CN114316022A

CN114316022A - Peptides that bind to PD-1 antibodies and uses thereof

Info

Publication number: CN114316022A
Application number: CN202111157785.XA
Authority: CN
Inventors: 夏瑜; 王忠民; 张鹏; 李百勇
Original assignee: Zhengda Tianqing Kangfang Shanghai Biomedical Technology Co ltd
Current assignee: Zhengda Tianqing Kangfang Shanghai Biomedical Technology Co ltd
Priority date: 2020-09-30
Filing date: 2021-09-30
Publication date: 2022-04-12
Also published as: WO2022068919A1

Abstract

The present invention relates to a peptide that binds to an anti-PD-1 antibody or an antigen-binding fragment thereof, comprising structural units 1 and 2, wherein structural unit 1 comprises the amino acids at positions 58-85 of the PD-1 protein and structural unit 2 comprises the amino acids at position 130-132 of the PD-1 protein.

Description

Peptides that bind to PD-1 antibodies and uses thereof

Technical Field

The present invention is in the field of immunology. In particular to a peptide for binding PD-1 antibody and application thereof.

Background

Cell surface glycans play an important role in cancer, such as cell signaling and communication, dissociation and invasion of tumor cells, cell-matrix interactions, tumor angiogenesis, immunomodulation and metastasis formation, and immune monitoring (Varki a. (2017). Glycobiology, 27, 3-49.). Glycosylation aids in tumor Cell escape immune surveillance (Okada m.et al (2017). Cell rep., 20, 1017-.

There are four reported glycosylation sites in the extracellular immunoglobulin variable (IgV) domain of PD-1, N49, N58, N74 and N116(Chen d.et al. (2019) iScience, 14, 113-. It has been reported that the N58 amino acid residue of PD-1 in mammalian (Tan sh.et al, (2017) Nat commun., 8, 14369) cells is located on the BC loop of PD-1, and its glycosylation is high, and in most cases, the glycans of the core portion consist of two N-acetylglucosamines (GlcNac, NAG) and one Fucose (Fucose, FUC). Fucosylation is associated with cancer (Pinho, 2015), and T cells depleted in tumors tend to be highly core fucosylated (Okada m.et al (2017): Cell rep., 20, 1017-. In some cancers, such as lung and breast cancers, overexpression of core fucosylation (FUT8) was observed (Liu cy. et al (2011). proc. natl. acad. sci. usa, 108, 11332-11337) (Potapenko. i.et a1.(2010). mol. oncol., 4, 98-118). Loss of core fucosylation reduces Cell surface PD-1 expression, thereby enhancing T Cell activation (Okada m.et al. (2017). Cell rep., 20, 1017-.

The glycosyl on the surface of PD-1 may interact with the anti-PD-1 antibody, enhancing the binding activity of the two, and further showing better clinical results (Fessas H.et al (2017). Semin. Oncol., 44, 136-140).

Disclosure of Invention

The inventors have found that amino acids T59, E61, S62, E84 and D85 of PD-1 protein (PD-1Genbank ID: NP-005009, SEQ ID NO: 1) interact with amino acids Y32, D33, S52, G54, Y57 and Y100 of anti-PD-1 antibody such as 14C12H1L1-Fab heavy chain, and amino acids P130, K131 and A132 of PD-1 protein interact with amino acids F325, E324 and D323 of anti-PD-1 antibody such as 14C12H1L1-Fab light chain. Thus, the present invention has been completed.

In particular, the present invention relates to the following aspects:

1. a peptide, preferably binding an anti-PD-1 antibody or antigen-binding fragment thereof, comprising structural unit 1 and structural unit 2, wherein structural unit 1 comprises a PD-1 protein fragment selected from the group consisting of: amino acids at positions 28-86 (shown as SEQ ID NO: 10), amino acids at positions 28-85 (shown as SEQ ID NO: 11), amino acids at positions 29-86 (shown as SEQ ID NO: 12), amino acids at positions 29-85 (shown as SEQ ID NO: 13), amino acids at positions 58-86 (shown as SEQ ID NO: 14), amino acids at positions 58-85 (shown as SEQ ID NO: 15), amino acids at positions 59-86 (shown as SEQ ID NO: 16) and amino acids at positions 59-85 (shown as SEQ ID NO: 17) of PD-1 protein,

structural unit 2 comprises a PD-1 protein fragment selected from the group consisting of: the amino acids at positions 127-133 (shown as SEQ ID NO: 18), the amino acids at positions 127-132 (shown as SEQ ID NO: 19), the amino acids at positions 128-133 (shown as SEQ ID NO: 20), and the amino acids at positions 128-132 (shown as SEQ ID NO: 21) of the PD-1 protein.

2. The peptide as claimed in claim 1, which comprises structural unit 1 and structural unit 2, wherein structural unit 1 comprises the amino acids from position 29 to 85 (as shown in SEQ ID NO: 11) or from position 58 to 85 (as shown in SEQ ID NO: 15) of the PD-1 protein, structural unit 2 comprises the amino acids from position 128 to 132 (as shown in SEQ ID NO: 21) or from position 130 to 132 (as shown in SEQ ID NO: 3) of the PD-1 protein, preferably structural unit 1 comprises the amino acids from position 58 to 85 (as shown in SEQ ID NO: 15) of the PD-1 protein, and structural unit 2 comprises the amino acids from position 130 to 132 (as shown in SEQ ID NO: 3) of the PD-1 protein.

3. The peptide of

item

1 or 2, wherein the amino acid sequence of the PD-1 protein is as set forth in SEQ ID NO: 1 is shown.

4. The peptide of any one of items 1-3, wherein structural unit 1 comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10-17, unit 2 comprises SEQ ID NO: 3 or SEQ ID NO: 18-21, preferably building block 1 comprises the sequence shown in any one of SEQ ID NO: 2, and building block 2 comprises SEQ ID NO: 3, and (b) is the sequence shown in the specification.

5. The peptide of any one of items 1 to 4, wherein the anti-PD-1 antibody binds to an antigenThe fragment is Fab fragment, Fab ', (Fab')₂Fab' -SH, Fab/c, Fv, single chain antibodies (e.g., scFv).

6. The peptide of item 5, wherein the heavy chain amino acid sequence of the Fab fragment is as set forth in SEQ ID NO: 4, and the light chain amino acid sequence is shown as SEQ ID NO: 5, respectively.

7. The peptide of any one of items 1 to 6, wherein the amino acids T59, E61, S62, E84, D85 of the PD-1 protein bind to amino acids Y32, D33, S52, G54, Y57, Y100 of the heavy chain of the anti-PD-1 antibody (e.g., 14C12H1L1-Fab), the amino acids P130, K131, A132 of the PD-1 protein bind to amino acids F325, E324, D323 of the light chain of the anti-PD-1 antibody, and the glycosylated side chain N58 of the PD-1 protein binds to amino acids S31, G53, G54, and R56 of the heavy chain of the anti-PD-1 antibody (e.g., 14C12H1L 1-Fab).

8. The peptide of item 7, wherein the glycosylated side chain comprises mannose, N-acetylglucosamine; fucose and β -D-mannose.

9. Use of a peptide according to any of items 1 to 8 for screening for an antibody or antigen-binding fragment thereof that binds to PD-1.

10, SEQ ID NO: 1, D29, T59, E61, S62, K78, E84, D85, L128, P130, a132, or a combination thereof in screening for an antibody or antigen-binding fragment thereof that binds to PD-1.

The term "antibody" as used herein refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2, and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). The VH and VL regions can also be subdivided into regions of high denaturation, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each VH and VL are composed of, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 are composed of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions (VH and VL) of each heavy/light chain pair form the antibody binding sites, respectively. The assignment of amino acids to the various regions or domains follows either Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J.mol.biol.196: 901-; chothia et al (1989) Nature 342: 878-883. The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody may be of a different isotype, for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on the antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are generally obtained using hybridoma technology first reported by Kohler et al (Nature, 256: 495, 1975), but can also be obtained using recombinant DNA technology (see, e.g., U.S. patent 4,816, 567).

As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or a portion of the CDR regions of a human immunoglobulin (recipient antibody) with the CDR regions of a non-human antibody (donor antibody), which may be a non-human (e.g., mouse, rat, or rabbit) antibody of the desired specificity, affinity, or reactivity. In addition, some amino acid residues of the Framework Region (FR) of the acceptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further refine or optimize the performance of the antibody. For more details on humanized antibodies, see, e.g., Jones et al, Nature, 321: 522525 (1986); reichmann et al, Nature, 332: 323329 (1988); presta, curr, op.struct.biol., 2: 593596 (1992); and Clark, immunol. today 21: 397402(2000).

In the present invention, antigen binding fragments include, but are not limited to: fab fragment, Fab ', (Fab')₂Fab/c, Fv, Fab' -SH, single chain antibodies (e.g., scFv).

As used herein, the term "Fab fragment" consists of one light chain and C_H1And the variable region of one heavy chain. The heavy chain of a Fab molecule is unable to form a disulfide bond with another heavy chain molecule.

As used herein, the term "Fab' fragment" contains one light chain and one heavy chain portion (which contains V)_HDomains and C_H1Domains and also C_H1And C_H2Part of the region between the domains) so that an interchain disulfide bond can be formed between the two heavy chains of the two Fab 'fragments to form F (ab')₂A molecule.

As used herein, the term "F (ab')₂Fragment "contains two light chains and two contain C_H1And C_H2Heavy chains that are part of a constant region between domains, such that an interchain disulfide bond is formed between the two heavy chains. F (ab')₂The fragment thus consists of two Fab' fragments held together by the disulfide bond between the two heavy chains.

As used herein, the term "Fv region" comprises the variable regions from the heavy and light chains, but lacks the constant regions.

As used herein, the term "Fab '-SH" is the designation herein for Fab', wherein one or more cysteine residues of the constant domain carry a free thiol group.

Description of the drawings:

FIG. 1: graph of the overall structure model of the PD-1 protein and 14C12H1L1-Fab complex. FAB-HC: 14C12H1L1-Fab heavy chain portion; FAB-LC: 14C12H1L1-Fab light chain portion.

FIG. 2: schematic representation of the interaction of PD-1 protein with the 14C12H1L1-Fab heavy chain amino acids. FAB-HC: 14C12H1L1-Fab heavy chain portion.

FIG. 3: schematic representation of the interaction of PD-1 protein with 14C12H1L1-Fab light chain amino acids. FAB-LC: 14C12H1L1-Fab light chain portion.

FIG. 4: schematic representation of the interaction of glycosylated side chain of Asn58 in PD-1 protein with amino acid of heavy chain of 14C12H1L 1-Fab. MAN: mannose, mannose; NAG: NAcetyl Glucosamine, acetylglucosamine; FUC: fucose, Fucose; BMA: beta-D-mannose, beta-D-mannose. FAB-HC: 14C12H1L1-Fab heavy chain portion; FAB-LC: 14C12H1L1-Fab light chain portion.

FIG. 5: PD-1 binds to the surface of 14C12H1L1-Fab, Pembrolizumab or nivolumab. In the PD-1 protein, the residues in contact with 14C12H1L1-Fab are the amino acid residues at positions E61 and S62, the residues in contact with nivolumab are the amino acid residues at positions V64, N66, Y68, Q75, T76, D77, K78 and P83, and the overlapping residue bound by 14C12H1L1-Fab and nivolumab is the amino acid residue at position E84. The residues that contact pembrolizumab are amino acid residues at positions L128, A129, and the overlapping residues that bind 14C12H1L1-FAB and pembrolizumab are amino acid residues at positions T59, P130, A131, K132. The amino acid residue at position D85 could not be revealed due to steric hindrance.

FIG. 6: the binding ELISA method was used to detect the binding activity of the anti-PD-1 antibody to human PD-1 protein mutant hPD1(D29A) -mFc and hPD1(E61A) -mFc.

FIG. 7: the binding ELISA method is used for detecting the binding activity of the anti-PD-1 antibody to human PD-1 protein mutant hPD1(K78A) -mFc and hPD1(E84A) -mFc.

FIG. 8: the binding ELISA method was used to detect the binding activity of the anti-PD-1 antibody to human PD-1 protein mutant hPD1(L128A) -mFc.

FIG. 9: and (3) detection results of kinetic characteristic parameters of the binding of the anti-PD-1 antibody and human PD-1 protein hPD 1-mFc.

FIG. 10: results of kinetic characteristic parameter detection of the binding of anti-PD-1 antibody and human PD-1 protein mutant hPD1(D29A) -mFc.

FIG. 11: results of kinetic characteristic parameter detection of the binding of anti-PD-1 antibody and human PD-1 protein mutant hPD1(E61A) -mFc.

FIG. 12: results of kinetic characteristic parameter detection of the binding of the anti-PD-1 antibody and the human PD-1 protein mutant hPD1(K78A) -mFc.

FIG. 13: results of kinetic characterization parameter detection of the binding of anti-PD-1 antibody to human PD-1 protein mutant hPD1(E84A) -mFc.

FIG. 14: results of kinetic characterization parameter detection of the binding of anti-PD-1 antibody to human PD-1 protein mutant hPD1(L128A) -mFc.

Detailed Description

Embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, but are conventional products available on the market.

Main instruments and reagents

PD-1-his (Kangfang biological production)

14C12H1L1-Fab-his (produced by Kangfang organism)

14C12H1L1(hG1TM) (Kangfang biological production)

hPD 1-mFc: kangfang biological production, batch number: 20181025 (Genbank ID of hPD-1: NP-005009, Genbank ID of mFc: P01863).

hPD1(D29A) -mFc: kangfang Bioproduction, lot number 20191113.

hPD1(E61A) -mFc: kangfang Bioproduction, lot number 20191113.

hPD1(K78A) -mFc: kangfang Bioproduction, lot number 20191113.

hPD1(E84A) -mFc: kangfang Bioproduction, lot number 20191113.

hPD1(L128A) -mFc: kangfang Bioproduction, lot number 20191113.

Preparation example 1 preparation of human PD1-his fusion protein

The sequence of human PD-1 (PD-1Genbank ID: NP-005009) was searched by NCBI protein database, and the amino acid sequence of the extracellular region of human PD-1 (amino acid at position 1 to amino acid at position 170) was designed by fusion with the his purification tag sequence (SEQ ID NO: 6), and the fusion protein was abbreviated as "PD 1-his" (SEQ ID NO: 7) and also as "hPD 1-his".

The cDNA sequence of the coded fusion protein is derived from the amino acid codon optimization and gene synthesis of organisms entrusted with Nanjing Kinshire, and the target gene is subcloned into a mammalian cell expression vector by adopting the standard molecular cloning technology such as PCR, enzyme digestion, gel recovery, ligation transformation, colony PCR or enzyme digestion identification and the like according to the standard technology introduced in molecular cloning instruction (second edition), and the target gene of the recombinant expression vector is further subjected to sequencing analysis. After the sequencing verification is correct, expression plasmids with endotoxin levels removed are prepared in large quantities and are transiently transfected into HEK293 cells for protein expression. After 7 days of culture, the cell culture broth was collected and subjected to affinity purification using MabSelect SuRe column (GE Healthcare).

Example 1 analysis of binding epitopes of human PD-1 antigen and anti-PD-1 antibody

The inventors employed a method of X-ray crystal diffraction based on the reported complex structures of PD-1-nivolumab Fab (Tan SH, et al (2017). Nat commun., 8, 14369) and PD-1-Pembrolizumab Fab (Horita S et al (2016). Sci rep., 6, 35297) to employ X-ray crystal diffraction

The interaction between the Fab part of the antibody 14C12H1L1 (14C12H1L1-Fab) and the PD-1 antigen was investigated and the differences were compared.

The amino acid sequence of the heavy chain portion of antibody 14C12H1L1-Fab is as follows, the underlined portion is CH1, the bold is the CDR region, the underlined bold italics is the histidine tag (his-tag):

the amino acid sequence of the light chain in antibody 14C12H1L1-Fab is as follows, CL is underlined, and CDR regions are bold:

the inventors have unexpectedly found that 14C12H1L1-Fab binds to PD-1 in a manner distinct from pembrolizumab and nivolumab. Although all three block the binding between PD-1 and PD-L1, Pembrolizumab and nivolumab were reported to bind to PD-1(Tan SH, et al (2017) Nat commu.s.8, 14369.) and PD-1-Pembrolizumab Fab (Horita S et al (2016): Sci rep.6, 35297), 14C12H1L1-Fab showed a number of interactions with the N58 linked sugar side chain on the BC loop of PD-1, in a glycosylation independent manner, according to literature.

The experimental method comprises the following steps: PD1 protein, such as PD-1-His, was mixed with 14C12H1L1-Fab-His at a molar ratio of 1: 2 and incubated on ice for 2 hours. The mixture was then purified by molecular sieve (Superdex 20010/300 column, GE Healthcare) to give a complex of PD-1 protein and 14C12H1L1-Fab-his in 20mM HEPES, pH 7.5, 100mM NaCl, 5mM DTT. The complex peak was harvested and concentrated by centrifugation (Millipore, MWCO 10kDa) to a final complex concentration of about 10 mg/ml. The concentrated compound was used for primary screening of crystals and left to stand in a 20 ℃ cell. After two weeks, the crystal plate was observed under a microscope, and the conditions of good crystal appearance were selected for repetition and optimization of crystallization conditions. The crystal was grown under PEG II sit F2(0.1M MES, pH 6.5, 20% PEG4000,0.6M sodium chloride) and 30% EG as an antifreeze. SDS-PAGE showed the crystal content to be PD1-His +14C12H1L1-Fab-His complex. After the available crystals are acquired, resolution data is collected as diffraction data by using the Shanghai synchrotron radiation light source

The diffraction pattern of (1).

The data analysis flow is as follows: and carrying out indexing and integration processing on the data by adopting DIALS. The data were analyzed by Aimless, combined and the 5% data was randomly selected for Rfree estimation. The phase solution was found in two steps using the Molrep molecular replacement procedure. Through sequence comparison, the 14C12H1L1-Fab has 85% sequence homology with 6foe in a PDB database, so that 6foe is used for searching a solution of 14C12H1L1-Fab-his, then the position of 14C12H1L1-Fab-his is fixed, and a structure of a PD-1 monomer (PDB: 3rre) is used for searching a solution of PD-1. The result of the analysis is that an asymmetric unit comprises a PD1-his monomer and a 14C12H1L1-Fab-his fragment. Model corrections were then performed in reciprocal space using REFMAC 5. And correcting the protein model in real space by adopting COOT. The model fits well with the electron density map, the crystallographic R-factors and Rfree were 0.21 and 0.27, respectively (fig. 5), and the stereochemical parameters of the structural model were within reasonable ranges.

The results of the structural analysis are shown in FIGS. 1-5.

Example 2 amino acid site-directed mutagenesis study of the 14C12H1L1(hG1TM) antigen-binding epitope of anti-PD-1 antibody

Human PD-1 protein was selected based on the binding site of the Nivolumab antibody in which 5 amino acid sites, aspartic acid at position 29, glutamic acid at position 61, lysine at position 78, glutamic acid at position 84 and leucine at position 128, were mutated to alanine [ Lee, J.Y., et al, Structural basis of checkpoint blockade by monoclonal antibodies in cancer immunology, 2016.7 (1): p.13354 ], which were respectively recorded as hPD1(D29A) -mFc, hPD1(E61A) -mFc, hPD1(K78A) -mFc, hPD1(E84A) -mFc, hPD1(L128A) -mFc, the binding activity of the anti-PD-1 antibody to these mutants was tested by binding ELISA and Fortebio Kinetics, and the experiment was compared with the unmutated PD1-mFc protein as a control.

1. Detection of binding Activity of anti-PD-1 antibody 14C12H1L1(hG1TM) with human PD-1 protein mutant by binding ELISA method

PD-1 antigen was diluted to 1. mu.g/mL with coating buffer, 50. mu.L per well was added to the microplate, and incubated overnight at 4 ℃. After washing the plates with PBST, 300. mu.L of 1% BSA (PBS) was added to each well and blocked at 37 ℃ for 2 h. After washing the plates, anti-PD-1 antibody (14C12H1L1(hG1TM)) was diluted to 0.3333. mu.g/mL with PBST as the starting concentration, in the presence of enzymeThe plate was diluted 1: 3 down to 7 gradient concentrations of 0.1111. mu.g/mL, 0.0370. mu.g/mL, 0.0123. mu.g/mL, 0.0041. mu.g/mL, 0.0014. mu.g/mL, 0.0005. mu.g/mL, and blank controls were added to make 2 duplicate wells of 100. mu.L per well, mixed and incubated at 37 ℃ for 30 min. Adding 50 μ L of horseradish peroxidase-labeled goat anti-human IgG (H + L) secondary antibody (Jackson) working solution into each well after washing the plate, incubating at 37 deg.C for 30min, washing the plate with PBST, adding 50 μ L of TMB into each well, incubating at room temperature in a dark place for 5min, and adding 50 μ L of stop solution (2M H) into each well₂SO₄Solution) to terminate the color reaction. Immediately putting the ELISA plate into an ELISA reader, and reading the OD value of each hole of the ELISA plate by selecting the wavelength of 450 nm. The data were analyzed using SoftMax pro6.2.1 software. The antibody concentration was plotted on the abscissa and the absorbance value on the ordinate as a 4-parameter fit curve to obtain the bound EC50 value of the antibody.

The detection results are shown in Table 1 and FIGS. 6-8, and the binding of five mutants D29A, E61A, K78A, E84A and L128A of human PD-1 protein and anti-PD-1 antibody with EC50 is 0.021nM and 2.47X 10 nM respectively¹²nM, 0.023nM, 0.022nM, 4.207 nM. The two mutants, E61A and L128A, had significantly reduced binding capacity to the anti-PD-1 antibody.

TABLE 1 binding of anti-PD-1 antibodies to human PD-1 mutants ELISAEC50 results

Detection of binding Activity of anti-PD-1 antibody 14C12H1L1(hG1TM) and human PD-1 protein mutant by Fortebio Kinetics method

The anti-PD-1 antibody is first diluted to 5 mu g/mL with PBS (containing 0.02% Tween-20, 0.1% BSA, pH7.4) and then fixed on the surface of an AHC sensor (Fortebio) for 120s, the sensor is equilibrated in a buffer for 60s, the anti-PD-1 antibody fixed on the sensor is combined with each PD1-mFc mutant at a concentration of 1.24-100nM (three-fold dilution) for 120s, and the protein is dissociated in the buffer for 300 s. The detection temperature was 37 deg.C, the detection frequency was 0.3Hz, and the sample plate vibration rate was 1000 rpm. The data were analyzed by fitting a 1: 1 model to obtain affinity constants.

The results of the binding of the anti-PD-1 antibody to each PD-1 mutant are shown in FIGS. 9-14 and Table 2, and the binding affinity constants of five mutants of human PD-1 protein, D29A, E61A, K78A, E84A and L128A to the anti-PD-1 antibody are 4.25E-10M, 3.30E-08M, 1.31E-10M, 6.65E-10M and 7.68E-09M, respectively. Consistent with the binding ELISA results, both the E61A and L128A mutants had significantly reduced binding ability to the anti-PD-1 antibody.

TABLE 2 Fortebio Kinetics results of anti-PD-1 antibodies with human PD-1 mutants

A sequence table:

<110> Zhengda Qing kang Fang (Shanghai) biological medicine science and technology Co., Ltd

<120> peptides binding to PD-1 antibody and uses thereof

<130>IB206166

<160>21

<170>PatentIn version 3.3

PD-1(SEQ ID NO: 1): human PD1(1-170) sequence

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV

Structural unit 1(SEQ ID NO: 2): human PD1(58-85)

NTSESFVLNWYRMSPSNQTDKLAAFPED

Building Block 1(SEQ ID NO: 10) human PD1(28-86)

PDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDR

Building Block 1(SEQ ID NO: 11) human PD1(28-85)

PDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPED

Building Block 1(SEQ ID NO: 12) human PD1(29-86)

DRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPFDR

Building Block 1(SEQ ID NO: 13) human PD1(29-85)

DRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPED

Building Block 1(SEQ ID NO: 14) human PD1(58-86)

NTSESFVLNWYRMSPSNQTDKLAAFPEDR

Building Block 1(SEQ ID NO: 15) human PD1(58-85)

NTSESFVLNWYRMSPSNQTDKLAAFPED

Building Block 1(SEQ ID NO: 16) human PD1(59-86)

TSESFVLNWYRMSPSNQTDKLAAFPEDR

Building Block 1(SEQ ID NO: 17) human PD1(59-85)

TSESFVLNWYRMSPSNQTDKLAAFPED

Structural unit 2(SEQ ID NO: 3): human PD1 (130-)

PKA

Structural unit 2(SEQ ID NO: 18): human PD1(127-

SLAPKAQ

Structural unit 2(SEQ ID NO: 19): human PD1(127-

SLAPKA

Structural unit 2(SEQ ID NO: 20): human PD1(128-133)

LAPKAQ

Structural unit 2(SEQ ID NO: 21): human PD1(128-132)

LAPKA

14C12H1L1(hG1TM)-Fab-HC-his(SEQ ID NO：4)：

EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGG SHHHHHH

Note: underlined are CDR sequences

14C12H1L1(hG1TM)-Fab-LC(SEQ ID NO：5)：

DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEOLKSGTASVVCLLNNFYPREAKV OWKVDNALOSGNSOESVTEODSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Note: underlined are CDR sequences

His-tag(SEQ ID NO：6)：

HHHHHH

Human PD1(1-170) -His 6 fusion protein sequence (SEQ ID NO: 7):

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVHHHHHH

amino acid sequence of the 14C12H1L1(hG1TM) heavy chain (SEQ ID NO: 8):

EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

amino acid sequence of 14C12H1L1(hG1TM) light chain (SEQ ID NO: 9):

DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

3. The peptide of claim 1 or 2, wherein the amino acid sequence of the PD-1 protein is as set forth in SEQ ID NO: 1 is shown.

4. The peptide of any one of claims 1-3, wherein structural unit 1 comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10-17, unit 2 comprises SEQ id no: 3 or SEQ ID NO: 18-21, preferably building block 1 comprises the sequence shown in any one of SEQ ID NO: 2, and building block 2 comprises SEQ ID NO: 3, and (b) is the sequence shown in the specification.

5. The peptide of any one of claims 1 to 4, wherein the antigen-binding fragment of the anti-PD-1 antibody is a Fab fragment, a Fab ', (Fab')₂Fab' -SH, Fab/c, Fv, single chain antibodies (e.g., scFv).

6. The peptide of claim 5, wherein the heavy chain amino acid sequence of the Fab fragment is as set forth in SEQ ID NO: 4, and the light chain amino acid sequence is shown as SEQ ID NO: 5, respectively.

7. The peptide of any one of claims 1 to 6, wherein amino acids T59, E61, S62, E84, D85 of the PD-1 protein bind to amino acids Y32, D33, S52, G54, Y57, Y100 of the heavy chain of the anti-PD-1 antibody (e.g., 14C12H1L1-Fab), amino acids P130, K131, A132 of the PD-1 protein bind to amino acids F325, E324, D323 of the light chain of the anti-PD-1 antibody, and the glycosylated side chain N58 of the PD-1 protein binds to amino acids S31, G53, G54, and R56 of the heavy chain of the anti-PD-1 antibody (e.g., 14C12H1L 1-Fab).

8. The peptide of claim 7, wherein the glycosylated side chain comprises mannose, N-acetylglucosamine; fucose and β -D-mannose.

9. Use of the peptide of any one of claims 1 to 8 for screening for an antibody or antigen-binding fragment thereof that binds to PD-1.