HK40015009B - Pd-l1 binding polypeptide or composite - Google Patents

Description

PD-L1 binding polypeptides or compounds

Technical Field

The invention relates to the field of medical biology, and discloses a high-resolution crystal structure of a compound of a PD-L1 blocking heavy chain single-domain antibody KN035 and PD-L1, and application of the crystal structure. The invention also relates to novel PD-L1 binding polypeptides or compounds developed based on the crystal structure and uses thereof.

Background

Programmed death-1 (PD-1) is a member of the CD28 receptor family, which includes CD28, CTLA-4, ICOS, PD-1 and BTLA. The original members of this family, CD28 and ICOS, were found to enhance T cell proliferation by addition of monoclonal antibodies (Hutloff et al (1999), nature 397:263-266; hansen et al (1980), immunogeneics 10:247-260). Two cell surface glycoprotein ligands for PD-1 have been identified, PD-L1 and PD-L2, which have been shown to down regulate T cell activation and cytokine secretion upon binding to PD-1 (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). PD-L1 (B7-H1) and PD-L2 (B7-DC) are both B7 homologs that bind to PD-1 but not to other CD28 family members (Blank et al 2004). Up-regulation of PD-L1 expression on the cell surface by IFN- γ stimulation has also been shown.

PD-L1 expression has been found in several murine and human cancers, including human lung Cancer, ovarian Cancer, colon Cancer, melanoma and various myelomas (Iwai et al (2002), PNAS 99:12293-7; ohigashi et al (2005), clin Cancer Res 11:2947-53). The existing results show that PD-L1, which is highly expressed by tumor cells, plays an important role in immune escape of tumors by increasing apoptosis of T cells. Researchers found that the P815 tumor cell line transfected with PD-L1 gene can resist the lysis of specific CTL in vitro, and has stronger tumorigenicity and invasiveness after being inoculated into mice. These biological properties can be reversed by blocking PD-L1. Mice knocked out of the PD-1 gene, which block the PD-L1/PD-1 pathway, were vaccinated with tumor cells and were unable to form tumors (Dong et al (2002), nat Med 8:793-800). PD-L1 has also been suggested to be likely to be associated with inflammation of the intestinal mucosa, and inhibition of PD-L1 prevented atrophy associated with colitis (Kanai et al (2003), J Immunol 171:4156-63).

Recently, immunotherapy using antibodies to block PD1/PD-L1 interactions has achieved dramatic clinical efficacy, showing sustained tumor suppression and improved patient survival. At least two PD1 antibodies (Optivo and Kytruda) have been batched and several PD-L1 antibodies have entered later clinical development. However, structural information on how these antibodies bind and block the PD1/PD-L1 interaction is very limited, preventing further development of the therapy.

Disclosure of Invention

The present invention provides an isolated polypeptide comprising the amino acid sequence shown in SEQ ID NO. 4, which polypeptide is capable of specifically binding to PD-L1 and blocking the interaction of PD-L1 and PD 1. In some embodiments, the polypeptide does not comprise the amino acid sequence of CDR1 and/or CDR2 of the antibody of SEQ ID NO. 1. The CDR1 amino acid sequence of the antibody of SEQ ID No. 1 may be selected from SEQ ID No. 2, 8 or 24 according to different definitions. The CDR2 amino acid sequence of the antibody of SEQ ID No. 1 may be selected from SEQ ID No. 3, 13 or 25 according to different definitions.

In some embodiments, the polypeptide consists of the amino acid sequence shown in SEQ ID NO. 4 (CDR 3 of the antibody of SEQ ID NO. 1).

As used herein, "PD-L1" or "hPD-L1" refers to human PD-L1. In some embodiments, it has the sequence of SEQ ID NO. 7.

The present invention provides a method of producing a PD-L1 binding polypeptide, the method comprising replacing CDR1 and/or CDR2 of an antibody of SEQ ID No. 1 with a CDR of an antibody that recognizes an additional target and/or a polypeptide that binds an additional target, thereby producing a polypeptide that binds PD-L1 and the additional target. Wherein the amino acid sequence of the CDR1 is shown as SEQ ID NO. 2, 8 or 24, and the amino acid sequence of the CDR2 is shown as SEQ ID NO. 3, 13 or 25.

As used herein, the term "additional target" refers to targets other than PD-L1, including but not limited to tumor antigens such as VEGFR, ERBB family proteins, CMET, or immune checkpoint related antigens such as CTLA 4.

The invention also provides a PD-L1 binding polypeptide which is a variant of the antibody of SEQ ID No. 1 and whose amino acid sequence corresponding to CDR1 and/or CDR2 of the antibody of SEQ ID No. 1 is replaced by a CDR of the antibody recognizing an additional target and/or a polypeptide binding to an additional target, said PD-L1 binding polypeptide being capable of binding to PD-L1 and said additional target. Wherein the amino acid sequence of the CDR1 is shown as SEQ ID NO. 2, 8 or 24, and the amino acid sequence of the CDR2 is shown as SEQ ID NO. 3, 13 or 25. The amino acid sequence of CDR3 of the antibody of SEQ ID No. 1 is shown as SEQ ID No. 4.

The invention also provides a method of producing a PD-L1 binding polypeptide, the method comprising grafting CDR3 of the antibody of SEQ ID No. 1 to an antibody that recognizes an additional target, thereby producing a polypeptide that binds PD-L1 and the additional target. Wherein the amino acid sequence of the CDR3 is shown as SEQ ID NO. 4. Many antibodies that recognize additional targets, such as VEGFR, CMET, CTLA4, or the like, are known in the art.

The invention also provides a method of producing a PD-L1 binding polypeptide, the method comprising grafting CDR3 of an antibody of SEQ ID No. 1 onto a non-immunoglobulin having a CDR loop-like structure, thereby enabling the non-immunoglobulin to bind PD-L1. Wherein the amino acid sequence of the CDR3 is shown as SEQ ID NO. 4. Such as CTLA4 proteins, fibronectin type III domains, etc., having a three-loop structure. In some embodiments, the CDR loop-like structure of the "non-immunoglobulin" is replaced with the CDR3 of the antibody of SEQ ID NO. 1.

The present invention also provides a method for producing a PD-L1 binding polypeptide comprising chemically modifying a polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 4 (corresponding to CDR3 of the antibody of SEQ ID NO. 1) so that it forms a stable helix. For example, the polypeptide may be chemically modified to form a stable helix structure as shown in CDR3 of the antibody of SEQ ID NO. 1 of the examples when it binds PDL 1. For example, the polypeptide may be formed into a helix by chemical coupling of TBMB.

The invention also provides a PD-L1 binding polypeptide produced by the method of the invention described above. In some embodiments, the PD-L1 binding polypeptides of the invention comprise the amino acid sequence of one of SEQ ID NOs 10, 12, 15-18, 20, 23.

The invention also provides a PD-L1 binding polypeptide that interacts (binds) with one or more of amino acid residues I54, Y56, E58, Q66, and R113 of PD-L1. In some embodiments, the binding polypeptide further interacts (binds) with one or more of amino acid residues D61, N63, V68, M115, S117, Y123, and R125 of PD-L1. In one embodiment, the PD-L1 binding polypeptide does not comprise SEQ ID NO. 2, 8 or 24, and/or SEQ ID NO. 3, 13 or 25, and/or SEQ ID NO. 4. In one embodiment, the PD-L1 binding polypeptide does not comprise SEQ ID NO. 1.

The invention also provides a crystal compound, which comprises an anti-PD-L1 single-domain antibody and an N-terminal immunoglobulin variable (IgV) domain of PD-L1, wherein the amino acid sequence of the anti-PD-L1 single-domain antibody is shown as SEQ ID NO. 1, and the amino acid sequence of the N-terminal immunoglobulin variable (IgV) domain of PD-L1 is shown as SEQ ID NO. 5. In some embodiments, the crystal complex belongs to space group P61 and the unit cell size isAnd α=β=90°, γ=120°.

The invention also provides a crystal of PD-L1, which belongs to the space group C2221 and has a unit cell size ofAnd α=β=γ=90°.

The invention also provides an atomic coordinate set or a subset thereof of the crystal structure of the above-mentioned crystal composite. In some embodiments, it is the set of atomic coordinates provided in appendix I or a subset thereof.

The present invention also provides a computer readable medium having recorded thereon data representing the atomic coordinates of the crystal structure of the above-described crystalline complex of the present invention or a subset thereof; or atomic coordinates provided in appendix I or a subset thereof; and/or a model generated using the atomic coordinates.

The present invention provides a computer-assisted method of identifying a compound that binds to PD-L1, the method comprising the steps of:

i) Interfacing the structure of the candidate compound with a structure defined by the atomic coordinates of the crystal structure of the invention or a subset thereof, or the atomic coordinates provided in appendix I, or a subset thereof, and

ii) identifying candidate compounds that can bind to PD-L1.

In some embodiments, the subset of atomic coordinates is the atomic coordinates corresponding to the N-terminal immunoglobulin variable (IgV) domain of PD-L1.

In some embodiments, the method further comprises synthesizing or obtaining the identified candidate compound, and determining whether the compound binds to PD-L1. Preferably, the compound blocks the binding of PD-L1 to PD 1.

The present invention provides a method of producing a compound that binds to PD-L1, comprising designing a compound molecule that binds to at least a portion of the interface defined by amino acid residues I54, Y56, E58, Q66, and R113 of PD-L1, synthesizing the compound molecule, and determining whether the compound binds to PD-L1. In some embodiments, the method comprises designing a compound molecule that binds to at least a portion of the interface defined by amino acid residues I54, Y56, E58, Q66, R113, D61, N63, V68, M115, S117, Y123, and R125 of PD-L1, synthesizing the compound molecule, and determining whether the compound binds to PD-L1. Preferably, the compound blocks the binding of PD-L1 to PD 1.

The present invention provides an anti-PD-L1 antibody that binds to a conformational epitope on PD-L1 defined by amino acid residues I54, Y56, E58, Q66, and R113. In some embodiments, the anti-PD-L1 antibody binds to a conformational epitope on PD-L1 defined by amino acid residues I54, Y56, E58, Q66, R113, D61, N63, V68, M115, S117, Y123, and R125.

Nucleic acids, vectors, and host cells

In another aspect, the invention relates to a nucleic acid molecule encoding a PD-L1 binding polypeptide of the invention. The nucleic acid of the invention may be RNA, DNA or cDNA. According to one embodiment of the invention, the nucleic acid of the invention is a substantially isolated nucleic acid.

The nucleic acids of the invention may also be in the form of a vector, may be present in and/or may be part of a vector, such as a plasmid, cosmid, or YAC. The vector may in particular be an expression vector, i.e. a vector which provides for expression of the PD-L1 binding polypeptide in vitro and/or in vivo (i.e. in a suitable host cell, host organism and/or expression system). The expression vector typically comprises at least one nucleic acid of the invention operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). The choice of the element and its sequence for expression in a particular host is common knowledge to the skilled person. Specific examples of regulatory elements and other elements useful or necessary for expression of the PD-L1 binding polypeptides of the invention, e.g., promoters, enhancers, terminators, integration factors, selectable markers, leader sequences, reporter genes.

The nucleic acids of the invention may be prepared or obtained by known means (e.g. by automated DNA synthesis and/or recombinant DNA techniques) based on information about the amino acid sequence of the polypeptides of the invention given herein and/or may be isolated from a suitable natural source.

In another aspect, the invention relates to a host cell expressing or capable of expressing one or more PD-L1 binding polypeptides of the invention and/or comprising a nucleic acid or vector of the invention. Preferred host cells of the invention are bacterial cells, fungal cells or mammalian cells.

Suitable bacterial cells include cells of gram-negative bacterial strains, such as e.g. Escherichia coli (Escherichia coli) strains, proteus (Proteus) and Pseudomonas (Pseudomonas) strains, and gram-positive bacterial strains, such as Bacillus (Bacillus) strains, streptomyces (Streptomyces) strains, staphylococcus (Staphylococcus) strains and Lactococcus (Lactococcus) strains.

Suitable fungal cells include cells of species of Trichoderma (Trichoderma), neurospora (Neurospora) and Aspergillus (Aspergillus); or cells of species including Saccharomyces (e.g., saccharomyces cerevisiae (Saccharomyces cerevisiae)), schizosaccharomyces (e.g., schizosaccharomyces pombe (Schizosaccharomyces pombe)), pichia (e.g., pichia pastoris (Pichia pastoris) and Pichia methanolica (Pichia methanolica)), and Hansen (Hansenula).

Suitable 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 used in the art for expression of heterologous proteins may also be used in the present invention.

The present invention also provides a method of preparing a PD-L1 binding polypeptide of the invention, which generally comprises the steps of:

-culturing a host cell of the invention under conditions allowing expression of a PD-L1 binding polypeptide of the invention; a kind of electronic device with high-pressure air-conditioning system

-recovering the PD-L1 binding polypeptide expressed by the host cell from the culture; a kind of electronic device with high-pressure air-conditioning system

-optionally further purifying and/or modifying the PD-L1 binding polypeptides of the invention.

In a preferred embodiment, the PD-L1 binding polypeptides of the invention are produced using mammalian cells.

The PD-L1 binding polypeptides of the invention can be produced in cells as described above in an intracellular manner (e.g., in the cytoplasm, in the periplasm or in inclusion bodies), then isolated from the host cell and optionally further purified; or it may be produced in an extracellular manner (e.g., in the medium in which the host cells are cultured), then isolated from the medium and optionally further purified.

Methods and reagents for recombinant production of polypeptides, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods of inducing protein expression, culture conditions, and the like are known in the art. Similarly, protein isolation and purification techniques suitable for use in methods of making the PD-L1 binding polypeptides of the invention are well known to those skilled in the art.

However, the PD-L1 binding polypeptides of the invention may also be obtained by other methods known in the art for producing proteins, such as chemical synthesis, including solid-phase or liquid-phase synthesis.

Immunoconjugates

In another aspect, the invention relates to PD-L1 binding polypeptides conjugated to a therapeutic moiety such as a cytotoxin, radioisotope, or biologically active protein. These conjugates are referred to herein as "immunoconjugates". Immunoconjugates comprising one or more cytotoxins are referred to as "immunotoxins". Cytotoxins include any agent that is detrimental to cells (e.g., killer cells). Examples include: paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, ipecine, mitomycin, epipodophyllotoxin glucopyranoside, epipodophyllotoxin thioglycoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrax-dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol and puromycin and their analogues or homologs.

Therapeutic agents useful for conjugation also include, for example: antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, amamide), alkylating agents (e.g., nitrogen mustard, chlorambucil, phenylalanine nitrogen mustard, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptavidin, mitomycin C and cisplatin (II) (DDP) cisplatin), anthranilotics (e.g., daunorubicin (formerly known as daunorubicin) and doxorubicin), antibiotics (e.g., actinomycin D (formerly known as actinomycin), bleomycin, mithramycin and Aflatoxin (AMC)), and antimitotics (e.g., vincristine and vinblastine).

Other preferred examples of therapeutic cytotoxins that can be conjugated to the PD-L1 binding polypeptides of the invention include sesquialter mycin, spinosad, maytansinoid, auristatin, and derivatives thereof.

Cytotoxins may be conjugated to PD-L1 binding polypeptides of the invention using linker techniques used in the art. Examples of types of linkers that have been used to conjugate a cytotoxin to a PD-L1 binding polypeptide include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. Alternatively, for example, a linker within the lysosomal compartment that is susceptible to cleavage by low pH or by a protease, such as a protease preferentially expressed in tumor tissue, such as a cathepsin (e.g., cathepsin B, C, D).

For further discussion of the types of cytotoxins, linkers and methods for conjugating therapeutic agents to antibodies, see Saito, g. Et al (2003) adv. Drug deliv. Rev.55:199-215; trail, p.a. et al (2003) cancer. Immunol. Immunother.52:328-337; payne, g. (2003) Cancer Cell 3:207-212; allen, t.m. (2002) nat.rev.cancer 2:750-763; pastan, i. and Kreitman, r.j. (2002) curr.opin.investig.drugs 3:1089-1091; senter, p.d. and Springer, c.j. (2001) adv. Drug deliv. Rev.53:247-264.

The PD-L1 binding polypeptides of the invention may also be conjugated to a radioisotope to produce a cytotoxic radiopharmaceutical, also known as a radioimmunoconjugate. Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine ¹³¹ Indium (indium) ¹¹¹ Yttrium ⁹⁰ And lutetium ¹⁷⁷ . Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, including ZevalinTM (IDEC Pharmaceuticals) and BexxarTM (Corixa Pharmaceuticals), and can be prepared using similar methods using the PD-L1 binding polypeptides of the present invention.

The PD-L1 binding polypeptides of the invention may also be conjugated to proteins having a desired biological activity and may be used to modify a particular biological response. Such biologically active proteins include, for example: toxins having enzymatic activity or active fragments thereof, such as abrin, ricin a, pseudomonas exotoxin or diphtheria toxin; proteins such as tumor necrosis factor or interferon-gamma; or a biological response modifier, such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), interleukin-10 ("IL-10"), granulocyte-macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other immune factors such as IFN, etc.

Techniques for conjugating such therapeutic moieties to antibody molecules are well known, see, e.g., arnon et al, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", monoclonal Antibodies And Cancer Therapy, reisfeld et al (ed.), pp.243-56 (Alan R.Lists, inc. 1985); hellstrom et al, "Antibodies For Drug Delivery", controlled Drug Delivery (2 nd Ed.), robinson et al (ed.), pp.623-53 (Marcel Dekker, inc. 1987); thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: a Review ", monoclonal Antibodies'84: biological And Clinical Applications, picchera et al (ed.), pp.475-506 (1985); "Analysis, results, and Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", monoclonal Antibodies For Cancer Detection And Therapy, baldwin et al (ed.), pp.303-16 (Academic Press 1985), and Thorpe et al, "The Preparation And Cytotoxic Properties Of Antibody-Toxin connections," immunol.Rev.,62:119-58 (1982).

Pharmaceutical composition

In another aspect, the invention provides a composition, e.g., a pharmaceutical composition, comprising one or a combination of a PD-L1 binding polypeptide of the invention and/or a compound that binds PD-L1 and/or an anti-PD-L1 antibody formulated with a pharmaceutically acceptable carrier. Such compositions may comprise one or a combination (e.g., two or more different) of the PD-L1 binding polypeptides or immunoconjugates of the invention. For example, the pharmaceutical compositions of the invention may contain a combination of antibody molecules that bind to different epitopes on a target antigen.

The pharmaceutical compositions of the invention may also be administered in combination therapy, i.e. in combination with other agents. For example, combination therapy may include a PD-L1 binding polypeptide or compound of the invention in combination with at least one other anti-neoplastic agent. For example, the PD-L1 binding polypeptides or compounds or antibodies of the invention may be used in combination with antibodies targeting other tumor-specific antigens. Such antibodies that target other tumor-specific antigens include, but are not limited to, anti-EGFR antibodies, anti-EGFR variant antibodies, anti-VEGFa antibodies, anti-HER 2 antibodies, or anti-CMET antibodies. Preferably, the antibody is a monoclonal antibody.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody molecule, immunoconjugate, may be encapsulated in a material to protect the compound from acids and other natural conditions that may inactivate the compound.

The pharmaceutical compositions of the present invention may comprise one or more pharmaceutically acceptable salts. By "pharmaceutically acceptable salt" is meant a salt that retains the desired biological activity of the parent compound without causing any undesirable toxicological effects (see, e.g., berge, S.M. et al (1977) J.Pharm. Sci.66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and non-toxic organic acids such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, as well as salts derived from nontoxic organic amines such as N, N' -dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) Water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) Oil-soluble antioxidants such as ascorbyl palmitate, butylated Hydroxyanisole (BHA), butylated Hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelators such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

These compositions may also contain adjuvants such as preserving, wetting, emulsifying and dispersing agents.

The prevention of the presence of microorganisms may be ensured by sterilization procedures or by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, and the like. In many cases, it is preferred to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium oxide in the composition. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion in the composition of delayed absorption agents, for example, monostearates and gelatins.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Conventional media or agents, except insofar as they are incompatible with the active compound, are possible in the pharmaceutical compositions of the present invention. Supplementary active compounds may also be incorporated into the compositions.

Therapeutic compositions must generally be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier may be a solvent or dispersant containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. For example, proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterile microfiltration. Generally, the dispersants are prepared by incorporating the active compound into a sterile carrier which contains a basic dispersion medium and the other required ingredients enumerated above. For sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) from a previously sterile-filtered solution thereof to yield a powder of the active ingredient plus any additional desired ingredient.

The amount of active ingredient that can be combined with the carrier material to prepare a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with the carrier material to prepare a single dosage form is generally the amount of the composition that produces a therapeutic effect. Typically, this amount ranges from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% of the active ingredient, on a 100% basis, in combination with a pharmaceutically acceptable carrier.

The dosage regimen can be adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus may be administered, several separate doses may be administered over time, or the dose may be proportionally reduced or increased as needed for the emergency of the treatment situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the desired pharmaceutical carrier. The specific description of dosage unit forms of the invention is limited to and directly depends on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) limitations inherent in the art for formulating such active compounds for use in the treatment of sensitivity in individuals.

For administration of the PD-L1 binding polypeptides or compounds or antibodies of the invention, the dosage range is about 0.0001 to 100mg/kg, more typically 0.01 to 20mg/kg of the recipient body weight. For example, the dosage may be 0.3mg/kg body weight, 1mg/kg body weight, 3mg/kg body weight, 5mg/kg body weight, 10mg/kg body weight or 20mg/kg body weight, or in the range of 1-20 mg/kg. Exemplary treatment regimens require weekly, biweekly, tricyclically, weekly, monthly, 3 months, 3-6 months, or a slightly shorter initial dosing interval (e.g., weekly to tricyclically) followed by longer post dosing intervals (e.g., monthly to 3-6 months).

Alternatively, the PD-L1 binding polypeptides or compounds or antibodies of the invention may be administered as sustained release formulations, in which case less frequent administration is required. Dosages and frequencies will vary depending on the half-life of the molecule in the patient. The dosage and frequency of administration will vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered at less frequent intervals over a long period of time. Some patients continue to receive treatment for the remainder of their lives. In therapeutic applications, it is sometimes desirable to administer higher doses at shorter intervals until the progression of the disease is reduced or stopped, preferably until the patient exhibits a partial or complete improvement in the symptoms of the disease. Thereafter, the patient may be administered a prophylactic regimen.

The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response to the particular patient, composition and mode of administration without toxicity to the patient. The dosage level selected will depend upon a variety of pharmacokinetic factors including the activity of the particular composition of the present invention or its esters, salts or amides, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular composition being used, the age, sex, weight, condition, general health and medical history of the patient undergoing treatment, and like factors well known in the medical arts.

The "therapeutically effective amount" of a PD-L1 binding polypeptide or compound or antibody of the invention preferably results in a decrease in severity of symptoms of the disease, an increase in the frequency and duration of the disease asymptomatic phase, or prevention of injury or disability due to suffering from the disease. For example, for the treatment of a PD-L1-associated tumor, a "therapeutically effective amount" preferably inhibits cell growth or tumor growth by at least about 10%, preferably by at least about 20%, more preferably by at least about 30%, more preferably by at least about 40%, more preferably by at least about 50%, more preferably by at least about 60%, more preferably by at least about 70%, more preferably by at least about 80% relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in an animal model system that predicts efficacy against human tumors. Alternatively, it may be assessed by examining the ability to inhibit cell growth, which inhibition may be determined in vitro by assays well known to those skilled in the art. A therapeutically effective amount of the therapeutic compound is capable of reducing tumor size or otherwise alleviating a symptom in a subject. Such amounts may be determined by one skilled in the art based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.

The compositions of the present invention may be administered by one or more routes of administration using one or more methods known in the art. Those skilled in the art will appreciate that the route and/or mode of administration will vary depending upon the desired result. Preferred routes of administration for the PD-L1 binding polypeptides or compounds or antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion. The phrase "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, typically injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

Alternatively, the PD-L1 binding polypeptides or compounds or antibodies of the invention may also be administered by a non-parenteral route, such as topical, epidermal or mucosal route, e.g., intranasal, oral, vaginal, rectal, sublingual or topical.

The active compounds can be prepared with carriers that protect the compound from rapid release, such as controlled release formulations, including implants, transdermal patches, and microcapsule delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid may be used. Many methods of preparing such formulations are patented or generally known to those skilled in the art. See, e.g., sustainedand controlled Release Drug Delivery Systems, j.r. robinson, ed., marcel Dekker, inc., new York,1978.

The therapeutic composition may be administered using medical devices known in the art. For example, in a preferred embodiment, the therapeutic compositions of the present invention may be administered using a needleless subcutaneous injection device, such as those described in U.S. Pat. Nos. 5,399,163;5,383,851;5,312,335;5,064,413;4,941,880;4,790,824; or 4,596,556. Examples of well known implants and modules that may be used in the present invention include: U.S. patent No.4,487,603, which discloses an implantable microinjection pump for dispensing a drug at a controlled rate; U.S. patent No.4,486,194, which discloses a therapeutic device for transdermal drug delivery; U.S. Pat. No.4,447,233, which discloses a medical infusion pump for delivering a drug at a precise infusion rate; U.S. Pat. No.4,447,224, which discloses a variable flow implantable infusion device for continuous delivery of a drug; U.S. Pat. No.4,439,196 discloses an osmotic drug delivery system having multiple compartments: and U.S. patent No.4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems and modules are known to those skilled in the art.

In certain embodiments, the PD-L1 binding polypeptides or compounds or antibodies of the invention may be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) prevents many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention are able to cross the BBB (if desired), they can be formulated, for example, in liposomes. As for the method of preparing liposomes, see, for example, U.S. Pat. nos. 4,522,811;5,374,548 and 5,399,331. Liposomes contain one or more targeting moieties that can be selectively transported into specific cells or organs, thereby enhancing targeted drug delivery (see, e.g., v.ranade (1989) j.clin.pharmacol.29:685). Examples of targeting moieties include folic acid or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al); mannosides (Umezawa et al (1988) biochem. Biophys. Res. Commun. 153:1038); antibody (P.G.Bloeman et al (1995) FEBS Lett.357:140; M.Owais et al (1995) Antimicrob.Agents chemther.39:180); surfactant protein A receptor (Briscoe et al (1995) am. J. Physiol. 1233:134); p120 (Schreier et al (1994) J.biol. Chem. 269:9090); see also k.keinanen; M.L.Laukkanen (1994) FEBS Lett.346:123; j. killion; fidler (1994) Immunomethods 4:273.

Disease prevention and treatment

In another aspect, the invention provides the use and method of the PD-L1 binding polypeptides or compounds or antibodies, nucleic acid molecules, host cells, immunoconjugates, and pharmaceutical compositions of the invention in the prevention and/or treatment of diseases associated with PD-L1. PD-L1-related diseases that can be prevented and/or treated with the PD-L1 binding polypeptides or compounds or antibodies of the invention are described in detail below.

Cancer of the human body

Blockade of PD-L1 by the PD-L1 binding polypeptides or compounds or antibodies of the invention can enhance the immune response to cancer cells in a patient. PD-L1 is enriched in a variety of human cancers (Dong et al (2002) Nat Med.8:78 7-9). The interaction of PD-1 with PD-L1 results in a decrease in lymphocytes that infiltrate the tumor, a decrease in T-cell receptor mediated proliferation, and immune escape of Cancer cells (Dong et al (2003) J Mol Med 81:281-7; blank et al (2004) Cancer Immunol Immunother [ epub ]; konishi et al (2004) Clin Cancer Res 10:5094-5100). Inhibiting the local interaction of PD-L1 with PD-1 can reverse immunosuppression, and the effect is synergistic when the interaction of PD-L2 with PD-1 is also blocked (Iwai et al (2002) PNAS 99:12293-7; brown et al (2003) J Immunol 170:1 257-66). The PD-L1 binding polypeptides or compounds or antibodies of the invention can be used alone to inhibit the growth of cancerous tumors. Alternatively, as described below, the PD-L1 binding polypeptides or compounds or antibodies of the invention may be used in combination with other anti-tumor therapies, for example, in combination with other immunogenic agents, standard cancer therapies, or other antibody molecules.

Accordingly, in one embodiment, the present invention provides a method of preventing and/or treating cancer comprising administering to the subject a therapeutically effective amount of a PD-L1 binding polypeptide or compound or antibody of the invention, inhibiting tumor cell growth in the subject.

Preferred cancers that may be prevented and/or treated using the PD-L1 binding polypeptides or compounds or antibodies of the invention include cancers that generally respond to immunotherapy. Non-limiting examples of preferred cancers that can be treated include lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma (e.g., metastatic malignant melanoma), kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, head and neck cancer, glioma, stomach cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine fibroids, and osteosarcoma. Examples of other cancers that may be treated with the methods of the invention include: bone cancer, pancreatic cancer, skin cancer, prostate cancer, skin or intraocular malignant melanoma, uterine cancer, anal region cancer, testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulval cancer, hodgkin's disease, non-hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, chronic or acute leukemia including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, childhood solid tumors, lymphocytic lymphomas, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central Nervous System (CNS) tumors, primary CNS lymphomas, tumor angiogenesis, spinal tumors, brain stem glioma, pituitary adenoma, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T cell lymphoma, environmentally induced cancers including asbestos-induced cancers, and combinations of said cancers. The invention is also useful for the treatment of metastatic cancers, in particular those expressing PD-L1 (Iwai et al (2005) Int Immunol 17:133-144).

Optionally, the PD-L1 binding polypeptides or compounds or antibodies of the invention can be used in combination with immunogenic agents such as cancer cells, purified tumor antigens (including recombinant proteins, peptides and carbohydrate molecules), cells transfected with genes encoding immunostimulatory cytokines (He et al (2004) J.Immunol 173:4919-28). Non-limiting examples of immunogenic agents that may be used include peptides of the melanoma antigen, such as gp100 peptide, MAGE antigen, trp-2, MART1 and/or tyrosinase, or tumor cells that express the cytokine GM-CSF after transfection.

In humans, some tumors have been shown to be immunogenic, such as melanoma. It is contemplated that tumor responses in a host may be activated by blocking PD-L1 using the PD-L1 binding polypeptides of the invention to promote T cell activation. When combined with tumor vaccination protocols, PD-L1 blockers (e.g., anti-PD-L1 antibodies, e.g., the PD-L1 binding polypeptides of the invention) are probably the most effective. Many experimental strategies for tumor vaccination have been devised (see Rosenberg, S.,2000,Development of Cancer Vaccines,ASCO Educational Book Spring:60-62;Logothetis,C,2000,ASCO Educational Book Spring:300-302;Khayat,D.2000,ASCO Educational Book Spring:414-428;Foon,K.2000,ASCO Educational Book Spring:730-738; see Restifo, N. And Sznol, M., cancer Vaccines, ch.61, pp.3023-3043, deVita, V., et al (eds.), fifth edition, 1997,Cancer:Principles and Practice of Oncology). In one of these strategies, autologous or allogeneic tumor cells are used to prepare the vaccine. These cell vaccines have proven to be most effective when tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a strong activator of antigen presentation for tumor vaccination (Dranoff et al (1993) Proa Natl. Acad. Sci U.S.A.90:3539-43).

Research into gene expression and large-scale gene expression patterns in various tumors identified a number of so-called tumor-specific antigens (Rosenberg, SA (1999) Immunity 10:281-7). In many cases, these tumor-specific antigens are differentiation antigens expressed in tumors and tumor-producing cells, such as gp100, MAGE antigen, and Trp-2. More importantly, many of these antigens were demonstrated to be targets for tumor-specific T cells found in the host. The PD-L1 binding polypeptides or compounds or antibodies of the invention may be used in combination with recombinantly produced tumor-specific proteins and/or peptides to generate immune responses against these proteins. These proteins are normally regarded by the immune system as self-antigens and are therefore tolerated. Tumor antigens may also include protein telomerase, which is essential for telomere synthesis of chromosomes and is expressed in more than 85% of human cancers, but only in a limited number of self tissues (Kim, N et al (1994) Science 266:2011-2013). The tumor antigen may also be a "neoantigen" expressed by cancer cells, for example, a fusion protein that alters the protein sequence due to somatic mutation or produces two unrelated sequences (e.g., bcr-abl in the Philadelphia chromosome).

Other tumor vaccines may include proteins from viruses associated with human cancers, such as Human Papilloma Virus (HPV), hepatitis virus (HBV and HCV), and Kaposi's Herpes Sarcoma Virus (KHSV). Another form of tumor-specific antigen that may be used in combination with a PD-L1 blocker (e.g., an anti-PD-L1 antibody, e.g., a PD-L1 binding polypeptide or compound or antibody of the invention) is a purified Heat Shock Protein (HSP) isolated from the tumor tissue itself. These heat shock proteins contain fragments of proteins from tumor cells, which HSPs are very effective in delivery to antigen presenting cells to elicit tumor immunity (Suot, R and Srivastava, P (1995) Science 269:1585-1588; tamura, Y. Et al (1997) Science 278:117-120).

Dendritic Cells (DCs) are strong antigen presenting cells that can be used to elicit antigen specific responses. DCs can be produced in vitro and carry various protein and peptide antigens and tumor cell extracts (Nestle, F. Et al (1998) Nature Medicine 4:328-332). DCs can also be transduced by genetic means to express these tumor antigens as well. DC have been fused directly to tumor cells for immunization (Kugler, A. Et al (2000) Nature Medicine 6:332-336). As an vaccination method, DC immunization may be effectively combined with a PD-L1 blocker (e.g. an anti-PD-L1 antibody, e.g. a PD-L1 binding polypeptide or compound or antibody of the invention) to activate a stronger anti-tumor response.

CAR-T, collectively known as chimeric antigen receptor T cell immunotherapy (Chimeric Antigen Receptor T-Cell Immunotherapy), is another effective cellular treatment of malignant tumors. Chimeric antigen receptor T cells (CAR-T cells) are obtained by coupling the antigen binding portion of an antibody recognizing a tumor antigen to the CD3- ζ chain or the intracellular portion of fceriγ in vitro to form a chimeric protein, and transfecting T cells of a patient by gene transduction to express the Chimeric Antigen Receptor (CAR). Meanwhile, a co-stimulatory molecule signal sequence can be introduced to improve the cytotoxic activity, proliferation and survival time of T cells and promote the release of cytokines. After the T cells of the patient are recoded, a large number of tumor-specific CAR-T cells can be generated by in vitro amplification and returned to the patient, so that the purpose of tumor treatment is realized. PD-L1 blockers (e.g., anti-PD-L1 antibodies, e.g., PD-L1 binding polypeptides or compounds or antibodies of the invention) can activate a stronger anti-tumor response in combination with CAR-T cell therapies.

The PD-L1 binding polypeptides or compounds or antibodies of the invention may also be combined with standard cancer therapies. The PD-L1 binding polypeptides or compounds or antibodies of the invention can be effectively combined with chemotherapeutic regimens. In these examples, it may reduce the dose of the chemotherapeutic agent administered (Mokyr, M.et al (1998) Cancer Research 58:5301-5304). An example of such a combination is the treatment of melanoma with a PD-L1 binding polypeptide or compound or antibody in combination with amiloride. Another example of such a combination is the treatment of melanoma with PD-L1 binding polypeptides or compounds or antibodies in combination with interleukin-2 (IL-2). The scientific principle of the combination of the PD-L1 binding polypeptides or compounds or antibodies of the invention and chemotherapy is cell death, which is the result of the cytotoxic effects of most chemotherapeutic compounds, should lead to elevated levels of tumor antigens in the antigen presentation pathway. Other combination therapies that may act synergistically with PD-L1 blockade through cell death are radiation therapy, surgery, and hormone deprivation. These protocols all produce a source of tumor antigen in the host. Angiogenesis inhibitors may also be combined with the PD-L1 binding polypeptides or compounds or antibodies of the invention. Inhibition of angiogenesis results in death of tumor cells, which can provide tumor antigens to the antigen presentation pathway of the host.

The PD-L1 binding polypeptides or compounds or antibodies of the invention may also be used in combination with antibodies targeting other tumor-specific antigens. Such antibodies that target other tumor-specific antigens include, but are not limited to, anti-EGFR antibodies, anti-EGFR variant antibodies, anti-VEGFa antibodies, anti-HER 2 antibodies, or anti-CMET antibodies. Preferably, the antibody is a monoclonal antibody.

The PD-L1 binding polypeptides or compounds or antibodies of the invention may also be used in combination with bispecific antigens that target fcα or fcγ receptor-expressing effector cells to tumor cells (see, e.g., US Patent nos.5,922.845 and 5,837,243). Bispecific antibodies can also be used to target two different antigens. For example, macrophages have been targeted to tumor sites using anti-Fc receptor/anti-tumor antigen (e.g., her-2/neu) bispecific antibodies. Such targeting can more effectively activate tumor-specific responses. The T cell aspect of these responses can be enhanced with PD-L1 blockers. Alternatively, the antigen may be delivered directly to the DCs using bispecific antibodies that bind to tumor antigens and dendritic cell-specific cell surface markers.

Tumors evade immune surveillance of the host by a variety of mechanisms. Many of these mechanisms can be overcome by inactivating tumor-expressed immunosuppressive proteins. TGF-beta (KehrL J. Et al (1986) J. Exp. Med. 163:1037-1050), IL-10 (Howard, M. And O' Garra, A. (1992) Immunology Today 13:198-200) and Fas ligand (Hahne, M. Et al (1996) Science 274:1363-1365) are among others included. Wherein each antibody may be used in combination with a PD-L1 binding polypeptide or compound or antibody of the invention to combat the effects of an immunosuppressant and to facilitate a tumor immune response in a host.

Other antibodies that may be used to activate a host immune response may be used in combination with the PD-L1 binding polypeptides or compounds or antibodies of the invention. anti-CD 40 antibodies are effective in replacing T cell helper activity (Ridge, J.et al (1998) Nature 393:474-478) and may be used in combination with PD-L1 binding polypeptides of the invention (Ito, N.et al (2000) Immunobiology 201 (5) 527-40). An activating antibody to a T cell co-stimulatory molecule such as OX-40 (Weinberg, A. Et al (2000) Immunol 164:2160-2169), 4-1BB (Melero, I. Et al (1997) Nature Medicine 3:682-685 (1997) and ICOS (Hutloff, A. Et al (1999) Nature 397:262-266) and an antibody blocking the activity of a negative co-stimulatory molecule such as CTLA-4 (e.g., U.S. Pat. No.5,811,097) or BTLA (Watanabe, N. Et al (2003) Natl Immunol 4:670-9), B7-H4 (Sica, GL et al (2003) Immunol 18:849-61) may also be combined in order to increase the level of T cell activation.

Bone marrow transplantation is currently used to treat a variety of tumors of hematopoietic origin. Graft versus host disease is a consequence of this treatment and the response of the graft to the tumor may be therapeutically beneficial. PD-L1 blockers can be used to increase the effectiveness of tumor-specific T cells. There are also several experimental treatment protocols involving ex vivo activation and expansion of antigen-specific T cells and adoptive transfer of these cells into recipients to combat tumors with antigen-specific T cells (Greenberg, r. And Riddell, s. (1999) Science 285:546-51). These methods can also be used to activate T cell responses to infectious agents such as CMV. Ex vivo activation in the presence of the PD-L1 binding polypeptides or compounds or antibodies of the invention is expected to increase the frequency and activity of adoptively transferred T cells. Accordingly, the invention also provides a method of activating immune cells (such as PBMCs or T cells) ex vivo comprising contacting the immune cells with a PD-L1 binding polypeptide or compound or antibody of the invention.

Infectious diseases

Other methods of the invention are useful for treating patients exposed to a particular toxin or pathogen. Accordingly, in a further aspect the present invention provides a method of preventing and/or treating an infectious disease in a subject comprising administering to the subject a PD-L1 binding polypeptide or compound or antibody of the invention such that the infectious disease in the subject is prevented and/or treated.

Similar to the use for tumors as described above, PD-L1 blockers can be used alone or as an adjuvant in combination with vaccines to stimulate an immune response to pathogens, toxins and self-antigens. Examples of pathogens for which the treatment method is particularly applicable include pathogens for which no effective vaccine is currently available, or pathogens for which conventional vaccines are not fully effective. Including but not limited to HIV, hepatitis virus (a, b, c), influenza virus, herpes virus, giardia, malaria, leishmania, staphylococcus aureus, pseudomonas aeruginosa. PD-L1 blockers are particularly useful against established infections by pathogens such as HIV, which present altered antigens during the course of infection. Upon administration of anti-human PD-L1 antibodies, these novel epitopes are recognized as foreign, thereby eliciting a strong T cell response that is unaffected by the negative signal of PD-L1.

Some examples of pathogen viruses that cause infectious diseases treatable with the methods of the invention include HIV, hepatitis (a, b, c), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II and CMV, EB virus), adenoviruses, influenza viruses, arboviruses, epox viruses, rhinoviruses, coxsackie viruses, coronaviruses, respiratory syncytial viruses, mumps viruses, rotaviruses, measles viruses, rubella viruses, parvoviruses, vaccinia viruses, HTLV viruses, dengue viruses, papillomaviruses, molluscs, polioviruses, rabies viruses, JC viruses, and arbovirus encephalitis viruses.

Some examples of pathogenic bacteria that cause infectious diseases treatable with the methods of the present invention include chlamydia, rickettsia, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and gonococci, klebsiella, proteus, ralstonia, pseudomonas, legionella, diphtheria, salmonella, bacillus, cholera, tetanus, botulinum, bacillus anthracis, plague, leptospira, and lyme disease bacteria.

Some examples of pathogenic fungi that cause infectious diseases treatable with the methods of the present invention include candida (candida albicans, candida krusei, candida glabrata, candida tropicalis, etc.), cryptococcus neoformans, aspergillus (aspergillus fumigatus, aspergillus niger, etc.), mucor (mucor, coluba, rhizopus), sporon sampsonii, blastomyces dermatitis, paracoccidiopsis brasiliensis, paracoccidiopsis crudus, and histoplasma membranaceus.

Some examples of pathogen parasites that cause infectious diseases treatable with the methods of the invention include Entamoeba histolytica, coccota ciliates, grignard, acanthamoeba, giardia, cryptosporidium, pycnopsis carinii, plasmodium vivax, babesia fructicola, trypanosoma brucei, trypanosoma cruzi, leishmania donovani, toxoplasma gondii, brazilian round-robinia.

In all of the above methods, the PD-L1 blocker may be combined with other forms of immunotherapy such as cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy that provides enhanced presentation of tumor antigens (see, e.g., holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; poljak (1994) Structure 2:1121-1123).

Autoimmune response

anti-PD-L1 antibodies can mount and amplify autoimmune responses. Thus, it is contemplated that vaccination protocols may be designed using the PD-L1 binding polypeptides or compounds or antibodies of the invention in combination with a variety of self-proteins to effectively generate an immune response against these self-proteins for use in the treatment of disease.

For example, alzheimer's disease involves inappropriate accumulation of aβ peptide in amyloid deposits in the brain; antibody responses against amyloid proteins are capable of scavenging these amyloid deposits (Schenk et al (1999) Nature 400:173-177). Other self-proteins can also be used as targets, such as IgE that is involved in the treatment of allergies and asthma, and tnfα that is involved in rheumatoid arthritis. Finally, PD-L1 binding polypeptides or compounds or antibodies can be used to induce antibody responses to various hormones. The response of neutralizing antibodies to reproductive hormones can be used for contraception. The response of neutralizing antibodies to hormones and other soluble factors required for growth of a particular tumor can also be considered as possible vaccination targets.

As described above, similar methods employing PD-L1 binding polypeptides or compounds or antibodies may be used to induce therapeutic autoimmune responses to treat patients with inappropriate autoantigen accumulation, such as amyloid deposits including aβ, cytokines such as tnfα and IgE in alzheimer's disease.

Chronic inflammatory diseases

The PD-L1 binding polypeptides or compounds or antibodies of the invention may also be used to treat diseases such as chronic inflammatory diseases, e.g., lichen planus, T cell mediated chronic inflammatory skin mucoses (Youngnak-Piconaratake et al (2004) Immunol Letters 94; 215-22). Accordingly, in one aspect, the invention provides a method of using T cells to eliminate chronic inflammatory disease comprising administering to a subject a PD-L1 binding polypeptide or compound or antibody of the invention.

Vaccine adjuvant

In one aspect, the invention provides the use of a PD-L1 binding polypeptide or compound or antibody of the invention as a vaccine adjuvant. By co-administering a PD-L1 binding polypeptide or compound or antibody and an antigen of interest (e.g., a vaccine), the PD-L1 binding polypeptide can be used to increase a specific immune response against the antigen.

Accordingly, in one aspect the invention provides a method of enhancing an immune response to an antigen in a subject comprising administering to the subject: (i) an antigen; and (ii) a PD-L1 binding polypeptide or compound or antibody of the invention such that the immune response to an antigen in the subject is enhanced. The antigen may be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen from a pathogen. Non-limiting examples of such antigens include those described in the section above, such as the tumor antigens (or tumor vaccines) described above, or antigens from the viruses, bacteria, or other pathogens described above.

Detection of

In another aspect the invention also provides a method of detecting the presence of PD-L1 or the expression level of PD-L1 in a biological sample, comprising contacting the biological sample and a control sample with a PD-L1 binding polypeptide or compound or antibody of the invention under conditions capable of forming a complex between the PD-L1 binding polypeptide or compound or antibody of the invention and PD-L1. Complex formation is then detected, wherein a difference in complex formation between the biological sample and the control sample is indicative of the presence of PD-L1 or the level of expression of PD-L1 in the sample.

PD-L1 has been found to be highly expressed in many tumors, or tumors or pathogens may cause immune cells near the site of infection by the tumor or pathogen to highly express PD-L1. Thus, the PD-L1 binding polypeptides or compounds or antibodies of the invention may be used to diagnose diseases associated with PD-L1, e.g., tumors or infectious diseases associated with high expression of PD-L1, such as viral infections.

In some embodiments, the PD-L1 binding polypeptides or compounds or antibodies of the invention are further conjugated with fluorescent dyes, chemicals, polypeptides, enzymes, isotopes, tags, and the like that are useful for detection or can be detected by other reagents.

Kit for detecting a substance in a sample

Also included within the scope of the invention are kits comprising a PD-L1 binding polypeptide or compound or antibody, immunoconjugate or pharmaceutical composition of the invention, and instructions for use. The kit may further comprise at least one additional agent or one or more additional PD-L1 binding polypeptides or compounds or antibodies of the invention (e.g., binding polypeptides that bind different epitopes of PD-L1). Kits typically include a label that indicates the intended use of the kit contents. The term label includes any written or recorded material provided on or with or otherwise with the kit.

Drawings

FIG. 1 shows the binding specificities of different members of the KN035 and B7/CD28 superfamily. EK293T cells were transfected with plasmids expressing PD-L1-EGFP, PD-L2-EGFP, mPD-L1-EGFP, B7H3-EGFP, ICOS-EGFP, B7H4-EGFP, respectively, and incubated with APC anti-human IgG Fc antibody or KN035-Fc+APC anti-human IgG Fc antibody. The signal was detected by flow cytometry. KN035 only showed high binding affinity to hPD-L1.

FIG. 2 shows the activity assay of KN 035. (A) Levels of INF-gamma secreted by CD4+ T cells after treatment with different concentrations of KN035 and Durvalumab. (B) Tumor inhibition activity of KN035 was evaluated by a transplanted mouse model. Where mice were vaccinated with a mixture of A375-hPD-L1 cells and PBMC (4:1 ratio), tumor volumes were measured continuously. KN035 showed strong antitumor effect at all three doses, whereas Durvalumab showed antitumor activity only at high concentrations (1 mg/kg). * p <0.05; ns, is not significant. KN035 as used herein is a fusion protein fused to an Fc domain.

FIG. 3 shows the general structure of KN 035/hPD-L1. Sequence and structure of (a) KN 035. The positions of CDR1, CDR2 and CDR3 and disulfide bridges (SS 1 and SS 2) are shown. (B) Structure of KN035/PD-L1 complex. PD-L1 is shown as a translucent surface. The secondary structures of PD-L1 and KN035 are also indicated on the figure.

FIG. 4 shows an overlap of the structure of the IgV domain of PD-L1. The PD-L1IgV domains from the PD-1/PD-L1 complex (PDB: 4ZQK, magenta) and KN035/PD-L1, the free PD-L1 structure resolved herein and the structure of PD-L1 reported previously (PDB: 5C 3T) overlap.

FIG. 5 shows the binding interface of KN035/PD-L1 complex. (A) Open view of KN035 (left) and PD-L1 (right) binding surfaces. (B) The electron cloud density map shows that the benzene ring of F101 residue KN035 and the aromatic rings of Y56 and F115 of PD-L1 overlap and form stable interactions with the surrounding hydrophobic residues. Affinity change of the C, PD-L1 mutant knot KN 035. Some specific KN035/PD-L1 interactions are shown as D, E, F. G, PD-L1 and PD-L2 sequence alignment based on three-dimensional crystal structure showed that PD1 and KN035 bind to the difference of P-L1 surface residues. Residues that bind PD1 are marked with open circles, those that bind KN035 are marked with solid inverted triangles, common residues are marked with triangular circles, and residues that bind PD1 are marked with open right triangles. H, the superimposed PD-L1/KN035 and PDL2 structures show that W110 of PD-L2 may hinder the binding of KN035 to PD-L2. .

FIG. 6 shows detailed binding interactions of KN035/PD-L1 and PD-1/PD-L1 interfaces.

FIG. 7 shows a comparison of the binding surfaces of PD-L1 and KN035 (A, C) and PD-L1 and PD1 (B, D).

FIG. 8 shows that chimeric antibodies m7 and m8 bind to human PDL1 protein.

Examples

Example 1 identification and structural analysis of PDL1 Single-Domain antibodies

Experimental materials and methods

Production of camelid single domain antibodies against hPD-L1

The PDL1-Fc fusion Protein for immunization was expressed by CHO cells (pCDNA 4, invitrogen, cat V86220) and purified by Protein A affinity chromatography. One Bactrian camel (Camelus bactrianus) from Xinjiang was selected for immunization. After 4 immunizations, lymphocytes from 100ml peripheral blood of camels were extracted and total RNA was extracted and reverse transcribed into cDNA using the Super-Script III FIRST STRANDSUPERMIX kit according to the instructions. Nucleic acid fragments encoding the variable regions of the heavy chain antibodies were then amplified using nested PCR to construct a phage display library of heavy chain single domain antibodies against PD-L1. The size of the storage capacity is 1.33 multiplied by 10 ⁸ The insertion rate reaches 100%.

Enrichment screening was performed against hPD-L1-Fc using PDL1-Fc fusion protein 10. Mu.g/well coated 96-well plates. High affinity phages were obtained after 4 rounds of screening. Monoclonal cells were randomly selected and amplified by culture. Positive clones verified by ELISA were sequenced, and clones with identical CDR1, CDR2, CDR3 sequences were regarded as the same antibody strain, while clones with different CDR sequences were regarded as different antibody strains. The obtained single domain antibody encoding gene was cloned into PET-32b (Novagen) and expressed and purified in E.coli. The blocking effect of PD-L1 single domain antibodies on PD-1 interaction with PD-L1 was investigated by competition ELISA.

hPD-L1 and preparation of a Complex with KN035

The gene encoding human PD-L1 amino acids 19-239 was cloned into pET-28a. The inclusion body-form protein with the C-terminal His tag (SEQ ID NO: 6) was expressed in E.coli BL21 (DE 3). Cells were cultured in LB at 37℃and induced with 0.5mM IPTG at OD600 of 1.0. After further incubation at 37℃for 16 hours, the cells were collected by centrifugation, resuspended in lysis buffer containing 20mM Tris-HCl pH7.4, 1% Triton X-100, 20mM EDTA and lysed by sonication. The inclusion bodies were recovered by centrifugation at 15000g for 10 min, washed 3 times with lysis buffer followed by washing with buffer without Triton X-100. The inclusion bodies were dissolved in 20mM Tris pH7.4 containing 6M guanidine hydrochloride, 500mM EDTA and 10mM DTT. The supernatant was collected by centrifugation and dialyzed against 10mM aqueous HCl. After dialysis, the sample was redissolved in 6M guanidine hydrochloride and added drop-wise to renaturation buffer (1M Arg hydrochloride,0.1M Tris pH8.0,2mM Na-EDTA,0.25mM oxidized glutathione and 0.25mM reduced glutathione). After overnight incubation at 4 ℃, the mixture was dialyzed against 10mm Tris ph8.0 and purified to homogeneity by a HisTrap Ni-Sepharose column, a hittrap SP ion exchange column and Superdex 75 (GE Healthcare). Other hPD-L1 variants, such as I54A, Y A, E A, D61A, N63A, Q A, V68A, R113A, M115A, S A, Y123A or R125A, are prepared by the same method.

To prepare the PD-L1/KN035 complex, the N-terminal IgV domain of hPD-L1 was similarly cloned into pET28a and expressed in E.coli as a C-terminal His-tagged protein (SEQ ID NO: 5). The renaturation is carried out in a renaturation buffer containing 0.1mg/ml KN035 protein. The PD-L1IgV domain/KN 035 complex (hereinafter referred to as PD-L1/KN035 complex) was then purified by ion exchange and gel filtration column (GE Healthcare).

hPD-L1 and crystals of a complex thereof with KN035

Purified PD-L1 and its complexes with KN035 were concentrated to approximately 15mg/ml and crystallization conditions were screened by drop-on vapor diffusion using commercially available buffers (Hampton Research, HR 2-110) in which 0.2. Mu.l of protein complex solution was mixed with 0.2. Mu.l of stock solution. After optimizing the conditions, the temperature was adjusted to 1.4M (NH ₄ )SO ₄ The 2M NaCl obtains PD-L1/KN035 crystal with diffraction quality. The crystals of PD-L1 were grown with 0.2mM ammonium acetate and 20% PEG3350 in precipitation solution.

Structure determination and refinement

The crystals were cryoprotected with 20% glycerol in the mother liquor and flash frozen in liquid nitrogen. Diffraction data were collected by X-ray diffraction and analyzed for structure.

Dissociation rate constant

Binding kinetics parameters of the hPD-L1 variant and KN035-Fc were measured by protein A sensor using a forte Bio Octet K2 device. All sensors were activated in PBS containing 0.1% w/v bovine serum albumin and agitated at 1000rpm in 96 well titer plates to minimize non-specific interactions. The final volume of the total solution was 200. Mu.l/well. The probe was saturated with 10. Mu.g/ml KN035 for 40s and then equilibrated in PBS+1% BSA for 60s. hPD-L1 variants were prepared as 2-fold serial dilutions (31.25, 62.5, 125, 250 and 500 nM) in 0.1% BSA and incubated with KN035 bound to the probe for 120s, respectively. The hPD-L1 variant was then dissociated for 320s, depending on the rate of dissociation observed. Baseline drift of all measurements was corrected by subtracting data from control sensors exposed only to running buffer. Data analysis and curve fitting were performed using Octet software. Since the affinity between hPD and hPD-L1 is very low (about 8. Mu.M), the affinity between PD-L1 variant and PD1 cannot be accurately measured.

Competitive and sandwich ELISA

ELISA plates were coated with hPD-L1-Fc dissolved in 50mM Na2CO3/NaHCO3, pH 9.6, at 2. Mu.g/ml. Plates were washed three times with PBST containing 0.05% Tween-20 and blocked with PBS containing 3% BSA for 1 hour, serial dilutions of KN035 were added to ELISA plates containing hPD-1-hIgG-biotin (10. Mu.g/ml) and incubated for 2h at 37 ℃. Binding was detected using horseradish peroxidase conjugated goat anti-human IgG, developed using TMB and the reaction stopped with sulfuric acid. The concentration was determined by measuring the absorbance at 450 nm.

Analysis of IFN-gamma production

PBMCs were obtained from heparinized peripheral blood samples of healthy donors by Ficoll-Hypaque density gradient centrifugation. After induction by TNF- α, mature dendritic cells were harvested and confirmed to be HLA-DR positive and PD-L1 positive by flow cytometry. Purified CD4T cells were grown at 10-20 in the presence of KN035 or Durvalumab: 1 was added to a U-bottom 96-well plate containing dendritic cells. Cells were incubated for 5 days. The supernatant was collected and the IFN-. Gamma.level was determined by ELISA kit according to the instructions.

In vivo study

To evaluate the in vivo antitumor effect of KN035, xenograft mouse models were prepared by subcutaneously inoculating a375 hPD-L1/human PBMC cells into NOD-SCID mice (6-12 weeks old, 6 per group). After 4 hours of tumor inoculation, KN035 antibody or Durvalumab was intraperitoneally administered, followed by once weekly administration for 4 weeks. Tumor size was measured along three perpendicular axes (a, b, and c) and calculated as tumor volume= (abc)/2. Tumor volume is greater than 2000mm ³ Is killed by carbon dioxide treatment.

Flow cytometry analysis

Binding of KN035-Fc and other B7/CD28 superfamily proteins was specifically assessed by flow cytometry. HEK293T cells were inoculated into complete DMEM medium in T75 flasks and transfected with plasmids expressing B7/CD28 superfamily proteins (PD-L1-EGFP, PD-L2-EGFP, mPD-L1-EGFP, B7H3-EGFP, ICOS-EGFP, B7H 4-EGFP), respectively. After 48 hours, cells were harvested and grouped. KN035-Fc was detected using an APC anti-human IgG Fc antibody. Data were acquired by BD FACSCalibur flow cytometer using BD Cellquest software. Data analysis was performed using FlowJo software.

Experimental results

Screening and identification of hPD-L1 single domain antibody KN035

A single domain antibody was identified, designated KN035, the sequence shown in SEQ ID NO:1. the antibody specifically bound to PD-L1 with a Kd value of 5.9nM, but did not specifically bind to PD-L2 or other members of this family (fig. 1). In the competition ELISA assay KN035 blocked the interaction of hPD-L1 and hPD1 with an EC50 of 420 ng/ml. KN035 was able to increase T cell responses and cytokine production in mixed lymphocyte reactions more effectively than Durvauumab when fused with Fc fragments (FIG. 2A). Whereas KN035 showed strong antitumor activity in the immune co-transplanted tumor model, and inhibited tumor growth more effectively than Durvalumab at lower concentrations (fig. 2B). These results show that KN035 is a potent inhibitor of the blockade of PD-1/PD-L1 interactions with strong antitumor activity.

Integral structure of KN035/PD-L1 complex

To further investigate the molecular mechanism of the PD-L1/KN035 interaction, we resolved the crystal structure of the complex of KN035 and the N-terminal immunoglobulin variable (IgV) domain of PD-L1 toWhereas free PD-L1 resolved to +.>Resolution of (a). Modeling and refinement to a good geometric model, results are shown in table 1. The crystal structure of the KN035/PD-L1 complex contains in asymmetric units the single N-terminal immunoglobulin variable (IgV) domains of KN035 and PD-L1 in a ratio of 1:1. Similar to other sdAb structures, KN035 has a typical IgV scaffold containing 4 Framework Regions (FR) forming the immunoglobulin domain core structure, and three hypervariable regions CDR1 (SEQ ID NO: 2), CDR2 (SEQ ID NO: 3) and CDR3 (SEQ ID NO: 4) loops consisting of 7, 3, and 18 amino acid residues, respectively (fig. 3A). The overall structure of KN035 was well overlapped with the previously disclosed sdAb structure (PDB: 1MEL, 1 HCV), and the Root Mean Square Deviation (RMSD) of the C.alpha.atom was +.>Similar to other sdabs from camels, KN035 has a conserved disulfide bond linking the B and F chains (SS 1: cys22-Cys 96). The CDR1 loop of KN035 forms a short alpha helix, while the CDR3 loop has a short alpha helix and a short 3 ₁₀ Helix, which is unique in sdabs. The short alpha helix of the CDR3 loop is held in the C chain of KN035 (FIG. 3) by an additional disulfide bond (SS 2: cys33-Cys 113), while the CDR3 loop is further stabilized by its hydrophobic interactions with the KN035 host.

KN035 binds to the IgV domain of PD-L1 through its CDR1 and CDR3, its CDR1 and CDR3 fill the CC' FG chain of PD-L1, burying the total surface area(FIG. 4). The binding of KN035 induces a slight conformational change in PD-L1 compared to the previously reported PD-L1 structure. In the KN035/PDL complex, the connecting loop connecting the C chain and the C' chain of PD-L1 was bent approximately +.>To interact with KN 035. Although the connecting loop connecting the C' chain and the D chain of PD-L1 is also offset by about +.>This is likely due to molecular packing in the crystal (fig. 4). These results are that the bonding surface of the surface PD-L1 is relatively rigid.

TABLE 1 crystallographic data collection and refinement statistics

KN035/PD-L1 interaction surface

The CDR1 and CDR3 loops of KN035 form a binding surface with a hydrophobic patch surrounded by a hydrophilic surface, complementary to PD-L1 (fig. 5, 6). A significant pi-pi stacking interaction was observed, in which the aromatic ring of Phe101 of KN035 was vertically stacked with the aromatic ring of Tyr56 of PD-L1 (fig. 5B), which was further stabilized by other hydrophobic residues (Val 109, leu108, ala114 and Phe115 in KN035CDR3 with Ile54, val68 and Met115 of PD-L1).

Mutation studies and subsequent affinity measurement experiments (fig. 5C) showed that the binding affinity to KN035 was reduced more than 200-fold after replacement of Tyr56 by Ala in PD-L1, whereas the affinity was reduced 40-fold after replacement of Ile54 by Ala in PD-L1 (fig. 5C, table 2). KN035 also formed 7 hydrogen bonds and 2 ionic bonds with PD-L1, involving 9 residues of KN035 and 6 residues of PD-L1 (Table 3). These polar interactions include a strong salt bridge between Asp99 of KN035 and Arg113 of PD-L1, the side chains of which are fully extended and stabilized by the action of surrounding residues (FIG. 5D). Substitution of Arg113 by Ala reduces the binding affinity between KN035 and PD-L1 by nearly 90-fold. The salt bridge of Arg113 is important for KN035, because the binding affinity of KN035 to mouse PD-L1, whose 113 position is Cys, is almost negligible. Glu58 of PD-L1 and Ser100 of KN035 form two hydrogen bonds (FIG. 5E), while the main chain or side chain of Gln66 and Thr105 of KN035 and Asp103 in the C' chain of PD-L1 form three hydrogen bonds (FIG. 5F). Similar substitutions of Glu58 and Gln66 in PD-L1 reduced KN035 binding affinities by 25-fold and 82-fold, respectively. Thus, these five residues (FIG. 5C) are likely to represent hot spot residues of the PD1/PD-L1 binding interface. Other residues in PD-L1 that are involved in the formation of hydrophobic or polar interactions also play an important role in stabilizing the KN035/PD-L1 complex With mutations at these residues all resulted in about 2-10 fold reduction in binding affinity (table 2). Interestingly, although residue Asp61 in the connecting loop between the C chain and C' chain of PD-L1 was shifted approximately towards KN035And the Ser29 and Ser30 residues in the CDR1 loop helix form hydrogen bonds, replacement of this residue with Ala only reduces affinity by a factor of 3.4. This means that the high binding affinity of KN035 to PD-L1 is mainly due to the interaction of CDR3 loop formation, while the interaction of CDR1 loop formation plays a secondary role.

Our preliminary screening showed that KN035 binds hPD-L1 with nanomolar affinity, but not hPD-L2. Based on the published structures of hPD1/hPD-L1, mPD 1/mPL 2 complexes and the KN035/PD-L1 complex structures shown herein, the sequences of hPDL1, hPDL2 and mPL 2 were aligned and residues involved in binding were highlighted (FIG. 5G). PDL2 has a shorter linker loop between the C and D chains of the IgV domain, whereas the corresponding longer linker loop in PDL1 forms the C and C' chains and is part of the binding surface of KN 035. The lack of this loop is expected to reduce the binding of PDL2 to KN 035. More importantly, when the structure of PDL2 and the structure of the PD-L1/KN035 complex are overlapped, it can be seen that Trp101 of PD-L2 (which is an important residue in the PD-1/PD-L2 binding interface, corresponding to Ala121 of PD-L1) will interfere with the CDR3 loop of KN035 and prevent the binding of PDL2 and KN035 due to its bulky side chain (fig. 5H). These results indicate that KN035 is a highly specific antibody against PD-L1 and will have less off-target effect in vivo.

TABLE 2 PD-L1 mutant and binding affinity

PD-1/PD-L1 structural comparison

Previous structural analysis has shown that PD1 has a topology of IgV type that binds PD-L1 through residues of the GFCC' chain (KN 035 through the CDR loop) (FIGS. 7A and B) with a total buried surface area ofHowever, PD1 binds to PD-L1 relatively weakly, with Kd of about 5. Mu.M, more than 800 times weaker than KN035. PD1 and KN035 interact similarly to the formation of a hotspot residue of PDL1, and their binding interface largely covers that of KN035 (FIGS. 7C and D). Arg113 of hPD-L1 and Glu136 of hPD1 form a salt bridge similar to the salt bridge with Asp99 of KN035 (FIGS. 5, 7B). However, this salt bridge in the hPD/hPD-L1 complex is relatively weak because the side chains of Arg113 and Glu136 are not well aligned (FIG. 6B). According to previous mutation studies on mouse PD1 and mouse PD-L1, the ionic interactions of this residue were not necessary in the mPD1/mPD-L1 interface, and the binding of the corresponding mutant (Cys 133 Tyr) to PD1 was increased by about 3-fold. Similarly, glu58, which provides about 25-fold binding affinity in hPD-L1 binding to KN035, is redundant or negative in mPD1 binding, and Glu58Ser mPD-L1 mutant binds to mPD1 about 3-fold more tightly. Likewise, the hydrophobic interaction between mPD1 and mPD-L1 appears to be predominantly at residue 115 (Met 115 in humans and Ile115 in mice) rather than Tyr56 as in PD-L1, the Ile115Ala mutant binds to mPD1 approximately 33-fold over wild-type mPD-L1, while the Tyr56Ser mutant binds to mPD1 identically to wild-type. In contrast, the key hydrophobic interactions at the KN035/hPD-L1 interface result from Tyr56, and similar hPD-L1 variants Met115Ala and Tyr56Ser reduced binding affinities to KN035 by a factor of 9 and 200, respectively. Furthermore, it has been shown that the hydrophobic interaction between mPD1 and mPD-L1 can be enhanced by a132L substitution in PD1, resulting in an increased binding affinity for both mPD-L1 and mPDL 2. Taken together, these results suggest that the binding interface of PD1 to PD-L1 is not as good as KN035.

Discussion of the invention

It is now clear that tumor cells often use immune checkpoint pathways as the primary mechanism of immune evasion, especially for T cells specific to tumor antigens. Because the ligand-receptor interactions of these checkpoints can be blocked by antibodies or recombinant ligands or receptors, the FDA has approved several antibodies against CTLA4 and PD-1 in these pathways for cancer immunotherapy, as well as many other antibodies under clinical investigation. However, there is no structural information on how these antibodies block these immune checkpoints. Herein, the co-crystallized structure of such anti-tumor antibodies KN035 and human PD-L1 upon complexation was reported for the first time, which paves the way for further development of antibodies of high binding affinity and specificity.

In previous structural studies on PD-1 and its ligands, the receptor/ligand binding surface was relatively flat (fig. 6B). We found that single domain antibody KN035 binds to the planar surface of PD-L1 primarily through its CDR3 loop, which forms an alpha helix and a unique short 3 ₁₀ The rotation angle of the spiral. The binding affinity of KN035 to PD-L1 nanomolar is mainly through hydrophobic and ionic interactions on its binding surface and fully utilizes all residues forming the binding interface. For example, residues Phe101 and Asp99 of KN035 are optimally aligned to interact with corresponding residues Tyr56 and Arg113 of the complementary binding surface of PD-L1, whereas the two residues of PD-L1 contribute little to the binding of PD-1 (fig. 6B and table 3). Another factor for the large difference in KN035 and PD1 binding affinities to PD-L1 is probably due to the flexibility of the CDR loops, which are adapted to interact with residues around the interface, whereas the binding surface of PD-1 is mainly formed by the beta-chain, the degree of freedom of which is limited. This may suggest that the interface between PD-1 and PD-L1 is not purposefully optimized for maximum in vivo binding affinity, whereas the intermediate binding affinity at the micromolar level between PD-1 and its ligand gives it optimal immune activation and inhibition.

Furthermore, the structure of the KN035 and PD-L1 complex can easily explain that KN035 cannot bind PDL2 because PDL2 has a short loop between the C chain and D chain and Tyr101 forms steric hindrance in PDL 2. The specific PD-L1 single domain antibodies can therefore be used in further studies to carefully analyze the role of PD-L1 and PD-L2 in tumors, which may be critical to direct the clinical use of different checkpoint blockers.

Although several crystal structures of PD1 and its ligand complex have been published, rational design for PD-1/PD-L1 surfaces has been rarely successful, largely due to the difficulty in targeting flat protein surfaces. The binding surface of KN035 identified herein will provide useful information for selecting peptides or chemical mimics based on the conformation of the CDR3 loop. More importantly, the semi-independent folding of the KN035CDR3 loop will be useful for the generation of bispecific or multispecific antibodies for combination immunotherapy.

Table 3 polar interaction between KNN035 and PD-L1 (distance. Ltoreq.))/>

Example 2 construction of variants of PDL1 Single-Domain antibodies based on structural analysis

Experimental materials and methods

Preparation of PDL1 Single-domain antibody mutant

Based on the crystal structure, the amino acid sequence (SEQ ID NO: 9) of the No. 10 single domain antibody (having a low affinity for human PDL 1) of patent application CN106397592A was used as a master, and the CDR1 and the immediately following cysteine residue sequences (SEQ ID NO: 8) and CDR3 (SEQ ID NO: 4) of the KN035 single domain antibody were replaced thereto to give mutant 1 (SEQ ID NO: 10). The amino acid sequence (SEQ ID NO: 11) of the 94 th single domain antibody (unable to block the interaction of human PD1 with PDL 1) of patent application CN106397592A was used as a master, and the CDR1 sequence (SEQ ID NO: 8) and CDR3 (SEQ ID NO: 4) of the KN035 single domain antibody were substituted thereto to obtain mutant 2 (SEQ ID NO: 12). The genes encoding these KN035 single domain antibody mutants were C-terminally added with His-tagged coding sequences and cloned into pCDNA4 mammalian expression vectors. The recombinant vector obtained was transiently transfected into suspension-cultured human HEK293 cells by PEI. After culturing for 6-7 days, taking culture supernatant, purifying by IMAC affinity chromatography in one step to obtain KN035 single domain antibody mutant protein.

With the sequence of KN035 single domain antibody (SEQ ID NO: 1) as master, the CDR2-KABAT sequence predicted from KABAT (SEQ ID NO: 13) was replaced with CDR2-KABAT (SEQ ID NO: 14) which was identical to the heavy chain of the Pertuzumab antibody of the VH3 subtype (US 7879325), to obtain a novel KN035 single domain antibody mutant sequence m3 (SEQ ID NO: 15). By the above method, an m3 mutant protein was further obtained.

The sequence (SEQ ID NO: 1) of KN035 single domain antibody is taken as a master, and the amino acid residues in the CDR2 (SEQ ID NO: 3) are replaced by Ala one by one, so as to obtain a series of mutant KN035 single domain antibody sequences m4, m5 and m6 (SEQ ID NO: 16-18). These mutant proteins were further obtained by the methods described above.

Preparation of PDL1 single domain antibody CDR3 chimeric antibody

The CDR3 sequence of KN035 single domain antibody (SEQ ID No: 4) was replaced with the framework of single domain antibody C38 (CN 201610332590.7) which did not recognize PDL1 (SEQ ID No: 19), to obtain a novel chimeric single domain antibody m7 (SEQ ID No: 20) in which KN035CDR3 sequence was chimeric. Or the CDR1+Cys (SEQ ID NO: 8) and CDR2-KABAT (SEQ ID NO: 13) sequences in the KN035 single domain antibody are replaced by the CDR1+Cys (SEQ ID NO: 21) and CDR2-KABAT (SEQ ID NO: 22) sequences in the C38 sequence to obtain the chimeric single domain antibody m8 (SEQ ID NO: 23). By the above method, a chimeric single domain antibody protein is further obtained.

Affinity between PD-L1 variant and PD 1.

The binding of the hPD-L1-Fc protein to each KN035 variant was measured using a forte Bio Octet K2 apparatus using the biological membrane interferometry technique (Bio-Layer Interferometry, BLI). The AHC sensor was used to cure PDL1-Fc protein in this experiment. The basic procedure is as described above, wherein AHC is cured Threshold 1nm and the control program is set to bind 60s and dissociate 100s. The dilution was 0.02PBST20% (pH 7.4), the regeneration solution was Glycine-HCl (pH 1.7), and the sample and regeneration solution were each 200. Mu.L in volume. The results obtained were analyzed on the Data using Data analysis 9.0 software. The KN035 variants were either prepared as 2-fold serial dilutions (31.25, 62.5, 125, 250 and 500 nM) or diluted directly to 100nM and 1. Mu.M depending on the nature.

Competitive ELISA investigating the blocking effect of KN035 variants on PD1-PDL1 interactions

ELISA plates were coated with hPD-L1-Fc dissolved in 50mM Na2CO3/NaHCO3, pH 9.6, at 2. Mu.g/ml. Plates were washed three times with PBST containing 0.05% Tween-20 and blocked with PBS containing 3% BSA for 1 hour, serial dilutions of KN035 variants were added to ELISA plates containing hPD-1-hIgG-biotin (10. Mu.g/ml) and incubated for 2h at 37 ℃. Binding was detected using horseradish peroxidase conjugated goat anti-human IgG, developed using TMB and the reaction stopped with sulfuric acid. The concentration was determined by measuring the absorbance at 450 nm.

Experimental results

Acquisition of KN035 single domain antibody CDR2 mutant and affinity and blocking function investigation.

A series of KN035 single domain antibody mutants with altered CDR2 regions are obtained by the transient expression of human HEK293 cells. The expression levels of these mutants were close to that of the wild-type KN035 single domain antibody. The purity of the protein obtained after the one-step purification of IMAC is more than 85% by SDS-PAGE non-reducing electrophoresis analysis. And then, investigating the binding condition of the mutants on the human PDL1 protein through fortibio, and comparing the obtained KD value with the wild type KN035, wherein the difference of the KD value and the wild type KN035 is within an order of magnitude. The activity of blocking human PDL1-PD1 interaction of the CDR2 mutants is examined by ELISA, and the proteins are found to have clear blocking functions, and the blocking activities of the proteins are between 70% and 130% relative to that of wild KN035 single domain antibodies.

Obtaining KN035CDR3 chimeric single domain antibody and binding condition with PDL1

Two KN035 single domain antibody CDR3 chimeras m7 and m8 were obtained by transient expression in human HEK293 cells. Fortibio analyzes its binding to human PDL1 protein, and clearly sees the binding and dissociation curves at high and low concentrations, thus demonstrating that both chimeras bind well to human PDL1 protein (FIG. 8).

Discussion of the invention

Co-crystallization results of KN035 single domain antibodies with its target protein PDL1 show that in KN035 single domain antibodies, the CDR3 sequence plays the most important role for PDL1 binding, whereas CDR2, including its surrounding amino acid residues (longer CDR2 region according to KABAT numbering, SEQ ID NO: 13) is essentially not involved in target binding.

The inventors examined a series of KN035 mutants and found that the substitution of the sequence of the CDR2 region under KABAT coding (SEQ ID NO: 13) with other single domain antibodies, with the CDR2 sequence of the heavy chain of the antibody, or directly with other non-functional amino acids, had substantially NO effect on the binding of the single domain antibody to PDL1 or on blocking of PDL1-PD 1. Similarly, the CDR1 sequences (including C-terminal Cys residues, SEQ ID NO: 8) and CDR3 sequences (SEQ ID NO: 4) of KN035 single domain antibodies can be substituted onto the backbones of other antibodies or similar antibodies, while maintaining their original activities. Although the dissociation constant KD values and blocking EC50 were slightly fluctuating, it was assumed that this was mainly due to the fluctuations in purity of the mutant proteins.

Considering that CDR3 is the main functional sequence of KN035 single domain antibodies, the inventors constructed CDR3 chimeric single domain antibodies and further examined their binding to PDL1 target protein. Both chimeric antibodies bind effectively to PDL1 protein.

Sequence listing

>SEQ ID NO:1 56

QVQLQESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLTTSGSTYLADSVKGRFTISQNNAKSTVYLQMNSLKPEDTAMYYCAADSFEDPTCTLVTSSGAFQYWGQGTQVTVSS

>SEQ ID NO:2

GKMSSRR

>SEQ ID NO:3

TTS

>SEQ ID NO:4

DSFEDPTCTLVTSSGAFQ

>SEQ ID NO:5Human PDL1-V

TVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNA

>SEQ ID NO:6Human PDL1-His

TVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTDKLAAALEHHHHHH

>SEQ ID NO:7 Human PDL1

TVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREENLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTD

>SEQ ID NO:8

GKMSSRRC

>SEQ ID NO:9 10

QVQLQESGGGSVQAGGSLRLSCAASGNIVSSYCMGWFRQAPGKERVGVAAIDSDGTTKYADSMKGRFTISKDNAKNTLDLQMNSLKPEDTAMYYCVARLNCPGPVDWVPMFPYRGQGTQVTVSS

>SEQ ID NO:10 m1

QVQLQESGGGSVQAGGSLRLSCAASGKMSSRRCMGWFRQAPGKERVGVAAIDSDGTTKYADSMKGRFTISKDNAKNTLDLQMNSLKPEDTAMYYCVADSFEDPTCTLVTSSGAFQYRGQGTQVTVSS

>SEQ ID NO:11 94

QVQLQESGGGSVQAGGSLRLSCAASLNIFSSYCMGWFRQAPGKQRVGVATIDSDGTTRYVDSVKGRFTISKDNAKNTLDLQMNSLKPEDTAMYYCAARLNCPGPVDWVPMFPYRGQGTQVTVSS

>SEQ ID NO:12 m2

QVQLQESGGGSVQAGGSLRLSCAASGKMSSRRCMGWFRQAPGKQRVGVATIDSDGTTRYVDSVKGRFTISKDNAKNTLDLQMNSLKPEDTAMYYCAADSFEDPTCTLVTSSGAFQYRGQGTQVTVSS

>SEQ ID NO:13

KLLTTSGSTYLADSVKG

>SEQ ID NO:14

DVNPNSGGSIYNQRFKG

>SEQ ID NO:15 m3

QVQLQESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVADVNPNSGGSIYNQRFKGRFTISQNNAKSTVYLQMNSLKPEDTAMYYCAADSFEDPTCTLVTSSGAFQYWGQGTQVTVSS

>SEQ ID NO:16 m4

QVQLQESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLATSGSTYLADSVKGRFTISQNNAKSTVYLQMNSLKPEDTAMYYCAADSFEDPTCTLVTSSGAFQYWGQGTQVTVSS

>SEQ ID NO:17 m5

QVQLQESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLTASGSTYLADSVKGRFTISQNNAKSTVYLQMNSLKPEDTAMYYCAADSFEDPTCTLVTSSGAFQYWGQGTQVTVSS

>SEQ ID NO:18 m6

QVQLQESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLTTAGSTYLADSVKGRFTISQNNAKSTVYLQMNSLKPEDTAMYYCAADSFEDPTCTLVTSSGAFQYWGQGTQVTVSS

>SEQ ID NO:19

QVQLQESGGGSVQAGGSLRLSCAASRYTASSNCMAWFRQAPGKEREGVATIYNGGGSTAYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCGAGSPRFCASATMTGGHHLFGYWGQGTQVTVSS

>SEQ ID NO:20 m7

QVQLQESGGGSVQAGGSLRLSCAASRYTASSNCMAWFRQAPGKEREGVATIYNGGGSTAYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCGAGSPRFCASDSFEDPTCTLVTSSGAFQYWGQGTQVTVSS

>SEQ ID NO:21

RYTASSNC

>SEQ ID NO:21

TIYNGGGSTAYADSVKG

>SEQ ID NO:23m8

QVQLQESGGGLVQPGGSLRLSCAASRYTASSNCMAWFRQAPGKERERVATIYNGGGSTAYADSVKGRFTISQNNAKSTVYLQMNSLKPEDTAMYYCAADSFEDPTCTLVTSSGAFQYWGQGTQVTVSS

>SEQ ID NO:24

GKMSSRRCMA

>SEQ ID NO:25

LTTSGS

Appendix I

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Sequence listing

<110> Kangangjie Biotech Co., ltd

<120> PD-L1 binding Polypeptides or Compounds

<130> P2018TC442

<160> 25

<170> PatentIn version 3.5

<210> 1

<211> 128

<212> PRT

<213> Artificial Sequence

<220>

<223> antibody Strain 56

<400> 1

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Lys Met Ser Ser Arg Arg

20 25 30

Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val

35 40 45

Ala Lys Leu Leu Thr Thr Ser Gly Ser Thr Tyr Leu Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asn Asn Ala Lys Ser Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Ala Asp Ser Phe Glu Asp Pro Thr Cys Thr Leu Val Thr Ser Ser

100 105 110

Gly Ala Phe Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 2

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 2

Gly Lys Met Ser Ser Arg Arg

1 5

<210> 3

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 3

Thr Thr Ser

1

<210> 4

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3

<400> 4

Asp Ser Phe Glu Asp Pro Thr Cys Thr Leu Val Thr Ser Ser Gly Ala

1 5 10 15

Phe Gln

<210> 5

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> Human PDL1-V

<400> 5

Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser Asn

1 5 10 15

Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu Ala

20 25 30

Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln Phe

35 40 45

Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg Gln

50 55 60

Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala Leu

65 70 75 80

Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys Met

85 90 95

Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val Asn

100 105 110

Ala

<210> 6

<211> 234

<212> PRT

<213> Artificial Sequence

<220>

<223> Human PDL1-His

<400> 6

Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser Asn

1 5 10 15

Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu Ala

20 25 30

Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln Phe

35 40 45

Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg Gln

50 55 60

Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala Leu

65 70 75 80

Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys Met

85 90 95

Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val Asn

100 105 110

Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro Val

115 120 125

Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys Ala

130 135 140

Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys Thr

145 150 155 160

Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr Ser

165 170 175

Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr Phe

180 185 190

Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile Pro

195 200 205

Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr Asp Lys Leu Ala

210 215 220

Ala Ala Leu Glu His His His His His His

225 230

<210> 7

<211> 221

<212> PRT

<213> Artificial Sequence

<220>

<223> Human PDL1

<400> 7

Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser Asn

1 5 10 15

Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu Ala

20 25 30

Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln Phe

35 40 45

Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg Gln

50 55 60

Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala Leu

65 70 75 80

Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys Met

85 90 95

Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val Asn

100 105 110

Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro Val

115 120 125

Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys Ala

130 135 140

Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys Thr

145 150 155 160

Thr Thr Thr Asn Ser Lys Arg Glu Glu Asn Leu Phe Asn Val Thr Ser

165 170 175

Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr Phe

180 185 190

Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile Pro

195 200 205

Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr Asp

210 215 220

<210> 8

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 8

Gly Lys Met Ser Ser Arg Arg Cys

1 5

<210> 9

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> antibody Strain 10

<400> 9

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asn Ile Val Ser Ser Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Val Gly Val

35 40 45

Ala Ala Ile Asp Ser Asp Gly Thr Thr Lys Tyr Ala Asp Ser Met Lys

50 55 60

Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Asp Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Val

85 90 95

Ala Arg Leu Asn Cys Pro Gly Pro Val Asp Trp Val Pro Met Phe Pro

100 105 110

Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 10

<211> 127

<212> PRT

<213> Artificial Sequence

<220>

<223> m1

<400> 10

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Lys Met Ser Ser Arg Arg

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Val Gly Val

35 40 45

Ala Ala Ile Asp Ser Asp Gly Thr Thr Lys Tyr Ala Asp Ser Met Lys

50 55 60

Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Asp Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Val

85 90 95

Ala Asp Ser Phe Glu Asp Pro Thr Cys Thr Leu Val Thr Ser Ser Gly

100 105 110

Ala Phe Gln Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 11

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> antibody Strain 94

<400> 11

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Leu Asn Ile Phe Ser Ser Tyr

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Val Gly Val

35 40 45

Ala Thr Ile Asp Ser Asp Gly Thr Thr Arg Tyr Val Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Asp Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Ala Arg Leu Asn Cys Pro Gly Pro Val Asp Trp Val Pro Met Phe Pro

100 105 110

Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 12

<211> 127

<212> PRT

<213> Artificial Sequence

<220>

<223> m2

<400> 12

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Lys Met Ser Ser Arg Arg

20 25 30

Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Val Gly Val

35 40 45

Ala Thr Ile Asp Ser Asp Gly Thr Thr Arg Tyr Val Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Asp Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Ala Asp Ser Phe Glu Asp Pro Thr Cys Thr Leu Val Thr Ser Ser Gly

100 105 110

Ala Phe Gln Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 13

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 13

Lys Leu Leu Thr Thr Ser Gly Ser Thr Tyr Leu Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 14

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2-KABAT of Pertuzumab antibody heavy chain

<400> 14

Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe Lys

1 5 10 15

Gly

<210> 15

<211> 128

<212> PRT

<213> Artificial Sequence

<220>

<223> m3

<400> 15

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Lys Met Ser Ser Arg Arg

20 25 30

Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val

35 40 45

Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asn Asn Ala Lys Ser Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Ala Asp Ser Phe Glu Asp Pro Thr Cys Thr Leu Val Thr Ser Ser

100 105 110

Gly Ala Phe Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 16

<211> 128

<212> PRT

<213> Artificial Sequence

<220>

<223> m4

<400> 16

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Lys Met Ser Ser Arg Arg

20 25 30

Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val

35 40 45

Ala Lys Leu Leu Ala Thr Ser Gly Ser Thr Tyr Leu Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asn Asn Ala Lys Ser Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Ala Asp Ser Phe Glu Asp Pro Thr Cys Thr Leu Val Thr Ser Ser

100 105 110

Gly Ala Phe Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 17

<211> 128

<212> PRT

<213> Artificial Sequence

<220>

<223> m5

<400> 17

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Lys Met Ser Ser Arg Arg

20 25 30

Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val

35 40 45

Ala Lys Leu Leu Thr Ala Ser Gly Ser Thr Tyr Leu Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asn Asn Ala Lys Ser Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Ala Asp Ser Phe Glu Asp Pro Thr Cys Thr Leu Val Thr Ser Ser

100 105 110

Gly Ala Phe Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 18

<211> 128

<212> PRT

<213> Artificial Sequence

<220>

<223> m6

<400> 18

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Lys Met Ser Ser Arg Arg

20 25 30

Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val

35 40 45

Ala Lys Leu Leu Thr Thr Ala Gly Ser Thr Tyr Leu Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asn Asn Ala Lys Ser Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Ala Asp Ser Phe Glu Asp Pro Thr Cys Thr Leu Val Thr Ser Ser

100 105 110

Gly Ala Phe Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 19

<211> 129

<212> PRT

<213> Artificial Sequence

<220>

<223> antibody C38

<400> 19

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Tyr Thr Ala Ser Ser Asn

20 25 30

Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Thr Ile Tyr Asn Gly Gly Gly Ser Thr Ala Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Gly Ala Gly Ser Pro Arg Phe Cys Ala Ser Ala Thr Met Thr Gly Gly

100 105 110

His His Leu Phe Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser

115 120 125

Ser

<210> 20

<211> 136

<212> PRT

<213> Artificial Sequence

<220>

<223> m7

<400> 20

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Tyr Thr Ala Ser Ser Asn

20 25 30

Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ala Thr Ile Tyr Asn Gly Gly Gly Ser Thr Ala Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Gly Ala Gly Ser Pro Arg Phe Cys Ala Ser Asp Ser Phe Glu Asp Pro

100 105 110

Thr Cys Thr Leu Val Thr Ser Ser Gly Ala Phe Gln Tyr Trp Gly Gln

115 120 125

Gly Thr Gln Val Thr Val Ser Ser

130 135

<210> 21

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> C38 CDR1 plus Cys

<400> 21

Arg Tyr Thr Ala Ser Ser Asn Cys

1 5

<210> 22

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> C38 CDR2-KABAT

<400> 22

Thr Ile Tyr Asn Gly Gly Gly Ser Thr Ala Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 23

<211> 128

<212> PRT

<213> Artificial Sequence

<220>

<223> m8

<400> 23

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Tyr Thr Ala Ser Ser Asn

20 25 30

Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val

35 40 45

Ala Thr Ile Tyr Asn Gly Gly Gly Ser Thr Ala Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asn Asn Ala Lys Ser Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Ala Asp Ser Phe Glu Asp Pro Thr Cys Thr Leu Val Thr Ser Ser

100 105 110

Gly Ala Phe Gln Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 24

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1

<400> 24

Gly Lys Met Ser Ser Arg Arg Cys Met Ala

1 5 10

<210> 25

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2

<400> 25

Leu Thr Thr Ser Gly Ser

1 5

Claims (3)

1. An isolated polypeptide comprising the amino acid sequence shown in SEQ ID NO. 4, said polypeptide being capable of specifically binding to human PD-L1 and blocking the interaction of PD-L1 and PD1,

wherein the polypeptide does not comprise the amino acid sequence of SEQ ID NO. 2 and/or SEQ ID NO. 3,

wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NOs:15-18, SEQ ID NO. 20 and SEQ ID NO. 23.

2. A crystalline complex comprising an anti-PD-L1 single domain antibody having an amino acid sequence as shown in SEQ ID No. 1 and an N-terminal immunoglobulin variable (IgV) domain of PD-L1 having an amino acid sequence as shown in SEQ ID No. 5, and wherein the crystalline complex belongs to space group P61 and has a unit cell size ofAnd α=β=90°, γ=120°.

A crystal of pd-L1 belonging to space group C2221 and having a unit cell size ofAnd α=β=γ=90°, the amino acid sequence of the PD-L1 is shown in SEQ id No. 6. / >