WO2022188801A1 - Pd-1 binding protein and pharmaceutical use thereof - Google Patents

Abstract

Provided are a PD-1 binding protein and a pharmaceutical use thereof. Specifically, provided are a PD -1 binding protein, a PD-1/LAG-3 binding protein, a method for treating cancer therefor, and a pharmaceutical use thereof.

Description

PD-1 binding protein and its medicinal use

This application claims the priority of Chinese patent application 2021102597905 filed on March 10, 2021 and Chinese patent application 2021102612286 filed on March 10, 2021.

technical field

The present disclosure belongs to the field of biomedicine and relates to PD-1 binding proteins and their uses for the treatment of diseases such as cancer.

Background technique

PD-1 (Programmed Cell death-1) belongs to the CD28 receptor family and is an immunosuppressive receptor (Riley et al. 2009, Immunol. Rev. 29:114-25). PD-1 is a type I transmembrane protein mainly expressed on activated B cells, T cells and myeloid cells (Chen et al. 2013, Nat. Rev. Immunol. 13:227-42), and there are two cell surface glycoproteins The ligands are PD ligand 1 (PD-L1, also known as CD274, B7-H1) and PD ligand 2 (PD-L2, also known as B7-DC). Neither PD-L1 nor PD-L2 bind to other CD28 receptor family members. PD-L1 is widely expressed in lymphocytes (such as CD4 ⁺ T cells and CD8 ⁺ T cells, macrophages, etc.) as well as in peripheral tissues, various tumor cells, and virus-infected cells. PD-L2 is mainly expressed on activated dendritic cells and macrophages (Dong et al. 1999, Nat. Med. 5:1365-9). After PD-1 binds to its ligand PD-L1 or PD-L2, it will down-regulate the function of T cells, including reducing T cell activation, differentiation and proliferation, and cytokine secretion. PD-L1 is highly expressed in a variety of human tumors, including melanoma, glioma, non-small cell lung cancer, head and neck cancer, leukemia, pancreatic cancer, kidney cancer, and liver cancer, etc. (Zou and Chen, 2008, Nat. Rev. Immunol. 8:467-77). PD-L1, which is highly expressed by tumor cells, downregulates T cell function, increases T cell apoptosis, and plays an important role in tumor immune escape (Freeman et al. 2000, J.Exp.Med.192:1027-34; Latchman et al. 2001, Nat. Immunol. 2:261-8; Cater et al. 2002, Eur. J. Immunol. 32:634-43; Ohigashi et al. 2005, Clin. Cancer Res. 11:2947-53). Blocking the interaction of PD-1 and PD-L1 can reverse immunosuppression, while simultaneous inhibition of PD-1 and PD-L1 and PD-L2 can act synergistically (Iwai et al. 2002, Proc.Natl.Acad. Sci. USA, 99: 12293-7; Brown et al. 2003, J. Immunol. 170: 1257-66).

LAG-3 (lymphocyte activation gene-3, also known as CD223) is also a member of the immunoglobulin superfamily, which can negatively regulate the functions and life cycle of immune cells. LAG-3 is mainly expressed on T lymphocytes, B lymphocytes, NK cells, Treg cells and DC cells (Proc Natl Acad Sci U S A, 1997, 94(11): 5744-9. Eur J Immunol, 2005, 35(7):2081-8; J Immunol, 2009, 182(4):1885-91). LAG-3 is a class of immunosuppressive molecules and one of the co-receptor components of TCR. It interferes with the TCR activation of T lymphocytes and plays a negative regulatory function in the activation of T lymphocytes. In some diseases, LAG-3 expression is elevated and corresponding immunosuppression occurs. Gandhi et al. found that in the blood and tumor tissues of patients with Hodgkin lymphoma, lymphocytes highly expressed LAG-3; the function of specific CD8+ T cells in tumor tissues was significantly impaired. Tumor function can be restored, and cytokine secretion is increased. Therefore, it is speculated that the expression of LAG-3 is related to the negative immune regulation function of specific T cells, and inhibiting the function of LAG-3 molecule can enhance the anti-tumor effect of T cells, which is a potential tumor immunotherapy target (Blood, 2006, 108(7):2280-9).

Camelids (such as camels and alpacas) produce a unique heavy chain antibody (HcAb) that lacks the light chain. The variable region fragment (VHH) derived from this antibody is called a single domain antibody (single domain antibody). sdAb). The molecular weight of single-domain antibody is only 12-15kDa, which is one tenth of that of traditional antibody (containing four chains). Its diameter is about 2.5nm and its length is about 4nm. Single domain antibodies also contain three CDRs, of which CDR3 plays a major role in affinity. Compared with human antibody VH, the CDR3 of single domain antibody is longer and can form a bulge loop structure, which can penetrate deep into the interior of the antigen to better bind the antigen. Therefore, VHH has the characteristics of high affinity and high specificity. In addition, the hydrophobic residues of FR2 in single-domain antibodies are replaced by hydrophilic residues, which are more water-soluble and less likely to form aggregates. Compared with traditional antibodies, single domain antibodies have many advantages such as high binding capacity, high specificity, high solubility, high stability and high expression.

At present, the single-domain antibodies against PD-1 are in the early stage of development worldwide. There is no single-domain antibody drug targeting PD-1, and no double-antibody drug targeting PD-1/LAG-3. in early development stage.

The present disclosure provides VHH antibodies that specifically bind PD-1, and bispecific antibodies that bind both PD-1 and LAG-3, the latter selectively targeting proteins expressing both PD-1 and LAG-3. cells, effectively block PD-1 and LAG-3 on T cells that overexpress both PD-1 and LAG-3, thereby reducing the side effects of LAG-3 antibodies and effectively treating tumors.

SUMMARY OF THE INVENTION

The present disclosure provides a PD-1 binding protein, more specifically, a PD-1/LAG-3 binding protein or a combination of a PD-1 binding protein and a LAG-3 binding protein, and medical uses thereof.

PD-1 binding protein

The present disclosure provides a PD-1 binding protein comprising at least one immunoglobulin single variable domain capable of specifically binding PD-1. In some embodiments, the PD-1 binding protein comprises an immunoglobulin single variable domain that specifically binds PD-1. In other embodiments, the PD-1 binding protein comprises 2, 3, 4 or more immunoglobulin single variable domains that specifically bind PD-1. In some embodiments, the PD-1 binding protein comprises two or more identical immunoglobulin single variable domains that specifically bind PD-1. In other embodiments, the PD-1 binding protein comprises two or more distinct immunoglobulin single variable domains that specifically bind PD-1. In some embodiments, the two or more immunoglobulin single variable domains that specifically bind PD-1 are directly linked. In other embodiments, the two or more immunoglobulin single variable domains that specifically bind PD-1 are linked by a linker. The linker may contain 1-20 or more amino acids and is a non-functional amino acid sequence. For example, the linker is a flexible linker such as _G4S , GS, GAP, ( _G4S ) _n , etc., wherein n is an integer between 1-8.

In some embodiments, the PD-1 binding proteins of the present disclosure comprise at least one immunoglobulin single variable domain comprising the amino acid sequence shown in DSVKGRFT or ASVKGRFA. In some specific embodiments, the immunoglobulin single variable domain comprises the three complementarity determining regions CDR1, CDR2, and CDR3, and DSVKGRFT or ASVKGRFA is located in CDR2.

In some embodiments, the PD-1 binding proteins of the present disclosure comprise, in order from the amino terminus to the carboxy terminus, three complementarity determining regions, CDR1, CDR2, and CDR3, spaced apart from each other. Described CDR1, CDR2, CDR3 are shown as CDR1, CDR2, CDR3 in any sequence of SEQ ID NO: 154-157, 7-33, 35-58, 123-128, CDR is according to Kabat, IMGT, Chothia, AbM or as defined by the Contact numbering system. In some embodiments, it is defined according to the Kabat numbering system.

In some embodiments, an immunoglobulin single variable domain of the present disclosure comprises (in order from amino-terminus to carboxy-terminus) three complementarity determining regions CDR1, CDR2 and CDR3, wherein:

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 62, CDR2 comprises the amino acid sequence shown in X ₁ IDSVGX ₂ TX ₃ YX ₄ X ₅ SVKG (SEQ ID NO: 115), wherein X ₁ is selected from S or T, X ₂ is selected from From T or A, X ₃ is selected from D, N or G, X ₄ is selected from T or A, X ₅ is selected from N or D, and CDR 3 comprises the amino acid sequence shown in SEQ ID NO: 64; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 81, CDR2 comprises the amino acid sequence shown in VVDRX ₂₄ GGX ₆ IYAX ₇ SVKX ₈ (SEQ ID NO: 116), wherein X ₂₄ is selected from Y or F, and X ₆ is selected from I or T, X ₇ is selected from A or D, X ₈ is selected from K or D, CDR3 comprises the amino acid sequence shown in GSYTX ₉ X ₁₀ X ₁₁ SCX ₁₂ PDAL (SEQ ID NO: 117), wherein X ₉ is selected from S or D , X ₁₀ is selected from A or D, X ₁₁ is selected from N or G, X ₁₂ is selected from Q or H; or

CDR1 comprises the amino acid sequence shown in YNX ₁₃ MX ₁₄ (SEQ ID NO: 118), wherein X ₁₃ is selected from F or Y, X ₁₄ is selected from S or T, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 66, and CDR3 comprises The amino acid sequence shown in SEQ ID NO: 67; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 84, CDR2 comprises the amino acid sequence shown in VINTGX ₁₅ NX ₁₆ TYYADSVKG (SEQ ID NO: 119), wherein X ₁₅ is selected from A or T, X ₁₆ is selected from S or T, CDR3 comprising the amino acid sequence shown in SEQ ID NO: 86; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 78, CDR2 comprises the amino acid sequence shown in X ₁₇ YPTAGX ₁₈ TYX ₁₉ X ₂₀ DSX ₂₁ KG (SEQ ID NO: 120), wherein X ₁₇ is selected from L or I, and X ₁₈ is selected from From R or K, X ₁₉ is selected from Y or F, X ₂₀ is selected from G or A, X ₂₁ is selected from M or V, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 80; or

CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 59, 60 and 61; or

CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 74, 75 and 76; or

CDR1, CDR2, and CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 88, 89, and 90;

Or, CDR1, CDR2, and CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 96, 97, and 98.

In some embodiments, the PD-1 binding proteins of the present disclosure comprise at least one immunoglobulin single variable domain comprising three complementarity determining regions CDR1, CDR2 and CDR3, wherein:

CDR1 comprises the amino acid sequence shown as X ₂₂ KCMG (SEQ ID NO: 152), wherein X ₂₂ is selected from N, D, E, F, G, H, I, K, L, M, P, Q, R or S;

CDR2 comprises the amino acid sequence shown in VVDRFGGTIYAX ₂₅ SVKG (SEQ ID NO: 204);

CDR3 comprises the amino acid sequence shown in GSYTSAX ₂₃ SCQPDAL (SEQ ID NO: 153), wherein X ₂₅ is selected from A or D, and X ₂₃ is selected from N, A, E, F, G, H, K, P, Q , R, or S.

In some specific embodiments, the PD-1 binding proteins of the present disclosure comprise any one selected from the group consisting of:

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 62, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 63, 68, 69, 70, 72, 77, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 64 or 73; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 81, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 71, 82, 91, 93, 94, CDR3 comprises the amino acid sequence shown in any of SEQ ID NO: 83, 92, 95 sequence; or

CDR1 comprises the amino acid sequence shown in any of SEQ ID NO: 65, 113, 114, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 66, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 67; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 84, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 85 and 102, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 86; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 78, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 79, 87, 99, 100, 101, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 80.

CDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 129-141, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 83; or

CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 81, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence set forth in any of SEQ ID NOs: 142-151; or

CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 129, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 145; or

CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 132, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 145; or

CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 129, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 146; or

CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 132, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 146; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 81, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 71 and 82, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 83.

In some specific embodiments, the PD-1 binding proteins of the present disclosure comprise any one selected from the group consisting of:

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 62, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 63, 68, 69, 70, 72, 77, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 64; or

CDR1, CDR2, and CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 62, 63, and 73; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 81, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 71 and 82, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 83; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 81, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 91 and 93, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 92; or

CDR1, CDR2, and CDR3 include the amino acid sequences shown in SEQ ID NOs: 81, 94, and 95, respectively.

In some embodiments, the immunoglobulin single variable domain of the PD-1 binding protein of the present disclosure comprises three complementarity determining regions CDR1, CDR2 and CDR3, wherein CDR3 is selected from SEQ ID NOs: 61, 64, 67, 73 , 76, 80, 83, 86, 90, 92, 95, 98 or the amino acid sequence with 3, 2 or 1 amino acid difference therefrom.

In some embodiments, in at least one immunoglobulin single variable domain in the PD-1 binding protein of the present disclosure:

(i) CDR1 comprises any amino acid sequence selected from the group consisting of SEQ ID NO: 59, 62, 65, 74, 78, 81, 84, 88, 113, 114, or has 3, 2, 1 amino acid difference therewith the amino acid sequence of ; and/or

(ii) CDR2 comprising selected from SEQ ID NO: 60, 63, 66, 68, 69, 70, 71, 72, 75, 77, 79, 82, 85, 87, 89, 91, 93, 94, 97, 99 , any amino acid sequence shown in 100, 101, 102, or an amino acid sequence with 3, 2, 1 amino acid difference; and/or

(iii) CDR3 comprises any amino acid sequence selected from the group consisting of SEQ ID NO: 61, 64, 67, 73, 76, 80, 83, 86, 90, 92, 95, 98, or has 3, 2, Amino acid sequence with 1 amino acid difference.

In some embodiments, one or more of the above-described CDRs are grafted onto a scaffold or FR (including, but not limited to, human-derived scaffolds, or non-immunoglobulin scaffolds). Scaffolds and techniques suitable for such CDR grafting are known in the art.

In some embodiments, the PD-1 binding protein of the present disclosure is an antibody that binds PD-1, or a conjugate, fusion protein comprising the antibody, antigen-binding fragment thereof.

In some specific embodiments, the antibody is a camelid antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or a fragment thereof. In some embodiments, the antigen-binding fragment is an sdAb or a bispecific, multispecific antibody.

In some embodiments, at least one immunoglobulin single variable domain in a PD-1 binding protein of the present disclosure is a VHH.

In some specific embodiments, the VHH comprises the amino acid sequence of any one of SEQ ID NOs: 7-33, or has at least 70%, at least 80%, at least 85%, at least Amino acid sequences of 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity.

In other embodiments, the VHH is a humanized VHH. The humanized VHH comprises the amino acid sequence of any of SEQ ID NOs: 154-157, 35-58, 123-128, or the amino acid sequence of any of SEQ ID NOs: 154-157, 35-58, 123-128 One has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, Amino acid sequences of at least 99% or 100% sequence identity. Alternatively, the amino acid sequence of the VHH comprises one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to any of SEQ ID NOs: 154-157, 7-33, 35-58, 123-128, for example, comprising 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions.

In some embodiments, the PD-1 binding proteins of the present disclosure are obtained by affinity maturation, eg, affinity maturation based on SEQ ID NOs: 154-157, 7-33, 35-58, 123-128. The affinity matured PD-1 binding protein can have one or more changes in one or more CDRs that result in an increased affinity for PD-1 compared to the parental PD-1 binding protein.

In some embodiments, the PD-1 binding proteins of the present disclosure, in addition to comprising at least one immunoglobulin single variable domain capable of specifically binding PD-1 or an epitope thereof, also comprise an Fc region.

Inclusion of an Fc region in the PD-1 binding proteins of the present disclosure allows the binding proteins to form dimeric molecules while extending the in vivo half-life of the binding proteins. Fc regions useful in the present disclosure can be from immunoglobulins of different subtypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtypes), IgAl, IgA2, IgD, IgE, or IgM. In general, an Fc region includes the hinge region or part of the hinge region of the constant region, the CH2 region and the CH3 region.

In some embodiments, mutations can be introduced in the wild-type Fc sequence to alter the relevant Fc-mediated activity. The mutations include, but are not limited to:

a) Mutations that alter (eg reduce) Fc-mediated CDC activity;

b) mutations that alter (eg reduce) Fc-mediated ADCC activity; or

c) Mutations that alter (eg increase) FcRn-mediated half-life in vivo.

Such mutations are described in: Leonard G Presta, Current Opinion in Immunology 2008, 20: 460-470; Esohe E. Idusogie et al. J Immunol 2000, 164: 4178-4184; RAPHAEL A. CLYNES et al. Nature Medicine, 2000, Volume 6, Number 4:443-446; Paul R. Hinton et al. J Immunol, 2006, 176:346-356. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids on the CH2 region can be used to increase or remove Fc-mediated ADCC or CDC activity or to enhance or attenuate FcRn Affinity. Additionally, protein stability can be increased by mutating 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the hinge region.

In some embodiments, mutations can be introduced in the Fc sequence to make the mutated Fc more likely to form homodimers or heterodimers. As mentioned in Ridgway, Presta et al. 1996 and Carter 2001, the knob-hole model using the steric interaction of amino acid side chain groups at the Fc contact interface makes it easier to form heterodimers between different Fc mutations; The charge of the amino acids at the Fc contact interface changes the ionic interaction force between the Fc contact interfaces, making it easier for different Fc mutation pairs to form heterodimers (see CN 102558355A), or Fc with the same mutation It is easier to form homodimers between them (see CN 103388013A).

The immunoglobulin Fc region is preferably a human immunoglobulin Fc region, eg, the Fc region of human IgGl Fc, human IgG4, human IgG4 (S228P). In some specific embodiments, the amino acid sequence of the immunoglobulin Fc region is as set forth in SEQ ID NOs: 103, 108 or has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity.

In some embodiments, in the PD-1 binding proteins of the present disclosure, the immunoglobulin single variable domain and the immunoglobulin Fc region are linked by a linker. The linker may be a non-functional amino acid sequence of 1-20 or more amino acids in length, and the linker itself does not form secondary or tertiary structure. For example, the linker is a flexible linker such as G4S, GS, GAP, (G4S) ₂ , ( _G4S ) ₃ , ( _{G4S )4 , (G4S)5 , ASGS} , etc. .

In some embodiments, the PD-1 binding proteins of the present disclosure comprise an immunoglobulin single variable domain linked directly or through a linker to the immunoglobulin Fc region. In some specific embodiments, the PD-1 binding proteins of the present disclosure comprise two immunoglobulin single variable domains linked directly or through a linker to an immunoglobulin Fc region that allows the PD-1 binding proteins form dimeric molecules comprising two immunoglobulin single variable domains. Such PD-1 binding proteins are also referred to as bivalent PD-1 binding proteins.

In some embodiments, the PD-1 binding proteins of the present disclosure comprise three or four immunoglobulin single variable domains and one immunoglobulin Fc region, directly or interconnected by a linker, the immunoglobulin Fc region The PD-1 binding protein is allowed to form a multimeric molecule comprising three or four immunoglobulin single variable domains. Such PD-1 binding proteins are also referred to as trivalent or tetravalent PD-1 binding proteins.

In other embodiments, the PD-1 binding protein comprises at least one PD-1 binding domain and at least one other antigen binding domain, eg, forms a heterodimer.

In some embodiments, the PD-1 binding protein comprising an immunoglobulin Fc region of the present disclosure comprises the amino acid sequence shown in any of SEQ ID NOs: 34, 104-107, 109-112, 200-203, or the same as SEQ ID NO. NO: 34, 104-107, 109-112, 200-203 any of at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity.

In some embodiments, the present disclosure provides PD-1 binding proteins capable of binding the same PD as a VHH consisting of the amino acid sequence of any of SEQ ID NOs: 154-157, 7-33, 35-58, 123-128 -1 epitope, or compete for binding to the same PD-1 epitope.

The PD-1 binding proteins of the present disclosure have at least one of the following characteristics:

(a) binds to human PD-1 or its epitope with a KD value of ≤10 ^-7 ;

(b) inhibiting the binding of PD-1 to PD-L1;

(c) inhibiting the binding of PD-1 to PD-L2;

(d) Inducing CD4+ T cells to secrete IFN-γ;

(e) enhance the activation of PBMC;

(f) enhancing the activation of T cells;

(g) Inhibition of tumor growth.

The PD-1 binding protein of the present disclosure may have a KD value of ≤ 1×10 ^-7 M for binding to PD-1, such as ≤ 1×10 ^-8 M, or ≤ 1×10 ^-9 M, or ≤ 1×10 ^{- 10M} .

In some embodiments, the PD-1 binding proteins of the present disclosure are capable of specifically binding human PD-1 and blocking the interaction of PD-1 and PD-L1, and/or the interaction of PD-1 and PD-L2.

The PD-1 binding proteins of the present disclosure are capable of inhibiting tumor growth by at least about 10%, eg, at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%.

In addition, the PD-1 binding protein of the present disclosure is resistant to heat treatment or has high stability. For example, treatment at 40°C for up to 30 days shows no significant aggregation or degradation and is stable at least at 60°C.

PD-1/LAG-3 binding protein

The present disclosure provides a PD-1/LAG-3 binding protein comprising a first antigen-binding domain that specifically binds PD-1 and a second antigen-binding domain that specifically binds LAG-3, the specificity The first antigen binding domain that binds PD-1 comprises at least one immunoglobulin single variable domain comprising three complementarity determining regions CDR1, CDR2 and CDR3, wherein:

CDR1 comprises the amino acid sequence shown as X ₂₂ KCMG (SEQ ID NO: 152), wherein X ₂₂ is selected from N, D, E, F, G, H, I, K, L, M, P, Q, R or S, CDR2 comprises the amino acid sequence shown in VVDRFGGTIYAX ₂₅ SVKG (SEQ ID NO: 204), CDR3 comprises the amino acid sequence shown in GSYTSAX ₂₃ SCQPDAL (SEQ ID NO: 153), wherein X ₂₅ is selected from A or D , X ₂₃ is selected from N, A, E, F, G, H, K, P, Q, R or S; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 62, CDR2 comprises the amino acid sequence shown in X ₁ IDSVGX ₂ TX ₃ YX ₄ X ₅ SVKG (SEQ ID NO: 115), wherein X ₁ is selected from S or T, X ₂ is selected from From T or A, X ₃ is selected from D, N or G, X ₄ is selected from T or A, X ₅ is selected from N or D, and CDR 3 comprises the amino acid sequence shown in SEQ ID NO: 64; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 81, CDR2 comprises the amino acid sequence shown in VVDRX ₂₄ GGX ₆ IYAX ₇ SVKX ₈ (SEQ ID NO: 116), wherein X ₂₄ is selected from Y or F, and X ₆ is selected from I or T, X ₇ is selected from A or D, X ₈ is selected from K or D, CDR3 comprises the amino acid sequence shown in GSYTX ₉ X ₁₀ X ₁₁ SCX ₁₂ PDAL (SEQ ID NO: 117), wherein X ₉ is selected from S or D , X ₁₀ is selected from A or D, X ₁₁ is selected from N or G, X ₁₂ is selected from Q or H; or

CDR1 comprises the amino acid sequence shown in YNX ₁₃ MX ₁₄ (SEQ ID NO: 118), wherein X ₁₃ is selected from F or Y, X ₁₄ is selected from S or T, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 66, and CDR3 comprises The amino acid sequence shown in SEQ ID NO: 67; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 84, CDR2 comprises the amino acid sequence shown in VINTGX ₁₅ NX ₁₆ TYYADSVKG (SEQ ID NO: 119), wherein X ₁₅ is selected from A or T, X ₁₆ is selected from S or T, CDR3 comprising the amino acid sequence shown in SEQ ID NO: 86; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 78, CDR2 comprises the amino acid sequence shown in X ₁₇ YPTAGX ₁₈ TYX ₁₉ X ₂₀ DSX ₂₁ KG (SEQ ID NO: 120), wherein X ₁₇ is selected from L or I, and X ₁₈ is selected from From R or K, X ₁₉ is selected from Y or F, X ₂₀ is selected from G or A, X ₂₁ is selected from M or V, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 80; or

CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 59, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 60, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 61; or

CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 74, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 75, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 76; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 88, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 89, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 90; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 96, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 97, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 98.

The PD-1/LAG-3 binding protein in some embodiments, wherein the immunoglobulin single variable domain in the first antigen binding domain that specifically binds PD-1 comprises the following CDR1 , CDR2 and CDR3:

CDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 129-141, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 83; or

CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 81, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence set forth in any of SEQ ID NOs: 142-151; or

CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 129, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 145; or

CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 132, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 145; or

CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 129, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 146; or

CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 132, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 146; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 62, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 63, 68, 69, 70, 72, 77, CDR3 comprises the amino acid sequence shown in SEQ ID NO: 64;

CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 62, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 63, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 73; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 81, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 71 and 82, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 83; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 81, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 91 and 93, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 92; or

CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 81, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 94, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 95; or

CDR1 comprises the amino acid sequence shown in any of SEQ ID NO: 65, 113, 114, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 66, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 67; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 84, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 85 and 102, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 86; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 78, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 79, 87, 99, 100, 101, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 80.

The PD-1/LAG-3 binding protein in some embodiments, wherein the immunoglobulin single variable domain in the first antigen binding domain that specifically binds PD-1 comprises as SEQ ID NOs: 154-157, 7 - An amino acid sequence represented by or at least 90%, at least 95%, at least 98%, at least 99% identical in sequence to any of 33, 35-58, 123-128.

The PD-1/LAG-3 binding protein in some embodiments, wherein the second antigen binding domain that specifically binds LAG-3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

or

VH comprises HCDR1, HCDR2, HCDR3 shown in SEQ ID NOs: 158-160, respectively, and VL comprises LCDR1, LCDR2, LCDR3 shown in SEQ ID NOs: 161-163, respectively.

The PD-1/LAG-3 binding protein in some embodiments, wherein the VH that specifically binds to the second antigen-binding domain of LAG-3 comprises or has at least 90 as set forth in any of SEQ ID NOs: 178-181 %, at least 95%, at least 98%, at least 99% amino acid sequence identity, VL comprising or having at least 90%, at least 95%, at least 98%, as shown in any of SEQ ID NOs: 182-186, amino acid sequences of at least 99% sequence identity; or

VH comprises an amino acid sequence as set forth in or at least 90%, at least 95%, at least 98%, at least 99% identical in sequence to any of SEQ ID NOs: 170-173 and VL comprises as SEQ ID NOs: 174-177 any amino acid sequence shown or having at least 90%, at least 95%, at least 98%, at least 99% sequence identity to it;

In some embodiments, VH comprises or is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 178 and VL comprises or is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 183.

The PD-1/LAG-3 binding protein in some embodiments, wherein the second antigen-binding domain that specifically binds LAG-3 comprises a full-length heavy chain (HC) and a full-length light chain (LC); e.g. , the full-length heavy chain is of the IgG1 or IgG4 isotype, and the full-length light chain is of the Kappa isotype; for example, the full-length heavy chain is shown in SEQ ID NO: 187 or has at least 90% sequence identity therewith, the full-length The light chain is set forth in SEQ ID NO: 188 or has at least 90% sequence identity therewith.

The PD-1/LAG-3 binding protein of some embodiments, wherein the second antigen binding domain that specifically binds LAG-3 comprises a heavy chain variable region (VH) and a light chain variable region (VL) ,in:

The immunoglobulin single variable domain that specifically binds the first antigen-binding domain of PD-1 is located in the heavy chain variable region or full-length heavy chain of the second antigen-binding domain that specifically binds LAG-3. N-terminal;

The immunoglobulin single variable domain that specifically binds the first antigen-binding domain of PD-1 is located in the heavy chain variable region or full-length heavy chain of the second antigen-binding domain that specifically binds LAG-3. C terminal;

The immunoglobulin single variable domain that specifically binds the first antigen-binding domain of PD-1 is located in the light chain variable region or full-length light chain of the second antigen-binding domain that specifically binds LAG-3. N-terminal; and/or

The immunoglobulin single variable domain that specifically binds the first antigen-binding domain of PD-1 is located in the light chain variable region or full-length light chain of the second antigen-binding domain that specifically binds LAG-3. C-terminal.

The PD-1/LAG-3 binding protein of some embodiments, wherein the immunoglobulin single variable domain that specifically binds the first antigen-binding domain of PD-1 and the second antigen that specifically binds LAG-3 The binding domains are connected directly or through a linker; for example, the linker has an amino acid sequence as shown in ( _G4S ) _x , wherein x is independently selected from an integer from 1-20; for example, the linker is Subsequences are the amino acid sequences represented by (G ₄ S) ₂ and (G ₄ S) ₃ .

A PD-1/LAG-3 binding protein in some embodiments comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising the amino acid sequence shown in any of SEQ ID NOs: 189-195 or an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99% sequence identity therewith, the second polypeptide chain comprising or at least 90% with the amino acid sequence shown in SEQ ID NO: 188 , amino acid sequences of at least 95%, at least 98%, at least 99% sequence identity.

The PD-1/LAG-3 binding protein in some embodiments has an activity selected from at least one of the following:

(a) binds to human _PD -1 or its epitope with a KD value of ≤10 ^-7 ;

(b) binds to human LAG-3 or its epitope with a _KD value of ≤10 ^-7 ;

(c) inhibiting the binding of PD-1 to PD-L1;

(d) inhibiting the binding of PD-1 to PD-L2;

(e) inhibiting the binding of LAG-3 to MHCII;

(f) inducing lymphocytes to secrete IFN-γ and/or IL-2;

(g) enhance the activation of PBMC;

(h) enhancing T cell activation, stimulating T cell responses, or stimulating T cell proliferation;

(i) Inhibit tumor growth and delay cancer development.

In some embodiments, the aforementioned PD-1/LAG-3 binding protein is an anti-PD-1/LAG-3 bispecific antibody.

In some embodiments, the anti-PD-1/LAG-3 bispecific antibody comprises an immunoglobulin single variable domain in the first antigen binding domain that specifically binds to PD-1 provided by the present disclosure, and the present The disclosure provides a heavy chain variable region (VH) and a light chain variable region (VL) in a second antigen binding domain that specifically binds LAG-3.

In some embodiments, in the anti-PD-1/LAG-3 bispecific antibody:

The first antigen-binding domain that specifically binds to PD-1 is a first antibody, which is a VHH, having the aforementioned CDR1, CDR2, CDR3 in the PD-1-binding protein provided by the present disclosure; and

The second antigen binding domain that specifically binds LAG-3 is a second antibody, which includes a heavy chain (HC) and a light chain (LC).

In some embodiments, the second antibody is any anti-LAG-3 antibody. WO2004/078928, WO2010/019570 (disclosing antibodies 25F7 and 26H10), US2011/070238, WO2014/008218, WO2015/138920 (eg BAP050), WO2011/140180, WO2015/116539, WO2016/0867628 , WO2016/200782, WO2017/015560, WO2019210848A, WO2019149716A, WO2019210848A, and LAG-3 antibodies in WO2017219995A.

In some embodiments, the VHH is located at the N-terminus and/or C-terminus of the heavy or light chain of the second antibody as the primary antibody.

In some embodiments, the anti-PD-1/LAG-3 bispecific antibody comprises 1 secondary antibody and 2 primary antibodies; the secondary antibody comprises two HCs and two LCs, one of the secondary antibodies The VH of the HC forms the antigen-binding site with the VL of one LC, and the VH of the other HC forms the antigen-binding site with the VL of the other LC.

In some embodiments, one of the anti-PD-1/LAG-3 bispecific antibodies is located at the N-terminus of the heavy or light chain of the second antibody, and the other first antibody is located on the heavy chain or the light chain of the second antibody. C-terminus of the light chain.

In some embodiments, each primary antibody in the anti-PD-1/LAG-3 bispecific antibody is located at the N-terminus of both heavy chains or both light chains of the secondary antibody, respectively; alternatively, each primary antibody located at the C-terminus of the two heavy chains or the two light chains of the second antibody, respectively.

In some embodiments, each primary antibody in the anti-PD-1/LAG-3 bispecific antibody is located at the N-terminus of the two heavy chains of the secondary antibody; alternatively, each primary antibody is located at the secondary antibody The C-termini of the two heavy chains;

In some specific embodiments, the second antibody in the anti-PD-1/LAG-3 bispecific antibody may be linked with 1, 2, 3, 4, 5, 6, 7, or 8 first antibodies, the first antibodies It can be the same or different, and both can be connected to the N-terminus of the heavy chain of the second antibody, or both can be connected to the C-terminus of the heavy chain of the second antibody, or both can be connected to the N-terminus of the light chain of the second antibody, or both are connected At the C-terminus of the light chain of the second antibody, or any combination of the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain, and the C-terminus of the light chain.

In some embodiments, the primary antibody in the anti-PD-1/LAG-3 bispecific antibody is linked directly or through a linker to the N-terminus or C-terminus of each heavy chain of the secondary antibody. The linker is selected from: an amino acid sequence as shown in (G _{m Sn} ) _x or a polyguanine (poly G), wherein m, n are each independently selected from an integer of 1-8 (for example, 1, 2 , 3, 4, 5, 6, 7, or 8), x is independently selected from an integer from 1 to 20 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19 or 20). For example, the linker is an amino acid sequence represented by G ₄ S, (G ₄ S) ₂ , (G ₄ S) ₃ , (G ₄ S) ₄ , (G ₄ S) ₅ , and (G ₄ S) ₆ .

In some embodiments, the heavy chain of the secondary antibody in the anti-PD-1/LAG-3 bispecific antibody comprises a heavy chain variable region (VH) and a heavy chain constant region (CH), and the light chain comprises a light chain variable region (VL) and light chain constant region (CL). The second antibody can be a full-length antibody.

In some embodiments, in the anti-PD-1/LAG-3 bispecific antibody, the heavy chain of the secondary antibody is of the IgG isotype, eg, IgGl, IgG2, IgG3, or IgG4, eg, of the IgGl isotype; and/or , the light chain of the second antibody is of the Kappa isotype.

In some embodiments, in the anti-PD-1/LAG-3 bispecific antibody, the two HCs comprise the same CDRs and/or the two LCs comprise the same CDRs. In some embodiments, the two HCs of the second antibody comprise the same VH and/or the two LCs comprise the same VL. In some embodiments, the two HCs of the second antibody have the same amino acid sequence and/or the two LCs have the same amino acid sequence.

In some embodiments, in the anti-PD-1/LAG-3 bispecific antibody, the two primary antibodies have the same or different amino acid sequences. For example, two of the first antibodies have the same amino acid sequence.

In some embodiments, the anti-PD-1/LAG-3 bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, wherein for each polypeptide chain:

a) the first polypeptide chains each independently comprise the heavy chain (HC) of the first antibody and the second antibody; and b) the second polypeptide chains each independently comprise the light chain (LC) of the second antibody; wherein VHH Connected to the N-terminus and/or C-terminus of the HC of the secondary antibody through a linker; or,

i) the first polypeptide chains each independently comprise the heavy chain (HC) of the second antibody; and ii) the second polypeptide chains each independently comprise the light chain (LC) of the first antibody and the second antibody; wherein VHH Linked directly or through a linker to the N-terminus and/or C-terminus of the LC of the secondary antibody.

In some embodiments, the anti-PD-1/LAG-3 bispecific antibody comprises two identical first polypeptide chains and two identical second polypeptide chains.

In some embodiments, a PD-1 binding protein, PD-1/LAG-3 binding protein, anti-PD-1 antibody, anti-PD-1/LAG-3 bispecific antibody of the present disclosure is provided that competitively binds to the same epitope of antibodies.

In some embodiments, the PD-1 binding protein, LAG-3 binding protein, PD-1/LAG-3 binding protein, anti-PD-1/LAG-3 bispecific antibody of the present disclosure further comprises a human immunoglobulin Fc region ; for example, the Fc region is the Fc region of human IgGl, IgG2 or IgG4. The Fc region may have mutations. Exemplary mutations are:

- K214T, E233P, L234A, L234V, L234F, L235E, L235A, G236 deletion, G237A, P238S, D265A, H268A, A327G, A330S, P331A, P331S, L358M, D365E on IgG1;

- V234A, G237A, P238S, H268A, H268Q, V309L, A330S, P331S on IgG2;

- Deletion of S228P, F234A, L235A, G236, G237A, P238S on IgG4;

- N297A on IgG1, IgG2, IgG3 or IgG4.

In some embodiments, the mutation in the Fc region is selected from: L234A/L235A on IgG1, V234A/G237A/P238S/H268A/V309L/A330S/P331S on IgG2, F234A/L235A on IgG4, S228P on IgG4 or S228P/F234A/L235A, N297A on IgG1, IgG2, IgG3 or IgG4, V234A/G237A on IgG2, K214T/E233P/L234V/L235A/G236 deletion on IgG1/A327G/P331A/D365E/L358MV309L/A330S/P33 , L234F/L235E/D265A on IgG1, L234A/L235A/G237A/P238S/H268A/A330S/P331S on IgG1, S228P/F234A/L235A/G237A/P238S on IgG4, and S228P/F234A/L235A on IgG4 G236 deletion/G237A/P238S. Hybrid IgG2/4 Fc domains can also be used, eg, an Fc with residues 117-260 from IgG2 and residues 261-447 from IgG4.

In some specific embodiments, the Fc region of the human IgG4 has S228P, F234A, L235A and/or K447A mutations. In some specific embodiments, the Fc region of the human IgGl has the L234A/L235A or L234A/L235A/P329G mutation.

Composition of PD-1 Binding Protein and LAG-3 Binding Protein

The present disclosure provides a composition comprising a PD-1-binding protein and a LAG-3-binding protein, wherein the PD-1-binding protein comprises the aforementioned first antigen-binding domain that specifically binds to PD-1 provided by the present disclosure, the LAG-3-binding protein A second antigen-binding domain that specifically binds to LAG-3 provided by the foregoing disclosure is included.

In some embodiments, the PD-1 binding protein in the composition comprises at least one immunoglobulin single variable domain comprising three complementarity determining regions CDR1, CDR2 and CDR3, the CDR1, CDR2, CDR3 are shown as CDR1, CDR2, CDR3 in any sequence of SEQ ID NO: 154-157, 7-33, 35-58, 123-128, CDR is according to Kabat, IMGT, Chothia, AbM or Contact The numbering system is defined, in some embodiments, according to the Kabat numbering system.

In some embodiments, in the immunoglobulin single variable domain:

CDR1, CDR2, CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 152, 204, 153, respectively; or

CDR1, CDR2, and CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 62, 115, and 64; or

CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 81, 116 and 117; or

CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 118, 66 and 67; or

CDR1, CDR2, and CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 84, 119, and 86; or

CDR1, CDR2, and CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 78, 120, and 80; or

CDR1, CDR2, CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 59-61; or

CDR1, CDR2, and CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 74-76; or

CDR1, CDR2, and CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 88-90; or

CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 96-98.

In some embodiments, CDR1 comprises the amino acid sequence shown in any of SEQ ID NOs: 129-141, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 83 amino acid sequence; or

CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 81, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence set forth in any of SEQ ID NOs: 142-151; or

CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 129, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 145; or

CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 132, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 145; or

CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 129, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 146; or

CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 132, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 146; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 62, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 63, 68, 69, 70, 72, 77, CDR3 comprises the amino acid sequence shown in SEQ ID NO: 64;

CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 62, 63 and 73; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 81, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 71 and 82, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 83; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 81, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 91 and 93, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 92; or

CDR1, CDR2, and CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 81, 94, and 95; or

CDR1 comprises the amino acid sequence shown in any of SEQ ID NO: 65, 113, 114, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 66, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 67; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 84, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 85 and 102, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 86; or

CDR1 comprises the amino acid sequence shown in SEQ ID NO: 78, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 79, 87, 99, 100, 101, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 80.

In some embodiments, the PD-1 binding protein comprises or has at least 90%, at least 95%, at least 98% as set forth in any of SEQ ID NOs: 154-157, 7-33, 35-58, 123-128 , an amino acid sequence of at least 99% sequence identity.

In some embodiments, the PD-1 binding protein is the amino acid sequence set forth in SEQ ID NOs: 200-203.

In some embodiments, the immunoglobulin single variable domain in the PD-1 binding protein is a VHH, eg, the VHH is a humanized and/or affinity matured VHH.

In some embodiments, the PD-1 binding protein is an antibody that specifically binds PD-1 or a fragment thereof; preferably, the antibody is a camelid antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. In some embodiments, the PD-1 binding protein further comprises a human immunoglobulin Fc region, eg, the Fc region of a human IgG1 or IgG4, eg, with mutations S228P, F234A, L235A, and/or K447A.

In some embodiments, the LAG-3 binding protein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: VH comprises HCDR1, HCDR2, HCDR3 as set forth in SEQ ID NOs: 164-166, VL comprises LCDR1, LCDR2, LCDR3 as shown in SEQ ID NOs: 167-169; or VH comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 158-160, VL comprises as SEQ ID NOs: 161-163 LCDR1, LCDR2, LCDR3 shown.

In some embodiments, the LAG-3 binding protein is a full-length antibody comprising a full-length heavy chain (HC) and a full-length light chain (LC), eg, of the IgGl or IgG4 isotype, a full-length light chain For example, the Kappa isoform.

In some specific embodiments, the LAG-3 binding protein comprises:

VH comprises an amino acid sequence as set forth in or at least 90%, at least 95%, at least 98%, at least 99% identical to any one of SEQ ID NOs: 178-181,

VL comprises an amino acid sequence as set forth in or at least 90%, at least 95%, at least 98%, at least 99% identical in sequence to any of SEQ ID NOs: 182-186; or

VH comprises an amino acid sequence as set forth in or at least 90%, at least 95%, at least 98%, at least 99% identical in sequence to any of SEQ ID NOs: 170-173,

VL comprises an amino acid sequence as set forth in or at least 90%, at least 95%, at least 98%, at least 99% identical in sequence to any of SEQ ID NOs: 174-177.

In some embodiments, VH comprises or is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 178, and VL comprises at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 183; In the scheme, VH and VL comprise the sequences shown in SEQ ID NOs: 178, 183, respectively.

In some specific embodiments, the full-length heavy chain of the LAG-3 binding protein is set forth in SEQ ID NO: 187 or has at least 80%, at least 90%, at least 95%, at least 98%, at least 99% sequence identity therewith , the full-length light chain is shown in SEQ ID NO: 188 or has at least 80%, at least 90%, at least 95%, at least 98%, at least 99% sequence identity therewith.

In some embodiments, for PD-1 binding proteins, LAG-3 binding proteins, PD-1/LAG-3 binding proteins, or anti-PD-1/LAG-3 bispecific antibodies defined in the present disclosure using sequence identity , the identity arises from conservative modifications or conservative substitutions to the amino acid sequence or the nucleotide sequence it encodes.

polynucleotide

The present disclosure provides polynucleotides encoding PD-1 binding proteins, LAG-3 binding proteins, PD-1/LAG-3 binding proteins, or anti-PD-1/LAG-3 bispecific antibodies of the present disclosure. The polynucleotides of the present disclosure can be RNA, DNA, or cDNA. According to some embodiments of the present disclosure, the nucleic acids of the present disclosure are substantially isolated nucleic acids.

The nucleic acids of the present disclosure may also be in the form of, be present in, and/or be part of a vector, such as a plasmid, cosmid, YAC, or viral vector. The vector may in particular be an expression vector, ie a vector that provides for expression of the PD-1 binding protein in vitro and/or in vivo (ie in a suitable host cell, host organism and/or expression system). The expression vector typically comprises at least one nucleic acid of the present disclosure operably linked to one or more suitable expression control elements (eg, promoters, enhancers, terminators, etc.). The selection of such elements and their sequences for expression in a particular host is within the general knowledge of those skilled in the art. Regulatory elements and other elements useful or necessary for the expression of the PD-1 binding proteins of the present disclosure are, for example, promoters, enhancers, terminators, integrons, selectable markers, leader sequences, reporter genes.

The nucleic acids of the present disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and/or recombinant DNA techniques) based on information on the amino acid sequences of the polypeptides of the present disclosure, and/or can be isolated from suitable natural sources.

host cell

The present disclosure provides expression of one or more PD-1 binding proteins, LAG-3 binding proteins, PD-1/LAG-3 binding proteins, anti-PD-1/LAG-3 bispecific antibodies of the present disclosure and/or containing Recombinant host cells of the polynucleotides or vectors of the present disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

Bacterial cells include, for example, gram-negative bacterial strains (eg, Escherichia coli, Proteus, and Pseudomonas strains) and gram-positive bacterial strains (eg, Bacillus (Bacillus), Streptomyces (Streptomyces), Staphylococcus (Staphylococcus) and Lactococcus (Lactococcus) cells).

Fungal cells include, for example, cells of species of Trichoderma, Neurospora, and Aspergillus; or Saccharomyces (eg, Saccharomyces cerevisiae), Schizosaccharomyces cerevisiae Genus Schizosaccharomyces (eg Schizosaccharomyces pombe), Pichia (eg Pichia pastoris and Pichia methanolica) and Hansen A cell of a species of the genus Hansenula.

Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

However, amphibian cells, insect cells, plant cells, and any other cell in the art for expressing heterologous proteins may also be used in the present disclosure.

Cells of the present disclosure cannot develop into completed plants or animals.

production or preparation method

The present disclosure provides methods of making the PD-1 binding proteins, LAG-3 binding proteins, PD-1/LAG-3 binding proteins, or anti-PD-1/LAG-3 bispecific antibodies of the present disclosure, the methods generally comprising The following steps:

- culturing the disclosed PD-1 binding protein, LAG-3 binding protein, PD-1/LAG-3 binding protein, or anti-PD-1/LAG-3 bispecific antibody under conditions that allow expression of the disclosed PD-1 binding protein, LAG-3 binding protein, PD-1/LAG-3 binding protein, or anti-PD-1/LAG-3 bispecific antibody host cells; and

- recovering from the culture the protein of interest expressed by the host cell; and

- Optionally, including further purification and/or modification of the protein of interest of the present disclosure.

The PD-1 binding proteins, LAG-3 binding proteins, PD-1/LAG-3 binding proteins, anti-PD-1/LAG-3 bispecific antibodies of the present disclosure can be administered intracellularly in cells as described above (eg, produced in the cytoplasm, in the periplasm, or in inclusion bodies) followed by isolation from the host cell and optionally further purification; or it can be produced extracellularly (eg, in the medium in which the host cell is cultured), followed by isolation from the medium and Optional further purification.

Methods and reagents for the recombinant production of polypeptides, such as specific suitable expression vectors, transformation or transfection methods, selectable markers, methods of inducing protein expression, culture conditions, etc., are known in the art. Similarly, protein isolation and purification techniques suitable for use in the methods of making the proteins of the present disclosure are well known to those of skill in the art. As an example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can stably transfect CHO cells. Mammalian-like expression systems result in glycosylation of the antibody, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones were expanded in serum-free medium in bioreactors for antibody production. The antibody-secreted culture medium can be purified and collected by conventional techniques. Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The obtained product should be frozen immediately, eg -70°C, or lyophilized.

However, the PD-1 binding proteins, LAG-3 binding proteins, PD-1/LAG-3 binding proteins, anti-PD-1/LAG-3 bispecific antibodies of the present disclosure can also be produced by other proteins known in the art obtained by methods such as chemical synthesis, including solid-phase or liquid-phase synthesis.

pharmaceutical composition

The present disclosure provides pharmaceutical compositions comprising a prophylactically or therapeutically effective amount of any one or a combination of the following: PD-1 binding protein, LAG-3 binding protein, PD-1/LAG of the present disclosure as described above -3 binding protein, anti-PD-1/LAG-3 bispecific antibody, polynucleotide encoding the above protein or antibody, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients .

In some specific embodiments, the pharmaceutical composition unit dose may contain 0.01 to 99% by weight of PD-1 binding protein, PD-1/LAG-3 binding protein or anti-PD-1/LAG-3 bispecific antibody . In other specific embodiments, the amount of PD-1 binding protein, PD-1/LAG-3 binding protein or anti-PD-1/LAG-3 bispecific antibody contained in a unit dose of the pharmaceutical composition is 0.1-2000 mg; some In a specific embodiment, it is 1-1000 mg.

Kit (or kit)

The present disclosure provides kits or kits comprising one or more containers each independently comprising any one or a combination selected from the group consisting of PD-1 binding proteins, LAG-3 binding proteins, PD-1 binding proteins of the present disclosure /LAG-3 binding protein, anti-PD-1/LAG-3 bispecific antibody, polynucleotide encoding the above protein or antibody.

In some embodiments, diagnostic reagents comprising the above-described polynucleotides are also provided, as well as related diagnostic uses.

Methods and pharmaceutical uses for the prevention and treatment of diseases

The present disclosure provides the PD-1/LAG-3 binding protein, anti-PD-1/LAG-3 bispecific antibody, PD-1 binding protein and LAG-3 binding protein compositions, polynucleotides, medicaments of the present disclosure Compositions for use and methods in the prevention and/or treatment of diseases, which may or may not be related to the PD-1 signaling pathway.

In some embodiments, the present disclosure provides a method of preventing and/or treating a disease associated with PD-1, the method comprising administering to a subject a prophylactically and/or therapeutically effective amount of a PD-1 binding protein of the present disclosure , or a pharmaceutical composition comprising the PD-1 binding protein of the present disclosure. And, the use of the PD-1-binding protein of the present disclosure in the preparation of a medicament for preventing and/or PD-1-related diseases is also provided.

The PD-1/LAG-3 binding protein, anti-PD-1/LAG-3 bispecific antibody, PD-1 binding protein and LAG-3 binding protein compositions, polynucleotides, pharmaceutical compositions of the present disclosure can be individually used, or in combination with other anti-tumor treatments (eg, in combination with other immunogenic agents, standard cancer therapies, or other antibody molecules), to inhibit the growth of cancerous tumors.

In some embodiments, the present disclosure provides a method of promoting T cell proliferation, in other embodiments, the present disclosure provides a method of benefiting a patient or subject from up-regulation of an immune response, in other embodiments, providing A method for promoting the expression of cytokines (such as INFγ, IL-2) in a subject or a patient, the methods all comprising administering to the patient or the subject a prophylactically and/or therapeutically effective amount of PD-1/2 of the present disclosure LAG-3 binding protein, anti-PD-1/LAG-3 bispecific antibody, PD-1 binding protein and LAG-3 binding protein composition, polynucleotide, pharmaceutical composition.

In some embodiments, the present disclosure provides a method of preventing and/or treating cancer, comprising administering to a patient or subject a prophylactically and/or therapeutically effective amount of a PD-1/LAG-3 binding protein, anti-PD -1/LAG-3 bispecific antibody, PD-1 binding protein and LAG-3 binding protein compositions, polynucleotides, pharmaceutical compositions inhibit tumor cell growth in a patient or subject. In some embodiments, the cancer is preferably, but not limited to, a cancer responsive to immunotherapy.

In the above methods, non-limiting examples of cancer include lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma (eg, metastatic malignant melanoma), kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancies disease, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, endometrial tumor and osteosarcoma. Examples of other cancers that can be treated with the methods of the present disclosure include: bone cancer, pancreatic cancer, skin cancer, prostate cancer, skin or intraocular malignant melanoma, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer Cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer , chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, Renal pelvis cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphomas, environmentally induced cancers, including asbestos-induced cancers, and combinations of such cancers. In some embodiments, the aforementioned cancer or tumor is metastatic.

In some embodiments, the present disclosure provides a method of PD-1 and/or LAG-3-related disorders and diseases, including autoimmune diseases, such as systemic lupus erythematosus (SLE), psoriasis, systemic scleroderma, autoimmune diabetes, etc., comprising administering an effective amount of the PD-1/LAG-3 binding protein, anti-PD-1/LAG-3 bispecific antibody, PD-1 binding protein and LAG- 3 Compositions of binding proteins, polynucleotides, and pharmaceutical compositions.

In addition, the present disclosure also provides a method of preventing and/or treating an infectious disease in a subject or patient, comprising administering to the subject or patient a PD-1 binding protein, PD-1/LAG- 3-binding protein, anti-PD-1/LAG-3 bispecific antibody, PD-1-binding protein and LAG-3-binding protein composition, polynucleotide, pharmaceutical composition, so that the infectious disease of the subject can be prevented and/or treatment. Similar to applications for tumors as described above, PD-1 binding proteins can be used alone or in combination with vaccines to stimulate immune responses to pathogens, toxins, and self-antigens. Examples of pathogens to which this method of treatment is particularly applicable include pathogens for which there is currently no effective vaccine, or pathogens for which conventional vaccines are not fully effective. These include but are not limited to HIV, Hepatitis Viruses (A, B, C), Influenza Virus, Herpes Virus, Giardia, Malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa.

Some examples of causative viruses of infectious diseases treatable by the methods of the present disclosure include HIV, hepatitis (A, B, C), herpes viruses (eg, VZV, HSV-1, HAV-6, HSV-II and CMV, Epstein-Barr virus) ), adenovirus, influenza virus, arbovirus, echo virus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus , vaccinia virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, polio virus, rabies virus, JC virus and arbovirus encephalitis virus.

Some examples of causative bacteria of infectious diseases treatable by the methods of the present disclosure include Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus and Neisseria Gonorrhoeae, Klebsiella Bacillus, Proteus, Ralstonia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Botox, Bacillus anthracis, Yersinia pestis, Leptospirosis, and Lyme disease bacteria.

Some examples of causative fungi of infectious diseases treatable by the methods of the present disclosure include Candida (C. albicans, C. krusei, C. glabrata, C. tropicalis, etc.), Cryptococcus neoformans, Aspergillus Genus (Aspergillus fumigatus, Aspergillus niger, etc.), Mucor (Mucor, Absidia, Rhizopus), Sporothrix schenckii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides spp. Plasma bacteria.

Some examples of causative parasites of infectious diseases that can be treated by the methods of the present disclosure include Entamoeba histolytica, pouch ciliates, Neglia flexneri, Acanthamoeba spp., Ramboja Parasites, Cryptosporidium species, Pneumocystis carinii, Plasmodium vivax, Babesia gondii, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, J. brasiliensis .

In some embodiments, the use of a combination of PD-1 binding protein and LAG-3 binding protein is provided for the preparation of a medicament for treating tumors, treating autoimmune diseases, treating infections, promoting T cell proliferation, enabling a test Or or patients benefit from up-regulation of immune response and/or promote the expression of cytokines (eg, INFγ, IL-2) in the subject or patient.

In some embodiments, the aforementioned PD-1 binding proteins are provided for use in combination with the aforementioned LAG-3 binding proteins for the treatment of autoimmune diseases, the treatment of infections, the promotion of T cell proliferation, the benefit of a subject or patient from up-regulation of an immune response And/or to promote the expression of cytokines (eg, INFγ, IL-2) in a subject or patient, the PD-1 binding protein and the LAG-3 binding protein are administered simultaneously or sequentially.

In some embodiments, the aforementioned LAG-3 binding proteins are provided for use in combination with the aforementioned PD-1 binding proteins for the treatment of autoimmune diseases, the treatment of infections, the promotion of T cell proliferation, the benefit of a subject or patient from up-regulation of an immune response And/or to promote the expression of cytokines (eg, INFγ, IL-2) in a subject or patient, the LAG-3 binding protein and the PD-1 binding protein are administered simultaneously or sequentially.

Description of drawings

Figure 1 is a graph showing the results of binding of PD-1 antibody to PD-1 on the cell line CHO-PD-1 that stably expresses high PD-1.

Figure 2 is a graph showing the results of blocking the binding of PD-L1 protein to PD-1 on the cell line CHO-PD-1 that stably expresses PD-1 highly by PD-1 antibody.

Figure 3 is a graph showing the immune activation results of PD-1 antibody releasing PD-1/PD-L1 blockade in vitro.

Figure 4 is a graph showing the results of in vitro activation of T cells and secretion of IFNγ by PD-1 antibodies numbered 7#, 32#, 32#_hu_3, 106#, and 107#.

FIG. 5 is a graph showing the results of in vitro activation of T cells and secretion of IFNγ by PD-1 single-domain antibodies numbered 32#_hu_3_hlgG4, 7#_hu_4_hlgG4, and 106#_hu_1_hlgG4.

6A to 6B are the results of inhibition of tumor growth of mouse M38 colon cancer by PD-1 antibody and mouse body weight.

Figure 7 shows the results of the binding of PD-1/LAG-3 double antibodies 2136#, 2138#, and 2140# to PD-1 on the surface of CHO-PD1 cells. The negative control is PBS, and the positive control is PD-1Ab646.

Figure 8 shows PD-1/LAG-3 double antibody 2140#, 2170#, 2171#, 2172#, 2173#, PD-1 antibody 106#_hu-1_hIgG4, 0076#_hIgG4 and PD-1 on the surface of CHO-PD1 cells Combined result graph.

Figure 9 shows the results of the binding of PD-1/LAG-3 double antibodies 2140#, 2170#, 2171#, 2172#, and 2173# to CHO-LAG-3 cells. The negative control is NC, and the positive control is LAG-3Ab303.

Figure 10 shows the results of PD-1/LAG-3 double antibody 2136#, 2138#, 2140# blocking the binding of PD-1 to PD-L1 on CHO-PD1 cells, using LAG-3Ab303 and PD-1Ab646 as controls .

Figure 11 shows that PD-1/LAG-3 double antibody 2140#, 2170#, 2171#, 2172#, 2173#, PD-1 antibody 106#_hu-1_hIgG4, 0076#_hIgG4 block PD-PD-1 on CHO-PD1 cells 1 Result plot of binding to PD-L1, using NC as a negative control.

Figure 12 is a graph showing the results of blocking the binding of LAG-3 to endogenously highly expressed MHCII on cell line A375 using PD-1/LAG-3 dual antibodies 2140#, 2170#, 2171#, 2172#, 2173#, using LAG -3Ab303 served as a control.

Figure 13 is a graph showing the immune activation results of PD-1/LAG-3 double antibody 2136#, 2138#, and 2140# all being able to relieve PD-1/PD-L1 blockade in vitro.

Figure 14 shows the results of immune activation that PD-1/LAG-3 double anti-2140#, 2170# and PD-1 antibodies 106#_hu-1_hIgG4, 0076#_hIgG4 can relieve PD-1/PD-L1 blockade in vitro .

Figures 15A to 15B show the results of PD-1/LAG-3 double antibody 2170#, RO7247669, PD-1 antibody 0076#_hlgg4, LAG-3Ab303, 0076#_hlgg4 combined with LAG-3Ab303 to activate PBMC to secrete IL-2 and IFNγ , using NC as a negative control.

Figure 16 shows that in the Staphylococcus aureus superantigen (SEB) stimulation experiment, PD-1/LAG-3 double antibody 2170#, RO7247669, PD-1 antibody 0076#_hIgG4, LAG-3Ab303, 0076#_hIgG4 combined with LAG-3Ab303 stimulated Result plot of IFNγ secretion by PBMC, using NC as a negative control.

Figure 17 shows the results of the combination of PD-1/LAG-3 double antibody 2170#, RO7247669, PD-1 antibody 0076#_hIgG4, LAG-3Ab303, 0076#_hIgG4 combined with LAG-3Ab303 in the tumor killing experiment of PBMC to promote PBMC to secrete IFNγ , using NC as a negative control.

Figures 18A to 18C show the results of inhibition of tumor growth by PD-1/LAG-3 double antibody 2136#, 2138#, and 2140# in a humanized mouse model, using PBS as a negative control. Figure 18A is tumor volume, Figure 18B is mouse body weight, and Figure 18C is human CD45 cell reconstitution level.

Figures 19A to 19B show the results of the inhibition of tumor growth by PD-1/LAG-3 double antibody 2136#, 2138#, and 2140# in a humanized mouse model, using PBS, PD-1Ab646, and LAG-3Ab303 as controls . Figure 19A is tumor volume and Figure 19B is mouse body weight.

Figures 20A to 20C show the results of anti-PD-1/LAG-3 2170#, PD-1 antibody 0076#_hlgg4, LAG-3Ab303, 0076#_hlgg4 combined with LAG-3Ab303 in inhibiting tumor growth in a humanized mouse model Figure, using NC as a negative control. Figure 20A is the tumor volume, Figure 20B is the mouse body weight, and Figure 20C is the reconstitution level of human CD45 cells.

Detailed ways

the term

For an easier understanding of the present disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined elsewhere in this disclosure, all other techniques and sciences used in this disclosure have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.

"Programmed death 1", "programmed cell death 1", "protein PD-1", "PD-1", "PDCD1" and "hPD-1" are used interchangeably and include variants of human PD-1 Isotypes, isotypes, interspecies homologs, and analogs that share at least one epitope with PD-1. The complete PD-1 sequence can be found from GenBank Accession No. U64863.

"Programmed death ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands of PD-1 (the other being PD-L2), which downregulates T cells when bound to PD-1 activation and cytokine secretion. "PD-L1" as used herein includes human PD-L1 (hPD-L1), variants, isoforms, and interspecies homologues of hPD-L1, and having at least one epitope in common with hPD-L1 analogs of . The complete hPD-L1 sequence can be found using GenBank accession number Q9NZQ7.

"LAG-3" refers to lymphocyte activation gene 3. The term "LAG-3" includes variants, isoforms, homologues, orthologs and paralogs. The term "human LAG-3" refers to the human sequence LAG-3, eg the complete amino acid sequence of human LAG-3 with Uniprot number: P18627. LAG-3 is also known in the art, eg CD223. The human LAG-3 sequence differs from the human LAG-3 of Uniprot No.: P18627 by having, for example, conservative mutations or mutations in non-conserved regions, and LAG-3 is substantially different from the human LAG-3 of Uniprot No.: P18627 the same biological function. For example, the biological function of human LAG-3 is to have an epitope in the extracellular domain of LAG-3 that is specifically bound by the antibodies of the present disclosure, or the biological function of human LAG-3 is to bind MHC class II molecules. The specific human LAG-3 sequence is generally at least 90% identical in amino acid sequence to human LAG-3 of Uniprot No.: P18627, and contains an amino acid sequence identified as human when compared to LAG-3 amino acid sequences of other species (e.g., murine) of amino acid residues. In certain instances, human LAG-3 may be at least 85% or even at least 95%, 96%, 97%, 98% or 99% identical in amino acid sequence to LAG-3 of Uniprot No.: P18627. In certain embodiments, the human LAG-3 sequence exhibits no more than 10 amino acid differences from the LAG-3 sequence of Uniprot No.: P18627. In certain embodiments, human LAG-3 may show no more than 5 or even no more than 4, 3, 2 or 1 amino acid difference from the LAG-3 sequence of Uniprot No.: P18627. Percent identity can be determined as described herein.

"Cytokines" are protein factors released by a population of cells that act as intercellular mediators on other cells, such as lymphokines, monokines, chemokines and traditional polypeptide hormones. Exemplary cytokines include: human IL-2, IFN-human, IL-6, TNF6, IL-17, and IL-5.

The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

"PD-1 binding protein" means any protein capable of specifically binding PD-1 or an epitope thereof, or any molecule comprising such a protein. PD-1 binding proteins may include antibodies against PD-1 as defined in the present disclosure, antigen-binding fragments thereof, or conjugates thereof. PD-1 binding proteins also encompass immunoglobulin superfamily antibodies (IgSF) or CDR grafting molecules. A "PD-1 binding protein" of the present disclosure may comprise at least one immunoglobulin single variable domain (eg, VHH) that binds PD-1. In some embodiments, a "PD-1 binding protein" may comprise 2, 3, 4, or more immunoglobulin single variable domains (eg, VHHs) that bind PD-1. In addition to binding to an immunoglobulin single variable domain comprising PD-1, the PD-1 binding proteins of the present disclosure may also contain linkers and/or moieties with effector functions, such as half-life extending moieties (eg, serum albumin-binding immunoglobulins) globulin single variable domains), and/or fusion partners (eg serum albumin) and/or conjugated polymers (eg PEG) and/or Fc regions. In some embodiments, "PD-1 binding proteins" of the present disclosure also encompass bi/multispecific antibodies comprising immunoglobulins that bind to different antigens (eg, a first antibody that binds a first antigen (eg, PD-1) and a second antibody that binds to a second antigen (eg, LAG-3, optionally, includes a third antibody that binds to a third antigen, and further optionally includes a fourth antibody that binds to a fourth antigen).

"LAG-3 binding protein" means any protein or any molecule comprising such a protein that is capable of specifically binding LAG-3 or an epitope thereof. LAG-3 binding proteins may include antibodies, antigen-binding fragments thereof, or conjugates thereof, as defined in the present disclosure, directed against LAG-3.

"PD-1/LAG-3 binding protein" means any protein or any molecule comprising said protein capable of specifically binding PD-1 or an epitope thereof and LAG-3 or an epitope thereof. PD-1/LAG-3 binding proteins may include antibodies, antigen-binding fragments thereof, or conjugates thereof, as defined in the present disclosure, against PD-1 and LAG-3.

"Binds to PD-1" means capable of interacting with PD-1 or a fragment or epitope thereof, which may be of human origin.

"Binds to LAG-3" means capable of interacting with LAG-3 or a fragment or epitope thereof, which may be of human origin.

"Antibody" or "immunoglobulin" broadly covers traditional antibodies (antibodies with a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds), as well as those with antigenic Binding active Fab, Fv, sFv, F(ab')2, linear antibody, single chain antibody, scFv, sdAb, sdFv, nanobody, peptibody, domain antibody (heavy chain (VH) antibody, light chain ( VL) antibodies) and multispecific antibodies (bispecific antibodies, diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv). Thus, "antibody" as used in this disclosure includes full-length antibodies, individual chains thereof, and any portion, domain or fragment thereof having antigen-binding activity, as well as individual chains thereof and any portion, structure comprising antigen-binding activity Domains or fragments of multispecific antibodies (including but not limited to antigen binding domains or fragments, eg, VHH domains or VH/VL domains, respectively). Traditional antibodies or immunoglobulins are usually tetrapeptide chains composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement sequence of the heavy chain constant region are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ, δ, and γ chains, respectively. , alpha chains, and epsilon chains. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into kappa chains or lambda chains by the difference in the constant region. Each of the five classes of Ig can have either a kappa (kappa) chain or a lambda (lambda) chain.

The "antibodies" of the present disclosure include, but are not limited to: (i) Fab fragments composed of VL, VH, CL and CH1 domains; (ii) Fd fragments composed of VH and CH1 domains; (iii) F(ab' )2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) a single-chain Fv molecule (scFv) in which the VH and VL domains are linked by a peptide linker that allows The two domains combine to form an antigen-binding site; (Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci U.S.A.)” 85:5879-5883) 242, herein incorporated by reference in its entirety); (iv) “diabodies” or “tribodies”, multivalent or multispecific fragments constructed by gene fusion (Tomlinson et al, 2000, Methods Enzymol. 326: 461-479; WO 94/13804; Holliger et al, 1993, Proceedings of the National Academy of Sciences 90: 6444-6448, all incorporated by reference in their entirety (v) "domain antibodies" or "dAbs" (sometimes referred to as "immunoglobulin single variable domains"), including immunoglobulin single variable domains from other species, such as rodents (eg as disclosed in WO00/29004), nurse shark and camelid VHH dAbs; (vi) SMIPs (small molecule immunopharmaceuticals), camelid antibodies, Nanobodies and IgNARs; (vii) (i)-(vi) above humanized antibodies.

The antibodies of the present disclosure may be polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic, or modified forms thereof, with monoclonal antibodies being particularly useful in various embodiments. Generally, the antibodies of the present disclosure are recombinant antibodies. "Recombinant" as used herein generally refers to a product such as a cell or nucleic acid, protein or vector, meaning that the cell, nucleic acid, protein or vector has been modified by introducing a heterologous nucleic acid or protein or altering a native nucleic acid or protein, or the Said cells are derived from cells so modified. For example, recombinant cells express genes that are not present in the native (non-recombinant) cellular form or express native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all.

A "domain" of a polypeptide or protein refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein, and in many cases can be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or the domain.

An "immunoglobulin domain" refers to a spherical region of an antibody chain (eg, a chain of a conventional tetrapeptide chain structure antibody or a chain of a heavy chain antibody), or to a polypeptide consisting essentially of such spherical regions. An immunoglobulin domain is characterized in that it maintains the immunoglobulin fold characteristics of an antibody molecule, e.g., in conventional tetrapeptide chain structure antibodies, it consists of two beta sheets linked by intrachain disulfide bonds of the heavy and light chains composed.

"Immunoglobulin variable domain" means consisting essentially of regions referred to in the art and hereinafter as "framework regions" and "CDR" regions, respectively, which contain "framework region 1" or "FR1", "framework region 2" or The four "framework regions" of "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", wherein said framework regions are defined by "complementarity determining region 1" or "CDR1", respectively ", "Complementarity Determining Region 2" or "CDR2", and "Complementarity Determining Region 3" or "CDR3" The three "complementarity determining regions" or "CDRs" are spaced apart. Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains confer antigen specificity by having an antigen binding site.

"Antibody framework (FR)" refers to a portion of a variable domain that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain.

For the determination or definition of "CDRs", definitive delineation of the CDRs and identification of residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and/or resolving the structure of the antibody-ligand complex. This can be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, Chothia numbering system, AbM numbering system, IMGT numbering system, contact definitions, conformational definitions. The Kabat numbering system is the standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, eg, Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8). The Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the positions of certain structural loop regions. (See eg, Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83). The AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group for modeling antibody structures (see, eg, Martin et al., 1989, ProcNatl Acad Sci (USA), 86:9268-9272; "AbMTM, A Computer Program for ModelingVariable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd). The AbM numbering system uses a combination of knowledge databases and de novo methods to model the tertiary structure of antibodies from basic sequences (see Samudrala et al., 1999, "Ab in PROTEINS, Structure, Function and Genetics Suppl., 3: 194-198" Initio Protein Structure Prediction Using a Combined Hierarchical Approach"). Contact definitions are based on analysis of complex crystal structures (see, eg, MacCallum et al., 1996, J. Mol. Biol., 5:732-45). In conformational definitions, the positions of the CDRs can be identified as residues that make enthalpy contributions to antigen binding (see, eg, Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166). Other CDR boundary definitions may not strictly follow one of the above approaches, but still overlap at least a portion of the Kabat CDRs, although they may be shortened or lengthened according to predictions or experimental results that specific residues or groups of residues do not significantly affect antigen binding. As used herein, a CDR can refer to a CDR defined by any method known in the art, including combinations of methods. The methods used herein may utilize CDRs defined according to any of these methods. For any given embodiment comprising more than one CDR, the CDR can be defined according to any of the Kabat, Chothia, Extended, AbM, IMGT, Contact and/or conformational definitions.

An "immunoglobulin single variable domain" is generally used to refer to a variable domain that can interact without interacting with other variable domains (eg, in the absence of a between the VH and VL domains as required for conventional four-chain monoclonal antibodies) In the case of VH/VL interactions), immunoglobulin variable domains (which may be heavy or light chain domains, including VH, VHH or VL domains) that form a functional antigen binding site. Examples of "immunoglobulin single variable domains" include Nanobodies (including VHHs, humanized VHHs and/or camelized VHs, eg camelized human VHs), IgNARs, domains, as VH domains or derived from VH Domain (single domain) antibodies (such as dAbs ^™ ) and (single domain) antibodies that are or are derived from VL domains (such as dAbs ^™ ). Immunoglobulin single variable domains based on and/or derived from heavy chain variable domains such as VH or VHH domains are generally preferred. A specific example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") as defined below.

"VHH domains", also known as heavy chain single domain antibodies, VHHs, VHH antibody fragments, VHH antibodies, Nanobodies, are antigen-binding immunoglobules known as "heavy chain antibodies" (ie, "light chain-deficient antibodies") Variable domains of proteins (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 ( 1993)). The term "VHH domain" is used to associate the variable domain with the heavy chain variable domains (which are referred to in this disclosure as "VH domains") and light chain variable domains present in conventional tetrapeptide chain structure antibodies. variable domains, which are referred to in this disclosure as "VL domains". The VHH domain specifically binds epitopes without the need for additional antigen binding domains (this is in contrast to the VH or VL domains in conventional tetrapeptide chain structure antibodies, in which case the epitope is recognized by the VL domain along with the VH domain) . VHH domains are small stable and efficient antigen recognition units formed from a single immunoglobulin domain. The terms "heavy chain single domain antibody", "VHH domain", "VHH", "VHH domain", "VHH antibody fragment", "VHH antibody" and "domain" ("Nanobody" are Ablynx N.V., Ghent , a trademark of Belgium) are used interchangeably. "VHH domain" includes, but is not limited to, natural antibodies produced by camelid animals, or antibodies produced by camelid animals and then humanized, or obtained by phage display technology.

As is known in the art for VH domains and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (ie one according to Kabat numbering or positions may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number 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 amino acid residues in the actual sequence. Other numbering systems or coding conventions include Chothia, IMGT, AbM. Unless otherwise specified, antibodies of the present disclosure generally use the Kabat numbering system. EU numbering in Kabat is also used for constant domains and/or Fc domains.

The total number of amino acid residues in a VHH domain will typically range from 110 to 120, often between 112 and 115. It should be noted, however, that smaller and longer sequences may also be suitable for the purposes described in this disclosure.

VHH domains (alone or as part of larger polypeptides) offer a number of significant advantages over the use of conventional VH and VL domains, scFvs or conventional antibody fragments (e.g. Fab- or F(ab')2-fragments):

- Only a single domain is required to bind antigen with high affinity and selectivity, so that neither the presence of two separate domains nor the need to ensure that the two domains are present in the appropriate spatial conformation and configuration (eg scFv typically requires using specially designed linkers);

- the VHH domain can be expressed by a single gene and does not require post-translational folding or modification;

- VHH domains can be easily engineered into multivalent and multispecific formats;

- The VHH domain is highly soluble and has no tendency to aggregate;

- VHH domains are highly stable to heat, pH, proteases and other denaturing agents or conditions, and thus can be prepared, stored or transported without the use of refrigeration equipment, resulting in cost, time and environmental savings;

- VHH domains are easy to prepare and relatively inexpensive, even at the scale required for production;

- The VHH domain is relatively small (about 15 kDa or 1/10 the size of conventional IgG) compared to conventional tetrapeptide chain structure antibodies or antigen-binding fragments thereof, and therefore compared to conventional tetrapeptide chain structure antibodies or antigen-binding fragments thereof , shows higher tissue penetration and can be administered at higher doses;

- The VHH domain can exhibit cavity-binding properties (especially due to its extended CDR3 loop compared to conventional VH domains), allowing access to targets and epitopes not accessible by conventional tetrapeptide chain structure antibodies or antigen-binding fragments thereof.

Methods for obtaining VHHs that bind specific antigens or epitopes have been previously disclosed in: R. van der Linden et al., Journal of Immunological Methods, 240(2000) 185-195; Li et al., J Biol Chem ., 287(2012) 13713-13721; Deffar et al., African Journal of Biotechnology Vol. 8(12), pp. 2645-2652, 17 June, 2009 and WO94/04678.

"Fc variant" or "variant Fc" means a protein comprising amino acid modifications in the Fc domain. The Fc variants of the present disclosure are defined in terms of the amino acid modifications that constitute them. Thus, for example, S228P or 228P is an Fc variant with a proline substitution at position 228 relative to the parent Fc polypeptide, wherein numbering is according to the EU index. The identity of the WT amino acid can be left unspecified, in which case the aforementioned variant is referred to as 228P.

Examples of "humanization" include those in which a VHH domain derived from the family Camelidae can be obtained by replacing the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in the VH domain of a human conventional tetrapeptide chain structure antibody "Humanization" of one or more amino acid residues in the VHH encompasses other modifications to the sequence, such as removal of potential post-translational modification sites, by one or more mutations that provide VHH-modifying properties. A humanized VHH domain can contain one or more fully human framework region sequences, and in some embodiments, can contain human framework region sequences of IGHV3. Yet another example of "humanization" includes the grafting of heterologous CDR sequences into human antibody variable region frameworks, ie antibodies produced in different types of human germline antibody framework sequences. The strong antibody variable antibody response induced by chimeric antibodies can be overcome because they carry a large number of heterologous protein components. Humanization methods such as protein surface amino acid resurfacing (resurfacing) and antibody humanization universal framework grafting (CDR Grafting to a universal framework), namely CDR "grafting" to other "scaffolds" (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Scaffolds and techniques suitable for such CDR transplantation are known in the art. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database, and in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. In addition, in order to avoid the decrease of the activity caused by the decrease of immunogenicity at the same time, the human antibody variable region framework sequence can be subjected to minimal back-mutation or back-mutation to maintain the activity.

An "affinity matured" PD-1 binding protein or PD-1 antibody has one or more changes in one or more CDRs that result in increased affinity for the antigen compared to its parent antibody. Affinity matured antibodies can be prepared, for example, by methods known in the art as described by Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813.; Shier et al., 1995, Gene 169:147-155; Yelton et al., 1995, Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310 -9; and Hawkins et al., 1992, J. MoI. Biol. 226(3): 889896; KS Johnson and RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press 1996.

Typically, the PD-1 binding proteins of the present disclosure will be present at preferably ^10-7 to ^10-10 moles/liter (M), more preferably ^10-8 to ^10-10 moles/liter as measured in Biacore or KinExA or Fortibio assays , even more preferably a dissociation constant (KD) of ^10-9 to ^10-10 or lower, and/or at least ^10-7 M, preferably at least ^10-8 M, more preferably at least ^10-9 M, more preferably at least An association constant (KA) of ^10-10 M binds the antigen to be bound (ie PD-1). Any _KD value greater than 10<" ⁴ >M is generally considered to be indicative of nonspecific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be assayed in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and/or competitive binding assays as described in this disclosure ( Examples are radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assays.

When "competing" is used in the context of antigen-binding proteins (eg, antigen-binding proteins or antibodies) that compete for the same epitope, it means competition between antigen-binding proteins, which is determined by the following assay: Binding of the antigen to be detected The protein (eg, antibody or immunologically functional fragment thereof) prevents or inhibits (eg, reduces) specific binding of the reference antigen binding protein (eg, ligand or reference antibody) to a common antigen (eg, PD-1 antigen or fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), Sandwich competition assay (see, eg, Stahli et al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (see, eg, Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid-phase 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); Solid Phase Direct Labeling with I-125 Label RIA (see, eg, Morel et al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (see, eg, Cheung, et al., 1990, Virology 176:546-552); and directly labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically the assay involves the use of purified antigen (either on a solid surface or on a cell surface) capable of binding with an unlabeled test antigen binding protein and a labeled reference antigen binding protein. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cells in the presence of the antigen binding protein to be tested. Typically, the antigen binding protein to be tested is present in excess. Antigen-binding proteins identified by competitive assays (competing antigen-binding proteins) include: antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein; Antigen-binding proteins that bind to epitopes that sterically prevent binding from occurring. Additional details regarding methods for determining competitive binding are provided in the Examples of the present disclosure. Typically when a competing antigen binding protein is present in excess, it will inhibit (eg decrease) by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70% -75% or 75% or more specific binding of the reference antigen binding protein to a common antigen. In certain instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

"Cross-reactivity" refers to the ability of the PD-1 binding proteins of the present disclosure to bind PD-1 or epitopes thereof from different species. For example, a single domain antibody or derived protein of the present disclosure that binds human PD-1 can also bind PD-1 of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays such as SPR and ELISA, or binding or functional interaction with cells that physiologically express PD-1. Methods to determine cross-reactivity include standard binding assays as described in the present disclosure, such as surface plasmon resonance (SPR) analysis, or flow cytometry.

"Inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition/blocking.

"Inhibition of growth" (eg, in relation to a cell) is intended to include any measurable reduction in cell growth.

"Epitope" or "antigenic determinant" used interchangeably refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopes typically contain chemically active surface groups of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. For example, epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which may be "linear" "Epitope" or "Conformational" Epitope. In a linear epitope, all points of interaction between a protein and an interacting molecule (eg, an antibody) exist linearly along the protein's primary amino acid sequence. In conformational epitopes, points of interaction exist across protein amino acid residues that are separated from each other. Epitopes of a given antigen can be identified using a number of epitope mapping techniques well known in the art (eg Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996), US4708871). Antibodies can be screened competitively for binding to the same epitope using routine techniques known to those of skill in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete for binding to the antigen (for high throughput screening methods see eg WO03/48731). Thus, antibodies and antigen-binding fragments thereof that compete with the antibody molecules of the present disclosure for binding to the same epitope on PD-1 can be obtained using conventional techniques known to those skilled in the art.

"Specific binding" and "selective binding" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, when recombinant human PD-1 or its epitope is used as the analyte and the antibody is used as the ligand, when measured by surface ^plasmon resonance (SPR) techniques in an instrument, the A small equilibrium dissociation constant (K _D ) binds to a predetermined antigen or its epitope, and its binding affinity to the predetermined antigen or its epitope is beyond its binding to the predetermined antigen (or its epitope) or to a closely related antigen The affinity of binding to non-specific antigens (such as BSA, etc.) is at least twice as high. "Antibody that recognizes an antigen" is used interchangeably herein with "antibody that specifically binds."

"Conservative modifications" or "conservative substitutions" apply to amino acid and nucleotide sequences. For a particular nucleotide sequence, conservative modifications or conservative substitutions refer to the mutual replacement of those nucleic acids encoding the same or substantially the same amino acid sequence, or, in the case of the nucleotides not encoding amino acid sequences, to substantially the same core nucleotide sequence. With regard to amino acid sequences, conservative modifications or conservative substitutions refer to the replacement of amino acids in a polypeptide by other amino acids with similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that frequent changes can be made And it has little or no effect on the function, activity or other biological properties of the polypeptide. Those skilled in the art are aware that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) MolecμLar Biology of the Gene, The Benjamin/Cummings Pub. Co., 224, (4th ed.).

The above conservative amino acid substitutions are well known in the art, for example conservative amino acid substitutions are preferably one amino acid within the following groups (i)-(v) replaced by another amino acid residue within the same group:

(i) smaller aliphatic non-polar or weakly polar residues: 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 non-polar 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 by Gly or Ser; Arg by Lys; Asn by Gln or His; Asp by Glu; Cys by Ser; Gln by Asn; Glu by Asp; Gly by Ala or Pro; His by Asn or Gln; Ile by Leu or Val; Leu by Ile or Val; Lys by Arg, Gln or Glu; Met by Leu, Tyr or Ile; Phe by Met, Leu or Tyr Substitution; Ser by Thr; Thr by Ser; Trp by Tyr; Tyr by Trp or Phe; Val by Ile or Leu.

"Amino acid mutation" includes amino acid substitutions, deletions, insertions, modifications, and any combination thereof, such that the final construct possesses desired properties, eg, increased stability, increased activity. Amino acid sequence deletions and insertions include amino- and/or carboxy-terminal deletions and amino acid insertions. Preferred amino acid mutations are amino acid substitutions. To alter, for example, the binding properties of an anti-PD-1 antibody, non-conservative amino acid substitutions, ie replacing one amino acid with another amino acid with different structural and/or chemical properties, can be made. Preferred amino acid substitutions include the replacement of hydrophobic amino acids with hydrophilic amino acids. Amino acid substitutions include substitutions from non-naturally occurring amino acids or from naturally occurring amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine) amino acid) replacement. Amino acid mutations can be generated using genetic or chemical methods known in the art, including methods such as site-directed mutagenesis, PCR, gene synthesis, chemical modification, and the like. Amino acid mutations can occur in the CDR, FR, or Fc regions of the antibody.

"Backmutation" refers to mutating the amino acid residues in the FR region of the human antibody to the amino acid residues in the corresponding positions of the original antibody, usually to avoid the decrease in immunogenicity caused by the humanized antibody and the decrease in activity. , the variable region of the humanized antibody can be minimally backmutated to maintain the activity of the antibody.

A "PD-1 binding protein" or "PD-1 antibody" of the present disclosure may comprise one or more effector molecules, eg, in a conjugated fashion. Such "effector molecules" include, for example, antineoplastic agents, drugs, toxins, biologically active proteins (eg enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof (eg DNA, RNA and fragments thereof) ), radionuclides, in particular radioiodides, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds detectable by NMR or ESR spectroscopic analysis. When the effector molecule is a polymer, it can generally be a synthetic or naturally occurring polymer, such as an optionally substituted linear or branched polyalkylene, polyalkenylene or polyoxyalkylene polymer or branched Polysaccharides or unbranched polysaccharides, such as homo- or hetero-polysaccharides. Specific optional substituents that may be present on the above-described synthetic polymers include one or more hydroxy, methyl, or methoxy groups. Specific examples of synthetic polymers include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, particularly optionally substituted poly(ethylene glycol) alcohol) such as methoxypoly(ethylene glycol) or derivatives thereof. Specific naturally occurring polymers include lactose, amylose, dextran, glycogen or derivatives thereof. The conjugation of the polymer to the PD-1 binding protein or PD-1 antibody can be achieved by conventional methods. In one embodiment, the polymer is albumin or a fragment thereof, eg, human serum albumin or a fragment thereof. In another embodiment, the PD-1 antibody of the present disclosure is pegylated (PEGylated) modified, eg, to enhance half-life. PEGs are linear or branched polyethers linked at one end to a hydroxyl group and have the following general structure: HO-( _{CH2CH2O ) n- CH2CH2 -} OH. To couple PEG to molecules (polypeptides, polysaccharides, polynucleotides, and small organic molecules), PEG can be activated by preparing derivatives of PEG with functional groups on some or both ends. A common approach to PEG conjugation of proteins is to activate PEG with functional groups suitable for reaction with lysine and N-terminal amino acid groups. In particular, a common reactive group involved in conjugation is the alpha or epsilon amino group of lysine. Reaction of the PEGylated linker with the protein can result in attachment of the PEG moiety primarily at the following sites: the alpha amino group at the N-terminus of the protein, the epsilon amino group on the side chain of a lysine residue, or a histidine residue imidazolyl on the side chain. Since most recombinant proteins have a single alpha and many epsilon amino and imidazole groups, many positional isomers can be generated depending on the chemistry of the linking group.

A "sequence" (eg, in the terms "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence", etc.) is generally understood to include both related amino acid sequences , which in turn includes the nucleic acid sequence or nucleotide sequence encoding the sequence, unless the disclosure requires a further defined interpretation.

"Polynucleotide" or "nucleic acid" refers to a chain of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or analogs thereof, or any substrate that can be incorporated into a chain by DNA or RNA polymerases. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and analogs thereof. Modifications to the nucleotide structure, if present, can be imparted before strand assembly or after strand assembly. Polynucleotides may also contain analogous forms of ribose or deoxyribose sugars generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'- Azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose, furanose, sedum heptulose ), acyclic analogs and abasic nucleoside analogs such as methyl riboside.

"Homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. Two DNA molecules are homologous when a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example if each position is occupied by an adenine, then the molecules are homologous at that position . The percent homology between the two sequences is a function of the number of matches or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, when sequences are optimally aligned, two sequences are 60% homologous if 6 of 10 positions in the two sequences are matched or homologous. In general, comparisons are made when the two sequences are aligned for the greatest percent homology.

"Nucleic acid molecule" is used interchangeably with "polynucleotide" and refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

"Vector" means a construct capable of delivering, and in some embodiments expressing, one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors bound to cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes , and certain eukaryotic cells such as producer cells.

A "host cell" includes each cell or cell culture that can be or has been a recipient of a vector for incorporation of a polynucleotide insert. A host cell includes the progeny of a single host cell, and the progeny may not necessarily be identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental or intentional mutation. Host cells include cells transfected and/or transformed in vivo with the polynucleotides of the present disclosure. "Cell," "cell line," and "cell culture" are used interchangeably and all such designations include their progeny; for example, including mutations that have the same function or biological activity as the parent cell screened in the original transformed cell offspring.

"Pharmaceutical composition" means a mixture containing one or more of the active ingredients described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, with other chemical components, as well as other components such as physiological/pharmaceutically acceptable Carriers and Excipients. The purpose of the pharmaceutical composition is to facilitate the administration to the organism, facilitate the absorption of the active ingredient and then exert the biological activity.

A "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate buffered saline, water, emulsions such as oil/water emulsions, and various types of wetting agents. In some embodiments, the diluent for aerosol or parenteral administration is phosphate buffered saline (PBS) or physiological (0.9%) saline. Compositions comprising such carriers are formulated by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and R Remington, The Science and Practice of Pharmacy 20th edition Mack Publishing, 2000).

"Cancer" and "cancerous" and "tumor" refer to or describe a physiological disorder in mammals that is usually characterized by unregulated cell growth. 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 carcinoma (eg, epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal carcinoma , liver cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, urinary tract cancer, breast cancer, colon cancer, rectal cancer, Colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma (superficial diffuse melanoma, lentigo maligna melanoma, acral melanoma, nodular melanoma), multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia , chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), and abnormalities associated with phakomatoses, edema (such as those associated with brain tumors), and Meigs' syndrome Vascular proliferation, brain tumors and brain cancer, and head and neck cancer, and associated metastases. In certain embodiments, cancers suitable for treatment by the PD-1 binding proteins of the present disclosure include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin's lymphoma tumor (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple Myeloma. In some embodiments, the cancer is selected from the group consisting of: non-small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer (eg, triple negative breast cancer), gastric cancer, colorectal cancer (CRC), and liver cell carcinoma. In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer (eg, triple negative breast cancer), including metastatic forms of those cancers.

A "proliferative disorder" refers to a disorder associated with some degree of abnormal cell proliferation. In one embodiment, the proliferative disorder refers to cancer.

"Tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. "Cancer," "cancerous," "proliferative disorder," and "tumor" are not mutually exclusive when referred to in this disclosure.

"Preventing cancer" means delaying, inhibiting, or preventing the onset of cancer in a subject in which the onset of cancer or the initiation of tumorigenesis has not been demonstrated, but has been identified, for example, by genetic screening or other methods cancer susceptibility. This also includes treating a subject with a precancerous condition to stop the progression of the precancerous condition to a malignancy or cause its regression.

"Administering," "administering," and "treating," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to exogenous drugs, therapeutic agents, diagnostic agents, or compositions that interact with the animal. , contact of humans, subjects, cells, tissues, organs or biological fluids, such as therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, wherein the fluids are in contact with cells. "Administering," "administering," and "treating" also mean in vitro and ex vivo treatment of, eg, cells by an agent, diagnostic, binding composition, or by another cell. When applied to human, veterinary or research subjects, it refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.

"Treatment" means administering an internal or external therapeutic agent, such as comprising any one of the antibodies of the present disclosure or a pharmaceutical composition thereof as a therapeutic agent, to a subject who has, is suspected of having, or is prone to have There are one or more proliferative diseases or symptoms thereof for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in a subject or population to be treated, whether by inducing regression of such symptoms or inhibiting the progression of such symptoms to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate symptoms of any particular disease (also referred to as a "therapeutically effective amount") may vary depending on a variety of factors, such as the subject's disease state, age, and weight, and the level of the drug that produces the desired effect in the subject. ability. Whether symptoms of a disease have been alleviated can be assessed by any clinical test commonly used by doctors or other health care professionals to assess the severity or progression of the symptoms. Although embodiments of the present disclosure (eg, methods of treatment or articles of manufacture) may be ineffective in alleviating symptoms of a target disease in a subject, the method of Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determine that it should reduce target disease symptoms in a statistically significant number of subjects.

An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or conditions of the medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a subject may vary depending on factors such as the condition being treated, the general health of the subject, the method, route and dosage of administration, and the severity of the side effect. An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects. Subjects of the present disclosure can be animal or human subjects.

"Optional" or "optionally" means that the subsequently described event or circumstance can, but need not, occur, and that the description includes instances where the event or circumstance occurs or instances where it does not.

"Subject", "patient" as used in the present disclosure means mammals, especially primates, especially humans.

The singular forms used in this disclosure also cover the plural forms unless otherwise indicated.

specific implementation

The following examples are used to further describe the present disclosure, but these examples do not limit the scope of the present disclosure. The experimental methods that do not specify specific conditions in the examples of the present disclosure generally follow conventional conditions, such as Cold Spring Harbor Antibody Technology Experiment Manual, Molecular Cloning Manual; or conditions suggested by raw material or commodity manufacturers. Reagents with no specific source indicated are conventional reagents purchased in the market.

Example 1: Preparation of PD-1 antigen and detection protein

PD-1 Antigen Design:

Using human PD-1 as the PD-1 template, the amino acid sequences of the PD-1 antigen and the detection protein were designed (the following PD-1 antigens without special description refer to human PD-1).

Human PD-1 full-length protein:

(Note: the double-dashed part is the signal peptide (Signal peptid); the underlined part is the PD-1 extracellular domain (Extracellular domain), of which positions 35-144 are Ig-like V-type 1Domain, and positions 70-77 are Interaction with CD274; the dashed line part is the transmembrane domain; the italic part is the intracellular domain (Cytoplasmic domain).)

SEQ ID NO: 1;

The full-length amino acid sequence of monkey PD-1:

(Note: the double-dashed part is the signal peptide; the dashed part is the extracellular region of PD-1, of which the 38-127 position is V-Set Domain, and the 39-125 position is Ig-like; the dot-dashed part is the transmembrane The region part (Transmembrane domain); the italicized part is the intracellular region (Cytoplasmic domain).)

SEQ ID NO: 2;

Human PD-1 antigen for screening and detection (as a commercial product (Sino Biological Cat.10377-H08H)):

(Note: The underlined part is the extracellular region of PD-1; the italicized part is the His-tag mark.)

SEQ ID NO: 3;

Human PD-1-Fc antigen for screening and detection (commercial product (Baiying Bio: B3789)):

(Note: The underlined part is the extracellular region; the italicized part is the human Fc marker.)

SEQ ID NO: 4;

Human PD-L1 antigen for detection (as a commercial product (Sino Biological cat : 10084-H08H-B)):

(Note: The underlined part is the extracellular region of PD-L1; the italicized part is the His-tag marker.)

SEQ ID NO: 5;

Human PD-L2 antigen for detection (as a commercial product (Sino Biological cat: 10292-H08H-B)):

(Note: The underlined part is the extracellular region of PD-L2; the italicized part is the His-tag marker.)

SEQ ID NO: 6.

Example 2. Screening of positive antibody sequences that specifically bind to human PD-1

Human PD-1 proteins (ACRO, Cat#PD-1-H5259 and ACRO, Cat#PD-1-H5221) were immunized with two Bactrian camels (Camelus dromedarius) respectively, and 5 mL of camel serum before immunization was taken and the serum was separated. After mixing Freund's complete adjuvant with antigen volume 1:1, the camels were subcutaneously immunized at multiple points (the immunization dose was 100 μg protein/head/each time, the first time was Freund's complete adjuvant, and the rest were Freund's adjuvant). incomplete adjuvant). Boosters were given every two weeks and titers were determined after four immunizations. Plates were coated with 5 μg/mL PD-1-his protein (100 μL/well) overnight at 4°C. The next day after washing, 4% nonfat dry milk was added for blocking, 37°C for 2h. After washing, different serial dilutions of camel serum were added and incubated at 37°C for 1 h. Negative controls were pre-immune serum (1:1000 dilution) and PBS solution. After incubation, wash with PBST three times, add HRP-anti-camel antibody (1:5000 dilution), and incubate at 37°C for 1 hour. Finally, wash and add alkaline phosphatase chromogenic solution, stop with 2M sulfuric acid, and read the absorption value at 450nm wavelength. 1: The titer was detected after 25600-fold dilution. If the titer is qualified, the peripheral blood of camel was collected for bank building.

Lymphocytes were isolated from camel peripheral blood, and the cell count was 1.2×10 ⁸ . Trizol reagent was added to resuspend (1×10 ⁷ cells/mL Trizol) to lyse the cells and placed on ice for 5 min; centrifuged at 13,000 rpm for 3 min, and the supernatant was collected and discarded. Precipitate; add 1/5 volume of chloroform, shake vigorously for 30-60s, let stand on ice for 2min; centrifuge at 13000rpm for 10min, suck the upper aqueous layer into a new 1.5mL tube; add an equal volume of isopropanol, mix well, -20℃ for 30min; centrifuge at 13000rpm for 10min, remove the supernatant, and keep the precipitate; add pre-cooled 75% ethanol to wash the precipitate, and leave it at room temperature for 5-10min; add 600μL of RNase-removed deionized water, reconstitute to obtain RNA, The cDNA was obtained by reverse transcription, and the phage library was constructed.

A single-domain antibody with high affinity to PD-1 antigen protein was obtained by screening the phage library, and 20 μg of PD-1-avi-biotin was combined with 1 mg of Dynabeads MyOne Streptavidin T1, and placed at 37°C for one hour Blocked with 2% skim milk at room temperature for 2 hours, added camel heavy chain single domain antibody phage display library, and acted at room temperature for 1 hour. Unbound phages were removed by washing 9 times with PBST (0.05% Tween-20) solution. Phages that specifically bind to PD-1 were eluted with 1 mg/mL trypsin and infected E. coli TG1 in log phase growth to produce and purify phages for the next round of screening. The same screening process was repeated after 2-3 rounds. Positive clones were enriched.

96 monoclonal colonies were picked from the screened and enriched positive clones and packaged into phage single chain antibodies for phage ELISA test. ELISA plates were coated with 2 μg/mL PD-1-his protein, and the phage supernatant diluted with blocking solution was added, and detected with anti-M13HRP. Clones with OD450 values greater than 0.5 in the ELISA binding test were sequenced, resulting in 51 specific sequences.

Example 3. Construction of intact monoclonal antibodies

The 51 specific sequences screened by the phage library in Example 2 were used to construct complete antibodies, and then 25 antibodies were determined to have strong binding ability and inhibit the interaction between PD-1 and PD-L1 through ELISA binding experiments and ELISA competition experiments. The results are shown in Table 1.

Table 1. ELISA test results of PD-1 antibody

Antibody number OD450 Antibody number OD450 Antibody number OD450 2# 1.71 56# 1.6633 109# 1.7868 4# 1.7036 59# 1.697 112# 1.6533 6# 1.8356 61# 1.7869 113# 1.4008

7# 1.7844 62# 1.7721 114# 1.5921 11# 1.5262 68# 1.6568 118# 1.5299 19# 1.6879 104# 1.765 122# 1.6316 32# 1.869 106# 1.7502 123# 1.5266 41# 1.4095 107# 1.659 Opdivo (positive control) 1.7387 54# 1.5173 108# 1.7068 PBS (negative control) 0.161

Its complete VHH sequence is as follows:

In the above sequence SEQ ID NO: 7-33, the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italic in the sequence is the FR sequence, and the underline is the CDR1, CDR2, CDR3 sequence respectively. The numbering rules of the PD-1 antibodies provided in the present disclosure are all Kabat, and the CDR sequences are summarized in Table 2.

Table 2. CDR sequences of PD-1 single domain antibodies

The antibody sequence was fused to a human IgG1-Fc (CH2-CH3) segment sequence and constructed into the PTT5 expression vector. The connected human IgG1-Fc sequence can be shown as follows:

The following is the entire protein sequence of the antibody sequence fused to the human Fc (CH2-CH3) segment, the single underline is the human IgG1-Fc (CH2-CH3) segment sequence (shown in SEQ ID NO: 103), and the double underline is the linker sequence. The protein sequence is as follows (take 32#, 7#, 106#, 107# as examples, the same is true for other PD-1 antibodies):

Example 4. Humanization of PD-1 antibody

Through three-dimensional structural homology modeling of selected specific PD-1 single-domain antibody molecules, combined with V-base human germline sequence database, IMGT human antibody heavy chain variable region germline gene database for alignment As a result, the heavy chain variable region germline gene with high homology to the screened antibody was selected as the template, and the CDR of the camel-derived single-domain antibody was transplanted into the corresponding human template to form the sequence of FR1-CDR1-FR2- Variable region sequences of CDR2-FR3-CDR3-FR4. The three-dimensional structure of the transplanted single-domain antibody was simulated and analyzed again, and the specific sites in the FR region that affected the structural shape of the CDR region were backmutated. The obtained humanized specific sequences are as follows:

As shown in the above sequence, in the process of humanization and back mutation, the CDRs of some antibodies changed. For example, 7#_Hu_5 has a T35S mutation, and the obtained CDR1 sequence shown in YNFMS (SEQ ID NO: 113); 7# _Hu_6 has mutations of F33Y and T35S, and the obtained CDR1 sequence shown in YNYMS (SEQ ID NO: 114); 106_hu_1 to 6 have the mutation of A61D, and the obtained CDR2 sequence shown in VVDRFGGTIYADSVKG (SEQ ID NO: 71); 112_hu_1 has Mutation of Y54F and A61D, the CDR2 sequence shown in VVDRFGGIIYADSVKG (SEQ ID NO: 93) was obtained.

Use the method in Example 4 to construct the complete protein sequence of the humanized PD-1 single domain antibody fused to the Fc (CH2-CH3) segment of hIgG1, and the single underline is the hIgG1-Fc (CH2-CH3) segment sequence (SEQ ID NO. : 103), the double underline is the linker sequence. The protein sequence is as follows (take 32_hu_3-IgG1 as an example, the same is true for other humanized PD-1 single domain antibodies):

Use the method in Example 4 to construct the complete protein sequence of humanized PD-1 single domain antibody fused to the Fc (CH2-CH3) segment of hIgG4, and the single underline is the hIgG4-Fc (CH2-CH3) segment sequence (SEQ ID NO. : 108).

The sequence of the linked human IgG4-Fc is shown below:

The obtained antibody sequences are as follows:

The plasmid was transfected into HEK293 cells. After 6 days, the expression supernatant was collected, centrifuged at high speed to remove impurities, and purified with Protein A column. Equilibrate with PBS until the A280 reading drops to baseline. The target protein was eluted with an acidic eluent at pH 3.0-3.5, and neutralized with 1M Tris-HCl, pH 8.0-9.0. After the eluted sample was properly concentrated, it was further purified by gel chromatography Superdex200 (GE) equilibrated with PBS to remove aggregates, and the monomer peaks were collected and separated for use. After detection, the PD-1 single domain antibody of the present disclosure is obtained.

Example 5. Glycosylation modification, expression and purification of PD-1 antibody

There are two N-glycosylation sites (NXC) in CDR1 ( N KCMG) and CDR3 (GSYTSA N SCQPDAL) in the 106#_hu_1_hIgG4 sequence, which are N ₃₁ KC and N ₁₀₄ SC, respectively. The specific method of 6) analysis shows that the ratio of glycosylation of NKC in CDR1 is 11%, and the ratio of glycosylation of NSC in CDR3 is 30%. Therefore, N ₃₁ is mutated with the following amino acids: N ₃₁ -D/ E/F/G/H/I/K/L/M/P/Q/R/S; N ₁₀₄ mutates the following amino acids: N ₁₀₄ -A/E/F/G/H/K/P/Q /R/S; SPR (Biacore T200) method (specific method in Example 7 below) was used to screen out sequences with little change in affinity, which were N ₃₁ -D/G and N ₁₀₄ -G/H, respectively.

106#_hu_1_hIgG4 (N ₃₁ -D, N ₁₀₄ -G) was named 0076#_hIgG4; 106#_hu_1_hIgG4 (N ₃₁ -G, N ₁₀₄ -G) was named 0077#_hIgG4; 106#_hu_1_hIgG4 (N ₃₁ -D, N ₁₀₄ -H) was designated as 0078#_hIgG4; 106#_hu_1_hIgG4 (N ₃₁ -G, N ₁₀₄ -H) was designated as 0079#_hIgG4. The sequences of CDR1 and CDR2 are summarized in Table 3.

Table 3. CDR1 and CDR2 sequences of glycosylated PD-1 antibody (the sequences of CDR2 are both VVDRFGGTIYADSVKG (SEQ ID NO: 71))

Accordingly, the present disclosure provides a PD-1 antibody whose CDR1 is X ₂₂ KCMG (SEQ ID NO: 152), wherein X ₂₂ is selected from N, D, E, F, G, H, I, K, L, M, P, Q, R or S; CDR2 is VVDRFGGTIYADSVKG (SEQ ID NO: 71); CDR3 is GSYTSAX ₂₃ SCQPDAL (SEQ ID NO: 153), wherein X ₂₃ is selected from N, A, E, F, G, H, K, P, Q, R or S.

An example of the antibody sequence obtained after glycosylation of 106#_hu_1 is as follows:

The constructed plasmid was transfected into Expi-CHO cells. After 9 days of culture, the expression supernatant was collected, centrifuged at high speed to remove impurities, and purified with a Protein A column. Equilibrate with PBS until the A280 reading drops to baseline. The target protein was eluted with an acidic eluent of pH 3.0-3.5, and neutralized with 1M Tris-HCl, pH 8.0-9.0. After the eluted sample was properly concentrated, it was further purified by gel chromatography Superdex200 (GE) equilibrated with PBS to remove aggregates, and the monomer peaks were collected and separated for use. After detection, the PD-1 antibody of the present disclosure is prepared.

Example 6. Mass spectrometry analysis and glycosylation analysis of PD-1 antibody

Instrument and equipment: American Agilent Q TOF 6530 mass spectrometer, equipped with Dual AJS ESI ion source and data analysis software Agilent MassHunter BioConfirm Software B.08.00, Agilent 1290Infinity high performance liquid chromatography system of American Agilent company.

Chromatographic conditions: Agilent AdvanceBio C18 (2.1x150mm, 2.7um) peptide mapping column; mobile phase: A phase is 100% H2O-0.1% FA, B phase is 100% ACN-0.1% FA; chromatographic elution Gradient 0-65min 3%-35%B; 65-68min 35%-95%B; 68-70min 95%B; 70-72min 95%-3%B; 72-75min 3%B; flow rate 0.4mL /min; column temperature 60°C; injection volume 20ul.

Mass spectrometry parameters: Mass spectrometry ion source is Dual AJS ESI electrospray ion source; ion spray voltage: 3.5KV; Gas temperature: 250℃; Sheath Gas temperature: 350℃; Sheath Gas Flow: 12l/min; Drying Gas: 10l/min; Nebulizer: 35psi; positive ion detection; mass range m/z 200-3000; acquisition rate 5 mass spectra per second; separation peak width: medium (about 4m/z).

Sample treatment: add a certain amount of guanidine hydrochloride to each sample, add reducing agent DTT to make the final concentration 20mmol/L, incubate at 60°C for 1h; add alkylating agent IAM to make the final concentration 40mmol/L, and react in the dark for 1h; then Dilute the samples respectively until the concentration of guanidine hydrochloride is below 2mol/L, add trypsin according to the mass ratio of protein:trypsin 25:1, and keep overnight at 37°C.

Data processing: The raw data collected in LC/MS/MS was processed using the data analysis software Agilent MassHunter BioConfirm Software B.08.00. Search results in mAb sequences, including alkylation (C), oxidation (M), deamidation (NQ), pyroglutamination (E) and glycosylation (N) various common modifications; The allowable error of mass spectrometry matching is ±20ppm, and the allowable error of MS/MS matching is ±50ppm. Two trypsin missed cleavage sites were allowed. The results are shown in Table 4.

The results showed that 106#_hu_1_hIgG4 had three glycosylation modifications, and the glycosylation ratios of the VHH segment were 11% and 30%, respectively, resulting in the heterogeneity of antibody proteins. The modified 0076#_hIgG4 antibody has only one glycosylation modification site, and the protein homogeneity is good.

Table 4. Glycosylation modification ratio of PD-1 antibody

Example 7. Affinity determination of PD-1 antibody binding to PD-1 protein

In order to detect the in vitro binding ability of the screened PD-1 single domain antibodies to human PD-1 protein and monkey PD-1, human PD-1 (Sino Biological Cat. 10377-H08H) and monkey PD-1 (Sino Biological Cat. 90311-C08H) was used for in vitro binding assays by ELISA binding experiments.

The negative control in this example is PBS, the positive control uses Opdivo (purchased from Shanghai chempartner (chempartner) lot: 180612001), and, in some experiments, IgG4 type PD-1Ab646 (hereinafter referred to as PD-1Ab646) in WO2017054646 is used as the positive control For comparison, the sequence is as follows:

PD-1 antibody heavy chain:

PD-1 antibody light chain:

Dilute the protein with PD-1 antibody to 2 μg/mL with pH 7.4 PBS buffer, add 100 μL/well to a 96-well microtiter plate (corning, 9018 25/box 96well clear flat bottom plate), at 4 ℃ placed overnight for 16-20 hours. After discarding the liquid, wash the plate three times with PBST (PH7.4, 0.05% Tween-20) buffer, add 2% BSA blocking solution (300 μL/well) diluted with PBS buffer, and incubate at 37°C for 2 hours to be closed. After blocking, discard the blocking solution and wash the plate three times with PBST buffer, add PD-1 antigen (Sino Biological Cat. 10377-H08H) protein at an initial concentration of 30 μg/mL, and dilute it three times with PBS buffer Eight gradients were placed in a 37°C incubator for 1 hour. After the incubation, discard the reaction solution in the ELISA plate, wash the plate 6 times with PBST, add 100 μL/HRP-labeled anti-his secondary antibody (Abcam ab1187) (1:5000 dilution) to each well, and incubate at 37°C for 1 hour . After washing the plate 6 times with PBST, add 100 μL of TMB chromogenic substrate, incubate at room temperature for 3-5 min, add 100 μL of 1M sulfuric acid to stop the reaction, read the absorbance at 450 nm with a SpectraMax M5 microplate reader, and calculate the EC ₅₀ value of the antibody binding to the antigen. . The _EC50 results of some of the antibodies are shown in Table 3. The results show that all of them have good binding ability with human and monkey PD-1 antigens.

Table 5. Binding EC ₅₀ (nM) of PD-1 antibody to human and monkey PD-1 antigen

Antibody number EC ₅₀ for binding to human PD-1 EC ₅₀ for binding to monkey PD-1 7# 1.86 2.2 32# 1.99 4.8 32#_hu_1 4.08 6.2 32#_hu_2 3.43 2.3 32#_hu_3 2.98 1.2 61# 1.85 /

106# 2.56 0.67 107# 3.14 2.9 112# 2.51 1.5 Positive control (Opdivo) 1.69 2.88 Negative control (PBS) 0 0

(Note: "/" means not detected)

In addition, the dissociation constant of PD-1 antibody and PD-1 protein was also determined by Biacore 8K (GE Healthcare) instrument. First, the anti-human IgG Fc antibody (GE Healthcare, Cat.#BR-1008-39) was covalently coupled to the CM5S series chip, and the PD-1 antibody to be detected was captured on the chip surface by affinity, and then flowed on the chip surface. For different concentrations of PD-1 protein (SEQ ID NO: 3), the Biacore instrument was used to detect the reaction signal in real time to obtain the binding and dissociation curve, and the binding force constant was obtained by fitting. The solution used in the experiment was HBS-P solution (10 mM HEPES, 150 mM NaCl, 0.005% P20, pH 7.4). At the end of each experimental cycle, the chip was washed and regenerated with 3M _MgCl2 solution. The affinity results of some antibodies are shown in Table 6. The results show that the affinity of the antibody obtained by the screening of the present disclosure to PD-1 is comparable to that of the positive control.

Table 6. Affinity of PD-1 antibody to PD-1

Antibody number antigen k _a (1/Ms) k _d (1/s) K _D (M) 7# PD-1 1.36E+05 2.81E-04 2.06E-09 32# PD-1 3.25E+05 2.07E-03 6.35E-09 32#_hu_1 PD-1 1.79E+05 2.91E-03 1.63E-08 32#_hu_2 PD-1 1.67E+05 1.56E-03 9.36E-09 32#_hu_3 PD-1 2.20E+05 2.01E-03 9.11E-09 32#_hu_4 PD-1 1.75E+05 3.53E-03 2.02E-08 32#_hu_5 PD-1 1.62E+05 3.19E-03 1.96E-08 61# PD-1 1.54E+05 8.19E-04 5.33E-09 61#_hu_1 PD-1 2.26E+05 4.61E-03 2.04E-08 106# PD-1 7.94E+04 4.77E-04 6.01E-09 107# PD-1 9.65E+04 7.82E-04 8.10E-09 Opdivo PD-1 5.91E+05 1.45E-03 2.45E-09

In addition, the dissociation constant of PD-1 antibody and PD-1 protein was also determined using Biacore T200 (GE Healthcare) instrument. Covalently coupled proteinA (Yaxin RSPA05) to the CM5S series chip, the antibody to be detected was captured to the chip surface by affinity, and then different concentrations of PD-1 protein (Sino Biological Cat.10377-H08H) flowed on the chip surface. , the reaction signal is detected in real time to obtain the binding dissociation curve, and the binding force constant is obtained by fitting. The solution used in the experiment was HBS-EP solution (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20, pH 7.4). At the end of each experimental cycle, the chip was washed and regenerated with a solution of 10 mM glycine, pH=1.5 (GE, BR-1003-54). See Table 7 and Table 8 for the results.

Table 7. Affinity K _D of PD-1 antibody to human PD-1

Antibody number k _a (1/Ms) k _d (1/s) K _D (M) 32#_hu_3_hIgG4 1.05E+05 2.01E-03 1.92E-08 7#_hu_4_hIgG4 4.72E+04 5.84E-03 1.24E-07

106#_hu_1_hIgG4 8.17E+03 7.05E-04 8.63E-08 107#_hu_4_hIgG4 9.40E+03 1.20E-03 1.28E-07 PD-1Ab646 6.18E+04 4.79E-04 7.75E-09

Table 8. Affinity K _D of PD-1 antibody to PD-1

Antibody number Affinity level 106#_hu_1_hIgG4 ++ 106#_hu_1_hIgG4(N ₃₁ -D) ++ 106#_hu_1_hIgG4(N ₃₁ -E) ++ 106#_hu_1_hIgG4(N ₃₁ -F) + 106# _{_hu_1_hIgG4} (N31-G) ++ 106#_hu_1_hIgG4(N ₃₁ -H) + 106#_hu_1_hIgG4(N ₃₁ -I) + 106#_hu_1_hIgG4(N ₃₁ -K) + 106#_hu_1_hIgG4(N ₃₁ -L) + 106#_hu_1_hIgG4(N ₃₁ -M) + 106#_hu_1_hIgG4(N ₃₁ -P) + 106#_hu_1_hIgG4(N ₃₁ -Q) + 106#_hu_1_hIgG4(N ₃₁ -R) + 106#_hu_1_hIgG4(N ₃₁ -S) + 106#_hu_1_hIgG4(N ₁₀₄ -A) + 106#_hu_1_hIgG4(N ₁₀₄ -E) + 106#_hu_1_hIgG4(N ₁₀₄ -F) + 106#_hu_1_hIgG4(N ₁₀₄ -G) ++ 106#_hu_1_hIgG4(N ₁₀₄ -H) ++ 106#_hu_1_hIgG4(N ₁₀₄ -K) + 106#_hu_1_hIgG4(N ₁₀₄ -P) + 106#_hu_1_hIgG4(N ₁₀₄ -Q) + 106#_hu_1_hIgG4(N ₁₀₄ -R) + 106#_hu_1_hIgG4(N ₁₀₄ -S) + 0076#_hIgG4 ++ 0077#_hIgG4 ++ 0078#_hIgG4 ++ 0079#_hIgG4 ++

(Note: "/" means that the specific value of the detection is not shown; the affinity grade "++" means <3.00E-07, and "+" means ≥3.00E-07)

Example 8. PD-1 antibody blocks the binding of PD-1 to PD-L1 and PD-L2

The functional experiments of PD-1 antibodies were detected by ELISA competition experiments that blocked the binding between PD-1 and PD-L1 and PD-L2.

The Fc-tagged PD-1 fusion protein was diluted with pH 7.4 PBS buffer to a concentration of 2 μg/mL, added to a 96-well microtiter plate at a volume of 100 μL/well, and placed at 4°C overnight for 16-20 hours. After discarding the liquid, the plate was washed three times with PBST (PH7.4, 0.05% Tween-20) buffer, then 300 μL/well of 2% BSA blocking solution diluted with PBS buffer was added, and incubated at 37°C for 2 hours for blocking. After blocking, discard the blocking solution and wash the plate three times with PBST buffer, add biotinylated PD-L1 and PD-L2 proteins at a protein concentration of 6 μg/mL, add 50 μL to each well, and then add the initial The PD-1 antibody protein at a concentration of 30 μg/mL was diluted three-fold with PBS buffer for 6 gradients, and incubated in a 37°C incubator for 1 hour. After the incubation, the reaction solution in the ELISA plate was discarded, the plate was washed 6 times with PBST, and 100 μL/well diluted (1:500) with PBS (0.5% BSA) HRP-labeled anti-SA secondary antibody (Peroxidase- conjugated Streptavidin, Jackson 136861), incubated at 37°C for 1 hour. After washing the plate 6 times with PBST, add 100 μL/well of TMB chromogenic substrate, incubate at room temperature for 3-5 min, add 1M sulfuric acid to stop the reaction, read the absorbance value at 450 nm with a SpectraMax M5 microplate reader, and calculate the antibody to the antigen. Binding _IC50 values. The _IC50 results of some of the antibodies are shown in Table 4. The results show that the antibodies can compete with PD-L1 and PD-L2 for binding to PD-1, the negative control is PBS, and the positive control uses Opdivo (Opdivo used in this disclosure is purchased from Shanghai Chempartner (chempartner) lot: 180612001) . The results of blocking the binding of PD-1 to PD-L1 by some antibodies are shown in Table 9 and Table 10.

Table 9. _IC50 (nM) of Different PD-1 Antibodies Competing PD-1 Antigen with PD-L1 and PD-L2

Table 10. _IC50 (nM) of different PD-1 antibodies competing against PD-1 antigen and PD-L1

Antibody number IC ₅₀ for blocking the binding of PD-1 to PD-L1 32#_hu_3_hIgG4 2.42 7#_hu_4_hIgG4 1.22 106#_hu_1_hIgG4 3.14 PD-1Ab646 2.79 Negative control (PBS) 9999

Example 9. In vitro cell binding assay of PD-1 antibody

The human PD-1 full-length gene was PCR-cloned into the mammalian cell expression vector pTargeT, and the linearized plasmid was used to electrotransfect CHO-S cells (preset CHO cell parameters that come with the electroporator), and 1mg/ml G418 was screened for 2 After 2 weeks, limited dilution was performed twice, and the PD-1 gene on the cell surface was detected by flow cytometry, and a monoclonal cell line with high expression of human PD-1 was selected. Named CHO-PD-1.

The cell line CHO-PD-1, which is stable and highly expressing PD-1, was collected at 5×10 ⁵ cells per well. Gradient dilution of PD-1 antibody to 16.67 μg/mL, 5.55 μg/mL, 1.85 μg/mL, 0.617 μg/mL, 0.205 μg/mL, 0.069 μg/mL, 0.023 μg/mL, with CHO-PD-1 on ice Incubate in the dark for 1 hour. After rinsing once with PBS, FITC anti-human IgG (1:100) was added to each well and incubated on ice for 1 hour in the dark. After rinsing again with PBS, resuspend in 100 μL of PBS per tube, and perform fluorescence detection on a BD C6Plus flow cytometer. Using Graphpad Prism9 software, curve fitting and graphing of the mean fluorescence intensity obtained from the treatment of each dose of antibody was performed to quantitatively analyze the binding of PD-1 antibody to CHO-PD-1 cells. The results showed that the binding strength of PD-1 antibody to CHO-PD-1 cells was in a dose-dependent manner.

The binding capacity EC ₅₀ results of some antibodies are shown in Table 11, Table 12 and Figure 1 . The results show that the antibodies (such as 2#, 32#, 32#_hu_1, 32#_hu_2, 32#_hu_3, 61#, 32#_hu_3_hIgG4, 7#_hu_4_hIgG4, 106#_hu_1_hIgG4, 107#_hu_4_hIgG4) and PD The binding capacity of -1 was significantly better than that of the positive control Opdivo. The binding ability of mutated 0076#_hlgg4 and 0078#_hlgg4 to PD-1 did not decrease compared with 106#_hu_1_hlgg4.

When the negative control of the present disclosure is NC, it is an antibody having the same constant region IgG4 as the PD-1 antibody of the present disclosure, but the variable region does not recognize the antigen PD-1. Opdivo was used as a positive control.

Table 11. EC ₅₀ of PD-1 antibody binding to cell surface antigen PD-1

Antibody _EC50 (nM) Negative Control (NC) 637030 Positive control (Opdivo) 66.3 2# 18.5 32# 2.9 32_hu_1# 7.3 32_hu_2# 4.8 32_hu_3# 6.1 61# 16.7 32#_hu_3_hIgG4 3.912 7#_hu_4_hIgG4 3.614 106#_hu_1_hIgG4 8.926 107#_hu_4_hIgG4 11.95

Table 12. EC ₅₀ of PD-1 antibody binding to cell surface antigen PD-1

Antibody number _EC50 (nM) 106#_hu_1_hIgG4 9.29 106# _{_hu_1_hIgG4} (N31-G) 4.76 106#_hu_1_hIgG4(N ₃₁ -D) 5.07 106#_hu_1_hIgG4(N ₁₀₄ -G) 5.21 106#_hu_1_hIgG4(N ₁₀₄ -H) 4.95 0076#_hIgG4 5.04 0078#_hIgG4 5.00

Example 10. Antibodies to PD-1 block the binding of PD-1 to PD-L1 on cells

The cell line CHO-PD-1, which is stable and highly expressing PD-1, was collected and adjusted to 5×10 ⁵ cells per well. Serially diluted PD-1 antibody to 50 μg/mL, 16.67 μg/mL, 5.55 μg/mL, 1.85 μg/mL, 0.617 μg/mL, 0.205 μg/mL, 0.069 μg/mL, 0.023 μg/mL, and CHO-PD -1 cells were incubated on ice for 1 hour. After rinsing once with PBS, add PD-L1-mIgG2a protein 1 μg/mL to each tube and incubate on ice for 1 hour, then wash with PBS again. After washing, add PE anti-mouse IgG2a (1:300) to each tube and incubate on ice for 1 hour. After rinsing once with PBS, resuspend in 100uL PBS per tube and perform fluorescence detection on BD C6Plus flow cytometer. Using Graphpad Prism9 software, curve fitting and graphing of the mean fluorescence intensity obtained from the treatment of each dose of antibody was performed to quantitatively analyze the binding of PD-1 to PD-L1 on cells blocked by PD-1 antibody.

The results showed that PD-1 antibody could block the binding of PD-L1 protein to CHO-PD-1 cells in a dose-dependent manner. The blocking _IC50 results of some antibodies are shown in Table 13, Table 14 and Figure 2. And, the antibodies of the present disclosure (eg, 7, 32, 32_hu_1, 32_hu_2, 32_hu_3, 106, 107, 112) have a stronger ability to block the binding of PD-L1 to PD-1 than the control Opdivo. Also, the ability of mutated molecules such as 0076#_hlgg4 and 0078#_hlgg4 to block the binding of PD-L1 to PD-1 was not reduced compared to 106#_hu_1_hlgg4.

Table 13. IC ₅₀ (nM) of PD-1 antibody blocking PD-L1 protein and cell surface antigen PD-1

Antibody number _IC50 (nM) 7# 3.8 32# 2.3 32#_hu_1 6.2 32#_hu_2 4.3 32#_hu_3 4.3 61# 5.0 61#_hu_1 197.20 61#_hu_2 334.80 106# 4.8 107# 5.0 112 6.1 Opdivo 43.9 Negative Control (NC) 9999

Table 14. IC ₅₀ (nM) of PD-1 antibody blocking PD-L1 protein and cell surface antigen PD-1

Antibody number IC50 ₍ nM) 106#_hu_1_hIgG4 9.38 106# _{_hu_1_hIgG4} (N31-G) 5.9 106#_hu_1_hIgG4(N ₃₁ -D) 6.1 106#_hu_1_hIgG4(N ₁₀₄ -G) 6.45 106#_hu_1_hIgG4(N ₁₀₄ -H) 6.47 0076#_hIgG4 4.5

0078#_hIgG4 5.5 Negative Control (NC) 9999

Example 11. Immune activation experiment of PD-1 antibody releasing PD-1/PD-L1 blockade in vitro

The CHO-PD-L1aAPC cell line (purchased from Promega, PD-1/PD-L1 Blockade Bioassay, J1252) with stable and high expression of PD-L1 and TCR activating molecules was collected, and PD-L1-negative CHO cells were used as a control. The complete medium was adjusted to a cell density of 4×10 ⁵ /mL, 100uL was added to each well, and placed in a 37°C 5% CO ₂ incubator for 20-24 hours.

On the day of the test, the PD-1 antibody was serially diluted to 1000, 250, 62.5, 15.6, 3.91, 0.98, 0.24, and 0.06 nM using assay medium, and 2 replicate wells were set for each concentration.

The effector cells Jurkat-PD-1 (purchased from Promega, PD-1/PD-L1 Blockade Bioassay, J1252) with stable and high expression of PD-1 were collected. The cells also stably expressed the luciferase reporter gene initiated by NFAT. The cell density was adjusted to 1.25 x ¹⁰⁶ /mL with assay medium.

Take out the culture plate inoculated with CHO-PD-L1aAPC cells the day before, discard the supernatant, add the diluted antibody and the adjusted density effector cell Jurkat-PD-1 to the cell culture plate at one time, add 40uL to each well, gently. Mix well and incubate in a 37°C 5% CO ₂ incubator for 6 hours.

During antibody incubation, the Bio-Glo ^™ Reagent (Promega) was removed and allowed to return to room temperature. After mixing and culturing, take out the cell culture plate and let it stand at room temperature for 5-10 minutes, then add 80uL Bio-Glo ^TM Reagent to each well, mix gently, and use the Molecular Device SpectraMax multi-function microplate reader to read the chemiluminescence value. The data were analyzed by curve fitting using Graphpad Prism9 software and plotted. Partial antibody results are shown in Table 15 and Figure 3 .

The results showed that both 0076#_hIgG4 and 106#_hu_1_hIgG4 could well relieve the immune activation of PD-1/PD-L1 blockade, and the effect of 0076#_hIgG4 was better than that of 106#_hu_1_hIgG4.

Table 15. _IC50 of PD-1 Antibody Removing PD-1/PD-L1 Blockade of Immune Activation

Antibody number _IC50 (nM) 106#_hu_1_hIgG4 29.18 0076#_hIgG4 14.25

Example 12. PD-1 antibody promotes cytokine secretion by mixed lymphocytes in vitro

Human fresh or thawed PBMCs were isolated from CD14 ⁺ monocytes by EasySep Human CD14 Positive Screening Kit (STEMCELL technologies, 17858). The isolated CD14 ⁺ cells were induced for 6 days by adding IL-4 and GM-CSF factors according to the method of the monocyte-derived dendritic cell differentiation kit (R&D system, CDK004), and then further induced by TNF-α for 3 days. , become a mature DC.

Human PBMCs were isolated from CD3 ⁺ T cells (different donor source from DC) by EasySep Human CD3 Positive Screening Kit (STEMCELL technologies, 18051). The isolated T and DC cells were mixed and cultured at a ratio of 10:1, and a low-endotoxin-controlled PD-1 antibody was added at the same time. After 5 days of culture, the human IFNγquantikine ELISA kit (R&D system, DIF50) was used to detect the IFNγ of activated T cells. secretion.

After the mixed culture, the amount of IFNγ secreted is shown in Table 16, Table 17, and Fig. 4 and Fig. 5 . The results show that multiple PD-1 antibodies obtained by screening can effectively enhance T cell activation and secrete IFNγ.

Table 16. PD-1 antibody promotes IFNγ secretion

Antibody number IFNγ secretion (pg/mL) 7# 963.5 32# 555.3 32#_hu_3 1031.5 106# 1164.2 107# 1776.6 Negative Control (NC) 49 Positive control (Opdivo) 1181.5

Table 17. PD-1 antibody promotes IFNγ secretion

Antibody number IFNγ secretion (pg/mL) 32#_hu_3_hIgG4 877.3 7#_hu_4_hIgG4 759.9 106#_hu_1_hIgG4 736.94 PD-1Ab646 549.8 Negative Control (NC) 163

Example 13. Antibodies to PD-1 inhibit tumor growth in a mouse colon cancer model

Animal experiments were completed by Shanghai Aifei Pharmaceutical Technology Co., Ltd., using HuPD-1 humanized transgenic mice, female, 6-8 weeks old, purchased from Nanjing Galaxy Bio-Pharmaceutical Co., Ltd.

The mouse colon cancer cell line MC38 cells resuspended in PBS were subcutaneously inoculated into the right flank of HuPD-1 humanized mice at a concentration of 5×10 ⁵ cells/0.1 mL and a volume of 0.1 mL/cell. When the average tumor volume reached 100 mm ³ (70-120 mm ³ ), mice with moderate tumor volume were selected into the group, and the tumor volume on the right side was the basis for grouping. The administration started on the day of the grouping, and the dosage was 0.3 mg/kg; the frequency of administration was once every three days, for a total of three weeks; the method of administration was intravenous injection.

The results of inhibition of mouse colon cancer tumor growth by PD-1 antibody are shown in Table 18 and Figures 6A and 6B. The results showed that on the 24th day, the tumor inhibition rate of the positive control was 47.3%; the tumor inhibition rate of 32#_hu_3_hIgG4 was 50.8%; the tumor inhibition rate of 7#_hu_4_hIgG4 was 68.4%; the tumor inhibition rate of 106#_hu_3_hIgG4 was 64.4%, Both can effectively inhibit tumor growth in mice.

Table 18. Results of PD-1 antibody inhibition of colon cancer tumor growth in mice

Example 14. Construction, Expression and Purification of PD-1/LAG-3 Bispecific Antibody

The PD-1 antibody of the present disclosure and the LAG-3 antibody were used to construct a bispecific antibody targeting PD-1/LAG-3.

Human LAG-3 full-length protein is shown in NCBI Reference Sequence: NP_002277.4. Human LAG-3 antigen, human LAG-3-Fc antigen and monkey LAG-3 antigen for screening and detection are all commercial products (Baiying Bio B2062; Sino Biological Cat. 16498-H02H; Sino Biological Cat: 90841-C08H). The sequence and preparation method of the LAG-3 antibody in WO2017219995A are incorporated herein in its entirety. The CDR sequences of the LAG-3 antibodies used in the present disclosure are shown in Table 19, and the numbering rule is Kabat.

Table 19. CDR sequences of LAG-3 antibodies

The heavy chain variable region (VH) and light chain variable region (VL) of the LAG-3 antibody are shown below:

Heavy chain sequence of exemplary LAG-3 antibody LAG-3Ab303:

Light chain sequence of LAG-3Ab303:

The construction method of the PD-1/LAG-3 bispecific antibody includes: linking the variable region sequence (ie VHH) of the PD-1 antibody of the present disclosure to the N-terminus of the heavy chain of the LAG-3 antibody, and connecting the LAG-3 antibody to the N-terminus of the LAG-3 antibody heavy chain. The C-terminus of the antibody heavy chain is linked, the N-terminus of the LAG-3 antibody light chain is linked, the C-terminus of the LAG-3 antibody light chain is linked, or a combination thereof. The linker connecting the PD-1 antibody and the LAG-3 antibody can be (G4S) ₂ , (G4S) ₃ , and (G4S) ₄ .

The sequences of bispecific antibodies 2140#, 2170#, 2171#, 2172#, 2173# constructed from 106#_hu_1 and its glycosylation variants 0076#, 0077#, 0078#, 0079# and LAG-3 antibody are as follows:

The first polypeptide chain of 2140#:

The first polypeptide chain of 2170#:

Heavy chain sequence of 2171#:

The first polypeptide chain of 2172#:

The first polypeptide chain of 2173#:

The second polypeptide chain sequences of 2140#, 2170#, 2171#, 2172#, and 2173# are all shown in SEQ ID NO: 188.

Another exemplary double antibody 2138#, 2136# sequence of 7#_hu_4, 32#_hu_4 and LAG-3 is as follows:

The first polypeptide chain of 2138#:

The first polypeptide chain of 2136#:

The second polypeptide chain sequences of 2138# and 2136# are also shown in SEQ ID NO: 188.

The bispecific antibody construct plasmid was transfected into Expi-CHO cells, and the expression supernatant was collected after 9 days of culture, and the impurities were removed by high-speed centrifugation, and purified with a Protein A column. Equilibrate with PBS until the A280 reading drops to baseline. The target protein was eluted with an acidic eluent at pH 3.0-3.5, and neutralized with 1M Tris-HCl, pH 8.0-9.0. The eluted sample was diluted with 25mM MES, pH 6.0, and purified by ion exchange chromatography with CIEX SP-HP column. The purified protein was concentrated and exchanged into PBS solution, and stored in aliquots. After detection, the PD-1/LAG-3 bispecific antibody of the present disclosure was obtained.

Example 15. Affinity determination of PD-1/LAG-3 double antibody binding to PD-1 and LAG-3 proteins

The affinity of bispecific antibodies to PD-1 and LAG-3 proteins was detected using the method of Biacore T200. The results are shown in Table 20 to Table 23. Compared with the anti-PD-1 antibody 0076#_hIgG4 alone and the anti-LAG-3 antibody mAb303-IgG4 alone, the affinity of the double antibody 2170# was not significantly reduced. The method is: using Biacore T200 (GE Healthcare) instrument to determine the dissociation constants of double antibody molecules with PD-1 protein and LAG-3 protein. Covalently couple proteinA (Yaxin RSPA05) to the CM5S series chip, the antibody to be detected is captured to the chip surface by affinity, and then different concentrations of PD-1 protein (Sino Biological Cat.10377-H08H) flow on the chip surface. and LAG-3 protein, real-time detection of the reaction signal to obtain the binding dissociation curve, and the binding force constant obtained by fitting. The solution used in the experiment was HBS-EP solution (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20, pH 7.4). At the end of each experimental cycle, the chip was washed and regenerated with a solution of 10 mM glycine, pH=1.5 (GE, BR-1003-54).

Table 20. Affinity of PD-1/LAG-3 dual antibody to human PD-1

Antibody number k _a (1/Ms) k _d (1/s) K _D (M) 106#_hu_1_hIgG4 3.79E+03 8.49E-04 2.24E-07 0076#_hIgG4 4.84E+03 1.11E-03 2.29E-07 2140# 6.19E+03 7.77E-04 1.53E-07

2170# 6.99E+03 9.17E-04 1.31E-07 2171# 5.66E+03 8.62E-04 1.52E-07 2172# 7.20E+03 1.13E-03 1.57E-07 2173# 5.73E+03 8.72E-04 1.52E-07

Table 21. Affinity of PD-1/LAG-3 dual antibody to monkey PD-1

Antibody number k _a (1/Ms) k _d (1/s) K _D (M) 0076#_hIgG4 6.04E+03 6.65E-04 1.10E-07 2170# 7.80E+03 5.33E-04 6.83E-08

Table 22. Affinity of PD-1/LAG-3 dual antibody to human LAG-3

Antibody number k _a (1/Ms) k _d (1/s) K _D (M) mAb303-IgG4 5.36E+06 9.94E-05 1.85E-11 2170# 5.28E+06 6.77E-05 1.28E-11

Table 23. Affinity of PD-1/LAG-3 dual antibody to monkey LAG-3

Antibody number k _a (1/Ms) k _d (1/s) K _D (M) mAb303-IgG4 1.33E+05 5.45E-03 4.09E-08 2170# 1.35E+05 4.73E-03 3.50E-08

Example 16. In vitro cell binding experiment of PD-1/LAG-3 double antibody

The negative control in this example was PBS, and the positive control was Opdivo. Part of the experiments used the IgG4-type PD-1 Ab646 in WO2017054646A1 as a positive control (as previously shown in SEQ ID NOs: 121 and 122). Part of the experiment used the IgG1 PD-1 and LAG-3 bispecific antibody (RO7247669) in WO2018185043A1 as a positive control, the sequence is as follows:

Antibody heavy chain 1 of RO7247669:

Antibody heavy chain 2 of RO7247669:

Antibody Light Chain 1 of RO7247669:

Antibody Light Chain 2 of RO7247669:

The method of Example 9 was used for detection, and the negative control used was NC. The binding _EC50 results for some of the antibodies are shown in Table 24, Table 25, Figure 7 and Figure 8. Among them, this example is to detect the double antibody, but it is the same for the binding of PD-1, so the concentration is the same as that in Example 9.

The results showed that the binding strength of the bispecific molecule to PD-1 on the cell surface was antibody dose-dependent, and compared with the anti-PD-1 antibody 0076#_hIgG4 or 106#_hu_1_hIgG4 alone, the double antibody (such as 2140#, 2170# ) was not significantly reduced.

Table 24. Binding EC ₅₀ of PD-1/LAG-3 dual antibody to cell surface antigen PD-1

Antibody number EC ₅₀ (ug/mL) 2136# 0.8367 2138# 0.7390 2140# 1.873 PD-1Ab646 0.2613

Table 25. Binding EC ₅₀ of PD-1/LAG-3 dual antibody to cell surface antigen PD-1

The CHO-LAG3 overexpression cell line was constructed by retrovirus infection. HEK293T (ATCC, CRL-3216) was used for virus packaging, and the pBABE expression vector containing the full-length LAG-3 gene and the helper vectors pVSV-G, pGag-pol were co-transfected into HEK293T, and the virus supernatant was collected and used to infect the cells. CHO-K1 (ATCC, CCL-61) cells were stained, monoclonal cell lines were obtained by limiting dilution, and the expression of LAG-3 on the cell membrane surface was detected by flow cytometry, and the cell lines with high expression of LAG-3 were selected and named CHO -LAG-3.

The cell line CHO-LAG-3 with stable high expression of LAG-3 was collected, the cell density was adjusted to 6×10 ⁶ /mL with PBS, and 50uL was added to each well. The serial dilution of LAG-3 antibody or double antibody was 111.1nM, 37.04nM, 12.3nM, 4.12nM, 1.37nM, 0.46nM, and 50uL was added to each well. Incubate with CHO-LAG-3 cells for 1 hour on ice. After rinsing once with PBS, add FITC anti-human IgG (1:100) to each tube and incubate on ice for 1 hour in the dark. After rinsing again with PBS, resuspend in 100uL PBS per tube, and perform fluorescence detection on BD C6Plus flow cytometer. Using Graphpad Prism9 software, curve fitting and graphing of the mean fluorescence intensity obtained from the treatment of each dose of antibody was performed to quantitatively analyze the binding of bispecific molecule or anti-LAG-3 antibody to CHO-LAG-3 cells. The negative control applied was NC.

The binding capacity EC ₅₀ results of some antibodies are shown in Table 26 and FIG. 9 . The results showed that the binding strength of bispecific molecule or anti-LAG-3 antibody to CHO-LAG-3 cells was in a dose-dependent manner. And compared with the single use of anti-LAG-3 antibody LAG-3Ab303, the binding capacity of bispecific molecules 2140# and 2170# was not significantly reduced.

Table 26. Binding EC ₅₀ of PD-1/LAG-3 dual antibody to cell surface antigen LAG-3

Antibody number _EC50 (nM) 2140# 1.888 2170# 2.322 2172# 2.064 2171# 2.085 2173# 1.665 LAG-3Ab303 0.4786

Example 17. PD-1/LAG-3 double antibody blocks the binding of PD-1 and PD-L1 on cells, and blocks the binding of LAG-3 to A735 which is highly expressed in MHCII

Detection was performed using the method in Example 10. The blocking IC ₅₀ results of some of the antibodies are shown in Table 27, Table 28, and Figures 10 and 11. The results showed that the double antibody could block the binding of PD-L1 protein to CHO-PD-1 cells in a dose-dependent manner. And compared with the single use of anti-PD-1 antibody 0076#_hIgG4 or 106#_hu_1_hIgG4, the ability of double antibody 2140# and 2170# to block the binding of PD-L1 protein to CHO-PD-1 cells was not significantly reduced.

Table 27. IC ₅₀ of PD-1/LAG-3 dual antibody blocking PD-L1 protein and cell surface antigen PD-1

Antibody number _IC50 (ug/mL) 2136# 0.7607 2138# 0.8715 2140# 1.839 PD-1Ab646 0.2722

Table 28. IC ₅₀ of PD-1/LAG-3 dual antibody blocking PD-L1 protein and cell surface antigen PD-1

The endogenous and stable MHCII-expressing cell line A375 was collected, and the cell density was adjusted to 10×10 ⁶ /mL with PBS, and 30 uL was added to each well. The serial dilution of LAG-3 antibody or bispecific antibody is 1000, 333.3, 111.1, 37.04, 12.3, 4.12, 1.37, 0.46nM, and 50uL is added to each well. At the same time, 20uL of 100ug/mL LAG-3-hIgG protein was added to each well, mixed well, incubated on ice for 1 hour, and rinsed once with PBS. After washing, add FITC anti-human IgG (1:100) to each tube and incubate on ice for 1 hour in the dark. After rinsing once with PBS, resuspend in 100uL PBS per tube and perform fluorescence detection on a BD C6Plus flow cytometer. . Using Graphpad Prism9 software, curve fitting and graphing of the mean fluorescence intensity obtained by each dose of antibody was used to quantitatively analyze the blocking of LAG-3 protein binding to MHCII on the cell surface by bispecific molecules or anti-LAG-3 antibody.

The blocking _IC50 results of some of the antibodies are shown in Table 29 and Figure 12. The results showed that the bispecific molecules 2140#, 2170#, 2171#, 2172#, and 2173# could block the binding of LAG-3 protein to MHCII on the cell surface, and compared with the anti-LAG-3 antibody LAG-3Ab303 alone , the double antibody function was not significantly reduced.

Table 29. _IC50 of PD-1/LAG-3 dual antibody blocking LAG-3 protein binding to cell surface MHCII

Antibody number _IC50 (nM) 2140# 109.6 2170# 72.04 2172# 86.46 2171# 99.53 2173# 106.4 LAG-3Ab303 66.10

Example 18. Immune activation experiment of PD-1/LAG-3 double antibody releasing PD-1/PD-L1 blockade in vitro

Collect the CHO-PD-L1aAPC cell line that stably and highly express PD-L1 and TCR activating molecules, and use PD-L1-negative CHO cells as a control, adjust the cell density to 4×10 ⁵ /mL with complete medium, each well Add 100uL and place in a 37°C 5% _CO2 incubator for 20-24 hours.

On the day of the test, the PD-1/LAG-3 double antibody was serially diluted to 1000, 250, 62.5, 15.6, 3.91, 0.98, 0.24, and 0.06 nM in assay medium, and two replicate wells were set for each concentration.

The effector cells Jurkat-PD-1 with high and stable expression of PD-1 were collected, and the cells also stably expressed NFAT-initiated luciferase reporter gene, and the cell density was adjusted to 1.25×10 ⁶ /mL with assay medium.

Take out the culture plate inoculated with CHO-PD-L1aAPC cells the day before, remove 95uL/well of the supernatant with a multi-channel pipette and discard, add the diluted antibody and effector cells with adjusted density to the cell culture plate at one time, each time Add 40uL to each well, mix gently, and incubate for 6 hours in a 37°C 5% CO2 incubator.

During antibody incubation, the Bio-Glo ^™ Reagent (Promega) was removed and allowed to return to room temperature. After mixing and culturing, take out the cell culture plate and let it stand at room temperature for 5-10 minutes, then add 80uL Bio-Glo ^TM Reagent to each well, mix gently, and use the Molecular Device SpectraMax multi-function microplate reader to read the chemiluminescence value. The data were analyzed by curve fitting using Graphpad Prism9 software and plotted.

The IC50 results of some of the antibodies are shown in Table 30, Table 31, and Figures 13 and 14. The results showed that the bispecific molecules 2136#, 2138# and 2140# could block the binding of PD-1 and PD-L1 on the cell surface, and relieve the immunosuppression caused by the binding of the two.

Table 30. _IC50 of PD-1/LAG-3 dual anti-PD-1/PD-L1 blocking immune activation

Antibody number _IC50 (nM) 2136# 5.481 2138# 59.83 2140# 16.26 PD-1Ab646 4.249 RO7247669 20.13

Table 31. IC50 of anti-PD-1/LAG-3 dual anti-PD-1/PD-L1 blocking immune activation

candidate antibody _IC50 (nM) 106#_hu_1_hIgG4 29.18 0076#_hIgG4 14.25 2140# 27.20 2170# 17.39 RO7247669 20.18

Example 19. PD-1/LAG-3 double antibody promotes the secretion of cytokines by mixed lymphocytes in vitro

Fresh or revived human PBMCs were isolated from monocytes by EasySep ^™ Human Monocyte Isolation Kit (STEMCELL technologies, 19359), and the isolated monocytes were separated by adding Differentiation Supplement according to the method of ImmunoCult ^™ Dendritic Cell Culture Kit (STEMCELL technologies, 19085). After induction of differentiation for 5 days, Maturation Supplement was added for further induction of maturation for 2 days to differentiate into mature DC cells. The cell density was adjusted to 1.25×10 ⁵ /mL with medium.

CD4+ T cells were isolated from fresh or thawed human PBMCs (different donor source than DC cells) by EasySep ^™ Human CD4 ⁺ T Cell Isolation Kit (STEMCELL technologies, 17952). After the isolated T cells were labeled and stained with 20 uM CFSE (Invitrogen 65-0850), the cell density was adjusted to 1×10 ⁶ /mL with medium, and 100 uL was added to each well. Then, 80uL of differentiated and mature DC cells were added to each well, and the two were mixed and cultured at a ratio of 10:1. At the same time, 20uL of low-endotoxin-controlled PD-1 or bispecific antibody was added to each well, and the treated cells were placed in an incubator at 37°C and 5% CO2.

On the 3rd and 5th day of mixed culture, the supernatant was taken to detect the IL-2 and IFNg secretion levels of activated T cells with HTRF (Cisbio 62HIL02PEG, 62HIFNGPEG), and the test was carried out according to the detection instructions, and Tecan Magellan Pro multifunctional enzyme label was used. The instrument reads the fluorescence value at 620nM and 665nM. And on the 5th day of culture, the proliferation of CFSE-labeled T cells was detected by BD C6Plus flow cytometer. The data obtained were analyzed by standard curve fitting with Graphpad Prism9 software and plotted.

The levels of IL-2 and IFNγ secretion are shown in Figure 15A and Figure 15B, respectively, compared with the negative control (NC) or the anti-LAG-3 antibody LAG-3Ab303 alone or the anti-PD-1 antibody 0076#_hIgG4 alone. Both anti-2170# and RO7247669 more significantly activated PBMC to secrete IFNγ, which was slightly better than the combined use of anti-LAG-3 antibody and anti-PD-1 antibody.

Table 32. Mixed Lymphocyte Response Stimulated Cytokine Secretion Levels

Example 20. Staphylococcus aureus superantigen stimulation experiment

Fresh or resuscitated human PBMCs were stimulated in vitro with medium containing 0.5 ng/mL superantigen Staphylococcus aureus (SEB) for 48 hours, and the pre-stimulated PBMCs were collected and rinsed twice with 1×PBS. Adjust the cell density to 5×10 ⁵ cells/mL with SEB medium containing 0.5 ng/mL, and add 100 μL to each well.

The antibodies were serially diluted with culture medium to 1000nM, 200nM, 40nM, 8nM, 1.6nM, 0.32nM and 0.064nM, and 2 replicate wells were set for each concentration. Add antibody to SEB pre-stimulated PBMC cells at 100 μL per well and mix gently. Place in a 37°C 5% CO ₂ incubator. After 48 hours of mixed culture, the supernatant was taken to detect the IFNγ secretion level of activated T cells (Cisbio 62HIFNGPEG) with HTRF, and the test was carried out according to the detection instructions, and the fluorescence values at 620nM and 665nM were read with a Tecan Magellan Pro multifunctional microplate reader. The data obtained were analyzed by standard curve fitting with Graphpad Prism9 software and plotted.

The results are shown in Figure 16. Compared with the negative control (NC) or the anti-LAG-3 antibody LAG-3Ab303 alone or the anti-PD-1 antibody 0076#_hIgG4 alone, the double antibody 2170# and RO7247669 both more significantly activated PBMC secretion IFNγ.

Example 21. Tumor killing experiment of PBMC

Fresh or thawed human PBMCs were cultured overnight using medium containing 20 IU/mL IL-2 (STEMCELL technologies, 78036) and Human CD3/CD28T Cell Activator (STEMCELL technologies, 10971). The activated PBMCs were collected the next day, and the cell density was adjusted to 1×10 ⁶ /mL with medium, and 100 uL was added to each well.

On the day of mixed culture, collect target cells, adjust the cell density to 1.25×10 ⁵ /mL with medium, add 80uL to each well, and mix and culture PBMC and target cells at a ratio of 10:1. At the same time, 20uL of low-endotoxin-controlled PD-1 or bispecific antibody was added to each well, and the treated cells were placed in an incubator at 37°C and 5% CO ₂ . After 48 hours of mixed culture, the supernatant was gently taken to detect the IFNγ secretion level of activated T cells (Cisbio 62HIFNGPEG) with HTRF, and the test was carried out according to the test instructions, and the Tecan Magellan Pro multifunctional microplate reader was used to read the fluorescence values at 620nM and 665nM, Standard curve fit analysis was performed and graphed.

The results are shown in Figure 17 and Table 33 below, compared with the negative control (NC) or with the anti-LAG-3 antibody LAG-3Ab303 alone or with the anti-PD-1 antibody 0076#_hIgG4 alone, or with the bispecific molecule RO7247669 , the bispecific molecule 2170# more significantly activated PBMC to secrete IFNγ.

Table 33. Tumor killing assay of PBMC stimulates the level of IFNγ secretion by T cells

Example 22. PD-1/LAG-3 dual antibody inhibits tumor growth in a humanized animal model

NOG mice (female, 6-8 weeks old, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were adaptively reared for one week. The numbers were weighed on the day of the experiment, and the human melanoma cell line A375 in the logarithmic growth phase was collected and subcutaneously inoculated into the right flank of mice at a dose of 6×10 ⁶ /cell (200 uL) per animal. Human PBMCs were recovered on the same day, placed in a 37°C 5% CO ₂ incubator for 1 hour, rinsed with PBS and adjusted to a cell density of 1 × 10 ⁷ /mL, inoculated with 5 × 10 ⁶ /cell per animal ( 500uL) for tail vein injection. 5-7 days after inoculation, after the tumor grows to ^50-90mm3 , remove the body weight, tumor too large or too small, and randomly group according to the following table 34-36, and inject the corresponding antibody intraperitoneally, twice a week, for a total of Dosing 6 times. Mice body weight and tumor volume were measured twice a week. The tumor volume was calculated as TV=L _long ×L _{short 2/2} ^. The tumor volume in each group was expressed as the mean ± standard deviation. Two-way ANOVA was used for statistical analysis, and the tumor inhibition rate (%TGI) was calculated. The calculation formula was %TGI=[1-(T-T0)/(C-C0) ] × 100%. At the end of the experiment, blood was collected, lysed red blood cells (BD 555899) were mixed with monoclonal antibodies targeting human and mouse CD45 (eBioscience ^TM 11-0451-82, 12-9459-42) and incubated for 40 minutes. The percentage of human CD45-positive cells in all lymphocytes in the blood of mice was detected by a cytometer, as the reconstitution level of human PBMC in this mouse model. The results are shown in Fig. 18A, Fig. 18B, Fig. 18C, Fig. 19A, Fig. 19B, Fig. 20A, Fig. 20B, Fig. 20C.

Table 34. PD-1/LAG-3 double-antibody dosing regimen and tumor inhibition effect in mouse model

grouping Dosage (mg/kg) Number of animals (only) Day 31 %TGI 2136# 12 12 80%**** 2138# 12 12 65%**** 2140# 12 12 86%**** Negative control (PBS) - 12 -

(Note: In the two-way ANOVA statistical analysis, compared with the PBS group, there was a significant difference in tumor size on day 31, when P<0.0001, marked as ****.)

Table 35. PD-1/LAG-3 double antibody dosing regimen and tumor inhibitory effect in mouse model

grouping Dosage (mg/kg) Number of animals (only) Day 24 %TGI 2136# 12 6 55%** 2138# 12 6 34% 2140# 12 6 93%**** PD-1Ab646 10 6 34% LAG-3Ab303 10 6 8% Negative control (PBS) - 6 -

(Note: In the two-way ANOVA statistical analysis, compared with the PBS group, the tumor size on day 24 was significantly different, when P<0.005, marked as **; when P<0.0001, marked as ****.)

Table 36. Dosing regimen of PD-1/LAG-3 double antibody and tumor inhibition effect in mouse model

(Note: In the two-way ANOVA statistical analysis, compared with the negative control group, the tumor size on the 30th day was significantly different, when P<0.005, marked as **; when P<0.0005, marked as ***.)

The results showed that at the same molar amount, compared with the negative control (PBS), double antibody 2136#, 2138# and 2140# could inhibit the growth of A375 tumor in the humanized model, and 2140# had the better tumor inhibition effect. For the first two, tumor inhibition rates at the endpoints of the two experiments were 86% (Table 34) and 93% (Table 35), respectively.

The results in Table 36 show that when the same amount of substance was administered, the bispecific molecule was compared to the negative control (NC) or the anti-LAG-3 antibody LAG-3Ab303 alone or the anti-PD-1 antibody 0076#_hIgG4 alone Both 2170# and combined administrations more significantly inhibited the growth of A375 tumor in the humanized model.

Although specific embodiments of the present disclosure have been described above, those skilled in the art should understand that these are merely illustrative, and various changes may be made to these embodiments without departing from the principles and spirit of the present disclosure. Revise. Accordingly, the scope of protection of the present disclosure is defined by the appended claims.

Claims (26)

A PD-1 binding protein comprising at least one immunoglobulin single variable domain comprising three complementarity determining regions CDR1, CDR2 and CDR3, wherein: CDR1, CDR2, and CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 152, 204, and 153, respectively. The PD-1 binding protein of claim 1, wherein: 1) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 129, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 145; or 2) CDR1 comprises the amino acid sequence shown in any of SEQ ID NO: 129-141, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 83; or 3) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 81, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence shown in any of SEQ ID NO: 142-151; or 4) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 132, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 145; or 5) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 129, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 146; or 6) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 132, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 146; or 7) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 81, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 83. The PD-1 binding protein of claim 1 or 2, wherein the immunoglobulin single variable domain is VHH; Preferably, the VHH is a humanized and/or affinity matured VHH; More preferably, the VHH is shown in any of SEQ ID NOs: 154-157 or has at least 90%, at least 95%, at least 98%, at least 99% sequence identity with any of SEQ ID NOs: 154-157 amino acid sequence. The PD-1 binding protein of any one of claims 1-3, which is an antibody that specifically binds PD-1 or a fragment thereof; Preferably, the antibody is a camelid antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. The PD-1 binding protein of any preceding claim, further comprising a human immunoglobulin Fc region; Preferably, the Fc region is the Fc region of human IgG1 or IgG4; More preferably, the Fc region of the human IgG4 has 228P, 234A, 235A and/or 447A mutations. The PD-1 binding protein of any preceding claim, comprising: An amino acid sequence set forth in or having at least 90%, at least 95%, at least 98%, at least 99% sequence identity to any of SEQ ID NOs: 200-203. PD-1/LAG-3 binding protein comprising: the first antigen-binding domain that specifically binds PD-1, and a second antigen-binding domain that specifically binds LAG-3, The first antigen binding domain that specifically binds to PD-1 comprises at least one immunoglobulin single variable domain comprising three complementarity determining regions CDR1, CDR2 and CDR3, wherein : 1) CDR1, CDR2 and CDR3 are CDR1, CDR2 and CDR3 as defined in claim 1; or 2) CDR1, CDR2 and CDR3 respectively comprise amino acid sequences as shown in SEQ ID NOs: 62, 115 and 64; or 3) CDR1, CDR2 and CDR3 respectively comprise amino acid sequences as shown in SEQ ID NO: 81, 116, 117; or 4) CDR1, CDR2 and CDR3 respectively comprise amino acid sequences as shown in SEQ ID NOs: 118, 66 and 67; or 5) CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 84, 119 and 86; or 6) CDR1, CDR2 and CDR3 respectively comprise amino acid sequences as shown in SEQ ID NOs: 78, 120 and 80; or 7) CDR1, CDR2 and CDR3 respectively comprise amino acid sequences as shown in SEQ ID NOs: 59, 60, 61; or 8) CDR1, CDR2 and CDR3 respectively comprise amino acid sequences as shown in SEQ ID NOs: 74, 75 and 76; or 9) CDR1, CDR2 and CDR3 respectively comprise amino acid sequences as shown in SEQ ID NO: 88, 89, 90; or 10) CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 96, 97 and 98. The PD-1/LAG-3 binding protein of claim 7, the immunoglobulin single variable domain comprising the following CDR1, CDR2 and CDR3: 1) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 129, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 145; or 2) CDR1 comprises the amino acid sequence shown in any of SEQ ID NO: 129-141, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 83; or 3) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 81, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence shown in any of SEQ ID NO: 142-151; or 4) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 132, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 145; or 5) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 129, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 146; or 6) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 132, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 146; or 7) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 62, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 63, 68, 69, 70, 72, 77, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 64; or 8) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 62, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 63, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 73; or 9) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 81, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 71 or 82, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 83; or 10) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 81, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 91 and 93, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 92; or 11) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 81, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 94, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 95; or 12) CDR1 comprises the amino acid sequence shown in any of SEQ ID NO: 65, 113 and 114, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 66, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 67; or 13) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 84, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 85 and 102, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 86; or 14) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 78, CDR2 comprises the amino acid sequence shown in any of SEQ ID NO: 79, 87, 99, 100, 101, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 80. The PD-1/LAG-3 binding protein of claim 7 or 8, wherein the immunoglobulin single variable domain comprises SEQ ID NOs: 154-157, 7-33, 35-58, 123-128 Any amino acid sequence shown or having at least 90%, at least 95%, at least 98%, at least 99% sequence identity therewith. The PD-1/LAG-3 binding protein of any one of claims 7 to 9, wherein the second antigen binding domain that specifically binds to LAG-3 comprises a variable region (VH) of a heavy chain and a variable region of a light chain. Variable region (VL), where: or VH comprises HCDR1, HCDR2, HCDR3 shown in SEQ ID NOs: 158-160, respectively, and VL comprises LCDR1, LCDR2, LCDR3 shown in SEQ ID NOs: 161-163, respectively. The PD-1/LAG-3 binding protein of claim 10, wherein: The VH comprises an amino acid sequence as set forth in or at least 90%, at least 95%, at least 98%, at least 99% identical in sequence to any of SEQ ID NOs: 178-181, and the VL comprises as SEQ ID NO: 182 -186 any of the amino acid sequences shown or having at least 90%, at least 95%, at least 98%, at least 99% sequence identity therewith; or VH comprises an amino acid sequence as set forth in or at least 90%, at least 95%, at least 98%, at least 99% identical in sequence to any of SEQ ID NOs: 170-173 and VL comprises as SEQ ID NOs: 174-177 any amino acid sequence shown or having at least 90%, at least 95%, at least 98%, at least 99% sequence identity to it; Preferably, VH comprises or is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 178 and VL comprises or is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 183. The PD-1/LAG-3 binding protein of claim 10 or 11, wherein the second antigen binding domain that specifically binds to LAG-3 comprises a full-length heavy chain (HC) and a full-length light chain ( LC); Preferably, the full-length heavy chain is of the IgGl or IgG4 isotype and the full-length light chain is of the Kappa isotype; More preferably, the full-length heavy chain is set forth in or has at least 90% sequence identity to SEQ ID NO: 187, and the full-length light chain is set forth in or has at least 90% sequence identity to SEQ ID NO: 188. The PD-1/LAG-3 binding protein of any one of claims 7 to 12, wherein: The immunoglobulin single variable domain that specifically binds the first antigen binding domain of PD-1 is located at the N-terminus of the heavy chain variable region that specifically binds the second antigen binding domain of LAG-3; The immunoglobulin single variable domain that specifically binds the first antigen-binding domain of PD-1 is located at the C-terminus of the heavy chain variable region that specifically binds the second antigen-binding domain of LAG-3; The immunoglobulin single variable domain that specifically binds the first antigen binding domain of PD-1 is located at the N-terminus of the light chain variable region of the second antigen binding domain that specifically binds LAG-3; and/ or The immunoglobulin single variable domain that specifically binds the first antigen binding domain of PD-1 is located C-terminal to the light chain variable region that specifically binds the second antigen binding domain of LAG-3. The PD-1/LAG-3 binding protein of claim 13, wherein the immunoglobulin single variable domain that specifically binds to the first antigen-binding domain of PD-1 and the first antigen-binding domain that specifically binds to LAG-3 The two antigen-binding domains are connected directly or through a linker; Preferably, the linker has an amino acid sequence shown as (G ₄ S) _x , wherein x is independently selected from an integer of 1-20; More preferably, the linker is an amino acid sequence represented by (G ₄ S) ₂ and (G ₄ S) ₃ . The PD-1/LAG-3 binding protein of any one of claims 7 to 14, comprising a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises the amino acid sequence shown in any of SEQ ID NOs: 189-195 or an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity therewith, The second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 188 or an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto. Anti-PD-1/LAG-3 bispecific antibody containing: an immunoglobulin single variable domain as defined in any one of claims 7 to 9, and heavy chain variable region (VH) and light chain variable region (VL) as defined in claim 10 or 11; Preferably, the anti-PD-1/LAG-3 bispecific antibody further contains the Fc region of human IgG1 or IgG4. A polynucleotide whose encoding is selected from any of the following: The PD-1 binding protein of any one of claims 1 to 6, The PD-1/LAG-3 binding protein of any one of claims 7 to 15, The anti-PD-1/LAG-3 bispecific antibody of claim 16. A host cell comprising the polynucleotide of claim 17. A method for preparing a PD-1 binding protein, a PD-1/LAG-3 binding protein, and an anti-PD-1/LAG-3 bispecific antibody, comprising the steps of: Culturing the host cell of claim 18; Recovery of the PD-1 binding protein of any one of claims 1 to 6, or the PD-1/LAG-3 binding protein of any one of claims 7 to 15, or the anti-PD- 1/LAG-3 bispecific antibody, and Optionally, the PD-1 binding protein, PD-1/LAG-3 binding protein, anti-PD-1/LAG-3 bispecific antibody is purified and/or modified. A composition or kit comprising a PD-1 binding protein and a LAG-3 binding protein, the PD-1 binding protein being the PD-1 binding protein of any one of claims 1 to 6; Preferably, the LAG-3 binding protein comprises a heavy chain variable region (VH) and a light chain variable region (VL) as defined in claim 10 or 11 , or a full length heavy chain as defined in claim 12 (HC) and full-length light chain (LC); PD-1 binding protein and LAG-3 binding protein are in the same or different containers. A pharmaceutical composition comprising: one or more pharmaceutically acceptable carriers, diluents, buffers or excipients, and Choose from any of the following: A therapeutically effective amount or a prophylactically effective amount of the PD-1 binding protein according to any one of claims 1 to 6, the PD-1/LAG-3 binding protein according to any one of claims 7 to 15, the PD-1/LAG-3 binding protein according to claim 16. The anti-PD-1/LAG-3 bispecific antibody, the composition or kit of claim 20, or a combination thereof. A method of treating cancer, comprising: The subject is administered a therapeutically effective amount of any one of the following: The PD-1 binding protein of any one of claims 1 to 6, the PD-1/LAG-3 binding protein of any one of claims 7 to 15, the anti-PD-1/LAG of claim 16 -3 bispecific antibody, the composition or kit of claim 20, the pharmaceutical composition of claim 21, or a combination thereof; Preferably, the cancer is selected from the group consisting of lung cancer, prostate cancer, breast cancer, head and neck cancer, esophagus cancer, stomach cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, Kidney cancer, squamous cell carcinoma, cancer of the blood system, or a disease or disorder characterized by uncontrolled cell growth. A method of promoting T cell proliferation or benefiting a subject from up-regulation of an immune response, comprising administering to the subject an effective amount of any one selected from the group consisting of: The PD-1 binding protein of any one of claims 1 to 6, the PD-1/LAG-3 binding protein of any one of claims 7 to 15, the anti-PD-1/LAG of claim 16 -3 bispecific antibody, the composition or kit of claim 20, the pharmaceutical composition of claim 21, or a combination thereof; Preferably, the subject has, is suspected of having, is susceptible to cancer; More preferably, the cancer is selected from the group consisting of lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma , kidney cancer, squamous cell carcinoma, hematological cancer, or a disease or disorder characterized by uncontrolled cell growth. Use of a PD-1-binding protein combined with a LAG-3-binding protein for preparing a drug for treating cancer, wherein the PD-1-binding protein is the PD-1-binding protein according to any one of claims 1 to 6, and the LAG- 3. The binding protein comprises a second antigen binding domain as defined in any one of claims 10 to 12; Preferably, the cancer is selected from the group consisting of lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, Kidney cancer, squamous cell carcinoma, cancer of the blood system, or a disease or disorder characterized by uncontrolled cell growth. A PD-1 binding protein for treating cancer in combination with a LAG-3 binding protein, the PD-1 binding protein and the LAG-3 binding protein being administered simultaneously or sequentially, the PD-1 binding protein being any one of claims 1 to 6 The PD-1 binding protein of item 1, the LAG-3 binding protein contains the second antigen binding domain as defined in any one of claims 10 to 12; Preferably, the cancer is selected from the group consisting of lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, Kidney cancer, squamous cell carcinoma, cancer of the blood system, or a disease or disorder characterized by uncontrolled cell growth. LAG-3 binding protein, which is used in combination with PD-1 binding protein for the treatment of cancer, LAG-3 binding protein and PD-1 binding protein are administered simultaneously or sequentially, and said PD-1 binding protein is any one of claims 1 to 6 The PD-1 binding protein of item 1, the LAG-3 binding protein contains the second antigen binding domain defined in any one of claims 10 to 12; Preferably, the cancer is selected from the group consisting of lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, Kidney cancer, squamous cell carcinoma, cancer of the blood system, or a disease or disorder characterized by uncontrolled cell growth.

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