cluster of differentiation 8) refers to a cell surface glycoprotein that is expressed predominantly on cytotoxic T lymphocytes, but also on subsets of dendritic cells, natural killer T cells, and γ δ T cells. Glycoproteins are composed of the two isoforms α and β (which are encoded by different genes) and are expressed as α α homodimers or α β heterodimers, the latter of which predominates. The CD8 co-receptor stabilizes the T cell receptor MHC-1 interaction and triggers intracellular signaling for activation by lymphocyte-specific protein tyrosine kinase (Lck) phosphorylation of the CD 3-associated Immunoreceptor Tyrosine Activation Motif (ITAM).

In the present application, the term "antigen" generally refers to any immunogenic fragment or determinant comprising the selected target, including a single epitope, multiple epitopes, single or multiple domains or the entire extracellular domain (ECD). The antigen can be an isolated full-length protein, a cell surface protein (e.g., immunized with cells expressing at least a portion of the antigen on their surface), or a soluble protein (e.g., immunized with an ECD portion having only the protein). The antigen may be produced in a genetically modified cell. Any of the above antigens may be used alone or in combination with one or more immunogenicity-enhancing adjuvants known in the art. The DNA encoding the antigen may be genomic or non-genomic (e.g., cDNA), and may encode at least a portion of the ECD sufficient to elicit an immunogenic response. Any vector can be used to transform the cells in which the antigen is expressed, including but not limited to adenoviral vectors, lentiviral vectors, plasmids, and non-viral vectors, such as cationic lipids. The term "antigen" as used herein includes, for example, proteins, different epitopes on proteins (as different antigens within the meaning of the invention) and polysaccharides. This includes mainly parts of bacteria, viruses and other microorganisms (shells, envelopes, cell walls, flagella, fimbrae and toxins). Lipids and nucleic acids are antigenic only when combined with proteins and polysaccharides. Non-microbial foreign (non-self) antigens may include pollen, egg white, and proteins from transplanted tissues and organs or proteins on the surface of blood cells that are transfused. Without limitation, the antigen may be selected from the group consisting of cytokines, cell surface proteins, enzymes and receptors.

As used herein, "tumor antigen" includes its meaning known in the art, which includes any molecule expressed on (or associated with the development of) a tumor cell that is known or believed to have an effect on the tumorigenic properties of the tumor cell. Many tumor antigens are known in the art. Whether a molecule is a tumor antigen can also be determined according to techniques and assays well known to those skilled in the art, such as clonogenic assays (clonogenic assays), transformation assays, in vitro or in vivo tumor formation assays, gel migration assays (gel migration assays), gene knock-out assays, and the like. In some embodiments, the term "tumor antigen" as used herein refers to a human transmembrane protein, i.e., a cell membrane protein anchored in a cellular lipid bilayer. As used herein, a human transmembrane protein will typically comprise an "extracellular domain" which may bind a ligand, a lipophilic transmembrane domain, a conserved intracellular domain, such as a tyrosine kinase domain, and a carboxy-terminal signaling domain having several tyrosine residues which may be phosphorylated. Tumor antigens include molecules such as EGFR, HER2/neu, HER3, HER4, epCAM, CEA, TRAIL receptor 1, TRAIL receptor 2, lymphotoxin-beta receptor, CCR4, CD19, CD20, CD22, CD28, CD33, CD40, CD80, CSF-1R, CTLA-4, fibroblast Activation Protein (FAP), hepsin, melanoma-associated chondroitin sulfate proteoglycan (MCSP), prostate Specific Membrane Antigen (PSMA), VEGF receptor 1, VEGF receptor 2, IGF1-R, TSLP-R, TIE-1, TIE-2, TNF-alpha, TNF-like weak inducer of apoptosis (TWEAK), or IL-1R.

The term "antigen binding protein" is used in its broadest sense and means a protein that comprises a moiety that binds to an antigen or target and optionally a framework or framework portion that allows the antigen binding moiety to adopt a configuration that facilitates binding of the antigen binding protein to the antigen. Examples of antigen binding proteins include human antibodies, humanized antibodies; a chimeric antibody; a recombinant antibody; a single chain antibody; a bifunctional antibody; a trifunctional antibody; a tetrafunctional antibody; a Fab fragment; f (ab') ₂ A fragment; an IgD antibody; an IgE antibody; an IgM antibody; an IgG1 antibody; an IgG2 antibody; an IgG3 antibody; or IgG4 antibodies and fragments thereof. Antigen binding proteins may include, for example, alternative protein frameworks or artificial frameworks with grafted CDRs or CDR derivatives. Such frameworks include, but are not limited to: an antibody-derived framework comprising mutations introduced to, for example, stabilize the three-dimensional structure of an antigen-binding protein; and fully synthetic frameworks comprising, for example, biocompatible polymers. See, e.g., korndorfer et al, 2003, proteins: structure, function, and Bioinformatics,53 (1): 121-129 (2003); roque et al, biotechnol. Prog.20:639-654 (2004). In addition, peptide antibody mimetics ("PAM") can be used, as can frameworks based on antibody mimetics that utilize a fibronectin component as a framework.

In the present application, the term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity (Milleretal (2003) journal. Of immunology170: 4854-4861). The antibody may be murine, human, humanized, chimeric, or derived from other species.

In the present application, the techniqueThe term "antigen-binding fragment" generally refers to a portion of an antibody molecule that comprises amino acids responsible for specific binding between an antibody and an antigen. The portion of the antigen specifically recognized and bound by the antibody is referred to as an "epitope" as described above. The antigen binding domain may typically comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not necessarily include both. Examples of antigen-binding fragments of antibodies include (1) Fab fragments, monovalent fragments with VL, VH, constant light Chain (CL) and CH1 domains; (2) F (ab') ₂ A fragment, a bivalent fragment having two Fab fragments linked by a disulfide bridge of the hinge region; (3) an Fd fragment having two VH and CH1 domains; (4) Fv fragments with VL and VH Domains of a Single arm of an antibody, (5) dAb fragments (Ward et al, "Binding Activities of a repertile of Single Immunoglobulin Variable Domains From Escherichia coli," Nature 341-546 (1989), which is incorporated herein by reference in its entirety), having a VH domain; (6) an isolated Complementarity Determining Region (CDR); (7) Single chain Fv (scFv), e.g.derived from a scFv-library. Although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by synthetic linkers that allow them to be prepared as a Single Protein Chain in which the VL and VH regions pair to form monovalent molecules (known as Single Chain Fv (scFv)) (see, e.g., huston et al, "Protein Engineering of Antibody Binding Sites: recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv antigen Produced in Escherichia coli," proc. Natl. Acad. Sci. Usa 85-5879-5883 (1988)); and (8) VHH, "VHH" relates to the variable antigen-binding domain of heavy chain antibodies from camelidae (camel, dromedary, llama, alpaca, etc.) (see Nguyen v.k. Et al, 2000, the EMBO journal,19, 921-930, muydermans S, 2001, j Biotechnol, 74, 277-302 and reviewed in Vanlandschoot p. Et al, 2011, antiviral Research 92, 389-407).

In the present application, the terms "heavy chain single domain antibody", "VHH domain", "VHH", "V _H H domain, VHH antibody fragment, VHH antibody and

and & ->

Domain "(" Nanobody "is a trademark of Ablynx n.v. company, ghent, belgium) is used interchangeably.

In the present application, the term "variable domain" generally refers to a variable domain of an antibody capable of specifically binding an epitope of an antigen. For example, antibody variable domains VH and VL (VH and VL domains). Another example of a variable domain is a "VHH domain" (or simply "VHH"). "VHH domain", also called heavy chain single domain antibody, VHH, V _H H domains, VHH antibody fragments and VHH antibodies, variable domains of antigen-binding immunoglobulins called "heavy chain antibodies" (i.e. "antibodies lacking light chain") (Hamers-Casterman C, atarhouch T, muylermans S, robinson G, hamers C, songa EB, bendahman N, hamers R.: naturally occuring antibodies void of light chains "; nature 363,446-448 (1993)). The term "VHH domain" is used to distinguish the variable domain from a heavy chain variable domain (which is referred to herein as a "VH domain") present in a conventional 4 chain antibody, and a light chain variable domain (which is referred to herein as a "VL domain") present in a conventional 4 chain antibody. The VHH domain specifically binds to an epitope without the need for an additional antigen binding domain (as opposed to the VH or VL domain in conventional 4 chain antibodies, in which case the epitope is recognized by the VL domain together with the VH domain).

In the present application, "variable domains" typically have the same general structure, each domain comprising 4 Framework (FR) regions highly conserved in sequence, wherein the FR regions include four "framework regions" of "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", three "complementarity determining regions" or "CDRs" of FR region "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3" are linked. The general structure or sequence of a variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Antibody variable domains confer specificity for an antigen by virtue of having an antigen binding site.

In this application, the term "CDR" generally refers to complementarity determining regions within the variable sequence of an antibody. There are 3 CDRs in each variable region of the heavy and light chains, which for each variable region are referred to as CDR1, CDR2 and CDR3. The exact boundaries of these CDRs have been defined differently for different systems. For example, for amino acid residues used in VHH domains of the Camelidae family, the FR regions of the humanized nanobody h-NbBcII10FGLA (JANUARY 30, 2009-VOLUME 284-NUMBER 5) may be numbered according to the general numbering of VH domains given by Kabat et al ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, publication No. 91). According to this numbering, FR1 comprises amino acid residues at positions 1-25, CDR1 comprises amino acid residues at positions 26-35, FR2 comprises amino acids at positions 36-49, CDR2 comprises amino acid residues at positions 50-59, FR3 comprises amino acid residues at positions 60-97, CDR3 comprises amino acid residues at positions 98-116, and FR4 comprises amino acid residues at positions 117-126. It should be noted, however, that the total number of amino acid residues in each CDR may be different and may not correspond to the total number of amino acid residues indicated by the Kabat numbering, as is well known in the art for VH and VHH domains (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.

h-NbBcII10FGLA:

QVQLVESGGGLVQPGGSLRLSCAASGGSEYSYSTFSLGWFRQAPGQGLEAVAAIASMGGLTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAVRGYFMRLPSSHNFRYWGQGTLVTVSS

Note: underlined are FR1 to FR4 regions

In the present application, the term "loop region" generally refers to a turn of more than 5 amino acid residues, also commonly referred to as a loop, and generally refers to a pattern less region linking two conventional structures. They are usually located in solvent exposed areas of the protein surface and often play an important role, such as interaction with other biomolecules. The protein structure can be divided into four levels of a primary structure, a secondary structure, a tertiary structure, a quaternary structure and the like. The primary structure is the amino acid sequence that makes up the protein. Secondary structures generally refer to local regular and ordered structures, for example, α -helix, β -sheet structures are connected in series by loop regions (usually containing one or more corners) to form tertiary structures. The quaternary structure refers to a complex structure with specific functions formed by interaction of multiple protein subunits. The protein loop region is usually located on the surface of the protein and is an important participant in the processes of signal transmission, protein-ligand recognition and the like. For example, the loop region may be involved in the formation of active sites and binding sites for proteins, regulating the binding of antigens to immunoglobulins. For example: the position where the antibody binds to the antigen consists of six loop regions, and the length and amino acid sequence of the loop regions vary from antibody to antibody. If the loop region is linked to two beta strands in different directions (antiparallels), the loop-region is called hairpin loops, and the position on the antibody where the antigen binds is generally the loop.

In the present application, the term "disulfide bond" refers to a chemical covalent bond that exists in the tertiary structure of a protein, typically one or more crosslinks between polypeptide chains formed by oxidation of cysteine residues or between portions of a polypeptide chain. The R group of cysteine is a sulfhydryl (-SH) group, and under oxidizing conditions two different cysteine side chains can form a covalent bond between each other, called a disulfide (-S-S-). Disulfide bonds can be cross-linked within a protein or between two proteins, and have thermal and mechanical stability, and furthermore can be broken by reducing a reducing agent in a protein solution.

In the present application, the term "monoclonal antibody" generally refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies in the population are identical except for possible small amounts of natural mutations. Monoclonal antibodies are typically highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which typically have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use herein can be prepared in hybridoma cells, or can be prepared by recombinant DNA methods.

The term "chimeric antibody" refers to an antibody comprising heavy and light chain variable region sequences from one species and constant region sequences from another species, e.g., an antibody having camelid heavy chain variable regions linked to human constant regions.

In the present application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR regions of a non-human antibody (e.g., an alpaca antibody) are replaced with corresponding amino acids derived from a human immunoglobulin. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions may also be permissible as long as they still retain the ability of the antibody to bind to a particular antigen. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region. "humanized antibodies" retain antigen specificity similar to the original antibody. "humanized" forms of non-human (e.g., alpaca) antibodies may be chimeric antibodies that minimally comprise sequences derived from non-human immunoglobulins. In some cases, CDR region residues in a human immunoglobulin (recipient antibody) can be replaced with CDR region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired properties, affinities and/or capabilities. In some cases, residues from the FR region of a human immunoglobulin may be replaced with corresponding non-human residues. In addition, humanized antibodies may comprise amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made in order to further improve the properties of the antibody, such as binding affinity.

In this application, the term "fully human antibody" generally refers to an antibody that is expressed by a genetically engineered antibody gene-deleted animal into which a human gene encoding the antibody has been transferred. All parts of the antibody (including the variable and constant regions of the antibody) are encoded by genes of human origin. The fully human antibody can greatly reduce the immune side reaction of the heterologous antibody to the human body. The method for obtaining fully human antibody in the field can be phage display technology, transgenic mouse technology, ribosome display technology, RNA-polypeptide technology and the like.

In the present application, the term "competition" when used in the context of antigen binding proteins that compete for the same epitope generally refers to competition between the antigen binding proteins as determined by an assay in which the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) being tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen (e.g., HER2 or fragment thereof). Many types of competitive binding assays can be used to determine whether one antigen binding protein competes with another, for example: solid phase direct or indirect Radioimmunoassays (RIA), solid phase direct or indirect Enzyme Immunoassays (EIA), sandwich competition assays (see, e.g., stahli et al, 1983, methods in enzymology 9; solid phase direct biotin-avidin EIA (see, e.g., kirkland et al, 1986, J.Immunol.137, 3614-3619) solid phase direct labeling assay, solid phase direct labeling sandwich assay (see, e.g., harlow and Lane,1988, antibodies, A Laboratory Manual, cold Spring Harbor Press); direct labeling of RIA using an I-125 labeled solid phase (see, e.g., morel et al, 1988, mol. Immunol.25; solid phase direct biotin-avidin EIA (see, e.g., cheung et al, 1990, virology 176; and direct labeling RIA (Moldenhauer et al, 1990, scand. J. Immunol.32. Typically, such assays involve the use of purified antigen bound to a solid surface or a unit carrying either of these, an unlabeled test antigen binding protein and a labeled reference antigen binding protein. Competitive inhibition is measured by determining the amount of label bound to a solid surface or unit in the presence of the test antigen binding protein. Typically, the test antigen binding protein is present in excess. Antigen binding proteins identified by competition assays (competing antigen binding proteins) include antigen binding proteins that bind the same epitope as the reference antigen binding protein and antigen binding proteins that bind adjacent epitopes sufficiently close to the epitope bound by the reference antigen binding protein to undergo steric hindrance. Typically, when the competing antigen binding protein is present in excess, it inhibits (e.g., reduces) specific binding of the reference antigen binding protein to the common antigen by at least about 40-45%, about 45-50%, about 50-55%, about 55-60%, about 60-65%, about 65-70%, about 70-75%, or about 75% or more. In some cases, binding is inhibited by at least about 80-85%, about 85-90%, about 90-95%, about 95-97%, or about 97% or more.

In the present application, the term "sequence identity" generally refers to a nucleic acid or amino acid sequence in which two or more aligned sequences are identical when aligned using a sequence alignment program. The term "% sequence identity" as used herein generally refers to the level of nucleic acid or amino acid sequence identity between two or more aligned sequences when aligned using a sequence alignment program. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program from the NCBI database can be used to determine identity. For the determination of sequence identity see, for example: computational Molecular Biology, lesk, a.m., ed., oxford University Press, new York,1988; biocomputing, information and Genome Projects, smith, D.W., ed., academic Press, new York,1993; computer Analysis of Sequence Data, part I, griffin, A.M., and Griffin, H.G., eds., humana Press, new Jersey,1994; sequence Analysis in Molecular Biology,20von Heinje, G., academic Press,1987 and Sequence Analysis Primer, gribskov, M.and Devereux, J., eds., M Stockton Press, new York,1991.

In the present application, amino acid residues will be represented according to the standard three-letter or one-letter amino acid code as is well known and agreed upon in the art. In comparing two amino acid sequences, the term "amino acid difference" generally refers to the specified number of amino acid residues at a position in the reference sequence compared to the other sequence, insertion, deletion or substitution. In some embodiments, the substitution is a conservative amino acid substitution, which refers to the replacement of an amino acid residue with another amino acid residue that is chemically similar in structure, and which has little or no effect on the function, activity, or other biological property of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example conservative amino acid substitutions are those in which one amino acid within the following groups (i) - (v) is replaced with another amino acid residue within the same group: (i) a less aliphatic non-polar or weakly polar residue: ala, ser, thr, pro, and Gly; (ii) polar negatively charged residues and their (uncharged) amides: asp, asn, glu and Gln; (iii) polar positively charged residues: his, arg and Lys; (iv) larger aliphatic apolar residues: met, leu, ile, val, and Cys; and (v) aromatic residues: phe, tyr, and Trp. Particularly preferred conservative amino acid substitutions are as follows: ala substituted by Gly or Ser; arg is replaced by Lys; asn is replaced by Gln or His; asp substituted by Glu; cys is substituted with Ser; gln is substituted by Asn; glu is substituted with Asp; gly by Ala or Pro; his is substituted with Asn or Gln; ile is substituted by Leu or Val; leu is substituted by Ile or Val; lys is substituted with Arg, gln, or Glu; met is substituted by Leu, tyr or Ile; phe is substituted by Met, leu or Tyr; ser substituted by Thr; thr is substituted by Ser; trp is substituted by Tyr; tyr is substituted with Trp or Phe; val is substituted by Ile or Leu. In some embodiments, the substitution is a non-conservative amino acid substitution, e.g., ala is substituted with Asp, asn, glu, or Gin.

In the present application, the term "affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a polypeptide or an antibody) and its binding partner (e.g., a target or an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, such as surface plasmon resonance, and also includes those methods reported herein. A higher affinity of molecule X for its binding partner Y can be seen in lower Kd values and/or EC50 values.

In the present application, the term "isolated" generally refers to a molecule (e.g., an antibody, a nucleic acid, etc.) that is at least partially separated from other molecules with which it is normally associated in its native state.

The terms "polynucleotide", "nucleic acid", "nucleotide" and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, or deoxyribonucleotides, ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may comprise modified nucleotides, such as methyl nucleotides and nucleotide analogs. Nucleotides, if present, may be modified before or after assembly of the polymer. The non-nucleotide component may interrupt the nucleotide sequence. The polynucleotide may be further modified after polymerization, such as by coupling with a labeling component.

In the present application, the term "vector" generally refers to a DNA or RNA molecule comprising a nucleotide sequence encoding a protein. A coding sequence or "coding nucleic acid sequence" may include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signals capable of directing expression in the cells of an individual to whom the nucleic acid molecule is administered. Examples include, but are not limited to, circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes comprising non-native nucleic acid sequences, and the like.

In the present application, the term "immunoconjugate" generally refers to a conjugate of an antibody or antibody fragment thereof linked to other active agents, such as chemotherapeutic agents, toxins, immunotherapeutic agents, radioactive elements, imaging probes, spectroscopic probes, and the like. The linkage may be a covalent bond, or a non-covalent interaction, such as by electrostatic force. A variety of linkers known in the art can be used to form immunoconjugates. The conjugate can deliver the additional agent to a target cell (e.g., a tumor cell) by specific binding of the antibody or antigen-binding fragment thereof to an antigen on the target cell. In addition, the immunoconjugate may be provided in the form of a fusion protein that may be expressed from a polynucleotide encoding the immunoconjugate.

In the present application, the term "chelating agent" generally refers to an organic molecule capable of forming a complex with a metal ion. Chelators are commonly used to label proteins or peptides. The end product of the metal ion conjugate is used for radioimmunoassay, radioimmunotherapy, magnetic resonance imaging, photodynamic therapy or other similar modalities. Non-limiting examples of chelating or complexing agents are DTPA (diethylenetriaminepentaacetic anhydride) and its derivatives, NOTA (1, 4, 7-triazacyclononane-N, N ', N "-triacetic acid) and its derivatives such as NODA-GA (NODAGA), maleimide-NODAGA, DOTA (1, 4,7, 10-tetraazacyclododecane-N, N ', N" ' -tetraacetic acid) (bound to radioactive metal ions) and its derivatives, TETA (1, 4,8, 11-tetraazacyclotetradecane-N, N ', N "' -tetraacetic acid) and its derivatives, DTTA (N- (p-isothiocyanatobenzyl) -diethylenetriamine-N, N ', N" ' -tetraacetic acid). These and other chelating agents are readily available from commercial sources.

In the present application, the term "detectable label" generally refers to a moiety having a detectable physical or chemical property, which label can produce a signal that can be detected by visual or instrumental means. Examples of labels for polypeptides include (but are not limited to) the following: radioisotopes or radionuclides, fluorescent labels (e.g., FITC, rhodamine (rhodamine), lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, β -galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotin groups (which can be detected by labeled avidin (e.g., molecules containing streptavidin moieties) and fluorescent labels or enzymatic activity that can be detected by optical methods or calorimetry), and predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).

In the present application, the term "pharmaceutically acceptable carrier" generally refers to one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient. Such formulations may routinely contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable formulations may also contain compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to humans. Other contemplated carriers, excipients, and/or additives that may be used in the formulations described herein include: for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, proteinaceous excipients (e.g., serum albumin, gelatin, casein), salt-forming counterions (e.g., sodium), and the like. These and additional known pharmaceutical carriers, excipients and/or additives suitable for use in The formulations described herein are known in The art, for example, as set forth in Remington's pharmaceutical sciences and practices (Remington: the Science & # x26; practice of Pharmacy), "21 st edition, lepidote Williams and Wilkins publishing company (Lippincott Williams & # x26; wilkins) (2005) and" physicians's desk Reference "(60 th edition, medical economic publications (Medical Economics), montmel (Montvale), new Jersey (2005). Pharmaceutically acceptable carriers may be routinely selected as appropriate for the desired or required mode of administration, solubility and/or stability.

In the present application, the term "administration (administer)" and similar terms are generally not limited to bodily administration, and suitable methods include in vitro, ex vivo, or in vivo methods. For example, any method of administration known to those skilled in the art for contacting a cell, organ or tissue with a composition may be employed. For example, the compounds may be introduced into the body of a subject in need of treatment by any route of introduction or delivery. In some embodiments, the compositions of the present application may be administered orally, topically, intranasally, intramuscularly, subcutaneously, intradermally, intrathecally, intraperitoneally, or transdermally.

In the present application, the term "contacting" generally means that two or more different types of substances are brought together in any order, in any manner, and for any length of time. The contacting can occur in vivo, ex vivo, or in vitro. In one embodiment, such contacting comprises direct injection of the cells by any method known in the art, such as microinjection. In another embodiment, the providing to the cell is indirect, e.g., by providing in a medium surrounding the cell, or administering to the subject, or by any route known in the art. In another embodiment, the term "contacting" means introducing a molecule of the invention into a subject receiving treatment and contacting the molecule with a cell in vivo. Each possibility represents a separate embodiment of the invention.

In the present application, the terms "ex vivo" and "in vitro" are interchangeable, and generally refer to activities performed in a controlled environment in cells, tissues and/or organs that have been removed from a subject.

In the present application, the term "diagnosing" generally refers to detecting a disease or condition, or determining the state or extent of a disease or condition. The term "diagnosing" may also include detecting a predisposition for a disease or condition, determining the therapeutic effect of a drug treatment, or predicting a response pattern to a drug treatment.

In the present application, the term "treatment" generally means: (i) Preventing the occurrence of a disease, disorder, or condition in a patient who may be predisposed to the disease, disorder, and/or condition, but has not yet been diagnosed as having the disease; (ii) Inhibiting, i.e., arresting the development of, the disease, disorder or condition; and (iii) ameliorating the disease, disorder, or condition, i.e., causing regression of the disease, disorder, and/or condition and/or symptoms associated with the disease, disorder, and/or condition.

In the present application, the terms "effective amount" or "effective dose" are used interchangeably and generally refer to an amount sufficient to achieve, or at least partially achieve, the desired therapeutic effect. The term "therapeutically effective dose" generally refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The amount effective for such use will depend on the severity of the infection and the overall state of the patient's own immune system.

In the present application, the terms "tumor" and "cancer" are used interchangeably and generally refer to neoplastic or malignant cell growth. The tumor of the present application may be benign or malignant. The tumors of the present application may be solid or non-solid. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, superficial diffuse melanoma, lentigo nevus melanoma, acromelanoma, melanoma, multiple myeloma and B-cell lymphoma, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and post-transplant lymphoproliferative disorder (ptphald), as well as associated with scarring (koospes), edema (such as associated with brain tumors) and proliferative brain (Meigs) cancer, and head and neck cancer. In certain embodiments, cancers suitable for treatment by the antibodies of the invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, kaposi's sarcoma, carcinoid carcinoma (carcinoid carcinosa), head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some embodiments, the cancer is selected from: non-small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple negative breast cancer), gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. Also, in some embodiments, the cancer is selected from: non-small cell lung cancer, colorectal cancer, glioblastoma and breast cancer (e.g., triple negative breast cancer), including metastatic forms of those cancers.

In the present application, the term "autoimmune disease" generally refers to a condition resulting from an autoimmune response. Autoimmune diseases are the result of inappropriate and excessive responses to self-antigens. Examples of autoimmune diseases include, but are not limited to, edison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, crohn's disease, diabetes mellitus, graves 'disease, guillain-Barre syndrome, hashimoto's disease, hemolytic anemia, neurolysis, hemolytic anemia, autoimmune hepatitis, autoimmune parotitis, crohn's disease, diabetes mellitus, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, graves' disease, guillain-Barr syndrome, hashimoto's disease, hemolytic anemia, neurolysis anemia systemic lupus erythematosus (systemic lupus erythematous), multiple sclerosis (multiple sclerosis), myasthenia gravis (myasthenia gravis), pemphigus vulgaris (pemphigus vulgaris), psoriasis (psoriasis), rheumatic fever (rhematic patient), rheumatoid arthritis (rhematic arthritis), sarcoidosis (sarcoidosis), scleroderma (scleroderma), sjogren's syndrome, spondyloarthritis (spinosynhymosis), thyroiditis (thyroiditis), vasculitis (vasculitis), typhoid fever (vitis), mucoadenoma, myxedema (myxedema), malignant epilepsy, ulcerative colitis (ulcerative colitis), and the like.

In the present application, the term "infectious disease" generally refers to an agent (such as a virus, fungus or bacterium) that is harmful to its host. In certain embodiments, a factor that is harmful to humans. "anti-infectious disease" means a therapeutic measure that prevents, ameliorates, or eradicates an infectious disease and/or its causative agent.

In the present application, the term "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, monkeys, etc.

In the present application, the term "about" generally refers to a variation in the range of 0.5% -10% above or below the specified value, such as a variation in the range of about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% above or below the specified value.

In this application, the terms "comprises," "comprising," and variations thereof, including "comprises," "comprising," and other variations thereof, are intended to cover a generic term for use herein, which comprises or includes other components, elements, values, steps, or the like.

In the present application, the term "substantially" generally refers to an inherent degree of uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation. The term "substantially" is also utilized herein to represent the degree by which a quantitative representation may vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

In this specification and the claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), and the terms "one or more" and "at least one" may be used interchangeably herein. For example, a cell may refer to a single cell or a population of cells.

Detailed Description

Antigen binding proteins

For example, the cysteine residue at the first position is native to the antigen binding protein, and the cysteine residue at the second position is mutated. For another example, the cysteine residue at the first position is mutated and the cysteine residue at the second position is self-contained by the antigen binding protein.

In certain embodiments, wherein the disulfide bond is capable of forming a sulfhydryl group upon reduction by a reducing agent, wherein the sulfhydryl group allows the antigen binding protein to be conjugated to a payload at the first position and/or the second position. Reducing agents include, but are not limited to, tris (2-carboxyethyl) phosphine, mercaptoethanol, dithiothreitol, cysteine, reduced glutathione, and the like.

For example, an antigen binding protein comprising the engineered cysteine residue has an affinity for the antigen of interest that is about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100% or more of that of the parent as compared to the parent.

In some embodiments, neither the first location nor the second location is in a loop region of the VHH.

In certain embodiments, wherein neither the first position nor the second position is adjacent to an amino acid residue of the internal disulfide bond of the VHH.

In certain embodiments, wherein the engineered cysteine does not pair with a self-contained cysteine residue of the antigen binding protein.

For example, the first position and the second position are located in the Framework Region (FR) of the VHH and neither the first position nor the second position is adjacent to an amino acid residue of the internal disulfide bond of the VHH.

In certain embodiments, wherein neither the first position nor the second position is adjacent to a cysteine residue carried by the VHH.

For example, the first and second positions are located in the Framework Region (FR) of the VHH, and neither the first nor second positions are adjacent to a cysteine residue carried by the VHH.

In the present application, the term "close to" generally means that the amino acid residues are spaced apart from C.beta. -C.beta.at a distance of about C.beta.

In scope, the term "not close to" generally means that the spatial distance of an amino acid residue (C β -C β) is greater than about->

The two cysteine residues are not close to each other and can not form disulfide bond pairing; the spatial distance of amino acid residues can be calculated predictively by Swiss-PdbViewer software.

For example, the first and second positions are located in the Framework Region (FR) of the VHH, and both the first and second positions have a cbp-cbp greater than about that of a cysteine residue carried by the VHH

In certain embodiments, wherein the cysteine at the first position and the cysteine at the second position have a C.beta. -C.beta.of no more than

For example, the first position and the second position are located in the Framework Region (FR) of the VHH, and the Cys at the first position and the Cys at the second position have a C.beta. -C.beta.of no more than about

For another example, the first position and the second position are located in the Framework Region (FR) of the VHH, and both the first position and the second position have a cbeta-cbeta of greater than about the cysteine residue carried by the VHH

And said cysteine at the first position is in contact with said the C beta-C beta of the cysteine in the second position does not exceed about->

In certain embodiments, wherein the cysteine at the first position and the cysteine at the second position are both located on the surface of the antigen binding protein.

Amino acid residues are distributed on the surface and in the inner core of the protein, the surface of the water-soluble protein comprises a plurality of polar amino acids, charged amino acids and non-polar amino acids, while the protein core mainly comprises non-polar amino acids, the non-polar amino acids of the protein are buried in the inner core during the folding process of the protein, and the rest amino acids are on the surface. In the present application, the term "antigen binding protein surface" generally refers to a region consisting of amino acid residues located on the surface of an antigen binding protein, for example, the region of the surface amino acid residues of the antigen binding protein may include a loop region, the N-terminus or the C-terminus. In some embodiments, whether an amino acid residue is located on the surface of an antigen binding protein can be distinguished by the relative solvent accessibility of the amino acid residue, e.g., an amino acid residue can be considered to be located on the surface of an antigen binding protein when the relative solvent accessibility (ASA%) of the amino acid residue is not less than about 20%; alternatively, amino acid residues may be considered to be located within the antigen binding protein when the relative solvent accessibility (ASA%) of the amino acid residues is less than about 20%. In this application, the term "internal disulfide bond" generally refers to a disulfide bond formed from an antibody itself bearing a cysteine in which at least one cysteine residue has solvent accessibility to ASA% < 20%. In the present application, the term "surface disulfide bond" generally means that the relative solvent accessibility (ASA%) of both cysteine residues that form a disulfide bond in an antigen binding protein is not less than about 20%, wherein at least one cysteine residue is engineered. Solvent accessibility of amino acid residues was predicted from http:// cib.cf.ocha.ac.jp/bitool/ASA/website or calculated by the Swiss-PdbViewer software.

In certain embodiments, the cysteine at the first position and the cysteine at the second position are capable of pairing to form a surface disulfide bond.

For example, the first position and the second position are located in the Framework Region (FR) of the VHH, the relative solvent accessibility (ASA%) of the amino acid residue at the first position to the amino acid residue at the second position is not less than about 20%, and the C β -C β of the cysteine at the first position and the cysteine at the second position is not more than about

For another example, the first position and the second position are located in the Framework Region (FR) of the VHH, the relative solvent accessibility (ASA%) of the amino acid residue at the first position to the amino acid residue at the second position is not less than about 20%, and the C β -C β of the cysteine residue at both the first position and the second position from the VHH is greater than about

And said cysteine at a first position does not exceed about ++ C β -C β +>

In certain embodiments, wherein the first location and the second location are each independently selected from the following locations: 3. 7, 9, 11, 12, 13, 17, 23, 39, 41, 66, 68, 83, 84, 87, 8, 16, 18, 25, 42, 45, 81, 82a, 82b, 85, 108, 110, 111, and 113; wherein the first location and the second location are different; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

For example, the first and second locations are each independently selected from the following locations: 3. 7, 9, 11, 12, 13, 17, 23, 39, 41, 66, 68, 83, 84, 87, 8, 16, 18, 25, 42, 45, 81, 82a, 82b, 85, 108, 110, 111, and 113; wherein the first location and the second location are different; wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering; the relative solvent accessibility (ASA%) of the amino acid residue at the first position to the amino acid residue at the second position is not less than about 20%, and the C β -C β of the cysteine at the first position to the cysteine at the second position is not more than about

In certain embodiments, wherein the first location is selected from: 3. 7, 9, 11, 12, 13, 17, 23, 39, 41, 66, 68, 83, 84, and 87; the second position is selected from: 8. 16, 18, 25, 42, 45, 81, 82a, 82b, 85, 108, 110, 111, and 113; the first and second positions are different; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

For example, wherein the first position is selected from: 3. 7, 9, 11, 12, 13, 17, 23, 39, 41, 66, 68, 83, 84, and 87; the second position is selected from: 8. 16, 18, 25, 42, 45, 81, 82a, 82b, 85, 108, 110, 111 and 113; the first and second positions are different; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering; the relative solvent accessibility (ASA%) of the amino acid residue at the first position to the amino acid residue at the second position is not less than about 20%, and the C β -C β of the cysteine at the first position to the cysteine at the second position is not more than about

In certain embodiments, wherein the combination of the first and second locations is selected from at least one of the following combinations: a) 66-82b; b) 3-25; c) 7-8; d) 84-113; e) 83-85; f) 68-81; g) 17-82a; h) 41-42; i) 9-108; j) 11 to 110; k) 87-111; l) 13 to 16; m) 12 to 18; n) 39-45; and o) 14 to 113; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, wherein the combination of amino acid residues at the first position and amino acid residues at the second position is selected from at least one of the following combinations: a) 66 (R/E) -82b (S/N/R/T/D/Y); b) 3 (Q) -25 (S); c) 7 (S) -8 (G); d) 84 (A/P/M) -113 (S); e) 83 (K/R/Q/S/T/E) -85 (E/D); f) 68 (T/V/A) -81 (Q/E/N); g) 17 (S) -82a (N/L/D); h) 41 (P/A/R) -42 (G); i) 9 (G) -108 (L/Q/H); j) 11 (S/L) -110 (T); k) 87 (T/A/L) -111 (V/D); l) 13 (Q/R) -16 (G); m) 12 (V/G) -18 (L); n) 39 (Q) -45 (R/C/L/H); and o) 14 (P) -113 (S); wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering and the amino acid residues in brackets refer to the type of amino acid residue at that position before the antigen binding protein is not mutated.

In certain embodiments, wherein said VHH comprises 2 pairs of cysteine residues, wherein the combination of said first and second positions in each pair of cysteine residues is selected from any two of the following combinations: a) 66-82b; b) 3-25; c) 7-8; d) 84-113; e) 83-85; f) 68-81; g) 17-82a; h) 41-42; i) 9-108; j) 11-110; k) 87-111; l) 13 to 16; m) 12 to 18; n) 39-45; and o) 14 to 113; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

For example, wherein the VHH comprises 2 pairs of cysteine residues, wherein the combination of the first and second positions in each pair of cysteine residues may be selected from: 66-82b and 3-25;66-82b and 7-8;66-82b and 84-113;66-82b and 83-85;66-82b and 68-81;66-82b and 17-82a;66-82b and 41-42;66-82b and 9-108;66-82b and 11-110;66-82b and 87-111;66-82b and 13-16;66-82b and 12-18;66-82b and 39-45;3-25 and 7-8;3-25 and 84-113;3-25 and 83-85;3-25 and 68-81;3-25 and 17-82a;3-25 and 41-42;3-25 and 9-108;3-25 and 11-110;3-25 and 87-111;3-25 and 13-16;3-25 and 12-18;3-25 and 39-45;7-8 and 84-113;7-8 and 83-85;7-8 and 68-81;7-8 and 17-82a;7-8 and 41-42;7-8 and 9-108;7-8 and 11-110;7-8 and 87-111;7-8 and 13-16;7-8 and 12-18;7-8 and 39-45;84-113 and 83-85;84-113 and 68-81;84-113 and 17-82a;84-113 and 41-42;84-113 and 9-108;84-113 and 11-110;84-113 and 87-111;84-113 and 13-16;84-113 and 12-18;84-113 and 39-45;83-85 and 68-81;83-85 and 17-82a;83-85 and 41-42;83-85 and 9-108;83-85 and 11-110;83-85 and 87-111;83-85 and 13-16;83-85 and 12-18;83-85 and 39-45;68-81 and 17-82a;68-81 and 41-42;68-81 and 9-108;68-81 and 11-110;68-81 and 87-111;68-81 and 13-16;68-81 and 12-18;68-81 and 39-45;17-82a and 41-42;17-82a and 9-108;17-82a and 11-110;17-82a and 87-111;17-82a and 13-16;17-82a and 12-18;17-82a and 39-45;41-42 and 9-108;41-42 and 11-110;41-42 and 87-111;41-42 and 13-16;41-42 and 12-18;41-42 and 39-45;9-108 and 11-110;9-108 and 87-111;9-108 and 13-16;9-108 and 12-18;9-108 and 39-45;11-110 and 87-111;11-110 and 13-16;11-110 and 12-18;11-110 and 39-45;87-111 and 13-16;87-111 and 12-18;87-111 and 39-45;13-16 and 12-18;13-16 and 39-45; and 12-18 and 39-45; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, wherein the VHH comprises 2 pairs of cysteine residues, wherein the combination of the first and second positions in each pair of cysteine residues is selected from any two of the following combinations: a) 66 (R/E) -82b (S/N/R/T/D/Y); b) 3 (Q) -25 (S); c) 7 (S) -8 (G); d) 84 (A/P/M) -113 (S); e) 83 (K/R/Q/S/T/E) -85 (E/D); f) 68 (T/V/A) -81 (Q/E/N); g) 17 (S) -82a (N/L/D); h) 41 (P/A/R) -42 (G); i) 9 (G) -108 (L/Q/H); j) 11 (S/L) -110 (T); k) 87 (T/A/L) -111 (V/D); l) 13 (Q/R) -16 (G); m) 12 (V/G) -18 (L); n) 39 (Q) -45 (R/C/L/H); and o) 14 (P) -113 (S); wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering and the amino acid residues in brackets refer to the type of amino acid residue at that position before the antigen binding protein is not mutated.

For example, wherein the VHH comprises 2 pairs of cysteine residues, wherein the combination of the first and second positions in each pair of cysteine residues may be selected from: 66 (R/E) -82b (S/N/R/T/D/Y) and 3 (Q) -25 (S); 66 (R/E) -82b (S/N/R/T/D/Y) and 7 (S) -8 (G); 66 (R/E) -82b (S/N/R/T/D/Y) and 84 (A/P/M) -113 (S); 66 (R/E) -82b (S/N/R/T/D/Y) and 83 (K/R/Q/S/T/E) -85 (E/D); 66 (R/E) -82b (S/N/R/T/D/Y) and 68 (T/V/A) -81 (Q/E/N); 66 (R/E) -82b (S/N/R/T/D/Y) and 17 (S) -82a (N/L/D); 66 (R/E) -82b (S/N/R/T/D/Y) and 41 (P/A/R) -42 (G); 66 (R/E) -82b (S/N/R/T/D/Y) and 9 (G) -108 (L/Q/H); 66 (R/E) -82b (S/N/R/T/D/Y) and 11 (S/L) -110 (T); 66 (R/E) -82b (S/N/R/T/D/Y) and 87 (T/A/L) -111 (V/D); 66 (R/E) -82b (S/N/R/T/D/Y) and 13 (Q/R) -16 (G); 66 (R/E) -82b (S/N/R/T/D/Y) and 12 (V/G) -18 (L); 66 (R/E) -82b (S/N/R/T/D/Y) and 39 (Q) -45 (R/C/L/H); 3 (Q) -25 (S) and 7 (S) -8 (G); 3 (Q) -25 (S) and 84 (A/P/M) -113 (S); 3 (Q) -25 (S) and 83 (K/R/Q/S/T/E) -85 (E/D); 3 (Q) -25 (S) and 68 (T/V/A) -81 (Q/E/N); 3 (Q) -25 (S) and 17 (S) -82a (N/L/D); 3 (Q) -25 (S) and 41 (P/A/R) -42 (G); 3 (Q) -25 (S) and 9 (G) -108 (L/Q/H); 3 (Q) -25 (S) and 11 (S/L) -110 (T); 3 (Q) -25 (S) and 87 (T/A/L) -111 (V/D); 3 (Q) -25 (S) and 13 (Q/R) -16 (G); 3 (Q) -25 (S) and 12 (V/G) -18 (L); 3 (Q) -25 (S) and 39 (Q) -45 (R/C/L/H); 7 (S) -8 (G) and 7 (S) -8 (G); 7 (S) -8 (G) and 84 (A/P/M) -113 (S); 7 (S) -8 (G) and 83 (K/R/Q/S/T/E) -85 (E/D); 7 (S) -8 (G) and 68 (T/V/A) -81 (Q/E/N); 7 (S) -8 (G) and 17 (S) -82a (N/L/D); 7 (S) -8 (G) and 41 (P/A/R) -42 (G); 7 (S) -8 (G) and 9 (G) -108 (L/Q/H); 7 (S) -8 (G) and 11 (S/L) -110 (T); 7 (S) -8 (G) and 87 (T/A/L) -111 (V/D); 7 (S) -8 (G) and 13 (Q/R) -16 (G); 7 (S) -8 (G) and 12 (V/G) -18 (L); 7 (S) -8 (G) and 39 (Q) -45 (R/C/L/H); 84 (A/P/M) -113 (S) and 83 (K/R/Q/S/T/E) -85 (E/D); 84 (A/P/M) -113 (S) and 68 (T/V/A) -81 (Q/E/N); 84 (A/P/M) -113 (S) and 17 (S) -82a (N/L/D); 84 (A/P/M) -113 (S) and 41 (P/A/R) -42 (G); 84 (A/P/M) -113 (S) and 9 (G) -108 (L/Q/H); 84 (A/P/M) -113 (S) and 11 (S/L) -110 (T); 84 (A/P/M) -113 (S) and 87 (T/A/L) -111 (V/D); 84 (A/P/M) -113 (S) and 13 (Q/R) -16 (G); 84 (A/P/M) -113 (S) and 12 (V/G) -18 (L); 84 (A/P/M) -113 (S) and 39 (Q) -45 (R/C/L/H); 83 (K/R/Q/S/T/E) -85 (E/D) and 68 (T/V/A) -81 (Q/E/N); 83 (K/R/Q/S/T/E) -85 (E/D) and 17 (S) -82a (N/L/D); 83 (K/R/Q/S/T/E) -85 (E/D) and 41 (P/A/R) -42 (G); 83 (K/R/Q/S/T/E) -85 (E/D) and 9 (G) -108 (L/Q/H); 83 (K/R/Q/S/T/E) -85 (E/D) and 11 (S/L) -110 (T); 83 (K/R/Q/S/T/E) -85 (E/D) and 87 (T/A/L) -111 (V/D); 83 (K/R/Q/S/T/E) -85 (E/D) and 13 (Q/R) -16 (G); 83 (K/R/Q/S/T/E) -85 (E/D) and 12 (V/G) -18 (L); 83 (K/R/Q/S/T/E) -85 (E/D) and 39 (Q) -45 (R/C/L/H); 68 (T/V/A) -81 (Q/E/N) and 17 (S) -82a (N/L/D); 68 (T/V/A) -81 (Q/E/N) and 41 (P/A/R) -42 (G); 68 (T/V/A) -81 (Q/E/N) and 9 (G) -108 (L/Q/H); 68 (T/V/A) -81 (Q/E/N) and 11 (S/L) -110 (T); 68 (T/V/A) -81 (Q/E/N) and 87 (T/A/L) -111 (V/D); 68 (T/V/A) -81 (Q/E/N) and 13 (Q/R) -16 (G); 68 (T/V/A) -81 (Q/E/N) and 12 (V/G) -18 (L); 68 (T/V/A) -81 (Q/E/N) and 39 (Q) -45 (R/C/L/H); 17 (S) -82a (N/L/D) and 41 (P/A/R) -42 (G); 17 (S) -82a (N/L/D) and 9 (G) -108 (L/Q/H); 17 (S) -82a (N/L/D) and 11 (S/L) -110 (T); 17 (S) -82a (N/L/D) and 87 (T/A/L) -111 (V/D); 17 (S) -82a (N/L/D) and 13 (Q/R) -16 (G); 17 (S) -82a (N/L/D) and 12 (V/G) -18 (L); 17 (S) -82a (N/L/D) and 39 (Q) -45 (R/C/L/H); 41 (P/A/R) -42 (G) and 9 (G) -108 (L/Q/H); 41 (P/A/R) -42 (G) and 11 (S/L) -110 (T); 41 (P/A/R) -42 (G) and 87 (T/A/L) -111 (V/D); 41 (P/A/R) -42 (G) and 13 (Q/R) -16 (G); 41 (P/A/R) -42 (G) and 12 (V/G) -18 (L); 41 (P/A/R) -42 (G) and 39 (Q) -45 (R/C/L/H); 9 (G) -108 (L/Q/H) and 11 (S/L) -110 (T); 9 (G) -108 (L/Q/H) and 87 (T/A/L) -111 (V/D); 9 (G) -108 (L/Q/H) and 13 (Q/R) -16 (G); 9 (G) -108 (L/Q/H) and 12 (V/G) -18 (L); 9 (G) -108 (L/Q/H) and 39 (Q) -45 (R/C/L/H); 11 (S/L) -110 (T) and 87 (T/A/L) -111 (V/D); 11 (S/L) -110 (T) and 13 (Q/R) -16 (G); 11 (S/L) -110 (T) and 12 (V/G) -18 (L); 11 (S/L) -110 (T) and 39 (Q) -45 (R/C/L/H); 87 (T/A/L) -111 (V/D) and 13 (Q/R) -16 (G); 87 (T/A/L) -111 (V/D) and 12 (V/G) -18 (L); 87 (T/A/L) -111 (V/D) and 39 (Q) -45 (R/C/L/H); 13 (Q/R) -16 (G) and 12 (V/G) -18 (L); 13 (Q/R) -16 (G) and 39 (Q) -45 (R/C/L/H); and 12 (V/G) -18 (L) and 39 (Q) -45 (R/C/L/H); wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering and the amino acid residues in brackets refer to the type of amino acid residue at that position before the antigen binding protein is not mutated.

For example, wherein the VHH may comprise 2 pairs of cysteine residues, wherein the combination of the first and second positions in each pair of cysteine residues may be selected from: a) 3-25 and 84-113; b) 7-8 and 3-25; c) 7-8 and 84-113; and d) 11-110 and 84-113; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

For example, wherein the VHH may comprise 2 pairs of cysteine residues, wherein the combination of the first and second positions in each pair of cysteine residues may be selected from: a) 3 (Q) -25 (S) and 84 (A/P/M) -113 (S); b) 7 (S) -8 (G) and 3 (Q) -25 (S); c) 7 (S) -8 (G) and 84 (A/P/M) -113 (S); and d) 11 (S/L) -110 (T) and 84 (A/P/M) -113 (S); wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering and the amino acid residues in brackets refer to the type of amino acid residue at that position before the antigen binding protein is not mutated.

In certain embodiments, wherein the antibody or antigen-binding fragment thereof comprises a VHH antibody or a heavy chain antibody.

For example, the antigen binding protein may be a VHH antibody, the VHH comprising one or more pairs of engineered cysteine residues, wherein each pair of the engineered cysteine residues independently comprises a cysteine residue at a first position and a cysteine residue at a second position, the cysteine residue at the first position being capable of pairing to form a disulfide bond with the cysteine residue at the second position; wherein at least one of the cysteine residue at the first position and the cysteine residue at the second position is mutated; wherein the combination of the first and second locations is selected from at least one of the following combinations: a) 66-82b; b) 3-25; c) 7-8; d) 84-113; e) 83-85; f) 68-81; g) 17-82a; h) 41-42; i) 9-108; j) 11-110; k) 87-111; l) 13 to 16; m) 12 to 18; n) 39-45; and o) 14 to 113; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

For example, the antigen binding protein may be a VHH antibody, the VHH comprising one or more pairs of engineered cysteine residues, wherein each pair of the engineered cysteine residues independently comprises a cysteine residue at a first position and a cysteine residue at a second position, the cysteine residue at the first position being capable of pairing to form a disulfide bond with the cysteine residue at the second position; wherein at least one of the cysteine residue at the first position and the cysteine residue at the second position is mutated; wherein the combination of the amino acid residue at the first position and the amino acid residue at the second position is selected from at least one of the following combinations: a) 66 (R/E) -82b (S/N/R/T/D/Y); b) 3 (Q) -25 (S); c) 7 (S) -8 (G); d) 84 (A/P/M) -113 (S); e) 83 (K/R/Q/S/T/E) -85 (E/D); f) 68 (T/V/A) -81 (Q/E/N); g) 17 (S) -82a (N/L/D); h) 41 (P/A/R) -42 (G); i) 9 (G) -108 (L/Q/H); j) 11 (S/L) -110 (T); k) 87 (T/A/L) -111 (V/D); l) 13 (Q/R) -16 (G); m) 12 (V/G) -18 (L); n) 39 (Q) -45 (R/C/L/H); and o) 14 (P) -113 (S); wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering and the amino acid residues in brackets refer to the type of amino acid residue at that position before the antigen binding protein is not mutated.

For example, the antigen binding protein may be a VHH antibody, the VHH comprising one or more pairs of engineered cysteine residues, wherein each pair of the engineered cysteine residues independently comprises a cysteine residue at a first position and a cysteine residue at a second position, the cysteine residue at the first position being capable of pairing to form a disulfide bond with the cysteine residue at the second position; wherein at least one of the cysteine residue at the first position and the cysteine residue at the second position is mutated; wherein said VHH may comprise 2 pairs of cysteine residues, wherein the combination of said first and second positions in each pair of cysteine residues is selected from the group consisting of: a) 3-25 and 84-113; b) 7-8 and 3-25; c) 7-8 and 84-113; and d) 11-110 and 84-113; wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering.

For example, the antigen binding protein may be a VHH antibody, the VHH comprising one or more pairs of engineered cysteine residues, wherein each pair of the engineered cysteine residues independently comprises a cysteine residue at a first position and a cysteine residue at a second position, the cysteine residue at the first position being capable of pairing to form a disulfide bond with the cysteine residue at the second position; wherein at least one of the cysteine residue at the first position and the cysteine residue at the second position is mutated; wherein the VHH comprises 2 pairs of cysteine residues, wherein the combination of the first and second positions in each pair of cysteine residues is selected from the group consisting of: a) 3 (Q) -25 (S) and 84 (A/P/M) -113 (S); b) 7 (S) -8 (G) and 3 (Q) -25 (S); c) 7 (S) -8 (G) and 84 (A/P/M) -113 (S); and d) 11 (S/L) -110 (T) and 84 (A/P/M) -113 (S); wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering and the amino acid residues in brackets refer to the type of amino acid residue at that position before the antigen binding protein is not mutated.

In another aspect, the present application provides a fusion polypeptide comprising an antigen binding protein described herein.

In the present application, the term "fusion protein" generally refers to a macromolecule formed by linking two different protein molecules by chemical and/or genetic fusion. The fusion protein can be an expression product obtained by recombining two genes by using a DNA recombination technology, and can also be a group of proteins for mediating the fusion of two cell plasma membranes. In the present application, the fusion protein may be a protein molecule having the above two-part domain, which is obtained by linking a gene of interest to the immunoglobulin Fc region or a variant fragment thereof gene at the gene level and expressing the protein molecule in eukaryotic or prokaryotic cells. In the present application, a fusion protein may comprise two polypeptide chains. In addition, fusion proteins can be produced by chemically linking two proteins.

anti-PD-L1 VHH antibodies

For example, the antigen binding protein may be an anti-PD-L1 VHH antibody, said VHH comprising one or more pairs of engineered cysteine residues, wherein each pair of said engineered cysteine residues independently comprises a cysteine residue at a first position and a cysteine residue at a second position, said cysteine residue at the first position being capable of pairing to form a disulfide bond with said cysteine residue at the second position; wherein at least one of the cysteine residue at the first position and the cysteine residue at the second position is mutated; wherein the combination of the first and second locations is selected from at least one of the following combinations: a) 66-82b; b) 3-25; c) 7-8; d) 84-113; e) 83-85; f) 68-81; g) 17-82a; h) 41-42; i) 9-108; j) 11-110; k) 87-111; l) 13 to 16; m) 12 to 18; n) 39-45; and o) 14 to 113; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

For example, the antigen binding protein may be an anti-PD-L1 VHH antibody, said VHH comprising one or more pairs of engineered cysteine residues, wherein each pair of said engineered cysteine residues independently comprises a cysteine residue at a first position and a cysteine residue at a second position, said cysteine residue at the first position being capable of pairing to form a disulfide bond with said cysteine residue at the second position; wherein at least one of the cysteine residue at the first position and the cysteine residue at the second position is mutated; wherein the combination of the amino acid residue at the first position and the amino acid residue at the second position is selected from at least one of the following combinations: a) 66 (R/E) -82b (S/N/R/T/D/Y); b) 3 (Q) -25 (S); c) 7 (S) -8 (G); d) 84 (A/P/M) -113 (S); e) 83 (K/R/Q/S/T/E) -85 (E/D); f) 68 (T/V/A) -81 (Q/E/N); g) 17 (S) -82a (N/L/D); h) 41 (P/A/R) -42 (G); i) 9 (G) -108 (L/Q/H); j) 11 (S/L) -110 (T); k) 87 (T/A/L) -111 (V/D); l) 13 (Q/R) -16 (G); m) 12 (V/G) -18 (L); n) 39 (Q) -45 (R/C/L/H); and o) 14 (P) -113 (S); wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering and the amino acid residues in brackets refer to the type of amino acid residue at that position before the antigen binding protein is not mutated.

For example, the antigen binding protein may be an anti-PD-L1 VHH antibody, said VHH comprising two pairs of engineered cysteine residues, said cysteine residue at a first position being capable of pairing to form a disulfide bond with said cysteine residue at a second position; wherein at least one of the cysteine residue at the first position and the cysteine residue at the second position is mutated; wherein the combination of the first and second positions in each pair of cysteine residues is selected from: a) 3-25 and 84-113; b) 7-8 and 3-25; c) 7-8 and 84-113; and d) 11-110 and 84-113; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

For example, the antigen binding protein may be an anti-PD-L1 VHH antibody, the VHH comprising two pairs of engineered cysteine residues, the cysteine residue at a first position being capable of pairing to form a disulfide bond with the cysteine residue at a second position; wherein the combination of the first and second positions in each pair of cysteine residues is selected from: a) 3 (Q) -25 (S) and 84 (A/P/M) -113 (S); b) 7 (S) -8 (G) and 3 (Q) -25 (S); c) 7 (S) -8 (G) and 84 (A/P/M) -113 (S); and d) 11 (S/L) -110 (T) and 84 (A/P/M) -113 (S); wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering and the amino acid residues in brackets refer to the type of amino acid residue at that position before the antigen binding protein is not mutated.

In another aspect, the present application provides an antigen binding protein that specifically binds PD-L1 comprising a variable antigen binding domain (VHH) of a heavy chain antibody, said VHH comprising a cysteine mutation compared to the sequence set forth in SEQ ID NO:49, the cysteine mutation position being selected from any one or more of the following: a) 66-82b; b) 3-25; c) 7-8; d) 84-113; e) 83-85; f) 68-81; g) 17-82a; h) 41-42; i) 9-108; j) 11-110; k) 87-111; l) 13 to 16; m) 12 to 18; n) 39-45; and o) 14-113; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, the VHH comprises a cysteine mutation compared to the sequence set forth in SEQ ID No. 49, at a position selected from any one or more of: a) 66 (R/E) -82b (S/N/R/T/D/Y); b) 3 (Q) -25 (S); c) 7 (S) -8 (G); d) 84 (A/P/M) -113 (S); e) 83 (K/R/Q/S/T/E) -85 (E/D); f) 68 (T/V/A) -81 (Q/E/N); g) 17 (S) -82a (N/L/D); h) 41 (P/A/R) -42 (G); i) 9 (G) -108 (L/Q/H); j) 11 (S/L) -110 (T); k) 87 (T/A/L) -111 (V); l) 13 (Q/R) -16 (G); m) 12 (V/G) -18 (L); n) 39 (Q) -45 (R/C/L/H); and o) 14 (P) -113 (S); wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering and the amino acid residues in brackets refer to the type of amino acid residue at that position before the antigen binding protein is not mutated.

In certain embodiments, the VHH comprises two pairs of cysteine mutations compared to the sequence set forth in SEQ ID No. 49, wherein the combination of the first and second positions in each pair of cysteine pairings is selected from the group consisting of: a) 3-25 and 84-113; b) 7-8 and 3-25; c) 7-8 and 84-113; and d) 11-110 and 84-113; wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, the VHH comprises two pairs of cysteine mutations, a) 3 (Q) -25 (S) and 84 (A/P/M) -113 (S), as compared to the sequence set forth in SEQ ID NO: 49; b) 7 (S) -8 (G) and 3 (Q) -25 (S); c) 7 (S) -8 (G) and 84 (A/P/M) -113 (S); and d) 11 (S/L) -110 (T) and 84 (A/P/M) -113 (S); wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering and the amino acid residues in brackets refer to the type of amino acid residue at that position before the antigen binding protein is not mutated.

In certain embodiments, the variable domain may comprise an amino acid sequence having one, two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or eleven, or twelve, or thirteen, or fourteen, or fifteen, or sixteen, or seventeen, or eighteen, or nineteen, or twenty amino acid substitutions, deletions, or additions relative to the amino acid sequence set forth in any of SEQ ID NOs 50-63.

In certain embodiments, it comprises an amino acid sequence set forth in any one of SEQ ID NOs 50-63 or a sequence having at least about 90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,99.1%,99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%, or 99.9% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs 50-63.

In certain embodiments, the amino acid sequence of the VHH may have one or several amino acid substitutions compared to the amino acid sequence of any one of SEQ ID NOs 50-63, and may include conservative and/or non-conservative substitutions. In some embodiments, the amino acid mutation can be in a CDR (e.g., a CDR1 region, a CDR2 region, or a CDR3 region) of the targeting moiety. In other embodiments, the amino acid change can be in a Framework Region (FR) of the targeting moiety (e.g., an FR1 region, an FR2 region, an FR3 region, or an FR4 region).

In certain embodiments, the substitution, deletion, or addition does not substantially reduce the ability of an anti-PD-L1 VHH antibody of the present application to specifically bind to PD-L1.

anti-HER 2 VHH antibodies

For example, the antigen binding protein may be an anti-HER 2 VHH antibody, said VHH comprising one or more pairs of engineered cysteine residues, wherein each pair of said engineered cysteine residues independently comprises a cysteine residue at a first position and a cysteine residue at a second position, said cysteine residue at the first position and said cysteine residue at the second position being capable of pairing to form a disulfide bond; wherein at least one of the cysteine residue at the first position and the cysteine residue at the second position is mutated; wherein the combination of the first and second locations is selected from at least one of the following combinations: a) 66-82b; b) 3-25; c) 7-8; d) 84-113; e) 83-85; f) 68-81; g) 17-82a; h) 41-42; i) 9-108; j) 11-110; k) 87-111; l) 13 to 16; m) 12 to 18; n) 39-45; and o) 14 to 113; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

For example, the antigen binding protein may be an anti-HER 2 VHH antibody, said VHH comprising one or more pairs of engineered cysteine residues, wherein each pair of said engineered cysteine residues independently comprises a cysteine residue at a first position and a cysteine residue at a second position, said cysteine residue at the first position and said cysteine residue at the second position being capable of pairing to form a disulfide bond; wherein at least one of the cysteine residue at the first position and the cysteine residue at the second position is mutated; wherein the combination of the amino acid residue at the first position and the amino acid residue at the second position is selected from at least one of the following combinations: a) 66 (R/E) -82b (S/N/R/T/D/Y); b) 3 (Q) -25 (S); c) 7 (S) -8 (G); d) 84 (A/P/M) -113 (S); e) 83 (K/R/Q/S/T/E) -85 (E/D); f) 68 (T/V/A) -81 (Q/E/N); g) 17 (S) -82a (N/L/D); h) 41 (P/A/R) -42 (G); i) 9 (G) -108 (L/Q/H); j) 11 (S/L) -110 (T); k) 87 (T/A/L) -111 (V/D); l) 13 (Q/R) -16 (G); m) 12 (V/G) -18 (L); n) 39 (Q) -45 (R/C/L/H); and o) 14 (P) -113 (S); wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering and the amino acid residues in brackets refer to the type of amino acid residue at that position before the antigen binding protein is not mutated.

For example, the antigen binding protein may be an anti-HER 2 VHH antibody, said VHH comprising two pairs of engineered cysteine residues, the cysteine residue at a first position being capable of pairing to form a disulfide bond with the cysteine residue at a second position; wherein at least one of the cysteine residue at the first position and the cysteine residue at the second position is mutated; wherein the combination of the first and second positions in each pair of cysteine residues is selected from: a) 3-25 and 84-113; b) 7-8 and 3-25; c) 7-8 and 84-113; and d) 11-110 and 84-113; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

For example, the antigen binding protein may be an anti-HER 2 VHH antibody, said VHH comprising two pairs of engineered cysteine residues, said cysteine residue at a first position being capable of pairing to form a disulfide bond with said cysteine residue at a second position; wherein the combination of the first and second positions in each pair of cysteine residues is selected from: a) 3 (Q) -25 (S) and 84 (A/P/M) -113 (S); b) 7 (S) -8 (G) and 3 (Q) -25 (S); c) 7 (S) -8 (G) and 84 (A/P/M) -113 (S); and d) 11 (S/L) -110 (T) and 84 (A/P/M) -113 (S); wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering and the amino acid residues in brackets refer to the type of amino acid residue at that position before the antigen binding protein is not mutated.

In certain embodiments, wherein the anti-HER 2 VHH antibody comprises:

In another aspect, the present application provides an antigen binding protein that specifically binds HER2 comprising a variable antigen binding domain (VHH) of a heavy chain antibody, said VHH comprising a cysteine mutation compared to the sequence shown in SEQ ID No. 24, at a position selected from any one or more of the following: a) 66-82b; b) 3-25; c) 7-8; d) 84-113; e) 83-85; f) 68-81; g) 17-82a; h) 41-42; i) 9-108; j) 11-110; k) 87-111; l) 13 to 16; m) 12 to 18; n) 39-45; and o) 14 to 113; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, the VHH comprises a cysteine mutation compared to the sequence set forth in SEQ ID No. 24 at a position selected from any one or more of: a) 66 (R/E) -82b (S/N/R/T/D/Y); b) 3 (Q) -25 (S); c) 7 (S) -8 (G); d) 84 (A/P/M) -113 (S); e) 83 (K/R/Q/S/T/E) -85 (E/D); f) 68 (T/V/A) -81 (Q/E/N); g) 17 (S) -82a (N/L/D); h) 41 (P/A/R) -42 (G); i) 9 (G) -108 (L/Q/H); j) 11 (S/L) -110 (T); k) 87 (T/A/L) -111 (V); l) 13 (Q/R) -16 (G); m) 12 (V/G) -18 (L); n) 39 (Q) -45 (R/C/L/H); and o) 14 (P) -113 (S); wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering and the amino acid residues in brackets refer to the type of amino acid residue at that position before the antigen binding protein is not mutated.

In certain embodiments, the VHH comprises two pairs of cysteine mutations compared to the sequence set forth in SEQ ID No. 24, wherein the combination of the first and second positions in each pair of cysteine pairings is selected from the group consisting of: a) 3-25 and 84-113; b) 7-8 and 3-25; c) 7-8 and 84-113; and d) 11-110 and 84-113; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, the VHH comprises two pairs of cysteine mutations, a) 3 (Q) -25 (S) and 84 (A/P/M) -113 (S), as compared to the sequence set forth in SEQ ID NO: 24; b) 7 (S) -8 (G) and 3 (Q) -25 (S); c) 7 (S) -8 (G) and 84 (A/P/M) -113 (S); and d) 11 (S/L) -110 (T) and 84 (A/P/M) -113 (S); wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering and the amino acid residues in brackets refer to the type of amino acid residue at that position before the antigen binding protein is not mutated.

In another aspect, the present application provides an antigen binding protein that specifically binds HER2 comprising a variable antigen binding domain (VHH) of a heavy chain antibody, said VHH comprising a cysteine mutation compared to the sequence shown in SEQ ID NO:67, the cysteine mutation position being selected from any one or more of the following: a) 66-82b; b) 3-25; c) 7-8; d) 84-113; e) 83-85; f) 68-81; g) 17-82a; h) 41-42; i) 9-108; j) 11-110; k) 87-111; l) 13 to 16; m) 12 to 18; n) 39-45; and o) 14 to 113; wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, the VHH comprises a cysteine mutation compared to the sequence set forth in SEQ ID NO 67 at a position selected from any one or more of: a) 66 (R/E) -82b (S/N/R/T/D/Y); b) 3 (Q) -25 (S); c) 7 (S) -8 (G); d) 84 (A/P/M) -113 (S); e) 83 (K/R/Q/S/T/E) -85 (E/D); f) 68 (T/V/A) -81 (Q/E/N); g) 17 (S) -82a (N/L/D); h) 41 (P/A/R) -42 (G); i) 9 (G) -108 (L/Q/H); j) 11 (S/L) -110 (T); k) 87 (T/A/L) -111 (V); l) 13 (Q/R) -16 (G); m) 12 (V/G) -18 (L); n) 39 (Q) -45 (R/C/L/H); and o) 14 (P) -113 (S); wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering and the amino acid residues in brackets refer to the type of amino acid residue at that position before the antigen binding protein is not mutated.

In certain embodiments, the VHH comprises two pairs of cysteine mutations compared to the sequence set forth in SEQ ID NO:67, wherein the combination of the first and second positions in each pair of cysteine pairings is selected from the group consisting of: a) 3-25 and 84-113; b) 7-8 and 3-25; c) 7-8 and 84-113; and d) 11-110 and 84-113; wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, the VHH comprises two pairs of cysteine mutations, a) 3 (Q) -25 (S) and 84 (A/P/M) -113 (S), as compared to the sequence set forth in SEQ ID NO: 67; b) 7 (S) -8 (G) and 3 (Q) -25 (S); c) 7 (S) -8 (G) and 84 (A/P/M) -113 (S); and d) 11 (S/L) -110 (T) and 84 (A/P/M) -113 (S); wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering and the amino acid residues in brackets refer to the type of amino acid residue at that position before the antigen binding protein is not mutated.

In certain embodiments, the variable domain may comprise an amino acid sequence having one, two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or eleven, or twelve, or thirteen, or fourteen, or fifteen, or sixteen, or seventeen, or eighteen, or nineteen, or twenty amino acid substitutions, deletions or additions relative to the amino acid sequence set forth in any of SEQ ID NOs 25-45, 68-84.

In certain embodiments, it comprises an amino acid sequence set forth in any of SEQ ID NOs 25-45, 68-83 or a sequence having at least about 90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,99.1%,99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%, or 99.9% sequence identity to an amino acid sequence set forth in any of SEQ ID NOs 25-45, 68-83.

In certain embodiments, the amino acid sequence of the VHH may have one or several amino acid substitutions compared to the amino acid sequence of any one of SEQ ID NOs 25-45, 68-84, and may include conservative and/or non-conservative substitutions. In some embodiments, the amino acid mutation can be in a CDR (e.g., a CDR1 region, a CDR2 region, or a CDR3 region) of the targeting moiety. In other embodiments, the amino acid change can be in a Framework Region (FR) of the targeting moiety (e.g., an FR1 region, an FR2 region, an FR3 region, or an FR4 region).

In certain embodiments, the substitution, deletion, or addition does not substantially reduce the ability of an anti-HER 2 VHH antibody of the present application to specifically bind to HER2.

In another aspect, the present application provides an antigen binding protein that specifically binds HER2 comprising CDR1, CDR2 and CDR3 of a variable antigen binding domain (VHH) of a heavy chain antibody, wherein the amino acid sequence of said VHH is set forth in any one of SEQ ID NOs 25-45, 68-84.

anti-CD 8 alpha VHH antibodies

For example, the antigen binding protein may be an anti-CD 8 α VHH antibody, said VHH comprising one or more pairs of engineered cysteine residues, wherein each pair of said engineered cysteine residues independently comprises a cysteine residue at a first position and a cysteine residue at a second position, said cysteine residue at the first position and said cysteine residue at the second position being capable of pairing to form a disulfide bond; wherein at least one of the cysteine residue at the first position and the cysteine residue at the second position is mutated; wherein the combination of the first and second locations is selected from at least one of the following combinations: a) 66-82b; b) 3-25; c) 7-8; d) 84-113; e) 83-85; f) 68-81; g) 17-82a; h) 41-42; i) 9-108; j) 11-110; k) 87-111; l) 13 to 16; m) 12 to 18; n) 39-45; and o) 14-113; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

For example, the antigen binding protein may be an anti-CD 8 α VHH antibody, said VHH comprising one or more pairs of engineered cysteine residues, wherein each pair of said engineered cysteine residues independently comprises a cysteine residue at a first position and a cysteine residue at a second position, said cysteine residue at the first position and said cysteine residue at the second position being capable of pairing to form a disulfide bond; wherein at least one of the cysteine residue at the first position and the cysteine residue at the second position is mutated; wherein the combination of the amino acid residue at the first position and the amino acid residue at the second position is selected from at least one of the following combinations: a) 66 (R/E) -82b (S/N/R/T/D/Y); b) 3 (Q) -25 (S); c) 7 (S) -8 (G); d) 84 (A/P/M) -113 (S); e) 83 (K/R/Q/S/T/E) -85 (E/D); f) 68 (T/V/A) -81 (Q/E/N); g) 17 (S) -82a (N/L/D); h) 41 (P/A/R) -42 (G); i) 9 (G) -108 (L/Q/H); j) 11 (S/L) -110 (T); k) 87 (T/A/L) -111 (V/D); l) 13 (Q/R) -16 (G); m) 12 (V/G) -18 (L); n) 39 (Q) -45 (R/C/L/H); and o) 14 (P) -113 (S); wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering and the amino acid residues in brackets refer to the type of amino acid residue at that position before the antigen binding protein is not mutated.

For example, the antigen binding protein may be an anti-CD 8 α VHH antibody, the VHH comprising two pairs of engineered cysteine residues, the cysteine residue at a first position being capable of pairing to form a disulfide bond with the cysteine residue at a second position; wherein at least one of the cysteine residue at the first position and the cysteine residue at the second position is mutated; wherein the combination of the first and second positions in each pair of cysteine residues is selected from: a) 3-25 and 84-113; b) 7-8 and 3-25; c) 7-8 and 84-113; and d) 11-110 and 84-113; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

For example, the antigen binding protein may be an anti-CD 8 α VHH antibody, the VHH comprising two pairs of engineered cysteine residues, the cysteine residue at a first position being capable of pairing to form a disulfide bond with the cysteine residue at a second position; wherein the combination of the first and second positions in each pair of cysteine residues is selected from: a) 3 (Q) -25 (S) and 84 (A/P/M) -113 (S); b) 7 (S) -8 (G) and 3 (Q) -25 (S); c) 7 (S) -8 (G) and 84 (A/P/M) -113 (S); and d) 11 (S/L) -110 (T) and 84 (A/P/M) -113 (S); wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering and the amino acid residues in brackets refer to the type of amino acid residue at that position before the antigen binding protein is not mutated.

In another aspect, the present application provides an antigen binding protein that specifically binds CD8 α, comprising a variable antigen binding domain (VHH) of a heavy chain antibody, said VHH comprising a cysteine mutation compared to the sequence set forth in SEQ ID No. 4, at a position selected from any one or more of the following: a) 66-82b; b) 3-25; c) 7-8; d) 84-113; e) 83-85; f) 68-81; g) 17-82a; h) 41-42; i) 9-108; j) 11-110; k) 87-111; l) 13 to 16; m) 12 to 18; n) 39-45; and o) 14 to 113; wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, the VHH comprises a cysteine mutation compared to the sequence set forth in SEQ ID No. 4, at a position selected from any one or more of: a) 66 (R/E) -82b (S/N/R/T/D/Y); b) 3 (Q) -25 (S); c) 7 (S) -8 (G); d) 84 (A/P/M) -113 (S); e) 83 (K/R/Q/S/T/E) -85 (E/D); f) 68 (T/V/A) -81 (Q/E/N); g) 17 (S) -82a (N/L/D); h) 41 (P/A/R) -42 (G); i) 9 (G) -108 (L/Q/H); j) 11 (S/L) -110 (T); k) 87 (T/A/L) -111 (V); l) 13 (Q/R) -16 (G); m) 12 (V/G) -18 (L); n) 39 (Q) -45 (R/C/L/H); and o) 14 (P) -113 (S); wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering and the amino acid residues in brackets refer to the type of amino acid residue at that position before the antigen binding protein is not mutated.

In certain embodiments, the VHH comprises two pairs of cysteine mutations compared to the sequence set forth in SEQ ID No. 4, wherein the combination of the first and second positions in each pair of cysteine pairings is selected from the group consisting of: a) 3-25 and 84-113; b) 7-8 and 3-25; c) 7-8 and 84-113; and d) 11-110 and 84-113; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

In certain embodiments, the VHH comprises two pairs of cysteine mutations, a) 3 (Q) -25 (S) and 84 (A/P/M) -113 (S), as compared to the sequence set forth in SEQ ID NO: 4; b) 7 (S) -8 (G) and 3 (Q) -25 (S); c) 7 (S) -8 (G) and 84 (A/P/M) -113 (S); and d) 11 (S/L) -110 (T) and 84 (A/P/M) -113 (S); wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering and the amino acid residues in brackets refer to the type of amino acid residue at that position before the antigen binding protein is not mutated.

In certain embodiments, the variable domain may comprise an amino acid sequence having one, two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or eleven, or twelve, or thirteen, or fourteen, or fifteen, or sixteen, or seventeen, or eighteen, or nineteen, or twenty amino acid substitutions, deletions or additions relative to the amino acid sequence set forth in any of SEQ ID NOs 5-18, 20.

In certain embodiments, it comprises an amino acid sequence set forth in any of SEQ ID NOs 5-18, 20 or a sequence having at least about 90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,99.1%,99.1%, 99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8% or 99.9% sequence identity to an amino acid sequence set forth in any of SEQ ID NOs 5-18, 20.

In certain embodiments, the amino acid sequence of the VHH may have one or several amino acid substitutions compared to the amino acid sequence of any one of SEQ ID NOs 5-18, 20, and may include conservative and/or non-conservative substitutions. In some embodiments, the amino acid mutation can be in a CDR (e.g., a CDR1 region, a CDR2 region, or a CDR3 region) of the targeting moiety. In other embodiments, the amino acid change can be in a Framework Region (FR) of the targeting moiety (e.g., an FR1 region, an FR2 region, an FR3 region, or an FR4 region).

In another aspect, the present application provides an antigen binding protein that specifically binds to CD8 a comprising CDR1, CDR2 and CDR3 of a variable antigen binding domain (VHH) of a heavy chain antibody, wherein the amino acid sequence of said VHH is as set forth in any one of SEQ ID NOs 50-63.

Payload

In the present application, the term "payload" denotes any molecule or combination of molecules that can be conjugated to an antigen binding protein of the present application. The term "payload" also refers to a moiety whose biological activity requires delivery to and/or localization in a cell or tissue. Payloads include, but are not limited to, markers, chemotherapeutic agents, anti-angiogenic agents, cytotoxins (e.g., pseudomonas exotoxin, ricin, abrin, diphtheria toxin (diptheria toxin), etc.), cytokines, prodrugs, enzymes, growth factors, transcription factors, drugs, radionuclides, ligands, antibodies or fragments thereof, liposomes, nanoparticles, viral particles, cytokines, and the like.

In certain embodiments, wherein the payload comprises a detectable label.

In certain embodiments, wherein the detectable label comprises a radionuclide. Radionuclides useful for conjugation may be those with energies between 20-4000KeV, including but not limited to ¹¹⁰ In、 ¹¹¹ In、 ¹⁷⁷ Lu、 ¹⁸ F、 ⁵² Fe、 ⁶² Cu、 ⁶⁷ Cu、 ⁶⁷ Ga、 ⁶⁸ Ga、 ⁶⁸ Ge、 ⁸⁶ Y、 ⁹⁰ Y、 ⁸⁹ Zr、 ^94m Tc、 ¹²⁰ I、 ¹²³ I、 ¹²⁴ I、 ¹²⁵ I、 ¹³¹ I、 ^154-158 Gd、 ³² P、 ¹¹ C、 ¹³ N、 ¹⁵ O、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ⁵¹ Mn、 ^52m Mn、 ⁷² As、 ⁷⁵ Br、 ⁷⁶ Br、 ^82m Rb、 ⁸³ Sr or other gamma-, beta-, or positron emitters.

For example, the radionuclide may be ^99m Tc、 ¹⁷⁷ Lu or ¹²⁵ I。

In certain embodiments, wherein the antigen binding protein is conjugated directly or indirectly to the detectable label. Methods for conjugating a detectable label to a polypeptide are well known to those skilled in the art. For example, the antigen binding protein may be conjugated to the detectable label by a chelator.

In certain embodiments, wherein the radionuclide is suitable for medical imaging and/or therapy.

Fluorescers that can be used for conjugation include, but are not limited to, isothionine fluorescein, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine; chemiluminescent agents that may be used for conjugation include, but are not limited to, luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters; bioluminescent agents that may be used for conjugation include, but are not limited to, luciferin, luciferase, and aequorin. Paramagnetic ions that can be used for conjugation include, but are not limited to, chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III), or are radiopaque materials such as dams, diatrizoate, ethiodide, gallium citrate, iocarmic acid, iodonatate, iododaramine, iodipamide, iopamoic acid, ioprotide, ioxadic acid, ioxofenac acid, iospiriaretic acid, iosulfotame, iophthalothiamide, iotisic acid, iothalamic acid, iotroxic acid, iopromic acid, meglumine, metridine, propidium iodide, and thallium oxide. Enzymes useful for conjugation include, but are not limited to, horseradish peroxidase and the like.

In certain embodiments, wherein the payload comprises a targeting moiety.

In certain embodiments, wherein the payload comprises a drug.

Connector

In this application, the terms "linker" and "linker" are used interchangeably and generally refer to any agent or molecule that connects an antigen binding protein to a payload. The linker forms a covalent or non-covalent bond with the antigen binding protein and the payload. The ideal linker may be any agent or molecule that remains stable, does not affect the functionality of the targeting polypeptide or pharmaceutically active moiety, or does not produce additional unwanted functions. The linker may be cleavable (cleavable) or non-cleavable (non-cleavable). Exemplary linkers include MC, MP, val-cit, ala-phe, PAB, SPP, SMCC, SIAB. In one embodiment, the linker is MC-vc-PAB.

The linker may comprise a cleavable unit (cleavable unit). In some such embodiments, the structure and/or sequence of the cleavable unit is selected such that it is cleavable under the action of an enzyme present at the target site (e.g., target cell). In other embodiments, cleavable units that are cleavable under changes in PH (e.g., labile to acids or bases), temperature, or radiation (e.g., labile to light) may also be used.

In some aspects, the cleavable unit is a peptide unit and is at least two amino acids long. Cleavage agents may include cathepsins B and D and plasmin (plasmins) (see, e.g., dubowchik and Walker,1999, pharm. Therapeutics 83. Most typically a cleavable unit that can be cleaved by an enzyme present in the cell, i.e., an enzyme cleavable linker. Thus the linker may be cleaved by intracellular peptidases (intracellular peptidases) or proteases, including lysosomal or endosomal proteases. For example, a linker that can be cleaved by the thiol-dependent protease cathepsin B, which is highly expressed in cancer tissues, can be used. (e.g., a linker comprising a phenylalanine-leucine peptide or a valine-citrullinated peptide or a valine-alanine peptide).

In one embodiment, wherein the linker comprises a cleavable unit that is cleaved by the action of an enzyme and a self-immolative group (self-immolative group) that releases the therapeutic agent after cleavage of the cleavable unit. In some embodiments, one end of the cleavable unit of the linker will be conjugated directly or indirectly to the therapeutic agent and the other end thereof will be conjugated directly or indirectly to the antibody. In some such embodiments, one end of the cleavable unit will be conjugated directly or indirectly (e.g., via a self-destructive or non-self-destructive spacer unit) to the therapeutic agent and the other end thereof will be conjugated directly or indirectly to the antibody via an extension unit (stretcher unit). An extension unit links the antibody to the remainder of the drug and/or drug linker. In one embodiment, the linkage between the antibody and the remainder of the drug and/or drug linker is through a maleimide group, for example through a maleimide hexanoyl linker. In some embodiments, the antibody will be linked to the drug via a disulfide (disulfide), such as the disulfide-linked maytansine conjugates SPDB-DM4 and SPP-DMI.

The linkage between the antibody and the linker can be through a number of different pathways, for example, through a thioether bond or through a disulfide bond. In one embodiment, the linkage between the VHH antibody and the linker is formed between a thiol group of a cysteine residue of the VHH antibody and a maleimide group of the linker. In some embodiments, the interchain bond of the antibody is converted to a free thiol group prior to reaction with the functional group of the linker. In some embodiments, a cysteine residue is introduced into the mutation site of the FR region of the VHH antibody and reacted with the linker.

For example, preferred linkers for modifying an antibody or antibody fragment to produce a disulfide-linked conjugate are N-succinimidyl 4- (2-pyridyldithio) valerate (SPP), N-succinimidyl 4_ (2-pyridyldithio) butyrate (STOB), or N-succinimidyl 4- (2-pyridyldithio) -2-sulfobutyrate (sulfonic acid-STOB).

For example, preferred linkers for modifying antibodies or antibody fragments to produce thioether-linked conjugates are 4- (maleimidomethyl) cyclohexanecarboxylic acid N-succinimidyl ester (SMCC), 4- (maleimidomethyl) cyclohexanecarboxylic acid N-succinimidyl ester Sulfonate (SMCC), 4- (iodoacetyl) -aminobenzoic acid N-succinimidyl ester (SIAB).

In certain embodiments, wherein the linker comprises a chelating agent.

For example, the chelating agent may be selected from DTPA, EDTA, NOTA, DOTA, TRAP, TETA, NETA, CB-TE2A, cyclen, cyclam, bispidine, TACN, ATSM, sarar, amBaSar, MAG3, MAG2, HYNIC, DADT, EC, NS3, H2dedpa, HBED, DFO, PEPA or HEHA and derivatives thereof.

Immunoconjugates

VHH-(L-D) _n (I)

wherein L is a linker; d is a payload; n is any number from 1 to 8; for example, n may be any integer of 1 to 8. Also for example, n can be 1, 2, 3, 4, 5, 6, 7, or 8.

In certain embodiments, the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs 5-18, 20, 25-45, 50-63, 68-95 or a sequence having at least about 90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,99.1%,99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8% or 99.9% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs 5-18, 20, 25-45, 50-63, 68-95.

In certain embodiments, wherein the VHH is attached to the linker through a thiol group of a cysteine at the first position and/or a thiol group of a cysteine at the second position.

In certain embodiments, wherein the linker comprises a cleavable (cleavable) or non-cleavable (non-cleavable).

In certain embodiments, wherein the linker is selected from the group consisting of: 6-Maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (alaphe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4- (2-pyridylthio) pentanoate (SPP), N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), (N-succinimidyl 4-iodo-acetyl) aminobenzoate (SIAB), SPDB, hydrazone, maleimidocaproyl and 6-maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB); or a branched linker comprising a peptide chain and derived from o-hydroxy p-aminobenzyl alcohol, wherein the peptide chain is linked to a benzene ring through a p-amino group, the payload is linked to a benzene ring through a benzyl alcohol moiety, and the antigen binding protein is linked to a benzene ring through an o-hydroxy group.

In certain embodiments, wherein the linker comprises a chelating agent.

In certain embodiments, wherein the chelator is selected from DTPA, EDTA, NOTA, DOTA, TRAP, TETA, NETA, CB-TE2A, cyclen, cyclam, bispidine, TACN, ATSM, sarAr, amBaSar, MAG3, MAG2, HYNIC, DADT, EC, NS3, H2dedpa, HBED, DFO, PEPA, or HEHA and derivatives thereof.

In another aspect, the present application provides an immunoconjugate comprising an anti-PD-L1 VHH antibody described herein, a payload, and a linker linking the payload to the antigen binding protein.

VHH-(L-D) _n (I)

wherein L is a linker; d is a payload; n is any number from 1 to 8; for example, n may be any integer of 1 to 8. For another example, n may be 2, 4, 6, or 8.

In certain embodiments, the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs 50-63 or a sequence having at least about 90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,99.1%,99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8% or 99.9% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs 5-18, 20, 25-45, 50-63, 68-95.

In another aspect, the present application provides an immunoconjugate comprising an anti-HER 2 VHH antibody described herein, a payload, and a linker linking the payload to the antigen binding protein.

VHH-(L-D) _n (I)

wherein L is a linker; d is a payload; n is any number from 1 to 8; for example, n may be any integer of 1 to 8. Also for example, n may be 2, 4, 6, or 8.

In certain embodiments, the VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs 25-45, 68-95 or a sequence having at least about 90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,99.1%,99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8% or 99.9% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs 25-45, 68-95.

In another aspect, the present application provides an immunoconjugate comprising an anti-CD 8 α VHH antibody described herein, a payload, and a linker linking the payload to the antigen binding protein.

VHH-(L-D) _n (I)

In certain embodiments, wherein the VHH comprises an amino acid sequence set forth in any one of SEQ ID NOs 5-18, 20 or a sequence having at least about 90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,99.1%,99.1%, 99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%, or 99.9% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs 5-18, 20.

In another aspect, the present application provides a method of a conjugate described herein, the method comprising: the antigen binding proteins described herein are conjugated to a payload.

VHH-(L-D)n(I)

In some embodiments, wherein the method comprises: i) Reducing open disulfide bonds formed by paired cysteines in the antigen binding protein to form free cysteines; ii) linking the sulfhydryl group of the free cysteine to a linker to form an antigen binding protein-payload conjugate.

In another aspect, the present application provides a method for preparing a radionuclide of the present application, e.g. ¹⁷⁷ A method of Lu-labeled immunoconjugate comprising 1) conjugating an antigen binding protein of the present application with a chelator to generate a conjugate precursor of the antigen binding protein and chelator; and 2) reacting the conjugate precursor of step 1) with a radionuclide such as ¹⁷⁷ Lu, whereby radionuclides such as ¹⁷⁷ Lu labels the antigen binding proteins of the present application by chelation by a chelator.

In some embodiments, the chelator is DOTA, and the precursor conjugate of the antigen binding protein and DOTA is generated in step 1) by reacting the antigen binding protein with p-SCN-Bn-DOTA or p-NH 2-Bn-DOTA.

The application may also employ IODO-BEADS solid phase labelling with radionuclides such as ¹²⁵ I labels the antigen binding proteins described herein.

Nucleic acids, vectors and cells

In another aspect, the present application provides an isolated nucleic acid molecule or molecules encoding an antigen binding protein described herein.

In another aspect, the present application provides a cell comprising a nucleic acid molecule as described herein or a vector as described herein.

Nucleic acids encoding the antigen binding proteins of the present application can be incorporated (linked) into vectors, which can be introduced into host cells by transfection, transformation, or transduction techniques. For example, a nucleic acid encoding a HER2 binding polypeptide of the application can be introduced into a host cell by retroviral transduction. In one embodiment, the host cell is eukaryotic, such as a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, sp20 cell). In one embodiment, the host cell is prokaryotic, such as an E.coli cell.

In another aspect, the present application provides a method of making an antigen binding protein as described herein, comprising culturing a cell as described herein under conditions that allow expression of the HER2 binding polypeptide.

The specific expression and purification conditions will vary depending on the expression system employed. For example, if a gene is expressed in E.coli, it is first cloned into an expression vector by placing the engineered gene downstream of a suitable bacterial promoter, such as Trp or Tac, and a prokaryotic signal sequence. In another example, if the engineered gene is to be expressed in eukaryotic cells, such as CHO cells, it is first inserted into an expression vector containing, for example, the appropriate eukaryotic promoter, secretion signal, enhancer, and various introns. The gene vector may be introduced into the host cell using transfection, transformation or transduction techniques.

In certain embodiments, the method further comprises recovering the antigen binding protein expressed by the cell.

In certain embodiments, the method further comprises purifying and/or modifying the antigen binding protein.

The antigen binding proteins of the present application may also be expressed in vivo, e.g., in a patient. For example, in certain embodiments, an antigen binding protein of the present application can be administered in the form of a nucleic acid encoding an antigen binding protein of the present application. The nucleic acid may be DNA or RNA. In some embodiments, the antigen binding proteins of the present application are encoded by modified mrnas, i.e., mrnas comprising one or more modified nucleotides. In some embodiments, the present application relates to gene therapy vectors comprising the modified mRNA. In some embodiments, the present application relates to gene therapy methods comprising said gene therapy vectors. In certain embodiments, the nucleic acid is in the form of an oncolytic virus, such as an adenovirus, reovirus, measles, herpes simplex, newcastle disease virus, or vaccinia.

ii) expressing the antigen binding protein.

Modification of the amino acid sequence can be achieved using any technique known in the art, such as site-directed mutagenesis or PCR-based mutagenesis. Such techniques are described, for example, in the following documents: sambrook et al, molecular Cloning, A Laboratory Manual, cold Spring Harbor Press, plainview, N.Y.,1989; and Ausubel et al, current Protocols in Molecular Biology, john Wiley & Sons, new York, N.Y.,1989.

Composition comprising a metal oxide and a metal oxide

Any of the compositions described herein can be administered to a subject as a component of a composition comprising a pharmaceutically acceptable carrier. Such compositions may optionally comprise suitable amounts of pharmaceutically acceptable excipients in order to provide a form for suitable administration.

Pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipient may be, for example, physiological saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliaries, stabilizers, thickeners, lubricants and colorants may be used. In one embodiment, the pharmaceutically acceptable excipient is sterile when administered to a subject. When any of the agents described herein are administered intravenously, water is a useful excipient. Physiological saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Further examples of suitable Pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R.Gennaro eds., 19 th edition, 1995), which is incorporated by reference herein.

The present application includes various formulation forms of the described compositions (and/or other therapeutic agents). Any of the inventive compositions (and/or other therapeutic agents) described herein can be in the form of a solution, suspension, emulsion, drop, tablet, pill, pellet, capsule, liquid-containing capsule, gelatin capsule, powder, sustained release formulation, suppository, emulsion, aerosol, spray, suspension, lyophilized powder, frozen suspension, dried powder, or any other suitable form. In one embodiment, the composition is in the form of a capsule. In another embodiment, the composition is in the form of a lozenge. In another embodiment, the composition is formulated in the form of a soft gel capsule. In another embodiment, the composition is formulated in the form of a gelatin capsule. In another embodiment, the composition is formulated as a liquid.

Any of the compositions described herein are formulated according to conventional procedures to be suitable for the mode of administration described herein.

Routes of administration include, for example: oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intrathecal, transdermal, rectal, by inhalation or topical. Administration may be local or systemic. In some embodiments, the administering is effected orally. In another embodiment, the administration is by parenteral injection. The mode of administration may be left to the discretion of the physician and will depend in part on the site of the medical condition.

In certain embodiments, the composition comprises an antigen binding protein described herein or an immunoconjugate described herein, which composition is a detection agent or a therapeutic agent.

In certain embodiments, wherein the detection agent is an agent for detecting a tumor antigen.

In certain embodiments, wherein the detection agent is a contrast agent.

In certain embodiments, wherein the contrast agent is a contrast agent for detecting a tumor antigen. For example, the contrast agent may be an ECT contrast agent. As another example, the ECT contrast agent may include a SPECT contrast agent or a PET contrast agent.

In certain embodiments, wherein the therapeutic agent is for treating a tumor.

Use of

In certain embodiments, the contacting is performed in vivo or in vitro.

In certain embodiments, the cell comprises a mammalian cell.

In certain embodiments, the cell comprises a tumor cell.

Method

In another aspect, the present application provides a method for screening a nanobody site-directed conjugation site, the method comprising: screening for amino acid residue pairing in VHH at C.beta. -C.beta.

In range (e.g., about +)>

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In certain embodiments, the method further comprises excluding amino acid residues having ASA% less than about 20% (e.g., about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10% or less) relative solvent accessibility.

In certain embodiments, the method further comprises excluding amino acid residues located in the CDR regions, or loop regions, or near the internal disulfide bonds of the single domain antibody of the VHH.

In certain embodiments, the method comprises:

1) Performing homologous modeling on the VHH;

2) Screening for amino acid residue pairing in VHH at C.beta. -C.beta.

Combinations within the ranges;

4) Amino acid residues located in the CDR regions of VHH, or loop regions, or near the internal disulfide bonds of single domain antibodies were excluded.

The present application also provides the following embodiments:

1. an antigen binding protein comprising a variable antigen binding domain (VHH) of a heavy chain antibody, wherein the VHH comprises one or more pairs of engineered cysteine residues, wherein each pair of the engineered cysteine residues independently comprises a cysteine residue at a first position and a cysteine residue at a second position, the cysteine residue at the first position and the cysteine residue at the second position being capable of pairing to form a disulfide bond; wherein at least one of the cysteine residue at the first position and the cysteine residue at the second position is mutated.

2. The antigen binding protein of embodiment 1, wherein the cysteine residue at the first position and the cysteine residue at the second position are both mutated.

3. The antigen binding protein of any one of embodiments 1-2, wherein the disulfide bond is capable of forming a sulfhydryl group upon reduction by a reducing agent, wherein the sulfhydryl group allows the antigen binding protein to be conjugated to a payload at the first location and/or the second location.

4. The antigen binding protein of any one of embodiments 1-3, wherein the engineered cysteine residue does not reduce the affinity of the antigen binding protein.

5. The antigen binding protein of embodiment 4, wherein the engineered cysteine residue does not pair with a cysteine residue carried by the antigen binding protein itself.

6. The antigen binding protein of any one of embodiments 1-5, wherein both the first position and the second position are located in the Framework Region (FR) of the VHH.

7. The antigen binding protein of any one of embodiments 1-6, wherein neither said first location nor said second location is located in a Complementarity Determining Region (CDR) of said VHH.

8. The antigen binding protein of any one of embodiments 1-7, wherein the cysteine residue at the first position and the cysteine residue at the second position have a C β -C β of no more than about

9. The antigen binding protein of any one of embodiments 1-8, wherein the relative solvent accessibility (ASA%) of the amino acid residue at the first position to the amino acid residue at the second position is not less than about 20%.

10. The antigen binding protein of any one of embodiments 1-9, wherein the first and second positions are each independently selected from the following positions: 3. 7, 9, 11, 12, 13, 17, 23, 39, 41, 66, 68, 83, 84, 87, 8, 16, 18, 25, 42, 45, 81, 82a, 82b, 85, 108, 110, 111, and 113; wherein the first location and the second location are different; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

11. The antigen binding protein of any one of embodiments 1-10, wherein the first position is selected from the group consisting of: 3. 7, 9, 11, 12, 13, 17, 23, 39, 41, 66, 68, 83, 84, and 87; the second position is selected from: 8. 16, 18, 25, 42, 45, 81, 82a, 82b, 85, 108, 110, 111 and 113; the first and second positions are different; wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

12. The antigen binding protein of any one of embodiments 1-11, wherein the combination of the first and second positions is selected from at least one of the following combinations:

p)66-82b；

q)3-25；

r)7-8；

s)84-113；

t)83-85；

u)68-81；

v)17-82a；

w)41-42；

x)9-108；

y)11-110；

z)87-111；

aa)13-16；

bb)12-18；

cc) 39-45; and

dd)14-113

wherein the numbers refer to amino acid residue positions of the VHH as defined by Kabat numbering.

13. The antigen binding protein according to any one of embodiments 1-12, wherein the combination of the amino acid residue at the first position and the amino acid residue at the second position is selected from at least one of the following combinations:

a)66(R/E)-82b(S/N/R/T/D/Y)；

b)3(Q)-25(S)；

c)7(S)-8(G)；

d)84(A/P/M)-113(S)；

e)83(K/R/Q/S/T/E)-85(E/D)；

f)68(T/V/A)-81(Q/E/N)；

g)17(S)-82a(N/L/D)；

h)41(P/A/R)-42(G)；

i)9(G)-108(L/Q/H)；

j)11(S/L)-110(T)；

k)87(T/A/L)-111(V/D)；

l)13(Q/R)-16(G)；

m)12(V/G)-18(L)；

n) 39 (Q) -45 (R/C/L/H); and

o)14(P)-113(S)。

14. The antigen binding protein of any one of embodiments 1-13, wherein said VHH comprises 1, 2, 3, or 4 cysteine residues.

15. The antigen binding protein according to any one of embodiments 1-14, wherein said VHH comprises 2 pairs of cysteine residues, wherein the combination of said first and second positions in each pair of cysteine residues is selected from any two of the following combinations:

a)66-82b；

b)3-25；

c)7-8；

d)84-113；

e)83-85；

f)68-81；

g)17-82a；

h)41-42；

i)9-108；

j)11-110；

k)87-111；

l)13-16；

m)12-18；

n) 39-45; and

o)14-113。

16. The antigen binding protein according to any one of embodiments 1-15, wherein said VHH comprises 2 pairs of cysteine residues, wherein the combination of said first and second positions in each pair of cysteine residues is selected from any two of the following combinations:

a)66(R/E)-82b(S/N/R/T/D/Y)；

b)3(Q)-25(S)；

c)7(S)-8(G)；

d)84(A/P/M)-113(S)；

e)83(K/R/Q/S/T/E)-85(E/D)；

f)68(T/V/A)-81(Q/E/N)；

g)17(S)-82a(N/L/D)；

h)41(P/A/R)-42(G)；

i)9(G)-108(L/Q/H)；

j)11(S/L)-110(T)；

k)87(T/A/L)-111(V/D)；

l)13(Q/R)-16(G)；

m)12(V/G)-18(L)；

n) 39 (Q) -45 (R/C/L/H); and

o)14(P)-113(S)。

wherein the numbers refer to the amino acid residue positions of the VHH as defined by Kabat numbering.

17. The antigen binding protein according to any one of embodiments 1-16, wherein said VHH comprises 2 pairs of cysteine residues, wherein the combination of said first and second positions in each pair of cysteine residues is selected from the group consisting of:

a) 3-25 and 84-113;

b) 7-8 and 3-25;

c) 7-8 and 84-113; and

d) 11-110 and 84-113;

18. The antigen binding protein according to any one of embodiments 1-17, wherein said VHH comprises 2 pairs of cysteine residues, wherein the combination of said first and second positions in each pair of cysteine residues is selected from the group consisting of:

a) 3 (Q) -25 (S) and 84 (A/P/M) -113 (S);

b) 7 (S) -8 (G) and 3 (Q) -25 (S);

c) 7 (S) -8 (G) and 84 (A/P/M) -113 (S); and

d) 11 (S/L) -110 (T) and 84 (A/P/M) -113 (S);

19. The antigen binding protein of any one of embodiments 1-18, wherein the antigen binding protein comprises an antibody or antigen binding fragment thereof.

20. The antigen binding protein of embodiment 19, wherein said antibody comprises a monoclonal antibody.

21. The antigen binding protein of any one of embodiments 19-20, wherein said antibody or antigen binding fragment thereof comprises a single domain antibody or a heavy chain antibody.

22. The antigen binding protein of any one of embodiments 19-21, wherein said antibody or antigen binding fragment comprises a chimeric antibody, a humanized antibody, and/or a fully human antibody.

23. The antigen binding protein of any one of embodiments 1-22, wherein said antigen binding protein is a humanized VHH antibody or a fully human VHH antibody.

24. The antigen binding protein of any one of embodiments 1-23, wherein said antigen binding protein specifically binds to a tumor antigen or a non-tumor antigen.

25. <xnotran> 24 , CD19, BCMA, TSHR, CD171, CS-1, CLL-1, GD3, tn Ag, FLT3, CD38, CD123, CD44v6, B7H3, B7H4, KIT, IL-13Ra2, IL-11Ra, PSCA, PSMA, PRSS21, EGFR2, lewisY, CD24, PDGFR-beta, SSEA-4, MUC1, EGFR, NCAM, CAIX, LMP2, ephA2, GM1, sLe, GM3, TGS5, HMWMAA, FOLR1, FOLR2, TEM1/CD248, TEM7R, CLDN6, CLDN18.2, GPRC5D, CXORF61, CD97, CD179a, ALK, , PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TAARP, WT1, ETV6-AML, SPA17, XAGE1, tie 2, MAD-CT-1, MAD-CT-2, FOSL1, hTERT, ML-IAP, ERG, NA17, PAX3, AR, B1, MYCN, rhoC, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD20, CD30, HER2, ROR1, TAAG72, CD22, CD33, GD2, gp100Tn, FAP, , EPCAM, CEA, IGF-1R, ephB2, , 17, CD32b, EGFRvIII, GPNMB, GPR64, HER3, LRP6, LYPD8, NKG2D, SLC34A2, SLC39A6, SLITRK6, GUCY2C, 5T4 / TACSTD2. </xnotran>

26. The antigen binding protein of any one of embodiments 1-25, wherein said antigen binding protein comprises an anti-PD-L1 VHH antibody, an anti-HER 2 VHH antibody, or an anti-CD 8 VHH antibody.

27. The antigen binding protein of any one of embodiment 26, wherein the anti-PD-L1 VHH antibody comprises: CDR1 of the amino acid sequence shown in SEQ ID NO. 46, CDR2 of the amino acid sequence shown in SEQ ID NO. 47 and CDR3 of the amino acid sequence shown in SEQ ID NO. 48.

28. The antigen binding protein of any one of embodiment 26, wherein the anti-HER 2 VHH antibody comprises: i) CDR1 of the amino acid sequence shown as SEQ ID NO. 21, CDR2 of the amino acid sequence shown as SEQ ID NO. 22 and CDR3 of the amino acid sequence shown as SEQ ID NO. 23; or ii) CDR1 of the amino acid sequence shown in SEQ ID NO. 64, CDR2 of the amino acid sequence shown in SEQ ID NO. 65 and CDR3 of the amino acid sequence shown in SEQ ID NO. 66.

29. The antigen binding protein of any one of embodiment 26, wherein the anti-CD 8 α VHH antibody comprises: CDR1 of the amino acid sequence shown in SEQ ID NO. 1, CDR2 of the amino acid sequence shown in SEQ ID NO. 2 and CDR3 of the amino acid sequence shown in SEQ ID NO. 3.

30. An antigen binding protein that specifically binds to PD-L1 comprising a variable antigen binding domain (VHH) of a heavy chain antibody, said VHH comprising a cysteine mutation compared to the sequence set forth in SEQ ID NO:49 at a position selected from any one or more of the following:

a)66-82b；

b)3-25；

c)7-8；

d)84-113；

e)83-85；

f)68-81；

g)17-82a；

h)41-42；

i)9-108；

j)11-110；

k)87-111；

l)13-16；

m)12-18；

n) 39-45; and

o)14-113；

31. The antigen binding protein of embodiment 30, wherein said VHH comprises a cysteine mutation at a position selected from any one or more of the following:

a)66(R/E)-82b(S/N/R/T/D/Y)；

b)3(Q)-25(S)；

c)7(S)-8(G)；

d)84(A/P/M)-113(S)；

e)83(K/R/Q/S/T/E)-85(E/D)；

f)68(T/V/A)-81(Q/E/N)；

g)17(S)-82a(N/L/D)；

h)41(P/A/R)-42(G)；

i)9(G)-108(L/Q/H)；

j)11(S/L)-110(T)；

k)87(T/A/L)-111(V)；

l)13(Q/R)-16(G)；

m)12(V/G)-18(L)；

n) 39 (Q) -45 (R/C/L/H); and

o)14(P)-113(S)

32. The antigen binding protein of any one of embodiments 30-31, comprising the amino acid sequence of any one of SEQ ID NOs 50-63.

33. An antigen binding protein that specifically binds PD-L1 comprising CDR1, CDR2 and CDR3 in the variable antigen binding domain (VHH) of a heavy chain antibody, wherein the amino acid sequence of said VHH is set forth in any one of SEQ ID NOS: 50-63.

34. An antigen binding protein that competes for binding to PD-L1 with the antigen binding protein of any one of embodiments 30-33.

35. An antigen binding protein that specifically binds to CD8 a comprising a variable antigen binding domain (VHH) of a heavy chain antibody, said VHH comprising a cysteine mutation compared to the sequence set forth in SEQ ID No. 4, the cysteine mutation position being selected from any one or more of the following:

a)66-82b；

b)3-25；

c)7-8；

d)84-113；

e)83-85；

f)68-81；

g)17-82a；

h)41-42；

i)9-108；

j)11-110；

k)87-111；

l)13-16；

m)12-18；

n) 39-45; and

o)14-113；

36. The antigen binding protein of embodiment 35, wherein said VHH comprises a cysteine mutation compared to the sequence set forth in SEQ ID No. 4 at a position selected from any one or more of the following:

a)66(R/E)-82b(S/N/R/T/D/Y)；

b)3(Q)-25(S)；

c)7(S)-8(G)；

d)84(A/P/M)-113(S)；

e)83(K/R/Q/S/T/E)-85(E/D)；

f)68(T/V/A)-81(Q/E/N)；

g)17(S)-82a(N/L/D)；

h)41(P/A/R)-42(G)；

i)9(G)-108(L/Q)；

j)11(S/L)-110(T)；

k)87(T/A/L)-111(V)；

l)13(Q/R)-16(G)；

m)12(V/G)-18(L)；

n) 39 (Q) -45 (R/C/L/H); and

o)14(P)-113(S)；

37. The antigen binding protein of embodiment 36, comprising the amino acid sequence of any one of SEQ ID NOs 5-18, 20.

38. An antigen binding protein that specifically binds to CD8 a comprising CDR1, CDR2 and CDR3 in the variable antigen binding domain (VHH) of a heavy chain antibody, wherein the amino acid sequence of said VHH is as set forth in any one of SEQ ID NOs 5-18, 20.

39. An antigen binding protein that competes for binding to CD8 a with the antigen binding protein of any one of embodiments 35-38.

40. An antigen binding protein that specifically binds to HER2 comprising a variable antigen binding domain (VHH) of a heavy chain antibody, said VHH comprising a cysteine mutation compared to the sequence set forth in SEQ ID NO:24 or 67 at a position selected from any one or more of the group consisting of:

a)66-82b；

b)3-25；

c)7-8；

d)84-113；

e)83-85；

f)68-81；

g)17-82a；

h)41-42；

i)9-108；

j)11-110；

k)87-111；

l)13-16；

m)12-18；

n) 39-45; and

o)14-113；

41. The antigen binding protein of embodiment 40, wherein said VHH comprises a cysteine mutation compared to the sequence set forth in SEQ ID NO. 24 or 67 at a position selected from any one or more of the following:

a)66(R/E)-82b(S/N/R/T/D/Y)；

b)3(Q)-25(S)；

c)7(S)-8(G)；

d)84(A/P/M)-113(S)；

e)83(K/R/Q/S/T/E)-85(E/D)；

f)68(T/V/A)-81(Q/E/N)；

g)17(S)-82a(N/L/D)；

h)41(P/A/R)-42(G)；

i)9(G)-108(L/Q/H)；

j)11(S/L)-110(T)；

k)87(T/A/L)-111(V)；

l)13(Q/R)-16(G)；

m)12(V/G)-18(L)；

n) 39 (Q) -45 (R/C/L/H); and

o)14(P)-113(S)；

42. The antigen binding protein of embodiments 40-41, comprising the amino acid sequence of any one of SEQ ID NOs 25-45, 68-83.

43. The antigen binding protein of embodiment 42, said VHH further being affinity optimized compared to the amino acid sequence of any one of SEQ ID NO 68-83.

44. The antigen binding protein of embodiment 42, comprising the amino acid sequence of any one of SEQ ID NOs 84-94.

45. An antigen binding protein that specifically binds HER2 comprising CDR1, CDR2 and CDR3 in the variable antigen binding domain (VHH) of a heavy chain antibody, wherein the amino acid sequence of said VHH is as set forth in any one of SEQ ID NOs: 25-45, 68-95.

46. An antigen binding protein that competes for binding to HER2 with the antigen binding protein of any one of embodiments 40-45.

47. A fusion polypeptide comprising the antigen binding protein of any one of embodiments 1-46.

48. An immunoconjugate comprising the antigen binding protein of any one of embodiments 1-46, a payload, and a linker linking the payload to the antigen binding protein.

49. The immunoconjugate according to embodiment 48, having the structure of formula I:

VHH-(L-D) _n (I)

wherein L is a linker; d is a payload; n is any number from 1 to 8.

50. The immunoconjugate according to any one of embodiments 48 to 49, wherein said VHH comprises the amino acid sequence set forth in any one of SEQ ID NOs 5-18, 20, 25-45, 50-63, 68-95.

51. The immunoconjugate according to any one of embodiments 48-50, wherein said VHH is linked to said linker by a cysteine residue at a first position and/or a cysteine residue at a second position.

52. The immunoconjugate according to any one of embodiments 48-51, wherein the surface disulfide bond formed by pairing a cysteine residue at a first position with a cysteine residue at a second position in said VHH is reduced to form a free cysteine, said free cysteine comprising a thiol group, said linker being linked to a thiol group.

53. The immunoconjugate of any one of embodiments 48-52, wherein said linker comprises cleavable or non-cleavable.

54. The immunoconjugate according to any one of embodiments 48-53, wherein said linker is selected from the group consisting of: 6-Maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (alaphe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4- (2-pyridylthio) pentanoate (SPP), N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), (N-succinimidyl 4-iodo-acetyl) aminobenzoate (SIAB),), SPDB, hydrazone, maleimidocaproyl and 6-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc-PAB); or a branched linker comprising a peptide chain and derived from o-hydroxy p-aminobenzyl alcohol, wherein the peptide chain is linked to a benzene ring through a p-amino group, the payload is linked to a benzene ring through a benzyl alcohol moiety, and the antigen binding protein is linked to a benzene ring through an o-hydroxy group.

55. The immunoconjugate of any one of embodiments 48-54, wherein said linker comprises a chelator.

56. The immunoconjugate according to embodiment 55, wherein the chelator is selected from DTPA, EDTA, NOTA, DOTA, TRAP, TETA, NETA, CB-TE2A, cyclen, cyclam, bispidine, TACN, ATSM, sarar, amBaSar, MAG3, MAG2, HYNIC, DADT, EC, NS3, H2dedpa, HBED, DFO, PEPA or HEHA and derivatives thereof.

57. The immunoconjugate of any one of embodiments 48-56, wherein said payload comprises a detectable label.

58. The immunoconjugate according to embodiment 57, wherein the detectable label is selected from the group consisting of: radionuclides, fluorescers, chemiluminescers, bioluminescent agents, paramagnetic ions and enzymes, and combinations thereof.

59. The immunoconjugate according to embodiment 58, wherein the detectable label comprises a radionuclide.

60. The immunoconjugate according to embodiment 59, wherein the radionuclide comprises ¹¹⁰ In、 ¹¹¹ In、 ¹⁷⁷ Lu、 ¹⁸ F、 ⁵² Fe、 ⁶² Cu、 ⁶⁷ Cu、 ⁶⁷ Ga、 ⁶⁸ Ga、 ⁶⁸ Ge、 ⁸⁶ Y、 ⁹⁰ Y、 ⁸⁹ Zr、 ^94m Tc、 ¹²⁰ I、 ¹²³ I、 ¹²⁴ I、 ¹²⁵ I、 ¹³¹ I、 ^154- ¹⁵⁸ Gd、 ³² P、 ¹¹ C、 ¹³ N、 ¹⁵ O、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ⁵¹ Mn、 ^52m Mn、 ⁷² As、 ⁷⁵ Br、 ⁷⁶ Br、 ^82m Rb、 ⁸³ Sr or other gamma-, beta-, or positron emitters.

61. The immunoconjugate according to any one of embodiments 57-60, wherein said radionuclide is linked to said antigen binding protein by a chelator.

62. The immunoconjugate of any one of embodiments 48-56, wherein said payload comprises a targeting moiety.

63. The immunoconjugate according to embodiment 62, wherein the targeting moiety is selected from the group consisting of: proteins, nucleic acids, lipids, carbohydrates, and combinations thereof.

64. The immunoconjugate of any one of embodiments 48-56, wherein said payload comprises a drug.

65. The immunoconjugate according to embodiment 64, wherein the drug is selected from the group consisting of: an anti-cancer therapeutic agent, an anti-inflammatory therapeutic agent, an anti-infection therapeutic agent, an anesthetic therapeutic agent, a cytotoxic therapeutic agent, a radionuclide, an immunomodulator, a cell signal peptide, a growth factor, an enzyme, an oligonucleotide, a photoactive therapeutic agent, and any combination thereof.

66. The immunoconjugate according to embodiment 65, wherein said anti-cancer therapeutic is selected from the group consisting of: cytostatics, cytotoxic nucleosides, tubulin binding agents, hormones and hormone antagonists, anti-angiogenic agents, enzyme inhibitors, gene modulators, proteasome inhibitors, pteridine, diyne, podophyllotoxin, auristatin, geldanamycin, calicheamicin, gramicin D, maytansine, neocarzinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, emetine, tinosporine, colchicine, dihydroxyanthracenedione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansine derivatives, anthracycline derivatives, bisphosphonate derivatives, tenmycin derivatives, streptomelanin derivatives, auristatin derivatives, duocarmycin derivatives, and any combination thereof.

67. The immunoconjugate according to any one of embodiments 65-66, wherein the cytostatic agent is selected from: anthraquinone, DNA synthesis inhibitors, DNA-intercalators, DNA-RNA transcription regulators, ansamycinquinone, quinone derivatives, busulfan, ifosfamide, mechlorethamine, triazone, diazinon, carbazolquinone, indoloquinone E09, diazaspinyl-benzoquinone methyl DZQ, triethylenephosphoramide, nitrosourea compounds, and any combination thereof.

68. The immunoconjugate according to any one of embodiments 65-67, wherein said cytotoxic nucleoside is selected from the group consisting of: vidarabine, cytarabine, 5-fluorouracil, fludarabine, fluorouracil nucleoside, ftoracure, 6-mercaptopurine, and combinations of any of these.

69. The immunoconjugate according to any one of embodiments 65-68, wherein said tubulin-binding agent is selected from the group consisting of: paclitaxel, nocodazole, rhizoxin, dolastatin, colchicine, combretastatin, vinca alkaloids, and any combination thereof.

70. The immunoconjugate according to any one of embodiments 65-69, wherein the hormone and hormone antagonist are selected from the group consisting of: corticosteroids, progestins, estrogens, antiestrogens, androgens, aromatase inhibitors, 17- (allylamino) -17-demethoxygeldanamycin, 4-amino-I, 8-naphthalimide, apigenin, brefeldin A, cimetidine, dichloromethylene diphosphonic acid, leuprolide, luteinizing hormone releasing hormone, pifithrin-a, rapamycin, sex hormone binding globulin, thapsigargin, and any combination thereof.

71. The immunoconjugate according to any one of embodiments 65-70, wherein the anti-angiogenic agent is selected from the group consisting of: angiostatin K1-3, DL- α -difluoromethyl-ornithine, endostatin, fumagillin, genistein, minocycline, staurosporine, (+) -thalidomide, and any combination thereof.

72. The immunoconjugate according to any one of embodiments 65-71, wherein the enzyme inhibitor is selected from the group consisting of: s (+) -camptothecin, curcumin, (-) -deguelin, 5, 6-dichlorobenzene-imidazole I-beta-D-ribofuranoside, etoposide, formestane, fosstricin, hispidin, 2-imino-1-imidazolidineacetic acid, mevinolin, trichostatin A, tyrphostin AG 34, tyrphostin AG 879, and any combination thereof.

73. The immunoconjugate according to any one of embodiments 65-72, wherein the gene modulator is selected from the group consisting of: 5-aza-2' -deoxycytidine, 5-azacytidine, cholecalciferol, 4-hydroxyttamoxifen, melatonin, mifepristone, raloxifene, trans retinal, retinoic acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol, tamoxifen, troglitazone, and any combination thereof.

74. The immunoconjugate according to any one of embodiments 65-73, wherein saidThe radionuclide comprises ¹¹⁰ In、 ¹¹¹ In、 ¹⁷⁷ Lu、 ¹⁸ F、 ⁵² Fe、 ⁶² Cu、 ⁶⁷ Cu、 ⁶⁷ Ga、 ⁶⁸ Ga、 ⁶⁸ Ge、 ⁸⁶ Y、 ⁹⁰ Y、 ⁸⁹ Zr、 ^94m Tc、 ¹²⁰ I、 ¹²³ I、 ¹²⁴ I、 ¹²⁵ I、 ¹³¹ I、 ^154-158 Gd、 ³² P、 ¹¹ C、 ¹³ N、 ¹⁵ O、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ⁵¹ Mn、 ^52m Mn、 ⁷² As、 ⁷⁵ Br、 ⁷⁶ Br、 ^82m Rb、 ⁸³ Sr or other gamma-, beta-, or positron emitters.

75. The immunoconjugate according to any one of embodiments 49-74, wherein said n is any integer from 1 to 8.

76. The immunoconjugate according to any one of embodiments 49-75, wherein said n is 2, 4, 6, or 8.

77. A pharmaceutical composition comprising an antigen binding protein of any one of embodiments 1-46 or a conjugate of any one of embodiments 48-76, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

78. An isolated nucleic acid molecule comprising a polynucleotide encoding an antigen binding protein of any one of embodiments 1-46 or a fusion protein of embodiment 47.

79. A vector comprising the nucleic acid molecule of embodiment 78.

80. A cell comprising the nucleic acid molecule of embodiment 78 and/or the vector of embodiment 79.

81. A method of making an antigen binding protein of any one of embodiments 1-46, the method comprising:

i) Site-directed mutagenesis of a nucleic acid sequence of a parent antibody, wherein the mutagenesis comprises substitution of one or more pairs of amino acid residues with cysteine residues; wherein each pair of said cysteine residues comprises a cysteine residue at a first position and a cysteine residue at a second position, said cysteine residue at the first position and said cysteine residue at the second position being capable of pairing to form a disulfide bond; and

ii) expressing the antigen binding protein.

82. The method of embodiment 81, wherein the method further comprises recovering the antigen binding protein.

83. The method of embodiment 82, wherein the method further comprises purifying the antigen binding protein.

84. The method of embodiment 83, wherein the mutagenesis further comprises affinity optimization of the antibody after substitution with a cysteine residue.

85. A method of making an antigen binding protein of any one of embodiments 1-46, the method comprising: comprising culturing the cell of embodiment 80 under conditions that allow expression of the antigen binding protein.

86. A method of making a conjugate of any of embodiments 48-76, the method comprising: conjugating an antigen binding protein of any one of embodiments 1-46 to a payload.

87. The method of embodiment 86, comprising: reacting at least one pair of cysteines of the antigen binding protein with the linker-payload intermediate to form an antigen binding protein-payload conjugate:

VHH-(L-D)n (I)

wherein L is a linker; d is a payload; n is any number from 1 to 8.

88. The method of embodiment 87, wherein n is any integer from 1 to 8.

89. The method of embodiment 88, wherein n is 2, 4, 6, or 8.

90. The method of embodiment 86, wherein the method comprises: i) Reducing open disulfide bonds formed by paired cysteine residues in the antigen binding protein to form free cysteines; ii) reacting the thiol group of the free cysteine with a linker-payload intermediate to form an antigen binding protein-payload conjugate.

91. The method of embodiment 90, wherein the method comprises:

i) Reducing open a disulfide bond formed by paired cysteine residues in the antigen binding protein to form free cysteines;

ii) linking the sulfhydryl group of the free cysteine to a linker to form an antigen binding protein-linker intermediate;

iii) Conjugating the antigen binding protein-linker intermediate to a payload to form an antigen binding protein-payload conjugate.

92. A method of inhibiting cell proliferation comprising contacting a cell with an effective amount of an antigen binding protein of any one of embodiments 1-46, a conjugate of any one of embodiments 48-76, or a pharmaceutical composition of embodiment 77.

93. The method of embodiment 92, wherein the contacting is performed in vivo or in vitro.

94. The method of embodiment 92, comprising including said contacting in a culture medium.

95. The method of embodiments 92-94, wherein the cell comprises a mammalian cell.

96. The method of embodiments 92-95, wherein the cell comprises a tumor cell.

97. A method of detecting, preventing and/or treating a tumor or other disease comprising administering to a subject in need thereof an antigen binding protein of any one of embodiments 1-46, a conjugate of any one of embodiments 48-76, or a pharmaceutical composition of embodiment 77.

98. The method of embodiment 97, wherein the tumor comprises a solid tumor and a non-solid tumor.

99. The method of embodiment 98, wherein the tumor is selected from any one or more of the group consisting of: lymphoma, multiple myeloma, breast cancer, ovarian cancer, kidney cancer, endometrial cancer, melanoma, pancreatic cancer, lung cancer, gastric cancer, liver cancer, mesothelioma, esophageal cancer, head and neck cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, thymus cancer, cervical cancer, brain cancer, prostate cancer, glioblastoma, squamous cell carcinoma, testicular cancer, nasopharyngeal cancer, and colorectal cancer.

100. The method of any one of embodiments 97-99, further comprising administering to the subject an additional therapy or drug.

101. The method of embodiment 100, wherein the other therapy is selected from the group consisting of: chemotherapy, radiation therapy, miRNA, and oligonucleotides.

102. The method of embodiment 97, wherein the other disease comprises an autoimmune disease or an infectious disease.

103. Use of the antigen binding protein of any one of embodiments 1-46, the conjugate of any one of embodiments 48-76, or the pharmaceutical composition of embodiment 77 for the preparation of a medicament for the treatment of a tumor or other disease.

104. Use of the antigen binding protein of any one of embodiments 1-46, the conjugate of any one of embodiments 48-76, or the pharmaceutical composition of embodiment 77 in combination with other therapies or drugs for the preparation of a medicament for the treatment of a tumor or other disease.

105. The use according to embodiment 104, wherein the other therapy is selected from the group consisting of: chemotherapy, radiation therapy, miRNA, and oligonucleotides.

106. The use of any one of embodiments 103-105, wherein the tumor comprises a solid tumor and a non-solid tumor.

107. The use of embodiment 106, wherein the tumor is selected from any one or more of the group consisting of: lymphoma, multiple myeloma, breast cancer, ovarian cancer, kidney cancer, endometrial cancer, melanoma, pancreatic cancer, lung cancer, gastric cancer, liver cancer, mesothelioma, esophageal cancer, head and neck cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, thymus cancer, cervical cancer, brain cancer, prostate cancer, glioblastoma, squamous cell carcinoma, testicular cancer, nasopharyngeal cancer, and colorectal cancer.

108. The use according to any one of embodiments 103-107, wherein the other disease comprises an autoimmune disease or an infectious disease.

109. A method of detecting a specific target for non-disease diagnostic purposes comprising using an antigen binding protein of any one of embodiments 1-46, a conjugate of any one of embodiments 48-76, or a pharmaceutical composition of embodiment 77.

110. A kit comprising an antigen binding protein of any one of embodiments 1-46, a conjugate of any one of embodiments 48-76, or a pharmaceutical composition of embodiment 77, and optionally instructions for use.

111. A kit comprising an antigen binding protein of any one of embodiments 1-46, a conjugate of any one of embodiments 48-76, or a pharmaceutical composition of embodiment 77, and optionally an administration device.

112. A method of screening for nanobody site-directed conjugation sites, the method comprising: screening for amino acid residues in VHH at about C.beta. -C.beta.

Combinations within the range.

113. The method of embodiment 112, further comprising excluding amino acid residues having ASA% less than about 20% relative solvent accessibility.

114. The method according to any one of embodiments 112 to 113, wherein the spatial distance and/or solvent accessibility of amino acid residues is calculated predictively according to the Swiss-PdbViewer software.

115. The method of any one of embodiments 112-114, further comprising excluding amino acid residues located in CDR regions, or loop regions, or near the internal disulfide bond of VHH.

116. The method according to any one of embodiments 112-115, further comprising homology modeling of VHHs prior to screening for amino acid residues.

117. The method of embodiment 116 wherein the homologous modeling is computed using modeler 9 software.

118. The method of any of embodiments 112-117, comprising:

1) Performing homologous modeling on the VHH;

2) Screening for amino acid residues in VHH at about C.beta. -C.beta.

Combinations within the ranges;

Without wishing to be bound by any theory, the following examples are only intended to illustrate the antigen binding proteins, preparation methods and uses, etc. of the present application, and are not intended to limit the scope of the invention of the present application.

Examples

As shown in FIG. 1, referring to the multiple sequence alignment data of Mitcell for 90 multiple single domain antibody sequences in PDB database, it was found that single domain antibodies are highly conserved in FR1, FR2, FR3 and FR4, with most amino acid positions being conserved to more than 80%, i.e., the sequences of the framework regions of single domain antibodies are highly conserved (Mitchell LS, colwell LJ. Comprehensive analysis of nanobody sequence and structure data. Proteins.2018 Jul;86 (7): 697-706.Doi

PDB modeling is carried out on the target nano antibody, and amino acid on the surface of the antibody is found through analysis of the model. These surface-located amino acids were analyzed, amino acids located in the CDR regions were excluded (CDR region amino acids may influence activity), and amino acids located in the FR regions (see FR region amino acid conservation analysis in the above figure) were retained. Theoretically, these amino acids can be substituted by cysteine and paired with each other to form disulfide bonds on the surface of the antibody, as long as the space distance is appropriate. When the coupling is needed, the corresponding reducing agent is used for reduction to open the disulfide bond, and the corresponding substance is coupled, so that the DAR value of the antibody coupled by the method is an even number theoretically. Can be represented by the general formula:

VHH-(L-D) _n

Wherein: VHH represents an antibody in which the surface amino acids of the FR region of the antibody are substituted with one or more cysteines; l is a small molecule linker; d is a drug that needs to be attached to an antibody, including but not limited to toxins, radionuclides, affinity ligands, and the like; n is the number of couplings and can be 2, 4, 6 and 8 (theoretically even).

The nano antibody related by the invention is randomly selected, in order to ensure the universality of a designed platform, three antibodies of three target points are randomly selected for verification, different micromolecular linkers and medicines (toxins or radionuclides) are coupled, and an in-vitro verification experiment is simultaneously carried out, so that the affinity of the nano antibody before and after the nano antibody is substituted by cysteine (a parent antibody and the modified antibody) is not obviously changed, the molecular weight of the modified antibody is consistent with the theory, and the surface disulfide bond is formed after the amino acid on the surface of an FR region is substituted by the cysteine. And simultaneously, carrying out affinity detection on the antibodies before and after coupling so as to ensure that the affinity of the coupled antibodies is not lost, and the method can be used for subsequent corresponding treatment or diagnosis and the like, and detecting the molecular weight of the coupled antibodies by using mass spectrometry so as to calculate the actual coupled DAR and the like.

The analysis method comprises the following steps:

amino acid residue pairing of single domain antibodies that can be engineered to facilitate disulfide bond formation can be found by the following several steps.

1. C37H,109, H2 and H1 are respectively subjected to homologous modeling, and Modeller9 is adopted by homologous modeling software.

2. All resolutions in the protein PDB database were less than

The disulfide bond in the three-dimensional data is used as a prediction model, and all amino acid residues are paired to form C beta-C beta in the three-dimensional structure data of nano antibodies such as C37H,109, H2, H1 and the like

All within the range are potential disulfide bond-forming combinations, the closer the distance theoretically, the easier it is to form disulfide bond pairings (Dombkowski A, crippen G M. Disufide recognition in an optimized cleaving potential [ J ]].Protein engineering,2000,13 (10):679-689.). And obtaining potential disulfide bond pairing through model prediction. The spatial distance of amino acid residues can be calculated predictively by Swiss-PdbViewer software.

3. And calculating the relative solvent accessibility (relative solvent accessibility) of the amino acids excluding fully masked amino acid residues based on the three-dimensional structure of the protein, theoretically ASA% less than 20% of the amino acids with relative solvent accessibility being non-fully exposed or non-exposed amino acids (Savojardo C, manfredi M, martelli P L, et al. Solvent accessibility of the remaining hydrophobic amino acids in humans: from protein structures to protein sequences [ J ]. Primers in molecular biology, 2021, 626363..

Solvent accessibility was either predicted according to http:// cib. Cf. Ocha. Ac. Jp/bitool/ASA/website or predicted by Swiss-PdbViewer software.

4. Finally, the final mutable amino acid pair is selected based on the structural and functional interaction of its single domain antibody. Specifically, these surface amino acids are analyzed for their position in the antibody sequence, excluding amino acid residues located in the CDR regions (which affect antibody antigen binding), or loop regions (which affect protein stability), of the antibody near the internal disulfide bonds of the single domain antibody. The newly engineered amino acid pairing residues should not participate (distint from) and not interfere with antigen binding, nor mismatch with the cysteines involved in disulfide bond formation.

5. Theoretically, the nano antibody is obtained by calculating that the ASA% value of the solvent accessibility of the amino acids in the FR region is more than or equal to 20%, the spatial position is proper, and the amino acid residues C beta-C beta for pairing are in

Mutations may be made to allow for disulfide bond pairing.

1. Sequence analysis and construction

1.1. Analysis of mutable sites

In order to analyze whether the mutation sites have universality, three antibodies (C37H, 109 and H2) of three targets (CD 8 alpha, PD-L1 and Her 2) are respectively selected for PDB modeling, amino acids on the surface of the antibodies are found (if the mutation amino acids are positioned in the antibodies, the reduction-coupling cannot be carried out on the mutation amino acids at a later stage), and the positions of the amino acids on the surface in the antibody sequences are analyzed, if the mutation sites are positioned in CDR regions, loop regions and the like of the antibodies, the biological activity of the antibodies can be influenced after mutation, if the mutation sites are too close to self-carrying disulfide bonds of the antibodies, mismatching is easy to occur in the expression process, and the like. By comprehensively analyzing the factors, the three antibodies respectively select some sites for mutation.

1.1.1 predictive analysis of the C37H antibody against the CD 8. Alpha. Target

PDB modeling was performed on the C37H antibody sequence, and analysis predicted that the C37H antibody itself had a total of 63 amino acids available for cysteine mutation. The specific sequence of C37H and the C β -C β distance between 63 pairs of sites at which mutations can be analyzed are as follows:

C37H：

QVQLVESGGGLVQPGGSLRLSCAASGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTYYADSVKGRFTISRDNSKNHVYLQMNSLRAEDTAVYYCAAGSYYSCSVYPAYDLDYWGKGTLVTVSS

among them, underlined portions indicate antibody CDR regions.

63 pairs of amino acids at specific positions which can undergo disulfide bond mutation

S49-I69	A33-Y97	V2-L27	V63-F67
				V12-L18	Q39-R45	I52a-R71	S96-D101
Y59-K64	I51-I69	A33-S100a	R38-I48
				R38-A88	F29-N76	E6-C92	S17-N82a
I34-V78	C50-V100b	I48-V63	I51-T57
				E6-T107	L82c-V111	Y32-W103	T28-D31
V2-Y102	L82c-D90	M82-L82c	F29-N73
				A40-A88	A33-V100b	A93-W103	R71-V78
R52-S100a	Y32-A94	R66-S82b	F67-M82
				Y52b-Y97	T87-V111	R38-Y90	R52-Y97
A33-R52	V100b-Y100f	A93-L100h	A84-S113
				F37-R45	Q3-S25	S7-G8	R83-E85
G9-L120	L11-T110	Q13-G16	T68-Q81
				Q3-G26	L4-G104	G8-G9	G9-G10
G9-T107	G10-L11	S7-S21	V12-G16
				G15-G16		G16-S17	A40-K43

Note: amino acid positions were named with reference to the Universal humanized Nanobody framework h-NbBcII10FGLA numbering (KABAT)

/>

Note: amino acid positions are named by referring to a universal humanized nano antibody framework h-NbBcII10FGLA number (KABAT), 63 pairs of mutable sites are analyzed, amino acids existing on the surface of the antibody are selected, the mutable sites are not located in a CDR region or a loop region of the antibody, the solvent accessibility of the mutable amino acids is theoretically more than or equal to 20%, and the self-carrying disulfide bond of the antibody is not involved. Finally, 14 pairs of sites which can be mutated are selected, and the specific mutated sequences are as follows:

(1)C37H-Q13-G16:

QVQLVESGGGLVCPGCSLRLSCAASGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTYYADSVKGRFTISRDNSKNHVYLQMNSLRAEDTAVYYCAAGSYYSCSVYPAYDLDYWGKGTLVTVSS

(2)C37H-T68-Q81:

QVQLVESGGGLVQPGGSLRLSCAASGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTYYADSVKGRFCISRDNSKNHVYLCMNSLRAEDTAVYYCAAGSYYSCSVYPAYDLDYWGKGTLVTVSS

(3)C37H-Q39-R45:

QVQLVESGGGLVQPGGSLRLSCAASGLTFSDYAIGWFRCAPGKECEGISCIRIYDGNTYYADSVKGRFTISRDNSKNHVYLQMNSLRAEDTAVYYCAAGSYYSCSVYPAYDLDYWGKGTLVTVSS

(4)C37H-V12-L18：

QVQLVESGGGLCQPGGSCRLSCAASGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTYYADSVKGRFTISRDNSKNHVYLQMNSLRAEDTAVYYCAAGSYYSCSVYPAYDLDYWGKGTLVTVSS

(5)C37H-R66-S82b：

QVQLVESGGGLVQPGGSLRLSCAASGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTYYADSVKGCFTISRDNSKNHVYLQMNCLRAEDTAVYYCAAGSYYSCSVYPAYDLDYWGKGTLVTVSS

(6)C37H-S17-N82a：

QVQLVESGGGLVQPGGCLRLSCAASGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTYYADSVKGRFTISRDNSKNHVYLQMCSLRAEDTAVYYCAAGSYYSCSVYPAYDLDYWGKGTLVTVSS

(7)C37H-Q3-S25：

QVCLVESGGGLVQPGGSLRLSCAACGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTYYADSVKGRFTISRDNSKNHVYLQMNSLRAEDTAVYYCAAGSYYSCSVYPAYDLDYWGKGTLVTVSS

(8)C37H-G9-L108：

QVQLVESGCGLVQPGGSLRLSCAASGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTYYADSVKGRFTISRDNSKNHVYLQMNSLRAEDTAVYYCAAGSYYSCSVYPAYDLDYWGKGTCVTVSS

(9)C37H-E6-T107：

QVQLVCSGGGLVQPGGSLRLSCAASGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTYYADSVKGRFTISRDNSKNHVYLQMNSLRAEDTAVYYCAAGSYYSCSVYPAYDLDYWGKGCLVTVSS

(10)C37H-A84-S113：

QVQLVESGGGLVQPGGSLRLSCAASGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTYYADSVKGRFTISRDNSKNHVYLQMNSLRCEDTAVYYCAAGSYYSCSVYPAYDLDYWGKGTLVTVSC

(11)C37H-S7-G8：

QVQLVECCGGLVQPGGSLRLSCAASGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTYYADSVKGRFTISRDNSKNHVYLQMNSLRAEDTAVYYCAAGSYYSCSVYPAYDLDYWGKGTLVTVSS

(12)C37H-R83-E85：

QVQLVESGGGLVQPGGSLRLSCAASGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTYYADSVKGRFTISRDNSKNHVYLQMNSLCACDTAVYYCAAGSYYSCSVYPAYDLDYWGKGTLVTVSS

(13)C37H-L11-T110：

QVQLVESGGGCVQPGGSLRLSCAASGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTYYADSVKGRFTISRDNSKNHVYLQMNSLRAEDTAVYYCAAGSYYSCSVYPAYDLDYWGKGTLVCVSS

(14)C37H-T87-V111：

QVQLVESGGGLVQPGGSLRLSCAASGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTYYADSVKGRFTISRDNSKNHVYLQMNSLRAEDCAVYYCAAGSYYSCSVYPAYDLDYWGKGTLVTCSS

(15)C37H-Q3-G26：

QVCLVESGGGLVQPGGSLRLSCAASCLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTYYADSVKGRFTISRDNSKNHVYLQMNSLRAEDTAVYYCAAGSYYSCSVYPAYDLDYWGKGTLVTVSS

in this case, the underlined region indicates the mutation site.

1.1.2 predictive analysis of H2 antibodies against Her2 targets

PDB modeling was performed on H2 antibody sequences, and analysis predicted that the H2 antibody itself had 64 pairs of amino acids available for cysteine mutation. The specific sequence of H2 and the C beta-C beta distance between 64 pairs of sites which can be mutated and the pairable sites are shown as follows:

H2：

QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCITSGGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASHYGLRVGTLCPETYEYDYWGQGTQVTVSS

In these, the underlined parts indicate the antibody CDR regions.

64 pairs of amino acids at specific positions that can undergo disulfide bond mutation

A33-V100a	A33-R100	R38-V48	L4-Y102
				S49-I69	L2-F27	T57-I69	F37-R45
E6-T107	R71-V78	P41-G42	V63-F67
				R38-A88	S17-N82a	T68-Q81	M82-L82c
T52-R100	R66-S82b	A24-L29	R19-Q81
				Y59-K64	Y32-A94	D30-N73	L29-N76
L82c-V111	T87-V111	R38-Y90	I51-T57
				Y32-S95	P14-G15	A33-S52a	H96-D101
A40-A88	V100a-L100d	A93-H96	H96-L99
				I51-I69	D58-R100	T52-V100a	L82c-D86
L2-Y102	S21-T77	Q39-R45	A23-T77
				F37-V48	V5-A23	N82a-S82b	Q3-S25
S7-G8	P84-S113	K83-E85	G9-Q108
				L11-T110	Q13-G16	L12-V18	G42-K43
Y91-G106	W103-G104	G104-Q105	Q105-G106
				G8-S21	A24-N76	D72-K75	K75-T77

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And analyzing 64 pairs of mutable sites, and selecting amino acids on the surface of the antibody, wherein the mutable sites are not positioned in a CDR region or a loop region of the antibody, and the self-carrying disulfide bond of the antibody is not involved. The solvent accessibility of the mutant amino acid is equal to or more than 20% theoretically, 20 pairs of sites which can be mutated are finally selected, and the specific mutated sequences are as follows:

(1)H2-Q39-R45：

QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRCAPGKECEGVSCITSGGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASHYGLRVGTLCPETYEYDYWGQGTQVTVSS

(2)H2-R66-S82b：

QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCITSGGSTDYADSVKGCFTISRDNAKNTVYLQMNCLKPEDTAVYYCAASHYGLRVGTLCPETYEYDYWGQGTQVTVSS

(3)H2-Q3-S25：

QLCLVESGGGLVQPGGSLRLSCAACGFTLDYYAIGWFRQAPGKEREGVSCITSGGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASHYGLRVGTLCPETYEYDYWGQGTQVTVSS

(4)H2-S7-G8：

QLQLVECCGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCITSGGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASHYGLRVGTLCPETYEYDYWGQGTQVTVSS

(5)H2-P84-S113：

QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCITSGGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKCEDTAVYYCAASHYGLRVGTLCPETYEYDYWGQGTQVTVSC

(6)H2-K83-E85：

QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCITSGGSTDYADSVKGRFTISRDNAKNTVYLQMNSLCPCDTAVYYCAASHYGLRVGTLCPETYEYDYWGQGTQVTVSS

(7)H2-G9-Q108：

QLQLVESGCGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCITSGGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASHYGLRVGTLCPETYEYDYWGQGTCVTVSS

(8)H2-T68-Q81：

QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCITSGGSTDYADSVKGRFCISRDNAKNTVYLCMNSLKPEDTAVYYCAASHYGLRVGTLCPETYEYDYWGQGTQVTVSS

(9)H2-L11-T110：

QLQLVESGGGCVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCITSGGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASHYGLRVGTLCPETYEYDYWGQGTQVCVSS

(10)H2-S17-N82a：

QLQLVESGGGLVQPGGCLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCITSGGSTDYADSVKGRFTISRDNAKNTVYLQMCSLKPEDTAVYYCAASHYGLRVGTLCPETYEYDYWGQGTQVTVSS

(11)H2-T87-V111：

QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCITSGGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDCAVYYCAASHYGLRVGTLCPETYEYDYWGQGTQVTCSS

(12)H2-E6-T107：

QLQLVCSGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCITSGGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASHYGLRVGTLCPETYEYDYWGQGCQVTVSS

(13)H2-Q13-G16：

QLQLVESGGGLVCPGCSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCITSGGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASHYGLRVGTLCPETYEYDYWGQGTQVTVSS

(14)H2-V12-L18：

QLQLVESGGGLCQPGGSCRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCITSGGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASHYGLRVGTLCPETYEYDYWGQGTQVTVSS

(15)H2-N82a-S82b：

QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCITSGGSTDYADSVKGRFTISRDNAKNTVYLQMCCLKPEDTAVYYCAASHYGLRVGTLCPETYEYDYWGQGTQVTVSS

(16)H2-P41-G42：

QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQCCGKEREGVSCITSGGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASHYGLRVGTLCPETYEYDYWGQGTQVTVSS

(17)H2-P14-G15：

QLQLVESGGGLVQCCGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCITSGGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASHYGLRVGTLCPETYEYDYWGQGTQVTVSS

(18)H2-V5-A23：

QLQLCESGGGLVQPGGSLRLSCCASGFTLDYYAIGWFRQAPGKEREGVSCITSGGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASHYGLRVGTLCPETYEYDYWGQGTQVTVSS

(19)H2-A23-T77：

QLQLVESGGGLVQPGGSLRLSCCASGFTLDYYAIGWFRQAPGKEREGVSCITSGGSTDYADSVKGRFTISRDNAKNCVYLQMNSLKPEDTAVYYCAASHYGLRVGTLCPETYEYDYWGQGTQVTVSS

(20)H2-R19-Q81：

QLQLVESGGGLVQPGGSLCLSCAASGFTLDYYAIGWFRQAPGKEREGVSCITSGGSTDYADSVKGRFTISRDNAKNTVYLCMNSLKPEDTAVYYCAASHYGLRVGTLCPETYEYDYWGQGTQVTVSS

in this case, the underlined region indicates the mutation site.

1.1.3 predictive analysis of the 109 antibody against the PD-L1 target

PDB modeling was performed on the 109 antibody sequence, and analysis predicted that the 109 antibody itself had 59 pairs of amino acids available for cysteine mutation. 109, and 59 pairs of sites that can be mutated and the distance between the mateable sites, C β -C β, are as follows:

109：

QVQLQESGGGSVQAGGSLRLSCTASGFSLDDSDMGWYRQARGNVCQLVSTIASDRSTYYTPSVKGRFTISHDRAKNTIYLQMNSLKPEDTAVYYCAAAPRLAYTTAMTCEGDFAYWGQGTQVTVSS

among them, underlined portions indicate antibody CDR regions.

59 specific positions of amino acids which can be subjected to disulfide bond mutation

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Note: amino acid positions are named by referring to the numbering of a universal humanized nano antibody framework h-NbBcII10FGLA

And analyzing the 59 pairs of mutable sites, selecting amino acids on the surface of the antibody, wherein the mutable sites are not positioned in a CDR region or a loop region of the antibody, the solvent accessibility of the mutable amino acids is not less than 20% theoretically, and the self-carrying disulfide bonds of the antibody are not involved. Finally, 14 pairs of sites which can be mutated are selected, and the specific mutated sequences are as follows:

(1)109-R66-S82b：

QVQLQESGGGSVQAGGSLRLSCTASGFSLDDSDMGWYRQARGNVCQLVSTIASDRSTYYTPSVKGCFTISHDRAKNTIYLQMNCLKPEDTAVYYCAAAPRLAYTTAMTCEGDFAYWGQGTQVTVSS

(2)109-Q3-S25：

QVCLQESGGGSVQAGGSLRLSCTACGFSLDDSDMGWYRQARGNVCQLVSTIASDRSTYYTPSVKGRFTISHDRAKNTIYLQMNSLKPEDTAVYYCAAAPRLAYTTAMTCEGDFAYWGQGTQVTVSS

(3)109-S7-G8：

QVQLQECCGGSVQAGGSLRLSCTASGFSLDDSDMGWYRQARGNVCQLVSTIASDRSTYYTPSVKGRFTISHDRAKNTIYLQMNSLKPEDTAVYYCAAAPRLAYTTAMTCEGDFAYWGQGTQVTVSS

(4)109-P84-S113：

QVQLQESGGGSVQAGGSLRLSCTASGFSLDDSDMGWYRQARGNVCQLVSTIASDRSTYYTPSVKGRFTISHDRAKNTIYLQMNSLKCEDTAVYYCAAAPRLAYTTAMTCEGDFAYWGQGTQVTVSC

(5)109-K83-E85：

QVQLQESGGGSVQAGGSLRLSCTASGFSLDDSDMGWYRQARGNVCQLVSTIASDRSTYYTPSVKGRFTISHDRAKNTIYLQMNSLCPCDTAVYYCAAAPRLAYTTAMTCEGDFAYWGQGTQVTVSS

(6)109-G9-Q108：

QVQLQESGCGSVQAGGSLRLSCTASGFSLDDSDMGWYRQARGNVCQLVSTIASDRSTYYTPSVKGRFTISHDRAKNTIYLQMNSLKPEDTAVYYCAAAPRLAYTTAMTCEGDFAYWGQGTCVTVSS

(7)109-S11-T110：

QVQLQESGGGCVQAGGSLRLSCTASGFSLDDSDMGWYRQARGNVCQLVSTIASDRSTYYTPSVKGRFTISHDRAKNTIYLQMNSLKPEDTAVYYCAAAPRLAYTTAMTCEGDFAYWGQGTQVCVSS

(8)109-T87-V111：

QVQLQESGGGSVQAGGSLRLSCTASGFSLDDSDMGWYRQARGNVCQLVSTIASDRSTYYTPSVKGRFTISHDRAKNTIYLQMNSLKPEDCAVYYCAAAPRLAYTTAMTCEGDFAYWGQGTQVTCSS

(9)109-E6-T107：

QVQLQCSGGGSVQAGGSLRLSCTASGFSLDDSDMGWYRQARGNVCQLVSTIASDRSTYYTPSVKGRFTISHDRAKNTIYLQMNSLKPEDTAVYYCAAAPRLAYTTAMTCEGDFAYWGQGCQVTVSS

(10)109-Q13-G16：

QVQLQESGGGSVCAGCSLRLSCTASGFSLDDSDMGWYRQARGNVCQLVSTIASDRSTYYTPSVKGRFTISHDRAKNTIYLQMNSLKPEDTAVYYCAAAPRLAYTTAMTCEGDFAYWGQGTQVTVSS

(11)109-V12-L18：

QVQLQESGGGSCQAGGSCRLSCTASGFSLDDSDMGWYRQARGNVCQLVSTIASDRSTYYTPSVKGRFTISHDRAKNTIYLQMNSLKPEDTAVYYCAAAPRLAYTTAMTCEGDFAYWGQGTQVTVSS

(12)109-T68-Q81：

QVQLQESGGGSVQAGGSLRLSCTASGFSLDDSDMGWYRQARGNVCQLVSTIASDRSTYYTPSVKGRFCISHDRAKNTIYLCMNSLKPEDTAVYYCAAAPRLAYTTAMTCEGDFAYWGQGTQVTVSS

(13)109-S17-N82a：

QVQLQESGGGSVQAGGCLRLSCTASGFSLDDSDMGWYRQARGNVCQLVSTIASDRSTYYTPSVKGRFTISHDRAKNTIYLQMCSLKPEDTAVYYCAAAPRLAYTTAMTCEGDFAYWGQGTQVTVSS

(14)109-R41-G42：

QVQLQESGGGSVQAGGSLRLSCTASGFSLDDSDMGWYRQACCNVCQLVSTIASDRSTYYTPSVKGRFTISHDRAKNTIYLQMNSLKPEDTAVYYCAAAPRLAYTTAMTCEGDFAYWGQGTQVTVSS

in which the underlined section marks the mutation site.

1.2. Analysis summarizes all amino acid positions located in the FR region that may be subject to mutation pairing

Analysis of three representative antibodies against the three targets selected above, along with analysis of other nanobody sequences (sequences not disclosed in this patent), resulted in the amino acid positions listed in table 1 in the FR region of the antibody that could be mutated to cysteine and disulfide-paired. In theory, all amino acids located in the FR region can undergo disulfide bond pairing, with only the steric positions being appropriate. If it is considered that the disulfide bond formed after mutation is located on the surface of the antibody to facilitate reduction, conjugation, etc., the solvent accessibility of the amino acid selected for mutation should be not less than 20%. The ASA% values for the amino acids in the FR region of the nanobody are shown in Table 2.

TABLE 1 amino acids that can be mutated and paired in the FR regions of Nanobody

3(Q)-25(S)	39(Q)-45(R/C/L/H)
		7(S)-8(G)	41(P/R/A)-42(G)
9(G)-108(Q/L/H)	66(R/E)-82b(S/N/T/R/D/Y)
		11(L/S)-110(T)	68(T/V/A)-81(Q/E/N)
12(V/G)-18(L)	83(R/K/Q/S/T/E)-85(E/D)
		13(Q/R)-16(G)	84(M/P/A)-113(S)
14(A/P/S)-113(S)	87(T/A/L)-111(V/D)
		17(S)-82a(N/L/D)

TABLE 2 amino acid solvent accessibility ASA% in the FR region of the Nanobody%

FR region amino acid solvent accessibility analysis is carried out on three representative nano antibodies of three targets, and the universality is found, for example, if the solvent accessibility of a certain amino acid in one nano antibody is less than 20%, the solvent accessibility of the amino acid at the same site in other nano antibodies is also less than 20% in a large probability, so that theoretically, the amino acid capable of combined mutation can be obtained through the value of the solvent accessibility ASA% and the spatial distance of the amino acid.

1.3. Plasmid construction of antibody for confirming mutation site

Through early analysis of three antibodies related to three targets, C37H antibody co-selection 14 of the CD8 alpha target is used for constructing a mutant antibody, H2 antibody co-selection 20 of the Her2 target is used for constructing the mutant antibody, and antibody 109 co-selection 14 of the PD-L1 target is used for constructing the antibody. Corresponding primers are respectively designed aiming at the mutation sites of the mutated antibodies, the nucleotide sequences of the antibodies are amplified by PCR by taking antibody bacteria liquid before mutation as a template, and then the antibodies are respectively cloned into PSNA008 [ pCDNA4 (Invitrogen, cat V86220) ] vectors, and the correctness of the obtained target clone gene sequences is determined by gene sequence determination, so that the subsequent experiments are promoted.

2. Mutant antibody expression and in vitro characterization

2.1. Production of post-mutation antibody proteins using mammalian cells

And subcloning a coding sequence of the single-domain antibody obtained by correct sequencing analysis into an expression vector, and transfecting HEK293 cells for antibody expression. Diluting the recombinant expression plasmid with Freestyle293 medium and adding PEI (Polyethylenimine) solution required for transformation, adding the plasmid/PEI mixture separately to the HEK293 cell suspension, standing at 37 deg.C, 8% CO ₂ Cultivation at 90 rpm. After four hours, the cells were supplemented with EX293 medium and cultured at 110 rpm. The next day VPA was added. After 6-7 days of culture, transient expression culture supernatant is collected and purified by corresponding fillers. Finally obtaining the antibody protein with the purity of more than 90 percent.

2.2. Detection of post-mutation antibody affinity by ELISA method

2.2.1 detection of binding of antibodies to CD8 alpha antigen after C37H mutation

The CD8 alpha-Fc protein is obtained by HEK293 transient expression and proteinA affinity chromatography purification. The resulting CD 8. Alpha. -Fc protein was diluted to 5ug/mL,100 uL/well was added to the ELISA plate, and coated overnight at 4 ℃. Discard the coating night the next day, add Blocking Buffer (3% BSA) for Blocking, add sample after Blocking, dilute each antibody after C37H control and mutation to 50ug/mL with 1ng/mL C37-chs in 1% BSA, dilute to 11 concentrations in 4-fold gradient, react at 37 ℃ for 1 hour. After washing, rb pAb to 6 × His tag (HRP) was added, diluted 10000-fold with 1% BSA, and reacted at 37 ℃ for 1 hour. After washing, a developing solution is added, and the absorption value is read at the wavelength of 450 nm. Data processing and mapping analysis are carried out by using software SotfMaxPro 6.5.1, a binding curve and an EC50 value of the antibody to CD8 alpha are obtained through four-parameter fitting, and the affinity capacity of the modified antibody to the CD8 alpha is reflected by taking the EC50 value of the C37H antibody before modification as a standard.

TABLE 3 ELISA results for CD8 alpha binding of sequences after C37H engineering

Name (R)	EC50(ng/mL)	Relative Activity (%)
			C37H (Standard)	60.68	100
C37H-Q3-S25	93.03	66
			C37H-S7-G8	50.34	122
C37H-E6-T107	86.85	71
			C37H (Standard)	64.92	100
C37H-Q39-R45	80.26	86
			C37H-Q13-G16	62.48	103
C37H-V12-L18	64.43	99
			C37H (Standard)	77.64	100
C37H-R66-S82b	76.53	96
			C37H-A84-S113	67.36	121
C37H-R83-E85	84.03	95
			C37H (Standard)	57.02	100
C37H-T87-V111	52.43	108
			C37H-Q3-G26	117	48
C37H (Standard)	5.094	100
			C37H-L11-T110	3.133	163
C37H (Standard)	9.389	100
			C37H-G9-L108	8.376	121.1

Note: ELISA detection only has partial data result, and target protein is not obtained due to too low expression level of the antibody after partial modification in the expression process

2.2.2 detection of binding of antibodies to Her2 antigen after H2 mutagenesis

The Her2-Fc protein is obtained by HEK293 transient expression and proteinA affinity chromatography purification. The resulting Her2-Fc protein was diluted to 1ug/mL,100 uL/well added to the ELISA plate and coated overnight at 4 ℃. The coating solution was discarded the next day, blocking was performed by adding Blocking Buffer (3% BSA), the sample was added after Blocking, and each of the H2 control and the post-mutation antibody was diluted to 50ug/mL with 20ng/mL H2-chs in 1% BSA, and 3-fold gradient dilution was performed to 11 concentrations, and the reaction was performed at 37 ℃ for 1 hour. After washing, rb pAb to 6 × His tag (HRP) was added, diluted 10000-fold with 1% BSA, and reacted at 37 ℃ for 1 hour. After washing, a developing solution is added, and the absorption value is read at the wavelength of 450 nm. Data processing and mapping analysis are carried out by using software SotfMaxPro 6.5.1, a binding curve and an EC50 value of the antibody to Her2 are obtained through four-parameter fitting, and the affinity of the modified antibody to Her2 is reflected by taking the EC50 value of the H2 antibody before modification as a standard.

TABLE 4 ELISA results for Her2 binding of sequences after H2 engineering

Name (R)	EC50(ng/mL)	Relative Activity (%)
			H2 (Standard sample)	92.69	100
H2-Q39-R45	509.1	18.2
			H2-L11-T110	57.59	160.9
H2 (Standard sample)	128.7	100
			H2-T87-V111	106.6	120.7
H2-E6-T107	104.4	123.3
			H2-Q13-G16	119.6	107.7
H2 (Standard sample)	114.0	100
			H2-P41-G42	95.85	118.9
H2-P14-G15	77.06	147.9
			H2-A23-T77	76.53	148.9
H2 (Standard sample)	933.4	100
			H2-K83-E85	888.9	105
H2-P84-S113	613.1	152.2
			H2-R66-S82b	1514	61.6
H2 (Standard sample)	90.16	100
			H2-V12-L18	104.4	86.4
H2-G9-Q108	61.48	146.7
			H2-Q3-S25	127.7	70.6
H2 (Standard sample)	96.81	100
			H2-S7-G8	141.7	68.3

Note: ELISA detection only has partial data result, and because the expression level of the antibody is too low after partial modification in the expression process, the target protein is not obtained

2.2.3 detection of binding of 109 mutated antibody to PD-L1 antigen

The PD-L1-Fc protein is obtained by HEK293 transient expression and proteinA affinity chromatography purification. The resulting PD-L1-Fc protein was diluted to 0.5ug/mL,50 uL/well was added to the ELISA plate, and coated overnight at 4 ℃. The coating solution was discarded the next day, blocking was performed by adding Blocking Buffer (3% BSA), the sample was added after Blocking, 109 control and after mutation, each antibody was diluted to 80ug/mL with 0.05ug/mL 109-chs in 1% BSA, diluted to 12 concentrations by 3-fold gradient, and reacted at 37 ℃ for 2 hours. After washing, rb pAb to 6 × His tag (HRP) was added, diluted 10000-fold with 1% BSA, and reacted at 37 ℃ for 1 hour. After washing, a developing solution is added, and the absorption value is read at the wavelength of 450 nm. Data processing and mapping analysis are carried out by using software SotfMaxPro 6.5.1, a binding curve and an EC50 value of the antibody to PD-L1 are obtained through four-parameter fitting, and the affinity capability of the modified antibody to PD-L1 is reflected by taking the EC50 value of the 109 antibody before modification as a standard.

TABLE 5 ELISA results for PD-L1 binding of the engineered antibodies

Name (R)	EC50(ng/mL)	Relative Activity (%)
			109 (Standard sample)	548.7	100
109-K83-E85	709.1	77.4
			109-P84-S113	514.4	106.7
109-V12-L18	581.3	94.4
			109 (Standard sample)	499.0	100
109-Q13-G16	505.8	98.7
			109-R66-S82b	950.5	52.5
109-S7-G8	434.6	114.8
			109 (Standard sample)	429.4	100
109-Q3-S25	313.7	136.9
			109 (Standard sample)	600.9	100
109-G9-Q108	811.1	74.1
			109-S11-T110	443.4	135.5
109-T87-V111	473.7	126.8

Through ELISA affinity detection of the modified antibodies of the three target points, the results show that the affinity of most of the modified antibodies of the three target points is not obviously reduced compared with that of the antibodies before modification, which indicates that the modified antibodies still keep good combination with the corresponding target point antigens, and also laterally proves that the selected mutation sites are representative, thereby providing a powerful basis for the application of cysteine mutation platforms on the surfaces of the antibodies.

2.3. SEC-HPLC and SDS-PAGE methods are adopted to detect the purity of the mutated antibody

The antibody was adjusted to an appropriate concentration with 1 XPBS (pH 7.4) and purity was checked by SEC-HPLC and SDS-PAGE, respectively. The detection methods of the related modified antibodies of the three targets are the same. SEC-HPLC adopts a chromatographic column of BioCoreSEC-150 to carry out detection, a protective column with corresponding specification is required to be matched, automatic integration is carried out according to a peak pattern diagram after the detection is finished, manual integration is carried out if the conditions of low separation degree or baseline disorder and the like occur, and after blank is deducted, the purity of the sample is calculated by an area normalization method (A%).

SDS-PAGE detection samples were treated with a non-returning buffer (containing no reducing agent) for the purpose of antibody (monomer) purity detection. The preparation method comprises the steps of selecting prefabricated gel (M00719) with 15% of Kinry concentration, loading about 10ug of each sample, dyeing the gel with Coomassie brilliant blue dyeing solution after the gel is run, and decolorizing until the bands are clearly visible. And finally, analyzing a target band and a miscellaneous band of the same antibody respectively by using related software to obtain an IntDen value of each band, wherein the percentage of the IntDen values of the target band in all the bands is the monomer purity of the antibody. Because the IntDen value of each band is obtained after manually framing the band, the method may have certain errors, but does not influence the analysis on whether a polymer is formed in the antibody re-expression process. Meanwhile, the comprehensive comparison can be carried out by referring to the result of SEC-HPLC.

The results are shown in tables 5-7, which directly show the percentage of each antibody monomer, e.g., the presence of a polymer, which can be removed during downstream purification.

TABLE 6 SEC-HPLC and SDS-PAGE detection of monomer purity of C37H engineered antibodies

TABLE 7 SEC-HPLC and SDS-PAGE detection of H2 engineered antibody monomer purity

Name of antibody	Percent monomer (SEC-HPLC)	Percent monomer (SDS-PAGE)
			H2-Q39-R45	100％	100％
H2-R66-S82b	100％	100％
			H2-S7-G8	100％	100％
H2-P84-S113	100％	53％
			H2-T87-V111	100％	100％
H2-E6-T107	100％	100％
			H2-Q13-G16	100％	100％
H2-P41-G42	98.63％	100％
			H2-P14-G15	97.48％	100％
H2-A23-T77	100％	100％
			H2-V12-L18	100％	100％
H2-K83-E85	68％	64％
			H2-G9-Q108	48％	53％
H2-Q3-S25	57％	50.4％
			H2-L11-T110	47％	57％

TABLE 8 SEC-HPLC and SDS-PAGE detection 109 engineered antibody monomer purity

The SEC-HPLC and SDS-PAGE are respectively carried out on the modified antibodies of the three targets to detect the purity (monomer content) of the antibodies, and the results show that most of the modified antibodies of the three targets exist in a monomer form after expression, and individual antibodies form polymers in the expression process (can be optimally removed in the later purification process). Lays a foundation for the smooth operation of the antibody post-coupling process and the application of the surface cysteine mutation platform.

2.4. Detection of antibody expression level after mutation

The expression amount detection adopts one-step affinity purification, and then the expression amount of the antibody is evaluated by calculating the obtained protein amount and the purified sample volume. Transient transfection expression of each antibody was consistent with the method mentioned in 2.1. After transient conversion, cell culture supernatant is collected and subjected to one-step affinity purification through protein A, the eluted target protein is subjected to A280 light absorption value detection, the extinction coefficient of each corresponding modified antibody is divided, the final antibody concentration is obtained, and the final protein amount is calculated through respective elution volumes, so that the expression amount of each antibody is evaluated. Because the cell state may be different at each transient transformation and there is some error in the purification process, the expression level will fluctuate in a small range, which is a normal phenomenon, and the antibody with low expression level can be improved by the late transient transformation optimization. Tables 9-11 show the expression levels (calculated as the results of one-shot purification) of the respective modified antibodies.

TABLE 9 expression levels of each of the modified antibodies of C37H

Name of antibody	Amount of expression (mg/L)	Name of antibody	Amount of expression (mg/L)
				C37H-V12-L18	10.8	C37H-Q13-G16	12.4
C37H-A84-S113	8.4	C37H-Q3-S25	3.4
				C37H-E6-T107	2.2	C37H-Q3-G26	3.0
C37H-S7-G8	5.1	C37H-R83-E85	2.0
				C37H-R66-S82b	9.2	C37H-G9-L108	0.9
C37H-T87-V111	4.1	C37H-Q39-R45	57
				C37H-L11-T110	4.4

TABLE 10 109 expression levels of each of the engineered antibodies

TABLE 11 expression levels of each of the engineered antibodies to H2

Name of antibody	Amount of expression (mg/L)	Name of antibody	Amount of expression (mg/L)
				H2-Q39-R45	22.2	H2-P41-G42	15.5
H2-R66-S82b	16.4	H2-P14-G15	5.4
				H2-S7-G8	30.8	H2-A23-T77	11.2
H2-P84-S113	9.7	H2-V12-L18	5.3
				H2-L11-T110	30.7	H2-K83-E85	3.9
H2-T87-V111	10.1	H2-G9-L108	28.3
				H2-E6-T107	3.7	H2-Q3-S25	26.8
H2-Q13-G16	22.8

2.5. Mass spectrometric characterisation of post-mutation antibody molecular weight

Because the surface amino acids selected by each antibody form corresponding disulfide bonds after cysteine mutation, and in order to detect whether the disulfide bonds are mismatched in the pairing process, each antibody adopts a mass spectrometry method to detect the complete molecular weight, and the actually measured molecular weight is compared with the theoretical molecular weight to evaluate whether the disulfide bonds are mismatched in the forming process. The actual molecular weight is matched with the theoretical molecular weight, so the specific results are not shown in detail.

2.6. Analysis of degree of solvent Exposure in mutant site ASA%

To further explore the effect of different amino acids on disulfide bond formation after mutation, we calculated the ratio of exposure of each mutated amino acid to the accessible surface of the protein solvent by correlation software, with 20% as the boundary, the higher the ASA%, the higher the exposure and the lower the degree of deregulation. And the amino acid with high exposure degree is selected for mutation, so that the disulfide bond on the surface of the antibody is more easily formed theoretically, and the reduction and the like in the later coupling process are facilitated.

2.7. Summary of in vitro characterization results of mutant antibodies

Through analysis of in vitro characterization results of each mutant antibody, the content of antibody monomers after mutation is defined to be more than or equal to 95%, the biological activity (relative activity) is more than or equal to 70%, the expression amount is more than or equal to 5mg/L, the solvent exposure degree ASA% is more than or equal to 20%, the measured molecular weight is consistent with the theoretical molecular weight, if the above criteria are met, the mutant antibody is a preferred antibody, the mutant site is a preferred site, otherwise, the mutant antibody is not an optimal antibody or an optimal mutant site. The specific results are shown in tables 12-14.

TABLE 12 summary of in vitro characterization results for each of the modified antibodies of C37H

Note: a marks the standard to reach the set standard, B marks the standard to not reach the set standard, C marks the poor characterization result

TABLE 13 summary of in vitro characterization results for each of the engineered antibodies

TABLE 14 summary of in vitro characterization results for each of the engineered antibodies for H2

The results show that most of the in vitro characterization results of the mutated antibodies can reach the set standard, the individual characterization results (such as expression quantity, monomer content and the like) of part of the mutated antibodies can not reach the set standard, and the related processes can be optimized and improved in the later period. The versatility and operability of the amino acid sites we have chosen are also reflected laterally.

3. Mutant antibody conjugation and characterization

"chelating agent", also referred to herein as a conjugated small molecule linker, generally refers to an organic molecule capable of forming a complex with a metal ion. Chelators are commonly used to label proteins or peptides. The end product of the metal ion conjugate is used for radioimmunoassay, radioimmunotherapy, magnetic resonance imaging, photodynamic therapy or other similar modalities. Chelating agents are of many kinds, such as DTPA (diethylenetriamine pentaacetic anhydride) and its derivatives, NOTA (1, 4, 7-triazacyclononane-N, N ', N "-triacetic acid) and its derivatives such as NODA-GA (NODAGA), maleimide-NODAA, DOTA (1, 4,7, 10-tetraazacyclododecane-N, N ', N", N ' "-tetraacetic acid) (binding radioactive metal ions) and its derivatives, TETA (1, 4,8, 11-tetraazacyclotetradecane-N, N ', N", N ' "-tetraacetic acid) and its derivatives, DTTA (N- (p-isothiocyanatobenzyl) -diethylenetriamine-N, N ', N", N ' "-tetraacetic acid). These and other chelating agents are readily available from commercial sources. The chelating agent for antibody coupling is conventional and universal, and has no other special modification. In addition, in addition to the selection of organic molecules capable of forming complexes with metal ions for conjugation, the present invention also selects covalent linkers (linkers) and cytotoxic small molecule loads (planoads) commonly used for ADC (Antibody-drug conjugates) drugs for conjugation, such as GGFG-DXD, CLA-SN38, or the like, based on GGFG tetrapeptide linkers. Wherein DXD and SN38 are topoisomerase I inhibitors.

3.1C 37H mutant conjugation and characterization

3.1.1 C37H mutant coupled NODA-GA

According to the reasons of affinity detection and antibody self-expression amount of the modified antibody in the early stage, the antibody protein after partial C37H mutation is selected to be uniformly mixed with TCEP (tris (2-carboxyethyl) phosphine) reducing agent in a molar ratio of 1.

3.1.2 Characterization detection of C37H mutant after coupling NODA-GA

And respectively carrying out ELISA (enzyme-linked immuno sorbent assay) affinity detection on the coupled antibodies to determine that the coupled antibodies do not lose biological activity. SEC-HPLC is used for determining whether the coupled antibody exists in a monomer form, and LC-MS and RP-SPLE are used for detecting the molecular weight of the coupled antibody, so as to calculate the DAR value of the coupled antibody, whether the coupling is successful and the like. The specific detection results are as follows:

TABLE 15 ELISA detection of CD8 alpha binding of antibodies after NODA-GA conjugation

TABLE 16 LC-MS detection of antibody molecular weight and DAR value after NODA-GA coupling

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TABLE 17 detection of antibody coupling ratio and monomer ratio after NODA-GA coupling by RP-HPLC and SEC-HPLC

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3.1.3 Summary of characterization results after C37H mutant coupling to NODA-GA

By analyzing the characterization result of the coupled antibody, the evaluation standard of the coupled antibody is defined as that the proportion of unconjugated protein is less than or equal to 10 percent, the proportion of DAR2/4 after coupling is more than or equal to 50 percent, the coupling rate is more than or equal to 90 percent, the proportion of monomers after coupling is more than or equal to 90 percent, and the biological activity of the coupled antibody is more than or equal to 70 percent (compared with that before coupling), and if the above standards are met, the mutation site of the antibody is the preferred site, the disulfide bond after mutation can be reduced and opened, and the antibody after reduction can be completely coupled.

TABLE 18 summary of characterization results after conjugation of various engineered antibodies to NODA-GA for C37H

Note: the mark A is up to the set standard, the mark B is not up to the set standard, and the mark C is poor in characterization result

Wherein, the two antibodies of the E6-T107 and T87-V111 combination can normally form disulfide bonds after mutation and carry out conventional transient expression to obtain proteins, which indicates that the spatial positions of the two E6-T107 and T87-V111 combinations are proper, so that disulfide bonds can be formed after mutation. However, the data after coupling is poor, the affinity after E6-T119 coupling is reduced by one time, and the coupling rate of T87-V111 is lower and is between 30% and 40% (from Table 2, the solvent accessibility ASA% of E6 and V111 is less than 20%). From this, it is understood that if the antibody after modification is subjected to further operations such as coupling, the spatial distance of the amino acids and the degree of exposure of the amino acids (solvent accessibility) should satisfy the requirements.

3.1.4 C37H mutant coupling GGFG-Dxd

According to the characterization result of a coupling small molecule chelating agent NODA-GA, the self affinity and the protein expression quantity of the modified antibody and other conditions, antibody protein after partial C37H mutation is selected to be uniformly mixed with TCEP (tris (2-carboxyethyl) phosphine) reducing agent in a molar ratio of 1.

3.1.5 Characterization and detection of C37H mutant after coupling GGFG-Dxd

And respectively carrying out ELISA affinity detection on the coupled antibodies to determine that the coupled antibodies do not lose biological activity. And simultaneously performing RP-HPLC and SEC-HPLC to detect the coupling rate and the monomer ratio of the coupled antibody, and detecting the molecular weight of the coupled antibody by LC-MS to calculate the DAR value of the coupled antibody, whether the coupling is successful or not and the like. The specific detection results are as follows:

TABLE 19 ELISA for detection of binding of antibodies to CD8 alpha after conjugation to GGFG-Dxd

TABLE 20 LC-MS measurement of antibody molecular weight and DAR value following GGFG-Dxd conjugation

TABLE 21 RP-HPLC and SEC-HPLC for detecting antibody coupling GGFG-Dxd rate and monomer ratio

Name of antibody	Coupling Rate% (RP-HPLC)	Percent monomer (SEC-HPLC)
			C37H-Q3-S25	33.57	58.18
C37H-L11-T110	65.06	85.01

3.1.6 Characterization of binding summary after C37H mutant conjugation to GGFG-Dxd

TABLE 22 summary of characterization results after conjugation of C37H modified antibodies to GGFG-Dxd

The results of the combined ELISA and LC-MS data show that most of the mutant antibodies meeting the conditions have no loss of biological activity or basically remain in a reasonable controllable range compared with the mutant antibodies before coupling after coupling, most of the mutant antibodies can be successfully coupled with two small molecules (NODAGA or GGFG-Dxd) by analyzing the molecular weight of the antibodies before and after coupling, and the partially incompletely coupled antibodies (the mixture of naked antibodies and DAR1 or DAR2 antibodies exists at the same time) can be completely coupled into DAR2 antibodies after deeply optimizing the coupling conditions.

3.2 H2 mutant coupling and characterization

3.2.1 H2 mutant coupling Mal-DOTA

According to the reasons of affinity detection and antibody self-expression amount of the modified antibody in the early stage, the antibody protein after partial H2 mutation is selected to be uniformly mixed with TCEP (tris (2-carboxyethyl) phosphine) reducing agent in a molar ratio of 1.

3.2.2 Characterization detection after coupling of H2 mutant with Mal-DOTA

And respectively carrying out ELISA (enzyme-linked immuno sorbent assay) affinity detection on the coupled antibodies to determine that the coupled antibodies do not lose biological activity. And (3) analyzing and evaluating the coupling rate of the coupled antibody and the existence of a polymer of the coupled antibody by PR-HPLC and SEC-HPLC detection, and detecting the molecular weight of the coupled antibody by LC-MS so as to calculate the DAR value of the coupled antibody, the success of coupling and the like. The specific detection results are as follows:

TABLE 23 ELISA detection of Her2 binding of antibody after conjugation to Mal-DOTA

Name (R)	EC50(ng/mL)	Relative Activity (%)
			H2-R66-S82b (Standard)	31.71	100
DOTA-H2-R66-S82b	74.35	42.6
			H2-Q3-S25 (Standard)	37.10	100
DOTA-H2-Q3-S25	26.59	139.5
			H2-S7-G8 (Standard)	55.29	100
DOTA-H2-S7-G8	60.92	90.8
			H2-P84-S113 (Standard)	5.972	100
DOTA-H2-P84-S113	19.67	30.4
			H2-K83-E85 (Standard)	11.06	100
DOTA-H2-K83-E85	23.07	47.9
			H2-L11-T110 (Standard)	35.94	100
DOTA-H2-L11-T110	29.94	120.0
			H2-V12-L18 (Standard)	12.67	100
DOTA-H2-V12-L18	25.74	49.2
			H2-G9-Q108 (Standard)	25.89	100
DOTA-H2-G9-Q108	19.88	130.2
			H2-T87-V111 (Standard sample)	11.77	100
DOTA-H2-T87-V111	26.55	44.3
			H2-E6-T107 (Standard)	29.16	100
DOTA-H2-E6-T107	15.93	183
			H2-Q13-G16 (Standard)	14.7	100
DOTA-H2-Q13-G16	24.25	60.6
			H2-Q39-R45 (Standard)	13.21	100
DOTA-H2-Q39-R45	11.44	115.5

TABLE 24 LC-MS detection of antibody molecular weight and DAR value after Mal-DOTA coupling

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TABLE 25 detection of antibody coupling ratio and monomer ratio after Mal-DOTA coupling by RP-HPLC and SEC-HPLC

Name of antibody	Coupling Rate% (RP-HPLC)	Percent monomer (SEC-HPLC)
			H2-V12-L18	0	100
H2-P84-S113	100	100
			H2-E6-T107	13.5	77.15
H2-S7-G8	100	100
			H2-R66-S82b	100	92.8
H2-T87-V111	61.2	59.8
			H2-Q13-G16	68	66.3
H2-Q3-S25	97.1	83.5
			H2-L11-T110	100	89.2
H2-K83-E85	100	100
			H2-G9-Q108	47.7	47.7
H2-Q39-R45	22	77.8

3.2.3 Summary of characterization results after coupling of H2 mutant to Mal-DOTA

By analyzing the characterization results of the coupled antibody, the results of the coupling of the H2 mutated antibody to Mal-DOTA are analyzed and summarized, the evaluation basis is 3.1.3, and the specific characterization evaluation results are shown in the following table.

TABLE 26 summary of characterization results after conjugation of each of the engineered antibodies to Mal-DOTA for H2

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Like the C37H mutant antibody, the two antibodies of the E6-T107 and T87-V111 combination can normally form disulfide bonds after mutation and carry out conventional transient expression to obtain proteins, which indicates that the two mutant combinations of E6-T107 and T87-V111 have proper spatial positions, so that disulfide bonds can be formed after mutation. However, the data after coupling is poor, the E6-T119 coupling rate is low, only about 13%, and the affinity after T87-V111 coupling is reduced by about 60% (see that ASA% < 20% of solvent accessibility of E6 and V111 in Table 2). Again, if the modified antibody needs further coupling and other operations, the spatial distance of the amino acid and the exposure degree (solvent accessibility) of the amino acid should meet the requirements.

3.2.4 H2 mutant coupling GGFG-Dxd

According to the characterization result of a coupled small molecule chelating agent Mal-DOTA, the affinity and protein expression quantity of the modified antibody, and the like, antibody protein after partial H2 mutation is selected to be uniformly mixed with TCEP (tris (2-carboxyethyl) phosphine) reducing agent in a molar ratio of 1.

3.2.5 Characterization detection of H2 mutant after coupling GGFG-Dxd

And respectively carrying out ELISA affinity detection on the coupled antibodies to determine that the coupled antibodies do not lose biological activity. And (3) analyzing and evaluating the coupling rate of the coupled antibody and the existence of a polymer of the coupled antibody by PR-HPLC and SEC-HPLC detection, and detecting the molecular weight of the coupled antibody by LC-MS so as to calculate the DAR value of the coupled antibody, the success of coupling and the like. The specific detection results are as follows:

TABLE 27 ELISA detection of Her2 binding of antibodies after conjugation to GGFG-Dxd

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TABLE 28 LC-MS detection of antibody molecular weight and DAR value after coupling to GGFG-Dxd

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TABLE 29 detection of antibody coupling ratio and monomer content after coupling of GGFG-Dxd by RP-HPLC and SEC-HPLC

Name of antibody	Coupling Rate% (RP-HPLC)	Percent monomer (SEC-HPLC)
			H2-S7-G8	51.68	100
H2-P84-S113	0	92.79
			H2-G9-Q108	72.8	51.2
H2-K83-E85	77	100
			H2-L11-T110	89.33	88.15
H2-Q3-S25	77.5	83.2
			H2-R66-S82b	100	100

3.2.6 Summary of characterization results of H2 mutant coupled to GGFG-Dxd

By analyzing the characterization results of the coupled antibody, the results of the coupling of the H2 mutated antibody to GGFG-Dxd are analyzed and summarized, the evaluation is according to reference 3.1.3, and the specific characterization evaluation results are shown in the following table.

TABLE 30 summary of characterization results after conjugation of each modified antibody of H2 to GGFG-Dxd

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The results of the data of ELISA, RP-HPLC, SEC-HPLC and LC-MS are integrated, so that most of the mutant antibodies have no loss of biological activity or basically remain in a reasonable and controllable range compared with the mutant antibodies before and after coupling, and most of the mutant antibodies can be successfully coupled with two small molecules (DOTA or GGFG-Dxd) by analyzing the molecular weight and the coupling rate of the antibodies before and after coupling, and the partially incompletely coupled antibodies (the mixture of naked antibodies and DAR1 or DAR2 antibodies exists at the same time) are considered to be completely coupled into the DAR2 antibodies after deeply optimizing the coupling conditions, so that the data of the part of the data are temporarily not reflected in the patent.

3.3.109 mutant conjugation and characterization

3.3.1 109 mutant conjugation GGFG-Dxd

According to the reasons of affinity detection and antibody self-expression amount of the modified antibody in the early stage, the antibody protein after mutation of the selection part 109 is uniformly mixed with TCEP (tris (2-carboxyethyl) phosphine) reducing agent in a molar ratio of 1.

3.3.2 109 mutant coupling GGFG-Dxd post characterization detection

And respectively carrying out ELISA affinity detection on the coupled antibodies to determine that the coupled antibodies do not lose biological activity. And detecting the coupling efficiency of the antibody and the monomer proportion of the coupled antibody by RP-HPLC and SEC-HPLC, and detecting the molecular weight of the coupled antibody by LC-MS so as to calculate the DAR value of the coupled antibody, whether the coupling is successful or not and the like. The specific detection results are as follows:

TABLE 31 ELISA for detection of binding of antibodies to PD-L1 after conjugation to GGFG-Dxd

Name (R)	EC50(ng/mL)	Relative Activity (%)
			109-Q3-25	91.35	100
Dxd-GGFG-109-Q3-S25	17.98	376.9
			109-S11-T110	77.69	100
Dxd-GGFG-109-S11-T110	33.66	146.5

TABLE 32 LC-MS detection of antibody molecular weight and DAR value after coupling to GGFG-Dxd

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TABLE 33 detection of antibody coupling ratio and monomer content after coupling of GGFG-Dxd by RP-HPLC and SEC-HPLC

Name of antibody	Coupling Rate% (RP-HPLC)	Percent monomer (SEC-HPLC)
			109-Q3-S25	60.27	59.6
109-S11-T110	74.43	68.17

3.3.3 Summary of characterization results after 109 mutant coupling to GGFG-Dxd

By analyzing the characterization results of the coupled antibody, the results of the coupling of the 109 mutant antibody to GGFG-Dxd are analyzed and summarized, the evaluation is according to reference 3.1.3, and the specific characterization evaluation results are shown in the following table.

TABLE 34 summary of characterization results after conjugation of each modified antibody to GGFG-Dxd

Surface cysteine transformation of relevant sites is carried out on three antibodies of three targets, affinity detection is carried out on the transformed antibodies, and the detection result shows that the affinity of most of the transformed antibodies has no obvious change compared with that before the transformation. Meanwhile, the coupling of a related metal chelator to the modified antibody and the coupling of an ADC-related linker-cytotoxic small molecule are respectively carried out, and a series of characterization detections are carried out on the coupled antibody, so that the fact that the affinity of most of the antibody is not obviously changed after the coupling, and the antibody can be coupled with 2 small molecule linkers (DAR 2) is found, and the expected result is consistent, and the initial assumption and the purpose of the invention can be realized.

4. Analysis of Universal mutation sites

Through analyzing three antibody mutation sites of three targets and corresponding experimental data after mutation, some mutation sites are found to be simultaneously suitable for three antibodies, and relevant detection after mutation and relevant characterization results after coupling are ideal, and the sites can be speculated to be suitable for site-specific coupling modification of a conventional nano antibody (the mutation sites summarized in the patent are most commonly used amino acids which can be replaced by cysteine, and the surface amino acids which are positioned in an FR region can be replaced by cysteine to form a new antibody according to a general formula), namely the antibody surface cysteine site-specific coupling technology. After cysteine mutation is carried out on the site, the affinity of the site is not obviously changed before mutation, coupling can be successfully carried out no matter coupling nuclide marker-related small molecule linker or coupling ADC drug-related linker and cytotoxin, and the affinity of the site is not obviously changed after coupling.

The framework regions of three selected nanobodies are divided and numbered by referring to a universal humanized nanobody framework h-NbBcII10FGLA (PDB number is 3 eak), and the universal sites which can be subjected to cysteine mutation are obtained as follows:

TABLE 35 Nanobody Universal site numbering

According to the previous experimental results, 15 pairs of amino acids located in the nano antibody frame region are selected, and the amino acids located on the surface of the nano antibody can be subjected to cysteine modification to form disulfide bonds. To facilitate surface-directed coupling. The method not only solves the problems that the DAR value of the conventional random coupling is not uniform, the process is responsible for and easily influences the biological activity, but also solves the problems that the DAR value of the end fixed-point coupling is low, a polymer is easily formed and the like, can improve the DAR value and can not influence the biological activity of the coupled antibody on the premise that the DAR value is controllable, and the process is relatively simple and controllable and the like. Provides a powerful foundation for the nanometer antibody to enter the fields of ADC, PDC, RDC and the like.

5. Universal mutation site validation

In order to verify the universality of the selected mutation sites again, and by utilizing the method, not only the transformation of the DAR2 antibody can be carried out, but also the construction of the DAR4 antibody can be carried out, so that another nano antibody H1-AN-TS of the Her2 target is selected for carrying out the transformation of the DAR2 and DAR4 antibodies, and meanwhile, the DAR4 antibody transformation is carried out again by using the C37H antibody of the CD8 alpha target and the H2 and H1-AN-TS antibodies of the Her2 target. Since the specific experimental methods of construction, expression, coupling, etc. have been introduced in the foregoing, only the corresponding detection and characterization results will be described in this section.

DAR4 antibody mutation by C37H and corresponding detection result

5.1.1 post-mutation sequence and ELISA detection results

According to the previous experimental results and the analysis of universal sites, 3 (Q) -25 (S) and 84 (A/P/M) -113 (S) are selected for combined mutation, the specific numbers of the corresponding C37H antibodies are Q3-S25 and A84-S113, and the specific sequences after mutation are as follows:

C37H-A84-S113+Q3-S25：

QVCLVESGGGLVQPGGSLRLSCAACGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTYYADSVKGRFTISRDNSKNHVYLQMNSLRCEDTAVYYCAAGSYYSCSVYPAYDLDYWGKGTLVTVSC

note: the mutation sites are underlined

And performing corresponding construction, expression and purification after mutation to obtain a corresponding target protein, performing ELISA detection, and verifying the affinity condition of the mutated antibody, wherein the specific detection result is as follows:

TABLE 36 ELISA detection of C37H DAR4 mutant antibody affinity

Name (R)	EC50(ng/mL)	Relative Activity (%)
			C37H (Standard sample))	51.06	100
C37H-A84-S113+Q3-S25	60.22	84.8

As can be seen from the ELISA detection results, the affinity of the antibody after modification is not obviously different from that before modification.

5.1.2 conjugation and characterization of C37H DAR4 antibodies

Mixing C37H-A84-S113+ Q3-S25 and TCEP according to a molar ratio of 1:10, mixing, reacting at 37 ℃ for 2h, dissolving NODA-GA in PBS, and mixing the reduced C37H-A84-S113+ Q3-S25 and NODA-GA according to a molar ratio of 1:20, mixing evenly, and reacting at 37 ℃ for 2h. And (3) after ultrafiltration, changing the solution to remove unconjugated small molecules, and simultaneously carrying out characterization on the conjugated antibody by ELISA and LC-MS, wherein the specific detection result is as follows:

TABLE 37 ELISA detection of affinity after C37H-A84-S113+ Q3-S25 coupling

Name (R)	EC50(ng/mL)	Relative Activity (%)
			C37H-A84-S113+ Q3-S25 (Standard)	60.22	100
NODA-GA-C37H-A84-S113+Q3-S25	52.06	157

TABLE 38 LC-MS detection of DAR values after C37H-A84-S113+ Q3-S25 coupling

TABLE 39 RP-HPLC and SEC-HPLC detection of C37H-A84-S113+ Q3-S25 coupling ratio and monomer ratio

Name of antibody	Coupling Rate% (RP-HPLC)	Percent monomer (SEC-HPLC)
			C37H-A84-S113+Q3-S25	94.04	100

Based on the C37H antibody, two pairs of mutable universal sites are selected to be combined to construct the DAR4 antibody, the affinity of the antibody after mutation is not obviously different from that before mutation, the C37H-A84-S113+ Q3-S25 antibody after mutation is coupled and characterized, and the result shows that the antibody after mutation can be completely coupled with 4 small molecule linkers, the coupling rate is high, the antibody after coupling basically exists in a monomer form, and the affinity after coupling is not influenced.

5.2.H2 DAR4 antibody mutation and corresponding detection results

5.2.1 post-mutation sequence and ELISA test results

Randomly selecting universal sites 3 (Q) -25 (S) and 7 (S) -8 (G) for combined mutation, wherein specific numbers corresponding to the H2 antibody are Q3-S25 and S7-G8, and specific sequences after mutation are as follows:

H2-S7-G8+Q3-S25：

QLCLVECCGGLVQPGGSLRLSCAACGFTLDYYAIGWFRQAPGKEREGVSCITSGGSTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASHYGLRVGTLCPETYEYDYWGQGTQVTVSS

note: the mutation sites are underlined

TABLE 40 ELISA detection of H2 DAR4 mutant antibody affinity

5.1.2 coupling and characterization of H2 DAR4 antibodies

Mixing H2-S7-G8+ Q3-S25 and TCEP according to a molar ratio of 1:10, uniformly mixing, reacting at 37 ℃ for 2H, and mixing the reduced H2-S7-G8+ Q3-S25 and Mal-DOTA according to the molar ratio of 1:20, mixing evenly, and reacting at 37 ℃ for 2h. And (3) ultrafiltering and replacing the solution into 0.25M sodium acetate solution, and performing detection ELISA and LC-MS characterization on the coupled antibody, wherein the specific detection result is as follows:

TABLE 41 ELISA detection of affinity after H2-S7-G8+ Q3-S25 coupling

Name (R)	EC50(ng/mL)	Relative Activity (%)
			H2-S7-G8+ Q3-S25 (Standard)	21.36	100
DOTA-H2-S7-G8+Q3-S25	65.45	32.6

TABLE 42 detection of DAR values after H2-S7-G8+ Q3-S25 coupling by LC-MS

TABLE 43 detection of H2-S7-G8+ Q3-S25 coupling ratio and monomer ratio by RP-HPLC and SEC-HPLC

According to ELISA detection results, the affinity of the antibody after coupling is slightly reduced compared with that before coupling, RP-HPLC and SEC-HPLC show that coupling is complete and all monomers are obtained after coupling, and LC-MS shows that the antibody is successfully coupled with 4 DOTA small molecules and coupling is complete.

5.3.H1-AN-TS DAR2 and DAR4 antibody mutation and corresponding detection result

Based on H1-AN-TS antibody, universal sites 11 (S/L) -110 (T), 7 (S) -8 (G), 3 (Q) -25 (S), 83 (K/R/Q/S/T/E) -85 (E/D), 84 (A/P/M) -113 (S) and 41 (A/P/R) -42 (G), 14 (P) -113 (S) sites are selected to carry out a pair of disulfide bond modification (DAR 2 antibody), and based on the constructed DAR2 antibody, two pairs of disulfide bond modification (DAR 4 antibody) are carried out by mutually combining and pairing, wherein the specific numbering and antibody sequence before and after the related H1-AN-TS mutation are as follows:

H1-AN-TS：

QLQLVESGGGLVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKPEDTAVYYCNTDPPWGDDPFERSASWGQGTQVTVSS

wherein the CDR regions are underlined

H1-L11-T110：

QLQLVESGGGCVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKPEDTAVYYCNTDPPWGDDPFERSASWGQGTQVCVSS

H1-Q3-S25：

QLCLVESGGGLVQPGGSLRLSCAACSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKPEDTAVYYCNTDPPWGDDPFERSASWGQGTQVTVSS

H1-S7-G8：

QLQLVECCGGLVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKPEDTAVYYCNTDPPWGDDPFERSASWGQGTQVTVSS

H1-P84-S113：

QLQLVESGGGLVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKCEDTAVYYCNTDPPWGDDPFERSASWGQGTQVTVSC

H1-K83-E85：

QLQLVESGGGLVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLCPCDTAVYYCNTDPPWGDDPFERSASWGQGTQVTVSS

H1-A41-G42：

QLQLVESGGGLVQPGGSLRLSCAASSSIFSVNNMGWYRQACCEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKPEDTAVYYCNTDPPWGDDPFERSASWGQGTQVTVSS

H1-L11-T110+R66-S82b：

QLQLVESGGGCVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGCVTISRDDAKSTVYLQMNCLKPEDTAVYYCNTDPPWGDDPFERSASWGQGTQVCVSS

H1-L11-T110+Q3-S25：

QLCLVESGGGCVQPGGSLRLSCAACSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKPEDTAVYYCNTDPPWGDDPFERSASWGQGTQVCVSS

H1-L11-T110+P84-S113：

QLQLVESGGGCVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKCEDTAVYYCNTDPPWGDDPFERSASWGQGTQVCVSC

H1-L11-T110+K83-E85：

QLQLVESGGGCVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLCPCDTAVYYCNTDPPWGDDPFERSASWGQGTQVCVSS

H1-L11-T110+A41-G42：

QLQLVESGGGCVQPGGSLRLSCAASSSIFSVNNMGWYRQACCEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKPEDTAVYYCNTDPPWGDDPFERSASWGQGTQVCVSS

H1-L11-T119+S7-G8：

QLQLVECCGGCVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKPEDTAVYYCNTDPPWGDDPFERSASWGQGTQVCVSS

H1-S7-G8+P84-S113：

QLQLVECCGGLVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKCEDTAVYYCNTDPPWGDDPFERSASWGQGTQVTVSC

H1-Q3-S25+S7-G8：

QLCLVECCGGLVQPGGSLRLSCAACSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKPEDTAVYYCNTDPPWGDDPFERSASWGQGTQVTVSS

H1-K83-E85+Q3-S25：

QLCLVESGGGLVQPGGSLRLSCAACSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLCPCDTAVYYCNTDPPWGDDPFERSASWGQGTQVTVSS

H1-Q3-S25+P84-S113：

QLCLVESGGGLVQPGGSLRLSCAACSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKCEDTAVYYCNTDPPWGDDPFERSASWGQGTQVTVSC

H1-L11-T110+P14-S113：

QLQLVESGGGCVQCGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKPEDTAVYYCNTDPPWGDDPFERSASWGQGTQVCVSC

Note: the mutation sites are underlined

Performing corresponding construction, expression and purification after mutation to obtain corresponding target protein, then performing ELISA to detect antibody affinity after mutation, SEC-HPLC and SDS-PAGE to detect monomer expression, and simultaneously verifying antibody expression amount after mutation, wherein the specific detection results are as follows:

TABLE 44 ELISA detection of H1-AN-TS DAR2/4 mutant antibody affinity

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TABLE 45 SEC-HPLC and SDS-PAGE detection of H1-AN-TS DAR2/4 mutant antibody monomer ratio

TABLE 46 expression level of each of the mutated antibodies H1-AN-TS DAR2/4

Name of antibody	Amount of expression (mg/L)	Name of antibody	Amount of expression (mg/L)
				H1-Q3-S25	74.2	H1-L11-T110+P84-S113	112
H1-S7-G8	41.6	H1-L11-T110+K83-E85	25
				H1-P84-S113	58.6	H1-L11-T110+A41-G42	18.2
HI-K83-E85	19.2	H1-S7-G8+P84-S113	51
				H1-L11-T110	20.8	H1-Q3-S25+P84-S113	21.6
H1-A41-G42	58.7	H1-Q3-S25+S7-G8	1.84
				H1-L11-T110+R66-S82b	90	H1-L11-T110+S7-G8	18
H1-L11-T110+Q3-S25	56	H1-K83-E85+Q3-S25	4

The mutation site solvent exposure degree ASA% is still predicted through a website or software, the molecular weight is estimated to be whether the theoretical molecular weight is consistent with the actually detected molecular weight through mass spectrum detection, other characterization results of the mutated antibody are analyzed, the results of the H1 mutated antibody are analyzed and summarized, the evaluation basis refers to 2.7, and the specific characterization evaluation results are shown in the following table.

TABLE 47 summary of in vitro characterization results for each of the engineered antibodies for H1

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Selecting DAR4 antibodies with relatively high DAR2 and DAR4 affinities for coupling (in order to not affect the coupling effect, if the antibody has a polymer, a monomer can be obtained by purification optimization firstly) and corresponding characterization, and mixing H1-L11-T110, H1-S7-G8 and H1-P84-S113 with TCEP according to the molar ratio of 1:5, uniformly mixing H1-L11-T110+ P84-S113 and H1-S7-G8+ P84-L113 with TCEP according to a molar ratio of 1:10, uniformly mixing, reacting at 37 ℃ for 2h, and mixing the reduced DAR2 and DAR4 with DOTA-Mal according to the molar ratio of 1:20, mixing evenly, and reacting at 37 ℃ for 2h. And (3) ultrafiltering and changing the solution into 0.25M sodium acetate solution, and subjecting the coupled antibody to detection ELISA, RP-HPLC, SEC-HPLC and LC-MS for characterization, wherein the specific detection results are as follows:

TABLE 48 ELISA detection of affinity after H1-DAR2/DAR4 coupling

Name (R)	EC50(ng/mL)	Relative Activity (%)
			H1-L11-T110 (Standard)	35.04	100
DOTA-H1-L11-T110	57.13	61.3
			H1-S7-G8 (Standard)	43.4	100
DOTA-H1-S7-G8	63.83	68
			H1-P84-S113 (Standard)	53.04	100
DOTA-H1-P84-S113	66.52	79.7
			H1-L11-T110+ P84-S113 (Standard)	117.6	100
DOTA-H1-L11-T110+P84-S113	135.2	87
			H1-S7-G8+ P84-S113 (Standard)	85.31	100
DOTA-H1-S7-G8+P84-S113	145.4	58.7

TABLE 49 LC-MS detection of DAR value after H1-DAR2/DAR4 coupling

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TABLE 50 detection of H1-DAR2/DAR4 coupling ratio and monomer condition by RP-HPLC and SEC-HPLC

By analyzing the characterization results of the coupled antibody, the results of the coupling of the H1 mutated antibody to Mal-DOTA are analyzed and summarized, the evaluation basis is 3.1.3, and the specific characterization evaluation results are shown in the following table.

TABLE 51 summary of characterization results after conjugation of each of the engineered antibodies to Mal-DOTA for H1

Another antibody H1-AN-TS selected for Her2 antigen was used for mutation site validation, we performed not only for the DAR2 site but also for the DAR4 site. According to the in vitro characterization of the mutated naked antibody and the characterization results of the coupled antibody, the selected site has universality and can be theoretically applied to the modification of any nano antibody, all the characterization results after modification are in line with expectations, the reduced antibody can be completely coupled with a corresponding small molecule linker to improve a corresponding DAR value, and all the characterization results of the coupled antibody are in line with expectations.

5.4. Affinity optimization for H1-DAR2/4 engineered antibodies

As can be seen from the in vitro characterization data before and after the mutation of H1-DAR2/4 and after the coupling of the antibody after the mutation, the modified antibody basically accords with the expectation from the aspects of affinity, expression quantity, coupling condition and the like. But we believe that by further sequence optimization, affinity also has room for improvement, and thus will have better effect on later stage antibodies, whether applied to targeting or therapy, so I chose to try optimization with the department H1-DAR2/4 antibody. The specific optimized sequence is as follows:

H1-L11-T110-SA：

QLQLVESGGGCVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKPEDTAVYYCNTDPPWGDDPFERSASWGQGTQVCVSSSA

H1-P84-S113-SA：

QLQLVESGGGLVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKCEDTAVYYCNTDPPWGDDPFERSASWGQGTQVTVSCSA

H1-P84-S113-GS：

QLQLVESGGGLVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKCEDTAVYYCNTDPPWGDDPFERSASWGQGTQVTVSCGS

H1-L11-T110+P84-S113-14A：

QLQLVESGGGCVQAGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKCEDTAVYYCNTDPPWGDDPFERSASWGQGTQVCVSC

H1-L11-T110+P84-S113-A：

QLQLVESGGGCVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKCEDTAVYYCNTDPPWGDDPFERSASWGQGTQVCVSCA

H1-L11-T110+P84-S113-GS：

QLQLVESGGGCVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKCEDTAVYYCNTDPPWGDDPFERSASWGQGTQVCVSCGS

H1-L11-T110+P84-S113-K：

QLQLVESGGGCVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKCEDTAVYYCNTDPPWGDDPFERSASWGQGTQVCVKC

H1-S7-G8+P84-S113-SA：

QLQLVECCGGLVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKCEDTAVYYCNTDPPWGDDPFERSASWGQGTQVTVSCSA

H1-S7-G8+P84-S113-K：

QLQLVECCGGLVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKCEDTAVYYCNTDPPWGDDPFERSASWGQGTQVTVKC

H1-S7-G8+P84-S113-GS：

QLQLVECCGGLVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKCEDTAVYYCNTDPPWGDDPFERSASWGQGTQVTVSCGS

H1-L11-T110+P14-S113-K

QLQLVESGGGCVQCGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKPEDTAVYYCNTDPPWGDDPFERSASWGQGTQVCVKC

Note: the mutation sites are underlined

Performing corresponding construction, expression and purification after optimization to obtain corresponding target protein, performing ELISA detection on the optimized antibody affinity, SEC-HPLC and SDS-PAGE detection monomer expression conditions, and verifying the mutated antibody expression quantity and the like, wherein the specific detection results are as follows:

TABLE 52 ELISA detection of H1-DAR2/4 optimized antibody affinities

TABLE 53 SEC-HPLC and SDS-PAGE detection of antibody monomer ratio after H1-DAR2/4 optimization

Name of antibody	Percent monomer (SEC-HPLC)	Percent monomer (SDC-PAGE)
			H1-L11-T110-SA	42	19.1
H1-P84-S113-SA	100	100
			H1-P84-S113-GS	100	100
H1-L11-T110+P84-S113-14A	42.73	30.65
			H1-L11-T110-P84-S113-A	41.1	23.95
H1-L11-T110+P84-S113-GS	37.93	21.04
			H1-L11-T110+P83-S113-K	74.68	35.05
H1-L11-T119+P14-S113-K	61.42	45.9
			H1-S7-G8+P84-S113-GS	100	99.15
H1-S7-G8+P87-S113-SA	100	100
			H1-S7-G8+P87-S122-K	100	99.14

TABLE 54 expression level of antibody after optimization of H1-DAR2/4

From each characterization result after optimization, the affinity of most antibodies after optimization is obviously improved compared with that before modification, and the expression level can be stably or partially improved compared with that before modification, which shows that even after the surface fixed-point coupling mutation modification is carried out on the nano antibody, the modified antibody can be further optimized according to the specific requirements of subsequent experiments, so as to achieve the expected result.

Through a series of mutation and verification experiments on different antibodies with different targets, a common universal site on the surface of the nano antibody, which can be used for cysteine mutation, is found and summarized. Theoretically, as long as the amino acid is positioned in the FR region, the accessibility of the amino acid solvent is more than or equal to 20 percent, and the spatial position is proper, the amino acid solvent can be mutated to form a disulfide bond and form an access site. This universal site can also be subjected to combinatorial mutation to give DAR4 or even DAR6 antibodies. The invention solves many disadvantages of random coupling and end fixed point coupling of nano antibody, provides a method with simple process, high DAR value and no influence on biological activity, combines the advantages of small molecular weight and strong penetrating power of nano antibody itself, and is believed to be widely applied to various treatment fields in the future.

Claims

2. The antigen binding protein of claim 1, wherein the cysteine residue at the first position and the cysteine residue at the second position are both mutated.

3. The antigen binding protein of any one of claims 1-2, wherein the disulfide bond is capable of forming a sulfhydryl group upon reduction by a reducing agent, wherein the sulfhydryl group allows the antigen binding protein to be conjugated to a payload at the first position and/or the second position.

4. The antigen binding protein of any one of claims 1-3, wherein the cysteine residue at the first position and the cysteine residue at the second position have a C β -C β of no more than about

5. The antigen binding protein of any one of claims 1-4, wherein the relative solvent accessibility (ASA%) of the amino acid residue at the first position to the amino acid residue at the second position is not less than about 20%.

6. The antigen binding protein of any one of claims 1-5, wherein the combination of the first and second positions is selected from at least one of the following combinations:

a)66-82b；

b)3-25；

c)7-8；

d)84-113；

e)83-85；

f)68-81；

g)17-82a；

h)41-42；

i)9-108；

j)11-110；

k)87-111；

l)13-16；

m)12-18；

n) 39-45; and

o)14-113

7. The antigen binding protein of any one of claims 1 to 6, wherein the antigen binding protein is a humanized VHH antibody or a fully human VHH antibody.

8. The antigen binding protein of any one of claims 1-7, wherein the antigen binding protein specifically binds to a tumor antigen or a non-tumor antigen.

9. A fusion polypeptide comprising the antigen binding protein of any one of claims 1-8.

10. An immunoconjugate comprising the antigen binding protein of any one of claims 1-8, a payload, and a linker connecting the payload to the antigen binding protein.