CN109721657A - Block the fusion protein and application thereof of PD-1/PD-L1 signal transduction path and activating T cell - Google Patents

Abstract

The present invention provides a kind of blocking PD-1/PD-L1 signal transduction path and the fusion proteins of activating T cell, and it includes the antigen-binding fragments that (i) is derived from anti-PD-1 antibody and/or anti-PD-L1 antibody；(ii) immunoglobulin Fc domain；(iii) CD80 extracellular domain (ECD).The present invention also provides the polynucleotides of encoding said fusion protein, the carrier comprising the polynucleotides, the host cell comprising the polynucleotides or carrier and the fusion proteins to treat in individual, prevents and/or diagnose to be activated the purposes being suppressed with T cell function in relevant disease to PD-1/PD-L1 signal transduction path.

Description

Block PD-1/PD-L1 signal transduction path and the fusion protein of activating T cell and its Purposes

Technical field

Present invention relates in general to technical field of pharmaceutical biotechnology.In particular it relates to be derived from anti-program comprising (i) Property (programmed death-1 (the PD-1)) antibody of death protein -1 and/or anti-death protein ligand 1 The antigen-binding fragment of (programmed death-1 ligand (PD-L1)) antibody；(ii) immunoglobulin Fc domain； The fusion protein of (iii) CD80 extracellular domain (ECD), encoding said fusion protein polynucleotides, include the multicore The carrier of thuja acid, the host cell comprising the polynucleotides or carrier and the fusion protein are treated in individual, are prevented And/or diagnosis is activated the purposes being suppressed in relevant disease with T cell function to PD-1/PD-L1 signal transduction path.

Background technique

Immunologic test point (immune checkpoint) is one kind inhibition signaling molecule present in immune system, is led to It overregulates the duration being immunoreacted in peripheral tissues and intensity avoids tissue damage, and participate in maintaining for the resistance to of autoantigen By (Pardoll DM., The blockade of immune checkpoints in cancer immunotherapy.Nat Rev Cancer,2012,12(4):252-264).The study found that tumour cell can escape vivo immuning system and increasing out of control The reason of growing first is that the inhibition signal path of immunologic test point is utilized, T lymphocyte activity is thereby inhibited, so that T Lymphocyte cannot effectively play lethal effect (Yao S, Zhu Y and Chen L., the Advances in tumour targeting cell surface signaling molecules for immune modulation.Nat Rev Drug Discov,2013,12(2):130-146)。

Death protein -1 (PD-1) is a kind of important immunologic test point albumen, at present and immunotherapy of tumors An important target.PD-1 was found for the first time in 1992, can after showing PD-1 activation to its gene cloning and expression Inducing T cell programmed death.There are PD-1 albumen for discovery in the T cell, B cell and myeloid cell of activation.PD-1 is also Inductivity it is expressed in macrophage, Dendritic Cells and monocyte.It is expressed in the lymphocytic cell surface of tranquillization without PD-1.

PD-1 is a kind of 55kDa I type transmembrane protein, and cytoplasmic domain contains an immunity receptor Tyrosine Inhibitory Motifs, with CD28 and CTLA-4 has homology.Two kinds of cell surface glycoprotein ligands of PD-1, respectively programmed death are identified Protein ligands 1 (PD-L1) and death protein ligand 2 (PD-L2).Matching for PD-1 is had found on many cancer cells Body surface reaches, including human lung cancer, oophoroma, colon cancer and a variety of myeloma.In addition, in all kinds of epitheliomas, blood cancer and other evils Property tumor cell surface on high expression PD-1 ligand.In tumor patient, the expression of the ligand of PD-1 such as PD-L1 is often and cancer Prognosis mala correlation (Iwai Y et al., Involvement of PD-L1on tumor cells in the escape From host immune system and tumor immunotherapy by PD-L1blockade, PNAS, 2002,99 (19):12293-7)。

The combination of the ligand of PD-1 and PD-1 is important for adjusting T lymphocyte activity and peripheral immune tolerance being maintained to play Effect.It can lead to t cell proliferation, immunological unresponsiveness, T cell " exhaustion " and secretion IL-10 after the ligand binding of PD-1 and PD-1 Deng.Therefore, PD-1 plays restricted T cells activation, inhibits T cell proliferation and improves the function to the tolerance of antigen.Activation The expression up-regulation of lymphocytic cell surface PD-1 can result in the inhibition to acquired or intrinsic immune response, consequently lead to (including T lymphocyte) is although tumor infiltrating lymphocyte has specific for tumour antigen, due to tumour cell The ligand of upper PD-1 produces in conjunction with the PD-1 on tumor infiltrating lymphocyte inhibits tumor infiltrating lymphocyte activation Signal, so that tumour cell can escape killing of the immune system to tumour cell.

Studies have shown that the tumor infiltrating lymphocyte of these expression PD-1 is dysfunction type lymphocyte, the leaching The antibody that the biological function of bar cell can pass through the ligand binding for blocking PD-1 and PD-1 restores.Currently, inhibit PD-1 with The antibody of the ligand binding of PD-1 mainly includes anti-PD-1 monoclonal antibody and anti-PD-L1 monoclonal antibody, but is also had for PD- The product of L2.

Currently, the anti-PD-1 antibody for studying comparative maturity has the Wu Dankang that receives of Bristol Myers Squibb (BMS) company (Nivolumab) and the pyridine aldoxime methyliodide (PAM) monoclonal antibody (Pembrolizumab) of Merck (Merck) company.Receive Wu Dankang (trade name) be full-length human IgG4 antibody molecule, pyridine aldoxime methyliodide (PAM) monoclonal antibody (trade name) it is humanization IgG4 antibody molecule.The anti-PD-1 monoclonal antibody in conjunction with the PD-1 in T lymphocyte after be able to suppress PD-1 and match with it Thus the combination of body PD-L1 and PD-L2 promote T lymphocyte activation, proliferation and generate immune activation cytokines such as IL- 2, and release inhibition of the PD-1 to T lymphocyte immunosurveillance with anti-tumor activity.U.S. Food and Drug Administration The military monoclonal antibody indication of receiving ratified at present includes: melanoma, non-small cell lung cancer, kidney, head and neck neoplasm etc.；Pyridine aldoxime methyliodide (PAM) monoclonal antibody Indication includes: head and neck neoplasm, non-small cell lung cancer, melanoma etc..It is researched and developed about anti-PD-L1 antibody, Roche (Roche) Avelumab, A Sili of atezolizumab, Merck KGaA (Merck KGaA) and Pfizer (Pfizer) cooperative development The durvalumab of Kang Yanfa also shows the therapeutic effect to tumour.

Although anti-PD-1 antibody, anti-PD-L1 antibodies on tumor have therapeutic effect, their average treated effects are only It is 20% or so, the five year survival rate of lung cancer only 16%.Still there is significant component of tumor patient to using anti-PD-1 antibody, anti- The treatment of PD-L1 antibody is unresponsive.Therefore, how to improve the validity of oncotherapy is still that current therapeutic field of tumor compels to be essential The problem to be solved.

On the other hand, the activation of T cell needs two signals model: the first signal is by T cell antigen receptor (TCR) and antigen Antigenic Peptide MHC (major histocompatibility complex) molecular complex on presenting cells (APC), which combines, to be provided, second signal by Costimulatory molecules on APC provide in conjunction with the respective ligand in T cell.In the costimulatory molecules for participating in t cell activation, T The combination of cell surface CD28 molecule and the surface APC respective ligand CD80 and CD86 play a significant role.But in tumour micro-loop CD80 and CD86 low expression or do not expressed in border, this is also to cause one of important mechanisms of immune escape.

CD80 and CD86 is transmembrane glycoprotein, belongs to immunoglobulin superfamily (IgSF) member.Mature CD80 molecule It is made of 254 amino acid, wherein 208 amino acid of extracellular domain (ECD), 25 amino acid of transmembrane domain and knot intracellular 21, structure domain amino acid.Similarly, mature CD86 molecule is made of 303 amino acid, wherein 222 amino of extracellular domain 61 acid, 20 amino acid of transmembrane domain and intracellular domain amino acid.The extracellular domain of CD80 and CD86 includes immune The area (IgV) globulin V and the area immunoglobulin C (IgC), CD80 and CD86 pass through immunoglobulin V (IgV) Qu Yuqi ligand CD28 and CTLA-4 is combined.In the situation of CD80 and CD86 in conjunction with CD28, CD80 and CD86 are living for antigen induced T-cell Change, the generation of proliferation and effector function have important adjustment effect, are positive regulating factors；And in CD80 and CD86 and CTLA-4 In conjunction with situation, it is negativity regulatory factor that CD80 and CD86, which lower immune response,.Therefore, CD80 and CD86 is that T lymphocyte is living Collaboration stimulating factor when change plays a significant role in autoimmunity monitoring, humoral immune response and allograft reaction.

In addition, CD80 can also be by blocking PD-1/PD-L1 to interact, to participate in siberian crabapple in conjunction with PD-L1 The activation of system.

In summary as it can be seen that influence of the CD80 protein to t cell response be it is complicated, influence in actual test Before be unpredictable (Salomon, B et al., Complexities of CD28/B7:CTLA-4 costimulatory Pathways in autoimmunity and transplantation.Annual review of immunology, 2001,19:225-252).

Since PD-1/PD-L1 signal transduction path is activated and T cell function is suppressed and all facilitates the generation of tumour And development, accordingly, there exist research and development to have interference, inhibit or block PD-1/PD-L1 signal transduction path and activating T cell this two The needs of the active albumen of kind.

The present inventor develops one group of interference, inhibition or blocks PD-1/PD-L1 signal transduction path by sharp study And the fusion protein of activating T cell, the fusion protein are able to suppress PD-1/PD-L1 signal transduction path and T can be induced thin Born of the same parents activation, thus, it is possible in individual treat, prevent and/or diagnosis with PD-1/PD-L1 signal transduction path be activated and T cell function is suppressed relevant disease, be particularly capable of for PD-1 antibody and/or PD-L1 Antybody therapy without anti- Answer or it is weakly reactive individual in improve tumor immune response.

Summary of the invention

The invention discloses a kind of novel fusion eggs for blocking PD-1/PD-L1 signal transduction path and activating T cell White, encoding said fusion protein polynucleotides, the carrier comprising the polynucleotides, comprising the polynucleotides or carrier Host cell and the fusion protein are treated in individual, prevent and/or are diagnosed and PD-1/PD-L1 signal transduction path quilt Activation and T cell function are suppressed the purposes in relevant disease.

Therefore, in one aspect, the present invention provides a kind of novel fusion protein, the fusion protein inhibits PD-1/ PD-L1 signal transduction path and can inducing T cell activation, it is anti-derived from anti-PD-1 antibody and/or anti-PD-L1 that it includes (i) The antigen-binding fragment of body；(ii) immunoglobulin Fc domain；(iii) CD80 extracellular domain (ECD).Implement at one In scheme, (i) of the fusion protein is Fab, Fab', F derived from anti-PD-1 antibody and/or anti-PD-L1 antibody (ab')₂, Fv, scFv.In one embodiment, (ii) of the fusion protein is human immunoglobulin(HIg) Fc structure Domain.In one embodiment, (iii) of the fusion protein includes people CD80ECD.

(i) antigen-binding fragment for including in the fusion protein can be derived from any anti-PD-1 antibody, as long as energy The enough antibody inhibited or reduce PD-1 and its ligand binding, including anti-PD-1 antibody well known in the prior art and future grind The anti-PD-1 antibody issued.In one embodiment, the antigen-binding fragment include selected from SEQ ID NO:1/2,3/4, 5/6,7/8,9/10,11/12,13/14,15/16,17/18,19/20,21/22,23/24 and 25/26 pairs of heavy chain variable region Whole 6 heavy chain CDR and light chain CDR contained in sequence/light-chain variable sequence, it is preferable that the antigen-binding fragment packet Containing selected from SEQ ID NO:1/2,3/4,5/6,7/8,9/10,11/12,13/14,15/16,17/18,19/20,21/22,23/ 24 and 25/26 pairs of weight chain variabl area sequence/light-chain variable sequence, or with the pairs of weight chain variabl area sequence/light chain Variable region sequences have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence The sequence of identity；It is highly preferred that the antigen-binding fragment includes selected from receiving Wu Dankang, pidilizumab and pyridine aldoxime methyliodide (PAM) monoclonal antibody Anti- PD-1 antibody heavy chain variable region and light chain variable region, particularly, the antigen-binding fragment be selected from receive Wu Dankang, Fab, Fab', F (ab') of pidilizumab and pyridine aldoxime methyliodide (PAM) monoclonal antibody₂, Fv, scFv.

(i) antigen-binding fragment for including in the fusion protein can also be derived from any anti-PD-L1 antibody, as long as It is able to suppress or reduces antibody of the PD-L1 in conjunction with its receptor (such as with PD-1 or CD80 (B7-1) or in conjunction with the two) i.e. Can, the anti-PD-L1 antibody developed including anti-PD-L1 antibody well known in the prior art and future.In one embodiment, The antigen-binding fragment includes pairs of weight chain variabl area sequence/light chain selected from SEQ ID NO:27/28,29/30 and 31/32 Whole 6 heavy chain CDR and light chain CDR contained in variable region sequences.Preferably, the antigen-binding fragment includes to be selected from SEQ Pairs of weight chain variabl area sequence/light-chain variable sequence of ID NO:27/28,29/30 and 31/32, or with the pairs of heavy chain Variable region sequences/light-chain variable sequence have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, the sequence of 99% or more sequence identity；It is highly preferred that the antigen-binding fragment be selected from atezolizumab, Fab, Fab', F (ab') of avelumab and durvalumab₂, Fv, scFv.

In one embodiment, the constant region of light chain type in the antigen-binding fragment can be κ type or λ type, excellent Selection of land is κ type.The κ type chain constant region amino acid sequence of exemplary anti-PD-1 antibody is shown in SEQ ID NO:33.SEQ The λ type chain constant region amino acid sequence of exemplary anti-PD-1 antibody is shown in ID NO:34.

(ii) immunoglobulin Fc domain for including in the fusion protein can be any immunoglobulin Fc structure Domain, particularly, (ii) are human immunoglobulin(HIg) Fc structural domains.In one embodiment, the immunoglobulin Fc knot Structure domain is the Fc structural domain of IgG class antibody, in particular IgG₁Subclass, IgG₂Subclass, IgG₄The Fc structural domain of Subclass Antibodies.? In one preferred embodiment, the immunoglobulin Fc domain being contained in fusion protein of the present invention is IgG₄Subclass The Fc structural domain of antibody, in particular human IgG₄The Fc structural domain of Subclass Antibodies.In one embodiment, the IgG₄Subclass Antibody includes amino acid replacement, especially amino acid replacement S228P at the position S228 (EU number) in the area Fc.SEQ ID Exemplary IgG is shown in NO:35₁The light chain constant region amino acid sequence of the anti-PD-1 antibody of subclass.It is shown in SEQ ID NO:36 Exemplary IgG is shown₂The light chain constant region amino acid sequence of the anti-PD-1 antibody of subclass.It is shown in SEQ ID NO:37 exemplary IgG₄The light chain constant region amino acid sequence of the anti-PD-1 antibody of subclass.Exemplary IgG is shown in SEQ ID NO:38₁Subclass is anti- The Fc domain amino acid sequence of PD-1 antibody.Exemplary IgG is shown in SEQ ID NO:39₂The Fc of the anti-PD-1 antibody of subclass Domain amino acid sequence.Exemplary IgG is shown in SEQ ID NO:40₄The Fc domain amino acid of the anti-PD-1 antibody of subclass Sequence.In some embodiments, (ii) immunoglobulin Fc domain for including in fusion protein be with SEQ ID NO:38, Amino acid sequence shown in 39 or 40 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Or more sequence identity Fc structural domain.

In one embodiment, (iii) CD80ECD for including in the fusion protein is the extracellular domain of CD80 A part.In one embodiment, the CD80ECD includes the area (CD80-IgV) CD80 immunoglobulin V (IgV).One In a embodiment, the CD80ECD includes the area CD80 immunoglobulin V and the area C (CD80-IgVIgC).In an embodiment party In case, the CD80ECD is people CD80ECD, and the preferably described CD80ECD includes people CD80IgV.In a specific embodiment In, the CD80-IgV has amino acid sequence shown in SEQ ID NO:41, or the amino acid sequence with SEQ ID NO:41 Amino acid at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity Sequence.In one embodiment, the CD80-IgVIgC has amino acid sequence shown in SEQ ID NO:42, or and SEQ The amino acid sequence of ID NO:42 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Or more identity amino acid sequence.

In one embodiment, the fusion protein also includes that the peptide between (i), (ii) and/or (iii) connects Head；Preferably, the peptide linker includes one or more amino acid, includes more preferably at least five amino acid, most preferably wraps Containing the peptide linker for being selected from SEQ ID NO:43-71.

In one embodiment, the fusion protein is from N-terminal to C-terminal with the sequence of (i), (ii) and (iii)；(iii), (i) and the sequence of (ii)；Or (iii), (ii) and sequence (i) effectively connect.

In one embodiment, the fusion protein includes

(a) anti-PD-1 antibody, anti-PD-L1 antibody or anti-PD-1 and PD-L1 bispecific antibody；With in the antibody The CD80ECD that the C-terminal of each heavy chain in two heavy chains effectively connects；

(b) anti-PD-1 antibody, anti-PD-L1 antibody or anti-PD-1 and PD-L1 bispecific antibody；The two of the antibody The CD80ECD that the N-terminal of each heavy chain in heavy chain effectively connects；With it is each light in the two light chains of the antibody The CD80ECD that the N-terminal of chain effectively connects；Or

(c)CD80ECD；In the immunoglobulin Fc domain for the dimeric forms that the C-terminal of CD80ECD effectively connects；With Anti- PD-1 antibody and/or anti-PD- are derived from what the C-terminal of the immunoglobulin Fc domain of the dimeric forms effectively connected The antigen-binding fragment of L1 antibody；

Preferably, the anti-PD-1 antibody is selected from and receives Wu Dankang, pidilizumab and pyridine aldoxime methyliodide (PAM) monoclonal antibody；The anti-PD-L1 is anti- Body is selected from atezolizumab, avelumab and durvalumab.Preferably, the antibody is IgG class antibody, in particular IgG₁Subclass, IgG₂Subclass, IgG₄Subclass Antibodies, more particularly IgG₄Subclass Antibodies；It is further preferred that the IgG₄Subclass Antibodies It include amino acid replacement, more preferably amino acid replacement S228P at the position S228 in Fc structural domain；Further preferably Ground, the light chain type of the antibody are κ type or λ type, it is therefore preferable to κ type.

In a specific embodiment, the fusion protein is the first subunit of fusion protein comprising SEQ ID NO:77 With the fusion protein of the second subunit of fusion protein of SEQ ID NO:79, hereinafter referred to fusion protein BY31.2, it includes anti- CD80IgV PD-1 antibody (IgG4, κ, S228P) and effectively connect by peptide linker with heavy chain of antibody C-terminal.

In a specific embodiment, the fusion protein is the first subunit of fusion protein comprising SEQ ID NO:81 With the fusion protein of the second subunit of fusion protein of SEQ ID NO:83, hereinafter referred to fusion protein BY31.3, it includes anti- CD80IgVIgC PD-1 antibody (IgG2, κ) and effectively connect by peptide linker with heavy chain of antibody C-terminal.

In a specific embodiment, the fusion protein is the first subunit of fusion protein comprising SEQ ID NO:85 With the fusion protein of the second subunit of fusion protein of SEQ ID NO:87, hereinafter referred to fusion protein BY31.7, it includes anti- It PD-1 antibody (IgG4, κ, S228P) and is effectively connect with the N-terminal of each light chain of antibody and each heavy chain by peptide linker CD80IgV。

In a specific embodiment, the fusion protein is the first subunit of fusion protein comprising SEQ ID NO:89 With the fusion protein of the second subunit of fusion protein of SEQ ID NO:91, hereinafter referred to fusion protein BY31.14, wherein described The first subunit of fusion protein includes CD80IgV, IgG4CH2 and CH3 structural domain effectively connected from N-terminal to C-terminal, peptide linker, resists PD-1 antibody light chain, second subunit of fusion protein include the heavy chain variable region of anti-PD-1 Fab fragments from N-terminal to C-terminal With CH1 structural domain, the anti-PD-1 antibody light chain part in second subunit and the first subunit of fusion protein BY31.14 passes through two Sulfide linkage connection.

In a specific embodiment, the fusion protein is the first subunit of fusion protein comprising SEQ ID NO:93 With the fusion protein of the second subunit of fusion protein of SEQ ID NO:95, hereinafter referred to fusion protein BY31.15, wherein described Variable region and constant region of the first subunit of fusion protein from N-terminal to C-terminal comprising anti-PD-1 antibody light chain, the fusion protein second CD80IgV, IgG4CH2 and CH3 structural domain, peptide linker, anti-PD-1 monoclonal antibody piece of the subunit from N-terminal to C-terminal comprising effectively connecting The heavy chain variable region and CH1 structural domain of section, heavy chain variable region and the CH1 knot of the PD-1 Fab fragments in second subunit Structure domain is connect with the first subunit by disulfide bond.

In a specific embodiment, the fusion protein includes

(a) the anti-PD-L1 antibody selected from atezolizumab, avelumab and durvalumab；Resist with described (optionally, effectively being connected by peptide linker) one that the C-terminal of each heavy chain in two heavy chains of PD-L1 antibody effectively connects A CD80 extracellular domain (ECD)；

(b) one is selected from the anti-PD-L1 antibody of atezolizumab, avelumab and durvalumab, in the anti-PD- The N-terminal of each heavy chain in two heavy chains of L1 antibody (optionally, effectively being connected by peptide linker) that effectively connects one What the N-terminal of CD80ECD and each light chain in the two light chains of the anti-PD-L1 antibody effectively connected (optionally, passes through What peptide linker effectively connected) CD80ECD；Or

(c)CD80ECD；In the immunoglobulin Fc domain for the dimeric forms that the C-terminal of CD80ECD effectively connects；With Anti- PD-L1 antibody is derived from what the C-terminal of the immunoglobulin Fc domain of the dimeric forms effectively connected The antigen-binding fragment of atezolizumab, avelumab or durvalumab.

Preferably, the CD80ECD is CD80IgV or CD80IgVIgC.

In one embodiment, the fusion protein specifically targets PD-1/PD-L1 signal transduction path and activation T cell.Fusion protein of the invention can not only high-affinity combination PD-1 and/or PD-L1, and can high-affinity combine The CD28 of constitutive expression on T cell surface.The structure of fusion protein designed by the present invention has fully ensured that the fusion egg The Desirable physical space length that Bai Yuqi target combines, one of the fusion protein of this structure and the target molecular specific Property combine after do not influence the specific bindings of other molecules in the fusion protein and target.

The present invention also provides the polynucleotides of coding fusion protein of the present invention, comprising encoding the more of fusion protein of the present invention The carrier of nucleotide, preferably expression vector, most preferably with the glutamine synthase expression carrier of double expression boxes.Another On one side, the present invention provides the host cells comprising polynucleotides of the present invention or carrier.In one embodiment, described Host cell is CHO, HEK293 or NSO cell.Present invention provides a kind of method for generating fusion protein of the present invention, Host cell of the invention is cultivated under conditions of being suitable for and expressing fusion protein of the present invention including step (i), and (ii) recycling is originally The fusion protein of invention.

In one aspect, the present invention provides diagnostic kits and pharmaceutical composition comprising fusion protein of the present invention.Into One step, additionally provides the purposes of fusion protein of the invention, diagnostic kit or pharmaceutical composition, for treat, prevent and/ Or diagnosis is activated to PD-1/PD-L1 signal transduction path and is suppressed relevant disease with T cell function, is particularly used for Treatment, prevention and/or diagnosis Cancerous disease (for example, solid tumor and soft-tissue tumor), be most specifically used in treatment, prevention and/or Diagnose melanoma, breast cancer, colon cancer, the cancer of the esophagus, gastrointestinal stromal tumors (GIST), kidney (for example, clear-cell carcinoma), liver Sick (the example of cancer, non-small cell lung cancer (NSCLC), oophoroma, cancer of pancreas, prostate cancer, head and neck neoplasm, gastric cancer, hematological malignancy Such as, lymthoma).

Unless otherwise defined, otherwise whole technologies used herein have with scientific term as of the art The normally understood identical meanings of those of ordinary skill.Whole publications, patent application mentioned by this paper, patent and other references Document is fully incorporated herein by reference by reference.In addition, material described herein, method and example are only It is illustrative and be not intended to be restrictive.Other features, objects, and advantages of the invention will be from this specification and attached drawing simultaneously And it is apparent from appended claims.

Brief description

When reading together in conjunction with the following drawings, it is better understood with preferred implementation side of the invention following detailed description of Case.For the purpose of illustrating the invention, currently preferred embodiment is shown in figure.It is, however, to be understood that the present invention is unlimited In the elaborate scheme and means of embodiment as shown in the figure.

Figure 1A, B and C provide the schematic diagram of fusion protein of the present invention, and wherein Figure 1A is instantiated from N-terminal to C-terminal comprising anti- The structural schematic diagram of the fusion protein of the antigen-binding fragment of body, immunoglobulin Fc domain and CD80ECD；Figure 1B is instantiated The structure of fusion protein from N-terminal to C-terminal comprising CD80ECD, the antigen-binding fragment of antibody and immunoglobulin Fc domain Schematic diagram；Fig. 1 C instantiates the antigen-binding fragment from N-terminal to C-terminal comprising CD80ECD, immunoglobulin Fc domain and antibody Fusion protein structural schematic diagram.

Fig. 2: show the fusion protein of the present invention for preparing and purifying in embodiment 2 in reducing agent (bis- sulphur of 5mM 1,4- Soviet Union Sugar alcohol) in the presence of by SDS-PAGE and with the result after Coomassie blue stain.Swimming lane 1: Protein Marker marker；Swimming Road 2: antibody BY18.1；Swimming lane 3: fusion protein BY31.2；Swimming lane 4: fusion protein BY31.3；Swimming lane 5: fusion protein BY31.7；Swimming lane 6: fusion protein BY31.14；Swimming lane 7: fusion protein BY31.15.

Fig. 3 A: the combination for instantiating the fusion protein BY31.2 and people CD28 of the present invention that prepare and purify in embodiment 2 is bent Line；Fig. 3 B: the binding curve of the fusion protein BY31.2 and human PD-L 1 of the present invention that prepare and purify in embodiment 2 are instantiated；Figure 3C: the binding curve of the fusion protein BY31.2 and people CTLA-4 of the present invention that prepare and purify in embodiment 2 are instantiated.

Fig. 4: show fusion protein BY31.2 of the invention in the animal model of embodiment 6 to experimental animal weight It influences.

Fig. 5: the internal antitumor work of fusion protein BY31.2 of the invention in the animal model of embodiment 6 is shown With.

Fig. 6: show fusion protein BY31.2 of the invention in the animal model of embodiment 7 to experimental animal weight It influences.

Fig. 7: it shows fusion protein BY31.2 and anti-PD-L1 monoclonal antibody of the invention in the animal model of embodiment 7 The schematic diagram that the Anticancer effect in vivo of Avelumab and anti-PD-1 monoclonal antibody Opdivo are compared.

Detailed description of the invention

The present invention provides interference, inhibition or the fusion eggs for blocking PD-1/PD-L1 signal transduction path and activating T cell White and pharmaceutical composition.The present invention also provides for generating the fusion protein method and the fusion protein in individual Treatment, prevention and/or diagnosis are activated to PD-1/PD-L1 signal transduction path and are suppressed relevant disease with T cell function In purposes.

Unless hereinafter in addition definition, otherwise the term in this specification is used as this field is conventionally used.

I. it defines

Term " about " meant when being used in combination with digital numerical value cover with smaller than designation number numerical value 5% lower limit and Digital numerical value in the range of bigger than designation number numerical value 5% upper limit.

As used herein, term "comprising" or " comprising " mean to include the element, integer or step, but do not arrange Except any other element, integer or step.

" PD-1 approach " refers to any Cellular Signaling Transduction Mediated approach caused and in conjunction with PD-1, including but unlimited In the intracellular letter that Cellular Signaling Transduction Mediated approach or PD-1 that PD-1 causes in conjunction with PD-L1 cause in conjunction with PD-L2 Number pathway or PD-1 and both PD-L1 and PD-L2 are combined and the Cellular Signaling Transduction Mediated approach of initiation.

" PD-L1 approach " refers to any Cellular Signaling Transduction Mediated approach caused and in conjunction with PD-L1, including but not It is limited to PD-L1 in conjunction with PD-1 and the Cellular Signaling Transduction Mediated approach or PD-L1 and CD80 (B7-1) that cause combine and initiation Both Cellular Signaling Transduction Mediated approach or PD-L1 and PD-1 and CD80 (B7-1) are combined and the Intracellular signals biography that causes Lead approach.

" interference " used herein, " inhibition " or " blocking " PD-1/PD-L1 signal transduction path may be used interchangeably, and be Refer to the interaction between (i) interference PD-1 and PD-L1；And/or (ii) leads to PD-1/PD-L1 signal transduction path at least A kind of inhibition of biological function.Caused by causing after fusion protein of the present invention and PD-1 and/or PD-L1 specific binding " interference ", " inhibition " or " blocking " PD-1/PD-L1 signal transduction path needs not be complete interference, inhibits or block.

As used herein, term " specific binding " mean the combination to antigen or molecules of interest have selectivity and can To be distinguished with undesired or non-specific interaction.The specific binding can pass through enzyme linked immunosorbent assay (ELISA) (ELISA) or other technologies familiar to those skilled in the art, such as surface plasma body resonant vibration (SPR) technology is (in BIAcore Analyzed on instrument) (Liljeblad et al., Analysis of agalacto-IgG in rheumatoid arthritis Using surface plasmon resonance, Glyco J., 2000,17,323-329) measurement.

" affinity " or " binding affinity " refer to reflection combine antithetical phrase member between interact inherently combine it is affine Power.Molecule X can be usually by dissociation constant (K to the affinity of its spouse's thing Y_D) represent, dissociation constant is dissociation rate constant It (is k respectively with association rate constants_offAnd k_on) ratio.Affinity can be measured by common methods known in the art.For A specific method for measuring affinity is surface plasmon resonance (SPR).

Term " antibody " is used herein with most wide meaning and including but not limited to monoclonal antibody, Anti-TNF-α Body, multi-specificity antibody (for example, bispecific antibody), as long as they show required antigen-binding activity.Antibody It can be the complete antibody of any type and hypotype (for example, IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgE, IgA1 and IgA2) (for example, there are two the light chain of overall length and the heavy chains of two overall lengths for tool).

Term " whole antibody ", " full length antibody ", " complete antibody " and " complete antibody " is used to refer to interchangeably herein A kind of antibody, the antibody have structure substantially similar with native antibody structure.

Term " heavy chain of antibody " refers to its naturally occurring conformation in the two types polypeptide chain present in antibody molecule The greater determines classification belonging to antibody under normal circumstances.

Term " antibody light chain " refers to its naturally occurring conformation in the two types polypeptide chain present in antibody molecule Smaller.κ light chain and lambda light chain refer to two main antibody light chain types.

" bispecific antibody " be tool there are two different heavy chain/light chain pair and tool there are two the people of different binding sites Work hybrid antibody.Bispecific antibody can be prepared by a variety of methods, the connection including hybridoma fusion or Fab ' segment.

" antigen-binding fragment " of term antibody is amino acid residue more less than complete or complete antibody or antibody chain A part of antibody or antibody chain or one section, can in conjunction with antigen or with complete antibody (i.e. with antigen-binding fragment institute source Complete antibody) competitive binding antigen.It can resist by recombinant DNA technology or by enzyme or the complete Antibody preparation of chemical cleavage Former binding fragment.Antigen-binding fragment includes but is not limited to Fab, Fab ', F (ab ')₂, Fv, scFv.The Fab segment is one Kind is by V_L、V_H、C_LThe monovalent fragment formed with CH1 structural domain, for example, can be obtained by papain digestion complete antibody Fab segment.In addition, digesting complete antibody below the disulfide bond of hinge area by pepsin generates F (ab')₂, it is Fab ' Dimer, be divalent fragments thereof.F(ab')₂It can be reduced in neutral conditions by the disulfide bond destroyed in hinge area, Therefore by F (ab')₂Dimer is converted into Fab' monomer.Fab' monomer is substantially to have the Fab segment of hinge area (other anti- The more detailed description of body segment refers to: basic immunology (Fundamental Immunology), W.E.Paul is edited, Raven Press,N.Y.(1993)).The Fv segment by antibody single armed V_LAnd V_HStructural domain composition.In addition, though Fv segment Two structural domain V_LAnd V_HIt is encoded by independent gene, but uses recombination method, it can be by them by the way that the two can be made The synthetic connector connection that structural domain is generated as single protein chain, the V in the single protein chain_LArea and V_HIt matches with shape in area At scFv.Can chemically, recombinant DNA method or protease digestion method obtain the antibody fragment.

Term " immunoglobulin " refers to the protein of the structure with naturally occurring antibody.For example, IgG immunoglobulin like protein By the heterotetrameric glycoproteins for about 150,000 dalton that the two light chains of disulfide-bonded and two heavy chains form.From N-terminal To C-terminal, every heavy chain immunoglobulin has a variable region (VH), also referred to as variable heavy chain domain or heavy-chain variable domains, It is followed by three constant domains (CH1, CH2 and CH3), also referred to as heavy chain constant region.Similarly, from N-terminal to C-terminal, every is exempted from Epidemic disease immunoglobulin light chains have a variable region (VL), also referred to as variable light domain or light variable domains, constant with the latter Light chain (CL) structural domain, also referred to as constant region of light chain.The heavy chain of immunoglobulin can belong to one of 5 classifications, referred to as α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), some of them classification can be further divided into subclass, such as γ₁ (IgG1)、γ₂(IgG2)、γ₃(IgG₃)、γ₄(IgG₄)、α₁(IgA₁) and α₂(IgA₂).The light chain of immunoglobulin can be based on The amino acid sequence of its constant domain and one of be divided into two kinds of types, referred to as κ and λ.Immunoglobulin is substantially by by exempting from Two Fab molecules of epidemic disease immunoglobulin hinge region connection and a Fc structural domain composition.

Term " Fc structural domain " or " area Fc " are used to define containing at least partially for heavy chain immunoglobulin herein The C-terminal region of constant region.The term includes the area native sequences Fc and the area variant Fc.Natural immunoglobulin " Fc structural domain " packet Containing two or three constant domains, i.e. CH2 structural domain, CH3 structural domain and optional CH4 structural domain.For example, in natural antibody In, immunoglobulin Fc domain includes the constant structure of second and third of two heavy chains from IgG, IgA and IgD class antibody Domain (CH2 structural domain and CH3 structural domain)；Or two articles of heavy chains comprising being originated from IgM and IgE class antibody second, third and the Four constant domains (CH2 structural domain, CH3 structural domain and CH4 structural domain).Unless other explanation herein, the otherwise area Fc or again Numbering amino acid residues in chain constant region are according to such as Kabat et al., Sequences of Proteins of Immunological Interes, the 5th edition, Public Health Service, National Institutes of EU number system described in Health, Bethesda, MD, 1991 (also referred to as EU index) is numbered.

" human immunoglobulin(HIg) " is such a immunoglobulin, possesses the immune ball corresponding to people or people's cell generation The amino acid sequence of albumen or from the inhuman source of the sequence using human immunoglobulin(HIg) library or other encoding human immunoglobulins It is derivative.

" homogeneity percentage (%) " of amino acid sequence refers to specific ammonia shown in candidate sequence and this specification After base acid sequence is compared and introduces vacancy Wei maximal sequence homogeneity percentage is reached if necessary, and not When considering a part of any conservative substitution as sequence identity, in candidate sequence with specific amino shown in this specification The identical amino acid residue percentage of the amino acid residue of acid sequence.

Term " effectively connection " means that specified each component is in a kind of pass for allowing them to work by way of expectations System.

" signal sequence " is the sequence for being connected to the amino acid of N- end part of protein, promotes Protein secretion to thin It is extracellular.The mature form of extracellular protein does not have signal sequence, is removed during secretion process.

Term " N-terminal " refers to that the most end amino acid of N-terminal, term " C-terminal " refer to the most end amino acid of C-terminal.

Term " fusion ", which refers to, to be directly connected to two or more components by peptide bond or effective by one or more peptide linkers Connection.

Term " host cell " refers to the cell for introducing exogenous polynucleotide thereto, the filial generation including this kind of cell. Host cell includes " transformant " and " cell of conversion ", this cell for including primary transformant and filial generation derived from it.Host Cell can be used to generate any kind of cell system of fusion protein of the present invention.Host cell includes the cell of culture, It also include transgenic animals, the plant tissue of genetically modified plants or culture or the cell inside animal tissue.

Term " individual " or " subject " are interchangeably used, and refer to mammal.Mammal includes but is not limited to tame and docile Change animal (for example, milk cow, sheep, cat, dog and horse), primate (for example, people and non-human primates such as monkey), rabbit and rodent (for example, mouse and rat).Particularly, individual is people.

Term " treatment " refers to the clinical intervention for being intended to change the natural process of disease in the individual for receiving treatment.Want Therapeutic effect include but is not limited to prevent disease occur or recurrence, mitigate symptom, reduce disease any direct or indirect disease Consequence of science prevents transfer, reduces disease progression rate, improves or eases the disease state, and alleviates or improve prognosis.One In a little embodiments, fusion protein of the invention is used to that disease is delayed to develop or for slowing down the progress of disease.

Term " antitumor action " refer to can by multiple means show biology effect, including but not limited to for example, Gross tumor volume is reduced, tumor cell number is reduced, tumor cell proliferation is reduced or tumor cell survival is reduced.Term " tumour " and " cancer " is used interchangeably herein, and covers solid tumor and liquid tumors.

II. fusion protein

The present invention provides a kind of novel fusion proteins, and it includes (i) to be derived from anti-PD-1 antibody and/or anti-PD-L1 The antigen-binding fragment of antibody；(ii) immunoglobulin Fc domain；(iii) CD80 extracellular domain (ECD).

In some embodiments, (i), (ii) and/or (iii) is effectively connected optionally by peptide linker.

In some embodiments, fusion protein of the invention is the first subunit of fusion protein by disulfide bonding and melts The heterotetrameric glycoproteins of the second subunit of hop protein composition.

Fusion protein blocking immunity checkpoint PD-1/PD-L1 signal transduction path of the invention and activating T cell.This melts The immunologic test point PD-1 approach that hop protein blocks is PD-1 and the signal transduction path that its ligand binding is mediated.The fusion egg The PD-L1 approach of white blocking is the signal transduction path that PD-L1 is mediated in conjunction with its receptor.The fusion protein is to T cell Activation is to realize by blocking immunity inhibition PD-1/PD-L1 access and by CD80ECD to the positive control of T cell.

In some embodiments, fusion protein of the invention is with 10^-8M or smaller, for example with 10^-9M to 10^-12The solution of M From constant (K_D) in conjunction with PD-1 or PD-L1；And with 10^-8M or smaller, for example with 10^-9M to 10^-12Dissociation constant (the K of M_D) with CD28 molecular specificity combines,

Antigen-binding fragment derived from anti-PD-1 antibody and/or anti-PD-L1 antibody

The antigen-binding fragment derived from anti-PD-1 antibody and/or anti-PD-L1 antibody for including in fusion protein of the present invention PD-1 and/or PD-L1 can be specifically bound；Or the PD-1 in conjunction with complete anti-PD-1 antibody and/or anti-PD-L1 antibody competition And/or PD-L1.The antigen-binding fragment includes but is not limited to Fab, Fab ', F (ab ')₂, Fv, scFv.

The antigen-binding fragment derived from anti-PD-1 antibody and/or anti-PD-L1 antibody for including in fusion protein of the present invention Enable fusion protein of the invention with high affinity, such as with 10^-8M or smaller, preferably with 10^-9M to 10^-12The K of M_D The signal for specifically binding with PD-1 and/or PD-L1, and thus PD-1 being blocked to be mediated in conjunction with ligand PD-L1/PD-L2 passes It leads approach and/or PD-L1 and receptor PD-1/CD80 (B7-1) is blocked to combine mediated signal transduction path.

It is provided in the following table 1 A in the antigen-binding fragment for the anti-PD-1 antibody for including in fusion protein of the present invention herein The example of pairs of heavy chain variable region (VH) and light chain variable region (VL).In addition, fusion of the present invention is provided in the following table 1 B herein The example of pairs of heavy chain variable region (VH) and light chain variable region (VL) in the antigen-binding fragment for the anti-PD-L1 antibody for including in albumen Son.In some embodiments, the antigen-binding fragment in fusion protein of the present invention include with shown in table 1A and/or table 1B The substantially same sequence of amino acid sequence, for example, with pairs of weight chain variabl area sequence/light chain shown in table 1A and/or table 1B Variable region sequences have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence The sequence of identity.

The example of heavy chain variable region and light-chain variable sequence in the antigen-binding fragment of the anti-PD-1 antibody of table 1A.

The example of heavy chain variable region and light-chain variable sequence in the antigen-binding fragment of the anti-PD-L1 antibody of table 1B.

In one embodiment, the antigen-binding fragment in fusion protein of the present invention include selected from SEQ ID NO:1/2, 3/4,5/6,7/8,9/10,11/12,13/14,15/16,17/18,19/20,21/22,23/24 and 25/26 pairs of heavy chain can Become whole 6 complementary determining region of heavy chain (CDR) and light chain CDR contained in region sequence/light-chain variable sequence.Implement at one In scheme, antigen-binding fragment in fusion protein of the present invention include selected from SEQ ID NO:27/28,29/30 and 31/32 at To whole 6 heavy chain CDR and light chain CDR contained in weight chain variabl area sequence/light-chain variable sequence.For identifying that heavy chain can Become the method and technique of the CDR in the amino acid sequence of area and light chain variable region to be as known in the art, and can be used for identifying CDR in the amino acid sequence of specific heavy chain variable region and/or light chain variable region disclosed herein.It can be used for identifying the boundary CDR Exemplary known technology include such as Kabat defines method, Chothia defines method and AbM defines method.See, e.g. Kabat,Sequences of Proteins of Immunological Interest,National Institutes of Health,Bethesda,Md.(1991)；Al-Lazikani et al., Standard conformations for the Canonical structures of immunoglobulins., J.Mol.Biol.273:927-948 (1997)；And Martin AC et al., Modeling antibody hypervariable loops:a combined algorithm, Proc.Natl.Acad.Sci.USA 86:9268-9272(1989)。

It can be with base as the anti-PD-1 antibody in the antigen-binding fragment source in fusion protein of the present invention or anti-PD-L1 antibody In its constant region of light chain amino acid sequence and be divided into κ type or λ type, preferably κ type.

The example of the amino acid sequence of the anti-antibody light chain constant region PD-1 is provided in the following table 2 herein.

The example of the constant light chain sequences of the anti-PD1 antibody of table 2.

It is based on as the anti-PD-1 antibody in the antigen-binding fragment source in fusion protein of the present invention or anti-PD-L1 antibody The amino acid sequence of heavy chain constant region is preferably IgG class antibody, in particular IgG₁Subclass, IgG₂Subclass, IgG₄Subclass is anti- Body, more particularly IgG₄Subclass Antibodies.Preferably, the IgG₄The anti-PD-1 antibody of subclass or anti-PD-L1 antibody are in Fc Qu Zhong The amino acid replacement of (arm-exchange) is exchanged at the position S228 comprising preventing arm, in particular amino acid replacement S228P。

The example of the amino acid sequence of anti-PD-1 heavy chain constant region is provided in the following table 3 herein.

The example of the heavy chain constant region sequence of the anti-PD1 antibody of table 3.

Immunoglobulin Fc domain

" immunoglobulin Fc domain " in fusion protein of the present invention includes naturally occurring immunoglobulin Fc structure Whole amino acid residues in domain or a part of amino acid residue comprising naturally occurring immunoglobulin Fc domain.Immune ball Albumen Fc structural domain provides advantageous pharmacokinetic properties, including but not limited to long half longevity of serum to fusion protein of the invention Phase.In addition, immunoglobulin Fc domain also to purify fusion protein of the invention for example, by protein A affinity chromatography and become It may.

Immunoglobulin Fc domain is usually dimer molecule, can be disappeared by papain digestion or trypsase Change complete (overall length) immunoglobulin to generate or can recombinate generations, it includes CH2 structural domains, CH3 structural domain and optionally CH4 structural domain.

In one embodiment, the area IgG Fc includes IgG CH2 structural domain and IgG CH3 structural domain.Preferably, it is immunized Immunoglobulin Fc domain is with amino acid sequence shown in SEQ ID NO:38-40 in table 4 or has and SEQ ID NO: Amino acid sequence shown in 38-40 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, The amino acid sequence of 99% or more identity.

The example of immunoglobulin Fc domain amino acid sequence in 4 fusion protein of table

Other than the sequence as defined in SEQ ID NO:38-40, the area IgG Fc can also be comprising to SEQ ID NO: 38-40 carries out the peptide sequence obtained after additional sequence modification, for example, to the amino acid residue in SEQ ID NO:38-40 into The peptide sequence obtained after capable one or more amino acid substitutions, missing or derivative.In one embodiment, in the area IgG Fc The amino acid replacement of (arm-exchange) is exchanged at the position S228 comprising preventing arm, in particular amino acid replacement S228P。

The extracellular domain (ECD) of-CD80

" extracellular domain (ECD) of CD80 " in fusion protein of the present invention includes the whole of naturally occurring CD80ECD Amino acid residue or a part of amino acid residue comprising naturally occurring CD80ECD.In some embodiments, described CD80ECD includes CD80IgV, it is preferable that the CD80 ECD includes people CD80 IgV, it is highly preferred that the CD80ECD has Amino acid sequence shown in SEQ ID NO:41 or 42 in table 5.

The example of CD80ECD amino acid sequence in 5 fusion protein of table

Other than the sequence as defined in SEQ ID NO:41 and 42, CD80ECD can also be comprising to SEQ ID NO: 41 and 42 carry out the peptide sequence obtained after additional sequence modification, for example, to the amino acid residue in SEQ ID NO:41 and 42 into The replacement of row one or more conservatives, missing or it is derivative after the peptide sequence that obtains, as long as having and unmodified peptide substantially phase Same activity or function.Modified peptide will retain activity relevant to unmodified peptide or function.Modified peptide is usual With the substantially homologous amino acid sequence of the amino acid sequence with unmodified sequence, for example, with the institute of SEQ ID NO:41 or 42 The amino acid sequence shown has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more The amino acid sequence of identity.

Peptide linker

Antigen binding fragment in fusion protein of the invention by (i) derived from anti-PD-1 antibody and/or anti-PD-L1 antibody Section；(ii) immunoglobulin Fc domain；" peptide linker " that (iii) CD80ECD is optionally effectively connected is one or more The peptide of amino acid, general about 2-20 amino acid.It is known in the art or there is described herein peptide linkers.

In some embodiments, the peptide linker includes at least five amino acid, preferably includes and is selected from AKTTPKLEEGEFSEAR (SEQ ID NO:43)；AKTTPKLEEGEFSEARV (SEQ ID NO:44)；AKTTPKLGG(SEQ ID NO:45)；SAKTTPKLGG (SEQ ID NO:46)；SAKTTP (SEQ ID NO:47)；RADAAP (SEQ ID NO:48)； RADAAPTVS (SEQ ID NO:49)；RADAAAAGGPGS (SEQ ID NO:50)；RADAAAA (SEQ ID NO:51)； SAKTTPKLEEGEFSEARV (SEQ ID NO:52)；ADAAP (SEQ ID NO:53)；DAAPTVSIFPP (SEQ ID NO: 54)；TVAAP (SEQ ID NO:55)；TVAAPSVFIFPP (SEQ ID NO:56)；QPKAAP (SEQ ID NO:57)； QPKAAPSVTLFPP (SEQ ID NO:58)；AKTTPP (SEQ ID NO:59)；AKTTPPSVTPLAP (SEQ ID NO:60)； AKTTAP (SEQ ID NO:61)；AKTTAPSVYPLAP (SEQ ID NO:62)；ASTKGP (SEQ ID NO:63)； ASTKGPSVFPLAP (SEQ ID NO:64)；GGGGSGGGGSGGGGS (SEQ ID NO:65)；GENKVEYAPALMALS(SEQ ID NO:66)；GPAKELTPLKEAKVS (SEQ ID NO:67)；GHEAAAVMQVQYPAS (SEQ ID NO:68)； GGGGSGGGGSGGGGSA (SEQ ID NO:69)；GQGTKVEIKRGGSGGGGSG (SEQ ID NO:70) and The peptide linker of GQGTLVTVSSGGGGSGGGGS (SEQ ID NO:71).

Fusion protein

There is provided herein in any order include the antigen binding of (i) derived from anti-PD-1 antibody and/or anti-PD-L1 antibody Segment；(ii) immunoglobulin Fc domain；The fusion protein of (iii) CD80ECD, including but not limited to fusion protein are from N End is to C-terminal with the sequence of (i), (ii) and (iii)；(iii), the sequence of (i) and (ii)；Or (iii), (ii) and (i) suitable Sequence effectively connects.

In one embodiment, the fusion protein from N-terminal to C-terminal comprising anti-PD-1 antibody, anti-PD-L1 antibody or Anti- PD-1 and PD-L1 bispecific antibody；One effectively connected with the C-terminal of each heavy chain in two heavy chains of the antibody A CD80 ECD.

In another embodiment, the fusion protein include anti-PD-1 antibody, anti-PD-L1 antibody or anti-PD-1 and PD-L1 bispecific antibody；The CD80 that the N-terminal of each heavy chain in two heavy chains of the antibody effectively connects ECD；The CD80 ECD effectively connected with the N-terminal of each light chain in the two light chains of the antibody.

In yet another embodiment, the fusion protein includes CD80 ECD from N-terminal to C-terminal；In the C-terminal of CD80 ECD The immunoglobulin Fc domain of the dimeric forms effectively connected；With the immunoglobulin Fc structure in the dimeric forms The antigen-binding fragment derived from anti-PD-1 antibody and/or anti-PD-L1 antibody that the C-terminal in domain effectively connects.

III. the production and purifying of fusion protein of the invention

Fusion protein of the invention can for example pass through solid-state peptide synthesis (such as Merrifield synthesis in solid state) or recombination Production obtains.For recombinant production, by the polynucleotides of the first subunit of encoding said fusion protein and/or the coding fusion The polynucleotides of second subunit of albumen are separated and are inserted into one or more carriers further to clone in host cell And/or expression.Using conventional method, the polynucleotides can be separated easily and are sequenced.In one embodiment, Provide the carrier comprising one or more polynucleotides of the invention, preferably expression vector.

Method well known to those skilled in the art can be used and carry out construction of expression vector.Expression vector includes but is not limited to disease Poison, plasmid, clay, λ bacteriophage or yeast artificial chromosome (YAC).In a preferred embodiment, it has used with double The glutamine synthelase efficient expression vector of expression cassette.

Once being prepared for the expression vector comprising one or more polynucleotides of the invention for expression, then may be used Expression vector is transfected or is introduced into suitable host cell.Multiple technologies can be used to realize this purpose, for example, primary Plast fusion, calcium phosphate precipitation, electroporation, the transduction of retrovirus, virus transfection, particle gun, the transfection based on liposome Or other routine techniques.

In one embodiment, the host cell comprising one or more polynucleotides of the present invention is provided.Some In embodiment, the host cell comprising expression vector of the present invention is provided.As used herein, refer to can be with for term " host cell " It is engineered to generate any kind of cell system of fusion protein of the invention.Suitable for replicating and supporting fusion egg of the invention The host cell of white expression is well known in the art.As needed, this kind of cell can be transfected or be transduceed with particular expression carrier, And the largely cell containing carrier can be cultivated to be used to be inoculated with large scale fermentation device to obtain the present invention fusion egg of sufficient amount It is white to be used for clinical application.Suitable host cell includes prokaryotic micro-organisms, such as Escherichia coli, eukaryotic microorganisms such as filamentous fungi or Yeast or various eukaryocytes, such as Chinese hamster ovary cell (CHO), insect cell.The culture that is suitable for suspending can be used Mammal cell line.The example of useful mammalian host cell line includes the monkey kidney CV1 system (COS-7) of SV40 conversion； Human embryo kidney (HEK) system (HEK 293 or 293F cell), baby hamster kidney cell (BHK), MK cells (CV1), African green monkey kidney cell (VERO-76), human cervical carcinoma cell (HELA), canine kidney cells (MDCK), the dirty cell of buffalo rat liver (BRL 3A), people's lung are thin Born of the same parents (W138), people's liver cell (Hep G2), Chinese hamster ovary celI, NSO cell, myeloma cell line such as YO, NS0, P3X63 and Sp2/0 Deng.Suitable for producing the summary of protedogenous mammalian host cell line see, for example, Yazaki and Wu, Methods in Molecular Biology, volume 248 (B.K.C.Lo writes, Humana Press, Totowa, NJ), the 255-268 pages (2003).In a preferred embodiment, the host cell is CHO, HEK293 or NSO cell.

The standard technique of the expression alien gene known in the art in these host cell systems.In an embodiment In, the method for generating fusion protein of the invention is provided, wherein the method includes being suitable for expressing the fusion protein Under the conditions of culture such as host cell provided herein, the host cell includes the polynucleotides of encoding said fusion protein, And the fusion protein is recycled from host cell (or host cell culture medium).

The fusion protein prepared as described herein can pass through the known prior art such as high performance liquid chromatography, ion exchange The purifying such as chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography.Also depend on for purifying the physical condition of specific protein In factors such as such as net charge, hydrophobicity, hydrophilies, and these it will be apparent to those skilled in the art that.

The purity of fusion protein of the invention can be determined by any method in a variety of known analysis methods, it is described Known analysis method includes gel electrophoresis, high performance liquid chromatography etc..Can by many measure method known in the art, identify, Screen or characterize the physical/chemical properties and/or biological activity of fusion protein provided herein.

IV. pharmaceutical composition and kit

On the other hand, the present invention provides composition, for example, pharmaceutical composition, the composition include with can medicine With carrier fusion protein as described herein formulated together.As used herein, " pharmaceutical acceptable carrier " includes physical compatibility Any and whole solvents, decentralized medium, isotonic agent and absorption delaying agent etc..Pharmaceutical composition of the invention is suitable for intravenous, flesh Interior, subcutaneous, parenteral, rectum, spinal cord or epidermis application (for example, passing through injection or infusion).

Composition of the invention may be at diversified forms.These forms for example including liquid, semisolid and solid dosage forms, Such as liquid solution agent (for example, injectable solution and infusible solutions agent), dispersion agent or suspension, Liposomal agents and bolt Agent.Preferred form depends on expected administration mode and therapeutical uses.Common preferred composition is in injectable solution Agent or infusible solutions dosage form formula.Preferred administration mode is parenteral (for example, intravenous, subcutaneous, abdominal cavity (i.p.), intramuscular) Injection.In a preferred embodiment, pass through intravenous infusion or injection application fusion protein.In another preferred implementation side In case, pass through intramuscular, abdominal cavity or subcutaneous injection application fusion protein.

Phrase " parenteral administration " as used herein and " parenteral modes application " mean to apply in addition to intestines and part is applied Administration mode except is usually applied by injection, and including but not limited to intravenous, intramuscular, intra-arterial, intradermal, abdomen Chamber, transtracheal, subcutaneous injection and infusion.

Therapeutic composition generally should be sterile and stable under conditions of manufacture and storage.Composition can be matched It is made as solution, microemulsion, dispersion, liposome or lyophilized form.Can by by reactive compound (i.e. fusion protein) to want The amount asked is added in suitable solvent, then filtering disinfection, prepares Sterile injectable solution.In general, by by the activity Compound is incorporated in sterile vehicle to prepare dispersion, and the sterile vehicle contains basic dispersion medium and other compositions.It can be with Use coating agent such as lecithin etc..In the case of a dispersion, the suitable of solution can be maintained by using surfactant Suitable mobility.It can cause injectable by the substance such as Monostearate and gelatin absorbed in the composition comprising delay The extension of composition absorbs.

In certain embodiments, fusion protein of the invention can be administered orally, such as with inert diluent or edible With carrier together oral administration.Fusion protein of the invention can also be enclosed in hard shell or soft shell gelatin capsules, compress in flakes Agent is directly incorporated into the diet of subject.Oral medication is applied, the compound can mix simultaneously with excipient And with ingestible tablet, cheek tablet, pastille (troche), capsule, elixir, suspension, syrup, wafer (wafer) etc. forms use.In order to apply fusion protein of the invention by method of administration outside parenteral, it may be necessary to will be described Fusion protein is co-administered with the material coating for preventing it from inactivating or with this material.Medical device known in the art can also be used Apply therapeutic combination.

Pharmaceutical composition of the invention may include the fusion of the present invention of " therapeutically effective amount " or " prevention effective dose " Albumen.The period that " therapeutically effective amount " refers to the dosage with needs and be continuously needed, the amount for the treatment of results needed for effectively realizing.It can To change therapeutically effective amount according to age, gender and weight of many factors such as morbid state, individual etc..Therapeutically effective amount is to appoint Amount of what toxic or illeffects not as good as treatment beneficial effect.Relative to untreated subject, " therapeutically effective amount " preferably Inhibit mensurable parameter (such as tumor growth rate) at least about 20%, more preferably at least about 40%, even more preferably at least About 60% and still more preferably at least about 80%.This hair can be evaluated in the animal model system of effect in indication human tumour Bright fusion protein inhibits the ability of mensurable parameter (for example, gross tumor volume).

The period that " prevention effective dose " refers to the dosage with needs and be continuously needed, prevention result needed for effectively realizing Amount.It uses, therefore prevents in subject before disease earlier stage or in disease earlier stage generally, due to preventative dosage Effective quantity is less than therapeutically effective amount.

Kit comprising fusion protein described herein is also in the scope of the present invention.Kit may include one or Multiple other elements, for example, operation instructions；Other reagents, such as marker or the reagent for coupling；Pharmaceutically acceptable load Body；With the device or other materials for being applied to subject.

V. the purposes of fusion protein

Fusion protein disclosed herein has in vitro and in vivo diagnostic uses and therapeutic and preventative purposes.For example, These molecules can be applied to external or in vitro culture cell or be applied to subject, for example, human experimenter, to control It treats, prevent and/or diagnoses a variety of be activated to PD-1/PD-L1 signal transduction path and be suppressed relevant disease with T cell function Disease, such as cancer.

In one aspect, the present invention provides detect biological sample, such as serum, sperm or urine or tissue in vitro or in vivo There are the diagnosis sides of PD-1, PD-L1, CD28 or CTLA-4 in biopsy samples (for example, from hyperproliferative or cancerous lesions) Method.The diagnostic method includes: (i) make under conditions of allowing and interacting and occur sample (and optionally, control sample) with such as Fusion protein contact as described herein applies the fusion protein and (ii) detection fusion protein and sample to subject The formation of compound between (and optionally, control sample).The formation of compound indicate there are PD-1, PD-L1, CD28 or CTLA-4, and can show the applicability or demand for the treatment of and/or prevention described herein.

In some embodiments, before treatment, for example, certain before initial treatment or after treatment interval is controlled Detection PD-1 or PD-L1, CD28, CTLA-4 before treating.The detection method that can be used includes immunohistochemistry, immunocyte Chemistry, FACS, ELISA measurement, PCR- technology (for example, RT-PCR) or animal imaging.Generally, detection in vivo and in vitro Fusion protein used in method is directly or indirectly marked with detectable substance to promote knot that is that detection combines or being not associated with Close object.Suitable detectable substance includes various biological organized enzyme, prothetic group, fluorescent material, luminescent substance, paramagnetic (for example, core Magnetic resonance active) substance and radioactive substance.

In some embodiments, the level and/or distribution of PD-1, PD-L1, CD28 or CTLA-4 are determined in vivo, for example, It is determined with non-intruding mode (for example, by using suitable imaging technique (for example, positron emission tomography (PET) is swept Retouch) detect the fusion protein of the present invention that can detect substance markers.In one embodiment, for example, passing through detection PET reagent (for example,¹⁸F- fluorodeoxyglucose (FDG)) with the fusion protein of the present invention of detectable mode label, in vivoassay PD-1, PD- The level and/or distribution of L1, CD28 or CTLA-4.

In one embodiment, the present invention provides the diagnosis examinations comprising fusion protein described herein and operation instructions Agent box.

On the other hand, the present invention relates to use to be used to treat or prevent in fusion protein body to need to increase in subject The disease of strong T cell activation and immune response, to inhibit or reduce the growth or appearance, transfer of related disease such as cancerous tumour Or recurrence.Fusion protein be can be used alone to inhibit the growth of cancerous tumour or prevent its appearance.Alternatively, fusion protein It can be administered in combination with other cancer therapeutic agents/prophylactic.When fusion protein of the invention and one or more other drugs groups When closing application, this combination can be applied or be administered simultaneously in any order.

Therefore, in one embodiment, the present invention provides a kind of method for inhibiting growth of tumour cell in subject, institute The method of stating includes that the fusion protein as described herein of therapeutically effective amount is applied to subject.In another embodiment, this hair It is bright provide it is a kind of prevent tumour cell in subject from occurring or the method for transfer or recurrence, the method includes to subject Apply the fusion protein as described herein of prevention effective dose.

In some embodiments, the cancer treated and/or prevented with fusion protein includes but is not limited to solid tumor, hematology Cancer (for example, leukaemia, lymthoma, myeloma, for example, Huppert's disease) and metastasis (metastases).In one embodiment, Cancer is solid tumor.The example of solid tumor includes malignant tumour, for example, the sarcoma and cancer of multiple tracts, such as invade lung, cream Room, ovary, lymph sample, (for example, the colon) of gastrointestinal tract, anus, genitals and genitourinary tract (for example, kidney, urothelium, Bladder cells, prostate), pharynx, CNS (for example, brain, nerve or Deiter's cells), head and neck, skin is (for example, melanocyte Tumor), those of nasopharynx (for example, differentiation or undifferentiated metastatic or local recurrence nasopharyngeal carcinoma) and pancreas cancer and gland cancer, wrap Malignant tumour is included, such as colon and rectum carcinoma, clear-cell carcinoma, liver cancer, non-small cell lung cancer, carcinoma of small intestine and cancer of the esophagus.Cancer can be with In early stage, mid-term or advanced stage or metastatic carcinoma.

In some embodiments, cancer is selected from melanoma, breast cancer, colon cancer, the cancer of the esophagus, gastrointestinal stromal tumors (GIST), kidney (for example, clear-cell carcinoma), liver cancer, non-small cell lung cancer (NSCLC), oophoroma, cancer of pancreas, prostate cancer, head Tumor colli, gastric cancer, hematological malignancy are sick (for example, lymthoma).

Following embodiment is described to assist the understanding of the present invention.It is not intended to and should not in any way illustrate implementation It is interpreted into and limits the scope of the invention.

Embodiment

The building of embodiment 1, glutamine synthelase efficient expression vector comprising target gene

(1) as the synthesis of the coding nucleotide of the anti-PD1 antibody BY18.1 of control and the building of expression vector

According to the Wu Dan that receives that number is 9623 in International Nonproprietary Name (INN) database Anti- amino acid sequence data is optimized for the following nucleotide sequence for being suitble to express in Chinese hamster ovary cancer cell (CHO), And Shanghai Jierui Biology Engineering Co., Ltd is entrusted to synthesize the nucleotide sequence.It is generated after the nucleotide sequence expression anti- PD1 antibody is denoted herein as antibody BY18.1.

Light chain (BY18.1L) nucleotide sequence (SEQ ID NO:72) of anti-PD1 antibody BY18.1:

CTCGAGGCCACCATGGAGACCGACACACTCCTCCTGTGGGTGCTGCTGCTGTGGGTGCCTGGCTCCACT GGCGAGATTGTGCTGACACAGTCCCCCGCTACTCTGAGCCTGAGCCCTGGCGAGAGGGCTACACTGTCTTGCAGAGC TTCTCAGTCCGTGTCTTCTTACCTCGCTTGGTATCAGCAGAAGCCCGGCCAGGCTCCAAGACTGCTGATCTATGACG CTTCTAACCGCGCTACAGGCATTCCTGCTAGGTTCAGCGGCAGCGGCTCTGGCACCGACTTCACACTCACAATTAGC TCTCTTGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTCTAGCAACTGGCCTAGAACATTCGGCCAGGGCAC TAAGGTGGAGATTAAGAGAACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTG GCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCC CTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCT GACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCG TGACCAAGAGCTTCAACCGGGGCGAGTGCTAAGAATTC

Light chain (BY18.1L) amino acid sequence (SEQ ID NO:73) of anti-PD1 antibody BY18.1:

METDTLLLWVLLLWVPGSTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC

Heavy chain (BY18.1H) nucleotide sequence (SEQ ID NO:74) of anti-PD1 antibody BY18.1:

TCTAGAGCCACCATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTCCTGTGGGTGCCTGGCTCCACAGGCCAGGT GCAGCTCGTGGAGTCCGGCGGCGGCGTGGTGCAGCCCGGCAGATCCCTCAGACTGGACTGCAAGGCATCCGGCATTA CATTCTCTAACTCTGGAATGCACTGGGTGAGACAGGCTCCTGGCAAGGGCCTGGAATGGGTGGCCGTGATTTGGTAC GACGGCTCTAAGAGATACTACGCTGACTCCGTGAAGGGCCGGTTCACAATTAGCAGAGACAACTCCAAGAACACTCT GTTCCTCCAGATGAACAGCCTGAGAGCCGAGGACACCGCTGTGTACTACTGCGCCACCAACGACGACTACTGGGGCC AGGGCACCCTCGTGACAGTGTCTTCCGCCTCCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCTCCCGCTCC ACCTCCGAGTCCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCCTGGAACTC CGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGG TGACCGTGCCTTCCTCCTCCCTGGGCACCAAGACCTACACCTGCAACGTGGACCACAAGCCTTCCAACACCAAGGTG GACAAGCGCGTGGAGTCCAAGTACGGCCCTCCTTGCCCTCCTTGCCCTGCCCCTGAGTTCCTGGGCGGCCCTTCCGT GTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGCACCCCTGAGGTGACCTGCGTGGTGGTGGACG TGTCCCAGGAGGACCCTGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCT CGCGAGGAGCAGTTCAACTCCACCTACCGCGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAA GGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCTCCATCGAGAAGACCATCTCCAAGGCCAAGGGCCAGC CTCGCGAGCCTCAGGTGTACACCCTGCCTCCTTCCCAGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTG GTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCAC CCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCCGCCTGACCGTGGACAAGTCCCGCTGGCAGGAGG GCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCCCTG GGCAAGTAA GTCGAC

Heavy chain (BY18.1H) amino acid sequence (SEQ ID NO:75) of anti-PD1 antibody BY18.1:METDTLLLWVLLLWVPGSTGQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSK RYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSES TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Wherein leukorrhagia dashed part "METDTLLLWVLLLWVPGSTG" it is signal peptide sequence.

Shanghai Jierui Biology Engineering Co., Ltd has synthesized above-mentioned BY18.1L coding nucleotide sequence and BY18.1H coding Nucleotide sequence.Respectively by BY18.1L coding nucleotide XhoI-EcoRI double digestion, by the glutamine with double expression boxes Synzyme efficient expression vector (patent authorization number: CN104195173B is obtained from Beijing than foreign Bioisystech Co., Ltd) is used XhoI-EcoRI double digestion, then by ligase by the BY18.1L coding nucleotide through XhoI-EcoRI double digestion connect into through The glutamine synthelase efficient expression vector with double expression boxes of XhoI-EcoRI double digestion, acquisition have imported The glutamine synthelase efficient expression vector with double expression boxes of BY18.1L coding nucleotide；Then, respectively will BY18.1H coding nucleotide XbaI-SalI double digestion, by imported BY18.1L coding nucleotide have double expression boxes Glutamine synthelase efficient expression vector XbaI-SalI double digestion, then by ligase will be through XbaI-SalI double digestion BY18.1H coding nucleotide connect into through XbaI-SalI double digestion to have imported having for BY18.1L coding nucleotide double The glutamine synthelase efficient expression vector of expression cassette, thereby is achieved and imported BY18.1L coding nucleotide and BY18.1H The glutamine synthelase efficient expression vector with double expression boxes of coding nucleotide, expresses after sequence verification is correct, obtains Obtain anti-PD1 antibody BY18.1.

Alternatively, BY18.1L coding nucleotide can also be connected into having imported having for BY18.1H coding nucleotide The glutamine synthelase efficient expression vector of double expression boxes expresses and obtains antibody BY18.1.

(2) synthesis and expression of the coding nucleotide of the fusion protein comprising anti-PD-1 antibody and CD80 extracellular domain carry The building of body

According to the chain constant of antibody in the heavy chain variable region of PD-1 antibody anti-in table 1A and light-chain variable sequence, table 2 The heavy chain constant region sequence of antibody in region sequence, table 3, in table 5 CD80 extracellular domain sequence and SEQ ID NO:43- 71 peptide linker sequence is optimized for the nucleotide sequence for being suitble to express in Chinese hamster ovary cancer cell (CHO), and entrusts Extra large JaRa bioengineering Co., Ltd synthesizes polynucleotide sequence shown in even-numbered in following SEQ ID NO:76-95.

The first subunit (BY31.2L) nucleotide sequence (SEQ ID NO:76) of fusion protein BY31.2 (κ, IgG4): CTCGAGGCCACCATGGAGACCGACACACTCCTCCTGTGGGTGCTGCTGCTGTGGGTGCCTGGCTCCACTGGCGAGAT TGTGCTGACACAGTCCCCCGCTACTCTGAGCCTGAGCCCTGGCGAGAGGGCTACACTGTCTTGCAGAGCTTCTCAGT CCGTGTCTTCTTACCTCGCTTGGTATCAGCAGAAGCCCGGCCAGGCTCCAAGACTGCTGATCTATGACGCTTCTAAC CGCGCTACAGGCATTCCTGCTAGGTTCAGCGGCAGCGGCTCTGGCACCGACTTCACACTCACAATTAGCTCTCTTGA ACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTCTAGCAACTGGCCTAGAACATTCGGCCAGGGCACTAAGGTGG AGATTAAGAGAACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCC AGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAG CGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGA GCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAG AGCTTCAACCGGGGCGAGTGCTAAGAATTC

The first subunit (BY31.2L) amino acid sequence (SEQ ID NO:77) of fusion protein BY31.2 (κ, IgG4):

METDTLLLWVLLLWVPGSTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC

The second subunit (BY31.2H) nucleotide sequence (SEQ ID NO:78) of fusion protein BY31.2 (κ, IgG4):

TCTAGAGCCACCATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTCCTGTGGGTGCCTGGCTCCACA GGCCAGGTGCAGCTCGTGGAGTCCGGCGGCGGCGTGGTGCAGCCCGGCAGATCCCTCAGACTGGACTGCAAGGCATC CGGCATTACATTCTCTAACTCTGGAATGCACTGGGTGAGACAGGCTCCTGGCAAGGGCCTGGAATGGGTGGCCGTGA TTTGGTACGACGGCTCTAAGAGATACTACGCTGACTCCGTGAAGGGCCGGTTCACAATTAGCAGAGACAACTCCAAG AACACTCTGTTCCTCCAGATGAACAGCCTGAGAGCCGAGGACACCGCTGTGTACTACTGCGCCACCAACGACGACTA CTGGGGCCAGGGCACCCTCGTGACAGTGTCTTCCGCCTCCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCT CCCGCTCCACCTCCGAGTCCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCC TGGAACTCCGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTC CTCCGTGGTGACCGTGCCTTCCTCCTCCCTGGGCACCAAGACCTACACCTGCAACGTGGACCACAAGCCTTCCAACA CCAAGGTGGACAAGCGCGTGGAGTCCAAGTACGGCCCTCCTTGCCCTCCTTGCCCTGCCCCTGAGTTCCTGGGCGGC CCTTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGCACCCCTGAGGTGACCTGCGTGGT GGTGGACGTGTCCCAGGAGGACCCTGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGA CCAAGCCTCGCGAGGAGCAGTTCAACTCCACCTACCGCGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTG AACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCTCCATCGAGAAGACCATCTCCAAGGCCAA GGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCCTTCCCAGGAGGAGATGACCAAGAACCAGGTGTCCCTGA CCTGCCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCTGAGAACAACTAC AAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCCGCCTGACCGTGGACAAGTCCCGCTG GCAGGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCC TGTCCCTGGGCGGCGGAGGATCTGGCGGCGGAGGCAGTGGAGGCGGCGGAAGTGTGATCCATGTAACAAAAGAAGTG AAGGAAGTGGCTACACTCTCTTGCGGCCACAACGTGTCCGTGGAGGAACTAGCTCAGACCCGGATCTATTGGCAGAA AGAAAAGAAGATGGTGCTGACCATGATGTCCGGCGACATGAACATTTGGCCAGAGTACAAGAACCGCACAATTTTCG ACATTACAAACAACCTCTCTATTGTGATTCTGGCTCTCAGGCCTAGCGACGAGGGCACATACGAGTGCGTGGTGCTC AAGTACGAGAAGGACGCTTTCAAGCGGGAGCACCTCGCTGAGGTGACCCTGTCCGTGAAGGCCGACTTCCCTACTCC ATCTTAAGTCGAC

The second subunit (BY31.2H) amino acid sequence (SEQ ID NO:79) of fusion protein BY31.2 (κ, IgG4):

METDTLLLWVLLLWVPGSTGQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVA VIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAP CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGGGGSGGGGSGGGGSVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTI FDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPS

The first subunit (BY31.3L) nucleotide sequence (SEQ ID NO:80) of fusion protein BY31.3 (κ, IgG2):

CTCGAGGCCACCATGGAGACCGACACACTCCTCCTGTGGGTGCTGCTGCTGTGGGTGCCTGGCTCCACT GGCGAGATCAAGCGGACCGTGGCCGCCCCATCCGTGTTCATTTTCCCACCTTCCGAGATTGTGCTGACACAGTCCCC CGCTACTCTGAGCCTGAGCCCTGGCGAGAGGGCTACACTGTCTTGCAGAGCTTCCAAGGGCGTGAGCACATCCGGCT ACTCCTACCTCCACTGGTATCAGCAGAAGCCAGGCCAGGCCCCAAGACTGCTGATATACCTCGCTTCTTACTTAGAG TCTGGCGTGCCCGCTCGGTTCAGCGGCTCCGGCTCTGGCACCGACTTCACCCTGACAATTTCTAGCCTGGAGCCCGA GGACTTCGCCGTGTACTACTGCCAGCACTCTAGGGACCTGCCTCTCACATTCGGCGGCGGCACTAAGGTGGAGATTA AGAGAACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTG GTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAA CAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGG CCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTC AACCGGGGCGAGTGCTAAGAATTC

The first subunit (BY31.3L) amino acid sequence (SEQ ID NO:81) of fusion protein BY31.3 (κ, IgG2):

METDTLLLWVLLLWVPGSTGEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPR LLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC

The second subunit (BY31.3H) nucleotide sequence (SEQ ID NO:82) of fusion protein BY31.3 (κ, IgG2):

TCTAGAGCCACCATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTCCTGTGGGTGCCTGGCTCCACA GGCCAGGTGCAGCTCGTGCAGTCTGGCGTGGAGGTGAAGAAGCCTGGCGCCTCTGTGAAGGTGTCTTGCAAGGCTTC CGGCTACACTTTCACTAACTACTACATGTACTGGGTGAGACAGGCTCCCGGCCAGGGCCTAGAGTGGATGGGCGGCA TTAACCCTAGCAACGGCGGCACAAACTTCAACGAGAAGTTCAAGAACCGCGTGACCCTGACCACAGACTCTAGCACA ACAACTGCTTACATGGAGCTGAAGTCTCTCCAGTTCGACGACACCGCTGTGTACTACTGCGCTCGGAGGGACTACAG ATTCGACATGGGCTTCGACTACTGGGGCCAGGGCACCACTGTGACAGTGTCTACAGCCTCCACCAAGGGCCCTTCCG TGTTCCCTCTGGCCCCTTGCTCCCGCTCCACCTCCGAGTCCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTC CCTGAGCCTGTGACCGTGTCCTGGAACTCCGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTC CTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCTTCCTCCAACTTCGGCACCCAGACATACACATGCAACG TGGACCACAAGCCTTCTAACACAAAGGTGGACAAGACCGTGGAGCGGAAGTGCTGCGTGGAGTGCCCACCTTGCCCC GCTCCTCCTGTGGCCGGCCCTTCTGTGTTCCTGTTCCCACCTAAGCCAAAGGACACACTCATGATCAGCAGAACCCC TGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGCAGTTCAACTGGTATGTGGACGGCGTGG AGGTGCACAACGCTAAGACCAAGCCTAGAGAAGAACAGTTCAACAGCACATTCAGAGTGGTGTCCGTGCTCACCGTG GTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCAGCCCCTATCGAAAA AACAATCAGCAAGACCAAGGGCCAGCCTAGAGAGCCTCAGGTGTACACACTGCCTCCATCTCGGGAAGAAATGACAA AGAACCAGGTGTCCCTCACATGCCTCGTGAAGGGCTTCTACCCATCCGACATCGCTGTGGAGTGGGAGTCTAACGGC CAGCCCGAGAACAACTACAAGACCACCCCTCCTATGCTCGACTCCGACGGCTCTTTCTTCCTGTACTCTAAGCTGAC CGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCTCTTGCAGCGTGATGCACGAGGCTCTCCACAACCACTACA CCCAGAAGTCCCTGAGCCTGTCTCCAGGCGGCGGAGGATCTGGCGGCGGAGGCAGTGGAGGCGGCGGAAGCGTCATT CACGTCACTAAGGAGGTCAAGGAGGTCGCAACCCTCAGTTGCGGACACAACGTCAGCGTGGAGGAGCTTGCACAGAC ACGCATCTACTGGCAGAAGGAGAAGAAGATGGTGCTGACCATGATGTCCGGCGATATGAACATTTGGCCAGAGTACA AGAATCGGACCATCTTCGATATTACAAATAACCTGTCCATCGTGATCCTCGCTCTGCGCCCTAGCGACGAGGGAACA TACGAGTGTGTGGTGCTGAAGTACGAGAAGGATGCATTTAAGCGCGAGCACCTGGCTGAGGTGACACTCTCCGTCAA GGCCGATTTTCCTACTCCTTCTATCTCCGACTTTGAGATTCCAACATCAAATATTAGGCGCATTATCTGTTCTACAT CCGGCGGATTCCCAGAGCCCCACCTCTCTTGGTTGGAGAACGGCGAGGAACTTAATGCTATCAATACAACCGTGTCT CAAGATCCCGAGACTGAGCTGTACGCCGTGTCTAGTAAGCTGGACTTTAACATGACTACCAATCACAGTTTCATGTG CCTGATTAAGTACGGCCACCTGCGGGTGAATCAGACCTTTAATTGGAATACTACCAAGCAGGAGCACTTCCCAGATA ACTAAGTCGAC

The second subunit (BY31.3H) amino acid sequence (SEQ ID NO:83) of fusion protein BY31.3 (κ, IgG2):

METDTLLLWVLLLWVPGSTGQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMG GINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSTASTKGP SVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTC NVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGGGGSGGGGSGGGGSVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPE YKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICS TSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFP DN

The first subunit (BY31.7L) nucleotide sequence (SEQ ID NO:84) of fusion protein BY31.7:

TCTAGAGCCACCATGGAGACCGACACACTCCTCCTGTGGGTGCTGCTGCTGTGGGTGCCTGGCTCCACTGGCGTCAT CCATGTGACCAAAGAGGTCAAAGAAGTCGCAACTCTGTCTTGCGGACACAACGTCTCCGTCGAAGAACTCGCCCAGA CTAGAATATATTGGCAGAAGGAAAAGAAGATGGTGCTCACCATGATGTCCGGCGATATGAACATTTGGCCTGAGTAC AAGAACCGGACAATCTTCGATATTACTAATAACCTGAGTATCGTGATTCTCGCTCTGCGCCCTAGCGACGAGGGCAC ATACGAGTGTGTGGTGCTGAAGTACGAGAAGGATGCATTCAAGCGGGAGCACCTCGCAGAGGTGACACTCTCCGTGA AGGCCGACTTCCCAACCCCATCTGGCCAGGGCACTAAGGTGGAGATTAAGAGAGGCGGCTCTGGCGGCGGAGGCAGT GGAGAGATTGTGCTGACACAGTCCCCCGCTACTCTGAGCCTGAGCCCTGGCGAGAGGGCTACACTGTCTTGCAGAGC TTCTCAGTCCGTGTCTTCTTACCTCGCTTGGTATCAGCAGAAGCCCGGCCAGGCTCCAAGACTGCTGATCTATGACG CTTCTAACCGCGCTACAGGCATTCCTGCTAGGTTCAGCGGCAGCGGCTCTGGCACCGACTTCACACTCACAATTAGC TCTCTTGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTCTAGCAACTGGCCTAGAACATTCGGCCAGGGCAC TAAGGTGGAGATTAAGAGAACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTG GCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCC CTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCT GACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCG TGACCAAGAGCTTCAACCGGGGCGAGTGCTAA GTCGAC

The first subunit (BY31.7L) amino acid sequence (SEQ ID NO:85) of fusion protein BY31.7:

METDTLLLWVLLLWVPGSTGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNI WPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSGQGTKVEIKRGGSG GGGSGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFT LTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The second subunit (BY31.7H) nucleotide sequence (SEQ ID NO:86) of fusion protein BY31.7:

CTCGAGGCCACCATGGAGACCGACACACTCCTCCTGTGGGTGCTGCTGCTGTGGGTGCCTGGCTCCACT GGCGTCATCCATGTGACCAAAGAGGTCAAAGAAGTCGCAACTCTGTCTTGCGGACACAACGTCTCCGTCGAAGAACT CGCCCAGACTAGAATATATTGGCAGAAGGAAAAGAAGATGGTGCTCACCATGATGTCCGGCGATATGAACATTTGGC CTGAGTACAAGAACCGGACAATCTTCGATATTACTAATAACCTGAGTATCGTGATTCTCGCTCTGCGCCCTAGCGAC GAGGGCACATACGAGTGTGTGGTGCTGAAGTACGAGAAGGATGCATTCAAGCGGGAGCACCTCGCAGAGGTGACACT CTCCGTGAAGGCCGACTTCCCAACCCCATCTGGCCAGGGCACACTCGTGACCGTGTCTAGCGGCGGCGGAGGCAGTG GCGGCGGAGGCAGTCAGGTGCAGCTCGTGGAGTCCGGCGGCGGCGTGGTGCAGCCCGGCAGATCCCTCAGACTGGAC TGCAAGGCATCCGGCATTACATTCTCTAACTCTGGAATGCACTGGGTGAGACAGGCTCCTGGCAAGGGCCTGGAATG GGTGGCCGTGATTTGGTACGACGGCTCTAAGAGATACTACGCTGACTCCGTGAAGGGCCGGTTCACAATTAGCAGAG ACAACTCCAAGAACACTCTGTTCCTCCAGATGAACAGCCTGAGAGCCGAGGACACCGCTGTGTACTACTGCGCCACC AACGACGACTACTGGGGCCAGGGCACCCTCGTGACAGTGTCTTCCGCCTCCACCAAGGGCCCTTCCGTGTTCCCTCT GGCCCCTTGCTCCCGCTCCACCTCCGAGTCCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTG TGACCGTGTCCTGGAACTCCGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTG TACTCCCTGTCCTCCGTGGTGACCGTGCCTTCCTCCTCCCTGGGCACCAAGACCTACACCTGCAACGTGGACCACAA GCCTTCCAACACCAAGGTGGACAAGCGCGTGGAGTCCAAGTACGGCCCTCCTTGCCCTCCTTGCCCTGCCCCTGAGT TCCTGGGCGGCCCTTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGCACCCCTGAGGTG ACCTGCGTGGTGGTGGACGTGTCCCAGGAGGACCCTGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCA CAACGCCAAGACCAAGCCTCGCGAGGAGCAGTTCAACTCCACCTACCGCGTGGTGTCCGTGCTGACCGTGCTGCACC AGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCTCCATCGAGAAGACCATC TCCAAGGCCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCCTTCCCAGGAGGAGATGACCAAGAACCA GGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCTG AGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCCGCCTGACCGTGGAC AAGTCCCGCTGGCAGGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAA GTCCCTGTCCCTGTCCCTGGGCAAGTAAGAATTC

The second subunit (BY31.7H) amino acid sequence (SEQ ID NO:87) of fusion protein BY31.7:

METDTLLLWVLLLWVPGSTGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNI WPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSGQGTLVTVSSGGGG SGGGGSQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTIS RDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Fusion protein BY31.14 the first subunit (attachment of PD-1 antibody light chain and CD80-Fc fusions C-terminal, that is, BY31.14L) nucleotide sequence (SEQ ID NO:88):

CTCGAGGCCACCATGGAGACCGACACACTCCTCCTGTGGGTGCTGCTGCTGTGGGTGCCTGGCTCCACT GGCGTCATCCATGTGACCAAAGAGGTCAAAGAAGTCGCAACTCTGTCTTGCGGACACAACGTCTCCGTCGAAGAACT CGCCCAGACTAGAATATATTGGCAGAAGGAAAAGAAGATGGTGCTCACCATGATGTCCGGCGATATGAACATTTGGC CTGAGTACAAGAACCGGACAATCTTCGATATTACTAATAACCTGAGTATCGTGATTCTCGCTCTGCGCCCTAGCGAC GAGGGCACATACGAGTGTGTGGTGCTGAAGTACGAGAAGGATGCATTCAAGCGGGAGCACCTCGCAGAGGTGACACT CTCCGTGAAGGCCGACTTCCCAACCCCATCTGACAAGCGCGTGGAGTCCAAGTACGGCCCTCCTTGCCCTCCTTGCC CTGCCCCTGAGTTCCTGGGCGGCCCTTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGC ACCCCTGAGGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAGGACCCTGAGGTGCAGTTCAACTGGTACGTGGACGG CGTGGAGGTGCACAACGCCAAGACCAAGCCTCGCGAGGAGCAGTTCAACTCCACCTACCGCGTGGTGTCCGTGCTGA CCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCTCCATC GAGAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCCTTCCCAGGAGGAGAT GACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGGAGTCCA ACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCCGC CTGACCGTGGACAAGTCCCGCTGGCAGGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCA CTACACCCAGAAGTCCCTGTCCCTGTCCCTGGGCGGCGGAGGATCTGGCGGCGGAGGCAGTGGAGGCGGCGGAAGTG AGATTGTGCTGACACAGTCCCCCGCTACTCTGAGCCTGAGCCCTGGCGAGAGGGCTACACTGTCTTGCAGAGCTTCT CAGTCCGTGTCTTCTTACCTCGCTTGGTATCAGCAGAAGCCCGGCCAGGCTCCAAGACTGCTGATCTATGACGCTTC TAACCGCGCTACAGGCATTCCTGCTAGGTTCAGCGGCAGCGGCTCTGGCACCGACTTCACACTCACAATTAGCTCTC TTGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTCTAGCAACTGGCCTAGAACATTCGGCCAGGGCACTAAG GTGGAGATTAAGAGAACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCAC CGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGC AGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACC CTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGAC CAAGAGCTTCAACCGGGGCGAGTGCTAAGAATTC

Fusion protein BY31.14 the first subunit (attachment of PD-1 antibody light chain and CD80-Fc fusions C-terminal, BY31.14L) amino acid sequence (SEQ ID NO:89):

METDTLLLWVLLLWVPGSTGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNI WPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSDKRVESKYGPPCPP CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGSGGGGSGGGG SEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS SLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Fusion protein BY31.14 the second subunit (heavy chain variable region and CH1 structural domain of PD-1 Fab fragments, that is, BY31.14H is connect with the PD-1 antibody light chain part in the first subunit of fusion protein BY31.14 by disulfide bond) nucleotides sequence It arranges (SEQ ID NO:90):

TCTAGAGCCACCATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTCCTGTGGGTGCCTGGCTCCACA GGCCAGGTGCAGCTCGTGGAGTCCGGCGGCGGCGTGGTGCAGCCCGGCAGATCCCTCAGACTGGACTGCAAGGCATC CGGCATTACATTCTCTAACTCTGGAATGCACTGGGTGAGACAGGCTCCTGGCAAGGGCCTGGAATGGGTGGCCGTGA TTTGGTACGACGGCTCTAAGAGATACTACGCTGACTCCGTGAAGGGCCGGTTCACAATTAGCAGAGACAACTCCAAG AACACTCTGTTCCTCCAGATGAACAGCCTGAGAGCCGAGGACACCGCTGTGTACTACTGCGCCACCAACGACGACTA CTGGGGCCAGGGCACCCTCGTGACAGTGTCTTCCGCCTCCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCT CCCGCTCCACCTCCGAGTCCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCC TGGAACTCCGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTC CTCCGTGGTGACCGTGCCTTCCTCCTCCCTGGGCACCAAGACCTACACCTGCAACGTGGACCACAAGCCTTCCAACA CCAAGGTGGACAAGCGCGTGGAGTCCTAAGTCGAC

The second subunit (BY31.14H) amino acid sequence (SEQ ID NO:91) of fusion protein BY31.14:

METDTLLLWVLLLWVPGSTGQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVA VIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAP CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVES

The first subunit (BY31.15L, that is, PD-1 antibody light chain) nucleotide sequence (SEQ ID of fusion protein BY31.15 NO:92):

TCTAGAGCCACCAATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTCCTGTGGGTGCCTGGCTCCAC AGGCGAGATTGTGCTGACACAGTCCCCCGCTACTCTGAGCCTGAGCCCTGGCGAGAGGGCTACACTGTCTTGCAGAG CTTCTCAGTCCGTGTCTTCTTACCTCGCTTGGTATCAGCAGAAGCCCGGCCAGGCTCCAAGACTGCTGATCTATGAC GCTTCTAACCGCGCTACAGGCATTCCTGCTAGGTTCAGCGGCAGCGGCTCTGGCACCGACTTCACACTCACAATTAG CTCTCTTGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGTCTAGCAACTGGCCTAGAACATTCGGCCAGGGCA CTAAGGTGGAGATTAAGAGAACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCT GGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGC CCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCC TGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCC GTGACCAAGAGCTTCAACCGGGGCGAGTGCTAAGTCGAC

The first subunit (BY31.15L) amino acid sequence (SEQ ID NO:93) of fusion protein BY31.15:

METDTLLLWVLLLWVPGSTGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC

The second subunit (BY31.15H, that is, CD80-Fc fusions C-terminal and PD-1 monoclonal antibody piece of fusion protein BY31.15 The heavy chain variable region of section and the attachment of CH1 structural domain) nucleotide sequence (SEQ ID NO:94):

CTCGAGGCCACCATGGAGACCGACACACTCCTCCTGTGGGTGCTGCTGCTGTGGGTGCCTGGCTCCACT GGCGTCATCCATGTGACCAAAGAGGTCAAAGAAGTCGCAACTCTGTCTTGCGGACACAACGTCTCCGTCGAAGAACT CGCCCAGACTAGAATATATTGGCAGAAGGAAAAGAAGATGGTGCTCACCATGATGTCCGGCGATATGAACATTTGGC CTGAGTACAAGAACCGGACAATCTTCGATATTACTAATAACCTGAGTATCGTGATTCTCGCTCTGCGCCCTAGCGAC GAGGGCACATACGAGTGTGTGGTGCTGAAGTACGAGAAGGATGCATTCAAGCGGGAGCACCTCGCAGAGGTGACACT CTCCGTGAAGGCCGACTTCCCAACCCCATCTGACAAGCGCGTGGAGTCCAAGTACGGCCCTCCTTGCCCTCCTTGCC CTGCCCCTGAGTTCCTGGGCGGCCCTTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGC ACCCCTGAGGTGACCTGCGTGGTGGTGGACGTGTCCCAGGAGGACCCTGAGGTGCAGTTCAACTGGTACGTGGACGG CGTGGAGGTGCACAACGCCAAGACCAAGCCTCGCGAGGAGCAGTTCAACTCCACCTACCGCGTGGTGTCCGTGCTGA CCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTTCCTCCATC GAGAAGACCATCTCCAAGGCCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCCTTCCCAGGAGGAGAT GACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCTTCCGACATCGCCGTGGAGTGGGAGTCCA ACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCCGC CTGACCGTGGACAAGTCCCGCTGGCAGGAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCA CTACACCCAGAAGTCCCTGTCCCTGTCCCTGGGCGGCGGAGGATCTGGCGGCGGAGGCAGTGGAGGCGGCGGAAGTC AGGTGCAGCTCGTGGAGTCCGGCGGCGGCGTGGTGCAGCCCGGCAGATCCCTCAGACTGGACTGCAAGGCATCCGGC ATTACATTCTCTAACTCTGGAATGCACTGGGTGAGACAGGCTCCTGGCAAGGGCCTGGAATGGGTGGCCGTGATTTG GTACGACGGCTCTAAGAGATACTACGCTGACTCCGTGAAGGGCCGGTTCACAATTAGCAGAGACAACTCCAAGAACA CTCTGTTCCTCCAGATGAACAGCCTGAGAGCCGAGGACACCGCTGTGTACTACTGCGCCACCAACGACGACTACTGG GGCCAGGGCACCCTCGTGACAGTGTCTTCCGCCTCCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCTCCCG CTCCACCTCCGAGTCCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCCTGGA ACTCCGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCC GTGGTGACCGTGCCTTCCTCCTCCCTGGGCACCAAGACCTACACCTGCAACGTGGACCACAAGCCTTCCAACACCAA GGTGGACAAGCGCGTGGAGTC

(PD-1 in the second subunit of BY31.15H, fusion protein BY31.15 is anti-for the second subunit of fusion protein BY31.15 The heavy chain variable region and CH1 structural domain of body Fab segment are connect with the first subunit of fusion protein BY31.15 by disulfide bond) amino Acid sequence (SEQ ID NO:95):

METDTLLLWVLLLWVPGSTGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNI WPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSDKRVESKYGPPCPP CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGSGGGGSGGGG SQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSK NTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES

Wherein amino acid sequence " METDTLLLWVLLLWVPGSTG " is signal peptide.

Using the identical method of above-described embodiment 1 (1), respectively by XhoI-EcoRI double digestion by BY31.2L, BY31.3L, BY31.7L, BY31.14L and BY31.15L coding nucleotide are connected to the synthesis of the glutamine with double expression boxes Enzyme efficient expression vector (patent authorization number: CN104195173B is obtained from Beijing than foreign Bioisystech Co., Ltd)；Pass through again XbaI-SalI double digestion clones BY31.2H, BY31.3H, BY31.7H, BY31.14H and BY31.15H coding nucleotide respectively The glutamine synthelase with double expression boxes to the coding nucleotide for having had connected another subunit of corresponding fusion protein is high Imitate expression vector；Or vice versa.By recombinant vector sequence verification it is correct after be used to express.Expressed fusion protein difference It is named as fusion protein BY31.2, BY31.3, BY31.7, BY31.14 and BY31.15.

The expression and purifying of embodiment 2, fusion protein

(1) transient expression of fusion protein

By 293F (being purchased from Invitrogen company, catalog number (Cat.No.): 11625-019) cell suspension cultures in serum-free CD 293 In culture solution (being purchased from Invitrogen company, catalog number (Cat.No.): 11913-019).It is heavy to obtain cell for centrifugation of cell cultures before transfecting It forms sediment, with fresh 293 culture solution suspension cell of serum-free CD, cell concentration is adjusted to 1 × 10⁶A cell/ml.By cell Suspension is placed in shaking flask.By taking 100ml cell suspending liquid as an example, the fusion protein BY31.2 that respectively prepares embodiment 1, Each DNA 250ug of each recombinant expression carrier plasmid and polyethyleneimine of BY31.3, BY31.7, BY31.14 and BY31.15 (polyethylenimine (PEI)) (Sigma, catalog number (Cat.No.): 408727) 500ug is added in 293 culture solution of 1ml serum-free CD It mixes, after being stored at room temperature 8 minutes, PEI/DNA suspension is added dropwise in the shaking flask for being placed with 100ml cell suspending liquid.Gently It is light to mix, it is placed in 5%CO₂, 37 DEG C of shaking table cultures (120 revs/min).Culture supernatant is collected after 5 days.

According to same method, transient expression generates the antibody BY18.1 as control.

(2) purifying of albumen is expressed

With HiTrap MabSelect SuRe 1ml column (the GE Healthcare Life of pH 7.4PBS solution equilibria Sciences product, catalog number (Cat.No.): 11-0034-93) it purifies and merges egg present in the culture supernatant that above-described embodiment 2 (1) is collected It is white.In brief, HiTrap MabSelect SuRe 1ml column, stream are balanced with 10 bed volumes with the PBS solution of pH 7.4 Speed is 0.5ml/ minutes；After 0.45 μm of membrane filtration of culture supernatant that above-described embodiment 2 (1) is collected, load sample is to using pH The HiTrap MabSelect SuRe 1ml column of 7.4PBS solution equilibria；After loading supernatant, which is used pH's 7.4 first PBS solution with the 5-10 bed volume of washing in flow velocity 0.5ml/ minutes, and then with 100mM citrate buffer solution (pH 4.0) with It elutes within flow velocity 0.5ml/ minutes.Eluting peak is collected, destination protein is present in eluting peak.

By SDS-PAGE and with Coomassie blue stain in the presence of reducing agent (5mM Isosorbide-5-Nitrae-dithiothreitol (DTT)), analysis is melted The purity and molecular weight of hop protein.As a result as shown in Figure 2.Molecular weight theoretical expectation values and actual measured value are shown in Table 6.Because of eukaryon table Up to the glycosylation existed in system to protein, therefore molecular weight actual measured value is slightly above theoretical expectation values.

The molecular size range of the purified expression albumen of table 6

Embodiment 3 is acted on using ELISA method detection specific binding

Respectively by recombined human CD28 (Sino Biological Inc.'s product, catalog number (Cat.No.): 50103-M08H), Recombined human PD-L1 (hundred Pu Saisi Biotechnology Co., Ltd of Beijing, catalog number (Cat.No.): PD1-H5229) and (north recombined human CTLA-4 The Divine Land Jing Yiqiao Bioisystech Co., Ltd product, catalog number (Cat.No.): 11159-H08H) be diluted to 0.5 μ g/ml, 0.25 μ g/ml and 1.0 μ g/ml are simultaneously coated with 96 hole elisa plate (Corning company, article No.s: 42592).The fusion that above-described embodiment 2 (2) is purified Protein B Y31.2, BY31.3, BY31.7, BY31.14 and BY31.15 are diluted to 2000 μ g/ml, then carry out 3 times and are serially diluted, 15-16 gradient is diluted altogether, and multiple holes detection is carried out to each concentration gradient.50 μ l of dilute sample is separately added into above-mentioned through recombinating In coated 96 orifice plate of people CD28 or recombined human PD-L1,37 DEG C are incubated for 2 hours.After washing 3 times, horseradish peroxidase is added Goat anti-Human's secondary antibody (Beijing Zhong Shan Golden Bridge Products, production number: ZDR-5301) of label, 37 DEG C are incubated for 1 hour.It washes After washing 3 times, 3,3', 5,5'- tetramethyl benzidine (TMB) substrate developing solutions are added, and (Beijing health is the limited public affairs of century biotechnology Department, production number: CW0050) 50 holes μ l/.After ten minutes, the H of 2N is added₂SO₄Color development stopping.Using ELISA readout instrument in 450nm Place measures the absorbance OD value in every hole.

ELISA the results show that fusion protein BY31.2, BY31.3, BY31.7, BY31.14 and BY31.15 of the invention both Recombined human PD-L1 and recombined human CD28 can specifically be combined；Also recombined human CTLA-4 can specifically be combined.Fig. 3 A, Fig. 3 B With fusion protein BY31.2 and recombined human CD28, recombined human PD-L1 and recombined human CTLA-4 specificity are set forth in Fig. 3 C In conjunction with binding curve.

Using GraphPadPrism5 software, by fusion protein BY31.2, BY31.3, BY31.7, BY31.14 and The protein concentration of BY31.15 maps to absorbance OD value, and fitting data is to generate fusion protein mediated specificity knot The half maximum effective concentration EC of cooperation₅₀Value.As a result as shown in table 7 below.

Combination of the fusion protein of the invention of table 7 to human PD-L 1, people CD28 and people CTLA-4

According to the result of table 7 as it can be seen that new fusion protein BY31.2, BY31.3, BY31.7 constructed by the present invention, BY31.14 and BY31.15 can specifically bind human PD-L 1, people CD28 and people CTLA-4.Fusion protein BY31.3 and people The binding ability of CD28, CTLA-4 and PD-L1 are better than fusion protein BY31.2 and fusion protein BY31.7, this may be because The area Zhong IgC CD80ECD is combined with certain stabilization to CD80 and CD28, CTLA-4 and PD-L1's.

Fusion protein BY31.14 and BY31.15 and the binding ability of human PD-L 1, people CD28 and people CTLA-4 are better than BY31.2, BY31.3 and BY31.7.It may be that CD80 extracellular domain (ECD) is placed in entirely by BY31.2, BY31.3 and BY31.7 Long antibody N-terminal or C-terminal have certain influence to respectively in connection with human PD-L 1, people CD28 and people CTLA-4.

In addition, similarly, having detected antibody BY18.1 and antigen PD-1 (Divine Land Yi Qiao, Beijing biology using ELISA method Technology Co., Ltd.'s product, catalog number (Cat.No.): 10377-H08H) specific binding effect EC₅₀Value is 3.154 μ g/ml.

Embodiment 4 measures fusion protein of the invention to the affinity of PD-1 using Biacore T100

?In 25 DEG C of progress tables on T100 instrument (GE Healthcare Biosciences AB, Sweden) Surface plasma resonance measurement.

Firstly, being coupled by amide by anti-igg antibody (GE Healthcare Life Sciences, catalog number (Cat.No.): BR- 1008-39) covalently it is fixed on CM5 chip.Use 60 μ l N- ethyl-N'- (3- dimethylaminopropyl) carbodiimide hydrochlorides Salt (EDC) and 60 μ l n-hydroxysuccinimides (NHS) activate CM5 chip, then add 95 μ l dilution slow 5 μ l anti-igg antibody Fliud flushing HBST (0.1M HEPES, 1.5MNaCl, pH7.4 add 0.005% polysorbas20) passes through amide after 0.2um membrane filtration Anti-igg antibody is covalently fixed on CM5 chip by coupling, generates the capture systems of about 9000-14000 resonance units (RU).Make CM5 chip is closed with 120 μ l ethanol amines.

Then, fusion protein of the invention and antibody BY18.1 prepared by embodiment 2 are diluted to 5 μ g/ml respectively, with stream Speed injection in the 10 μ L/ minutes dilution 2 minutes, fusion protein and antibody of the invention prepared by the embodiment 2 of 1600RU BY18.1 is noncovalently captured on CM5 chip surface by the respective area Fc.It is resulting to stablize by being crosslinked with EDC/NHS Compound, to avoid the baseline drift during measurement and regeneration.

Antigen PD-1 (Sino Biological Inc.'s product, catalog number (Cat.No.): 10377-H08H) is formulated as Following concentration gradient: 7nM, 22nm, 66nM, 200nM, 600nM.By with 30 μ of flow velocity injection in l/ minutes each concentration 180 seconds, Dissociation time 600 seconds, measurement combined.By with 3M MgCl₂Solution makes surface regeneration in 30 seconds with washing in 10 μ of flow velocity L/ minutes.Make With BIA evaluation software, (BIAevaluation 4.1software comes from GE Healthcare Biosciences AB, auspicious Allusion quotation) data analysis is carried out, obtain affinity data shown in the following table 8.

The combination of table 8 each protein and PD-1

Protein title	ka(1/Ms)	kd(1/s)	KD(M)
				Fusion protein BY31.2	6.66E+05	1.58E-03	2.37E-09
Fusion protein BY31.3	5.62E+05	5.66E-04	1.01E-09
				Fusion protein BY31.7	8.56E+05	3.55E-03	4.15E-09
Fusion protein BY31.14	5.04E+05	3.55E-03	7.04E-09
				Fusion protein BY31.15	1.56E+04	8.18E-04	5.24E-08
Antibody BY18.1	1.76E+05	5.18E-04	2.94E-09

According to data shown in table 8 as it can be seen that fusion protein BY31.2, BY31.3 and BY31.7 of the invention can be with height Affinity combination PD-1, affinity (KD) respectively reach 2.37 × 10^-9M、1.01×10^-9M and 4.15 × 10^-9M.Antibody BY18.1 is with 2.94 × 10^-9The KD value combination PD-1 of M.

The ability of fusion protein BY31.14 and BY31.15 combination PD-1 is weaker than BY31.2, BY31.3 and BY31.7.It may To be due to the antigen-binding fragment in fusion protein BY31.14 and BY31.15 be positioned at C-terminal Fab structure, compared with whole antibody Combination has certain decrease.Fusion protein BY31.14 combination PD-1 is better than fusion protein BY31.15, and KD value can reach 10^-9M It is horizontal.

The influence of fusion protein of the present invention to IL-2 and IFN-γ secretion in embodiment 5, mixed lymphocyte reaction (MLP) (MLR)

CD4 is obtained from Beijing Shi He Biotechnology Co., Ltd⁺T lymphocyte and Dendritic Cells (DC), the CD4⁺T Lymphocyte and Dendritic Cells (DC) derive from different Healthy Peoples.By CD4⁺T lymphocyte and Dendritic Cells (DC) point It An 1 × 10⁵A cells/well and 1 × 10⁴A cells/well cover plant is in 96 porocyte culture plates.Experiment is divided into 6 groups, respectively Blank control group, BY18.1 group, BY31.2 group, BY31.3 group, BY31.7 group and BY31.14 group, every group of 6 multiple holes.Except blank Outside control group, other groups are separately added into antibody or fusion protein by amount shown in table 9.It finally adds containing 10% fetal calf serum 1640 culture mediums, making final volume is 200 μ l.37 DEG C, 5%CO₂Culture.

After culture 5 days, compared with the control group, each experimental group packed cell is more, and has substantial portion of fusiform and patch Parietal cell occurs.Culture supernatant is taken, uses the IL-2 kit (article No. EH002-96) of Yi Kesai Biotechnology Co., Ltd respectively Each group IL-2 and IFN-γ expression are detected with IFN-γ kit (article No.: EH008-96ELISA).With antibody BY18.1 group It compares, BY31.2 group, BY31.3 group, BY31.7 group and BY31.14 group have been significantly increased the expression water of IL-2 and IFN-γ Flat (P < 0.01), wherein with IL-2 in BY31.14 group supernatant and IFN-γ expression quantity highest.

Influence of each fusion protein of table 9 to IL-2 and IFN-γ secretion

Antibody or protein title	μg/ml	IL-2(pg/ml)	IFN-γ(pg/ml)
				Antibody BY18.1	5.0	485±59.4	9552±754
Fusion protein BY31.2	6.0	775±143.6	26431±812
				Fusion protein BY31.7	6.9	903±186.5	21651±1890
Fusion protein BY31.14	6.0	1896±266.2	36378±1531
				Blank control	0	55.4±10.6	63.5±4.8

The Anticancer effect in vivo of embodiment 6, fusion protein of the invention in B-hPD-1 humanized mouse model

The present embodiment has studied the internal antitumor work of fusion protein of the present invention using B-hPD-1 humanized mouse model With.

B-hPD-1 humanization mouse (is obtained from hundred Olympic Competition figure Genetic Biotechnologies Co., Ltd of Beijing, product number: B-CM- It 001) is a kind of people PD-1 (hPD-1) to be knocked in into the mouse obtained after C57BL/6 mouse genome.It is small in B-hPD-1 humanization People PD-1 is able to detect that in mouse⁺Cell.

By 5 × 10 in 0.1mL DMEM culture medium⁵A MC38 mouse colon cancer cell (being obtained from ATCC, the U.S.) is inoculated in about 18g, 6 week old female B-hPD-1 humanization right side of mice fore flank flanks are subcutaneous.Tumour is grown up in the Mice Body.To tumour Volume reaches about 108mm³When tumor-bearing mice is grouped at random, every group 6, totally 3 groups, be respectively as follows: PBS solvent control group, fusion Protein B Y31.2 group (6.0mg/kg) and antibody BY18.1 group (5mg/kg), each administration group dosage is with the dosage of antibody BY18.1 group For standard, institute's applied dose is equivalent in mole for fusion protein BY31.2 group and antibody BY18.1 group.It will The time of administration is set as the 0th day for the first time.All groups of administration routes are abdominal cavity (i.p.) injection, are administered once every three days, even Terminate experiment after continuous administration 6 times, last dose 3 days.Measure gross tumor volume and mouse weight 2 times weekly, record mouse weight and Gross tumor volume.At the end of experiment, by animal euthanasia, strips tumour weighing, takes pictures, calculating inhibition rate of tumor growth (TumorGrowth INhibition%).Calculating the formula that TGI% is used is: [1- (mean value/PBS of administration group tumor volume change The mean value of solvent control group tumor volume change)] x 100%.The experiment is in the hundred limited public affairs of Olympic Competition figure Genetic Biotechnologies of Beijing Department implements.

In whole experiment process, all animal mental states are good, no animal dead.At the end of experiment (after first administration The 21st day), groups of animals weight average is about 19g.It is compareed by fusion protein BY31.2 group of the invention and as drug Antibody BY18.1 group with PBS solvent control group animal carry out weight compared with, without significant difference (P > 0.05), show animal To fusion protein BY31.2 well-tolerated (Fig. 4) of the invention.

At the end of experiment, PBS solvent control group mean tumour volume ± standard is mistaken for 1386 ± 170mm³, fusion protein BY31.2 group mean tumour volume ± standard is mistaken for 953 ± 166mm³, the inhibition rate of tumor growth of fusion protein BY31.2 group It (TGI%) is 31.2%, showing fusion protein BY31.2 has technical effect (Fig. 5) antitumor in vivo.

In addition, at the end of experiment, the antibody BY18.1 group mean tumour volume ± standard as drug control is mistaken for 739 ± 128, TGI% 46.9%, show that the antibody BY18.1 compareed as drug passes through specific binding B-hPD-1 humanization mouse In hPD-1⁺Cells play Anticancer effect in vivo.This is consistent with report in the prior art, that is, military monoclonal antibody of receiving is one Kind is directed to the monoclonal antibody specific of people PD-1 molecule, by blocking people PD-1 points in conjunction with people's PD-1 molecular specificity The inhibition biological effect that son is mediated plays antitumor action accordingly, for the human patients of expression PD-1.

Fusion protein BY31.2 inhibits the effect of tumour growth not have relatively compared with the antibody BY18.1 compareed as drug Significant difference, this may be and to melt because only anti-PD-1 antibody moiety can play a role in fusion protein BY31.2 People CD80ECD in hop protein BY31.2 is not tied with the mouse CD28 of B-hPD-1 humanization mouse, mouse CTLA-4 and mouse PD-L1 phase It closes；In addition, it could also be because that the molecular weight of fusion protein BY31.2 is greater than control antibodies BY18.1, applied with equivalent mole Used time, it is less also related (being hypertonic environment inside tumor tissues) that fusion protein BY31.2 penetrates into the amount in tumor tissues.

Embodiment 7, fusion protein of the present invention are in people CD34⁺Internal antitumor work in the NSG mouse model of HSC transplanting With

The present embodiment by by MDA-MB-231 people's triple negative breast cancer cell line (that is, estrogen receptor, progesterone receptor It is negative breast cancer cell line with HER-2, is obtained from ATCC, the U.S.) it is seeded to people CD34⁺The NSG mouse model of HSC transplanting Have studied the Anticancer effect in vivo of fusion protein of the present invention.

People CD34⁺The NSG mouse (being obtained from Beijing Ai Demo Bioisystech Co., Ltd) of HSC transplanting is a kind of by people CD34⁺Candidate stem cell (HSC) is transplanted to the NOD/SCID/IL2r- γ of immune deficiency^null(NSG) in Mice Body, make NSG mouse Human immune system is rebuild to a kind of mouse model of the humanization in terms of immunity of organism obtained.

The present embodiment is in people CD34⁺The tumor cell line of source of people is inoculated on the basis of the NSG mouse model of HSC transplanting, that is, MDA-MB-231 people's triple negative breast cancer cell line, is finally obtained in terms of tumour and is the small of humanization in immunology Mouse.Such mouse due to having the immune system and source of people tumor tissues of people simultaneously, can real simulation people it is immune The process of system and tumour interaction, is for evaluating the validity to tumour immunotherapy and the ideal animals mould of safety Type.

By 5 × 10⁶The amount of a MDA-MB-231 people's triple negative breast cancer cell/mouse is by MDA-MB-231 cell inoculation To weight about 23g, the people CD34 of 28-32 week old⁺HSC transplants successful NSG female mice, and inoculation position is skin after back is right Under.When gross tumor volume reaches about 150mm³, tumor-bearing mice is grouped, every group 5 at random, totally 4 groups, is respectively as follows: PBS solvent pair According to group, fusion protein BY31.2 group (5.8mg/kg), anti-PD-L1 monoclonal antibody Avelumab group (10mg/kg, the still strong biological skill in Beijing The preparation of art Co., Ltd) and anti-PD-1 monoclonal antibody Opdivo group (preparation of the Divine Land 10mg/kg, Yi Qiao Bioisystech Co., Ltd, batch Number: MB09MA1201), wherein the application mole of fusion protein BY31.2 group is that anti-PD-1 monoclonal antibody Opdivo group, anti-PD-L1 are mono- 1/2 application mole of anti-Avelumab group.By first time, the time of administration is set as the 0th day.All groups of administration routes are Intraperitoneal injection, is administered once, successive administration 4 times for every 5 days.Gross tumor volume and mouse weight 3 times are measured weekly, record mouse weight And gross tumor volume.At the end of experiment, by animal euthanasia, strips tumour weighing, takes pictures, calculate inhibition rate of tumor growth (TGI%).The experiment is implemented in Beijing Ai Demo (IDMO) Bioisystech Co., Ltd.

Fig. 6 shows the changes of weight of animal after administration.At the 25th day, by fusion protein BY31.2 group of the invention with The animal of PBS solvent control group carries out weight comparison, and significant difference (P > 0.05) is not observed, shows fusion of the invention Protein B Y31.2 does not have overt toxicity to animal.

Fig. 7 shows that the gross tumor volume of animal after administration changes with time situation.At the 25th day, PBS solvent control Group mean tumour volume ± standard is mistaken for 1287.11 ± 184.71mm³, anti-PD-L1 monoclonal antibody Avelumab group be 964.25 ± 20.01mm³, anti-PD-1 monoclonal antibody Opdivo group is 1354.62 ± 126.65mm³, fusion protein BY31.2 group be 773.14 ± 310.66mm³。

Relative to PBS solvent control group, the tumor control rate (TGI%) of anti-PD-L1 monoclonal antibody Avelumab group is 25.08%, the TGI% of anti-PD-1 monoclonal antibody Opdivo group is -5.24%, and the TGI% of fusion protein BY31.2 group is 39.93%.

By the above experimental result as it can be seen that compared with PBS solvent control group, fusion protein BY31.2 of the invention is being used Molar dose compared with the existing technology in anti-PD-L1 monoclonal antibody, anti-PD-1 monoclonal antibody molar dose halve in the case where, also can Inhibit tumour growth (P < 0.05) significantly.

In addition, it is surprising that having the gross tumor volume of a mouse at the 25th day in fusion protein BY31.2 group When, narrow down to only 60mm³, and the gross tumor volume of PBS solvent control group be up to 1287.11 at the 25th day ± 184.71mm³, show this mouse and complete response (CR, complete response) produced to tumour.In anti-PD-L1 In monoclonal antibody Avelumab group and anti-PD-1 monoclonal antibody Opdivo group, the mouse of complete response is not observed.

While developing such from Beijing Ai Demo (IDMO) Bioisystech Co., Ltd the immune system with people with And since the effect experiment of the mouse implementation antineoplastic of source of people tumor tissues, tumour is complete to be observed to tested antineoplastic Response mouse is very rare situation.Fusion protein of the invention the molar dose used compared with the existing technology in it is anti- PD-L1 monoclonal antibody, anti-PD-1 monoclonal antibody molar dose halve in the case where, still be able to observe the complete response to tumour, by This, obtains unexpected technical effect.

Anti- PD-1 monoclonal antibody Opdivo group absolutely not antitumous effect in this mouse model.Anti- PD-L1 monoclonal antibody Although with certain tumor inhibition effect, (gross tumor volume of PBS solvent control group is Avelumab in this mouse model 1287.11±184.71mm³, the gross tumor volume of anti-PD-L1 monoclonal antibody Avelumab group is 964.25 ± 20.01mm³), still, do not have Have the antitumous effect of fusion protein BY31.2 of the invention it is significant (gross tumor volume of fusion protein BY31.2 group for 773.14 ± 310.66mm³).This demonstrate fusion protein BY31.2 of the invention interacts in real simulation human immune system and tumour The antitumous effect of highly significant can be generated in the mouse model of process.

Claims (18)

1. a kind of fusion protein for blocking PD-1/PD-L1 signal transduction path and activating T cell, it includes (i) derived from anti- The antigen-binding fragment of PD-1 antibody and/or anti-PD-L1 antibody；(ii) immunoglobulin Fc domain；(iii) CD80 is extracellular Structural domain (ECD).

2. fusion protein according to claim 1, wherein (i) is anti-derived from anti-PD-1 antibody and/or anti-PD-L1 Fab, Fab', F (ab') of body₂, Fv, scFv；Preferably, (i) includes to be selected from SEQ ID NO:1/2,3/4,5/6,7/ 8,9/10,11/12,13/14,15/16,17/18,19/20,21/22,23/24 and 25/26 pairs of weight chain variabl area sequence/ Whole 6 heavy chain CDR and light chain CDR contained in light-chain variable sequence, it is preferable that (i) includes anti-PD-1 antibody Selected from SEQ ID NO:1/2,3/4,5/6,7/8,9/10,11/12,13/14,15/16,17/18,19/20,21/22,23/24 With 25/26 pairs of weight chain variabl area sequence/light-chain variable sequence, or can with the pairs of weight chain variabl area sequence/light chain Becoming region sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence same The sequence of one property；It is highly preferred that (i) includes selected from receiving Wu Dankang (Nivolumab), pidilizumab and pyridine aldoxime methyliodide (PAM) monoclonal antibody (Pembrolizumab) heavy chain variable region and light chain variable region of anti-PD-1 antibody；And/or (i) is anti-comprising anti-PD-L1 It is contained in pairs of weight chain variabl area sequence/light-chain variable sequence selected from SEQ ID NO:27/28,29/30 and 31/32 of body All 6 heavy chain CDR and light chain CDR；Preferably, (i) includes to be selected from SEQ ID NO:27/28,29/30 and 31/32 Pairs of weight chain variabl area sequence/light-chain variable sequence, or with the pairs of weight chain variabl area sequence/light-chain variable sequence Sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity Column.

3. fusion protein according to claim 1 or 2, wherein (ii) is human immunoglobulin(HIg) Fc structural domain；It is preferred that Ground, (ii) are the Fc structural domain of human IgG1, IgG2, IgG3 or IgG4；It is highly preferred that (ii) includes SEQ ID NO: 38, the Fc structural domain of amino acid sequence shown in 39 or 40, or comprising with amino acid sequence shown in SEQ ID NO:38,39 or 40 Fc at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity Structural domain.

4. fusion protein according to any one of claim 1-3, wherein (iii) includes people CD80ECD；It is preferred that Ground, (iii) include people CD80IgV or people CD80IgVIgC；It is highly preferred that (iii) have SEQ ID NO:41 or Amino acid sequence shown in 42 or with amino acid sequence shown in SEQ ID NO:41 or 42 have at least 90%, 91%, 92%, 93%, the amino acid sequence of 94%, 95%, 96%, 97%, 98%, 99% or more identity.

5. fusion protein described in any one of -4 according to claim 1, also include (i), (ii) and/or (iii) it Between peptide linker；Preferably, the peptide linker includes one or more amino acid, more preferably includes at least five amino acid, most Preferably include the peptide linker selected from SEQ ID NO:43-71.

6. fusion protein according to any one of claims 1-5, wherein the fusion protein from N-terminal to C-terminal with (i), (ii) and the sequence of (iii)；(iii), the sequence of (i) and (ii)；Or (iii), (ii) and sequence (i) effectively connect.

7. fusion protein according to claim 6, it includes

(a) anti-PD-1 antibody, anti-PD-L1 antibody or anti-PD-1 and PD-L1 bispecific antibody；With at two of the antibody The CD80ECD that the C-terminal of each heavy chain in heavy chain effectively connects；

(b) anti-PD-1 antibody, anti-PD-L1 antibody or anti-PD-1 and PD-L1 bispecific antibody；In two weights of the antibody The CD80ECD that the N-terminal of each heavy chain in chain effectively connects；With each light chain in the two light chains of the antibody The CD80ECD that N-terminal effectively connects；Or

(c)CD80ECD；In the immunoglobulin Fc domain for the dimeric forms that the C-terminal of CD80ECD effectively connects；With institute State the C-terminal of the immunoglobulin Fc domain of dimeric forms effectively connect it is anti-derived from anti-PD-1 antibody and/or anti-PD-L1 The antigen-binding fragment of body；

Preferably, the antibody is IgG class antibody, in particular IgG₁Subclass, IgG₂Subclass, IgG₄Subclass Antibodies, more particularly It is IgG₄Subclass Antibodies；It is further preferred that the IgG₄Subclass Antibodies are set at the position S228 comprising amino acid in Fc structural domain It changes, more preferably amino acid replacement S228P；It is further preferred that the light chain type of the antibody be κ type or λ type, preferably For κ type.

8. fusion protein described in any one of -7 according to claim 1, wherein the anti-PD-1 antibody be selected from receive Wu Dankang, Pidilizumab and pyridine aldoxime methyliodide (PAM) monoclonal antibody；The anti-PD-L1 antibody is selected from atezolizumab, avelumab and durvalumab.

9. fusion protein according to claim 1 to 8, is selected from

(1) the second subunit of fusion protein of the first subunit of fusion protein comprising SEQ ID NO:77 and SEQ ID NO:79 melt Hop protein；

(2) the second subunit of fusion protein of the first subunit of fusion protein comprising SEQ ID NO:81 and SEQ ID NO:83 melt Hop protein；

(3) the second subunit of fusion protein of the first subunit of fusion protein comprising SEQ ID NO:85 and SEQ ID NO:87 melt Hop protein；

(4) the second subunit of fusion protein of the first subunit of fusion protein comprising SEQ ID NO:89 and SEQ ID NO:91 melt Hop protein；

(5) the second subunit of fusion protein of the first subunit of fusion protein comprising SEQ ID NO:93 and SEQ ID NO:95 melt Hop protein.

10. polynucleotides encode fusion protein of any of claims 1-9.

11. carrier, preferably expression vector, described most preferably with the glutamine synthase expression carrier of double expression boxes Carrier includes polynucleotides described in any one of claim 10.

12. host cell, it includes carriers described in polynucleotides described in any one of claim 10 or claim 11, it is preferable that The host cell is CHO, HEK293 or NSO cell.

13. the method for generating fusion protein of any of claims 1-9, the method includes the steps (i) to exist Suitable for cultivating host cell described in claim 12, and (ii) recycling fusion egg under conditions of the expression fusion protein It is white.

14. pharmaceutical composition, it includes fusion protein of any of claims 1-9 and pharmaceutical acceptable carrier.

15. the purposes of pharmaceutical composition described in fusion protein of any of claims 1-9, claim 14 is used The drug of disease relevant to PD-1 activity, PD-L1 activity and CD28 activity is treated or prevented in individual in preparation, preferably Ground, the disease are Cancerous disease (for example, solid tumor and soft-tissue tumors), more preferably melanoma, breast cancer, colon cancer, The cancer of the esophagus, gastrointestinal stromal tumors (GIST), kidney (for example, clear-cell carcinoma), liver cancer, non-small cell lung cancer (NSCLC), ovary Cancer, cancer of pancreas, prostate cancer, head and neck neoplasm, gastric cancer, hematological malignancy are sick (for example, lymthoma)；Particularly, the disease It is colon cancer or triple negative breast cancer；Preferably, wherein the individual is mammal, more preferably people.

16. diagnostic kit, it includes fusion protein of any of claims 1-9 and optionally markers or use In the reagent of coupling.

17. diagnostic kit according to claim 16, it includes with the detectable mark of positron emission tomography Remember the fusion protein of any of claims 1-9 of substance markers, it is preferable that the marker is¹⁸F- fluorine deoxyglucose Sugar.

18. the purposes of diagnostic kit described in claim 16 or 17, be used to prepare in individual diagnosis and PD-1 activity, The reagent of PD-L1 activity and the relevant disease of CD28 activity, it is preferable that the disease is Cancerous disease (for example, solid tumor and soft Histioma), more preferably melanoma, breast cancer, colon cancer, the cancer of the esophagus, gastrointestinal stromal tumors (GIST), kidney (for example, Clear-cell carcinoma), liver cancer, non-small cell lung cancer (NSCLC), oophoroma, cancer of pancreas, prostate cancer, head and neck neoplasm, gastric cancer, blood Liquid malignant diseases (for example, lymthoma)；Preferably, wherein the individual is mammal, more preferably people.