阅读说明：本技术 重组抗pd-l1单克隆抗体 (Recombinant anti-PD-L1 monoclonal antibody ) 是由 聂丽 刘广洛 于 2018-09-06 设计创作，主要内容包括：一种重组抗PD-L1单克隆抗体,涉及生物医药技术领域,用于给晚期或转移性癌症患者提供有效的治疗药物,尤其是现有抗PD-L1药物治疗无效或耐药的患者。该抗体的互补决定区具有SEQ ID NO:1到SEQ ID NO:6所列的序列。与现有抗PD-L1药物相比,该抗体具有独特结合表位,并且对人PD-L1亲和性更好、抑瘤效果更佳。(A recombinant anti-PD-L1 monoclonal antibody relates to the technical field of biological medicines and is used for providing effective treatment medicines for patients with advanced or metastatic cancers, in particular to patients who have ineffective or resistant treatment of the existing anti-PD-L1 medicines. The complementarity determining regions of the antibody have the sequences set forth in SEQ ID NO 1 through SEQ ID NO 6. Compared with the existing anti-PD-L1 drug, the antibody has unique binding epitope, and has better affinity to human PD-L1 and better tumor inhibition effect.)

1. A recombinant anti-PD-L1 monoclonal antibody is characterized in that heavy chain CDR1, CDR2 and CDR3 regions respectively contain sequences of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.

2. The antibody of claim 1, wherein the heavy chain variable region comprises the sequence of SEQ ID NO. 7.

3. The antibody of claim 1, wherein the heavy chain variable region comprises the sequence of SEQ ID NO 10.

4. The antibody of claim 1, wherein the light chain CDR1, CDR2, and CDR3 regions comprise the sequences of SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, respectively.

5. The antibody of claim 4, wherein the light chain variable region comprises the sequence of SEQ ID NO 8 or SEQ ID NO 9.

6. The antibody of claim 4, wherein the light chain variable region comprises the sequence of SEQ ID NO. 11.

7. The antibody of claim 1, 2, 4 or 5, which is an antibody of the human IgG1 kappa subtype.

8. The antibody of claim 1, 3, 4 or 6, which is an antibody of murine IgG1 kappa subtype.

9. Use of the antibody of claim 1, 2, 4, or 5 in the manufacture of a medicament for treating or preventing cancer, an immune disease.

10. Use of the antibody of claim 1, 3, 4, or 6 for cancer or immune disease detection, or for prediction of drug efficacy.

Technical Field

The invention relates to the technical field of biological medicines, and particularly discloses a monoclonal antibody suitable for treating or detecting cancers and immune diseases.

Background

PD-L1 (Programmed Cell Death 1 ligand 1), also known as surface antigen cluster of differentiation 274 (CD 274) or B7 homolog 1 (B7 homolog 1, B7-H1), is a transmembrane Protein of 40kDa in size, is one of 2 ligands of PD-1 (Programmed Cell Death Protein 1), is highly expressed in heart, skeletal muscle, placenta, lung, is lowly expressed in thymus, spleen, kidney, liver, is also expressed in immune cells such as activated T cells, B cells, dendritic cells, and is widely expressed on various tumor cells.

After being combined with PD-1, PD-L1 can transmit an immunosuppressive signal and reduce the proliferation of T cells, and an Immune checkpoint (Immune checkpoint) mechanism can be utilized by cancer cells to realize Immune escape, so that PD-1 and PD-L1 become targets for cancer immunotherapy, and a plurality of anti-PD-1 or anti-PD-L1 monoclonal antibodies enter clinical trials. In 2016, 5 months, recombinant anti-PD-L1 humanized monoclonal antibody Atezolizumab was approved by the U.S. Food and Drug Administration (FDA) for treatment of locally advanced or metastatic urothelial cancer, under the tradename Tecntriq, the first therapeutic antibody to be obtained as a target against PD-L1 that was licensed worldwide. In the same year of 10 months, the indication of Tecntriq increases metastatic non-small cell lung cancer, and the anti-PD-L1 antibody is the first approval for lung cancer treatment in the world.

In a clinical trial with 310 people attending Tecentriq for locally advanced or metastatic urothelial cancer, the Overall Response Rate (ORR) was 26.0% in patients with PD-L1 expression ≧ 5%, with a complete response rate (CR) of only 12.0%. In a clinical trial with Tecentriq for metastatic non-small cell lung cancer with 287 patients, the overall response rate was 15%, with a complete response rate of only 0.7%, and median survival of 12.6 months in patients, which was 2.9 months longer than the control group with docetaxel. Therefore, it can be seen that more than half of patients do not have obvious response to anti-PD-L1 treatment (which may be related to factors such as individual gene diversity, tumor type and variability), the improvement on the survival time of patients is limited compared with that of chemotherapeutic drugs, and the effective rate and curative effect of anti-PD-L1 treatment are still to be further improved. The discovery and screening of the anti-PD-L1 monoclonal antibody with new pharmacodynamic and pharmacokinetic characteristics, such as the monoclonal antibody with new binding epitope, binding/dissociation kinetics, tissue permeability and stability, is a way to provide diversified alternative choices for patients and improve the treatment effectiveness of the anti-PD-L1.

The antibody drug is also called Therapeutic Antibodies (Therapeutic Antibodies), belongs to targeted drugs and is generally IgG type immunoglobulin. The basic structure of an immunoglobulin of the IgG type is composed of four peptide chains, namely two identical peptide chains of smaller molecular weight, called light chains (L chains), and two identical peptide chains of larger molecular weight, called heavy chains (H chains). In most cases the H chain plays a more important role in binding to the antigen. The amino-terminal (N-terminal) amino acid sequence of H chain or L chain varies widely, and this region is called variable region (V), and V regions of L chain and H chain are called VL and VH, respectively; the carboxy terminus (C-terminus) is relatively stable and varies little, this region being termed the constant region (C), and the C regions of the L and H chains being termed CL and CH, respectively.

Certain local regions in the VL and VH, which regions have a higher degree of variation in amino acid composition and arrangement, are called hypervariable regions (HVRs). The amino acid set faces and alignment of non-HVR sites in the V region are relatively conserved, called the Framework region (Framework). Analysis by X-ray crystallography has shown that the hypervariable regions are indeed the sites where the antibody binds to the antigen and are therefore called Complementarity Determining Regions (CDRs). VL and VH each have three HVR regions, designated HVR1, HVR2 and HVR3, respectively, and may also be referred to as CDR1, CDR2 and CDR3, with CDR3 generally having a higher degree of hypervariability.

CL and CH have genetic markers of partial allotypes. The CH2 region of IgG-type immunoglobulins has a complement binding site that activates the classical pathway of complement activation, inducing CDC effects. The CH3 region has the function of binding to receptors of Fc segment of monocytes, macrophages, granulocytes, B cells and NK cells, and can induce ADCC effect. Aspartic acid (Asn 297) at position 297 of the H chain constant region has a conserved N-linked glycosylation site, and different forms of glycosylation modification have important influences on the properties of CDC effect, ADCC effect, immunogenicity, half-life and the like of the antibody.

Disclosure of Invention

In order to provide effective treatment drugs for patients with advanced or metastatic cancers, particularly to provide new drug choices for patients with Tecntriq treatment ineffectiveness or drug resistance, the invention provides a recombinant anti-PD-L1 humanized monoclonal antibody and a murine anti-PD-L1 monoclonal antibody, and the technical scheme is as follows:

the heavy chain CDR1, CDR2 and CDR3 regions of the recombinant anti-PD-L1 monoclonal antibody respectively contain the sequences of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.

The heavy chain variable region of the antibody contains a sequence of SEQ ID NO. 7 (humanized antibody) or a sequence of SEQ ID NO. 10 (murine antibody).

The light chain CDR1, CDR2 and CDR3 regions of the antibody respectively contain the sequences of SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.

The variable region of the light chain of the antibody contains a sequence of SEQ ID NO. 8 or SEQ ID NO. 9 (humanized antibody), or contains a sequence of SEQ ID NO. 11 (murine antibody).

The humanized antibody is an antibody of human IgG1 subtype.

The humanized antibody is used for manufacturing a therapeutic drug or a preventive drug for cancer and immune diseases.

The murine antibody is used for detecting cancer or immune diseases and predicting drug effect.

Specifically, the names and sequences of the humanized antibodies are shown in the following table:

TABLE 1 sequences of recombinant anti-PD-L1 humanized monoclonal antibodies T0004-BC and T0004-C

The names and sequences of the murine antibodies are shown in the following table:

TABLE 2 sequence of murine anti-PD-L1 monoclonal antibody T0004

Characterization of antibodies	Amino acid (or nucleotide) sequence	SEQ ID NO:
			Heavy chain CDR1	SAYIWH	1
Heavy chain CDR2	YIHYSTITKYNPSLKS	2
			Heavy chain CDR3	EGNDYGGFAY	3
Heavy chain variable region	DVQLQESGPDLVKPSQSLSLTCTVTGYSITSAYIWHWIRQFPGNKLEWMGYIHYSTITKYNPSLKSRFSITRDTSKNQFFLQLSSVTTEDTATYYCAREGNDYGGFAYWGQGTLVTVSA	10
			Light chain CDR1	KASQSVSNGVA	4
Light chain CDR2	YASNRYT	5
			Light chain CDR3	QQDYSSPYT	6
Light chain variable region	SIVMTQTPKFLLVSAGDRVTITCKASQSVSNGVAWYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPYTFGGGTRLEIKR	11

Surface plasmon resonance detection shows that the murine antibody T0004 has extremely high affinity to human PD-L1, and the equilibrium dissociation constant (KD value) is less than 1 pM (the detection result is 0.817 pM, which is beyond the detection range of the instrument); the humanized antibodies T0004-BC (KD value 1.008E-11M) and T0004-C (KD value 1.250E-11M) have higher affinity for human PD-L1 than Tecntriq (KD value 2.720E-11M); and T0004-BC (Kd value 4.452E-5), T0004-C (Kd value 4.852E-5) bind to human PD-L1 (Kd value 1.410E-4) with higher stability than Tecntriq.

Enzyme-linked immunosorbent assay and flow cytometry detection show that T0004-BC and T0004-C can compete with Tecntriq for binding to soluble or membrane surface human PD-L1, and the fact that the three antibodies are relatively close to binding epitopes on human PD-L1 or have a space blocking effect is proved. However, T0004-BC and T0004-C can be combined with D49Y and V111E mutants of human PD-L1 on the surface of a cell membrane, and Tecntriq can not be combined with the mutants, so that the binding epitopes of the T0004-BC and T0004-C are different, and the binding epitopes of the T0004-BC and T0004-C on human PD-L1 have certain uniqueness compared with a commercial anti-PD-L1 antibody.

In an animal experiment taking a mouse as a model, T0004-BC and T0004-C can obviously inhibit the volume increase of tumors formed by MC38 colorectal cancer cells, and the tumor inhibition effect is superior to that of a commercial anti-PD-L1 antibody.

Since murine antibody T0004 has the same CDR regions as humanized antibodies T0004-BC, T0004-C and competes with human PD-1 for binding to human PD-L1, it is presumed that they have the same binding epitope as human PD-L1. And the murine antibody T0004 has extremely high affinity to human PD-L1, and is expected to be used for cancer or immune disease detection and drug effect prediction.

In conclusion, the invention provides the recombinant anti-PD-L1 humanized monoclonal antibody which has better affinity to human PD-L1, unique binding epitope and better tumor inhibition effect, and the murine antibody which has extremely high affinity to human PD-L1 and can be used for cancer or immune disease detection and drug effect prediction.

Drawings

FIG. 1: the binding capacity of the murine antibody T0004 antibody to cell membrane surface PD-L1 was tested by flow cytometry.

FIG. 2: the enzyme-linked immunosorbent assay detects the binding capacity of the murine antibody T0004 antibody and soluble PD-L1.

FIG. 3: the surface plasmon resonance detects the affinity of the murine antibody T0004 with human PD-L1.

FIG. 4: the enzyme-linked immunoassay detects the condition that the murine antibody T0004 competes with PD-1 to bind to PD-L1.

FIG. 5: the enzyme-linked immunoassay detects the condition that the murine antibody T0004 competes with Tecntriq for binding to PD-L1.

FIG. 6: murine antibody T0004 heavy chain variable region A, B, C, D four humanization schemes.

FIG. 7: murine antibody T0004 light chain variable region A, B, C, D four humanization protocols.

FIG. 8: differential scanning calorimetry measured the thermostability of the humanized antibody T0004-BC.

FIG. 9: differential scanning calorimetry measured the thermostability of the humanized antibody T0004-C.

FIG. 10: flow cytometry detection of binding of humanized antibody T0004-BC to membrane PD-L1.

FIG. 11: flow cytometry detection of binding of humanized antibody T0004-C to membrane PD-L1.

FIG. 12: results of flow cytometry detection of the binding of commercial antibody Tecentriq (control) to membrane PD-L1.

FIG. 13: and (3) detecting the binding capacity of T0004-BC and T0004-C and soluble PD-L1 by an enzyme-linked immunosorbent assay.

FIG. 14: surface plasmon resonance detects the affinity of the humanized antibody T0004-BC to human PD-L1.

FIG. 15: surface plasmon resonance detects the affinity of the humanized antibody T0004-C with human PD-L1.

FIG. 16: surface Plasmon Resonance (SPR) detects the affinity of the commercial antibody Tecntriq (control) to human PD-L1.

FIG. 17: flow cytometry was used to detect the ability of T0004-BC to compete with Tecntriq for binding to cell membrane surface PD-L1.

FIG. 18: flow cytometry was used to detect the ability of T0004-C to compete with Tecntriq for binding to cell membrane surface PD-L1.

FIG. 19: flow cytometry tested Tecentriq (control) for its ability to compete with Tecentriq for binding to cell membrane surface PD-L1.

FIG. 20: the enzyme-linked immunosorbent assay detects the capacity of T0004-BC and T0004-C to compete with Tecntriq for binding to soluble PD-L1.

FIG. 21: flow cytometry was used to detect the ability of T0004-BC to compete with PD-1 for binding to PD-L1 on the cell membrane surface.

FIG. 22: flow cytometry was used to detect the ability of T0004-C to compete with PD-1 for binding to PD-L1 on the cell membrane surface.

FIG. 23: flow cytometry tested the ability of Tecentriq (control) to compete with PD-1 for binding to cell membrane surface PD-L1.

FIG. 24: and (3) detecting the capacity of competitive binding of T0004-BC and T0004-C and PD-1 to soluble PD-L1 by an enzyme-linked immunosorbent assay.

FIG. 25: reporter gene assay detects the biological activity of humanized antibody T0004-BC activated TCR.

FIG. 26: reporter gene assay detects the biological activity of humanized antibody T0004-C to activate TCR.

FIG. 27 is a schematic view showing: reporter gene assay measures the biological activity of Tecentriq (control) activated TCR.

FIG. 28: the reporter gene method detects the capacity of the humanized antibody T0004-BC to induce ADCC effect.

FIG. 29: the reporter gene method detects the capacity of the humanized antibody T0004-C to induce ADCC effect.

FIG. 30: the reporter gene method measures the ability of Tecentriq (control) to induce ADCC effects.

FIG. 31: the lactate dehydrogenase method is used for detecting the CDC effect inducing ability of the humanized antibodies T0004-BC and T0004-C.

FIG. 32: the binding capacity of T0004-BC and T0004-C to the human PD-L1 mutant strain was examined by flow cytometry.

FIG. 33: study of tumor suppressor ability of humanized antibodies T0004-BC and T0004-C in animals.

Detailed Description